The Impact And Mechanism Of Slit2Protein On Proliferation And Migration Of Rat Thoracic Aortic Vascular Smooth Muscle Cells Stimulated By TNF-α

Slit protein family, a kind of secreted glycoprotein,were originallyidentified in Drosophila by Nusslein-Volhard in1984. Slit in mammals havethree subtypes：Slit1, Slit2and Slit3. Slit1protein is mainly expressed innervous tissues, Slit2and Slit3can also expressed in Lung, kidney, liver,breast, heart and retina. Slit contains four leucine-rich regions, nine epidermalgrowth factor repeats, a laminin G-domain and a C-terminal cysteine-richdomain. Slit in the process of hydrolysis can be cleaved into the N-andC-terminal fragments,the N-terminal fragment are longer than C-terminalfragments isoforms, the N-terminus of Slit can bind cell membrane receptorRobos then working，but C-terminal fragments have no biological activities.It is proved that Slit-Robo signals repel longitudinal axons away fromthe ventral midline and may have a role in the formation of thehypothalamic-pituitary axis; Slit-Robo signals can inhibit CXCL12-inducedand CXCR4-mediated T cell and monocyte chemotaxis in inflammatoryresponse; in tumours from nude mice injected with Slit2expressing cells，expression of antiapoptotic Bcl-xl and the proliferation-associated cell cycleproteins Cdk6and Cyclin D1was reduced and the proliferation rate was alsolower in Slit2expressing hepatocellular carcinoma cells. Slit-Robo can alsoguide migrating cardioblasts and pericardial cells, hinder E-cadherin activityand this is crucial to the formation of the lumen in the Drosophila heart.Recent years, LIU find that Slit2-N inhibits PDGF-Mediated Migration of HASMCs by Suppressing Small GTPase RAC1activation, but had no effecton PDGF-Mediated growth of HASMCs. In the Slit family, Slit2is one of themost studied subtypes. Morlot find that Slit2D4forms a homodimer using theconserved residues on its concave face and can also bind to heparan sulphate.In the end, Slit2D4enhance the combination of Slit2D2area and Robo.Slit2-Robo1signaling has been proved to be repulsive of chemotaxis ofneutrophils，which can inhibit migration of neutrophils, lymphocytes andmacrophages in inflammatory responses. Treatment of ApoE-/-mice with Slit2potently elevated HDL and drastically reduced atherosclerosis，it proved thatSlit-Robo signaling may critically regulate the pathogenesis ofatherosclerosis.TNF-α is an inflammation factor, which promote migration andproliferation of VSMCs and involve in the formation and development ofcoronary atherosclerosis. An experiment has proved that TNF-α upregulatedSlit2mRNA expression in A375. Therefore We have chosen Slit2for thestudy, observe the influence of Slit2on the proliferation and migration ofVSMCs under the effection of the TNF-α. For now, the report about TNF-αand Slit-Robo signal have not been reported, we cultivate VSMCs in vitro,examine the expression of Slit2protein in VSMCs and observe expression ofSlit2protein and Slit2mRNA under different concentration of TNF-α. Toobserve potential effects of Slit2on VSMCs proliferation and migration andexplore its possible mechanism, so as to provide experimental evidence instudying functions of Slit-Robo signaling pathways in coronaryatherosclerosis.Objective:1. To culture VSMCs in rats by tissue explant, examineexpression of Slit2protein and Slit2mRNA in Rat VSMCs.2. To examine expression of Slit2mRNA and secretion of Slit2protein, using the differentconcentration of TNF-α to stimulate VSMCs. Explore potential effects ofdifferent concentration of TNF-α in proliferation and migration of VSMCs.3.To observe the effect of Slit2-Robo signaling pathway on VSMCsproliferation and migration, trying to clarify its possible mechanism.Methods:1. VSMCs were isolated from rats carotid artery by tissueexplant.2. To examine expression of Slit2protein and mRNA in VSMCs byimmunol Fluorence and RT-PCR.3. Rat VSMCs are divided into two groups,normal control group(ordinary culture)and experimental groups(culture with0.01,0.1,1,10,100ng/ml TNF-α respectively). To detect the potential effectsof cell solution in regulating proliferation and migration of VSMCs by CCK-8and transwell experiment; to detect the expression of Slit2mRNA in VSMCsby real time PCR technology and secretion of Slit2protein in cell superate byELISA kit respectively.4. Rat VSMCs are divided into normal controlgroup(ordinary culture) and experimental groups (culture with50,75,100,125,150ng/mlSlit2respectively). To detect potential effects of two groupscell solution in regulating proliferation and migration of VSMCs by CCK-8and transwell experiment.5. Rat VSMCs are divided into normal controlgroup(ordinary culture), postive control group(culture with10ng/mlTNF-α)and experimental groups (culture with10ng/ml TNF-α and50,75,100,125,150ng/mlSlit2respectively). To detect potential effects of two groupscell solution in regulating proliferation and migration of VSMCs by CCK-8and transwell experiment.6. Rat VSMCs are divided into normal controlgroup(ordinary culture); TNF-α group(culture with10ng/ml TNF-α); Slit2group(culture with100ng/ml Slit2) and TNF-α+Slit2group(culture with10ng/ml TNF-α and100ng/ml Slit2). To examine expression of RAC1by Werstern Blot and real time PCR.Results:1. Slit2mRNA expression was assessed by RT-PCR, Slit2immunofluorescence staining showed that VSMCs cytoplasm was positive.2.In proliferative assay, the difference of OD value increasing has statisticalsignificance(P<0.05), the effective was maximum at10ng/ml TNF-α. Inmigration assay, the experimental groups at1,10,100ng/ml TNF-α comparedwith normal control group have statistically significant difference (P<0.05),the effect on migration was most obvious while TNF-α reach10ng/ml, butnumbers of VSMCs at0.01,0.1ng/ml groups have no statistically significantdifference (P>0.05). Five experimental groups compared with normal controlgroup, the difference of grey value increasing has statistical significance(P<0.05), and meanwhile statistical analysis shows that the difference of ODvalue increasing has statistical significance(P<0.05). Expression of Slit2mRNA and secretion of Slit2protein have maximum level at1ng/ml TNF-α.3.Results of Slit2on proliferation and migration of VSMCs showed that thedifference has no statistical significance between experimental groups andnormal control (P>0.05).4. Results of Slit2on VSMCs proliferation andmigration stimulated by TNF-α showed that experimental groups and positivecontrol group compared with normal control group, the difference of ODvalue has statistical significance(P<0.05), but there was no significantdifference between experimental groups and positive control group (P>0.05);the difference of the number of VSMCs migration being statisticallysignificant between groups (P<0.05).5. Detection of RAC1proteins: RAC1mRNA and protein levels in TNF-α group and TNF-α+Slit2group weresignificantly higher than normol control group(P<0.05)，there are significantdifferences between TNF-α group and TNF-α+Slit2group (P<0.01), there was no significant difference between Slit2group and normol control group.Conclusion:1. There are expression and release of Slit2in culturedVSMCs.2. With TNF-α stimulates VSMCs, expression of Slit2mRNAincreases, at the same time, secretion of Slit2protein also increases, Slit2protein secretion has maximum level at1ng/ml TNF-α.3. TNF-α promotedthe proliferation and migration of VSMCs.4.100ng/ml Slit2protein caninhibit migration of VSMCs mediated by10ng/ml TNF-α，whereas it has noeffect on proliferation of VSMCs.5.10ng/mlTNF-α can increased theexpression of RAC1;100ng/ml Slit2can inhibits the expression of RAC1induced by TNFα.