Effect And Mechanism Of Placental Growth Factor On Pulmonary Angiogenesis And Alveolarization Of Hyperoxia-induced Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia(BPD)is a common respiratory disease in premature infants,especially extremely preterm infants.BPD is one of the three high-risk factors affecting the long-term death or disability of very preterm infants.The pathophysiology of BPD is complex and may result from multifactors,including perinatal infection,mechanical ventilation and hyperoxia exposure.In recent years,with the improvement of neonatal intensive care,the survival rate of premature infants,especially very low birth weight infants,has increased,and the incidence of BPD has also increased.The incidence of BPD in extremely-low birth weight infants has reached 30%.Not only the lung function of BPD children is seriously damaged,but also their physical development and nervous system development are greatly affected,which is resulting in a heavy burden on the family and society.Therefore,there is an urgent need to understand the mechanism of BPD so as to find effective measures for prevention BPD in premature infants,and ultimately improve the long-term quality of life of premature infants.It has been found that vascular growth is closely related to alveolar development.Inhibiting the normal growth of blood vessels directly results in the obstruction of alveolarization.Abman et al proposed the "vascular hypothesis" of lung development.It can be seen that pulmonary vascular development disorder and alveolar block are the key events in the development of BPD.At present,it is considered that pulmonary vascular development and alveolarization are related to many factors,including gene expression regulation,growth factor expression,extracellular matrix production and maturation,and the synergistic effect of respiratory movement.Growth factors are believed to play a key role in pulmonary vascular development and alveolarization.Various growth factor signals have been found in late pulmonary development and BPD development.Placental growth factor(P1GF)is a secretory homodimer glycoprotein and a member of the vascular endothelial growth factor family.P1GF is mainly expressed in placenta,thyroid,lung and other tissues.It binds to vascular endothelial growth factor receptor 1 and regulates vascular development.P1GF only participates in neovascularization in pathological state,but it has little effect on normal vascular development.P1GF can induce leukocyte infiltration,tumor growth,stromal cell migration and vascular recanalization in ischemic tissues;P1GF can recruit monocytes and macrophages,and activate macrophages to release inflammatory factors.The macrophage infiltration and inflammation in the tissues of mice with defective expression of P1GF decrease.In addition,inhibiting the activity of P1GF can reduce the severity of fibrosis and inflammation in mice with liver cirrhosis.In animal models of cancer,anti-placental growth factor monoclonal antibodies can inhibit tumor growth,angiogenesis and macrophage aggregation without affecting healthy tissues.Normally,the expression of P1GF and Flt-1 increased in the last three months of human pregnancy,i.e.during the alveolar and alveolar stages of lung development,but decreased rapidly during the formation of pulmonary septum and alveolarization.Accordingly,we speculate that P1GF overexpresses in the injured lung in late stage of development,and P1GF promotes the activation,proliferation and migration of pulmonary vascular endothelial cells,which is leading to pulmonary vascular disorders,and activating macrophages to release inflammatory factors.At the same time,we hypothesize that anti-PIGF monoclonal antibodies may play a role in blocking the abnormal proliferation of pulmonary vessels in the animal model of BPD in premature infants.Part 1 Effects of overexpression of P1GF on the model of hyperoxia exposed lung epithelial cellsObjectives:To investigate the effect of hyperoxia or placental growth factor(P1GF)overexpression on pulmonary microvascular endothelial cells(PMEC)and type II alveolar epithelial cell(AEC-?).Methods:1.The plasmid and bacterial strain that expressing P1GF were obtained by DNA synthesis.The recombinant plasmid and pHelper packaging plasmid were cotransfected into HEK293a cells.The one and second-generation adenovirus were isolated.Viruses were purified by ultracentrifugation.The titers were determined by cytopathic effect(CPE)on HEK293a cells in a 96-well plate by a fluorescence microscope.2.The negative control(NC)empty Adenovirus were named as Ad-NC.The Adenovirus expressing P1GF were named as Ad-P1GF.PMEC and AEC-II cells were cultured under hyperoxia to construct hyperoxic model.PMEC and AEC-II cells were infected with adenovirus expressing P1GF(Ad-PIGF)to construct Ad-P1GF group.The cells that were cultured under normal condition or infected with negative control adenovirus(Ad-NC)were as control groups.3.ELISA was used to examine the level of ICAM-1/CD54 and VEGFR1 in each group.Transwell was used to detect the migration capacity of each group cells.4.qPCR and western blot were used to detect the expression level of E-cadherin,COL1A1,FN and a-SMA in each group.Results:1.The virus titer was 2×1010 PFU/ml.The results of immunofluorescence showed that both hyperoxia and Ad-P1GF infection could increase P1GF expression level in PMEC and AEC-II,indicating that models were successfully constructed and could be used for the following assays.2.The results of ELISA showed that both hyperoxia and P1GF overexpression could induce the upregualtion of ICAM-1/CD54 and VEGFR1.The results of transwell showed that both hyperoxia and P1GF overexpression could increase the migration capacity of PMEC and AEC-II cells.3.The results of qPCR and western blot showed that both hyperoxia and P1GF overexpression