Abstract
Purpose :
A key feature of age-related macular degeneration (AMD) is atrophic pathology in the choriocapillaris (CC). It could be considered that loss of the CC, at least in part, relates to the age-related decline in endogenous vascular repair mechanisms creating hypoxia in the outer retina. We hypothesised that the CC harbours vessel-resident endothelial progenitors which could become senescent and dysfunctional with age and impair normal homeostasis and repair. Recently, the endothelial protein C receptor (EPCR) has been identified as a marker of progenitors known as endothelial colony forming cells (ECFCs). This study aimed to investigate EPCR and its ligand, APC, in ECFCs in the context of the CC.
Methods :
Mouse choroidal flatmounts were investigated for presence of EPCR. Western blot, RT-PCR, flow cytometry, 3D-tube formation in matrigel, choroidal explant, immunofluorescence for Lectin/CD31/CD201, b-galactosidase staining, xCelligence system. For multiple comparisons, we used one-way analysis of variance (ANOVA) and paired two tailed t-test analysis. p≤0.05 were considered significant.
Results :
EPCR was present in sub-populations of cells in the murine CC and in choroidal explants. ECFCs senescence lead a significant decrease in the gene and protein expression of EPCR (p=0.0002). ECFCs exposed to hypoxia (1% and 5% O2) showed a marked decrease in the EPCR gene and protein expression when compared to normoxic conditions (p=0.002). APC treatment in prevented hypoxia-induced decrease in ECFC tubulogenesis in and improved barrier function (p≤0.05). The knockdown of EPCR by siRNA lead a significant reduction in tubulogenesis (p<0.0001), and this was also accompanied by decreased expression of eNOS and CCBN2 (p<0.0001). We also observed an alteration of quiescent markers but not the senescence pathway. Choroidal explants exposed to EPCR siRNA showed a significant decrease of the sprouting in the matrigel and the treatment with APC re-established the vessel growth.
Conclusions :
EPCR is present in the CC which may relates to ECFC-like cells resident in these vessels. Regulation of the EPCR-APC pathway impact on progenitor function and could be a potential therapeutic avenue to enhance endogenous repair of the CC, especially in the context of AMD.
This is a 2021 ARVO Annual Meeting abstract.