The Preventive Roles And Mechanisms Of Estrogen On Hypoxic Pulmonary Hypertension In Rats

Background:Hypoxic pulmonary hypertension (HPH) is one of subtypes of pulmonary hypertension(PH). HPH is common in those patients of chronic obstructive pulmonary disease (COPD)and residents in high altitude. HPH is characterized by pulmonary arterial thickening andremodeling, which pathophysiological changes including hypoxic pulmonaryvasoconstriction (HPV), pulmonary arterial smooth muscle cells hypertrophy andproliferation, pulmonary vascular remodeling (PVR), and increase of pulmonary arterialpressure in late phase. PVR and right ventricle failure during late phase is the major factorcuases deterioration or death in those HPH patients. Thickening of arterial media andmuscularization of the normally distal pulmonary arteries are the main pathologicalchanges in HPH.Early researches found there exist significant gender differences in HPH morbidity,which is much lower in women than men. The female HPH patients’ clinical symptoms are relative less severe than male ones. Later researches reported that estrogen possessesimportant effects such as vasodilation and lowering pulmonary arterial pressure. Therefore,further exploring the effects of estrogen on HPH and the mechanisms underlying,searching safe and effective exogenous estrogen for alleviating HPH will provide newtargets and methods for early treatment. This study focused on the whole animal, organ,and cell levels to study effects of endogenous estrogen, exogenous estrogen, andphytoestrogen on HPH, and to further explore the mechanisms underlying.Objective:To study the preventive effects of endogenous estrogen, exogenous estrogen, andphytoestrogen on HPH on three levels of whole animal, organ, and cell, respectively. Tofurther explore cell cycle, inflammation, and oxidative stress etc. participate inmechanisms of estrogen on preventing HPH.1) Further to study the protective effects of endogenous estrogen and its mechanismsunderlying during the development of HPH.2) Investigate the roles of AKT/Skp2/P27kip1pathway in the preventive effects ofexogenous estrogen on HPH.3) Explore the roles of the phytoestrogen on attenuating HPH and the underlyingmechanisms.Methods:1. For observing the effects of endogenous estrogen on HPH, we performedexperiments on rats, pulmonary arterial rings, and PASMCs, respectively. Female, male,pregnant, and ovariectomized SD (Sprague-Dawley) rats were randomly separated intonormoxic and hypoxic groups (total8groups). The normoxic groups were kept at ambientbarometric pressure of about718mmHg (pO2of Xi’an area≈150.6mmHg). The hypoxicgroups were housed at hypobaric hypoxia chamber depressurized to380mmHg (equals analtitude of5000metres, and pO2≈79.6mmHg) for continuing21days to duplicate HPHmodels of rat. Then we observed the hemodynamic indexes of right ventricular systolicpressure (RVSP), mean pulmonary arterial pressure (mPAP), and pathological changes inrat lung circulation. Next, the third-division (external diameter <300μm) pulmonary arteries were obtained from rats of male and different estrous cycle female afteranesthetization. PA rings were immersed in glass waterbaths containing Krebs-Henseleitsolution continuing bubbled with95%O2-5%CO2at37°C for60min, and then changedthe gas into95%N2-5%O2. An estrogen-modulator, raloxifene and U0126(MAPKinhibitor) were added. Radio-immuno assays were adopted to detect the estrogen levels ofeach group rats, and further to observe the protective effects of endogenous estrogen onacute HPV. Last, pulmonary arteries were isolated from different sex’s rats and differentestrous cycle, and pulmonary arterial smooth muscle cells were cultured by explantsmethod and exposed under normoxic or hypoxic condition. Raloxifene and U0126wereadded respectively, and [3H]-thymidine incorporation assays were employed to observethe effects of endogenous estrogen on hypoxia-induced PASMCs proliferation.2. To investigate the effects of exogenous estrogen on HPH, the HPH models of ratswere duplicated, and exogenous estrogen β-estradiol was administered. The hemodynamicindexes and pathological changes of pulmonary arterioles of rats were detected. Westernblot and RT-PCR assays were adopted to investigate the key proteins (AKT/Skp2/P27kip1)of cell cycle in rat lungs and PASMCs. After observing the effects of β-estradiol onPASMCs proliferation under hypoxia exposure, we further explored if the protectiveeffects of exogenous estrogen were via modulating the cell cyle during HPH.3. For searching effective phytoestrogen with fewer side effects, we further observed theeffects of a phytoestrogen, resveratrol on preventing HPH. In vivo, HPH modles of maleSD rats were duplicated as mentioned above, and resveratrol (40mg/kg/day, gavageadministration) was given. The hemodynamic indexes, together with the pathologicalchanges of pulmonary arterioles were also observed. Immunohistochemial staining wasperformed to detect inflammatory cells infiltration around the rat pulmonary