Alterations Of Endogenous Nitric Oxide And Endothelin Cascade In Rat With High Flow Induced Pulmonary Hypertension And The Effects Of Intervention With Nebulization Of Nitroglycerin

Congenital heart disease with increased pulmonary blood flow commonly leads to the development of pulmonary hypertension and increased vascular reactivity. This abnormal postnatal hemodynamic state results in progressive structural and functional abnormalities of the pulmonary vascular bed. If uncorrected, these vascular changes result in obliteration of the pulmonary vascular bed and death secondary to severe cyanosis and heart failure. To date, nitric oxide (NO) and endothelin-1 (ET-1) have been shown to be the major endothelium-dependent vasomediators. NO is a potent endogenous vasodilator synthesized by the action of the enzyme endothelial nitric oxide synthase (eNOS) in the lungs and other tissues, while ET-1 is a potent vasoconstrictive peptide. Despite significant progress, alterations of endogenous nitric oxide and endothelin cascade with high flow induced pulmonary hypertension still remain unclear.Inhaled nitric oxide (iNO) has been used successfully in children with advanced pulmonary hypertension secondary to congenital heart disease with increased pulmonary blood flow. Unfortunately, the technology can not be universally generalized for needing mechanical ventilation and strict monitor system, plus, its potent toxicity and the cost are also worrisome. Recently reports show that nebulization of nitroglycerin (NTG), one of NO donors, can selectively reduce pulmonary hypertension. However, there is no report about the effect of nebulization of NTG on pulmonary hypertension at the start of the hemodynamic change and the related mechanism especially the alternations of NO/ET cascade. In this study, we investigated the alternations of NO/ET cascade by a rat model undergoing abdominal aortocaval shunt to increase pulmonary blood, and the effects of nebulization of NTG on early stage after shunt operation.Part I Establishment of the Rat Model of high flow induced pulmonary hypertensionObjectiveTo establish a rat model with high flow induced pulmonary hypertension mimic the hemodynamic changes in congenital heart disease with left to right shunt.Materials and methodsTen healthy SD rats (150-200g) underwent abdominal aortocaval shunt to increase pulmonary blood flow for 12 weeks. Control group underwent a sham operation. In both test group (Group S) and control group (Group C) , hemodynamic parameters such as pulmonary velocity (PV), right ventricular ejection period (RPEP), acceleration time (AT), and RPEP/AT were measured by echocardiography. Right ventricular systolic pressure (RVSP) was evaluated by a catheter introducing into right ventricle directly through chest under monitoring with echocardiography. The masses of LV (left ventricle), RV (right ventricle), and septum (S) were weighed and the ratio of RV/(LV+S)was calculated.Results1. Group S presented a significant slowly weight gain during the 12 weeks after shunt operation as compared with group C.2. Tricuspid reguigitation was detected in all of group S and 50% of group C, while pulmonary regurgitation in 60% in group S, and none in group C. In group S, PV (0.83±0.08m/s) and the ratio of RPEP/AT (0.80±0.03) are higher than what are in group C (PV0.76±0.08 m/s,RPEP/AT0.56±0.07) (P<0.05)o3. At 12 weeks after shunt operation, RVSP and RV/(LV+S) in group S were 28.63±2.91 mmHg and 0.273±0.023 respectively, which were both significantly higher than those of group C (RVSP=21.30±4.89 mmHg, and RV/(LV + S)=0.218±0.012(p<0.01).ConclusionRat model of high flow induced pulmonary hypertension was successfully established by the abdominal aortocaval shunt in our study.Part II Alterations of Endogenous Nitric Oxide and Endothelin Cascade in rat with High Flow Induced Pulmonary HypertensionObjectiveTo observe the changes of NO/ET cascade in rat with high flow inducedpulmonary hypertension.Materials and methodsThe animal groups and the methods to achieve pulmonary hypertension were the same as shown in Part I . At 12 weeks after the shunt operation hemodynimic parameters and ratio of RV/(LV+S) were measured as Part I . Pulmonary vascular micro-structures were examined by a light microscope. The degree of pathology changes were graded according to Heath-Edwards' classification of pulmonary hypertension. The degrees of muscularization were studied by vascular wall thickness/vascular diameter (WT/D) and vascular area/total vascular area (WA%). The concentration of plasma NO and ET-1 were measured by spectrophotometry and radioimmunoassay. The expression of endothelial NO synthase (eNOS), ET_aR, ET_bR by pulmonary arteries were detected by immunohistochemistry, respectively.Results1. The plasma concentration of both NO (29.56 ±5.61 μmol/L) and ET-1 (49.95±5.05 pg/ml) increased in group S as compared with group C (NO=21.83 ±5.54 