A Combination Of ENOS Gene Transfer And Sildenafil Prevents Hypoxic Pulmonary Hypertension In Rats

ObjectivePulmonary hypertension (PH) is a variety of disease states characterized by increased pulmonary artery pressure and/or pulmonary vascular resistance. It is a common complication of the heart and lung disease and also a quiz during cardiovascular surgery perioperative period. The severity of PH always determines the prognosis of patients. Although the pathogenetic factors differs, they all lead to pulmonary vasoconstriction and structural remodeling (e.g. intimal fibrosis, medial hypertrophy, adventitial proliferation, extracellular matrix augment) and ultimately increase PVR. It has been demonstrated, endothelial dysfunction plays a key role in the pathogenesis of PH. Under the normal condition, pulmonary vascular endothelial cells make the pulmonary circulation low resistance and high capacity state by keeping the balance among endothelin (ET) pathway, NO/cGMP pathway and prostacycline/cGMP pathway. The break of the balance leads to the pathogensis of PH.It has been demonstrated, the relative deficiency of NO/cGMP under the patho-condition is the key pathogenetic element of PH. Accordingly, NO inhalation has been successfully used for the treatment of PH, and has made satisfactory results. Though NO inhalation can selectively and efficiently relax pulmonary vessels, improve the oxygenation with no systematic side-effect. It is expensive, unconvenient to be used for requiring a fairly sophisticated delivery system. Furthermore, it has short half-life and can easily produce drug resistance and rebound PH after it's withdraw. As NO is produced by the catalysis of nitric oxide synthase(NOS), and among the three submits of NOS, which comprise nNOS, eNOS and iNOS, eNOS derived NO plays the key role in maintaining the pulmonary resistance. So, eNOS can be produced by eNOS gene transfer to elevate endogenous NO, which can treat PH without side-effect accompanied NO inhalation.As NO relax pulmonary vessels through the second messenger cGMP. Intracellular cGMP level plays the key role in treating PH. It has been showed, the intracellular cGMP level depends on the balance between it's production and degradation. cGMP is hydrolyzed by phosphodiesterases (PDEs). In mammals, PDEs comprises 11 families and 50 submits. PDE5, which is the main PDE in lungs, abounds in lung and corpus cavemousum. In PH animal models, PDE5 expression and activity in pulmonary vessels upregulates, which indicate PDE5 may participate in the pathogensis of PH. So, selective inhibiton of PDE5 can act by slowing down the degradation of cGMP and hence augment the role of NO/cGMP, which provide a novel strategy in treating PH.So, we hypothesis that a combination of eNOS gene transfer and oral sildenafil( a highly selective PDE5 inhibitor) can produce a synergistic effect in preventing PH by promoting the production of cGMP and decreasing its degradation. The effect is more powerful than anyone used alone and can avoid the side-effect accompanied at the same time.As one of the factors among the progression of PH is hypoxia in alveoli, and hypoxia can induce PH. So, in our experiment, we establish PH model in rats, and transfer eNOS gene through recombinant adenovirus AdCMVceNOS, combined oral sildenafil. Through hemodynamic, histological, biochemical and molecular biological measures, we investigate the feasibility, therapeutic effect and accompanied side-effect of preventing acute hypoxic pulmonary vasoconstriction(HPV) and chronic hypoxia induced pulmonary hypertension(CHPH) by a combination of eNOS gene transfer and sildenafil. Providing a novel strategy in treating PH.Materials1. Experiment materialsAdCMVceNOS DNA (Dr Rober Gerard), 293cells and pXC1 vector (Canada Microbix Biosystems company), primier (Takara company), 