周期蛋白D1在香烟烟雾致人肺动脉平滑肌细胞增殖中作用及其与蛋白激酶Cα关系的研究
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摘要
第一部分蛋白激酶Cα和周期蛋白D1在吸烟患或不患慢性阻塞性肺疾病患者肺血管中的表达变化
目的检测蛋白激酶Cα(PKCα)和周期蛋白D1 (cyclinD1)在吸烟患或不患慢性阻塞性肺疾病(COPD)患者肺血管中的表达变化并探讨两者在吸烟导致的肺血管重构中的作用。方法将35例因肺癌而行肺叶切除术患者的外周肺组织标本分为三组：不吸烟肺功能正常组(正常组)10例,吸烟肺功能正常组(吸烟组)14例,吸烟伴COPD组(COPD组)11例。采用苏木精-伊红(HE)染色观察肺小动脉形态学改变,应用免疫组织化学染色法检测肺小动脉平滑肌细胞中α-平滑肌肌动蛋白(α-SMA)、增殖细胞核抗原(PCNA)、PKCα和cyclinD1蛋白表达,以PCNA阳性细胞百分率表示平滑肌细胞增殖指数(PI),采用实时荧光PCR法检测肺小动脉平滑肌细胞中]PKCa mRNA、cyclin D1 mRNA的表达。结果(1)肺小动脉血管壁面积占血管总面积的百分比值(WA%)在吸烟组和COPD组均高于正常组。(2)吸烟组和COPD组PI均高于正常组(P<0.01)。(3)吸烟组、COPD组PKCa蛋白和cyclinD1蛋白表达吸光度(A)值较正常组均显著升高(P<0.01)。(4)吸烟组、COPD组PKCa mRNA和cyclinD1 mRNA表达水平较正常组均显著升高(P<0.01)。(5)PKCa蛋白表达水平分别与WA%、PI呈正相关(P<0.01),cyclinD1蛋白表达水平分别与WA%、PI呈正相关(P<0.01),PKCa与cyclin D1在蛋白和mRNA表达水平均呈明显正相关(P<0.01)。结论在吸烟者及早期COPD患者肺血管中PKCa和cyclinD1表达均显著上调,二者可能参与肺血管重构中肺动脉平滑肌细胞的异常增殖。
第二部分周期蛋白D1在香烟烟雾提取物促进人肺动脉平滑肌细胞增殖中的作用
目的本文旨在探讨周期蛋白D1(cyclinD1)在香烟烟雾提取物(cigarette smoke extract,CSE)所致人肺动脉平滑肌细胞(HPASMCs)增殖中的作用。方法用不同浓度的CSE(1%,2.5%,5%,10%,20%)干预同步化的HPASMCs。构建反义cyclin D1基因真核表达载体(pIRES2-EGFP-ascyclin D1),采用脂质体介导基因转染法将空载体(pIRES2-EGFP)和pIRES2-EGFP-ascyclin D1导入正常HPASMCs后,分别进行CSE(5%)干预。细胞随机分为6组：对照组、空载体组、反义cyclinD1组、5%CSE组、空载体+5%CSE组、反义cyclin D1+5% CSE组。用台盼蓝染色法检测不同浓度CSE干预的HPASMCs活细胞率,采用四甲基偶氮唑盐比色法(MTT)、流式细胞术、增殖细胞核抗原(PCNA)染色法测定细胞增殖能力,用实时荧光RT-PCR和Western blot法分别检测cyclin D1 mRNA和蛋白的表达。结果低浓度的CSE(1%,2.5%,5%,10%)促进HPASMCs增殖,且浓度5%时细胞增殖能力最强,高浓度的CSE(20%)对HPASMCs具有毒性作用,抑制细胞增殖。反义cyclin D1基因真核表达载体pIRES2-EGFP-ascyclin D1成功构建,并成功转染入HPASMCs,转染后HPASMCs中cyclin D1的mRNA和蛋白表达水平较对照组均显著下降(P<0.05)。与对照组比较,5%CSE组cyclin D1的nRNA和蛋白表达水平均明显升高(P<0.05),细胞增殖能力显著增强(P<0.05)。与5%CSE组比较,反义cyclin D1+5% CSE组cyclin D1 mRNA和蛋白表达水平均明显下降(P<0.05),细胞增殖能力显著降低(P<0.05)。结论CSE可通过上调cyclin D1表达促进HPASMCs增殖,反义cyclin D1基因真核表达载体可抑制CSE介导的HPASMCs增殖,提示cyclin D1在CSE促进HPASMCs增殖中发挥重要的正向调控作用。
第三部分蛋白激酶Cα和周期蛋白D1在香烟烟雾提取物促进人肺动脉平滑肌细胞增殖中的作用和关系
目的研究蛋白激酶Cα(PKCα)和周期蛋白D1(cyclin D1)在香烟烟雾提取物(CSE)促进人肺动脉平滑肌细胞(HPASMCs)增殖中的作用和关系。方法用5%浓度的CSE干预同步化的HPASMCs。通过Western blot法检测PKCα蛋白在胞浆和胞膜中的表达。采用小干扰RNA (siRNA)技术分别剔除PKCα和cyclin D1基因表达。用实时荧光RT-PCR法检测cyclin D1 mRNA表达,Western blot法检测PKCα和cyclin D1蛋白的表达。采用四甲基偶氮唑盐比色法(MTT)和细胞计数法测定细胞增殖,流式细胞术观察细胞周期变化。结果5%CSE可激活PKCα,PKCα蛋白由胞浆转移至胞膜表达。通过Go6976抑制PKCα活性或采用siRNA技术剔除PKCα基因表达可显著抑制CSE介导的HPASMCs增殖和细胞周期的G1期向S期转换。5%CSE促进HPASMCs增殖中,cyclin D1mRNA和蛋白表达水平均明显升高。转染cyclin D1 siRNA可抑制CSE介导的HPASMCs增殖。此外,使用Go6976或转染PKCa siRNA可显著抑制CSE介导的HPASMCs中cyclin D1 mRNA和蛋白表达水平升高。结论香烟烟雾提取物通过蛋白激酶Cα途径上调周期蛋白D1表达促进人肺动脉平滑肌细胞增殖。
Background and Objective
Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease, its prevalence rate is high, and so does the mortality it caused. Pulmonary vascular remodeling hinders the respiration process and may result in pulmonary hypertension, which is one of the principal factors on reducing the survival rate of COPD patients. Controlling pulmonary vascular remodeling can effectively prevent the formation of pulmonary hypertension, which is of great significance in the clinical treatment of COPD and cor pulmonale. Previous researchers mainly focused on the pathogenesis of hypoxic pulmonary hypertension and deem that hypoxia-induced pulmonary vasoconstriction and pulmonary vascular remodeling lead to the formation of pulmonary hypertension. In recent studies, it has been reported that pulmonary vascular remodeling occurs in smokers with normal lung functions and mild COPD patients without hypoxemia. Exposure to cigarette smoke for 6 months produces pulmonary hypertension in guinea pigs. These findings suggest that cigarette smoke may also promote pulmonary vascular remodeling, leading to pulmonary hypertension. The abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of major morphological features of pulmonary vascular remodeling induced by smoking. Cigarette smoke can result in an abnormal proliferation of pulmonary artery smooth muscle cells. However, the intrinsic mechanism has not been thoroughly understood yet.
The proliferation of cells is closely related with cell cycle changes. While in a cell, cyclin plays a very important role in the regulation of cell cycle. In the Gl phase, cyclin D combines with cyclin-dependent kinase 4 (CDK4) or CDK6 to form complexes. These complexes phosphorylate retinoblastoma protein (Rb) and other family members. Phosphorylation of Rb activates the transcription factors and mediates the transcription of genes required for the cell to progress into S phase, which leads to the cell proliferation. Cyclin D1 is one of the major subtypes of cyclin D, and it regulates the G1/S phase transition. Cyclin D1 has been proven to be related to the proliferation of carcinoma cells. So far, few studies have been reported on the role of cyclin D1 in the vascular smooth-muscle cell proliferation. The effect of cyclin D1 on the proliferation of PASMCs remains to be understood.
Protein kinase C (PKC), which is one of crucial members in serine-threonine kinase family, is a key intracellular signal transduction molecule. At present, PKC has been found with at least 12 isoforms, and functions of PKC isoforms in different cells vary greatly. Among the PKC isoforms, PKCαis generally reported to be a positive mediator for the vascular smooth muscle cells proliferation. Our previous study has shown that PKCαwas closely associated with the abnormal proliferation of the rat pulmonary arterial smooth muscle cells induced by cigarette smoking extract. However, the downstream mechanism of PKCαhas remained unclear so far. PKCαhas been implicated in the up-regulation of the cyclin D1 expression in several types of cultured cells. In HPASMCs, however, the existence of a similar mechanism needs to be proven.
In this study, we investigate the pathobiology of human pulmonary artery smooth muscle cell proliferation induced by cigarette smoke from the perspective of the cell cycle control and the intracellular signal transduction. Our study may provide the basis for a new therapeutic approach for addressing COPD associated with pulmonary hypertension.
Part one The changes of protein kinase Ca and cyclin D1 expressions in pulmonary artery from smokers with and without chronic obstructive pulmonary disease
Objective The purpose of this study was to investigate the changes of protein kinase Ca (PKCa) and cyclin D1 expressions in pulmonary artery from smokers with normal lung function and smokers with mild to moderate chronic obstructive pulmonary disease (COPD). Methods The peripheral lung tissues were obtained from 10 nonsmokers with normal lung function (nonsmoker group),14 smokers with normal lung function (smoker group),11 smokers with mild to moderate COPD (COPD group). The morphological changes of pulmonary arteries were observed by hematoxylin-eosin staining. The expressions of a-smooth muscle actin (a-SMA), proliferating cell nuclear antigen (PCNA), PKCa and cyclin D1 proteins in pulmonary artery smooth muscle (PASM) were determined by immunohistochemical staining. The percentages of PCNA-positive cells were considered as the smooth muscle cells proliferation index (PI). The mRNA expressions of PKCa and cyclin D1 in PASM were evaluated by real-time fluorescence PCR. Results Morphometry analysis showed that the ratio of pulmonary artery wall area to total area (WA%) in smoker group and COPD group was significantly greater than that in nonsmoker group (P< 0.01). The PASMCs proliferation index in smoker group and COPD group was obviously higher than that in nonsmoker group (P< 0.01). The protein levels of PKCa and cyclin D1 in PASMCs were markedly increased in smoker group and COPD group as compared with nonsmoker group (P< 0.01). The mRNA expressions of PKCa and cyclin D1 in PASMCs were significantly elevated in smoker group and COPD group as compared with nonsmoker group(P< 0.01). Significant correlations were found between PKCa protein and WA% or PI (P< 0.01). Correlations between cyclin D1 protein and WA% or PI also existed (P< 0.01). The expression of PKCa positively correlated with the expression of cyclin D1 at both protein and mRNA levels (P< 0.01). Conclusions Increased expressions of PKCa and cyclin D1 might be involved in the pathogenesis of abnormal proliferation of PASMCs in smokers with normal lung function and smokers with mild to moderate COPD.
