Protective Role And Mechanisms Of ALDH2(Aldehyde Dehydrogenase2) On Hypoxia-Induced Pulmonary Hypertension

BackgroundPulmonary hypertension's diagnostic standard is that the average pressure of pulmonary artery measured by right cardiac catheter is>25mmHg at sea level.Clinically,pulmonary hypertension is often divided into five categories:arterial pulmonary hypertension;pulmonary hypertension caused by left heart disease;pulmonary hypertension caused by hypoxia or pulmonary disease;chronic thromboembolic pulmonary hypertension;pulmonary hypertension with unknown mechanism.The symptoms of patients with pulmonary hypertension are non-specific,and the early symptoms are not obvious.However,with the progress of the disease and the progressive increase of pulmonary circulation resistance,they may have dyspnea,shortness of breath after activity,fatigue,syncope,angina or chest pain.In the later stage,they may have related symptoms of right heart failure.The global incidence of pulmonary hypertension is relatively low,but Chinese is a large population.Even though the prevalence of PH is still not accurate,PH patients are not rare.The diagnosis and classification of pulmonary hypertension is little complicated.The most common types of pulmonary hypertension are caused by left heart disease and pulmonary disease or hypoxia.Pulmonary hypertension is difficult to treat and has a poor prognosis.Therefore,the field of pulmonary hypertension is still worthy of further study.There are many smokers in our country.In addition,in recent years,the air pollution is serious,and more and more patients suffer from lung diseases Hypoxia-induced pulmonary hypertension(HPH)is the third kind of pulmonary hypertension in clinic.The etiology include chronic obstructive pulmonary disease,interstitial pulmonary disease,bronchiectasis and so on.Chronic alveolar hypoxia can lead to pulmonary vasoconstriction and remodeling,which leads to the increase of pulmonary vascular resistance and pulmonary artery pressure.If things continue this way,it will gradually develop into pulmonary heart disease and right heart failure,So the difficulty of treatment and poor prognosis is undisputed.Many studies have shown that chronic hypoxia can induce vasoconstriction,proliferation of smooth muscle cells,and muscularization of anterior arterioles of capillaries,which are the pathophysiological mechanisms of HPH.HIF-1? plays an important role in the development of HPH.HIF-1 is a nuclear protein with transcription activity,and HIF-la is the active subunit of HIF-1.Under the condition of constant oxygen,the special residues of HIF-1?,prolinyl and aspartyl,will be hydroxylated,resulting in their inactivation and degradation,while under the condition of low oxygen,the degradation will be inhibited,resulting in the increase of HIF-1? level,which can up regulate the expression of genes.Some studies suggested that HIF-la can promote the proliferation of PASMCs and aggravate the pulmonary hypertension induced by hypoxia.Hypoxia can lead to producing a large number of reactive oxygen species(ROS)in mitochondria,then ROS reacts with phospholipids,enzymes and polyunsaturated fatty acids,nucleic acids and other macromolecules related to membrane receptors,which promote the oxidation of lipids,especially polyunsaturated fatty acids.This process is called lipid peroxidation.Lipid peroxidation can produce many oxidation products,such as malonaldehyde(MDA)and 4-hydroxynonenal(4-HNE),which interact with nucleic acids and proteins in cells and form adducts,resulting in DNA damage and proteins inactivation.A study in 2016 found that 4-HNE can promote the proliferation of smooth muscle cells,however,whether toxic aldehydes can play a role in HPH has not been exploredAldehyde dehydrogenase 2(ALDH2)is a kind of aldehydes,which catalyzes the oxidation of acetaldehyde to acetic acid,and is an important anti-alcoholic enzyme.It is mainly located in mitochondria and can metabolize 4-hydroxynonenaldehyde,malondialdehyde and other toxic aldehydes.There are many studies on