Study Of The Molecular Mechanism Of VSMC Proliferation And Migration Induced By Apelin

Objective: Vascular smooth muscle cells (VSMCs) have differentiatedand proliferating phenotypes, and phenotypic switch between these twophenotypes is an important base of cardiovascular diseases, such ashypertension, atherosclerosis and restenosis. Orphan G protein coupledreceptor APJ and its endogenous ligand apelin in the body have importantphysiological functions. Pleiotropic roles of the apelin/APJ system have beenelucidated in different tissues and organs, including modulation of thecardiovascular system, fluid homeostasis, metabolic pathway and vascularformation. In blood vessels, apelin and APJ expression are spatiotemporallyregulated in endothelial cells (ECs) during angiogenesis. In vitro analysisrevealed that the apelin/APJ system regulates angiogenesis by the induction ofproliferation, migration and cord formation of cultured ECs. Moreover, apelinseems to stabilize cell-cell junctions of ECs. In addition, geneticallyengineered mouse models suggest that apelin/APJ system regulates vascularstabilization and maturation in physiological and pathological angiogenesis.Apelin is secreted as a77amino acid pre-proprotein, an immature peptide,which is cleaved by protease to form C-terminal products, including apelin-13,apelin-17and apelin-36, and apelin-36is the main form. These isoforms havedistinct activities, and the shorter isoform seems to be the more potentactivator for APJ. And apelin-13showed more potent biological activity. Sothis research mainly observed the effect of apelin-13on VSMC proliferationand migration, and investigated its action mechanism.Methods: VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats. In all experiments, only cells of passages3~5were used. Theexpressions of mmp-2, NF-κB, cyclin D1and cyclin E1were examined bywestern blotting. Cell proliferation was examined by a cck-8kit. Wound healing assay was used to detect VSMC migration, and TRITC-phalloidinstaining was used to detect the changing of cell morphology and stress fibers.Results：1Apelin-13promotes VSMC proliferation and migrationVSMCs were treated with0,0.1,0.5and1μM apelin-13, respectively,for24h, then the CCK–8was added for2h, and optical density at450nmwas detected with spectrophotometer. Results showed that apelin-13stimulation might induce VSMC proliferation in a concentration-dependentmanner.Wounding cell migration assay showed that, when VSMCs were treatedwith0.1μM apelin-13for24h, VSMC migration significantly increased asdetermined by Hematoxylin-eosin (HE) staining.2Apelin-13upregulates expression of cyclin D1, cyclin E1, NF-κB andmmp-2Western blot analysis showed that, after VSMCs were incubated withapelin-13for24h at concentrations of0,0.1,0.5and1μM, the expression ofcyclin D1, cyclin E1, NF-κB and mmp-2, compared with the control,markedly increased.To examine the time effect of apelin-13on expression of cyclin D1, NF-κB and mmp-2, VSMCs were incubated with0.1μM apelin-13for0,6,12and24h. Western blot analysis showed that apelin-13promoted theexpression of cyclin D1, NF-κB and mmp-2in time-dependent manner. Theabove results show that apelin-13can promote the proliferation-related geneexpression in time-dependent and in concentration-dependent manner.3Apelin-13inhibits VSMC differentiationTo further test the effect of apelin-13on VSMC differentiation, wedetected the protein level of VSMC differentiation markers SM22α andα-actin and the changes of VSMC cytoskeleton. The results showed that theapelin-13led to decrease in the level of SM22α and α-actin. TRITC-phalloidinstaining indicated that apelin-13stimulation could affect VSMC morphologyand stress fiber organization. VSMC morphology was changed from spindle into polygon, the cytoskeleton became irregular, and myofilamentsignificantly reduced. These results suggest that apelin-13inhibits VSMCdifferentiation.4Akt/ERK signaling pathways mediate VSMC proliferation induced byapelin-13To examine the effect of apelin-13on expression of phospho-Akt (p-Akt)and phospho-ERK (p-ERK), VSMCs were incubated with0.1μM apelin-13for0min to6h. Western blot analysis showed that apelin-13promoted theexpression of phospho-Akt and phospho-ERK in time-dependent manner.p-Akt expression level peaked at30min, and p-ERK expression levelwas in peaked at4h, and subsequently declined, but still higher by6h thanthat of the control group.The influence of Akt inhibitor LY294002on p-Akt level was examined.After pretreated with50μM LY294002for1h, VSMCs were stimulated with0.1μM apelin-13for30min. Western blot results revealed that the expressionof phospho-Akt, and cyclin D1was decreased.The influence of ERK inhibitor PD98059on p-ERK level was alsoexamined. After pretreated with20μM PD98059for1h, VSMCs wereincubated with0.1μM apelin-13for4h. Western blot results revealed that theexpression of phospho-ERK1/2and cyclin D1was decreased.Conclusions：1Apelin-13promotes VSMC proliferation and migration.2Apelin-13promotes VSMC proliferation and migration by inducingexpression of cyclin D1, cyclin E1, NF-ΚB and mmp-2.3Apelin-13inhibits VSMC differentiation.4Akt/ERK signaling pathways mediate VSMC proliferation induced byapelin-13.