Study On The Signaling Transduction Mechanism Of Smooth Muscle Cell Migration Induced By Rho GTPases

Background:Intimal hyperplasia is the principal cause of restenosis of blood vessels following vascular intervention or injury. Migration from the media to intima and subsequent proliferation of vascular smooth muscle cells(VSMC) are key events in the early development of the intimal hyperplastic lesion. Thus the actin-based structures which have tight relation with the migration of VSMC take part in the development of restenosis. These structures include filopodia, lamellipodia, stress fiber and focal adhesion. Recent studies showed that Rho GTPase played a key role in the reorganization of cytoskeleton and in Swiss 3T3 cells, Cdc42, Rac1 and RhoA, three members of Rho superfamily of small GTPase, regulated the polymerization of actin to produce filopodia , lamellipodia, stress fiber and focal adhension, respectively. However, different cell types may undergo different signaling pathways. So the study of cytoskeleton change in human VSMCs induced by Rho GTPase is essential to clarify the signaling mechanism of migration of human VSMCs in vascular restenosis.Part Ⅰ Reorganization of cytoskeleton in human vascular smooth muscle cell after induction of L-1α, PDGF-BB and LPAObjectives: To study the reorganization of cytoskeleton in human VSMCs induced by Rho GTPase-activating cytokines.Methods: Firstly, the primary human vascular smooth muscle cells were cultured. After inducement Rho GTPase-activating cytokines including IL-1α, PDGF-BB and LPA, F-actin was stained by FITC-labeled Phalloidine to visualize actin-based structure including filopodia, lamellipodia and stress fiber. And vinculin distribution of focal adhesion were visualized through indirect immunofluorescence. These cytoskeletons of VSMC were then observed with confocal laser scanning microscope at different time points. Results: The filopodia, lamelloipodia, stress fibers and focal adhensions in VSMC appeared at 10min, 20min and 30min after inducement of IL-1α. respectively. PDGF-BB induced the production lamellipoddia, stress fibers at 5min and 15min respectively. And focal adhensions accompanied with stress fiber was observed 20min after inducement of LPA.Conclusions: Rho GTPase-activating cytokines induced the reorganization of cytoskeleton in human VSMCs, which indicated inner activating sequence among members of Rho superfamily of small GTPase.