Figure 7.

Vascular endothelial growth factor (VEGF)-dependent activation of VEGF receptors (VEGFRs) contributes minimally to the bioactivity of proliferative vitreoretinopathy (PVR) vitreous. A: RT-PCR quantification of VEGFR2, VEGFR1, and platelet-derived growth factor alpha (PDGFRα) expression in human ARPE-19α cells, primary human corneal fibroblasts (HCFs), and primary human umbilical vascular endothelial cells (HUVECs). Resulting data show absolute mRNA copy number per 100 ng of total RNA, and were determined on the basis of comparison to standard curves using purified plasmids for each receptor type. ARPE-19α expresses both VEGFRs and PDGFRαs (the latter is overexpressed), whereas HCFs and HUVECs express only PDGFRα or VEGFRs, respectively. B: The kinase activity of VEGFRs expressed on ARPE-19α cells does not contribute to vitreous-induced reduction of TP53. Cells were starved and treated in parallel for 10 minutes (to assess VEGFR activation) or 2 hours (to assess TP53 reduction) with no treatment (----), 20 ng/mL VEGF, or RV-PVR in either the absence (--) or presence (+) of yrosine kinase inhibitor (VR-inh), which was added 30 minutes before treatment and used at a concentration of 360 nmol/L, sufficient to neutralize both VEGFR isoforms.²⁸ After treatment, cells were lysed and subjected to Western blot analysis using the indicated antibodies. The signal intensity of the resulting immunoblots was quantified by densitometry, and ratios representing band intensities normalized to non-stimulated cells are shown under the immunoblot. C: Vitreous VEGF protects from apoptosis through its canonical ability to activate VEGFRs and its noncanonical ability to promote indirect activation of PDGFRα. ARPE-19α cells were starved and treated as in B, except treatment was maintained for 72 hours instead of 2 hours, with starvation media plus supplements replaced daily, including VR-Inh. At 96 hours, cells were subjected to TUNEL analysis. The data shown represent the means ± SD of three independent experiments. Data were compared using a paired t-test. D: Vitreous VEGF protects from senescence primarily by its noncanonical ability to promote indirect activation of PDGFRα. ARPE-19α cells were starved 24 hours and then treated as in C. The percentage of senescent cells was determined at 96 hours. The data shown represent the means ± SD from three independent experiments, and were statistically compared as in C. ^∗P < 0.05. DMSO, dimethyl sulfoxide.