| Background Idiopathic pulmonary fibrosis is a chronic, relentlessly progressive fibrosing disease of the lung of unknown etiology. The prognosis of idiopathic pulmonary fibrosis, pathologically characterised by UIP, is not improved by available therapy. The investigation of the mechanism on medical therapy will provide us with new ideas for the clinical treatment of pulmonary fibrosis. Data have demonstrated that the expression of VEGF exists in a body of tissues, including chronic inflammatory tissue, fibrotic lung and cancers. VEGF is a homodimeric glycoprotein that is mitogenic for endothelial cells and is an angiogenic factor that acts via the endothelial-specific receptor tyrosine kinases (VEGF receptors, VEGFRs) VEGFR-1 (Fit-1) and VEGFR-2 (Flk-1). The development of blood vessels in the embryo is dependent on VEGF as the formation of vessels in mouse embryos heterozygous for a disrupted VEGF gene was aberrant and resulted in embryonic lethality. VEGF is also a potent inducer of vascular permeability. Additionally, VEGF can stimulate the expression of integrin and TGF-β1, and promote the recruitment of macrophage. The activity of VEGF remains to be investigated in pulmonary fibrosis. The application of SU5416, a VEGFR inhibitor, could contribute to determining the role of VEGF signaling in pulmonary fibrosis.Objective The aims of this study were: to investigate the expression of VEGF/VEGFR in murine BPF lung, to analyse pathologic characteristic of BPF lung tissue, to study the effect of SU5416 on fibrosis in BPF mice, and to demonstrate its mechanism by exploring the relationship between VEGF/VEGFR signaling and angiogenesis, TGF-β1/p-Smad3 signaling and recruitment of inflammatory cells.Methods Pulmonary fibrosis was induced by intratracheal administration of bleomycin in male KUNMING mice. Paraffin-embeded slices from the murine lung tissues were stained for the expression of VEGF, Flk-1 and CD31 (angiogenesis) by means of immunohistochemistry. The abundance of VEGF and Flk-1 mRNA was tested by real time quantative RT-PCR. The contents of p-Smad3 from lung tissues were evaluated by Western-blotting, and those of VEGF and TGF-β1 in BALF were determined by ELISA. The activity of LDH was tested by IFCC assay and the differential white cell counts were analyzed by Wright-Gimosa stainging in BALF. The contents of hydroproline were detected by spectrophotometry.Results (1) Hematoxylin and eosin staining showed that the Ashcroft scoresincreased in BPF mice compared with control, which could be inhibited by early SU5416 treatment.(2) Spectrophotometry and masson staining indicated that the contents of hydroxyproline and collagen increased in BPF mice compared with control, and could be inhibited by early SU5416 treatment.(3) IFCC assay data showed that the highest activity of LDH was tested in BPF mice on Day 7 compared with control, and gradually reduced on Day 14 and 28 (P <0.01). The high activity of LDH was significantly lowered by treatment with early SU5416 administration (P <0.01).(4) Western-blotting demonstrated that the highest contents of p-Smad3 were found in BPF mice on Day 7 compared with control, and gradually stepped down on Day 14 and 28 (P <0.01). The p-Smad3 was downregulated by early administration of SU5416 in BPF mice (P <0.01).(5) Immunohistochemistry and real time quantitative RT-PCR indicated that VEGF and Flk-1 overexpressed in BPF mice on Day 7. The abundance of mRNA and expression of protein were depressed, while the Ashcroft score, the contents of cllagen and hydroproline increased in BPF lung in a time-dependent manner (P <0.01). The VEGF and Flk-1 were significantly ameliorated in BPF mice treated with early SU5416 administration (P <0.01). Immunohistochemistry suggested that the highest angiogenesis (CD31) existed in BPF mice on Day 7, which was inhibited by early SU5416 administration, and gradually reduced on Day 14 and 28 (P <0.01).(6) ELISA assay demonstrated that the highest contents of VEGF and TGF-β1 were found in BPF mice on Day 7 compared with control, and gradually reduced on Day 14 and 28 (P<0.01). These proteins was downregulated by early administration of SU5416 in BPF mice (P<0.01).(7) Wright-Gimosa staining showed that the highest counts of WBC, neutrophil and macrophage in BPF mice ocurred on Day 7 compared with control, and gradually reduced on Day 14 and 28 (P<0.01). The high counts were significantly inhibited by early SU5416 administration (P<0.01).Conclusions (1) A pulmonary fibrosis model, that mimics IPF, is successively induced by intratracheal instillation of bleomycin and the overexpression of VEGF and Flk-1 is observed in bleomycin-induced pulmonary fibrosis on Day 7 compared with control.(2) Early administration of SU5416 significantly downregulates VEGF/VEGFR signaling in murine bleomycin-induced pulmonary fibrosis.(3) The Ashcroft scores, contents of collagen and hydroproline attenuated by early SU5416 administration are associated with downregulation of VEGF/VEGFR signaling.(4) These findings suggests that VEGF signaling contributes to the onset and development of pulmonary fibrosis, and demonstrates that this mechanism might be ideal strategies for targeted therapy of pulmonary fibrosis. The data suggest that the inhibtion of VEGF signaling may play a pivotal role in the progression of bleomycin-induced pulmonary fibrosis in mice via inhibiting TGF-β1/p-Smad3 signaling and recruitment of inflammatory cells.
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