The Role Of Tadalafil In Pulmonary Vascular Remodeling And Myocardial Cx43Phosphorylation Of Shunt-induced Pulmonary Hypertension In Rats

Part Ⅰ：ET-1and cGMP participated in the process of lung pulmonaryhypertensionObjective: Pulmonary arterial hypertension (PAH) is described as akind of pathophysiological syndrome characterized by a progressiveraised pulmonary arterial pressure and pulmonary vascular resistance,resulting in right heart failure or even death. This study was designed toestablish the abdominal aorta-inferior vena cava shunt model in rats andto investigate the morphological and molecular mechanisms ofpulmonary vascular remodeling and right ventricular hypertrophy.Method:72male SD rats were randomly divided into two groups: thesham operation (Sham, S) group and the fistula (Fistula，F) group. Accordingto Garcia’s method, a midline abdominal incision was made and thedescending aorta above the renal bifurcation was cleared of adjacent tissuesand a snugger was placed. The infra-renal portion of the aorta and inferiorvena cava were exposed at a site where the two vessels share a common fascia.The supra-renal portion of the abdominal aorta was then occluded with asnugger to control bleeding. The shunt was then created with an18Gangiocatheter inserted between the sutures on the anterior surface of the aorta.Following release of the snugger in thirty seconds, we observed mixing ofarterial and venous blood in the IVC, with distention of and pulsations in theIVC. Sham animals were treated the same way except for IVC puncture. Ratswere anesthetized again to measure the pressure of pulmonary artery and rightventricular (RV) four or six weeks after the corresponding treatment via amicro-catheter in the right ventricular outflow tract placed by thoracotomy.The pulmonary morphological changes were observed by HE staining of the right lower lobe tissue of the lung. The relative weight of ventricular wasdetermined and the changes of myocardial collagen were identified by Massonspecial staining.Results:1. There is no significantly difference of pulmonary arterypressure between two groups (P>0.05). Compared with S group, the meanpulmonary artery pressure (mPAP), the right ventricular systolic pressure(RVSP) and the maximal positive velcity of right ventricular pressure(+RVdP/dtmax) were increased in F group.2. Compared with S group, thepercentage of wall thickness (WT%) and the wall-to-total area ratio (WA%)were increased duing the process of the model (P<0.05).3. The two kindsmuscular artery which extremely diameter limited to50~100μm respectivelywere highly increased in F4and F6rats.4The RV/body weight ratio (RV/BW)and the percentage of (left ventricle+interventricular septum)/body weight(LV+S)/BW were markedly increased in F groups compared with that of S rats.5Compared with S4and S6, the myocardial collagen volume fraction (CVF)increased in F4and F6groups. Moreover, the CVF of F6rats was much higherthan that in F4.Conclusions:1Arterio-venous shunt induced pulmonary hypertensionand right ventricular hypertrophy model can be successfully established in ratsby the abdominal aorta and inferior vena cava fistula surgery.2The rightventricular hypertrophy was detected at2w after aorto-caval shunt. After8wof the aorto-caval shunt, discompensation of cardiac hypertrophy was detectedin model rats.3The concentration of ET-1and cGMP was increased with theprocess of the pulmonary hypertension.Part Ⅱ：The effect of Tadalafil on pulmonary vascular remodeling inpulmonary hypertension model caused by left to right shuntObjective: To investigate the effects of Tadalafil, a Phosphodiesterase5inhibitor, on the pulmonary vascular remodeling in shunt-induced pulmonaryhypertension via establishing shunt pulmonary hypertension model in rats.Method: Male Spague-Dawley rats (250-290g) were randomly dividedinto the following six groups (n=8): S4, S6(four or six weeks after sham operation), F4, F6(four or six weeks after shunt), T4and T6group (treatmentwith four or six weeks after shunt). The rats in T4and T6group were givenTadalafil (10mg/kg/d) by gavage four weeks after the aorta-caval fistulasurgery. The remaining rats were received the same volume of saline.Therefore, the S4, F4and T4groups received gavage for one week, while theS6, F6and T6groups for three weeks. The rats were anesthetized to measurethe mapped RVSP according to the above mentioned method after thecorresponding treatment. The pulmonary morphological changes wereobserved by HE staining of the right lower lobe tissue of the lung. ELISA isused to determinate serum content of ET-1and cGMP. The expression ofETAR and ETBR was detected with Immunohistochemistry staining.Results:1Compared with S4and S6, the mPAP, sPAP and+PAdP/dtmaxof F4and F6group increased significantly (P<0.05). The mPAP, sPAP and+PAdP/dtmax in T4and T6rats were higher even than that of F4and F6group(P<0.05).2The serum concentration of cGMP increased in T4and T6ratscompared with that of F4and F6groups (P<0.05).3The concentration of ET-1in serum of F4and F6groups is higher than that of S4and S6rats (P<0.05).Compared with the F4and F6group, ET-1concentrations in serum