The Mechanism Of Baicalin Inhibits The Migration Of Vascular Smooth Muscle Cells In Vitro And In Vivo

Objective: The migration of vascular smooth muscle cells (VSMCs) isthe physiological and pathological basis of atherosclerosis lesion andrestenosis. Platelet-derived growth factor BB (PDGF-BB) is the most potentmitogenic factor and chemoattractant, and plays an important role inmigration.Baicalin, an herb-derived flavonoid compound, with a broad spectrum ofbiological activities, including anti-inflammatory, antioxidant andanti-proliferation, shows a stronger cardio-protective effect. In previousstudies, we had showed that baicalin had an inhibitory effect on theproliferation of VSMCs, but little was known about the role of baicalin inregulating VSMC migration. Smooth muscle22alpha (SM22α) is adifferentiation marker of VSMCs, suppressing the phenotypic remodelingfrom contractile to synthetic state. Another study showed that SM22α preventstumor metastasis via inhibiting the expression of MMP-9, a migration-relatedprotein. Based on these findings, we investigated the effect of baicalin onVSMC migration in vitro and in vivo and the role of SM22α in this progress.Methods and results:1The effect of PDGF-BB on VSMC lamellipodia formation andmigrationQuiescent VSMCs were treated with PDGF-BB (20ng/ml and50ng/ml)for different times (0,2,5,10,30and60min). Cells were stained byTRITC-phalloidin, and then the lamellipodia formation of VSMCs wasobserved by laser scanning confocal microscope following PDGF-BBstimulation for5-10min.2Baicalin inhibits PDGF-BB-induced VSMC lamellipodia formationVSMCs were pretreated with baicalin followed by stimulation with PDGF-BB for5min. Baicalin inhibited the PDGF-BB-induced lamellipodiaformation and inhibited PDGF-BB-induced the Ras-ERK axis activation.3Baicalin inhibits the expression of MMP-9in carotid arteries afterballoon injuryImmunohistochemical staining showed that there were a lot of MMP-9positive cells in balloon-injured carotid arteries, and the intimal thickness andMMP-9expression were significantly reduced in injury plus baicalin-treatedgroup compared with the injured alone.4SM22α inhibits PDGF-BB-induced VSMC lamellipodia formationVSMCs were effectively infected with Ad-SM22α. Western blot showedthat overexpression of SM22α inhibited PDGF-BB-induced the Ras-ERK axisactivation. siSM22α was used to silence endogenous SM22α expression inVSMCs. TRITC-phalloidin staining showed that knockdown of SM22αenhanced lamellipodia formation induced by PDGF-BB. Furthermore, theactivation of Ras-ERK axis was increased in siSM22α-transfected cells thanthat in siCon-transfected.5Baicalin inhibits PDGF-BB-induced phosphorylation of SM22αBased on three potential phosphorylation sites in SM22α,immunoprecipitation assay was performed to examine the phosphorylation ofSM22α upon PDGF-BB. The result showed that phosphorylated SM22α wasaccumulated in the short-term PDGF-BB-stimulated VSMCs without affectingits overall expression. Overexpression of SM22α non-phosphorylation mutant,S181A, inhibited PDGF-BB-induced lamellipodia formation and migration,and inhibited PDGF-BB-induced Ras-ERK axis activation. These resultssuggested that SM22α phosphorylation mediated formation of pseudopodiainduced by PDGF-BB. Furthermore, Baicalin might inhibitedPDGF-BB-induced VSMC migration via blockade of SM22α phosphorylation.Conclusions:1Baicalin inhibits PDGF-BB-induced VSMC lamellipodia formation.2SM22α inhibits lamellipodia formation via suppression of Ras-ERK axisactivation. 3Baicalin inhibits VSMC migration and neointimal formation through thesuppression of SM22α phosphorylation and MMP-9expression.