Objective The apoptosis of vascular smooth muscle cells(VSMCs)influenced by abnormal cyclic stretch is crucial for vascular remodeling during hypertension.We explored that the causes of mechano-responsive lamin A/C ch...Objective The apoptosis of vascular smooth muscle cells(VSMCs)influenced by abnormal cyclic stretch is crucial for vascular remodeling during hypertension.We explored that the causes of mechano-responsive lamin A/C changingin aonormai cyclic stretcn and its roles in VSMC apoptosis.Methods and results Our previous vascular proteomics study revealed that LaminA/C is mechano-sensitive molecule.When VSMCs are subjected to cyclic stretch,the expression of LaminA/C is significantly changed which participates dysfunctions of VSMCs during hypertension.However,the molecular mechanism involved in regulation of LaminA/C expression and the role of LaminA/C in the VSMC apoptosis during cyclic stretch application are still unclear.In the present study,VSMCs were subjected to different amplitudes of cyclic steetch in vitro:5%cyclic stretch(physiological strain)or 15%cyclic stretch(pathological strain).The expression of 2 different selective cleavage isomers of LaminA/C,i.e.LaminA and LaminC,and the apoptosis of VSMCs were detected.The results showed that compared with 5%group,15%cyclic stretch significantly decreased the expression of LaminA and LaminC,and promoted the apoptosis of VSMCs.Using specific small interfering RNA(siRNA)transfection which targets on LMNA the encoding gene of LaminA/C,the expression of LaminA and LaminC in VSMCs was significantly decreased,and the apoptosis was significantly increased.In order to study the molecular mechanism involved in cyclic stretch regulating the expression of LaminA/C,we focused on the microRNA(miR).Bioinformatics analysis showed that the 3’untranslated region(3’UTR)of LMNA has two potential binding sites to miR-124-3p.Double luciferase reported system revealed that both sites have binding abilities to miR-124-3p.Under static condition,miR-124-3p inhibitor significantly up-regulated the expression levels of LaminA and LaminC,while the miR-124-3p mimics significantly down-regulated them.RT-PCR results showed that 15%cyclic stretch significantly up-regulated the expression of miR-124-3p compared with 5%cyclic stretch.Furthermore,in order to study the role of changeed LaminA/C in VSMC apoptosis,LMNA-specific siRNA was transfected to repress the expression of LaminA/C in VSMCs,and Protein/DNA microarray was used to detecte the activity of transcription factors.The transcription factors whose activity were changed significantly(increase or decrease more than 2 times)were analyzed by cluster analysis and ingenurity pathway analysis(IPA).Six transcription factors associated with apoptosis were screened,in which TP53 was activated by the specific siRNA transfection and the other 5 were inavtived,including TP53,CREB1,MYC,STAT1/5/6 and JUN.Using abdominal aorta coarctation hypertensive model,the change of miR-124-3p in VSMCs was explored in vivo.A marked increase of miR-124-3p in thoracic aorta was revealed compared with the sham-operated controls,and in situ FISH revealed that this increase was mainly in the VSMCs.Conclusions The present study suggest that abnormally increased cyclic stretch(15%)up-regulates the expression of miR-124-3p in VSMCs,which subsequently targets on the 3’UTR of LMNA and decreases the expression of nuclear envelope protein LaminA/C;the repressed LaminA/C may play an important role in the apoptosis of VSMCs by regulating the activity of virious transcription factors,such as TP53,CREB1,MYC,STAT1/5/6 and JUN.The present study may provide a new insight into understanding the molecular mechanisms of vascular remodeling.展开更多
Vascular remodeling is the essential pathogenic process of various cardiovascular disorders,including hypertension,atherosclerosis,stroke,and restenosis after vein graft.The main characterization of vascular remodelin...Vascular remodeling is the essential pathogenic process of various cardiovascular disorders,including hypertension,atherosclerosis,stroke,and restenosis after vein graft.The main characterization of vascular remodeling is abnormal variations of vascular cell phenotype,morphological structure and functions such as migration,hypertrophy,proliferation and apoptosis.Numerous researches revealed that mechanical stress,including shear stress and cyclic stretch,participates in physiological vascular homeostasis,or pathophysiological vascular remodeling.The understanding of mechanobiological mechanism in vascular remodeling will play a unique role in understanding human physiology and disease,and will generate important