Advances in research on notch signaling pathway in pulmonary vascular remodeling of pulmonary arterial hypertension / 中国药理学通报

Pulmonary hypertension is a cardiovascular disease characterized by persistent elevation of pulmonary artery pressure and pulmonary vascular resistance, causing pulmonary vascular remodeling. Notch signaling pathway is a highly conserved signaling pathway involved in the occurrence and development of cardiovascular, neurological and tumor diseases mainly by regu-lating cell proliferation, differentiation and apoptosis. TTiis paper mainly reviews the molecular mechanism of Notch signaling path way involved in pulmonary vascular remodeling, contributing to provide a new strategy for the treatment of pulmonary arterial hypertension.

Effects of siRNA on EPAS1 activity and proliferation of hypoxic pulmonary artery smooth muscle cells in rats / 解放军医学杂志

Objective This study explored whether EPAS1 gene is involved in the proliferation of pulmonary arterial smooth muscle cells (PASMCs) during hypoxia when EPAS1 gene expression was interfered by small interfering RNA (siRNA).Methods The rat primary pulmonary artery smooth muscle cells were cultured and identified by immunofluorescence.The specific lipidosomes of EPAS1 siRNA were constructed and transfected into PASMCs,and the best targets were selected from the three interfering targets.The transfected PASMCs were cultured in hypoxia (37℃,5％ CO2,2％ O2) or normoxia (37℃,5％ CO2,20％ O2) for 24h,48h and 72h,respectively.The PASMCs proliferation was detected by CCK-8 assay.The protein expression of EPAS 1 and vascular endothelial growth factor (VEGF) were determined by Western blotting to investigate the effect of silencing EPAS1 gene expression on the proliferation of PASMCs under hypoxic condition.Results The specific liposomes ofEPAS1 siRNA were successfully constructed and transfected into PASMCs,and the best interfering target were selected from the three interference targets.The proliferation of PASMCs was increased and the protein expression of VEGF was up-regulated in the PASMCs under hypoxic condition.Under hypoxic or normoxic condition,PASMCs proliferation and VEGF protein expression of PASMCs were suppressed by EPAS 1 siRNA.Conclusion EPAS 1 gene might be involved in the proliferation of rat PASMCs by regulating VEGF protein level under hypoxic condition.

Research progress on role of TGF-β1 in pulmonary artery hypertension / 中国药理学通报

Pulmonary artery hypertension is a disease with complicated pathogenesis, which is characterized by enhanced pulmonary artery constriction and arterial wall remodeling, leading to progressive increase of pulmonary vascular resistance and pulmonary artery pressure, then resulting in right heart failure.Many studies have shown that transforming growth factor-β1(TGF-β1) plays an important role in the development of various diseases, especially in cardiovascular and cerebrovascular diseases.TGF-β1 is involved in multiple cellular responses including cell proliferation, differentiation, migration and apoptosis.TGF-β1 participates in pulmonary artery hypertension mainly via promoting the proliferation of pulmonary artery smooth muscle cells as well as inducing the deposition of extracellular matrix and endothelial-to-mesenchymal transition(EndMT) through many signaling, which is mainly dominated by pulmonary artery smooth muscle cells and pulmonary artery endothelial cells.This review mainly introduces the role of TGF-β1 in pulmonary artery hypertension in order to provide potential drug targets and therapeutic strategies for pulmonary artery hypertension.

Effect of autophagy inhibitor 3-methyladenine on phenotype transformation and proliferation of pulmonary arterial smooth muscle cells / 实用医学杂志

Objective To evaluate the effect of autophagy inhibitor 3-methyladenine (3-MA) on phenotype transformation and proliferation of rat pulmonary arterial smooth muscle cells (PASMCs).Methods Cultured PASMCs were treated with different concentrations of 3-MA (low-dose group,2.0 mmol/L;middle-dose group,4.0 mmol/L;high-dose group,8.0 mmol/L;control group,0 mmol/L).The protein expression of LC3 Ⅱ,OPN and Vimentin was detected by Western blotting.Cell proliferation was detected by MTT assay.Results The autophagy of PASMCs was decreased with the increase of the concentration of 3-MA.Compared with the control group,significantly down-regulated protein expression of LC3 Ⅱ,OPN and Vimentin was observed in 3-MA-treated cells,with significantly lower proliferation activity.Conclusion The autophagy inhibitor 3-MA significantly down-regulated the expression of LC3 Ⅱ,OPN and Vimentin in PASMCs,with inhibiting the proliferation of PASMCs.

