【作者】 李刚；

【导师】 刘迎龙；

【作者基本信息】 北京协和医学院 ， 外科学， 2012， 博士

【摘要】 背景和目的致病因素的作用下,肺循环调节功能的失衡启动一系列病理生理改变,包括内皮功能障碍、炎症激活等,引起肺血管异常收缩,进而平滑肌细胞增殖、血管重构,最终导致肺动脉高压。肾素血管紧张素系统是心血管系统的重要调节系统,它在肺动脉高压发病过程中发挥重要作用,但是干预经典的肾素血管紧张素系统(ACE-Ang Ⅱ-AT1R轴)治疗肺动脉高压的效果欠佳。血管紧张素转化酶2(ACE2)及其参与的ACE2-Ang-(1-7)-Mas轴是肾素血管紧张素系统的新成员,起着拮抗经典肾素血管紧张素轴的作用,而且在呼吸系统疾病中发挥着广泛的作用。本研究应用ACE2激动剂Resorcinolnaphthalein(Rec)干预野百合碱联合肺叶切除构建重度肺动脉高压模型,探讨ACE2激活在预防和逆转重度肺动脉高压中的作用及其机制。方法1.实验动物和分组：SD大鼠,随机分为6组：空白对照组,ACE2对照组,肺动脉高压组,ACE2预防组,ACE2治疗组,Mas受体拮抗组(Rec+A-779)。左肺切除后1周,皮下注射野百合碱诱导重度肺动脉高压。ACE2对照和ACE2预防组在野百合碱注射后第1天开始持续注射Rec, ACE2治疗组第14天开始注射Rec, Mas受体拮抗组第1天开始同时注射Rec和Mas受体拮抗剂A-779。2.干预2周后,除ACE2治疗组外(干预42天),其余各组取8只,心导管测定平均肺动脉压和动脉收缩压,分离右心室测定右心室肥厚指数,弹力纤维染色(Verhoeff-铁苏木素染色)后分析肺动脉(100-200gm)中膜厚度和肺小动脉(50-100gm)内膜梗阻性增生程度。3.内皮功能检测：肺动脉内注射不同浓度的乙酰胆碱和硝普钠,检测内皮依赖的血管舒张反应的变化。4.活性荧光共振能量转移法检测肺组织ACE2酶活性。5.生存分析：采用Kaplan-Meier生存分析图分析。6. ELISA检测肺组织Ang Ⅱ和Ang-(1-7)浓度,Western blot检测肺组织ACE, ACE2, AT1R, Mas表达水平。7.免疫组化检测直径50-100μm肺动脉管壁平滑肌抗体(a-SMA)和增殖细胞核抗原表达,分析肺动脉平滑肌细胞增殖情况。8. RT-PCR检测肺组织炎症因子IL-6, MCP-1, TNF-α, IL-10。结果1.Rec对大鼠肺组织ACE2酶活性的影响ACE2对照组ACE2酶活性是正常的1.74倍；肺动脉高压组ACE2酶活性下降38.2%,ACE2预防和治疗组ACE2酶活性则分别比肺动脉高压组升高1.87和1.78倍,提示Rec可以激活大鼠肺组织ACE2。2.ACE2激活对PAH大鼠生存率的影响实验终点时,肺动脉高压大鼠和Mas受体拮抗组大鼠全部死亡,ACE2预防组未出现死亡,ACE2治疗组生存率为80%。统计学分析,ACE2治疗组和预防组与肺动脉高压组差异显著。3.ACE2激活对血液动力学和右心室的影响大鼠左肺切除联合野百合碱注射2周后,肺动脉压力升高,右心室肥大；ACE2对照组肺动脉压力没有明显变化。ACE2激活可以防止肺动脉高压和右心室肥厚,同样ACE2治疗4周也可以降低肺动脉压力,但是无法逆转肥厚的右心室。各处理组大鼠动脉收缩压没有变化。4.ACE2激活对肺动脉内皮细胞功能的影响在相同的基础肺动脉压力下,肺动脉高压大鼠乙酰胆碱诱导的内皮依赖的舒张反应减弱,ACE2不但可以抑制它的减弱,而且可以修复受损的舒张反应。硝普钠诱导的肺内皮依赖的舒张反应各组之间没有明显差异。5.ACE2激活对肺动脉中膜肥厚和内膜增生的影响肺动脉高压大鼠的直径为100-200gm的肺小动脉主要表现为中膜肥厚,ACE2激活可以抑制和逆转中膜肥厚。直径为50-100μm的肺小动脉主要表现为内膜增生,梗阻明显,ACE2预防组大鼠则主要表现为中膜肥厚,ACE2治疗组大鼠小动脉梗阻程度也显著下降。6.Mas受体对ACE2效果的影响同时应用ACE2激活剂Rec和Mas受体阻断剂A-779,肺动脉高压预防作用消失,包括改善生存率,降低肺动脉压力,抑制右心室肥厚,肺小动脉中膜肥厚和内膜增生。7.ACE2激活对肾素血管紧张素系统各主要成份的影响肺动脉高压形成以后,ACE和AT1R上升为正常的2.49和2.75倍,Ang IⅡ则由25.06±3.81pg/mg上升到91.00±6.70pg/mg,ACE2激活可抑制ACE(78.5％), AT1R(78.9%)和Ang Ⅱ(46.60±10.00pg/mg)的上升,但是肺动脉形成以后再激活ACE2,ACE和AT1R下降不明显,仅能降低Ang Ⅱ(52.01±7.61pg/mg,).肺动脉高压大鼠肺组织ACE2降低为正常的41.67%,而预防和治疗组ACE2则达到肺动脉高压大鼠的2.52和2.35倍。肺动脉高压对Mas和Ang-(1-7)的影响不大,ACE2激活后,Mas则分别升高了的2.43和2.51倍,Ang-(1-7)也相应升高。肺动脉高压大鼠肺组织ACE/ACE2和AT1R/Mas比值明显升高,而预防组和治疗组这两个比值则降低。8.ACE2激活对肺动脉平滑肌细胞增殖的影响各组直径为50-100gm肺小动脉壁均广泛表达平滑肌细胞抗体,提示平滑肌细胞在其病变中占重要地位。肺动脉高压大鼠肺动脉增殖细胞核抗原阳性率显著升高,ACE2激活后可以降低平滑肌细胞的增殖细胞核抗原阳性率,抑制增殖。9.ACE2激活对肺组织炎症因子的影响大鼠肺动脉高压形成后,IL-6上升47倍,MCP-1上升31倍,TNF-a上升16倍,IL-10下降79%；而预防组IL-6降低85%,MCP-1降低86%, TNF-α降低82%,IL-10上升11倍,治疗组则IL-6降低82%,MCP-1降低80%, TNF-α降低78%,IL-10升高9倍。结论1.Rec持续注射可以在大鼠体内有效的激活肺组织ACE22.ACE2激活可预防左肺切除联合野百合碱注射诱导的大鼠重度肺动脉高压形成3.ACE2激活一定程度上逆转大鼠重度肺动脉高压的血液动力学和病理学改变4.ACE2激活通过调控肾素血管紧张素系统内缩血管／促增殖轴(ACE-AngⅡ-AT1R轴)和血管保护轴(ACE2-Ang-(1-7)-Mas轴)之间的平衡起作用。5.在大鼠肺动脉高压的形成和进展过程中激活ACE2,可修复内皮依赖的血管舒张反应,改善内皮功能；可减少新生内膜中平滑肌细胞PCNA的表达,抑制平滑肌细胞增生；可降低肺组织促炎因子IL-6, MCP-1, TNF-α,升高抗炎因子IL-10,从而减轻炎症反应。更多还原