1125-cGMP RIA kit (Shanghai traditional medicine university), eNOS monoclone antibody (BD company), NO kit (Nanjing jiancheng biocompany), immunohistochemical kit and elastic fiber staining kit (Beijing zhongshan biotech company), Sildenafil citrate (Pfizer company).2. Experiment instrumentsPCR instrument (Germany Biometra company), 721 spectrophotometer(Shanghai jingmi scientific instrument limited company), Animal ventilator(Jiangxi teli anesthesia respiratory instrument limited company).3. Experiment animals126 Male Wistar rats, provided by Experiment Animal Center, General Hospital of Shenyang Military Area.Methods1. Part 1(1)DNA of virus AdCMVceNOS was transferred into 293 cells by liposome, and packaged inside. After being confirmed by PCR analysis, the virus was amplified in 293 cells and purified by discontinous CsCl gradient. Viral titer was determined with TCID50 method.(2)54 Male Wistar rats were randomized to control(C)group, Ad-LacZ group and Ad-eNOS group(n=18 each), then Rats were given 600μl of virus conservation solution, AdCMVLacZ or AdCMVceNOS with titer of 5×10⁹pfu/ml by intratracheal instillation respectively. HR and SAP of 6 rats from each group were masured 3, 5 and 17 days after adenovirus transfection. After that, X-gal staining, eNOS immunohistochemical staining, HE staining, eNOS Westernblot of rat lung were performed and cGMP, NO content were measured. X-gal staining of rat liver, spleen and renal were also performed 5 days after virus infection.2. Part 2Thirty six male Wistar rats were randomized to normoxia(N) group, hypoxia(H) group, hypoxia LacZ(H-LacZ) group, hypoxia eNOS(H-eNOS) group, hypoxia sildenafil(H-S) group, and hypoxia eNOS sildenafil(H-eNOS-S) group(n=6). Gene transfer via airway were performed(N, H and H-S group received virus conservation solution, H-LacZ group received 5×10⁹PFU/ml AdCMVLacZ 600μl, H-eNOS and H-eNOS-S group received 5×10⁹PFU/ml AdCMVceNOS 600μl) on the 1^st day. Three days after adenovirus administration, rats from N group were ventilated with room air for 30min, and rats from other group with 10%O₂. 30min before hypoxia, rats from H-S and H-eNOS-S group were gavaged with 25mg/kg of 0.3% sildenafil. Other rats were gavaged with 8.33ml/kg of NS. SAP, HR and MPAP were measured at the end of hypoxia, arterial blood gas analysis were performed, cGMP and NO content in rat lung were measured.3. Part 3Thirty six male Wistar rats were randomized to normoxia(N) group, hypoxia(H) group, hypoxia LacZ(H-LacZ) group, hypoxia eNOS(H-eNOS) group, hypoxia sildenafil(H-S) group, and hypoxia eNOS sildenafil(H-eNOS-S) group(n=6). Gene transfer via airway were performed(N, H and H-S group received virus conservation solution, H-LacZ group received 5×10⁹PFU/ml AdCMVLacZ 600μl, H-eNOS and H-eNOS-S group received 5×10⁹PFU/ml AdCMVceNOS 600μl) on the 1^st day. After that, rats were raised three days in room air, gavaged once a day. From the 4^th day, rats apart from N group began hypoxia(10% O₂) exposure 8 hours a day for 2 weeks. Rats from N group were kept in the same room adjacent to hypobaric chamber. Rats were gavaged 30 minutes before hypoxia(normoxia) exposure, H-S and H-eNOS-S group with 0.3%sildenafil 25mg/kg, other groups with 8.33ml/kg of NS. At the end of hypoxia exposure, hemodynamics measurement, Hct, cGMP and NO level in rats lung, pulmonary vascular remodeling index, RV/(LV+S), and eNOS Westemblot of rats lung were made.Results1. Part 1 (1)DNA of virus AdCMVceNOS was transferred into 293 cells and integrated virus was made. Ater being confirmed and amplified, the virus was quantified to be 1.58×10¹⁰PFU/ml.