Part two The role of cyclin D1 in human pulmonary artery smooth muscle cells proliferation induced by cigarette smoke extract
Objective The present study was aimed to investigate the role of cyclin D1 in human pulmonary artery smooth muscle cells (HPASMCs) proliferation induced by cigarette smoke extract (CSE). Methods Synchronized HPASMCs were treated with different concentrations of CSE (1%,2.5%,5%,10%,20%). The antisense eukaryotic expression vector of cyclin D1 gene (pIRES2-EGFP-ascyclin D1) was recombinated. The recombinant and empty vector were separately transfected into normal HPASMCs using liposome. Then the cells were treated with or without 5% CSE. The cells were randomly divided into six groups:control group, vector group, antisense cyclin D1 group,5% CSE treated group, vector+5% CSE treated group and antisense cyclin Dl+5% CSE treated group. The proliferation of HPASMCs was examined by cell cycle analysis, MTT assay and proliferation cell nuclear antigen (PCNA) immunocytochemical staining. The expressions of cyclin D1 mRNA and protein were detected by real-time fluorescence RT-PCR and Western blot, respectively. Results Low concentrations of CSE (1%,2.5%,5%,10%) stimulated proliferation of HPASMCs, with its maximal effect at concentration of 5%. On the contrary, high concentrations of CSE (20%) were the inhibitory of cell proliferation as a result of cytotoxicity. The antisense eukaryotic expression vector of cyclin D1 gene was constructed and transfected into HPASMCs successfully. The cyclin D1 mRNA and protein levels in antisense cyclin D1 group were significantly lower than those in control group (P < 0.05). In 5% CSE group, the cyclin D1 mRNA and protein levels were elevated significantly compared with those in control group (P< 0.05), and the indicators of cell proliferation were increased as well (P<0.05). The cyclin D1 mRNA and protein levels, the indicators of cell proliferation in antisense cyclin D1+5% CSE group were remarkably lower than those in 5% CSE group (P< 0.05). Conclusions CSE could promote HPASMCs proliferation through up-regulation of cyclin D1 expression. PIRES2-EGFP-ascyclin D1 could attenuate CSE-induced proliferation of HPASMCs by suppressing the expression of cyclin D1, which implicates that cyclin D1 might be involved in the process of HPASMCs proliferation stimulated by CSE.
Part three Cigarette smoke extract promotes human pulmonary artery smooth muscle cells proliferation through protein kinase Cα-dependent induction of cyclin D1
Objective Exposure to cigarette smoke stimulates the proliferation of human pulmonary artery smooth muscle cells (HPASMCs) in vivo and in vitro. However, the molecular mechanism remains unclear. This study was aimed to investigate the role of signaling pathways involving protein kinase C alpha (PKCα) and cyclin D1 in the cigarette smoke extract (CSE)-induced HPASMCs proliferation. Methods Synchronized HPASMCs were treated with 5% CSE. Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and cell counting. Cell cycle was analyzed by flow cytometry with propidium iodide staining. Activation of PKCαwas measured by detecting the expression of PKCαprotein in the cytosolic and membrane fractions using Western blot analysis. Small interference RNA (siRNA) was used to knockdown PKCa and cyclin D1. The cyclin D1 mRNA was assessed by real-time RT-PCR. The PKCa and cyclinDl protein levels were detected by Western blotting. Results CSE (5%) led to PKCa activation. Inhibition of PKCa activity by using Go 6976 or siRNA-mediated knockdown of PKCa significantly attenuated CSE-induced cell proliferation and G1/S transition. Cyclin D1, one of key regulators of G1/S transition, was found to be upregulated by CSE (5%) at both mRNA and protein levels. CSE-stimulated cell proliferation and G1/S transition was markedly abolished by cyclin Dl siRNA. Moreover, Go 6976 or PKCa siRNA significantly suppressed CSE-induced upregulation of cyclin Dl at both mRNA and protein levels. Conclusions Cigarette smoke extract promotes HPASMCs proliferation through protein kinase C alpha-dependent induction of cyclin D1
目的检测蛋白激酶Cα(PKCα)和周期蛋白D1 (cyclinD1)在吸烟患或不患慢性阻塞性肺疾病(COPD)患者肺血管中的表达变化并探讨两者在吸烟导致的肺血管重构中的作用。方法将35例因肺癌而行肺叶切除术患者的外周肺组织标本分为三组：不吸烟肺功能正常组(正常组)10例,吸烟肺功能正常组(吸烟组)14例,吸烟伴COPD组(COPD组)11例。采用苏木精-伊红(HE)染色观察肺小动脉形态学改变,应用免疫组织化学染色法检测肺小动脉平滑肌细胞中α-平滑肌肌动蛋白(α-SMA)、增殖细胞核抗原(PCNA)、PKCα和cyclinD1蛋白表达,以PCNA阳性细胞百分率表示平滑肌细胞增殖指数(PI),采用实时荧光PCR法检测肺小动脉平滑肌细胞中]PKCa mRNA、cyclin D1 mRNA的表达。结果(1)肺小动脉血管壁面积占血管总面积的百分比值(WA%)在吸烟组和COPD组均高于正常组。(2)吸烟组和COPD组PI均高于正常组(P<0.01)。(3)吸烟组、COPD组PKCa蛋白和cyclinD1蛋白表达吸光度(A)值较正常组均显著升高(P<0.01)。(4)吸烟组、COPD组PKCa mRNA和cyclinD1 mRNA表达水平较正常组均显著升高(P<0.01)。(5)PKCa蛋白表达水平分别与WA%、PI呈正相关(P<0.01),cyclinD1蛋白表达水平分别与WA%、PI呈正相关(P<0.01),PKCa与cyclin D1在蛋白和mRNA表达水平均呈明显正相关(P<0.01)。结论在吸烟者及早期COPD患者肺血管中PKCa和cyclinD1表达均显著上调,二者可能参与肺血管重构中肺动脉平滑肌细胞的异常增殖。
第二部分周期蛋白D1在香烟烟雾提取物促进人肺动脉平滑肌细胞增殖中的作用
目的本文旨在探讨周期蛋白D1(cyclinD1)在香烟烟雾提取物(cigarette smoke extract,CSE)所致人肺动脉平滑肌细胞(HPASMCs)增殖中的作用。方法用不同浓度的CSE(1%,2.5%,5%,10%,20%)干预同步化的HPASMCs。构建反义cyclin D1基因真核表达载体(pIRES2-EGFP-ascyclin D1),采用脂质体介导基因转染法将空载体(pIRES2-EGFP)和pIRES2-EGFP-ascyclin D1导入正常HPASMCs后,分别进行CSE(5%)干预。细胞随机分为6组：对照组、空载体组、反义cyclinD1组、5%CSE组、空载体+5%CSE组、反义cyclin D1+5% CSE组。用台盼蓝染色法检测不同浓度CSE干预的HPASMCs活细胞率,采用四甲基偶氮唑盐比色法(MTT)、流式细胞术、增殖细胞核抗原(PCNA)染色法测定细胞增殖能力,用实时荧光RT-PCR和Western blot法分别检测cyclin D1 mRNA和蛋白的表达。结果低浓度的CSE(1%,2.5%,5%,10%)促进HPASMCs增殖,且浓度5%时细胞增殖能力最强,高浓度的CSE(20%)对HPASMCs具有毒性作用,抑制细胞增殖。反义cyclin D1基因真核表达载体pIRES2-EGFP-ascyclin D1成功构建,并成功转染入HPASMCs,转染后HPASMCs中cyclin D1的mRNA和蛋白表达水平较对照组均显著下降(P<0.05)。与对照组比较,5%CSE组cyclin D1的nRNA和蛋白表达水平均明显升高(P<0.05),细胞增殖能力显著增强(P<0.05)。与5%CSE组比较,反义cyclin D1+5% CSE组cyclin D1 mRNA和蛋白表达水平均明显下降(P<0.05),细胞增殖能力显著降低(P<0.05)。结论CSE可通过上调cyclin D1表达促进HPASMCs增殖,反义cyclin D1基因真核表达载体可抑制CSE介导的HPASMCs增殖,提示cyclin D1在CSE促进HPASMCs增殖中发挥重要的正向调控作用。
第三部分蛋白激酶Cα和周期蛋白D1在香烟烟雾提取物促进人肺动脉平滑肌细胞增殖中的作用和关系
目的研究蛋白激酶Cα(PKCα)和周期蛋白D1(cyclin D1)在香烟烟雾提取物(CSE)促进人肺动脉平滑肌细胞(HPASMCs)增殖中的作用和关系。方法用5%浓度的CSE干预同步化的HPASMCs。通过Western blot法检测PKCα蛋白在胞浆和胞膜中的表达。采用小干扰RNA (siRNA)技术分别剔除PKCα和cyclin D1基因表达。用实时荧光RT-PCR法检测cyclin D1 mRNA表达,Western blot法检测PKCα和cyclin D1蛋白的表达。采用四甲基偶氮唑盐比色法(MTT)和细胞计数法测定细胞增殖,流式细胞术观察细胞周期变化。结果5%CSE可激活PKCα,PKCα蛋白由胞浆转移至胞膜表达。通过Go6976抑制PKCα活性或采用siRNA技术剔除PKCα基因表达可显著抑制CSE介导的HPASMCs增殖和细胞周期的G1期向S期转换。5%CSE促进HPASMCs增殖中,cyclin D1mRNA和蛋白表达水平均明显升高。转染cyclin D1 siRNA可抑制CSE介导的HPASMCs增殖。此外,使用Go6976或转染PKCa siRNA可显著抑制CSE介导的HPASMCs中cyclin D1 mRNA和蛋白表达水平升高。结论香烟烟雾提取物通过蛋白激酶Cα途径上调周期蛋白D1表达促进人肺动脉平滑肌细胞增殖。
Background and Objective
Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease, its prevalence rate is high, and so does the mortality it caused. Pulmonary vascular remodeling hinders the respiration process and may result in pulmonary hypertension, which is one of the principal factors on reducing the survival rate of COPD patients. Controlling pulmonary vascular remodeling can effectively prevent the formation of pulmonary hypertension, which is of great significance in the clinical treatment of COPD and cor pulmonale. Previous researchers mainly focused on the pathogenesis of hypoxic pulmonary hypertension and deem that hypoxia-induced pulmonary vasoconstriction and pulmonary vascular remodeling lead to the formation of pulmonary hypertension. In recent studies, it has been reported that pulmonary vascular remodeling occurs in smokers with normal lung functions and mild COPD patients without hypoxemia. Exposure to cigarette smoke for 6 months produces pulmonary hypertension in guinea pigs. These findings suggest that cigarette smoke may also promote pulmonary vascular remodeling, leading to pulmonary hypertension. The abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of major morphological features of pulmonary vascular remodeling induced by smoking. Cigarette smoke can result in an abnormal proliferation of pulmonary artery smooth muscle cells. However, the intrinsic mechanism has not been thoroughly understood yet.
