【作者】 范右飞；

【导师】 王玉林； 荆志成；

【作者基本信息】 山东大学 ， 儿科学， 2014， 博士

【摘要】 肺动脉高压(pulmonary arterial hypertension, PAH)是一类多病因、进展性的疾病,其定义为静息状态右心导管(RHC)测定平均肺动脉压大于等于25mmHg,最终进展为右心衰竭,直至死亡。肺动脉高压是多种生理途径和细胞类型引起的多因素的病理生理改变,包括肺血管收缩、增殖,血管阻塞性重构,炎性浸润及原位血栓形成等机制。越来越多的研究表明,氧化应激参与了肺动脉高压的发病过程。氧化应激被定义为氧化物的增加(如过氧化氢、超氧阴离子等)伴或不伴有抗氧化物或抗氧化酶(如超氧化物歧化酶)的降低。增加的氧化物不但能够通过氧化特定的生物分子,直接损伤组织器官,包括脂质过氧化和DNA氧化,而且可以与一氧化氮(NO)结合,导致毒性分子过氧亚硝酸的产生和内皮一氧化氮合成酶(endothelial nitric oxide synthase, eNOS)的脱偶联。在过去几年里,肺动脉高压的药物治疗有了很大进展,文献报导显示使用靶向药物治疗的肺动脉高压患者比安慰剂组的死亡率下降了43%,住院率下降了61%31。目前应用于肺动脉高压治疗的靶向药物有以下3类：1)前列环素类似物；2)内皮素受体拮抗剂；3)5-磷酸二酯酶抑制剂。其中5-磷酸二酯酶抑制剂包括西地那非、伐地那非和他达拉非,5-磷酸二酯酶抑制剂在肺血管系统中大量表达。5-磷酸二酯酶抑制剂通过NO/cGMP通路起到扩张血管和抗增殖作用。5型磷酸二酯酶(phosphodiesterase-type5, PDE5)是在肺组织中广泛表达的的磷酸二酯酶异构酶,它能够在鸟苷酸环化酶(guanylate cyclase)的作用下降解环磷酸鸟苷(cyclic guanosine monophosphate, cGMP),引起皿官收缩。PDE5抑制剂,包括西地那非(sildenafil)和他达拉非(tadalafil),目前已经被批准用于肺动脉高压的临床治疗。伐地那非(vadenafil),属于新型PDE5抑制剂。与传统的PDE5抑制剂相比,它完全溶于水和乙醇,对PDE5的特异性作用更强。2011年,通过一项临床双盲、随机、安慰剂实验,研究者发现伐地那非能够有效的改善PAH患者的运动能力、血流动力学指数和心功能,但机制尚不明确。在勃起功能障碍的基础和临床研究中,研究人员发现除了PDE5抑制功能,PDE5抑制剂还能够通过抑制氧自由基形成,降低NADPH氧化酶起到强大的抗氧化作用,从而缓解内皮功能障碍和血管重构,但其他PAH靶向药物,如依前列醇(epoprostenol)并没有此功能。Ghofrani等报道给予PAH患者短期伐地那非治疗后,显示虽然伐地那非能引起肺血管收缩,但其对肺循环血管缺乏特异性,另外,伐地那非中长期治疗也有个案报道,显示其对PAH的治疗并没有特殊效果。因此,伐地那非治疗PAH的有效性及耐受性仍值得进一步商榷。2009年,荆志成教授及其课题组,通过对45名PAH患者的研究,显示1年的伐地那非治疗能够有效地治疗PAH。2011年,该课题组继续进行了一项随机、双盲、安慰剂-对照及多中心的研究。结果显示与西地那非和他达拉非不同,伐地那非单药治疗即能够有效的延缓PAH患者的临床恶化程度,改善其血流动力学变化。然而伐地那非治疗肺动脉高压的具体机制仍不明确。鉴于目前伐地那非治疗PAH功能研究的缺乏,本课题收入15名特发性PAH患者,同时以野百合碱诱导的PAH大鼠为体内模型,以H202诱导的氧化应激内皮细胞为体外模型,研究伐地那非治疗PAH的机制研究和可能涉及的信号通路,为PAH的治疗提供了依据。研究分以下三部分：第一部分伐地那非改善氧化应激并治疗特发性肺动脉高压的临床研究本研究选择特发性肺动脉高压患者作为研究对象,分析了伐地那非对特发性肺动脉高压患者的治疗作用,及其对患者体内氧化应激水平的改善作用。1)我们选取了EVALUATION研究中的15名特发性肺动脉高压的患者,并选择了年龄及性别与之相匹配的健康人群作为对照。入选患者给予伐地那非每次5mg,每日1次口服1个月,若患者未出现严重不良反应,则加量至每次5mg,每日2次继续口服2个月。2)我们于基线及3个月后评估完成以下内容：标准病史与体格检查,6分钟步行距离,WHO肺动脉高压功能分级,血生化指标,心电图,肺功能检查,和完整的右心导管检查所测血流动力学资料。结果显示3个月的伐地那非治疗能显著改善特发性肺动脉高压患者WHO功能分级、六分钟步行距离、心指数、肺血管阻力等指标。尽管跟用药前相比较,患者平均肺动脉压力、肺毛细血管楔压、平均右房压力较前有降低,但并没有统计学差异。以上结果说明伐地那非能够改善肺动脉高压患者临床症状及血流动力学变化。3)随后我们采用商品化试剂盒测量了肺动脉高压患者及健康对照组血液中一氧化氮、丙二醛、8-异前列素F2α、3-硝基酪氨酸、超氧化物歧化酶等氧化应激及氮化应激相关产物的水平。发现肺动脉高压患者血浆中氧化损伤产物丙二醛、8-异前列素F2α、3-硝基酪氨酸明显高于正常人,氧化应激保护性产物一氧化氮、超氧化物歧化酶明显低于正常对照者。伐地那非治疗3个月后,氧化损伤产物8-异前列素F2α及3-硝基酪氨酸水平均较前降低,但仍高于正常人,而丙二醛的水平并没有明显改变。血管扩张产物一氧化氮较治疗前明显升高,基本恢复正常水平。抗氧化酶超氧化物歧化酶较治疗前升高,与正常人相比,仍处于较低水平。以上以上结果显示,肺动脉高压患者体内存在较高的氧化应激水平,抗氧化能力明显较正常人减弱。而伐地那非治疗能够降低患者的氧化应激水平,改善其抗氧化能力,起到保护性作用。第二部分伐地那非改善野百合碱诱导肺动脉高压大鼠氧化应激水平的研究本研究选择了34只雄性SPF级Sprague-Dawley (SD)大鼠,体重200-220g,随机分为正常对照组、野百合碱模型组以及伐地那非治疗组。给予50mg/kg野百合碱21天后,伐地那非治疗组大鼠予1mg/kg/d灌胃共21天。之后进行如下研究：1)应用右心导管检查术测量大鼠的血流动力学指标,发现MCT组大鼠的mPAP、RVSP和PVR明显高于正常对照组,而CO则明显低于正常对照组。给予伐地那非治疗后,伐地那非组大鼠的mPAP、RVSP和PVR较MCT组分别降低41%、25%和26%。以上结果与我们临床试验结果相一致,进一步验证了伐地那非对血流动力学的改善作用。2)为研究伐地那非对血流动力学改善的原因,我们对大鼠肺组织进行了组织学染色并应用相关软件对血管组织进行测量。与正常对照组相比,野百合碱组大鼠的%WT和%WA明显增高。而给予伐地那非治疗后,伐地那非组的%WT和%WA明显降低。以上结果在<50μm-100μm、100-200μm直径的肺血管中均有所体现。之后我们通过a-SMA抗体染色,检测大鼠的血管肌化程度。跟正常对照组相比,野百合碱组血管肌化程度显著增加,镜下观察平滑肌细胞明显肥大增生。给予伐地那非治疗后,伐地那非组肌化程度较MCT组降低了39%,平滑肌细胞增生肥大有所改善。最后我们对肺血管平滑肌细胞进行了PCNA及TUNEL染色,发现伐地那非对血管厚度的改善是通过诱导大鼠肺动脉平滑肌细胞的增值,同时促进其凋亡而实现的。