heparitin-sulfate and Fibrosis

Heparanase (HPSE) is a multitasking protein characterized by enzymatic and non-enzymatic activities. By means of its enzymatic activity, HPSE catalyzes the cutting of the side chains of heparan sulfate (HS) proteoglycans, thereby inducing the remodeling of the extracellular matrix and basement membranes. Thanks to the cleavage of HS, HPSE also promotes the release and diffusion of several HS-linked molecules such as growth factors, cytokines and enzymes. In addition to degrading HS chains, HPSE has non-enzymatic functions that trigger several signaling pathways. This signaling activity is achieved by interacting with transmembrane proteins, activating kinases such as Akt and Src, or modulating the activity of factors such as FGF-2 and TGF-β. Several studies have recently highlighted a possible intracellular activity for HPSE, particularly at nuclear level. While HPSE activity is quite limited in physiological conditions, its demonstrated increasing involvement in various pathological conditions, such as in tumor progression and renal disease, have attracted the attention of a growing number of researchers. The fact that no other molecule is capable of performing the same function as HPSE makes this enzyme an attractive potential target of medical treatment. With this short conceptual overview, we aim to provide an update on current knowledge concerning the HPSE protein in the experimental and clinical settings, paying particular attention to its role in fibrosis, inflammation and cancer.

Topics: Epithelial-Mesenchymal Transition; Fibrosis; Glucuronidase; Heparitin Sulfate; Humans; Inflammation; Kidney; Models, Biological; Neoplasms; Signal Transduction

Interferon-gamma is a T cell secreted cytokine. It has a great number of biological activities, among which is the down regulation of collagen molecules. IFN-gamma acts through cell surface receptors, but also binds to heparan sulfate proteoglycans. In vivo, it is postulated that the heparan sulfate/IFN-gamma interaction can modulate the activity and availability of the cytokine.

Topics: Animals; Extracellular Matrix; Fibrosis; Heparitin Sulfate; Humans; Interferon-gamma; Protein Binding

Heparan sulfate (HS) as a mediator is usually involved in both inflammation and fibrosis. Besides, pre-fibrils are the intermediates of amyloid fibrils that usually cause cell death and tissue damage, like the amyloid-β in Alzheimer's disease, α-synuclein in Parkinson disease and islet amyloid polypeptide in type II diabetes mellitus. However, the related study was involved rarely in breast. Therefore, the combing technologies including hematoxylin-eosin staining and thioflavin S staining were used to investigate the influence of HS on breast amyloidosis. To further study the toxicity of the pre-fibrils formed by β-casein on the HC11 cells and the breast mammary gland, the combing technologies including pentamer formyl thiophene acetic acid fluorescence analysis, MTT assay, Annexin V/PI staining and hematoxylin-eosin staining were performed. The results demonstrated that HS, acted as an endogenous molecule, induced the inflammation and amyloid fibril formation at the same time, and there was a close relationship between inflammation and fibrosis of breast. In addition, the pre-fibrils formed by β-casein were toxic because they induced the death and apoptosis of HC11 cells, as well as the inflammation of mammary gland of rats. Therefore, the early examination and identify of the pre-fibrils in the breast were worth considering to prevent the disease development, and it was interesting to explore the HS mimetics to impair the breast amyloidosis and attenuate the inflammatory response in the future.

Topics: Amyloid; Amyloidosis; Animals; Caseins; Diabetes Mellitus, Type 2; Eosine Yellowish-(YS); Fibrosis; Hematoxylin; Heparitin Sulfate; Inflammation; Rats

