The Role Of Sildenafil: Both In Cardio-pulmonary Remodeling And Changes Of Mmp-2, MMP-9/TIMP-1in Volume Overload Rats

Sildenafil, a selective inhibitor of cGMP specific PDE-5, is now providedas a novel drug therapy for pulmonary hypertension. By inhibiting thehydrolytic breakdown of cGMP, sildenafil prolongs the action of intracellularcGMP, which relaxes vascular smooth muscle, resulting in vasodilation, andsildenafil can also suppress cell proliferation via NO-cGMP signaling pathway.Because the over-expression of PDE-5was detected in hypertrophicmyocardium, cGMP as a signaling molecule in the NO-sGC-cGMP-cGKpathway, is proposed as a latent therapeutic target to myocardial hypertrophyand heart failure. However, there are also several clinical trials related tosildenafil in cardiovascular disease exhibit a conflict consequence. Furtherresearches about NO-sGC-cGMP-cGK signaling pathway, either in theregulatory mechanisms at molecular level or various pathologic stimulatingfactors altering the signal transduction, also including the recognition of itscomplex influence on extracellular matrix metalloproteinases and tissueinhibitors system （MMPs/TIMP）, have finally leaded to some doubts on therole of NO-cGMP in regulating vascular proliferative diseases. Furthermore, ithas been already reported that the increase of cGMP, induced by exogenousNO or phosphodiesterase （PDE） inhibitors, could enhance the expression ofTIMP-1which as a fibrotic factor resulting in tissue fibrosis. Thus, it isindefinable that how sildenafil impacts MMPs/TIMP by the NO-cGMPpathway. And the effects of long-term application of sildenafil oncardiovascular remodeling under pathologic state are still unclear. So, in thistest, we use volume overload rats as an experimental animal model toinvestigate the role of sildenafil on ventricular and pulmonary arterialremodeling through NO-cGMP pathway, and also to explore their correlationswith MMPs/TIMP. Objective:（1） To observe the effects of sildenafil on ventricular and pulmonaryarterial remodeling in volume overload rats.（2） To observe the effects of sildenafil on MMP-2, MMP-9, TIMP-1andPCNA expressions of ventricle and pulmonary artery in volume overload rats.（3） To explore the influence of sildenafil on the correlations ofmyocardium and pulmonary artery remodeling with the changes ofMMPs/TIMP, by NO-cGMP signaling pathway.Methods:（1） For pulmonary artery hypertension models induced by aortocavalfistula （ACF）. In Sprague-Dawley rats, pulmonary artery hypertension wasinduced by ACF operation. Forty ACF-operated animals were randomlydivided into four groups: ACF group, ACF+Sildenafil group,ACF+Sildenafil+L-NAME group, and ACF+L-NAME group. Subcutaneousinjections of sildenafil (10mg kg^-1 d^-1), L-NAME (20mg kg^-1 d^-1) or theircombination were initiated the second day after ACF for7weeks. Twentysham-operated rats were randomly divided into two groups: Sham-operatedcontrol group and Sham+Sildenafil group. The Sham+Sildenafil group wastreated with sildenafil-injection (10mg kg^-1 d^-1) for7weeks. The rats in ACFgroup and Sham group were received0.9%saline-injections.（2） For observation Index. At the end of the seven-week periodadministration, rats were anesthetized with intraperitoneal pentobarbital（45mg kg-1）. Then blood was collected from the rats for determination ofserum NO and iNOS. Meanwhile, hearts and lungs harvested usingasepsis-treated tools and then the ratios of cardiac ventricle （%body weight）,right ventricular hypertrophy index （RV/LV+SP wet weight ratio） and relativeright ventricular area （%body weight） were calculated. Isolated inferior lobeof left lung and middle lobe of right lung were fixed in10%formaldehyde forParaffin Embedding and sectioning. Then with hematoxylin and eosin （H&E）staining and Van Gieson collagen staining, morphological changes and fibrosisextent of pulmonary arterioles were observed under light microscopy. Based on digital analysis of these images, data of relative medial thickness （RMT）,relative medial area （RMA） and relative lumen area （RLA） was obtained.Western blotting was carried out for the determination of MMP-2, MMP-9,TIMP-1and PCNA expression in pulmonary artery and myocardial tissues.mRNA levels of MMP-9and TIMP-1in pulmonary artery were detected byReal-time PCR.Results:（1） Effects of sildenafil on ventricle remodeling. The decentered rightventricular hypertrophy was exhibited in ACF group rats. As compared withsham-operated controls, there was a significant increase in RV/（LV+SP）,CV/BW and RVA/BW in ACF rats （P<0.01, P<0.05, P<0.01）.There were alsoan obvious decentered right ventricular dilation and myocardial hypertrophy inACF+Sildenafil group, of which the RV/LV+SP, CV/BW and RVA/BW werenot significantly different from ACF group. However, the ventricle remodelingin rats from ACF+Sildenafil+L-NAME group and ACF+L-NAME group bothtreated with L-NAME for7weeks, had been reduced, and RV/（LV+SP）,RVA/BW were significantly decreased compared with ACF andACF+Sildenafil group （P<0.05）, and CV/BW was significantly decreasedcompared with ACF+sildenafil group（P<0.01）.