HMGB1 Promotes the Development of Pulmonary Arterial Hypertension in Rats

<div><p>Rationale</p><p>Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance leading to right ventricular failure and death. Recent studies have suggested that chronic inflammatory processes are involved in the pathogenesis of PAH. However, the molecular and cellular mechanisms driving inflammation have not been fully elucidated.</p><p>Objectives</p><p>To elucidate the roles of high mobility group box 1 protein (HMGB1), a ubiquitous DNA-binding protein with extracellular pro-inflammatory activity, in a rat model of PAH.</p><p>Methods</p><p>Male Sprague-Dawley rats were administered monocrotaline (MCT). Concentrations of HMGB1 in bronchoalveolar lavage fluid (BALF) and serum, and localization of HMGB1 in the lung were examined over time. The protective effects of anti-HMGB1 neutralizing antibody against MCT-induced PAH were tested.</p><p>Results</p><p>HMGB1 levels in BALF were elevated 1 week after MCT injection, and this elevation preceded increases of other pro-inflammatory cytokines, such as TNF-α, and the development of PAH. In contrast, serum HMGB1 levels were elevated 4 weeks after MCT injection, at which time the rats began to die. Immunohistochemical analyses indicated that HMGB1 was translocated to the extranuclear space in periarterial infiltrating cells, alveolar macrophages, and bronchial epithelial cells of MCT-injected rats. Anti-HMGB1 neutralizing antibody protected rats against MCT-induced lung inflammation, thickening of the pulmonary artery wall, and elevation of right ventricular systolic pressure, and significantly improved the survival of the MCT-induced PAH rats.</p><p>Conclusions</p><p>Our results identify extracellular HMGB1 as a promoting factor for MCT-induced PAH. The blockade of HMGB1 activity improved survival of MCT-induced PAH rats, and thus might be a promising therapy for the treatment of PAH.</p></div

Anti-HMGB1 antibody dampens lung inflammation.

<p>BALF samples were collected from MCT-induced PAH rats treated with anti-HMGB1 IgY or control IgY at 3 weeks after MCT challenge. The number of leukocytes (A) and the concentrations of endothelin-1 (B), TNF-α, MCP-1, and IL-1β (C) in BALF were measured (n = 6–7 per group). All data are expressed as mean ± SEM. *<i>P</i><0.05 and **<i>P</i><0.01.</p

HMGB1 is translocated from the nucleus to the cytoplasm of periarterial infiltrating leukocytes, alveolar macrophages, and bronchial epithelial cells in MCT-injected rats.

<p>Lung samples were collected from rats 1 week after vehicle-injection (A, D, H) or MCT-injection (B, C, E–G, I). (C) and (F) are magnified views of the insets shown in (B) and (E), respectively. The localization of HMGB1 was assessed by immunohistochemistry. Nuclear protein HMGB1 was translocated to the cytoplasm of periarterial infiltrating cells, alveolar macrophages (arrowheads), and bronchial epithelial cells (arrows) in MCT-induced PAH rats. Representative images of n = 5–6. Scale bars represent 50 µm (A–E, H, I) and 20 µm (F, G).</p

Anti-HMGB1 antibody prevents wall thickening of pulmonary arterioles in MCT-injected rats.

<p>(A) H&E staining (upper panels) and Elastica van Gieson staining (lower panels) of pulmonary arteries of MCT-induced PAH rats treated with anti-HMGB1 IgY or control IgY at 3 weeks after MCT challenge. Representative images of n = 6. Scale bars = 20 µm. (B) Pulmonary artery wall thickness were measured in the PAH rats treated with anti-HMGB1 IgY or control IgY at 3 weeks after MCT challenge (n = 6 per group).The external diameter and medial wall thickness of the pulmonary arteries were measured in 20 muscular arteries (ranging in size from 25–100 µm in external diameter) on Elastica van Gieson–stained sections. For each artery, medial wall thickness was expressed as follows: % wall thickness = [(medial thickness×2)/external diameter]×100. All data are expressed as mean ± SEM. **<i>P</i><0.01.</p

Anti-HMGB1 antibody prevents the development of PAH.

