The Role And Mechanism Of Calcium-sensing Receptors In Hypoxia Combined With SU5416-induced Pulmonary Vein Remodeling In Mice

【BACKGROUND】Pulmonary hypertension(PH)is a refractory and fatal respiratory and circulatory disease.It is a pathophysiological change of pulmonary vessels caused by a variety of heterogenous diseases and different pathogenesis.The two main pathogenesis are continuous pulmonary vasoconstriction and irreversible pulmonary vascular remodeling.The pathological manifestations included abnormal proliferation of extracellular matrix of adventitia,excessive proliferation of media smooth muscle,formation of new endothelium and intimal fibrosis,narrowing of vascular lumen and even occlusion.Finally,it leads to pulmonary vasoconstriction and pulmonary vascular remodeling,which leads to the occurrence of PH,in which smooth muscle proliferation is the key to vascular remodeling.In recent years,the prevalence rate of pulmonary hypertension caused by chronic lung diseases is increasing year by year,and people pay more and more attention to it,but most of these research objects are arteries,while veins,as an important component of pulmonary circulation,are relatively scarce.At present,more and more evidence supports that vascular remodeling of PH occurs not only in arteries,but also in veins,manifested as contraction remodeling of pulmonary veins,increased venous resistance,and decreased compliance of pulmonary veins.Our previous study found that pulmonary vein remodeling may be related to the enhanced expression and function of Ca SR,and TRPC6 is also involved.Therefore,Therefore,the hypoxia combined with SU5416 mouse model,which can develop plexiform lesions and represent severe pulmonary hypertension,can be used to further study the specific mechanism of Ca SR in the development of PH and to find new targets for the treatment of PH.【OBJECTS】1.To determine whether chronic hypoxia combined with SU5416 can enhance pulmonary vein remodeling in mice.2.To observe the effect of chronic hypoxia on pulmonary vein remodeling in mice with chronic hypoxia combined with SU5416 pulmonary hypertension,and to explore the expression of Ca SR and its mechanism in pulmonary vein remodeling in hypoxic pulmonary hypertension.【METHODS】1.Effects of chronic hypoxia combined with SU5416 on pulmonary vein remodeling in mice1.1 Twenty male C57BL/6 mice were randomly divided into Normoxia(Nor)group and hypoxia combined with SU5416(Hy Su)group.On the first day,the hypoxia combined with SU5416 group was subcutaneously injected with SU5416 once a week for three weeks.The hypoxia group was exposed to hypoxia device box(temperature 22℃,humidity 50-60%,oxygen concentration 10%)for 4 weeks,and the normoxia group was exposed to SPF normoxia environment.1.2 Right ventricular systolic pressure(RVSP)was measured by right cardiac catheterization to evaluate the changes of hemodynamics in mice,and right ventricle hypertrophy index(RVHI)and right ventricular weight to body weight ratio(RV/BW)were calculated to assess changes in right ventricular remodeling.1.3 The pulmonary veins were identified according to the anatomical structure of pulmonary vessels and airways and Weigert elastic fiber staining.The morphological changes of pulmonary walls of veins were observed by HE staining and the thickening of smooth muscle was observed by α-SMA immunohistochemical staining.Pulmonary vein media area ratio(MA%),media thickness(MT)and media thickness percentage(MT%)were measured and calculated to evaluate vascular remodeling.2.Mechanism of pulmonary vein remodeling induced by calcium-sensing receptors in mice2.1 Forty C57BL/6 male mice were randomly divided into four groups: normoxic group(Nor),normoxic+NPS2143 group(Nor+N),hypoxia combined with SU5416group(Hy Su)and hypoxia combined with SU5416+NPS2143 group(Hy Su+N).The mice in Nor+N and Hy Su+N groups were treated with intraperitoneal injection of Ca SR antagonist NPS2143 once a day for 4 weeks.2.2 The following indexes were measured: RVSP,RVHI and RV/BW to evaluate the changes of hemodynamics and cardiac hypertrophy in mice.2.3 The pulmonary vein was identified according to the anatomical location and Weigert elastic fiber staining.The morphological changes of pulmonary vein wall were observed by HE staining,the smooth muscle thickness and smooth muscle proliferation index were observed by α-SMA and Ca SR immunohistochemical and immunofluorescence double staining of both to evaluate vascular remodeling.2.4 The lung tissue proteins of the four groups were extracted and the expression of Ca SR,TRPC4 and TRPC6 was detected by Western-blot.