Endostatin, an angiogenesis inhibitor, ameliorates bleomycin-induced pulmonary fibrosis in rats

Abstract

Background: Recent evidence has demonstrated the role of angiogenesis in the pathogenesis of pulmonary fibrosis. Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis. The aim of our study was to assess whether endostatin has beneficial effects on bleomycin (BLM)-induced pulmonary fibrosis in rats.

Methods: The rats were randomly divided into five experimental groups: (A) saline only, (B) BLM only, (C) BLM plus early endostatin treatment, (D) BLM plus late endostatin treatment, and (F) BLM plus whole-course endostatin treatment. We investigated the microvascular density (MVD), inflammatory response and alveolar epithelial cell apoptosis in rat lungs in each group at different phases of disease development.

Results: Early endostatin administration attenuated fibrotic changes in BLM-induced pulmonary fibrosis in rats. Endostatin treatment decreased MVD by inhibiting the expression of VEGF/VEGFR-2 (Flk-1) and the activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). Endostatin treatment also decreased the number of inflammatory cells infiltrating the bronchoalveolar lavage fluid during the early inflammatory phase of BLM-induced pulmonary fibrosis. In addition, the levels of tumour necrosis factor-α (TNF-α) and transforming growth factor β1 (TGF-β1) were reduced by endostatin treatment. Furthermore, endostatin decreased alveolar type II cell apoptosis and had an epithelium-protective effect. These might be the mechanism underlying the preventive effect of endostatin on pulmonary fibrosis.

Conclusions: Our findings suggest that endostatin treatment inhibits the increased MVD, inflammation and alveolar epithelial cell apoptosis, consequently ameliorating BLM-induced pulmonary fibrosis in rats.

Figure 1

Effect of endostatin on BLM-induced pulmonary fibrosis. (A) Representative images of hematoxylin and eosin (H&E) and (B) Masson trichrome –stained sections of rats in each experimental group on day 28 (×200). (C) Comparison of the Ashcroft score among the experimental groups. (D) Collagen deposition was assessed by measuring the hydroxyproline content. Bar = 100μm. Results are expressed as mean ± SD, n = 5 in each group, *P < 0.001 vs SA group; #P < 0.05 vs BLM group; △P < 0.01 vs BLM group; +P < 0.001 vs BLM group.

Figure 2

Effect of endostatin on the inflammatory response in BLM-induced pulmonary fibrosis. Changes in (A) total cell count, (B) neutrophils, (C) lymphocytes, (D) macrophages and (E) TNF-α expression in BALF of rats at each stage were presented. (F) Representative PPI change, calculated as BALF total protein/plasma total protein × 100. Results are expressed as mean ± SD, n = 4 or 5 in each group, *P < 0.001 vs SA group; $P < 0.05 vs SA group; #P < 0.05 vs BLM group; +P < 0.001 vs BLM group.

Figure 3

Effect of endostatin on MVD in BLM-induced pulmonary fibrosis. Immunohistochemical staining of CD31 in lung sections in SA (A), BLM (B) and E-ES (C) groups on day 7 (×200). (D) MVD was assayed by counting the number of the microvessels per high power field in lung sections stained with antibody CD31. Bar = 100μm. *P < 0.001 vs SA group; #P < 0.01 vs BLM group; +P < 0.001 vs BLM group.

Figure 4

Effect of endostatin on VEGF/Flk-1 mRNA expression in BLM-induced pulmonary fibrosis. (A) VEGF mRNA and (B) Flk-1 mRNA expression were measured by real-time PCR. Results are expressed as mean ± SD, n = 5 in each group, *P < 0.001 vs SA group; $P < 0.05 vs SA group; #P < 0.05 vs BLM group; +P < 0.001 vs BLM group.

Figure 5

Effect of endostatin on VEGF/Flk-1 protein expression in BLM-induced pulmonary fibrosis. (A) VEGF concentration in lung tissue lysates and Flk-1 immunostaining scores (B) among different experimental groups were presented. Immunohistochemical staining of Flk-1 in lung sections in BLM (C) and E-ES (D) groups on day 7 (×200) were presented. Results are expressed as mean ± SD, n = 5 in each group, *P < 0.001 vs SA group; $P < 0.05 vs SA group; △P < 0.01 vs BLM group; #P < 0.05 vs BLM group; +P < 0.001 vs BLM group.

Figure 6

Effect of endostatin on expresion of TGF-β1 in lung tissue lysates. TGF-β1 concentrations in lung tissue lysates among different experimental groups were presented. Results are expressed as mean ± SD, n = 5 in each group, *P < 0.001 vs SA group; #P < 0.05 vs BLM group; +P < 0.001 vs BLM group.

Figure 7

Endostatin suppressed the ERK1/2 phosphorylation. (A) Western blot showed pERK1/2 protein expression among different experimental groups on days 3, 7, 14 and 28. (B) Quantitative densitometry analysis of western blot analysis for pERK1/2. Data are presented as the ratio pERK1/2 and ERK. Results are expressed as mean ± SD, n = 3 in each group, *P < 0.01 vs SA group; $P < 0.05 vs SA group; #P < 0.05 vs BLM group; +P < 0.001 vs BLM group.

Figure 8

Effect of endostatin on alveolar epithelial cell apoptosis. (A-D) Representative co-immunofluorescence for TUNEL (A), SP-C (B) and DAPI (C) identifies apoptotic alveolar type II cells (D, merge) from a lung section. (E-G) Representative fluorescent micrographs for in SA (E), BLM (F) and E-ES (G) groups on day 7 (×400) are shown. (F) Quantitative assessment of alveolar type II cell apoptosis among different experimental groups on days 7 and 28 were presented. Bar = 100μm. Results are expressed as mean ± SD, n = 5 in each group, *P < 0.001 vs SA group; #P < 0.05 vs BLM group; +P < 0.001 vs BLM group.