Effects Of Hypoxia/Selenium Deficiency On Expressions Of Endothelev-1and Its Receptors In The Lungs Of Broiler Chickens

Pulmonary hypertension (PH) followed by pulmonary vascular remolding (PVR) and ascites (As) has been recognized as a serious cause of economic loss in commercial broiler production. Now it is believed that hypoxic PH is the central part of pathological mechanisms in broilers As. Various factors leading to hypoxia make the body depleted of oxygen with increased cardiac output and enhanced heart and lung functioning result into PH. Right ventricle first undergoes functional compensatory mechanism by induced PH with the involvement of cardiopulmonary system function by sustained PH. The continuous development of PH leads to right heart failure and structural remodeling, proliferation, migration of vascular smooth muscle cells, and deposition of vascular matrix thus eventually resulting into the occurrence of As.Endothelin-1(ET-1), a21-amino-acid isopeptide generated by/the vascular endothelium and characterized by sustained and potent vasoconstrictor action, acts through specific receptors termed ETA and ETB. ETA receptors are represented only on smooth muscle cells and mediate contractions and promote growth. In contrast, ETB receptors are located both on smooth muscle cells to evoke contractions and on endothelial cells to induce relaxation by production of nitric oxide (NO). ET-1, a potent vasoconstrictor, stimulates pulmonary artery smooth muscle cell proliferation. Mammalian models and clinical data have demonstrated the expressions of ET-1and its receptor abnormalities closely associated with the development of PH and PVR. Studies have shown that, plasma levels of ET-1were significantly increased in PH mouse, while its ET-1expression in lung tissue was also increased. Foreign clinical data showed that an ET-1level was increased in pulmonary circulation of patients with PH, and an increase in ET-1degree exerts positive correlation with PH. These observations suggest that ET-1and its receptor play an important role in the pathogenesis of PH.However, the above study was mainly focused on organ level, and about ET-1and its receptor dynamic expression in the process of broiler PH and PVR rarely reported. Based on the above background, we tried to explore the patterns of ET-1and its receptor gene expression in the lungs, pulmonary vascular cells (pulmonary artery smooth muscle cells and lung microvascular endothelial cells) of broiler chickens under hypoxic/Se-deficient conditions from the animal level, cellular, molecular and protein levels. 1. Effect of High Altitude Hypoxia on PH and Expressions of Endothelin-1and Its Receptors in the Lungs of Broiler ChickensTo investigate the influence of exposure to high altitude (HA) hypoxia on the expressions of endothelin-1(ET-1), ET type A (ETA) and B (ETB) receptors in broiler chickens, qRT-PCR and Western blot studies were performed in the lungs. Six hundred1-day-old male broiler chickens were randomly divided into two groups:group A, birds maintained under rich oxygen conditions (the oxygen content is21%); and group B, birds exposed to HA hypoxia (the oxygen content is17%). Our data showed that exposure to altitude elevated ET-1(P<0.05, P<0.05) and ETA (P<0.01, P<0.01) gene expressions at21and28days of age when compared with the rich oxygen group. Meanwhile, a marked increase in ETB mRNA expression was observed at21days of age in the course of HA, then ETB mRNA expression was decreased at28days of age, although there were no significant changes (P>0.05) at7and14days of age. The increased response was accompanied by adverse effects on weekly body weight gain (P<0.05) and ascites mortality (P<0.05). These observations suggested that ET-1, ETA and ETB genes are normally expressed in the lungs of birds. It is probably that increased levels of ET-1and ETA and decreased ETB gene expression in the lungs are involved in the lung dysfunction of broiler chickens with developmental As.2.Cultivation and characterization of pulmonary artery smooth muscle cells from chick embryosIn this study, tissue culture method was used to cultivate pulmonary artery smooth muscle cells (PASMCs) from19-day embryonated eggs. The cultured cells were identified by cellular morphology and immunocytochemistry. Results revealed that a few cells migrated out of tissue pieces at48h of cultured, and underwented extensive proliferation after72h. The cultured cells exhibited typical morphology of vascular smooth cells under inverted microscope, and significantly, these cultured cells stained positive for a-SMA. The protocol provides a relative simple, reliable method of isolating PASMCs in vitro culture conditions, which could be used for study in avian cadiovascular disease such as pulmonary hypertension, ascites syndrome and pulmonary vascular structural remodelling. 