<p>RV systolic pressure (A) and RV weight (B–C) of the PAH rats treated with anti-HMGB1 IgY or control IgY at 3 weeks after MCT challenge (n = 6–12 per group). All data are expressed as mean ± SEM. (D) Kaplan-Meier survival curves of the PAH rats treated with either anti-HMGB1 IgY or control IgY (n = 13 per group). *<i>P</i><0.05 and **<i>P</i><0.01.</p

Nuclear protein HMGB1 is released to the bronchoalveolar space during the early stage of PAH.

<p>(A–B) BALF samples were collected from MCT- or vehicle-injected rats at the indicated time points. The levels of HMGB1, MCP-1, and TNF-α (A), and the number of leukocytes (B) in BALF were measured. (C) Serum samples were collected from MCT- or vehicle-injected rats, and the levels of HMGB1 and MCP-1 were measured (n = 6–13 per group). All data are expressed as mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001.</p

Adaptive Iterative Dose Reduction Using Three Dimensional Processing (AIDR3D) Improves Chest CT Image Quality and Reduces Radiation Exposure

<div><p>Objective</p><p>To assess the advantages of Adaptive Iterative Dose Reduction using Three Dimensional Processing (AIDR3D) for image quality improvement and dose reduction for chest computed tomography (CT).</p><p>Methods</p><p>Institutional Review Boards approved this study and informed consent was obtained. Eighty-eight subjects underwent chest CT at five institutions using identical scanners and protocols. During a single visit, each subject was scanned using different tube currents: 240, 120, and 60 mA. Scan data were converted to images using AIDR3D and a conventional reconstruction mode (without AIDR3D). Using a 5-point scale from 1 (non-diagnostic) to 5 (excellent), three blinded observers independently evaluated image quality for three lung zones, four patterns of lung disease (nodule/mass, emphysema, bronchiolitis, and diffuse lung disease), and three mediastinal measurements (small structure visibility, streak artifacts, and shoulder artifacts). Differences in these scores were assessed by Scheffe's test.</p><p>Results</p><p>At each tube current, scans using AIDR3D had higher scores than those without AIDR3D, which were significant for lung zones (<i>p</i><0.0001) and all mediastinal measurements (<i>p</i><0.01). For lung diseases, significant improvements with AIDR3D were frequently observed at 120 and 60 mA. Scans with AIDR3D at 120 mA had significantly higher scores than those without AIDR3D at 240 mA for lung zones and mediastinal streak artifacts (<i>p</i><0.0001), and slightly higher or equal scores for all other measurements. Scans with AIDR3D at 60 mA were also judged superior or equivalent to those without AIDR3D at 120 mA.</p><p>Conclusion</p><p>For chest CT, AIDR3D provides better image quality and can reduce radiation exposure by 50%.</p></div

Correlations between quantitative image noise and body weight^*.

<p><i>Definition of abbreviations</i>: LPV: lower pulmonary vein; NS: not significant.</p><p>* Spearman rank correlation analysis was used to evaluate correlations between image noise and body weight. Correlation coefficient (ρ) and p values are shown.</p

Axial plain chest CT images at the upper lung zone (60-year-old male weighing 76 kg).

<p>These images were created from scan data at 240 mA (<b>A, D</b>), 120 mA (<b>B, E</b>) and 60 mA (<b>C, F</b>). The three upper images (<b>A–C</b>) were reconstructed using AIDR3D and the three lower images (<b>D–F</b>) were reconstructed using a conventional reconstruction mode (Boost3D). Each image pair at the same tube current was created from single row data. Image noise was obviously reduced on images with AIDR3D, particularly at lower tube currents.</p

Axial plain chest CT images with a solid lung mass in the right middle lobe (75-year-old female weighing 56 kg).

<p>Images are arranged as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105735#pone-0105735-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105735#pone-0105735-g002" target="_blank">2</a>. Spiculae were found on all images, while density heterogeneity inside the mass was severe on images at 60 mA without AIDR3D (<b>F</b>).</p