μmol/L, p<0.01; ET-1=22.94 ±5.15 pg/ml, p=0.03).2. Group S showed severe pathological changes on pulmonary arteries which labeled to a high grade (III—IV) of Heath-Edwards's classification. In group S, WT/D (10.21%±0.83%) and WA% (22.34±2.51 %) were both much higher than those of group C (WT/A=5.33%±0.47%; WA%= 14.01 ±1.03%).3. The immunohistochemical staining score for ET_AR, ET_bR and eNOS showed that the expression of all these three proteins elevated in rats with shunt (p<0.01) as compared with control. [ET_AR (84.68±8.46) vs (33.51±4.03) ], [ET_BR (84.54±7.71) vs (32.71±2.94) ], [eNOS (44.87±9.1) vs (32.01±4.11) ].Conclusions1. Both NO and ET cascade up-regulated during the process of high flow induced pulmonary hypertension.2. The elevation of ET cascade may play the most important role in the development of pulmonary hypertension after hemodynimic change. Both ET_aR and ET_bR are involved in the pathogenesis.3. The up-regulation of endogenous NO cascade may be secondary to the elevation of ET cascade. But this change still can not compensate for the unbalance of NO/ET.Part III The Effects of Nebulization of Nitroglycerin on high flow induced pulmonary hypertension and the potential mechanismObjectiveTo evaluate the effects of nebulization of nitroglycerin on treatment and prevention of high flow induced pulmonary hypertension and investigate the potential mechanism.Materials and methodsForty healthy SD rats (150-200g) were randomly divided into four groups as listed below.Group S (n=10): Operation was performed as shown in Part I . Nebulization of NS was administered for 20 minutes once a day from the 2^nd day after shunt-operation for 12 weeks.Group Sa (n=10): Operation was performed as shown in Part I . Nebulization of NTG was administered for 20 minutes once a day for 8 weeks from the 2^nd day after shunt-operation and then, nebulization of N.S. for 20 minutes once a day for 4 weeks subsequently.Group Sb (n=10): Operation was performed as shown in Part I . Nebelization of NS was administered for 20 minutes once a day for 8 weeksrom the 2^nd day after shunt-operation and then, nebelization of NTG for 20 minutes twice a day for 4 weeks subsequently.Group C (n=10): A sham operation was performed as shown in Part I . Nebelization of NS was administered for 20 minutes once a day for 12 weeks.Mean artery pressure (MAP) was measured by catheter introducing into carotid artery. Blood was collected for measuring methemoglobin (MetHb). Other parameters were measured as shown in Part IIResults1. There was no significant difference in incidence of TR and PR in group Sa and group Sb as compared with group C (P>0.05). In group Sa and Sb, The ratio of RPEP/AT, the pulmonary velocity (PV) were all decreased as compared with group S (p<0.05).2. RVSP in group Sa and group Sb were lower than that in group S (p<0.01).RV/(LV+S) in group Sa decreased significantly as compared with group S. There was no difference in MSP among group S, group Sa and group Sb.3. Plasma concentration of ET and NO in group Sa and group Sb were lower than that of group S significantly (p<0.01). No difference was observed in MetHb among these four groups.4. The grades of pulmonary artery damage were from mild to serious, which were shown as group C < group Sa < group Sb < group S. Group Sa showed a low ratio in both WT/D (6.02±0.72%) and WA% ( 16.23±2.09) as compared with group Sb (WT/D8.53±0.67%, WA%19.03±2.01%) and group S (WT/D 10.21 ±0.83%, WA%22.34±2.51) .5. Expression of ET_aR, ET_bR increased in both group Sa (48.93±14.09 and 44.76±9.10) and group Sb (49.42±2.75 and 58.82±17.31) as compared with group S(84.68±8.46 and 84.54±7.71 ) (p<0.05). The expression of ET_BR in group Sa was even higher than that of group Sb (p<0.05).6. Expression of eNOS did not show any difference among group S (44.87±9.1), group Sa (47.79±15.29) and group Sb (48.39±1.73) (p>0.05).Conclusions1. Nebulization of NTG can decrease pulmonary hypertension selectively without adverse effect on systematic pressure.2. Nebulization of NTG may ameliorate the remodeling of pulmonary vascularity. The effect can be more effective if nebulization is administered on the early stage of hemodynamic change.3. Nebulization of NTG may inhibit the over-expression of ET cascade. Nebulization on the early stage may enhance this effect.4. Nebulization of NTG dose not inhibit the expression of eNOS in lungs.5. Nebulization of NTG doesn't cause methemoglobinemia.SummaryThe up-regulation of ET cascade may be the most important change on the early stage of the development of high flow induced pulmonary hypertension. ETaR, ETbR may play an important role in the process. Endogenous NO cascade is up-regulated, which is secondary to the alternation of ET cascade. Nebulization of NTG is safe, effective and can be used to treat the pulmonary hypertension and protects the pulmonary vessels from remodeling. Nebulization at the early stage may be more effective.