摘要
研究背景
吸烟不仅可引起慢性支气管炎、慢性阻塞性肺气肿,而且还可进一步导致肺动脉高压和慢性肺源性心脏病。肺动脉高压与慢性阻塞性肺病(COPD)患者的临床预后恶化、生存率降低密切相关,目前尚缺乏特异而有效的治疗。既往对COPD合并肺动脉高压发病机制的研究多集中于缺氧性肺血管收缩和缺氧性肺血管重建。近年发现吸烟可直接导致肺血管重建、参与肺动脉高压的形成,其主要机制可能是吸烟促进肺动脉平滑肌细胞(PASMC)的增殖,但具体机制尚不清楚。细胞内信号转导通路的激活、生长因子的产生和释放在吸烟致PASMC增殖中可能起着重要作用。
蛋白激酶C(PKC)是细胞内生物信号转导途径中的一类关键酶,参与调控血管平滑肌细胞的增殖、凋亡、分化、迁移和基因表达等。我们实验室以往的研究发现PKC的活化,PKCα亚型的表达上调在低氧致PASMC增殖中起重要作用。前期研究工作还发现无论是慢性吸烟大鼠,还是吸烟非COPD患者和吸烟COPD患者其肺小动脉PKC-αmRNA及蛋白质的表达都增加,提示PKC-α可能参与了慢性吸烟所致的肺血管重建。但PKC特别是PKC-α在吸烟对PASMC增殖的影响中所起的作用有待在细胞培养水平进一步予以研究。
碱性成纤维细胞生长因子(bFGF)作为一种生长因子和丝裂原,可促进肺动脉内皮细胞增殖,外膜成纤维细胞生长,强烈地刺激中膜平滑肌细胞增殖、迁移、合成分泌胶原和纤维连接蛋白等细胞外基质,在低氧和野百合碱所致的肺血管重建和肺动脉高压中发挥着重要作用。有研究发现,在吸烟合并COPD的患者中,bFGF在肺小动脉平滑肌细胞的表达增加,可能参与了吸烟所致的肺血管重建,但目前尚无相关的动物在体实验和离体实验的研究。
本研究从PKC信号转导通路和bFGF两个方面探讨吸烟致PASMC增生和肺血管重建的可能机制,为COPD合并肺动脉高压的发病机制研究和防治提供基础资料。
第一部分香烟烟雾提取物对肺动脉平滑肌细胞增殖的影响及蛋白激酶C相关作用的研究
目的:探讨香烟烟雾提取物(CSE)对体外培养的大鼠和人PASMC增殖的影响及PKC信号转导途径在其中所起的作用。
方法:组织块法培养正常Wistar大鼠PASMC,复苏培养人PASMC细胞系。(1)不同浓度的CSE作用于PASMC 24h,四甲基偶氮唑盐(MTT)比色法检测PASMC增殖,台盼蓝排斥法检测活细胞率。(2)PASMC与PKC激活剂12-肉豆蔻酰-13-乙酸佛波酯(PMA)预孵育24h下调PKC或与PKC抑制剂马来酰亚胺甲磺酸盐(Ro31-8220)预孵育30min然后暴露于5%CSE,MTT法、流式细胞术和增殖细胞核抗原(PCNA)染色等方法观察细胞增殖的变化。(3)5%CSE作用PASMC 24h,逆转录-聚合酶链反应(RT-PCR)和Western Blot分别检测PKC-αmRNA和蛋白质表达量的变化。免疫荧光细胞化学及激光共聚焦扫描检测大鼠PASMC内PKC-α的分布。(4)大鼠PASMC暴露于5%CSE前6h转染PKC-α的反义寡核苷酸,观察PKC-α蛋白表达量和细胞增殖的变化。
结果:( 1)20%和30%CSE对PASMC有细胞毒性作用,5%和10%CSE不影响PASMC的活细胞率。5%CSE组PASMC MTT的吸光度(A)值较对照组明显增高。(2)5%CSE组PASMC的MTT A值、增殖指数(PI)、S期细胞分数(SPF)和PCNA染色平均光密度值增高,与对照组比较差异有统计学意义(P<0.05)。PMA+5%CSE组和Ro+5%CSE组以上指标均降低,与5%CSE组比较,差异有统计学意义(P<0.05),与对照组比较,差异无显著性。(3)5%CSE组PKC-αmRNA和蛋白表达量增加,与对照组比较,差异有统计学意义(P<0.01)。5%CSE处理后,大鼠PASMC中PKC-α由细胞浆转位至细胞核周或核内(。4)PKC-α反义寡核苷酸转染组大鼠PASMC中PKC-α蛋白质表达量降低(转染前后分别为1.180±0.056、0.713±0.047),MTT A值降低(0.252±0.041),与5%CSE组(0.302±0.021)比较,差异有统计学意义(P<0.01),与对照组(0.244±0.016)比较,差异无显著性。
结论:高浓度的CSE对大鼠和人PASMC有细胞毒性作用,低浓度(5%)的CSE则能促进大鼠和人PASMC的增殖。PKC特别是PKC-α在CSE促PASMC增殖的信号转导机制中起重要作用。
第二部分香烟烟雾对大鼠肺动脉平滑肌细胞碱性成纤维细胞生长因子表达的影响
分题1烟雾暴露大鼠肺动脉bFGF表达的变化及其与肺血管重建的关系
目的:探讨烟雾暴露对大鼠肺动脉bFGF表达的影响及这种影响与肺血管重建的关系。