(2)At each time point, the eNOS protein, cGMP and NO content of Ad-eNOS rats lung were higher than those of C and Ad-LacZ rats (p<0.01). Immunohistochemical staining showed diffused transgenic expression in bronchi epithelial cells , alveoli lining cells, and endothelial cells in medium- and small-sized pulmonary vessels. In C and Ad-LacZ rats, eNOS, cGMP and NO content of rats lung were similar(p>0.05) and transgenic expression were only observed in endothelial cells in large-sized pulmonary vessels.(3)Five days after adenovirus infection, liver, spleen and renal of Ad-LacZ rats showed no transgenic expression as demonstrated by X-gal staining. However, one from six rats showed diffused inflammation in lung.(4)At each time point, there were no difference in rats weight, heart rate and systematic arterial pressure among 3 group rats(p>0.05).2. Part 2(1)MPAP of H-eNOS and H-S rats were lower than those of H and H-LacZ rats, higher than those of H-eNOS-S and N rats(p<0.01). There was no difference in MPAP between H-eNOS-S and N rats(p>0.05).(2)SAP, PaO₂, PaCO₂ of N rats were higher and heart rate was lower than other rats(p<0.05). There was no difference among each hypoxia group(p>0.05).(3)NO content of N rats lung was higher than that of H, H-LacZ and H-S rats, lower than that of H-eNOS and H-eNOS-S rats(p<0.05). There was no difference among H, H-lacZ and H-S group, and also between H-eNOS and H-eNOS-S group(p> 0.05).(4)cGMP content of H and H-LacZ rats lung was lower than that of N rats(p< 0.05). cGMP of H-eNOS and H-S rats was higher than that of N rats(p<0.05), lower than that of H-eNOS-S rats(p<0.01). 3. Part 3(1)Weight of N rats was higher and lung weight/body weight ratio was lower than those of other rats(p<0.01). HR, SAP, liver weight/body weight ratio and renal weight/body weight ratio were similar among all rats(p>0.05).(2)MPAP, CMA/MA and RV/(LV+S) of N rats were lower than those of other rats(p<0.01), with H-eNOS and H-S rats lower than H and H-LacZ rats(p<0.01), higher than H-eNOS-S rats(p<0.01). There was no difference between H, H-LacZ group and also between H-eNOS, H-S group(p>0.05).(3)Hct of H-eNOS, H-S and H-eNOS-S rats was higher than that of N rats(p< 0.05), lower than that of H and H-LacZ rats(p<0.05).(4)eNOS level of H, H-LacZ and H-S rats lung was higher than that of N rats(p< 0.01), lower than that of H-eNOS and H-eNOS-S rats(p<0.01).(5)NO level of N rats lung was higher than that of H, H-LacZ and H-S rats(p< 0.05), lower than that of H-eNOS and H-eNOS-S rats(p<0.01).(6)cGMP level of H-eNOS-S rats lung was higher than that of other rats(p<0.01), with N, H and H-LacZ group lower than H-eNOS and H-S group(p<0.01). cGMP of N, H and H-LacZ rats lung was similar(p>0.05).Conclusions1. Recombinant adenovirus can mediate eNOS gene express efficiently in rat airway. Transgenic expression diffused widely (including bronchi epithelial cells , alveoli lining cells, and endothelial cells in medium- and small-sized pulmonary vessels) without distant organ expression. Transgenic expression can last at least 17 days. The expression of eNOS gene upregulate NO and cGMP level in rats lung.2. Transfer recombinant adenovirus via airway showed no interference to rats vital sign, but do has the possibility of inducing immunoreactivity. So the dose and titer of virus used should be limited while reaching the curative effect.3. Thirty minutes exposure to hypoxia can lower SAP, elevate HR of rats, and induce HPV. The pathogenesis is related to downregulation of NO/cGMP level in rats lung.4. Chronic hypoxia exposure can elevate MPAP and Hct, induce pulmonary vascular remodeling, right ventricular hypertrophy, which accompanied with the upregulation of eNOS, NO and cGMP level in rats lung.5. Transfect with AdCMVceNOS and oral sildenafil, can increase cGMP level of lung, reduce rats acute HPV, reduce the elevation of MPAP, Hct, pulmonary vascular remodeling, right ventricular hypertrophy induced by chronic hypoxia. A combination of the two measures can act synergisticly, but cannot utterly prohibit the pathogenesis of CHPH.