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Joanna Wisniewska-Kruk, Ingeborg Klaassen, Ilse M Vogels, Cornelis J Van Noorden, Reinier O Schlingemann, Ocular Angiogenesis Group; PLVAP Modulates Angiogenesis By Tuning VEGF Signaling In Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2241. doi: https://doi.org/.
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Plasmalemma vesicle associated protein (PLVAP) is an endothelial cell-specific protein, detected by the PAL-E antibody that has been used for decades as endothelial marker. PLVAP is expressed in a large part of the vascular system, but is absent from blood vessels with an intact blood-retinal barrier. However, in pathological conditions associated with neovascularization in the central nervous system, as occurs in diabetic retinopathy, PLVAP expression significantly increases. In this study, we investigated the role of PLVAP in angiogenesis and the potential molecular mechanisms that underlie its pro-angiogenic activity.
To elucidate the role of PLVAP in new blood vessel formation in vivo, we used the oxygen-induced retinopathy mouse model and injected siRNA intraocularly. Five days after injection, retinas were flat-mounted and the vasculature was analyzed. Furthermore, the possible pro-angiogenic function of PLVAP was investigated in in vitro models, including the aortic ring assay, the endothelial spheroid-based assay and migration assays. The silencing efficiency of siRNA and lentiviral delivered shRNA in all experiments was confirmed on mRNA and protein levels. The expression of VEGF receptors and its co-receptors after Plvap silencing in endothelial cells was investigated using western blot, immunohistochemistry and flow cytometry analysis.
Silencing of Plvap in the oxygen-induced retinopathy model resulted in decreased vascular and neovascular areas, and increased avascular areas in the retinas. Plvap inhibition in cultured endothelial cells exhibited decreased sprout formation, reduced numbers of endothelial tip cells and diminished cell migration. However, no effect on cell viability or cell proliferation was observed after Plvap silencing in these cells. Reduced angiogenic capacity of cells that lack PLVAP expression was linked to reduced VEGFR2 protein levels, selective downregulation of VEGF co-receptors and VEGFR2 down-stream signaling kinases.
Our data highlight PLVAP as an important endothelium-specific cofactor for physiological and pathological angiogenesis through a VEGFR2 dependent mechanism. Therefore, PLVAP may serve as a potential therapeutic target in proliferative vascular eye disorders, such as diabetic retinopathy and retinopathy of prematurity.
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