Increased S100A4 expression in the vasculature of human COPD lungs and murine model of smoke-induced emphysema - PubMed (original) (raw)

doi: 10.1186/s12931-015-0284-5.

Ludger Fink 2 3, Jochen Wilhelm 4, Julia Hoffmann 5, Mariola Bednorz 6, Michael Seimetz 7, Isabel Dessureault 8, Roger Troesser 9, Bahil Ghanim 10, Walter Klepetko 11, Werner Seeger 12, Norbert Weissmann 13, Grazyna Kwapiszewska 14 15

Affiliations

Increased S100A4 expression in the vasculature of human COPD lungs and murine model of smoke-induced emphysema

Sebastian Reimann et al. Respir Res. 2015.

Abstract

Background: Chronic obstructive lung disease (COPD) is a common cause of death in industrialized countries often induced by exposure to tobacco smoke. A substantial number of patients with COPD also suffer from pulmonary hypertension that may be caused by hypoxia or other hypoxia-independent stimuli - inducing pulmonary vascular remodeling. The Ca(2+) binding protein, S100A4 is known to play a role in non-COPD-driven vascular remodeling of intrapulmonary arteries. Therefore, we have investigated the potential involvement of S100A4 in COPD induced vascular remodeling.

Methods: Lung tissue was obtained from explanted lungs of five COPD patients and five non-transplanted donor lungs. Additionally, mice lungs of a tobacco-smoke-induced lung emphysema model (exposure for 3 and 8 month) and controls were investigated. Real-time RT-PCR analysis of S100A4 and RAGE mRNA was performed from laser-microdissected intrapulmonary arteries. S100A4 immunohistochemistry was semi-quantitatively evaluated. Mobility shift assay and siRNA knock-down were used to prove hypoxia responsive elements (HRE) and HIF binding within the S100A4 promoter.

Results: Laser-microdissection in combination with real-time PCR analysis revealed higher expression of S100A4 mRNA in intrapulmonary arteries of COPD patients compared to donors. These findings were mirrored by semi-quantitative analysis of S100A4 immunostaining. Analogous to human lungs, in mice with tobacco-smoke-induced emphysema an up-regulation of S100A4 mRNA and protein was observed in intrapulmonary arteries. Putative HREs could be identified in the promoter region of the human S100A4 gene and their functionality was confirmed by mobility shift assay. Knock-down of HIF1/2 by siRNA attenuated hypoxia-dependent increase in S100A4 mRNA levels in human primary pulmonary artery smooth muscle cells. Interestingly, RAGE mRNA expression was enhanced in pulmonary arteries of tobacco-smoke exposed mice but not in pulmonary arteries of COPD patients.

Conclusions: As enhanced S100A4 expression was observed in remodeled intrapulmonary arteries of COPD patients, targeting S100A4 could serve as potential therapeutic option for prevention of vascular remodeling in COPD patients.

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Figures

Fig. 1

Fig. 1

S100A4 expression in mouse lungs after smoke-exposure. Immunohistochemical staining of S100A4 protein in a intrapulmonary vessels, b mononuclear cells, c smooth muscle cells of bronchus. d negative control. Bars represent 50 μm and 100 μm. e real-time PCR analysis of S100A4 from lung homogenates after three and eight months of smoke exposure compared to three and eight months control animals. 3 m-three months, 8 m-eight months, SE-smoke exposure

Fig. 2

Fig. 2

S100A4 localization and mRNA expression in intrapulmonary vessels in mouse lungs after smoke-exposure. Immunohistochemical staining of a S100A4, b α smooth muscle actin in serial section from mouse lungs, c negative control. Bars represent 50 μm. d real-time PCR analysis of S100A4 from laser-microdissected arteries after three and eight months of smoke exposure compared to control animals. 3 m-three months, 8 m-eight months, SE-smoke exposure. The two _p_-values indicate the results from the linear hypothesis about difference between SE and controls at eight months tested within a 2-factorial model (p = 0.123) and the result from testing the difference between SE at 8 month against the pooled controls (p = 0.023)

Fig. 3

Fig. 3

S100A4 protein expression in intrapulmonary vessels in mouse lungs after smoke-exposure. Representative images of intrapulmonary arteries from a control animals, b smoke exposed animals. Bars represent 50 μm and 200 μm. c semi-quantitative analysis of S100A4 protein expression in intrapulmonary arteries. Δ Intensity- color intensity of S100A4 protein after immunohistochemical staining. 8 m-eight months, SE-smoke exposure

Fig. 4

Fig. 4

S100A4 localisation and expression in human intrapulmonary arteries. Immonohistochemical staining of S100A4, α smooth muscle actin and negative control in intrapulmonary vessels of COPD patients and donor lungs. Bars represent 100 μm. b S100A4 expression in laser-microdissected arteries from donor and COPD lungs

Fig. 5

Fig. 5

S100A4 protein expression in intrapulmonary vessels of donor and COPD lungs. Representative images of intrapulmonary arteries from a control lungs, b COPD patients. Bars represent 100 μm and 500 μm. c semi-quantitative analysis of S100A4 protein expression in intrapulmonary arteries. Δ Intensity- color intensity of S100A4 protein after immunohistochemical staining

Fig. 6

Fig. 6

RAGE mRNA and protein expression in murine emphysema model and human COPD. a Representative images of intrapulmonary arteries from control and from 3 and 8 month smoke exposed animals. b Representative images of intrapulmonary arteries from control and COPD patients. Bars represent 100 μm. c RAGE expression in laser-microdissected arteries from donor and COPD lungs

Fig. 7

Fig. 7

Hypoxia-dependent S100A4 expression in human primary pulmonary arterial cells. a Real-time analysis of S100A4 expression in human PASMC after normoxia and hypoxia exposure (24 h). b Representative images of S100A4 immunofluorescence labeling in human PASMC after normoxia and hypoxia treatment. c Schematic representation of potential HRE in upstream and downstream sequence from S100A4 coding site. d EMSA analyses of potential HREs [HRE1:-4694, HRE2:857, HRE3:1359]. Slots were loaded as follows: 1: Labeled Probe only, 2: Labeled Probe and Nuclear extract (24 h normoxia), 3 Labeled Probe, Nuclear extract (24 h hypoxia), 4: Labeled Probe, Nuclear extract (24 h hypoxia) and Competitor. e Supershift analysis: 1: Labeled Probe only, 2: Labeled Probe and Nuclear extract (24 h hypoxia), 3 Labeled Probe, Nuclear extract (24 h hypoxia) and Competitor, 4: Labeled Probe, Nuclear extract (24 h hypoxia) and Antibody against HIF-1, 5: Labeled Probe, Nuclear extract (24 h hypoxia) and Antibody against HIF-2. Arrows represent specific band. f Real-time analysis of S100A4 expression after pretreatment of human PASMC with siRNA against HIF1, HIF2 or EGR1. ΔCt- relative expression of S100A4 to reference gene. siR- siRandom. g Immunofluorescence labeling of S100A4, SMC-actin and DAPI

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