theoretical and clinical significance [2].Non-coding RNAs are newly recognized RNAs which cannot be translated into proteins but are involved in epigenetic modification of gene regulation.The studies revealed that non-coding RNAs,such as microRNAs(miRNAs)and long noncoding RNAs(long ncRNAs,IncRNA),as well as small interfering RNAs(siRNAs),piwi-interacting RNAs(piRNAs),small nucleolar RNAs(snoRNAs),play essential roles in the regulation of various processes,such as metabolism,development,cell proliferation,cell apoptosis,cell differentiation,oncogenesis and vascular homeostasis[5].However,the roles of non-coding RNAs in the cardiovascular system under mechanical stresses are still not clarified.Our recent researches detected the mechanical regulation of IncRNAs and miRNAs in vascular remodeling.LncRNAs are non-protein-coding transcripts that are longer than 200 nucleotides(nt),which is an arbitrary cut-off value that distinguishes these transcripts from other small RNAs.Unlike the well-established mechanism of microRNA action,the functional mode of IncRNAs is not fully understood.Increasing evidence shows that IncRNAs modulate gene expression via a multilevel-regulated pathway.Given their large number and complicated functional modes,lncRNAs are emerging as important regulators of a variety of cellular responses,developmental processes and diseases.Using a gene microarray,we screened the differences in the IncRNAs and mRNAs between spontaneously hypertensive rats(SHR)and Wistar Kyoto rats(WKY).The results showed that 68 IncRNAs and 255 mRNAs were up-regulated in the aorta of SHR,while 167 IncRNAs and 272 mRNAs were down-regulated.Expressions of the screened IncRNAs,including XR007793,were validated by real-time PCR.A co-expression network was composed,and gene function was analysed using Ingenuity Pathway Analysis.In vitro,vascular smooth muscle cells(VSMCs)were subjected to cyclic stretch at a magnitude of 5%(physiological normotensive cyclic stretch)or 15%(pathological hypertensive cyclic stretch)by Flexercell-5000TM.15%-cyclic-stretch increased XR007793 expression.XR007793 knockdown attenuated VSMC proliferation and migration and inhibited co-expressed genes such as signal transducers and activators of transcription 2(stat2),LIM domain only 2(lmo2)and interferon regulatory factor 7(irf7)[4].Illuminating the role of IncRNAs in vascular remodeling induced by hyper mechanical stretch may provide deeper insight into the mechanobiological mechanism underlying hypertension,and contribute to identifying potential targets for hypertension therapy.miRNAs are endogenous,non-coding,single-stranded RNAs of 18-22 nucleotides that constitute a novel class of gene regulators.miRNAs bind to their target genes within their 3’-untranslated regions(3’-UTRs),leading to direct degradation of mRNA or translational repression by a complete,i.e.in plants,or incomplete,i.e.in animals,complement respectively.Our resent works revealed several important mechano-responsive miRNA and their potential effects in vascular remodeling.Forexample,miRNA-33 is regulated by cyclic stretch in the grafted vessels,which targets to BMP3 and subsequent modulates smad signaling pathway.The miRNA-33-BMP3-smad pathway protects against venous VSMC proliferation in response to arterial cyclic stretch.Therefore,miRNA-33 may be a potential therapeutic target in autologous vein grafted surgery,and locally overexpression of miR-33 may attenuates neointimal hyperplasia of grafted human saphenous vein [3].The unpublished data revealed that 15%cyclic stretch also significantly elevated the expression of miRNA-124-3p which bound to the 3’UTR of Lmna mRNA,and then negatively regulated protein expression of lamin A/C which is the important skeletal proteins in nucleus.In addition to primary intracellular locations of miRNAs,our recent study showed that miRNAs can be secreted and protected extracellularly via inclusion into membrane-derived vesicles including microparticles.Microparticles are extracellular vesicles ranging from 0.1 to 1μm in size and have been shown to deliver various bioactive molecules,i.e.,chemokines,enzymes and miRNAs,to recipient cells.Increasing evidence shows that microparticles play a pivotal role in many pathological processes,such as cancer,inflammatory diseases and cardiovascular disease.Our present study showed that platelet-derived microparticles(PMPs),which are released by active platelets,are important vehicles for communication and play crucial roles in inducing abnormal EC proliferation in hypertension.In briefly,EC proliferation was increased in renal hypertensive rats established by abdominal aortic coarctation compared to control