heparitin-sulfate and Disease-Models--Animal

Topics: Aged, 80 and over; Aging; Air Pollution; Animals; Cadmium; COVID-19; Cytokine Release Syndrome; Disease Models, Animal; Disease Susceptibility; Endothelium, Vascular; Environmental Pollutants; Female; Gene Expression Regulation, Enzymologic; Glucuronidase; Glycosaminoglycans; Heparitin Sulfate; Humans; Lung; Male; Prognosis; Pulmonary Embolism; SARS-CoV-2; Venous Thromboembolism; Venous Thrombosis

Sanfilippo syndrome or mucopolysaccharidosis III is a lysosomal storage disorder caused by mutations in genes responsible for the degradation of heparan sulfate, a glycosaminoglycan located in the extracellular membrane. Undegraded heparan sulfate molecules accumulate within lysosomes leading to cellular dysfunction and pathology in several organs, with severe central nervous system degeneration as the main phenotypical feature. The exact molecular and cellular mechanisms by which impaired degradation and storage lead to cellular dysfunction and neuronal degeneration are still not fully understood. Here, we compile the knowledge on this issue and review all available animal and cellular models that can be used to contribute to increase our understanding of Sanfilippo syndrome disease mechanisms. Moreover, we provide an update in advances regarding the different and most successful therapeutic approaches that are currently under study to treat Sanfilippo syndrome patients and discuss the potential of new tools such as induced pluripotent stem cells to be used for disease modeling and therapy development.

Topics: Acetyltransferases; Animals; Disease Models, Animal; Enzyme Replacement Therapy; Genetic Therapy; Heparitin Sulfate; Humans; Hydrolases; Mucopolysaccharidosis III; Mutation; Stem Cell Transplantation

Heparan sulfate (HS) is an essential component of cell surface and matrix proteoglycans (HS-PGs) that include syndecans and perlecan. Because of their unique structural features, the HS chains are able to specifically interact with signaling proteins -including bone morphogenetic proteins (BMPs)- via their HS-binding domain, regulating protein availability, distribution and action on target cells. Hereditary Multiple Exostoses (HME) is a rare pediatric disorder linked to germline heterozygous loss-of-function mutations in EXT1 or EXT2 that encode Golgi-resident glycosyltransferases responsible for HS synthesis, resulting in a systemic HS deficiency. HME is characterized by cartilaginous/bony tumors -called osteochondromas or exostoses- that form within perichondrium in long bones, ribs and other elements. This review examines most recent studies in HME, framing them in the context of classic studies. New findings show that the spectrum of EXT mutations is larger than previously realized and the clinical complications of HME extend beyond the skeleton. Osteochondroma development requires a somatic "second hit" that would complement the germline EXT mutation to further decrease HS production and/levels at perichondrial sites of osteochondroma induction. Cellular studies have shown that the steep decreases in local HS levels: derange the normal homeostatic signaling pathways keeping perichondrium mesenchymal; cause excessive BMP signaling; and provoke ectopic chondrogenesis and osteochondroma formation. Data from HME mouse models have revealed that systemic treatment with a BMP signaling antagonist markedly reduces osteochondroma formation. In sum, recent studies have provided major new insights into the molecular and cellular pathogenesis of HME and the roles played by HS deficiency. These new insights have led to the first ever proof-of-principle demonstration that osteochondroma formation is a druggable process, paving the way toward the creation of a clinically-relevant treatment.

Topics: Animals; Bone Morphogenetic Proteins; Disease Models, Animal; Exostoses, Multiple Hereditary; Heparitin Sulfate; Humans; Mice; Mutation; N-Acetylglucosaminyltransferases; Signal Transduction

A number of neurodegenerative disorders have been linked directly to the accumulation of amyloid fibres. These fibres are made up of proteins or peptides with altered structures and which join together in vivo in association with heparan sulphate-type polysaccharides.. To examine the most recent concepts in the biology of heparan sulphates and their role in the aggregation of the peptide Abeta, of tau protein, of alpha-synuclein and of prions. The study also seeks to analyse their implications in neurodegenerative disorders such as Alzheimer's and Parkinson's disease and prion diseases.. In vitro, heparan sulphates have played an important role in the process of oligomerisation and fibrillation of amyloidogenic proteins or peptides, in the stabilisation of these bodies and their resistance to proteolysis, thereby participating in the formation of a wide range of amyloid fibres. Heparan sulphates have also been related to the internalisation of pro-amyloid fibres during the process of intercellular propagation (spreading), which is considered to be crucial in the development of proteinopathies, the best example of which is Alzheimer's disease.. This study suggests that the fine structures of heparan sulphates, their localisation in cells and tissues, together with their local concentration, may regulate the amyloidosis processes. The advances made in the understanding of this area of glyconeurobiology will make it possible to improve the understanding of the cell and molecular mechanisms underlying the neurodegenerative process.. Heparan sulfatos, amiloidosis y neurodegeneracion.. Introduccion. Numerosos trastornos neurodegenerativos se han asociado directamente a la acumulacion de fibras amiloides. Estas fibras estan formadas por proteinas o peptidos con conformaciones alteradas y que se agregan in vivo en asociacion con polisacaridos de tipo heparan sulfatos. Objetivos. Examinar los conceptos mas recientes sobre la biologia de los heparan sulfatos y su papel en la agregacion del peptido Abeta, de la proteina tau, de la alfa-sinucleina y de los priones, y analizar sus implicaciones en trastornos neurodegenerativos como las enfermedades de Alzheimer y de Parkinson y las enfermedades prionicas. Desarrollo. In vitro, los heparan sulfatos han desempeñado un papel importante en el proceso de oligomerizacion y fibrilacion de proteinas o peptidos amiloidogenos, en la estabilizacion de estos cuerpos y su resistencia a la proteolisis, participando asi en la formacion de una gran variedad de fibras amiloides. Los heparan sulfatos se han relacionado tambien con el proceso de internalizacion de fibras proamiloides durante el proceso de propagacion intercelular (spreading) considerado como central en la evolucion de las proteinopatias, cuyo mejor ejemplo es la enfermedad de Alzheimer. Conclusion. Este trabajo sugiere que las estructuras finas de los heparan sulfatos, sus localizaciones celulares y tisulares, asi como sus concentraciones locales, pueden regular los procesos de amiloidosis. Avances en la comprension de esta area de la gliconeurobiologia permitiran mejorar la comprension de los mecanismos celulares y moleculares del proceso neurodegenerativo.

Topics: Alzheimer Disease; Amyloidosis; Animals; Disease Models, Animal; Heparitin Sulfate; Humans; Neurodegenerative Diseases; Parkinson Disease; Prion Diseases

Heparan sulfate (HS) proteoglycans (PGs) are ubiquitously expressed on cell surfaces and in the extracellular matrix of most animal tissues, having essential functions in development and homeostasis, as well as playing various roles in disease processes. The functions of HSPGs are mainly dependent on interactions between the HS-side chains with a variety of proteins including cytokines, growth factors, and their receptors. In a given HS polysaccharide, negatively charged sulfate and carboxylate groups are arranged in various types of domains, generated through strictly regulated biosynthetic reactions and with enormous potential for structural variability. The mode of HS-protein interactions is assessed through binding experiments using saccharides of defined composition in vitro, signaling assays in cell models where HS structures are manipulated, and targeted disruption of genes for biosynthetic enzymes in animals (mouse, zebrafish, Drosophila, and Caenorhabditis elegans) followed by phenotype analysis. Whereas some protein ligands appear to require strictly defined HS structure, others bind to variable saccharide domains without apparent dependence on distinct saccharide sequence. These findings raise intriguing questions concerning the functional significance of regulation in HS biosynthesis and the potential for development of therapeutics targeting HS-protein interactions.

Topics: Animals; Disease; Disease Models, Animal; Heparitin Sulfate; Humans; Models, Molecular; Protein Binding; Proteins

Frequent benign outgrowths from bone known as osteochondromas, exhibiting typical endochondral ossification, are reported from single to multiple lesions. Characterised by a high incidence of osteochondromas and skeletal deformities, multiple hereditary exostoses (MHE) is the most common inherited musculoskeletal condition. While factors for severity remain unknown, mutations in exostosin 1 and exostosin 2 genes, encoding glycosyltransferases involved in the biosynthesis of ubiquitously expressed heparan sulphate (HS) chains, are associated with MHE. HS-binding bone morphogenetic proteins (BMPs) are multifunctional proteins involved in the morphogenesis of bone and cartilage. HS and HS proteoglycans are involved in BMP-mediated morphogenesis by regulating their gradient formation and activity. Mutations in exostosin genes disturb HS biosynthesis, subsequently affecting its functional role in the regulation of signalling pathways. As BMPs are the primordial morphogens for bone development, we propose the hypothesis that BMP signalling may be critical in osteochondromas. For this reason, the outcomes of exostosin mutations on HS biosynthesis and interactions within osteochondromas and MHE are reviewed. Since BMPs are HS binding proteins, the interactions of HS with the BMP signalling pathway are discussed. The impact of mouse models in the quest to better understand the cell biology of osteochondromas is discussed. Several challenges and questions still remain and further investigations are needed to explore new approaches for better understanding of the pathogenesis of osteochondromas.

Topics: Animals; Bone Morphogenetic Proteins; Bone Neoplasms; Disease Models, Animal; Heparitin Sulfate; Humans; Mice; Mutation; N-Acetylglucosaminyltransferases; Osteochondroma; Signal Transduction

Heparan sulfates (HSs) are N- and O-sulfated polysaccharide components of proteoglycans, which are important constituents of the cell surface as well as the extracellular matrix. Heparin, with extensive clinical application as an anticoagulant, is a highly sulfated form of HS present within the granules of connective tissue type mast cells. The diverse functions of HS, which include the modulation of growth factor/cytokine activity, interaction with matrix proteins and binding of enzymes to cell surfaces, depend greatly on the presence of specific, high affinity regions on the chains. N-acetylglucosamine N-deacetylase/N-sulfotransferases, NDSTs, are an important group of enzymes in HS biosynthesis, initiating the sulfation of the polysaccharide chains and thus determining the generation of the high affinity sites. Here, we review the role of the four vertebrate NDSTs in HS biosynthesis as well as their regulated expression. The main emphasis is the phenotypes of mice lacking one or more of the NDSTs.

Topics: Amidohydrolases; Animals; Carbohydrate Sequence; Disease Models, Animal; Gene Expression Regulation, Enzymologic; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Isoenzymes; Mice; Protein Biosynthesis; Sulfotransferases; Transcription, Genetic

Heparin-induced thrombocytopenia (HIT), a drug-induced immunohaematological adverse reaction, is a rare but potentially very severe condition. The main problem for this complex syndrome is its recognition and management, which should be as early as possible to avoid the development of life-threatening complications. Most studies have reported heterogeneous populations of patients with other diseases that potentially induce thrombocytopenia. There is no gold standard diagnostic criteria, and we have established a score with anamnestic criteria that allows us to evaluate the likelihood of HIT. In clinical practice, the diagnosis is based on the analysis of clinical features and laboratory tests. Platelet aggregation test (PAT) and an ELISA test (heparin platelet-induced antibodies) are generally performed by expert laboratories to confirm the occurrence of HIT. In our experience, both tests are concordant in the majority of patients. PAT seems to correlate better with the clinical features while ELISA appears more specific. Regarding their limits, both are complementary in the determination of HIT diagnosis coupled to the clinical score system. The treatment often requires a multidisciplinary approach. Danaparoid (Orgaran) or lepirudin (Refludan) are the two alternative treatments for HIT patients with marketing approval. To avoid further exposure to heparin, every HIT patient should carry a written document that confirms the immunoallergy.

Topics: Animals; Anticoagulants; Chondroitin Sulfates; Dermatan Sulfate; Disease Models, Animal; Drug Combinations; Fibrinolytic Agents; Heparin; Heparitin Sulfate; Hirudin Therapy; Hirudins; Humans; Immunoassay; Platelet Activation; Recombinant Proteins; Thrombocytopenia

Beckwith-Wiedemann syndrome (BWS), a human overgrowth syndrome with a complex genetic basis, is caused by alterations to chromosome 11p15, a region subject to genomic imprinting. These alterations include translocations, duplications, single gene mutations of p57KIP2, and increased expression of insulin-like growth factor 2 (IGF2). A phenotypically related X-linked overgrowth syndrome, Simpson Golabi Behmel syndrome (SGBS), is caused by alterations in glypican-3 (GPC3), a molecule that may interact with the gene products identified to be important in generating the BWS phenotype, that is, IGF2 and p57KIP2. The crucial defect in these overgrowth syndromes is likely to be an imbalance in contributions of growth-promoting genes versus growth-inhibitory genes in critical tissues at specific developmental stages. Murine models have been used to study the effects of targeted deletions of the genes p57KIP2 and GPC3, as well as overexpression of IGF2. At this time, there are still many issues which remain to be explored before we can fully understand the molecular basis of BWS and SGBS.

Topics: Animals; Beckwith-Wiedemann Syndrome; Chromosome Mapping; Chromosomes, Human, Pair 11; Cyclin-Dependent Kinase Inhibitor p57; Disease Models, Animal; Genomic Imprinting; Glypicans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Mice; Nuclear Proteins; Proteoglycans

Topics: Animals; Antibodies, Antinuclear; Autoantibodies; Disease Models, Animal; DNA Topoisomerases, Type I; Heparitin Sulfate; Humans; Mice; Mice, Inbred Strains; Receptors, Fc; RNA Polymerase I; Scleroderma, Systemic

We have described the age-related deposition of fibrillar material in brains of B6 mice and SAM. Since in other inbred strains similar deposits were absent or occurred only occasionally and only in aged individuals, a genetic predisposition of B6 mice and SAM to accumulate the fibrillar material is suggested. The deposits are mostly associated with astrocytic processes and have been referred to as astrocytic inclusions. HSPG- and laminin-like molecules have been identified as components of the fibrillar material. The deposits have similarities with CA in humans, but they also show some important differences; thus there is presently insufficient evidence to consider the deposits the murine equivalent of CA. Although the physiological significance of the fibrillar material is not yet clear, the awareness of the deposits appears pertinent because they might contribute to various aspects of CNS function of susceptible strains of mice, and therefore could lead to possible misinterpretations of the results of studies employing these strains. Future directions of our research will determine the potential of the murine deposits to model aspects of human neuropathology, in particular, whether the deposits may mimic the deposition of ECM molecules as an early-event in the pathogenesis of amyloid plaque formation.

Topics: Aging; Animals; Biomarkers; Brain Diseases; Central Nervous System Diseases; Disease Models, Animal; Female; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Laminin; Male; Mice; Mice, Inbred Strains; Neurofibrils; Proteoglycans; Species Specificity

Sanfilippo disease, caused by mutations in the genes encoding heparan sulfate (HS) (a glycosaminoglycan; GAG) degradation enzymes, is a mucopolysaccharidosis (MPS), which is also known as MPS type III, and is characterized by subtypes A, B, C, and D, depending on identity of the dysfunctional enzyme. The lack of activity or low residual activity of an HS-degrading enzyme leads to excess HS in the cells, impairing the functions of different types of cells, including neurons. The disease usually leads to serious psychomotor dysfunction and death before adulthood. In this work, we show that the use of molecules known as dietary (poly)phenolic antioxidants and other natural compounds known as autophagy activators (genistein, capsaicin, curcumin, resveratrol, trehalose, and calcitriol) leads to accelerated degradation of accumulated HS in the fibroblasts of all subtypes of MPS III. Both the cytotoxicity tests we performed and the available literature data indicated that the use of selected autophagy inducers was safe. Since it showed the highest effectivity in cellular models, resveratrol efficacy was tested in experiments with a mouse model of MPS IIIB. Urinary GAG levels were normalized in MPS IIIB mice treated with 50 mg/kg/day resveratrol for 12 weeks or longer. Behavioral tests indicated complete correction of hyperactivity and anxiety in these animals. Biochemical analyses indicated that administration of resveratrol caused autophagy stimulation through an mTOR-independent pathway in the brains and livers of the MPS IIIB mice. These results indicate the potential use of resveratrol (and possibly other autophagy stimulators) in the treatment of Sanfilippo disease.

Topics: Animals; Antioxidants; Autophagy; Disease Models, Animal; Heparitin Sulfate; Mice; Mucopolysaccharidosis III; Phenols; Resveratrol

Sepsis is a lethal syndrome manifested by an unregulated, overwhelming inflammation from the host in response to infection. Here, we exploit the use of a synthetic heparan sulfate octadecasaccharide (18-mer) to protect against sepsis. The 18-mer not only inhibits the pro-inflammatory activity of extracellular histone H3 and high mobility group box 1 (HMGB1), but also elicits the anti-inflammatory effect from apolipoprotein A-I (ApoA-I). We demonstrate that the 18-mer protects against sepsis-related injury and improves survival in cecal ligation and puncture mice and reduces inflammation in an endotoxemia mouse model. The 18-mer neutralizes the cytotoxic histone-3 (H3) through direct interaction with the protein. Furthermore, the 18-mer enlists the actions of ApoA-I to dissociate the complex of HMGB1 and lipopolysaccharide, a toxic complex contributing to cell death and tissue damage in sepsis. Our study provides strong evidence that the 18-mer mitigates inflammatory damage in sepsis by targeting numerous mediators, setting it apart from other potential therapies with a single target.

Topics: Animals; Apolipoprotein A-I; Disease Models, Animal; Endotoxemia; Heparitin Sulfate; HMGB1 Protein; Lipopolysaccharides; Mice; Sepsis

There is an urgent need for therapies that target the multicellular pathology of central nervous system (CNS) disease. Modified, nonanticoagulant heparins mimic the heparan sulfate glycan family and are known regulators of multiple cellular processes. In vitro studies have demonstrated that low sulfated modified heparin mimetics (LS-mHeps) drive repair after CNS demyelination. Herein, we test LS-mHep7 (an in vitro lead compound) in experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination. In EAE, LS-mHep7 treatment resulted in faster recovery and rapidly reduced inflammation which was accompanied by restoration of animal weight. LS-mHep7 treatment had no effect on remyelination or on OLIG2 positive oligodendrocyte numbers within the corpus callosum in the cuprizone model. Further in vitro investigation confirmed that LS-mHep7 likely mediates its pro-repair effect in the EAE model by sequestering inflammatory cytokines, such as CCL5 which are upregulated during immune-mediated inflammatory attacks. These data support the future clinical translation of this next generation modified heparin as a treatment for CNS diseases with active immune system involvement.

Topics: Animals; Central Nervous System Diseases; Corpus Callosum; Cuprizone; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Heparitin Sulfate; Mice; Mice, Inbred C57BL; Myelin Sheath; Oligodendroglia; Sulfates

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder characterized by an accumulation of glycosaminoglycans (GAGs), including heparan sulfate, in the body. Major manifestations involve the central nerve system (CNS), skeletal deformation, and visceral manifestations. About 30% of MPS II is linked with an attenuated type of disease subtype with visceral involvement. In contrast, 70% of MPS II is associated with a severe type of disease subtype with CNS manifestations that are caused by the human iduronate-2-sulfatase (IDS)-Pro86Leu (P86L) mutation, a common missense mutation in MPS II. In this study, we reported a novel Ids-P88L MPS II mouse model, an analogous mutation to human IDS-P86L. In this mouse model, a significant impairment of IDS enzyme activity in the blood with a short lifespan was observed. Consistently, the IDS enzyme activity of the body, as assessed in the liver, kidney, spleen, lung, and heart, was significantly impaired. Conversely, the level of GAG was elevated in the body. A putative biomarker with unestablished nature termed UA-HNAc(1S) (late retention time), one of two UA-HNAc(1S) species with late retention time on reversed-phase separation,is a recently reported MPS II-specific biomarker derived from heparan sulfate with uncharacterized mechanism. Thus, we asked whether this biomarker might be elevated in our mouse model. We found a significant accumulation of this biomarker in the liver, suggesting that hepatic formation could be predominant. Finally, to examine whether gene therapy could enhance IDS enzyme activity in this model, the efficacy of the nuclease-mediated genome correction system was tested. We found a marginal elevation of IDS enzyme activity in the treated group, raising the possibility that the effect of gene correction could be assessed in this mouse model. In conclusion, we established a novel Ids-P88L MPS II mouse model that consistently recapitulates the previously reported phenotype in several mouse models.

Topics: Animals; Biomarkers; Disease Models, Animal; Heparitin Sulfate; Humans; Iduronate Sulfatase; Iduronic Acid; Mice; Mucopolysaccharidosis II; Mutation

Mucopolysaccharidosis IIIC (MPSIIIC) is one of four Sanfilippo diseases sharing clinical symptoms of severe cognitive decline and shortened lifespan. The missing enzyme, heparan sulfate acetyl-CoA: α-glucosaminide-N-acetyltransferase (HGSNAT), is bound to the lysosomal membrane, therefore cannot cross the blood-brain barrier or diffuse between cells. We previously demonstrated disease correction in MPSIIIC mice using an Adeno-Associated Vector (AAV) delivering HGSNAT via intraparenchymal brain injections using an AAV2 derived AAV-truetype (AAV-TT) serotype with improved distribution over AAV9.. Here, intraparenchymal AAV was delivered in sheep using catheters or Hamilton syringes, placed using Brainlab cranial navigation for convection enhanced delivery, to reduce proximal vector expression and improve spread.. Hamilton syringes gave improved AAV-GFP distribution, despite lower vector doses and titres. AAV-TT-GFP displayed moderately better transduction compared to AAV9-GFP but both serotypes almost exclusively transduced neurons. Functional HGSNAT enzyme was detected in 24-37% of a 140g gyrencephalic sheep brain using AAV9-HGSNAT with three injections in one hemisphere.. Despite variabilities in volume and titre, catheter design may be critical for efficient brain delivery. These data help inform a clinical trial for MPSIIIC.

Topics: Acetyltransferases; Animals; Brain; Dependovirus; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Heparitin Sulfate; Mucopolysaccharidoses; Mucopolysaccharidosis III; Sheep

Mucopolysaccharidosis type IIIB (MPS IIIB) is a rare and devastating childhood-onset lysosomal storage disease caused by complete loss of function of the lysosomal hydrolase α-N-acetylglucosaminidase. The lack of functional enzyme in MPS IIIB patients leads to the progressive accumulation of heparan sulfate throughout the body and triggers a cascade of neuroinflammatory and other biochemical processes ultimately resulting in severe mental impairment and early death in adolescence or young adulthood. The low prevalence and severity of the disease has necessitated the use of animal models to improve our knowledge of the pathophysiology and for the development of therapeutic treatments. In this study, we took a systematic approach to characterizing a classical mouse model of MPS IIIB. Using a series of histological, biochemical, proteomic and behavioral assays, we tested MPS IIIB mice at two stages: during the pre-symptomatic and early symptomatic phases of disease development, in order to validate previously described phenotypes, explore new mechanisms of disease pathology and uncover biomarkers for MPS IIIB. Along with previous findings, this study helps provide a deeper understanding of the pathology landscape of this rare disease with high unmet medical need and serves as an important resource to the scientific community.

Topics: Acetylglucosaminidase; Adult; Animals; Child; Disease Models, Animal; Heparitin Sulfate; Humans; Hydrolases; Mice; Mucopolysaccharidosis III; Proteomics; Young Adult

Heparan sulfate proteoglycans (HSPGs) are at the forefront of host-microbe interactions. Cell surface HSPGs are thought to promote infection as attachment and internalization receptors for many bacterial pathogens and as soluble inhibitors of host immunity when released from the cell surface by ectodomain shedding. However, the importance of HSPG-pathogen interactions in vivo has yet to be clearly established. Here we describe several representative methods to study the role of HSPGs in systemic bacterial infections, such as bacteremia and sepsis. The overall experimental strategy is to use mouse models to establish the physiological significance of HSPGs, to determine the identity of HSPGs that specifically promote infection, and to define key structural features of HSPGs that enhance bacterial virulence in systemic infections.

Topics: Animals; Bacterial Infections; Cell Membrane; Disease Models, Animal; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Mice; Sepsis

Evaluating prospective anticoagulant therapies in animal thrombosis and bleeding models are standard pre-clinical approaches. Mice are frequently used for initial evaluations because a variety of models have been developed in this well-characterized species, and mice are relatively inexpensive to maintain. Because mice seem to be resistant to forming "spontaneous" thrombosis, vessel injury is used to induce intravascular clot formation. For the purpose of testing heparin-based drugs, we adapted a well-established model in which thrombus formation in the carotid artery is induced by exposing the vessel to ferric chloride. For studying anticoagulant effects on venous thrombosis, we use a model in which the inferior vena cava is ligated and the size of the resulting clots are measured. The most common adverse effect of anticoagulation therapy is bleeding. We describe a simple tail bleeding time that has been used for many years to study the effects of anticoagulants on hemostasis. We also describe a more reproducible, but more technically challenging, saphenous vein bleeding model that is also used for this purpose.

Topics: Animals; Anticoagulants; Disease Models, Animal; Hemorrhage; Heparitin Sulfate; Mice; Prospective Studies; Thrombosis

The emergence of new epidemic variants of alphaviruses poses a public health risk. It is associated with adaptive mutations that often cause increased pathogenicity. Getah virus (GETV), a neglected and re-emerging mosquito-borne alphavirus, poses threat to many domestic animals and probably even humans. At present, the underlying mechanisms of GETV pathogenesis are not well defined. We identified a residue in the E2 glycoprotein that is critical for viral adsorption to cultured cells and pathogenesis

Topics: Alphavirus; Alphavirus Infections; Amino Acid Substitution; Animals; Binding Sites; Cells, Cultured; Disease Models, Animal; Heparitin Sulfate; Humans; Mice; Viral Envelope Proteins

Hereditary multiple exostoses (HME) is a rare, pediatric disorder characterized by osteochondromas that form along growth plates and provoke significant musculoskeletal problems. HME is caused by mutations in heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. Seemingly paradoxically, osteochondromas were found to contain excessive extracellular heparanase (Hpse) that could further reduce HS levels and exacerbate pathogenesis. To test Hpse roles, we asked whether its ablation would protect against osteochondroma formation in a conditional HME model consisting of mice bearing floxed Ext1 alleles in Agr-CreER background (Ext1

Topics: Animals; Bone Neoplasms; Child; Disease Models, Animal; Exostoses, Multiple Hereditary; Glucuronidase; Heparitin Sulfate; Humans; Mice; Mutation; N-Acetylglucosaminyltransferases; Osteochondroma; Retinoids; Tamoxifen; X-Ray Microtomography

Mucopolysaccharidosis type IIIA (MPS-IIIA) is an autosomal recessive disorder caused by mutations in SGSH involved in the degradation of heparan sulfate. MPS-IIIA presents severe neurological symptoms such as progressive developmental delay and cognitive decline, for which there is currently no treatment. Brain targeting represents the main challenge for therapeutics to treat MPS-IIIA, and the development of small-molecule-based treatments able to reach the CNS could be a relevant advance for therapy. Using cell-based high content imaging to survey clinically approved drugs in MPS-IIIA cells, we identified fluoxetine, a selective serotonin reuptake inhibitor. Fluoxetine increases lysosomal and autophagic functions via TFEB activation through a RagC-dependent mechanism. Mechanistically, fluoxetine increases lysosomal exocytosis in mouse embryonic fibroblasts from MPS-IIIA mice, suggesting that this process may be responsible for heparan sulfate clearance. In vivo, fluoxetine ameliorates somatic and brain pathology in a mouse model of MPS-IIIA by decreasing the accumulation of glycosaminoglycans and aggregated autophagic substrates, reducing inflammation, and slowing down cognitive deterioration. We repurposed fluoxetine for potential therapeutics to treat human MPS-IIIA disease.

Topics: Animals; Disease Models, Animal; Fibroblasts; Fluoxetine; Heparitin Sulfate; Hydrolases; Mice; Mucopolysaccharidosis III

Alzheimer's disease (AD) is a neurodegenerative disease characterized by memory loss and cognitive impairments. Amyloid-β (Aβ) deposition and neurotoxicity play important roles in AD. It has been widely reported that heparan sulfate (HS) proteoglycans play a nonnegligible role in the release, uptake and misfolding of Aβ, resulting in the discovery of HS as a therapeutic drug for AD. In this manuscript, HS from porcine mucosa could promote Aβ fibrosis and improve the cognitive defects of APPswe/PS1ΔE9 mice. Furthermore, HS enhanced the phagocytosis of neutrophils to clear Aβ

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Disease Models, Animal; Heparitin Sulfate; Mice; Mice, Transgenic; Mucous Membrane; Neurodegenerative Diseases; Swine

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B; OMIM #252920) is a lethal, pediatric, neuropathic, autosomal recessive, and lysosomal storage disease with no approved therapy. Patients are deficient in the activity of N-acetyl-alpha-glucosaminidase (NAGLU; EC 3.2.150), necessary for normal lysosomal degradation of the glycosaminoglycan heparan sulfate (HS). Tralesinidase alfa (TA), a fusion protein comprised of recombinant human NAGLU and a modified human insulin-like growth factor 2, is in development as an enzyme replacement therapy that is administered via intracerebroventricular (ICV) infusion, thus circumventing the blood brain barrier. Previous studies have confirmed ICV infusion results in widespread distribution of TA throughout the brains of mice and nonhuman primates. We assessed the long-term tolerability, pharmacology, and clinical efficacy of TA in a canine model of MPS IIIB over a 20-month study. Long-term administration of TA was well tolerated as compared with administration of vehicle. TA was widely distributed across brain regions, which was confirmed in a follow-up 8-week pharmacokinetic/pharmacodynamic study. MPS IIIB dogs treated for up to 20 months had near-normal levels of HS and nonreducing ends of HS in cerebrospinal fluid and central nervous system (CNS) tissues. TA-treated MPS IIIB dogs performed better on cognitive tests and had improved CNS pathology and decreased cerebellar volume loss relative to vehicle-treated MPS IIIB dogs. These findings demonstrate the ability of TA to prevent or limit the biochemical, pathologic, and cognitive manifestations of canine MPS IIIB disease, thus providing support of its potential long-term tolerability and efficacy in MPS IIIB subjects. SIGNIFICANCE STATEMENT: This work illustrates the efficacy and tolerability of tralesinidase alfa as a potential therapeutic for patients with mucopolysaccharidosis type IIIB (MPS IIIB) by documenting that administration to the central nervous system of MPS IIIB dogs prevents the accumulation of disease-associated glycosaminoglycans in lysosomes, hepatomegaly, cerebellar atrophy, and cognitive decline.

Topics: Animals; Brain; Child; Disease Models, Animal; Dogs; Enzyme Replacement Therapy; Glycosaminoglycans; Heparitin Sulfate; Humans; Mucopolysaccharidosis III

Mucopolysaccharidosis type II (MPS II) is a neurometabolic disorder, due to the deficit of the lysosomal hydrolase iduronate 2-sulfatase (IDS). This leads to a severe clinical condition caused by a multi-organ accumulation of the glycosaminoglycans (GAGs/GAG) heparan- and dermatan-sulfate, whose elevated levels can be detected in body fluids. Since 2006, enzyme replacement therapy (ERT) has been clinically applied, showing efficacy in some peripheral districts. In addition to clinical monitoring, GAG dosage has been commonly used to evaluate ERT efficacy. However, a strict long-term monitoring of GAG content and composition in body fluids has been rarely performed. Here, we report the characterization of plasma and urine GAGs in Ids knock-out (Ids-ko) compared to wild-type (WT) mice, and their changes along a 24-week follow-up, with and without ERT. The concentration of heparan-sulfate (HS), chondroitin-sulfate (CS), and dermatan-sulfate (DS), and of the non-sulfated hyaluronic acid (HA), together with their differentially sulfated species, was quantified by capillary electrophoresis with laser-induced fluorescence. In untreated Ids-ko mice, HS and CS + DS were noticeably increased at all time points, while during ERT follow-up, a substantial decrease was evidenced for HS and, to a minor extent, for CS + DS. Moreover, several structural parameters were altered in untreated ko mice and reduced after ERT, however without reaching physiological values. Among these, disaccharide B and HS 2s disaccharide showed to be the most interesting candidates as biomarkers for MPS II. GAG chemical signature here defined provides potential biomarkers useful for an early diagnosis of MPS II, a more accurate follow-up of ERT, and efficacy evaluations of newly proposed therapies. KEY MESSAGES : Plasmatic and urinary GAGs are useful markers for MPS II early diagnosis and prognosis. CE-LIF allows GAG structural analysis and the quantification of 17 different disaccharides. Most GAG species increase and many structural features are altered in MPS II mouse model. GAG alterations tend to restore to wild-type levels following ERT administration. CS+DS/HS ratio, % 2,4dis CS+DS, and % HS 2s are potential markers for MPS II pathology and ERT efficacy.

Topics: Animals; Biomarkers; Body Fluids; Dermatan Sulfate; Disaccharides; Disease Models, Animal; Enzyme Replacement Therapy; Glycosaminoglycans; Heparitin Sulfate; Mice; Mice, Knockout; Mucopolysaccharidosis II

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by N-sulfoglucosamine sulfohydrolase (SGSH) deficiency. SGSH removes the sulfate from N-sulfoglucosamine residues on the nonreducing end of heparan sulfate (HS-NRE) within lysosomes. Enzyme deficiency results in accumulation of partially degraded HS within lysosomes throughout the body, leading to a progressive severe neurological disease. Enzyme replacement therapy has been proposed, but further evaluation of the treatment strategy is needed. Here, we used Chinese hamster ovary cells to produce a highly soluble and fully active recombinant human sulfamidase (rhSGSH). We discovered that rhSGSH utilizes both the CI-MPR and LRP1 receptors for uptake into patient fibroblasts. A single intracerebroventricular (ICV) injection of rhSGSH in MPS IIIA mice resulted in a tissue half-life of 9 days and widespread distribution throughout the brain. Following a single ICV dose, both total HS and the MPS IIIA disease-specific HS-NRE were dramatically reduced, reaching a nadir 2 weeks post dose. The durability of effect for reduction of both substrate and protein markers of lysosomal dysfunction and a neuroimmune response lasted through the 56 days tested. Furthermore, seven weekly 148 μg doses ICV reduced those markers to near normal and produced a 99.5% reduction in HS-NRE levels. A pilot study utilizing every other week dosing in two animals supports further evaluation of less frequent dosing. Finally, our dose-response study also suggests lower doses may be efficacious. Our findings show that rhSGSH can normalize lysosomal HS storage and markers of a neuroimmune response when delivered ICV.

