Role Of Heat Shock Protein 27 Phosphorylation In Migration Of Vascular Smooth Muscle Cells

Objective: To investigate the role of heat shock protein 27 (HSP27) phosphorylation in the migration of vascular smooth muscle cells(VSMCs) from spontaneously hypertensive rats(SHR) induced by angiotensin II(AngII) and platelet derived growth factor-BB(PDGF-BB).Methods: Cultured VSMCs derived from SHR thoracic aorta were used. The activity of HSP27 was evaluated by Western blot with phospho-HSP27 antibody.The effect of F-actin polymerization was detected by FITC-Phalloidine staining and examined by confocal microscopy. Modified Boyden chamber technique was employed for VSMCs migration accessment.Results:1.The phosphorylation of HSP27 was increased by AngII and PDGF-BB in a concentration-dependent manner in VSMCs.Treatment with AngII and PDGF-BB resulted in a substantial increase in the number of stress fibers and rearrangement of these structures into ordered parallel arrays.The migration of VSMCs was promoted by AngII and PDGF-BB.The peak of HSP27 phosphorylation stimulated by AngII(10-7mol/L)and PDGF-BB(30ng/ml)was at 30 min. The activity of HSP27 in response to 10-5mol/L AngII and 100ng/ml PDGF-BB were at 338.9% and 159.1% compared with control(P<0.01). The phosphorylation of HSP27 induced by AngII and PDGF-BB was blocked by the selective HSP inhibitor Quercetin in a concentration-dependent manner, with maximal inhibition rate at 71.7% and 66.7% respectively(P<0.01). Reorganization of actin cytoskeleton stimulated by AngII and PDGF-BB was inhibited in 100umol/L Quercetin pretreated group.The inhibition rates of 100umol/L Quercetin on migration of VSMCs induced by AngII and PDGF-BB were at 55.3% and 53.6%(P<0.01).2.The activity of HSP27 and migration of VSMCs induced by AngII and PDGF-BB were decreased by the specific inhibitor of P38MAPK, PI3K and ERK1/2. Within a given concentration , the phosphorylation of HSP27 induced by AngII and PDGF-BB was blocked by the specific P38MAPK inhibitor SB202190, the specific PI3K inhibitor LY294002 and the specific ERK1/2 inhibitor U0126 in a concentration-dependent manner,with a peak inhibition rate at 87.2%, 78.4% and 37.3% respectively induced by AngII(P<0.01),with a peak inhibition rate at 85.0%, 55.3% and 41.0% respectively induced by PDGF-BB(P<0.01).The migration of VSMCs induced by AngII and PDGF-BB was inhibited by 100umol/L SB202190, 30umol/L LY294002 and 30umol/L U0126, with a inhibition rate at 60.1%, 71.7% and 47.3% respectively provoked by AngII(P<0.01),with a inhibition rate at 55.3%, 55.6% and 38.1% respectively provoked by PDGF-BB(P<0.01).3.The phosphorylation of HSP27 induced by AngII was inhibited by AT1 receptor antagonist candesartan. The activity of HSP27 stimulated by PDGF-BB was significantly blocked by trapidil,one of PDGF receptor antagonist. The phosphorylation of HSP27 in response to AngII and PDGF-BB was suppressed by Fluvastatin in a dose-dependent manner,and maximal inhibition rates were at 42.1% and 58.5% with 10-5mol/L Fluvastatin respectively(P<0.01).The maximal inhibition rates of 10-4mol/L sodium ferulate on HSP27 phosphorylation induced by AngII and PDGF-BB were at 39.0%(P<0.05)and 56.8%(P<0.01)respectively.Conclusions:1.HSP27 phosphorylation plays an important role in mediating the rearrangement of F-actin and migration of VSMCs induced by AngII and PDGF-BB.2.P38MAPK and PI3K/AKT are important pathways that contribute to the phosphorylation of HSP27 and migration of VSMCs in response to AngII and PDGF-BB. ERK1/2 might be involved in HSP27 phosphorylation and migration of VSMCs provoked by AngII and PDGF-BB. 3. Fluvastatin and sodium ferulate influenced the migration of VSMCs in part by inhibiting HSP27 phosphorylation.