Vitronectin Expression in the Airways of Subjects with Asthma and Chronic Obstructive Pulmonary Disease

<div><p>Vitronectin, a multifunctional glycoprotein, is involved in coagulation, inhibition of the formation of the membrane attack complex (MAC), cell adhesion and migration, wound healing, and tissue remodeling. The primary cellular source of vitronectin is hepatocytes; it is not known whether resident cells of airways produce vitronectin, even though the glycoprotein has been found in exhaled breath condensate and bronchoalveolar lavage from healthy subjects and patients with interstitial lung disease. It is also not known whether vitronectin expression is altered in subjects with asthma and COPD. In this study, bronchial tissue from 7 asthmatic, 10 COPD and 14 control subjects was obtained at autopsy and analyzed by immunohistochemistry to determine the percent area of submucosal glands occupied by vitronectin. In a separate set of experiments, quantitative colocalization analysis was performed on tracheobronchial tissue sections obtained from donor lungs (6 asthmatics, 4 COPD and 7 controls). Vitronectin RNA and protein expressions in bronchial surface epithelium were examined in 12 subjects who undertook diagnostic bronchoscopy. Vitronectin was found in the tracheobronchial epithelium from asthmatic, COPD, and control subjects, although its expression was significantly lower in the asthmatic group. Colocalization analysis of 3D confocal images indicates that vitronectin is expressed in the glandular serous epithelial cells and in respiratory surface epithelial cells other than goblet cells. Expression of the 65-kDa vitronectin isoform was lower in bronchial surface epithelium from the diseased subjects. The cause for the decreased vitronectin expression in asthma is not clear, however, the reduced concentration of vitronectin in the epithelial/submucosal layer of airways may be linked to airway remodeling.</p></div

Stereological measurements of cadaveric bronchial tissues according to histopathological diagnosis.

<p>Quantitative variables are expressed as medians (interquartile range)</p><p>^a Statistically significant values by Kruskall-Wallis H test.</p><p>^b Values expressed as absolute and relative (in parenthesis) frequencies.</p><p>Stereological measurements of cadaveric bronchial tissues according to histopathological diagnosis.</p

Vitronectin expression in human bronchial tissues.

<p>Expression in submucosal glands (<b>a</b>), and in respiratory surface epithelium (<b>b</b>). The measurements included 7 healthy controls (HC), 6 asthmatic and 4 COPD subjects. For submucosal glands 42 control, 41 asthmatic and 19 COPD acinar structures were evaluated; whereas for respiratory surface epithelium 37 control, 32 asthmatic and 16 COPD tissue structures were assessed. GEA, glandular epithelial area; SEA, surface epithelium area.</p

Colocalization of MUC5B or MUC5AC and vitronectin.

<p>Confocal images showing localization of MUC5B (<b>a</b>) and MUC5AC (<b>b</b>) (green fluorescence), as markers of mucous cells in submucosal glands and epithelial goblet cells, respectively and vitronectin (red fluorescence). Overlay of green and red channels showed that vitronectin is not present in cells producing MUC5B (R_p 0.16, IQR 0.057–0.42) or MUC5AC (R_p 0.04; IQR 0.02–0.13). Nuclei were counterstained with DAPI (blue fluorescence). <i>xy</i> and xz views are shown to provide sufficient details of spatial distribution in the 3D subcellular space. The colocalization maps (scatter plots) of voxels are shown. MUC5B and MUC5AC emission intensities are plotted on the x-axis, while vitronectin is plotted on the y-axis. R_p, Pearson´s colocalization coefficient; IQR, interquartile range.</p

(a) Agarose gel electrophoresis of the amplification product for vitronectin cDNA from bronchial brushings.

<p>The amplified fragment corresponds to a band size of 151bp. MW, molecular weight marker, Bb, bronchial brushing, and NC, negative control or “non-template control” of qPCR. The number accompanying bronchial brushings corresponds to the number assigned to different patients. <b>(b) Agarose gel electrophoresis of the amplification product for vitronectin cDNA from cultured cells</b>. Molecular weight marker (MW), normal human bronchial epithelial cells (NHBE), cells from adenocarcinoma of epithelial origin (NCL-H23), bronchial brushing (Bb), extraction and retro-transcription control (RT control) or “no reverse-transcriptase control”, and negative control (NC).</p

Localization of vitronectin in human bronchial epithelium.

<p>Indirect immunoperoxidase staining of paraffin sections from cadaver tissue (healthy control). <b>a</b>) Vitronectin expression in submucosal glands of a mainstem bronchus (arrows), magnification x100. <b>b</b>). Presence of vitronectin in the bronchial surface epithelium (arrows), magnification x100. <b>c-d</b>). Enlarged sections from panels <b>a</b> and <b>b</b>, magnification x400.</p

Lung structural change in asthma and COPD.

<p><b>a</b>) Haematoxylin and eosin staining of bronchial tissues from asthmatic subjects. Arrows point to thickened smooth muscle cell bundles. <b>b</b>) Same staining as in panel <b>a</b>, for tissues from COPD subjects. Arrows point to emphysematous alveolus space.</p

Vitronectin expression in healthy, asthmatic, and COPD individuals.

<p>Immunohistochemical staining shows vitronectin expression in airway tissues (arrows) from the control (<b>a</b>), asthmatic (<b>b</b>) and COPD (<b>c</b>) individuals.</p

Pearson's Correlation Coefficients for colocalization assays.

<p>The plot shows Pearson´s correlation coefficients for the colocalization analysis. For the total sample, medians of R_p values for vitronectin and lactoferrin (0.55, IQR 0.47–0.67), vitronectin and MUC5B (0.16, IQR 0.06–0.42), and vitronectin and MUC5AC (0.04, IQR 0.02–0.13 indicate that colocalization was only significant for the marker of serous cell and vitronectin. The Kruskall-Wallis H Test was used for calculating statistical differences. SC, serous cells; GMC, glandular mucous cells; GC, goblet cells.</p

Western analysis of vitronectin isoforms expression in bronchial surface epithelium from bronchial brushings.

<p><b>(a)</b> Immunoblots of vitronectin expression in control individuals (number 10, 15 and 17) and diseased subjects (numbers 12, 13, 16, 18, 20, 22, 23, 24 and 25). 65- and 75-kDa vitronectin isoforms are presents in all subjects. β-actin was used as a control for protein loading. <b>(b-c) Quantification by densitometry of 65- and 75-kDa vitronectin isoforms.</b> 65-kDa vitronectin expression was lower in diseased subjects when compared with control individuals (p = 0.0412). The vitronectin/β-actin ratio is represented on the y axes for controls and diseased subjects. Error bars represent the standard error of means.</p