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Matthew B. Neu, Sergio Li Calzi, Maria Grant, Daniel L. Purich; Enhanced Actin-Based Motility and Leading-Edge VASP Recruitment in Human Microvascular Endothelial Cells Treated with Sphingosine-1 Phosphate. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4859.
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We previously showed in human retinal endothelial cells (HRECs) that the excitatory gases nitric oxide and carbon monoxide alter the rate of actin-based chemotaxis with concomitant changes in the site-specific phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Herein, we investigated the effects of the upstream modulator sphingosine-1 phosphate (S1P), a key regulator of endothelial progenitor cell recruitment and differentiation that has been shown to be critical in the pathogenesis of diabetic retinopathy.
Human microvascular endothelial cell (HMEC) migration towards increasing concentrations of S1P was assessed using modified Boyden chambers. Immunofluorescence imaging was used to examine VASP redistribution following S1P treatment using FITC-labeled antibodies. Site-specific VASP phosphorylation was assessed by Western Blotting Analysis.
S1P treatment stimulated VASP recruitment to the leading edge, with the attendant activation of actin-based cellular locomotion on the same time-scale that we previously showed for CO- or NO-stimulated phosphorylation. Western blotting with phosphorylation site-specific anti-VASP antibodies indicated that phosphorylation occurred at Ser-239, suggesting that S1P mainly signals through NO-sensitive reactions.
We now suggest a multi-site model for VASP, where (a) prior to S1P treatment, VASP is situated in focal adhesions as well as sites very near the leading edge, and (b) upon S1P activation of actin-based motility, VASP is recruited to advancing filopodia and lamellipodia at the leading edge itself. Blockade of S1P may provide a critical strategy to inhibit migration of EPCs resulting in pathological neovascularization and Diabetic Retinopathy.
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