Topics: Animals; Brain; Brain Diseases; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Heparitin Sulfate; Humans; Hydrolases; Lysosomes; Mice; Mucopolysaccharidosis III; Pilot Projects

Topics: Animals; Disease Models, Animal; Endothelial Cells; Female; Glycocalyx; Heparitin Sulfate; Liver; Lung; Male; Mice; Mice, Inbred C57BL; Neutrophil Activation; Neutrophils; Sepsis; Staphylococcus aureus

Signaling from multiple receptor tyrosine kinases (RTK) contributes to therapeutic resistance in glioblastoma (GBM). Heparan sulfate (HS), present on cell surfaces and in the extracellular matrix, regulates cell signaling via several mechanisms. To investigate the role for HS in promoting RTK signaling in GBM, we generated neural progenitor cells deficient for HS by knockout of the essential HS-biosynthetic enzyme

Topics: Animals; Cell Proliferation; Disease Models, Animal; ErbB Receptors; Glioblastoma; Heparitin Sulfate; Humans; Mice; N-Acetylglucosaminyltransferases; Receptor Protein-Tyrosine Kinases; Signal Transduction

Mucopolysaccharidosis (MPS) IIIB is a neuropathic lysosomal storage disease characterized by the deficient activity of a lysosomal enzyme obligate for the degradation of the glycosaminoglycan (GAG) heparan sulfate (HS). The pathogenesis of neurodegeneration in MPS IIIB is incompletely understood. Large animal models are attractive for pathogenesis and therapeutic studies due to their larger size, outbred genetics, longer lifespan, and naturally occurring MPS IIIB disease. However, the temporospatial development of neuropathologic changes has not been reported for canine MPS IIIB. Here we describe lesions in 8 brain regions, cervical spinal cord, and dorsal root ganglion (DRG) in a canine model of MPS IIIB that includes dogs aged from 2 to 26 months of age. Pathological changes in the brain included early microscopic vacuolation of glial cells initially observed at 2 months, and vacuolation of neurons initially observed at 10 months. Inclusions within affected cells variably stained positively with PAS and LFB stains. Quantitative immunohistochemistry demonstrated increased glial expression of GFAP and Iba1 in dogs with MPS IIIB compared to age-matched controls at all time points, suggesting neuroinflammation occurs early in disease. Loss of Purkinje cells was initially observed at 10 months and was pronounced in 18- and 26-month-old dogs with MPS IIIB. Our results support the dog as a replicative model of MPS IIIB neurologic lesions and detail the pathologic and neuroinflammatory changes in the spinal cord and DRG of MPS IIIB-affected dogs.

Topics: Animals; Brain; Disease Models, Animal; Dog Diseases; Dogs; Heparitin Sulfate; Mucopolysaccharidoses; Mucopolysaccharidosis III

The main pathophysiological mechanism of acute respiratory distress syndrome (ARDS) invovles the increase in alveolar barrier permeability that is primarily caused by epithelial glycocalyx and tight junction (TJ) protein destruction. This study was performed to explore the effects of the alveolar epithelial glycocalyx on the epithelial barrier, specifically on TJ proteins, in ARDS. We used C57BL/6 mice and human lung epithelial cell models of lipopolysaccharide (LPS)-induced ARDS. Changes in alveolar permeability were evaluated via pulmonary histopathology analysis and by measuring the wet/dry weight ratio of the lungs. Degradation of heparan sulfate (HS), an important component of the epithelial glycocalyx, and alterations in levels of the epithelial TJ proteins (occludin, zonula occludens 1, and claudin 4) were assessed via ELISA, immunofluorescence analysis, and western blotting analysis. Real-time quantitative polymerase chain reaction was used to detect the mRNA of the TJ protein. Changes in glycocalyx and TJ ultrastructures in alveolar epithelial cells were evaluated through electron microscopy. In vivo and in vitro, LPS increased the alveolar permeability and led to HS degradation and TJ damage. After LPS stimulation, the expression of the HS-degrading enzyme heparanase (HPA) in the alveolar epithelial cells was increased. The HPA inhibitor N-desulfated/re-N-acetylated heparin alleviated LPS-induced HS degradation and reduced TJ damage. In vitro, recombinant HPA reduced the expression of the TJ protein zonula occludens-1 (ZO-1) and inhibited its mRNA expression in the alveolar epithelial cells. Taken together, our results demonstrate that shedding of the alveolar epithelial glycocalyx aggravates the epithelial barrier and damages epithelial TJ proteins in ARDS, with the underlying mechanism involving the effect of HPA on ZO-1.

Topics: A549 Cells; Alveolar Epithelial Cells; Animals; Blood-Air Barrier; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Glycocalyx; Heparitin Sulfate; Humans; Male; Mice, Inbred C57BL; Permeability; Respiratory Distress Syndrome; Tight Junctions; Zonula Occludens-1 Protein

Mucopolysaccharidosis II (MPS II), a lysosomal storage disease caused by mutations in iduronate-2-sulfatase (IDS), is characterized by a wide variety of somatic and neurologic symptoms. The currently approved intravenous enzyme replacement therapy with recombinant IDS (idursulfase) is ineffective for CNS manifestations due to its inability to cross the blood-brain barrier (BBB). Here, we demonstrate that the clearance of heparan sulfate (HS) deposited in the brain by a BBB-penetrable antibody-enzyme fusion protein prevents neurodegeneration and neurocognitive dysfunctions in MPS II mice. The fusion protein pabinafusp alfa was chronically administered intravenously to MPS II mice. The drug reduced HS and attenuated histopathological changes in the brain, as well as in peripheral tissues. The loss of spatial learning abilities was completely suppressed by pabinafusp alfa, but not by idursulfase, indicating an association between HS deposition in the brain, neurodegeneration, and CNS manifestations in these mice. Furthermore, HS concentrations in the brain and reduction thereof by pabinafusp alpha correlated with those in the cerebrospinal fluid (CSF). Thus, repeated intravenous administration of pabinafusp alfa to MPS II mice decreased HS deposition in the brain, leading to prevention of neurodegeneration and maintenance of neurocognitive function, which may be predicted from HS concentrations in CSF.

Topics: Administration, Intravenous; Animals; Antibodies; Blood-Brain Barrier; Brain; Disease Models, Animal; Glycoproteins; Heparitin Sulfate; Humans; Iduronate Sulfatase; Immunoglobulin G; Mice; Mucopolysaccharidosis II; Neurocognitive Disorders; Receptors, Transferrin; Recombinant Fusion Proteins; Recombinant Proteins; Spatial Learning

Mucopolysaccharidosis IIIB (MPS IIIB, Sanfilippo syndrome type B) is caused by a deficiency in α-N-acetylglucosaminidase (NAGLU) activity, which leads to the accumulation of heparan sulfate (HS). MPS IIIB causes progressive neurological decline, with affected patients having an expected lifespan of approximately 20 years. No effective treatment is available. Recent pre-clinical studies have shown that intracerebroventricular (ICV) ERT with a fusion protein of rhNAGLU-IGF2 is a feasible treatment for MPS IIIB in both canine and mouse models. In this study, we evaluated the biochemical efficacy of a single dose of rhNAGLU-IGF2 via ICV-ERT in brain and liver tissue from Naglu

Topics: Acetylglucosaminidase; Animals; Animals, Newborn; Disease Models, Animal; Dogs; Enzyme Replacement Therapy; Heparitin Sulfate; Humans; Infusions, Intraventricular; Insulin-Like Growth Factor II; Mice; Mice, Knockout; Mucopolysaccharidosis III; Nervous System Diseases; Recombinant Fusion Proteins

Respiratory syncytial virus (RSV) has been reported to use CX3CR1

Topics: Animals; Antibodies, Viral; Binding Sites; Cell Line; CX3C Chemokine Receptor 1; Disease Models, Animal; Epithelial Cells; Heparitin Sulfate; Humans; Mutation; Receptors, Virus; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; Respiratory System; Sigmodontinae; Viral Envelope Proteins; Virus Replication

Corneal transparency relies on the precise arrangement and orientation of collagen fibrils, made of mostly Type I and V collagen fibrils and proteoglycans (PGs). PGs are essential for correct collagen fibrillogenesis and maintaining corneal homeostasis. We investigated the spatial and temporal distribution of glycosaminoglycans (GAGs) and PGs after a chemical injury. The chemical composition of chondroitin sulfate (CS)/dermatan sulfate (DS) and heparan sulfate (HS) were characterized in mouse corneas 5 and 14 days after alkali burn (AB), and compared to uninjured corneas. The expression profile and corneal distribution of CS/DSPGs and keratan sulfate (KS) PGs were also analyzed. We found a significant overall increase in CS after AB, with an increase in sulfated forms of CS and a decrease in lesser sulfated forms of CS. Expression of the CSPGs biglycan and versican was increased after AB, while decorin expression was decreased. We also found an increase in KS expression 14 days after AB, with an increase in lumican and mimecan expression, and a decrease in keratocan expression. No significant changes in HS composition were noted after AB. Taken together, our study reveals significant changes in the composition of the extracellular matrix following a corneal chemical injury.

Topics: Alkalies; Animals; Biomarkers; Burns, Chemical; Corneal Diseases; Dermatan Sulfate; Disease Models, Animal; Extracellular Matrix; Eye Burns; Fluorescent Antibody Technique; Gene Expression; Glycosaminoglycans; Heparitin Sulfate; Keratan Sulfate; Mice; Proteoglycans

Heparan sulfate proteoglycans (HSPGs) consist of a core protein with side chains of the glycosaminoglycan heparan sulfate (HS). We have previously identified (i) the HSPGs syndecan-1 (SDC1), and collagen type XVIII (COL18) inside mouse and human islet beta cells, and (ii) a critical role for HS in beta cell survival and protection from reactive oxygen species (ROS). The objective of this study was to investigate whether endoplasmic reticulum (ER) stress contributes to oxidative stress and type 2 diabetes (T2D) by depleting beta cell HSPGs/HS. A rapid loss of intra-islet/beta cell HSPGs, HS and heparanase (HPSE, an HS-degrading enzyme) accompanied upregulation of islet ER stress gene expression in both young T2D-prone db/db and Akita Ins2WT/C96Y mice. In MIN6 beta cells, HSPGs, HS and HPSE were reduced following treatment with pharmacological inducers of ER stress (thapsigargin or tunicamycin). Treatment of young db/db mice with Tauroursodeoxycholic acid (TUDCA), a chemical protein folding chaperone that relieves ER stress, improved glycemic control and increased intra-islet HSPG/HS. In vitro, HS replacement with heparin (a highly sulfated HS analogue) significantly increased the survival of wild-type and db/db beta cells and restored their resistance to hydrogen peroxide-induced death. We conclude that ER stress inhibits the synthesis/maturation of HSPG core proteins which are essential for HS assembly, thereby exacerbating oxidative stress and promoting beta cell failure. Diminished intracellular HSPGs/HS represent a previously unrecognized critical link bridging ER stress, oxidative stress and beta cell failure in T2D.

Topics: Activating Transcription Factors; Animals; Cell Survival; Cells, Cultured; Diabetes Mellitus, Type 2; Disease Models, Animal; Endoplasmic Reticulum Stress; Glucuronidase; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Hydrogen Peroxide; Insulin-Secreting Cells; Lactones; Mice; Mice, Inbred C57BL; Mice, Obese; Oxidative Stress; Sesquiterpenes; Transcription Factor CHOP; Up-Regulation

Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function. Hyperdopaminergia and autistic-like behaviours are corrected by the dopamine D1-like receptor antagonist SCH-23390, providing a potential alternative strategy to the D2-like antagonist haloperidol that has only minimal therapeutic effects in MPS-IIIA. These findings identify embryonic dopaminergic neurodevelopmental defects due to altered function of HS leading to autistic-like behaviours in MPS-II and MPS-IIIA and support evidence showing that altered HS-related gene function is causative of autism.

Topics: Animals; Autism Spectrum Disorder; Benzazepines; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Dopamine; Dopamine Antagonists; Dopaminergic Neurons; Heparitin Sulfate; Lysosomal Storage Diseases; Mesencephalon; Mice; Mucopolysaccharidosis III; Receptors, Dopamine D1

Dengue virus (DENV) is the most prevalent pathogen of the

Topics: Animals; Binding Sites; Brain; Cell Communication; Cell Line; Dengue; Dengue Virus; Disease Models, Animal; Female; Genotype; Heparin; Heparitin Sulfate; Host-Pathogen Interactions; Humans; Mice; Mice, Inbred BALB C; Molecular Docking Simulation; Phenotype; Phylogeny; Protein Conformation; Viral Envelope Proteins; Virulence; Virus Attachment

While malignant pleural effusion (MPE) is a common and significant cause of morbidity in patients with cancer, current treatment options are limited. Human heparanase, involved in angiogenesis and metastasis, cleaves heparan sulfate (HS) side chains on the cell surface.. To explore the coagulation milieu in MPE and infectious pleural effusion (IPE) focusing on the involvement of heparanase.. Samples of 30 patients with MPE and 44 patients with IPE were evaluated in comparison to those of 33 patients with transudate pleural effusions, using heparanase ELISA, heparanase procoagulant activity assay, thrombin and factor Xa chromogenic assays and thromboelastography. A cell proliferation assay was performed. EMT-6 breast cancer cells were injected to the pleural cavity of mice. A peptide inhibiting heparanase activity was administered subcutaneously.. Levels of heparanase, factor Xa and thrombin were significantly higher in exudate than transudate. Thromboelastography detected almost no thrombus formation in the whole blood, mainly on MPE addition. This effect was completely reversed by bacterial heparinase. Direct measurement revealed high levels of HS chains in pleural effusions. Higher proliferation was observed in tumour cell lines incubated with exudate than with transudate and it was reduced when bacterial heparinase was added. The tumour size in the pleural cavity of mice treated with the heparanase inhibitor were significantly smaller compared with control (p=0.005).. HS chains released by heparanase form an anticoagulant milieu in MPE, preventing local thrombosis and enabling tumour cell proliferation. Inhibition of heparanase might provide a therapeutic option for patients with recurrent MPE.

Topics: Animals; Anticoagulants; Biomarkers, Tumor; Blood Coagulation; Case-Control Studies; Cell Proliferation; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Glucuronidase; Heparitin Sulfate; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; Pleural Effusion, Malignant; Reference Values; Statistics, Nonparametric; Thrombelastography; Thrombosis; Tumor Cells, Cultured

With the increasing applications of 3D printing technology in biomedical field, the composition or additives of the related materials has become critical for the next development. In the current study, we have prepared 3D printed polycaprolactone-hydroxyapatite (PCL-HA) porous scaffolds with loaded heparan sulfate (HS), in order to reveal the reparative effect of different concentrations of HS on the healing of bone defects. As a result, the scaffold itself showed sound compression resistance, air porosity and good biocompatibility. From both in vitro and in vivo experiments, the scaffold with low concentration of HS led to positive effects in promoting osteoblast maturation and accelerating bone defect repair. Moreover, scaffold with high concentration of HS showed notable inhibitive effort on the proliferation of osteoblasts, yet it still brought about positive effects in repairing bone defects in organisms. Thus, with good structural properties and biocompatibility, 3D printed PCL-HA-HS composite scaffold can facilitate and accelerate the repairing of biological bone defects, demonstrating as an effective biomaterial for bone defect repair.

Topics: Animals; Biocompatible Materials; Bone and Bones; Cell Adhesion; Cell Differentiation; Cell Proliferation; Disease Models, Animal; Durapatite; Heparitin Sulfate; Microscopy, Electron, Scanning; Osteoblasts; Osteogenesis; Polyesters; Porosity; Printing, Three-Dimensional; Rabbits; Tissue Scaffolds; X-Ray Microtomography

Syndecan-4 (sdc4) is a cell-anchored proteoglycan that consists of a transmembrane core protein and glucosaminoglycan (GAG) side chains. Binding of soluble factors to the GAG chains of sdc4 may result in the dimerisation of sdc4 and the initiation of downstream signalling cascades. However, the question of how sdc4 dimerisation and signalling affects the response of cells to inflammatory stimuli is unknown.. Sdc4 immunostaining was performed on rheumatoid arthritis (RA) tissue sections. Interleukin (IL)-1 induced extracellular signal-regulated kinases (ERK) phosphorylation and matrix metalloproteinase-3 production was investigated. Il-1 binding to sdc4 was investigated using immunoprecipitation. IL-1 receptor (IL1R1) staining on wild-type, sdc4 and IL1R1 knockout fibroblasts was performed in fluorescence-activated cell sorting analyses. A blocking sdc4 antibody was used to investigate sdc4 dimerisation, IL1R1 expression and the histological paw destruction in the human tumour necrosis factor-alpha transgenic mouse.. We show that in fibroblasts, the loss of sdc4 or the antibody-mediated inhibition of sdc4 dimerisation reduces the cell surface expression of the IL-1R and regulates the sensitivity of fibroblasts to IL-1. We demonstrate that IL-1 directly binds to sdc4 and in an IL-1R-independent manner leads to its dimerisation. IL-1-induced dimerisation of sdc4 regulates caveolin vesicle-mediated trafficking of the IL1R1, which in turn determines the responsiveness to IL-1. Administration of antibodies (Ab) against the dimerisation domain of sdc4, thus, strongly reduces the expression IL1R1 on arthritic fibroblasts both in vitro and an animal model of human RA.. Collectively, our data suggest that Ab that specifically inhibit sdc4 dimerisation may support anti-IL-1 strategies in diseases such as inflammatory arthritis.

Topics: Animals; Antibodies, Blocking; Arthritis, Rheumatoid; Dimerization; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Gene Knockout Techniques; Heparitin Sulfate; Hindlimb; Humans; Interleukin-1; Interleukin-1beta; MAP Kinase Signaling System; Matrix Metalloproteinase 3; Mice; Mice, Transgenic; NIH 3T3 Cells; Osteoarthritis; Phosphorylation; Protein Transport; Receptors, Interleukin-1 Type I; Signal Transduction; Syndecan-4; Synovial Membrane; Tumor Necrosis Factor-alpha

Osteoarthritis (OA) is a progressive degenerative disease of the articular cartilage caused by an unbalanced activity of proteases, cytokines and other secreted proteins. Since heparan sulfate (HS) determines the activity of many extracellular factors, we investigated its role in OA progression.. To analyze the role of the HS level, OA was induced by anterior cruciate ligament transection (ACLT) in transgenic mice carrying a loss-of-function allele of Ext1 in clones of chondrocytes (Col2-rtTA-Cre;Ext1. All investigated mouse strains showed reduced OA scores (Col2-rtTA-Cre;Ext1. A decreased HS content or a reduced sulfation level protect against OA progression by regulating protease activity rather than expression.

Topics: Aggrecans; Animals; Anterior Cruciate Ligament; Blotting, Western; Cartilage, Articular; Chondrocytes; Disease Models, Animal; Disease Progression; Heparitin Sulfate; Loss of Function Mutation; Matrix Metalloproteinase 2; Mice; Mice, Transgenic; N-Acetylglucosaminyltransferases; Osteoarthritis; Real-Time Polymerase Chain Reaction; Sulfotransferases

Enterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine,

Topics: Animals; Antiviral Agents; Capsid Proteins; Cell Line; Cinnamates; Depsides; Disease Models, Animal; Drug Evaluation, Preclinical; Enterovirus A, Human; Enterovirus Infections; Heparitin Sulfate; Humans; Jurkat Cells; Membrane Glycoproteins; Mice; Mutation; Plant Extracts; Protein Binding; Rosmarinic Acid; Salvia miltiorrhiza; Static Electricity; Virulence Factors

Myelin destruction is followed by resident glia activation and mobilization of endogenous progenitors (OPC) which participate in myelin repair. Here we show that in response to demyelination, mature oligodendrocytes (OLG) bordering the lesion express Ndst1, a key enzyme for heparan sulfates (HS) synthesis. Ndst1+ OLG form a belt that demarcates lesioned from intact white matter. Mice with selective inactivation of Ndst1 in the OLG lineage display increased lesion size, sustained microglia and OPC reactivity. HS production around the lesion allows Sonic hedgehog (Shh) binding and favors the local enrichment of this morphogen involved in myelin regeneration. In MS patients, Ndst1 is also found overexpressed in oligodendroglia and the number of Ndst1-expressing oligodendroglia is inversely correlated with lesion size and positively correlated with remyelination potential. Our study suggests that mature OLG surrounding demyelinated lesions are not passive witnesses but contribute to protection and regeneration by producing HS.

Topics: Animals; Corpus Callosum; Demyelinating Diseases; Disease Models, Animal; Gene Deletion; Hedgehog Proteins; HEK293 Cells; Heparitin Sulfate; Humans; Lysophosphatidylcholines; Macrophage Activation; Mice, Transgenic; Microglia; Multiple Sclerosis; Oligodendroglia; Remyelination; Sulfotransferases; Up-Regulation

Osteoprotegerin (OPG), a secreted decoy receptor for receptor activator of nuclear factor B ligand (RANKL), plays an essential role in regulating bone resorption. While much is known about the function of the N-terminal domains of OPG, which is responsible for binding to RANKL, the exact biological functions of the three C-terminal domains of OPG remain uncertain. We have previously shown that one likely function of the C-terminal domains of OPG is to bind cell surface heparan sulfate (HS), but the in vivo evidence was lacking. To investigate the biological significance of OPG-HS interaction in bone remodeling, we created OPG knock-in mice (

Topics: Animals; Binding Sites; Bone and Bones; Bone Density Conservation Agents; Bone Resorption; Cell Line; Disease Models, Animal; Female; Heparitin Sulfate; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Osteoblasts; Osteoclasts; Osteogenesis; Osteoporosis; Osteoprotegerin; RANK Ligand; Transcriptome

To determine if endothelial dysfunction in a mouse model of diet-induced obesity and in obese humans is mediated by the suppression of endothelial Kir (inwardly rectifying K. We conclude that obesity-induced impairment of flow-induced vasodilation is attributed to the loss of flow-sensitivity of endothelial Kir channels and propose that the latter is mediated by the biophysical alterations of the glycocalyx.

Topics: Adult; Animals; Cells, Cultured; Diet, High-Fat; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Female; Glycocalyx; Heparitin Sulfate; Humans; Male; Mechanotransduction, Cellular; Membrane Potentials; Mesenteric Arteries; Mice; Middle Aged; Obesity; Potassium Channels, Inwardly Rectifying; Regional Blood Flow; Vasodilation

We recently developed a blood-brain barrier (BBB)-penetrating enzyme transport vehicle (ETV) fused to the lysosomal enzyme iduronate 2-sulfatase (ETV:IDS) and demonstrated its ability to reduce glycosaminoglycan (GAG) accumulation in the brains of a mouse model of mucopolysaccharidosis (MPS) II. To accurately quantify GAGs, we developed a plate-based high-throughput enzymatic digestion assay coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure heparan sulfate and dermatan sulfate derived disaccharides in tissue, cerebrospinal fluid (CSF) and individual cell populations isolated from mouse brain. The method offers ultra-high sensitivity enabling quantitation of specific GAG species in as low as 100,000 isolated neurons and a low volume of CSF. With an LOD at 3 ng/mL and LLOQs at 5-10 ng/mL, this method is at least five times more sensitive than previously reported approaches. Our analysis demonstrated that the accumulation of CSF and brain GAGs are in good correlation, supporting the potential use of CSF GAGs as a surrogate biomarker for brain GAGs. The bioanalytical method was qualified through the generation of standard curves in matrix for preclinical studies of CSF, demonstrating the feasibility of this assay for evaluating therapeutic effects of ETV:IDS in future studies and applications in a wide variety of MPS disorders.

Topics: Animals; Biomarkers; Blood-Brain Barrier; Brain; Chromatography, Liquid; Dermatan Sulfate; Disaccharides; Disease Models, Animal; Glycosaminoglycans; Heparitin Sulfate; Humans; Iduronate Sulfatase; Mice; Mucopolysaccharidosis II; Tandem Mass Spectrometry

Exostosin-1 (EXT1) and EXT2 are the major genetic etiologies of multiple hereditary exostoses and are essential for heparan sulfate (HS) biosynthesis. Previous studies investigating HS in several mouse models of multiple hereditary exostoses have reported that aberrant bone morphogenetic protein (BMP) signaling promotes osteochondroma formation in Ext1-deficient mice. This study examined the mechanism underlying the effects of HS deficiency on BMP/Smad signaling in articular cartilage in a cartilage-specific Ext. We generated mice with a conditional Ext1 knockout in cartilage tissue (Ext1-cKO mice) using Prg4-Cre transgenic mice. Structural cartilage alterations were histologically evaluated and phospho-Smad1/5/9 (pSmad1/5/9) expression in mouse chondrocytes was analyzed. The effect of pharmacological intervention of BMP signaling using a specific inhibitor was assessed in the articular cartilage of Ext1-cKO mice.. Hypertrophic chondrocytes were significantly more abundant (P = 0.021) and cartilage thickness was greater in Ext1-cKO mice at 3 months postnatal than in control littermates (P = 0.036 for femur; and P < 0.001 for tibia). However, osteoarthritis did not spontaneously occur before the 1-year follow-up. matrix metalloproteinase (MMP)-13 and adamalysin-like metalloproteinases with thrombospondin motifs(ADAMTS)-5 were upregulated in hypertrophic chondrocytes of transgenic mice. Immunostaining and western blotting revealed that pSmad1/5/9-positive chondrocytes were more abundant in the articular cartilage of Ext1-cKO mice than in control littermates. Furthermore, the BMP inhibitor significantly decreased the number of hypertrophic chondrocytes in Ext1-cKO mice (P = 0.007).. HS deficiency in articular chondrocytes causes chondrocyte hypertrophy, wherein upregulated BMP/Smad signaling partially contributes to this phenotype. HS might play an important role in maintaining the cartilaginous matrix by regulating BMP signaling.

Topics: ADAMTS5 Protein; Animals; Bone Morphogenetic Proteins; Cartilage, Articular; Chondrocytes; Disease Models, Animal; Heparitin Sulfate; Hypertrophy; Matrix Metalloproteinase 13; Mice; Mice, Knockout; Mice, Transgenic; N-Acetylglucosaminyltransferases; Osteoarthritis, Knee; Pyrazoles; Pyrimidines; Smad1 Protein; Smad5 Protein; Smad8 Protein

Sanfilippo syndrome is an untreatable form of childhood-onset dementia. Whilst several therapeutic strategies are being evaluated in human clinical trials including i.v. delivery of AAV9-based gene therapy, an urgent unmet need is the availability of non-invasive, quantitative measures of neurodegeneration. We hypothesise that as part of the central nervous system, the retina may provide a window through which to 'visualise' degenerative lesions in brain and amelioration of them following treatment. This is reliant on the age of onset and the rate of disease progression being equivalent in retina and brain. For the first time we have assessed in parallel, the nature, age of onset and rate of retinal and brain degeneration in a mouse model of Sanfilippo syndrome. Significant accumulation of heparan sulphate and expansion of the endo/lysosomal system was observed in both retina and brain pre-symptomatically (by 3 weeks of age). Robust and early activation of micro- and macroglia was also observed in both tissues. There was substantial thinning of retina and loss of rod and cone photoreceptors by ~ 12 weeks of age, a time at which cognitive symptoms are noted. Intravenous delivery of a clinically relevant AAV9-human sulphamidase vector to neonatal mice prevented disease lesion appearance in retina and most areas of brain when assessed 6 weeks later. Collectively, the findings highlight the previously unrecognised early and significant involvement of retina in the Sanfilippo disease process, lesions that are preventable by neonatal treatment with AAV9-sulphamidase. Critically, our data demonstrate for the first time that the advancement of retinal disease parallels that occurring in brain in Sanfilippo syndrome, thus retina may provide an easily accessible neural tissue via which brain disease development and its amelioration with treatment can be monitored.

Topics: Animals; Asymptomatic Diseases; Brain; Disease Models, Animal; Endosomes; Genetic Therapy; Heparitin Sulfate; Humans; Hydrolases; Lysosomes; Mice; Microglia; Mucopolysaccharidosis III; Neurodegenerative Diseases; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B) is an autosomal recessive lysosomal storage disorder caused by the deficiency of alpha-N-acetylglucosaminidase activity, leading to increased levels of nondegraded heparan sulfate (HS). A mouse model has been useful to evaluate novel treatments for MPS IIIB, but has limitations. In this study, we evaluated the naturally occurring canine model of MPS IIIB for the onset and progression of biochemical and neuropathological changes during the preclinical stages (onset approximately 24-30 months of age) of canine MPS IIIB disease. Even by 1 month of age, MPS IIIB dogs had elevated HS levels in brain and cerebrospinal fluid. Analysis of histopathology of several disease-relevant regions of the forebrain demonstrated progressive lysosomal storage and microglial activation despite a lack of cerebrocortical atrophy in the oldest animals studied. More pronounced histopathology changes were detected in the cerebellum, where progressive lysosomal storage, astrocytosis and microglial activation were observed. Microglial activation was particularly prominent in cerebellar white matter and within the deep cerebellar nuclei, where neuron loss also occurred. The findings in this study will form the basis of future assessments of therapeutic efficacy in this large animal disease model.

Topics: Acetylglucosaminidase; Animals; Astrocytes; Cerebellum; Cerebral Cortex; Disease Models, Animal; Disease Progression; Dog Diseases; Dogs; Female; Heparitin Sulfate; Histocytochemistry; Humans; Lysosomes; Male; Microglia; Mucopolysaccharidosis III; Neurons; Prosencephalon; White Matter

The endothelial glycocalyx and its regulated shedding are important to vascular health. Endo-β-D-glucuronidase heparanase-1 (HPSE1) is the only enzyme that can shed heparan sulfate. However, the mechanisms are not well understood. We show that HPSE1 activity aggravated Toll-like receptor 4 (TLR4)-mediated response of endothelial cells to LPS. On the contrary, overexpression of its endogenous inhibitor, heparanase-2 (HPSE2) was protective. The microfluidic chip flow model confirmed that HPSE2 prevented heparan sulfate shedding by HPSE1. Furthermore, heparan sulfate did not interfere with cluster of differentiation-14 (CD14)-dependent LPS binding, but instead reduced the presentation of the LPS to TLR4. HPSE2 reduced LPS-mediated TLR4 activation, subsequent cell signalling, and cytokine expression. HPSE2-overexpressing endothelial cells remained protected against LPS-mediated loss of cell-cell contacts. In vivo, expression of HPSE2 in plasma and kidney medullary capillaries was decreased in mouse sepsis model. We next applied purified HPSE2 in mice and observed decreases in TNFα and IL-6 plasma concentrations after intravenous LPS injections. Our data demonstrate the important role of heparan sulfate and the glycocalyx in endothelial cell activation and suggest a protective role of HPSE2 in microvascular inflammation. HPSE2 offers new options for protection against HPSE1-mediated endothelial damage and preventing microvascular disease.

Topics: Animals; Disease Models, Animal; Endothelial Cells; Glucuronidase; Glycocalyx; Heparitin Sulfate; Humans; Lipopolysaccharides; Male; Mice; Microfluidic Analytical Techniques; Sepsis; Signal Transduction; Toll-Like Receptor 4

Lack of the N-alpha-acetylglucosaminidase gene is responsible for the occurrence of a rare disease - the Sanfilippo syndrome, type B. The result of this gene knock-out is accumulation of glycosaminoglycans (GAGs) - more specifically heparan sulfate - a sulfate rich macromolecule. The sulfur oxidative pathway is involved in the sulfate groups' turnover in the cells. In contrast, the non-oxidative sulfur pathway leads mostly to formation of sulfane sulfur-containing compounds. The aim of our research was to observe an interaction between MPS IIIB and non-oxidative sulfur metabolism. In this work, we examined selected tissues (livers, kidneys, hearts and spleens) of 3 month old mice with confirmed accumulation of GAGs. The activity and expression of three sulfurtransferases (components of non-oxidative sulfur metabolism): rhodanese, 3-mercaptopyruvate sulfurtransferase and cystathionine γ-lyase was determined, as well as the sulfane sulfur level and the level of other low molecular sulfur-containing compounds (reduced and oxidized glutathione, cysteine and cystine). In all tested tissues, the sulfane sulfur and/or sulfurtransferases' activities, as well as the cysteine content, underwent statistically significant changes. These correlations were also related to the sex of the tested animals. The obtained results indicated that accumulation of incompletely degraded GAGs in the tissues had affected the non-oxidative sulfur metabolism.

Topics: Animals; Cystathionine gamma-Lyase; Disease Models, Animal; Glycosaminoglycans; Heparitin Sulfate; Humans; Kidney; Liver; Mice; Mucopolysaccharidosis III; Myocardium; Oxidative Stress; Spleen; Sulfur; Sulfurtransferases; Thiosulfate Sulfurtransferase

Aldosterone contributes to end-organ damage in heart failure and chronic kidney disease. Mineralocorticoid-receptor inhibitors limit activation of the receptor by aldosterone and slow disease progression, but side effects, including hyperkalemia, limit their clinical use. Damage to the endothelial glycocalyx (a luminal biopolymer layer) has been implicated in the pathogenesis of endothelial dysfunction and albuminuria, but to date no one has investigated whether the glomerular endothelial glycocalyx is affected by aldosterone. In vitro, human glomerular endothelial cells exposed to 0.1 nM aldosterone and 145 mMol NaCl exhibited reduced cell surface glycocalyx components (heparan sulfate and syndecan-4) and disrupted shear sensing consistent with damage of the glycocalyx. In vivo, administration of 0.6 μg/g/d of aldosterone (subcutaneous minipump) and 1% NaCl drinking water increased glomerular matrix metalloproteinase 2 activity, reduced syndecan 4 expression, and caused albuminuria. Intravital multiphoton imaging confirmed that aldosterone caused damage of the glomerular endothelial glycocalyx and increased the glomerular sieving coefficient for albumin. Targeting matrix metalloproteinases 2 and 9 with a specific gelatinase inhibitor preserved the glycocalyx, blocked the rise in glomerular sieving coefficient, and prevented albuminuria. Together these data suggest that preservation of the glomerular endothelial glycocalyx may represent a novel strategy for limiting the pathological effects of aldosterone.