ALDH2.Some studies have confirmed that ALDH2 can play a role in myocardial protection through scavenging toxic aldehydes in ischemia-reperfusion injury.Some studies have also confirmed the protective effect of ALDH2 on stroke.In the field of pulmonary hypertension,it has been confirmed that activation of ALDH2 can alleviate monocrotaline induced pulmonary hypertension in rats,but the effect and potential mechanism of ALDH2 on hypoxia induced pulmonary hypertension have not been explored.Objectives1.To explore the changes of lipid peroxidation in hypoxia induced pulmonary hypertension model.2.To explore the effect of LPO products on hypoxia induced pulmonary hypertension.3.To explore the effect of ALDH2 on pulmonary hypertension induced by hypoxia4.To explore whether ALDH2 affects the proliferation of smooth muscle cells and the underlying molecular mechanisms.Methods1.Establishment of experimental animal model(1)Acquisition of genetically engineered mice:ALDH2 knockout mice(ALDH2-/-)and ALDH2 transgenic overexpression mice(ALDH2-Tg)were obtained by purchase or donation,and were bred in the laboratory animal room of our team.(2)The model of pulmonary hypertension induced by hypoxia was established in mice,and the adult male mice about 10 weeks old were selected to make the model,and the model group was fed in hypoxia tank(10%O2)for 4 weeks.The control group was fed in air condition for 4 weeks.2.Establishment and experiment of cell modelHuman pulmonary artery smooth muscle cells(HPASMCs)were cultured in the special medium.Alda-1,Daidzin or adenovirus overexpressing ALDH2 were used in the experiment and HPASMCs were cultured for 0,24,48 and 72 hours under the condition of normal oxygen(21%O2)or low oxygen(2%O2)respectively.3.To explore whether chronic hypoxia can increase the production of 4-HNE and other lipid peroxidation productsWe randomly divided the mice into two groups:?C57BL/6J mice+normoxia for 4 weeks;?C57BL/6J mice+hypoxia for 4 weeks.At the end of the experiment,the indexes of pulmonary hypertension were detected and a-SMA immunohistochemical staining was performed.The levels of 4-HNE and 8-iso-PGF2a in plasma,MDA in plasma and tissues,4-HNE and three lipoxygenase(LO)in mouse tissues were detected by ELISA,TBA,Western blot,respectively.The level of ROS was detected by DHE staining.4.To explore the changes of enzymes that metabolize toxic aldehydes in HPHWe sent the lung tissue of the model mice to the company for transcriptome sequencing,and drew the thermogram of the enzymes metabolising aldehydes with statistical significance,and verified the change of ALDH2 by q-PCR and Western blot methods,and carried out the double staining of the lung tissue immunofluorescence(a-SMA and ALDH2).5.To explore the effect of ALDH2 overexpression on HPHWe divided the mice into four groups:?ALDH2-Wt mice+normoxia for 4 weeks;?DALDH2-Wt mice+hypoxia for 4 weeks;?ALDH2-Tg mice+normoxia for 4 weeks;?ALDH2-Tg mice+hypoxia for 4 weeks.In order to explore the effect of activating ALDH2 on hypoxia induced pulmonary hypertension,we divided the mice into four groups:?C57BL/6J mice+intraperitoneal DMSO(4 weeks of normoxia);?C57BL/6J mice+intraperitoneal DMSO(4 weeks of hypoxia);?C57BL/6J mice+ intraperitoneal Alda-1(4 weeks of normoxia);?C57BL/6J mice+intraperitoneal Alda-1(4 weeks of hypoxia).At the end of the experiment,the related indexes of pulmonary hypertension were detected and ?-SMA immunohistochemical staining was performed.Western blot was used to detect the levels of 4-HNE,PCNA and HIF-la in the lung tissues of mice.6.To explore the effect of ALDH2 Knockout on HPHWe divided the mice into 4 groups:?ALDH2+/+mice+ normoxia for 4 weeks;?ALDH2-/-mice+normoxia for 4 weeks;?ALDH2+/+mice+hypoxia for 4 weeks;?ALDH2-/-mice+ hypoxia for 4 weeks.At the end of the experiment,the indexes of pulmonary hypertension were detected and ?