P-417: Multiple roles for rho kinase in cultured human vascular smooth muscle cells

Rho kinase (ROCK) is a direct downstream effector of the monomeric GTPase rho and has recently been shown to a play an important role in neointimal formation in response to vessel injury, adding to the role in vessel contractility established earlier. This study, using human vascular smooth muscle cells (SMCs), examined the functional involvement of ROCK in the mitogenic and chemotactic processes which underlie vessel wound healing and remodelling. Cultured saphenous vein SMCs were derived from patients undergoing bypass surgery using an explant method. Mitogenesis, chemotaxis, cell morphology and the distribution pattern of F-actin were determined using labelled thymidine incorporation, blind-well migration assays, phase contrast microscopy and fluorescence microscopy of cells stained with FITC-labelled phalloidin respectively. Treatment of cells with the specific ROCK inhibitor Y27632 (1μM-30μM) dose-dependently inhibited DNA synthesis in response to platelet-derived growth factor-BB (PDGF-BB, 10 ng/ml) and foetal calf serum (FCS, 15% vol/vol). DNA synthesis in response to PDGF-BB and FCS displayed a Y27632 insensitive component of 31±7% and 42±5% (mean±standard error of the mean, n=4 cell strains) of the untreated control values respectively. In contrast, peak stimulated chemotaxis in response to PDGF-BB (2 ng/ml) was completely abolished (n=4) at a concentration of 10μM Y27632. When cells were plated sparsely (1000/cm2) to allow full spreading, subsequent addition of Y27632 (10μM and 30μM) resulted in time-dependent alterations in cell morphology. The most prominent feature was the development of neuron-like protrusions, appearing over a timescale of 1-4 hours after addition of ROCK inhibitor. These protusions were found to contain elongated actin filaments, indicating that ROCK inhibition could inititiate actin polymerisation. It is concluded that ROCK has multiple functional roles in human SMCs. DNA synthesis proceeds via ROCK dependent and independent pathways whereas chemotaxis is critically dependent on ROCK activity. Active ROCK plays an important role in the maintenance of normal cell morphology, at least in part by regulation of actin polymerisation and organisation. Inhibition of ROCK activity, leading to altered regulation of the actin cytoskeleton and loss of control of morphology could explain the Y27632 induced abolition of chemotaxis, which requires a co-ordinated series of morphological rearrangements to effect mobility. Highly localised