Topics: Albuminuria; Aldosterone; Animals; Cell Line; Disease Models, Animal; Endothelial Cells; Glycocalyx; Heparitin Sulfate; Humans; Kidney Glomerulus; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Renal Insufficiency, Chronic; Sodium Chloride; Syndecan-4

Plasma-based resuscitation showed protective effects on the endothelial glycocalyx compared with crystalloid resuscitation. There is paucity of data regarding the effect of coagulation factor concentrates (CFC) on the glycocalyx in hemorrhagic shock (HS). We hypothesized that colloid-based resuscitation supplemented with CFCs offers a therapeutic value to treat endothelial damage following HS.. Eighty-four rats were subjected to pressure-controlled (mean arterial pressure (MAP) 30-35 mm Hg) and lab-guided (targeted cutoff: lactate >2.2. mmol/L and base deficit > 5.5 mmol/L) HS. Animals were resuscitated with fresh frozen plasma (FFP), human albumin (HA) or Ringer's lactate (RL) and RL or HA supplemented with fibrinogen concentrate (FC) or prothrombin complex concentrate (PCC). Serum epinephrine and the following markers of endothelial damage were assessed at baseline and at the end-of-observation (120 min after shock was terminated): syndecan-1, heparan sulfate, and soluble vascular endothelial growth factor receptor 1 (sVEGFR 1).. Resuscitation with FFP had no effect on sVEGFR1 compared with crystalloid-based resuscitation (FFP: 19.3 ng/mL vs. RL: 15.9 ng/mL; RL+FC: 19.7 ng/mL; RL+PCC: 18.9 ng/mL; n.s.). At the end-of-observation, syndecan-1 was similar among all groups. Interestingly, HA+FC treated animals displayed the highest syndecan-1 concentration (12.07 ng/mL). Resuscitation with FFP restored heparan sulfate back to baseline (baseline: 36 ng/mL vs. end-of-observation: 36 ng/mL).. The current study revealed that plasma-based resuscitation normalized circulating heparan sulfate but not syndecan-1. Co-administration of CFC had no further effect on glycocalyx shedding suggesting a lack of its therapeutic potential.. VExperimental in vivo study.

Topics: Animals; Biomarkers; Blood Coagulation Factors; Crystalloid Solutions; Disease Models, Animal; Heparitin Sulfate; Humans; Male; Rats; Rats, Sprague-Dawley; Resuscitation; Shock, Hemorrhagic; Syndecan-1

Familial members of urolithiasis have high risk for stone development. We observed the low sulfated glycosaminoglycan (GAG) excretion in urolithiasis patients and their descendants. In this study, we investigated urinary excretion of sulfated GAG, chondroitin sulfate (CS), heparan sulfate (HS) and hyaluronic acid (HA) in urolithiasis and their children, and explored the effect of CS and HA supplement in urolithic hyperoxaluric rats. The 24-hour urines were collected from urolithiasis patients (28) and their children (40), as well as healthy controls (45) and their children (33) to measure urinary sulfated GAG, CS, HS and HA excretion rate. Our result showed that urinary sulfated GAG and CS were diminished in both urolithiasis patients and their children, while decreased HS and increased HA were observed only in urolithiasis patients. Percentage of HS per sulfated GAG increased in both urolithiasis patients and their children. In hyperoxaluric rats induced by ethylene glycol and vitamin D, we found that CS supplement could prevent stone formation, while HA supplement had no effect on stone formation. Our study revealed that decreased urinary GAG and CS excretion are common in familial members of urolithiasis patients, and CS supplement might be beneficial in calcium oxalate urolithiasis prophylaxis for hyperoxaluric patients.

Topics: Adult; Animals; Child; Chondroitin Sulfates; Creatinine; Dietary Supplements; Disease Models, Animal; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Kidney; Male; Middle Aged; Rats; Rats, Wistar; Urolithiasis

Mucopolysaccharidosis III type C is a lysosomal storage disorder caused by the accumulation of heparan sulfate in lysosomes. The disorder occurs due to Heparan Acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT) deficiency, an enzyme which typically catalyzes the transmembrane acetylation of heparan sulfate, a basement membrane component. When the gene encoding this enzyme is mutated, it cannot perform the processing of heparan sulfate, leading to un-acetylated heparan sulfate build-up in the lysosomes of cells, causing a storage disorder. This defect has been studied primarily in brain and liver cells, but its effect on the structural integrity of the glomerulus is poorly known. The present study focuses on the effect of Hgsnat gene inactivation and heparan sulfate toxicity on the integrity of the renal corpuscle. This cortical structure was chosen because of its abundance of basement membranes and heparan sulfate as well as the renal corpuscle's physiological importance in glomerular filtration. Light microscopy, electron microscopy, and immunocytochemistry of genetically modified mice revealed a buildup of lysosomes in the podocytes, suggesting that these cells are responsible for the processing of glomerular basement membranes.

Topics: Acetyltransferases; Animals; Basement Membrane; Disease Models, Animal; Glycosaminoglycans; Heparitin Sulfate; Immunohistochemistry; Kidney; Kidney Glomerulus; Lysosomes; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mucopolysaccharidosis III; Podocytes

Here, we sought to elucidate the role of CAR (a cyclic peptide) in the accumulation and distribution of fasudil, a drug for pulmonary arterial hypertension (PAH), in rat lungs and in producing pulmonary specific vasodilation in PAH rats. As such, we prepared liposomes of fasudil and CAR-conjugated liposomal fasudil and assessed the liposomes for CAR conjugation, physical properties, entrapment efficiencies, in vitro release profiles, and stabilities upon incubation in cell culture media, storage, and aerosolization. We also studied the cellular uptake of fasudil in different formulations, quantified heparan sulfate (HS) in pulmonary arterial smooth muscle cells (PASMCs), and investigated the distribution of the liposomes in the lungs of PAH rats. We assessed the drug accumulation in a close and recirculating isolated perfused rat lung model and studied the pharmacokinetics and pharmacological efficacy of the drug and formulations in Sugen/hypoxia-induced PAH rats. The entrapment efficiency of the liposomal fasudil was 95.5 ± 4.5%, and the cumulative release was 93.95 ± 6.22%. The uptake of CAR liposomes by pulmonary arterial cells and their distribution and accumulation in the lungs were much greater than those of no-CAR-liposomes. CAR-induced increase in the cellular uptake was associated with an increase in HS expression by rat PAH-PASMCs. CAR, when conjugated with liposomal fasudil and given via an intratracheal instillation, extended the elimination half-life of the drug by four-fold compared with fasudil-in-no-CAR-liposomes given via the same route. CAR-conjugated liposomal fasudil, as opposed to fasudil-in-no-CAR-liposomes and CAR pretreatment followed by fasudil-in-no-CAR-liposomes, reduced the mean pulmonary arterial pressure by 40-50% for 6 h, without affecting the mean systemic arterial pressure. On the whole, this study suggests that CAR aids in concentrating the drug in the lungs, increasing the cellular uptake, extending the half-life of fasudil, and eliciting a pulmonary-specific vasodilation when the peptide remains conjugated on the liposomal surface, but not when CAR is given as a pretreatment or alone as an admixture with the drug.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Disease Models, Animal; Half-Life; Heparitin Sulfate; Humans; Hypertension, Pulmonary; Liposomes; Lung; Male; Myocytes, Smooth Muscle; Peptides, Cyclic; Pulmonary Artery; Rats; Time Factors; Vasodilation; Vasodilator Agents

Topics: Animals; Antibodies, Monoclonal; Brain; Disease Models, Animal; Female; Heparitin Sulfate; Humans; Hydrolases; Immunoglobulin G; Liver; Male; Mice; Mice, Knockout; Mucopolysaccharidosis III; Receptors, Transferrin; Recombinant Fusion Proteins

Ischemia-reperfusion (I/R) injury is a major clinical problem linked to vascular surgery. Currently, no drugs to prevent or to treat I/R injury are approved for clinical use. C1 inhibitor (C1 INH) is known to reduce activation of the plasma cascade systems that are involved in the pathophysiologic process of I/R injury. The aim of this study was therefore to investigate the effect of C1 INH on complement deposition and endothelial cell activation in a rat model of hind limb I/R injury.. Male Wistar rats (wild type, bred at the central animal facility, University of Bern), weighing 250 to 320 g, were used. The rats underwent 2-hour ischemia and 24-hour reperfusion by unilateral clamping of the femoral artery and additional use of a tourniquet. Five groups were divided according to intravenous treatment 5 minutes before ischemia: 50 IU/kg C1 INH (n = 5); 100 IU/kg C1 INH (n = 7); vehicle control (n = 5); nontreated control (n = 7); and normal, healthy control without intervention (n = 4). At the end, muscle edema, tissue viability, and histologic features were assessed. Deposition of immunoglobulin M, C1r, C4d, and fibrin and expression of plasminogen activator inhibitor 1, heparan sulfate (HS), E-selectin, and vascular cell adhesion molecule 1 were evaluated by fluorescence staining. In addition, high-mobility group box 1 protein was measured in plasma.. Edema formation was reduced by C1 INH at two dosages, mirrored by improved histologic injury scores and preserved muscle viability. Deposition of immunoglobulin M, C4d, and fibrin was significantly decreased by 100 IU/kg C1 INH compared with nontreated controls. Pretreatment with 100 IU/kg C1 INH also significantly reduced HS shedding and expression of plasminogen activator inhibitor 1 as well as plasma levels of high-mobility group box 1 protein.. Pretreatment with both 50 and 100 IU/kg C1 INH attenuated reperfusion injury of rat hind limbs. Pretreatment with 100 IU/kg also preserved the endothelial HS layer as well as the natural, profibrinolytic phenotype of the endothelium. Prevention of endothelial cell activation by C1 INH may therefore be a promising strategy to prevent I/R injury in the clinical setting of peripheral vascular diseases and elective surgery on extremities.

Topics: Animals; Complement Activation; Complement C1 Inhibitor Protein; Complement C1r; Complement C4b; Complement Inactivating Agents; Disease Models, Animal; E-Selectin; Edema; Endothelial Cells; Fibrin; Heparitin Sulfate; Hindlimb; HMGB1 Protein; Immunoglobulin M; Male; Muscle, Skeletal; Peptide Fragments; Plasminogen Activator Inhibitor 1; Rats, Wistar; Reperfusion Injury; Tissue Survival; Vascular Cell Adhesion Molecule-1

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder resulting from the deficit of the N-sulfoglucosamine sulfohydrolase (SGSH) enzyme that leads to accumulation of partially-degraded heparan sulfate. MPS IIIA is characterized by severe neurological symptoms, clinically presenting as Sanfilippo syndrome, for which no effective therapy is available. The lysosomal SGSH enzyme is conserved in Drosophila and we have identified increased levels of heparan sulfate in flies with ubiquitous knockdown of SGSH/CG14291. Using neuronal specific knockdown of SGSH/CG14291 we have also observed a higher abundance of Lysotracker-positive puncta as well as increased expression of GFP tagged Ref(2)P supporting disruption to lysosomal function. We have also observed a progressive defect in climbing ability, a hallmark of neurological dysfunction. Genetic screens indicate proteins and pathways that can functionally modify the climbing phenotype, including autophagy-related proteins (Atg1 and Atg18), superoxide dismutase enzymes (Sod1 and Sod2) and heat shock protein (HSPA1). In addition, reducing heparan sulfate biosynthesis by knocking down sulfateless or slalom expression significantly worsens the phenotype; an important observation given that substrate inhibition is being evaluated clinically as a treatment for MPS IIIA. Identifying the cellular pathways that can modify MPS IIIA neuropathology is an essential step in the development of novel therapeutic approaches to prevent and/or ameliorate symptoms in children with Sanfilippo syndrome.

Topics: Age Factors; Animals; Animals, Genetically Modified; Autophagy; Brain; Disease Models, Animal; Drosophila; Drosophila Proteins; Gene Expression Regulation; Green Fluorescent Proteins; Heparitin Sulfate; Hydrolases; Larva; Locomotion; Mucopolysaccharidosis III; Mutation; Neurons; Psychomotor Disorders; RNA Interference; RNA, Messenger

Bone remodeling is a highly coordinated process involving bone formation and resorption, and imbalance of this process results in osteoporosis. It has long been recognized that long-term heparin therapy often causes osteoporosis, suggesting that heparan sulfate (HS), the physiological counterpart of heparin, is somehow involved in bone mass regulation. The role of endogenous HS in adult bone, however, remains unclear. To determine the role of HS in bone homeostasis, we conditionally ablated Ext1, which encodes an essential glycosyltransferase for HS biosynthesis, in osteoblasts. Resultant conditional mutant mice developed severe osteopenia. Surprisingly, this phenotype is not due to impairment in bone formation but to enhancement of bone resorption. We show that osteoprotegerin (OPG), which is known as a soluble decoy receptor for RANKL, needs to be associated with the osteoblast surface in order to efficiently inhibit RANKL/RANK signaling and that HS serves as a cell surface binding partner for OPG in this context. We also show that bone mineral density is reduced in patients with multiple hereditary exostoses, a genetic bone disorder caused by heterozygous mutations of Ext1, suggesting that the mechanism revealed in this study may be relevant to low bone mass conditions in humans.

Topics: Adult; Animals; Bone and Bones; Bone Density; Bone Diseases, Metabolic; Bone Resorption; Cell Differentiation; Cells, Cultured; CHO Cells; Coculture Techniques; Cricetulus; Disease Models, Animal; Exostoses, Multiple Hereditary; Female; HEK293 Cells; Heparitin Sulfate; Humans; Male; Mice; Mice, Knockout; Mutagenesis, Site-Directed; Mutation; N-Acetylglucosaminyltransferases; Osteoblasts; Osteoclasts; Osteoprotegerin; Primary Cell Culture; Recombinant Proteins

Certain recessively inherited diseases result from an enzyme deficiency within lysosomes. In mucopolysaccharidoses (MPS), a defect in glycosaminoglycan (GAG) degradation leads to GAG accumulation followed by progressive organ and multiple system dysfunctions. Current methods of GAG analysis used to diagnose and monitor the diseases lack sensitivity and throughput. Here we report a LC-MS method with accurate metabolite mass analysis for identifying and quantifying biomarkers for MPS type I without the need for extensive sample preparation. The method revealed 225 LC-MS features that were >1000-fold enriched in urine, plasma and tissue extracts from untreated MPS I mice compared to MPS I mice treated with iduronidase to correct the disorder. Levels of several trisaccharides were elevated >10000-fold. To validate the clinical relevance of our method, we confirmed the presence of these biomarkers in urine, plasma and cerebrospinal fluid from MPS I patients and assessed changes in their levels after treatment.

Topics: Animals; Biomarkers; Chromatography, Liquid; Disease Models, Animal; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Iduronidase; Male; Mice; Mucopolysaccharidosis I; Trisaccharides

Multiple osteochondromas (MO) syndrome is a dominant autosomal bone disorder characterized by the formation of cartilage-capped bony outgrowths that develop at the juxtaposition of the growth plate of endochondral bones. MO has been linked to mutations in either EXT1 or EXT2, two glycosyltransferases required for the synthesis of heparan sulfate (HS). The establishment of mouse mutants demonstrated that a clonal, homozygous loss of Ext1 in a wild type background leads to the development of osteochondromas. Here we investigate mechanisms that might contribute to the variation in the severity of the disease observed in human patients. Our results show that residual amounts of HS are sufficient to prevent the development of osteochondromas strongly supporting that loss of heterozygosity is required for osteochondroma formation. Furthermore, we demonstrate that different signaling pathways affect size and frequency of the osteochondromas thereby modulating the severity of the disease. Reduced Fgfr3 signaling, which regulates proliferation and differentiation of chondrocytes, increases osteochondroma number, while activated Fgfr3 signaling reduces osteochondroma size. Both, activation and reduction of Wnt/β-catenin signaling decrease osteochondroma size and frequency by interfering with the chondrogenic fate of the mutant cells. Reduced Ihh signaling does not change the development of the osteochondromas, while elevated Ihh signaling increases the cellularity and inhibits chondrocyte differentiation in a subset of osteochondromas and might thus predispose osteochondromas to the transformation into chondrosarcomas.

Topics: Animals; beta Catenin; Cell Differentiation; Chondrocytes; Disease Models, Animal; Exostoses, Multiple Hereditary; Growth Plate; Hedgehog Proteins; Heparitin Sulfate; Humans; Loss of Heterozygosity; Mice; N-Acetylglucosaminyltransferases; Receptor, Fibroblast Growth Factor, Type 3; Signal Transduction; Wnt Signaling Pathway

Peripheral arterial disease is a major cause of limb loss and its prevalence is increasing worldwide. As most standard-of-care therapies yield only unsatisfactory outcomes, more options are needed. Recent cell- and molecular-based therapies that have aimed to modulate vascular endothelial growth factor-165 (VEGF

Topics: Animals; Disease Models, Animal; Heparitin Sulfate; Hindlimb; Human Umbilical Vein Endothelial Cells; Humans; Ischemia; Mice; RAW 264.7 Cells

Infection by enterovirus 71 (EV71) is affected by cell surface receptors, including the human scavenger receptor B2 (hSCARB2), which are required for viral uncoating, and attachment receptors, such are heparan sulfate (HS), which bind virus but do not support uncoating. Amino acid residue 145 of the capsid protein VP1 affects viral binding to HS and virulence in mice. However, the contribution of this amino acid to pathogenicity in humans is not known. We produced EV71 having glycine (VP1-145G) or glutamic acid (VP1-145E) at position 145. VP1-145G, but not VP1-145E, enhanced viral infection in cell culture in an HS-dependent manner. However, VP1-145G virus showed an attenuated phenotype in wild-type suckling mice and in a transgenic mouse model expressing hSCARB2, while VP1-145E virus showed a virulent phenotype in both models. Thus, the HS-binding property and

Topics: Amino Acid Substitution; Animals; Capsid Proteins; Cell Line; Central Nervous System; Disease Models, Animal; Endothelial Cells; Enterovirus A, Human; Enterovirus Infections; Glycine; Heparitin Sulfate; Humans; Lysosomal Membrane Proteins; Mice; Mice, Transgenic; Mutation; Receptors, Scavenger; Viral Load; Virus Attachment

Mucopolysaccharidosis II (MPS II) is caused by a deficiency of iduronate-2-sulfatase that results in accumulation of glycosaminoglycans (GAG), including heparan sulfate (HS), which is considered to contribute to neuropathology. We examined the efficacy of intracerebroventricular (ICV) enzyme replacement therapy (ERT) of idursulfase-beta (IDS-β) and evaluated the usefulness of HS as a biomarker for neuropathology in MPS II mice. We first examined the efficacy of three different doses (3, 10, and 30 μg) of single ICV injections of IDS-β in MPS II mice. After the single-injection study, its long-term efficacy was elucidated with 30 μg of IDS-β ICV injections repeated every 4 weeks for 24 weeks. The efficacy was assessed by the HS content in the cerebrospinal fluid (CSF) and the brain of the animals along with histologic examinations and behavioral tests. In the single-injection study, the 30 μg of IDS-β ICV injection showed significant reductions of HS content in brain and CSF that were maintained for 28 days. Furthermore, HS content in CSF was significantly correlated with HS content in brain. In the long-term repeated-injection study, the HS content in the brain and CSF was also significantly reduced and correlated. The histologic examinations showed a reduction in lysosomal storage. A significant improvement in memory/learning function was observed in open-field and fear-conditioning tests. ICV ERT with 30 μg of IDS-β produced significant improvements in biochemical, histological, and functional parameters in MPS II mice. Furthermore, we demonstrate for the first time that the HS in the CSF had significant positive correlation with brain tissue HS and GAG levels, suggesting HS in CSF as a useful clinical biomarker for neuropathology.

Topics: Animals; Biomarkers; Blood-Brain Barrier; Disease Models, Animal; Enzyme Replacement Therapy; Heparitin Sulfate; Iduronate Sulfatase; Infusions, Intraventricular; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucopolysaccharidosis II

Mucopolysaccharidosis type II (MPS II or Hunter syndrome) is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS), an enzyme that catabolizes glycosaminoglycans (GAGs) including heparan sulfate (HS) and dermatan sulfate (DS). GAG accumulation leads to severe neurological and somatic impairments. At present, the most common treatment for MPS II is intravenous enzyme replacement therapy; however, the inability of recombinant IDS to cross the blood-brain barrier (BBB) restricts therapeutic efficacy for neurological manifestations. We recently developed a BBB-penetrating IDS fusion protein, JR-141, and demonstrated its ability to reduce GAG accumulation in the brain of human transferrin receptor knock-in and Ids knock-out mice (TFRC-KI/Ids-KO), an animal model of MPS II, following intravenous administration. Given the impossibility of measuring GAG accumulation in the brains of human patients with MPS II, we hypothesized that GAG content in the cerebrospinal fluid (CSF) might serve as an indicator of brain GAG burden. To test this hypothesis, we optimized a high-sensitivity method for quantifying HS and DS in low-volume samples by combining acidic methanolysis and liquid chromatography-tandem mass spectrometry (LC/MS/MS). We employed this method to quantify HS and DS in samples from TFRC-KI/Ids-KO mice and revealed that HS but not DS accumulated in the central nerve system (CNS). Moreover, concentrations of HS in CSF correlated with those in brain. Finally, intravenous treatment with JR-141 reduced levels of HS in the CSF and brain in TFRC-KI/Ids-KO mice. These results suggest that CSF HS content may be a useful biomarker for evaluating the brain GAG accumulation and the therapeutic efficacy of drugs in patients with MPS II.

Topics: Animals; Biomarkers; Blood-Brain Barrier; Brain; Chromatography, Liquid; Dermatan Sulfate; Disease Models, Animal; Heparitin Sulfate; Humans; Iduronate Sulfatase; Mice; Mice, Knockout; Mucopolysaccharidosis II; Nervous System Diseases; Receptors, Transferrin; Tandem Mass Spectrometry

Sanfilippo syndrome, MPS IIIA-D, results from deficits in lysosomal enzymes that specifically degrade heparan sulfate, a sulfated glycosaminoglycan. The accumulation of heparan sulfate results in neurological symptoms, culminating in extensive neurodegeneration and early death. To study the impact of storage in postnatal neurodevelopment, we examined murine models of MPS IIIA, which lack the enzyme sulfamidase. We show that changes occur in excitatory postsynaptic structure and function in the somatosensory cortex prior to signs of neurodegeneration. These changes coincide with accumulation of heparan sulfate with characteristic non-reducing ends, which is present at birth in the mutant mice. Accumulation of heparan sulfate was also detected in primary cultures of cortical neural cells, especially astrocytes. Accumulation of heparan sulfate in cultured astrocytes corresponded with augmented extracellular heparan sulfate and glypican 4 levels. Heparan sulfate from the cerebral cortex of MPS IIIA mice showed enhanced ability to increase glutamate AMPA receptor subunits at the cell surface of wild type neurons. These data support the idea that abnormalities in heparan sulfate content and distribution contribute to alterations in postsynaptic function. Our findings identify a disease-induced developmental phenotype that temporally overlaps with the onset of behavioral changes in a mouse model of MPS IIIA.

Topics: Animals; Cerebral Cortex; Disease Models, Animal; Heparitin Sulfate; Mice; Mice, Knockout; Mucopolysaccharidosis III; Receptors, AMPA

Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths-called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant Ext1f/f;Col2-CreER or Ext1f/f;Aggrecan-CreER mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -collagen 2 and collagen X- within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional Ext1-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and Chordin, Fgf9 and Fgf18 expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention.

Topics: Animals; Bone Morphogenetic Proteins; Cervical Cord; Chondrogenesis; Disease Models, Animal; Embryonic Development; Exostoses, Multiple Hereditary; Growth Plate; Heparitin Sulfate; Humans; Magnetic Resonance Imaging; Mice; Mice, Knockout; Mutation; N-Acetylglucosaminyltransferases; Osteochondroma; Pyrazoles; Pyrimidines; Smad1 Protein; Tomography, Emission-Computed

Intra-cerebrospinal fluid (CSF) injection of recombinant human lysosomal enzyme is a potential treatment strategy for several neurodegenerative lysosomal storage disorders including Sanfilippo syndrome (Mucopolysaccharidosis type IIIA; MPS IIIA). Here we have utilised the MPS IIIA Huntaway dog model to compare the effectiveness of the repeated intermittent bolus injection strategy being used in the trials with an alternate approach; slow, continual infusion of replacement enzyme (recombinant human sulphamidase; rhSGSH) into the spinal CSF using a SynchroMed II® pump attached to a spinal infusion cannula. The ability of each enzyme delivery strategy to ameliorate lesions in MPS IIIA brain was determined in animals treated from ∼three- to six-months of age. Controls received buffer or no treatment. Significant reductions in heparan sulphate (primary substrate) were observed in brain samples from dogs treated via either cisternal or lumbar spinal CSF bolus injection methods and also in slow intra-spinal CSF infusion-treated dogs. The extent of the reduction differed regionally. Pump-delivered rhSGSH was less effective in reducing secondary substrate (G

Topics: Animals; Cerebral Cortex; Cerebrospinal Fluid; Disease Models, Animal; Dogs; Enzyme Replacement Therapy; Heparitin Sulfate; Humans; Hydrolases; Lumbar Vertebrae; Mucopolysaccharidosis III; Neurodegenerative Diseases; Recombinant Proteins

The recycling, S-nitrosylated heparan sulfate (HS) proteoglycan glypican-1 releases anhydromannose (anMan)-containing HS chains by a nitrosothiol-catalyzed cleavage in endosomes that can be constitutive or induced by ascorbate. The HS-anMan chains are then transported to the nucleus. A specific nuclear target for HS-anMan has not been identified. We have monitored endosome-to-nucleus trafficking of HS-anMan by deconvolution and confocal immunofluorescence microscopy using an anMan-specific monoclonal antibody in non-growing, ascorbate-treated, and growing, untreated, wild-type mouse embryonic fibroblasts and hypoxia-exposed Alzheimer mouse Tg2576 fibroblasts and human U87 glioblastoma cells. In all cells, nuclear HS-anMan targeted a limited number of sites of variable size where it colocalized with DNA and nucleolin, an established marker for nucleoli. HS-anMan also colocalized with ethynyl uridine-tagged nascent RNA and two acetylated forms of histone H3. Acute hypoxia increased the formation of HS-anMan in both Tg2576 and U87 cells. A portion of HS-anMan colocalized with nucleolin at small discrete sites, while most of the nucleolin and nascent RNA was dispersed. In U87 cells, HS-anMan, nucleolin and nascent RNA reassembled after prolonged hypoxia. Nucleolar HS may modulate synthesis and/or release of rRNA.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Cell Nucleus; Disease Models, Animal; Endosomes; Glypicans; Heparitin Sulfate; Humans; Mannose; Mice; Microscopy, Confocal; Nitric Oxide; Nucleolin; Phosphoproteins; RNA-Binding Proteins

The genetic metabolic disease mucopolysaccharidosis III type C (MPS IIIC, Sanfilippo disease type C) causes progressive neurodegeneration in infants and children, leading to dementia and death before adulthood. MPS IIIC stands out among lysosomal diseases because it is the only one caused by a deficiency not of a hydrolase but of HGSNAT (heparan--glucosaminide N-acetyltransferase), which catalyzes acetylation of glycosaminoglycan heparan sulfate (HS) prior to its hydrolysis.

Topics: Animals; Autophagy; Brain; Cell Death; Disease Models, Animal; Heparitin Sulfate; Humans; Lysosomes; Mice; Mice, Knockout; Mitochondria; Mucopolysaccharidosis III; Neurons

To prolong the release of a heparan sulfate binding peptide, G2-C, using a commercially available contact lens as a delivery vehicle and to demonstrate the ability of the released peptide to block herpes simplex virus-1 (HSV-1) infection using in vitro, ex vivo, and in vivo models of corneal HSV-1 infection.. Commercially available contact lenses were immersed in peptide solution for 5 days prior to determining the release of the peptide at various time points. Cytotoxicity of the released samples was determined by MTT and cell cycle analysis, and the functional activity of the released samples were assessed by viral entry, and viral spread assay using human corneal epithelial cells (HCE). The ability to suppress infection in human and pig cornea ex vivo and mouse in vivo models were also assessed.. Peptide G2-C was released through the contact lens. Following release for 3 days, the peptide showed significant activity by inhibiting HSV-1 viral entry and spread in HCE cells. Significant suppression of infection was also observed in the ex vivo and in vivo experiments involving corneas.. Extended release of an anti-HS peptide through a commercially available contact lens can generate significant anti-HSV-1 activity and provides a new and effective way to control corneal herpes.

Topics: Animals; Contact Lenses; Cornea; Delayed-Action Preparations; Disease Models, Animal; Eye Infections, Viral; Female; Heparitin Sulfate; Herpesvirus 1, Human; Humans; Keratitis, Herpetic; Male; Mice; Mice, Inbred BALB C; Swine

There is a functional relationship between the heparan sulfate proteoglycan glypican-1 and the amyloid precursor protein (APP) of Alzheimer disease. In wild-type mouse embryonic fibroblasts, expression and processing of the APP is required for endosome-to-nucleus translocation of anhydromannose-containing heparan sulfate released from S-nitrosylated glypican-1 by ascorbate-induced, nitrosothiol-catalyzed deaminative cleavage. In fibroblasts from the transgenic Alzheimer mouse Tg2576, there is increased processing of the APP to amyloid-β peptides. Simultaneously, there is spontaneous formation of anhydromannose-containing heparan sulfate by an unknown mechanism. We have explored the effect of hypoxia on anhydromannose-containing heparan sulfate formation in wild-type and Tg2576 fibroblasts by deconvolution immunofluorescence microscopy and flow cytometry using an anhydromannose-specific monoclonal antibody and by (35)SO4-labeling experiments. Hypoxia prevented ascorbate-induced heparan sulfate release in wild-type fibroblasts, but induced an increased formation of anhydromannose-positive and (35)S-labeled heparan sulfate in Tg2576 fibroblasts. This appeared to be independent of glypican-1 S-nitrosylation as demonstrated by using a monoclonal antibody specific for S-nitrosylated glypican-1. In hypoxic wild-type fibroblasts, addition of nitrite to the medium restored anhydromannose-containing heparan sulfate formation. The increased release of anhydromannose-containing heparan sulfate in hypoxic Tg2576 fibroblasts did not require addition of nitrite. However, it was suppressed by inhibition of the nitrite reductase activity of xanthine oxidoreductase/aldehyde oxidase or by inhibition of p38 mitogen-activated protein kinase or by chelation of iron. We propose that normoxic Tg2576 fibroblasts maintain a high level of anhydromannose-containing heparan sulfate production by a stress-activated generation of nitric oxide from endogenous nitrite. This activation is enhanced by hypoxia.

Topics: Alzheimer Disease; Animals; Antibodies, Monoclonal; Ascorbic Acid; Cell Hypoxia; Deferoxamine; Disease Models, Animal; Enzyme Inhibitors; Fibroblasts; Glypicans; Heparitin Sulfate; Humans; Iron Chelating Agents; Mannose; Mice; Mice, Transgenic; Microscopy, Fluorescence; Nitric Oxide; Nitrite Reductases; Nitrites; Oxidation-Reduction; Oxygen; p38 Mitogen-Activated Protein Kinases; Primary Cell Culture

Previous research has shown that podocytes unable to assemble heparan sulfate on cell surface proteoglycan core proteins have compromised cell-matrix interactions. This report further explores the role of N-sulfation of intact heparan chains in podocyte-matrix interactions. For the purposes of this study, a murine model in which the enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) was specifically deleted in podocytes and immortalized podocyte cell lines lacking NDST1 were developed and used to explore the effects of such a mutation on podocyte behavior in vitro. NDST1 is a bifunctional enzyme, ultimately responsible for N-sulfation of heparan glycosaminoglycans produced by cells. Immunostaining of glomeruli from mice whose podocytes were null for Ndst1 (Ndst1(-/-)) showed a disrupted pattern of localization for the cell surface proteoglycan, syndecan-4, and for α-actinin-4 compared with controls. The pattern of immunostaining for synaptopodin and nephrin did not show as significant alterations. In vitro studies showed that Ndst1(-/-) podocytes attached, spread, and migrated less efficiently than Ndst1(+/+) podocytes. Immunostaining in vitro for several markers for molecules involved in cell-matrix interactions showed that Ndst1(-/-) cells had decreased clustering of syndecan-4 and decreased recruitment of protein kinase-Cα, α-actinin-4, vinculin, and phospho-focal adhesion kinase to focal adhesions. Total intracellular phospho-focal adhesion kinase was decreased in Ndst1(-/-) compared with Ndst1(+/+) cells. A significant decrease in the abundance of activated integrin α5β1 on the cell surface of Ndst1(-/-) cells compared with Ndst1(+/+) cells was observed. These results serve to highlight the critical role of heparan sulfate N-sulfation in facilitating normal podocyte-matrix interactions.

Topics: Actinin; Animals; Cell Adhesion; Cell Movement; Cells, Cultured; Cytoskeleton; Disease Models, Animal; Extracellular Matrix; Focal Adhesions; Glomerular Basement Membrane; Heparitin Sulfate; Integrin alpha5beta1; Mice; Mice, Transgenic; Podocytes; Sulfotransferases; Syndecan-4

Mucopolysaccharidosis type IIIA (MPS IIIA) is predominantly a disorder of the central nervous system, caused by a deficiency of sulfamidase (SGSH) with subsequent storage of heparan sulfate-derived oligosaccharides. No widely available therapy exists, and for this reason, a mouse model has been utilized to carry out a preclinical assessment of the benefit of intraparenchymal administration of a gene vector (AAVrh10-SGSH-IRES-SUMF1) into presymptomatic MPS IIIA mice. The outcome has been assessed with time, measuring primary and secondary storage material, neuroinflammation, and intracellular inclusions, all of which appear as the disease progresses. The vector resulted in predominantly ipsilateral distribution of SGSH, with substantially less detected in the contralateral hemisphere. Vector-derived SGSH enzyme improved heparan sulfate catabolism, reduced microglial activation, and, after a time delay, ameliorated GM3 ganglioside accumulation and halted ubiquitin-positive lesion formation in regions local to, or connected by projections to, the injection site. Improvements were not observed in regions of the brain distant from, or lacking connections with, the injection site. Intraparenchymal gene vector administration therefore has therapeutic potential provided that multiple brain regions are targeted with vector, in order to achieve widespread enzyme distribution and correction of disease pathology.

