Notch Signaling And Bleomycin-induced Pulmonary Fibrosis In Rats

Background：Pulmonary microvascular endothelial cells (PMVECs), an importantcomponent of the air-blood barrier, play an important role in maintainingfunctional vascular structure and repair after lung injury. In the process ofpulmonary fibrosis (PF), the activation of fibroblasts (Fbs) and myofibroblasts(MFbs) play a critical role in the excessive deposition of extracellular matrixcomponents. It has been confirmed that the lung MFbs had four sources: themesenchymal Fbs transform into MFbs by inflammatory cells secretingcytokines; bone marrow stem cells transition; alveolar epithelial cellsmesenchymal transition (EMT) and vascular endothelial mesenchymal transition(EndoMT). The role of EndoMT in pulmonary fibrosis has been widelyconcerned by now.Previous studies showed that PF patients and experimental animal modelsof lung fibrosis demonstrated an uneven distribution of microvasculature:abnormal neovascularizition around the fibrotic foci and regression in thecentral area. The proliferative PMVECs expressed an insufficient differentiation and poor perfusion. Our previous studies showed that PMVECs secretepro-fibrotic cytokines, such as transforming growth fctor β1(TGF-beta1) andconnective tissue growth factor (CTGF), and induce the expression ofalpha smooth muscle actin (α-SMA) of MFbs. The regulation of this processwas still unknown.The Notch signainling pathway was widely involved in organismdevelopment, cell differentiation and homeostasis. Discovered in recent years,Notch played a crucial role in the regulation of ECs’ proliferation anddifferentiation through inhibiting the expression of vascular endothelial cellreceptor2(VEGF2/Flk-1/KDR). There was also reported that Notch signallingrelated to EndoMT.In summary, MFbs, the landmark of pulmonary fibrosis, had diversedsources including EndoMT. Based on the role of Notch signaling in theregulation of microvascular, we assumed that BLM may reduce or injure Notchsignaling in multiple cells. This may also be the premise and basis for EndoMTand MFbs differentiation of PMVECs and Fbs.Method and Purpose:In this study, we injected BLM-intratracheal induced PF model of rats andinjected with sterile saline as the control animals. After euthanized at7,14dayspost-instillation, the peripheral lung tissue was cut for primary cell culture ofPMVECs and Fbs. The remanent lung tissue was prepaired for histologicalanalysis.Lung tissue was detected in the immunohistochemical staining of Hes1andKDR; immunofluorescence tested the expression of PCNA in PMVECs;Western blot detected Notch1, Hes1, Jag1, KDR and Dll4proteins expression;real-time quantitative PCR (Real-time PCR) detected the mRNA level of Notch1, Hes1, KDR, Dll4, Jag1ssand CTGF, combined with the expression of PCNA,CTGF and-SMA protein. We performed to analyze the role of the Notchsignaling pathway in the process of pulmonary fibrosis and the effect onproliferation, secretion, and differentiation of PMVECs and Fbs.Result:1. Immunofluorescence and Western Blot revealed a high expression of PCNAand-SMA protein in PMVECs in BLM group and co-expression ofα-SMA andVWF in lung tissue, confirmed the proliferative status and phenotype change ofPMVECs.2. Immunohistochemistry, Western blot and Real-time PCR showed that thePMVECs in BLM group downregulated Hes1and Dll4; upregulated KDR andJag1, which indicated the repression of Notch signaling pathway and abnormalexpression of Notch ligands in PMVECs in pulmonary fibrosis.3. Western blot revealed downregulation of Hes1and upregulation of Jag1,accompanied with high expression of PCNA and-SMA, indicated theinhibition of Notch signaling might related to the production of MFbs.Conclusion:1. EndoMT of PMVECs and differentiation of MFbs in Fbs might be one ofthe important reasons of BLM-induced pulmonary fibrosis in rats.Inhibition ofNotch signaling pathway might be the basis of abnormal proliferative cells(PMVECs and Fbs) transforming into MFbs.2. The overexpression of Jag1and low expression of Dll4might be the causeof abnormal VEGF-Notch-KDR negative feedback regulatory pathway ofPMVECs in pulmonary fibrosis.3. The high expression level of Jag1might related to the proliferation anddifferentiation of Fbs.