Effects And Mechanism Of ACE2,PLGF And SFlt-1 On Acute Lung Injury In Mice

Acute respiratory distress syndrome(ARDS)is a disease caused by acute lung injury(ALI),and the affected population in the United States can reach200000 each year.Clinical studies showed that about 60% ARDS patients were present as pulmonary fibrosis,which brings about a serious economic burden to the society.It is generally accepted that the Angiotensin(ANG)system disorder is closely related to the ARDS/ALI onset process.Its excessive activation can induce pulmonary endothelial cell apoptosis and casue the change of capillary membrane permeability in the alveolar.The Angiotensin II(ANGII)is the most important effect factor for the RAS system,which is distributed in the fluid circulatory system and several important organs,including lung tissue,and several studies confirmed the increased expression of ANGII was associated with multiple pathological events such as oxidative stress,vascular permeability changes,and inflammatory cell infiltration.High level of ANGII can be detected in both animal models and in patients with ALI disease.As another important member of the RAS system,the catalytic active region of angiotensin-converting enzyme 2(ACE2)shares the 61% homology with ACE,but its biological activities differ from the ACE.ACE can cause decompositon of ANGI and form ANGII.ANGII and its specific receptor AT1 A combine together and trigger several biological effects including vascular contraction,inflammatory response,cell proliferation and apoptosis.Furthermore ACE2 could promote the degradation of ANGII and exert the protection effect on Ang-(1-7),which confirm the important regulatory function of ACE2 in ALI.Placental growth factor(PLGF)is a glycoprotein which shares highly homologous with vascular endothelial growth factor(VEGF).It is widely expressed in many organs including lung tissue,and has the function ofpromoting endothelial cell proliferation and differentiation,boosting angiogenesis,facilitating inflammatory factor release and resisting apoptosis.Vascular endothelial growth factor receptor-1(VEGF receptor 1,VEGFR1 or Flt-1)is a key receptors of PLGF,while soluble vascular endothelial growth factor receptor 1(soluble Flt-1,sFlt-1)is the negative regulatory factor of endogenous angiogenesis pathway,which functions as a competitive inhibitor of Flt1.Previous studies have shown that the expression of VEGF in adult lung tissue is significantly higher than in the other organs,while in the lung tissue of ali/ards patients,VEGF levels can be further increased two-fold as compared with the normal group,accompanied with the release of the alveolar active components and increasing vascular permeability of the lungs,which could boost VEGF expression and induce the vicious cycle of Ali/ards pathological feature.Animal studies revealed that sFlt-1 intervention can effectively improve the pulmonary fibrosis degree and alleviate the inflammatory response,suggesting that VEGF family members may have an important regulatory role in ali/ards process.Given the regulatory role of ACE2 and PLGF/Flt-1 in the alveolar-capillary regulation process,our study focus on protective effects of ACE2 in acute lung injury models to provide a new pathway for the treatment of acute lung injury.This topic is divided into three parts,specific as follows:Part one Effects of ACE2 in BLM-induced Acute Lung Injury ModelObjective: ALI model was established to explore the protective effect of ACE2 in the course of acute lung injury.Methods: Specific pathogen free(SPF)Balb/c mice(aged 10 weeks,weight 18-22 g)were supplied by Laboratory Animal Center of Shanghai Academy of Sciences,Chinese Academy of Sciences,China.After acclimatization for 1 week,a group of mice were used as the no-injury group and received no treatments(NT).The lung injury model was induced in another groups of mice,by intraperitoneal administration of the bleomycin solution;Lung tissue function were evaluated by HE staining,blood Gasanalyzer.MDA and SOD contents in lung tissue were assessed by spectrophotometric determination;Evans Blue method were used to evaluate the capillary permeability in the lung tissue of each group.TNF-α,IL-6 and CXCL-1 protein expression were determined by Western-blot analysis.Results:1.General situationThe mice in the control group had better postoperative recovery,presenting as lively and agile behavior.The mice in ALI group were in bad mental state,manifested as sluggishness and drowsiness,with thinning of hair,which was not found in ACE2-treated groups..2.Lung pathological examination and lung injury scoreHE staining showed that BLM could induce acute lung injury in mice,the lung injury score was significant higher than that of the control group.The mortality of ALI mice were decreased after ACE2 treatment.The histology