| ObjectivePulmonary arterial hypertension(PAH) is characterized by a sustained and progressive elevation of pulmonary arterial pressure leading to right ventricular hypertrophy,right heart failure and ultimately to death.The cause remains unknown and available treatments are limited,expensive,and often associated with significant side effects.PAH has a complex pathobiology involving pulmonary vasoconstriction, vascular remodeling,inflammation and microthrombosis.The pulmonary vascular remodeling in PAH is characterized by changes in pulmonary vascular structure associated with medial hypertrophy,which is mainly caused by imbalanced proliferation and apoptosis in pulmonary artery smooth muscle cells(PASMCs).It has also been demonstrated that either increased proliferation or decreased apoptosis of PASMCs contribute to pulmonary vascular medial hypertrophy and vascular remodeling.More effective therapies for PAH will have to directly and selectively target the mechanisms leading to the pro-proliferative and anti-apoptotic environment in the vascular wall.Recently,"apoptosis-based therapeutic strategies" to reduce pulmonary vascular thickening gained attention and have been successful in experimental animals.Thus,inhibition of PASMCs growth and augmentation of PASMCs apoptosis could serve as therapeutic approaches for patients with PAH. However,the precise mechanisms involved in the regulation of PASMCs proliferation and apoptosis in PAH are still incompletely understood.It has been recently discovered that Kv channels also participate in vascular remodeling by regulating cell proliferation and apoptosis.From the variety of Kv channels expressed in PASMCs,special interest has been paid to Kvl.5.The decreased Kv channels expression and activity not only causes pulmonary vasoconstriction by inducing membrane depolarization and increases in cytoplasmic Ca2+ concentration([Ca2+]cyt) in PASMCs but also contributes to pulmonary vascular medial hypertrophy by inhibiting apoptotic cell shrinkage and apoptosis.However, up-regulation of Kvl.5 correlates with an increase in apoptosis/proliferation ratio and inhibition of PAH.The resistance to apoptosis is further enhanced by the selective downregulation of Kv channels that has been shown in human and animal models of PAH.Endogenous substances,e.g.vasoactive agonists,growth factors and cytokines, regulate PASMCs proliferation and apoptosis.Among these substances,serotonin (5-hydroxytryptamine,5-HT) is a potent vasoconstrictor and mitogen that has been implicated in the pathophysiology of PAH.Serotonin mediated proliferative signaling pathways via serotonin transporter(5-HTT) and serotonin receptors on PASMCs. Furthermore,the expression of 5-HTT in the pulmonary artery is increased in patients with pulmonary arterial hypertension.Exposure of PASMCs to hypoxia results in a rapid increase in the level of 5-HTT mRNA.It has been shown that 5-HT and 5-HTT play a central role in the pathogenesis of pulmonary artery smooth muscle cell proliferation,not only in experimental PAH but also in human PAH,whether idiopathic (iPAH) or associated with various diseases(aPAH).Selective serotonin re-uptake inhibitors(SSRIs),which are used clinically as antidepressants,such as fluoxetine,sertraline and paroxetine etc.,block serotonin internalization by inhibiting 5-HTT.We previously reported that fluoxetine decreased serotonin mediated proliferation of PASMCs in vitro.It has been shown that fluoxetine inhibited the effects of 5-HT on Kv currents in PASMCs and pulmonary vasoconstriction.Furthermore,fluoxetine also protected rats from both chronically hypoxic and monocrotaline(MCT)-induced pulmonary vascular remodeling and pulmonary arterial hypertension.However,it is not clear whether the beneficial effects of fluoxetine on pulmonary artery remodeling is relevant to its effects on Kv channel,PASMCs proliferation and apoptosis.Therefore,the present study was designed to investigate the roles of apoptosis and Kvl.5 channels in MCT-induced PAH, and if these