| Pulmonary arterial hypertension(PAH)is a chronic progressive disease characterized by progressive increase of pulmonary arterial pressure(PAP)and pulmonary vascular resistance due to vascular dysplasia,contraction,remodeling and thrombosis.Its severe symptoms can lead to right heart failure,poor prognosis and high mortality.At present,it has been listed as the third common cardiovascular disease after coronary heart disease and hypertension.Traditional drugs for the treatment of PAH include anticoagulants,diuretics,calcium channel blockers,etc.these drugs can improve the symptoms of patients with PAH,but can not inhibit or reverse the progress of PAH.Some drugs,including prostacyclin and its analogues,ET-1 receptor antagonists,phosphodiesterase-5(PDE-5)inhibitors,can improve the clinical symptoms and hemodynamic indicators,but still can not be cured,and the mortality of PAH is still very high.With the in-depth study of the molecular and cellular biological mechanism of PAH,new PAH targeted therapeutic drugs including soluble guanylate cyclase agonists,Rho kinase inhibitors,5-hydroxytryptamine receptor antagonists and protease activated receptor 2 inhibitors have gradually become a research hotspot in the field of PAH.In this study,the left to right shunt PAH rat model was established by common carotid artery internal jugular vein anastomosis.This method can effectively control myocardial hypertrophy and hemodynamic changes by controlling the shunt flow,and simulate the early and middle stage PAH caused by clinical left to right shunt congenital heart disease.Riociguat,an agonist of guanylate cyclase,was used to intervene the early and middle stages of PAH in rats,the pressure changes of right ventricle and pulmonary artery were observed;the levels of ET-1,NO and cGMP in serum and lung tissue were detected;the protein and mRNA contents of ?-smooth muscle actin(?-SMA),osteopontin(OPN)and integrin ?3 were detected,to explore the possible mechanism of riociguat from the perspective of pulmonary vascular remodeling and right ventricular hypertrophy in histology and molecular biology,to provide experimental and theoretical basis for clinical drug treatment of PAH.Part I:Guanosine cyclophosphate and endothelin participate in the morphological changes of cardiopulmonary function in rats with shunt pulmonary hypertensionObjective:In this study,we established a rat model of common carotid artery external jugular vein shunt,observed the related hemodynamic changes and pathological characteristics,and explored the process and possible mechanism of pulmonary vascular remodeling and right ventricular hypertrophy within 3-10 weeks of arteriovenous shunt from the functional and morphological aspects,as well as the role of cGMP and endothelin in the morphological changes of cardiopulmonary function of shunt PAH.Methods:Sixty healthy male Sprague Dawley rats weighing 180-220 g,aged 6 weeks,were randomly divided into two groups:shunt(F)group and sham operation(S)group.According to the methods of Zhao Keyan(2014),the rat model of left to right shunt PAH was established.After anesthesia,the rats were fixed in supine position with a 2 cm incision in the middle of the neck.In F group,the right external jugular vein was exposed and the distal end was ligated;the right common carotid artery was blocked with a small vascular clamp,and the common carotid artery was ligated and cut off about 0.5 cm below the bifurcation of internal carotid artery and external carotid artery;A small vascular clamp was used to block the proximal end of the external jugular vein.The incision of 0.2 cm was made on the medial side of the external jugular vein.The end to side anastomosis of the jugular artery and vein was performed with 8-0 prolene line.When the vascular clamp was released,the proximal end of the external jugular vein was filled and pulsatile blood flow appeared,which proved that the anastomosis was unobstructed.In S group,the common carotid artery and external jugular vein were exposed only through neck incision,and ligation was performed without anastomosis.At the 3rd,4th,6th,8th and 10th week after operation,F3,F4,F6,F8,F10 and S3,S4,S6,S8 and S10 groups,respectively,there were 6 rats in each group.Hemodynamic parameters were measured and recorded:maximum rate of right ventricular systolic pressure rise(dp/dtmax),RVSP,mPAP and mSAP;The related indexes of ventricular hypertrophy:right ventricular mass/body weight(RV/BW),(left ventricular+interventricular septum)mass/body weight[(LV+S)/BW]and right ventricular hypertrophy index=right ventricular mass/(left ventricular+interventricular septum)mass[RV/(LV+S)];The contents of ET-1,NO and cGMP in serum and lung tissue were detected;the morphological changes of pulmonary artery were observed by HE staining;the morphological changes of right ventricular fibrosis and myocardial cells were observed by Masson staining.Results:Hemodynamic changes of rats in each group at 3-10 weeks:mPAP,RVSP and dp/dtmax levels in F group were significantly higher than those in S group(P<0.05 or P<0.01);mPAP was not remarkably different at 3-6 weeks(P>0.05),but increased at 8th and 10th week(P<0.05).The contents of ET-1,cGMP and no in serum of rats were