-SMA immunohistochemical staining was performed.Western blot was used to detect the levels of 4-HNE,PCNA and HIF-la in the lung of mice.7.To explore whether ALDH2 affects HPH through endothelial cells or smooth muscle cellsWe divided the mice into four groups:?C57BL/6J mice+AAV1-ALDH2 virus;?C57BL/6J mice+AAV1-GFP virus;?C57BL/6J mice+AAV2-ALDH2 virus;?C57BL/6J mice+AAV2-GFP virus.After the establishment of HPH model,the indexes of pulmonary hypertension were detected and a-SMA immunohistochemical staining was performed.8.To explore whether NAC or exogenous 4-HNE can affect the severity of HPHIn order to explore whether 4-HNE is the key factor to promote hypoxia induced pulmonary hypertension,we divided the mice into two groups.The administration way was micro-pumping,and the dose was 0.19mg/kg/h,which is the maximum dose that micro-pumping could achieve within the solubility range of 4-HNE:?C57BL/6J mice+vehicle;?C57BL/6J mice+4-HNE.NAC is an antioxidant,which can clear toxic aldehydes.In order to explore whether NAC can reduce pulmonary hypertension induced by hypoxia,we divided the mice into five groups:?C57BL/6J mice+vehicle(normal saline);?C57BL/6J mice+NAC;?ALDH2+/+mice+vehicle(normal saline);?ALDH2-/-mice+ vehicle(normal saline);?ALDH2-/-mice+NAC.The HPH model was established in mice.After the experiment,the related indexes of pulmonary hypertension were detected.The levels of 4-HNE and HIF-la were detected by Western blot.9.To explore whether ALDH2 affects the proliferation of HPASMCsTo investigate whether the activation or inhibition of ALDH2 can affect the proliferation of HPASMCs under normoxia or hypoxia,the cells were divided into six groups:?DMSO+normoxia;?Alda-1+normoxia;?Daidzin+ normoxia;?DMSO+hypoxia;?Alda-1+hypoxia;?Daidzin+hypoxia.In order to investigate whether the over expression of ALDH2 affects the proliferation of HPASMCs,the cells were divided into four groups:?ALDH2 adenovirus+normoxia;?GFP adenovirus+normoxia;?ALDH2 adenovirus+hypoxia;?GFP adenovirus+hypoxia.In order to investigate whether 4-HNE can stimulate the proliferation of HPASMCs,we gave cell concentration-gradient stimulation(0?M,0.1?mM,1?M,10?M),and added Alda-1 or Daidzin to the cells in advance after determining the intermediate concentration of 1?M.CCK8 and Edu were used to detect cell proliferation.The wound healing plug-in was used for scratch test to detect the migration of HPASMCs.The changes of 4-HNE and ALDH2 were detected by Western blot.10.To explore the effect and mechanism of ALDH2 on mitochondrial fissionTo investigate the effects of hypoxia or 4-HNE stimulation on mitochondria of HPASMCs,cells were divided into four groups:?normoxia group;?hypoxia group;?DMSO group;.4-HNE group.In order to investigate whether mdivi-1,an inhibitor of Drp1,affects mitochondrion division and cell proliferation induced by hypoxia or 4-HNE,the cells were divided into the following six groups:?normoxia group;?hypoxia group;?hypoxia+mdivi-1 group;?DMSO group;?4-HNE group;?4-HNE+mdivi-1 group.In order to investigate whether Alda-1 can alleviate the mitosis induced by hypoxia or 4-HNE,cells were divided into four groups:?hypoxia+DMSO group;?hypoxia+4-HNE group;?4-HNE group;?4-HNE+Alda-1 group.To detect the morphology of mitochondria,cells need to be planted on 24 well cell slides.After being stained with mitotracker,they can be observed for confocal photography.We stimulated HPASMCs with hypoxia or 4-HNE,and set up time gradient(0 hour,0.5 hour,1 hour,6 hour,12 hour).Western blot was used to detect the expression levels of OPA1,MFN1,MFN2,FIS1,Drp1,Drpl ser616,Drpl ser637,HIF-la.Results1.Chronic hypoxia can increase the production of 4-HNEThe levels of 4-HNE and MDA in plasma,4-HNE in lung tissue and 8-iso-PGF2a in plasma were increased in HPH model mice.However,the levels of 4-HNE and MDA in the heart,liver and arterial tissues of mice did not change.DHE staining showed that ROS level in lung tissue of HPH model mice increased.The expression of three lipoxygenases also increased under hypoxia.2.Down regulation of ALDH2 expression in lung tissue of HPH model miceWe selected five kinds of enzymes related to acetaldehyde metabolism(aldehyde dehydrogenase,alcohol dehydrogenase,aldehyde reductase,aldehyde-ketone