Fibroblast growth factor 10 attenuates chronic obstructive pulmonary disease by protecting against glycocalyx impairment and endothelial apoptosis

Abstract

Background: The defects and imbalance in lung repair and structural maintenance contribute to the pathogenesis of chronic obstructive pulmonary diseases (COPD), yet the molecular mechanisms that regulate lung repair process are so far incompletely understood. We hypothesized that cigarette smoking causes glycocalyx impairment and endothelial apoptosis in COPD, which could be repaired by the stimulation of fibroblast growth factor 10 (FGF10)/FGF receptor 1 (FGFR1) signaling.

Methods: We used immunostaining (immunohistochemical [IHC] and immunofluorescence [IF]) and enzyme-linked immunosorbent assay (ELISA) to detect the levels of glycocalyx components and endothelial apoptosis in animal models and in patients with COPD. We used the murine emphysema model and in vitro studies to determine the protective and reparative role of FGF10/FGFR1.

Results: Exposure to cigarette smoke caused endothelial glycocalyx impairment and emphysematous changes in murine models and human specimens. Pretreatment of FGF10 attenuated the development of emphysema and the shedding of glycocalyx components induced by CSE in vivo. However, FGF10 did not attenuate the emphysema induced by endothelial-specific killing peptide CGSPGWVRC-GG-_D(KLAKLAK)₂. Mechanistically, FGF10 alleviated smoke-induced endothelial apoptosis and glycocalyx repair through FGFR1/ERK/SOX9/HS6ST1 signaling in vitro. FGF10 was shown to repair pulmonary glycocalyx injury and endothelial apoptosis, and attenuate smoke-induced COPD through FGFR1 signaling.

Conclusions: Our results suggest that FGF10 may serve as a potential therapeutic strategy against COPD via endothelial repair and glycocalyx reconstitution.

Keywords: Chronic obstructive pulmonary diseases (COPD); Endothelial apoptosis; Fibroblast growth factor 10 (FGF10); Fibroblast growth factor receptor 1 (FGFR1); Glycocalyx.

Fig. 1

Exposure to cigarette smoke causes endothelial apoptosis, glycocalyx impairment and emphysema in mouse. Mice were exposed to air or cigarette smoke for 1 month or 3 months. A Representative haematoxylin and eosin (H&E) staining images of airspace. Alveolar size was measured by mean linear intercept (MLI) (n = 6). Scale bar = 100 μm. B Representative immunofluorescence staining images of cleaved caspase-3 in mice lungs. Nuclei were visualized with 4ʹ,6-diamidino-2-phenylindole (DAPI). White arrowheads indicate the apoptotic endothelial cells. Quantitative analysis of percentage of apoptotic endothelial cells was performed (n = 6). Scale bar = 20 μm. C Representative immunofluorescence staining images of mouse lung sections. Chondroitin sulfate and syndecan-1 antibodies were used as glycocalyx specific antigens, and CD31 was used as the mark of endothelial cell. Nuclei were visualized with DAPI. Scale bar = 20 μm. D Quantitative analysis of fluorescence intensity for heparan sulfate, chondroitin sulfate, syndecan-1 and syndecan-4 (n = 6). *P < 0.05, **P < 0.01. n.s., not significant

Fig. 2

Endothelial glycocalyx impairment is involved in SU5416-induced rat emphysema model. Male Sprague-Dawley rats (~ 300 g) were treated with VEGF-R antagonist SU5416 (20 mg/kg) subcutaneously 3 times per week for 3 weeks. A Representative haematoxylin and eosin (H&E) staining images of airspace. Scale bar = 50 μm. Alveolar size was measured by mean linear intercept (MLI) (n = 5). B Cleaved caspase-3 antibody was used as the mark of cell apoptosis. Group data of cleaved caspase-3 positive cells per field (n = 5). Scale bar = 50 μm. C Representative immunofluorescence staining images of rat lung sections. Heparan sulfate and Chondroitin sulfate antibodies were used as glycocalyx specific antigens, and CD31 was used as the mark of endothelial cell. Nuclei were visualized with 4ʹ,6-diamidino-2-phenylindole (DAPI). Scale bar = 20 μm. Quantitative analysis of fluorescence intensity for heparan sulfate, chondroitin sulfate and syndecan-1 (n = 5). D ELISA analysis of blood and BALF samples (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant

Fig. 3

Endothelial glycocalyx is impaired in COPD patients. A Representative immunofluorescence staining images of heparan sulfate and chondroitin sulfate in non-COPD and COPD lungs. Heparan sulfate and chondroitin sulfate antibodies were used as glycocalyx specific antigens, and CD31 was used as the mark of endothelial cell. Nuclei were visualized with 4ʹ,6-diamidino-2-phenylindole (DAPI). Scale bar = 50 μm. Quantitative analysis of fluorescence intensity was performed (n = 7–10). B Serum heparan sulfate and chondroitin sulfate in subjects detected by ELISA analysis (n = 25 in non-COPD, n = 14 in COPD, n = 26 in non-smoker, n = 13 in smoker). Correlations of circulating heparan sulfate and chondroitin sulfate levels with lung function parameters (the ratio of FEV1/FVC and FEV1% predicted) in all subjects (n = 39) were analysed using Pearson correlation test. *P < 0.05. n.s., not significant

Fig. 4

FGF10 attenuates the development of emphysema induced by cigarette smoke extract in mice. A Male C57BL/6 mice (6–8 weeks) were treated with 400 μl cigarette smoke extract (CSE) intraperitoneally once a week for 4 weeks, with different doses of FGF10 administration (10 μg/kg, 100 μg/kg, 1 mg/kg, 2.5 mg/kg) intravenously 24 h before CSE injection. All mice were sacrificed at Day 28. Representative haematoxylin and eosin (H&E) staining images of airspace. Scale bar = 50 μm. Alveolar size was evaluated by mean linear intercept (MLI) (n = 10). B Mice were administered standard saline solution or 240 μg of KLAKLAK₂ intraperitoneally once per week for 4 weeks, with different doses of FGF10 administration intravenously 24 h prior to CSE injection. Representative H&E staining images of airspace. Scale bar = 50 μm. Alveolar size was evaluated by mean linear intercept (MLI), (n = 8–10). C Serum glycocalyx component levels in mice from each group detected by ELISA (n = 6–8). *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant

Fig. 5

FGF10 increases the intensity of glycocalyx components in murine COPD model. Representative immunofluorescence staining images of heparan sulfate (A), chondroitin sulfate (C) and syndecan-1 (E) in mice lungs. Heparan sulfate, chondroitin sulfate and syndecan-1 antibodies were used as glycocalyx specific antigens, and CD31 was used as the mark of endothelial cell. Nuclei were visualized with 4ʹ,6-diamidino-2-phenylindole (DAPI). Scale bar = 50 μm. B, D, F Quantitative analysis of fluorescence intensity was performed (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant

Fig. 6

FGF10 attenuates cigarette smoke-induced endothelial apoptosis and glycocalyx repair through FGFR1 signaling. A The human pulmonary microvascular endothelial cells (hPMVECs) were treated with CSE (24 h, 2%), with or without pretreatment of FGF10 (2 h prior to CSE stimulation, 50 ng/ml) and the FGFR1 inhibitor, AZD4547 (24 h prior to CSE stimulation, 20 nM). TNF-α + SM-164 serves as positive controls (24 h, 1:500). After 24 h stimulation of 2% CSE, hPMVECs were performed for flow cytometry (n = 3). Annexin V/PI-stained cells showing apoptotic rates. FITC, fluorescein isothiocyanate; PI, propidium iodide. B Relative FGFR1 mRNA expression in hPMVECs from each group (n = 3). C Relative mRNA expression of FGFR1 and FGFR2 in hPMVECs (n = 3). D Representative western blots and corresponding group data showing expression levels of the total and phosphorylation of FGFR1. E Representative western blots and corresponding group data showing expression levels of the total and phosphorylation of ERK and AKT in hPMVECs. F Venn diagram of overlapping putative genes related both to cell apoptosis pathway and COPD, from “GO_Apoptosis_Signaling” (http://www.broadinstitute.org/gsea/index.jsp), and “New loci”, “Loci previously described” and “Candidates”. G Relative SOX9 mRNA expression in hPMVECs from each group (n = 3). H Endothelial cells were transfected with si-control and si-SOX9 for 48 h, stimulated beforehand with FGF10 (100 ng/ml) for 2 h, and then with CSE (2%) for another 24 h. Endothelial cells were performed for flow cytometry (n = 3). I Prediction of the combination site of SOX9 on the promoter region of HS6ST1. Relative HS6ST1 mRNA expression in hPMVECs from each group (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant

Fig. 7

Schematic concept of the FGF10/FGFR1/ERK/SOX9/HS6ST1/HS loop in glycocalyx synthesis and lung repair. A model could be suggested in which cigarette smoke impairs endothelial glycocalyx and FGFR1 signaling, while FGF10 attenuates the development of emphysema and the shedding of glycocalyx induced by CSE in animal models. Mechanistically, FGF10 alleviates smoke-induced endothelial apoptosis and glycocalyx repair through a FGFR1/ERK/SOX9/HS6ST1 loop