heparitin-sulfate and Macular-Degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness in developed nations and has been associated with complement dysregulation in the central retina. The Y402H polymorphism in the complement regulatory protein factor H (CFH) can confer a >5-fold increased risk of developing AMD and is present in approximately 30% of people of European descent. CFH, in conjunction with other factors, regulates complement activation in host tissues, and the Y402H polymorphism has been found to alter the protein's specificity for heparan sulphate (HS) - a complex polysaccharide found ubiquitously in mammals. HS, which is present on the cell surface and also in the extracellular matrix, exhibits huge structural diversity due to variations in the level/pattern of sulphation, where particular structures may act as 'ZIP codes' for different tissue/cellular locations. Recent work has demonstrated that CFH contains two HS-binding domains that each recognize specific HS ZIP codes, allowing differential recognition of Bruch's membrane (in the eye) or the glomerular basement membrane (in the kidney). Importantly, the Y402H polymorphism impairs the binding of CFH to the HS in Bruch's membrane, which could result in increased complement activation and chronic local inflammation (in 402H individuals) and thereby contribute to AMD pathology.

Topics: Animals; Bruch Membrane; Complement Activation; Complement Factor H; Genetic Predisposition to Disease; Heparitin Sulfate; Humans; Macular Degeneration; Polymorphism, Genetic; Protein Binding; Protein Interaction Domains and Motifs; Retina

Heparan sulfate (HS), a ubiquitous and structurally diverse cell surface polysaccharide and extracellular matrix component, is a factor common to several major eye pathologies. Its multitude of functions and variable distribution among the different ocular tissues makes it an important contributor to a variety of disease states. Although HS facilitates the pathogenesis of many disorders, its role in each varies. Unique functions of HS have been particularly noted in viral and bacterial keratitis and age-related macular degeneration. Combined, these pathologies comprise a large portion of conditions leading to visual impairment worldwide. Given this prevalence of diseases facilitated by HS, it is prudent to take an in-depth look at this compound in the context of these pathologic states. While the initial part of the review will discuss the pathogenic aspects of HS, it is also important to consider the wider implications of such roles for HS. The remainder of the article will specifically address one such implication, the possibility for future use of novel HS-based therapeutics to combat these eye pathologies.

Topics: Animals; Corneal Neovascularization; Corneal Ulcer; Eye Diseases; Heparitin Sulfate; Humans; Keratitis, Herpetic; Macular Degeneration

Choroidal endothelial cells play a central role in the pathogenesis of age-related macular degeneration (AMD). Protocols for isolating primary choroidal endothelial cells have been described but require access to human donor eyes, which is a limiting factor. Therefore, a conditionally immortalized choroidal endothelial cell (ciChEnC) line has been established.. Choroidal endothelial cells were selected by magnetic-activated cell sorting and conditionally immortalized using temperature-sensitive simian virus 40 large T antigen and human telomerase. The cell line obtained was characterized based on expression of endothelial marker proteins and endothelial cell-specific responses to various stimuli. Binding of AMD-associated and non-AMD variants of complement factor H in the context of a recombinant CCP6-8 (complement control protein domains 6-8) construct was determined using ELISA.. ciChEnCs maintained morphology and von Willebrand factor and vascular endothelial cadherin expression for up to 27 passages. The cells internalized acetylated low-density lipoprotein, formed tubes on Matrigel, and increased intercellular adhesion molecule-1 expression in response to tumor necrosis factor-α. Cells grew into dense monolayers with barrier function and showed characteristics of choriocapillary cells, such as expression of plasmalemma vesicle-associated protein, human leukocyte antigen ABC, carbonic anhydrase IV, and membrane indentations reflecting fenestrations. ciChEnCs synthesized glycosaminoglycans chondroitin sulfate and the complement factor H ligand heparan sulfate. Interestingly, binding of the AMD-associated 402H variant of factor H to ciChEnC was significantly decreased compared to the 402Y variant.. A novel ciChEnC cell line with choriocapillary characteristics has been established and should greatly facilitate investigation of the pathogenesis of AMD in the context of the choriocapillary microenvironment.