The extracellular matrix crosslinking enzyme transglutaminase 2 (TG2) is highly implicated in tissue fibrosis that precedes end-stage kidney failure. TG2 is unconventionally secreted through extracellular vesicles in a way that depends on the heparan sulphate (HS) proteoglycan syndecan-4 (Sdc4), the deletion of which reduces experimental kidney fibrosis as a result of lower extracellular TG2 in the tubule-interstitium. Here we establish a model of TG2 externalisation in NRK-52E tubular epithelial cells subjected to glucose stress. HS-binding TG2 mutants had reduced extracellular TG2 in transfected NRK-52E, suggesting that TG2-externalisation depends on an intact TG2 heparin binding site. Inhibition of N-ethylmaleimide sensitive factor (NSF) vesicle-fusing ATPase, which was identified in the recently elucidated TG2 kidney membrane-interactome, led to significantly lower TG2-externalisation, thus validating the involvement of membrane fusion in TG2 secretion. As cyclin-G-associated kinase (GAK) had emerged as a further TG2-partner in the fibrotic kidney, we investigated whether glucose-induced TG2-externalisation was accompanied by TG2 phosphorylation in consensus sequences of cyclin-dependent kinase (CDK). Glucose stress led to intense TG2 phosphorylation in serine/threonine CDK-target. TG2 phosphorylation by tyrosine kinases was also increased by glucose. Although the precise role of glucose-induced TG2 phosphorylation is unknown, these novel data suggest that phosphorylation may be involved in TG2 membrane-trafficking.

Topics: Animals; Binding Sites; Cell Line; Cyclins; Epithelial Cells; Extracellular Matrix; Fibrosis; Glucose; GTP-Binding Proteins; Heparitin Sulfate; Kidney; Kidney Tubules; Membrane Fusion; Protein Glutamine gamma Glutamyltransferase 2; Protein Serine-Threonine Kinases; Protein Transport; Rats; Syndecan-4; Transglutaminases

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

Selective delivery of drugs and biotherapeutics to the site of disease (synaphic targeting) has a number of advantages. First, the enhanced accumulation of the therapeutic compound at the target tissue increases drug efficacy without increasing side effects. Alternatively, the dose of the drug can be lowered to reduce the side effects. On the practical level, when a drug is difficult or expensive to make, being able to lower the dose may be the key to commercial viability. Certain targeting systems can change the distribution of the drug in a beneficial way. Examples include wider distribution and deeper penetration of the drug in the target tissue, active intracellular targeting when desirable, and even targeting to a particular subcellular organelle. In this chapter, we illustrate these principles by describing the development of a targeting system for an antifibrotic biotherapeutic, decorin. The system is based on vascular homing peptide (sequence: CARSKNKDC; referred to as CAR) that specifically recognizes angiogenic blood vessels in injured (regenerating) and inflammatory tissues and can deliver a payload to such tissues with high selectivity. So far, the CAR-targeted decorin has been shown to promote tissue repair with reduced scarring in a skin wound model, but this biotherapeutic can potentially be used in other injuries and in various fibrotic diseases.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Proliferation; Cells, Cultured; Cloning, Molecular; Decorin; Drug Delivery Systems; Drug Design; Fibrosis; Heparitin Sulfate; Humans; Male; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Peptide Fragments; Peptide Library; Protein Binding; Recombinant Fusion Proteins; Sequence Analysis, DNA; Skin; Wound Healing

To investigate the contribution of heparan sulfate proteoglycan and Ras/MEK/ERK to the overexpression of profibrotic proteins and the enhanced contractile ability of dermal fibroblasts from patients with systemic sclerosis (SSc; scleroderma).. The effects of the MEK/ERK inhibitor U0126, the heparan sulfate side chain formation inhibitor beta-xyloside, and soluble heparin on the overexpression of profibrotic genes were compared in fibroblasts from lesional skin of patients with diffuse SSc and fibroblasts from healthy control subjects. Identified protein expressions were compared with the contractile abilities of fibroblasts while they resided within a collagen lattice. Forces generated were measured using a culture force monitor.. Inhibiting MEK/ERK with U0126 significantly reduced expression of a cohort of proadhesive and procontractile proteins that normally are overexpressed by scleroderma fibroblasts, including integrin alpha4 and integrin beta1. Antagonizing heparan sulfate side chain formation with beta-xyloside or the addition of soluble heparin prevented ERK activation, in addition to reducing the expression of these proadhesive/contractile proteins. Treatment with either U0126, beta-xyloside, or heparin resulted in a reduction in the overall peak contractile force generated by dermal fibroblasts. Blocking platelet-derived growth factor receptor with Gleevec (imatinib mesylate) reduced overall contractile ability and the elevated syndecan 4 expression and ERK activation in SSc fibroblasts.. The results of this study suggest that heparan sulfate-dependent ERK activation contributes to the enhanced contractile ability demonstrated by dermal fibroblasts from lesional skin of patients with scleroderma. These results are consistent with the notion that the MEK/ERK procontractile pathway is dysregulated in scleroderma dermal fibroblasts. Additionally, the results suggest that antagonizing the MEK/ERK pathway is likely to modulate heparan sulfate proteoglycan activity, which in turn may have a profound effect on the fibrotic response in SSc.