Effect of SKF96365 and NiCl2 on cyclopiazonic acid induced intracellular calcium cation concentration increase in rat distal pulmonary arterial smooth muscle cells / 重庆医学

Objective To study the effect of SKF96365 and NiCl2 on cyclopiazonic acid (CPA) induced intracellular calcium cation concentration ([Ca2+ ]i ) change in rat distal pulmonary arterial smooth muscle cells (PASMC) .Methods The rat distal PASMC were isolated and cultured .The effects of CPA ,SKF96365 and NiCl2 on [Ca2+ ]i in PASMC were tested by fluorescence microscope and InCyte [Ca2+ ]i measurement system .Results PASMC were incubated with Ca2+‐free Krebs solution containing 5μmol/L nifedipine ,10 μmol/L CPA caused a small transient increase in [Ca2+ ]i ;after restoration of extracellular Ca2+ to 2 .5 mmol/L ,10 μmol/L CPA caused marked increases in [Ca2+ ]i in PASMC incubated with Krebs solution containing 5 μmol/L nife‐dipine .Both 50 μmol/L SKF96365 and 500 μmol/L NiCl2 distinctly attenuated the increases in [Ca2+ ]i caused by 10 μmol/L CPA in PASMC .However ,neither 50 μmol/L SKF96365 nor 500 μmol/L NiCl2 affected the increases in [Ca2+ ]i caused by 60 mmol/L KCl in PASMC .Conclusion CPA induced increases in [Ca2+ ]i may related to Ca2+ release from sarcoplasmic reticulum and the in‐flux of Ca2+ through store‐operated Ca2+ channels (SOCC) in rat distal PASMC .Both SKF96365 and NiCl2 could selectively block SOCC and attenuated the influx of Ca2+ through SOCC in PASMC .

Effect of chloride channel blocker on pulmonary hypertension induced by high pulmonary blood flow in rats / 中华实用儿科临床杂志

Objective To study the effect of chloride channel blocker(niflumic acid,NFA) on pulmonary hypertension induced by high pulmonary blood flow in rats.Methods Fifty male or female Sprague-Dawley rats were randomly divided into 5 groups:normal group,sham group,model group,drug 1 group,and drug 2 group,with 10 rats in each group.After subjected to an abdominal aorta-inferior vena cava shunt,all the rats were reared under the same condition for 11 weeks.Then,mean pulmonary artery pressure(mPAP) and right ventricular hypertrophy index(RVHI) of each rat were measured.In addition,arterial wall area/vessel area (W/V) and arterial wall thickness/vessel external diameter(T/D) of each rat were also measured.Results 1.The mPAP of model group [(25.79 ± 4.03) mmHg,1 mmHg =0.133 kPa] was significantly higher than those of normal group [(16.48 ± 1.70) mmHg],sham group [(17.03 ± 2.01) mmHg],drug 1 group [(21.78 ± 2.77) mmHg] and drug 2 group [(20.31 ± 2.15) mmHg] (F =18.983,P ＜0.01).Although the mPAP of drug 1 group was a little higher than drug 2 group,there was no significant difference (P ＞ 0.05).Compared with normal group and sham group,the mPAP of drug 1 group and drug 2 group increased(P ＜0.01,respectively).2.The W/V and T/D of model group were significantly higher than those of normal group,sham group,drug 1 group and drug 2 group (F =26.135,15.527,all P ＜ 0.001).The W/V and T/D of two drug groups showed no significant difference,but they were higher than those of normal group and sham group (P ＜ 0.01,respectively).Conclusions Chloride channel blocker NFA partly decrease mPAP of pulmonary hypertension indnced by high pulmonary blood flow in rats,and inhibit proliferation of vascular smooth muscle cells.These results suggest that NFA had part of therapeutic effect to pulmonary hypertension induced by high pulmonary blood flow.

Role of ERK1/2 and PI3K/PKB signaling pathway in expression of extracellular matrix gene in pulmonary arterial smooth muscle cells in rat / 临床儿科杂志

