Background and Objective In-stent restenosis(ISR)remains a major limitation of percutaneous coronary intervention despite improvements in stent design and pharmacological agents,whereas the mechanism of ISR has not be...Background and Objective In-stent restenosis(ISR)remains a major limitation of percutaneous coronary intervention despite improvements in stent design and pharmacological agents,whereas the mechanism of ISR has not been fully clarified.In the present study,we sought to investigate the potential association of serum soluble TREM-1(sTREM-1)levels with the incidence of ISR.The role of TREM-1 was evaluated in cultured vascular smooth muscle cells(VSMCs).展开更多
Instruction Shear stress,caused by the parallel frictional drag force of blood flow,is a biomechanical force which plays an important role in the control of blood vessels growth and functions [1]. Clinical researches ...Instruction Shear stress,caused by the parallel frictional drag force of blood flow,is a biomechanical force which plays an important role in the control of blood vessels growth and functions [1]. Clinical researches had found out that atherosclerotic le-展开更多
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
Observation of stilbene dropping pill and yiqi drug-containing serum influence mechanism of vascular smooth muscle proliferation， cell cycle and Cyclin D1 and CDK4Choose male SD rats were randomly divided into 2 gr...Observation of stilbene dropping pill and yiqi drug-containing serum influence mechanism of vascular smooth muscle proliferation， cell cycle and Cyclin D1 and CDK4Choose male SD rats were randomly divided into 2 groups， lavage qishen yiqi pill and the gastric saline group，extract the drug-containing serum and normal serum；To set the two groups of serum respectively different concentrations，concentration in different time by CCK8 detection effects on vascular smooth muscle cell proliferation， select best concentration and action time.Flow cytometry instrument and high-throughput screening detect serum medicated effect on vascular smooth muscle cell cycle；Western blot detect the drug-containing serum of cell cycle protein Cyclin D1 and CDK4 expression.Result is 5%， 10% medicated serum inhibits cell proliferation significantly higher than the normal serum concentrations of same within 24 h， 48 h.G1 phase cells 5% medicated serum group was obviously higher than that of 5% in normal group (P<005)， serum and cell proliferation index significantly less than 5% normal serum group (P<005)，At the same time， Cyclin D1 and CDK4 expression significantly less than 5% normal serum group (P<005).Conclusion serum of qishen yiqi pill can inhibit vascular smooth muscle cell proliferation， may be through inhibiting cell cycle protein Cyclin D1 and CDK4 expression， block the cell cycle G1 process is closely related to the role.展开更多
Purpose To assess the maximum uptake of Iododeo xyur idine (IUdR) by proliferating smooth muscle cells in vitro to determine the opti mal concentration to be administrated in an in vivo experiment. The long-term g oal...Purpose To assess the maximum uptake of Iododeo xyur idine (IUdR) by proliferating smooth muscle cells in vitro to determine the opti mal concentration to be administrated in an in vivo experiment. The long-term g oal is to utilize radioactive IUdR to inhibit smooth muscle cell proliferation a nd restenosis of arteries after balloon angioplasty in vivo. Methods Porcine smooth muscle cells (SMCs) were cultured in 5% FBS medium and stim ulated to proliferate by the addition of medium containing 10% FBS and insulin. IUdR was added at 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, respectively, in prolif erating SMCs with control for 1, 3, 5, 7 day incubation. Fluorescence Activated Cell Scanning (FACS) was performed after the SMCs were harvested and double-sta ined with FITC-conjugated anti-IUdR antibody (B44) and propidium iodide (PI). The ratio of IUdR-labeled cells to total cell population for each IUdR concentr ation and duration was determined by FACS. All data were repeated three times at each time point. The doubling times, growth curve and cell density of the proli ferating SMCs were investigated using Beckman Coulter Particle Counter and digit al microscopy. Results The percentage of proliferating SMCs uptaking IUdR incr eased from 1 to 5 days incubation with all concentrations of IUdR; In day 5, the uptake rate reached the peak value, then decreased by 7 days. IUdR uptake on d ay 5 was higher with concentrations of 10 μM and 20 μM. The doubling times of the SMCs were prolonged with IUdR concentration increasing, while the proliferat ing cell number and density compared with control decreased obviously by day 5 ( P<0.05).Conclusion The peak time to uptake IUdR was 5 days and optimal concentration of IUdR was between10 μM to 20 μM for proliferating SMCs to upta ke in vitro. IUdR itself could inhibit the SMCs’ proliferation and the inhibito ry effect was related to the concentration.[展开更多
