BS09 ROCK1, and not RhoA, is a critical modulator of human saphenous vein smooth muscle cell phenotype

Introduction Vascular smooth muscle cells (SMCs) are the principal cell type in the vascular wall. Their ability to switch between a differentiated contractile and dedifferentiated synthetic phenotype according to external cues, makes them a study target in understanding their role in cardiovascular disease pathogenesis. RhoA is a small G protein that regulates cell morphology, migration, and proliferation. Deregulation of this protein is likely to alter SMC phenotype and function and its roles are yet to be fully elucidated. Of particular interest, RhoA activity is modulated by statins, which are widely prescribed in cardiovascular disease. The interlinks between statins, RhoA and vascular SMCs are as yet unknown. Methods Human primary SMC were transfected with siRNA against RhoA, the downstream effector ROCK1 (Rho kinase) or scrambled negative control. RhoA activity was also indirectly inhibited by fluvastatin. siRNA efficiency was measured using qPCR and western blotting. SMC phenotypic parameters including morphology was measured by light microscopy and ImageJ analysis, cytoskeletal arrangement by immunofluorescence, and proliferation by cell counts. Results Direct inhibition of RhoA by siRNA had no impact on cell morphology, the organisation of the cytoskeleton, or proliferation. In contrast, ROCK1 inhibition markedly increased both spread cell area (20%) and proliferation (18%) and caused disorganisation of the cytoskeleton. Interestingly, these effects were also apparent in statin-treated SMC where spread cell area was increased by 67% and proliferation by 14.7%. Conclusion Despite the well-recognised multiple roles of RhoA in morphology and cytoskeletal arrangement in other cell types, direct manipulation by siRNA had no impact on vascular SMC phenotype. In contrast, inhibition of ROCK1 resulted in the acquisition of a dedifferentiated synthetic phenotype suggesting that ROCK1 is more important in controlling SMC differentiation than RhoA. Furthermore, statins also caused a dedifferentiation of SMC suggesting they may be acting by pathways other than inhibition of RhoA. Conflict of Interest NA