Objective To explore the effects of the extracellular regulated protein kinase’s (ERK1/2) inhibitor PD98059 and ino-sitol triphosphate kinase (PI3K/PKB) signaling pathway’s inhibitor LY294002 on extracellular matrix (ECM) deposition in pulmonary arterial smooth muscle cells (PASMCs) stimulated by connective tissue growth factor (CTGF). Methods PASMCs of SD rat were cul-tured in vitro. The PASMCs were divided into control group, CTGF group, CP (CTGF+PD98059) group, CL (CTGF+LY294002) group and CPL (CTGF+PD98059+LY294002) group. Real-time lfuorescent quantitative RT-PCR was used to detect the expression of colla-gen III and ifbronectin mRNA of PASMCs, and the expression of collagenШprotein of PASMCs was detected by immunohistochem-istry and western-blot. Results The expressions of collagenШand ifbronectin mRNA of PASMCs stimulated with CTGF (50 ng/ml) for 48 h were signiifcantly higher than those in control group, and the collagen proteinШof PASMCs was decreased signiifcantly after stimulation with CTGF (50 ng/ml) for 72 h (P<0.05). The expressions of collagenШand ifbronectin mRNA in PASMCs cultured with PD98059 (20μmol/L) and/or LY294002 (10μmol/L) for 48 h was signiifcantly lower than those in CTGF group (P<0.05). The collagen proteinШin PASMCs cultured with PD98059 (20μmol/L) and/or LY294002 (10μmol/L) for 72 h was increased (P<0.05). The expres-sions of collagenШand ifbronectin mRNA of PASMCs stimulated with both PD98059 and LY294002 were more signiifcant. Conclu-sions CTGF may increase the expression of collagenШand ifbronectin mRNA in PASMCs, which may contribute to the deposition of ECM in PASMCs during pulmonary vascular remodeling. PD98059 and LY294002 may repress ERK1/2 and PI3K/PKB signaling pathways and interfere with the biological effect of CTGF.

Fluoxetine Protects against Big Endothelin-1 Induced Anti-Apoptosis by Rescuing Kv1.5 Channels in Human Pulmonary Arterial Smooth Muscle Cells

PURPOSE: Pulmonary Kv channels are thought to play a crucial role in the regulation of cell proliferation and apoptosis. Previous studies have shown that fluoxetine upregulated the expression of Kv1.5 and prevented pulmonary arterial hypertension in monocrotaline-induced or hypoxia-induced rats and mice. The current study was designed to test how fluoxetine regulates Kv1.5 channels, subsequently promoting apoptosis in human PASMCs cultured in vitro. MATERIALS AND METHODS: Human PASMCs were incubated with low-serum DMEM, ET-1, and fluoxetine with and without ET-1 separately for 72 h. Then the proliferation, apoptosis, and expression of TRPC1 and Kv1.5 were detected. RESULTS: In the ET-1 induced group, the upregulation of TRPC1 and down regulation of Kv1.5 enhanced proliferation and anti-apoptosis, which was reversed when treated with fluoxetine. The decreased expression of TRPC1 increased the expression of Kv1.5, subsequently inhibiting proliferation while promoting apoptosis. CONCLUSION: The results from the present study suggested that fluoxetine protects against big endothelin-1 induced anti-apoptosis and rescues Kv1.5 channels in human pulmonary arterial smooth muscle cells, potentially by decreasing intracellular concentrations of Ca2+.

Effects of Gax gene transfection on proto-oncogene expression and proliferation of PASMCs in rats under hypoxia conditions / 中国病理生理杂志

AIM:To explore the effects of Gax gene transfection on expressions of c-fos and c-jun mRNA and proliferation of pulmonary arterial smooth muscle cells (PASMCs) in rat under hypoxia. METHODS: PASMCs were transfected with Gax gene by Ad-Gax. Under normal oxygentention (21% O2) or hypoxia (2.5% O2) for 12 h condition, expressions of Gax mRNA and protein in PASMCs were detected by RT-PCR and immunocytochemistry. The expressions of c-fos and c-jun mRNA were evaluated by RT-PCR. [3H]-TdR incorporation was used to measure the PASMCs proliferation. RESULTS: The Gax overexpression in transfection group was confirmed by RT-PCR and immunocytochemistry. Under normal oxygentention or hypoxia, the c-fos and c-jun mRNA levels in transfection group were lower than those in the non-transfection group, respectively (P

Effect of chronic cigarette smoking on BK_(Ca) and Kv1.5 expression in rat pulmonary arterial smooth muscle cells / 中国病理生理杂志

AIM:To investigate the role of potassium channel expression alteration in chronic cigarette smoking-induced increase in pulmonary vascular responsiveness,the effect of chronic cigarette smoking on large-conductance calcium-activated potassium channel(BKCa) and voltage-dependent delayed rectifier potassium channel(Kv1.5) expression in rat pulmonary smooth muscle cells were investigated in vivo.METHODS: HE staining,immuno-histochemistry and in situ hybridization techniques were used.RESULTS:(1) Chronic cigarette smoking downregulates the protein and mRNA expression of BKCa in pulmonary arterial smooth muscles.(2) Chronic cigarette smoking downregulated the protein and mRNA expression of Kv1.5 in pulmonary arterial smooth muscles.(3) In big artery,BKCa decreased more makedly than Kv1.5,but in small artery,both of them decreased equally.CONCLUSION: Chronic cigarette smoking downregulates the levels of BKCa and Kv1.5 in rat pulmonary arterial smooth muscle cells in vivo,which maybe contribute to the mechanism of cigarette smoking-induced increase in pulmonary vascular responsiveness.