The proliferation of cells is closely related with cell cycle changes. While in a cell, cyclin plays a very important role in the regulation of cell cycle. In the Gl phase, cyclin D combines with cyclin-dependent kinase 4 (CDK4) or CDK6 to form complexes. These complexes phosphorylate retinoblastoma protein (Rb) and other family members. Phosphorylation of Rb activates the transcription factors and mediates the transcription of genes required for the cell to progress into S phase, which leads to the cell proliferation. Cyclin D1 is one of the major subtypes of cyclin D, and it regulates the G1/S phase transition. Cyclin D1 has been proven to be related to the proliferation of carcinoma cells. So far, few studies have been reported on the role of cyclin D1 in the vascular smooth-muscle cell proliferation. The effect of cyclin D1 on the proliferation of PASMCs remains to be understood.
Protein kinase C (PKC), which is one of crucial members in serine-threonine kinase family, is a key intracellular signal transduction molecule. At present, PKC has been found with at least 12 isoforms, and functions of PKC isoforms in different cells vary greatly. Among the PKC isoforms, PKCαis generally reported to be a positive mediator for the vascular smooth muscle cells proliferation. Our previous study has shown that PKCαwas closely associated with the abnormal proliferation of the rat pulmonary arterial smooth muscle cells induced by cigarette smoking extract. However, the downstream mechanism of PKCαhas remained unclear so far. PKCαhas been implicated in the up-regulation of the cyclin D1 expression in several types of cultured cells. In HPASMCs, however, the existence of a similar mechanism needs to be proven.
In this study, we investigate the pathobiology of human pulmonary artery smooth muscle cell proliferation induced by cigarette smoke from the perspective of the cell cycle control and the intracellular signal transduction. Our study may provide the basis for a new therapeutic approach for addressing COPD associated with pulmonary hypertension.
Part one The changes of protein kinase Ca and cyclin D1 expressions in pulmonary artery from smokers with and without chronic obstructive pulmonary disease
Objective The purpose of this study was to investigate the changes of protein kinase Ca (PKCa) and cyclin D1 expressions in pulmonary artery from smokers with normal lung function and smokers with mild to moderate chronic obstructive pulmonary disease (COPD). Methods The peripheral lung tissues were obtained from 10 nonsmokers with normal lung function (nonsmoker group),14 smokers with normal lung function (smoker group),11 smokers with mild to moderate COPD (COPD group). The morphological changes of pulmonary arteries were observed by hematoxylin-eosin staining. The expressions of a-smooth muscle actin (a-SMA), proliferating cell nuclear antigen (PCNA), PKCa and cyclin D1 proteins in pulmonary artery smooth muscle (PASM) were determined by immunohistochemical staining. The percentages of PCNA-positive cells were considered as the smooth muscle cells proliferation index (PI). The mRNA expressions of PKCa and cyclin D1 in PASM were evaluated by real-time fluorescence PCR. Results Morphometry analysis showed that the ratio of pulmonary artery wall area to total area (WA%) in smoker group and COPD group was significantly greater than that in nonsmoker group (P< 0.01). The PASMCs proliferation index in smoker group and COPD group was obviously higher than that in nonsmoker group (P< 0.01). The protein levels of PKCa and cyclin D1 in PASMCs were markedly increased in smoker group and COPD group as compared with nonsmoker group (P< 0.01). The mRNA expressions of PKCa and cyclin D1 in PASMCs were significantly elevated in smoker group and COPD group as compared with nonsmoker group(P< 0.01). Significant correlations were found between PKCa protein and WA% or PI (P< 0.01). Correlations between cyclin D1 protein and WA% or PI also existed (P< 0.01). The expression of PKCa positively correlated with the expression of cyclin D1 at both protein and mRNA levels (P< 0.01). Conclusions Increased expressions of PKCa and cyclin D1 might be involved in the pathogenesis of abnormal proliferation of PASMCs in smokers with normal lung function and smokers with mild to moderate COPD.
Part two The role of cyclin D1 in human pulmonary artery smooth muscle cells proliferation induced by cigarette smoke extract
Objective The present study was aimed to investigate the role of cyclin D1 in human pulmonary artery smooth muscle cells (HPASMCs) proliferation induced by cigarette smoke extract (CSE). Methods Synchronized HPASMCs were treated with different concentrations of CSE (1%,2.5%,5%,10%,20%). The antisense eukaryotic expression vector of cyclin D1 gene (pIRES2-EGFP-ascyclin D1) was recombinated. The recombinant and empty vector were separately transfected into normal HPASMCs using liposome. Then the cells were treated with or without 5% CSE. The cells were randomly divided into six groups:control group, vector group, antisense cyclin D1 group,5% CSE treated group, vector+5% CSE treated group and antisense cyclin Dl+5% CSE treated group. The proliferation of HPASMCs was examined by cell cycle analysis, MTT assay and proliferation cell nuclear antigen (PCNA) immunocytochemical staining. The expressions of cyclin D1 mRNA and protein were detected by real-time fluorescence RT-PCR and Western blot, respectively. Results Low concentrations of CSE (1%,2.5%,5%,10%) stimulated proliferation of HPASMCs, with its maximal effect at concentration of 5%. On the contrary, high concentrations of CSE (20%) were the inhibitory of cell proliferation as a result of cytotoxicity. The antisense eukaryotic expression vector of cyclin D1 gene was constructed and transfected into HPASMCs successfully. The cyclin D1 mRNA and protein levels in antisense cyclin D1 group were significantly lower than those in control group (P < 0.05). In 5% CSE group, the cyclin D1 mRNA and protein levels were elevated significantly compared with those in control group (P< 0.05), and the indicators of cell proliferation were increased as well (P<0.05). The cyclin D1 mRNA and protein levels, the indicators of cell proliferation in antisense cyclin D1+5% CSE group were remarkably lower than those in 5% CSE group (P< 0.05). Conclusions CSE could promote HPASMCs proliferation through up-regulation of cyclin D1 expression. PIRES2-EGFP-ascyclin D1 could attenuate CSE-induced proliferation of HPASMCs by suppressing the expression of cyclin D1, which implicates that cyclin D1 might be involved in the process of HPASMCs proliferation stimulated by CSE.
Part three Cigarette smoke extract promotes human pulmonary artery smooth muscle cells proliferation through protein kinase Cα-dependent induction of cyclin D1
Objective Exposure to cigarette smoke stimulates the proliferation of human pulmonary artery smooth muscle cells (HPASMCs) in vivo and in vitro. However, the molecular mechanism remains unclear. This study was aimed to investigate the role of signaling pathways involving protein kinase C alpha (PKCα) and cyclin D1 in the cigarette smoke extract (CSE)-induced HPASMCs proliferation. Methods Synchronized HPASMCs were treated with 5% CSE. Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and cell counting. Cell cycle was analyzed by flow cytometry with propidium iodide staining. Activation of PKCαwas measured by detecting the expression of PKCαprotein in the cytosolic and membrane fractions using Western blot analysis. Small interference RNA (siRNA) was used to knockdown PKCa and cyclin D1. The cyclin D1 mRNA was assessed by real-time RT-PCR. The PKCa and cyclinDl protein levels were detected by Western blotting. Results CSE (5%) led to PKCa activation. Inhibition of PKCa activity by using Go 6976 or siRNA-mediated knockdown of PKCa significantly attenuated CSE-induced cell proliferation and G1/S transition. Cyclin D1, one of key regulators of G1/S transition, was found to be upregulated by CSE (5%) at both mRNA and protein levels. CSE-stimulated cell proliferation and G1/S transition was markedly abolished by cyclin Dl siRNA. Moreover, Go 6976 or PKCa siRNA significantly suppressed CSE-induced upregulation of cyclin Dl at both mRNA and protein levels. Conclusions Cigarette smoke extract promotes HPASMCs proliferation through protein kinase C alpha-dependent induction of cyclin D1
引文
1. Santos S, Peinado VI, Ramirez J, et al. Characterization of pulmonary vascular remodelling in smokers and patients with mild COPD. Eur Respir J,2002,19(4): 632-638.
2. Wright JL, Churg A. Effect of long-term cigarette smoke exposure on pulmonary vascular structure and function in the guinea pig. Exp lung Res,1991,17(6): 997-1009.
3. Ferrer E, Peinado VI, Diez M, et al. Effects of cigarette smoke on endothelial function of pulmonary arteries in the guinea pig. Respir Res,2009,10:76.
4. Wang S, Desai D, Wright G, et al. Effects of protein kinase C alpha overexpression on A7r5 smooth muscle cell proliferation and differentiation. Exp Cell Res,1997,236(1): 117-126.
5. Grillo M, Bott MJ, Khandke N, et al. Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells:Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression. Breast Cancer Res Treat,2006,95(2):185-194.
6. Qiao LF, Xu YJ, Liu XS, et al. Role of protein Ca and cyclin Dl in the profileration of airway smooth muscle in asthmatic rats. Chin Med J,2008,121(20):2070-2076.
7. Hu J, Xu YJ, Zhang ZY, et al. Effect of cigarette smoke extract on proliferation of rat pulmonary artery muscle cells and relevant roles of protein kinase C. Chin Med J,2007, 120(17):1523-1528.
8. Chronic obstructive pulmonary disease study group in Chinese Society of Respiratory Diseases. Diagnosis and treatment guidelines of chronic obstructive pulmonary disease. Chin J Intern Med (Chinese),2007,46(3):254-261.
9. Chaouat A, Naeije R, Weitzenblum E. Pulmonary hypertension in COPD. Eur Respir J, 2008,32(5):1371-1385.
10. Liang W, Ray JB, He JZ, et al. Regulation of proliferation and membrane potential by chloride currents in rat pulmonary artery smooth muscle cells. Hypertension,2009, 54(2):286-93.
11. Mandegar M, Fung YC, Huang W, et al. Cellular and molecular mechanisms of pulmonary vascular remodeling:role in the development of pulmonary hypertension. Microvasc Res,2004,68(2):75-103.
12. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature,1988,334(6184):661-665.
13. Malhotra A, Kang BP, Opawumi D, et al. Molecular biology of protein kinase C signaling in cardiac myocytes. Mol Cell Biochem,2001,225(1-):97-107.
