Dynamic Study On The Expressions Of VEGF And PDGF In Chronic Lung Disease Induced By Hyperoxia In Newborn Rats And The Involved Mechanisms Of Lung Development

Bronchopulmonary Dysplasia ( BPD) is the most common form of chronic lung disease (CLD) in infancy. The clinical, radiological, and pathological features of BPD were first described a little more than three decaded ago. The disease was seen in large preterm infants with severe respiratory distress syndrome who had been treated with high inspired oxygen concentrations with high positive airway pressures. With the widespread use of antenatal steroids and surfactant treatment, CLD is still one of the major complications in mechanically ventilated premature infants. The incidence of CLD reaches 30% - 40% a-broad, which reduces survivors and the life quality of prematurity. Topics on CLD become the most challenging in neonatal field.So far the mechanisms research on CLD has been one of the hot topics all a-round the world. Although a great deal of animal experiments and clinical researches have been done on inflammation, apoptosis, oxidative stress and arrested lung development induced by the lack of some growth factors to clarity the incidence and the development of CLD, the accurate mechanism of CLD has not identified. Most of the previous studies have demonstrated the mechanisms of vescular differentiation disease. The development of lung includes two equal important aspects: alveolarization and vascularization. At the mean time vescular differentiation the microvessel extension and develop to effective air -blood exchange barrier. The pathway of the pneumoangiogram formation at least include two kind vasculogenesis is the phase of interstitial blood cell different to be thevessel. Angiogenesis is the phase of existence vessel become to new vessel by the method of budding. There are many agents which can adjust the lung vessel. Among them, the most important is VEGF and Endostatin. There are many a-gents which can adjust the lung development. Among them, the most important is PDGF. VEGF is a homodimeric peptide that has important angiogenic actions in addition to including endothelial cell proliferation. VEGF is the key adjustment at the phase of lung development. Animal test verify that VEGF decrease at the damage phase in the lung development. With the rebuild of the normal lung development at restoration period, VEGF is up - regulate. After that the premature with BPD has less VEGF compare with the neonate of restoration period. Bhatt find that the maker PEC AM -1 protein and mRNA decrease in the endothelial cell, VEGF mRNA and its receptor Fit -1 was also decrease. So he concluded that the abnormal generation of the vessel of lung is the key factor of BPD. Endostatin, a proteolytic derivative of the carboxy terminal domain of basement membrane type X V ID collagen, has potent antiangiogenic properties. Endostatin has a specific inhibitory effect on endothelial cell proliferation. PDGF is a mitogen for cells of mesenchymal origin. PDGF is important for both alveogenesis and angiogenesis of the normally developing lung. Stimulation of PDGF receptors by PDGF ligands induced lung development. About of the dynamic change of VEGF, Endostatin, PDGF - A, PDGF - B, PDGFR - a, PDG-FR - p in CLD had not been systematic studied.Based on the characters that the sequence and the relative timing of alveo-larization in rat resembles human lung development, in the study we prepared an animal model of CLD with newborn rats by continuously inhaling high concentration of oxygen (hyperoxia). Using immunohistochemistry and reverse transcription polymerase chain reaction ( RT - PCR) to observe the rule of the lung tissue pathology of the neonate rat and also measure the VEGF, Endostatin, PDGF -A, PDGF - B, PDGF - p at the protein and molecular level and study the rule of them in the episode process of CLD, which can clarify the abnormal alteration and pathogenesis of CLD induced by hyperoxia. We try to provide more information of pathophysiological mechanism of CLD.Materials and Methods1. Animal modelWithin 12 hours of birth, pups were randomly redistributed to the newly delivered mothers. One - half of the newborn pups were placed in hyperoxia, and another one -? half remained in room air. Hyperoxia exposures were done in Plexiglas chamber into which oxygen was continuously delivered to achieve a constant level of 90 ~ 95% oxygen monitored daily with an oxygen monitor ( OM -25ME, USA). Oxygen and room air were filtered through Sodalime to keep CO2 levels below 0. 