OBJECTIVE Pulmonary artery hypertension(PAH)is a severe disease characterized by the mean pulmonary artery pressure exceeding 25 mm Hg at rest.PAH could induce right heart failure and has a very high mortality rate.At...OBJECTIVE Pulmonary artery hypertension(PAH)is a severe disease characterized by the mean pulmonary artery pressure exceeding 25 mm Hg at rest.PAH could induce right heart failure and has a very high mortality rate.At present,several kinds of drugs have been used in the treatment of PAH.However,most of these drugs aim to relax pulmonary arteries and do not inhibit the injury of vessels.In other words,the drugs available for PAH treatment do not improve the survival rate of PAH patients and cannot satisfy the needs in clinic.To discover and develop novel candidate compounds effective on the treatment of pulmonary artery injury and remodeling will be very important.Based on these background,the present study aimed to study the protective effect of two novel Rho-kinases(Rho-associated coiledcoil forming protein serine/threonine kinase,ROCK)inhibitors,DL0805 derivatives(DL0805-1and DL0805-2),on pulmonary arterial cells and further evaluate the underlying mechanisms and the possibility of DL0805 derivatives become therapeutic drugs for PAH.METHODS The primary cultured pulmonary arterial cells including human pulmonary artery endothelium cells(HPAECs)and human pulmonary artery smooth muscle cells(HPASMCs)were used in this study.HPAECs were injured under hypoxia environment(1%O2)and treated with or without DL0805 derivatives.After 48 h,the proliferation and oxidative stress were observed.CCK8 was used to detect cell viability.DCFH-DA was used as probe for reactive oxygen species(ROS)under fluorescence imaging system.HPASMCs was stimulated by growth factors including platelet-derived growth factor-BB(PDGF-BB)and Fetal Bovine Serum(FBS).The proliferation was observed in the cells treated with or without DL0805 derivatives.HPASMCs treated with or without DL0805 derivatives were further incubated with endothelin(ET-1),the proliferation and cytoskeleton remodeling of cells were detected by immunofluorescence assay.At last,Western blotting(WB)and immunofluorescence assay were employed to analysis the underlying mechanisms in the above experiments.RESULTS 10μmol·L-1DL0805-2 could inhibit the proliferation of HPAECs induced by hypoxia.Each concentration of DL0805-1 and DL0805-2attenuated the production of ROS in HPAECs.Results from WB indicated that DL0805 derivatives decreased the injury of HPAECs induced by hypoxia through the inhibition of the expression of Rho A and the activity of ROCK.On HPASMCs,DL0805 derivatives reduced the proliferation induced by PDGF-BB and FBS and inhibited cytoskeleton remodeling induced by ET-1.Immunofluorescence assay showed that DL0805 derivatives inhibited ROCK activity and down regulated the phosphorylation levels of ROCK substrates.CONCLUSION DL0805derivatives exhibited protective effect on pulmonary arterial cells including endothelium cells and smooth muscle cells.Among the above experiments,DL0805-2 showed stronger potency than DL0805-1.These two compounds might protect the cells through the inhibition of Rho A/ROCK pathway and they probably have the potential in the treatment of PAH and deserve further evaluation.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
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
OBJECTIVE To explore the role of calpain in in pulmonary vascular remodeling in hypoxia induced pulmonary hypertension and the underlying mechanism.METHODS Sprague-Dawley rats were randomly divided into hypoxia group ...OBJECTIVE To explore the role of calpain in in pulmonary vascular remodeling in hypoxia induced pulmonary hypertension and the underlying mechanism.METHODS Sprague-Dawley rats were randomly divided into hypoxia group and normoxia control group.Right ventricular systolic pressure(RVSP)and mean pulmonary artery pressure(m PAP)were monitored by the method of right external jugular vein cannula.Right ventricular hypertrophy index was expressed as the ratio of right ventricular weight to left ventricular weight(left ventricle plus septum weight).Level of calpain-1,calpain-2and calpain-4 m RNA in pulmonary artery trunk were determined by real-time PCR.Expression of calpain-1,calpain-2 and calpain-4 protein was determined by Western Blot.Primary rat pulmonary arterial smooth muscle cells(PASMCs)were divided into 4 groups:normoxia control group,normoxia+MDL28170 group,hypoxia group and hypoxia+MDL28170 group.Cell proliferation was detected by MTS and flow cytometry.Level of Ki-67 and PCNA m RNA were determined by real-time PCR.RESULTS RVSP,m PAP and right ventricular remodeling index were significantly higher in the hypoxia group than those in the normoxia group.In the hypoxia group,pulmonary vascular remodeling occurred,and the expression of calpain-1,calpain-2 and calpain-4 m RNA and protein expression was increased in the pulmonary artery.MDL28170 significantly inhibited hypoxia-induced proliferation of PASMCs accompanied with decreased Ki-67and PCNA m RNA expression.CONCLUSION Calpain mediated vascular remodeling via promoting proliferation of PASMCs in hypoxia induced pulmonary hypertension.展开更多