14. Berkenbosch JW, Baribeau J, Ferretti E, et al. Role of protein kinase C and phosphatases in the vasculature of neonatal piglets. Crit Care Med,2001,29(6): 1229-1233.
15. Dempsey EC, Frid MG, Aldashev AA, et al. Heterogeneity in the proliferative response of bovine pulmonary artery smooth muscle cells to mitogens and hypoxia:importance of protein kinase C. Can J Physiol Pharmacol,1997,75 (7):936-944.
16. Okazaki J, Mawatari K, Liu B, et al. The effect of protein kinase C and its alpha subtype on human vascular smooth muscle cell proliferation, migration and fibronectin production. Surgery,2000,128(2):192-197.
17. Grillo M, Bott MJ, Khandke N, et al. Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells:Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression. Breast Cancer Res Treat,2006,95(2):185-194.
18. Dyson N. The regulation of E2F by pRB-family proteins. Genes Dev,1998,12(15): 2245-2262.
19. Imoto M, Tanabe K, Simizu S, et al. Inhibition of cyclin D1 expression and induction of apoptosis by inostamycin in small cell lung carcinoma cells. Jpn J Cancer Res,1998, 89(3):315-322.
20. Marsit CJ, Black CC, Posner MR, et al. A genotype-phenotype examination of cyclin D1 on risk and outcome of squamous cell carcinoma of the head and neck. Clin Cancer Res,2008,14(8):2371-2377.
21. Zhang YM, Wang KQ, Zhou GM, et al. Endothelin-1 promoted proliferation of vascular smooth muscle cell through pathway of extracellular signal-regulated kinase and cyclin D1. Acta Pharmacol Sin,2003,24(6):563-568.
22. Lee SD, Lee DS, Chun YG, et al. Cigarette smoke extract induces endothelin-1 via protein kinase C in pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol,2001,281(2):L403-L411.
23. Black JD. Protein kinase C-mediated regulation of the cell cycle. Front Biosci,2000,5: D406-D423.
24. Soh JW, Weinstein IB. Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin Dl and related genes. J Biol Chem,2003,278(36): 34709-34716.
1. Barbera JA, Peinado VI, Santos S. Pulmonary hypertension in chronic obstructive pulmonary disease. Eur Respir J,2003,21(5):892-905.
2. Santos S, Peinado VI, Ramirez J, et al. Characterization of pulmonary vascular remodelling in smokers and patients with mild COPD. Eur Respir J,2002,19(4): 632-638.
3. Hu J, Xu YJ, Zhang ZX, et al. Effect of cigarette smoke extract on proliferation of rat pulmonary artery smooth muscle cells and the relevant roles of protein kinase C. Chin Med J,2007,120(17):1523-1528.
4. Wright JL, Tai H, Churg A. Vasoactive mediators and pulmonary hypertension after cigarette smoke exposure in the guinea pig. J Appl Physiol,2006,100(2):672-678.
5. Ferrer E, Peinado VI, Diez M, et al. Effects of cigarette smoke on endothelial function of pulmonary arteries in the guinea pig. Respir Res,2009,10:76.
6. Nakagawa M, Ohno T, Maruyama R, et al. Sesquiterpene lactone suppresses vascular smooth muscle cell proliferation and migration via inhibition of cell cycle progression. Biol Pharm Bull,2007,30(9):1754-1757.
7. Su Y, Han W, Giraldo C, et al. Effect of cigarette smoke extract on nitric oxide synthase in pulmonary artery endothelial cells. Am J Respir Cell Mol Biol,1998,19(5): 819-825.
8. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods,2001,25(4):402-408.
9. Sobin SS, Chen PC. Ultrastructural changes in the pulmonary arterioles in acute hypoxic pulmonary hypertention in the rat. High Alt Med Biol,2000,1(4):311-322.
10. Wright JL, Churg A. Effect of long-term cigarette smoke exposure on pulmonary vascular structure and function in the guinea pig. Exp lung Res,1991,17(6):997-1009.
11. Janoff A, Pryor WA, Bengali ZH. Effects of tobacco smoke components on cellular and biochemical processes in the lung. Am Rev Respir Dis,1987,136(4):1058-1064.
12. Lan MY, Ho CY, Lee TC, et al. Cigarette smoke extract induces cytotoxicity on human nasal epithelial cells. Am J Rhinol,2007,21(2):218-223.
13. Gelbman BD, Heguy A, O'Connor TP, et al. Upregulation of pirin expression by chronic cigarette smoking is associated with bronchial epithelial cell apoptosis. Respir Res,2007,8:10.
14. Liu X, Togo S, Al-Mugotir M, et al. NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract. Respir Res,2008,9:66.
15. Cucina A, Fuso A, Coluccia P, et al. Nicotine inhibits apoptosis and stimulates proliferation in aortic smooth muscle cells through a functional nicotinic acetylcholine receptor. J Surg Res,2008,150(2):227-235.
16. Villablanca AC. Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro. J Appl Physiol,1998,84(6):2089-2098.
17. Kamer AR, El-Ghorab N, Marzec N, et al.Nicotine induced proliferation and cytokine release in osteoblastic cells. Int J Mol Med,2006,17(1):121-127.
18. Baldin V, Lukas J, Marcote MJ, et al. yclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev,1993,7(5):812-821.
19. Karpurapu M, Wang D, Quyen DV, et al. Cyclin D1 is a bona fide target gene of NFATcl and is sufficient in the mediation of injury-induced vascular wall remodeling. J Biol Chem,2010,285(5):3510-3523.
20. Chen L, Wang WY, Wang YP. Inhibitory effects of lithospermic acid on proliferation and migration of rat vascular smooth muscle cells. Acta Pharmacol Sin,2009,30(9): 1245-1252.
21. Liang KW, Yin SC, Ting CT, Lin SJ, Hsueh CM, Chen CY, Hsu SL. Berberine inhibits platelet-derived growth factor-induced growth and migration partly through an AMPK-dependent pathway in vascular smooth muscle cells. Eur J Pharmacol,2008, 590:343-354.
1. Wright JL, Tai H, Churg A. Vasoactive mediators and pulmonary hypertension after cigarette smoke exposure in the guinea pig. J Appl Physiol 2006; 100:672-678.
2. Hu J, Xu YJ, Zhang ZX, et al. Effect of cigarette smoke extract on proliferation of rat pulmonary artery smooth muscle cells and the relevant roles of protein kinase C. Chin Med J,2007,120:1523-1528.
3. Musashi M, Ota S, Shiroshita N. The role of protein kinase C isoforms in cell proliferation and apoptosis. Int J Hematol,2000,72:12-19.
4. Haller H, Lindschau C, Quass P, et al. Differentiation of vascular smooth muscle cells and the regulation of protein kinase C-alpha. Circ Res,1995,76:21-29.
5. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science,1992,258:607-614.
6. Okazaki J, Mawatari K, Liu B, et al. The effect of protein kinase C and its alpha subtype on human vascular smooth muscle cell proliferation, migration and fibronectin production. Surgery,2000,128:192-197.
7. Leszczynski D, Joenvaara S, Foegh ML. Protein kinase C-alpha regulates proliferation but not apoptosis in rat coronary vascular smooth muscle cells. Life Sci,1996,58: 599-606.
8. Itoh H, Yamamura S, Ware JA, et al. Differential effects of protein kinase C on human vascular smooth muscle cell proliferation and migration. Am J Physiol Heart Circ Physiol,2001,281:H359-H370.
9. Sherr CJ. Mammalian G1 cyclins. Cell,1993,73:1059-1065.
10. Resnitzky D, Gossen M, Bujard H, et al. Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol,1994,14: 1669-1679.
11. Quasnichka H, Slater SC, Beeching CA, et al. Regulation of smooth muscle cell proliferation by beta-catenin/T-cell factor signaling involves modulation of cyclin D1 and p21 expression. Circ Res,2006,99:1329-1337.
12. Pestana IA, Vazquez-Padron RI, Aitouche A, et al. Nicotinic and PDGF-receptor function are essential for nicotine-stimulated mitogenesis in human vascular smooth muscle cells. J Cell Biochem,2005,96:986-995.
13. Li F, Li L, Qin X, et al. Apelin-induced vascular smooth muscle cell proliferation:the regulation of cyclin D1. Front Biosci,2008,13:3786-3792.
14. Qiao LF, Xu YJ, Liu XS, et al. PKC promotes proliferation of airway smooth muscle cells by regulating cyclinD1 expression in asthmatic rats. Acta Pharmacol Sin,2008,29: 677-686.
15. Okumura M, Yamamoto M, Sakuma H, et al. Leptin and high glucose stimulate cell proliferation in MCF-7 human breast cancer cells:reciprocal involvement of PKC-alpha and PPAR expression. Biochim Biophys Acta,2002,1592:107-116.
16. Bogatkevich GS, Gustilo E, Oates JC, et al. Distinct PKC isoforms mediate cell survival and DNA synthesis in thrombin-induced myofibroblasts. Am J Physiol Lung Cell Mol Physiol,2005,288:L190-L201.
17. Su Y, Han W, Giraldo C, et al. Effect of cigarette smoke extract on nitric oxide synthase in pulmonary artery endothelial cells. Am J Respir Cell Mol Biol,1998,19:819-825.
18. Lee SD, Lee DS, Chun YG, et al. Cigarette smoke extract induces endothelin-1 via protein kinase C in pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol,2001,281:L403-L411.
19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods,2001,25:402-408.
20. Shin VY, Wu WK, Chu KM, et al. Functional role of beta-adrenergic receptors in the mitogenic action of nicotine on gastric cancer cells. Toxicol Sci,2007,96:21-29.
21. Santos S, Peinado VI, Ramirez J, et al. Characterization of pulmonary vascular remodelling in smokers and patients with mild COPD. Eur Respir J,2002,19: 632-638.
22. Wright JL, Churg A. Effect of long-term cigarette smoke exposure on pulmonary vascular structure and function in the guinea pig. Exp lung Res,1991,17:997-1009
23. Ferrer E, Peinado VI, Diez M, et al. Effects of cigarette smoke on endothelial function of pulmonary arteries in the guinea pig. Respir Res,2009,10:76.
24. Newton AC. Regulation of the ABC kinases by phosphorylation:protein kinase C as a paradigm. Biochem J,2003,370 (Pt 2):361-371.
25. Park JW, Kim HP, Lee SJ, et al. Protein kinase C alpha and zeta differentially regulate death-inducing signaling complex formation in cigarette smoke extract-induced apoptosis. J Immunol,2008,180:4668-4678.
26. Chu M, Guo J, Chen CY. Long-term exposure to nicotine, via ras pathway, induces cyclin D1 to stimulate G1 cell cycle transition. J Biol Chem,2005,280:6369-6379.
27. Hu H, Zhang S, Zhu S. Influence of aspirin and cigarette smoke extract on the expression of cyclin D1 and effects of cell cycle in esophageal squamous cell carcinoma cell line. Dis Esophagus,2009,22:310-316.
