| Background: Dysregulated metabolism and insulin resistance is important risk factors of pulmonary artery hypertension(PAH).Many PAH patients showed insulin resistance.Insulin modulates the function of liver and fat tissue to maintain the homeostasis of lipid and lipoproteins.Insulin resistance of PAH patients normally showed as increased triglyceride(TG)/ High-density lipoprotein related cholesterol(HDL-C)ratio and dyslipidemia,followed by increased circulating level of free fatty acid.Moreover,metabolism is a very important mechanism that participated in right heart failure.During right heart failure,as a consequence of hypoxia,the cardiomyocytes transfer to using glycolysis as a main energy source instead of fueling fatty acids.Thus,large amounts of fatty acids or their related lipid metabolites accumulate inside the cardiomyocyte,leading to heart lipid deposition and dysfunction.Among all these lipids,TG have a very important role.Apolipoprotein A5(apo A5)is a significant regulator of TG in circulation as well as several organs.Our team has showed that apo A5 could be uptake by the heart and could inhibit the obese related heart lipid deposition and dysfunction.Objective: To examine the role of apo A5 in pulmonary arterial hypertension and right heart failure and to investigate its underlying mechanism.Methods:1.Expression level of apo A5 in PAH patients and animal modelPAH patients were recruited following the inclusion and exclusion criteria.Meantime,20 healthy controls were collected.Characteristic data,liver and renal function,serum lipids and NT-pro BNP data were collected from the medical record system.Serum apo A5 level were measured by ELISA.Use MCT to induce the pulmonary hypertension(PH)animal model.Set up 2 groups for control(Ctrl group)and MCT induced PAH(MCT group).14 SD rats weighed around 200 g were randomly distributed to these 2 groups.For MCT group,rats received intraperitoneal injection of60mg/kg MCT for once.For Ctrl group,same amount saline was injected.After 21 days,RVSP and RVHI were measured.Serum apo A5 and NT-pro BNP were measured by ELISA.Association of these 2 parameters were analyzed.Serum TG and right ventricle TG were measured by enzymic method.Use oil-red staining to further investigate the level and distribution of TG inside right ventricle.Apo A5 expression level in right ventricle and liver were measured by western blot.Liver RNA were extracted and genes that modulate the expression of apo A5 were measured to investigate the related mechanisms.2.Overexpression of apo A5 alleviates MCT induced PH and right heart failureRats met body weight and health standards were divided into three groups by a random number table:(a)Control(Ctrl)group(n = 9): rats received intraperitoneal(ip)injection of physiological saline for parallel control of other groups;(b)MCT + green fluorescent protein(GFP)group(n = 9): rats received ip injection of monocrotaline(MCT;60mg/kg)and subsequent inject control GFP adenovirus trough tail vein at Day 7 and Day 14.(c)MCT + apo A5 group(n = 9): rats received ip injection of MCT(60 mg/kg)and a subsequent inject apo A5 adenovirus trough tail vein at Day 7 and Day 14.Treatments in all groups were lasted for 4 weeks to reach the maximum effect of PH model establishing.At day 28,echocardiography was used to measure the heart function of each group.The following day,RVSP and RVHI were measured.Serum apo A5 were measured by ELISA.TG were measured by enzymic method.Use oil-red staining to further investigate the level and distribution of TG inside right ventricle.Apo A5 and NT-pro BNP level in right ventricle were measured by western blot and RT-PCR.Pathology and fibrosis of the ventricle were showed by HE,masson and sirus red staining.Primary heart fibroblasts were isolated using trypsin and purified by different adherent time.After GFP or apo A5 were overexpressed,TGFβ1was added to induce fibrosis.CCK8,Ed U incorporation assay and cell scratching assay were used to detect the proliferation,migration.Western blot and immunofluorescence(IF)staining were used to measure protein level of cell proliferation and fibrosis.3.Overexpression of apo A5 alleviates MCT induced PH