Prevention Of Hypoxic Pulmonary Hypertension By Overexpression Of Angiotensin Converting Enzyme2

Background and objective:Hypoxic pulmonary hypertension is one of the common diseases in respiratory system that has a poor prognosis for its lacking of ideal treatment modality. Hypoxic pulmonary vasoconstriction and pulmonary vascular remodeling are the two major mechanisms in the pathogenesis of hypoxic pulmonary hypertension. Hypoxic pulmonary vasoconstriction, as an acute reversible process, happens at the early stage of hypoxia, while chronic hypoxia usually leads to irreversible pulmonary vascular remodeling which plays an important role in hypoxic pulmonary hypertension and has become the focus of study. Pulmonary vascular remodeling has been defined as the condition of muscularization of pulmonary arteriolae that attribute to its smooth muscle cell's migration and proliferation. Hypoxia would promotes vascular remodeling via its up-regulation of angiotensin converting enzyme (ACE) which facilitate the pulmonary smooth muscle cell's migration and proliferation. Angiotensin converting enzyme2(ACE2), a newly discovered isozyme of ACE, would counteract the effect of ACE via its degradation of Ang Ⅱ through the promotion of Ang-(1-7) synthesis. Imbalance of ACE to ACE2ratio may lead to pathological conditions which the expression of ACE is up-regulated as the ACE2always insufficient. Preliminary studies have showed hypoxia could lead to the imbalance of ACE to ACE2ratio, and ACE2could restrain the hypoxia induced smooth muscle cell's migration and proliferation, which indicated that ACE2may inhibit the pulmonary vascular remodeling. Based on these rationales, we presume that locally expressed ACE2could inhibit the pulmonary vascular remodeling which has a profound potential in hypoxic pulmonary hypertension treatment. Our research using the lenti-virus as expression vector, plans to locally express ACE2, observe its inhibition of vascular remodeling and remission of pulmonary hypertension, so as to initiate a new modality for hypoxic pulmonary hypertension treatment via regulating ACE2expression.Methods:Rat primary PASMCs were exposed to hypoxia in a hypoxia incubator (1%O2,94%N2, and5%CO2) for0h,6h,12h,24h,48h respectively, and then ACE and ACE2expression were detected by RT-PCR and Western blot, and the ratio of ACE/ACE2was figured out. The rat model of hypoxic pulmonary hypertension was set up. We detected ACE and ACE2expression of the lungs of the two group rats: Normoxia group and Hypoxia group. PASMCs were transfected with overexpressed ACE2gene, mediated by Lenti-viral, and then the effect on proliferation and migration ability of cells under normoxia or hypoxia conditions was examined by CCK-8Kit and Transwell chamber assay. In addition, the overexpressed ACE2gene was transfected to the rats by endotracheal drip.2weeks later, the rats were feeded in hypoxic or normoxic conditions.4weeks later, the RVSP, RVHI, vascular morphological characteristics were measured and analyzed. The role of ACE2transfer in protecting from pulmonary hypertention and vascular remodeling was assessed. Results:(1) The expression levels of ACE mRNA and protein of primary PASMCs from rats were increased when the hypoxia culture time prolonged, lasting to48h (P<0.01); The expression levels of ACE mRNA and protein reached to the highest levels at12h and24h after hypoxia culture, respectively, and then decreased. There was no significant difference in the ratio of mRNA/protein expression in both ACE and ACE2among the different periods before48h after hypoxia culture, while the ratio increased significantly at48h (P<0.05).(2) Compared with the lung tissue from the control group, the expression of ACE mRNA in lung tissue from hypoxia culture increased notably (P<0.01); no significance in mRNA expression between the two groups was observed; the ratio of mRNA expression of ACE/ACE2was increased (P<0.05). No statistical significance in ACE protein expression between the two groups was observed; the ACE2protein expression in hypoxia culture group was significantly lower than that in the control group (P<0.05); the ratio of ACE/ACE2in hypoxia culture group was significantly higher than that in the control group (P<0.05).(3) The results from CCK-8cell proliferation showed that the proliferation of PASMCs transfected with ACE2gene was significantly decreased compared with the PASMCs transfected with the control virus (P<0.05); Transell migration experiment showed that the migration ability in experimental group was weaker than that in the control group (P<0.01).(4) The RSVP in the group treated with hypoxia and ACE2transfection was lower than that in the group treated with hypoxia and control virus transfection [(36.45±0.64) mmHg VS (41.21±1.26) mmHg, P<0.05]; its RVHI was notably lower than that in control group (0.24±0.01VS0.32±0.03, P<0.05); The relative thickness of Vascular wall WT%in the group treated with hypoxia and ACE2transfection was decreased compared with that in the group treated with hypoxia and control virus transfection [(25.68±2.07)%VS (41.58±2.81)%, P<0.01]; The relative area of Vascular wall WA%in the group treated with hypoxia and ACE2transfection was decreased compared with that in the group treated with hypoxia and control virus transfection [(32.22±3.24)%VS (44.08±4.96)%,P<0.05].Conclusion:The study shows that:(1) hypoxia can induce the imbalance of expression of ACE/ACE2in the primary cultured pulmonary artery smooth muscle cells, and the ACE/ACE2expression in the lung tissue from hypoxic pulmonary hypertension rat model is also impaired, indicating that ACE2is related to the formation of pulmonary vascular remodeling and pulmonary hypertension.(2) Over-expression of ACE2gene by transfection leads to the inhibition of PASMCs proliferation and migration. Based on the Lenti-viral as carrier, transferring the over-expressed ACE2gene to lung tissue of rats by pulmonary airway injection can lead to the high expression of ACE2in local area of lung tissue, and transformation of over-expressed ACE2gene to lung tissue before pulmonary hypertension induced by hypoxia can prevent increase in the pulmonary artery pressure, hypertrophy of the right ventricle, and pulmonary vascular remodeling. The results suggest that ACE2can be to a powerful target for the gene therapy of hypoxic pulmonary arterial hypertension.