The Investigation Of Mechanism Underlying The Increase Of Pulmonary Arterial Pressure Induced By Smoking

【Background】Pulmonary arterial hypertension （PAH） is a group of diseases characterized bypulmonary vascular wall proliferation and remodeling,as well as progressiveincrease of pulmonary arterial pressure that causes right ventricular hypertrophy,right ventricular failure and finally death. PAH is an important complications ofchronic obstructive pulmonary disease （COPD）, and an independent risk factor forCOPD patients. The incidence of mild to moderate pulmonary arterial hypertension（PAH） is highly prevalent, reaching to50%in advanced chronic obstructivepulmonary disease （COPD）. The sequence of changes that lead to PH in COPDbegins at early disease stages by the impairment of endothelial function, which isassociated with impaired release of endothelium-derived vasodilating （nitric oxide,prostacyclin） and vasoconstrictive agents （endothelin-1） and imbalance amongthem.PAH in COPD is caused by vasoconstriction and remodelling of pulmonaryarteries, which is characterized by the intimal proliferation of poorly differentiatedsmooth muscle cells and the deposition of elastic and collagen fibres. Hypoxia,inflammation and toxic effects of cigarette smoke, independently or additivelyinteracting, are confirmed factors leading to PAH. Smoking not only causes chronicbronchitis, chronic obstructive pulmonary disease, but also contribute to PAH andChronic cor pulmonale.The research has suggested that smoking is one of the main cause of COPD and PAH. In the past, most studies concerning the pathogenesis ofCOPD-related pulmonary arterial hypertension were focused on hypoxia-inducedpulmonary vasoconstriction and pulmonary remodeling. However, recent human andanimal experimental data show that smoking can act directly in pulmonary vascularsystem and eventually leads to vascular remodeling and a series of vascularphysiological reactions. However, the specific mechanism of smoking induced PAHremains unclear.General increases in intracellular Ca²⁺concentration play an essential role inpulmonary artery constriction. migration and proliferation. we and other teams havepreviously found that the subunit of store-operated calcium channel （SOCC）receptor operated calcium channel （ROCC） protein was composed of canonicaltransient receptor potential（TRPC）gene encoding the protein, with store-operatedcalcium entry （SOCE） to promote PASMCs proliferation, which leads to pulmonaryvascular wall hypertrophy and pulmonary vascular tone thickening. TRPC whichlocates on the cell membrane is a kind of important cationic channel superfamily,and plays a important role in the pulmonary artery smooth muscle cell contraction.In vascular smooth muscle, potassium channels have four channels:Voltage-gatedpotassium channel （Kv）, calcium activate potassium channel （KCa）, ATP-dependentpotassium channel （KATP） and double holed potassium channel. Of these,voltage-gated K+（Kv） channels play the most important role in regulatingmembrane potential, cytoplasmic free Ca²⁺concentration, and vasomotor tone.Dysfunction or downregulation of K+channels leads to depolarization of cellmembrane and contributes to sustained elevation of [Ca²⁺]_iby:1) activatingvoltage-dependent calcium channel （VDCC）,2) facilitating the production of inositol1,4,5-trisphosphate, which stimulates the release of sarcoplasmic reticulum Ca²⁺intothe cytoplasm,and3) promoting Ca²⁺entry through the reverse mode of Na⁺/Ca²⁺exchange,finally inducing persisted vasoconstriction, which is one of thepathological causes of PAH. 【Objective】In our experiment, rat model of chronic exposure to cigarette smoke andprimary cell culture model were established to investigate the expression of TRPCchannel and Kv channel expression in rat pulmonary arterial smooth muscle; on theother hand, Kv1.5and Kv2.1mRNA expression of PASMCs treated with nicotinewas studied. Thus, the hypothetic mechanism of the increase of pulmonary arterialpressure induced by cigarette smoke was explored, with its clinical significance tobe revealed.【Methods】Rat model by chronic exposure to cigarette smoke was established:（1） usingright heart catheterization and HE stainingto detectright ventricular systolic pressure（RVSP）, mean pressure （mPAP）, right ventricular hypertrophy index [RV/（LV+S）] as well as lung histological changes after smoking exposure for1month,3months and6months, the effect of smoking exposure on rat hemodynamics andpulmonary arterial remodeling were determined （2）Real-time quantitative PCR wasused: to detect the effect of smoke exposure on rat pulmonary artery smooth muscleTRPC1,TRPC6mRNA and protein and Kv1.5,Kv2.1mRNA expression at differenttime points（1month,3months and6months）.Model of primary culture of PASMCs was established:（1）Rat distal PASMCswere isolated and cultured after digestion by collagenase; rat distal PASMCs basic[Ca²⁺]_iand SOCE were measured using Incyte intracellular Ca²⁺concentrationsystem.（2） Kv1.5and Kv2.1mRNA expression of rat PASMCs treated by nicotinewas detected by real-time quantitative PCR.【Results】RVSP、mPAP and RV/（LV+S） were increased in rats chonically exposed tocigarette smoke for6months.Vascular wall of pulmonary artery in rats chonicallyexposed to cigarette smoke for3months has thickened, which occurred to aseverer stage after chronic exposure for6months; Chonic exposure to cigarette smoke for1month,3months and6months led to significant elevation of mRNA andprotein expression of TRPC1and TRPC6, while Kv1.5and Kv2.1mRNAexpression was downregulated after exposure.In primarily cultured PASMCs isolated from3-month and6-month smokingexposed rats, basal [Ca²⁺]_iand SOCE were higher than those in PASMCs from thecorresponding control rats. Moreover, the rise of basal [Ca²⁺]_iand SOCE inPASMCs from rats after6-month exposure were more significant than those inPASMCs from rats after3-month exposure. On the other hand, Nicotine inhibitsKv1.5and Kv2.1mRNA expression in rat PASMCs.【Conclusion】1. Pulmonary arterial pressure rised after chronic exposure to cigarette smoke.2. Chonic exposure to cigarette smoke for1month,3months and6months led tosignificant elevation of mRNA and protein expression of TRPC1and TRPC6,while Kv1.5and Kv2.1mRNA expression was downregulated after exposure, whichinduced increasing Ca²⁺influx through SOCC, finally the increase of intracellularCa²⁺concentration.3. Nicotine inhibited Kv1.5and Kv2.1mRNA expression in rat PASMCs.