Topics: Biomarkers; Cell Line; Chondroitin Sulfates; Choroid; Complement Factor H; Electric Impedance; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Glycosaminoglycans; Heparitin Sulfate; Humans; Immunomagnetic Separation; Intercellular Adhesion Molecule-1; Macular Degeneration; Microscopy, Electron, Scanning; Middle Aged

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The polymorphism rs10490924 in the ARMS2 gene is highly associated with AMD and linked to an indel mutation (del443ins54), the latter inducing mRNA instability. At present, the function of the ARMS2 protein, the exact cellular sources in the retina and the biological consequences of the rs10490924 polymorphism are unclear.. Recombinant ARMS2 was expressed in Pichia pastoris, and protein functions were studied regarding cell surface binding and complement activation in human serum using fluoresence-activated cell sorting (FACS) as well as laser scanning microscopy (LSM). Biolayer interferometry defined protein interactions. Furthermore, endogenous ARMS2 gene expression was studied in human blood derived monocytes and in human induced pluripotent stem cell-derived microglia (iPSdM) by PCR and LSM. The ARMS2 protein was localized in human genotyped retinal sections and in purified monocytes derived from AMD patients without the ARMS2 risk variant by LSM. ARMS2 expression in monocytes under oxidative stress was determined by Western blot analysis.. Here, we demonstrate for the first time that ARMS2 functions as surface complement regulator. Recombinant ARMS2 binds to human apoptotic and necrotic cells and initiates complement activation by recruiting the complement activator properdin. ARMS2-properdin complexes augment C3b surface opsonization for phagocytosis. We also demonstrate for the first time expression of ARMS2 in human monocytes especially under oxidative stress and in microglia cells of the human retina. The ARMS2 protein is absent in monocytes and also in microglia cells, derived from patients homozygous for the ARMS2 AMD risk variant (rs10490924).. ARMS2 is likely involved in complement-mediated clearance of cellular debris. As AMD patients present with accumulated proteins and lipids on Bruch's membrane, ARMS2 protein deficiency due to the genetic risk variant might be involved in drusen formation.

Topics: Age Factors; Aged; Aged, 80 and over; Animals; CHO Cells; Complement System Proteins; Cricetulus; Female; Heparitin Sulfate; Humans; Hydrogen Peroxide; Immunologic Factors; Macular Degeneration; Male; Microglia; Middle Aged; Monocytes; Polymorphism, Single Nucleotide; Properdin; Protein Binding; Proteins; Retina; Young Adult

Heparan sulfate (HS) has been implicated in age-related macular degeneration (AMD), since it is the major binding partner for complement factor H (CFH) in human Bruch's membrane (BrM), and CFH has a central role in inhibiting complement activation on extracellular matrices. The aim was to investigate potential aging changes in HS quantity and composition in human BrM.. Postmortem human ocular tissue was obtained from donors without known retinal disease. The HS was purified from BrM and neurosensory retina, and after digestion to disaccharides, fluorescently labeled and analyzed by reverse-phase HPLC. The HS and heparanase-1 were detected by immunohistochemistry in macular tissue sections from young and old donors, and binding of exogenously applied recombinant CCP6-8 region of CFH (402Y and 402H variants) was compared.. Disaccharide analysis demonstrated that the mean quantity of HS in BrM was 50% lower (P = 0.006) in old versus young donors (average 82 vs. 32 years). In addition, there was a small, but significant decrease in HS sulfation in old BrM. Immunohistochemistry revealed approximately 50% (P = 0.02) less HS in macular BrM in old versus young donors, whereas heparanase-1 increased by 24% in old macular BrM (P = 0.56). In young donor tissue the AMD-associated 402H CCP6-8 bound relatively poorly to BrM, compared to the 402Y form. In BrM from old donors, this difference was significantly greater (P = 0.019).. The quantity of HS decreases substantially with age in human BrM, resulting in fewer binding sites for CFH and especially affecting the ability of the 402H variant of CFH to bind BrM.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Aging; Bruch Membrane; Cadaver; Chromatography, Reverse-Phase; Female; Heparitin Sulfate; Humans; Macular Degeneration; Male; Middle Aged; Polysaccharide-Lyases; Retina

