The Role Of TWIST1 In Smooth Muscle Cell Phenotypic Transition And Underlying Mechanism

Research BackgroundThe vascular smooth muscle cell(vascular smooth muscle cell,VSMC)is an important component of the vascular wall,which performs contraction and extracellular matrix synthesis.Adult vascular smooth muscle cells are quiescent,expressing a repertoire of contractile proteins,many of which are also commonly used as differentiation markers.However,different from terminally differentiated cardiomyocytes or skeletal muscle cells,VSMCs retain remarkable plasticity,which allows them to respond to various microenvironmental stimuli accordingly.To adapt to changes in local environmental cues such as vascular injury,differentiated VSMCs can undergo comprehensive structural,functional and molecular changes,rapidly and reversibly switching from quiescent and contractive state to migrative,proliferative or synthetized phenotype,which is called phenotypic transition.The plasticity is critical for vascular repair and maintaining hemodynamic stability while aberrant phenotypic plasticity can also lead to irreversible vascular structural and functional disorders.The abnormal environmental stimulation or disturbed signal transduction pathway may continuously activate proliferation,extracellular matrix synthesis or pro-inflammatory cytokines production,which plays a key role in pathogenesis and/or progression of multiple vascular diseasesTWIST1 is a crucial transcription factors for cell differentiation,which has been demonstrated to involve in vascular diseases in human studies and animal experiments.Our previous study also confirmed that specific knockout of TWIST1 in smooth muscle cells could prevent pulmonary vascular remodeling,but the mechanism is still obscure.Based on those background,we aimed to explore whether TWIST1 is involved in phenotypic transition of VSMCs and vascular remodeling,thus promoting the occurrence and development of vascular diseases,in order to provide possible therapy target for vascular diseases.Research Methods1.PDGF-BB induced phenotypic transition of vascular smooth muscle cells in vitro.The human aortic smooth muscle cells were induced to dedifferentiat by culturing with platelet-derived growth factor-BB(platelet derived growth factor-BB,PDGF-BB).The expression of TWIST1 was then detected by cellular immunofluorescence and western blot.2.To verify the relationship between TWIST1 and phenotypic transition of smooth muscle cells in vitro.Firstly,the cell models of TWIST1 gene silencing and overexpression were established by transfection of VSMCs with TWIST1 si RNA virus and TWIST1 c DNA virus respectively.Secondly,the expression of VSMCs differentiation marker proteins was examined by western blot and cell morphology and cytoskeleton were observed by cell immunofluorescence and F-actin labeling in TWIST1 silencing and overexpression VSMCs and their corresponding control cells.In addition,harmine,a TWIST1 inhibitor was also used to treat VSMCs and then the expression of cell differentiation markers was tested.3.To verify the relationship between TWIST1 and phenotypic transition of smooth muscle cells in the rat carotid artery injury model.In the experimental group,the acute carotid artery injury was caused by balloon embolectomy catheter.In the control group,the incision was made at the same position of the carotid artery without damaging the intima.After the model was established,the mice were sacrificed at pre-designed time point,and the injured carotid artery tissues were prepared for tissue protein extraction or pathological examination or stored at-80?.Immunofluorescence and tissue protein western blot were used to compare the expression of TWIST1 or differentiated markers in vascular wall and smooth muscle cells between the control group and the experimental group.4.To explore the mechanism of TWIST1 affecting the phenotypic transition of VSMCs.(1)Bioinformatics search was performed to find out whether there is a direct interaction between TWIST1 and SMA promoter and whether it directly regulates the transcription of differentiation marker protein smooth muscle ?-actin(smooth muscle ?-actin,SMA).(2)In the cultured VSMCs,RT-q PCR and western blot were performed to identify the relationship between TWIST1 and pro-differentiation micro RNA cluster,micro RNA143/145 and a crucial protein for maintaining contractile phenotype,p68.