5% ( Dapex Gas Monitor, USA). Temperature and humidity were maintained at 24 ~ 26*C and 60% ~ 70% respectively. Chamber was opened for 0. 5h daily to switch nursing mother and change water, add food, clean dirty cages, record survival and body weight daily. Nursing mothers were rotated between oxygen exposed and room air litters every 24h to avoid oxygen toxicity in the mothers and to eliminate maternal effects between groups.2. Sample collection and treatmentEach group of hyperoxia as well as air group rats was anesthetized at each time point. The thorax was opened and lung tissue collected and stored respectively according to the following methods:2. 1 The samples of the left lung are fixed in 4% formaldehydum polymer-isatum which contains 0. 1% DEPC, then were dehydrated, embedded in wax within 24 hours.2. 2 The samples of the upper lobe of the right lung are fixed in 2.5% gl-utaraldehyde.2. 3 The samples of the residual lobe of the right lung are put in the Rnase -free Eppendorf tubes, and then put in — 80*C freezer.3. Experimental methods and Analysis marker 3.1 The changes of the lung pathology3. 1. 1 Microscope exam of section stained with HE;lung histological study.3.1.2 Radical alveolar counts (RAC) : value on the development of thelung.3. 1. 3 Ultrastructure:the structures of the mitochondrion, endoplasmic, reticulum in lung vessel endothelial cell and the basement member of the capillary.3. 2 Immunohistochemistry: Use to measure the expression levels of VEGF, Endostatin, PDGF, PDGFR proteins in the samples of both groups.3.3 Western - blotting: Use to measure the expression levels of Endostatin , PDGF - A, PDGF - B proteins in the samples of both groups.3.4 RT - PCR: Use to measure the expression levels of VEGF, PDGF -A, PDGF - B mRNAs in the samples of both groups.4. Statistical analysisAll the data were expressed as the means ± SD. All results were normally distributed and analyzed by pair t test with SPSS 11.0 statistical software. Statistical significance was achieved at P <0.05.Results1. General status of neonate rats in two groupsIn the air group: neonate rats are in good situation, sensitive, skin red and weight increased, but in the hyoeroxia group: the rats are in bad situation, and weight decreased. After being detached from higher level of supplement oxygen, some rats were at lOd light dyspnea, almost all presented dyspnea of various degrees at the 14d and all had serious dyspnea with cyanosis through the body at the 21 d. Such symptoms can be relieved gradually after being put into oxygen chamber again.2. Morphologic changes in neonate rats with hyperoxia - induced CID2.1 Pathological morphology: Inflammation cells infiltration were seen in alveolar space and interstitial area on day 3, alveolar number and septum were decreased, interstitial cells and alveolar internal surface area were increased after day 7 and more pronounced until day 21.2.2 RAC increased with days of rats in air groups, while they began to decrease from 7d for hyperoxia rats compared to air group ( P < 0. 05 ) , whichwas more evident on 14d and 21 d ( P < 0.001).2.3 The ultrastructure of the lung capillary endothelial cell In the air group, the nuclei was recognized clearly, cytoplasm near the nuclei can find rich of mitochondrion, endoplasmic, reticulum and the basement member of the capillary is even. Hyperoxia group show swelled mitochondrion and endoplasmic in the day 1 and day 3, basement member of the capillary is not even. At the 14d, 21 d, the sweller severisely.3. The expression of VEGF and Endostatin3.1 The expression of VEGF and Endostatin proteinsThe study showed that the VEGF protein was mainly expressed in the epithelial of the conductive airway on the day 1 and gradually increased in the epithelial cells after the 3 th day in the normal neonate rat's lung. But in the hyperoxia group, the expression level of protein was pretty lower than that of the air group on the 7th day. It was hardly to see the VEGF positive cells on 14d. There was no significant difference between the two group on Id, 3d P>0.05, but7dP< 0.05,14d, 21d, P< 0.01.In the air group, the expression of endostatin protein had