（2） Effects of sildenafil on the expression of MMP-2, MMP-9, TIMP-1and PCNA in myocardium. Western bolting was used for the determination ofprotein levels. As compared with sham controls, the expression of TIMP-1was significantly increased （P<0.05） while MMP-2significantly decreased（P<0.05） in the myocardium of ACF rats, and MMP-9protein levels had atendency to decrease but without significance; There was no significantdifference between ACF and ACF+Sildenafil group in the expression ofMMP-2, MMP-9, and TIMP-1. However, the protein level of MMP-2in ratsof ACF+L-NAME and ACF+Sildenafil+L-NAME group was significantlyincreased compared with ACF and ACF+Sildenafil group （P<0.01or P<0.05）.L-NAME treatment could decrease TIMP-1protein level in bothACF+Sildenafil+L-NAME and ACF+L-NAME groups. And TIMP-1in ACF+L-NAME group was significantly less than the ACF andACF+Sildenafil group （P<0.01, P<0.05）. The results indicate that ACFinduces a decrease of MMP-2, conversely an increase of TIMP-1in ratmyocardium, which may lead to collagen deposition and myocardial fibrosis.But, sildenafil treatment can not attenuate the ACF-induced changes ofMMP-2and TIMP-1, while L-NAME does.The expression of PCNA in rat myocardium from ACF andACF+L-NAME group was increased, compared with Sham andACF+Sildenafil group （P<0.05, P<0.05）（3） Effects of sildenafil on morphological changes in pulmonaryarterioles.HE staining: The thickened medial layer and narrowed lumen ofpulmonary arterioles of rats in ACF group were showed under lightmicroscope. Compared with sham group, there were dramatic increase inRMT and RMA, and an obvious decrease in RLA （P<0.05, P<0.05, P<0.01）.Moreover, sildenafil and L-NAME both significantly blunted ACF-inducedmorphological changes in RMT, RMA and RLA （P<0.01or P<0.05）. Theircombination also significantly decreased RMT and RMA compared with ACFgroup （P<0.05）, while there was an increased tendency of RLA withoutsignificance.Van Gieson staining: In ACF group, hyperplastic collagen fibers aroundthe vascular wall were shown under light microscope. Sildenafil promoted thecollagen hyperplasia and contributed to collagen accumulation inACF+Sildenafil group. Mono-treated with L-NAME or its combination withsildenafil could inhibit the collagen accumulation.（4） Effects of sildenafil on the expressions of MMP-2, MMP-9, TIMP-1and PCNA. Western bolting was used for the determination of MMP-2,MMP-9, TIMP-1and PCNA at protein levels. MMP-9and TIMP-1mRNAlevels were determined by Real time PCR. Either at protein level or at mRNAlevel, the expression of TIMP-1was significantly increased while MMP-9significantly decreased in the pulmonary arteries of ACF rats, compared with sham group （P<0.01or P<0.05）. At the same time, the expression of TIMP-1in ACF+Sildenafil group was even higher than ACF group, and MMP-9evensignificantly lower （P<0.01）. The results indicate that the increase of collagenin pulmonary artery may attribute to the changes of MMP-9/TIMP-1. Whentreated with L-NAME, TIMP-1expression levels were significantly reduced inACF+Sildenafil+L-NMAE group and ACF+L-NAME group, compared withACF group and ACF+Sildenafil group（P<0.01or P<0.05）, and MMP-9wassignificantly increased compared with ACF+Sildenafil （P<0.01）.ACF induced a significant increase in MMP-2protein level of pulmonaryartery compared with Sham-operated controls. None of the treatments ofSidenafil, L-NAME or their combination could significantly affect the proteinlevels compared with ACF group.As compared with Sham-operated controls, ACF induced a significantincrease in PCNA protein level of pulmonary artery. Both Sidenafil andL-NAME could significantly blunt the protein increase of pulmonary arterytissues, but the effect of their combination was not so significant.（5） Effects of sildenafil on serum NO and iNOS levels of volumeoverload rats.As compared with Sham-operated controls, serum NO levels weresignificantly increased （P<0.01） in ACF group. Serum NO levels ofACF+Sildenafil group and ACF+Sildenafil+L-NAME group were alsoincreased. While there is a very significantly decrease in serum NO levels ofACF+L-NAME group compared with ACF and ACF+Sildenafil group（P<0.01） and the serum iNOS activities of ACF+L-NAME group were alsosignificantly decreased compared with ACF+Sildenafil group.Sub-Total:1The volume overload model was successfully established withaortocaval fistula treatment. In myocardium and pulmonary artery, theexpression of MMP-2and MMP-9was down-regulated while TIMP-1wasup-regulated, which caused by the ACF-induced volume overload for7weeks.According to these results, we supposed that the volume overload-induced remodeling may turn into a fibrosis stage, and further studies needs to confirmthe conjectures.2Sildenafil failed to blunt or reverse volume overload-inducedventricular remodeling of ACF rats. However, sildenafil did reduce the medialthickness of pulmonary arterioles and blunt the lumen narrowness, otherwisecause a collagen accumulation. These may relate to the decrease of MMP-2orMMP-9and the increase of TIMP-1caused by sildenafil in myocardium andpulmonary artery of ACF rats.3Low dose of L-NAME could attenuate ACF-induced ventricle andpulmonary arterioles remodeling, and partly reversed the changes of MMP-2,MMP-9and TIMP-1expressions induced by ACF or sildenafil.4All the results above indicate that NO-cGMP pathway may participatein the ACF-induced ventricle and pulmonary artery remodeling, and sildenafilmay affect the expressions of MMP-2, MMP-9and TIMP-1by increasingcGMP levels.Conclusion:Sildenafil application for7weeks did not blunt or reverse themyocardium weight increase and right ventricle decentered hypertrophy involume overload rats. Sildenafil induced the high expression of TIMP-1,which had a bidirection regulation in the attenuation of pulmonary arteryremodeling and the promotion of collagen fibrosis. To identify that thedown-regulation of MMP-2or MMP-9and the up-regulation of TIMP-1caused by excess cGMP, whether was an adaptive response induced bysildenafil or a promoter of tissue fibrosis in ventricle and pulmonary artery,still needs prolonged observations.