Pharmacology Study of the Multiple Angiogenesis Inhibitor RC28-E on Anti-Fibrosis in a Chemically Induced Lung Injury Model

Abstract

Background: Disease-related injury in any organ triggers a complex cascade of cellular and molecular responses that culminate in tissue fibrosis, inflammation, and angiogenesis simultaneously. Multiple cell angiogenesis is an essential part of the tissue damage response, which is involved in fibrosis development. RC28-E is a novel recombinant dual decoy receptor lgG1 Fc-fusion protein that can block vascular endothelial growth factor (VEGFA), platelet-derived growth factor (PDGF), and fibroblast growth factor-2 (FGF-2) simultaneously. This protein has stepped into clinical trials (NCT03777254) for the treatment of pathological neovascularization-related diseases. Here, we report on the role of RC28-E during anti-fibrosis and its potential multitarget function in regulating fibrosis.

Methods: A bleomycin-induced pulmonary fibrosis C57BL/6 mouse model was established. Hematoxylin and eosin staining (HE) and Masson staining (Masson's) were performed to evaluate the pulmonary fibrosis based on the scoring from, Ashcroft score. Fibrosis related factors and inflammatory cytokines including HYP, α-SMA, procollagen, ICAM, IL-6, IL-1, and TNF-α were also determined at the protein and mRNA levels to characterize the fibrosis. Both mRNA and protein levels of VEGF, FGF, and transforming growth factor (TGF)-β were detected by quantitative real-time PCR (qRT-PCR) and immunohistochemical (IHC) analysis, respectively. Pulmonary fibrosis and related cytokines were re-evaluated in vivo after 3 doses of RC28-E (5 mg/kg, 15 mg/kg, and 50 mg/kg, ip. Tiw × 9) in comparison with a mono-target antagonist treatment (VEGF or FGF blocking). RC28-E attenuated the activation of TGF-β induced fibroblasts in vitro. Expression levels of α-SMA and collagen I, as well as proliferation and migration, were determined with the human skin fibroblast cell line Detroit 551 and primary murine pulmonary fibroblast cells. The mechanism of RC28-E via the TGF-β/Smad pathway was also investigated.

Results: RC28-E exhibits significant anti-fibrosis effects on Idiopathic pulmonary fibrosis (IPF) in vivo. Moreover, TGF-β induced fibroblast activation in vitro via the inhibition of the TGF-β downstream Smad pathway, thus providing potential therapeutics for clinical disease-related fibrosis-like IPF as well as chemotherapy-induced fibrosis in cancer therapy.

Keywords: FGF; VEGF; angiogenesis; anti-fibrosis; lung injury; pharmacology study.

Conflict of interest statement

The authors declare that they have no conflict of interest concerning this article.

Figures

Figure 1

Schematic diagram of RC28 molecular design. VEGFR1: vascular endothelial growth factor receptor 1; FGFR1: fibroblast growth factor receptor 1.

Figure 2

Pathological changes in lung tissue and the level of inflammatory cytokine in model of mice after bleomycin-induced pulmonary fibrosis formation. (A) The lung section from the mice that have undergone different treatments were stained by hematoxylin–eosin (HE) (n = 4 per group). (B) Lung sections from various treatment groups were subjected to Masson trichrome staining (n = 4 per group). (C) The expression of α-SMA was determined by using immunohistochemical analysis. Scale bar = 200 μm for each picture (original magnification: ×100, n = 4 per group). (D) The lung fibrosis score based on the scoring from Ashcroft score. (E) Quantitative analysis of α-SMA positive area. (F) Hydroxyproline concentration in lung tissue was detected by using alkaline hydrolysis (n = 4 per group). (G) Change of α-SMA, procollagen, and ICAM transcripts were analyzed by qRT-PCR (n = 6 per group). (H) The expression of IL-6, IL-1, and TNF-α was examined by using ELISA in the lung and serum. All data are expressed as the mean ± SD. #p < 0.05, ##p < 0.01 versus control.

Figure 3

Expression levels of VEGF, FGF, and TGF-β in lung of mice after bleomycin-induced pulmonary fibrosis formation. (A–C) The VEGF, FGF, and TGF-β expression levels in the control mice and modeling mice were determined by immunohistochemistry assay. Scale bar = 200 μm for each picture (original magnification: X100, n = 4 per group). Quantitative analysis of VEGF, FGF, and TGF-β positive area. Densitometry data are shown as mean ± SD. (D) The levels of VEGF, FGF, and TGF-β mRNA in lung tissue were detected by qRT-PCR (n = 6 per group). Results are shown as mean ± SD. #p < 0.05, ##p < 0.01 versus control.