方法:健康雄性Wistar大鼠30只,随机分为正常对照组(C组)、烟雾暴露组(S2w组、S4w组、S8w组、S12w组)。取动脉血检测血氧分压(PaO2),右心导管法测定平均肺动脉压(mPAP)。α平滑肌肌动蛋白(α-SM-actin)染色显示肺血管的重建,PCNA染色检测细胞增殖。RT-PCR检测肺动脉bFGF mRNA表达,免疫组织化学染色检测bFGF蛋白表达。
结果:与C组大鼠比较,烟雾暴露各组大鼠PaO2和mPAP无明显改变。S4w组、S8w组和S12w组大鼠肺泡管旁肺小动脉肌化比例和烟雾暴露各组大鼠肺小血管壁细胞的增殖指数明显高于C组(P<0.05)。烟雾暴露各组大鼠肺动脉bFGF mRNA[(0.327±0.076)、(0.483±0.131)、(0.828±0.207)、(0.743±0.159)]和蛋白的表达[(0.184±0.012)、(0.230±0.017)、(0.292±0.013)、(0.325±0.020)]均较C组明显增强(P<0.05)。大鼠肺小动脉肌化比例、肺小血管壁细胞的增殖指数与bFGF mRNA和蛋白表达水平成显著正相关。
结论:烟雾暴露能诱导肺小血管壁细胞增殖,导致肺血管重建,其程度随暴露时间延长而加重,bFGF在此过程中可能起重要作用。
分题2香烟烟雾提取物通过PKC途径促进大鼠肺动脉平滑肌细胞bFGF表达
目的:研究CSE对大鼠PASMC中bFGF表达的影响及PKC途径在其中所起的作用。
方法:组织块法培养正常Wistar大鼠PASMC。5%CSE作用于PASMC不同时间或PASMC先与PKC特异性抑制剂Ro31-8220预孵育,然后再暴露于5%CSE,RT-PCR检测PASMC bFGF mRNA表达,免疫细胞化学检测bFGF蛋白表达。
结果:对照组PASMC中有少量bFGF mRNA和蛋白表达(分别为0.093±0.034,0.142±0.013)。暴露于5%CSE后4h细胞内bFGF mRNA和蛋白表达即增加,mRNA于8h达高峰(0.436±0.103,P<0.01),蛋白于12h达高峰(0.237±0.031,P<0.01),此后逐渐下降,但24h都仍高于对照组。Ro31-8220预孵育可显著抑制5%CSE诱导的bFGF mRNA和蛋白表达增加。
结论:5%CSE可通过激活PKC途径促进大鼠PASMC bFGF的表达。
Background
Cigarette smoking is not only the major cause of chronic bronchitis and emphysema, but also can lead to pulmonary hypertension and cor pulmonale. The presence of pulmonary hypertension is associated with worse clinical outcomes and less survival rates. At the present time, there is no specific and effective treatment for pulmonary hypertension in chronic obstructive pulmonary disease (COPD). In the past, most of the studies on the pathogenesis of COPD-related pulmonary hypertension were focused on hypoxic pulmonary vasoconstriction and hypoxic pulmonary remodeling. However, recent studies suggest that cigarette smoke has a direct effect on the pulmonary vasculature and this effect may lead eventually to pulmonary vascular remodeling and pulmonary hypertension. Smoke-induced proliferation of pulmonary artery smooth muscle cells (PASMC) plays a vital role in the process, but the underlying molecular mechanisms remain largely unclear. Activation of several intracellular pathways and production of numerous growth factors may be involved in the growth-stimulating effect of cigarette smoke on PASMC.
