| Background and objective:Vascular smooth muscle cells (VSMCs)proliferative and migration disorders are the most common causes of severecardiovascular diseases, a common molecular mechanism remains elusive.The primary function of adult arterial SMCs is to contract. However, duringvascular disease VSMCs can be called on to perform noncontractilefunctions. In response to vascular stress, VSMCs specialized to contract canbe induced to lose their contractile function and acquire synthetic functions,including replication, migration, and elaboration and degradation ofextracellular matix proteins. VSMCs form an intercellular layer in arteriesby changing from a differentiated, contractile phenotype to adedifferentiated, migratory, and proliferative phenotype. This conceptforms an important hypothesis of the response to injury mechanism ofatherosclerosis,restonsis and other vascular diseases. The human cellular repressorof E1A-stimulated genes was reportedoriginally as a secreted glycoprotein, which contributes to thetranscriptional control of cell growth and differentiation. CREG inhibitscell growth depending on the mannose-6-phosphate/insulin-like growthfactor II receptor (M6P/IGF2R). Recently, a uniquely responsiveVSMCs-HITASY was successfully cloned in our lab from a single humanartery that is capable of reversible converting between a noncontractile statewith migratory, proliferative, and synthetic properties and awell-differentiated state that contracts in response to vasomotor agonists.Furthermore, overexpressing CREG was cloned from HITASY withdifferentiated state using PCR-differentiated display technology. It washypothesized that CREG expression would regulate smooth muscle cellsdifferentiation. Foregoing studies suggest that CREG acts as atranscriptional factor and interacts with serum response factor to promoteVSMCs differentiation through enhancing SM α-actin promoter activity. Inthe model of vascular restenosis established by balloon injury of rat carotidarteries we also observed that CREG participates in regulation of theproliferation of VSMCs. In this study, our aim was to understand the roleand the possible mechanism of CREG on differentiation,proliferation andmigration of SMCs-HITASY. Methods:(1)To study the effect of the cellular repressor of CREG ondifferentiation,proliferation and migration of SMCs -HITASY, the fulllength human sense and antisepses-CREG cDNA were cloned to retroviralvectors, pLNCX2 and pLXSN. (2)HITASY was transfected with retrovirusvectors pLNCX2(+)/CREG,pLXSN(-)/CREG in vitro. Stably infection ofHITASY cells was obtained by G418 selection. Expression and location ofCREG was detected in differential phenotypes VSMCs. The location ofCREG in HITASY cells was observed by immunoflourescence assay.RT-PCR and Western blot analyzed the changes in VSMCs aftertransfection. (3) Western blot and immunoflourescence analysis wereused to investigate the expression of CREG and SM α-actin in HITASY. (4)A 5-bromo-2'-deoxyuridine (BrdU) labeling method was used to determinethe rate of SMC proliferation in the neointimal cells. (5) The migration ofHITASY was showed by scrape-wounding and time-lapse analysis.Moreover, the secretion of MMPs in HITASY was detected by Westernblot. Gelatin SDS-PAGE zymography analysis was used to reveal theactivities of MMPs in HITASY. The expression of MAPK was detected byWestern blot analysis. Result:(1) The full length human sense and antisense-CREG vectorwas constructed as pLNCX2(+)/CREG and pLXSN(-)/CREG. After DNAsequencing, BLAST analysis revealed that the nucleotide sequencesmatched with the published sequence in GenBank database completely. (2)Western blot and immunoflourescence analysis showed that the expressionof CREG and SMα-actin was increased in HITASY after infection withpLNCX2( + )/CREG and decreased in HITASY infection withpLXSN(-)/CREG. (3) Measurement of cell counter analysis,BrdU labelingand Western blot analysis showed that over-expression of CREG inhibitedVSMCs proliferation through ERK1/2. (4) The migration of HITASY cellsinfected with pLNCX2...
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