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

K6F-induced HGF migration and its inhibition by p38 MAPK and JNK1/2 inhibitors.

<p>A: Cells were seeded into the upper chamber of a transwell apparatus equipped with a 6.5-mm polycarbonate filter (8-μm pore) layered with 40 μg of Matrigel. Lower wells contained 500 μl of control medium supplemented with 1 μg/ml of K6F or ScK6F. The Matrigel chambers were incubated for 12 or 24 h in the presence or absence of 1 μM SB203580 or 1 μM SP600125 in the upper chamber. Cells in the top well were completely removed and cells on the underside of the membrane were counterstained with Mayer’s hematoxylin. B: Images were captured and cells were counted at ×40 magnification. The migration-enhancing effect is shown as the relative invasion capacity of the number of migratory cells in the sample versus that in control media. Data are the mean ± SD (n = 3) of two independent experiments. ‘NT’ denotes no treatment. <i>*P<</i>0.05 <i>vs</i>. control. <i>**P</i><0.05 <i>vs</i>. K6F alone.</p

Rat GEC cytokeratin-6 degradation by Kgp.

<p>A: Cells were either untreated (control, top), treated with 50 nM Kgp for 6 h or treated with Kgp inactivated by TLCK. After treatment, cells were stained with anti-cytokeratin 6 antibody (FITC, green) and Concanavalin A (ConA-Alexa 543, red), followed by DNA staining with DAPI (blue). Images were obtained by a fluorescence microscope at 400× magnification. Arrowhead indicates degraded cytokeratin-6 protein in rat GECs. Scale bar = 30 μm. B: Images were captured and fluorescence intensity was quantitated using Image J software. The percentage fluorescence intensity relative to control is shown and expressed as mean ± SD (n = 3). C: Flow cytometry of cytokeratin-6 protein is shown as the geometric mean of the fluorescence intensity (x-axis). The shaded histograms denote fluorescence in Kgp-treated cells before the assay. Data are representative of three independent experiments. *<i>P</i><0.05.</p

Effects of Kgp on cytokeratin-6 in the cytoskeleton fraction and K6F in rat GEC culture supernatants.

<p>A: Cytoskeletal protein was extracted from rat GECs treated with 10 nM Kgp, and cytokeratin-6 levels were measured by immunoblotting. Bands were quantified by densitometry. B: Cells were treated with <i>P</i>. <i>gingivalis</i> (<i>P</i>.<i>g</i>), <i>A</i>. <i>actinomycetemcomitans</i> (<i>A</i>.<i>a</i>) LPS or RgpB for 6 h before measurement of cytokeratin-6 levels in the cytoskeleton fraction. Vimentin was used as a loading control. Data are representative of three independent experiments. C: K6F in rat GEC supernatant after incubation with 10 nM Kgp as measured by ELISA. D: <i>P</i>.<i>g</i>., <i>A</i>.<i>a</i>. LPS or RgpB were used instead of Kgp. Data are the mean ± SD (n = 3).<i>*P<</i>0.05.</p

MALDI-TOF mass spectrometry profiles of GCF and rat GEC culture supernatants and K6F concentrations in GCF from PD patients and healthy volunteers.

<p>A: The proteins in GCF were extracted from the strips into the assay buffer and analyzed by MALDI-TOF mass spectrometry in arbitrary units (a.u.). The profile of GCF from PD patients on K6F peptides (red line) was superimposed onto that of healthy subjects (blue line). B: Profile of K6F detected in rat GEC supernatants. Activated gingipains were incubated with primary cultures of rat GECs, and supernatants were collected. A peak of K6F was detected in supernatants from rat GECs treated with Kgp (red line) at a molecular weight of 2217. C: GCF samples were collected and K6F titers were quantitatively measured by ELISA. Chart shows a dot plot of K6F levels in individual patients. Experiments were performed two times in triplicate. *<i>P</i><0.001 versus PD.</p