我们应用werstern blot的方法及商品化试剂盒,研究了伐地那非治疗肺动脉高压的相关机制。与正常对照组相比,野百合碱组大鼠血浆及肺组织内NO的浓度降低,.给予伐地那非治疗后,跟野百合碱组相比,伐地那非组血浆及肺组织内NO水平均较前恢复。原因是通过伐地那非上调eNOS蛋白的表达而实现的。之后我们发现伐地那非能够改善大鼠体内氧化应激水平,与我们临床试验结果相一致。为了进一步研究可能的机制,我们选择了氧化应激过程中主要的酶,NADPH氧化酶和超氧化物歧化酶,进行下一步研究。结果显示与正常对照组相比,野百合碱大鼠大鼠体内NADPH氧化酶催化亚基,NOX2和NOX4,的水平明显上调,但激活亚基RAC1水平并没有明显变化。伐地那非治疗能显著改善NOX2和NOX4的表达水平,却同样不影响RAC1亚基的表达水平。相反,MCT组大鼠体内SOD活性较正常对照组相比明显降低,而伐地那非治疗后,SOD活性得到了显著改善。以上结果提示,伐地那非对氧化应激水平的改善,其中一部分是通过改善NADPH氧化酶和超氧化物歧化酶而实现的。第三部分伐地那非通过BMP信号通路改善内皮细胞氧化损伤的机制研究通过第一部分和第二部分的体内研究,我们明确了伐地那非能改善氧化应激同时治疗肺动脉高压,但具体机制仍不清楚。因此第三部分,我们应用CRL-1730内皮细胞系为研究模型,给予不同剂量外源性H202造成其氧化应激损伤,进一步研究了伐地那非对氧化应激损伤保护性作用的具体机制。1)通过MTT及TUNEL的方法,我们发现与对照组比较,H202组细胞活力显著降低,伐地那非各剂量组与H202组比较,细胞活力显著升高,且具有明显的剂量依赖性。与对照组比较,H202组细胞凋亡率显著增高,给予伐地那非处理后,细胞凋亡率较前明显改善,且具有剂量依赖性。以上结果于体内试验结果相互印证：伐地那非对内皮细胞起到了强烈的保护性作用,且该作用呈剂量依赖性。2)之后我们检测了骨形态发生蛋白(bone morphogenetic proteins,BMPs)信号通路在各组细胞中的表达,BMP信号通路广泛参与了肺动脉高压、动脉粥样硬化等血管疾病。结果显示,氧化应激随时能够上调BMP受体1A、BMP受体1B、BMP受体2,同时上调胞浆内p-smad水平,最后进入细胞核内,上调转录因子MSX2的表达。而伐地那非能够改善上述通路,起到保护性作用。3)最后我们应用BMP信号通路的抑制剂,Dorsomorphin干预细胞,并通过TUNEL检测细胞凋亡情况。我们发现,与对照组比较,H202组细胞内细胞凋亡率显著增高,伐地那非组与H202组比较,细胞凋亡率显著下降,而与伐地那非组比较,Dorsomorphin干预组细胞凋亡率明显上升。说明Dorsomorphin能抑制伐地那非对内皮细胞的保护性作用,进一步说明BMP信号通路参与了伐地那非对内皮细胞氧化应激损伤的改善。综上所述,我们通过临床研究、动物实验及体外细胞培养,证明了伐地那非对PAH的治疗作用以及对PAH患者体内氧化应激水平的改善作用,及其相关的机制。更多还原

【Abstract】 Pulmonary arterial hypertension (PAH) is characterized by a multifactorial, progressive disease with a sustained increase in pulmonary pressure ultimately leading to right ventricular failure and death. Remodeling of pulmonary vessels is a frequent structural change in chronic PAH, which involves a proliferative and anti-apoptosis state of pulmonary arterial smooth muscle cells (PASMCs) within the vessel wall. Oxidative stress is mainly caused by an imbalance between the activity of endogenous pro-oxidative enzymes, such as NADPH oxidase, and the anti-oxidative enzymes, such as superoxide Dismutase (SOD), leading to superoxide anion (#O2-) accumulation. The excessive·O2-reacts with nitric oxide (NO), resulting in toxic molecule peroxynitrite production and endothelial nitric oxide synthase (eNOS) uncoupling. Furthermore,·O2-itself also attack the lipid, protein and DNA, causing the damage of tissues in vivo. This, in turn, causes impaired smooth muscle dysfunction and subsequent pulmonary vascular remodeling. Recently, evidence suggests that increased oxidative stress may be important in the pathogenesis of PAH in animal models and in humans. In contract, PAH could be improved or even reversed by anti-oxidant treatment.There are three main classes of drugs in use for the treatment of PAH: prostanoids, endothelin-1(ET-1) receptor antagonists, and phosphodiesterase-type5(PDE5) inhibitors. PDE5is a phosphodiesterase isoenzyme that abundant in lung tissue, and degrades cyclic guanosine monophosphate (cGMP) causing constriction of blood vessel walls. The PDE5inhibitors, including sildenafil and tadaladil, were approved for the treatment of PAH in2005and2009respectively. Previous studies suggest that PDE5inhibitors may actually exert direct