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D.C. Darland, P.A. D'Amore; Pericyte Expression of VEGF in Developing Retinal Vasculature . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2889.
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Purpose: Pericytes are thought to play a role in vessel formation, but less is known about the influence of pericytes on vessel stabilization. Pericyte loss in non-proliferative diabetic retinopathy, for example, precedes vessel dysfunction and appearance of acellular capillaries. Vascular endothelial growth factor (VEGF), which is required for endothelial cell (EC) survival and angiogenesis, may also affect vessel stabilization. We hypothesized that pericyte precursors differentiate upon contact with EC, express VEGF and provide a local source of survival factor. We wanted to test the hypothesis that pericyte production of VEGF is contact-dependent and contributes to vessel stability. Methods: We used postnatal day 10.5 mice that express lacZ (nuclear) under control of the endogenous VEGF promoter. Eyes were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24h at 4°C. Retinas were removed from fixed tissue and stained for ß-galactosidase (ß-gal) histochemistry (Stratagene kit) or treated for 24h at 4 °C with blocking solution (4% donkey serum, 3% goat serum, 1% saponin, 0.1% triton X-100 in PBS). Retinas were stained with primary antibodies to pericyte- (NG2 and SMA) and EC-specific (PECAM) proteins in combination with ß-gal. Primary antibody incubations were 24h at 4 °C followed by repeated PBS washes. Fluorescent-conjugated secondary antibody (Alexafluor488, cy3 or cy5) incubations were 4h at rm temp followed by repeated PBS washes. Retinas were flat-mounted and visualized with standard light or confocal microscopy. Results: Analysis of retinal whole mounts revealed that ß-gal activity was present in the nuclei of perivascular cells associated with developing retinal microvasculature. The ß-gal positive nuclei were confirmed to be pericytes; ß-gal immunoreactivity co-localized with NG2 and SMA, proteins associated with pericyte differentiation. A few ß-gal positive nuclei were observed in some cells not directly contacting the endothelial cells both in the microvascular bed and in the avascular zone in the periphery of the retina. The highest density of ß-gal positive nuclei were observed at the periphery of the retinal vasculature, the site of active angiogenesis. PECAM-positive EC processes projected into the avascular zone and were closely associated with the ß-gal positive nuclei. Conclusions: We find that as new vessels are added at the retinal periphery, VEGF expression is induced in differentiating pericytes. These results indicate that contact-dependent pericyte production of VEGF may be an important component of vessel formation and stabilization during retinal development.
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