Fig. 7.

Transfer of cPWWP2A between pericyte and EC regulates pericyte–EC crosstalk. (A) Pericytes and ECs were sorted from nondiabetic and diabetic retinas (after 3-mo diabetes induction). cPWWP2A expression in pericytes and ECs was determined by qRT-PCRs (n = 5 per group; *P < 0.05 versus nondiabetic group). (B) Pericytes and ECs were sorted from nondiabetic retinas, and then were exposed to 30 mM glucose for 24 h and 48 h, or left untreated (Ctrl). cPWWP2A expression in pericytes and ECs was determined by qRT-PCRs (n = 5 per group). (C) Representative CD31 staining (green) or NG2 staining (green) with FISH detection of cPWWP2A expression (red) in nondiabetic and diabetic retinas. (Scale bar, 100 μm.) (D) The medium of high glucose (HG)-treated pericytes was pretreated with or without apoptosis inhibitor Z-VAD-FMK (ZVAD) or N-SMase inhibitor GW4869. ECs were then incubated with the vesicles isolated from the above-mentioned medium. cPWWP2A expression in ECs was determined by qRT-PCRs (n = 4, *P < 0.05 versus HG). (E–G) Pericytes were incubated with the medium containing 5 mM glucose (Ctrl) or 30 mM glucose (HG) for 24 h. Pericyte medium with or without cPWWP2A antisense transcript was then incubated with cPWWP2A^−/− ECs. EC proliferation was detected at 48 h postincubation using Ki67 staining (E). EC migration and tube formation was detected at 8 h postincubation using a Transwell assay (F) and growth factor-reduced Matrigel (G). The significant difference was evaluated by one-way ANOVA followed by Bonferroni’s post hoc test.