Topics: Cell Movement; Cells, Cultured; Dermis; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblasts; Fibrosis; Gene Expression; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; MAP Kinase Signaling System; Phenotype; Platelet-Derived Growth Factor; RNA, Messenger; Scleroderma, Systemic; Syndecan-4

The chemokine monocyte chemoattractant protein (MCP)-1 has been implicated in the monocyte/macrophage infiltration that occurs during tubulointerstitial nephritis (TIN). We investigated the role of MCP-1 in rats with TIN by administering a neutralizing anti-MCP-1 antibody (Ab). We observed significantly reduced macrophage infiltration and delayed neutrophil clearance in the kidneys of TIN model rats treated with the anti-MCP-1 Ab. To exclude the possibility that an observed immune complex could affect the resolution of apoptotic neutrophils via the Fc receptor, TIN model rats were treated with a peptide-based MCP-1 receptor antagonist (RA). The MCP-1 RA had effects similar to those of the anti-MCP-1 Ab. In addition, MCP-1 did not affect macrophage-mediated phagocytosis of neutrophils in vitro. Deposition of the anti-MCP-1 Ab in rat kidneys resulted from its binding to heparan sulfate-immobilized MCP-1, as demonstrated by the detection of MCP-1 in both pull-down and immunoprecipitation assays. We conclude that induction of chemokines, specifically MCP-1, in TIN corresponds with leukocyte infiltration and that the anti-MCP-1 Ab formed an immune complex with heparan sulfate-immobilized MCP-1 in the kidney. Antagonism of MCP-1 in TIN by Ab or RA may alter the pathological process, most likely through delayed removal of apoptotic neutrophils in the inflammatory loci.

Topics: Animals; Antibodies; Antigen-Antibody Complex; Apoptosis; Chemokine CCL2; Chemokines; Chemokines, CXC; Chemotaxis; Cytokines; Escherichia coli; Fibrosis; Heparitin Sulfate; Intercellular Signaling Peptides and Proteins; Kidney; Macrophages, Peritoneal; Nephritis, Interstitial; Neutrophil Infiltration; Neutrophils; Peptide Fragments; Phagocytosis; Rats; Rats, Inbred BN; Receptors, CCR2; Receptors, Chemokine; Time Factors

Topics: Antibodies, Monoclonal; Binding Sites; Fibroblast Growth Factor 2; Fibrosis; Glomerulosclerosis, Focal Segmental; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunoenzyme Techniques; Kidney; Proteoglycans

Mast cells are resident in tissues, particularly in association with endothelial and epithelial cell basement membranes, and increase at sites of inflammation, injury, and fibrosis. Although mast cells are known to both release and generate proinflammatory molecules in response to inflammatory stimuli, little is known about their normal biologic function. Here we demonstrate that IL-3-dependent mouse PT18 mast cells, mouse bone marrow-derived mast cells, and rat basophilic leukemia cells express large amounts of mRNA for collagen IV, laminin, and heparan sulfate proteoglycan. Western blot analysis confirmed that mast cells synthesize and secrete significant amounts collagen IV and laminin B1 and B2 chains. These data suggest that mast cells may contribute to normal tissue repair and/or the early overproduction of basement membrane components seen in a variety of fibrotic conditions.

Topics: Actins; Animals; Basement Membrane; Blotting, Northern; Blotting, Western; Collagen; Extracellular Matrix Proteins; Fibrosis; Heparitin Sulfate; Interleukin-3; Laminin; Mast Cells; Mice; Proteoglycans; RNA, Messenger; Transcription, Genetic