28. Soh JW, Weinstein IB. Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes. J Biol Chem,2003,278: 34709-34716.
1. Fabbri LM, Hurd SS. GOLD scientific committee. Global strategy for the diagnosis, management and prevention of COPD:2003 update. Eur Respir J,2003,22(1):1-2.
2. Barbera, JA, Peinado, VI, Santos, S. Pulmonary hypertension in chronic obstructive pulmonary disease. Eur Respir J,2003,21(5):892-905.
3. Kubo K, Ge RL, Koizumi T, et al. Pulmonary artery remodeling modifies pulmonary hypertension during exercise in severe emphysema. Respir Physiol,2000,120(1):71-79.
4. Hislop A, Reid L. New findings in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension. Br J Exp Pathol,1976,57(5):542-554.
5. Meyrick B, Reid L. Hypoxia-induced structural changes in the media and adventitia of the rat hilar pulmonary artery and their regression. Am J Pathol,1980,100(1):151-178.
6. Meyrick B, Reid L. Hypoxia and incorporation of 3H-thymidine by cells of the rat pulmonary arteries and alveolar wall. Am J Pathol,1979,96(1):51-70.
7. Yi ES, Kim H, Ahn H, et al. Distribution of obstructive intimal lesions and their cellular phenotypes in chronic pulmonary hypertension. Am J Respir Crit Care Med, 2000,162(4 Pt1):1577-1586.
8. Yu Y, Sweeney M, Zhang S, et al. PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression. Am J Physiol Cell Physiol,2003, 284(2):C316-C330.
9. Tanabe Y, Saito M, Ueno A, et al. Mechanical stretch augments PDGF receptor beta expression and protein tyrosine phosphorylation in pulmonary artery tissue and smooth muscle cells. Mol Cell Biochem,2000,215(1-2):103-113.
10. Ambalavanan N, Bulger A, Philips III JB. Hypoxia-induced release of peptide growth factors from neonatal porcine pulmonary artery smooth muscle cells. Biol Neonate, 1999,76(5):311-319.
11. Perros F, Montani D, Dorfmuller P, et al. Platelet-derived growth factor expression and function in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med, 2008,178(1):81-88.
12. Tantini B, Manes A, Fiumana E, et al. Antiproliferative effect of sildenafil on human pulmonary artery smooth muscle cells. Basic Res Cardiol,2005,100(2):131-138.
13. Oqawa A, Firth AL, Yao W, et al. Prednisolone inhibits PDGF-induced nuclear translocation of NF-kappaB in human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol,2008,295(4):L648-L657.
14. Fehrenbach H, Kasper M, Haase M, et al. Differential immunolocalization of VEGF in rat and human adult lung, and in experimental rat lung fibrosis:light, fluorescence, and electron microscopy. Anat Rec,254(1):61-73.
15. BelAlba RS, Diordjevic T, Bonello S, et al. Redox-sensitive regulation of the HIF pathway under non-hypoxic conditions in pulmonary artery smooth muscle cells. Biol Chem,2004,385(3-4):249-257.
16. Santos S, Peinado VI, Ramirez J, et al. Enhanced expression of vascular endothelial growth factor in pulmonary arteries of smokers and patients with moderate chronic obstructive pulmonary disease. Am J Respir Crit Care Med,2003,167(9):1250-1256.
17.颜浩,陈文彬,程德云,等.碱性成纤维细胞生长因子在低氧性肺动脉高压大鼠肺内表达及分布的观察.中华结核和呼吸杂志,1997,20(6)：344-346.
18. Wedgwood S, Devol JM, Grobe A, et al. Fibroblast growth factor-2 expression is altered in lambs with increased pulmonary blood flow and pulmonary hypertension. Pediatr Res,2007,61(1):32-36.
19. Black SM, DeVol JM, Wedgwood S. Regulation of fibroblast growth factor-2 expression in pulmonary arterial smooth muscle cells involves increased reactive oxygen species generation. Am J Physiol Cell Physiol,2008,294(1):C345-C354.
20. Birker-Robaczewska M, Studer R, Haenig B, et al. bFGF induces S1P1 receptor expression and functionality in human pulmonary artery smooth muscle cells. J Cell Biochem,2008,105(4):1139-1145.
21. Ambalavana N, Bulger A, Philips III JB. Hypoxia-induced release of peptide growth factors from neonatal porcine pulmonary artery smooth muscle cells. Biol Neonate, 1999,76(5):311-319.
22. Feng X, Cai Y. Hypoxia and endothelin-1 stimulate DNA synthesis of pulmonary artery smooth muscle cells. Chin Med Sci J,1996,11(1):28-31.
23. Yamanaka R, Otsuka F, Nakamura K, et al. Involvement of the bone morphogenetic protein system in endothelin-and aldosterone-induced cell proliferation of pulmonary arterial smooth muscle cell isolated from human patients with pulmonary arterial hypertension. Hypertens Res,2010, Epub ahead of print.
24.解卫平,王虹,齐栩,等.新型ATP敏感性钾通道开放剂埃他卡林对兔肺动脉平滑肌细胞增殖的影响.中华结核和呼吸杂志,2004,27(11)：752-755.
25. Zamora MA, Dempsey EC, WalchakSJ, et al. BQ123, an ETA receptor antagonist, inhibits endothelin-1-mediated proliferation of human pulmonary artery smooth muscle cells. Am J Respir Cell Mol Biol,1993,9(4):429-433.
26. Davie N, Haleen SJ, Upton PD, et al. ET(A) and ET(B) receptors modulate the proliferation of human pulmonary artery smooth muscle cells. Am J Respir Crit Care Med,2002,165(3):398-405.
27. Li H, Chen SJ, Chen YF, et al. Enhanced endothelin-1 and endothelin receptor gene expression in chronic hypoxia. J Appl Physiol,1994,77(3):1451-1459.
28. Fredenburgh LE, Liang OD, Macias AA, et al. Absence of cyclooxygenase-2 exacerbates hypoxia-induced pulmonary hypertension and enhances contractility of vascular smooth muscle cell. Circulation,2008,117(16):2114-2122.
29. Wort SJ, Woods M, Warner TD, et al. Cyclooxygenase-2 acts as an endogenous brake on endothelin-1 release by human pulmonary artery smooth muscle cells:implications for pulmonary hypertension. Mol Pharmacol,2002,62(5):1147-1153.
30. Lee SL, Wang WW, Lanzillo JJ, et al. Serotonin produces both hyperplasia and hypertrophy of bovine pulmonary artery smooth muscle cells in culture. Am J Physiol, 1994,266 (1 Pt 1):L46-L52.
31. Fanburg BL, Lee SL. A new role for an old molecule:Serotonin as a mitogen. Am J Physiol,1997,272 (5 Pt 1):L795-L806.
32. Eddahibi S, Fabre V, Boni C, et al. Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells. Relationship with the mitogenic action of serotonin. Circ Res,1999,84(3):329-336.
33. Lee SL, Wang WW, Moore BJ, et al. Dual effect of serotonin on growth of bovine pulmonary artery smooth muscle cells in culture. Circ Res,1991,68(5):1362-1368.
34. Pitt BR, Weng W, Steve AR, et al. Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture. Am J Physiol,1994,266 (2Pt1):L178-L186.
35. Eddahibi S, Hanoun N, Lanfumey L, et al. Attenuated hypoxia pulmonary hypertension in mice lacking the 5-hydroxytryptamine transporter gene. J Clin Invest,2000,105(11): 1555-1562.
36. Eddahibi S, Adnot S, Frisdal E, et al. Dexfenfluramine-associated changes in 5-hydroxytryptamine transporter expression and development of hypoxic pulmonary hypertension in rats. J Pharmacol Exp Ther,2001,297(1):148-154.
37. MacLean MR, Deuchar GA, Hicks MN, et al. Overexpression of the 5-hydroxytryptamine transporter gene:effect on pulmonary hemodynamics and hypoxia-induced pulmonary hypertension. Circulation,2004,109(17):2150-2155.
38. Fagan KA, Tyler RC, Fouty BW, et al. Relative contribution of endothelial, inducible and neuroal NOS to tone in the murine pulmonary circulation. Am J Phsiol,1999,277 (3 Pt 1):472-478.
39. Ambalavanan N, Mariani G, Bulger A, et al. Role of nitric oxide in regulating neonatal porcine pulmonary artery smooth muscle cell proliferation. Biol Neonate,1999,76(5): 291-300.
40. Thomae KR, Nakayama DK, Billiar TR, et al. The effect of nitric oxide on fetal pulmonary artery smooth muscle growth. J Surg Res,1995,59(3):337-343.
41. Giaid A, Saleh D. Reduced expression of endothelial nitric oxide in the lung of patients with pulmonary hypertension. N Engl J Med,1995,333(4):214-221.
42.丁毅鹏,徐永健,张珍祥.蛋白激酶C对低氧猪肺动脉平滑肌细胞结构型一氧化氮合酶mRNA表达作用的探讨.中华结核和呼吸杂志,2001,24(10)：588-591.
43. Horstman DJ, Frank DU, Rich GF. Prolonged in haled NO attenuates hypoxia, but not monocrotaline-induced, pulmonary vascular remodeling in rats. Anesth Analg,1998, 86(1):74-81.
44. Mitani Y, Maruyama K, Sakurai M. Prolonged administration of L-arginine ameliorates chronic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation,1997,96(2):689-697.
45. Puikuan K, Chunyu Z, Jin F, et al. Inhalation of nebulized nitroglycerin, a nitric oxide donor, for the treatment of pulmonary hypertension induced by high pulmonary blood flow. Heart Vessels,2006,21(3):169-179.
46. Touyz R M, Berry C. Recent advances in angiotensin II signaling. Braz J Med Biol Res, 2002,35(9):1001-1015.
47.金立军,杨沙宁,黄从新,等.血管紧张素Ⅱ在肺动脉高压形成中的作用及其临床意义.临床荟萃,2001,16：264-265.
48. Zhang R, Wu Y, Zhao M, et al. Role of HIF-1 alpha in the regulation ACE and ACE2 expression in hypoxic human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol,2009,297(4):L631-640.
49. Yu TZ, Ma CT. Effects of angiotensin-converting enzyme and angiotensin II on hypoxia-induced proliferation of cultured intra-pulmonary artery smooth muscle cells. Acta Pharmacol Sin,2000,21(4):381-384.
50. Han SX, He GM, Wang T, et al. Losartan attenuates chronic cigarette smoke exposure-induced pulmonary arterial hypertension in rats:Possible involvement of angiotensin-converting enzyme-2. Toxicol Appl Pharmacol.2010, Epub ahead of print.