of lung in ALI+ACE2 group showed the significiant decreased congestion and cellular infiltration.3.Arterial blood gas analysis in each groupAfter BLM stimulation the pO2 values ??were(75.18±6.70)mmHg and(62.17±9.39)mmHg in mice 3 and 7 days,which were significantly lower than that in the control group(P<0.05).After treated with ACE2,the p O2 values were(84.08±4.83)mmHg and(79.11±8.46)mmHg in 3 d and 7 d.Compared with the model group,the p O2 values in ACE2 intervention group were significantly increased.Moreover,ACE2 treatment significantly increased the arterial oxygen saturation(95.62±0.93)% after 3 days intervention,but had no significant effect on blood oxygen saturation.4.Lung wet/dry weight ratio in each groupAfter BLM stimulation the wet/dry weight ratios of lungs tissue were(6.80±0.68)and(7.65±0.62)in days and 7 days,respectively,which were significantly higher than those as compared with the control group(P<0.05).After ACE2 intervention,the wet/dry weight ratio of lungs tissue was decreased to(5.81±0.58)and(6.88±0.48),respectively.5.SOD and MDA content in lung tissueThe MDA contents of lung tissue in ALI model group were(9.31±1.35)nmol/mg prot and(13.45±4.40)nmol/mg prot in 3 days and 7 days,which were significantly increased as compared with those in the control group(P <0.05).After ACE2 intervention,the MDA content decreased to(6.35±2.27)nmol/mg prot and(9.10±2.89)nmol/mg prot,respectively.The activity of SOD in the lung tissue of control group was(10.07±1.10)U/mg prot and(9.72±0.92)U/mg prot,respectively.Compared with the control group,the activity of SOD were significantly reduced in ALI model mice.ACE2 administration had no significant affect on the SOD activity.6.The changes of capillary permeabilityAfter BLM stimulation the Evans blue content in lung tissue were(157.43±12.45)ng/mg and(193.17±17.89)ng/mg in 3 days and 7 days,which were significantly higher than that of control group(58.93±4.67)ng/ml.In ACE2 intervention group,Evans blue content in lung tissue were decreased to(77.33±21.14)ng/mg and(129.92±10.34)ng/mg.7.The expression of TNF-α,IL-6 and CXCL-1 protein in lung tissue of each groupCompared with the control group,the expression of TNF-α,IL-6 and CXCL-1 protein in the lung tissue were significantly increased after BLM intervention for 7 days(P<0.01).ACE2 treatment reduce the expression of TNF-α,IL-6 and CXCL-1.Summary:1.BLM can induce the acute lung injury in mice,manifested as increased lung permeability.Blood gas analysis results in a decrease in oxygen partial pressure and oxygen saturation.The lung injury is further confirmed by increased pathological score and elevated expression of TNF-α,IL-6,and CXCL-1 protein.2.ALI induces the oxidative stress injury in lung tissue,manifested as decreased SOD activity and increased MDA content.3.Exogenous ACE2 treatment reduces the lung injury degree,decreasesthe recruitment of neutrophils and block the secretion of TNF-α,IL-6,and CXCL-1 in lung tissue decreased,the MDA content decreased,and lungs are suppressed.Tissue inflammation and oxidative damage demonstrate that ACE2 has a protective effect on acute lung injury in mice.Part two Effects of ACE2 on mitochondrial function in ALI modelObjective: ALI model was established to explore the protective effect of ACE2 on mitochondrial function in mice.Methods: The lung injury model were induced by intraperitoneal administration of the bleomycin solution.The mice were further randomly divided into groups to receive single tail vein injection of either saline,or ACE2 at a dose of 1 mg/kg body weight.Mitochondria were isolated from lung tissue for further analysis.The mitochondrial membrane potential were detected,and Western-blot analysis were used to evaluate the cytoplasm and mitochondria cytochrome C protein expression.Results:1.The change of mitochondrial membrane potentialCompared with the control group,the mitochondrial membrane potential in ALI model group were significantly reduced,suggesting that BLM could damage the mitochondrial membrane structure integrity.The mitochondrial membrane potential were increased after ACE2 administration,indicating that ACE2 can reverse the mitochondrial damage induced by BLM.2.The change of cytochrome C protein expressionThe cytochrome C protein expression in mitochondria were significantly down-regulated after BLM intervention,accompanied by increased cytoplasmic cytochrome C content.ACE2 could restore the mitochondrial cytochrome C content,and down-regulate the cytochrome C protein distribution in cytoplasm.Summary:1.BLM stimulation damages the mitochondrial membrane integrity in lung tissue.ACE2 treatment could alleviate the mitochondrial injury inducedby BLM.2.BLM stimulation could trigger the intrinsic mitochondrial pathway releasing pro-apoptotic molecules such as cytochrome C into the cytoplasm,which can