PKC is a crucial family of serine-threonine kinases in the intracellular signal transduction pathway. It has been implicated in a variety of cellular functions including proliferation, apoptosis, differentiation of vascular smooth muscle cells. Previous studies in our laboratory showed that activation of PKC and upregulation of PKC-αisozyme play an important role in hypoxia-induced proliferation of PASMC. Recent work within our group also found that PKC-αmRNA and protein expression in small intrapulmonary arteries were markedly increased either in the smoke-exposed animals or in COPD patients and non-COPD smokers, suggesting a possible role for PKC-αin smoke-induced pulmonary vascular remodeling. However, the role of PKC and PKC-αin smoke-induced PASMC proliferation is yet to be investigated in vitro.
As an important growth factor and mitogen, bFGF has been reported to stimulate the proliferation of pulmonary artery endothelial cells and adventitial fibroblasts, enable the medial smooth muscle cells to migrate, proliferate and synthesize extracellular matrix such as collagen and fibronectin. It contributes to pulmonary vascular remodeling in rats with hypoxia-induced or monocrotaline-induced pulmonary hypertension. Studies in smokers with COPD showed an unequivocal increase in bFGF expression in vascular smooth muscle of small pulmonary vessels with a luminal diameter under 200μm. The data suggest a role of bFGF in the pathogenesis of COPD-associated vascular remodeling. However, to our knowledge, no studies have examined the involvement of bFGF in smoke-induced PASMC proliferation in animal models and in PASMC cell culture model.
In the present study, we investigated the roles of PKC and bFGF in the proliferation of PASMC and pulmonary vascular remodeling caused by cigarette smoke.
PartⅠEffect of Cigarette Smoke Extract on Proliferation of Pulmonary Artery Smooth Muscle Cells and the Relevant Roles of Protein Kinase C
Objective To investigate the effects of cigarette smoke extract on proliferation of rat and human PASMC and to evaluate the relevant roles of protein kinase C (PKC).
Methods Cultured rat and human PASMC were studied in the following conditions: (1) PASMC were exposed to different concentrations of CSE for 24h, then MTT colorimetric assay was used for detection of cell proliferation. Cell viability was assessed by trypan blue exclusion. (2) PASMC were pre-incubated with PMA for 24h or Ro31-8220 for 30min before exposure to 5% CSE for 24h. Cell proliferation was examined by MTT colorimetric assay, cell cycle analysis and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. (3) PASMC were exposed to 5% CSE for 24h, then PKC-αmRNA expression was detected by Reverse transcription-polymerase chain reaction(RT- PCR)and protein expression by Western Blot. In rat PASMC, intracellular distribution of PKC-αwas also observed by immunofluorescence staining and confocal microscopy. (4) Rat PASMC were transfected with specific antisense oligodeoxynucleotides against PKC-α6h before exposure to 5% CSE for 24h. PKC-αprotein expression and cell proliferation were detected by methods described previously.
Results (1) At low concentrations (5%) CSE increased proliferation of PASMC, whereas high concentrations (20%, 30%) were inhibitory as a result of cytotoxicity. (2) The absorbance (A) value, proliferation index (PI), S-phase cell fraction (SPF ) and average optical density of PCNA staining in PASMC from 5% CSE exposure group were significantly increased compared to those of control group (P<0.05). PKC down-regulation by PMA pretreatment or PKC inhibition by Ro31-8220 pre-incubation abolished the effect of 5% CSE on PASMC proliferation. (3) After exposure to 5% CSE for 24h, PKC-αmRNA and protein expression in PASMC (1.054±0.078, 1.185±0.041, respectively) were much higher than in untreated cells (0.573±0.054, 0.671±0.055, respectively) (P<0.01). Moreover, in rat PASMC, 5% CSE induced a translocation of PKC-αfrom cytoplasm toward the perinuclear area and into the nucleus. (4) Specific antisense oligodeoxynucleotides against PKC-αreduced 5% CSE-induced expression of PKC-αprotein (0.713±0.047 vs 1.180±0.056), also abolished the effect of 5% CSE on rat PASMC proliferation significantly.
Conclusions CSE can be cytotoxic at high concentrations. But at low concentrations, it makes a mitogenic effect on cultured rat and human PASMC. PKC, especially its alpha isozyme, seems to play an important role in CSE-induced proliferation of both rat PASMC and human PASMC.
PartⅡSection 1 Basic fibroblast growth factor expression and pulmonary vascular remodeling after cigarette smoke exposure in the rat
Objective To investigate basic fibroblast growth factor expression and pulmonary vascular remodeling in rats exposed to cigarette smoke.
