Effect And Mechanism Of TGF-β₁ Inducing Human Aortic Vascular Smooth Muscle Cell Phenotype Switch

Background:Thoracic aortic dissection (TAD) results from varieties causes of bursts in thoracic aortic intima,with blood rushing from a break into the middle of intercellar layer and outer layer,and finally forms a false lumen,which is a serious life-threatening aortic disease.This disease appears fatal and has a high mortality,while pathogenesis is still unclear partly.It is known that the morphological changes of TAD are primarily in the media of the vessel wall and vascular smooth muscle cell(VSMC) forms the main structure of the media of aorta.Recently,found that phenotype switch of VSMC will weaken functions of the vessel wall,which will make thoracic aortic wall break easily.Under normal circumstances,contractile VSMC can mainly maintain the vascular tone and homeostasis of the blood vessel wall.However,in TAD,found most VSMCs switch from contractile to synthetic phenotypes.This finding suggests that phenotypic switch may play a important role in the occurrence of TAD,but detailed mechanism of phenotypic switch is not clear.VSMCs in TAD exhibit two different phenotypes,i.e.,a contractile phenotype and a synthetic phenotype.The former has a high level of differentiation,a weak ability of proliferation and migration;its specific marker molecules include smooth muscle a-actin(a-SMA) and smooth muscle 22a(SM22a),etc.While the latter has a low level of differentiation,a stronger proliferation and migration ability;its specific marker molecules include Osteopontin(OPN) and epidermal growth factor Epiregulin,etc.The VSMC which switches to the synthetic phenotype leads to the increased expressions of markers for this phenotype,while as contractile phenotype switches leads to the increased expressions of markers for contractile phenotype.Under normal circumstances,contractile VSMC can maintain the vascular tone and homeostasis of the blood vessel wall.However,different pathological and physiological stimuli may cause the VSMC phenotypic switch,leading to proliferation,migration and synthesis of excessive extracellular matrix.These effects result in pathological and physiological changes to tissues of aortic vessel wall.Recently found that VSMC switches from contractile to synthetic phenotype in the media of vessel wall of TAD,which reveals that phenotypic switch may be an important factor in the occurrence of TAD.Further studies have shown that TGF-β can induce VSMC proliferation and migration,and promote VSMC switch from a resting contractile phenotype to active synthetic phenotype,which involve in vascular remodeling.TGF-β1 is an important factor that leads to the VSMC phenotypic switch. Various signaling pathways are involved in TGF-β1-induced switching.TGF-β functions by activating downstream signaling pathways,which are divided into two categories:1)Smad-dependent classic signaling pathways:TGF-β is usually combined with the TGF-P receptor-Ⅱ,so as to activate TGF-P receptor-I,and then make the Smad2 and Smad3 protein phosphorylation,next form Smad2/3-Smad4 complex, through adjusting the position of transcription factors in the nuclei and activate gene transcription targeting TGF-β,which ultimately affect cell proliferation,growth,development and death;2)Smad-independent signaling pathways,such as PI3K/AKT,p38,and ERK,etc.In recent years,PI3K/AKT and other Smad-independent signalings have received increasing attentions,and those signaling pathways may play a regulatory role independently or in cooperation with Smad-dependent signaling pathways.PI3K is an intracellular signaling protein with catalytic activities and can regulate the level of AKT phosphorylation to initiate the PI3K/AKT signaling pathway.By transducing stimulatory extracellular signals to the nucleus,the PI3K/AKT signaling pathway can trigger a series of biological reactions,including cell proliferation,differentiation and apoptosis and plays