【Abstract】 [Background and objective]The imbalance of the physiological regulation caused by the etiological factors in the pulmonary circulation initiating a cascade of pathophysiological events including endothelial dysfunction and inflammation lead to abnormal vasoconstriction and proliferation of vascular cells, which results in vascular remodeling and pulmonary arterial hypertension (PAH). Previous data have shown the involvement of classic rennin-angiotensin axis [angiotensin-converting-enzyme (ACE)-angiotensin Ⅱ (Ang Ⅱ)-angiotensin Ⅱ receptor1(AT1R)] which is one of the most important regulation system of cardiovascular function during the pathogenesis of pulmonary arterial hypertension, but the results of pharmacological blockade with ACE inhibitors or AT1R antagonists in medical use were unsatisfactory. A recent discovered ACE homologue, angiotensin-converting enzyme2(ACE2), has opposite effects on classical RAS and was deeply involved in lung disease, indicating a promising agent against PAH. In current study, we evaluated the activation of ACE2by a small synthetic molecule resorcinolnaphthalein (Rec) in preventing and reversing severe PAH induced by monocrotaline injection following left pneumonectomy in rats and further explored its underlying mechanisms.[Methods]1. Treatment and groupsEighty male SD rats were randomly divided into the following six treatment groups. Placebo control group (n=8), ACE2control group (n=8), PAH group (n=18), ACE2preventive group (n=18), ACE2therapeutic group (n=10), Mas receptor blocked group (n=18). The rats in the two control group received sham operation and vehicle; the other rats were injected monocrotaline one week following left pneumonectomy for inducing severe PAH. Rec was started injection with the dose of120μg/kg at the first day after MCT injection in ACE2control group and ACE2preventive group and at the fourteenth day in ACE2therapeutic group. The rats in Mas receptor blocked group received simultaneous injection of Rec and A-779both at the dose of120μg/kg since the first day after MCT injection.2. The hemodynamics including mean pulmonary arterial pressure (mPAP) and systolic blood pressure (SBP) were measured by catheterization after two weeks treatment in8rats in the all the groups except ACE2therapeutic group (6weeks treatment). The ratio of right ventricular to left ventricular plus septum weight (RV/LV+S) was calculated for each animal as an index of right ventricular hypotrophy (RVHI). The pulmonary arteries were stained for elastic lamina by Verhoeff iron hematoxylin method, then the percent of wall thickness (WT%) were calculated to analyze media hypertrophy in pulmonary arteries with the diameter of100-200μm and mean vascular obstruction score (mVOS) were calculated as an index of neointima in arteries with the diameter of50-100μm.3. Endothelium-dependent vasorelaxation was assessed by the reductions in mPAP in response to bolus injection of acetylcholine (0.1,0.3,1.0μg/Kg) and sodium nitroprusside (5,10,15μg/Kg) into pulmonary artery. In order to keep similar baseline of pulmonary arterial pressure, the arteries were preconstricted by a continuous infusion of U46619, if necessary4. The enzymatic activity of ACE2in lungs was examined by its ability to cleave the fluorescent peptide substrate Mca-Ala-Pro-Lys(Dnp)-OH.5. Survival analysis:The differences of survival were analyzed by Kaplan-Meier survival plot among the ACE2preventive group (the rest of the10rats), Mas receptor blocked group (the rest of the10rats), PAH group and ACE2therapeutic group。