or indirect effect beyond PDE5inhibition, such as anti-oxidative stress, anti-inflammation, and anti-proliferation. They could decrease the·O2-formation and Nox2expression both in vitro and in vivo, improving the endothelial dysfunction caused by oxidative stress, which subsequently leads to vascular remodeling.Vardenafil is a PDE5inhibitor that was approved for the treatment of erectile dysfunction in2005. Pharmacodynamics of this agent has shown that it to be more potent than sildenafil and tadalafil in inhibiting PDE5. IIn this study, we used zebrafish as an in vivo model to investigate the function of SLC33A1in the development of zebrafish embryos, especially in the motor neuron development. Ghofrani et al reported that with short-term use in patients with PAH, vardenafil appeared to lack specificity for the pulmonary circulation despite inducing significant pulmonary vasorelaxation.In adiition, the efficacy and safety of mid-to long-term vardenafil treatment in patients with PAH has only been examined in case reports,which had insufficient power and sample size. Consequently, it is still unclear whether vardenafil is an effective and well-tolerated treatment for PAH.In2009, our group enrolled45PAH patients and demonstrated that1-year’streatment with vardenafil had sustained beneficial effects on PAH. In2011, the Jing and his coworkers started a randomized, double-blind, placebo-controlled, multicenter study. The results showed that, unlike sildenafil and tadalafil, vardenafil mono-therapy delayed clinical worsening in PAH patients. However, how vardenafil works in vivo is still unclear. We hypothesized here that vardenafil can improve PAH and vascular remodeling via oxidative stress pathway.In this study, we enrolled15idiopathic PAH patients, used monocrotiline-induced PAH rats as an in vivo model and H2O2induced oxidative stress injury as an in vitro model to investigate the protective effect of vadenafil on the development of PAH.Part I. Vardinafil improved PAH and oxidative stress level in IPAH patientsIn order to investigate the protective role of vadinafil in vivo, we first enrolled15idiopathic PAH (IPAH) and examined their hemodynamic parameters and oxidative stress levels at baseline as well as afrer3-month vadinafil treatment.1) Patients from the Efficacy and Safety of Vardenafil in the Treatment of Pulmonary Arterial Hypertension (EVALUATION) Study Group with idiopathicPAH (IPAH) who were World Health Organization (WHO) functional classes II or III were enrolled in this study (n=15). Patients were given vardenafil5mg orally daily for1month after which the dosage was increased to5mg orally twice daily if no significant adverse events occurred.2) Patients who met the inclusion criteria were hospitalised in one of the EVALUATION study, which consisted of history, physical examination, echocardiography, ECG, chest x-ray examination, nuclear ventilation perfusion scan, chest computed tomography scan and a6min walk test. All patients underwent diagnostic cardiac catheterisation. A total