reductase,glutathione-S-transferase)from the sequencing results,and selected statistically significant results for heat map.The results showed that compared with the normoxia group,hypoxia reduced the expression of 13 genes including ALDH2 and increased the expression of 9 genes including ALDH1A1.Western blot and q-PCR confirmed that hypoxia did reduce the expression of ALDH2.Immunofluorescence also confirmed it.3.Over expression of ALDH2 can attenuate HPHCompared with the control mice,RVSP,RVH and the ratio of muscularized vessels decreased in the mice with over-expression of ALDH2 under hypoxia condition,but there was no change in the two kinds of mice under normoxia condition.Compared with the control group,the severity of HPH in mice injected with Alda-1 also decreased significantly.Western blot showed that hypoxia increased the levels of 4-HNE,PCNA and HIF-la,while over-expression of ALDH2 decreased the levels of 4-HNE,PCNA and HIF-la4.ALDH2 knockout can aggravate HPHCompared with the control mice,RVSP,RVH and the ratio of muscularized vessels in ALDH2 knockout mice increased under hypoxia condition,while the indexes of the two groups did not change under normoxia condition.Western blot showed that hypoxia increased the levels of 4-HNE,PCNA and HIF-1?,while ALDH2 knockout further increased the levels of 4-HNE,PCNA and HIF-la.5.ALDH2 affects HPH through smooth muscle cells rather than endothelial cellsCompared with AAV2-GFP mice,the indexes of pulmonary hypertension(RVSP,RVH,ratio of muscularized vessels)in AAV2-ALDH2 mice decreased.Compared with AAV1-GFP mice,the indexes of pulmonary hypertension in AAV1-ALDH2 mice did not change.6.NAC can attenuate HPH in ALDH2 knockout miceCompared with the control mice,the indexes of pulmonary hypertension(RVSP,RVH,ratio of muscularized vessels)in NAC mice were decreased.Meanwhile,the levels of 4-HNE and HIF-1? in lung tissues were also decreased.Furthermore,the severity of pulmonary hypertension in ALDH2 knockout mice are also decreased after gavage of NAC.7.Activation of ALDH2 can reduce the proliferation of HPASMCs induced by hypoxia or 4-HNEThe results of CCK8 and Edu showed that activation or inhibition of ALDH2 did not change the proliferation of HPASMCs under normoxia condition,while activation of ALDH2 could reduce the proliferation of HPASMCs under hypoxia condition,and inhibition of ALDH2 could accelerate the proliferation of HPASMCs.4-HNE can stimulate the proliferation of HPASMCs in a concentration dependent manner,and the activation or inhibition of ALDH2 can also interfere with the proliferation of HPASMCs induced by 4-HNE.Similarly,the over-expression of ALDH2 after adenovirus transfection can reduce the proliferation of HPASMCs induced by hypoxia.Scratch test results showed that activation of ALDH2 could reduce the migration of HPASMCs induced by hypoxia.Western blot showed that hypoxia decreased the expression of ALDH2 and increased the level of 4-HNE in a time-dependent manner.8.ALDH2 can regulate mitochondrial fission induced by hypoxia or 4-HNEThe confocal results showed that hypoxia or 4-HNE could induce mitochondrial fission,but the inhibition of Drpl by adding Mdivi-1 could inhibit mitochondrial fission,and Alda-1 could also alleviate mitochondrial fission induced by hypoxia or 4-HNE.The results of CCK8 showed that Mdivi-1 could reduce the cell proliferation induced by hypoxia or 4-HNE.Western blot showed that when HPASMCs cells were stimulated with time gradient hypoxia or 4-HNE,the protein levels of Drp1,Drp1 ser616 and HIF-1? increased,while the protein levels of OPA1,MFN1,MFN2,FIS1 and Drp1ser637 did not change.However,activation of ALDH2 can alleviate hypoxia or 4-HNE induced increase of HIF-1? and phosphorylation of Drp1 ser616.Conclusions1.Hypoxia can increase the level of lipid peroxidation,and the increased lipid peroxidation can promote the development of HPH.2.ALDH2 can attenuate the severity of HPH.3.ALDH2 regulates mitochondrial fission and smooth muscle cell proliferation through 4-HNE/HIF-1?/Drp1 signaling pathway.