The rat was sacrificed when it lose more than20%of its weight. Both the sacrificed and died rats during the42days treatment were included in the analysis.6. Ang Ⅱ and Ang-(1-7) levels in lungs of the rats were determined by ELISA; ACE, ACE2, AT1R, Mas protein expression were detected by Western blot analysis.7. The proliferation of pulmonary smooth muscle cells were analyzed by stain for a-smooth muscle actin and proliferating cell nuclear antigen with immunohistochemical method in small pulmonary arterial in diameter of50-100μm.8. Real-time quantitative polymerase chain reaction (RT-PCR) was used for measuring tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1(MCP-1), interleukin-6(IL-6), and IL-10gene levels in the lungs.[Result]1. The activation of ACE2by Rec in lungs The ACE2activity increased by1.74folds in ACE2control group when compared the values in Placebo control group, while the enzyme activity decreased38.2%in PAH group (P<0.05). Compared to PAH group, ACE2activity respectively increased1.87fold and1.78fold in ACE2preventive group (P<0.05) and ACE2therapeutic group (P<0.05)2. The activation of ACE2improved survival rates The rats in PAH group and Mas receptor blocked group were sacrificed or died before the experimental endpoint without exception (survival,0), whereas rats in ACE2preventive group were active and without death in the42days experimental duration (survival,100%). Rats in ACE2therapeutic group showed a low mortality (survival,80%). These data indicated that Rec treatment resulted in an increased survival in severe PAH rats (ACE2preventibe group vs PAH group, ACE2therapeutic group vs PAH group, all p<0.05).3. The effects of ACE2activation on PAH Compared to Placebo control group (15±2mmHg), the mPAP was similar in ACE2control group (17±2mmHg) but increased in PAH group (45±6mmHg)(p<0.05). The mPAP in ACE2preventive group (22±5mmHg) and ACE2therapeutic group (30±5mmHg) were significant lower than the values in PAH group (45±6mmHg). Similarly, ACE2activation prevented the increase in RVHI (ACE2preventive group,0.36±0.06vs PAH group,0.59±0.12, p<0.05) but did not reverse the increased RVHI (ACE2therapeutic group,0.53±0.05vs PAH group,0.59±0.12, p>0.05). The systolic blood pressure was not changed in rats among the groups.4. The effects of ACE2activation on endothelial function in pulmonary arteries The decrease of mPAP in response to acetylcholine injection under the similar initiating pressure conditions were smaller in PAH group than in placebo control group (p<0.05) and the values in both ACE2preventive group and ACE2therapeutic group were significantly increased when compared to PAH group. However, the decrease of mPAP in response to sodium nitroprusside injection was similar among the groups.5. The effects of ACE2activation on pulmonary vascular remodeling The pulmonary arteries with the diameter between100-200μm from PAH rats showed media hypertrophy with higher WT%than the values in control rats (Placebo control group,16.37±3.20%vs PAH group,52.13±11.06%, P<0.05). In contrast, WT%in ACE2preventive group and ACE2therapeutic group were decreased (PAH group,52.13±11.06%μs ACE2preventive group,31.38±8.49%, P<0.05; PAH group,52.13±11.06%vs ACE2therapeutic group,36.13±4.39%, P<0.05). Neointima formed in small pulmonary arteries in diameter