Roles Of Gap Junctions In Hcy And TGF Beta1Mediated Vascular Smooth Muscle Cell Proliferation In The Rats And Its Mechanism

Objective: We explore the roles of gap junctions in homocysteine （Hcy） and transforming growthfactor β₁（TGF-β₁）-mediated vascular smooth muscle cells（VSMC） proliferation in the rats.Methods:（1） The VSMC were got from thoracic aorta and cultured by explanted rat aortic wall tissue,trypsin digestion of cell subculture. The expression of smooth muscle α-actin in VSMC was used as theirverification. The cultured VSMC of third to fifth were used. The VSMC proliferations were detected withdifferent concentrations of Hcy （0,0.01,0.025,0.05,0.1,0.2,0.5mmol/L）and TGF-β₁（0,0.01,0.1,1,10ng/mL）at different time（0,12,24,48,72h）by MTT, so the concentrations and working times of Hcy and TGF-β₁were decided. Then VSMC which were normally growing were divided into eight groups: control group（normal rat VSMC）, Hcy group, TGF-β₁group,18α-GA group, TGF-β₁+18α-GA, Hcy+18α-GAgroup,Hcy+TGF-β₁group and Hcy+TGF-β₁+18α-GA group.（2） MTT and flow cytometry were used toexplore the influence of different treatment factors on the VSMC proliferation.（3） The Cx43and Cx40proteins expression and co-localization in rats VSMC were detected by immunofluorescence.（4） Theeffects of different treatment factors on Cx43, Cx40proteins expression in VSMC were detected byWestern blotting.（5） The molecular dye transfer method （scrape dye transfer method） was applied to detectthe influence of different treatment factors on VSMC gap junction function.（6） Data were reported asM±SD, the single factor analysis of variance was used to compare between groups and statisticalcomparisons were made using Student-Newman-Keulsa（SNK）.All data were analyzed in SPSS13.0. Adifference was considered significant at P<0.05.Results:（1） The expression of Cx43and Cx40proteins in rats VSMC was positive, confocal microscopysupported the co-localization of Cx43and Cx43in the cytoplasm.（2） The results of MTT with differentconcentrations and different working times of Hcy and TGF-β₁were as fellows: the proliferation of VSMCstimulated by Hcy was time dependent, the proliferation of VSMC increased when working time prolonged.Wihle the different concentrations of Hcy had complex effect to the proliferation of VSMC. Comparedwith the control group,0.01mmol/L Hcy group decreased, the proliferation of VSMC of0.025mmol/L Hcygroup increased, the proliferation of VSMC of0.05mmol/L Hcy group slowed down, the proliferation ofVSMC of0.1mmol/L Hcy group was highest. Then the proliferation of VSMC increased slowly at higherconcentration of Hcy（0.2mmol/L、0.5mmol/L）. The proliferation of VSMC in0.1mmol/L group hadsignificant differences with all the other groups at24h. The proliferation of VSMC stimulate by TGF-β₁was time and dose dependent. Compared with the control group, the proliferation of VSMC at theconcentrations of0.01ng/ml and0.1ng/ml TGF-β₁groups had no differences at all times （12,24,48,72h）（P>0.05）,1ng/ml and10ng/ml TGF-β₁groups increased at all times （12,24,48,72h）（P<0.05）.Compared with the1ng/ml TGF-β₁group, the proliferation of VSMC10ng/ml TGF-β₁group increased at24h and48h（P<0.05）.（3） The results of MTT with different treated groups were as fellows: compared withthe control group, the A₅₇₀values of Hcy group and TGF-β₁group increased （P<0.05）, the A₅₇₀value of18α-GA group decreased （P<0.05）. Compared with the Hcy group and TGF-β₁group respectively, the A₅₇₀value of Hcy+18α-GA group and TGF-β₁+18α-GA group significantly decreased respectively（P<0.05）. Compared with Hcy group and TGF-β₁group, the A₅₇₀values of Hcy+TGF-β₁group decreased（P>0.05）.（4） The results of cell cycle were as follows: compared with the control group, the S value ofHcy group and significantly increased （P<0.05）,18α-GA group decreased （P<0.05）; TGF-β₁group had nosignificant differences though it had the trend of higher value （P>0.05）. Compared with the Hcy group andTGF-β₁group respectively, the S value of Hcy+18α-GA group and TGF-β₁+18α-GA group significantlydecreased respectively （P<0.05）.（5） The results of Western blotting were as follows:①The changes ofCx40expression: compared with the control group, the expression of Cx40protein in Hcy group andTGF-β₁group increased （P<0.05）,18α-GA group had no significant differences （P>0.05）. Compared withHcy group and TGF-β₁group respectively, Hcy+18α-GA group and TGF-β+18α-GA group decreasedrespectively（P<0.05）, and Hcy+TGF-β₁group decreased （P<0.05）. Compared with Hcy+TGF-β₁group, theHcy+TGF-β₁+18α-GA decreased （P<0.05）.②The changes of Cx43expression: compared with the controlgroup, the expression of Cx43protein in Hcy group and TGF-β₁group increased （P<0.05）,18α-GA grouphad no significant difference （P>0.05）. Compared with Hcy group and TGF-β₁group respectively,Hcy+18α-GA group and TGF-β₁+18α-GA group decreased respectively（P<0.05）, and Hcy+TGF-β₁groupdecreased （P<0.05）. Compared with Hcy+TGF-β₁group, Hcy+TGF-β₁+18α-GA decreased （P<0.05）.③Thechanges of phosphorylated-and non-phosphorylated-Cx43ratio: Compared with the control group, theratio in Hcy group had no significant differences （P>0.05）,18α-GA group and TGF-β₁group decreased（P<0.05）; compared with TGF-β₁group, TGF-β₁+18α-GA group decreased （P<0.05）.（6） The results of dyetransfer assay scratch marks as follows: compared with the control group, the gap junction functions of Hcygroup enhanced （P<0.05）,18α-GA group significant weakened （P<0.01）, TGF-β₁group had no significantdifference （P>0.05）; Compared with Hcy group and TGF-β₁group respectively, the gap junction functionsof Hcy+18α-GA group and TGF-β₁+18α-GA group significantly decreased respectively （P<0.05）. The gapjunction functions of Hcy+TGF-β₁group decreased （P<0.05） and had no significant difference whencompared with the Hcy group and TGF-β₁group respectively, Compared with the Hcy+TGF-β₁group, thegap junction functions of Hcy+TGF-β₁+18α-GA group decreased （P<0.05）.Conclusions:（1） TGF-β₁, mainly through increasing Cx40and Cx43protein expression in VSMC, andinhibiting phosphorylation of Cx43, thereby contributes to the proliferation of rats VSMC.（2） Hcy canpromote the rats VSMC proliferations, and the mechanism may involve the effect of enhancing gapjunctional intercellular communication caused by up-regulating the expression of Cx43and Cx40proteinsin rats VSMC.（3）It has negative cooperation to the proliferation of rat VSMC mainly throughdown-regulating the expression of Cx40and Cx43protein when Hcy and TGF-β₁work together.