Role of Rho kinase in sphingosine 1-phosphate-mediated endothelial and smooth muscle cell migration and differentiation.

The role of sphingosine 1-phosphate (S1P)-induced Rho kinase (ROCK) activation in the angiogenic responses of pulmonary artery-derived endothelial cells (PAEC) and smooth muscle cells (PASMC) was examined. S1P, a biologically active phospholipid that regulates angiogenesis, promoted PAEC chemotaxis and capillary morphogenesis; furthermore, this activity was unaltered by pretreatment with the pharmacological inhibitor of ROCK, H1152. In contrast, S1P (500 nM) significantly inhibited spontaneous PASMC chemotaxis and differentiation; however, this inhibition was eradicated upon H1152 pretreatment. Similarly, PASMCs transfected with ROCK II siRNA diminished S1P-induced inhibition of the development of multi-cellular structures. Analysis by RT-PCR identified the presence of S1P(1) and S1P(3) receptors on both PAECs and PASMCs, while S1P(2) receptor expression was confined to only PASMCs. Consistent with this observation, the S1P(1) and S1P(3) receptor antagonist, VPC23019, virtually abolished the S1P-initiated PAEC differentiation but did not impede the S1P-induced inhibition of PASMC differentiation. However, the S1P(2) receptor antagonist, JTE013, had no effect on S1P-mediated differentiation of PAECs but abolished the S1P-induced inhibition of PASMC function. Co-cultured endothelial and smooth muscle cells differentiated into "neovascular-like" networks, which were significantly inhibited by S1P. The inhibition of co-culture differentiation in both PAECs and PASMCs was negated by H1152 pretreatment. However, when smooth muscle cells were added to S1P-initiated endothelial cell networks, additional S1P treatment did not inhibit the cellular networks generated by these cells. In conclusion, S1P-induced PAEC angiogenic responses are regulated by S1P(1) and/or S1P(3) receptors independent of Rho kinase activation, whereas S1P(2) receptor-mediated curtailment of PASMC function by S1P.