51. Morrell NW, Upton PD, Higham MA, et al. Angiotensin Ⅱ stimulates proliferation of human pulmonary artery smooth muscle cells via the ATI receptor. Chest,1998,114(1 Suppl):90S-91S.
52.涂明利,王汉琴,雷怀定,等.腺病毒介导反义血管紧张素Ⅱ1型受体cDNA对人肺动脉平滑肌细胞生物学行为的影响.中华结核和呼吸杂志,2005,28(4)：263-267.
53.易斌,钱桂生,白莉,等.低氧致大鼠肺动脉平滑肌细胞增殖过程中PTEN／Akt1表达的变化.中华结核和呼吸杂志,2008,31(8)：586-590.
54. Wang GS, Qian GS, Zhou DS, et al. JAK-STAT signaling pathway in pulmonary arterial smooth muscle cells is activated by hypoxia. Cell Biol Int,2005,29(7): 598-603.
55. Bai L, Yu Z, Qian G, et al. SOCS3 was induced by hypoxia and suppressed STAT3 phosphorylation in pulmonary arterial smooth muscle cells. Respir Physiol Neurobiol, 2006,152(1):83-91.
56. Wang C, Li JF, Zhao L, et al. Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells. Respir Res,2009,10(1):123.
57. Chen XY, Dun JN, Miao QF, et al. Fasudil hydrochloride hydrate, a Rho-kinase inhibitor, suppresses 5-hydroxytryptamine-induced pulmonary artery smooth muscle cell proliferation via JNK and ERK1/2 pathway. Pharmacology,2009,83(2):67-79.
58. Zhu Z, Xu Y, Zou H, Zhang Z, Ni W, Chen S. Effects of puerarin on pulmonary vascular remodeling and protein kinase C-alpha in chronic cigarette smoke exposure smoke-exposed rats. J Huazhong Univ Sci Technolog Med Sci,2008,28(1):27-32.
59. Burg ED, Remillard CV, Yuan JX. Potassium channels in the regulation of pulmonary artery smooth muscle cell proliferation and apoptosis:pharmacotherapeutic implications. Br J Pharmacol,2008,153(Suppl 1):S99-S111.
60. Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol,1995,268(4 Pt 1):C799-C822.
61. Muraki S, Tohse N, Seki S, et al. Decrease in the Ca2+-activated K+current of pulmonary arterial smooth muscle in pulmonary hypertension rats. Naunyn Schmiedebergs Arch Pharmacol,2001,364(3):183-192.
62. Wang J, J uhaszova M, Rubin LJ, et al. Hypoxia inhibits gene expression of voltage-gated K+channel asubunits in pulmonary artery smooth muscle cells. J Clin Invest,1997,100(9):2347-2353.
63. Yuan JX, Aldinger AM, Juhaszova M, et al. Dysfunctional voltage-gated K+channels in pulmonary artery smooth muscle cells of patients with primary pulmonary hypertension. Circulation,1998,98(14):1400-1406.
64. Platoshyn O, Golovina VA, Bailey CL, et al. Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation. Am J Physiol Cell Physiol,2000, 279(5):C1540-C1549.
65. Baldin V, Lukas J, Marcote MJ, et al. Cyclin Dl is a nuclear protein required for cell cycle progression in G1. Genes Dev,1993,7(5):812-821.
66. Kadowaki M, Mizuno S, Demura Y, et al. Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation:the role of p27kip1. Respir Res,2007,8:77.
67. Yu L, Quinn DA, Garg HQ et al. Gene expression of cyclin-dependent kinase inhibitors and effect of heparin on their expression in mice with hypoxia-induced pulmonary hypertension. Biochem Biophys Res Commun.2006,345(4):1565-1572.
68. Chattergoon NN, D'Souza FM, Deng W, et al. Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol,2005,288(1):L202-L211.
69. Yu L, Quinn DA, Garg HG, et al. Cyclin-dependent kinase inhibitor p27Kip1, but not p21WAF1/Cip1, is required for inhibition of hypoxia-induced pulmonary hypertension and remodeling by heparin in mice. Circ Res,2005,97(9):937-945.
70. Lin CL, Zhang ZX, Xu YJ, et al. Focal adhesion kinase antisense oligodeoxynucleotides inhibit human pulmonary artery smooth muscle cells proliferation and promote human pulmonary artery smooth muscle cells apoptosis. Chin Med J,2005,118(1):20-26.
2. Wright JL, Churg A. Effect of long-term cigarette smoke exposure on pulmonary vascular structure and function in the guinea pig. Exp lung Res,1991,17(6): 997-1009.
3. Ferrer E, Peinado VI, Diez M, et al. Effects of cigarette smoke on endothelial function of pulmonary arteries in the guinea pig. Respir Res,2009,10:76.
4. Wang S, Desai D, Wright G, et al. Effects of protein kinase C alpha overexpression on A7r5 smooth muscle cell proliferation and differentiation. Exp Cell Res,1997,236(1): 117-126.
5. Grillo M, Bott MJ, Khandke N, et al. Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells:Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression. Breast Cancer Res Treat,2006,95(2):185-194.
6. Qiao LF, Xu YJ, Liu XS, et al. Role of protein Ca and cyclin Dl in the profileration of airway smooth muscle in asthmatic rats. Chin Med J,2008,121(20):2070-2076.
7. Hu J, Xu YJ, Zhang ZY, et al. Effect of cigarette smoke extract on proliferation of rat pulmonary artery muscle cells and relevant roles of protein kinase C. Chin Med J,2007, 120(17):1523-1528.
8. Chronic obstructive pulmonary disease study group in Chinese Society of Respiratory Diseases. Diagnosis and treatment guidelines of chronic obstructive pulmonary disease. Chin J Intern Med (Chinese),2007,46(3):254-261.
9. Chaouat A, Naeije R, Weitzenblum E. Pulmonary hypertension in COPD. Eur Respir J, 2008,32(5):1371-1385.
10. Liang W, Ray JB, He JZ, et al. Regulation of proliferation and membrane potential by chloride currents in rat pulmonary artery smooth muscle cells. Hypertension,2009, 54(2):286-93.
11. Mandegar M, Fung YC, Huang W, et al. Cellular and molecular mechanisms of pulmonary vascular remodeling:role in the development of pulmonary hypertension. Microvasc Res,2004,68(2):75-103.
12. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature,1988,334(6184):661-665.
13. Malhotra A, Kang BP, Opawumi D, et al. Molecular biology of protein kinase C signaling in cardiac myocytes. Mol Cell Biochem,2001,225(1-):97-107.
14. Berkenbosch JW, Baribeau J, Ferretti E, et al. Role of protein kinase C and phosphatases in the vasculature of neonatal piglets. Crit Care Med,2001,29(6): 1229-1233.
15. Dempsey EC, Frid MG, Aldashev AA, et al. Heterogeneity in the proliferative response of bovine pulmonary artery smooth muscle cells to mitogens and hypoxia:importance of protein kinase C. Can J Physiol Pharmacol,1997,75 (7):936-944.
16. Okazaki J, Mawatari K, Liu B, et al. The effect of protein kinase C and its alpha subtype on human vascular smooth muscle cell proliferation, migration and fibronectin production. Surgery,2000,128(2):192-197.
17. Grillo M, Bott MJ, Khandke N, et al. Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells:Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression. Breast Cancer Res Treat,2006,95(2):185-194.
18. Dyson N. The regulation of E2F by pRB-family proteins. Genes Dev,1998,12(15): 2245-2262.
19. Imoto M, Tanabe K, Simizu S, et al. Inhibition of cyclin D1 expression and induction of apoptosis by inostamycin in small cell lung carcinoma cells. Jpn J Cancer Res,1998, 89(3):315-322.
20. Marsit CJ, Black CC, Posner MR, et al. A genotype-phenotype examination of cyclin D1 on risk and outcome of squamous cell carcinoma of the head and neck. Clin Cancer Res,2008,14(8):2371-2377.
21. Zhang YM, Wang KQ, Zhou GM, et al. Endothelin-1 promoted proliferation of vascular smooth muscle cell through pathway of extracellular signal-regulated kinase and cyclin D1. Acta Pharmacol Sin,2003,24(6):563-568.
22. Lee SD, Lee DS, Chun YG, et al. Cigarette smoke extract induces endothelin-1 via protein kinase C in pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol,2001,281(2):L403-L411.
23. Black JD. Protein kinase C-mediated regulation of the cell cycle. Front Biosci,2000,5: D406-D423.
24. Soh JW, Weinstein IB. Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin Dl and related genes. J Biol Chem,2003,278(36): 34709-34716.
1. Barbera JA, Peinado VI, Santos S. Pulmonary hypertension in chronic obstructive pulmonary disease. Eur Respir J,2003,21(5):892-905.
2. Santos S, Peinado VI, Ramirez J, et al. Characterization of pulmonary vascular remodelling in smokers and patients with mild COPD. Eur Respir J,2002,19(4): 632-638.
3. Hu J, Xu YJ, Zhang ZX, et al. Effect of cigarette smoke extract on proliferation of rat pulmonary artery smooth muscle cells and the relevant roles of protein kinase C. Chin Med J,2007,120(17):1523-1528.
4. Wright JL, Tai H, Churg A. Vasoactive mediators and pulmonary hypertension after cigarette smoke exposure in the guinea pig. J Appl Physiol,2006,100(2):672-678.
5. Ferrer E, Peinado VI, Diez M, et al. Effects of cigarette smoke on endothelial function of pulmonary arteries in the guinea pig. Respir Res,2009,10:76.
6. Nakagawa M, Ohno T, Maruyama R, et al. Sesquiterpene lactone suppresses vascular smooth muscle cell proliferation and migration via inhibition of cell cycle progression. Biol Pharm Bull,2007,30(9):1754-1757.
7. Su Y, Han W, Giraldo C, et al. Effect of cigarette smoke extract on nitric oxide synthase in pulmonary artery endothelial cells. Am J Respir Cell Mol Biol,1998,19(5): 819-825.
8. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods,2001,25(4):402-408.
9. Sobin SS, Chen PC. Ultrastructural changes in the pulmonary arterioles in acute hypoxic pulmonary hypertention in the rat. High Alt Med Biol,2000,1(4):311-322.
10. Wright JL, Churg A. Effect of long-term cigarette smoke exposure on pulmonary vascular structure and function in the guinea pig. Exp lung Res,1991,17(6):997-1009.
11. Janoff A, Pryor WA, Bengali ZH. Effects of tobacco smoke components on cellular and biochemical processes in the lung. Am Rev Respir Dis,1987,136(4):1058-1064.
12. Lan MY, Ho CY, Lee TC, et al. Cigarette smoke extract induces cytotoxicity on human nasal epithelial cells. Am J Rhinol,2007,21(2):218-223.
