Abstract
Purpose :
Our previous studies have demonstrated that Prominin-1 (Prom1/CD133)-dependent autophagy contributes to the cytoprotection of RPE cells against stress-induced mitochondrial damage and apoptosis. Although oxidative stress to RPE upregulates the secretion of pro-angiogenic VEGF and contributes to the progression of neovascularization, it is unknown whether Prom1 is a regulator of stress-induced VEGF expression and its secretion by the RPE.
Methods :
A Prom1 lentiviral construct was used to overexpress wildtype (WT) and CRISPR/Cas9 lentivirus was used to knockout (KO) Prom1 in ARPE19 cells. Cells were polarized in transwell inserts and transepithelial resistance was measured with an ohmmeter. Polarized RPE monolayers were treated with moderate hypoxia (8% oxygen). Western blotting was used to quantify levels of intracellular and apical and basolateral secretion of VEGF-A and –C. Co-immunoprecipitation was used to detect the interaction between VEGF and Prom1.
Results :
Overexpression of Prom1 increased intracellular expression of VEGF-C in non-polarized ARPE-19 cells. Prom1 immunoprecipitates from WT cell lysates contained increased amounts of VEGF-C in the immune-complex compared to control ARPE-19 cells, which decreased in KO cells. Secretion of VEGF-A and –C by polarized ARPE-19 cells increased in response to moderate hypoxia, and this stimulation was higher on the apical than the basolateral side. Hypoxia decreased transepithelial resistance and polarity in control ARPE-19 cells, but had no effect in WT cells. Although basal secretion of VEGF-C significantly decreased in WT cells, KO of Prom1 further potentiated hypoxia-induced VEGF-C secretion on the apical side, which is a driving signal for choroidal neovascularization (CNV) development.
Conclusions :
Given the known role of VEGF in development and progression of CNV, our data demonstrate a new role of Prom1 in modulating VEGF expression and its secretion by the RPE through its ability to interact with VEGF. This new finding may be of importance in understanding pathological angiogenesis in the retina given our recent report that Prom1 plays a central role in regulating autophagy and its homeostasis in the RPE and under conditions of stress.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.