factors are involved in the protective effect against PAH by fluoxetine.Methods1.Animal Models and Experimental DesignEighty male Wistar rats(160±10g) from Animal Resource Center,China Medical University(Certificate No:Liaoning 034) were randomly divided into four groups,i.e. control,MCT,MCT plus fluoxetine 2mg/kg(MCT + F2) and MCT plus fluoxetine 10 mg/kg(MCT + F10).Rats in the MCT group and two fluoxetine-treated groups were treated with a single intraperitoneal injection of MCT,and rats in the control group were received an intraperitoneal injection of vehicle.Rats in MCT + F2 and MCT + F10 groups were further treated with intragastric administration of fluoxetine 2 mg/kg and 10 mg/kg once a day for 3 weeks,respectively.Meanwhile,rats in the control and MCT groups were treated with vehicle only.And then these rats were fed with solid food and water ad lib in an alternating 12 h light/dark cycle under controlled temperature(18-22℃) and humidity(50%-70%) for 3 weeks.2.Assessment of pulmonary arterial hypertensionAfter 3 weeks,rats in all groups were intraperitoneal anaesthetized with 3% pentobarbital sodium(40 mg/kg).A polyethylene catheter was inserted into the right carotid artery to measure systemic arterial pressure(SAP).Another polyvinyl PV-1 catheter was introduced into the right jugular vein and pushed through the right ventricle into the pulmonary artery for measurement of pulmonary arterial pressure (PAP).After the measurements had been completed,rats were sacrificed with overdose of pentobarbital sodium.Heart tissue was separated as right ventricle(RV) and left ventricle plus septum(LV + S),right ventricular index was calculated by RV/(LV + septum).3.Morphometric AnalysisThe right lower lobes of the rat lungs were cut and fixed with formalin buffer. After paraffin embedding,5μm-thick lung sections were stained with hematoxyline-eosine(HE).The morphologic alterations of the pulmonary arteries were observed under optical microscope.Three rats per group were studied and from each rat at least 2 separate lung sections were examined.Resistance pulmonary arteries(PAs)(50-200mm) chosen randomly from low power fields(magnification,×10) were analyzed(approximately 60 arteries/group;2-3 slides/rat;3 rats/group) by 2 blinded investigators.Pulmonary arteries remodeling was measured as percent medial thickness.Medial wall thickness of pulmonary arteries was calculated and expressed as follows:index(%) =[(external diameter-internal diameter)/external diameter]x 100.4.Protein expression of Bcl-2,Bcl-xl and Cleaved caspase-3 were detected by Western Blot5.Expression of Kvl.5 mRNA was evaluated by reverse transcfiption-polymerase chain reaction(RT-PCR)6.Protein expression of Kv 1.5 was detected by Western blot7.Proliferative and apoptotic assessmentProliferating cells were evaluated by using proliferating cell nuclear antigen immunohistological staining;apoptotic cells were detected by using the terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end-labeling(TUNEL) method according to the manufacture's instruction of the in situ cell death detection kit. The number of PCNA- and TUNEL-positive cells in 10 fields for each section of small to moderate size pulmonary arteries was quantitatively evaluated as a percent of total SMCs at a magnification of 400×in a blind manner. 8.Statistical analysisAll data are expressed as mean±SD.Statistical analyses were performed by one-way ANOVA.A value of P＜0.05 was considered statistically significant.Results1.Hemodynamics assessmentCompared with the control group,mean PAP in the MCT group was significantly elevated from 17.8±1.2 to 32.7±5.7 mmHg(P＜0.01 vs control),but was reduced from 32.7±5.7 to 25.8±5.4 mmHg by fluoxetine 10mg/kg(P＜0.05,vs MCT).Yet, Mean SAP did not differ significantly in all groups.2.Right ventricular indexRVI(%) was significantly increased by MCT treatment from 34.1±2.1 to 51.3±8.8(P＜0.01 vs control) and decreased in MCT + F2 and MCT + F10 groups from 51.3±8.8 to 45.2±5.3(P＜0.05 vs MCT) and 43.4±3.3(P＜0.01,vs