(3)To explore the mechanism how TWIST1 regulate p68 expression.Co-immunoprecipitation assay was firstly used to explore the protein-protein interaction between TWIST1 and p68 in human aortic smooth muscle cells.Secondly,proteasome inhibitor was used to treat VSMCs to test whether the expression of p68 was affected by TWIST1 through TWIST1 proteasome activity.Finally,the VSMCs transfected with TWIST1 si RNA virus were re-transfected with p68 si RNA virus or control GFP si RNA virus.After that,the expressions of micro RNA-143/145 and differentiation markers were examined and the cell morphology and cell skeletal were detected.Research Results1.PDGF-BB-driven VSMCs phenotypic transition experiment in vitroThe cultured vascular smooth muscle cells underwent phenotypic transition after PDGF-BB stimulation,characterized by decreased expression of differentiation marker protein and morphological change.Meanwhile,up-regulation of TWIST1 expression was also detected.Furthermore,the upregulation of TWIST1 magnified with the increase of stimulation time and PDGF-BB concentration.2.To verify he relationship between TWIST1 and phenotypic transition of smooth muscle cells in vitro.In VSMCs treated with TWIST1 inhibitor or TWIST1 gene silencing,the expression of differentiation marker protein including SMA,smooth muscle myosin heavy chain(SMMHC),troponin(calponin)and transgelin(Tagln)increased.Meanwhile,the cells showed contractile spindle shape.In VSMCs with TWIST1 gene overexpression,the expression of differentiation marker protein decreased and the spindle shape changed into more round.3.The rat carotid artery injury vascular remodeling modelCompared with the control group,the injured vascular wall was thickened.Immunofluorescence showed that the media layer where was mainly VSMCs was thickened,the intracellular SMA expression decreased,and the expression of TWIST1 increased in the thickened intima.4.The mechanism of TWIST1 on phenotypic transition of VSMCs(1)Bioinformatics search of Cistrome DB database indicates that TWIST1 is not a transcription factor of SMA and does not directly regulate the its transcription.(2)TWIST1 was negatively correlated with micro RNA-143/145 and p68,that is,in TWIST1 overexpression model,the transcription level of micro RNA-143/145 and p68 decreased,and the expression of p68 protein decreased.In TWIST1 silencing model,the transcription level of micro RNA-143/145 and p68 increased and the expression of p68 protein increased,the same as the effect of TWIST1 inhibitor on VSMCs.(3)Bioinformatics search of Cistrome DB database suggests that TWIST1 does not directly regulate the transcription of p68 as its transcription factor.Co-immunoprecipitation assay in vascular smooth muscle cells showed that TWIST1 could bind to p68.After treated with proteasome inhibitor MG132,the expression of p68 increased in VSMCs with TWIST1 overexpression and the control VSMCs.Moreover,the inhibitor could almost reverse the down-regulation effect of TWIST1 on p68 in TWIST1 overexpression VSMCs,indicating that TWIST1 can down-regulate p68 through its proteasome activity.(4)Compared with TWIST1 silenced VSMCs transfected with GFP si RNA virus,p68 si RNA virus could partially reverse the effect of TWIST1 gene silencing on VSMCs.It inhibited the VSMC differentiation marker protein,prevented morphological changes and reduced the biogenesis of micro RNA143/145.Conclusion1.PDGF-BB can induce phenotypic transition of in cultured VSMCs and the TWIST1 is up-regulated in this process.2.In cultured VSMCs,TWIST1 inhibited the expression of the VSMC differentiation marker protein,promoted the change of contractile morphology and promoted differentiation.The expression of TWIST1 was up-regulated in the process of vascular medial layer thickening and phenotypic transition of smooth muscle cells induced by acute carotid artery injury in rats.3.TWIST1 has no direct transcriptional regulation on SMA or p68 but induce phenotypic transition of the VSMCs through downregulation p68 and micro RNA-143/145.TWIST1 inhibits the expression of p68 and micro RNA-143/145 by down-regulating p68 transcription and accelerate p68 proteasome-dependent degradation.