of15patients with IPAH (11women and4men, median age29years) were treated with vardenafil. The median6MWD was increased from baseline by79m in patients treated with vardenafil, indicating the improvement of exercise capacity. Therewas a significant decrease in PVR from baseline, along with a significant increase in CI.3) The levels of different oxidative damage products (8-Iso-PGF2a,3-nitrotyrosine and MDA), SOD activity and NO levels in the plasma of humans were evaluated using commercial kits. Compared with the saline group, NO levels were markedly decreased in the saline MCT group). In patients, vardenafil significantly increased NO levels in the plasma of patients who had decreased NO levels owing to PAH. Similar results were seen in oxidative damage products:after3months of treatment with vardenafil, levels of8-Iso-PGF2a and3-nitrotyrosine in plasma were significantly decreased from baseline.Part Ⅱ. Vardenafil reverses pulmonary arterial hypertension through reducing oxidative stress in monocrotaline-induced PAH ratsIn order to study the mechanism of vardinafil treatment, we take monocrotaline (MCT)-induced PAH rats as in vivo models. A total of34adult male Sprague-Dawley rats (body weights,200-220g) were divided into two groups:control rats that received a single subcutaneous injection of saline (n=10; saline group) and rats that received a single subcutaneous injection of MCT(50mg kg-1) to induce PAH (n=24). Three weeks later, rats with induced PAH were randomly and equally divided into two groups for oral treatment with either vardenafil (lmg kg-1day-1; vardenafil MCT group) or vehicle (an equal volumeof saline; saline MCTgroup) for a further21days.1) The haemodynamic parameters of rats were measured by a polygraph system. Rats challenged with MCT consistently developed significant PAH, with higher mPAP, RVSP, and PVR, and lower CO, than the saline group. Vardenafil treatment resulted in a marked reduction in mPAP, RVSP, and PVR by41,25, and26%, respectively, compared with the saline MCT group. Accordingly, vardenafil significantly increased CO without affecting heart rate2) Then we use Integrated Performance Primitives version5.0software to analyse the morphometric changes in lung tissue of rats. The pulmonary vessel wall was markedly increased and appeared stenosedor occluded in the saline MCT group compared with the vardenafil MCT group or the saline group. The medial vessel wall thickness was markedly reduced in the vardenafil MCT group compared with the saline MCT group. The vardenafil MCT group had a significant reduction compared with the saline MCT group in medial vessel wall thickness and area at all sizes. In addition, vardenafil reduced muscularization of the distal pulmonary arteries compared with the saline MCT group, which may caused by the modulation of proliferation and apoptosis.3) At last, we ues western blotting method and commercial kits to evaluate the levels of oxidative stress and the key enzymes during oxidative pathway, including NADPH oxidase and SOD. Compared with