Effects Of Bosentan On The Therapy Of Chronic High-altitude Environment-induced Pulmonary Hypertension And Its Underlying Mechanism

Background and objectiveHigh-altitude environment-induced pulmonary hypertension (HAEPH) is defined that hypobaric hypoxia in the plateau environment leads to the change of pulmonary artery in its function and structure as well as the increase of pulmonary arterial pressure. High-altitude environment-induced pulmonary hypertension at its early stage is mainly caused by pulmonary arterial contraction due to hypobaric hypoxia. Long-term hypobaric hypoxia induces the remodeling of the pulmonary vessels and the heart , which finally leads to the incurable and obstructive pulmonary hypertension. So far, the pathogenesis of HAEPH has not been elucidated thoroughly, and there is still no ideal therapeutic strategy for HAEPH. Therefore, the investigation of the pathogenesis and the therapeutic strategy of HAEPH has become a hot point in the international medical studies. Bosentan is a dual antagonist of ETA and ETB receptors, with a slightly higher affinity for the ETA receptor. Bosentan is commercially called Tracleer, which is produced cooperatively by a Canadian company called as Acetlion and an American company called as Genentetch Inc. Nowadays, bosentan is used to treat the patients inflicted with the primary pulmonary hypertension or the pulmonary hypertension related to the collagen vascular disease. However, it has not been reported that bosentan can reverse the established high-altititude environment-induced pulmonary hypertension which is induced by long-term hypobaric hypoxia. The effects of bosentan on the reversal of the established HAEPH can reflect the pharmaceutical role of bosentan in the treatment of chronic HAEPH.Serotonin (5-HT) is a vasoconstrictive and mitogenic factor in the pulmonary tissue. Activation of 5-HT1B receptor by serotonin in vascular smooth muscle cells increases the activities of phosphodiesterase, which decrease intracellular cAMP levels. The decrease of cAMP levels induces the contraction of pulmonary vessels. Binding of serotonin into 5-HT1B receptor and/or serotonin transporter (5-HTT) promotes te proliferation and the hypertrophy of pulmonary vascular smooth muscle cells, which finally cause the remodeling of pulmonary vessels. It has been proved that both of 5-HT1B receptor and 5-HTT play an important role in the development of the primary pulmonary hypertension and the hypoxia-induced pulmonary hypertension. However, the changes of 5-HTT and 5-HT1B receptor in the development of HAEPH have not been reported and the effects of bosentan on these changes are poorly understood.We performed this experiment for the first time to investigate: (1) The effects of bosentan on the established chronic HAEPH and its mechanisms. (2) The changes of 5-HT,5-HTT and 5-HT1B receptor in the development of chronic HAEPH and the effects of bosentan on these changes.1 Effects of bosentan on the established chronic HAEPH(1) Methods1) Rats were exposed to low-pressure and low-oxygen condition in an auto-modulating hypobaric and hypoxic cabin (air pressure 50 kPa) to establish HAEPH animal model. Sixty Male Sprague–Dawley rats were randomly divided into 6 groups: control group, 3w group (hypobaric hypoxia for 3 weeks), 4w group (hypobaric hypoxia for 4 weeks), 6w group (hypobaric hypoxia for 6 weeks), placebo group, and bosentan group. The rats in control group stayed in orthobaric environment. The rats in experimental groups had been kept in hypobaric chamber. In placebo group and bosentan group, the 3-weeks administration of sodium chloride or bosentan in hypobaric chamber commenced 3 weeks after hypobaric hypoxic environment.2) A micro-catheter was inserted into right ventricle and pulmonary artery through right external jugular vein, and the mean pulmonary arterial pressure (mPAP) and right ventricular systolic pressure (RVSP) were measured. Electrocardiogram (ECG) and heart rate (HR) were simultaneously recorded by the polygraph (RM-6280).3) Right ventricle (RV),left ventricle (LV),septum (S) and pulmonary tissue were isolated. After the absorbing of water using filter paper, RV,LV+S and pulmonary tissue were weighed. The RV/(LV+S) ratio was calculated and used as the index for right ventricular hypertrophy. The PM/BM ratio was used as the index for pneumonedema.4) Morphologic changes of peripheral pulmonary artery were detected by light microscope, and the microscopic images were analyzed using a computerized morphometric system. The external diameter (ED) and medial wall thickness (WT)of peripheral pulmonary artery were measured. The ratio of vascular medial wall thickness to external diameter (WT%) and muscularization of non-muscularited peripheral small pulmonary arterioles(diameter: 40～60μm) were calculated to assess the degree of pulmonary artery remodeling.5) Concentrations of NO and ET in blood and their contents in pulmonary tissues were detected by nitrate reductase assay and radio immunoassay.6) The activities of cNOS and total NOS in the pulmonary tissue were detected.