The Role Of Lamin A In Pathological Vascular Remodeling

Nucleus in a eukaryotic cell is the main control center for various life activities of the cell.It is not only a place for storage,replication and transcription of genetic material,but also can regulate abundant cell functions such as metabolism,growth,differentiation,and et al.Nuclear skeleton protein plays a decisive role in supporting the nucleus as the inner skeleton of the nucleus.The previous results based on proteomics research in our laboratory revealed that the nuclear skeleton protein lamin A and is a mechanically sensitive molecule.Different magnitudes of cyclic strain induce the expression of lamin A which participates in the dysfunction of vascular smooth muscle cells(VSMCs).However,the molecular mechanism by which cyclic strain regulates lamin A expression and the role of altered lamin A in vascular remodeling are still unclear.Here we explored the changes of the nuclear skeleton protein,lamin A,in VSMCs during vascular remodeling induced by hyper-cyclic strain of hypertension and intimal injury.Firstly,using an abdominal aorta coarctation hypertensive animal model,frozen section-Hematoxylin-Eosin(HE)staining revealed that compared with the sham operation control,thoracic aorta wall was significantly thickened in hypertensive rats,which suggested the occurring of vascular remodeling.Cleved-Caspase 3 ELISA showed that the apoptosis of aortic VSMCs in hypertensive group was significantly increased,while the expression of lamin A and and its alternative spliceosome lamin C,were decreased.Using FX^TM5000 strain system,5%cyclic strain(mimics physical strain under normotension)and 15%cyclic strain(mimics pathological strain under hypertension)were subjected to cultured VSMCs in vitro.The results showed that compared with the 5%group,15%cyclic strain significantly reduced the expression of the nuclear skeleton protein lamin A and lamin C and promoted the apoptosis of VSMCs.When transfected with si RNA targetting lmna,the m RNA of lamin A and lamin C,the expression of lamin A and lamin C in VSMCs was significantly reduced,while apoptosis was significantly increased.In order to study the underlying molecular mechanism,we first detected the expression of lmna by q PCR.The results showed that compared with the 5%group,15%cyclic strain did not change the expression of lmna,suggested that the change in lamin A and lamin C expression may not occurred at transcriptional level;we then investigated the degradation of proteins regulated by autophagy,and the results showed that although chloroquine(CQ)and Bafilomycin A1(Baf)effectively blocked cell autophagy,lamin A and lamin C expression was not changed significantly;furthermore,the role of micro RNAs(mi Rs)in lamin A and lamin C expression was demonstrated.Bioinformatics analysis showed that there were 2 potential binding sites on 3'untranslated regions(3'UTR)of lmna with mi R-124-3p.The binding ability of mi R-124-3p to these 2 sites and the repression of lmna translation were verified using dual luciferase reporter.The results showed that compared with negative control(NC)group,mi R-124-3p mimics effectively reduced the expression of luciferase,indicating that mi R-124-3p binded to lmna 3'UTR and inhibited its translation;subsequently,respective single mutation or a double mutation were co-transfected,and the results showed that compared with the wild type control,all mutations revesed the effect of mi R-124-3p,indicating that both sites on lmna 3'UTR contributed to the binding of mi R-124-3p and were necessary to inhibit lamin A and lamin C expression.Under static conditions,mi R-124-3p inhibitor significantly increased the expression of lamin A and lamin C,while mi R-124-3p mimics significantly decreased lamin A and lamin C.RT-PCR results showed that compared to 5%physiological cyclic strain,15%cyclic strain significantly up-regulated mi R-124-3p expression.Then,lmna-specific interference fragments were used to inhibit lamin A and lamin C expression in VSMCs,and Protein/DNA array was used to detect the activity of transcription factors.Ingenuity Pathway Analysis(IPA)and cluster analysis were used to screen the transcription factors whose activity was regulated by reduced lamin A and lamin C expression,and to obtain the potential transcription factors related to apoptosis.In addition to the effects of mechanical factors,many cardiovascular diseases or interventional procedures will damage endothelial cells(ECs)lined inside the blood vessels,and expose the collagen layer under the ECs.It had been reported that the exposure of collagen activates platelets which subsequently release a large number of platelet-derived microvesicles(PMVs).Whether the contaction of PMVs with VSMCs affect the expression or post-translational modifications of nuclear skeleton proteins,and modulate VSMC functions have not been reported.Using rat carotid artery intimal injury model,transmission electron microscopy showed that a large number of microvesicles were enriched in the intimal layer of the vascular injury.After 2 weeks'injury,VSMCs were remarkably proliferated and the nuclei of the proliferative VSMCs were significantly deformed.In vitro,PMVs and VSMCs adhesion was observed with a fluorescence confocal microscope.The PMVs obviously adhered to the membranes of VSMCs.After 24 hours of stimulation,PMVs induced the deformation of VSMC nucleus which was similar to that in vivo,indicating that PMVs can cause abnormalities in certain nuclear skeleton proteins.RT-PCR and immunoblot were used to analyze the transcription and expression of different subtypes of lamin A.It was found that among different subtypes,i.e.,laimn A,lamin C,progerin,pre-lamin A,lamin B1,and lamin B2,onlypre-lamin A,one immature subtype of lamin A that carrys a farnesyl modification,was abnormally accumulated.In summary,pathological cyclic strain and PMVs induce the abnormal expressions of lamin A and its spliceosome lamin C and abnormal farnesyl modification of lamin A,which play important roles in the regulation of VSMC functions.Under hypertensive cyclic strain conditions,mechanical factors increase the expression of mi R-124-3p in VSMCs,and subsequently inhibit the protein expression of lamin A and lamin C,which then induce apoptosis of VSMCs.Whereas under intimal injury conditions,activated platelets release PMVs and subsequently cause the accumulation of pre-lamin A in VSMCs,which induces deformation in the nucleus of VSMCs.The nuclear skeleton protein and the related molecules suggested in this study may provide potential targets for translational medicine such as diagnosis,clinical treatment and efficacy evaluation of vascular pathological remodeling during different cardiovascular diseases.