13. Gelbman BD, Heguy A, O'Connor TP, et al. Upregulation of pirin expression by chronic cigarette smoking is associated with bronchial epithelial cell apoptosis. Respir Res,2007,8:10.
14. Liu X, Togo S, Al-Mugotir M, et al. NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract. Respir Res,2008,9:66.
15. Cucina A, Fuso A, Coluccia P, et al. Nicotine inhibits apoptosis and stimulates proliferation in aortic smooth muscle cells through a functional nicotinic acetylcholine receptor. J Surg Res,2008,150(2):227-235.
16. Villablanca AC. Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro. J Appl Physiol,1998,84(6):2089-2098.
17. Kamer AR, El-Ghorab N, Marzec N, et al.Nicotine induced proliferation and cytokine release in osteoblastic cells. Int J Mol Med,2006,17(1):121-127.
18. Baldin V, Lukas J, Marcote MJ, et al. yclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev,1993,7(5):812-821.
19. Karpurapu M, Wang D, Quyen DV, et al. Cyclin D1 is a bona fide target gene of NFATcl and is sufficient in the mediation of injury-induced vascular wall remodeling. J Biol Chem,2010,285(5):3510-3523.
20. Chen L, Wang WY, Wang YP. Inhibitory effects of lithospermic acid on proliferation and migration of rat vascular smooth muscle cells. Acta Pharmacol Sin,2009,30(9): 1245-1252.
21. Liang KW, Yin SC, Ting CT, Lin SJ, Hsueh CM, Chen CY, Hsu SL. Berberine inhibits platelet-derived growth factor-induced growth and migration partly through an AMPK-dependent pathway in vascular smooth muscle cells. Eur J Pharmacol,2008, 590:343-354.
1. Wright JL, Tai H, Churg A. Vasoactive mediators and pulmonary hypertension after cigarette smoke exposure in the guinea pig. J Appl Physiol 2006; 100:672-678.
2. Hu J, Xu YJ, Zhang ZX, et al. Effect of cigarette smoke extract on proliferation of rat pulmonary artery smooth muscle cells and the relevant roles of protein kinase C. Chin Med J,2007,120:1523-1528.
3. Musashi M, Ota S, Shiroshita N. The role of protein kinase C isoforms in cell proliferation and apoptosis. Int J Hematol,2000,72:12-19.
4. Haller H, Lindschau C, Quass P, et al. Differentiation of vascular smooth muscle cells and the regulation of protein kinase C-alpha. Circ Res,1995,76:21-29.
5. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science,1992,258:607-614.
6. Okazaki J, Mawatari K, Liu B, et al. The effect of protein kinase C and its alpha subtype on human vascular smooth muscle cell proliferation, migration and fibronectin production. Surgery,2000,128:192-197.
7. Leszczynski D, Joenvaara S, Foegh ML. Protein kinase C-alpha regulates proliferation but not apoptosis in rat coronary vascular smooth muscle cells. Life Sci,1996,58: 599-606.
8. Itoh H, Yamamura S, Ware JA, et al. Differential effects of protein kinase C on human vascular smooth muscle cell proliferation and migration. Am J Physiol Heart Circ Physiol,2001,281:H359-H370.
9. Sherr CJ. Mammalian G1 cyclins. Cell,1993,73:1059-1065.
10. Resnitzky D, Gossen M, Bujard H, et al. Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol,1994,14: 1669-1679.
11. Quasnichka H, Slater SC, Beeching CA, et al. Regulation of smooth muscle cell proliferation by beta-catenin/T-cell factor signaling involves modulation of cyclin D1 and p21 expression. Circ Res,2006,99:1329-1337.
12. Pestana IA, Vazquez-Padron RI, Aitouche A, et al. Nicotinic and PDGF-receptor function are essential for nicotine-stimulated mitogenesis in human vascular smooth muscle cells. J Cell Biochem,2005,96:986-995.
13. Li F, Li L, Qin X, et al. Apelin-induced vascular smooth muscle cell proliferation:the regulation of cyclin D1. Front Biosci,2008,13:3786-3792.
14. Qiao LF, Xu YJ, Liu XS, et al. PKC promotes proliferation of airway smooth muscle cells by regulating cyclinD1 expression in asthmatic rats. Acta Pharmacol Sin,2008,29: 677-686.
15. Okumura M, Yamamoto M, Sakuma H, et al. Leptin and high glucose stimulate cell proliferation in MCF-7 human breast cancer cells:reciprocal involvement of PKC-alpha and PPAR expression. Biochim Biophys Acta,2002,1592:107-116.
16. Bogatkevich GS, Gustilo E, Oates JC, et al. Distinct PKC isoforms mediate cell survival and DNA synthesis in thrombin-induced myofibroblasts. Am J Physiol Lung Cell Mol Physiol,2005,288:L190-L201.
17. Su Y, Han W, Giraldo C, et al. Effect of cigarette smoke extract on nitric oxide synthase in pulmonary artery endothelial cells. Am J Respir Cell Mol Biol,1998,19:819-825.
18. Lee SD, Lee DS, Chun YG, et al. Cigarette smoke extract induces endothelin-1 via protein kinase C in pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol,2001,281:L403-L411.
19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods,2001,25:402-408.
20. Shin VY, Wu WK, Chu KM, et al. Functional role of beta-adrenergic receptors in the mitogenic action of nicotine on gastric cancer cells. Toxicol Sci,2007,96:21-29.
21. Santos S, Peinado VI, Ramirez J, et al. Characterization of pulmonary vascular remodelling in smokers and patients with mild COPD. Eur Respir J,2002,19: 632-638.
22. Wright JL, Churg A. Effect of long-term cigarette smoke exposure on pulmonary vascular structure and function in the guinea pig. Exp lung Res,1991,17:997-1009
23. Ferrer E, Peinado VI, Diez M, et al. Effects of cigarette smoke on endothelial function of pulmonary arteries in the guinea pig. Respir Res,2009,10:76.
24. Newton AC. Regulation of the ABC kinases by phosphorylation:protein kinase C as a paradigm. Biochem J,2003,370 (Pt 2):361-371.
25. Park JW, Kim HP, Lee SJ, et al. Protein kinase C alpha and zeta differentially regulate death-inducing signaling complex formation in cigarette smoke extract-induced apoptosis. J Immunol,2008,180:4668-4678.
26. Chu M, Guo J, Chen CY. Long-term exposure to nicotine, via ras pathway, induces cyclin D1 to stimulate G1 cell cycle transition. J Biol Chem,2005,280:6369-6379.
27. Hu H, Zhang S, Zhu S. Influence of aspirin and cigarette smoke extract on the expression of cyclin D1 and effects of cell cycle in esophageal squamous cell carcinoma cell line. Dis Esophagus,2009,22:310-316.
28. Soh JW, Weinstein IB. Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes. J Biol Chem,2003,278: 34709-34716.
1. Fabbri LM, Hurd SS. GOLD scientific committee. Global strategy for the diagnosis, management and prevention of COPD:2003 update. Eur Respir J,2003,22(1):1-2.
2. Barbera, JA, Peinado, VI, Santos, S. Pulmonary hypertension in chronic obstructive pulmonary disease. Eur Respir J,2003,21(5):892-905.
3. Kubo K, Ge RL, Koizumi T, et al. Pulmonary artery remodeling modifies pulmonary hypertension during exercise in severe emphysema. Respir Physiol,2000,120(1):71-79.
4. Hislop A, Reid L. New findings in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension. Br J Exp Pathol,1976,57(5):542-554.
5. Meyrick B, Reid L. Hypoxia-induced structural changes in the media and adventitia of the rat hilar pulmonary artery and their regression. Am J Pathol,1980,100(1):151-178.
6. Meyrick B, Reid L. Hypoxia and incorporation of 3H-thymidine by cells of the rat pulmonary arteries and alveolar wall. Am J Pathol,1979,96(1):51-70.
7. Yi ES, Kim H, Ahn H, et al. Distribution of obstructive intimal lesions and their cellular phenotypes in chronic pulmonary hypertension. Am J Respir Crit Care Med, 2000,162(4 Pt1):1577-1586.
8. Yu Y, Sweeney M, Zhang S, et al. PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression. Am J Physiol Cell Physiol,2003, 284(2):C316-C330.
9. Tanabe Y, Saito M, Ueno A, et al. Mechanical stretch augments PDGF receptor beta expression and protein tyrosine phosphorylation in pulmonary artery tissue and smooth muscle cells. Mol Cell Biochem,2000,215(1-2):103-113.
10. Ambalavanan N, Bulger A, Philips III JB. Hypoxia-induced release of peptide growth factors from neonatal porcine pulmonary artery smooth muscle cells. Biol Neonate, 1999,76(5):311-319.
11. Perros F, Montani D, Dorfmuller P, et al. Platelet-derived growth factor expression and function in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med, 2008,178(1):81-88.
12. Tantini B, Manes A, Fiumana E, et al. Antiproliferative effect of sildenafil on human pulmonary artery smooth muscle cells. Basic Res Cardiol,2005,100(2):131-138.
13. Oqawa A, Firth AL, Yao W, et al. Prednisolone inhibits PDGF-induced nuclear translocation of NF-kappaB in human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol,2008,295(4):L648-L657.
14. Fehrenbach H, Kasper M, Haase M, et al. Differential immunolocalization of VEGF in rat and human adult lung, and in experimental rat lung fibrosis:light, fluorescence, and electron microscopy. Anat Rec,254(1):61-73.
15. BelAlba RS, Diordjevic T, Bonello S, et al. Redox-sensitive regulation of the HIF pathway under non-hypoxic conditions in pulmonary artery smooth muscle cells. Biol Chem,2004,385(3-4):249-257.
16. Santos S, Peinado VI, Ramirez J, et al. Enhanced expression of vascular endothelial growth factor in pulmonary arteries of smokers and patients with moderate chronic obstructive pulmonary disease. Am J Respir Crit Care Med,2003,167(9):1250-1256.
17.颜浩,陈文彬,程德云,等.碱性成纤维细胞生长因子在低氧性肺动脉高压大鼠肺内表达及分布的观察.中华结核和呼吸杂志,1997,20(6)：344-346.
18. Wedgwood S, Devol JM, Grobe A, et al. Fibroblast growth factor-2 expression is altered in lambs with increased pulmonary blood flow and pulmonary hypertension. Pediatr Res,2007,61(1):32-36.
19. Black SM, DeVol JM, Wedgwood S. Regulation of fibroblast growth factor-2 expression in pulmonary arterial smooth muscle cells involves increased reactive oxygen species generation. Am J Physiol Cell Physiol,2008,294(1):C345-C354.
20. Birker-Robaczewska M, Studer R, Haenig B, et al. bFGF induces S1P1 receptor expression and functionality in human pulmonary artery smooth muscle cells. J Cell Biochem,2008,105(4):1139-1145.