pulmonary vascular remodeling and its underlying mechanismUsing the lung tissue from each group for HE,immunohistochemistry(IHC)as well as IF to measure the remodeling of lung vasculature and apo A5 expression.Using tissue plant method to isolate rat PASMCs.After overexpress GFP and apo A5,PDGF-BB were used for inducing PASMC proliferation.CCK8 and Ed U incorporation assay were used to detect the proliferation of PASMCs.Coimmunoprecipitation-mass spectrum(co IP-MS)was used to determine the function of proteins that combine and react with apo A5,to evaluate the cellular mechanisms.After that,western blot was used to verify the findings.Later,Scramble si RNA and GRP78 si RNA were used transfect into the PASMCs to knockdown GRP78 expression.Then,CCK8,Ed U incorporation assay and western blot were demonstrate again to examine the blocking effects.Results: 1.Expression level of apo A5 in PAH patients and animal model(1)Compared with healthy control,PAH patients showed lower serum apo A5(102878±5978 vs 89136±2649 pg/ml)(P<0.01)and there was no difference in serum TG.Serum apo A5 level in PAH patients have no association with TG but have negative association with NT-pro BNP(R=-0.360,P<0.01).(2)Compared with Ctrl group,MCT group showed a lower serum apo A5 level(105.20±12.48 vs 72.67±7.16 ng/ml)(P<0.01)and there was no difference in serum TG.There was no significance between serum apo A5 and NT-pro BNP(R=-0.495,P>0.05).(3)Western blot showed a lower apo A5(P<0.05)and no difference of TG in right ventricle of MCT group.(4)Westernblot and RT-PCR indicates decreased apo A5 synthesis and secretion from liver.Genes modulating apo A5 expression in liver such as CREBH and SREBP1 c were decreased about to 50% and 40% of Ctrl group respectively(all P<0.05).2.Overexpression of apo A5 alleviates MCT induced PH and right heart failure(1)Level of apo A5 showed increase in the serum and right ventricle of MCT+apo A5 group(all P<0.05).(2)MCT+GFP group showed a sharp increase of RVSP and RVHI while MCT+apo A5 group were lower than that(RVSP 62.33±3.16 vs46.14±1.47 mm Hg;RVHI 0.58±0.05 vs 0.38±0.02)(all P<0.05).(3)MCT+GFP group had an increase of RV size and echocardiography showed worse TAPSE and PAAT.This group also have a higher m RNA expression of NT-pro BNP in RV.Compared with MCT+GFP group,MCT+apo A5 group showed decreased RV size,improved TAPSE.The expression level of NT-pro BNP decreased to 1/3of MCT+GFP group(all P<0.05).(4)MCT+GFP group have a higher RV fibrosis while apo A5 overexpression helps to prevent it(P<0.05).(5)TGFβ1 induces proliferation and migration of primary cardiac fibroblast while apo A5 overexpression could inhibit this effect(P<0.05).(6)TGFβ1 induces expression α-SMA,synthesis and secretion of collagen of primary cardiac fibroblast and apo A5 overexpression could ameliorate these effects.3.Overexpression of apo A5 alleviates MCT induced PH pulmonary vascular remodeling and its underlying mechanism(1)IF staining indicates apo A5 is mainly uptake by pulmonary smooth muscle cell and western blots of PASMC showed decreased expression of apo A5 and its receptor(all P<0.05).(2)MCT+GFP group showed a higher medial wall thickness and muscularization and MCT+apo A5 group showed improvement of that(medial wall thickness 55.95±1.09 vs 41.97±1.24 %;fully muscularization vessel percentage 27.45±4.42 vs 14.90±2.08 %)(all P<0.05).(3)Overexpression of apo A5 could inhibit PDGF-BB induced PASMC proliferation(all P<0.05).(4)GO analysis showed that protein that combine to and interact with apo A5 were enriched to protein folding and protein stabilization pathway.(5)Overexpression of apo A5 induce expression of GRP78,and inhibit the activation of ER stress related protein PERK,IRE1α and ATF6 as well as PASMC proliferation that leading by PDGF-BB.(6)After knocking down GRP78,the ER stress and PASMC proliferation inhibiting effects of apo A5 were blocked.Conclusion:1.Overexpression of apo A5 alleviates MCT induced PH and right heart failure.2.Overexpression of apo A5 alleviates MCT induced PH pulmonary vascular remodeling and PDGF-BB induced PASMC proliferation by inhibiting ER stress. |
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