Cytoskeletal Rearrangements via Vasodilator-Stimulated Phosphoprotein (VASP) Phosphorylation and Redistribution in Human Retinal Endothelial Cells (HREC), Endothelial Precursor Cells(EPC) and Platelets: Implications for Retinal Vessel Repair

Abstract

Purpose:: The actin motor protein VASP powers cell migration, vasodilatation, and inhibition of platelet aggregation- essential processes for maintaining retinal vascular health. VASP has three phosphorylation sites (Ser-157, Ser-239, and Thr-278) that are regulated by cGMP and cAMP protein kinases. Therefore, we examined the effect of the vasoactive angiogenic agents CO, NO and stromal derived factor-1 (SDF-1) on cell migration, VASP phosphorylation, and VASP localization.

Methods:: CD34+ EPCs were isolated from human peripheral blood using magnetic microbeads and platelets using Opti-Prep^TM. HREC were prepared as previously described (Grant MB 1991). After 1, 5 and 15 min exposure to the NO donor DETA/NO, the CO donor Ru(II)Cl₂(CO)₃ dimer or SDF-1, VASP phosphorylation in platelets and endothelial cells was evaluated by Western analysis. FACS analysis was used to evaluate EPCs. Phosphospecific anti-pSer-157 antibody or anti-pSer-239 antibody was used for analysis. Immunohistochemistry was performed to evaluate VASP redistribution in HREC and EPCs using anti-VASP antibody following exposure to CO, NO or SDF-1. Migration to CO, NO and SDF-1 was performed using the modified Boyden chamber assay in HREC and EPC.

Results:: Upon CO stimulation in human platelets, VASP was phosphorylated on Ser-157, but not Ser-239, while NO exposure resulted in phosphorylation mainly of Ser-239. In endothelial cells, VASP is phosphorylated on Ser-239 in response to both CO and NO exposure. In EPC and HREC, VASP was redistributed to filopodia after incubation with either CO, NO or SDF-1. At the concentrations tested, all three agents induced EPC and endothelial cell migration in a dose-dependent manner.

Conclusions:: In the retinal vasculature, NO, CO and SDF-1 may compensate for one another or work together to promote cytoskeletal changes through site-specific phosphorylation of VASP. Vasoactive agents may promote retinal vascular repair and improved tissue perfusion by increasing EPC and endothelial cell migration and by preventing platelet aggregation .

Keywords: nitric oxide • differentiation • vascular cells