CTRP9 Relieves Hypoxia-induced Pulmonary Hypertension And Underlying Mechanisms

?Introduction?Pulmonary hypertension(PH)is a complex disease that causes significant morbidity and mortality.PH leads to increase of pulmonary artery pressure progressively followed by right ventricle hypertrophy,ultimately right heart failure and death.Hypoxia-induced pulmonary hypertension is one of the most common subcategories of PH in clinical,which imitates closely PH in aspects of hemodynamics,histopathology and motality.The pathophysiology of HPH is very complex and has not been well understood resulting in limited and scare treatment strategy and a worsen outcome.Most PH patients have to accept lung transplantation.Hypoxia impairs the pulmonary artery endothelium and makes endothelial dysfunction.Pulmonary artery endothelium lesion and endothelial dysfunction is indicated by the disordered expression of vasoactive molecules,growth factor,cytockines and inflammatory factors and the increase of reactive oxygen species production in pulmonary artery endothelial cells and the apoptosis of pulmonary artery endothelial cells.C1q/TNF-related protein 9(CTRP9)is a new adipokine secreted by adipose tissue and functions in lipid metabolism.CTRP9 acts as a vasodilator to systematic artery by increasing the NO production.It also play important roles in anti-inflammatory,anti-arteriosclerosis and anti-platelet aggregation induced by ADP and inhibits the endothelium impairment followed by vascular remodeling,perivascular inflammatory triggered by inflammatory factors and leukocyte adhesion.Although CTRP9 presents multiple benefits in systematic vascular,its roles in regulating pulmonary vascular endothelium dysfunction remain largely unknown.Therefore,this study was aimed to explore whether CTRP9 influences the pulmonary endothelial impairment and plays a protective role in HPH development.?Aims?(1)To clarify the impact of CTRP9 on PMVECs dysfunction resulted from hypoxia stimulator,and to further elucidate its underlying mechanisms.(2)To clarify whether overexpression of CTRP9 in lung tissue in vivo improves pulmonary microvascular endothelial dysfunction and relieves HPH.?Methods and Results?(1)Separated and cultured primary PMVECs,72 hours later,the morphologic appearance of PMVECs under invert microscope was presented like polygon,oval or paving stones shape,the cell was transparent,the outline was clear and the junction between cells was closely,which confirmed the cells as PMVECs.(2)Primary PMVECs were cultured under normoxia,hypoxia(50ml/L O2),hypoxia with CTPR9 treatment or Compound C treatment 30 min before hypoxia and CTRP9 treatment,the cell culture supernatant were harvested for detecting the level of NO by nitrate reductase method and ET-1 by ELISA,the cell lysate were used to detect the level of AMPK/p-AMPK?e NOS/p-e NOS?ERK1/2/p-ERK1/2 by western blot.The results demonstrated that CTRP9 may inhibit the decrease of hypoxia induced AMPK and e NOS phosphorylation and NO production,and the increase of hypoxia induced ERK1/2 phosphorylation and ET-1 production.The effects of CTRP9 could be interrupted by AMPK inhibitors.(3)The further study based on HPAECs investigated and verified the effects of CTRP9 on NO and ET-1 production and the mechanisms.We chose lentivirus as a vector to interrupt the gene for expressing AMPK(Lv-PRKAA1-RNAi).We showed that Lv-PRKAA1-RNAi reduced the effects of CTRP9 on NO and ET-1 production with down regulation of p-AMPK and p-e NOS level and up regulation of p-ERK1/2.(4)Primary PMVECs were cultured under normoxia,hypoxia(50ml/L O2)or hypoxia with CTPR9 treatment.m RNA of IL-6 and TNF-? in the cell lysate were determined by RT-PCR,cell culture supernatant IL-6 and TNF-? were measured by ELISA.The results demonstrated that the expressions of IL-6 and TNF-? in PMVECs increased significantly under hypoxia environment and CTRP9 treatment effectively inhibited the increase.(5)Primary PMVECs were cultured under normoxia,hypoxia(50ml/L O2)or hypoxia with CTPR9 treatment.The reactive oxygen species(ROS)and apoptosis of PMVECs were determined by flow cytometry.The results demonstrated that the production of ROS and the apoptosis ratio in PMVECs increased significantly under hypoxia environment and CTRP9 treatment effectively inhibited the increase.(6)SD rats were randomly divided into AAV-Control group(14)and AAV-CTRP9 group(14)and administrated AAV by intra-tracheal instillation.Two weeks later,each group was randomly divided into normoxic subgroup(6)and hypoxic subgroup(8),the rats in the hypoxic group were exposed to hypobaric and hypoxic condition in an auto-modulating hypobaric and hypoxic cabin(air pressure 55 k Pa,oxygen concentration 10%)for 28 days to establish the animal model of hypoxic pulmonary hypertension(HPH).Record of the survival and the death number were kept.The transfection and expression effect of AAV in lung were evaluated by fluorescence microscope.The histopathological change,the hemodynamic index and the right ventricular hypertrophy index of rats were detected.Serum CTRP9 and ET-1 were measured by ELISA while NO were measured by nitrate reductase.Two rats died in the AAV-Control and hypoxia treatment group during the process of HPH while all animals survived in other groups.In the AAV-Control and hypoxia treatment group,the right ventricular systolic pressure,the right ventricular hypertrophy index and the thickness of pulmonary arterial wall were increased compared with those in the AAV-Control and normoxia group,the serum CTRP9 and NO reduced while ET-1 enhanced;AAV-CTRP9 administration effectively inhibited those change induced by hypobaric and hypoxic condition;the mean carotid artery pressure was the same among all groups.?Conclution? In hypoxia condition,CTRP9 restores the imbanlance between e NOS/NO and ERK1/2/ET-1 and improves the pulmonary vesscular endothelial function;The anti-inflammatory,anti-oxidative stress and anti-apoptosis effects may also play important roles in protecting pulmonary vascular endothelium.Overexpression of CTRP9 by adeno-associated virus in lung significantly relieves HPH development and right ventricle hypertrophy through improving pulmonary vascular endothelial functions.These results provide direct evidence for understanding the molecular pathogenesis of HPH and using CTRP9 as a novel potential therapy to prevent and limit the development of HPH.