The tight regulation of innate immunity on extracellular matrix (ECM) is a vital part of immune homeostasis throughout the human body, and disruption to this regulation in the eye is thought to contribute directly to the progression of age-related macular degeneration (AMD). The plasma complement regulator factor H (FH) is thought to be the main regulator that protects ECM against damaging complement activation. However, in the present study we demonstrate that a truncated form of FH, called FH-like protein 1 (FHL-1), is the main regulatory protein in the layer of ECM under human retina, called Bruch's membrane. Bruch's membrane is a major site of AMD disease pathogenesis and where drusen, the hallmark lesions of AMD, form. We show that FHL-1 can passively diffuse through Bruch's membrane, whereas the full sized, glycosylated, FH cannot. FHL-1 is largely bound to Bruch's membrane through interactions with heparan sulfate, and we show that the common Y402H polymorphism in the CFH gene, associated with an increased risk of AMD, reduces the binding of FHL-1 to this heparan sulfate. We also show that FHL-1 is retained in drusen whereas FH coats the periphery of the lesions, perhaps inhibiting their clearance. Our results identify a novel mechanism of complement regulation in the human eye, which highlights potential new avenues for therapeutic strategies.

Topics: Bruch Membrane; Complement Activation; Complement C3b Inactivator Proteins; Complement Factor H; Extracellular Matrix; Gene Expression Regulation; Glycosylation; Heparitin Sulfate; Homeostasis; Humans; Immunity, Innate; Macular Degeneration; Protein Binding; Protein Transport; Retina; Retinal Drusen; Signal Transduction

Complement factor H (CFH) regulates complement activation in host tissues through its recognition of polyanions, which mediate CFH binding to host cell surfaces and extracellular matrix, promoting the deactivation of deposited C3b. These polyanions include heparan sulfate (HS), a glycosaminoglycan with a highly diverse range of structures, for which two regions of CFH (CCP6-8 and CCP19-20) have been implicated in HS binding. Mutations/polymorphisms within these glycosaminoglycan-binding sites have been associated with age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome. In this study, we demonstrate that CFH has tissue-specific binding properties mediated through its two HS-binding regions. Our data show that the CCP6-8 region of CFH binds more strongly to heparin (a highly sulfated form of HS) than CCP19-20, and that their sulfate specificities are different. Furthermore, the HS binding site in CCP6-8, which is affected by the AMD-associated Y402H polymorphism, plays the principal role in host tissue recognition in the human eye, whereas the CCP19-20 region makes the major contribution to the binding of CFH in the human kidney. This helps provide a biochemical explanation for the genetic basis of tissue-specific diseases such as AMD and atypical hemolytic uremic syndrome, and leads to a better understanding of the pathogenic mechanisms for these diseases of complement dysregulation.

Topics: Adult; Aged; Aged, 80 and over; Amino Acid Substitution; Atypical Hemolytic Uremic Syndrome; Autopsy; Binding Sites; Complement Activation; Complement Factor H; Escherichia coli; Eye; Female; Hemolytic-Uremic Syndrome; Heparitin Sulfate; Humans; Kidney; Macular Degeneration; Male; Middle Aged; Mutation; Organ Specificity; Protein Binding; Recombinant Proteins

Age-related macular degeneration (AMD) is the predominant cause of blindness in the industrialized world where destruction of the macula, i.e. the central region of the retina, results in loss of vision. AMD is preceded by the formation of deposits in the macula, which accumulate between the Bruch's membrane and the retinal pigment epithelium (RPE). These deposits are associated with complement-mediated inflammation and perturb retinal function. Recent genetic association studies have demonstrated that a common allele (402H) of the complement factor H (CFH) gene is a major risk factor for the development of AMD; CFH suppresses complement activation on host tissues where it is believed to bind via its interaction with polyanionic structures. We have shown previously that this coding change (Y402H; from a tyrosine to histidine residue) alters the binding of the CFH protein to sulfated polysaccharides. Here we demonstrate that the AMD-associated polymorphism profoundly affects CFH binding to sites within human macula. Notably, the AMD-associated 402H variant binds less well to heparan sulfate and dermatan sulfate glycosaminoglycans within Bruch's membrane when compared with the 402Y form; both allotypes exhibit a similar level of binding to the RPE. We propose that the impaired binding of the 402H variant to Bruch's membrane results in an overactivation of the complement pathway leading to local chronic inflammation and thus contributes directly to the development and/or progression of AMD. These studies therefore provide a putative disease mechanism and add weight to the genetic association studies that implicate the 402H allele as an important risk factor in AMD.

