Study On The Dynamic Changes Of VEGF, VEGFRs, HIF-1α, ANG-1, TIE-2及PECAM-1 In Hyperoxia-treated Newborn Rat Lungs

Recently, medical and technical advances have enabled more infants with respiratory distress syndrome to survive and have reduced mortality for very low birth weight (VLBW) infants, but have also resulted in a corollary increase in survivors with Chronic Lung Disease ( CLD) at the same time. CLD is a major cause for premature infants to be in hospital chronically, to grow slowly and to get respiratory diseases repeatedly. With the rise of survival rate in VLBW,the morbidity of CLD has reached 30 ~40% abroad, and has a rising tendency in our country year by year. The CLD group demonstrated deficits in intelligence, reading, mathematics, and gross motor skills compared with term children. Its pathogenic mechanisms were not fully understood. There have been no effective preventions and curable measures until now. Previous studies showed that there were a variety of factors that could result in CLD, including hyperoxia, ventilation , chronic inflammation, abnormal repairing processes, insufficient of the an-ti - oxidant system and lack of surfactant proteins in the premature lung. Hyperoxia is considered the most important cause of CLD during the perineonate period at present.Most of the previous studies have demonstrated the mechanisms of decreased alveolarization caused by hyperoxia. Lung volume and surface area dramatically increase during the late canalicular and early saccular stages of the normal intrauterine development. Secondary septa form new alveoli in the late fetus and newborn stages. Alveolarization continues at a slower rate during the first 2-3 yr of life. Parallel vascular growth is closely synchronized with alveo-larization during the same time periods. But the signal molecules that link distal airspace growth with angiogenesis are uncertain. Of several angiogenic factors, vascular endothelial growth factor ( VEGF) families and angiopoietin families have been considered to be the most important. VEGF has been shown to play a central role in the process of vascular development. VEGF is a potent endothelial cell - specific mitogen and survival factor. It stimulates angiogenesis, promotes vessel remodeling, and enhances endothelial survival. VEGF signaling is absolutely critical to vascular development and embryonic survival. It appears to protect against hyperoxia or cytokine - induced endothelial cell injury. Whether disruption of VEGF signaling system would impair lung vascular growth and contributes to the pathogenesis of CLD has been uncertain.Lung VEGF expression was decreased in infants with CLD. Because the premature lung circulation may be particularly susceptible to injury, it has recently been speculated that the inhibition of vascular growth itself may, directly impair alveolarization process. Treatment of newborn rats with a VEGF receptor inhibitor, SU5416, or with less specific antiangiogenesis drugs (thalidomide and fumagiLlin) inhibited vascular growth and decreased alveolarization. These findings suggested that angiogenesis was necessary for alveolarization during normal lung development, and that injury to the developing pulmonary circulation during the critical period of lung development also contributed to lung hypoplasia.Lung development includes two equal important aspects: alveolarization and vascularization. The effect of hyperoxia on abnormal microvascular development of animal lung remains poorly understood. There is little knowledge about the changes of the expressions of hyperoxia induced factor - la ( HIF - la) -VEGF - VEGFR system, ANG - 1 and TIE -2—a ANG - 1 receptor and the microvascular density represented by platelet endothelial cell adhesive molecule - 1 (PEC AM - 1 ) in newborn rat lung during sustaining hyperoxia exposure. We try to elucidate the mechanisms of abnormal vascularization in hyperoxia -induced neonate rat CLD model and to provide more information for the precaution and treatment of CLD in the future.Material and Methods1. Animal modelsSeveral litters of neonate Sprague - Dawley pups were pooled together within 12 hours after birth and randomly redistributed to surrogate mother rats. Then half of the pups were placed in 0. 14 - m3 Plexiglas exposure chamber containing>95% 02 into which oxygen was continuously delivered at 3.0 L/min to maintain a constant level of >95% oxygen and to keep CO2 levels below 0.5% and the other half in 21% O2 ( room air/ normoxia) from days 1 to 21 of their postnatal life. O2 concentrations were monitored daily. Temperature and humidity were maintained at 26°C and 60 - 70% , respectively. The chamber was opened for< 20 min daily to switch dams between air and O2 environments for avoiding the dams oxygen toxicity, to clean dirty cages, to allow provision of fresh food, water and to weigh of the pups.2. Preparation of Lung SamplesPups from each exposure group were killed on days 1, 4, 7, 10, 14, and 21 with an intraperitoneal overdose of pentobarbital sodium (150mg. kg" ). The lungs were removed. The left lungs were fixed in formaldehydum polymerisatum for immunohistochemistry tests; the right was immediately stored at - 80 °C freezer for RT -PCR tests in the RNase -free Eppendorf tubes.3. Experimental methods and Analysis marks(1) To observe the pathology of the lungs of the pups from both groups with a light microscope.(2) Immunohistochemistry: Use to measure the expression levels of VEGF, VEGFR1, VEGF2, PEC AM - 1, TIE -2 proteins in the samples of both groups.(3) RT - PCR: Use to measure the expression levels of VEGF, HIF - la, ANG -1 mRNAs.