Topics: Animals; Antibodies, Neutralizing; Autophagy; Biomarkers; Brain; Dependovirus; Disease Models, Animal; DNA-Binding Proteins; Endosomes; Enzyme Activation; Female; G(M3) Ganglioside; Genetic Therapy; Genetic Vectors; Glial Fibrillary Acidic Protein; Heparitin Sulfate; High Mobility Group Proteins; Humans; Hydrolases; Lysosomes; Male; Mice; Mucopolysaccharidosis III; Saccharomyces cerevisiae Proteins; Transduction, Genetic

Accumulation of amyloid-β (Aβ) peptide in the brain is the first critical step in the pathogenesis of Alzheimer's disease (AD). Studies in humans suggest that Aβ clearance from the brain is frequently impaired in late-onset AD. Aβ accumulation leads to the formation of Aβ aggregates, which injure synapses and contribute to eventual neurodegeneration. Cell surface heparan sulfates (HSs), expressed on all cell types including neurons, have been implicated in several features in the pathogenesis of AD including its colocalization with amyloid plaques and modulatory role in Aβ aggregation. We show that removal of neuronal HS by conditional deletion of the Ext1 gene, which encodes an essential glycosyltransferase for HS biosynthesis, in postnatal neurons of amyloid model APP/PS1 mice led to a reduction in both Aβ oligomerization and the deposition of amyloid plaques. In vivo microdialysis experiments also detected an accelerated rate of Aβ clearance in the brain interstitial fluid, suggesting that neuronal HS either inhibited or represented an inefficient pathway for Aβ clearance. We found that the amounts of various HS proteoglycans (HSPGs) were increased in postmortem human brain tissues from AD patients, suggesting that this pathway may contribute directly to amyloid pathogenesis. Our findings have implications for AD pathogenesis and provide insight into therapeutic interventions targeting Aβ-HSPG interactions.

Topics: Aged, 80 and over; Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Animals; Disease Models, Animal; Heparitin Sulfate; Hippocampus; Humans; Inflammation; Mice; Neurons; Protein Aggregation, Pathological

The endothelial glycocalyx plays an essential role in many physiological functions and is damaged after hemorrhage. Fluid resuscitation may further change the glycocalyx after an initial hemorrhage-induced degradation. Plasma levels of syndecan-1 and heparan sulfate have been used as indirect markers for glycocalyx degradation, but the extent to which these measures are representative of the events in the microcirculation is unknown. Using hemorrhage and a wide range of resuscitation fluids, we studied quantitatively the relationship between plasma biomarkers and changes in microvascular parameters, including glycocalyx thickness. Rats were bled 40% of total blood volume and resuscitated with seven different fluids (fresh whole blood, blood products, and crystalloids). Intravital microscopy was used to estimate glycocalyx thickness in >270 postcapillary venules from 58 cremaster preparations in 9 animal groups; other microvascular parameters were measured using noninvasive techniques. Systemic physiological parameters and blood chemistry were simultaneously collected. Changes in glycocalyx thickness were negatively correlated with changes in plasma levels of syndecan-1 (r = -0.937) and heparan sulfate (r = -0.864). Changes in microvascular permeability were positively correlated with changes in both plasma biomarkers (r = 0.8, P < 0.05). Syndecan-1 and heparan sulfate were also positively correlated (r = 0.7, P < 0.05). Except for diameter and permeability, changes in local microcirculatory parameters (red blood cell velocity, blood flow, and wall shear rate) did not correlate with plasma biomarkers or glycocalyx thickness changes. This work provides a quantitative framework supporting plasma syndecan-1 and heparan sulfate as valuable clinical biomarkers of glycocalyx shedding that may be useful in guiding resuscitation strategies following hemorrhage.

Topics: Abdominal Muscles; Animals; Biomarkers; Capillary Permeability; Dextrans; Disease Models, Animal; Fluorescein-5-isothiocyanate; Glycocalyx; Heparitin Sulfate; Male; Plasma Substitutes; Proteolysis; Rats, Sprague-Dawley; Resuscitation; Shock, Hemorrhagic; Syndecan-1; Venules

In cancer, proteoglycans have been found to play roles in facilitating the actions of growth factors, and effecting matrix invasion and remodeling. However, little is known regarding the genetic and functional importance of glycan chains displayed by proteoglycans on dendritic cells (DCs) in cancer immunity. In lung carcinoma, among other solid tumors, tumor-associated DCs play largely subversive/suppressive roles, promoting tumor growth and progression. Herein, we show that targeting of DC glycan sulfation through mutation in the heparan sulfate biosynthetic enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1) in mice increased DC maturation and inhibited trafficking of DCs to draining lymph nodes. Lymphatic-driven DC migration and chemokine (CCL21)-dependent activation of a major signaling pathway required for DC migration (as measured by phospho-Akt) were sensitive to Ndst1 mutation in DCs. Lewis lung carcinoma tumors in mice deficient in Ndst1 were reduced in size. Purified CD11c+ cells from the tumors, which contain the tumor-infiltrating DC population, showed a similar phenotype in mutant cells. These features were replicated in mice deficient in syndecan-4, the major heparan sulfate proteoglycan expressed on the DC surface: Tumors were growth-impaired in syndecan-4-deficient mice and were characterized by increased infiltration by mature DCs. Tumors on the mutant background also showed greater infiltration by NK cells and NKT cells. These findings indicate the genetic importance of DC heparan sulfate proteoglycans in tumor growth and may guide therapeutic development of novel strategies to target syndecan-4 and heparan sulfate in cancer.

Topics: Animals; Cell Movement; Cell Proliferation; Chemokines; Dendritic Cells; Disease Models, Animal; Endothelial Cells; Heparitin Sulfate; Humans; Immunophenotyping; Mice; Mice, Transgenic; Mutation; Neoplasms; Phenotype; Proteoglycans; Syndecan-4; Tumor Burden

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by the deficient activity of iduronate 2-sulfatase (IDS), which is involved in the lysosomal catabolism of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. Such a deficiency leads to the accumulation of undegraded GAGs in some organs. Although enzyme replacement therapy is available as a treatment of MPS II, there are some limitations, such as the requirement of weekly administration for whole life. To avoid such limitations, hematopoietic cell transplantation (HSCT) is a possible alternative. In fact, some report suggested positive effects of HSCT for MPS II. However, HSCT has also some limitations. Strong conditioning regimens can cause severe side effects. For overcome this obstacle, we studied the efficacy of ACK2, an antibody that blocks KIT, followed by low-dose irradiation as a preconditioning regimen for HSCT using a murine model of MPS II. This protocol achieves 58.7±4.92% donor chimerism at 16weeks after transplantation in the peripheral blood of recipient mice. GAG levels were significantly reduced in liver, spleen, heart and intestine. These results indicated that ACK2-based preconditioning might be one of the choices for MPS II patients who receive HSCT.

Topics: Animals; Antibodies, Anti-Idiotypic; Bone Marrow Transplantation; Dermatan Sulfate; Disease Models, Animal; Glycoproteins; Heparitin Sulfate; Humans; Lysosomes; Mice; Mice, Knockout; Mucopolysaccharidosis II; Proto-Oncogene Proteins c-kit

Transglutaminase-2 (TG2) is a new anti-fibrotic target for chronic kidney disease, for its role in altering the extracellular homeostatic balance leading to excessive build-up of matrix in kidney. However, there is no confirmation that TG2 is the only transglutaminase involved, neither there are strategies to control its action specifically over that of the conserved family-members. In this study, we have profiled transglutaminase isozymes in the rat subtotal nephrectomy (SNx) model of progressive renal scarring. All transglutaminases increased post-SNx peaking at loss of renal function but TG2 was the predominant enzyme. Upon SNx, extracellular TG2 deposited in the tubulointerstitium and peri-glomerulus via binding to heparan sulphate (HS) chains of proteoglycans and co-associated with syndecan-4. Extracellular TG2 was sufficient to activate transforming growth factor-β1 in tubular epithelial cells, and this process occurred in a HS-dependent way, in keeping with TG2-affinity for HS. Analysis of heparin binding of the main transglutaminases revealed that although the interaction between TG1 and HS is strong, the conformational heparin binding site of TG2 is not conserved, suggesting that TG2 has a unique interaction with HS within the family. Our data provides a rationale for a novel anti-fibrotic strategy specifically targeting the conformation-dependent TG2-epitope interacting with HS.

Topics: Animals; Binding Sites; Cell Line; Disease Models, Animal; Gene Expression Regulation; Glomerulonephritis; GTP-Binding Proteins; Heparitin Sulfate; Kidney Function Tests; Mice; NIH 3T3 Cells; Protein Glutamine gamma Glutamyltransferase 2; Rats; Syndecan-4; Transforming Growth Factor beta1; Transglutaminases

Injection of lysosomal enzyme into cisternal or ventricular cerebrospinal fluid (CSF) has been carried out in 11 lysosomal storage disorder models, with each study demonstrating reductions in primary substrate and secondary neuropathological changes, and several reports of improved neurological function. Whilst acute studies in mucopolysaccharidosis (MPS) type II mice revealed that intrathecally-delivered enzyme (into thoraco-lumbar CSF) accesses the brain, the impact of longer-term treatment of affected subjects via this route is unknown. This approach is presently being utilized to treat children with MPS types I, II and III. Our aim was to determine the efficacy of repeated intrathecal injection of recombinant human sulfamidase (rhSGSH) on pathological changes in the MPS IIIA dog brain. The outcomes were compared with those in dogs treated via intra-cisternal or ventricular routes. Control dogs received buffer or no treatment. Significant reductions in primary/secondary substrate levels in brain were observed in dogs treated via all routes, although the extent of the reduction differed regionally. Treatment via all CSF access points resulted in large reductions in microgliosis in superficial cerebral cortex, but only ventricular injection enabled amelioration in deep cerebral cortex. Formation of glutamic acid decarboxylase-positive axonal spheroids in deep cerebellar nuclei was prevented by treatment delivered via any route. Anti-rhSGSH antibodies in the sera of some dogs did not reduce therapeutic efficacy. Our data indicates the capacity of intra-spinal CSF-injected rhSGSH to circulate within CSF-filled spaces, penetrate into brain and mediate a significant reduction in substrate accumulation and secondary pathology in the MPS IIIA dog brain.

Topics: Animals; Chromatography, Liquid; Disease Models, Animal; Dogs; Enzyme-Linked Immunosorbent Assay; Heparitin Sulfate; Humans; Hydrolases; Immunohistochemistry; Injections, Spinal; Mass Spectrometry; Mucopolysaccharidoses; Recombinant Proteins

Evaluating anticoagulants in animal thrombosis models is a standard component of preclinical drug testing. Mice are frequently used for these initial evaluations because a variety of thrombosis models have been developed and are well characterized in this species, and the animals are relatively inexpensive to maintain. Because mice have a natural resistance to forming intravascular thrombi, vessel injury is required to induce intravascular clot formation. Several methods have been established for inducing arterial or venous thrombosis in mice. For the purpose of testing heparin-based drugs, we adapted a well-established model in which thrombus formation in the carotid artery is induced by exposing the vessel to ferric chloride. For studying anticoagulant effects on venous thrombosis, we use a model in which the inferior vena cava is ligated and the size of the resulting clots is measured. The most common adverse effect of anticoagulation therapy is bleeding. The effect of heparin-based anticoagulants can be tested in mice in a simple tail bleeding assay.

Topics: Animals; Anticoagulants; Chlorides; Disease Models, Animal; Ferric Compounds; Hemorrhage; Heparitin Sulfate; Mice, Inbred C57BL; Tail; Thrombosis; Vena Cava, Inferior

Intracerebrospinal fluid (CSF) infusion of replacement enzyme is under evaluation for amelioration of disease-related symptoms and biomarker changes in patients with the lysosomal storage disorder mucopolysaccharidosis type IIIA (MPS IIIA; www.clinicaltrials.gov ; NCT#01155778; #01299727). Determining the optimal dose/dose-frequency is important, given the invasive method for chronically supplying recombinant protein to the brain, the main site of symptom generation. To examine these variables, we utilised MPS IIIA Huntaway dogs, providing recombinant human sulphamidase (rhSGSH) to young pre-symptomatic dogs from an age when MPS IIIA dog brain exhibits significant accumulation of primary (heparan sulphate) and secondary (glycolipid) substrates. Enzyme was infused into CSF via the cisterna magna at one of two doses (3 mg or 15 mg/infusion), with the higher dose supplied at two different intervals; fortnightly or monthly. Euthanasia was carried out 24 h after the final injection. Dose- and frequency-dependent reductions in heparan sulphate were observed in CSF and deeper layers of cerebral cortex. When we examined the amount of immunostaining of the general endo/lysosomal marker, LIMP-2, or quantified activated microglia, the higher fortnightly dose resulted in superior outcomes in affected dogs. Secondary lesions such as accumulation of GM3 ganglioside and development of GAD-reactive axonal spheroids were treated to a similar degree by both rhSGSH doses and dose frequencies. Our findings indicate that the lower fortnightly dose is sub-optimal for ameliorating existing and preventing further development of disease-related pathology in young MPS IIIA dog brain; however, increasing the dose fivefold but halving the frequency of administration enabled near normalisation of disease-related biomarkers.

Topics: Animals; Biomarkers; Brain; Disease Models, Animal; Dogs; Drug Administration Schedule; Drug Dosage Calculations; Enzyme Replacement Therapy; Glycolipids; Heparitin Sulfate; Hydrolases; Infusions, Intraventricular; Lysosomal-Associated Membrane Protein 2; Mucopolysaccharidosis III; Recombinant Proteins; Time Factors

Malfunctioning of the intestinal microcirculation secondary to severe acute pancreatitis (SAP) can cause injuries to the intestinal mucosal barrier, translocation of gut flora, and sepsis. The glycocalyx on the vascular endothelium helps maintain its normal function through multiple mechanisms, including regulation of vascular permeability and inhibition of intercellular adhesion. It is unknown that whether pancreatitis inflicts injuries to the intestinal mucosal barrier through damaging glycocalyx or stabilizing glycocalyx can be a potential therapeutic target in maintaining the integrity of the intestinal mucosal barrier during SAP. Injecting sodium taurocholate into the pancreatic duct of Sprague-Dawley rats induced SAP. Intestinal perfusion, changes in endothelial glycocalyx, and the associated molecular mechanisms were assessed by laser Doppler velocimetry, electron microscopy, and the levels of heparan sulfate, syndacan-1, and tumor necrosis factor-α (TNF-α) in the superior mesenteric vein. Protective effects of hydrocortisone treatment in the intestinal microcirculation during SAP were evaluated. Degradation of the glycocalyx in intestinal vascular endothelium developed 3 h after the onset of SAP in rats. By 12 h, significant reduction of intestinal perfusion was observed. The concomitant elevated levels of TNF-α in the superior mesenteric vein suggest that TNF-α is involved in the degradation of the glycocalyx. With the use of hydrocortisone, intestinal perfusion was improved and the degradation of glycocalyx was reduced. The degradation of glycocalyx is involved in the malfunction of the intestinal microcirculation. The massive release of TNF-α participates in this process and leads to glycocalyx degradation. Hydrocortisone may be a good therapy to prevent this process.

Topics: Animals; Cell Adhesion; Disease Models, Animal; Endothelium, Vascular; Glycocalyx; Heparitin Sulfate; Hydrocortisone; Intestines; Male; Mesenteric Veins; Microcirculation; Pancreatitis; Perfusion; Permeability; Rats; Rats, Sprague-Dawley; Sepsis; Syndecan-1; Taurocholic Acid; Time Factors; Tumor Necrosis Factor-alpha

Circulation of leukocytes via blood, tissue and lymph is integral to adaptive immunity. Afferent lymphatics form CCL21 gradients to guide dendritic cells and T cells to lymphatics and then to draining lymph nodes (dLN). Vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 (VEGFR-3) are the major lymphatic growth factor and receptor. We hypothesized these molecules also regulate chemokine gradients and lymphatic migration.. CD4 T cells were injected into the foot pad or ear pinnae, and migration to afferent lymphatics and dLN quantified by flow cytometry or whole mount immunohistochemistry. Vascular endothelial growth factor receptor 3 or its signaling or downstream actions were modified with blocking monoclonal antibodies (mAbs) or other reagents.. Anti-VEGFR-3 prevented migration of CD4 T cells into lymphatic lumen and significantly decreased the number that migrated to dLN. Anti-VEGFR-3 abolished CCL21 gradients around lymphatics, although CCL21 production was not inhibited. Heparan sulfate (HS), critical to establish CCL21 gradients, was down-regulated around lymphatics by anti-VEGFR-3 and this was dependent on heparanase-mediated degradation. Moreover, a Phosphoinositide 3-kinase (PI3K)α inhibitor disrupted HS and CCL21 gradients, whereas a PI3K activator prevented the effects of anti-VEGFR-3. During contact hypersensitivity, VEGFR-3, CCL21, and HS expression were all attenuated, and anti-heparanase or PI3K activator reversed these effects.. Vascular endothelial growth factor C/VEGFR-3 signaling through PI3Kα regulates the activity of heparanase, which modifies HS and CCL21 gradients around lymphatics. The functional and physical linkages of these molecules regulate lymphatic migration from tissues to dLN. These represent new therapeutic targets to influence immunity and inflammation.

Topics: Adoptive Transfer; Animals; Antibodies, Monoclonal; CD4-Positive T-Lymphocytes; Chemokine CCL21; Chemotaxis, Leukocyte; Dermatitis, Contact; Dinitrofluorobenzene; Disease Models, Animal; Enzyme Activation; Enzyme Activators; Glucuronidase; Heparitin Sulfate; Lymph Nodes; Lymphatic Vessels; Mice, Inbred C57BL; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Signal Transduction; Skin; Vascular Endothelial Growth Factor C; Vascular Endothelial Growth Factor Receptor-3

MPS IIIA is an inherited neurodegenerative lysosomal storage disorder characterized by cognitive impairment, sleep-wake cycle disturbance, speech difficulties, eventual mental regression and early death. Neuropathological changes include accumulation of heparan sulfate and glycolipids, neuroinflammation and degeneration. Pre-clinical animal studies indicate that replacement of the deficient enzyme, sulfamidase, via intra-cerebrospinal fluid (CSF) injection is a clinically-relevant treatment approach, reducing neuropathological changes and improving symptoms. Given that there are several routes of administration of enzyme into the CSF (intrathecal lumbar, cisternal and ventricular), determining the effectiveness of each injection strategy is crucial in order to provide the best outcome for patients. We delivered recombinant human sulfamidase (rhSGSH) to a congenic mouse model of MPS IIIA via each of the three routes. Mice were euthanized 24h or one-week post-injection; the distribution of enzyme within the brain and spinal cord parenchyma was investigated, and the impact on primary substrate levels and other pathological lesions determined. Both ventricular and cisternal injection of rhSGSH enable enzyme delivery to brain and spinal cord regions, with the former mediating large, statistically significant decreases in substrate levels and reducing microglial activation. The single lumbar CSF infusion permitted more restricted enzyme delivery, with no reduction in substrate levels and little change in other disease-related lesions in brain tissue. While the ventricular route is the most invasive of the three methods, this strategy may enable the widest distribution of enzyme within the brain, and thus requires further exploration.

Topics: Animals; Brain; Cisterna Magna; Disease Models, Animal; Drug Administration Routes; Enzyme Replacement Therapy; Heparitin Sulfate; Humans; Hydrolases; Infusions, Intraventricular; Infusions, Spinal; Injections; Injections, Intraventricular; Mice; Mice, Congenic; Mice, Inbred C57BL; Mucopolysaccharidosis III; Recombinant Proteins

Islet transplantation is a promising therapy for patients with diabetes, but its long-term success is limited by many factors, including the formation of islet amyloid deposits. Heparin is employed in clinical islet transplantation to reduce clotting but also promotes fibrillization of amyloidogenic proteins. We hypothesized that heparin treatment of islets during pre-transplant culture may enhance amyloid formation leading to beta cell loss and graft dysfunction. Heparin promoted the fibrillization of human islet amyloid polypeptide (IAPP) and enhanced its toxicity to INS-1 beta cells. Heparin increased amyloid deposition in cultured human islets, but surprisingly decreased islet cell apoptosis. Treatment of human islets with heparin prior to transplantation increased the likelihood of graft failure. Removal of islet heparan sulfate glycosaminoglycans, which localize with islet amyloid deposits in type 2 diabetes, by heparinase treatment decreased amyloid deposition and protected against islet cell death. These findings raise the possibility that pretransplant treatment of human islets with heparin could potentiate IAPP aggregation and amyloid formation and may be detrimental to subsequent graft function.

Topics: Amyloid; Animals; Apoptosis; Cells, Cultured; Diabetes Mellitus, Experimental; Disease Models, Animal; Dose-Response Relationship, Drug; Graft Rejection; Heparin; Heparin Lyase; Heparitin Sulfate; Humans; Islet Amyloid Polypeptide; Islets of Langerhans; Islets of Langerhans Transplantation; Mice, Inbred NOD; Mice, SCID; Streptozocin

Islet beta cells in situ express intracellular heparan sulfate (HS), a property previously shown in vitro to be important for their survival. We report that HS levels inside islet beta cells correlate with the novel intracellular localization of the HSPG core proteins for collagen type XVIII (Col18), a conventional extracellular matrix component. Syndecan-1 (Sdc1) and CD44 core proteins were similarly localized inside beta cells. During isolation, mouse islets selectively lose HS to 11-27% of normal levels but retain their HSPG core proteins. Intra-islet HS failed to recover substantially during culture for 4 days and was not reconstituted in vitro using HS mimetics. In contrast, significant recovery of intra-islet HS to ∼40-50% of normal levels occurred by 5-10 days after isotransplantation. Loss of islet HS during the isolation procedure is independent of heparanase (a HS-degrading endoglycosidase) and due, in part, to oxidative damage. Treatment with antioxidants reduced islet cell death by ∼60% and increased the HS content of isolated islets by ∼twofold compared to untreated islets, preserving intra-islet HS to ∼60% of the normal HS content of islets in situ. These findings suggest that the preservation of islet HS during the islet isolation process may optimize islet survival posttransplant.

Topics: Animals; Biopsy, Needle; Cell Survival; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; Flow Cytometry; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunohistochemistry; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity

The glomerular filtration barrier consists of podocytes, the glomerular basement membrane, and endothelial cells covered with a glycocalyx. Heparan sulphate (HS) in the glomerular filtration barrier is reduced in patients with proteinuria, which is associated with increased expression of the HS-degrading enzyme heparanase. Previously, we showed that heparanase is essential for the development of proteinuria in experimental diabetic nephropathy. Vitamin D supplementation reduces podocyte loss and proteinuria in vitro and in vivo. Therefore, we hypothesize that vitamin D reduces proteinuria by reducing glomerular heparanase. Adriamycin-exposed rats developed proteinuria and showed increased heparanase expression, which was reduced by 1,25-dihydroxyvitamin D3 (1,25-D3) treatment. In vitro, adriamycin increased heparanase mRNA in the podocyte, which could be corrected by 1,25-D3 treatment. In addition, 1,25-D3 treatment reduced transendothelial albumin passage after adriamycin stimulation. In line with these results, we showed direct binding of the vitamin D receptor to the heparanase promoter, and 1,25-D3 dose-dependently reduced heparanase promoter activity. Finally, 1,25-D3-deficient 25-hydroxy-1α-hydroxylase knockout mice developed proteinuria and showed increased heparanase, which was normalized by 1,25-D3 treatment. Our data suggest that the protective effect of vitamin D on the development of proteinuria is mediated by inhibiting heparanase expression in the podocyte.

Topics: Animals; Calcitriol; Chromatin Immunoprecipitation; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Glucuronidase; Heparitin Sulfate; Mice; Mice, Knockout; Podocytes; Proteinuria; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction

Cigarette smoke induces injury and neutrophilic inflammation in the airways of smokers. The stability and activity of inflammatory effectors, IL8 and neutrophil elastase (NE), can be prolonged by binding to airway heparan sulfate (HS)/syndecan-1, posing risk for developing chronic obstructive pulmonary disease(COPD). We hypothesize that antagonizing HS/syndecan-1 binding of the inflammatory effectors could reduce smoking-related neutrophil-mediated airway inflammation. Analysis of bronchoalveolar lavage fluid(BALF) of COPD patients found both total and unopposed NE levels to be significantly higher among smokers with COPD than non-COPD subjects. Similar NE burden was observed in smoke-exposed rats compared to sham air controls. We chose sulfated-maltoheptaose(SM), a heparin-mimetic, to antagonize HS/sydecan-1 binding of the inflammatory mediators in airway fluids and lung tissues of the smoke-exposed rat model. Airway treatment with SM resulted in displacement of CINC-1 and NE from complexation with bronchio-epithelial HS/syndecan-1, dissipating the chemokine gradient for neutrophil flux across to the bronchial lumen. Following SM displacement of NE from shed HS/syndecan-1 in bronchial fluids, NE became accessible to inhibition by α1-antitrypsin endogenous in test samples. The antagonistic actions of SM against syndecan-1 binding of NE and CINC-1 in smoke-exposed airways suggest new therapeutic opportunities for modulating airway inflammation in smokers with SM delivery.

Topics: Aged; alpha 1-Antitrypsin; Animals; Bronchi; Bronchoalveolar Lavage Fluid; Case-Control Studies; Chemokine CXCL1; Chitosan; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Glucans; Heparitin Sulfate; Humans; Inflammation; Inflammation Mediators; Leukocyte Elastase; Male; Middle Aged; Neutrophils; Peroxidase; Pulmonary Disease, Chronic Obstructive; Rats; Rats, Sprague-Dawley; Smoking; Syndecan-1

The identification and validation of gene-gene interactions is a major challenge in human studies. Here, we explore an approach for studying epistasis in humans using a Drosophila melanogaster model of neonatal diabetes mellitus. Expression of the mutant preproinsulin (hINS(C96Y)) in the eye imaginal disc mimics the human disease: it activates conserved stress-response pathways and leads to cell death (reduction in eye area). Dominant-acting variants in wild-derived inbred lines from the Drosophila Genetics Reference Panel produce a continuous, highly heritable distribution of eye-degeneration phenotypes in a hINS(C96Y) background. A genome-wide association study (GWAS) in 154 sequenced lines identified a sharp peak on chromosome 3L, which mapped to a 400-bp linkage block within an intron of the gene sulfateless (sfl). RNAi knockdown of sfl enhanced the eye-degeneration phenotype in a mutant-hINS-dependent manner. RNAi against two additional genes in the heparan sulfate (HS) biosynthetic pathway (ttv and botv), in which sfl acts, also modified the eye phenotype in a hINS(C96Y)-dependent manner, strongly suggesting a novel link between HS-modified proteins and cellular responses to misfolded proteins. Finally, we evaluated allele-specific expression difference between the two major sfl-intronic haplotypes in heterozygtes. The results showed significant heterogeneity in marker-associated gene expression, thereby leaving the causal mutation(s) and its mechanism unidentified. In conclusion, the ability to create a model of human genetic disease, map a QTL by GWAS to a specific gene, and validate its contribution to disease with available genetic resources and the potential to experimentally link the variant to a molecular mechanism demonstrate the many advantages Drosophila holds in determining the genetic underpinnings of human disease.

Topics: Alleles; Animals; Animals, Genetically Modified; Diabetes Mellitus; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Epistasis, Genetic; Eye; Female; Gene Expression; Gene Knockdown Techniques; Genetic Variation; Genome-Wide Association Study; Heparitin Sulfate; Humans; Introns; Male; Mutation; Phenotype; Proinsulin; Protein Folding; RNA Interference; Sulfotransferases

Growth factors (GFs) are critical in tissue repair, but their translation to clinical use has been modest. Physiologically, GF interactions with extracellular matrix (ECM) components facilitate localized and spatially regulated signaling; therefore, we reasoned that the lack of ECM binding in their clinically used forms could underlie the limited translation. We discovered that a domain in placenta growth factor-2 (PlGF-2(123-144)) binds exceptionally strongly and promiscuously to ECM proteins. By fusing this domain to the GFs vascular endothelial growth factor-A, platelet-derived growth factor-BB, and bone morphogenetic protein-2, we generated engineered GF variants with super-affinity to the ECM. These ECM super-affinity GFs induced repair in rodent models of chronic wounds and bone defects that was greatly enhanced as compared to treatment with the wild-type GFs, demonstrating that this approach may be useful in several regenerative medicine applications.

Topics: Animals; Becaplermin; Bone Morphogenetic Protein 2; Disease Models, Animal; Extracellular Matrix; Extracellular Matrix Proteins; Heparitin Sulfate; Humans; Intercellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Placenta Growth Factor; Pregnancy Proteins; Protein Engineering; Protein Structure, Tertiary; Proto-Oncogene Proteins c-sis; Vascular Endothelial Growth Factor A; Wound Healing

Determination of genotype can be difficult, especially during the early stages of developing an animal model, e.g. when PCR primers are not yet available. An increase or decrease in specific metabolites can be used as a surrogate marker for genotype; for instance, in homozygous MPS IIIA mice heparan sulphate (HS) is increased.. A simple method was developed for extracting and depolymerising HS from mouse toe tissue using methanolysis under acidic conditions. The sample was lyophilised and resuspended in methanolic HCl. The reaction products are desulphated disaccharides and readily analysable by liquid chromatography/tandem mass spectrometry (LC/MS/MS) in positive ion multiple reaction monitoring mode. Measurements were normalised to a spiked deuterated HS internal standard and to endogenous chondroitin sulphate (CS).. HS was measured in toe tissue taken from 30 mice in three groups of 10 (normal controls, MPS IIIA homozygotes and heterozygotes). A significant difference was observed between the MPS IIIA homozygotes and the other two groups, making it possible to identify mice with the MPS IIIA genotype based on the measurement of HS. Normalisation to CS was shown to correct for sample variability and reaction efficiency.. Analysis of toe tissue provides a simple and rapid way of determining a storage phenotype at 5 to 7 days of age. Significantly, this method does not require any additional samples to be taken from animals, as it utilises tissue that is a by-product of toe clipping, a method that is routinely used to permanently identify mice.

Topics: Animals; Chromatography, Liquid; Disease Models, Animal; Heparitin Sulfate; Heterozygote; Homozygote; Linear Models; Mice; Mucopolysaccharidosis III; Phenotype; Reproducibility of Results; Tandem Mass Spectrometry; Toes

The glomerular endothelial glycocalyx is postulated to be an important modulator of permeability and inflammation. The glycocalyx consists of complex polysaccharides, the main functional constituent of which, heparan sulfate (HS), is synthesized and modified by multiple enzymes. The N-deacetylase-N-sulfotransferase (Ndst) enzymes initiate and dictate the modification process. Here we evaluated the effects of modulation of HS in the endothelial glycocalyx on albuminuria and glomerular leukocyte influx using mice deficient in endothelial and leukocyte Ndst1 (TEKCre+/Ndst1flox/flox). In these mice, glomerular expression of a specific HS domain was significantly decreased, whereas the expression of other HS domains was normal. In the endothelial glycocalyx, this specific HS structure was not associated with albuminuria or with changes in renal function. However, glomerular leukocyte influx was significantly reduced during antiglomerular basement membrane nephritis, which was associated with less glomerular injury and better renal function. In vitro decreased adhesion of wild-type and Ndst1-deficient granulocytes to Ndst1-silenced glomerular endothelial cells was found, accompanied by a decreased binding of chemokines and L-selectin. Thus, modulation of HS in the glomerular endothelial glycocalyx significantly reduced the inflammatory response in antiglomerular basement membrane nephritis.

Topics: Animals; Anti-Glomerular Basement Membrane Disease; Autoantibodies; Cell Adhesion; Cell Line; Chemokines; Chemotaxis, Leukocyte; Coculture Techniques; Disease Models, Animal; Down-Regulation; Endothelial Cells; Female; Glycocalyx; Heparitin Sulfate; Kidney Glomerulus; L-Selectin; Leukocytes; Male; Mice, Inbred C57BL; Mice, Knockout; RNA Interference; Signal Transduction; Sulfotransferases; Time Factors; Transfection

Identification of carbohydrate sequences that determine affinity to specific chemokines is a critical step for strategies to interfere with chemokine-mediated leukocyte trafficking. Here, we first characterized the development of allergic asthma in Tie2-dependent and inducible Ext1-knockout (Tie2-Ext1(iKO)) mice. We showed that heparan sulfate is essential for leukocyte recruitment in the peribronchial region and bronchoalveolar lavage fluid (BALF), and is crucial for induction of airway hyperresponsiveness. Our glycan microarray showed a unique affinity profile of chemokine CCL20 to substructures of heparin and heparin-like oligo/di/monosaccharides. Among them, we identified a synthetic and not naturally occurring monosaccharide, 2,4-O-di-sulfated iduronic acid (Di-S-IdoA), as a potential inhibitor for CCL20-heparan sulfate interaction. Mice injected with Di-S-IdoA via tail vain or nasal inhalation showed attenuated leukocyte recruitment into inflammatory sites and BALF. These results demonstrate a critical role of chemokine-heparan sulfate interaction in the asthma development and Di-S-IdoA as a potential drug for asthma treatment.

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Carbohydrate Sequence; Chemokine CCL20; Chemotaxis; Disease Models, Animal; Eosinophils; Heparitin Sulfate; Iduronic Acid; Lung; Mice; Mice, Knockout; N-Acetylglucosaminyltransferases; Ovalbumin; Polysaccharides; Receptor, TIE-2; Sulfates; T-Lymphocytes

Heparan sulfate (HS) plays a crucial role in the fibrosis associated with chronic allograft dysfunction by binding and presenting cytokines and growth factors to their receptors. These interactions critically depend on the distribution of 6-O-sulfated glucosamine residues, which is generated by glucosaminyl-6-O-sulfotransferases (HS6STs) and selectively removed by cell surface HS-6-O-endosulfatases (SULFs). Using human renal allografts we found increased expression of 6-O-sulfated HS domains in tubular epithelial cells during chronic rejection as compared with the controls. Stimulation of renal epithelial cells with TGF-β induced SULF2 expression. To examine the role of 6-O-sulfated HS in the development of fibrosis, we generated stable HS6ST1 and SULF2 overexpressing renal epithelial cells. Compared with mock transfectants, the HS6ST1 transfectants showed significantly increased binding of FGF2 (p = 0.0086) and pERK activation. HS6ST1 transfectants displayed a relative increase in mono-6-O-sulfated disaccharides accompanied by a decrease in iduronic acid 2-O-sulfated disaccharide structures. In contrast, SULF2 transfectants showed significantly reduced FGF2 binding and phosphorylation of ERK. Structural analysis of HS showed about 40% down-regulation in 6-O-sulfation with a parallel increase in iduronic acid mono-2-O-sulfated disaccharides. To assess the relevance of these data in vivo we established a murine model of fibrosis (unilateral ureteric obstruction (UUO)). HS-specific phage display antibodies (HS3A8 and RB4EA12) showed significant increase in 6-O-sulfation in fibrotic kidney compared with the control. These results suggest an important role of 6-O-sulfation in the pathogenesis of fibrosis associated with chronic rejection.

