TIMP3 Aggregates in Endothelial Cells Expressing Sorsby Fundus Dystrophy Associated TIMP3 Mutations is Regulated by Glycosylation.

Abstract

Purpose : Purpose: Sorsby’s fundus dystrophy (SFD) is an autosomal dominant macular disorder that closely resembles age-related macular degeneration (AMD). SFD is caused by mutations in tissue Inhibitor of metalloproteinase-3 (TIMP3) gene and is of considerable interest due to early onset of choroidal neovascularization (CNV). The objective of this study is to investigate the molecular mechanism(s) by which TIMP3 mutants regulate choroidal neovascularization

Methods : Methods: Porcine aortic endothelial (PAE) cells lines that express wild-type TIMP3, three unique SFD mutant TIMP3 genes (S156C-TIMP3, Y168C-TIMP3 and S181C-TIMP3) and N184Q-SFD-TIMP3 mutants and RPE-choroid tissue from S156C-TIMP3 knock-in mouse and AMD eyes were analyzed for TIMP3 isoforms and VEGF-induced signaling.

Results : Results: During the analysis of these cell lines we determined that compared to wild-type TIMP3 in endothelial cells, mutant TIMP3 was more glycosylated and showed small increases in aggregates. To evaluate if the glycosylation state of TIMP3 contributed to the aggregation of TIMP3 we evaluated double mutant lines with an additional mutation at Asn¹⁸⁴ (the only potential N-glycosylation site on TIMP3 [23]). We observed that upon deglycosylation of TIMP3 there was a dramatic increase in high molecular weight species of aggregated mutant TIMP3 (resistant to reducing agents) as well as increased angiogenesis in response to VEGF.

Conclusions : Conclusion: The crosslinking and aggregate formation of mutant TIMP3 is inversely proportional to the glycosylation of TIMP3, which contributes to increased angiogenesis and CNV.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.