Methods Thirty male Wistar rats were randomly divided into five groups: control group(C group), smoke exposure groups (S2w, S4w, S8w, S12w group). Mean pulmonary artery pressure (mPAP) was measured by right cardiac catheterization. The percentage of muscularised small pulmonary arteries adjacent to alveolar ducts was determined byα-SM-actin staining and vascular cell proliferation by proliferating cell nuclear antigen staining. RT- PCR and immunohistochemistry staining analysis were used for the detection of bFGF mRNA and protein expression in pulmonary arteries.
Results Compared to control group, artery oxygen partial pressure and mPAP were not altered in smoke exposure groups. The percentage of completely muscularised small vessels adjacent to alveolar ducts was significantly increased in S4w, S8w and S12w group. Cigarette smoke caused marked proliferation of pulmonary vascular cells. The expressions of bFGF mRNA (0.327±0.076, 0.483±0.131, 0.828±0.207, 0.743±0.159, respectively) and protein (0.184±0.012, 0.230±0.017, 0.292±0.013, 0.325±0.020, respectively) in pulmonary arteries in smoke exposure groups were higher than those of C group (P<0.05). The percentage of muscularised vessels, proliferation index of vascular wall cells correlated strongly with levels of bFGF mRNA expression and the values of average optical density of bFGF protein.
Conclusions These data suggest that smoke causes marked production of bFGF in pulmonary arteries. bFGF appears to play an important role in the pathogenesis of smoke-induced vascular remodeling.
Section 2 Cigarette smoke extract induces bFGF expression via protein kinase C in rat pulmonary artery smooth muscle cells
Objective To investigate the effect of CSE on bFGF expression in rat PASMC and the possible role of PKC in this process.
Methods Cultured PASMC were prepared by an explant method from intrapulmonary artery in normal male Wistar rats. Sub-confluent PASMC were serum-starved to induce quiescence, then were exposed to 5%CSE for different time periods. Some PASMC were pre-incubated for 30min with PKC inhibitor Ro31-8220 before exposure to 5%CSE for 12h. RT-PCR and immuocytochemistry staining were performed for the detection of bFGF mRNA and protein expression in PASMC.
Results The control PASMC expressed bFGF mRNA and protein at low levels (0.093±0.034, 0.142±0.013, respectively). PASMC treated with 5%CSE for various times showed a time-dependent increase in bFGF expression. Increased bFGF mRNA and protein expression were evident at 4h after CSE treatment. Maximal increase in bFGF mRNA occurred at 8h (0.436±0.103, P<0.01), while peak expression of bFGF protein was at 12h (0.237±0.031, P<0.01). Then bFGF mRNA and protein expression were slowly declined and were still elevated at 24h. Pretreatment with Ro31-8220 nearly completely inhibited CSE-induced bFGF mRNA and protein expression.
Conclusion These results indicate that 5%CSE stimulates bFGF mRNA and protein expression in cultured rat PASMC. The induction of bFGF seems to occur through activation of the PKC pathway.
引文
1. Wright JL, Farmer SG, Churg A. A neutrophil elastase inhibitor reduces cigarette smoke-induced remodelling of lung vessels. Eur Respir J, 2003, 22: 77 - 81.
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: 632 - 638.
3.丁毅鹏,徐永健,张珍祥.蛋白激酶C-αmRNA在低氧猪肺动脉平滑肌细胞的表达及对细胞增殖的作用.中华结核和呼吸杂志, 2001, 24: 174-175.
4. Guldemeester A, Stenmark KR, Brough GH, et al. Mechanisms regulating cAMP-mediated growth of bovine neonatal pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 1999, 276: 1010-1017.
5. Kashyap R, Floreani AA, Heires AJ, et al. Protein kinase C-alpha mediates cigarette smoke extract- and complement factor 5a-stimulated interleukin-8 release in human bronchial epithelial cells. J Investig Med, 2002, 50: 46-53.
6. 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.
7. 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: 936-944.
8. Oltmanns U, Chung KF, Walters M, et al. Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle. Respir Res, 2005, 6: 74-83.
9. Lampasso JD, Marzec N, Margarone 3rd J, et al. Role of protein kinase C alpha in primary human osteoblast proliferation. J Bone Miner Res, 2002, 17: 1968-76.
10. Shapiro SD. Smoke Gets in Your Cells. Am J Respir Cell Mol Biol, 2004, 31: 481- 482.
11. Luppi F, Aarbiou J, Wetering SV, et al. Effect of cigarette smoke condensate on proliferation and wound closure of bronchial epithelial cells in vitro: role of glutathione. Respir Res, 2005, 6: 140-151.
