The Preventive And Therapeutic Effects Of ACE2Activation On Severe Pulmonary Arterial Hypertension In Rats And Its Underlying Mechanisms

[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.