Effect Of ABCA1 On Hypoxia-induced Pulmonary Arterial Hypertension And The Underlying Mechanisms

BackgroundHypoxia pulmonary arterial hypertension(HPAH)is a subclass of pulmonary arterial hypertension(PAH)and is characterized by the abnormal proliferation of pulmonary arterial smooth muscle cells(PASMCs)of the distal pulmonary vasculature,and resulting the vascular stenosis.So,the abnormal proliferation of PASMCs is a key point of the development of HPAH.This paper will use the PASMCs as the research object.ATP-binding cassette transporterAl(ABCA1)is a member of ATP-binding cassette transporter superfamily.It could transport cholesterol and phospholipids from the plasma membrane of peripheral cells to lipid-free or lipid-poor apolipoprotein,and reduce the accumulation of lipid on the vascular wall,suppress the formation of foam cells and the vascular inflammatory response.Moreover,ABCA1 play an important role in cardiovascular disease.Recent years,the disorders of lipids metabolism in the progression of PAH and pulmonary vascular remodeling attracted more attention.ABCA1 could regulate the production of serum high density lipoprotein(HDL).Whereas HDL was reported to regulate the proliferation and migration of PASMCs.So,we speculate that ABCA1 may play an important effect on the progression of PAH.It was reported microRNA(miRNA)was dysregulated expression under the condition of hypoxia.These miRNAs could regulate the PASMCs proliferation,apoptosis,migration and were involved in the process of PAH.Previous study reported that miR-361-5p was up-regulated in the blood of PAH patient.The software of microRNA.org-Targets and Expression predicated that ABCA1 was a potential target of miR-361-5p.But,the exact effect of miR-361-5p on the progression of PAH and the underlying mechanism still remain unknown.ObjectiveThe aim of this study was to analyze the effect of ABCA1 on the hypoxia induced PASMCs proliferation,migration and the expression of inflammmatory factors.Furthermore,the effect of miR-361-5p on the progression of PAH and the underlying mechanism will be investigated.MethodsIn order to simulate the HPAH in vitro,hPASMCs was cultured under the hypoxia condition(37?,5%CO2,93%N2)or normoxia condition(37?,5%CO2,95%O2).Then the experiments was as follows:(1)In order to analyze the effect of ABCA1 on hypoxia induced hPASMCs,ABCA1 expression under hypoxia was first examined using Real-time PCR and Western blot.Then,pcDNA-ABCA1 or sh-ABCA1 and their relative control was transfected into cells according to the instructions of Lipofectamine 3000 and cultured under hypoxia condition.The groups were as follows:normoxia,hypoxia,hypoxia+pcDNA-ABCA1,hypoxia+pcDNA-vector;hypoxia+sh-ABCA1,hypoxia+shRNA-control.(2)In order to analyze the effect of miR-361-5p on hypoxia induced PASMCs,anti-miR-361-5p and miR-361-5p mimic and their relative control was transfected into cells.The groups were as follows:normoxia,hypoxia,hypoxia+anti-miR-361-5p,hypoxia+anti-NC,hypoxia+ miR-361-5p mimic,hypoxia+mimic control.After all the aforementioned treatments,the expression of ABCA1 in each group was measured by Real-time PCR and Western blot.The cell proliferation was measured by CCK-8 method.The cell migration was measured by Transwell method.The expression of IL-6,IL-1?,TNF-?,p-JAK2 and p-STAT3 was measured by Western blot.Hypoxia induced PAH rat model was used for the in vivo study.Ad-ABCA1 and antagomiR-361-5 with their matched negative controls(Ad-control or antagomiR-NC)were injected into the rat by tail vain injection,and then were exposed to hypoxia to induce PAH.The groups were designed as follows:(1)pAd-ABCA1 were injected into the rat by tail vain injection,the groups were:Control,PAH,PAH+Ad-control,PAH+Ad-ABCA1.(2)antagomiR-361-5p and negative control were injected into PAH rat by tail vain injection,the groups were:control,PAH,PAH+antagomiR-361-5p,PAH+antagomiR-NC,PAH+antagomiR-361-5p+Ad-ABCA1.The pulmonary hemodynamics,right heart hypertrophy index were measured.ABCA1 and miR-361-5p levels were determined and their association with pulmonary hemodynamics,pulmonary smooth muscle hypertrophy,inflammation level and p-JAK2,p-STAT3 expression were evaluated.Results(1)Compared with normoxia group,the expression of ABCA1 was significantly decreased with a time dependent manner after cells were treated with hypoxia for 24,48 and 72 h.(2)hPASMCs were transfected with pcDNA-ABCA1 and exposed to hypoxia for 24,48 and 72 h,cell proliferation was dramatically decreased compared to pcDNA-vector group.After 24 h of transfection,cell migration and production of inflammatory mediators(IL-6,IL-1? and TNF-?)were significantly suppressed.While,the transfection of sh-ABCA1 exhibited adverse effects on cell proliferation,migration and inflammation against pcDNA-ABCA1 group.(3)After hypoxia induced PASMCs were transfected with pcDNA-ABCAl for 48 h,the expression of IL-6,IL-1?,TNF-a,p-JAK2 and p-STAT3 was noticeably decreased.Whereas sh-ABCA1 showed the opposite effect.(4)Compared with pcDNA-ABCA1 group,AG490+pcDNA-ABCA1 could significantly downregulated the expression of p-JAK2 and p-STAT3.Compared with sh-ABCA1 group,AG490+ABCA1-shRNA could reverse the effect of sh-ABCA1 on p-JAK2 and p-STAT3.(5)Compared with normoxia group,expression level of miR-361-5p was significantly increased after cells were treated with hypoxia for 24,48 and 72 h.The result of luciferase activity assay showed that miR-361-5p could target regulate the expression of ABCA1.In addition,after hypoxia induced cells were transfected with antimiR-361-5p,the cell proliferation and migration was suppressed,and the expression of IL-6,IL-1?,TNF-?,p-JAK2 and p-STAT3 was decreased.pcDNA-ABCA1 could enhanced anti-miR-361-5p effect.However,miR-361-5p mimic showed the opposite effect,pcDNA-ABCA1 could reverse its effect.(6)After PAH rats were injected with pAd-ABCAl,the mean pulmonary arterial pressure,right ventricular systolic pressure,right ventricular hypertrophy index was dramatically decreased;the pulmonary artery hypertrophy was decreased;the expression of IL-6,IL-1?,TNF-?,p-JAK2 and p-STAT3 was decreased.(7)After PAH rats were injected with antagomiR-361-5p,the mean pulmonary arterial pressure,right ventricular systolic pressure,right ventricular hypertrophy index was dramatically decreased;the proliferation of pulmonary artery smooth muscle was decreased;the expression of IL-6,IL-1?,TNF-?,p-JAK2 and p-STAT3 was decreased.Ad-ABCA1 could enhanced antagomiR-361-5p effect.ConclusionThe present study showed that ABCA1 was down-regulated in hypoxia induced hPASMCs and PAH rat models.Overexpression of ABCA1 inhibited hypoxia induced PASCMs proliferation,migration and the expression of inflammatory factor through regulating JAK2/STAT3 signaling pathway.In addition,ABCA1 was the direct target of miR-36 1-5p.Inhibition of miR-36 1-5p attenuated the progression of PAH through increasing ABCA1 expression.