12. Carty CS, Huribal M, Marsan BU, et al. Nicotine and its metabolite cotinine are mitogenic for human vascular smooth muscle cells. J Vasc Surg, 1997, 25: 682-688.
13. Ambalavanan N, Carlo WF, Bulger A, et al. Effect of cigarette smoke extract on neonatal porcine vascular smooth muscle cells. Toxicol Appl Pharmacol, 2001, 170: 130-136.
14. Zhu BH, Yao ZX, Luo SJ, et al. Effects of antisense oligonucleotides of PKC-alpha on proliferation and apoptosis of HepG2 in vitro. Hepatobiliary Pancreat Dis Int, 2005; 4: 75-79.
15. Eude I, Dallot E, FerréF, et al. Protein kinase C-αis required for endothelin-1-induced proliferation of human myometrial cells. Biol Reprod, 2002; 66: 44-49.
16. Buchner K. The role of protein kinase C in the regulation of cell growth and in signalling to the cell nucleus. J Cancer Res Clin Oncol, 2000; 126: 1-11.
17. Zini N, Martelli AM, Neri LM, et al. Immunocytochemical evaluation of protein kinase C translocation to the inner nuclear matrix in 3T3 mouse fibroblasts after IGF-I treatment. Histochem Cell Biol, 1995; 103: 447-457.
1. Wright JL, Farmer SG, Churg A. A neutrophil elastase inhibitor reduces cigarette smoke-induced remodelling of lung vessels. Eur Respir J, 2003, 22: 77 - 81.
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: 632 - 638.
3. Wright J L, Levy RD, Churg A. Pulmonary hypertension in chronic obstructive pulmonary disease: current theories of pathogenesis and their implications for treatment. Thorax, Jul 2005, 60: 605 - 609.
4. 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.
5. Nana-Sinkam SP, Lee JD, Sotto-Santiago S, et al. Prostacyclin Prevents Pulmonary Endothelial Cell Apoptosis Induced by Cigarette Smoke. Am J Respir Crit Care Med, 2007, 175(7): 676 - 685.
6. Tuder RM, Wood K, Taraseviciene L, et al. Cigarette Smoke Extract Decreases the Expression of Vascular Endothelial Growth Factor by Cultured Cells and Triggers Apoptosis of Pulmonary Endothelial Cells. Chest, 2000, 117:241-242.
7. Luppi F, Aarbiou J, Wetering SV, et al. Effect of cigarette smoke condensate on proliferation and wound closure of bronchial epithelial cells in vitro: role of glutathione.Respir Res, 2005, 6: 140-151.
8.王起恩,樊晶光,刘世杰.烟溶液与温石棉对细胞增殖活性的影响.中华预防医学杂志,1999, 33(4): 231-233.
9. Carty CS, Huribal M, Marsan BU, et al. Nicotine and its metabolite cotinine are mitogenic for human vascular smooth muscle cells. J Vasc Surg, 1997, 25: 682-688.
10. Ambalavanan N, Carlo WF, Bulger A, et al. Effect of cigarette smoke extract on neonatal porcine vascular smooth muscle cells. Toxicol Appl Pharmacol, 2001, 170: 130-136.
11. Nakashima S. Protein kinase Cα(PKCα): Regulation and Biological Function. J Biochem (Tokyo), 2002, 132: 669 - 675.
12.丁毅鹏,徐永健,张珍祥.蛋白激酶C-αmRNA在低氧猪肺动脉平滑肌细胞的表达及对细胞增殖的作用.中华结核和呼吸杂志, 2001, 24: 174-175.
13.朱朝霞,徐永健,邹晖等.吸烟和慢性阻塞性肺疾病患者肺血管重建及蛋白激酶C-α表达的研究中国组织化学与细胞化学杂志,2005, 14(3): 322-326.
14.朱朝霞,徐永健,邹晖等.烟雾暴露和低氧单独及联合作用对大鼠肺血管重建及蛋白激酶C-α表达的影响.中华结核和呼吸杂志, 2005, 28(12): 825-829.
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.颜浩,陈文彬.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.中华结核和呼吸杂志, 1997,20(6): 344-346.
3. Arcot SS, Fagerland JA, Lipke DW, et al. Basic fibroblast growth factor alterations during development of monocrotaline-induced pulmonary hypertension in rats.Growth Factors,1995,12(2): 121-30.
4. Wright JL, Farmer SG, Churg A. A neutrophil elastase inhibitor reduces cigarette smoke-induced remodelling of lung vessels. Eur Respir J, 2003, 22: 77 - 81.
