Study On The Action Of Small G Proteins And MAP Kinase To Vascular Smooth Muscle Cell Migration Stimulated By Lipoprotein(a)

Part one Cytoskeleton reorganization and cell migration in human vascular smooth muscle cell stimulated by Lp(a)Objectives: To study the reorganization of cytoskeleton and cell migration in human vascular smooth muscle cell (VSMC) stimulated by lipoprotein(a)[Lp(a)]. Methods: Firstly, the primary human VSMCs were cultured. Migration assays were applied to VSMC stimulated by Lp(a) of different concentrations. After VSMCs had been stimulated by Lp(a), F-actin was stained by FITC-labeled phalloidin to visualize actin-based structure including filopodia and stress fiber at different time points. And vinculin distribution of focal adhesion was visualized through indirect immunofluorescence at different time points too. These cytoskeletons of VSMC were then observed with confocal laser scanning microscope. Results: When the final concentration of Lp(a) was confined from 40μg/ml to 640μg/ml, the number of migrating VSMCs added while the final concentration was increased with significant difference (P<0.05) . The filopodia, stress fibers and focal adhesions in VSMC appeared at 10min after stimulation of Lp(a). The stress fibers and focal adhesions appeared remarkably at 20min. There was no evident change of these structures at 30min. Conclusion: Lp(a) induced the reorganization of cytoskeleton in human VSMC and stimulated cell migration of VSMC.Part two The role of Rac1 and RhoA in cell migration of human VSMCs stimulated by Lp(a)Objectives: To verify the effect of Rac1 and RhoA in cytoskeleton reorganization and cell migration of human VSMCs stimulated by Lp(a). Methods: The siRNA was introduced into cultured human VSMCs by lipofectamine to block the expression ofRac1 gene. Then VSMCs were stimulated by Lp(a). Reverse-transcription polymerase chain reaction (RT-PCR) and western blot were applied to detect the efficacy of RNA interference. The cytoskeletons of these VSMCs were then observed with confocal laser scanning microscope. The number of migrated cells was determined by migration assays. C3 exoenzyme was introduced into VSMCs by lipofectamine to block the activity of RhoA. Then VSMCs were stimulated by Lp(a). The cytoskeletons of these VSMCs were then observed with confocal laser scanning microscope and the number of cells that had migrated was determined by migration assays. Results: The Rac1 siRNA demonstrated the efficiency in the blocking of Rac1, with significant difference from the control (P<0.01) . After VSMCs were stimulated by Lp(a) for 20min, there was no filopodia, stress fibers and focal adhesions in VSMC which Rac1 siRNA had been introduced into. The number of VSMCs that had migrated in the RNAi group was fewer than the control groups with significant difference (P<0.01) . After VSMCs were stimulated by Lp(a) for 20min, there was no filopodia, stress fibers and focal adhesions in VSMC which C3 exoenzyme had been introduced into. The number of VSMCs that had migrated in the C3 exoenzyme group was fewer than the control groups with significant difference (P<0.01) . Conclusion: Stimulation of VSMC cytoskeleton reorganization and migration by Lp(a) is dependent on Rac1 and RhoA.Part three The role of ERK and p38 in cell migration of human VSMCs stimulated by Lp(a)Objectives: To verify the effect of ERK and p38 in cytoskeleton reorganization and cell migration of human VSMCs stimulated by Lp(a). Methods: The ERK kinase inhibitor PD98059 of different concentrations and p38 kinase inhibitor SB202190 of different concentrations were applied to inhibit activation of ERK and p38 in human VSMCs. Then VSMCs were stimulated by Lp(a). Western blot were applied to detect the expression of P-ERK and P-p38. The cytoskeletons of these VSMCs were then observed with confocal laser scanning microscope. The number of cells that had migrated was determined by migration assays. Results: The expression of P-ERK wasdecreasing while the final concentration of PD98059 was increased with significant difference (P<0.01) , when the final concentration of PD98059 was confined from 20μmol/L to 40μmol/L. The expression of P-p38 was decreasing while the final concentration of SB202190 was increased with significant difference (P<0.01) ,when the final concentration of SB202190 was confined from 5μmol/L to 20μmol/L. After VSMCs were stimulated by Lp(a) for 20min, there was no filopodia, stress fibers and focal adhesions in VSMCs which had been treated with PD98059 or SB202190. The number of VSMCs that had migrated in the PD98059 group and SB202190 group was fewer than the control groups with significant difference (P<0.01) . Conclusion: Stimulation of VSMC cytoskeleton reorganization and migration by Lp(a) is dependent on activation of ERK and p38.