could decrease the mRNA and protein expression level of epithelial-mesenchymal transition(EMT)associated protein E-cadherin,and increase the mRNA and protein expression level of fibrotic markers COL1A1,FN and a-SMA.Conclusions:Both hyperoxia and P1GF overexpression could increase angiogenic ability and migration capacity of PMEC and AEC-II cells,and induce EMT and fibrosis.Part 2 Effects of anti-PIGF monoclonal antibody on hyperoxia-induced bronchopulmonary dysplasia in ratObjectives:To investigate the effect of anti-PIGF antibody on the hyperoxia-induced lung injury newborn rats and whether P1GF overexpression can induce lung injury of newborn rats.Methods:1.The full optimized CDS sequence of P1GF was insert into prokaryotic expression vector PGEX-6p-1 by DNA synthesis.The recombinant plasmid was transformed into prokaryotic expression host bacterium BL21(DE3).SDS-PAGE was used to identify P1GF protein expression.The P1GF protein was purified by high affinity Ni resin and renatured using urea.Purified P1GF protein was used to immune emale New Zealand white rabbits Fourteen days later after last mmunity,serum was obtained and anti-PIGF antibody was purified.2.After partum,the newborn rats were randomly divided into normoxia group,Normoxia+Ad-NC,Normoxia+Ad-PIGF,Hyperoxia group and Hyperoxia+anti-PIGF group.3.The pathological changes of lung tissue were observed by HE staining.Ultrastructure of lung cells was observed by transmission electron microscopy.4.The expression of P1GF and receptor flt-1 was detected by qPCR and western blot.The levels of TNF-a and IL-6 were detected by ELISA.5.Cell suspension of bronchoalveolar lavage(BAL)was prepared for smear,and cell number was counted after hematoxylin staining.Changes in CD34 were detected by immunohistochemistry.Results:1.The titer of P1GF polyclonal antibody is 1:243,000,which meets the requirements of subsequent experiments.After modeling,no abnormal findings were found in each group.No abnormal reactions were observed in the animals after the intervention of adenovirus and anti-PIGF antibody.A t the end of the experiment,no animals died abnormally.2.The results of HE staining and transmission electron microscopy showed that both P1GF overexpression adenovirus injection and hyperoxia can cause obvious lung injury in newborn rats,and the injection of anti-PIGF antibody can significantly improve the lung tissue injury caused by hyperoxia.3.The results of qPCR and western blot indicated that the expression level of P1GF and receptor Flt-1 mRNA and protein in lung tissues was significantly increased by the injection of P1GF overexpression adenovirus.Compared with normoxia group,the expression levels of P1GF and receptor flt-1 protein were significantly increased in both Hyperoxia group and Hyperoxia+anti-PIGF group.4.ELISA results showed that both P1GF overexpression adenovirus injection and Hyperoxia significantly increased TNF-a and il-6 expression levels in BAL.The expression levels of TNF-a and IL-6 were significantly reduced in BAL of Hyperoxia+anti-PIGF group compared with Hyperoxia group.The results of hematoxylin staining showed that both P1GF overexpression adenovirus injection and hyperoxia could decrease the number of monoytes and neutrophils.There were no significant changes in monocytes and neutrophils in the Hyperoxia+anti-PIGF group compared with the Hyperoxia group.5.Immunohistochemical results showed that both P1GF overexpression adenovirus injection and hyperoxia treatment significantly increased CD34 expression.The expression of CD34 was significantly decreased in the Hyperoxia+anti-PIGF group compared with the hyperoxia group.Conclusions:1.Hyperoxia can increase the mRNA and protein expression levels of P1GF and its receptor Flt-1 in lung tissues of newborn rats.The lung injury of newborn rats in the Hyperoxia group was aggravated,the expression levels of TNF-a and 11-6 in BAL were significantly increased,and the expression level of CD34 was significantly increased.The lung tissue of newborn rats injected with P1GF overexpressed lentivirus also showed similar changes to that of the Hyperoxia group.This suggests that P1GF plays a regulatory role in Hyperoxia-induced lung injury in newborn rats.2.The injection of anti-PIGF antibody could not reduce the abnormally high expression of P1GF and its receptor Flt-1 induced by hyperoxia,but could significantly improve the injury of lung tissues,reduce the expression levels of TNF-a and il-6 in BAL and CD34 in lung tissues.The results suggest that blocking the action of P1GF can improve the hyperoxia exposure injuryPart 3 Association between umbilical cord blood P1GF level and bronchopulmonary dysplasia in preterm infantsObjectives:To investigate the association between umbilical cord blood P1GF and BPD,and the value of umbilical cord blood P1GF in predicting bronchopulmonary dysplasia in premature infants.Methods:This prospective cohort study was performed from 2015 to 2017 with preterm neonates of birth weight ?1500 g.We obtained heparinized umbilical cord blood samples.The blood samples were immediately centrifuged at 3000 ×g for 10min at 4?C to obtain plasma and then stored at-80? The P1GF levels in plasma were measured using a quantitative colorimetric sandwich enzyme-linked immunosorbent assay kit(RayBio(?)Human P1GF ELISA Kit Protocol)in accordance with themanufacturer's instructions.Results:111 preterm infants were studied.BPD was identified in 30 of these infants.After adjusting for potential confounders,serum P1GF concentrations were found to be elevated in BPD infants relative to non-BPD infants.When cord blood P1GF>9.854 pg/ml,the diagnostic sensitivity and specificity were 93.3%and 71.5%respectively,and the AUC value was 0.9177.Conclusions:Cord blood P1GF level is closely related to BPD and can be used as a biomarker for early prediction of BPD.