【RESULTS】1.Effects of chronic hypoxia combined with SU5416 on pulmonary vein remodeling in mice1.1 A mouse model of pulmonary hypertension was successfully established.RVSP,RVHI and RV/BW in hypoxia combine SU5416 group were significantly higher than those in normoxia group after chronic hypoxia combined with SU5416 exposure for 4 weeks.1.2 According to the anatomical position of the lung tissue section and Weigert elastic fiber staining in the normoxic group,it was observed that the pulmonary artery was closely associated with the airway,while the pulmonary vein was far away from the airway,and the pulmonary elastic fibreboard was only single layer or discontinuous or even missing,while the pulmonary artery had multiple layers of continuous internal elastic fibreboard.In addition,the results of HE and α-SMA immunohistochemical staining showed that the venous wall and smooth muscle layer were thinner,while the arterial wall and smooth muscle layer were thicker.1.3 Weigert elastic fiber staining,HE staining and α-SMA immunohistochemical staining showed that 4 weeks after hypoxia combined with SU5416 exposure,vascular remodeling occurred in the distal pulmonary veins in the hypoxia combined with SU5416 group,which showed thickening of the smooth muscle layer of the distal pulmonary veins(the diameter of the vessels was in the range of 0-100 μm),but there was no difference in the remodeling of the proximal pulmonary vein(the diameter of the vessels > 100 μm).2.Mechanism of pulmonary vein remodeling induced by calcium-sensing receptors in mice2.1 After 4 weeks of chronic hypoxia combined with SU5416 exposure,RVSP,RVHI and RV/BW in hypoxia combined with SU5416 group were significantly higher than those in normoxic group,which was consistent with the results of the first part,while the increase of RVSP,RVHI and RV/BW in hypoxia combined with SU5416+NPS2143 group was significantly lower than that in hypoxia combined with SU5416 group.2.2 Through Weigert elastic fiber staining,HE staining,and α-SMA and Ca SR immunohistochemical and immunofluorescence double staining of both,it was observed that there were significant vascular remodeling in the distal pulmonary veins in the hypoxia combined with SU5416 group compared with the normoxic group,while the pulmonary veins remodeling in the Hy Su+NPS group treated with NPS2143 were not significant.In the proximal pulmonary veins with a diameter of more than100 μm,there was no difference in the degree of vascular remodeling among the four groups.2.3 Western-blot analysis showed that compared with the normoxic group,the protein levels of Ca SR and TRPC6 in the lung tissue of mice in the hypoxia group combined with SU5416 group were significantly up-regulated,while those in the NPS2143 group were significantly down-regulated,and there were statistical differences.There was no significant difference in the expression of TRPC4 among the groups.【CONCLUSIONS】1.Chronic hypoxia combined with SU5416 exposure could induce pulmonary hypertension and vascular remodeling of distal pulmonary vein in mice,but had no effect on proximal pulmonary vein.2.Chronic hypoxia combined with SU5416 can up-regulate the expression of Ca SR and TRPC6 in lung tissue of mice.3.Chronic hypoxia combined with SU5416 up-regulates the expression of TRPC6 by up-regulating the expression of Ca SR in the smooth muscle of distal pulmonary vein,which leads to pulmonary vein remodeling,which may be involved in the pathogenesis of pulmonary hypertension.