(4) Statistical analysisAll dada were expressed as means ± SD. All results were normally distributed and were analyzed by one - way ANOVA, where variance was distributed ac-cording to treatment and time. When a significant F value was found, LSD and Dunnett T3 post hoc test was used to determine significance with SPSS 11.5 statistical software. Statistical significance was achieved at P <0.05.Results1. General status of neonate ratsWithin 10 days, the animal s general status and weight increases in both groups were in good condition, we did not notice significant difference in their appearance. But after 14 test days, the rats from the hyperoxia group were not in so good condition as the animals of the control group: the weight of the rats under hyperoxia treatment increased slower than the control's; Dyspnea appeared after detached from hyperoxia surroundings; on days 21, the rats from hyperoxia group moved slower, dyspnea became more severe, all had evident cyanosis through their bodies. Those signs can be relieved gradually while being put under the hyperoxia chamber again.2. The lung morphologyThe alveolar - like structures in full - term newborn rat lung at birth was irregular , there were a few number of septa; On days 4, the alveolar appeared, the septa increased and became thinner; After the 7th day, the alveoli were regular, uniformly distributed. In the hyperoxia -treated group: the morphology of lung appeared similar to that of the control on day 1; there were red blood cells in some air space, inflammatory response in interstitial tissue and fewer alveoli compared with the control group rat lungs on days 4; on the 7th day, inflammation and hemorrhage subsidized, the alveolar numbers started to became fewer, the air space larger compared with those of the control at the same age; On days 14, single air space became even much larger, the alveolar numbers were much fewer, and a few of alveolar walls interrupted, the numbers of cells in the interstitial tissue increased, the lung structure became abnormal; On days 21, the normal alveolar structure disappeared, fibrosis was evident.3. Expression of PECAM - 1 proteinExpression of PECAM -1 protein in control group started to increase on the4th day, but P >0.05 compared with Id, increased significantly on the 7th day ( P <0. 01, compared with Id) , which reached its peak on days 21 P <0.001, compared with Id. In hyperoxia group, the expression of PEC AM - 1 protein didnt increased with age, the levels of Id, 4d were not significantly different from those of the control with P >0.05; on days 7 , the level started to be lower than that of the control group; the levels of expression of PECAM - 1 protein at 7d,10d,14d,21d in hyperoxia - treated rat lungs were significantly lower than those of the control group with P <0.05 , P <0. 01, P <0.001, P <0.001, respectively.4. Expression of VEGF proteinOur study showed that the VEGF protein was mainly expressed in the epithelium of the conductive airway on the day 1 and gradually increased in distant epithelial cells after the 4th day in the normal neonate rat lungs. But in the chronically hyperoxia - treated rat's lungs, the expression level of protein was pretty lower than that of the control group on the 7 th day and to the lowest level on days 10; It was hardly to see the VEGF positive cells on lOd ,14d; There was no significant difference between the two group on Id, 4d P >0.05, but on 7d,10d,14d,21d, P<0.001.5. Expression of VEGFR1 proteinThe expression of VEGFR1 protein was located in the microvascular endo-thelium cells in distant alveolar interstitial tissue and bronchial epithelium but not in large vascular endothelium. In control group, there was a little positive cells in the bronchial epithelium and in distant interstitial tissue on day 1; After the 4th day, more positive cells could be seen in distant alveolar wall and interstitial tissue. At this point, it was consistent with the expression model of VEGF protein. But hyperoxia exposure made expression of VEGF protein reduced with age. There were no significant difference between the two groups on daysld, 4d, P>0.05; But7dP<0.05; lOd and 14d P <0.001 ;21d P <0.01. The location of the expression of VEGFR1 protein was alike in both groups.6. Expression of VEGFR2 proteinThe location and model of the expression of VEGFR2 protein was similar to those of VEGFR1 in the control group. It was gradually reduced for the hyperox-ia exposed rats to express VEGFR2 protein in their lungs; There were no significant difference on Id, 4d with P>0.05 ; but on 7d P<0.01; lOd, 14d, 21 d P<0.001.7. Expression of VEGF mRNAIn the rat lungs of control group, the expression of VEGF mRNA was increased with age and reached its peak level at 14th day, consistent with the expression of VEGF protein. But in he hyperoxia exposed rat lungs, the expression of VEGF mRNA was reduced with age; on Id and 4d P >0.05; 7d, lOd, 14d, 21d P <0. 001, respectively.8. Expression of HIF - la mRNAIn the control group rat lungs, the expression of HIF - 1 a mRNA seemed to be no evident change with age. However, on the lth day, the level of expression of HIF -1 a mRNA was reduced significantly compared with that of the control group at the same time point, with P=0.01 ; 4d,7d,10d,14d P<0.001; 21 d P <0. 01, respectively.9. Expression of TIE - 2 proteinIn the newborn rat lungs of control group, the expression of TIE - 2 protein was mainly located in the distant alveolar interstitial tissue, in microvascular en-dothelium around the bronchial inner lining, which seemed to maintain a relatively constant extent through days 1 to 21 after birth; there were no significant differences among each time point ( P > 0. 05 ). But the expression level of TIE- 2 protein became less than that of the control group after the 7 th day hyperoxia treatment in the rat lungs; compared with the same time points, on Id and 4d P>0.05, buton7dP<0.05,10 P <0.01,14 P <0.01, 21dP<0.05, respectively.10. Expression of ANG - 1 mRNAIn control group lungs, the relative values of the expression of ANG - 1 mRNA did not change markedly through Id to 21 d after birth, there were no significant differences among all time points with P >0.05. Hyperoxia exposure did not make the level of the expression of ANG - 1 mRNA evidently changed during the test period; there were no statistical differences between the two groups at any time point.