Background and Aim Vascular smooth muscle cell （SMC） phenotype change is a hallmark of vascu-lar remodeling, which can be regulated via MicroRNAs （miRNAs）-dependent mechanism. We recently identified Asymmetric dim...Background and Aim Vascular smooth muscle cell （SMC） phenotype change is a hallmark of vascu-lar remodeling, which can be regulated via MicroRNAs （miRNAs）-dependent mechanism. We recently identified Asymmetric dimethylarginine （ADMA） positively correlates to vascular remodeling-based diseases. Here, we hy-pothesized that ADMA induces SMC phenotypic change via a miRNA-dependent mechanism. Methods and Results Microarray analysis enabled the identification of 7 deregulated microRNAs in ADMA-treated human aortic artery smooth muscle cells （hASMCs）. miR-182 was validated by real-time-PCR. Isobaric tags for relative and absolute quantitation （iTRAQ） based analysis of the hASMC proteome revealed that transfection of an miR-182 inhibitor sig- nificantly increased myeloid-associated differentiation marker （MYADM）, which was verified using Western blot and reporter activity quantization with the MYADM 3＇-UTR dual-luciferase reporter system, miR-182 knockdown further repressed Sprouty2 and enhanced MYADM, leading to ERICZMAP kinase-dependent and MYADM-depend- ent hASMC phenotypic change including proliferation, migration and differentiation marker gene expression change. In vivo, adeno-miR-182 markedly suppressed carotid neointimal formation by using balloon-injured rat carotid artery model, specifically via decreased MYADM expression. Atherosclerotic lesions from patients with high ADMA plas- ma levels exhibited decreased miR-182 expression levels and elevated MYADM expression levels. In patients with coronary heart disease （n- 164）, the miR-182 expression level in plasma was negatively correlated with the plas- ma ADMA levels. Conclusions miR-182 is a novel SMC phenotypic modulator by targeting MYADM and can be a potential therapeutic target combating vascular remodeling-associated diseases. Reduced plasma miR-182 levels might be a new predictor of high vascular remodeling risk especially in patient with coronary heart disease.展开更多
目的探讨肌微管素相关微蛋白7(myotubularin related protein 7,MTMR7)在肺动脉高压中的作用及机制。方法健康♂小鼠、Mtmr7转基因(Mtmr7-Tg)小鼠各20只,分为对照组、Mtmr7-Tg组、野百合碱(monocrotaline,MCT)组、MCT+Mtmr7-Tg组,超声...目的探讨肌微管素相关微蛋白7(myotubularin related protein 7,MTMR7)在肺动脉高压中的作用及机制。方法健康♂小鼠、Mtmr7转基因(Mtmr7-Tg)小鼠各20只,分为对照组、Mtmr7-Tg组、野百合碱(monocrotaline,MCT)组、MCT+Mtmr7-Tg组,超声测量肺动脉血流加速时间(pulmonary artery acceleration time,PAT)和射血时间(pulmonary artery ejection time,PET),取材时分离右心室游离壁,计算右心肥厚指数,免疫染色观察肺小动脉重构。将小鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)以低氧处理,观察其增殖和迁移情况。结果MTMR7在肺血管中表达,并且MCT处理之后,Mtmr7-Tg小鼠的PAT/PET比值降低(P<0.05),右心肥厚指数升高(P<0.01)。以Mtmr7基因过表达腺病毒转染PASMCs,可以抑制其增殖和迁移。用chemerin-9恢复p-ERK1/2的活性,过表达MTMR7对PASMCs增殖、迁移抑制作用消失。结论过表达MTMR7可减轻低氧诱导的小鼠PASMCs增殖和迁移,从而逆转肺动脉高压,机制与MTMR7降低ERK1/2磷酸化密切相关。展开更多
文摘Background and Objective In-stent restenosis(ISR)remains a major limitation of percutaneous coronary intervention despite improvements in stent design and pharmacological agents,whereas the mechanism of ISR has not been fully clarified.In the present study,we sought to investigate the potential association of serum soluble TREM-1(sTREM-1)levels with the incidence of ISR.The role of TREM-1 was evaluated in cultured vascular smooth muscle cells(VSMCs).
基金supported by grants from the National Natural Science Foundation of China,Nos10732070,10702043,30970703,10972140 and 30470432
文摘Instruction Shear stress,caused by the parallel frictional drag force of blood flow,is a biomechanical force which plays an important role in the control of blood vessels growth and functions [1]. Clinical researches had found out that atherosclerotic le-
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.