wereincreased obviously in T4and T6rats (P<0.05).4Compared with the F4andF6groups, the WT%and WA%of pulmonary arterial were much lower in T4and T6rats (P<0.01). The expression of ETAR was mainly observed in theSMC of the pulmonary arteries. However, the expression of ETBR wasobserved in endothelial cells and partially in SMC of the pulmonary arteries.The amount of ETAR expression was increased in F group than that in controlgroup (P<0.01). However, in T group the expression of ETRA was obviouslydecreased compared with the control (P<0.01). Furthermore, the amount ofETRB expression was reduced significantly in F group than that in controlgroup (P<0.01). On the contrary, the expression of ETRB was obviouslyhigher in T group than F group (P<0.01), but was still lower than controlgroup (P<0.05).Conclusions:1Tadalafil can effectively increase cGMP concentrations in pulmonary vascular smooth muscle by inhibiting widespread pulmonaryvascular phosphodiesterase-5ring guanosine monophosphate (cGMP)degradation.2The decrease of cGMP can effectively reduce the shunt withpulmonary hypertensionrat pulmonary artery pressure through the nitric oxide(NO) pathway.3Tadalafil can increase ET-1levels by adjusting pulmonaryvasoconstriction/diastolic balance.Part Ⅲ：The role of Tadalafil on the Cx43phosphorylation in left-rightshunt pulmonary hypertensionObjective: To investigate the effects of phosphodiesterase-5inhibitorTadalafil on cardiac hypertrophy and the phosphorylation of Cx43in shunt-induced pulmonary hypertension via establishing shunt pulmonaryhypertension model in rats.Method: Male Spague-Dawley rats (250-290g) were randomly dividedinto the following six groups (n=8): S4, S6(four or six weeks after shamoperation), F4, F6(four or six weeks after shunt), T4and T6group (treatmentwith four or six weeks after shunt). Masson special staining was carried out toobserve the changes of myocardial collagen. The expression of Cx43and thephosphorylation Cx43in heart were determined with immunohistochemicalstaining and Western blot.Results:1Compared with the F4and F6groups, the RV/BW,(LV+S)/BW and RV/(LV+S) decreased in T4and T6rats (P<0.05).2Compared with F4and F6groups, the CVF in T4and T6groups wassignificantly decreased (P<0.01).3Compared with S4rats, the mean opticaldensity (OD) of the brown positive staining particles of Cx43or pCx43inmyocardium was increased significantly in F4group (P<0.05). There was nosignificant difference between F6and S4or between F6and F4(P>0.05).Compared with the F groups, the OD value of T groups remained unchanged,however, the ratio of the intercalated disc OD to total OD increasedsignificantly (P<0.05),4Western-blot: Compared with the S4group, theexpression of Cx43and pCx43was upregulated in F4group (P<0.05). Therewas no significant difference between F6and S4or between F6and F4(P> 0.05).Compared with F4group, Cx43and pCx43expression in T4group hasno significant changes (P>0.05). Compared with the F6group, the expressionof Cx43and pCx43decreased (P<0.05).Conclusions:1Tadalafil, the Phosphodiesterase5inhibitors, can notonly reduce pulmonary artery pressure load by expensing the pulmonaryartery smooth muscle, but also attenuate the stimulating effects of beta-adrenergic and postpone the cardiac hypertrophy and remodeling viaincreasing cGMP levels.2Tadalafil can prompt the translocation of Cx43andpCx43from the intercalated disc to the cytoplasm, i.e. preventing loss ordestruction.Part Ⅳ： Coumestrol inhibits carotid sinus baroreceptor activity bycAMP/PKA depended nitric oxide release in anesthetized male ratsObjective: Although evidences suggest that phytoestrogens offermultiple beneficial effects on the cardiovascular system, but the effects ofcoumestrol (CMT), one of the well-known phytoestrogens, on arterialbaroreceptors are still unknown.Method: In this study we examine the effects of CMT on arterialbaroreceptors activity (CBA) for the first time by isolated carotid sinusperfusion, sinus nerve afferent discharge recording and western blot.Results: The results showed that CMT inhibited the CBA, which shiftedthe functional curve of the carotid baroreceptor to the right and downward,with a marked decrease in the peak slope and the peak integral value of carotidsinus nerve discharge in a concentration dependent manner. Also, the possiblemechanisms underlying these effects were examined. We found thatpretreatment with an estrogen-receptor antagonist, tamoxifen, did not affectthe inhibition of CMT on CBA, while pretreatment with NG-nitro-L-argininemethyl ester, an inhibitor of nitric oxide (NO) synthase, could completelyabolish the effects of CMT, moreover, a NO donor, SIN-1, could potentiatethese inhibitory effects. In addition, we also found that the intracellular cAMPlevels and phosphorylation of Ser1176-eNOS protein expression in carotidbifurcation tissue could be increased dose-dependently by CMT, and the phosphorylation of Ser1176-eNOS was blocked by a highly selective PKAinhibitor H89.Conclusions: These findings indicate that CMT could inhibit CBA viathe local release of NO, which were mediated by the cAMP/PKA pathway andwere unrelated to the estrogenic effect.