The role of IL-6 in pathogenesis of abdominal aortic aneurysm in mice.

Although the pathogenesis of abdominal aortic aneurysm (AAA) remains unclear, evidence is accumulating to support a central role for inflammation. Inflammatory responses are coordinated by various soluble cytokines of which IL-6 is one of the major proinflammatory cytokines. In this study we examined the role of IL-6 in the pathogenesis of experimental AAA induced by a periaortic exposure to CaCl2 in mice. We now report that the administration of MR16-1, a neutralizing monoclonal antibody specific for the mouse IL-6 receptor, mildly suppressed the development of AAA. The inhibition of IL-6 signaling provoked by MR16-1 also resulted in a suppression of Stat3 activity. Conversely, no significant changes in either NFκB activity, Jnk activity or the expression of matrix metalloproteinases (Mmp) -2 and -9 were identified. Transcriptome analyses revealed that MR16-1-sensitive genes encode chemokines and their receptors, as well as factors that regulate vascular permeability and cell migration. Imaging cytometric analyses then consistently demonstrated reduced cellular infiltration for MR16-1-treated AAA. These results suggest that IL-6 plays an important but limited role in AAA pathogenesis, and primarily regulates cell migration and infiltration. These data would also suggest that IL-6 activity may play an important role in scenarios of continuous cellular infiltration, possibly including human AAA

MRTF-A promotes angiotensin II-induced inflammatory response and aortic dissection in mice.

Aortic dissection (AD) is a major cause of acute aortic syndrome with high mortality due to the destruction of aortic walls. Although recent studies indicate the critical role of inflammation in the disease mechanism of AD, it is unclear how inflammatory response is initiated. Here, we demonstrate that myocardin-related transcription factor A (MRTF-A), a signal transducer of humoral and mechanical stress, plays an important role in pathogenesis of AD in a mouse model. A mouse model of AD was created by continuous infusion of angiotensin II (AngII) that induced MRTF-A expression and caused AD in 4 days. Systemic deletion of Mrtfa gene resulted in a marked suppression of AD development. Transcriptome and gene annotation enrichment analyses revealed that AngII infusion for 1 day caused pro-inflammatory and pro-apoptotic responses before AD development, which were suppressed by Mrtfa deletion. AngII infusion for 1 day induced pro-inflammatory response, as demonstrated by expressions of Il6, Tnf, and Ccl2, and apoptosis of aortic wall cells, as detected by TUNEL staining, in an MRTF-A-dependent manner. Pharmacological inhibition of MRTF-A by CCG-203971 during AngII infusion partially suppressed AD phenotype, indicating that acute suppression of MRTF-A is effective in preventing the aortic wall destruction. These results indicate that MRTF-A transduces the stress of AngII challenge to the pro-inflammatory and pro-apoptotic responses, ultimately leading to AD development. Intervening this pathway may represent a potential therapeutic strategy

Effect of MR16-1 on Mcp-1 expression and calcification.

<p>(A) Photomicrographs are shown for Mcp-1 staining of aortic tissue with sham operation, 1 week after CaCl₂ exposure with saline treatment (CaCl₂+NaCl), and 1 week after CaCl₂ exposure with MR16-1 treatment (CaCl₂+MR). Expression of Mcp-1 was evaluated by calculating the ratio of Mcp-1-positive area to the tissue area (Mcp-1 area). (B) Von Kossa staining was performed for aortic tissue with sham operation (sham), 6 weeks after CaCl₂ exposure with saline treatment (CaCl₂+NaCl), and 6 weeks after CaCl₂ exposure with MR16-1 treatment (CaCl₂+MR). The extent of aortic tissue calcification was evaluated by calculating the ratio of the calcified (brown) area to the tissue area (Calcified area). Symbols in graphs indicate individual data and bars indicate means ± standard errors. The numbers of mice for observation are indicated in parentheses. *p<0.05, **p<0.01, and ***p<0.001. Bars denote 200 μm.</p

Effect of MR16-1 on inflammatory signaling in AAA.

<p>Molecular activities of inflammatory signaling molecules in aortic tissue. (A) Immunoblots for phospho-Jnk (P-Jnk), total Jnk, phospho-Stat3 (pStat3), total Stat3, and lysyl oxidase are shown. Gelatin zymograms are also shown for Mmp-9 and Mmp-2. Expression of β-actin served as an internal loading control. (B) Quantitative analysis of immunoblots and gelatin zymograms are shown in which a sham-operated aorta was assigned a value of 1. Symbols indicate individual data and bars indicate means ± standard errors from 8 independent observations in each experimental group. *p<0.05, **p<0.01, and ***p<0.001.</p

Effect of MR16-1 on cellular signaling.

<p>(A) Representative photomicrographs are shown for 3-color immunofluorescence staining of aortic tissue without (sham) or with CaCl₂ exposure followed by saline injection (NaCl) or control rat IgG injection (IgG). Blue and red colors indicate nuclear and SMA stainings, respectively. Green color indicate NFκB, pStat3 or pSmad2. All samples are shown with the luminal side up. A bar denote 50 μm. (B) Scattergrams of imaging cytometric analyses are shown for nuclear signals of NFkB, pStat3 and pSmad2, together with cytosolic signal of SMA. Cell percentages for each quadrant are shown in black. The percentages of active vs. inactive NFκB, pStat3 and pSmad2 are indicated by blue numbers. The percentages of SMA-negative and -positive populations are shown in red numbers. The counts of total cells are shown in parenthesis below each panel. Data were obtained from 4 mouse aortae in each experimental group.</p

Effect of MR16-1 on serum cytokine profiles.

<p>Serum concentrations of cytokines are shown for mice without (sham) and with AAA, treated with physiological saline (NaCl), non-specific rat IgG (IgG), and MR16-1 (MR), measured 1 week after CaCl₂ exposure. Symbols indicate individual data and bars indicate means ± standard errors from 8 independent observations in each experimental group. *p<0.05.</p