| BackgroundsBronchopulmonary dysplasia (BPD) is a chronic lung disease(CLD) duing to the inhalation of high concentration of oxygen, injury caused by using long term mechanical ventilation, infection,which is common in preterm infants, especially in VLBWI (very low birth weight infant, VLBWI) and ELBWI.(extremely low birth weight infant, ELBWI). Along with ventilation strategy changed, glucocorticosteroid used perinatal,and lung surfactant applicated postnatal,more and more premature and extremely low birth weight infants can survive,but the incidence of new BPD is also to be growing year by year. The survivors’lung function and gas exchange are usually abnormal,who need long term trearment of oxygen and mechanical ventilation and bring grave economics and psychic burden to families and the society, affecting the patients’life quality seriously in future. Because BPD pathogenesis is complicated. At present no therapeutic strategy has been proven clinically effective.Therefore, to investigate how to prevent BPD and reduce the incidence is the important topic attecting the economic development of the society,which can protect our limit medic resources BPD is classified two types based on the pathological feature:classic or "old BPD"and "new BPD". The "old BPD"was first described by Northway and Colleagues in1967. Classic BPD is characterized by lung inflammation, parenchyma fibrosis due to hyperinflation and pulmonary atelectasis; New BPD is a milder form and be much more frequent today. The essential features are both lung microvascular and alveolarization dysplasia or abnormal, lung inflammation and parenchyma fibrosis are usually milder.The pathogenesy of new BPD is extremely complex,which are involved preterm, oxidative stress, prenatal infection,postpartum Inflammary and pulmonary dysplasia etc.However,the pathogenesy of BPD is not definite. The reports about the new BPD pathophysiology were mainly on plumonary interstitial fibrosis and pulmonary alveoli dysplasia,which achieved no advancement in the past. As the Abnormality of pulmonary vascular is essential to lung dysplasia, genes and proteins which could control lung angiogenesis and vasculogenesis are investigated recentantly. Abnormal lung microvascular can directly induce BPD. If the expressions of these genes are illegitimate,the normal development of lung vascular and alveolization will be interfered.It has important effect on the pathophysiology of new BPD.Epidermal growth factor-like domain7is a protein discovered recently that is exclusively secreted by endothelial cells.EGFL7is one of epidermal growth factor-like domain growth factors family,which contains two epidermal growth factor-like domain and be evolutively conservative.indicating it may participate in important vital activity of interaction of proterns in different species.The expression of HGFL7gene is special, which mainly exist in lung,heart,kidney and placenta. CGFL7is secreted by endothelial cells to extracellular matrix and then may promote to angiogenesis by autocrining.Importantly,EGFL7is highly expressed in process of growth in lungs of embryons and newborns. The distinct pattern of expression reveals that EGFL7may be an important factor controlling lung angiogenesis and vasculogenesis. Accordingly, EGFL7is regarded as an important factor in lung angiogenesis and vasculogenesis. Whether EGFL7is participated in the development of new BPD and the relation of EGFL7and pathogenesy of new BPD is unknown.Hyperoxia is one of the major contributors to the development of BPD. Hyperoxia-exposured model is often used in studing of pathogenesis of BPD. Neonatal hyperoxic lung injury is a complex pathophysiological process, the mechanism of injury involving the oxidant/antioxidant imbalance, inflammatory edema, angiogenesis, extracellular matrix remodeling, abnormal tissue repair, apoptosis and signal conduction system abnormalities and so on. Using a hyperoxia-induced lung injury model of neonatal rat, which mimics some aspects of the pathology of BPD, we have demonstrated that EGFL7expression is significantly decreased during the neonatal rat lung injury caused by hyperoxia.In virto experiments, Overexpression of hEGFL7protects endothelial cells from hyperoxia-induced cell death, which may be due to inhibition of mitochondria-dependent, intrinsic apoptotic pathway.However,mechanism of vascular endothelial cell apoptosis in hyperoxia lung injury is unclear completely,thus how EGFL7protect endothelial cells from hyperoxia-induced cell death needs be researched further. In vivo experiments, whether EGFL7protect pulmonary microvascular endothelial cell apoptosis from hyperocia-induced cell death and take part in the development