Extracellular matrix defects in aneurysmal Fibulin-4 mice predispose to lung emphysema

Background: In this study we set out to investigate the clinically observed relationship between chronic obstructive pulmonary disease (COPD) and aortic aneurysms. We tested the hypothesis that an

Extracellular Matrix Defects in Aneurysmal Fibulin-4 Mice Predispose to Lung Emphysema

<div><p>Background</p><p>In this study we set out to investigate the clinically observed relationship between chronic obstructive pulmonary disease (COPD) and aortic aneurysms. We tested the hypothesis that an inherited deficiency of connective tissue might play a role in the combined development of pulmonary emphysema and vascular disease.</p><p>Methods</p><p>We first determined the prevalence of chronic obstructive pulmonary disease in a clinical cohort of aortic aneurysms patients and arterial occlusive disease patients. Subsequently, we used a combined approach comprising pathological, functional, molecular imaging, immunological and gene expression analysis to reveal the sequence of events that culminates in pulmonary emphysema in aneurysmal Fibulin-4 deficient (Fibulin-4^R) mice.</p><p>Results</p><p>Here we show that COPD is significantly more prevalent in aneurysm patients compared to arterial occlusive disease patients, independent of smoking, other clinical risk factors and inflammation. In addition, we demonstrate that aneurysmal Fibulin-4^R/R mice display severe developmental lung emphysema, whereas Fibulin-4^+/R mice acquire alveolar breakdown with age and upon infectious stress. This vicious circle is further exacerbated by the diminished antiprotease capacity of the lungs and ultimately results in the development of pulmonary emphysema.</p><p>Conclusions</p><p>Our experimental data identify genetic susceptibility to extracellular matrix degradation and secondary inflammation as the common mechanisms in both COPD and aneurysm formation.</p></div

Clinical characteristics of patients with aortic aneurysm (AA) or arterial occlusive disease (AOD).

<p>Clinical characteristics of patients with aortic aneurysm (AA) or arterial occlusive disease (AOD).</p

Higher MMP and NE activity in Fibulin-4^+/R and Fibulin-4^R/R lungs.

<p><i>In</i> (A) and (C) <i>ex vivo</i> imaging of excised lungs using Odyssey shows increased activity of MMP and NE respectively in Fibulin-4^+/R (n = 7) and Fibulin-4^R/R lungs (observed for n = 5, but two animals died during the procedure) as compared to Fibulin-4^+/+ lungs (n = 5), with a significant upregulation for Fibulin-4^R/R lungs (**p<0.01). (B) Open-chest registration of NE activity with Neutrophil Elastase FAST 680 probes shows increased activity in Fibulin-4^+/R and Fibulin-4^R/R lungs as compared to Fibulin-4^+/+ lungs. (D) Elastin staining of Fibulin-4^+/+, Fibulin-4^+/R and Fibulin-4^R/R lungs (n = 3, n = 3, n = 3) shows fragmented elastin layers in Fibulin-4^+/R and Fibulin-4^R/R lungs, indicated by arrows. Magnification 40x. Scale bar 10 µm.</p

Increased and prolonged inflammatory response in LPS exposed Fibulin-4^+/R mice.

<p>Quantification of immune cells shows significantly increased F4/80+ cells after 18 hours of LPS exposure in Fibulin-4^+/R lungs (n = 4) and a significantly higher number of Gr1+, CD11c+, and CD3+ cells after 72 hours of LPS exposure as compared to Fibulin-4^+/+ lungs (n = 4, *p<0.05).</p

Enlarged alveolar airspaces in lungs of Fibulin-4 knockdown mice.