MCT),respectively.3.Morphological analysis of pulmonary arteriesPulmonary arteries in the MCT group exhibited obvious medial wall thickening. The percentage of medial wall thickness of pulmonary arteries was significantly increased in the MCT group from 26.9±7.1 to 50.5±10.8(P＜0.01 vs control). Compared with the MCT group,the percentage of medial wall thickness of pulmonary arteries in MCT + F2 and MCT + F10 groups were reduced from 50.5±10.8 to 46.0±8.2(P＜0.05 vs MCT) and 36.6±7.0(P＜0.01 vs MCT),respectively.These results indicated that MCT significantly promotes pulmonary vascular remodeling,whereas fluoxetine attenuates the effects of MCT.4.Protein expression of Bcl-2,Bcl-xl and Cleaved caspase-3Protein expression of Bcl-2,Bcl-xl and Cleaved caspase-3 were measured by Western blot.Compared with the control group,the levels of Bcl-2,Bcl-xl in the MCT group were significantly increased from 1.22±0.10 and 1.86±0.25 to 2.89± 0.82 and 2.91±0.57(both P＜0.05 vs control),respectively.Fluoxetine inhibited MCT induced increase of these anti-apoptotic genes in a dose dependent manner.In MCT + F2 group,these levels were decreased to 2.65±0.97 and 2.32±0.45, respectively,although there was no statistical significance compared with the MCT group.And in MCT±F10 group,these levels were significantly decreased to 1.55±0.60 and 2.01±0.37(both P＜0.05 vs MCT),respectively.It was also found that Cleaved caspase-3 expression decreased in the MCT group from 1.49±0.10 to 0.68±0.18(P＜0.01 vs control).And it was increased to 1.20±0.27(P＜0.05 vs MCT) and 1.33±0.24(P＜0.01 vs MCT) in MCT + F2 group and MCT + F10 group, respectively.5.Expression of Kv1.5 by RT-PCR and Western blotSemiquantitative RT-PCR analysis was utilized to detect changes in mRNA expression of Kv1.5 in pulmonary arteries of MCT-induced PAH rats.The PCR products of the expected sizes for Kv1.5 andβ-actin were 267bp and 612bp, respectively.MCT significantly decreased the expression of Kv1.5 mRNA levels compared with the control group(1.23±0.42 vs 2.00±0.55;P＜0.05).Compared with the MCT group,the level of Kv1.5 mRNA was not obviously changed in MCT+ F2 group,yet it was increased significantly from 1.23±0.42 to 1.90±0.38 in MCT + F10 group(P＜0.05 vs MCT).Results from Western blot analysis also demonstrated that the levels of relative Kv1.5 expression decreased in the MCT group compared with the control group(0.70±0.06 vs 1.23±0.22;P＜0.01),and it was increased in MCT + F2 and MCT + F10 groups(1.02±0.14 vs 0.70±0.06,1.09±0.14 vs 0.70±0.06,respectively;P＜0.05 vs MCT).6.Effect of fluoxetine treatment on PASMCs proliferationFluoxetine treatment suppressed proliferation and enhanced apoptosis of PASMCs in MCT-induced PAH rats.The percentage of PCNA-positive PASMCs in small to moderate sized pulmonary arteries was significantly increased in the MCT group compared with the control group(15.7±3.2%vs 3.7±1.2%;P＜0.01).Then,it was decreased in MCT + F10 group(8.2±2.8%vs 15.7±3.2%;P＜0.01 vs MCT).7.Effect of fluoxetine treatment on PASMCs apoptosisThe percentage of TUNEL-positive cells was slightly decreased in the MCT group compared with the control group(2.9±1.1%vs 3.3±1.3%;P＜0.05),which was increased in MCT + F2 and MCT + F10 groups(7.9±1.9%vs 2.9±1.1%,11.7±2.5% vs 2.9±1.1%,respectively;P＜0.01 vs MCT).Conclusion1.Fluoxetine protected against MCT-induced pulmonary arterial hypertension by decreasing pulmonary arterial pressure,reducing right ventricular index and inhibiting pulmonary artery remodeling.2.Fluoxetine protected against vascular remodeling by suppressing PASMCs proliferation and inducing PASMCs apoptosis which might be related to decreasing expression of Bcl-2 and Bcl-xl protein,increasing expression of Cleaved caspase-3 protein.3.Fluoxetine enhanced expression of Kvl.5 protein and mRNA levels in pulmonary arteries of MCT-induced PAH rats.These findings suggested that fluoxetine protected against MCT-induced PAH,in which up-regulating Kvl.5 channels was considered to be involved.
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