of pulmonary vessels is worthy of further study.Accordingly, a recent study confirmed a new role of EGFL7.The study shows that:EGFL7prevents nuclear factor-κB nuclear localization and suppresses intercellular adhesion molecule1expression in response to hypoxia/reoxygenation injury in human coronary artery endothelial cells. Nuclear factor-kappa B is a multifunctional nuclear transcription factor, involved in immune inflammatory reaction, cell cycle regulation, apoptosis and cell adhesion process by regulation of downstream target genes transcription.It suggests EGFL7may be a new anti-inflammatory factors, playing an impotant role in response to inflammatory actuating link. Research shows that:the NF kappa B mediated microtubule endothelial cell injury, which is mainly associated with inflammatory infiltration induced damage and vascular endothelial cells apoptosis when NF-kb is activated.The inflammatory response is an important factor in the development of BPD,wich can damage the lung microvascular endothelial cells, lead to endothelial cells apoptosis or necrosis and block the development of pulmonary microvascular. Whether can EGFL7inhibit the development of lung local inflammatory reaction,protect pulmonary microvascular endothelial cells from apoptosis and reversal and reversal the pathlolgical process of pulmonary microvascular dysplasia by effecting of NF-κB signal pathway?The pathogenesis of BPD is closely related with pulmonary microvascular dysplasia. Abnormal gene expression regulating pulmonary microvascular development directly affects the normal development of the pulmonary microvascular.We have found that:60%oxygen resulted changes in lung tissue of neonatal rats,which is similar to the pathlolgical changes of BPD. At the same time, Egfl7expression is significantly decreased during the neonatal rat lung injury caused by hyperoxia.In summary,we believe that:EGFL7involve in the development of new RPD,however,the mechanisim of new BPD pathogenesis is unclear. Whether EGFL7can inhibit the development of lung local inflammatory reaction,protect pulmonary microvascular endothelial cells from apoptosis and reversal and reversal the pathlolgical process of pulmonary microvascular dysplasia by effecting of NF-κB signal pathway is worth of further study.This research attempts to to construct eukaryotic expression vector of human EGFL7, transfect it to EA.hy926cells and establish of hyperoxia-exposured cell model,discusses that Whether EGFL7can inhibit the development of lung local inflammatory reaction,protect pulmonary microvascular endothelial cells from apoptosis and reversal and reversal the pathlolgical process of pulmonary microvascular dysplasia by effecting of NF-κB signal pathway,which provide new intervention targets for the effective prevention or delay the occurrence and development of BPD.Methods1. cell culture:culture EA.hy926and the HUVEC cell separately using the DMEM and RPMI-1640nutrient fluid and10%newborn cowblood serum,50μg/ml penicillin and50μg/ml streptomyc in the cell generatin37℃and in5%CO2saturated humidity’s incubator.Replace culture medium interval2to3days. 2. Construction of eukaryotic expression vector of human EGFL7and establishment of stably transfected EA.hy926cell line:The open reading frame(ORF)of EGFL7was amplified from human umbilical vein endothelial cells (HUVEC) by RT-PCR. After being digested by HindⅢ and BamHI, the products were subcloned into the eukaryotic expression vector pEGFP-N1.The recombined plasmid pEGFP-Nl-EGFL7was indentified by sequencing and transfected into EA.hy926cell by electroporators. After screening culture by G418,stable tranfected EA,hy926cell line was established, and the expression of EGFL7was detected by real time PCR and western bloting.3. Establishment of hyperoxia-exposured cell model:The human endothelial cell line EA.hy926, EA.hy926-pEGFP-N1and EA.hy926-pEGFP-EGFL7were randomly divided into normoxic exposure group and yperoxic exposure group.ALL of them were grown in DMEM containing10%fetal bovine serum,50μg/ml penicillin and50μg/ml streptomycin in5%CO2at37℃. Normoxic exposure of the cells was conducted under room air and5%CO2in a humidified cell culture incubator at37℃. Hyperoxic exposure of the cells was conducted in a humidified chamber and the chamber was flushed with60%02-5%CO2-35%N2at a flow rate of10L·min-1for15min before incubation at37℃. Setting three time points (24h,48h,72h), we observe the protein expression of EGFL7,apoptotic cell death and NF-KB nuclear activation.3.1The mRNA and protein expression of EGFL7were determined by fluorescent quantitation real time PCR and Western blotting.3.2Apoptotic cell death was measured by flow cytometry analysis, protein