3. Cultivation and characterization of pulmonary microvascular endothelial cells from chick embryosA method of cultivating avian pulmonary microvascular endothelial cells (PMVECs) from chick embryos is described. In the present study, cultivation of cells was based on tissue culture method modified from the techniques previously used for mammalian cells. Cells isolated from chick embryos were identified by cellular morphology and immunocytochemistry. Our findings showed that the cultured cells exhibited a typical cobblestone morphology viewed under an inverted microscope, and they bound with Bandeiraea simplicifolia lectin and stained positive for CD31and factor Ⅶ-related antigen. These observations suggested that the isolated cells manifested typical endothelial cells (ECs) characteristics. The protocol for the isolation and cultivation of PMVECs may allow more detailed analysis of their biological properties, and provide a valuable model for studying pathological processes including pulmonary hypertension (PH), ascites and pulmonary vascular remodeling in broiler chickens.4. Effects of Hypoxia on Expressions of ET-1、ETA and ETB in Avian PASMCsAbnormal proliferation of PASMCs is pathological basis for PH, As and PVR, furthermore, ET-1has a strong ability to induce proliferation of smooth muscle cells. In the current study, expressions of ET-1and its receptors mRNA and protein in hypoxia-treated PASMCs of broiler chicken was detected by qRT-PCR and Western blot. Compared with the control group, our results showed that the expressions of ET-1mRNA and protein in PASMCs which hypoxia for24h and48h were confirmed to increase (P<0.05, P<0.01) by qRT-PCR and Western blot. At12h,24h and48h after hypoxia, expression levels of ETA mRNA and protein were significantly up-regulated (P<0.05, P<0.01, P<0.05) in PASMCs. As for ETB, our data showed that expression levels of ETB mRNA and protein were noticeably increased (P<0.05, P<0.01) in PASMCs after6h and12h hypoxia, however, at48h after hypoxia, ETB gene expression levels were significantly decreased (P<0.05) in PASMCs. These findings suggested that the increased expressions of ET-1and ETA and decreased of ETB in hypoxia-treated PASMCs might play an important role in PH、As and PVR of broiler chickens. 5. Effects of Salidroside on Expressions of ET-1、ETA and ETB in Hypoxia-treated PASMCsPH is a severe condition characterized by vascular proliferation and remodeling. Emerging evidence has shown the association and/or causation between the expression level of ET-1and the progression of PVR in animals and humans models. Salidroside (SDS), a traditional Tibetan medicine, was known as the main active ingredient in the root of Rhodiola rosea L.(Crassulaceae). It has been demonstrated to have various pharmacological properties including anti-hypoxia, anti-fatigue, enhancing work performance, and is especially famous in the treatment of mountain mal-hypoxia in Tibet. However, the effects of SDS on expressions of ET-1and its receptors in chicken embryonic PASMCs exposed to hypoxic conditions is still unclear. The present study was therefore designed to evaluate the cardioprotective role of SDS and the underlying mechanisms in hypoxia-treated PVR. Chicken embryonic PASMCs pretreated with and without SDS for1h were then exposed to hypoxic condition for24h. The expressions of ET-1and its receptors were examined by qRT-PCR and Western blot. Compared with the hypoxia-treated group, our findings showed that among the various concentrations of SDS pretreatment, the levels of ET-1gene expression were markedly decreased (P<0.01, P<0.05) in both the moderate dose group (80μg/ml) and the high dose group (120μg/ml) respectively, but no obvious decrease was found in the low dose group (40μg/ml). Meanwhile, pretreatment with SDS (40,80,120μg/ml) was found to be effective in reducing the expression levels of ETA(P<0.01, P<0.01, P<0.05, respectively) in PASMCs exposed to hypoxic conditions in vitro. Moreover, pretreatment with SDS markedly attenuated (P<0.01, P<0.01) ETB mRNA expression levels in both the low dose group (40μg/ml) and the moderate dose group (80μg/ml) respectively, however, there was no obvious decrease in high dose group (120μg/ml). Results obtained in this study indicate that the effect of SDS anti-altitude may be achieved by reducing abnormally high expressions of PASMCs ET-1, ETA and ETB under hypoxic conditions.6. Effects of Selenium Supplementation on Expressions of Endothelin-1and its Receptors in Pulmonary Microvascular Endothelial Cells from Chick EmbryosThe aim of the current study was to explor the influence of supplemental selenium (Se) on expressions of ET-1、ETA and ETB in cultured chick embryos PMVECs. To accomplish this, PMVECs were treated in Se-deficient or Se-supplement (12,24,50,100ng/ml) culture medium for48h. Low Se medium was achieved by reducing serum concentrations and the essential growth factors were added. After the incubation, the effects of supplemental Se on ET-1、ETA and ETB gene expressions were assessed by qRT-PCR. Compared with the control group, the results showed that among the different concentrations of Se supplement, the levels of ET-1gene expression treated with both the moderate Se doses (24,50ng/ml, P<0.01, P<0.01, respectively) and the high doses (100ng/ml, P<0.05) were noticeably decreased (P<0.01), although there was no change in the low dose group (12ng/ml). Meanwhile, Se supplement (24,50,100ng/ml, P<0.01, P<0.05, P<0.05, respectively) was found to be effective in reducing the expression levels of ETA in chick embryos PMVECs grown in low Se medium. However, there were no significant changes (P>0.05) in ETB mRNA levels during the cell proliferation. These observations indicated that Se may play both direct and indirect role in the regulation of ET-1and its receptors gene expression and their production in avian PMVECs. Se supplement decrease in ET-1and ETA production in Se-deficient PMVECs may partly explain the mechanism of the protective effects of the Se on the cardiovascular system.