5. Wright JL, Churg A. Effect of long-term cigarette smoke exposure on pulmonaryvascular structure and function in the guinea pig. Exp Lung Res, 1991,17(6): 997-1009.
6. 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.
7. Redington AE, Roche WR, Madden J, et al. Basic fibroblast growth factor in asthma: measurement in bronchoalveolar lavage fluid basally and following allergen challenge.J Allergy Clin Immunol, 2001,107(2): 384-7.
8. Carty CS, Soloway PD, Kayastha S, et al. Nicotine and cotinine stimulate secretion of basic fibroblast growth factor and affect expression of matrix metalloproteinases in cultured human smooth muscle cells. J Vasc Surg, 1996, 24(6): 927-34.
9.蒋凌志,吴尚洁.碱性成纤维细胞生长因子与低氧性肺血管重构.国外医学呼吸系统分册, 2005, 25(10): 759-761.
1. Wright JL, Farmer SG, Churg A. A neutrophil elastase inhibitor reduces cigarette smoke-induced remodelling of lung vessels. Eur Respir J, 2003, 22(1): 77 - 81.
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. Wright JL, Tai H, Churg A. Vasoactive mediators and pulmonary hypertension aftercigarette smoke exposure in the guinea pig. J Appl Physiol, 2006, 100(2): 672 - 678.
4. Kranenburg AR, De Boer WI, Van Krieken JH, et al. Enhanced expression of fibroblast growth factors and receptor FGFR-1 during vascular remodeling in chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol, 2002, 27(5): 517-525.
5.张焕萍,徐永健,张珍祥,等.蛋白激酶C-核因子κB信号转导通道对人肺动脉平滑肌细胞增殖和血管内皮生长因子表达的影响.中华结核和呼吸杂志, 2004, 27(4): 218-223.
6. Oltmanns U, Chung KF, Walters M, et al. Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle. Respir Res, 2005, 6(1): 74-83.
7. Quinn TP, Schlueter M, Soifer SJ, et al. Mechanotransduction in the Lung: Cyclic mechanical stretch induces VEGF and FGF-2 expression in pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol, 2002, 282(5): L897 - L903.
8.李丰,张燕,车东媛,等.缺氧内皮细胞介导肺动脉平滑肌细胞血小板源性生长因子mRNA表达.中华病理学杂志, 1998, 27(6): 425-428.
9. Wort SJ, Woods M, Warner TD, et al. Endogenously released endothelin-1 from human pulmonary artery smooth muscle promotes cellular proliferation: relevance to pathogenesis of pulmonary hypertension and vascular remodeling. Am J Respir Cell Mol Biol, 2001, 25(1): 104–110.
10.蒋凌志,吴尚洁.碱性成纤维细胞生长因子与低氧性肺血管重构.国外医学呼吸系统分册,2005, 25(10): 759-761.
11. Cucina A, Sapienza P, Corvino V, et al. Nicotine-induced smooth muscle cell proliferation is mediated through bFGF and TGF-beta 1. Surgery, 2000, 127(3): 316-322.
12. Kashyap R, Floreani AA, Heires AJ, et al. Protein kinase C-alpha mediates cigarette smoke extract- and complement factor 5a-stimulated interleukin-8 release in human bronchial epithelial cells. J Investig Med, 2002, 50(1): 46-53.
13. 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 MolPhysiol, 2001, 281 (2): L403-L411.
14. Ali S, Becker MW, Davis MG, et al. Dissociation of vasoconstrictor-stimulated basic fibroblast growth factor expression from hypertrophic growth in cultured vascular smooth muscle cells. Relevant roles of protein kinase C. Circ Res, 1994, 75(5): 836-843.
15. Alberts GF, Hsu DK, Peifley KA, et al. Differential regulation of acidic and basic fibroblast growth factor gene expression in fibroblast growth factor-treated rat aortic smooth muscle cells. Circ Res, 1994, 75(2): 261-267.
16. Shibata F, Baird A, Florkiewicz RZ. Functional characterization of the human basic fibroblast growth factor gene promoter. Growth Factors, 1991, 4(4): 277-287.
17. Lee YJ, Galoforo SS, Berns CM, et al. Effect of ionizing radiation on AP-1 binding activity and basic fibroblast growth factor gene expression in drug-sensitive human breast carcinoma MCF-7 and multidrug-resistant MCF-7/ADR cells. J Biol Chem, 1995, 270(48): 28790-28796.
