| Obstructive sleep apnea syndrome (OSAS), a common disease affectingup to2%-4%of people, is strongly associated with many cardiovasculardiseases. Animals when exposed to intermittent hypoxia for several hours perday to mimic OSA developed sustained pulmonary hypertension andpulmonary vascular remodeling within a few weeks. Therefore, intermittenthypoxia is a risk factor for cardiovascular disorders and in some cases iscomplication of pulmonary hypertension, as the endothelium is affected.Studies have shown that intermittent hypoxia is associated with elevatedplasma ET-1level, endothelial dysfunction and augmented vasoconstriction.Endothelium may play an important local regulatory role by producing a largenumber of biologically active substances that participate in the regulation ofvascular tone, cell growth, inflammation, and thrombosis, such as ET-1andnitric oxide. Diminished production of NO and exaggerated release of ET-1are believed to be key initiators of endothelial injury.In this study, we observed the impaired endothelium-dependentvasodilation and an increased ET-1responsiveness induced by CIH inpulmonary arteries of rats. Meanwhile, we tried to explain this phenomenonaccording to changes of vessel tension and ET receptors expression.Objective: We simulated OSA by exposing rats to cyclicintermittent hypoxia (CIH) to investigate the effect of CIH on pulmonaryvascular endothelial dysfunction.Methods: Male Sprague-Dawley rats (180-190g) were purchased fromthe Laboratory Animal Center (Hebei Medical University, China) and housedin rust-free cages at20°C~22°C temperature,45%~55%relative humidity on a12h light–dark cycle. The rats were divided into two groups: normoxiaand CIH. During hypoxic exposure, animals were placed daily in commercialhypoxic chambers that were flushed with100%N2to inspired O2fraction(FIO2) nadir of9%for1min. The FIO2gradually returned to21%over theremainder of each cycle. The exposure cycle was repeated every2min for8h/day,7days/wk for3wk during the animal's sleeping hours. Normoxicanimals underwent identical handling and exposure, but chambers wereflushed with room air rather than N2. After the exposure cycle was completed,animals were randomly assigned to either physiological investigation ormolecular studies.The indices as follow:1Vessel tension studyIn this study the pulmonary artery were divided into three groups:normoxia and endothelium-intact, normoxia and endothelium-denuded, CIH.The tests include relaxation to ACh (10-6mol/L) in pulmonary artery ringscontracted with PE (10-6mol/L), concentration-response curves about ET-1onpulmonary artery, effect of antagonists (BQ123--an antagonist of ETA receptor,and BQ788--an antagonist of ETB receptor) or inhibitors (L-NAME, aninhibitor of eNOS) of vascular responsiveness to ET-1.2Histopathological examinations of pulmonary arteriesHistopathological examinations included hematoxylin-eosin (HE)staining and transmission electron microscopy (TEM).3Reverse Transcription Polymerase Chain Reaction (RT-PCR)Total RNA was extracted from fresh-frozen pulmonary arteries using theTrizol Reagent. Expressions of ET-1, ETAreceptor, ETBreceptor and GAPDHin pulmonary arteries were examined by RT-PCR.4Immunohistochemical analysisThe expression and localization of ET-1, ETAreceptor and ETBreceptorwere analyzed by immunohistochemistry5Western BlottingThe expression of eNOS protein was determined by Western Blotting. 