Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The Y402H polymorphism in complement factor H (CFH) is a common and important risk factor, where CFH is an inhibitor of the alternative complement pathway. The disease-associated protein variant (CFH402H) binds poorly to aged human macular Bruch’s membrane (BM), a site of AMD formation. Heparan sulphate (HS) is the major binding site for CFH in this extracellular matrix. Unlike CFH402Y, CFH402H binds poorly to lowly sulphated HS. The aim of this research was to investigate the presence and distribution of proteoglycan (PG) core proteins and glycosaminoglycans (GAGs) in the normal adult human macula, and to analyse potential changes with age in the quantity and composition of HS and other potential molecular determinants of disease in BM. Post mortem human eye tissue was obtained from consenting donors (age range 18-93 years), and either dissected into tissue layers or used to produce frozen macular tissue sections. Proteomic analysis of different retinal tissue layers was performed by tandem mass spectrometry. Immunofluorescence microscopy was undertaken on the macular tissue sections. Compositional analysis of HS in BM was performed by 2-aminoacridone labelling of HS disaccharides and reverse phase high performance liquid chromatography against reference HS disaccharide standards. PG core proteins were identified in BM and other macular tissue layers, including members of the basement membrane, hyalectan and short leucine-rich repeat PG families. HS, chondroitin sulphate, dermatan sulphate and hyaluronan were present throughout the retina and choroid, but keratan sulphate only in the sclera. The mean quantity of HS in BM was 47% lower (p=0.006) in old donors (n=13, 64-92 years), compared to young donors (n=6; 26-39 years). The mean level of HS sulphation was also lower in old donors, e.g. 34% vs. 39% (p=0.02) N-sulphated HS. The mean level of HS in macular BM by immunohistochemistry was approximately 50% lower (p=0.02) in old donors (n=10, 18-93 years), and the mean level of the HS PG core protein perlecan was reduced by 85% (p=0.01; n=18, 27-90 years). High levels of complement activation (C3b and membrane attack complex) were observed in some young donors. Reduced HS was associated with increased complement activation in some donors (r2 0.30). A combination of proteomics and immunohistochemistry approaches has provided the first comprehensive analysis of the presence and distribution of PG core proteins and their associated GAG chains throughout the macular layers of the normal adult human retina. These demonstrate a differential distribution according to PG core protein, GAG class and GAG sulphation state. The quantity of HS decreases substantially with age in human BM, and its sulphation level also decreases. The presence of less HS in old BM would make fewer binding sites available for CFH, and could contribute to AMD pathogenesis through increased complement activation. This idea is supported by the observation that reduced HS is associated in some individuals with increased C3b in BM. These findings have important implications for unravelling mechanisms of ocular disease and planning novel therapeutic strategies, particularly in the case of AMD.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:607094 |
| Date | January 2013 |
| Creators | Keenan, Tiarnan Daniel |
| Contributors | Bishop, Paul; Day, Tony |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/proteoglycans-of-the-human-macula-normal-distribution-and-agerelated-changes(61ab7a93-ea71-44fa-a2d5-3006e619105d).html |
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