Abstract 1912: Nitric Oxide Activates the Vav3-Rac1 Pathway via RhoA Phosphorylation to Promote Arterial Smooth Muscle Cell Migration
Nitric oxide (NO) is well known for its ability to induce angiogenesis and arteriogenesis. Although numerous studies have described its effects on endothelial cells, very little is known about its role on vascular smooth muscle cells (VSMC) migration. We have previously shown that under physiological conditions, in VSMC, RhoA is phosphorylated on Ser188 by NO-stimulated cGMP-dependent kinase (PKG). This Ser188 phosphorylation of RhoA leads to inhibition of RhoA-Rho kinase pathway. Here we hypothesize that in addition to this inhibition, cytosolic Ser188-phosphorylated RhoA has its own functions. To address this hypothesis we analyzed VSMC functions in cells stimulated by the PKGactivator 8pCPTcGMP, or in cells expressing wild-type RhoA (WT), constitutively active (Q63L), phosphoresistant (S188A), phosphomimetic (S188E) and double-mutants Q63L-S188A and Q63L-188E. Using a scratch-wound repair assay in VSMC monolayers, we observed that phosphoresistant mutants reduced the wound-closure (37.5±1.4% for Q63L-S188A compared to 49.0±3.1% for Q63L; p<0.01; n=5) while phosphomimetic mutants greatly accelerated it (91.5±3.8% Q63L-S188E; p<0.01, n=5). The NO donor sodium nitroprusside (100 μM) or 8pCPTcGMP (100 μM) accelerated the wound-closure in VSMC expressing the WT RhoA (77.5±4.3% and 90.0±8.6% respectively vs 57.5±2.6%; p<0.01, n=3) but not in VSMC expressing the phosphoresistant mutant. As Rac1 is well known to regulate migration, we looked at its activation by measuring PAK phosphorylation. 8pCPTcGMP induced Rac1 translocation to the plasma membrane and PAK phosphorylation in VSMC expressing the WT RhoA (3.4±0.8 fold compared to non stimulated cells ; p<0.05; n=3) but not in cells expressing the S188A. Expression of Q63L-S188E was sufficient to induce Rac1 activation, and using silencing RNA we shown that the Rho exchange factor Vav3 is necessary for this activation. As expected, 8pCPTcGMP- or Q63L-S188E-induced wound healing was inhibited by a dominant negative Rac1 mutant or by Vav3 siRNA. In conclusion, our work demonstrates that the effect of NO on arteriogenesis, may result not only on its effect on the endothelium but also, through RhoA phosphorylation and activation of the Vav3-Rac1 pathway, on positive effect on VSMC migration.