Effect Of Lung Specific VEGF Inhibition On Lung Development And Function

VEGF-A,a member of VEGF family,functions by binding to its receptors VEGFR-1 and VEGFR-2.VEGF regulates microvascular endothelial cells proliferation and differentiation.It contributes to angiogenesis and survival of endothelial cells.Pulmonary is an organ with abundant in blood vessels and essential for adult life.In the study,a mouse model,phSPC-rtTRtg/+/VEGFtetO/tetO is generated and allows us to reversibly inhibit VEGF transcription in the lung through doxycycline food.Tissue-specific inhibition of VEGF is used to mimic abnormal expression of VEGF in lung.We divided mice into two groups,developing mice and adult mice,then each group subdivided into two small groups(doxycycline food and regular food).After several days of feeding,we determined relative transcript level by real time PCR.In adult lung,rtTR ?Ct gene expression level is tg(tg/+): 22.05±0.14,wt(+/+): 25.86±0.03,P(0.028)<0.05,vegf?Ct expression level is(tg/+)1: 23.79± 0.02,(tg/+)2: 22.47±0.036,wt(+/+): 21.93±0.006,P1(0.004)<0.01,P2(0.005)<0.01.VEGF protein level was determined by western blot.It is down regulated comparing with wild type mice but no inhibition in kidney.In developing mice lung,vegf expression level is tg1: 22.01±0.085,tg2: 21.86±0.071,wt: 21.6± 0.02,P1(0.055)>0.05,P2(0.176)>0.05.According to this data,we can sure phSPC-rtTRtg/+/ VEGFtetO/tetO model inhibits VEGF specifically in lung and mimic abnormal biological situation of lung.We analyzed other relative gene expression by Real time PCR with VEGF down regulation.In adult mice lung,vegfr-1 ?Ct expression level is tg(tg/+)1: 24.41±0.178,tg(tg/+)2: 24.98±0.004,wt(+/+):24.15±0.06,P1(0.462)>0.05,P2(0.043)<0.05.vegfr-1 expression is decreased with VEGF inhibition,but there exists individual variation.vegfr-2 ? Ct expression is not affected.Sp-c expression level is tg(tg/+)1:18.89±0.018,tg(tg/+)2:16.63±0.012,wt(+/+): 15.8±0.113,P1(0.027)<0.05,P2(0.077)>0.05,it is also affected by VEGF inhibition,but there exists individual variation.In developing mice lung,vegfr-1 expression is not changed,but vegfr-2 expression is decreased and the level is tg(tg/+): 24.43±0.025,wt(+/+): 23.95±0.022,P(0.004)<0.05).Sp-c expression is not affected by VEGF inhibition.Histological analysis(H&E)staining showed some disturbance in alveolarization in mature lung,and increasing lung mesenchymal cells,incrassation of alveolar septum and enlargement of air space.VEGF down regulation makes bronchus epithelial cells maldistribution and villus be short.It may indicate VEGF participates in maintaining of mature pulmonary structure.In developing mice lung,compared with control group,we find that transgenic mice lung alveolarization is more sufficient and bronchus is well developed and it's epithelial cells is equally distributed,villus is short and Vascular muscle layer is thickened.It suggests VEGF can limit lung development.Moreover,we tested phSPC-rtTRtg/+/VEGFtetO/tetO mice respiratory rate and we observed that the mice breath becomes shortness and increases breathing rate.It shows that VEGF is contributing to respiratory.Conclusion.(1)When VEGF is inhibited,there are some structural changes in histology.It means VEGF is a significant factor to lung development and maturity,and the function of VEGF is different with the developing stage.Thus we can prognosis some disease based on VEGF expression level.(2)VEGF affects mouse respiratory system and may be associated with airway inflammations.