an important regulatory role in these processes.Recent studies have shown that the PI3K/AKT signaling pathway is related to the contractile function of vascular smooth muscle,contributing to VSMC dysfunction, vasoconstriction and vascular remodeling,etc.At the same time,it was reported that PI3K/AKT is involved in regulating the VSMC phenotypic switch,acting through an unidentified downstream transcription factor.In recent research abroad,ID2 as a transcriptional regulatory factor plays an important regulatory role in muscle cell growth,proliferation and differentiation.An important feature of the VSMC phenotypic switch is the enhancement of cell migration and proliferation and ID2 is very likely to be an important regulatory factor in this switch.Recent data showed that ID2,ZEB1 and other transcription factors play an important role in the phenotype transformation of smooth muscle cell.Moreover,our previous study (data not shown) found that the expressions of ID2 was significantly up-regulated in the aortic wall of patients with TAD compared to unaffected individuals and the VSMCs of aortic wall in patients with TAD showed clear signs of phenotypic switching.These datas suggest that ID2 is involved in the phenotypic switch.Although the above evidences suggest that ID2 may be involved in VSMC phenotypic switch,its specific role and the connection to PI3K/AKT signaling remain unclear.Through detecting specific marker molecules of each phenotype and analyse proliferation,migration ability and morphology of VSMCs,we can confirm their phenotype and if phenotype switch has happened,and explore downstream signaling pathways of TGF-β1 regulating PI3K/AKT,then clarify the molecular mechanism of VSMC phenotype switch.This result will broaden our horizon in the process of exploring the pathogenesis of TAD,help to determine the operation method and time of TAD,and can provide possible new target in the research of medicine for the prevention and treatment of TAD,even can explore new medicine,and provide theoretical evidence for further gene therapy of patients with TAD.Part One:Objective:To study the effect of transforming growth factor-β1 (TGF-β1) to human aortic vascular smooth muscle cells (HA-VSMCs) proliferations and migrations.Methods:HA-VSMCs were cultured in vitro,then treated with recombinant human TGF-β1 of different concentrations(0、0.05、0.1、0.5、1、2、5、10ng/ml);Cell proliferations of different time(0、6、12、24、36h) were detected via CCK-8 test and cell migrations after 24h were detected via Transwell assay,respectively.Results:When concentrations of TGF-β1 were less than 5ng/ml,as TGF-β1 concentrations increased,levels of HA-VSMCs proliferations gradually increased,while in 10ng/ml,levels of cells proliferations decreased.Different concentrations of TGF-β1(0、0.05、0.1、0.5、1、2、5、10ng/ml) treated cells,as time passed,and levels of cells proliferations increased,which reached a peak value at 24h,then gradually reduced.Among them,levels of cells proliferations were the highest when the concentration of TGF-β1 was 5ng/ml and the time of treatment was 24h(OD value:0.843±0.016) (P<0.01).When concentrations of TGF-β1 were less than 5ng/ml or 5ng/ml(0、0.05、0.1、0.5、1、2、5ng/ml) and the time of treatment was 24h,as TGF-β1 concentrations increased,levels of HA-VSMCs migrations gradually increased and reached a peak value when the concentration of TGF-β1 was 5ng/ml (migrations numbers:84.667±0.577);while in 10ng/ml,levels of cells migrations decreased(migrations numbers:69.667±1.528) (P<0.01).Conclusion:5ng/ml of TGF-β1 can stimulate more human aortic vascular smooth muscle cells proliferations and migrations after 24h.Part Two:Test One:Objective:To study the effect of PI3K/AKT signaling pathway in TGF-β1 inducing human aortic vascular smooth muscle cells (HA-VSMCs) phenotype switch.Methods:HA-VSMCs were divided into five groups:①Blank control group; ②TGF-β1 group;③TGF-β1 inhibitor group;④AKT inhibitor group;⑤Combined inhibitors