Abstract 1003: Nitric Oxide-Dependent Phosphorylation of Vasodilator-Stimulated Phosphoprotein Regulates Vascular Smooth Muscle Cell Contraction, Adhesion and Spreading
Background: Vasodilator-stimulated phosphoprotein (VASP) belongs to the enabled (Ena)/VASP family, which appears to regulate actin filament formation. VASP can be phosphorylated at three sites (S157, S239, and T278) with varying selectivity. cGMP-dependent protein kinase (PKG) preferentially phosphorylates S239, whereas S157 is the preferred phosphorylation site for cAMP-dependent protein kinase. Our recent studies in smooth muscle cells (SMCs) indicate that phosphorylation of S239 modulates the growth inhibitory effects of NO, since inhibition of S239 phosphorylation prevents the NO effect. In this study, we have investigated the role of S239 phosphorylation in SMC contraction, adhesion, and spreading, all important processes in the pathogenesis of atherosclerosis and restenosis after angioplasty.
Methods: Aortic SMCs derived from VASP-deficient mice were stably transfected with retroviral vectors encoding either wild-type VASP or VASP mutants (S239A-VASP and S239D-VASP), in which serine 239 was replaced by either the non-phosphorylatable amino acid, alanine, or the phospho-mimic amino acid, aspartic acid. SMC contraction of type I collagen gels was measured after serum stimulation and was defined as the percentage of retraction of the collagen gel. SMC adhesion and spreading were studied in thick collagen gels as well as on collagen bound to plastic wells. Organization of F-actin filaments was investigated by fluorescent staining.
Results: After 24 hours, gel contraction by wt-VASP-SMCs (90 ± 2 %, n=7) was significantly inhibited by the addition of an NO donor (100 μM deta NONOate) (71 ± 3 %; n=3). Compared to wt-VASP-SMCs, S239D-VASP-SMCs exhibited lower gel contraction (78 ± 4 % at 24 hours, n= 5), and the addition of NO did not further inhibit gel contraction. Spreading of S239D-VASP-SMCs within collagen gels was impaired compared to wt-VASP-SMCs, and addition of the NO donor only inhibited spreading of wt-VASP-SMCs. Similar results were observed for adhesion to a monolayer of polymeric collagen.
Conclusions: Our data suggest that phosphorylation of VASP at S239 is critical for the inhibitory effects of NO on SMC contraction, adhesion and spreading.