be inhibited by ACE2 treatment,demonstrating the protective function of it to the acute lung injury induced by BLM.Part three Crosstalk between ACE2 and PLGF regulates vascular perme--ability during acute lung injuryObjective: To investigate the relationship between ACE2,PLGF and sFlt-1 in acute lung injury.Methods: Balb/c mice were randomly divided into 4 Group: PLGF control group(n=40),sFlt-1 control group(n=40),ACE2+PLGF intervention group(n=40)and sFlt-1 intervention group(n=40).The changes of capillary permeability in lung tissue were evaluated by Evans Blue method.The airway resistance were determined.PLGF and VEGFR-1 levels were evaluated by Western-blot analysis.Results:1.ACE2 does not alter PLGF levels in ALI mouse lungCompared to the mice without treatment.We found that ALI inducedsignificantly increases in PLGF and VEGFR-1.While ACE2 treatment did not alter PLGF and VEGFR-1levels.2.Exogenous PLGF can be antagonistic ACE2 to ALI improved vascular permeability in model lung tissueIn order to confirm that ACE2 may antagonize the effects of PLGF-mediated increases in lung vessel permeability,we implanted a releasing pump of PLGF at the time of ACE2 treatment(ALI+ACE2+PLGF pump).Mini-pumps containing PBS only were implanted in control mice(ALI+ACE2+CTL pump).We found that provision of ectogenic PLGF abolished the antagonizing effects of ACE2 on the vessel permeability against PLGF,and then the effects of ACE2 on lung function protection in a methacholine response test for lung resistance.Hence,this loss-of-functionexperiment suggests that ectogenic PLGF may abolish the antagonizing effects of ACE2 on the vessel permeability against PLGF,resulting in aggravated lung function.3.Exogenous PLGF can be antagonistic ACE2 to ALI model lung function improvementThe airway resistance of the various groups is increased gradually after the stimulation of the acetylcholine-methyl choline,and the dose-effect relationship with the dosage of acetylcholine is definite.The airway resistance of mice in Micro-osmotic pump injection PLGF Group was larger than that of control group,and the rate of change was significantly accelerated,which indicate that exogenous supplement PLGF could suppress the effect of ACE2 the on the airway reactivity in mice.4.Exogenous sFlt-1 improve the vascular permeability in ALI modelCompared with the ALI model group,the content of Evans blue in the lung tissue after sFlt-1 injection for 3 d and 7 d were significantly reduced,indicating that sFlt-1 supplementation can improve the vascular permeability in ALI mice.5.Exogenous sFlt-1 improve the lung function in ALI modelThe airway resistance of the various groups is increased gradually after the stimulation of the acetylcholine-methyl choline,while the airway resistance of mice in sFlt-1 injection Group was lower than that of control group,and the rate of change was significantly decreased.Summary:1.Exogenous ACE2 supplementation has no obvious affect on PLGF and VEGFR-1 expression in ALI model.2.Exogenous PLGF can antagonize the protective effects of ACE2 on pulmonary fuction,manifested as increased vascular permeability changes and airway resistance.3.Exogenous sFlt-1 supplementation ACE2 may antagonize the BLM-mediated increases in lung vessel permeability,resulting in improvement of lung function.4.Exogenous ACE2 and sFlt-1 can improve the respiratory function in ALI model.Exogenous PLGF can antagonize the protective effects of ACE2 on lung tissue.ACE2 does not directly affect the expression of PLGF and VEGF-1,suggeating that the antagonism of ACE2 by PLGF is achieved via downstream signaling.Conclusions: Based on the above three experiments,the conclusions are as follows:1.ALI are severe forms of diffuse lung disease,presented as increased inflammatory injury and oxidative stress,decreased oxygen partial pressure and oxygen saturation.2.Exogenous ACE2 can improve the respiratory function in ALI model by reducing alveolar-capillary permeability,inhibiting lung inflammation and oxidative damage.3.ALI can disrupt the mitochondrial membrane integrity of mice by activating the mitochondrial apoptotic pathway and further influence the mitochondrial membrane permeability.ACE2 mainly participates in anti-apoptosis by increasing mitochondrial cytochrome C expression in ALI lung tissue,and ACE2 does not affect the expression of PLGF and VEGFR-1in lung tissue of ALI model mice.4.PLGF can antagonize the protective effect of ACE2 on pulmonary arteries in ALI mice,which is characterized by changes in vascular permeability and airway resistance.The specific mechanism may be through intervention of downstream signaling.5.ACE2 and sFlt-1 can antagonize pulmonary vascular injury in ALI mice,and can improve lung function in ALI model mice,but exogenous PLGF can antagonize the protective effect of ACE2 on lung tissue.