expression of caspase-3was determined by Western blotting.3.3NF-κB nuclear activation were measured by Western blotting and immuno--fluorescence.3.4The protein expression of TNF-a and ICAM-1were determined by Western blotting.4. Statistics analysis:All data were analyzed using the SPSS13.0.All data were expressed as mean±standard deviation(X±S);the fluorescence quantitation PCR result’s is used2-ΔΔCT value to analyze,date uses one-sample T test between EA.hy926and N1-EA.hy926,date uses independent-samples T test between Nl-EA.hy926and hEGFL7-EA.hy926;The Western blot data is used ratio which is a value by analyzing with the ImageJ sofeware, date uses the completely randomized disigned One-Way ANOVA during EA.hy926、N1-EA.hy926and hEGFL7-EA.hy926cell line; In hyperoxia-exposured cell model,date of the protein expression of EGFL7and NF-kb use independent-samples T test during group at each time point; In overexpression of hEGFL7of hyperoxia-exposured cell model,date of the protein expression of EGFL7,caspase-3,NF-kb and apoptosis rate uses factorial analysis of variance text; P<0.05presents statistics different.Results1、The recombinant vector pEGFP-Nl-EGFL7was successfully constructed and established EA.hy926cell line stably expressing EGFL7.The EGFL7overexpression was detected.2、EA.hy926cell model of hyperoxia-exposured were successfully established.2.1The dynamic changes of EGFL7mRNA and protein level in EA.hy926cell exposed to60%oxygen2.1.1The mRNA expression of EGFL7in EA.hy926cell:EGFL7mRNA expression was high in control group, no significant difference in different time point(F=0.159, P=0.925). Compared with the control group,the mRNA expression of EGFL7was significantly decreased in treated group.The fluctuation was from43.65%-79.92%and the differences had statistical significance (t=-12.167、-29.611、23,871, P=0.007、0.001、0.002)2.1.2The protein expression of EGFL7in EA.hy926cell:The protein expression of EGFL7was high in control group, no significant difference in different time point (F=0.459, P=0.584). Compared with the control group, The protein expression of EGFL7was significantly decreased in treated group.The fluctuation was from27.23%-75.89%and the differences had statistical significance (t=3.075、7.570、 9.250, P=0.037、0.002、0.01)2.2The dynamic changes of apoptotic cell death in EA.hy926cell exposed to60%oxygen2.2.1Apoptotic rate measured by flow cytometry analysis:Hyperoxia significantly increased necrotic cell death EA.hy926cells. Compared with the control group,apoptotic rate was significantly increeased in treated group. The fluctuation was from3.69%、7.00%to30.66%、33.73%and the differences had statistical significance (t=-14.017、-35.175, P=<0.001、<0.001).2.2.2The protein expression of caspase-3in EA.hy926cell2.2.2.1The protein expression of procaspase-3in EA.hy926cell:The protein expression of procaspase-3was high in control group, no significant difference in different time point (F=0.899, P=0.371). Compared with the control group, The protein expression of procaspase-3was significantly decreased in treated group.The fluctuation was from46.68%-75.30%and the differences had statistical significance (t=12.422、47.539, P=<0.001、<0.001)2.2.2.2The protein expression of cleaved caspase-3in EA.hy926cell:The protein expression of cleaved caspase-3was low in control group, no significant difference in different time point (F=41.825, P<0.001). Compared with the control group, The protein expression of cleaved caspase-3was significantly increased in treated group.The fluctuation was from1.15-3.60times and the differences had statistical significance (t=-41.633、-39.190, P=<0.001、<0.001)2.3The dynamic changes of NF-κB nuclear activation in EA.hy926cell exposed to60%oxygen2.3.1The protein expression of NF-κB in Cytoplasm and nucleus of EA.hy926cell:The results show NF-κB nuclear localization.2.3.1.1The protein expression of iκb-α in Cytoplasm of EA.hy926cell:The protein expression of iκb-α was high in control group, no significant difference in different time point (P=0.513). Compared with the control group, The protein expression of iκb-a was significantly decreased in treated group.The fluctuation was from35.36%-44.51%and the differences had statistical significance (t=4.985、9.400、19.044, P=0.036、0.001、<0.001)2.3.1.2The protein expression of NF-κB p65in Cytoplasm of EA.hy926cell: The protein expression of NF-κB p65was high in control group, no significant difference in different time point (F=2.256, P=0.186). Compared with the control group, The protein expression of NF-κB p65was significantly decreased in treated group The fluctuation was from12.98%-64.40%and the differences had statistical significance (t=3.378、6.053、10.635, P=0.029、0.004、<0.001)2.3.1.3The