Topics: Animals; Chronic Disease; Disease Models, Animal; Fibroblast Growth Factor 2; Fibrosis; Graft Rejection; Heparitin Sulfate; Humans; Kidney; Kidney Transplantation; Male; Mice; Mice, Inbred C57BL; Recombinant Proteins; Signal Transduction; Sulfatases; Sulfates; Sulfotransferases

In severe acute pancreatitis, the administration of fluids in the presence of positive fluid responsiveness is associated with better outcome when compared to guiding therapy on central venous pressure. We compared the effects of such consequent maximization of stroke volume index with a regime using individual values of stroke volume index assessed prior to severe acute pancreatitis induction as therapeutic hemodynamic goals.. Prospective, randomized animal study.. University animal research laboratory.. Thirty domestic pigs.. After randomization, fluid resuscitation was started 2 hours after severe acute pancreatitis induction and continued for 6 hours according to the respective treatment algorithms. In the control group, fluid therapy was directed by maximizing stroke volume index, and in the study group, stroke volume index assessed prior to severe acute pancreatitis served as primary hemodynamic goal.. Within the first 6 hours of severe acute pancreatitis, the study group received a total of 1,935.8 ± 540.7 mL of fluids compared with 3,462.8 ± 828.2 mL in the control group (p < 0.001). Pancreatic tissue oxygenation did not differ significantly between both groups. Vascular endothelial function, measured by flow-mediated vasodilation before and 6 hours after severe acute pancreatitis induction, revealed less impairment in the study group after treatment interval (-90.76% [study group] vs -130.89% [control group]; p = 0.046). Further, lower levels of heparan sulfate (3.41 ± 5.6 pg/mL [study group] vs 43.67 ± 46.61 pg/mL [control group]; p = 0.032) and interleukin 6 (32.18 ± 8.81 pg/mL [study group] vs 77.76 ± 56.86 pg/mL [control group]; p = 0.021) were found in the study group compared with control group. Histopathological examination of the pancreatic head and corpus at day 7 revealed less edema for the study group compared with the control group (1.82 ± 0.87 [study group] vs 2.89 ± 0.33 [control group, pancreatic head]; p = 0.03; 2.2 ± 0.92 [study group] vs 2.91 ± 0.3 [control group, pancreatic corpus]; p = 0.025).. Individualized optimization of intravascular fluid status during the early course of severe acute pancreatitis, compared with a treatment strategy of maximizing stroke volume by fluid loading, leads to less vascular endothelial damage, pancreatic edema, and inflammatory response.

Topics: Acute Disease; Animals; Disease Models, Animal; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Fluid Therapy; Glycocalyx; Hemodynamics; Heparitin Sulfate; Inflammation; Pancreatitis; Prospective Studies; Random Allocation; Severity of Illness Index; Stroke Volume; Swine; Syndecan-1

The temporal relationship between the onset of clinical signs in the mucopolysaccharidosis type IIIA (MPS IIIA) Huntaway dog model and cerebellar pathology has not been described. Here we sought to characterize the accumulation of primary (heparan sulfate) and secondary (G(M3)) substrates and onset of other changes in cerebellar tissues, and investigate the relationship to the onset of motor dysfunction in these animals. We observed that Purkinje cells were present in dogs aged up to and including 30.9 months, however by 40.9 months of age only ~12% remained, coincident with the onset of clinical signs. Primary and secondary substrate accumulation and inflammation were detected as early as 2.2 months and axonal spheroids were observed from 4.3 months in the deep cerebellar nuclei and later (11.6 months) in cerebellar white matter tracts. Degenerating neurons and apoptotic cells were not observed at any time. Our findings suggest that cell autonomous mechanisms may contribute to Purkinje cell death in the MPS IIIA dog.

Topics: Animals; Asymptomatic Diseases; Cell Death; Cerebellar Nuclei; Cerebellum; Disease Models, Animal; Dogs; Heparitin Sulfate; Humans; Inflammation; Motor Activity; Mucopolysaccharidosis III; Purkinje Cells; White Matter

The first step in attachment of Chlamydia to host cells is thought to involve reversible binding to host heparan sulfate proteoglycans (HSPGs), polymers of variably sulfated repeating disaccharide units coupled to diverse protein backbones. However, the key determinants of HSPG structure that are involved in Chlamydia binding are incompletely defined. A previous genome-wide Drosophila RNAi screen suggested that the level of HSPG 6-O sulfation rather than the identity of the proteoglycan backbone maybe a critical determinant for binding. Here, we tested in mammalian cells whether SULF1 or SULF2, human endosulfatases, which remove 6-O sulfates from HSPGs, modulate Chlamydia infection. Ectopic expression of SULF1 or SULF2 in HeLa cells, which decreases cell surface HSPG sulfation, diminished C. muridarum binding and decreased vacuole formation. ShRNA depletion of endogenous SULF2 in a cell line that primarily expresses SULF2 augmented binding and increased vacuole formation. C. muridarum infection of diverse cell lines resulted indownregulation of SULF2 mRNA. In a murine model of acute pneumonia, mice genetically deficient in both endosulfatases or in SULF2 alone demonstrated increased susceptibility to C. muridarum lung infection. Collectively, these studies demonstrate that the level of HSPG 6-O sulfation is a critical determinant of C. muridarum infection in vivo and that 6-O endosulfatases are previously unappreciated modulators of microbial pathogenesis.

Topics: Animals; Bacterial Adhesion; Chlamydia Infections; Chlamydia muridarum; Disease Models, Animal; Disease Susceptibility; HeLa Cells; Heparitin Sulfate; Humans; Mice; Mice, Knockout; Pneumonia, Bacterial; Sulfatases; Sulfotransferases

Heparan sulfate proteoglycans (HSPGs) are glycoproteins consisting of a core protein to which linear heparan sulfate (HS) side chains are covalently attached. These HS side chains mediate a variety of biologic functions involved in inflammation. Radionuclide imaging of HS side chains in tissues with inflammation may be used for the stratification of patients who would most likely benefit from HSPG-targeting therapy. The goal of this study was to evaluate the feasibility of in vivo radionuclide imaging of HS side chain expression in a mouse model of asthma using the recombinant eosinophil cationic protein (rECP).. rECP was radioiodinated with (125)I or (123)I using the Chloramine-T method. The 50% inhibitory concentration value for (125)I-labeled rECP was determined in a competitive cell-binding assay using Beas-2B cells. The binding of radiolabeled rECP to HS side chains was evaluated both in vitro and in vivo. The biodistribution of radiolabeled rECP was assessed in asthma mice or in control mice using SPECT imaging, ex vivo biodistribution measurements, and microautoradiography.. The 50% inhibitory concentration value for (125)I-rECP was 7.4 ± 0.1 nM. The loss of HS side chains substantially inhibited the cellular and tissue uptake of (125)I- or (123)I-rECP, indicating that HS side chains of HSPGs are required for (125)I- or (123)I-eosinophil cationic protein binding and uptake both in vitro and in vivo. SPECT imaging demonstrated an appreciably higher accumulation of radioactivity in the lungs of asthma mice than in those of control mice. Ex vivo biodistribution studies also confirmed that there was at least a 4-fold increase in the lung-to-muscle ratio of asthma mice, compared with control mice. The accumulation of radiolabeled rECP was linearly correlated with leukocyte infiltration.. This study illustrates the feasibility of using radiolabeled rECP for the visualization of HS side chains of HSPGs and the evaluation of allergic lung inflammation in living subjects. Our data indicate that radiolabeled rECP is a novel imaging agent for HS side chains of HSPGs in predicting allergic lung inflammation in living mice.

Topics: Allergens; Animals; Asthma; Biological Transport; Cell Line, Tumor; Disease Models, Animal; Drug Stability; Eosinophil Cationic Protein; Female; Gene Expression Regulation; Heparitin Sulfate; Humans; Hypersensitivity; Inflammation; Iodine Radioisotopes; Isotope Labeling; Leukocytes; Mice; Molecular Imaging; Multimodal Imaging; Positron-Emission Tomography; Radiochemistry; Recombinant Proteins; Tomography, X-Ray Computed

Recently, we compared amino acid sequences of the E2 glycoprotein of natural North American eastern equine encephalitis virus (NA-EEEV) isolates and demonstrated that naturally circulating viruses interact with heparan sulfate (HS) and that this interaction contributes to the extreme neurovirulence of EEEV (C. L. Gardner, G. D. Ebel, K. D. Ryman, and W. B. Klimstra, Proc. Natl. Acad. Sci. U. S. A., 108:16026-16031, 2011). In the current study, we have examined the contribution to HS binding of each of three lysine residues in the E2 71-to-77 region that comprise the primary HS binding site of wild-type (WT) NA-EEEV viruses. We also report that the original sequence comparison identified five virus isolates, each with one of three amino acid differences in the E2 71-to-77 region, including mutations in residues critical for HS binding by the WT virus. The natural variant viruses, which possessed either a mutation from lysine to glutamine at E2 71, a mutation from lysine to threonine at E2 71, or a mutation from threonine to lysine at E2 72, exhibited altered interactions with heparan sulfate and cell surfaces and altered virulence in a mouse model of EEEV disease. An electrostatic map of the EEEV E1/E2 heterotrimer based upon the recent Chikungunya virus crystal structure (J. E. Voss, M. C. Vaney, S. Duquerroy, C. Vonrhein, C. Girard-Blanc, E. Crublet, A. Thompson, G. Bricogne, and F. A. Rey, Nature, 468:709-712, 2010) showed the HS binding site to be at the apical surface of E2, with variants affecting the electrochemical nature of the binding site. Together, these results suggest that natural variation in the EEEV HS binding domain may arise during EEEV sylvatic cycles and that this variation may influence receptor interaction and the severity of EEEV disease.

Topics: Amino Acid Substitution; Animals; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; DNA Mutational Analysis; Encephalitis Virus, Eastern Equine; Encephalomyelitis, Equine; Heparitin Sulfate; Lysine; Mice; Mutagenesis, Site-Directed; Protein Binding; Receptors, Virus; Static Electricity; Viral Envelope Proteins; Virus Attachment

Mucopolysaccharidosis type IIIA (MPSIIIA) is a lysosomal storage disorder caused by mutations in N-sulfoglucosamine sulfohydrolase (SGSH), resulting in heparan sulfate (HS) accumulation and progressive neurodegeneration. There are no treatments. We previously demonstrated improved neuropathology in MPSIIIA mice using lentiviral vectors (LVs) overexpressing SGSH in wild-type (WT) hematopoietic stem cell (HSC) transplants (HSCTs), achieved via donor monocyte/microglial engraftment in the brain. However, neurological disease was not corrected using LVs in autologous MPSIIIA HSCTs. To improve brain expression via monocyte/microglial specificity, LVs expressing enhanced green fluorescent protein (eGFP) under ubiquitous phosphoglycerate kinase (PGK) or myeloid-specific promoters were compared in transplanted HSCs. LV-CD11b-GFP gave significantly higher monocyte/B-cell eGFP expression than LV-PGK-GFP or LV-CD18-GFP after 6 months. Subsequently, autologous MPSIIIA HSCs were transduced with either LV-PGK-coSGSH or LV-CD11b-coSGSH vectors expressing codon-optimized SGSH and transplanted into MPSIIIA mice. Eight months after HSCT, LV-PGK-coSGSH vectors produced bone marrow SGSH (576% normal activity) similar to LV-CD11b-coSGSH (473%), but LV-CD11b-coSGSH had significantly higher brain expression (11 versus 7%), demonstrating improved brain specificity. LV-CD11b-coSGSH normalized MPSIIIA behavior, brain HS, GM2 ganglioside, and neuroinflammation to WT levels, whereas LV-PGK-coSGSH partly corrected neuropathology but not behavior. We demonstrate compelling evidence of neurological disease correction using autologous myeloid driven lentiviral-HSC gene therapy in MPSIIIA mice.

Topics: Animals; Brain; CD11b Antigen; Cell Line; Disease Models, Animal; Female; Genetic Therapy; Genetic Vectors; Green Fluorescent Proteins; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Heparitin Sulfate; Humans; Hydrolases; Lentivirus; Leukocytes; Lysosomes; Mice; Mice, Inbred C57BL; Microglia; Mucopolysaccharidosis III; Myeloid Cells; Organ Specificity; Promoter Regions, Genetic

To evaluate the effects of hyperprolactinemia on the sulfated glycosaminoglycans (GAGs) of the murine uterus.. Experimental research.. University biochemistry laboratory.. Eighty female mice were divided into two groups of 40 animals each and treated with 0.2 mL of saline solution (controls, Ctr) and 200 μg of metoclopramide (experimental, HPrl). Treatments lasted for 50 consecutive days. The animals were divided into four subgroups of 10 animals each per treatment (Ctr and HPrl) and sacrificed according to the phase of the estrous cycle. The uterine horns were removed for biochemical analyses, and blood samples were collected for hormone measurements.. Induced hyperprolactinemia.. To quantify the sulfated GAGs, and PRL and sex steroid levels.. The endometrium during the estrus phase was significantly thicker in the HPrl animals than in the Ctr mice. The levels of chondroitin and dermatan sulfate were significantly increased in the HPrl group than in the Ctr group during all phases except metestrus. The amounts of heparan sulfate were lower during estrus and diestrus and higher in the metestrus phase in HPrl than in Ctr animals. Serum PRL levels were increased whereas the levels of E2 and P were decreased in all phases in the HPrl group than in the Ctr group.. The hyperprolactinemia changed the amounts of uterine sulfated GAGs. Our data suggest that these changes may not be correlated with ovarian steroid levels.

Topics: Animals; Biomarkers; Chondroitin Sulfates; Dermatan Sulfate; Disease Models, Animal; Estradiol; Estrous Cycle; Female; Glycosaminoglycans; Heparitin Sulfate; Hyperprolactinemia; Metoclopramide; Mice; Progesterone; Time Factors; Uterus

Entamoeba histolytica infection is associated with considerable morbidity and mortality in the form of intestinal and extraintestinal amoebiasis. No vaccine is yet available for amoebiasis. Heparan Sulphate Binding Proteins (HSBPs) from E. histolytica were evaluated for immunogenicity and protective efficacy in a Guinea pig model. Animals were immunized subcutaneously with 30μg of HSBP by three weekly inoculations. The immunogenicity of HSBP was determined by antibody response (IgG, IgM and IgA), splenocyte proliferation assay and in vitro direct amoebicidal assay with splenic lymphocytes and monocytes from vaccinated and control animals. The efficacy of the vaccine was evaluated by challenge infection to vaccinated and control animals by intra-caecal inoculation of E. histolytica trophozoites and comparing gross and histopathological findings in caeca of these animals. HSBP was found to induce specific anti-amoebic response as seen by specific antibody production and direct amoebicidal activity of splenocytes. The vaccine also showed partial protection against challenge infection in vaccinated animals as shown by mild/absent lesions and histopathological findings.

Topics: Animals; Antibodies, Protozoan; Carrier Proteins; Cecum; Disease Models, Animal; Dysentery, Amebic; Entamoeba histolytica; Guinea Pigs; Heparitin Sulfate; Immunity, Cellular; Immunoglobulins; Lymphocytes; Male; Monocytes; Protozoan Proteins; Protozoan Vaccines; Spleen; Vaccination

Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by mutations in lysosomal enzymes involved in degradation of glycosaminoglycans (GAGs). Patients with MPS grow poorly and become physically disabled due to systemic bone disease. While many of the major skeletal effects in mouse models for MPS have been described, no detailed analysis that compares GAGs levels and characteristics of bone by micro-CT has been done. The aims of this study were to assess severity of bone dysplasia among four MPS mouse models (MPS I, IIIA, IVA and VII), to determine the relationship between severity of bone dysplasia and serum keratan sulfate (KS) and heparan sulfate (HS) levels in those models, and to explore the mechanism of KS elevation in MPS I, IIIA, and VII mouse models. Clinically, MPS VII mice had the most severe bone pathology; however, MPS I and IVA mice also showed skeletal pathology. MPS I and VII mice showed severe bone dysplasia, higher bone mineral density, narrowed spinal canal, and shorter sclerotic bones by micro-CT and radiographs. Serum KS and HS levels were elevated in MPS I, IIIA, and VII mice. Severity of skeletal disease displayed by micro-CT, radiographs and histopathology correlated with the level of KS elevation. We showed that elevated HS levels in MPS mouse models could inhibit N-acetylgalactosamine-6-sulfate sulfatase enzyme. These studies suggest that KS could be released from chondrocytes affected by accumulation of other GAGs and that KS could be useful as a biomarker for severity of bone dysplasia in MPS disorders.

Topics: Animals; Biomarkers; Bone and Bones; Bone Density; Bone Diseases, Developmental; Chondrocytes; Disease Models, Animal; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Keratan Sulfate; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucopolysaccharidoses; Spinal Canal; X-Ray Microtomography

BTBR T+tf/J (BTBR) mice have emerged as strong candidates to serve as models of a range of autism-relevant behaviors, showing deficiencies in social behaviors; reduced or unusual ultrasonic vocalizations in conspecific situations; and enhanced, repetitive self-grooming. Recent studies have described their behaviors in a seminatural visible burrow system (VBS); a Social Proximity Test in which avoidance of a conspecific is impossible; and in an object approach and investigation test evaluating attention to specific objects and potential stereotypies in the order of approaching/investigating objects. VBS results confirmed strong BTBR avoidance of conspecifics and in the Social Proximity Test, BTBR showed dramatic differences in several close-in behaviors, including specific avoidance of a nose-to-nose contact that may potentially be related to gaze-avoidance. Diazepam normalized social avoidance by BTBRs in a Three-Chamber Test, and some additional behaviors - but not nose to nose avoidance - in the Social Proximity Test. BTBR also showed higher levels of preference for particular objects, and higher levels of sequences investigating 3- or 4-objects in the same order. Heparan sulfate (HS) associated with fractal structures in the subventricular zone of the lateral ventricles was severely reduced in BTBR. HS may modulate the functions of a range of growth and guidance factors during development, and HS abnormalities are associated with relevant brain (callosal agenesis) and behavioral (reductions in sociality) changes; suggesting the value of examination of the dynamics of the HS system in the context of autism.

Topics: Animals; Autistic Disorder; Disease Models, Animal; Exploratory Behavior; Grooming; Heparitin Sulfate; Humans; Male; Mice; Mice, Inbred Strains; Social Behavior

Heparan sulfate proteoglycans (HSPGs) are considered important in maintaining physiological homeostasis in many systems. Their expression is altered greatly in several pathophysiological conditions. Herein, we assess the expression and cellular localization of HSPGs in two murine models of human inflammatory bowel disease (IBD).. Expression and localization of HSPGs, syndecans, and HS epitopes were examined in the colon of 129SvEv interleukin 10 knockout (IL10(-/-)), C3Bir IL10(-/-), and their genetic control (IL10(+/+)) counterparts (129SvEv; C3H/HeJ). mRNA expression of syndecans and heparan sulfate biosynthesis enzymes were evaluated by real-time polymerase chain reaction (PCR). Localization of HSPGs was determined by immunofluorescence.. mRNA for all syndecans was detected and expression in colonic tissues altered in IL10(-/-) mice. Syndecan-1 protein was expressed in the intestinal epithelium and on lamina propria cells of IL10(-/-) and control mice but was significantly reduced on the intestinal epithelial cells of IL10(-/-), mice particularly with severe colitis. Syndecan-2 was not detected, whereas syndecan-3 immunoreactivity was localized in the lamina propria but did not differ between control and IL10(-/-) mice. Syndecan-4 was present on epithelial cells of all mice but was significantly reduced in IL10(-/-) mice. Differences in the expression of HS epitopes between control and IL10(-/-) mice were also confirmed.. The study has revealed altered expression of syndecan-1 and -4 and HS epitopes in the gut of mice with an IBD-like gut disorder. The IL10(-/-) mouse is a useful model for further study of the functional role of HSPGs in chronic inflammation and in maintaining healthy gut barrier.

Topics: Animals; Blotting, Western; Cells, Cultured; Colitis; Colon; Disease Models, Animal; Female; Fluorescent Antibody Technique; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Inflammation; Interleukin-10; Intestinal Mucosa; Mice; Mice, Inbred C3H; Mice, Knockout; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Syndecan-1; Syndecan-2; Syndecan-3; Syndecan-4; Syndecans

Multiple studies converge to implicate alterations of the hippocampus and amygdala in the pathology of autism. We have previously reported anatomical alterations of the meninges, vasculature and fractones, the specialized extracellular matrix (ECM) of the subventricular zone, in the forebrain of adult BTBR T+ tf/J mice, animal model for autism. Here, we used bisbenzidine cell nucleus staining and dual immunofluorescence histochemistry for laminin and N-sulfated heparan sulfate proteoglycans (NS-HSPG) to examine a series of brain sections containing the amygdala and hippocampus in the adult BTBR T+ tf/j mouse. We observed an excessive separation of the two hippocampi, a modified trajectory of the meninges leading to a shrunken choroid plexus in the lateral ventricle, a shorter granular layer of the dentate gyrus, and a reduced size of the amygdala nuclei. The lateral ventricle near the amygdala, and the third ventricle were shrunken. The number and size of fractones, and their immunoreactivity for NS-HSPG, were reduced throughout the third and lateral ventricles walls. Enlarged blood vessels were found at the endopiriform cortex/amygdala interface. These results show anatomical alterations of the hippocampal/amygdala that are associated with defects of the choroid plexus/ventricular system and the ECM in the BTBR T+ TF/J mouse. Similar alterations of the hippocampus/amygdala axis in humans with autism to these observed in BTBR T+ tf/J mice make this animal model highly valuable for the study of autism. Moreover, the meningo/vascular and ECM alterations in BTBR T+ Tf/J mice suggest a possible role of the brain connective tissue in autism.

Topics: Amygdala; Animals; Autistic Disorder; Cerebral Ventricles; Disease Models, Animal; Extracellular Matrix; Female; Fluorescent Antibody Technique; Heparitin Sulfate; Hippocampus; Male; Mice; Mice, Neurologic Mutants

BTBR T+tf/J (BTBR) mice show abnormal social, communicatory, and repetitive/stereotyped behaviors paralleling many of the symptoms of autism spectrum disorders. BTBR also show agenesis of the corpus callosum (CC) suggesting major perturbations of growth or guidance factors in the dorsal forebrain [1]. Heparan sulfate (HS) is a polysaccaride found in the brain and other animal tissues. It binds to a wide variety of ligands and through these ligands modulates a number of biological processes, including cell proliferation and differentiation, migration and guidance. It is aggregated on fractal-like structures (fractones) in the subventricular zone (SVZ), that may be visualized by laminin immunoreactivity (LAM-ir), as well as by HS immunoreactivity (HS-ir). We report that the lateral ventricles of BTBR mice were drastically reduced in area compared to C57BL/6J (B6) mice while the BTBR SVZ was significantly shorter than that of B6. In addition to much smaller fractones for BTBR, both HS and LAM-ir associated with fractones were significantly reduced in BTBR, and their anterior-posterior distributions were also altered. Finally, the ratio of HS to LAM in individual fractones was significantly higher in BTBR than in B6 mice. These data, in agreement with other findings linking HS to callosal development, suggest that variations in the quantity and distribution of HS in the SVZ of the lateral ventricles may be important modulators of the brain structural abnormalities of BTBR mice, and, potentially, contribute to the behavioral pathologies of these animals.

Topics: Analysis of Variance; Animals; Autistic Disorder; Brain; Corpus Callosum; Disease Models, Animal; Heparitin Sulfate; Lamins; Lateral Ventricles; Male; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Phenotype

Alzheimer's disease (AD) is characterized by extracellular cerebral accumulation of amyloid β peptide (Aβ). Heparan sulfate (HS) is a glycosaminoglycan that is abundant in the extracellular space. The state of sulfation within the HS chain influences its ability to interact with a variety of proteins. Highly sulfated domains within HS are crucial for Aβ aggregation in vitro. Here, we investigated the expression of the sulfated domains and HS disaccharide composition in the brains of Tg2576, J20, and T41 transgenic AD mouse models, and patients with AD. RB4CD12, a phage display antibody, recognizes highly sulfated domains of HS. The RB4CD12 epitope is abundant in the basement membrane of brain vessels under physiological conditions. In the cortex and hippocampus of the mice and patients with AD, RB4CD12 strongly stained both diffuse and neuritic amyloid plaques. Interestingly, RB4CD12 also stained the intracellular granules of certain hippocampal neurons in AD brains. Disaccharide compositions in vessel-enriched and nonvasculature fractions of Tg2576 mice and AD patients were found to be comparable to those of non-transgenic and non-demented controls, respectively. The RB4CD12 epitope in amyloid plaques was substantially degraded ex vivo by Sulf-1 and Sulf-2, extracellular HS endosulfatases. These results indicate that formation of highly sulfated HS domains may be upregulated in conjunction with AD pathogenesis, and that these domains can be enzymatically remodeled in AD brains.

Topics: Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Cerebral Cortex; Disaccharides; Disease Models, Animal; Evidence-Based Medicine; Female; Heparitin Sulfate; Hippocampus; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Weight; Plaque, Amyloid; Recombinant Proteins; Sulfatases; Sulfotransferases; Up-Regulation

Mucopolysaccharide diseases (MPS) are caused by deficiency of glycosaminoglycan (GAG) degrading enzymes, leading to GAG accumulation. Neurodegenerative MPS diseases exhibit cognitive decline, behavioural problems and shortened lifespan. We have characterised neuropathological changes in mouse models of MPSI, IIIA and IIIB to provide a better understanding of these events.Wild-type (WT), MPSI, IIIA and IIIB mouse brains were analysed at 4 and 9 months of age. Quantitative immunohistochemistry showed significantly increased lysosomal compartment, GM2 ganglioside storage, neuroinflammation, decreased and mislocalised synaptic vesicle associated membrane protein, (VAMP2), and decreased post-synaptic protein, Homer-1, in layers II/III-VI of the primary motor, somatosensory and parietal cortex. Total heparan sulphate (HS), was significantly elevated, and abnormally N-, 6-O and 2-O sulphated compared to WT, potentially altering HS-dependent cellular functions. Neuroinflammation was confirmed by significantly increased MCP-1, MIP-1α, IL-1α, using cytometric bead arrays. An overall genotype effect was seen in all parameters tested except for synaptophysin staining, neuronal cell number and cortical thickness which were not significantly different from WT. MPSIIIA and IIIB showed significantly more pronounced pathology than MPSI in lysosomal storage, astrocytosis, microgliosis and the percentage of 2-O sulphation of HS. We also observed significant time progression of all genotypes from 4-9 months in lysosomal storage, astrocytosis, microgliosis and synaptic disorganisation but not GM2 gangliosidosis. Individual genotype*time differences were disparate, with significant progression from 4 to 9 months only seen for MPSIIIB with lysosomal storage, MPSI with astrocytocis and MPSIIIA with microgliosis as well as neuronal loss. Transmission electron microscopy of MPS brains revealed dystrophic axons, axonal storage, and extensive lipid and lysosomal storage. These data lend novel insight to MPS neuropathology, suggesting that MPSIIIA and IIIB have more pronounced neuropathology than MPSI, yet all are still progressive, at least in some aspects of neuropathology, from 4-9 months.

Topics: Animals; Carrier Proteins; Cytokines; Disease Models, Animal; Disease Progression; Female; G(M2) Ganglioside; Glycosaminoglycans; Heparitin Sulfate; Homer Scaffolding Proteins; Immunohistochemistry; Lysosomes; Male; Mice; Mucopolysaccharidosis I; Mucopolysaccharidosis III; Neurons; Parietal Lobe; Somatosensory Cortex; Vesicle-Associated Membrane Protein 2

Vascular leakage after ischemia-reperfusion (IR) is largely attributed to the destruction of the endothelial barrier and its associated negatively charged glycocalyx. In vitro, sevoflurane attenuates these changes. Therefore, we compared sevoflurane with propofol with regard to the protection of the glycocalyx and the release of negatively charged substances in vivo.. After surgical preparation under midazolam-fentanyl, nine pigs each received either propofol or sevoflurane. Ischemia of 90 min was induced by a balloon catheter in the thoracic aorta. After 120 min of reperfusion, the anesthetics were changed back to midazolam-fentanyl. Five animals, each without aortic occlusion, served as time controls. Blood electrolyte parameters were measured, from which the strong ion gap (SIG) was calculated. Serum heparan sulfate concentrations and immunohistology served as a marker of glycocalyx destruction.. Immediately after reperfusion, SIG increased significantly only in the propofol group (+6.7 mEq/l versus baseline; p < .05), remaining stable in sevoflurane and both time-controlled groups. Initially, heparan sulfate concentration increased comparably in both experimental groups, but after 120 min, it became stable in sevoflurane-anesthetized animals, while increasing further in the propofol group (p < .05).. Unmeasured anions, predictive of negative outcome in previous studies, did not increase significantly in sevoflurane-anesthetized animals. Additionally, there was less heparan sulfate shedding over time, signaling less destruction of the glycocalyx. Therefore, in this in-vivo situation, sevoflurane proves to be superior to propofol in protecting the endothelium from IR injury.

Topics: Acid-Base Equilibrium; Anesthetics; Animals; Capillary Permeability; Disease Models, Animal; Endothelium, Vascular; Female; Glycocalyx; Heparitin Sulfate; Male; Methyl Ethers; Propofol; Reperfusion Injury; Sevoflurane; Sus scrofa

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

Topics: Acute Lung Injury; Adoptive Transfer; Animals; Cell Adhesion; Disease Models, Animal; Endothelium; Endotoxemia; Enzyme Activation; Gene Expression Regulation; Glucuronidase; Glycocalyx; Heparitin Sulfate; Humans; Intercellular Adhesion Molecule-1; Intestinal Perforation; Lipopolysaccharides; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Pulmonary Alveoli; Receptors, Tumor Necrosis Factor, Type I; Respiratory Insufficiency; Tumor Necrosis Factor-alpha; Ventilator-Induced Lung Injury

Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disorder caused by a deficiency in the activity of the lysosomal hydrolase, sulphamidase, an enzyme involved in the degradation of heparan sulphate. MPS IIIA patients exhibit progressive mental retardation and behavioural disturbance. While neuropathology is the major clinical problem in MPS IIIA patients, there is little understanding of how lysosomal storage generates this phenotype. As reduced neuronal communication can underlie cognitive deficiencies, we investigated whether the secretion of neurotransmitters is altered in MPS IIIA mice; utilising adrenal chromaffin cells, a classical model for studying secretion via exocytosis. MPS IIIA chromaffin cells displayed heparan sulphate storage and electron microscopy revealed large electron-lucent storage compartments. There were also increased numbers of large/elongated chromaffin granules, with a morphology that was similar to immature secretory granules. Carbon fibre amperometry illustrated a significant decrease in the number of exocytotic events for MPS IIIA, when compared to control chromaffin cells. However, there were no changes in the kinetics of release, the amount of catecholamine released per exocytotic event, or the amount of Ca(2+) entry upon stimulation. The increased number of large/elongated granules and reduced number of exocytotic events suggests that either the biogenesis and/or the cell surface docking and fusion potential of these vesicles is impaired in MPS IIIA. If this also occurs in central nervous system neurons, the reduction in neurotransmitter release could help to explain the development of neuropathology in MPS IIIA.

Topics: Adrenal Glands; Analysis of Variance; Animals; Calcium; Carbon; Carbon Fiber; Catecholamines; Cells, Cultured; Chromaffin Cells; Disease Models, Animal; Exocytosis; Heparitin Sulfate; Lysosomes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Electron, Transmission; Mucopolysaccharidosis III; Statistics, Nonparametric

Topics: Animals; Autistic Disorder; Disease Models, Animal; Heparitin Sulfate; Mice; Mice, Inbred Strains

Heat stroke is a condition characterized by high body temperature that can lead to hemorrhage and necrosis in multiple organs. Anticoagulants, such as danaparoid sodium (DA), inhibit various types of inflammation; however, the anti-inflammatory mechanism of action is not well understood. Given that heat stroke is a severe inflammatory response disease, we hypothesized that DA could inhibit inflammation from heat stress and prevent acute heat stroke.. Male Wistar rats were given a bolus injection of saline or DA (50 U/kg body weight) into the tail vein just prior to heat stress (42 °C for 30 min). Markers of inflammation were then determined in serum and tissue samples.. In rats pretreated with DA, induction of cytokines (interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α), nitric oxide (NO), and high mobility group box 1 (HMGB1) protein were reduced compared with saline-treated rats. Histologic changes observed in lung, liver, and small intestine tissue samples of saline-treated rats were attenuated in DA-treated rats. Moreover, DA pretreatment improved survival in our rat model of heat stress-induced acute inflammation.. Collectively, our findings demonstrate that DA pretreatment may have value as a new therapeutic tool for heat stroke.

Topics: Acute Disease; Animals; Anticoagulants; Antithrombin III; Chondroitin Sulfates; Cytokines; Dermatan Sulfate; Disease Models, Animal; Fibrin Fibrinogen Degradation Products; Heat Exhaustion; Heat Stroke; Heparitin Sulfate; HMGB1 Protein; Inflammation; Intestine, Small; Liver; Lung; Male; Nitrates; Nitric Oxide; Nitrites; Peptide Hydrolases; Rats; Rats, Wistar; Survival Rate

The primary pathology in mucopolysaccharidosis (MPS) IIIB is lysosomal storage of heparan sulfate (HS) glycosaminoglycans, leading to complex neuropathology and dysfunction, for which the detailed mechanisms remain unclear. Using antibodies that recognize specific HS glycoforms, we demonstrate differential cell-specific and domain-specific lysosomal HS-GAG distribution in MPS IIIB mouse brain. We also describe a novel neuron-specific brain HS epitope with broad, non-specific increase in the expression in all neurons in MPS IIIB mouse brain, including cerebellar granule neurons, which do not exhibit lysosomal storage pathology. This suggests that biosynthesis of certain HS glycoforms is enhanced throughout the CNS of MPS IIIB mice. Such a conclusion is further supported by demonstration of increased expression of multiple genes encoding enzymes essential in HS biosynthesis, including HS sulfotransferases and epimerases, as well as FGFs, for which HS serves as a co-receptor, in MPS IIIB brain. These data suggest that lysosomal storage of HS may lead to the increase in HS biosyntheses, which may contribute to the neuropathology of MPS IIIB by exacerbating the lysosomal HS storage.

