Expression Of Survivin In Pulmonary Tissue Exposed To Hypoxia And Its Efect On Expression Of Kv1.5and Kv2.1

Pulmonary artery vasoconstriction and media hypertrophy are cardinalmanifestation of hypoxic pulmonary hypertension. The imbalance betweenproliferation and apoptosis in PASMCs can induce pulmonary vascularremodeling, and play an essential role in the development of hypoxicpulmonary hypertension (HPH). The drug treatment of PHP oftenemphasize on dilated pulmonary artery, and the high pressure of pulmonaryartery has been formed, the main pathological changes are proliferation orapoptosis of PASMCs, the role of vasodilator drugs for remodelingpulmonary artery is very little. And the vasodilator drugs have no specificeffect on pulmonary circulation, which may lead to poor treatment of HPH.Survivin is a member of the inhibitor of apoptosis protein (IAP) family andhas been involved in both suppression of apoptosis and regulation ofmitosis. Survivin does not express in most of normal tissues, but it isoverexpressed in many malignancies, including leukemia, breast, lung,colon, pancreas, liver, and head and neck cancers. Chronic hypoxia caninhibit voltage-gated potassium current of PASMCs. On the one hand,inhibition of Kv opening induced by hypoxia cause decreased membranepotential and depolarization of PASMCs.The voltage-gated calciumchannel opens as membrane potential reach a certain threshold, which canlead to pulmonary vasoconstriction. On the other hand, the inhibition of Kvinduced by chronic hypoxia leads to the increased intracellular calciumion.The concentration of intracellular K+has elevated, which result indecreased activity of caspase. Finally, the apoptosis of PASMCs isinhibited by hypoxia.The above research showed that survivin can regulate both proliferation and contraction of PASMCs.Then, this experiment intendto explain whether survivin cooperates with Kv1.5to regulate proliferationof PASMCs, and we want to analyse the molecular mechanisim of thesynergy of Kv1.5and survivin. Based on this, the study aims to clarify therole of Survivin and Kv in the occurrence and development of chronicpulmonary hypertension from the gene and protein level and provide theexperimental bases for the pathogenesis of HPH.The experiment steps: Eighteen age and body weight matched maleWistar rats were randomly divided into control, chronic hypoxic group for4weeks and chronic hypoxic group for4weeks treated with YM155(anovel small-molecule survivin suppressant) group. The expression anddistribution of survivin were examined along the axial pathway of thepulmonary arteries of three group rats using immunohistochemical method.Survivin mRNA expression of lung tissue of three group rats were analyzedby real-time polymerase chain reaction. The expression of mRNA andprotein of Kv1.5, Kv2.1(the two mainly members of voltage-gatedpotassium channels family) in pulmonary artery smooth muscle of ratswere detected with real time-PCR, immunohistochemical technique andWestern Blotting.The results of experiments:(1) After treating with4weeks of chronic hypoxia, mPAP and RV/(LV+S) of hypoxic rats were increased from (16.20±0.70) to (29.40±1.70)and from (0.21±0.01) to (0.49±0.02), and the weight of RV has beenincreased from (0.15±0.01) to (0.29±0.02)(P<0.01) These data showedthat the model of HPH has been established successfully.(2) After treating with YM155, mPAP of hypoxia rats was declinedfrom (29.4±1.7) mmHg to (19.6±1.3) mmHg (P<0.01), and the weight ofRV was declined from (0.2±90.02) g to (0.1±90.02) g (P<0.01).(3) In normal condition, Survivin was not expressed in the pulmonaryarteries neither in the proximal nor in distal segments. Following exposure to hypoxia, the distribution of Survivin localized predominately in themedia of the distal segments of pulmonary artery, whereas Survivin wasnot almost expressed in the intima of the distal segments (P<0.01). Aftertreating with YM155, the expression of Survivin was down-regulated,especially in the media of the distal segments of pulmonary artery afterfour weeks exposure to chronic hypoxia (P<0.01).(4) Under normoxic condition, the mRNA expression of Survivin inrat pulmonary tissues was not detected, after4weeks hypoxia, the mRNAexpression of Survivin in pulmonary tissues was significantly increased.When treated with YM155,the mRNA expression of Survivin in hypoxicrat pulmonary tissues was remarkable decreased from（27989.55±15130.29） to (5406.11±1554.20)(P<0.01).(5) Following4weeks exposure to hypoxia, the mRNA and proteinexpression of Kv1.5and Kv2.1of pulmonary artery smooth muscle weredown-regulated compared with control group (P<0.01) Kv1.5mRNA of Cgroup:1.000，Kv1.5mRNA of H group：(0.408±0.091)；Kv2.1mRNAof C group：(1.000)， Kv2.1mRNA of H group：(0.450±0.149)；Kv1.5protein of C group：(0.410±0.029)，Kv1.5protein of H group：(0.204±0.026)；Kv2.1protein of C group：(0.850±0.073)，Kv2.1proteinof H group：(0.228±0.021）. Contrarily, after treating with YM155, themRNA expression of Kv1.5and Kv2.1of pulmonary artery smooth muscleof hypoxic rats were up-regulated, and the protein level of Kv1.5andKv2.1expression of pulmonary artery smooth muscle of hypoxic rats wereboth also up-regulated compared with the hypoxic group (P<0.01). What’smore, using immunohistochemical technique, we found that proteinexpression of Kv1.5and Kv2.1in distal pulmonary arteries of rats incontrol group was consistent with the expression in HY group.The results show that: Survivin is highly expressed in the pulmonaryarteries of rats exposed to chronic hypoxia, its distribution in the media isin the distals segments.YM155, a novel small-molecule Survivin suppressant can not only down-regulate the expression of Survivin in thedistal pulmonary artery and lung tissue of rats exposed to chronic hypoxiabut also reduce mPAP and RVH, and reverse HPH of the rats. YM155canpromote apoptosis of PASMC according to changing factors of theintracellular signal transduction pathway, which indirectly promote theexpression of Kv, indicating that Kv may play an important role in cellapoptosis mediated by Survivin.