Figure 4

Effect of RC28-E on VEGF, FGF, and TGF-β expression in bleomycin-induced fibrosis formation. (A–C) Immunohistochemistry was used to examine VEGF, FGF, and TGF-β expression in lung tissues. Scale bar = 200 μm for each picture (original magnification: ×100). (D–F) The change of VEGF, FGF, and TGF-β transcripts were analyzed by qRT-PCR. (n = 4 per group). Results are shown as mean ± SD. * p < 0.05, ** p < 0.01 versus model alone. #p < 0.05, ##p < 0.01 versus control.

Figure 5

The effect of RC28-E on the development of fibrosis in a therapeutic mouse bleomycin model. (A) The lung sections from the mice that have undergone different treatments were stained by hematoxylin–eosin (HE). (B) Lung sections from various treatment groups were subjected to Masson trichrome staining. (C) Immunohistochemistry was used to examine α-SMA expression in pulmonary tissues. Scale bar = 200 μm for each picture (original magnification: ×100, n = 4 per group). (D) The lung fibrosis score based on the scoring from Ashcroft score. (E) Quantitative analysis of α-SMA positive area. (F) The effect of RC28-E on hydroxyproline concentration in lung tissue. (G–I) The expression of α-SMA, procollagen, and ICAM mRNA were detected by qRT-PCR. (J–L) IL-1, IL-6, and TNF-α ELISA kit were used to detect the IL-1, IL-6, and TNF-α expression in the bleomycin induced lung tissue. Results are shown as mean ± SD. * p < 0.05, ** p < 0.01 versus model alone. #p < 0.05, ##p < 0.01 versus control.

Figure 6

RC28-E attenuates TGF-β-induced fibroblast activation. (A–D) Human skin fibroblasts line Detroit 551 were treated with RC28-E (2400 nM) or 2400 nM RC28-C1 (VEGF-trap) and RC28-C2 (FGF-trap) in the presence hTGF-β1 (10 ng/mL) for 72 h. (A) The protein expression levels of α-SMA and collagen I were examined by western blot and GAPDH as loading control. Quantitative data were from western blot analysis (n = 3). (B) The expression of α-SMA was detected by immunofluorescence assay. Nuclei were stained with DAPI (blue). Scale bar = 275 μm for each picture (original magnification: ×200). (C,D) Changes of α-SMA and procollagen transcripts were analyzed by qRT-PCR. (E) cells were treated with 1200, 1600, 2000, and 2400 nM RC28-E or 1200, 1600, 2000, and 2400 nM RC28-C1 (VEGF-trap) or 1200, 1600, 2000, and 2400 nM RC28-C2 (FGF-trap), with hTGF-β1 (10 ng/mL) as the control, for 48 h. Relative cell proliferation was determined by using cell counting kit-8. (F) Transwell was used to migration analysis, the migrated cells were stained with crystal violet. Results are shown as mean ± SD. * p < 0.05, ** p < 0.01 versus hTGF-β1 alone. #p < 0.05, ##p < 0.05 versus control (n = 3).

Figure 7

RC28-E downregulated protein levels of VEGF and FGF and inhibited the phosphorylation activity of the downstream signaling molecules Smad in human skin fibroblasts. (A–E) Human skin fibroblasts Detroit 551 were pretreated with RC28-E (2400 nM) for 1 h, followed by the addition of hTGF-β1 into the culture medium to a final concentration of 10 ng/mL and incubation for 72 h. (A,B) The protein levels of VEGF and FGF were examined by ELISA. (C,D) The expression of VEGF and FGF mRNA were analyzed by qRT-PCR. (E) The expression of P-smad2, P-smad3, and smad2/3 was determined using western blot and GAPDH as loading control. Representative gel electrophoresis bands are shown, and the expression levels of proteins were quantified by densitometry and normalized to the expression of smad2/3. Densitometry data are shown as mean ± SD. * p < 0.05, ** p < 0.01 versus hTGF-β1 alone. #p < 0.05, ##p < 0.05 versus control (n = 3).