saline control rats and control participants, levels of8-Iso-PGF2a, MDA, and3-nitrotyrosine, were higher in the lung tissue of rats treated with MCT plus saline. Vardenafil treatment significantly reduced levels of8-Iso-PGF2a and3-nitrotyrosine in MCT-treated rats. Compared with the saline group, protein expression of the catalytic subunits NOX2and NOX4was strongly up-regulated in the saline MCT group, whereas the expression of the activator subunit RAC1did not vary between groups. NOX2and NOX4expression were markedly decreased in the vardenafil MCT group compared with the saline MCT group, but the expression of the RAC1subunits remained unchanged. The SOD activity in lung tissue from saline MCT rats was significantly lower than that in lung tissue from the saline group When rats with MCT-induced PAH were treated with vardenafil, SOD activity was increased. Taken together, the vadenafil could improve the oxidative stress levels of MCT-induced PAH rats via NADPH oxidase and SOD.Part III. Vadenafil protected H2O2-induced oxidative injury via BMP pathway in endothelial cells.After the studies of Part I and Part II, we confirmed that vardenafil could improved PAH as well as redued oxidative stress. However, how vardenafil works is still unclear. We take endothelial cells as in vitro models and hypothesized here that vardenafil can alleviate oxidative injury via bone morphogenetic proteins (BMP) pathway.1) We first use MTT method to detect cell viability of endothelial cells. Compared with control group, the cell viability of H2O2group significantly lower. Whereas the cell viability in different vardenafil groups are dose-dependent higher than H202group. By means of TUNEL method, we deternmined the apoptosis rate in every group. Compared with control group, the apoptosis rate in H2O2group significantly increased. Conversely, the apopsosisi rate decreased after vardenafil treatment.2) Second, we determined BMP pathway, which take important role in the course of PAH, via western bloting and real-time PCR methods. Compared with control group, H2O2could up-regulate the protein expression of BMP receptor (BMPR), including BMPRIA, BMPRIB and BMPR2. In addition, H2O2could increase the levels of p-smad in cytoplasm and mRNA expression in nucleus. Given vardenafil treatment, the BMP pathway have been nomolized, suggesting the protective effect on oxidative injury in endothelial cells.3) At last, in order to learn more about vardenafil and BMP pathway, we added the inhibitor of BMPR(Dorsomorphinthe), and evaluated the apoptosis rate in every groups. Compared with control group, the apoptosis rate in H2O2group significantly increased. Therefore, compared with vardenafil group, Dorsomorphin group’s apoptosis rate significantly increased, indicating the important role of vardenafil in protection of oxidative injury.In summary, we used clinical study, MCT-induced PAH rats and endothelial cells culture study, to confirm the protection of vardenafil on PAH and oxidative stress, as well as the relative mechamism.更多还原