Carbon Monoxide and Nitric Oxide Mediate Cytoskeletal Reorganization in Microvascular Cells via Vasodilator–Stimulated Phosphoprotein (VASP) Phosphorylation: Evidence for Blunted Responsiveness in Diabetes

Abstract

Objective: We examined the effect of the vasoactive agents carbon monoxide (CO) and nitric oxide (NO) on the phosphorylation and intracellular redistribution of VASP, a critical actin motor protein required for cell migration that also controls vasodilation and platelet aggregation.

Research Design: We examined the effect of donor-released CO and NO in Endothelial Progenitor Cells (EPC) and platelets from nondiabetics and diabetics and in Human Microvascular Endothelial Cells (HMEC) cultured under low- (5.5 mM) or high- (25 mM) glucose conditions. VASP phosphorylation was evaluated using phosphorylation site-specific antibodies.

Results: In control platelets, CO selectively promotes phosphorylation at VASP Ser-157, while NO promotes phosphorylation primarily at Ser-157 and also Ser-239, with maximal responses at 1 min with both agents on Ser-157 and at 15 min on Ser-239 with NO treatment. In diabetic platelets, neither agent resulted in VASP phosphorylation. In nondiabetic EPCs, NO and CO increased phosphorylation at Ser-239 and Ser-157, respectively, but this response was markedly reduced in diabetic EPCs. In endothelial cells cultured at low glucose, both CO and NO induced phosphorylation at Ser-157 and Ser-239; however, this response was completely lost when cells were cultured at high glucose. In control EPCs and in HMEC exposed to low glucose, VASP was redistributed to filopodia-like structures following CO or NO exposure, however, redistribution was dramatically attenuated in high glucose.

Conclusions: Both vasoactive gases promote cytoskeletal changes through site- and cell type-specific VASP phosphorylation and in diabetes blunted responses to these agents may lead to reduced vascular repair and tissue perfusion.