a tendency of decrease with age. In the hyperoxia group, the expression of "endostatin declined gradually. Among.the 3d and 21 d the expression in the hyperoxia group are all lower than the air group. There was no significant difference between the two group on Id P>0.05, 3dP<0.05, 7d,14d,21d P <0.01.3.2 The expression of VEGF mRNAIn the rat lungs of air group, the expression of VEGF mRNA was increased with age and reached its peak level at 14d, consistent with the expression of VEGF protein. But in the hyperoxia exposed rat lungs, the expression of VEGF mRNA was decreased with age: on Id and 3d P >0.05;7d P < 0.05, 14d, 21 d P< 0.01, respectively.04. The expression of PDGF - A and PDGF - B4.1 The expression of PDGF - A and PDGF - B protein In the newborn rat lungs of air group, the expression of PDGF - A protein was mainly located in airway epithelium and parenchymal tissue. In the air group, the expression of PDGF - A was increased with age. But in the hyperoxiaexposed rat lungs, the expression of PDGF - A had no significant difference. There was no significant difference between the two group on Id P >0.05, 3d P <0.05, 7d,14dand21dP<0.01.In the newborn rat lungs of air group, the expression of PDGF - B protein was mainly located in airway epithelium and airway and alveolar epithelium. In the air group, the expression of PDGF - B was increased with age. But in the hyperoxia exposed rat lungs, the expression of PDGF - B was increased with age in 7d and decreased with age after 7d. There was no significant difference be-tweenthe two group on Id, 3d J d P <0.05, 14d,21d P >0.05.4.2. The expression of PDGFR - a and PDGFR - p proteinIn the newborn rat lungs of air group, the expression of PDGFR - a protein was mainly located in airway epithelium and parenchymal tissue. In the air group, the expression of PDGFR - a did not change significantly through 1 d to 21 d after birth. In the hyperoxia exposed rat lungs, the expression of PDGFR -a had no significant difference. There was no significant difference between the two groupsP >0.05.In the newborn rat lungs of air group, the expression of PDGFR - p protein was mainly located in airway epithelium and parenchymal tissue, the expression of PDGFR - p was increased with age. But in the hyperoxia exposed rat lungs, the expression of PDGFR - (3 was increased with age in 7d and decreased after 7d compared with air group Id P >0.05, 3d,7d P < 0.05,14d and 21d P > 0. 05.4.3 The expression of PDGF - A and PDGF - B mRNAIn the newborn rat lungs of air group, the expression of PDGF - A mRNA was increased with age. But in the hyperoxia exposed rat lungs, the expression of PDGF - A mRNA had no significant difference. There was no significant difference between the two group on Id, 3d P <0.05, 7d,14d,21d P <0.01.In the air group, the expression of PDGF - B was increased with age. But in the hyperoxia exposed rat lungs, the expression of PDGF - B mRNA was increased with age in 7d and decreased with age after 7d. Compared with air group ldP>0.05, 3d,7dP< 0.01,14d and21d P> 0.05.Conclusions1. Pathological findings charactered by the arrested alveolar development and lung capillary endothelial cell damage in newborn rats after prolonged hyperoxia are similar to those of CLD of prematurity.2. Hyperoxia caused the lung microvessel endothelial cell of newborn rat damage, which suggested that hyperoxia attend the process to injure the lung vessel.3. Hyperoxia caused the decrease of expression of VEGF and Endostatin in the rat lung.4. The growth of the lung vessel is the key period of the lung development, which suggest that vessel growth factor would be one of the mechanisms of doing to the lung vessel dysplasia induced by hyperoxia.5. PDGF - A is a key factor in alveogenesis in two weeks after birth. Hyperoxia suppressed product of PDGF - A to lead to pulmonary branching and septation and the number of alveoli decreased. These suggested that PDGF - A would participate in the process of lung dysplasia in CLD induced by Hyperoxia.6. PDGF - B is a key effect in lung mass growth after birth. Overexpres-sion of PDGF - B induced by hyperoxia lead to pulmonary tissue cell damage and contributed to abnormal lung proliferation to affect lung tissue whole development eventually. These suggested that PDGF - B would participate in the process of lung dysplasia in CLD induced by Hyperoxia.