Topics: Animals; Brain; Carbohydrate Epimerases; Disease Models, Animal; Fibroblast Growth Factors; Heparitin Sulfate; Lysosomes; Mice; Mice, Knockout; Mucopolysaccharidosis III; Neurons; Protein Isoforms; Sulfotransferases; Tissue Distribution

Heparan sulfate (HS) is present on the surface of virtually all mammalian cells and is a major component of the extracellular matrix (ECM), where it plays a pivotal role in cell-cell and cell-matrix cross-talk through its large interactome. Disruption of HS biosynthesis in mice results in neonatal death as a consequence of malformed lungs, indicating that HS is crucial for airway morphogenesis. Neonatal mortality (~50%) in newborns with congenital diaphragmatic hernia (CDH) is principally associated with lung hypoplasia and pulmonary hypertension. Given the importance of HS for lung morphogenesis, we investigated developmental changes in HS structure in normal and hypoplastic lungs using the nitrofen rat model of CDH and semi-synthetic bacteriophage ('phage) display antibodies, which identify distinct HS structures.. The pulmonary pattern of elaborated HS structures is developmentally regulated. For example, the HS4E4V epitope is highly expressed in sub-epithelial mesenchyme of E15.5 - E17.5 lungs and at a lower level in more distal mesenchyme. However, by E19.5, this epitope is expressed similarly throughout the lung mesenchyme.We also reveal abnormalities in HS fine structure and spatiotemporal distribution of HS epitopes in hypoplastic CDH lungs. These changes involve structures recognised by key growth factors, FGF2 and FGF9. For example, the EV3C3V epitope, which was abnormally distributed in the mesenchyme of hypoplastic lungs, is recognised by FGF2.. The observed spatiotemporal changes in HS structure during normal lung development will likely reflect altered activities of many HS-binding proteins regulating lung morphogenesis. Abnormalities in HS structure and distribution in hypoplastic lungs can be expected to perturb HS:protein interactions, ECM microenvironments and crucial epithelial-mesenchyme communication, which may contribute to lung dysmorphogenesis. Indeed, a number of epitopes correlate with structures recognised by FGFs, suggesting a functional consequence of the observed changes in HS in these lungs. These results identify a novel, significant molecular defect in hypoplastic lungs and reveals HS as a potential contributor to hypoplastic lung development in CDH. Finally, these results afford the prospect that HS-mimetic therapeutics could repair defective signalling in hypoplastic lungs, improve lung growth, and reduce CDH mortality.

Topics: Animals; Carbohydrate Conformation; Disease Models, Animal; Epigenomics; Epitopes; Female; Fibroblast Growth Factors; Heparitin Sulfate; Herbicides; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Lung; Mice; Molecular Sequence Data; Morphogenesis; Phenyl Ethers; Pregnancy; Rats; Rats, Sprague-Dawley

Mucopolysaccharide (MPS) diseases are characterized by accumulation of glycosaminoglycans (GAGs) due to deficiencies in lysosomal enzymes responsible for GAG breakdown. Using a murine model of MPSI Hurler (MPSIH), we have quantified the heparan sulfate (HS) accumulation resulting from α-l-iduronidase (Idua) deficiency. HS levels were significantly increased in liver and brain tissue from 12-week-old Idua(-/-) mice by 87- and 20-fold, respectively. In addition, HS chains were shown to contain significantly increased N-, 2-O-, and 6-O-sulfation. Disaccharide compositional analyses also uncovered an HS disaccharide uniquely enriched in MPSIH, representing the terminal iduronic acid residue capping the non-reducing end of the HS chain, where no further degradation can occur in the absence of Idua. Critically, we identified that excess HS, some of which is colocalized to the Golgi secretory pathway, acts as a positive regulator of HS-sulfation, increasing the N-sulfotransferase activity of HS-modifying N-deacetylase/N-sulfotransferase enzymes. This mechanism may have severe implications during disease progression but, now identified, could help direct improved therapeutic strategies.

Topics: Animals; Disease Models, Animal; Golgi Apparatus; Heparitin Sulfate; Humans; Iduronic Acid; Iduronidase; Mice; Mice, Knockout; Mucopolysaccharidosis I; Sulfotransferases

Mucopolysaccharide (MPS) diseases are lysosomal storage disorders caused by deficiencies of enzymes catabolising glycosaminoglycans (GAGs). Abnormal GAG accumulation leads to symptoms including severe progressive neurological decline, skeletal deformities, organomegally, respiratory compromise and premature death. Treatment is available for some MPS diseases; enzyme replacement therapy for MPS I, II and VI, and haematopoietic stem cell transplantation for MPS I, VI and VII. These treatments are reliant on early diagnosis of the disease and accurate monitoring of treatment outcomes. Blood enzyme levels and total urinary GAGs are commonly used biomarkers in diagnosis of MPS but are not good measures of treatment outcome. Serum heparin cofactor II-thrombin complex (HCII-T), which is a GAG regulated serpin-protease complex, has recently been identified as a promising biomarker for MPS diseases. Here we present an assessment of the HCII-T biomarker in mouse models of MPS I, IIIA and IIIB, which suggests that HCII-T is a reliable marker for MPS I when measured in serum or dried blood spots stored for over a year at 4 degrees C, but that murine MPS IIIA and IIIB cannot be reliably detected using this biomarker. We also show that HCII-T formation in vivo is dependent on the presence of excess intravenous dermatan sulphate (DS), whilst intravenous heparan sulphate (HS), does not promote complex formation effectively. This suggests that HCII-T will prove effective as a biomarker for MPS I, II, VI and VII diseases, storing dermatan sulphate but may not be as appropriate for MPS III, storing heparan sulphate. With careful sample preparation, HCII-T ELISA could prove to be a useful biomarker for both newborn screening and measurement of treatment outcomes in selected MPS diseases.

Topics: Animals; Biomarkers; Blood Specimen Collection; Dermatan Sulfate; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Heparin Cofactor II; Heparitin Sulfate; Humans; Mice; Mice, Inbred C57BL; Mucopolysaccharidosis I; Mucopolysaccharidosis III; Thrombin

Dendrimers are hyperbranched synthetic well-defined molecules with a number of potential applications, especially in relation to the need for new antiviral agents. One subclass of dendrimers are peptide-derivatized dendrimers which consist of a peptidyl branching core and covalently attached surface peptide functional units. Few studies have addressed the potential uses of peptide dendrimers as direct-acting antiviral agents. Here, we report on the ability of two peptide dendrimers, SB105 and SB105_A10, to directly and almost completely inhibit human cytomegalovirus (HCMV) replication in both primary fibroblasts and endothelial cells; the agents were also found to inhibit murine CMV replication, whereas they were not able to inhibit adenovirus or vesicular stomatitis virus. The peptide dendrimers prevented adsorption of the HCMV to cells at 4 degrees C, whereas SB104, a dendrimer with a different amino acid sequence within the functional group and minimal anticytomegaloviral activity, was ineffective in blocking HCMV attachment. In effect, SB105_A10 bound to human cells through an interaction with cell surface heparan sulfate and thereby blocked virion attachment to target cells. These results indicate that the SB105 and SB105_A10 dendrimers could provide a useful starting point for the development of novel molecules to block HCMV infection.

Topics: Adenoviridae; Adenoviridae Infections; Animals; Antiviral Agents; Cell Line; Cells, Cultured; Cytomegalovirus; Dendrimers; Disease Models, Animal; Endothelial Cells; Fibroblasts; Heparitin Sulfate; Herpesviridae Infections; Humans; Mice; Muromegalovirus; Peptides; Rhabdoviridae Infections; Vesiculovirus; Virus Attachment; Virus Replication

Human antibodies specific for glycoprotein C (gC1) of herpes simplex virus type 1 (HSV-1) neutralized the virus infectivity and efficiently inhibited attachment of HSV-1 to human HaCaT keratinocytes and to murine mutant L cells expressing either heparan sulfate or chondroitin sulfate at the cell surface. Similar activities were observed with anti-gC1 monoclonal antibody B1C1. In addition to HaCaT and L cells, B1C1 antibody neutralized HSV-1 infectivity in simian GMK AH1 cells mildly pre-treated with heparinase III. Human anti-gC1 antibodies efficiently competed with the binding of gC1 to B1C1 antibody whose epitope overlaps a part of the attachment domain of gC1. Human anti-gC1 and B1C1 antibodies extended survival time of mice experimentally infected with HSV-1. We conclude that in HaCaT cells and in cell systems showing restricted expression of glycosaminoglycans, human and some monoclonal anti-gC1 antibodies can target the cell-binding domain of this protein and neutralize viral infectivity.

Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Binding Sites; Cell Line; Chlorocebus aethiops; Disease Models, Animal; Epitopes; Heparitin Sulfate; Herpes Simplex; Herpesvirus 1, Human; Humans; Keratinocytes; Mice; Neutralization Tests; Survival Analysis; Viral Envelope Proteins; Virus Attachment

Heparan sulfate (HS) is a structurally complex polysaccharide that interacts with a broad spectrum of extracellular effector ligands and thereby is thought to regulate a diverse array of biologic processes. The specificity of HS-ligand interactions is determined by the arrangement of sulfate groups on HS, which creates distinct binding motifs. Biologically important HS motifs are expected to exhibit regulated expression, yet there is a profound lack of tools to identify such motifs; consequently, little is known of their structures and functions. We have identified a novel phage display-derived antibody (NS4F5) that recognizes a highly regulated HS motif (HS(NS4F5)), which we have rigorously identified as (GlcNS6S-IdoA2S)(3). HS(NS4F5) exhibits a restricted expression in healthy adult tissues. Blocking HS(NS4F5) on cells in culture resulted in reduced proliferation and enhanced sensitivity to apoptosis. HS(NS4F5) is up-regulated in tumor endothelial cells, consistent with a role in endothelial cell activation. Indeed, TNF-α stimulated endothelial expression of HS(NS4F5), which contributed to leukocyte adhesion. In a mouse model of severe systemic amyloid protein A amyloidosis, HS(NS4F5) was expressed within amyloid deposits, which were successfully detected by microSPECT imaging using NS4F5 as a molecularly targeted probe. Combined, our results demonstrate that NS4F5 is a powerful tool for elucidating the biological function of HS(NS4F5) and can be exploited as a probe to detect novel polysaccharide biomarkers of disease processes.

Topics: Amyloidogenic Proteins; Amyloidosis; Animals; Antibodies, Monoclonal; Biomarkers; Carbohydrate Sequence; Cell Proliferation; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Endothelial Cells; Female; Heparitin Sulfate; Humans; Male; Mice; Neoplasms; Rats; Rats, Wistar; Single-Chain Antibodies; Tumor Necrosis Factor-alpha

Neurodegenerative metabolic disorders such as mucopolysaccharidosis IIIB (MPSIIIB or Sanfilippo disease) accumulate undegraded substrates in the brain and are often unresponsive to enzyme replacement treatments due to the impermeability of the blood brain barrier to enzyme. MPSIIIB is characterised by behavioural difficulties, cognitive and later motor decline, with death in the second decade of life. Most of these neurodegenerative lysosomal storage diseases lack effective treatments. We recently described significant reductions of accumulated heparan sulphate substrate in liver of a mouse model of MPSIIIB using the tyrosine kinase inhibitor genistein.. We report here that high doses of genistein aglycone, given continuously over a 9 month period to MPSIIIB mice, significantly reduce lysosomal storage, heparan sulphate substrate and neuroinflammation in the cerebral cortex and hippocampus, resulting in correction of the behavioural defects observed. Improvements in synaptic vesicle protein expression and secondary storage in the cerebral cortex were also observed.. Genistein may prove useful as a substrate reduction agent to delay clinical onset of MPSIIIB and, due to its multimodal action, may provide a treatment adjunct for several other neurodegenerative metabolic diseases.

Topics: Animals; Blood-Brain Barrier; Brain; Disease Models, Animal; Gene Expression Regulation; Genistein; Heparitin Sulfate; Heterozygote; Immunohistochemistry; Liver; Lysosomes; Mice; Mice, Inbred C57BL; Mucopolysaccharidosis III; Neurodegenerative Diseases; Neurons; Protein Kinase Inhibitors

Systemic inflammatory response syndrome (SIRS) is responsible for pancreatitis-associated mortality, but its initiating events are poorly understood. Possible candidates may be endogenous substances, which have previously been shown to mediate inflammatory responses. The aim of this study was to investigate whether SIRS could be exaggerated by heparan sulfate (HS) in acute pancreatitis (AP).. AP was induced in mice by cerulein injection and HS was administered one hour after the final cerulein injection. The severity of pancreatitis was assessed by serum amylase activity, pancreatic edema, and pancreatic myeloperoxidase (MPO) activity. Systemic inflammation was evaluated by assessing lung injury and by measuring serum levels of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. Cytokine levels were also measured in pancreas and lung tissues.. HS did not worsen the pancreatic injury induced by cerulein. In contrast, HS exacerbated the systemic inflammation as measured by augmented lung MPO activity, increased lung TNF-alpha and IL-6 levels, and elevated serum IL-6 levels.. Our results indicate a potential role for HS in propagating pancreatic inflammation from a local process to a systemic response and thus suggest the possibility that blockade of HS might improve the outcome of SIRS in AP.

Topics: Animals; Biopsy, Needle; Ceruletide; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Heparitin Sulfate; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Pancreatic Function Tests; Pancreatitis, Acute Necrotizing; Peroxidase; Probability; Random Allocation; Sensitivity and Specificity; Statistics, Nonparametric; Survival Analysis; Systemic Inflammatory Response Syndrome; Tumor Necrosis Factor-alpha

Mucopolysaccharidosis type IIIA is a neurodegenerative lysosomal storage disorder characterized by progressive loss of learned skills, sleep disturbance and behavioural problems. Absent or greatly reduced activity of sulphamidase, a lysosomal protein, results in intracellular accumulation of heparan sulphate. Subsequent neuroinflammation and neurodegeneration typify this and many other lysosomal storage disorders. We propose that intra-cerebrospinal fluid protein delivery represents a potential therapeutic avenue for treatment of this and other neurodegenerative conditions; however, technical restraints restrict examination of its use prior to adulthood in mice. We have used a naturally-occurring Mucopolysaccharidosis type IIIA mouse model to determine the effectiveness of combining intravenous protein replacement (1 mg/kg) from birth to 6 weeks of age with intra-cerebrospinal fluid sulphamidase delivery (100 microg, fortnightly from 6 weeks) on behaviour, the level of heparan sulphate-oligosaccharide storage and other neuropathology. Mice receiving combination treatment exhibited similar clinical improvement and reduction in heparan sulphate storage to those only receiving intra-cerebrospinal fluid enzyme. Reductions in micro- and astrogliosis and delayed development of ubiquitin-positive lesions were seen in both groups. A third group of intravenous-only treated mice did not exhibit clinical or neuropathological improvements. Intra-cerebrospinal fluid injection of sulphamidase effectively, but dose-dependently, treats neurological pathology in Mucopolysaccharidosis type IIIA, even when treatment begins in mice with established disease.

Topics: Analysis of Variance; Animals; Antibodies; Body Weight; Brain; Chromatography, High Pressure Liquid; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Routes; Exploratory Behavior; Heparitin Sulfate; Hydrolases; Lysosomal Storage Diseases; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucopolysaccharidosis III; Necrosis; Proteins; Tandem Mass Spectrometry; Time Factors

Mucopolysaccharidosis type IIIA (MPS IIIA) is an inherited neurodegenerative lysosomal storage disorder characterised by progressive loss of learned skills, sleep disturbance and behavioural problems. Reduced activity of sulphamidase (SGSH; EC 3.10.1.1) results in intracellular accumulation of heparan sulphate (HS), with the brain the primary site of pathology. We have used a naturally-occurring MPS IIIA mouse model to determine the effectiveness of SGSH replacement via the cerebrospinal fluid (CSF) to decrease neuropathology. This is a potential therapeutic option for patients with this disorder. Mice received intra-CSF injections of recombinant human SGSH (30, 50 or 70 mug) fortnightly from six-18 weeks of age, and the cumulative effect on neuropathology was examined and quantified. Anti-SGSH antibodies detected in plasma at euthanasia did not appear to impact upon the health of the mice or the experimental outcome, with significant, but region- and dose-dependent reductions in an HS-derived oligosaccharide observed in the brain and spinal cord using tandem mass spectrometry. SGSH infusion reduced the number of storage inclusions observed in the brain when visualised using electron microscopy and this correlated with a significant decrease in the immunohistochemical staining of a lysosomal membrane marker (LIMP-II). Reduced numbers of activated isolectin-B4-positive microglia and GFAP-positive astrocytes were seen in many, but not all, brain regions. Significant reductions in the number of ubiquitin-positive intracellular inclusions were also observed. These outcomes demonstrate the effectiveness of this method of enzyme delivery in reducing the spectrum of neuropathological changes in murine MPS IIIA brain.

Topics: Animals; Brain; Disease Models, Animal; Heparitin Sulfate; Humans; Hydrolases; Male; Mice, Transgenic; Mucopolysaccharidosis III; Nervous System Diseases

Heparan sulfate (HS) has been proposed to be antiatherogenic through inhibition of lipoprotein retention, inflammation, and smooth muscle cell proliferation. Perlecan is the predominant HS proteoglycan in the artery wall. Here, we investigated the role of perlecan HS chains using apoE null (ApoE0) mice that were cross-bred with mice expressing HS-deficient perlecan (Hspg2(Delta3/Delta3)). Morphometry of cross-sections from aortic roots and en face preparations of whole aortas revealed a significant decrease in lesion formation in ApoE0/Hspg2(Delta3/Delta3) mice at both 15 and 33 weeks. In vitro, binding of labeled mouse triglyceride-rich lipoproteins and human LDL to total extracellular matrix, as well as to purified proteoglycans, prepared from ApoE0/Hspg2(Delta3/Delta3) smooth muscle cells was reduced. In vivo, at 20 minutes influx of human (125)I-LDL or mouse triglyceride-rich lipoproteins into the aortic wall was increased in ApoE0/Hspg2(Delta3/Delta3) mice compared to ApoE0 mice. However, at 72 hours accumulation of (125)I-LDL was similar in ApoE0/Hspg2(Delta3/Delta3) and ApoE0 mice. Immunohistochemistry of lesions from ApoE0/Hspg2(Delta3/Delta3) mice showed decreased staining for apoB and increased smooth muscle alpha-actin content, whereas accumulation of CD68-positive inflammatory cells was unchanged. We conclude that the perlecan HS chains are proatherogenic in mice, possibly through increased lipoprotein retention, altered vascular permeability, or other mechanisms. The ability of HS to inhibit smooth muscle cell growth may also influence development as well as instability of lesions.

Topics: Actins; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Aorta; Apolipoproteins B; Apolipoproteins E; Atherosclerosis; Capillary Permeability; Cell Proliferation; Crosses, Genetic; Disease Models, Animal; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Inflammation; Lipoproteins, LDL; Mice; Mice, Knockout; Myocytes, Smooth Muscle; Protein Binding; Triglycerides

Topics: Animals; Disease Models, Animal; Heparitin Sulfate; Humans; Intercellular Junctions; Interferon-gamma; Intestinal Mucosa; Mice; Protein-Losing Enteropathies; Syndecan-1; Tumor Necrosis Factor-alpha

Asbestosis is a form of interstitial lung disease caused by the inhalation of asbestos fibers, leading to inflammation and pulmonary fibrosis. Inflammation and oxidant/antioxidant imbalances are known to contribute to the disease pathogenesis. Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that has been shown to protect the lung from oxidant-mediated damage, inflammation, and interstitial fibrosis. Extracellular matrix (ECM) components, such as collagen and glycosaminoglycans, are known to be sensitive to oxidative fragmentation. Heparan sulfate, a glycosaminoglycan, is highly abundant in the ECM and tightly binds EC-SOD. We investigated the protective role of EC-SOD by evaluating the interaction of EC-SOD with heparan sulfate in the presence of reactive oxygen species (ROS). We found that ROS-induced heparin and heparan sulfate fragments induced neutrophil chemotaxis across a modified Boyden chamber, which was inhibited by the presence of EC-SOD by scavenging oxygen radicals. Chemotaxis in response to oxidatively fragmented heparin was mediated by Toll-like receptor-4. In vivo, bronchoalveolar lavage fluid from EC-SOD knockout mice at 1, 14, and 28 days after asbestos exposure showed increased heparan sulfate shedding from the lung parenchyma. We demonstrate that one mechanism through which EC-SOD inhibits lung inflammation and fibrosis in asbestosis is by protecting heparin/heparan sulfate from oxidative fragmentation.

Topics: Animals; Asbestosis; Chemotaxis, Leukocyte; Disease Models, Animal; Extracellular Matrix; Heparin; Heparitin Sulfate; Humans; Lung; Mice; Mice, Inbred C57BL; Neutrophils; Pulmonary Fibrosis; Superoxide Dismutase; Superoxides

Amyloid beta-peptide (Abeta) plaques, one of the major neuropathological lesions in Alzheimer's disease (AD), can be broadly subdivided into two morphological categories: neuritic and diffuse. Heparan sulfate (HS) and HS proteoglycans (HSPGs) are codeposits of multiple amyloidoses, including AD. Although HS has been considered a limiting factor in the initiation of amyloid deposition, the pathological implications of HS in Abeta deposits of AD remain unclear. In this study, immunohistochemistry combined with fluorescence and confocal microscopy was employed to gain deeper insight into the accumulation of HS with Abeta plaques in sporadic and familial AD. Here we demonstrate that HS preferentially accumulated around the Abeta40 dense cores of neuritic plaques, but was largely absent from diffuse Abeta42 plaques, suggesting that Abeta42 deposition may occur independently of HS. A codeposition pattern of HS with Abeta deposits in Tg2576 mice was also examined. We identified the membrane-bound HSPGs, glypican-1 (GPC1) and syndecan-3 (SDC3), in glial cells associated with Abeta deposits, proximal to sites of HS accumulation. In mouse primary glial cultures, we observed increased levels of GPC1 and SDC3 following Abeta stimulation. These results suggest that HS codeposits with Abeta40 in neuritic plaques and is mainly derived from glial cells.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Animals; Biomarkers; Brain; Cells, Cultured; Disease Models, Animal; Extracellular Matrix; Female; Glypicans; Heparitin Sulfate; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Neuroglia; Peptide Fragments; Plaque, Amyloid; Syndecan-3; Up-Regulation

One of the major complicating factors in insulin-dependent diabetes mellitus is nephropathy. Several investigators have linked heparan sulfate (HS) alterations in the glomerular basement membrane (GBM) with albuminuria as a marker of abnormal blood filtration and the subsequent progression to renal failure. In this study, we examined the fine structure of HS in the glomerulus and the GBM isolated from the kidneys of rats injected with streptozotocin. Using fluorophore-assisted carbohydrate electrophoresis, we obtained disaccharide composition analyses for HS. In a time course study, we observed that normal rat HS isolated from the GBM becomes more N-sulfated as the glomeruli mature over a period of 8 weeks. Diabetic rats injected with streptozotocin at the beginning of this period showed a reversal of this trend. Using a graded sieve technique, we found that two different sizes of glomeruli could be isolated from the rat kidneys and that there was a significant difference in the HS disaccharide content between these two pools of glomeruli. Only the larger sized glomeruli had less N-sulfation of HS as a result of insulin-dependent diabetes mellitus. This change in the fine structure of HS was localized to the GBM and was not associated with cell surface HS. We also generated oligosaccharides of HS that portray fine structural alterations in the diabetic rats indicative of a loss of the sulfation of N-acetylglucosamine.

Topics: Acetylglucosamine; Albuminuria; Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disaccharides; Disease Models, Animal; Heparitin Sulfate; Kidney Glomerulus; Male; Oligosaccharides; Polysaccharide-Lyases; Rats; Rats, Sprague-Dawley; Sulfates

At present, there is no widely available, safe and effective treatment for lysosomal storage disorders (LSD) that affect the brain. We have used a naturally occurring mouse model of mucopolysaccharidosis type IIIA (MPS IIIA) or Sanfilippo syndrome, to evaluate the effect of repeated injection of recombinant human sulfamidase (rhSGSH) into the cerebrospinal fluid via the cisterna magna (CM) on central nervous system (CNS) pathology and behavioral function. Mice received up to seven injections of rhSGSH (5-20 microg rhSGSH per injection) or vehicle on a fortnightly or monthly basis. A dose-dependent reduction in the level of a heparan sulfate-derived monosulfated disaccharide was observed within the brain (up to 62% reduction compared with vehicle-treated MPS IIIA mice) and spinal cord (up to 71% reduction). Ultrastructural examination revealed a reduction in lysosomal vesicle formation in various cell types and fewer (ubiquitin-positive) axonal spheroids were observed in several brain regions. The biochemical changes were accompanied by improved behavior, particularly in mice-treated more frequently. A humoral immune response to rhSGSH was observed in treated animals. Intra-CM injection of lysosomal enzyme may therefore represent an immediately applicable method of treating the CNS effects of this and potentially other LSD that affect the brain.

Topics: Animals; Antibodies, Heterophile; Behavior, Animal; Brain; Cisterna Magna; Disease Models, Animal; Heparitin Sulfate; Humans; Hydrolases; Immunohistochemistry; Injections, Intraventricular; Male; Mice; Mice, Mutant Strains; Microscopy, Electron; Mucopolysaccharidosis III; Recombinant Proteins

Sulfated polyanions, including pentosan polysulfate (PPS) and heparan mimetics, number among the most effective drugs that have been used in experimental models of prion disease and are presumed to act in competition with endogenous heparan sulfate proteoglycans as co-receptors for prion protein (PrP) on the cell surface. PPS has been shown to prolong the survival of animals after intracerebral perfusion and is in limited use for the experimental treatment of human transmissible spongiform encephalopathies (TSEs). Here, PPS is compared with CR36, a new heparan mimetic. Ex vivo, CR36 was more efficient than PPS in reducing PrPres in scrapie-infected cell cultures and showed long-lasting activity. In vivo, CR36 showed none of the acute toxicity observed with PPS and reduced PrPres accumulation in spleens, but had only a marginal effect on the survival time of mice infected with bovine spongiform encephalopathy. In contrast, mice treated with PPS that survived the initial toxic mortality had no detectable PrPres in the spleens and lived 185 days longer than controls (+55%). These results show, once again, that anti-TSE drugs cannot be encouraged for human therapeutic trials solely on the basis of in vitro or ex vivo observations, but must first be subjected to in vivo animal studies.

Topics: Animals; Cell Line; Disease Models, Animal; Female; Heparitin Sulfate; Humans; Mice; Mice, Inbred C57BL; Pentosan Sulfuric Polyester; Prion Diseases; Prions; Spleen; Survival Analysis

Glutaric aciduria type 1 (GA1) is a childhood metabolic disorder associated with crises that lead to striatal necrosis. Although the disorder can be controlled with diet, there is no current treatment to ameliorate the neurodegeneration following a metabolic crisis. We hypothesized that heparan sulfate (HS) administration would stimulate neural stem cell proliferation by dimerizing with FGF-2 and binding to the FGF-2 receptor on neural stem cells, thus enhancing the number of newly generated neurons to repair damage following a metabolic crisis. In addition, FGF-2 is known to exert neuroprotective effects independent of neurogenesis, so HS may also have neuroprotective activities. To test these hypotheses, ibotenic acid was injected into the striatum of adult mice, mimicking the metabolic crisis and damage caused by glutaric aciduria. Daily doses of HS and bromodeoxyuridine (BrdU) or BrdU alone were administered starting 1 day after the ibotenic acid lesion. BrdU was used to label dividing cells. Fluorescent immunohistochemistry was used to quantify the lesion size and evaluate the phenotype of BrdU-positive cells. Intrastriatal administration of ibotenic acid resulted in a substantial striatal lesion that occupied 18.5% of the ipsilateral brain hemisphere. In contrast, animals treated with HS exhibited a lesion volume representing <1% of the ipsilateral brain hemisphere (ANOVA; p < 0.0001). Increased neurogenesis, however, was not observed in this group. These results suggest that HS administration 2 days after a "metabolic crisis" can ameliorate brain injury in an animal model of GA1. The neuroprotective mechanisms of HS, however, remain to be elucidated but may exert their actions indirectly through binding with FGF-2.

Topics: Amino Acid Metabolism, Inborn Errors; Animals; Antimetabolites; Brain; Bromodeoxyuridine; Child; Disease Models, Animal; Excitatory Amino Acid Agonists; Female; Glutarates; Heparitin Sulfate; Humans; Ibotenic Acid; Mice; Neuroprotective Agents

Many viruses, including flaviviruses, display affinity for cell surface heparan sulfate (HS) proteoglycans with biological relevance in virus attachment/entry. This raises the possibility of the application of HS mimetics in antiviral therapy. We have evaluated the antiviral effect of the sulfated polysaccharides, suramin, pentosan polysulfate (PPS) and PI-88, which are currently approved or in trial for clinical use, against dengue virus (DEN) and the encephalitic flaviviruses, Japanese encephalitis virus, West Nile virus, and Murray Valley encephalitis virus. A flow cytometry-based method for the measurement of inhibition of virus infectivity was developed, which showed the in vitro antiviral activity of the three compounds, albeit with differences in efficiency which were virus-dependent. The 50% effective concentration (EC(50)) values for DEN inhibition were in the order: PPS

Topics: Animals; Antiviral Agents; Cell Line; Dengue; Dengue Virus; Disease Models, Animal; Drug Evaluation, Preclinical; Encephalitis Viruses, Japanese; Encephalitis, Arbovirus; Female; Flavivirus Infections; Heparitin Sulfate; Injections, Intraperitoneal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oligosaccharides; Pentosan Sulfuric Polyester; Suramin; Treatment Outcome

Heparan sulfate proteoglycans are integral components of the extracellular matrix that surrounds all mammalian cells. In addition to providing structural integrity, they act as a storage depot for a variety of heparan sulfate (HS)-binding proteins, including growth factors and chemokines. Heparanase is a matrix-degrading enzyme that cleaves heparan sulfate side chains from the core proteoglycans, thus liberating such HS-binding proteins, as well as potentially contributing to extracellular matrix degradation. Here, we report that heparanase mRNA and protein expression are increased in the neoplastic stages progressively unfolding in a mouse model of multistage pancreatic islet carcinogenesis. Notably, heparanase is delivered to the neoplastic lesions in large part by infiltrating Gr1+/Mac1+ innate immune cells. A sulfated oligosaccharide mimetic of heparan sulfate, PI-88, was used to inhibit simultaneously both heparanase activity and HS effector functions. PI-88 had significant effects at distinct stages of tumorigenesis, producing a reduction in the number of early progenitor lesions and an impairment of tumor growth at later stages. These responses were associated with decreased cell proliferation, increased apoptosis, impaired angiogenesis, and a substantive reduction in the number of invasive carcinomas. In addition, we show that the reduction in tumor angiogenesis is correlated with a reduced association of VEGF-A with its receptor VEGF-R2 on the tumor endothelium, implicating heparanase in the mobilization of matrix-associated VEGF. These data encourage clinical applications of inhibitors such as PI-88 for the many human cancers where heparanase expression is elevated or mobilization of HS-binding regulatory factors is implicated.

Topics: Amino Acid Sequence; Animals; Capillaries; Cattle; Disease Models, Animal; Endothelium, Vascular; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glucuronidase; Heparitin Sulfate; Islets of Langerhans; Mice; Molecular Sequence Data; Neovascularization, Pathologic; Pancreatic Neoplasms; Peptide Fragments; Polymerase Chain Reaction

Sanfilippo syndrome type B is caused by alpha-N-acetylglucosaminidase (Naglu) enzyme deficiency leading to an accumulation of undegraded heparan sulfate, a glycosaminoglycan (GAG). Cell therapy is a promising new treatment and human umbilical cord blood (hUCB) cell transplantation may be preferred for delivery of the missing enzyme. We investigated the ability of mononuclear hUCB cells administered into the lateral cerebral ventricle to ameliorate/prevent histopathological changes in mice modeling Sanfilippo syndrome type B. These are the first results supporting enzyme replacement by administered hUCB cells. In vivo, transplanted hUCB cells survived long-term (7 months), migrated into the parenchyma of the brain and peripheral organs, expressed neural antigens, and exhibited neuron and astrocyte-like morphology. Transplant benefits were also demonstrated by stable cytoarchitecture in the hippocampus and cerebellum, and by reduced GAGs in the livers of treated mutant mice. A hUCB cell transplant may be an effective therapeutic strategy for enzyme delivery in Sanfilippo syndrome type B.

Topics: Animals; Cell Differentiation; Cell Movement; Cell Survival; Cell Transplantation; Cerebellum; Disease Models, Animal; Fetal Blood; Glycosaminoglycans; Heparitin Sulfate; Hippocampus; Humans; Immunohistochemistry; Leukocytes, Mononuclear; Liver; Mice; Mucopolysaccharidosis III; Neurons; Umbilical Veins

Mucopolysaccharidosis I (MPS I) is a lysosomal disorder characterized by a deficiency of the enzyme alpha-L: -iduronidase (IDUA), which is responsible for the degradation of glycosaminoglycans (GAGs). This deficiency leads to the accumulation of dermatan and heparan sulphate in lysosomes. Presently available treatments include bone marrow transplantation and enzyme replacement therapies, both of which are limited in their effects. In this work, knockout (KO) MPS I mice were treated with a nonviral vector containing the human IDUA cDNA. KO mice were transfected by hydrodynamic injection of pRIDUA in the caudal vein (i.v., n = 3) or by intraperitoneal injection of pRIDUA/Superfect complexes (i.p., n = 3). GAG concentration and IDUA activity were analysed in the kidneys, spleen, lungs, brain and liver. The expression of IDUA in the organs of i.v.- and i.p.-treated mice was also analysed by real-time reverse-transcription (RT) PCR and compared by relative quantification. The concentration of GAGs in the organs differed between KO and wild-type mice. In the spleen and liver, GAG levels were lower in i.v.- and i.p.-treated KO mice than in control nontreated animals. Real-time RT-PCR showed that the transgene is expressed in all the analysed organs of i.p.- and i.v.-treated KO mice. Enzyme activity was similarly observed in all the organs analysed. Our data suggest that this kind of transfection may be a useful tool for studies of nonviral protocols for gene therapy of MPS.

Topics: Animals; Bone Marrow Transplantation; Dermatan Sulfate; Disease Models, Animal; DNA, Complementary; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Glycosaminoglycans; Heparitin Sulfate; Humans; Lysosomes; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucopolysaccharidosis I; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Distribution; Transduction, Genetic; Transfection

The accumulation of PrP(res), the protease-resistant abnormal form of the host-encoded cellular prion protein, PrP(C), plays a central role in transmissible spongiform encephalopathies. Human contamination by bovine spongiform encephalopathy (BSE) has propelled many scientific teams on a highway for anti-prion drug development. This study reports that heparan sulfate mimetics (HMs), developed originally for their effect on tissue regeneration, abolish prion propagation in scrapie-infected GT1 cells. PrP(res) does not reappear for up to 50 days post-treatment. When tested in vivo, one of these compounds, HM2602, hampered PrP(res) accumulation in scrapie- and BSE-infected mice and prolonged significantly the survival time of 263K scrapie-infected hamsters. Interestingly, HM2602 is an apparently less toxic and more potent inhibitor of PrP(res) accumulation than dextran sulfate 500, a molecule known to exhibit anti-prion properties in vivo. Kinetics of PrP(res) disappearance in vitro and unaffected PrP(C) levels during treatment suggest that HMs are able to block the conversion of PrP(C) into PrP(res). It is speculated that HMs act as competitors of endogenous heparan sulfates known to act as co-receptors for the prion protein. Since these molecules are particularly amenable to drug design, their anti-prion potential could be developed further and optimized for the treatment of prion diseases.