基金The project supported by Central Public Scientific Research Institution Fundamental Project(2016CX09)by National Natural Science Foundation of China(81573645)
文摘OBJECTIVE Pulmonary artery hypertension(PAH)is a severe disease characterized by the mean pulmonary artery pressure exceeding 25 mm Hg at rest.PAH could induce right heart failure and has a very high mortality rate.At present,several kinds of drugs have been used in the treatment of PAH.However,most of these drugs aim to relax pulmonary arteries and do not inhibit the injury of vessels.In other words,the drugs available for PAH treatment do not improve the survival rate of PAH patients and cannot satisfy the needs in clinic.To discover and develop novel candidate compounds effective on the treatment of pulmonary artery injury and remodeling will be very important.Based on these background,the present study aimed to study the protective effect of two novel Rho-kinases(Rho-associated coiledcoil forming protein serine/threonine kinase,ROCK)inhibitors,DL0805 derivatives(DL0805-1and DL0805-2),on pulmonary arterial cells and further evaluate the underlying mechanisms and the possibility of DL0805 derivatives become therapeutic drugs for PAH.METHODS The primary cultured pulmonary arterial cells including human pulmonary artery endothelium cells(HPAECs)and human pulmonary artery smooth muscle cells(HPASMCs)were used in this study.HPAECs were injured under hypoxia environment(1%O2)and treated with or without DL0805 derivatives.After 48 h,the proliferation and oxidative stress were observed.CCK8 was used to detect cell viability.DCFH-DA was used as probe for reactive oxygen species(ROS)under fluorescence imaging system.HPASMCs was stimulated by growth factors including platelet-derived growth factor-BB(PDGF-BB)and Fetal Bovine Serum(FBS).The proliferation was observed in the cells treated with or without DL0805 derivatives.HPASMCs treated with or without DL0805 derivatives were further incubated with endothelin(ET-1),the proliferation and cytoskeleton remodeling of cells were detected by immunofluorescence assay.At last,Western blotting(WB)and immunofluorescence assay were employed to analysis the underlying mechanisms in the above experiments.RESULTS 10μmol·L-1DL0805-2 could inhibit the proliferation of HPAECs induced by hypoxia.Each concentration of DL0805-1 and DL0805-2attenuated the production of ROS in HPAECs.Results from WB indicated that DL0805 derivatives decreased the injury of HPAECs induced by hypoxia through the inhibition of the expression of Rho A and the activity of ROCK.On HPASMCs,DL0805 derivatives reduced the proliferation induced by PDGF-BB and FBS and inhibited cytoskeleton remodeling induced by ET-1.Immunofluorescence assay showed that DL0805 derivatives inhibited ROCK activity and down regulated the phosphorylation levels of ROCK substrates.CONCLUSION DL0805derivatives exhibited protective effect on pulmonary arterial cells including endothelium cells and smooth muscle cells.Among the above experiments,DL0805-2 showed stronger potency than DL0805-1.These two compounds might protect the cells through the inhibition of Rho A/ROCK pathway and they probably have the potential in the treatment of PAH and deserve further evaluation.
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.[
基金The project supported by National Natural Science Foundation of China(81273512,81460010)by Natural Science Foundation of Jiangxi province(20142BAB215035)
文摘OBJECTIVE To explore the role of calpain in in pulmonary vascular remodeling in hypoxia induced pulmonary hypertension and the underlying mechanism.METHODS Sprague-Dawley rats were randomly divided into hypoxia group and normoxia control group.Right ventricular systolic pressure(RVSP)and mean pulmonary artery pressure(m PAP)were monitored by the method of right external jugular vein cannula.Right ventricular hypertrophy index was expressed as the ratio of right ventricular weight to left ventricular weight(left ventricle plus septum weight).Level of calpain-1,calpain-2and calpain-4 m RNA in pulmonary artery trunk were determined by real-time PCR.Expression of calpain-1,calpain-2 and calpain-4 protein was determined by Western Blot.Primary rat pulmonary arterial smooth muscle cells(PASMCs)were divided into 4 groups:normoxia control group,normoxia+MDL28170 group,hypoxia group and hypoxia+MDL28170 group.Cell proliferation was detected by MTS and flow cytometry.Level of Ki-67 and PCNA m RNA were determined by real-time PCR.RESULTS RVSP,m PAP and right ventricular remodeling index were significantly higher in the hypoxia group than those in the normoxia group.In the hypoxia group,pulmonary vascular remodeling occurred,and the expression of calpain-1,calpain-2 and calpain-4 m RNA and protein expression was increased in the pulmonary artery.MDL28170 significantly inhibited hypoxia-induced proliferation of PASMCs accompanied with decreased Ki-67and PCNA m RNA expression.CONCLUSION Calpain mediated vascular remodeling via promoting proliferation of PASMCs in hypoxia induced pulmonary hypertension.