Topics: Alleles; Amino Acid Substitution; Bruch Membrane; Complement Activation; Complement Factor H; Heparitin Sulfate; Humans; Macula Lutea; Macular Degeneration; Mutation, Missense; Protein Binding; Retinal Pigment Epithelium; Risk Factors

An imbalance between activation and inhibition of the complement system has been implicated in the etiologies of numerous common diseases. Allotypic variants of a key complement fluid-phase regulatory protein, complement factor H (CFH), are strongly associated with age-related macular degeneration (AMD), a leading cause of worldwide visual dysfunction, although its specific role in AMD pathogenesis is still not clear. CFH was isolated from individuals carrying combinations of two of the nonsynonymous coding variants most strongly associated with AMD risk, V62/H402 (risk haplotype variants), I62/Y402 (nonrisk haplotype variants), and V62/Y402. These proteins were used in two functional assays (cell surface- and fluid-phase-based) measuring cofactor activity of CFH in the factor I-mediated cleavage of C3b. Although no variant-specific differences in the cofactor activity were detected, when heparan sulfate (HS) was added to these assays, it accelerated the rate of C3b cleavage, and this effect could be modulated by degree of HS sulfation. Bruch's membrane/choroid, a site of tissue damage in AMD, contains high concentrations of glycosaminoglycans, including HS. Addition of human Bruch's membrane/choroid to the fluid-phase assay accelerated the C3b cleavage, and this effect was lost posttreatment of the tissue with heparinase III. Binding of CFH variants to Bruch's membrane/choroid isolated from elderly, non-AMD donor eyes, was similar, as was the functional activity of bound CFH. These findings refine our understanding of interactions of HS and complement and support the hypothesis that these interactions play a role in the transition between normal aging and AMD in Bruch's membrane/choroid.

Topics: Adult; Aged; Aged, 80 and over; Bruch Membrane; Complement C3b; Complement Factor H; Complement Pathway, Alternative; Female; Heparitin Sulfate; Humans; Macular Degeneration; Male; Microscopy, Electron, Transmission; Middle Aged; Protein Isoforms

Age-related macular degeneration is one of the major causes of severe visual loss is elderly individuals. However, relatively little is known about its etiology. The disease may be associated with senescence. Ultrastructural and immunohistochemical studies on SAM (senescence accelerated mouse) eyes were carried out to learn details of aging changes in the retinal pigment epithelium (RPE) and Bruch's membrane. SAM P 1 mice aged 2, 10, 14 months were examined in this study. The eyes were analysed for type IV collagen and heparan sulfate proteoglycan (HSPG) by the avidin-biotin-peroxidase complexes (ABC) method and post-embeddig immunolocalization with colloidal gold. With the ABC method, the basement membranes of both the RPE and the choriocapillaris showed markedly positive staining when treated with anti-type IV collagen antibody and moderately positive staining when treated with anti-HSPG antibody. In ultrastructural immunolocalization, both basement membranes showed fairly heavy labeling in response to the antibodies to type IV collagen, and moderate labeling in response to the antibodies to HSPG. With aging, the thickness of the basement membrane of the choriocapillaris and gold particle labeling by the antibodies to type IV collagen increased. The gold particle labeling by the antibodies to HSPG increased slightly, but was distributed sparsely. These results showed the advancing process of senescence changes in Bruch's membrane.

Topics: Aging; Animals; Bruch Membrane; Collagen; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunohistochemistry; Macular Degeneration; Mice; Proteoglycans