protein expression of NF-κB p65in nucleus of EA.hy926cell:The protein expression of NF-κB p65was low in control group, no significant difference in different time point (F=4.341, P=0.068). Compared with the control group, The protein expression of NF-κB p65was significantly increased in treated group.The fluctuation was from1.40-2.90times and the differences had statistical significance (t=-20.543、-29.678、-15.031, P=0.002、<0.001、<0.001)2.3.2Immunofluorescence shows NF-κB nuclear localization.2.4The dynamic changes of TNF-a and ICAM-1protein level in EA.hy926cell exposed to60%oxygen2.4.1The protein expression of TNF-a in EA.hy926cell:The protein expression of TNF-a was low in control group, no significant difference in different time point (F=4.341, P=0.068). Compared with the control group, The protein expression of TNF-α was significantly increased in treated group.The fluctuation was from0.41-1.08times and the differences had statistical significance (t=-5.251、-10.528、-8.762、P=0.006、0.001、0.001)2.4.2The protein expression of ICAM-1in EA.hy926cell:The protein expression of ICAM-1was low in control group, no significant difference in different time point (F=0.057, P=0.945). Compared with the control group, The protein expression of ICAM-1was significantly increased in treated group.The fluctuation was from4.09-8.69and the differences had statistical significance (t=-12.809、-42.097、-34.023, P=0.006、<0.001、<0.001)3、Epidermal growth factor-like domain7protects endothelial cells from hyperoxia-induced cell death3.1Apoptotic rate measured by flow cytometry analysis: Hyperoxia significantly increased necrotic cell death in both Neo-EA.hy926and hEgfl7-EA.hy926cells. The necrotic cell death was increased from5.45%,14.63%to45.82%,57.94%in Neo-EA.hy926cells and from2.05%,11.71%to17.93%,30.89%in hEgfl7-EA.hy926cells after48h,72h hyperoxic exposure and the differences had statistical significance (F=80.441, P<0.001). Compared with hyperoxia-treated Neo-EA.hy926cells, the apoptotic cell death was significantly reduced in hyperoxia-treated hEgfl7-EA.hy926cells.3.2The protein expression of caspase-3in Neo-EA.hy926and hEgfl7-EA.hy926cells.3.2.1The protein expression of procaspase-3: Hyperoxia significantly decreased the protein expression of procaspase-3in both Neo-EA.hy926and hEgfl7-EA.hy926cells. The protein expression of procaspase-3was significantly decreased in treated group.The fluctuation was from65.11%-92.92%in Neo-EA.hy926cells and from34.33%-38.61%in hEgfl7-EA.hy926cells after48h,72h hyperoxic exposure and the differences had statistical significance (F=63.452, P<0.001). Compared with hyperoxia-treated Neo-EA.hy926cells, the decrease of the protein expression of procaspase-3was significantly reduced in hyperoxia-treated hEgfl7-EA.hy926cells and the differences had statistical significance (F=5.841, P=0.028)3.2.2The protein expression of cleaved caspase-3: Hyperoxia significantly increased the protein expression of cleaved caspase-3in both Neo-EA.hy926and hEgfl7-EA.hy926cells. The protein expression of cleaved caspase-3was significantly increased in treated group.The fluctuation was from2.72-2.98times in Neo-EA.hy926cells and from0.21-0.32times in hEg/l7-EA.hy926cells after48h,72h hyperoxic exposure and the differences had statistical significance (F=284.093, P<0.001). Compared with hyperoxia-treated Neo-EA.hy926cells, the increase of the protein expression of cleaved caspase-3was significantly reduced in hyperoxia-treated hEgfl7-EA.hy926cells and the differences had statistical significance (F=126.659, P<0.001)4、Epidermal growth factor-like domain7protects endothelial cells from hyperoxia-induced NF-κB nuclear inversion4.1The protein expression of iκb-α in Cytoplasm of Neo-EA.hy926and hEgfl7-EA.hy926cells: Hyperoxia significantly decreased the protein expression of iKb-a in both Neo-EA.hy926and hEgfl7-EA.hy926cells. The protein expression of iκb-awas significantly decreased in treated group.The fluctuation was from55.54%-55.57%in Neo-EA.hy926cells and from4.98%-23.05%in hEgfl7-EA.hy926cells after48h,72h hyperoxic exposure and the differences had statistical significance (F=42.815, P<0.001). Compared with hyperoxia-treated Neo-EA.hy926cells, the decrease of the protein expression of iKb-a was significantly reduced in hyperoxia-treated hEgfl7-EA.hy926cells and the differences had statistical significance (F=17.114, P=0.001)4.2The protein expression of NF-kB p65in Cytoplasm of Neo-EA.hy926and hEgfl7-EA.hy926cells: Hyperoxia significantly decreased the protein expression of NF-κB p65in both Neo-EA.hy926and hEgfl7-E A.hy926cells. The protein expression of NF-kB p65was significantly decreased in treated group.The fluctuation was