1. Weitzenblum E, Sautegeau A, Ehrhart M, et al. Long-term oxygen therapy can reverse the progression of pulmonary hypertension in patients with chronic obstructive pulmonary disease. Am Rev Respir Dis, 1985, 131(4): 493-8.
2. Scharf SM, Iqbal M, Keller C, et al. Hemodynamic characterization of patients with severe emphysema. Am J Respir Crit Care Med 2002, 166: 314–322.
3. Barbera JA, Riverola A, Roca J, et al. Pulmonary vascular abnormalities and ventilation-perfusion relationships in mild chronic obstructive pulmonary disease. Am J Respir Crit Care Med, 1994, 149: 423–429.
4. Wright JL, Petty TL, Thurlbeck WM. Analysis of the structure of the muscular pulmonary arteries in patients with pulmonary hypertension and COPD: National Institutes of Health Nocturnal Oxygen Therapy Trial. Lung, 1992, 170:109–24.
5. Jeffery TK, Wanstall JC. Pulmonary vascular remodeling: a target for therapeutic intervention in pulmonary hypertension. Pharmacol Ther, 2001, 92(1): 1-20.
6. Magee F, Wright JL, Wiggs BR, et al. Pulmonary vascular structure and function in chronic obstructive pulmonary disease. Thorax, 1988, 43: 183–189.
7. Hale KA, Niewoehner DE, Cosio MG. Morphologic changes in the muscular pulmonary arteries: Relationship to cigarette smoking, airway disease, and emphysema. Am Rev Respir Dis, 1980, 122: 273–27
8. 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.
9. Wilkinson M, Langhorne CA, Heath D, et al. A pathophysiological study of 10 cases of hypoxic cor pulmonale. Q J Med, 1988, 249: 65–85.
10. Peinado VI, Barbera JA, Ramirez J, et al. Endothelial dysfunction in pulmonary arteries of patients with mild COPD. Am J Physiol, 1998, 274: L908–L913.
11. Wright JL, Tai H, Churg A. Cigarette smoke induces persisting increases of vasoactive mediators in pulmonary arteries. Am J Respir Cell Mol Biol, 2004, 31:501–509.
12. Wright JL, Lawson L, Pare PD, et al. The structure and function of the pulmonary vasculature in mild chronic obstructive pulmonary disease. Am Rev Respir Dis, 1983, 128:702–707.
13. 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, 167:1250–1256.
14. Hale KA, Ewing SL, Gosnell BA, et al. Lung disease in long-term cigarette smokers with and without chronic air-flow obstruction. Am Rev Respir Dis, 1984; 130:716–721.
15. Peinado VI, Barbera JA, Abate P, et al. Inflammatory reaction in pulmonary muscular arteries of patients with mild chronic obstructive pulmonary disease. Am J Respir Crit Care Med, 1999, 159: 1605–1611.
16.杨光田,王迪浔.吸烟对肺血管的作用.国外医学:生理、病理科学与临床分册, 1989, (1): 6-8.
17. Barbera JA, Peinado VI, Santos S, et al. Reduced expression of endothelial nitric oxide synthase in pulmonary arteries of smokers. Am J Respir Crit Care Med, 2001, 164: 709–713.
18. Hutchison SJ, E. Sievers R, Zhu BQ, et al. Secondhand Tobacco Smoke Impairs Rabbit Pulmonary Artery Endothelium-Dependent Relaxation. Chest, 2001, 120(6): 2004-2012.
19. Barbera JA, Peinado VI, Santos S, et al. Reduced expression of endothelial nitric oxide synthase in pulmonary arteries of smokers. Am J Respir Crit Care Med, 2001, 164: 709–713.
20. Giaid A, Yanagisawa M, Langleben D, et al. Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med, 1993, 328: 1732–1739.
21. Roland M, Bhowmik A, Sapsford RJ, et al. Sputum and plasma endothelin-1 levels in exacerbations of chronic obstructive pulmonary disease. Thorax, 2001, 56: 30–35.
22. Nana-Sinkam SP, Lee JD, Sotto-Santiago S, et al. Prostacyclin prevents pulmonary endothelial cell apoptosis induced by cigarette smoke. Am J Respir Crit Care Med, 2007, 175(7): 676 - 685.
23. 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, 2001, 281: L403-411.
24. 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.
25. Wright JL, Tai H, Dai J, et al. Cigarette smoke induces rapid changes in gene expression in pulmonary arteries. Lab Invest, 2002, 82:1391–1398.
26.焦云根,刘乃丰.吸烟与血管内皮功能不全.心血管康复医学杂志,2004, 13(2):190-193.