21. Ambalavana N, Bulger A, Philips III JB. Hypoxia-induced release of peptide growth factors from neonatal porcine pulmonary artery smooth muscle cells. Biol Neonate, 1999,76(5):311-319.
22. Feng X, Cai Y. Hypoxia and endothelin-1 stimulate DNA synthesis of pulmonary artery smooth muscle cells. Chin Med Sci J,1996,11(1):28-31.
23. Yamanaka R, Otsuka F, Nakamura K, et al. Involvement of the bone morphogenetic protein system in endothelin-and aldosterone-induced cell proliferation of pulmonary arterial smooth muscle cell isolated from human patients with pulmonary arterial hypertension. Hypertens Res,2010, Epub ahead of print.
24.解卫平,王虹,齐栩,等.新型ATP敏感性钾通道开放剂埃他卡林对兔肺动脉平滑肌细胞增殖的影响.中华结核和呼吸杂志,2004,27(11)：752-755.
25. Zamora MA, Dempsey EC, WalchakSJ, et al. BQ123, an ETA receptor antagonist, inhibits endothelin-1-mediated proliferation of human pulmonary artery smooth muscle cells. Am J Respir Cell Mol Biol,1993,9(4):429-433.
26. Davie N, Haleen SJ, Upton PD, et al. ET(A) and ET(B) receptors modulate the proliferation of human pulmonary artery smooth muscle cells. Am J Respir Crit Care Med,2002,165(3):398-405.
27. Li H, Chen SJ, Chen YF, et al. Enhanced endothelin-1 and endothelin receptor gene expression in chronic hypoxia. J Appl Physiol,1994,77(3):1451-1459.
28. Fredenburgh LE, Liang OD, Macias AA, et al. Absence of cyclooxygenase-2 exacerbates hypoxia-induced pulmonary hypertension and enhances contractility of vascular smooth muscle cell. Circulation,2008,117(16):2114-2122.
29. Wort SJ, Woods M, Warner TD, et al. Cyclooxygenase-2 acts as an endogenous brake on endothelin-1 release by human pulmonary artery smooth muscle cells:implications for pulmonary hypertension. Mol Pharmacol,2002,62(5):1147-1153.
30. Lee SL, Wang WW, Lanzillo JJ, et al. Serotonin produces both hyperplasia and hypertrophy of bovine pulmonary artery smooth muscle cells in culture. Am J Physiol, 1994,266 (1 Pt 1):L46-L52.
31. Fanburg BL, Lee SL. A new role for an old molecule:Serotonin as a mitogen. Am J Physiol,1997,272 (5 Pt 1):L795-L806.
32. Eddahibi S, Fabre V, Boni C, et al. Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells. Relationship with the mitogenic action of serotonin. Circ Res,1999,84(3):329-336.
33. Lee SL, Wang WW, Moore BJ, et al. Dual effect of serotonin on growth of bovine pulmonary artery smooth muscle cells in culture. Circ Res,1991,68(5):1362-1368.
34. Pitt BR, Weng W, Steve AR, et al. Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture. Am J Physiol,1994,266 (2Pt1):L178-L186.
35. Eddahibi S, Hanoun N, Lanfumey L, et al. Attenuated hypoxia pulmonary hypertension in mice lacking the 5-hydroxytryptamine transporter gene. J Clin Invest,2000,105(11): 1555-1562.
36. Eddahibi S, Adnot S, Frisdal E, et al. Dexfenfluramine-associated changes in 5-hydroxytryptamine transporter expression and development of hypoxic pulmonary hypertension in rats. J Pharmacol Exp Ther,2001,297(1):148-154.
37. MacLean MR, Deuchar GA, Hicks MN, et al. Overexpression of the 5-hydroxytryptamine transporter gene:effect on pulmonary hemodynamics and hypoxia-induced pulmonary hypertension. Circulation,2004,109(17):2150-2155.
38. Fagan KA, Tyler RC, Fouty BW, et al. Relative contribution of endothelial, inducible and neuroal NOS to tone in the murine pulmonary circulation. Am J Phsiol,1999,277 (3 Pt 1):472-478.
39. Ambalavanan N, Mariani G, Bulger A, et al. Role of nitric oxide in regulating neonatal porcine pulmonary artery smooth muscle cell proliferation. Biol Neonate,1999,76(5): 291-300.
40. Thomae KR, Nakayama DK, Billiar TR, et al. The effect of nitric oxide on fetal pulmonary artery smooth muscle growth. J Surg Res,1995,59(3):337-343.
41. Giaid A, Saleh D. Reduced expression of endothelial nitric oxide in the lung of patients with pulmonary hypertension. N Engl J Med,1995,333(4):214-221.
42.丁毅鹏,徐永健,张珍祥.蛋白激酶C对低氧猪肺动脉平滑肌细胞结构型一氧化氮合酶mRNA表达作用的探讨.中华结核和呼吸杂志,2001,24(10)：588-591.
43. Horstman DJ, Frank DU, Rich GF. Prolonged in haled NO attenuates hypoxia, but not monocrotaline-induced, pulmonary vascular remodeling in rats. Anesth Analg,1998, 86(1):74-81.
44. Mitani Y, Maruyama K, Sakurai M. Prolonged administration of L-arginine ameliorates chronic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation,1997,96(2):689-697.
45. Puikuan K, Chunyu Z, Jin F, et al. Inhalation of nebulized nitroglycerin, a nitric oxide donor, for the treatment of pulmonary hypertension induced by high pulmonary blood flow. Heart Vessels,2006,21(3):169-179.
46. Touyz R M, Berry C. Recent advances in angiotensin II signaling. Braz J Med Biol Res, 2002,35(9):1001-1015.
47.金立军,杨沙宁,黄从新,等.血管紧张素Ⅱ在肺动脉高压形成中的作用及其临床意义.临床荟萃,2001,16：264-265.
48. Zhang R, Wu Y, Zhao M, et al. Role of HIF-1 alpha in the regulation ACE and ACE2 expression in hypoxic human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol,2009,297(4):L631-640.
49. Yu TZ, Ma CT. Effects of angiotensin-converting enzyme and angiotensin II on hypoxia-induced proliferation of cultured intra-pulmonary artery smooth muscle cells. Acta Pharmacol Sin,2000,21(4):381-384.
50. Han SX, He GM, Wang T, et al. Losartan attenuates chronic cigarette smoke exposure-induced pulmonary arterial hypertension in rats:Possible involvement of angiotensin-converting enzyme-2. Toxicol Appl Pharmacol.2010, Epub ahead of print.
51. Morrell NW, Upton PD, Higham MA, et al. Angiotensin Ⅱ stimulates proliferation of human pulmonary artery smooth muscle cells via the ATI receptor. Chest,1998,114(1 Suppl):90S-91S.
52.涂明利,王汉琴,雷怀定,等.腺病毒介导反义血管紧张素Ⅱ1型受体cDNA对人肺动脉平滑肌细胞生物学行为的影响.中华结核和呼吸杂志,2005,28(4)：263-267.
53.易斌,钱桂生,白莉,等.低氧致大鼠肺动脉平滑肌细胞增殖过程中PTEN／Akt1表达的变化.中华结核和呼吸杂志,2008,31(8)：586-590.
54. Wang GS, Qian GS, Zhou DS, et al. JAK-STAT signaling pathway in pulmonary arterial smooth muscle cells is activated by hypoxia. Cell Biol Int,2005,29(7): 598-603.
55. Bai L, Yu Z, Qian G, et al. SOCS3 was induced by hypoxia and suppressed STAT3 phosphorylation in pulmonary arterial smooth muscle cells. Respir Physiol Neurobiol, 2006,152(1):83-91.
56. Wang C, Li JF, Zhao L, et al. Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells. Respir Res,2009,10(1):123.
57. Chen XY, Dun JN, Miao QF, et al. Fasudil hydrochloride hydrate, a Rho-kinase inhibitor, suppresses 5-hydroxytryptamine-induced pulmonary artery smooth muscle cell proliferation via JNK and ERK1/2 pathway. Pharmacology,2009,83(2):67-79.
58. Zhu Z, Xu Y, Zou H, Zhang Z, Ni W, Chen S. Effects of puerarin on pulmonary vascular remodeling and protein kinase C-alpha in chronic cigarette smoke exposure smoke-exposed rats. J Huazhong Univ Sci Technolog Med Sci,2008,28(1):27-32.
59. Burg ED, Remillard CV, Yuan JX. Potassium channels in the regulation of pulmonary artery smooth muscle cell proliferation and apoptosis:pharmacotherapeutic implications. Br J Pharmacol,2008,153(Suppl 1):S99-S111.
60. Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol,1995,268(4 Pt 1):C799-C822.
61. Muraki S, Tohse N, Seki S, et al. Decrease in the Ca2+-activated K+current of pulmonary arterial smooth muscle in pulmonary hypertension rats. Naunyn Schmiedebergs Arch Pharmacol,2001,364(3):183-192.
62. Wang J, J uhaszova M, Rubin LJ, et al. Hypoxia inhibits gene expression of voltage-gated K+channel asubunits in pulmonary artery smooth muscle cells. J Clin Invest,1997,100(9):2347-2353.
63. Yuan JX, Aldinger AM, Juhaszova M, et al. Dysfunctional voltage-gated K+channels in pulmonary artery smooth muscle cells of patients with primary pulmonary hypertension. Circulation,1998,98(14):1400-1406.
64. Platoshyn O, Golovina VA, Bailey CL, et al. Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation. Am J Physiol Cell Physiol,2000, 279(5):C1540-C1549.
65. Baldin V, Lukas J, Marcote MJ, et al. Cyclin Dl is a nuclear protein required for cell cycle progression in G1. Genes Dev,1993,7(5):812-821.
66. Kadowaki M, Mizuno S, Demura Y, et al. Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation:the role of p27kip1. Respir Res,2007,8:77.
67. Yu L, Quinn DA, Garg HQ et al. Gene expression of cyclin-dependent kinase inhibitors and effect of heparin on their expression in mice with hypoxia-induced pulmonary hypertension. Biochem Biophys Res Commun.2006,345(4):1565-1572.
68. Chattergoon NN, D'Souza FM, Deng W, et al. Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol,2005,288(1):L202-L211.
69. Yu L, Quinn DA, Garg HG, et al. Cyclin-dependent kinase inhibitor p27Kip1, but not p21WAF1/Cip1, is required for inhibition of hypoxia-induced pulmonary hypertension and remodeling by heparin in mice. Circ Res,2005,97(9):937-945.
70. Lin CL, Zhang ZX, Xu YJ, et al. Focal adhesion kinase antisense oligodeoxynucleotides inhibit human pulmonary artery smooth muscle cells proliferation and promote human pulmonary artery smooth muscle cells apoptosis. Chin Med J,2005,118(1):20-26.