(2) Results1) Pulmonary hypertension established after 3 weeks of hypobaric hypoxic environment in rats. Compared with normoxic control group, mPAP,RVSP,WT%,RV/(LV+S) ratio and PM/BM ratio of 3w group all increased significantly. Thickened wall and reduced lumen of peripheral pulmonary artery were detected by light microscope. The number of muscularited peripheral pulmonary arterioles increased significantly. There were more changes of pulmonary hypertension following prolonged hypobaric hypoxia, above changes were more significant at the end of 6 weeks. But parameters reflecting systemic circulation such as HR did not change.2) After 6 weeks, compared with control group, the levels of mPAP and RVSP in placebo group increased significantly. bosentan group showed a significant decrease in mPAP and RVSP, which were not significantly different with those in normoxic group.3) Both of the RV/(LV+S) ratio and the PM/BM ratio in bosentan group decreased significantly compared with placebo group. Compared with placebo group, both of WT% and muscularization of non-muscularited peripheral pulmonary arterioles in bosentan group decreased significantly, which were not different significantly with those in normoxic group. Detected by light microscope, proliferation and migration of pulmonary arterial smooth muscle cells as well as thickness of pulmonary artery wall were reduced significantly in bosentan group.4) Compared with normoxic group, NO level decreased while ET-1 level increased remarkably in both blood and pulmonary tissue of rats in placebo group. In bosentan group, the concentration of NO was higher and the concentration of ET-1 was lower than those of placebo group, although the concentration of ET-1 in blood was higher than that of placebo group.5) cNOS and total NOS activities in pulmonary tissue of placebo group rats were lower than those of normoxic group rats. Compared with placebo group, cNOS and total NOS activities in pulmonary tissue of bosentan group rats were higher.(3) Conclusion1) Bosentan effectively reduces the increased mPAP, lowers right ventricular hypertrophy and pulmonary edema and reverses the established remodeling of pulmonary vessels of the chronic HAEPH rats. This suggests that bosentan could be used for the therapy of chronic HAEPH.2) Bosentan significantly increases the activities of cNOS and total NOS and decreases ET-1 contents in pulmonary tissues of chronic HAEPH rats, which leads to the increase of NO contents in blood and pulmonary tissues of chronic HAEPH rats. It may be one of the mechanisms underlying the therapeutic effect of bosentan. 2 The effects of bosentan on the 5-HT levels and the expressions of 5-HTT and 5-HT1B receptor in pulmonary artery of chronic HAEPH rat(1) Methods1) Thirty Male Sprague–Dawley rats were randomly divided into 3 groups: control group,placebo group and bosentan group. The rats in control group stayed in orthobaric environment. The rats in experimental altitude groups had been kept in hypobaric chamber. In placebo group and bosentan group, the 3-weeks administration of sodium chloride or bosentan in hypobaric chamber commenced 3 weeks after hypobaric hypoxic environment.2) 5-HT levels in blood and pulmonary tissues of chronic HAEPH rats were detected by enzyme-linked immunosorbent assay (ELASA).3) The immunohistochemistry and Western blot were used to observe the expressions of 5-HTT and 5-HT1B receptor in pulmonary tissues of each group rats.(2) Results1) Compared with control group, 5-HT levels in blood and pulmonary tissues of placebo group significantly increased. After the 3-weeks administration of bosentan in the established chronic HAEPH rats, 5-HT levels in blood and pulmonary tissues were significantly lower than those of placebo group.2) The expressions of 5-HTT and 5-HT1B receptor in pulmonary tissues of placebo group rats were significantly higher than those of control group. However, compared with placebo group, the expressions of 5-HTT and 5-HT1B receptor in pulmonary tissues of bosentan rats were significantly lower. (3) Conclusion1) Hypobaric hypoxia not only increases 5-HT levels in blood and pulmonary tissues, but also promotes the over-expression of 5-HTT and 5-TH1B receptor in pulmonary tissues of rats. This proves that the activation of 5-HT system takes an effect on the development of chronic HAEPH.2) Bosentan reduces 5-HT levels in blood and pulmonary tissues and decreases the expressions of 5-HTT and 5-HT1B receptor in pulmonary tissues of the established chronic HAEPH rats. This reveals that bosentan maybe have a new mechanism in its therapy of chronic HAEPH—affect the activiation of 5-HT system.