27. Ota Y, Kugiyama K, Sugiyama S, et al. Impairment of endothelium-dependent relaxation of rabbit aortas by cigarette smoke extract--role of free radicals and attenuation by captopril. Atherosclerosis, 1997, 131(2): 195-202.
28. Wright JL, Tai H, Wang R, et al. Cigarette smoke upregulates pulmonary vascular matrix metalloproteinases via TNF-alpha signaling. Am J Physiol Lung Cell Mol Physiol. 2007, 292(1): L125-133.
29. Wright JL, Farmer SG, Churg A. A neutrophil elastase inhibitor reduces cigarette smoke-induced remodelling of lung vessels. Eur Respir J, 2003, 22:77–81.
30. Ranganna K, Yousefipour Z, Nasif R, et al. Acrolein activates mitogen-activated protein kinase signal transduction pathways in rat vascular smooth muscle cells. Mol Cell Biochem, 2002, 240(1-2): 83-98.
31.朱朝霞,徐永健,邹晖等.烟雾暴露和低氧单独及联合作用对大鼠肺血管重建及蛋白激酶C-α表达的影响.中华结核和呼吸杂志, 2005, 28(12): 825-829.
32.朱朝霞,徐永健,邹晖等.吸烟和慢性阻塞性肺疾病患者肺血管重建及蛋白激酶C-α表达的研究.中国组织化学与细胞化学杂志, 2005, 14(3): 322-326.
33. Racke K, Schworer H, G Simson. Effects of cigarette smoking or ingestion of nicotine on platelet 5-hydroxytryptamine (5-HT) levels in smokers and non-smokers. Clin Investig, 1992, 70(3-4): 201-204.
34. Kitamura S. Effects of cigarette smoking on metabolic events in the lung. Environ Health Perspect, 1987, 72: 283-96.
35. Eddahibi S, Chaouat A, Morrell N, et al. Polymorphism of the Serotonin Transporter Gene and Pulmonary Hypertension in Chronic Obstructive Pulmonary Disease. Circulation, 2003, 108: 1839-1844.
36. Yu XL, Jin XR, Wang DX. Effects of cigarette smoking on the function of metabolizing arachidonic acid and angiotensin I in the isolated perfused rat lungs. J Tongji Med Univ, 1992, 12(4): 201-204.
37. Sugiyama Y, Yotsumoto H, Takaku F. Increase of serum angiotensin-converting enzyme level after exposure to cigarette smoke and nicotine infusion in dogs. Respiration, 1986, 49(4): 292-295.
38. 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: 1250–1256.
39. Tuder RM, Wood K, Taraseviciene L, et al. Cigarette smoke extract decreases the expression of vascular endothelial growth factor by cultured cells and triggers apoptosis of pulmonary endothelial cells. Chest, 2000, 117: 241-242.
40. Carty CS, Soloway PD, Kayastha S, et al. Nicotine and cotinine stimulate secretion of basic fibroblast growth factor and affect expression of matrix metalloproteinases in cultured human smooth muscle cells. J Vasc Surg, 1996, 24(6): 927-34.
41. Kranenburg AR, De Boer WI, Van Krieken JH, et al. Enhanced expression of fibroblast growth factors and receptor FGFR-1 during vascular remodeling in chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol, 2002, 27(5): 517-525.
42. Liu SQ, Fung YC. Changes in the structure and mechanical properties of pulmonary arteries of rats exposed to cigarette smoke. Am Rev Respir Dis, 1993, 148: 768–77.
43. 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.
44. Wright JL. Glutathione levels play a role in cigarette smoke-induced cell proliferation in the rat lung. Inhalation Toxicology, 1998, 10: 969-994.
45. Cucina A, Sapienza P, Corvino V, et al. Nicotine-induced smooth muscle cell proliferation is mediated through bFGF and TGF-beta 1. Surgery, 2000, 127(3): 316-322.
46. Cowan KN, Jones PL, Rabinovitch M. Regression of hypertrophied rat pulmonary arteries in organ culture is associated with suppression of proteolytic activity, inhibition of tenascin-C, and smooth muscle cell apoptosis. Circ Res, 1999, 84: 1223-1233.
47. Lee JH, Lee DS, Kim EK, et al. Simvastatin inhibits cigarette smoking–induced emphysema and pulmonary hypertension in rat lungs. Am J Respir Crit Care Med, 2005,172: 987 - 993.
48. Rubin LJ. Therapy of Pulmonary Hypertension: The Evolution from Vasodilators to Antiproliferative Agents. Am J Respir Crit Care Med, 2002, 166: 1308-1309.
