Glycosaminoglycan-omics of Bruch’s membrane reveals elevated levels of heparan sulfate in early and intermediate Age-related Macular Degeneration

Abstract

Purpose : Several lines of evidence suggest that Bruch’s membrane (BrM) plays a central role in Age-related Macular Degeneration (AMD) pathogenesis. A major component of BrM is glycosaminoglycans (GAG). However, the content and composition of GAGs in BrM in AMD has not been investigated. Using glycan reductive isotope labelling combined with liquid chromatography and mass spectrometry (GRIL-LC/MS) our group analyzed the content and composition of GAGs in BrM in post-mortem AMD tissue.

Methods : Donor eyes were procured from the San Diego Eye Bank from ages 25 – 45 years old and 65-90 years old, including patients with AMD (N=34). A short (<12-hour) death-receiving interval was used to ensure GAG stability. After dissection, high-resolution, color and digital fundus photographs were taken. AMD grading was performed according to the Minnesota Grading scale. Macular and peripheral punches of the RPE/BrM/choroid complex was performed. BrM was isolated from the RPE/BrM/choroid complex by microscopic dissection. GAGs were quantified by GRIL-LC/MS. Briefly, for heparan sulfate (HS), proteins, DNA, and unwanted GAGs were digested and then HS was isolated by anion-exchange chromatography. Heparan lyase-generated disaccharides were tagged with [¹²C]aniline. Samples are mixed with [¹³C_]aniline-tagged HS disaccharide standards and quantified with LC/MS. ANOVA statistical analysis was performed to correct for multiple comparisons.

Results : Overall, 6 young patients, 10 patients with early and intermediate AMD and 18 age-matched control patients were included. Our results show that HS is the predominant GAG present in BrM (216 ± 12 ng/BrM vs 38 ± 19 ng/BrM, chondroitin sulfate) in the macula. With age total HS was unchanged but in patients with early/intermediate AMD macular HS was significantly increased (216 ± 12 ng/BrM vs 367 ± 57 ng/BrM (p < 0.05)). Compositional analysis of macular HS chains did not reveal any significant change in disaccharide composition, but the increase in HS content indicates AMD BrM results in a higher exposure to N-, 2-O and 6-O sulfated domains in AMD (p < 0.05).

Conclusions : Higher content of BrM HS was associated with early and intermediate AMD. Given the affinity of highly sulfated HS for apolipoproteins these findings suggest that elevated levels of BrM HS may contribute to the lipoprotein aggregation and drusen formation in AMD.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.