文摘Observation of stilbene dropping pill and yiqi drug-containing serum influence mechanism of vascular smooth muscle proliferation， cell cycle and Cyclin D1 and CDK4Choose male SD rats were randomly divided into 2 groups， lavage qishen yiqi pill and the gastric saline group，extract the drug-containing serum and normal serum；To set the two groups of serum respectively different concentrations，concentration in different time by CCK8 detection effects on vascular smooth muscle cell proliferation， select best concentration and action time.Flow cytometry instrument and high-throughput screening detect serum medicated effect on vascular smooth muscle cell cycle；Western blot detect the drug-containing serum of cell cycle protein Cyclin D1 and CDK4 expression.Result is 5%， 10% medicated serum inhibits cell proliferation significantly higher than the normal serum concentrations of same within 24 h， 48 h.G1 phase cells 5% medicated serum group was obviously higher than that of 5% in normal group (P<005)， serum and cell proliferation index significantly less than 5% normal serum group (P<005)，At the same time， Cyclin D1 and CDK4 expression significantly less than 5% normal serum group (P<005).Conclusion serum of qishen yiqi pill can inhibit vascular smooth muscle cell proliferation， may be through inhibiting cell cycle protein Cyclin D1 and CDK4 expression， block the cell cycle G1 process is closely related to the role.
文摘Purpose To assess the maximum uptake of Iododeo xyur idine (IUdR) by proliferating smooth muscle cells in vitro to determine the opti mal concentration to be administrated in an in vivo experiment. The long-term g oal is to utilize radioactive IUdR to inhibit smooth muscle cell proliferation a nd restenosis of arteries after balloon angioplasty in vivo. Methods Porcine smooth muscle cells (SMCs) were cultured in 5% FBS medium and stim ulated to proliferate by the addition of medium containing 10% FBS and insulin. IUdR was added at 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, respectively, in prolif erating SMCs with control for 1, 3, 5, 7 day incubation. Fluorescence Activated Cell Scanning (FACS) was performed after the SMCs were harvested and double-sta ined with FITC-conjugated anti-IUdR antibody (B44) and propidium iodide (PI). The ratio of IUdR-labeled cells to total cell population for each IUdR concentr ation and duration was determined by FACS. All data were repeated three times at each time point. The doubling times, growth curve and cell density of the proli ferating SMCs were investigated using Beckman Coulter Particle Counter and digit al microscopy. Results The percentage of proliferating SMCs uptaking IUdR incr eased from 1 to 5 days incubation with all concentrations of IUdR; In day 5, the uptake rate reached the peak value, then decreased by 7 days. IUdR uptake on d ay 5 was higher with concentrations of 10 μM and 20 μM. The doubling times of the SMCs were prolonged with IUdR concentration increasing, while the proliferat ing cell number and density compared with control decreased obviously by day 5 ( P<0.05).Conclusion The peak time to uptake IUdR was 5 days and optimal concentration of IUdR was between10 μM to 20 μM for proliferating SMCs to upta ke in vitro. IUdR itself could inhibit the SMCs’ proliferation and the inhibito ry effect was related to the concentration.[
文摘Background and Aim Vascular smooth muscle cell （SMC） phenotype change is a hallmark of vascu-lar remodeling, which can be regulated via MicroRNAs （miRNAs）-dependent mechanism. We recently identified Asymmetric dimethylarginine （ADMA） positively correlates to vascular remodeling-based diseases. Here, we hy-pothesized that ADMA induces SMC phenotypic change via a miRNA-dependent mechanism. Methods and Results Microarray analysis enabled the identification of 7 deregulated microRNAs in ADMA-treated human aortic artery smooth muscle cells （hASMCs）. miR-182 was validated by real-time-PCR. Isobaric tags for relative and absolute quantitation （iTRAQ） based analysis of the hASMC proteome revealed that transfection of an miR-182 inhibitor sig- nificantly increased myeloid-associated differentiation marker （MYADM）, which was verified using Western blot and reporter activity quantization with the MYADM 3＇-UTR dual-luciferase reporter system, miR-182 knockdown further repressed Sprouty2 and enhanced MYADM, leading to ERICZMAP kinase-dependent and MYADM-depend- ent hASMC phenotypic change including proliferation, migration and differentiation marker gene expression change. In vivo, adeno-miR-182 markedly suppressed carotid neointimal formation by using balloon-injured rat carotid artery model, specifically via decreased MYADM expression. Atherosclerotic lesions from patients with high ADMA plas- ma levels exhibited decreased miR-182 expression levels and elevated MYADM expression levels. In patients with coronary heart disease （n- 164）, the miR-182 expression level in plasma was negatively correlated with the plas- ma ADMA levels. Conclusions miR-182 is a novel SMC phenotypic modulator by targeting MYADM and can be a potential therapeutic target combating vascular remodeling-associated diseases. Reduced plasma miR-182 levels might be a new predictor of high vascular remodeling risk especially in patient with coronary heart disease.