of50-100μm from PAH rats with increased mVOS when compared to control rats (Placebo control group vs PAH group, P<0.05). The mVOS in ACE2preventive group and ACE2therapeutic group were lower than in PAH group (P<0.05).6. The Role of Mas in the effect of ACE2activation on PAH The beneficial effects of ACE2activation on survival, mPAP, RVHI, WT%and mVOS in PAH rats were abolished by simultaneous injection of Mas receptor inhibitor A-779.7. The change of the renin-angiotensin system components in the lung The PAH caused2.49folds and2.75folds increase in ACE and ATIR protein expression in lungs, and increased Ang Ⅱ from25.06±3.81pg/mg to91.00±6.70pg/mg, while ACE2activation inhibited the increase in ACE(78.5%), ATIR (78.9%) and Ang Ⅱ(46.68±10.00pg/mg). However, ACE2activation had insignificant effect on ACE and ATIR except for the significant decrease in Ang Ⅱ (52.01±7.61pg/mg).The protein level of ACE2in PAH group decreased to41.67%of the values in Placebo control group while increased to2.52and2.35folds of the values in PAH group. The levels of Mas increased to2.43and2.51folds of PAH group in ACE2preventive group and ACE2therapeutic group respectively, and Ang-(1-7) increased from32.74±4.72pg/mg in PAH group to59.28±8.91pg/mg in ACE2preventive group and53.10±7.34pg/mg in ACE2therapeutic group.The ratios of ACE/ACE2and ATIR/Mas were significant higher in PAH group than in Placebo control group, but markedly lower in ACE2preventive group and ACE2therapeutic group than PAH group.8. The effects of ACE2activation on pulmonary smooth muscle proliferation The a-SMA were widely expressed on small pulmonary arteries with the diameter between50-100μm, indicating the critical role of smooth muscle cells in the pathological change during the development of PAH. The PCNA positive rate were higher in PAH group (13.63±3.42%) than in placebo control group (3.88±1.36%)(p<0.05), but the rate decreased in both ACE2preventive group (3.38±0.92%) and ACE2therapeutic group(4.75±1.04%)group when compared the values in PAH group (p<0.05).9. ACE2activation mediated the levels of cytokinesThe gene levels of IL-6, MCP-1and TNF-α were increased47folds,31folds,16folds of the values in placebo control group in PAH group (p<0.05), while IL-10decreased79%. In contrast, the levels of IL-6, MCP-1and TNF-α decreased85%,86%,82%but IL-10was increased11folds in ACE2preventive group respectively, when compared to PAH group, and in ACE2therapeutic group, IL-6decreased82%, MCP-1decreased80%, TNF-a decreased78%, but IL-10increased9folds(p<0.05). [Conclusions]1. Rec infusion effectively activated ACE2in lungs from rats.2. ACE2activation prevented the development of severe PAH induced by momocrotaline injection following left pneumonectomy in rats.3. ACE2activation partially reversed the hemodynamics and pathological change in rats with severe PAH.4. ACE2activation shifted the vasoconstrictive proliferative axis (ACE-Ang II-AT1R) toward vasoprotective axis (ACE2-Ang-(1-7)-Mas) in RAS.5. ACE2activation improved endothelial function, inhibited the proliferation of pulmonary smooth muscle cells and mediated inflammation by decreasing IL-6, MCP-1,TNF-α and increasing IL-10in both preventive and therapeutic protocols.更多还原