determined:compared with S group,the level of ET-1 in F group was significantly higher and the level of NO was remarkably lower at 3-10 weeks(all P<0.01),cGMP levels were observably lower at 6-10 weeks(P<0.05 or P<0.01).The contents of ET-1,cGMP and no in lung tissue were measured:compared with S group,the level of ET-1 in F group increased significantly at 4-10 weeks,while the level of cGMP decreased prominently at 6-10 weeks(P<0.05 or P<0.01);The level of NO in F group was remarkably lower than that in S group at 3-10 weeks(all P<0.01).There was no accidental death in rats of two groups,and the body weight gradually increased with time in 3-10 weeks,and the increasing trend was consistent,there was no difference(P>0.05).Compared with S group,the RV/BW value in F group increased prominently at 3-10 weeks(P<0.05 or P<0.01),and the(LV+S)/BW value increased significantly at 6-10 weeks(P<0.05 or P<0.01),RV/(LV+S)increased remarkably at 8th and 10th week(P<0.05 or P<0.01),suggesting that the change of right ventricular hypertrophy was greater than that of left ventricle in the late stage of PAH.Under light microscope,HE staining showed that the wall of pulmonary artery was thin,the intima was smooth,and the endothelial cells were flat;In F group,the intima and media of pulmonary artery began to thicken from the 4th week,accompanied by inflammatory cell infiltration,showing mild proliferation of endothelial cells,cell body changed from flat to high columnar,and cell nucleus enlarged,smooth muscle cells proliferated,arranged disorderly,and cell nucleus enlarged;Compared with S group,the wall of pulmonary artery in F group was thickened,the lumen became smaller,the smooth muscle layer of media was thickened,the diameter of smooth muscle fiber was thickened,the number of cells was increased,and the WT%and WA%of pulmonary artery with diameter of 50-150?m were significantly increased(P<0.01).Under the light microscope,the myocardial fibers in S group were arranged orderly,intercalated discs were clear,and collagen tissue between muscle fibers was less;In F group,after 4 weeks of shunt,the myocardial fibers became thicker and disordered,and the collagen tissue between muscle fibers increased.After 8 weeks of shunt,the myocardial fibers were disordered,the intercalated discs were irregularly arranged,and the collagen tissue between muscle fibers increased prominently.CVF%in group F was significantly higher than that in S group(P<0.05 or P<0.01).Conclusion:The results showed that the left to right shunt rat PAH model constructed by common carotid artery external jugular vein shunt can successfully simulate the early and middle pathophysiological state of PAH caused by left to right shunt in congenital heart disease;Right ventricular hypertrophy began to appear 3rd week after shunt,and aggravated 4-8 weeks later;The changes of ET-1,NO and cGMP in serum and lung tissue were related to the pathophysiological changes in the early and middle stages of PAH.Part II:The effect and mechanism of Riociguat on pulmonary vascular remodeling in rats with left to right shunt pulmonary hypertensionObjective:In this study,a shunt PAH model was established by common carotid artery external jugular vein anastomosis to simulate the early and mid-term PAH caused by clinical left to right shunt congenital heart disease,to investigate the effects of riociguat,a guanylate cyclase agonist,on pulmonary vascular remodeling in rats with PAH at early and middle stages and its possible mechanism.Methods:Thirty six male SD rats weighing 180-220 g,aged 6 weeks,were randomly divided into sham operation(S)group,Fistula(F)group and Riociguat(R)group,with 12 rats in each group.F group and R group underwent common carotid artery external jugular vein fistulation to construct left to right shunt PAH rat model,and S group was treated the same as the first part.From the 7th week,rats in R Group were given riociguat(10 mg/kg)by gavage every day,while rats in the other two groups were given normal saline every day for 4 weeks.At the end of the 10th week,the indexes of all experimental groups were measured.The pressure measurement is the same as the first part;The contents of ET-1 and cGMP in serum and lung tissue were determined by ELISA,and the contents of NO were determined by Griess method;The pathological changes of pulmonary arterioles were observed by HE staining;The expression of ?-SMA protein in lung tissue was detected and the expression of ?-SMA mRNA was detected.Results:Hemodynamic changes:the levels of dp/dtmax,mPAP and RVSP in F group were remarkably higher than those in S group(P<0.05);There were significant differences in dp/dtmax,mPAP and RVSP between R group and F group(P<0.05);There was no obvious difference in mS AP among the groups.The contents of ET-1,cGMP and NO in serum and lung tissue were determined:compared with S group,the levels of ET-1 in F group were prominently higher,while the levels of NO and cGMP were significantly lower(P<0.01);Compared with F group,the level of ET-1 in R group decreased remarkably,the level of NO increased observably,and the level of cGMP increased significantly(all P<0.05),and there was no obvious difference in the level of cGMP between R group and S group,suggesting that the level of cGMP in serum and lung tissue of rats can be increased after the intervention of riociguat.Result of the expression of ?