Topics: Animals; Anti-Infective Agents; Cattle; Cells, Cultured; Cricetinae; Disease Models, Animal; Disease Progression; Drug Design; Encephalopathy, Bovine Spongiform; Female; Heparitin Sulfate; Injections, Intraperitoneal; Mice; Mice, Inbred C57BL; Molecular Structure; PrPC Proteins; PrPSc Proteins; Scrapie; Time Factors

Basic fibroblast growth factor (or FGF-2) has been shown to be a potent stimulator of retinal ganglion cell (RGC) axonal growth during development. Here we investigated if FGF-2 upregulation in adult RGCs promoted axon regrowth in vivo after acute optic nerve injury. Recombinant adeno-associated virus (AAV) was used to deliver the FGF-2 gene to adult RGCs providing a sustained source of this neurotrophic factor. FGF-2 gene transfer led to a 10-fold increase in the number of axons that extended past 0.5 mm from the lesion site compared to control nerves. Detection of AAV-mediated FGF-2 protein in injured RGC axons correlated with growth into the distal optic nerve. The response to FGF-2 upregulation was supported by our finding that FGF receptor-1 (FGFR-1) and heparan sulfate (HS), known to be essential for FGF-2 signaling, were expressed by adult rat RGCs. FGF-2 transgene expression led to only transient protection of injured RGCs. Thus the effect of this neurotrophic factor on axon extension could not be solely attributed to an increase in neuronal survival. Our data indicate that selective upregulation of FGF-2 in adult RGCs stimulates axon regrowth within the optic nerve, an environment that is highly inhibitory for regeneration. These results support the hypothesis that key factors involved in axon outgrowth during neural development may promote regeneration of adult injured neurons.

Topics: Animals; Cell Differentiation; Cell Survival; Disease Models, Animal; Female; Fibroblast Growth Factor 2; Gene Expression Regulation, Developmental; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Growth Cones; Heparitin Sulfate; Nerve Regeneration; Optic Nerve Injuries; Rats; Rats, Sprague-Dawley; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptors, Fibroblast Growth Factor; Retina; Retinal Ganglion Cells; Up-Regulation

Sanfilippo syndrome type B (MPS III B) is a neurodegenerative disorder characterized by profound mental retardation and early death. It is caused by deficiency of a lysosomal enzyme involved in heparan sulfate (HS) degradation. Because HS accumulation can be a major feature of this disease, we have examined crucial molecular systems associated with HS function. Using a knockout mouse with disruption of the gene responsible for HS degradation, we evaluated the effects of possible HS accumulation on neuroplasticity that are within the spectrum of action of fibroblast growth factors (FGFs) and their receptor (FGFR). We found that levels of mRNA for the FGFR-1 were attenuated in the mutant mice by the age of 6 months, whereas the mRNAs for FGF-1 and FGF-2 were reduced or unchanged in the brain regions tested. Neurogenesis, in which FGF-2 is involved, was inhibited in the MPS III B mouse brain at both young and adult ages. We also examined the expression of the glial fibrillary acidic protein (GFAP) gene and GFAP-positive cell density in both normal and injured conditions to study the functional response of astrocytes to insult. We found that, although the mutation alone caused drastic induction of reactive astrocytes, acute injury to the mutant brains failed to induce additional reactive astrocytes. Our results showed important alterations in the expression of several genes involved in the maintenance of neuroplasticity in the MPS III B. This in turn may result in reduction of neuronal health and brain function.

Topics: Acetylglucosaminidase; Animals; Astrocytes; Cell Division; Cell Survival; Disease Models, Animal; Fibroblast Growth Factors; Glial Fibrillary Acidic Protein; Heparitin Sulfate; Male; Mice; Mice, Knockout; Mucopolysaccharidosis III; Neuronal Plasticity; Neurons; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptors, Fibroblast Growth Factor; Stem Cells

Hereditary multiple exostoses (HME), a dominantly inherited disorder characterized by multiple cartilaginous tumors, is caused by mutations in the gene for, EXT1 or EXT2. Recent studies have revealed that EXT1 and EXT2 are required for the biosynthesis of heparan sulfate and exert maximal transferase activity as a complex. The Drosophila homologue of EXT1 (tout-velu) regulates the movement and signaling of Hedgehog protein, which plays an important role in the regulation of chondrocyte differentiation and bone development. In this study, to investigate the biological role of EXT2 in bone development in vivo and the pathological role of HME mutations in the development of exostoses, we generated transgenic mice expressing EXT2 or mutant EXT2 in developing chondrocytes. Histological analyses and micro-CT scanning showed that the biosynthesis of heparan sulfate and the formation of trabeculae were upregulated in EXT2-transgenic mice, but not in mutant EXT2-transgenic mice. The expression of EXT1 is concomitantly upregulated in EXT2-transgenic and even mutant EXT2-transgenic mice, suggesting an interactive regulation of EXT1 and EXT2 expression. These findings support that the EXT2 gene encodes an essential component of the glycosyltransferase complex required for the biosynthesis of heparan sulfate, which may eventually modulate the signaling involved in bone formation.

Topics: Animals; beta-Galactosidase; Cartilage; Chondrocytes; Disease Models, Animal; Embryo, Mammalian; Exostoses, Multiple Hereditary; Gene Expression; Genes, Reporter; Glycosyltransferases; Heparitin Sulfate; Immunohistochemistry; Mice; Mice, Transgenic; Mutation; N-Acetylglucosaminyltransferases; Osteogenesis; Protein Biosynthesis; Proteins; Signal Transduction; Tomography, X-Ray Computed; Transgenes; Up-Regulation

Cell-surface heparan sulphate proteoglycans (HSPGs) are ubiquitous and abundant receptors/co-receptors of extracellular ligands, including many microbes. Their role in microbial infections is poorly defined, however, because no cell-surface HSPG has been clearly connected to the pathogenesis of a particular microbe. We have previously shown that Pseudomonas aeruginosa, through its virulence factor LasA, enhances the in vitro shedding of syndecan-1-the predominant cell-surface HSPG of epithelia. Here we show that shedding of syndecan-1 is also activated by P. aeruginosa in vivo, and that the resulting syndecan-1 ectodomains enhance bacterial virulence in newborn mice. Newborn mice deficient in syndecan-1 resist P. aeruginosa lung infection but become susceptible when given purified syndecan-1 ectodomains or heparin, but not when given ectodomain core protein, indicating that the ectodomain's heparan sulphate chains are the effectors. In wild-type newborn mice, inhibition of syndecan-1 shedding or inactivation of the shed ectodomain's heparan sulphate chains prevents lung infection. Our findings uncover a pathogenetic mechanism in which a host response to tissue injury-syndecan-1 shedding-is exploited to enhance microbial virulence apparently by modulating host defences.

Topics: Animals; Animals, Newborn; Bacterial Adhesion; Disease Models, Animal; Heparin; Heparitin Sulfate; Lung; Lung Diseases; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Protein Structure, Tertiary; Proteoglycans; Pseudomonas aeruginosa; Pseudomonas Infections; Syndecan-1; Syndecans; Virulence

Propagation of the flavivirus tick-borne encephalitis virus in BHK-21 cells selected for mutations within the large surface glycoprotein E that increased the net positive charge of the protein. In the course of 16 independent experiments, 12 different protein E mutation patterns were identified. These were located in all three of the structural domains and distributed over almost the entire upper and lateral surface of protein E. The mutations resulted in the formation of local patches of predominantly positive surface charge. Recombinant viruses carrying some of these mutations in a defined genetic backbone showed heparan sulfate (HS)-dependent phenotypes, resulting in an increased specific infectivity and binding affinity for BHK-21 cells, small plaque formation in porcine kidney cells, and significant attenuation of neuroinvasiveness in adult mice. Our results corroborate the notion that the selection of attenuated HS binding mutants is a common and frequent phenomenon during the propagation of viruses in cell culture and suggest a major role for HS dependence in flavivirus attenuation. Recognition of this principle may be of practical value for designing attenuated flavivirus strains in the future.

Topics: Adaptation, Physiological; Animals; Binding Sites; Cell Line; Disease Models, Animal; Encephalitis Viruses, Tick-Borne; Encephalitis, Tick-Borne; Heparitin Sulfate; Mice; Models, Molecular; Mutation; Viral Envelope Proteins; Viral Plaque Assay; Virulence

The Steno hypothesis (Deckert et al. ) states that in diabetes mellitus (DM), changes in vascular heparan sulfate proteoglycan (HSPG) expression are involved in systemic endothelial dysfunction and increased capillary permeability. In diabetes-induced glomerular capillary leakage, loss of HSPG and its side chains has been documented. This study aimed to investigate whether microvascular leakage in diabetic retinopathy (DR) is also associated with altered expression of HSPG in retinal microvessels.. Serial cryosections of post-mortem eyes of 22 subjects with DM and 7 controls were stained with antibodies against the core proteins of the basement membrane HSPGs agrin (Abs Bl31 and JM72) and perlecan (Ab 1948), and four antibodies against heparan sulfate side chains (HS) (Abs JM403, HepSS1, JM13, 3G10). Moreover, we investigated Cynomolgus monkey eyes injected with vascular endothelial growth factor (VEGF)-A, as a model of retinal microvas-cular leakage. The endothelial antigen PAL-E was used to detect microvascular leakage.. In the retina of all controls and DM cases, agrin and perlecan core proteins and HS as recognized by JM403 and 3G10 were expressed in the walls of microvessels. Staining for JM13 was variable between cases, but unrelated to microvascular leakage as determined by PAL-E. Staining for HepSS1 was absent in all human retinal microvessels. In monkey retinas, HSPG staining was identical to that in human retinal tissues, except for the staining for HepSS1, which was found absent in control monkey eyes but which was positive in VEGF-injected eyes.. Increased microvascular permeability in human DR is not associated with changes in expression of the HSPGs studied, whereas high amounts of VEGF may induce increased expression of the HS side chain epitope recognized by HepSS1. These results suggest that the mechanism underlying retinal leakage is different from diabetic glomerular capillary leakage.

Topics: Aged; Aged, 80 and over; Agrin; Animals; Antibody Specificity; Blood-Retinal Barrier; Capillary Permeability; Diabetes Mellitus; Diabetic Retinopathy; Disease Models, Animal; Endothelial Growth Factors; Fluorescent Antibody Technique, Indirect; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunoenzyme Techniques; Macaca fascicularis; Middle Aged; Retinal Vessels; Vascular Endothelial Growth Factor A

Danaparoid and heparin, on the basis of anti-activated factor X (anti-FXa) activity, were equipotent in accelerating the rate of interaction of FXa and antithrombin III. In rat tissue factor-induced disseminated intravascular coagulation (DIC) models, an intravenous administration of danaparoid inhibited the decrease in plasma fibrinogen and platelet counts and the increase in serum fibrinogen degradation products. Expressed on the basis of anti-FXa activity, these effects were comparable with those of dalteparin and heparin. In rat mesenteric small artery and vein, less bleeding was observed after intravenous administration of danaparoid than after dalteparin or heparin. Danaparoid did not affect adenosine diphosphate- or collagen-induced platelet aggregation, and showed weaker inhibitory effects on aggregation induced by thrombin, or collagen + thrombin, than did dalteparin or heparin. These findings suggest that danaparoid may be useful for the prevention of DIC and has less tendency to cause bleeding than dalteparin or heparin, probably as a result of its weaker ability to inhibit platelet aggregation.

Topics: Animals; Anticoagulants; Antithrombin III; Bleeding Time; Chondroitin Sulfates; Dalteparin; Dermatan Sulfate; Disease Models, Animal; Disseminated Intravascular Coagulation; Drug Combinations; Drug Evaluation, Preclinical; Enzyme Inhibitors; Factor Xa; Factor Xa Inhibitors; Heparin; Heparitin Sulfate; Kinetics; Male; Platelet Aggregation; Rats; Rats, Wistar; Risk Assessment; Thromboplastin

The Sanfilippo syndrome type B (MPS III B) is an autosomal recessive disease caused by deficiency of alpha-N-acetylglucosaminidase (EC 3. 2.1.50), one of the lysosomal enzymes required for the degradation of heparan sulfate. The disease is characterized by profound neurodegeneration but relatively mild somatic manifestations, and is usually fatal in the second decade. A mouse model had been generated by disruption of the Naglu gene in order to facilitate the study of pathogenesis and the development of therapy for this currently untreatable disease. Recombinant human alpha-N-acetylglucosaminidase (rhNAGLU) was prepared from secretions of Lec1 mutant Chinese hamster ovary cells. The enzyme, which has only unphosphorylated high-mannose carbohydrate chains, was endocytosed by mouse peritoneal macrophages via mannose receptors, with half-maximal uptake at ca. 10(-7) M. When administered intravenously to 3 month-old mice, rhNAGLU was taken up avidly by liver and spleen but marginally if at all by thymus, lung, kidney, heart, and brain (in order of diminishing uptake). The half-life of the enzyme was 2.5 days in liver and spleen. Immunohistochemistry and electron microscopy showed that only macrophages were involved in enzyme uptake and correction in these two organs, yet the storage of glycosaminoglycan was reduced to almost normal levels. The results show that the macrophage-targeted rhNAGLU can substantially reduce the body burden of glycosaminoglycan storage in the mouse model of Sanfilippo syndrome III B.

Topics: Acetylglucosaminidase; Animals; Disease Models, Animal; Endocytosis; Gene Deletion; Glycosaminoglycans; Half-Life; Heparitin Sulfate; Humans; Immunohistochemistry; Injections, Intravenous; Liver; Macrophages, Peritoneal; Mice; Mice, Knockout; Microscopy, Electron; Mucopolysaccharidosis III; Recombinant Proteins; Spleen

Mucopolysaccharidosis type IIID (MPS IIID) is a lysosomal storage disorder resulting from lack of activity of the lysosomal hydrolase N-acetylglucosamine 6-sulfatase (6S) (EC 3.1.6.14). The syndrome is associated with systemic and central nervous system (CNS) heparan sulfate glycosaminoglycan (HS-GAG) accumulation, secondary storage of lipids, and severe, progressive dementia. In this investigation, caprine MPS IIID, established as a large animal model for the human disease, was used to evaluate the efficacy of enzyme replacement therapy (ERT). Recombinant caprine 6S (rc6S) (1 mg/kg/dose) was administered intravenously to one MPS IIID goat kid at 2, 3, and 4 wks of age. Five days after the last dose, the uronic acid (UA) content and the composition of uncatabolized HS-GAG fractions in the brain of the ERT-treated MPS IIID kid were similar to those from a control, untreated MPS IIID animal. However, hepatic uronic acid levels in the treated MPS IIID kid were approximately 90% lower than those in the untreated MPS IIID control; whereas the composition of the residual hepatic HS-GAG was identical to that in the untreated animal. Marked reduction of lysosomal storage vacuoles in hepatic cells of the treated MPS IIID kid was observed, but ERT had no effect on CNS lesions. No residual 6S activity was detected in brain or liver. This preliminary investigation indicates that other treatment regimens will be necessary to ameliorate MPS III-related CNS lesions.

Topics: Animals; Brain; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Female; Glycosaminoglycans; Goats; Heparitin Sulfate; Hepatocytes; Liver; Male; Mucopolysaccharidosis III; Neurons; Recombinant Proteins; Sulfatases; Treatment Outcome; Uronic Acids

Heparins are useful for the protection of residual renal function in several nephropathies, but the anticoagulant action and the need of parenteral administration are two main drawbacks limiting their use in chronic renal failure patients. Heparan sulphate (HS) is a heparin-like mucopolysaccharide devoid of anticoagulant action and active orally. In this study, the effects of HS oral administration have been evaluated in 18 subtotally nephrectomized rats;18 untreated remnant kidney rats served as control. No mortality was observed in the HS-treated rats, whereas in the control rats the survival rate was 72.2% at 18 weeks. At the end of the study, HS-treated rats showed lower urinary protein excretion (44 +/- 22 vs. 80 +/- 54 mg/24 h, p < 0.01), lower urea plasma levels (75 +/- 34 vs. 134 +/- 105 mg/dl, p < 0.01) and higher creatinine clearance (66 +/- 15 vs. 47 +/- 21 ml/min. 10(2), p < 0.05) than control rats. Remnant kidney weight (2.3 +/- 1.1 vs. 1.3 +/- 0.2 g, p < 0.01) and heart weight (1.3 +/- 0.2 vs. 1.1 +/- 0.1 g, p < 0.05) were greater in the control than in the HS-treated rats, as well as the systemic blood pressure values (167 +/- 19 vs. 115 +/- 32 mm Hg, respectively, p < 0.001). The remnant kidney histological examination in the HS-treated rats showed a lower prevalence of glomerular sclerosis, mesangial proliferation, and a much less evident tubulointerstitial damage than in controls. The antiproliferative and anti-inflammatory actions of HS together with its protective action on the endothelium are the putative mechanisms that could account for our findings. In conclusion, the present study supports evidence of an antiproteinuric and a renoprotective effect of orally administered HS in subtotally nephrectomized rats. This is in keeping with the well-known effects exerted also by other heparins, but the effectiveness of an orally available heparin-like product in this animal model could suggest the possibility of a clinical use also in progressing chronic renal failure patients.

Topics: Administration, Oral; Animals; Disease Models, Animal; Heparitin Sulfate; Kidney; Kidney Failure, Chronic; Male; Nephrectomy; Proteinuria; Rats; Rats, Wistar

Invasive growth of cancer cells is a complex process involving specific interactions between tumour cells and the orderly, integrated complexes of the extracellular matrix. Basement membranes have been proposed as one constituent of extracellular matrix which carries responsibility for regulating invasion and metastasis. Using a chemically induced rat tongue carcinoma model, it has been shown that components of the basement membrane and its overall structure are altered during tumour invasion, and methods have been developed to quantitate some of these differences. Since the basement membrane can be specifically characterized by its fibrous protein network of Type IV collagen and laminin, which is embedded in a heparan sulphate-rich proteoglycan matrix, these components have been targeted. In particular, the current paper presents results in the context of current concepts of early changes in neoplastic invasion of underlying connective tissues. In consequence, further elaboration of the underlying mechanisms of epithelial migration in oral cancer may allow an exploration of the use of alterations in expression of basement membrane components as prognostic indicators.

Topics: 4-Nitroquinoline-1-oxide; Animals; Basement Membrane; Carcinogens; Carcinoma; Cell Movement; Collagen; Coloring Agents; Connective Tissue; Disease Models, Animal; Epithelium; Extracellular Matrix; Extracellular Matrix Proteins; Heparitin Sulfate; Immunohistochemistry; Laminin; Neoplasm Invasiveness; Polylysine; Proteoglycans; Rats; Tongue Neoplasms

Mucopolysaccharidosis type III A (MPS III A, Sanfilippo syndrome) is a rare, autosomal recessive, lysosomal storage disease characterized by accumulation of heparan sulfate secondary to defective function of the lysosomal enzyme heparan N- sulfatase (sulfamidase). Here we describe a spontaneous mouse mutant that replicates many of the features found in MPS III A in children. Brain sections revealed neurons with distended lysosomes filled with membranous and floccular materials with some having a classical zebra body morphology. Storage materials were also present in lysosomes of cells of many other tissues, and these often stained positively with periodic-acid Schiff reagent. Affected mice usually died at 7-10 months of age exhibiting a distended bladder and hepatosplenomegaly. Heparan sulfate isolated from urine and brain had nonreducing end glucosamine- N -sulfate residues that were digested with recombinant human sulfamidase. Enzyme assays of liver and brain extracts revealed a dramatic reduction in sulfamidase activity. Other lysosomal hydrolases that degrade heparan sulfate or other glycans and glycosaminoglycans were either normal, or were somewhat increased in specific activity. The MPS III A mouse provides an excellent model for evaluating pathogenic mechanisms of disease and for testing treatment strategies, including enzyme or cell replacement and gene therapy.

Topics: Animals; Brain; Disease Models, Animal; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Hydrolases; Liver; Lysosomes; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Microscopy, Electron; Mucopolysaccharidosis III; Myocardium; Spleen; Urinary Bladder

The Sanfilippo syndrome type B is an autosomal recessive disorder caused by mutation in the gene (NAGLU) encoding alpha-N-acetylglucosaminidase, a lysosomal enzyme required for the stepwise degradation of heparan sulfate. The most serious manifestations are profound mental retardation, intractable behavior problems, and death in the second decade. To generate a model for studies of pathophysiology and of potential therapy, we disrupted exon 6 of Naglu, the homologous mouse gene. Naglu-/- mice were healthy and fertile while young and could survive for 8-12 mo. They were totally deficient in alpha-N-acetylglucosaminidase and had massive accumulation of heparan sulfate in liver and kidney as well as secondary changes in activity of several other lysosomal enzymes in liver and brain and elevation of gangliosides G(M2) and G(M3) in brain. Vacuolation was seen in many cells, including macrophages, epithelial cells, and neurons, and became more prominent with age. Although most vacuoles contained finely granular material characteristic of glycosaminoglycan accumulation, large pleiomorphic inclusions were seen in some neurons and pericytes in the brain. Abnormal hypoactive behavior was manifested by 4.5-mo-old Naglu-/- mice in an open field test; the hyperactivity that is characteristic of affected children was not observed even in younger mice. In a Pavlovian fear conditioning test, the 4.5-mo-old mutant mice showed normal response to context, indicating intact hippocampal-dependent learning, but reduced response to a conditioning tone, perhaps attributable to hearing impairment. The phenotype of the alpha-N-acetylglucosaminidase-deficient mice is sufficiently similar to that of patients with the Sanfilippo syndrome type B to make these mice a good model for study of pathophysiology and for development of therapy.

Topics: Acetylglucosaminidase; Animals; Base Sequence; Behavior, Animal; Brain Chemistry; Disease Models, Animal; Female; Gangliosides; Glycosaminoglycans; Heparitin Sulfate; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Sequence Data; Mucopolysaccharidosis III

Previous reports strongly suggest that, in addition to the nicotinic acetylcholine receptor, rabies virus can use other, as-yet-unidentified receptors. We found that laboratory cell lines susceptible to rabies virus infection express the neural cell adhesion molecule (NCAM) (CD56) on their surface, whereas resistant cells do not, supporting the idea that NCAM could be a rabies virus receptor. We observed that (i) incubation with rabies virus decreases the surface expression of NCAM; (ii) treatment of susceptible cells with heparan sulfate, a ligand for NCAM, or with NCAM antibodies significantly reduces the rabies virus infection; and (iii) preincubation of rabies virus inoculum with soluble NCAM protein as a receptor decoy drastically neutralizes the capacity of rabies virus to infect susceptible cells. Moreover, we demonstrated that transfection of resistant L fibroblasts with the NCAM-encoding gene induces rabies virus susceptibility whereas absence of NCAM in the primary cortical cell cultures prepared from NCAM-deficient mice reduces the rabies virus infection and virus production. This provides evidence that NCAM is an in vitro receptor for the rabies virus. Moreover, the in vivo relevance for the use of NCAM as a receptor was demonstrated by the infection of NCAM-deficient mice, in which rabies mortality was delayed and brain invasion by rabies virus was drastically restricted. Our results showed that NCAM, which is expressed mainly in the adult nervous system, plays an important role in rabies infection. However, it cannot be excluded that receptors other than NCAM are utilized. Thus, the description of NCAM as a new rabies virus receptor would be another example of the use by viruses of more than one receptor to gain entry into the host.

Topics: Animals; Antibodies, Monoclonal; Brain; CD56 Antigen; Cell Line; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; DNA, Complementary; Heparitin Sulfate; Mice; Neutralization Tests; Rabies; Rabies virus; Receptors, Virus; Solubility; Transfection

A decrease in anionic change and the loss of heparan sulfate proteoglycan have previously been observed in the glomerular basement membrane (GBM) during diabetic glomerulosclerosis. We studied the chronological changes in the anionic character and the glycosaminoglycan content in the GBM of WBN/ Kob rats with spontaneous diabetes. Two types of cationic probes were used: polyethyleneimine (PEI) and cationic colloidal gold (CCG). Immunogold labeling was performed with anti-monoclonal-heparan-sulfate-glycosaminoglycan (HS-GAG) and anti-chondroitin-sulfate-glycosaminoglycan (CS-GAG) antibodies. The GBM width, the anionic sites and the GAG sites were investigated in diabetic WBN/Kob rats at 2, 10 and 19 months, compared with control rats. Diabetes was confirmed in WBN/Kob rats after 8 months in this study. The GBM width gradually thickened with age. The PEI anionic sites significantly decreased in the lamina rara externa (LRE) at 19 months (vs. 2 and 10 months). The HS-GAG sites also significantly decreased in the LRE at 10 and 19 months (vs. 2 months). However, the CCG anionic sites and the CS-GAG sites significantly increased in the LRE and the lamina densa at 10 months (vs. 2 months) and, after 19 months, returned to the level seen at 2 months. Results indicate that there is an early transient increase in CS-GAG in the GBM while HS-GAG decreases. We noticed a transient increase in the CCG anionic sites at this early stage of diabetic glomerulosclerosis as well. The increase in CS-GAG may provide a marker for early diabetic changes in the GBM.

Topics: Animals; Anions; Antibodies, Monoclonal; Basement Membrane; Chondroitin Sulfates; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Heparitin Sulfate; Immunohistochemistry; Kidney Glomerulus; Male; Rats; Rats, Wistar

To evaluate the interaction in the renal tubules between calcium oxalate microliths and glycosaminoglycans, especially heparan sulfate, we planned an experimentally-induced calcium oxalate microlith-producing model in rats. We found by immunofluorescence study and immunoelectron microscopy using the heparan sulfate antibody that heparan sulfate was produced in the renal tubular cells in the formation of calcium oxalate microliths and that heparan sulfate exists in the microliths. Heparan sulfate/crystal interaction might play a role as a nucleating agent and promote stone enlargement.

Topics: Animals; Calcium Oxalate; Crystallization; Disease Models, Animal; Fluorescent Antibody Technique; Heparitin Sulfate; Kidney Tubules; Male; Microscopy, Electron, Transmission; Microscopy, Immunoelectron; Rats; Rats, Wistar; Urolithiasis

An essential and distinguishing feature of all amyloids is the presence of fibrillar structures of approximately 10-nm width. The precise nature of the fibril is not yet clearly understood, particularly in situ, and the ultrastructure of isolated fibrils differs significantly from that of fibrils observed in situ. The fibrils are generally believed to be composed of a protein specific to each type of amyloid, but increasing evidence suggests additional associations with other components such as heparan sulfate proteoglycan (HSPG) and amyloid P component (AP). Experimental AA amyloidosis was induced in mice by amyloid enhancing factor and an inflammatory stimulus (subcutaneous AgNO3); fibrils were thereafter examined in detail. Particular attention was paid to ultrastructural characteristics known to represent particular molecular components of basement membranes such as HSPG and AP. Additionally, rabbit anti-mouse AA antisera was used with 5-nm and 1-nm gold particles to establish the location of the AA protein in-situ. Amyloid fibrils could be identified in their mature form as well as at apparent intermediate stages of formation. The fibril contained an apparent core which is composed of an assembly of 3.5-nm wide pentosomal particles having the characteristics of AP. Wound around the AP assembly in a helical fashion is a "double tracked" ribbon-like entity, 3 nm wide, having the morphologic characteristics of chondroitin sulfate proteoglycan (CSPG). Covering the surface of this structure is a second ribbon-like double track structure, but this one is wider (4.6 nm vs 3.0 nm) than the CSPG. These have the ultrastructural characteristics of HSPG. Routine fixation and tissue preparation techniques that usually remove HSPG from microfibrils did not do so with amyloid fibrils, suggesting an alteration in affinity between these components. The AA protein could be identified as a 1 - to 2-nm filament network on the most exterior surface of the fibril. The ultrastructure of AA amyloid fibrils in situ resembles that of connective tissue microfibrils, and, in addition to AA protein, is likely composed of HSPG, CSPG, and AP. Amyloid fibrils can be distinguished from microfibrils by the apparently stronger binding of HSPG to the surface of the amyloid fibril and the presence of the AA filaments. A model of the in situ organization of AA amyloid fibrils is proposed.

Topics: Amyloidosis; Animals; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Female; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Mice; Microscopy, Electron; Microscopy, Immunoelectron; Proteoglycans; Rabbits; Serum Amyloid A Protein; Serum Amyloid P-Component; Splenic Diseases

In this study, we carried out an immunohistochemical investigation of time-dependent alterations in the distribution of proteoglycans, and the proliferation profiles of hepatocytes and fat-storing cells (FSCs) in the livers of rats intoxicated with D-galactosamine (GalN). The proliferative cells were analyzed by proliferative cell nuclear antigen (PCNA) staining. In untreated rats, heparan sulfate, dermatan sulfate, and chondroitin/chondroitin sulfate were detected within the portal spaces and the central veins, and, with the exception of chondroitin, also within the reticular fibers. After administration of GalN, the number of PCNA-positive cells (FSCs and hepatocytes) and FSCs increased, reaching maximal on the 2nd and 3rd days, respectively. Heparan sulfate showed complicated changes. Dermatan sulfate decreased in portal spaces from the 2nd to the 3rd day, and in reticular fibers from 12 h to the 6th day. Chondroitin/chondroitin sulfate staining was observed from 2 h to the 6th day in the sinusoidal endothelia, which suggests that the sinusoidal endothelia may produce chondroitin/chondroitin sulfate transiently during liver damage as part of the mechanism of regeneration. Heparan sulfate and chondroitin/chondroitin sulfate were detected in necrotic regions, but dermatan sulfate was not. These observations suggest that heparan sulfate and chondroitin/chondroitin sulfate are involved in cell proliferation or morphogenesis and that the dermatan sulfate plays a role in the differentiation or functional maintenance of cells in liver regeneration.

Topics: Alanine Transaminase; Animals; Chemical and Drug Induced Liver Injury; Dermatan Sulfate; Disease Models, Animal; Extracellular Matrix; Galactosamine; Heparitin Sulfate; Immunohistochemistry; Liver; Liver Diseases; Male; Microscopy, Electron; Proliferating Cell Nuclear Antigen; Proteoglycans; Rats; Rats, Sprague-Dawley

Intra-ocular fibrin deposition following ocular surgery is a serious postoperative complication. The current management of severe postoperative fibrin response is usually ineffective. A quantitative model of fibrin deposition in the rabbit anterior chamber was used to assess the efficacy of Suleparoide I.N.N. (HHS-5) in preventing fibrin formation and in promoting the clearance of fibrinous membranes. Citrated human plasma was injected intracamerally after paracentesis to induce the formation of fibrin clots: 10 min or 24 h after plasma injection, solutions of HHS-5 at different concentrations were injected into the anterior chamber of rabbits. Intra-ocular HHS-5 injection 10 min after plasma injection prevented the formation of fibrinous membranes in a dose-dependent fashion in almost all treated animals. When HHS-5 was injected at 24 h after fibrin clot formation, a reduction of clot areas was observed but the membranes did not completely disappear. No evidence of ocular toxicity was detected by slit-lamp biomicroscopy, intra-ocular pressure and corneal thickness measurements. On the basis of the results obtained with the present animal model, HHS-5 appears as an effective and safe agent for the prevention of fibrinous membrane formation. The data suggest a potential use of HHS-5 for the prophylaxis of fibrin formation in human eyes undergoing extensive intraocular surgery, or in diabetic patients where a hypercoagulable state may be present.

Topics: Analysis of Variance; Animals; Anterior Chamber; Disease Models, Animal; Dose-Response Relationship, Drug; Fibrinolysis; Fibrinolytic Agents; Follow-Up Studies; Heparitin Sulfate; Intraocular Pressure; Male; Rabbits

Co-infusion of the specific heparan sulfate proteoglycan (HSPG), perlecan, and beta-amyloid protein (A beta) into rodent hippocampus leads to a consistent animal model to study the effects of fibrillar A beta amyloid in brain [Snow, A.D. et al. (1994) Neuron 12, 219-234]. In the present study, we describe our rapid novel method of perlecan isolation. The isolation method does not require cesium chloride centrifugation and exploits a newly discovered aggregating property of a approximately 220 kDa PG observed during gel filtration chromatography, which allowed it to be affectively separated from non-aggregating perlecan. Fifty or 100 g of EHS tumor were routinely extracted using 4 M guanidine-HCl, followed by anion-exchange and gel filtration chromatography. SDS-PAGE (before and after digestion with heparitinase/heparinase or nitrous acid) followed by staining with silver demonstrated no other contaminating proteins in the perlecan preparations. Western blots using a specific perlecan core protein antibody (HK-102) following heparitinase digestion showed a characteristic doublet at 400 and 360 kDa indicative of intact perlecan core protein. Absence of contamination by other basement membrane components produced by the EHS tumor was confirmed by absence of immunoreactive bands on Western blots using antibodies against laminin, fibronectin, or type IV collagen. One week continuous co-infusion of perlecan obtained from this methodology, with A beta (1-40) into rodent hippocampus, led to deposition of fibrillar A beta amyloid in 100% (10 of 10) of animals. The detailed protocol for isolation and characterization of perlecan from EHS tumor ensures perlecan of the highest quality, and maximizes the potential effects of A beta amyloid deposition/persistence in brain using the animal model. High quality perlecan obtained from this novel isolation method will also allow future studies utilizing in vitro assays to determine the potential interactions of this specific HSPG with other macromolecules.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloidosis; Animals; Blotting, Western; Brain; Chromatography, Gel; Chromatography, Ion Exchange; Disease Models, Animal; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Hippocampus; Humans; Male; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Proteoglycans; Rats; Rats, Sprague-Dawley; Staining and Labeling

Ultrafiltration failure has been one of the major causes of drop out from CAPD treatment. The present study was designed to develop an ultrafiltration failure model in the rat and to assess whether various agents could prevent a decrease in ultrafiltration capacity in this rat model. Peritoneal hyperpermeability was induced by repeated intraperitoneal injection of 15 ml of 4.25% dextrose dialysate for 7 days. Some of the rats received agents, such as phosphatidylcholine, chondroitin sulfate, siliac acid, heparan sulfate, keratan sulfate, sodium sulfate, sodium phosphate, and heparin sodium, simultaneously administered with 4.25 % dextrose dialysate for the same duration. Ultrafiltration volume, D4/D0 ratio of glucose and peritoneal net fluid absorption were evaluated by 4-hour dwelling of 30 ml of 2.5% dextrose dialysate. Sodium phosphate and heparan sulfate prevented peritoneal hyperpermeability due to repeated injection of hypertonic dialysate. Sodium phosphate, sodium sulfate, heparin and heparan sulfate suppressed peritoneal net fluid absorption, resulting in an increase in ultrafiltration. These findings may be applicable to CAPD patients with ultrafiltration loss.