<p>(A) Expression levels of Fibulin-4 in lungs isolated from newborn (n = 4, n = 4, n = 3) and adult (n = 4, n = 4, n = 4) Fibulin-4^+/+, Fibulin-4^+/R and Fibulin-4^R/R mice relative to Fibulin-4^+/+ lungs (**p<0.01). (B) Mean peak inspiratory flow (PIF) and peak expiratory flow (PEF) values for Fibulin-4^+/+ (n = 4), Fibulin-4^+/R (n = 4) and Fibulin-4^R/R mice (observed for n = 4, but two animals died during the procedure) at 3-minute intervals. After a 9 minute adaptation period (the first three time intervals), PIF follows similar trends in Fibulin-4^+/+, Fibulin-4^+/R and Fibulin-4^R/R mice, while Fibulin-4^R/R mice show a decrease in PEF. (C) HE stained sections of formalin fixed lungs of male mice. Enlarged alveolar airspaces are observed in Fibulin-4^+/R (middle, n = 3) and Fibulin-4^R/R lungs (right, n = 3), with the latter being more pronounced, compared to Fibulin-4^+/+ (n = 3). Enlarged alveolar airspaces are already present in Fibulin-4^R/R newborn lungs (n = 3), while lungs of Fibulin-4^+/R littermates (n = 5) show no difference compared to Fibulin-4^+/+ lungs (n = 4). Scale bar 100 µm. Magnification 10x. (D) D₂ quantification (see methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106054#pone.0106054.s001" target="_blank">Figure S1</a> for further explanation) of the alveolar airspaces revealed a significant difference between adult Fibulin-4^+/+ and Fibulin-4^+/R (*p<0.05) and between adult Fibulin-4^+/+ and Fibulin-4^R/R lungs (**p<0.01) as well as between newborn Fibulin-4^+/+ and Fibulin-4^R/R lungs (*p<0.05).</p

Increased inflammation in Fibulin-4^R/R lungs compared to Fibulin-4^+/+ and Fibulin-4^+/R lungs.

<p>(A) Flow cytometric analysis revealed more Gr1+ granulocytes and CD19+ B-cells in BAL samples from Fibulin-4^R/R (n = 4) compared to Fibulin-4^+/+ mice (n = 4, *p<0.05) and (B) increased numbers of F4/80 macrophages in Fibulin-4^R/R lungs (n = 4, *p<0.05). (C) HE stained sections from adult (n = 4, n = 4, n = 4) Fibulin-4^+/+, Fibulin-4^+/R and Fibulin-4^R/R lungs showing focal infiltrations around vessels and airways in Fibulin-4^R/R lungs (black arrows). (D) Staining for T-cells (CD3+) and (E) dendritic cells (CD11c+) points to the presence of inflammatory cells within the focal infiltrations. Magnification 20x. Scale bar 50 µm. (F) ELISA analysis showing increased IL-1β levels in Fibulin-4^R/R lungs (n = 4, *p<0.05).</p

Prevalence and severity of COPD in patients with an aortic aneurysm (AA) or arterial occlusive disease (AOD).

<p>(A) The prevalence of COPD in all GOLD classes was higher in AA (n = 614) compared to AOD patients (n = 779, ***p<0.001). (B) Serum high-sensitivity CRP levels according to severity of COPD in patients with AA or AOD. There was no significant difference between patients with and without COPD (p for trend = 0.123).</p

Increased TGF-β signaling in Fibulin-4^+/R and Fibulin-4^R/R lungs.

<p>Immunoblot analysis of pSmad2 in lung homogenates shows an increase in the amount of pSmad2 (A) and pSmad3 (B) in Fibulin-4^+/R (n = 3) and Fibulin-4^R/R (n = 3) lungs, compared to the total amount of Smad, and to their Fibulin-4^+/+ control (n = 3). (C) Increased pSmad2 staining of inflammatory and endogenous cells on Fibulin-4^+/R (n = 3) and Fibulin-4^R/R (n = 3) lung sections. Magnification 2.5x (scale bar 1 mm) upper panel and 20x (scale bar 200 µm) lower panel. (D) Ingenuity pathway explorer showed MMP9 as the shortest connection between Fibulin-4 and SERPINA1. SERPINA1 inhibits neutrophil elastase, which affects elastin. MMP9 itself was not deregulated in Fibulin-4^R/R lungs (n = 4), but could be connected to 16 deregulated genes in Fibulin-4^R/R compared to Fibulin-4^+/+ lungs (n = 4, red, up-regulated; green, downregulated), suggestion altered MMP9 activity. Black arrows indicate the connection between Fibulin-4 (EFEMP2), MMP9, SERPINA1, elastase and ELN. Grey arrows indicate the connection of these genes with deregulated genes between Fibulin-4^+/+ and Fibulin-4^R/R lungs.</p