-SMA protein in lung tissue:the expression of?-SMA protein in F group was prominently higher than that in S group(P<0.05);the expression of ?-SMA protein in R group was observably lower than that in F group(P<0.01).Immunohistochemical observation:the positive expression of ?-SMA protein in pulmonary artery smooth muscle cells showed brown granules.The expression of ?-SMA mRNA in lung tissue:the relative content of ?-SMA mRNA in F group was observably higher than that in S group(P<0.05);The relative content of ?-SMA mRNA in R group was prominently lower than that in F group(P<0.05).HE staining of pulmonary vascular tissue under light microscope showed that the wall of pulmonary artery in S group was thin,the intima was smooth,and the endothelial cells were flat;In F group,pulmonary artery wall thickening,lumen stenosis,intima and media thickening,smooth muscle cell proliferation,arrangement disorder and inflammatory cell infiltration were observed;Compared with F group,the pathological changes of lung tissue in R group were significantly improved.The WT%and WA%of F group and R group were significantly higher than those of S group(P<0.05);the WT%and WA%of R group were observably lower than those of F group(P<0.05).Conclusion:The results showed that the levels of ET-1 in serum and lung tissue of PAH rats were decreased,while the levels of NO and cGMP were increased by riociguat,which may be related to the changes of pulmonary vascular remodeling and the regulation of pulmonary artery pressure;Riociguat may inhibit pulmonary vascular remodeling by inhibiting the expression of ?-SMA mRNA and protein to reduce the activation,proliferation and fibrosis of pulmonary vascular smooth muscle cells and fibroblasts.Part ?:The effect and mechanism of Riociguat on right ventricular hypertrophy in rats with left to right shunt pulmonary hypertensionObjective:On the basis of the successful establishment of PAH rat model,the changes of osteopontin and integrin ?3 on the surface of cardiac myocytes in the early and middle stage of shunt PAH and the intervention of riociguat were studied to explore the mechanism of riociguat in improving cardiac hypertrophy.Methods:The experimental animals and model preparation were the same as the second part.The indexes of all experimental groups were measured at the end of the 10th week.The related indexes of ventricular hypertrophy:RV/BW,(LV+S)/BW and RV/(LV+S);Masson staining was used to observe the morphological changes of right ventricular myocytes;Western blot was used to detect the expression of osteopontin and integrin ?3 in right ventricular myocardium;RT-PCR was used to detect the expression of osteopontin mRNA and integrin ?3 mRNA in right ventricular myocardium.Results:The body weight(BW)of the three groups increased gradually with time within 3-10 weeks,and the increasing trend was consistent,and there was no significant difference in body weight(P>0.05).At the end of the experiment,the results were as follows.Results of right ventricular hypertrophy indexes were measured:RV/(LV+S),RV/BW and(LV+S)/BW ratio in F group were observably higher than those in S group(P<0.05);RV/(LV+S),RV/BW and(LV+S)/BW ratio in R group were prominently lower than those in F group(P<0.05).Masson staining and light microscope observation:in S group,the myocardial fibers were arranged orderly,intercalated discs were clear,and the collagen tissue between muscle fibers was less;In F group,the myocardial fibers were obviously thickened and disordered,the intercalated discs were irregularly arranged,and the collagen tissue between myocardial fibers was increased;The pathomorphological changes of right ventricular myocytes in R group were significantly less than those in F group,the myocardial structure and fiber thickness were between the two groups.Expression of OPN and integrin ?3 in right ventricle of rats:the expression of OPN and integrin ?3 protein in F group was remarkably higher than that in S group(P<0.05),and the expression of OPN and integrin ?3 protein in R group was significantly lower than that in F group(P<0.05).Expression of OPN mRNA and integrin ?3 mRNA in right ventricle of rats:the expression of OPN mRNA and integrin ?3 mRNA in F group was prominently higher than that in S group(P<0.05,P<0.01);The expression of OPN mRNA in R group was observably lower than that in F group(P<0.05),but there was no significant difference in the expression of integrin ?3 mRNA between F group and R group(P>0.05).Conclusion:PAH induced right ventricular hypertrophy is related to the changes of osteopontin and integrin ?3 in myocardium;Riociguat can reduce the level of OPN protein and mRNA in right ventricular myocardium,reduce myocardial fibrosis,inhibit myocardial hypertrophy and ventricular remodeling,improve the right ventricular systolic function in early and middle stage of PAH,and improve the effect of myocardial dynamics;The expression of integrin ?3 protein in right ventricular myocardium was decreased,but the change of integrin ?3 mRNA was not obvious. |
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