rats and that elevated thrombin in plasma promoted platelet activation,which may induce the release of PMPs.miRNA array and qPCR revealed a higher level of miRNA-142-3p in platelets and PMPs.In vitro,PMPs delivered miRNA-142-3p into ECs and enhanced EC proliferation via Bcl-2-associated transcription factor 1(BCLAF1)and its downstream genes.These results indicated that PMPs deliver miRNA-142-3p from activated platelets into ECs and that miRNA-142-3p may play important roles in EC dysfunction under hypertensive conditions and might be a novel therapeutic target for maintaining EC homeostasis in hypertension[1].These results provide possible mechanisms by which non-coding RNAs regulate cellular functions under different mechanical stresses,and suggest a novel potential therapeutic approach for vascular remodeling.The further studies on noncoding RNAs may provide new insight into understanding the mechanism of vascular remodeling in different various cardiovascular disorders,and may provide novel targets for the maintenance of vascular homeostasis.展开更多
基金supported by grants from the National Natural Science Foundation of China( 11572199 and 11625209)
文摘Objective The apoptosis of vascular smooth muscle cells(VSMCs)influenced by abnormal cyclic stretch is crucial for vascular remodeling during hypertension.We explored that the causes of mechano-responsive lamin A/C changingin aonormai cyclic stretcn and its roles in VSMC apoptosis.Methods and results Our previous vascular proteomics study revealed that LaminA/C is mechano-sensitive molecule.When VSMCs are subjected to cyclic stretch,the expression of LaminA/C is significantly changed which participates dysfunctions of VSMCs during hypertension.However,the molecular mechanism involved in regulation of LaminA/C expression and the role of LaminA/C in the VSMC apoptosis during cyclic stretch application are still unclear.In the present study,VSMCs were subjected to different amplitudes of cyclic steetch in vitro:5%cyclic stretch(physiological strain)or 15%cyclic stretch(pathological strain).The expression of 2 different selective cleavage isomers of LaminA/C,i.e.LaminA and LaminC,and the apoptosis of VSMCs were detected.The results showed that compared with 5%group,15%cyclic stretch significantly decreased the expression of LaminA and LaminC,and promoted the apoptosis of VSMCs.Using specific small interfering RNA(siRNA)transfection which targets on LMNA the encoding gene of LaminA/C,the expression of LaminA and LaminC in VSMCs was significantly decreased,and the apoptosis was significantly increased.In order to study the molecular mechanism involved in cyclic stretch regulating the expression of LaminA/C,we focused on the microRNA(miR).Bioinformatics analysis showed that the 3’untranslated region(3’UTR)of LMNA has two potential binding sites to miR-124-3p.Double luciferase reported system revealed that both sites have binding abilities to miR-124-3p.Under static condition,miR-124-3p inhibitor significantly up-regulated the expression levels of LaminA and LaminC,while the miR-124-3p mimics significantly down-regulated them.RT-PCR results showed that 15%cyclic stretch significantly up-regulated the expression of miR-124-3p compared with 5%cyclic stretch.Furthermore,in order to study the role of changeed LaminA/C in VSMC apoptosis,LMNA-specific siRNA was transfected to repress the expression of LaminA/C in VSMCs,and Protein/DNA microarray was used to detecte the activity of transcription factors.The transcription factors whose activity were changed significantly(increase or decrease more than 2 times)were analyzed by cluster analysis and ingenurity pathway analysis(IPA).Six transcription factors associated with apoptosis were screened,in which TP53 was activated by the specific siRNA transfection and the other 5 were inavtived,including TP53,CREB1,MYC,STAT1/5/6 and JUN.Using abdominal aorta coarctation hypertensive model,the change of miR-124-3p in VSMCs was explored in vivo.A marked increase of miR-124-3p in thoracic aorta was revealed compared with the sham-operated controls,and in situ FISH revealed that this increase was mainly in the VSMCs.Conclusions The present study suggest that abnormally increased cyclic stretch(15%)up-regulates the expression of miR-124-3p in VSMCs,which subsequently targets on the 3’UTR of LMNA and decreases the expression of nuclear envelope protein LaminA/C;the repressed LaminA/C may play an important role in the apoptosis of VSMCs by regulating the activity of virious transcription factors,such as TP53,CREB1,MYC,STAT1/5/6 and JUN.The present study may provide a new insight into understanding the molecular mechanisms of vascular remodeling.