from39.97%-49.51%in Neo-EA.hy926cells and from17.50%-20.81%in hEgfl7-EA.hy926cells after48h,72h hyperoxic exposure and the differences had statistical significance (F=37.245, P<0.001). Compared with hyperoxia-treated Neo-EA.hy926cells, the decrease of the protein expression of NF-kB p65was significantly reduced in hyperoxia-treated hEgfl7-EA.hy926cells and the differences had statistical significance (F=6.348, P=0.023)4.3The protein expression of NF-kB p65in nucleus of Neo-EA.hy926and hEgfl7-EA.hy926cells: Hyperoxia significantly increased the protein expression of NF-κB p65in both Neo-EA.hy926and hEgfl7-EA.hy926cells. The protein expression of NF-kB p65was significantly increased in treated group.The fluctuation was from0.44-0.79times in Neo-EA.hy926cells and from0.09-0.25times in hEgfl7-EA.hy926cells after48h,72h hyperoxic exposure and the differences had statistical significance (F=231.699, P<0.001). Compared with hyperoxia-treated Neo-EA.hy926cells, the increase of the protein expression of NF-κB p65was significantly reduced in hyperoxia-treated hEgfl7-EA.hy926cells and the differences had statistical significance (F=119.950, P<0.001)5、Overexpression of hEGFL7effective on the protein expression of TNF-α and ICAM-15.1The protein expression of TNF-α in Neo-EA.hy926and hEgfl7-EA.hy926cells: Hyperoxia significantly increased the protein expression of TNF-α in both Neo-EA.hy926and hEgfl7-EA.hy926cells. The protein expression of TNF-α was significantly increased in treated group.The fluctuation was from1.60-1.48times in Neo-EA.hy926cells and from0.18-0.5times in hEgfl7-EA.hy926cells after48h,72h hyperoxic exposure and the differences had statistical significance (F=282.220, P<0.001).Compared with hyperoxia-treated Neo-EA.hy926cells, the increase of the protein expression of TNF-a was significantly reduced in hyperoxia-treated hEgfl7-EA.hy926cells and the differences had statistical significance (F=66.812, P<0.001)5.2The protein expression of ICAM-1in Neo-EA.hy926and hEgfl7-EA.hy926cells: Hyperoxia significantly increased the protein expression of ICAM-1in both Neo-EA.hy926and hEgfl7-EA.hy926cells. The protein expression of ICAM-1was significantly increased in treated group.The fluctuation was from0.96-0.99times in Neo-EA.hy926cells and from0.03-0.34times in hEgfl7-EA.hy926cells after48h,72h hyperoxic exposure and the differences had statistical significance (F=152.768, P<0.001). Compared with hyperoxia-treated Neo-EA.hy926cells, the increase of the protein expression of ICAM-1was significantly reduced in hyperoxia-treated hEgfl7-EA.hy926cells and the differences had statistical significance (F=154.854, P<0.001)Conclusions1、The recombinant vector pEGFP-N1-EGFL7was successfully constructed and established EA.hy926cell line stably expressing EGFL7.The EGFL7overexpression was detected. The construction of the eukaryotic expression vector pEGFP-N1-EGFL7will provide solid experimental foudation for further studies on the function of EGFL7gene in bronchopulmonary dysplasia of preterm infants.2、In hyperoxia-exposured EA.hy926cell model, the expression of EGFL7mRAN and protein is decreased by exposured by60%oxygen.It shows it is feasible to establish of hyperoxia-exposured cell model by60%oxygen and EGFL7may be involved in the occurrence and development of the new BPD. Compared with the control group,apoptotic rate was significantly increeased in treated group,implying that new BPD may be related with pulmonary microvascular endothelial cell apoptosis caused by hyperoxia.In treated group, NF-κB nuclear translocation and the protein expression of TNF-a and ICAM-1increased significantly,which is in according with increase of apoptotic rate.In short, it implys that hyperoxia-induced endothelial cell apoptosis may be associated with the process of imflammatory reaction mediated by NF-KB Signal pathway.3、High expression of EGFL7protects endothelial cells from hyperoxia-induced cell death and protects NF-κB from nuclear inversion.It is infered that Epidermal growth factor-like domain7protects endothelial cells from hyperoxia-induced cell death by inhibiting nuclear factor-KB activation.Accordingly, high expression of HGFL7decreses the protein expression of TNF-a and ICAM-1,which are important factors in the process of inflammatory response.In short, EGFL7can inhibit the development of lung local inflammatory reaction,protect pulmonary microvascular endothelial cells from apoptosis and reversal and reversal the pathlolgical process of pulmonary microvascular dysplasia by effecting of NF-κB signal pathway,which provide new intervention targets for the effective prevention or delay the occurrence and development of BPD. |
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