arterioles.Realtime-PCR assays were adopted to test the mRNA levels of those inflammatory factorssuch as IL-6, TNF-α etc. The key factors related with inflammation and oxidative stress ofHIF-1α, NF-κB, Nrf-2/Trx-1, H2O2, GSH, and SOD expression were detected by WBassays and other methods. Primary PASMCs were cultured under normoxic or hypoxiccondition, and different concentrations of resveratrol were administered, together with estrogen receptor inhibitor ICI182780administration. WST-1assays were performed toexplore the roles of phytoestrogen on hypoxia-induced PASMCs proliferation. HIF-1αinhibitor KC7F2, AKT inhibitor LY294002, and ERK inhibitor PD98059were added tofurther reveal the anti-inflammation and anti-oxidative stress effects of resveratrol on HPHand hypoxia-induced PASMCs proliferation.Results:1. Physiological changes of endogenous estrogen level affected the development ofHPH. Those rats with higher level of endogenous estrogen showed less elevation in RVSP,mPAP, and pulmonary arterial thickening. Compared with the male groups, the femalegroups exhibited lighter changes after hypoxia exposure, and the pregnant group with thehighest estrogen level showed the lightest hypoxic changes. Moreover, ovariectomizedrats showed nearly the same estrogen level as the male ones, and the changes of RVSP,mPAP in those ovariectomized rats were as same as the male ones. Isolated pulmonaryarteries vasoconstriction assays showed that the arteies from the proestrus rats with higerestrogen level exhibited less contraction compared with male group and diestrus onesunder acute hypoxia exposure. Treated with an estrogen modulator, raloxifene enhancedthe dilation of pulmonary arteries in proestrus group. However, a MAPK inhibitor, U0126increased the contraction reaction of pulmonary arteries under acute hyupoxia in theproestrus group. The in vitro assasys revealed that PASMCs from the proestrus groupexhibited less proliferation compared with those male or diestrus groups after hypoxiaexposure. Raloxifene and U0126treatment to some extent increased the PASMCsproliferation. Raloxifene and U0126revealed a synergistic effect on PASMCs. Treatedwith both raloxifene and U0126, PASMCs exhibited significant proliferation after hypoxiaexposure.2. Hypoxia resulted in significant elevation of RVSP, RVHI, and thickening ofpulmonary arterial wall. Hypoxia also induced significant decrease of cell cycle inhibitorP27kip1, and increase of its specific degradation enzyme, S phase kinase associatedprotein2(Skp-2) in rat lungs. The phosphorylated AKT was also showed significantlyelevation after hypoxia exposure. Exogenous estrogen β-estradiol effectively attenuated hypoxia-iuduced right ventricular remodeling, decreased pulmonary arterial pressure andpulmonary arterial remodeling. Moreover, β-estradiol treatment significantly decreasedthe elevated expression of phosphorylated AKT and Skp-2induced by hypoxia.Meanwhile, β-estradiol treatment notably increased the expression of P27kip1afterhypoxia exposure. In vitro experiments further verified the in vivo results, anddemonstrated that β-estradiol greatly decrased hypoxia-induced proliferation of PASMCs.3. Phytoestrogen resveratrol significantly inhibited rat HPH, attenuated RVSP, haltedright ventricular remodeling, and inhibited hypoxia-induced PASMCs proliferation.Resveratrol also decreased HIF-1α and phosphorylated NF-κB expression, lessenedinfiltration of inflammatory cells around pulmonary arteries, and reduced the productionof H2O2in rat lungs. Moreover, resveratrol notably attenuated increased expression ofmRNA level of inflammatory factors in rat lungs such as IL6, and TNF-α etc. In the sametime, resveratrol enhanced expression of those key factors related with anti-oxidativestress such as Nrf-2, Trx-1, GSH, and SOD. In vitro experiments revealed that resveratrolattenuated HIF-1α expression in PASMCs after hypoxia exposure in a dose-dependentway. Inhibition of HIF-1α inhibited proliferation of PASMCs under hypoxia exposure.Treated with the estrogen receptors blocker, ICI182780could not oppose theanti-proliferation effect of resveratrol. Additionally, resveratrol significantly attenuatedPASMCs proliferation, along with inhibition of expression of phosphorylated AKT andERK.Conclusions1) Endogenous estrogen may exert inhibiting effects during the development of HPH inrats. The inhibiting effects may through both non-genomic (by attenuating HPV viaGPR30receptor pathway), and genomic (by inhibiting PVR and PASMCs proliferation)mechanisms.2) Exogenous estrogen β-estradiol effectively attenuated PVR and HPH of rat models. Theunderlying mechanism may be through inhibiting PASMCs proliferation via regulatingAKT/Skp-2/P27kip1signaling pathway.3) Phytoestrogen resveratrol may attenuate rat HPH through inhibiting HIF-1α signaling pathway and inflammatory response, and decreasing oxidative stress via modulating theNrf-2/Trx-1axis. The protective effects of resveratrol may through inhibiting AKT andERK signaling pathway, but an ER-independent way.