Topics: Absorption; Animals; Ascitic Fluid; Depression, Chemical; Disease Models, Animal; Heparitin Sulfate; Male; Peritoneal Dialysis, Continuous Ambulatory; Peritoneum; Permeability; Phosphates; Rats; Rats, Sprague-Dawley; Ultrafiltration

Bone resorption resulting from the metastatic human melanoma cell line (A375) was investigated morphologically using an experimental model of bone metastases in nude mice. An injection of A375 (1 x 10(5)) in the left ventricle produced multiple osteolytic lesions. Many TRAPase-positive multinucleated cells, identified by EM as osteoclasts, were observed on the bone surface at the site of metastases. The findings suggest that bone resorption was caused by osteoclasts developed in the presence of tumor cells. Even where tumor cells were juxtaposed to bone surface, small and flat TRAPase-positive cells were shown to exist on the bone surface. Thus, bone resorption was mainly associated with the occurrence of osteoclasts. A large number of osteoclast progenitor cells were also observed adjacent to tumor cells and/or stromal cells located apart from bone, indicating possible participation of tumor cells and/or stromal cells in the differentiation of osteoclasts. Ultrastructurally, stromal cells and/or extracellular matrices were present between tumor cells and osteoclast progenitor cells. Immunohistochemical observation clarified the localization of heparan sulfate proteoglycan (HSPG) and fibronectin (FN) around osteoclast progenitor cells. These findings suggest that they play an important role in providing a microenvironment favorable for osteoclast differentiation and activation. The immunohistochemical localization of IL-6, PGE2, and TGF-alpha also indicates that they are involved in osteoclast differentiation and activation. In conclusion, bone resorption at the metastatic sites of A375 is mediated via osteoclasts and A375 cells may be involved in the differentiation and activation of osteoclasts in association with stromal cells, extracellular matrices (HSPG, FN) and osteotropic cytokines (IL-6, PGE2, TGF-alpha).

Topics: Acid Phosphatase; Animals; Bone Neoplasms; Bone Resorption; Cell Differentiation; Dinoprostone; Disease Models, Animal; Fibronectins; Giant Cells; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunohistochemistry; Interleukin-6; Male; Melanoma, Amelanotic; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Electron; Osteoclasts; Proteoglycans; Stem Cells; Stromal Cells; Transforming Growth Factor alpha; Tumor Cells, Cultured

Soluble dextran polymer derivatives (CMDBSs) are originally synthesized as heparin-like plasma substitutes. Some of them mimic heparin in its interactions and stabilize, protect and facilitate actions of heparin binding growth factors. The wound healing activity of one specific CMDBS was studied in a model of corneal ulcer on the rabbit eye and compared with the activity of basic fibroblast growth factors (bFGF) added alone or in association with CMDBS. Total reepithelization was observed with bFGF + CMDBS, bFGF alone and CMDBS alone after, respectively, 3.8 +/- 0.78, 4.3 +/- 0.67 and 4.4 +/- 0.51 days. All treatments were efficient if compared with eyes treated with saline (p < 0.0001). The grade of significance of the applied treatments was as follows: bFGF + CMDBS > bFGF > CMDBS > saline. Our study pinpoints that some specific CMDBS are as potent agents as bFGF for corneal ulcer healing, and can therefore be proposed for therapeutic use.

Topics: Animals; Cell Line; Cells, Cultured; Cornea; Corneal Ulcer; Cricetinae; Dextrans; Disease Models, Animal; Drug Therapy, Combination; Epithelium; Fibroblast Growth Factor 2; Fibroblasts; Heparitin Sulfate; Lung; Male; Ophthalmic Solutions; Rabbits; Wound Healing

Topics: Animals; Dietary Proteins; Disease Models, Animal; Extracellular Matrix Proteins; Gene Expression Regulation; Glomerulosclerosis, Focal Segmental; Glycoproteins; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kidney Glomerulus; Metalloendopeptidases; Nephrectomy; Proteinuria; Proteoglycans; Puromycin; Rats; RNA, Messenger; Tissue Inhibitor of Metalloproteinases

Topics: Administration, Oral; Animals; Anticoagulants; Blood Coagulation Tests; Disease Models, Animal; Fibrinolytic Agents; Glycosaminoglycans; Heparitin Sulfate; Humans; Injections, Intravenous; Macaca mulatta; Male; Rabbits

This study was designed to assess how the expression of genes for components of the extracellular matrix is altered in a model of focal glomerular sclerosis. In this model, a unilateral nephrectomy combined with injections of puromycin aminonucleoside induces a much higher incidence of focal glomerular sclerosis. Rats received puromycin aminonucleoside on days 0, 27, 34, and 41 and underwent unilateral nephrectomy on day 22. Control rats received physiologic saline injections with and without unilateral nephrectomy. Rats from each group were killed on days 48, 60, and 80. The steady-state levels of glomerular mRNA encoding type IV collagen, the B1 and B2 chains of laminin, heparan sulfate proteoglycan, and type I and type III collagens were compared in both the puromycin aminonucleoside-treated and the control glomeruli. The mRNA levels encoding type IV collagen and laminin B1 and B2 were increased three-, two-, and twofold, respectively, on day 48 of focal glomerular sclerosis. These transcripts were further increased eight-, seven-, and eightfold, respectively, on day 80 compared with the control glomeruli (P < 0.01). In contrast, heparan sulfate proteoglycan mRNA levels were not increased on day 48 when the animals had marked proteinuria. However, the heparan sulfate proteoglycan mRNA levels did become elevated by day 60 and remained elevated thereafter. The expression of type I and type III collagen mRNA was increased 12- and 7-fold, respectively (P < 0.01), on day 80 in focal glomerular sclerosis rats compared with the controls. An immunofluorescence study revealed the accumulation of immunoglobulin M, C3, type IV collagen, laminin, heparan sulfate proteoglycan, and type I and type III collagens in the sclerotic area. These data indicate that changes in the mRNA levels for components of the basement membrane and interstitial collagen are associated with the development of glomerular sclerosis.

Topics: Animals; Collagen; Disease Models, Animal; Extracellular Matrix; Extracellular Matrix Proteins; Gene Expression; Glomerulosclerosis, Focal Segmental; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kidney Glomerulus; Laminin; Male; Nephrectomy; Proteoglycans; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; RNA, Messenger

In chronic murine schistosomiasis, extramedullar myelopoiesis was observed, with proliferation of myeloid cells in liver parenchyma and in periovular granulomas. We have studied the question of whether cells obtained from granulomatous connective tissue may act as myelopoietic stroma, supporting long-term myeloid proliferation. Primary cell lines (GR) were obtained in vitro from periovular granulomas, induced in mouse livers by Schistosoma mansoni infection. These cells were characterized as myofibroblasts, and represent liver connective tissue cells involved in fibro-granulomatous reactions. They were able to sustain survival and proliferation of the multipotent myeloid cell lines FDC-P1 and DA-1 (dependent on interleukin-3 and/or granulocyte-macrophage colony stimulating factor, GM-CSF) without the addition of exogenous growth factors. This stimulation was dependent upon myeloid cell attachment to the GR cell layer; GR cell-conditioned medium had no activity. Primary murine skin fibroblasts could not sustain myelopoiesis. The endogenous growth-factor was identified as GM-CSF by neutralization assays with monoclonal antibodies. The stimulation of myelopoiesis occurred also when GR cells had been fixed with glutardialdehyde. The observed stimulatory activity was dependent upon heparan sulphate proteoglycans (HSPGs) associated with GR cell membranes. It could be dislodged from the cell layer with heparin or a high salt buffer. Our results indicate a molecular interaction between endogenous growth-factor and HSPGs; this interaction may be responsible for the stabilization and presentation of growth factors in myelopoietic stromas, mediating extramedullar proliferation of myeloid cells in periovular granulomas.

Topics: Animals; Cell Membrane; Disease Models, Animal; Granulocyte-Macrophage Colony-Stimulating Factor; Granuloma, Foreign-Body; Heparitin Sulfate; Liver; Macrophages; Mice; Mice, Inbred C3H; Myeloproliferative Disorders; Ovum; Proteoglycans; Schistosomiasis mansoni

Cross-reactivity between anti-DNA antibodies and heparan sulfate (HS)/heparan sulfate-proteoglycan (HS-PG) of glomerular basement membrane has been previously reported. Conceivably, this determines the final outcome of glomerular injury in lupus nephritis.. We investigated the status of glomerular injury in NZB/NZW F1 mice after the administration of rabbit anti-HS-PG antibody (experiment group). The controls received normal rabbit IgG only.. All experimental animals became proteinuric 2 weeks after the administration of anti-HS-PG. The animals of the older age group (16 weeks) had significant hematuria as well. Their glomeruli exhibited hypercellularity with a heavy influx of polymorphonuclear leukocytes and monocytes into their capillaries, and some of them exhibited crescentic changes. Electron-dense deposits were present in subepithelial, subendothelial, and mesangial regions of the glomeruli. The control group had normocellular glomeruli with a few mesangial deposits. Mouse IgG and C3 displayed a granular pattern of immunofluorescence in the experimental group. Anti-rabbit IgG titers in the serum were higher in the control group, which lower in the renal glomerular eluates. No significant differences were observed in the concentrations of anti-dsDNA and -ssDNA either in the sera or in the eluates. There was also no difference between the control and experimental group in terms of antibody synthesis by the splenic lymphocytes and their proliferation subsequent to antigenic challenge.. Data suggest that administration of anti-HS-PG accentuates the glomerular injury during the natural course of lupus nephritis in (NZB/NZW F1 mice; seemingly these two antibodies (anti-HS-PG and -DNA) do not competitively inhibit the binding of the other to the same anionic sites of glomerular basement membrane enriched with heparan sulfate in vivo.

Topics: Animals; Antibodies; Antibodies, Antinuclear; Antibody Affinity; Antibody Formation; Disease Models, Animal; Female; Fluorescent Antibody Technique; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunoglobulin G; In Vitro Techniques; Kidney Glomerulus; Lupus Nephritis; Lymphocyte Activation; Mice; Proteinuria; Proteoglycans; Rabbits

Expansion of the mesangial extracellular matrix (ECM) with subsequent glomerular sclerosis is a prominent finding in most progressive renal diseases. To investigate the chronology of accumulation of ECM components as it relates to previously described cellular events, biopsies were obtained from rats at various times following 5/6-nephrectomy as well as from sham-operated controls. The biopsies were stained with PAS as well as immunostained for PCNA (a cell proliferation marker), monocytes/macrophages, types I and IV collagen, laminin, s-laminin, fibronectin, heparan sulfate proteoglycan and entactin/nidogen. Immunostaining of biopsies obtained from 5/6 nephrectomized rats demonstrated an early glomerular cell proliferation, peaking at week 2. Expansion of the glomerular tuft area with rare glomeruli demonstrating focal sclerosis were also seen at week 2. Glomerular macrophage influx correlated with later ECM expansion and glomerulosclerosis. A progressive accumulation of all ECM proteins investigated was noted in the pathological mesangial matrix at week 2 and later time points. Northern analysis of total glomerular RNA at weeks 2 and 6 after 5/6 nephrectomy showed de novo expression type I collagen mRNA as well as small increases of glomerular mRNA levels for type IV collagen (1.2- and 1.4-fold over control RNA) and laminin (1.3- and 1.5-fold) but not s-laminin (1.1- and 0.9-fold). We conclude that cellular events including glomerular cell proliferation and macrophage influx are associated with increased gene and protein expression by ECM proteins in the remnant kidney model and may contribute to the development of sclerosis.

Topics: Animals; Cell Division; Collagen; Disease Models, Animal; Extracellular Matrix Proteins; Fibronectins; Glomerulosclerosis, Focal Segmental; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Histocytochemistry; Immunohistochemistry; Kidney Failure, Chronic; Kidney Glomerulus; Laminin; Male; Membrane Glycoproteins; Nephrectomy; Proteoglycans; Rats; RNA, Messenger

The present study describes the development of membranous glomerulopathy (MGP) with high proteinuria in DZB rats exposed to mercuric chloride (HgCl2). IgG1 and IgG2a antibodies, eluted from glomeruli with subepithelial immune deposits, bind to the interface of the GBM and epithelial cells. High reactivity to GBM was demonstrated by ELISA and Western blotting, which could be absorbed for 30% by laminin or laminin-associated extracellular matrix components. No reactivity was found with type IV collagen, fibronectin, heparan sulfate proteoglycans, or tubular brush border antigens. Absorption to GBM removed the reactivity to renal antigens. Passively transferred eluted antibodies bind in a predominantly linear pattern along the GBM, causing focal ultrastructural transformations of the podocytes. These results suggest that this type of HgCl2-induced MGP, associated with epithelial cell injury and proteinuria, is caused by autoantibodies to basement membrane components which are located at the epithelial cell-basement membrane interface and may be involved in cell-matrix binding.

Topics: Animals; Antibody Specificity; Autoantibodies; Blotting, Western; Collagen; Complement C3; Complement C9; Complement Membrane Attack Complex; Cross Reactions; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fibronectins; Glomerulonephritis, Membranous; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunoglobulin E; Immunoglobulin G; Immunoglobulin M; Immunohistochemistry; Kidney Glomerulus; Laminin; Mercuric Chloride; Microscopy, Electron; Proteinuria; Proteoglycans; Rats; Rats, Inbred Strains; Time Factors

Renal glomerular basement membranes (GBMs) exhibit a charge-selective barrier, consisting of heparan sulfate proteoglycan (HSPG) that restricts the passage of anionic molecules into the urine. Previous efforts to localize the HSPG core protein within various layers of the GBM have been contradictory. Furthermore, attempts to correlate proteinuria in several disease states with a decrease in anionic sites of HSPG core protein have yielded conflicting results. When antibodies to HSPG from the EHS tumor matrix [anti-(EHS) HSPG] and GBMs [anti-(GBM) HSPG] were used together with immunogold to label renal tissues from puromycin aminonucleoside nephrotic (PAN) rats, immunolabeling results indicated that a portion of the protein core recognized by anti-(EHS) HSPG was significantly reduced, while immunolabeling with anti-(GBM) HSPG was only slightly reduced in early PAN. Anionic sites (stained with the cationic probe, polyethyleneimine) within the lamina rara externa of the GBM remained unaltered throughout the course of PAN.

Topics: Animals; Basement Membrane; Disease Models, Animal; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunohistochemistry; Kidney Glomerulus; Male; Nephrosis; Proteoglycans; Puromycin Aminonucleoside; Rats; Rats, Inbred Strains

Previous histochemical studies have suggested a close temporal relationship between the deposition of highly sulfated glycosaminoglycans (GAGs) and amyloid during experimental AA amyloidosis. In the present investigation, we extended these initial observations by using specific immunocytochemical probes to analyze the temporal and ultrastructural relationship between heparan sulfate proteoglycan (HSPG) accumulation and amyloid deposition in a mouse model of AA amyloidosis. Antibodies against the basement membrane-derived HSPG (either protein core or GAG chains) demonstrated a virtually concurrent deposition of HSPGs and amyloid in specific tissue sites regardless of the organ involved (spleen or liver) or the induction protocol used (amyloid enhancing factor + silver nitrate, or daily azocasein injections). Polyclonal antibodies to AA amyloid protein and amyloid P component also demonstrated co-localization to sites of HSPG deposition in amyloid sites, whereas no positive immunostaining was observed in these locales with a polyclonal antibody to the protein core of a dermatan sulfate proteoglycan (known as "decorin"). Immunogold labeling of HSPGs (either protein core or GAG chains) in amyloidotic mouse spleen or liver revealed specific localization of HSPGs to amyloid fibrils. In the liver, heparan sulfate GAGs were also immunolocalized to the lysosomal compartment of hepatocytes and/or Kupffer cells adjacent to sites of amyloid deposition, suggesting that these cells are involved in HSPG production and/or degradation. The close temporal and ultrastructural relationship between HSPGs and AA amyloid further implies an important role for HSPGs during the initial stages of AA amyloidosis.

Topics: Amyloidosis; Animals; Disease Models, Animal; Female; Heparitin Sulfate; Immunohistochemistry; Liver; Mice; Mice, Inbred CBA; Microscopy, Electron; Serum Amyloid A Protein; Serum Amyloid P-Component; Spleen

This study deals with the quantification of mRNA of basement membrane components (laminin, type IV collagen, heparan sulfate proteoglycan) and type I collagen in focal glomerular sclerosis induced by the aminonucleoside of puromycin (PAN) in rats. PAN (15 mg/100 g B.W.) was injected intraperitoneally to male Sprague-Dawley rats on day 0. On day 22, the right kidney was removed from group II and III. Rats in group III received injections of PAN (5 mg/100 g B.W.) on day 27, 34 and 41. Rats in group II received injections of 0.9% NaCl instead of PAN. Remnant kidneys were removed on days 48, 60 and 80 and processed for RNA isolation and histopathological study. Glomerular RNAs were isolated using guanidinium thiocyanate and then dotted onto nylon membrane. Filters were hybridized with specific cDNA probes and exposed to film for analysis by densitometer. FGS was detected in 70% of glomeruli on day 80 in group II. All the basement membrane components and type I collagen were accumulated in the sclerotic areas. The mRNA coding for laminin and type IV collagen continued to increase in group III till day 80. The mRNA for HSPG decreased when the urinary protein excretion was maximum on day 48, then increased with the remission of proteinuria. The type I collagen mRNA also increased during the course of the FGS. We suggest that decrease of mRNA for HSPG may play an important role in the development of proteinuria in PAN nephrosis and increase of mRNA coding for laminin, type IV collagen and type I collagen may be involved in focal glomerular sclerosis.

Topics: Animals; Basement Membrane; Chondroitin Sulfate Proteoglycans; Collagen; Disease Models, Animal; Gene Expression; Glomerulosclerosis, Focal Segmental; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Laminin; Male; Puromycin; Rats; Rats, Inbred Strains; RNA, Messenger

Two experimental thrombosis models in rats have been compared with regard to the composition of the formed thrombi and the effects of various treatments on thrombus formation. In the first model thrombosis is induced in the vena cava by a combination of venous stasis and hypercoagulability; these thrombi consist merely of red cells and fibrin with only a few platelets. In the second model thrombosis is induced in an arterio-venous shunt in which the formed thrombi consist of red cells, fibrin and a large amount of platelet aggregates adhering to the foreign material. Antiplatelet serum and acetylsalicylic acid, which reduce blood platelet activity, inhibited thrombus formation only in the arteriovenous shunt model. Dicumoxane, an oral anticoagulant, was active in both models. The glycosaminoglycans heparin, Org 10172, Fragmin and the pentasaccharide, representing the AT-III binding sequence of heparin, were active in both models. However, there were qualitative and quantitative differences between the effects of the glycosaminoglycans suggesting differences in their modes of action.

Topics: Animals; Arteriovenous Shunt, Surgical; Aspirin; Blood Coagulation Factors; Blood Platelets; Chondroitin Sulfates; Coumarins; Dermatan Sulfate; Disease Models, Animal; Fibrin; Fibrinolytic Agents; Glycosaminoglycans; Heparin; Heparin, Low-Molecular-Weight; Heparitin Sulfate; Immune Sera; Ligation; Male; Oligosaccharides; Rats; Thrombosis; Venae Cavae; Vitamin K

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Hemostasis; Heparin; Heparitin Sulfate; Male; Rats; Rats, Inbred Strains; Thrombophlebitis

The glomerular capillary wall functions as both a size-selective and charge-selective barrier. Heparan sulfate is known to be an important component of the charge-selective barrier to filtration of polyanions. We studied the alterations in both the charge and size selectivity barriers in a model of experimental membranous nephropathy in the rabbit. The fractional clearance of both charged and uncharged dextrans compared to inulin was measured. Sulfate incorporation into glycosaminoglycans was measured and the glomerular heparan sulfate was isolated and biochemically characterized. Membranous nephropathy in the rabbit was induced with daily injections of cationic bovine serum albumin. After three weeks of injection animals had 735 +/- 196 mg/24 hours of protein excretion. There was no change in [35S] incorporation in 24 hours by experimental animals, 440 +/- 91 DPM/mg dry weight of glomeruli, N = 9 versus 410 +/- 98, N = 11 in controls. The percentage of [35S] incorporated into heparan sulfate versus chondroitin sulfate was decreased, 60% +/- 3 versus 79% +/- 2, P less than 0.001. Heparan sulfate from membranous nephropathy eluted from ion exchange chromatography in a lower molarity salt, indicating a lower effective charge. Fractional clearance of neutral dextrans was significantly increased in membranous nephropathy for dextrans greater than 48 A, while fractional clearance of dextran sulfates was significantly increased compared to controls for dextrans greater than 32 A. Thus, in membranous nephropathy there is loss of both charge selectivity and size selectivity. The loss of charge selectivity correlated with a change in the structure of the glomerular heparan sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Basement Membrane; Capillary Permeability; Dextrans; Disease Models, Animal; Glomerular Filtration Rate; Glycosaminoglycans; Heparitin Sulfate; Inulin; Kidney Glomerulus; Proteinuria; Rabbits

Antibodies to glomerular basement membrane, heparan sulfate-proteoglycans are nephrotoxic but possess a weak nephritogenic potential. In order to enhance the nephritogenic potential, the antibodies were intravenously administered into rats presensitized with heterologous rabbit IgG. This resulted in the integration of heterologous and autologous phases, the two phases characteristic of the traditional model of nephrotoxic serum nephritis. The presensitization caused a dramatic shift in the binding characteristics of the heterologous antibodies between the kidney and lymphoid tissues. A proliferative form of immune complex glomerulonephritis associated with a remarkable proteinuric response was observed. In addition, a moderate degree of hematuria was noted as well. The proteinuria was largely complement-dependent and may possibly be cell-mediated as well. The proteinuria became severe with increasing production of host IgG antibodies and with their subsequent sequestration in the glomeruli. The predominant glomerular lesions were in the form of epimembranous/subepithelial immune deposits, which became more frequent with timely increasing titer of host autologous IgG antibodies. These findings indicate that antibodies to heparan sulfate-proteoglycan, an authentic component of the basement membrane, are capable of mediating a glomerular injury with acquisition of nephritogenic potential in an appropriate environment of the host. At present, it seems that this is the sole constituent of the basement membrane whose antibodies are capable of inducing an immune complex nephritis.

Topics: Animals; Antigen-Antibody Complex; Basement Membrane; Binding Sites, Antibody; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Female; Glomerulonephritis, Membranoproliferative; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immune Complex Diseases; Immunization; Immunoglobulin G; Kidney Glomerulus; Proteoglycans; Rats; Rats, Inbred F344

Administration of antibody, directed against glomerular basement membrane (GBM) heparan sulfate-proteoglycan, into a presensitized rat results in the induction of membranous nephropathy with subepithelial immune-complex deposits. In this investigation, we examined the mechanisms responsible for the formation of subepithelial immune-complex deposits in the anti-HS-PG model. In initial experiments, the intravenously administered radioiodinated antibody was seen exclusively localized in the regions of the glomerular capillary wall where the subepithelial deposits were observed. To determine their exclusive localization in the subepithelial space, kinetics of movement of the intravenously administered antibody was investigated. The antibody localized in the inner layers of the GBM within a few minutes after its administration. It equilibrated in the inner and outer layers of the GBM in a matter of a few hours. Then, after 24 hours, it gradually disappeared from the inner layers of the GBM and persisted in the outer layers only. The ready clearance of the antibody from the inner layers may be related to the differential in the kinetics of lateral intrinsic plasma fluid currents within the GBM. The persistence of heterologous antibody exclusively in the outer layers and the availability of host autologous antibodies probably resulted in the development of immune complex deposits in the subepithelial space. The glomeruli devoid of plasma water currents showed no change in the concentration of the antibody in the inner and outer layers of the GBM or mesangial matrix. Also, no antibody binding was observed with the plasmalemma of either the foot processes or visceral epithelia. The data suggest that the biochemical-biophysical properties of the glomerular capillary wall, in concert with its intraglomerular hemodynamics, most likely played a significant role in the development of subepithelial immune-complex deposits in this model.

Topics: Animals; Antigen-Antibody Complex; Basement Membrane; Binding Sites, Antibody; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Female; Glomerulonephritis, Membranoproliferative; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immune Complex Diseases; Immunization; Immunoglobulin G; Kidney Glomerulus; Proteoglycans; Rats; Rats, Inbred F344

Basement membranes surround the renal tubules and have been shown to limit their distension in vitro. Therefore, it has been postulated that a defect in a basement membrane component(s) underlies the pathogenesis of polycystic kidney disease. Here we have studied a murine model of congenital polycystic kidney disease and found by immunohistology, that the components of the peri-cyst basement membrane appeared to diminish with time. We also measured mRNA levels for collagen IV and laminin, and found a different pattern than in the normal mouse kidney. In normal kidneys, mRNA levels for the B1 and B2 chains of laminin were maximal at birth, and at 1 week for the alpha 1(IV) chain of collagen IV. With all three chains, the levels then rapidly declined. In contrast, mRNA for the alpha 1(IV) chain in congenital polycystic kidneys was half normal 1 week after birth and then increased. Laminin B1 and B2 chain mRNA's were 80% of normal at 1 week but were maintained at that level. As a control, beta-actin mRNA was examined and found to remain constant in both normal and diseased kidneys. In situ hybridization of cRNA probes for the alpha 1(IV) chain confirmed that cells associated with cysts were the principal source of expression of these basement membrane mRNAs. Thus, there exists an abnormal regulation of basement membrane gene expression in congenital polycystic kidney disease. The first stage is characterized by reduced levels of expression. In the second stage, the levels are abnormally high, perhaps representing a compensatory synthesis of basement membrane as cysts enlarge.

Topics: Animals; Basement Membrane; Chondroitin Sulfate Proteoglycans; Collagen; Disease Models, Animal; DNA; Fluorescent Antibody Technique; Gene Expression Regulation; Genes; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kidney; Laminin; Mice; Mice, Inbred C57BL; Nucleic Acid Hybridization; Organ Size; Polycystic Kidney Diseases; RNA, Messenger

Though the presence of glycosaminoglycans in amyloid deposits has been recognized for a long time their role in the pathogenesis of the disorder has remained elusive. As shown here, liver and spleen of human patients with secondary amyloidosis contain 5 to 10 times the amount of glycosaminoglycans as normal organs. Of the three major glycosaminoglycans measured, the heparan sulfate fraction showed the largest increase. In mice where amyloidosis was induced by the injection of casein and enhancing factor (accelerated model) 35SO4-labeled, or Alcian blue stained glycosaminoglycans appeared as early as and at the same location as proteins detected by Congo red staining which was about 2 days after initiation of the procedure. When glycosaminoglycan synthesis was followed in liver and spleen slices of control and experimental animals a significant increase in rate was found in the spleen of the experimental mice. Though there was an increase in heparan sulfate synthesis the major contribution to the overall increase was made by the chondroitin sulfates in the accelerated as well as in the standard induction model. In addition, unlike in the human disorder the chondroitin sulfates were the major glycosaminoglycans which had accumulated in the spleens of animals which had amyloidosis induced by the long term standard procedure (6 weeks) as measured by isolation and uronic acid analysis. The data presented here show that glycosaminoglycans appear to play an important and perhaps direct role in the process of amyloid deposition in the human disease as well as in the experimentally induced disorder in animals.

Topics: Amyloidosis; Animals; Disease Models, Animal; Glycosaminoglycans; Heparitin Sulfate; Humans; In Vitro Techniques; Liver; Liver Cirrhosis, Alcoholic; Mice; Spleen

The behaviour of extracellular matrix glycoproteins (fibronectin, laminin, basement membrane heparan-sulphate proteoglycan, type III, IV and V collagens) has been investigated in a sequential model of experimental hepatic fibrosis, using an immunofluorescence technique. The presence of some basement membrane macromolecules (such as type IV and V collagens, laminin and basement membrane heparan-sulphate proteoglycan) is detectable only in the early stages of septa formation, while type III collagen and fibronectin persist in late septa. These data suggest that hepatic fibroplasia proceeds through different steps in which stromal glycoproteins are preferentially engaged, as happens during organogenesis.

Topics: Animals; Basement Membrane; Chondroitin Sulfate Proteoglycans; Collagen; Disease Models, Animal; Extracellular Matrix; Fibronectins; Fluorescent Antibody Technique; Glycoproteins; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Laminin; Liver Cirrhosis; Male; Rats; Rats, Inbred Strains; Swine

This report presents the neurochemical findings on the first dog to die with deficiency of alpha-L-iduronidase (mucopolysaccharide alpha-L-iduronohydrolase; EC 3.2.1.76). The principal findings were (a) markedly increased glycosaminoglycan content in all neural tissues examined (from threefold in sciatic nerve to 15-fold in brainstem), (b) a modest increase in levels of gangliosides GM2, GM3, and GD3, particularly in gray matter, (c) excessive accumulation of glycosaminoglycans in the CSF, (d) the increased glycosaminoglycans were dermatan sulfate and heparan sulfate, and (e) the molecular weights of the liver glycosaminoglycans were shifted toward smaller sizes, indicating partial degradation. The canine disorder thus resembles mucopolysaccharidosis I in all aspects.

Topics: Animals; Brain Chemistry; Brain Stem; Dermatan Sulfate; Disease Models, Animal; Dog Diseases; Dogs; Electrophoresis, Cellulose Acetate; Female; Gangliosides; Glycosaminoglycans; Glycoside Hydrolases; Heparitin Sulfate; Iduronidase; Molecular Weight; Mucopolysaccharidoses; Sciatic Nerve

This unit has in the past evaluated the Nb rat prostatic adenocarcinoma model with respect to chemotherapies. Recently, this unit has been evaluating agents that may have a role in decreasing metastatic rate. The three androgen-insensitive tumors, Nb Pr A.I. I, II, III, have been evaluated herein. Agents that have been used include indomethacin, heparin, heparin plus cortisone, and heparan sulfate (SP54). It has been shown that these agents do play a role in reducing the metastasis. In evaluation of Nb Pr A.I, I, control animals had a metastatic rate of 57%. In treatment with indocin, only 21% of the animals, three of 14 animals treated, had a metastasis, and treatment with heparin and cortisone resulted in one of 14 animals having metastasis. Similar observations were seen when treatment with SP54 and heparin was evaluated in the Nb Pr A.I. III; 18 and 27% of treatment group animals had metastasis, whereas 55% of control groups had metastasis. Similarly, in the Nb Pr A.I. II evaluation, control animals having a metastatic rate of 43% had heparin plus cortisone and heparin alone, and this particular tumor model revealed complete resolution with no animals having metastatic disease. The majority of these agents have not effected tumor volume in terms of reduction as much as the best chemotherapeutic agents in this model system include cyclophosphamide and cis-platinum.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Cortisone; Disease Models, Animal; Drug Therapy, Combination; Heparin; Heparitin Sulfate; Indomethacin; Male; Neoplasm Metastasis; Prostatic Neoplasms; Rats; Rats, Inbred Strains

Endotoxin-induced lung injury has previously been shown to produce lesions that resemble emphysema morphologically and biochemically as demonstrated by the reduction in the content of lung elastin. The purpose of this study was to define the changes in one other connective tissue component, glycosaminoglycans, during the acute phase of the lung injury. Intravenous administration of a single dose of endotoxin in rats resulted in an increase in the total synthesis of glycosaminoglycans by the pulmonary parenchyma. There was a significant increase in the proportion of dermatan sulfate synthesized during the first 48 hr and a concomitant decrease in heparin/heparan sulfate synthesis. At 48 hr the increased synthesis of dermatan sulfate had reached 7.3 times control values and began to decline, whereas the synthesis of chondroitin-4-sulfate rose from 4.1 to 10.7 times control values between 48 and 72 hr. Analysis of the rates of synthesis revealed that the total amount of heparin/heparan sulfate remained constant while the synthesis of chondroitin-6-sulfate increased proportionally to the overall synthesis of glycosaminoglycans. These findings indicate that dramatic changes in glycosaminoglycan synthesis are an integral part of endotoxin lung injury.

Topics: Animals; Chondroitin Sulfates; Dermatan Sulfate; Disease Models, Animal; Endotoxins; Female; Glycosaminoglycans; Heparin; Heparitin Sulfate; Lung; Pulmonary Emphysema; Rats; Rats, Inbred Strains; Time Factors

The pharmacological profile of Org 10172, a mixture of sulphated glycosaminoglycorunans derived from hog intestinal mucosa, has been assessed in experimental thrombosis and bleeding models in rats and compared with heparin USP. Org 10172 inhibited thrombus formation in arterio-venous shunts dose dependently, the dose required for 50% inhibition (ID50) of thrombus formation was 40 anti-Xa units/kg i.v. The ID50 for heparin USP was 70 anti-Xa units/kg i.v. Org 10172 hardly increased bleeding in doses upto 1600 anti-Xa units/kg i.v., whereas heparin USP dose dependently increased bleeding from 90 anti-Xa units/kg i.v. onwards. The benefit (anti-thrombotic)/risk (bleeding) ratio of Org 10172 was therefore considerably better than that of heparin USP. The improved profile of Org 10172 towards bleeding might be caused by differences in the interaction with blood platelets in comparison with heparin USP. Org 10172 had less effect on the platelet content in thrombi than heparin USP. Org 10172 did not inhibit collagen induced release of serotonin in contrast to heparin USP. Org 10172 inhibited factor Xa induced aggregation of rabbit platelets but only at anti-Xa levels which were fifteen times higher than for heparin USP. In contrast to heparin USP Org 10172 had only a very weak effect on the activated partial thromboplastin time (APTT) ex vivo.

Topics: Animals; Blood Coagulation; Blood Platelets; Chondroitin Sulfates; Dermatan Sulfate; Disease Models, Animal; Fibrinolytic Agents; Glycosaminoglycans; Heparin; Heparinoids; Heparitin Sulfate; Male; Platelet Aggregation; Rabbits; Rats; Rats, Inbred Strains; Thrombophlebitis

Topics: Animals; Carbon Radioisotopes; Disease Models, Animal; Embolism, Fat; Glycosaminoglycans; Heparin; Heparitin Sulfate; Injections, Intravenous; Lipase; Lung; Mice; Pulmonary Embolism; Triolein