The Recombinant Adenovirus-mediated Human Endothelial Nitric Oxide Synthase Gene Therapy On Chronic Hypoxic Pulmonary Hypertension Rats

ObjectivePulmonary arterial hypertension (PAH) is the complication of many heart and pulmonary vascular diseases. PAH results in a progressive increase in pulmonary vascular resistance and, ultimately, right ventricular failure and death. It occurs in cardiovascular surgery and with a high mortality rate during the perioperation. Chronic hypoxemia is a considerable factor in procedure of initiating PAH. There are three main factors to be thought to cause the chronic hypoxic pulmonary hypertension(CHPH) that characterized this disease: pulmonary vasoconstriction, remodeling of the pulmonary vessel wall,and thrombosis in situ. As the Endothelium Derived Relaxing Factor (EDRF), Nitric Oxide (NO) plays an important role in endothelial vasodilatation. NO can regulate the vascular tone and the mesenchymal cell growth, keep the pulmonary circulation stable. The production and activity of NO is closely related to the etiology of PAH,so it has considerable meaning re-establish the function of NO. At present, NO is approved officially in the therapy of PAH by FDA. However, the short biological half-life,the quick forming toleration, and the obviously side effect and unsuitable utilization make the clinical application of NO limited. Following the progresses of gene enfineering and molecular biology research, it is possible for using the recombinant adeno virus-mediated endothelial nitric oxide synthase gene to therapy the PHA. Many experiment has demonstrated that gene transfer of eNOS in vivo and in vitro with overproduction of endogenous eNOS may be a promising approach to ameliorate pulmonary hypertension without those tedious side effects. So far,the ways to treat PAH through which exogeoous gene are transferred include vascular endothelium,adventitia and airway epithelia. To provide data of gene therapy for PAH, study the feasibility, the effect and the mechanism of PAH transgene therapy, adenovirus-mediated human endo-thelial nitric oxide synthase gene was transfered to the chronic hypoxic pulmonary hypertension (CHPH) rats through airway.Materials1. Experimental aniamals:150 male Wistar rats, provided by Experimental Animal Center, General Hospital of Shenyang Command of PLA.2. Experimental instruments: Animal ventilator(Harvard 638, U. S. A.),Olympus light microscope(Japan), PTC-100 PCR amplification device (U. S. A.), Sorvall ST-70 low temperature and high speed centrifuger(U. S. A), Chem image 5500 gel image analyzing system (U. S. A), PE 7000 automatic fluorescent quantitation PCR device(U. S. A)3. Chemical and reagents; cDNA plasmid of virus AdCMVceNOS(Dr Robert Gerard),293 cells(Canada Microbix Biosystems company),NO kit (Nangjing), fluorescent probe (Shanghai), dNTPs (Sigma), Taq enzyme (ABI), monoclonal antibody (BD company)Methods1. The changes of eNOS/NO system in CHPH: 50 Wistar rats were randomly allocated to one of the five experimental groups, N group: normoxia group;H7d group:the rats underwent hypoxia 7 days;H14d group: the rats underwent hypoxia 14 days;H21d group: the rats underwent hypoxia 21 days;H28d group: the rats underwent hypoxia 28 days. The rats in hypoxia group were put into the normal pressure hypoxia case 8 hours daily. The mixed gas(90%N₂ and 10% O₂) blowed into the case. Under the gas monitoring, the oxygen concentration maintained in 10. 0±0. 5) % and carbon dioxide concentration maintained in (3. 0±0. 5)%. The external carotid artery and pulmonary artery pressure were measured respectively. After measured, 2 ml pulmonary artery blood was withdraw for the measurement of NO content. The expression of eNOS protein was detected by Western blot. The pneumoangiogram architeconic change was determined by elastic fibers stain.2. The transfection of adenovirus mediated endothelial nitric oxide synthase gene: 60 male Wistar rats were randomly divided into N group and T groups. Vector conservation solution or 600μl AdCMVceNOS with concentration of 5×10⁹pfu/ml was administered by intratracheal instillation into rat lungs from two groups respectively. T groups were redivided into T2d group,T5d group,T7d group,T14d group and T21d group. The NO content, the expression of eNOS protein and the eNOS total RNA content were performed on the 2nd,5th,7th, 14th and 21st day.3. The adenovirus mediated human endothelial nitric oxide synthase gene therapy on chronic hypoxic pulmonary hypertension rats. After 3 weeks chronic hypoxia,40 male Wistar rats were randomly divided into H group and T groups. T groups were redivided into three groups: low dose (TL) group,middle dose(TM) group and high dose(TH) group. Vector conservation solution or 600μl AdCMVceNOS with different concentration were injected into rat lungs by intratracheal instillation.Results1. There were no significant differences in mCAP among these groups (P＞0. 05).2. Contrast to normoxia group, The mPAP and the expression of eNOS protein in H14d,H21d and H28d group increased significantly (P＜0. 05). The expression of eNOS protein A value in H21d and H28d group increased significantly (P＜0. 05). The NO content in H14d,H21d and H28d group decreased significantly (P＜0. 05). The CMA percentage in H21d and H28d group were increased compared with N group(P＜0. 05). 3. The level of NO, eNOS protein,eNOS total RNA were increased in T2d,T5d,T7d,T14d and T21d group compared with N group(P＜0.05), and there had significant changes between T2d group and T7d group.4. Contrast to H group, the mPAP in TM and TH group decreased significantly(P＜0. 05). The level of NO, eNOS protein,eNOS total RNA were increase in TM and TH group compared with H group (P＜0. 05). The NMA percentage in TM and TH group were increased compared with H group(P＜0. 05).5. There was almost no eNOS expression in liver,spleen and kidney.6. There was inflammatory infiltration phenomenon in lung of TH group.Conclusion1. The dysfunction in eNOS/NO system caused by eNOS defect was the major cause of the CHPH.2. Intratracheal administration of recombinant adenovirus can implement the expression of exogenous eNOS gene in trachea, tracheal epithelium and alveolar.3. Transgene expression was quickly,just afer the administration of virus 2 days. Peak expression occurred on the 7th day,and lasted at least 21 days.4. Transgene expression has high target, without distant organ expression.5. The product of transgene expression was stable and had normal enzyme activity. Adenovirus-mediated eNOS gene transfection by intratracheal instillation had the effect of restitution eNOS/NO system function.6. At certain dosage range,the transfer efficiency was correlated with the dose of viral vector administered. The suitable dose of viral vector was 3×10⁹ pfu. At this dose, the transfer efficiency was high and no adverse reaction. 7. Adenovirus-mediated eNOS gene transfection by intratracheal instillation had the effect of CHPH therapy.8. The therapy effect of eNOS gene transfection was mediated by NO. Through restitution eNOS/NO system function, it could lessen the pulmonary vasoconstriction and pulmonary vascular remodeling.