基金supported by grants from the National Natural Science Foundation of China ( 11625209,11572199,31670958)
文摘Vascular remodeling is the essential pathogenic process of various cardiovascular disorders,including hypertension,atherosclerosis,stroke,and restenosis after vein graft.The main characterization of vascular remodeling is abnormal variations of vascular cell phenotype,morphological structure and functions such as migration,hypertrophy,proliferation and apoptosis.Numerous researches revealed that mechanical stress,including shear stress and cyclic stretch,participates in physiological vascular homeostasis,or pathophysiological vascular remodeling.The understanding of mechanobiological mechanism in vascular remodeling will play a unique role in understanding human physiology and disease,and will generate important theoretical and clinical significance [2].Non-coding RNAs are newly recognized RNAs which cannot be translated into proteins but are involved in epigenetic modification of gene regulation.The studies revealed that non-coding RNAs,such as microRNAs(miRNAs)and long noncoding RNAs(long ncRNAs,IncRNA),as well as small interfering RNAs(siRNAs),piwi-interacting RNAs(piRNAs),small nucleolar RNAs(snoRNAs),play essential roles in the regulation of various processes,such as metabolism,development,cell proliferation,cell apoptosis,cell differentiation,oncogenesis and vascular homeostasis[5].However,the roles of non-coding RNAs in the cardiovascular system under mechanical stresses are still not clarified.Our recent researches detected the mechanical regulation of IncRNAs and miRNAs in vascular remodeling.LncRNAs are non-protein-coding transcripts that are longer than 200 nucleotides(nt),which is an arbitrary cut-off value that distinguishes these transcripts from other small RNAs.Unlike the well-established mechanism of microRNA action,the functional mode of IncRNAs is not fully understood.Increasing evidence shows that IncRNAs modulate gene expression via a multilevel-regulated pathway.Given their large number and complicated functional modes,lncRNAs are emerging as important regulators of a variety of cellular responses,developmental processes and diseases.Using a gene microarray,we screened the differences in the IncRNAs and mRNAs between spontaneously hypertensive rats(SHR)and Wistar Kyoto rats(WKY).The results showed that 68 IncRNAs and 255 mRNAs were up-regulated in the aorta of SHR,while 167 IncRNAs and 272 mRNAs were down-regulated.Expressions of the screened IncRNAs,including XR007793,were validated by real-time PCR.A co-expression network was composed,and gene function was analysed using Ingenuity Pathway Analysis.In vitro,vascular smooth muscle cells(VSMCs)were subjected to cyclic stretch at a magnitude of 5%(physiological normotensive cyclic stretch)or 15%(pathological hypertensive cyclic stretch)by Flexercell-5000TM.15%-cyclic-stretch increased XR007793 expression.XR007793 knockdown attenuated VSMC proliferation and migration and inhibited co-expressed genes such as signal transducers and activators of transcription 2(stat2),LIM domain only 2(lmo2)and interferon regulatory factor 7(irf7)[4].Illuminating the role of IncRNAs in vascular remodeling induced by hyper mechanical stretch may provide deeper insight into the mechanobiological mechanism underlying hypertension,and contribute to identifying potential targets for hypertension therapy.miRNAs are endogenous,non-coding,single-stranded RNAs of 18-22 nucleotides that constitute a novel class of gene regulators.miRNAs bind to their target genes within their 3’-untranslated regions(3’-UTRs),leading to direct degradation of mRNA or translational repression by a complete,i.e.in plants,or incomplete,i.e.in animals,complement respectively.Our resent works revealed several important mechano-responsive miRNA and their potential effects in vascular remodeling.Forexample,miRNA-33 is regulated by cyclic stretch in the grafted vessels,which targets to BMP3 and subsequent modulates smad signaling pathway.The miRNA-33-BMP3-smad pathway protects against venous VSMC proliferation in response to arterial cyclic stretch.Therefore,miRNA-33 may be a potential therapeutic target in autologous vein grafted surgery,and locally overexpression of miR-33 may attenuates neointimal hyperplasia of grafted human saphenous vein [3].The unpublished data revealed that 15%cyclic stretch also significantly elevated the expression of miRNA-124-3p which bound to the 3’UTR of Lmna mRNA,and then negatively regulated protein expression of lamin A/C which is the important skeletal proteins in nucleus.In addition to primary intracellular locations of miRNAs,our recent study showed that miRNAs can be secreted and protected extracellularly via inclusion into membrane-derived vesicles including microparticles.Microparticles are extracellular vesicles ranging from 0.1 to 1μm in size and have been shown to deliver various bioactive molecules,i.e.,chemokines,enzymes and miRNAs,to recipient cells.Increasing evidence shows that microparticles play a pivotal role in many pathological processes,such as cancer,inflammatory diseases and cardiovascular disease.Our present study showed that platelet-derived microparticles(PMPs),which are released by active platelets,are important vehicles for communication and play crucial roles in inducing abnormal EC proliferation in hypertension.In briefly,EC proliferation was increased in renal hypertensive rats established by abdominal aortic coarctation compared to control rats and that elevated thrombin in plasma promoted platelet activation,which may induce the release of PMPs.miRNA array and qPCR revealed a higher level of miRNA-142-3p in platelets and PMPs.In vitro,PMPs delivered miRNA-142-3p into ECs and enhanced EC proliferation via Bcl-2-associated transcription factor 1(BCLAF1)and its downstream genes.These results indicated that PMPs deliver miRNA-142-3p from activated platelets into ECs and that miRNA-142-3p may play important roles in EC dysfunction under hypertensive conditions and might be a novel therapeutic target for maintaining EC homeostasis in hypertension[1].These results provide possible mechanisms by which non-coding RNAs regulate cellular functions under different mechanical stresses,and suggest a novel potential therapeutic approach for vascular remodeling.The further studies on noncoding RNAs may provide new insight into understanding the mechanism of vascular remodeling in different various cardiovascular disorders,and may provide novel targets for the maintenance of vascular homeostasis.
