Abstract
Purpose :
Current treatments for diabetic retinopathy (DR) focus on end-stage disease. Targeting vaso and neurodegeneration in early DR could delay or prevent progression to the sight-threatening end-stages. Endothelial colony forming cells (ECFCs) have vasoreparative properties in the retina. ECFCs (and endogenous vascular stem cells in the retina) express unusually high levels of endothelial protein C receptor (EPCR) and when triggered by Activated Protein C (APC) can evoke a range of vasoprotective responses. We sought to determine if modulation of the APC/EPCR signalling pathway has a potential to enhance the vasoreparative function of endothelial progenitor cells in DR.
Methods :
siRNA knockdown of EPCR was used to determine the effect on ECFC functionality in tubulogenics, clonogenics and migration assays. We assessed the effect of diabetes-like culture conditions on EPCR levels using flow cytometry and on soluble EPCR levels using ELISA. The xCELLigence RTCA System was used to measure the ability of APC to protect the ECFCs barrier from a thrombin or VEGF induced insult. The effect of APC on maintaining ECFC tight junctions was evaluated by immunocytochemistry. Downstream signalling effects of APC were investigated by Western blot. Murine choroidal explants were used as an ex vivo model of sprouting angiogenesis.
Results :
EPCR knockdown impaired normal ECFC function as determined by significantly reduced tubulogenesis, clonogenicity and migration. Diabetes-like culture conditions significantly reduced membrane-bound EPCR levels, although soluble EPCR levels were not significantly affected. APC treatment prevented hypoxia-induced decrease in tubulogenesis, while pre-treatment with APC protected against thrombin or VEGF induced endothelial cell barrier disruption and loss of tight junction integrity (p<0.05-0.001). Downregulation of pERK, and upregulation of pVEGFR2 and pFAK was observed following APC exposure. EPCR knockdown in choroidal explants showed increased vascular sprouting (p<0.05).
Conclusions :
Our results suggest that EPCR is important for normal ECFC function and that the APC/EPCR pathway is a potential therapeutic target to promote vasoprotective effects in diabetic retinopathy. We continue to investigate the effect of APC/EPCR pathway modulation in endothelial cells in vitro, and the potential of APC treatment to overcome diabetes-induced vascular dysfunction in vivo.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.