group;Cell proliferations and migrations of each group were detected via CCK-8 and Transwell assay,respectively;Cytoskeleton shape of groups were observed via coomassie brilliant blue staining;Western Blot assay were used to detect the expression of Phosphoinositide-3-kinase(PI3K),Protein kinase B(AKT),Phosphorylat-ed Phosphoinositide-3-kinase(p-PI3K),Phosphorylated Protein kinase B(p-AKT),DNA binding inhibitory factor 2(ID2),Smooth muscle 22a(SM22a),a-smooth muscle actin(a-SMA) and Osteopontin(OPN),respectively. Quantitative Real-time PCR(qRT-PCR) were used to detect the expression of PI3K,AKT,ID2,SM22a,a-SMA and OPN,respectively.Results.Cell proliferations and migrations of TGF-β1 group were increased compared with Blank control group,while Combined inhibitors group were decreased compared with Blank control group obviously.Coomassie brilliant blue staining found that cells in TGF-β1 group had hypertrophic apprearance and "hill and valley" growth;other Groups had spindle elongated morphology,which was the most obvious in Combined inhibitors group.TGF-β1 promoted the expressions of PI3K,p-PI3K,AKT,p-AKT,ID2 and OPN protein and suppressed the expressions of a-SMA and SM22a protein;the opposite results were observed for Combined inhibitors group.After the stimulation of TGF-β1 signaling, the mRNA levels of PI3K,AKT,ID2,and OPN were the highest, while the mRNA levels of a-SMA and SM22a were the lowest;the opposite results were found in the same group above.Conclusion:PI3K/AKT/ID2 signaling pathway is involved in TGF-β1-mediated human aortic VSMC phenotypic switching,that is from a contractile to synthetic phenotype,and Combined inhibitors(SB525334+MK2206-2HCL) was more effective in inhibiting the phenotypic switch than a single inhibitor(SB525334 or MK2206-2HCL).The Combined inhibitors experiments may provide new avenues for the prevention and treatment of thoracic aortic dissection(TAD) that are based on the pathological effects of phenotypic switching.Test Two:ObjectiverTo study the effect of DNA binding inhibitory factor 2 (ID2) in human aortic vascular smooth muscle cells (HA-VSMCs) phenotype switch.Methods:Design and compound siRNA targeting ID2 and transfect human aortic smooth muscle cells (HA-VSMCs),Quantitative Real-time PCR(qRT-PCR) select a siRNA which has the most efficient silence ability.After biosynthesis of ID2 gene sequences, insert it into pIRES2-ZsGreenl plasmid vector, build the ID2 recombinant overexpression plasmid vector pIRES2-ZsGreenl-ID2 and identify it. HA-VSMC is divided into five groups:blank control group、empty plasmid control group、ID2 overexpression group、siRNA control group、ID2 siRNA group.qRT-PCR and Western Blot assay were used to detect the expression of DNA binding inhibitory factor 2(ID2)、smooth muscle 22a(SM22a)、α-smooth muscle actin(a-SMA) and osteopontin(OPN),respectively.Results:Successfully designed and compounded the siRNA targeting ID2 with higher silence ability, and constructed overexpression plasmid pIRES2-ZsGreenl-ID2.Compared with blank control group、empty plasmid control group and siRNA control group;ID2 overexpression group ID2、OPN mRNA and protein expression were significantly higher[ID2 mRNA:2.637±0.053;OPN mRNA:1.692±0.086;ID2:0.580±0.017;OPN:0.460±0.015],SM22、α-SMA mRNA and protein expression were significantly lower[SM22a mRNA:0.367±0.048;a-SMA mRNA:0.428±0.031;SM22a:0.100±0.004;a-SMA:0.063±0.010](P<0.01);While ID2 siRNA group ID2、OPN mRNA and protein expression were significantly lower[ID2 mRNA:0.322±0.046;OPN mRNA:0.250±0.056;ID2:0.061±0.007;OPN:0.066±0.006], SM22、α-SMA mRNA and protein expression were significantly higher[SM22amRNA:1.827±0.041;a-SMA RNA:1.653±0.088;SM22a:0.547±0.009; a-SMA:0.671±0.051](P<0.01).Conclusion:Overexpression of ID2 gene can induce HA-VSMCs to switch from a contractile to synthetic phenotype,while silence the gene can inhibit this process.ID2 as a direct signaling involves in the regulation of HA-VSMCs phenotype switch.