6Measurement of NO in pulmonary artery.Results:1Effects of CIH on Endothelial FunctionIn every group pulmonary arteries, ACh all produced a relaxation to PEinduced contraction, however, ACh only cause a faint relaxation fromendothelium-denuded normoxic rats (3.03±2.52%, n=6). In pulmonaryarteries from CIH rats (53.3±4.31%, n=6), the ACh-induced relaxation wassignificantly decreased when compared with endothelium-intact normoxic rats(93.3±4.30%, n=6), this indicated that endothelial function was damaged. Ofcourse, it was greater than that in the endothelium-denuded normoxic rats.2Effects of CIH on ET-1Mediated ConstrictionIn pulmonary arteries from endothelium-denuded and endothelium-intactnormoxic rats and CIH rats, ET-1(0.01–100nmol/L) produced aconcentration-dependent contraction. Compared with endothelium-intactaortas from normoxic rats (EC50=0.718±0.008nM, n=4), the absolutecontraction caused by ET-1in pulmonary artery from CIH rats (EC50=0.575±0.003nM, n=4, P <0.01) was markedly increased (P <0.01), but it wasdecreased compared with endothelium-denuded pulmonary artery fromnormoxic rats (EC50=0.493±0.004nM, n=4, P <0.01).3Effects of CIH on ET_Aand ET_BReceptor Antagonists to ET-1-InducedContractionET_Areceptor antagonist BQ123almost completely inhibitedET-1-mediated contraction in pulmonary arteries from endothelium-denudedand endothelium-intact normoxic rats and CIH rats, and the vasoconstrictionpercentages of BQ123to precontract with ET-1were no significantlydifference among groups. ET_Breceptor antagonist BQ788also significantlyinhibited ET-1-mediated contractions in pulmonary arteries fromendothelium-intact (44.13±7.04%, n=6) and endothelium-denuded (72.59±5.17%, n=6) normoxic rats and CIH rats (52.79±1.95%, n=6). Comparedwith the endothelium-intact normoxic rat pulmonary arteries, the inhibitionwas markedly decreased both in the normoxic endothelium-denuded (P <0.01) and CIH group (P <0.01), while the decrease of the endothelium-denudedrings was larger.4Effects of CIH on L-NAME to ET-1-Induced ContractionPretreatment with L-NAME significantly augment the contraction causedby ET-1in the normoxic endothelium-intact rings (115.68±7.13%, n=6).However, after the removal of the endothelium, the contraction evoked byET-1was not affected by L-NAME (100.83±6.61%, n=6). Surprisingly,L-NAME markedly inhibited the contrction induced by ET-1in CIH rats'pulmonary arteries (79.72±8.33%, n=6).5Effects of CIH on morphological and histological changesA normal tissue architecture of the endothelium was seen in the normoxiagroup, comprised of complete endothelial monolayer, regularly shaped andoriented endothelial cells, normal mitochondria. Rat pulmonary arteries in theCIH group exhibited mild histopathological changes of endothelial monolayerwith cellular enlargement and edema, shrinkage of endochylema, anddenudation of some endothelial cells. Large defects of the endothelial layer,celluer vacuolation and mitochondrial damage could be observed by TEM.6Effects of CIH on expressions and localization of ET-1, ET_Areceptor andET_BreceptorThe level of ET-1and ET_Areceptor mRNA showed a significant increasein the CIH group compared with the normoxia group (P <0.01), whereas ET_BmRNA was remarkably decreased in the CIH group (P <0.01).Immunoreactivity for ET-1increased both in the media and intima of thepulmonary artery after exposure to CIH. In normal rats, ET_Areceptor waslocalized in the media of the pulmonary artery with predominant distribution,and there was faint distribution in the intima of the pulmonary artery.Immunoreactivity for ET_Areceptor increased in the media of the pulmonaryartery after exposure to CIH. Also, it showed positive staining for ET_Breceptors in the intima and weak staining in the media of pulmonary artery.Immunoreactivity for ET_Breceptor decreased in the intima and media of thepulmonary artery after exposure to CIH. 7Effects of CIH on NO production and eNOS expressionThe levels of NO in pulmonary artery in CIH rats were dramaticallyreduced (P <0.05), compared with the normoxia group. The expression ofeNOS in pulmonary artery of CIH rats was also significantly reduced,compared with that of the normoxia group (P <0.05).Conclusions: Endothelium-dependent vasodilation was impaired and thevasoconstrictor responsiveness was increased by CIH. The increasedresponsiveness to ET-1induced by intermittent hypoxia in pulmonary arteriesof rats may due to both increased expression of ET_Areceptor and decreasedexpression of endothelial ET_Breceptor. |
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