Toll-like receptor 2 activation and serum amyloid A regulate smooth muscle cell extracellular matrix.

Smooth muscle cells contribute to extracellular matrix remodeling during atherogenesis. De-differentiated, synthetic smooth muscle cells are involved in processes of migration, proliferation and changes in expression of extracellular matrix components, all of which contribute to loss of homeostasis accompanying atherogenesis. Elevated levels of acute phase proteins, including serum amyloid A (SAA), are associated with an increased risk for atherosclerosis. Although infection with periodontal and respiratory pathogens via activation of inflammatory cell Toll-like receptor (TLR)2 has been linked to vascular disease, little is known about smooth muscle cell TLR2 in atherosclerosis. This study addresses the role of SAA and TLR2 activation on smooth muscle cell matrix gene expression and insoluble elastin accumulation. Cultured rat aortic smooth muscle cells were treated with SAA or TLR2 agonists and the effect on expression of matrix metallopeptidase 9 (MMP9) and tropoelastin studied. SAA up-regulated MMP9 expression. Tropoelastin is an MMP9 substrate and decreased tropoelastin levels in SAA-treated cells supported the concept of extracellular matrix remodeling. Interestingly, SAA-induced down-regulation of tropoelastin was not only evident at the protein level but at the level of gene transcription as well. Contributions of proteasomes, nuclear factor κ B and CCAAT/enhancer binding protein β on regulation of MMP9 vs. tropoleastin expression were revealed. Effects on Mmp9 and Eln mRNA expression persisted with long-term SAA treatment, resulting in decreased insoluble elastin accumulation. Interestingly, the SAA effects were TLR2-dependent and TLR2 activation by bacterial ligands also induced MMP9 expression and decreased tropoelastin expression. These data reveal a novel mechanism whereby SAA and/or infection induce changes in vascular elastin consistent with atherosclerosis

SAA decreases tropoelastin expression.

<p>SMCs were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171711#pone.0171711.g001" target="_blank">Fig 1A</a> for the indicated time (A) or for 24 hours with the indicated dose (B) and Western blot analysis performed with antibodies directed against tropoelastin and tubulin. SMCs were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171711#pone.0171711.g001" target="_blank">Fig 1A</a> (C-E). <i>Eln</i> mRNA (C, E) and hnRNA (D) levels are expressed relative to the 10-hour control-treated sample ± SD (n = 3) (C) or 0-hour control-treated sample ± SD (n = 3) (D, E). SMCs were co-transfected with 216-luc and a <i>Renilla</i> construct, the latter to normalize for transfection efficiency (F). Cells were treated (or control-treated) with SAA for 6 or 24 hours. Data are expressed as luciferase/<i>Renilla</i> ± SD (n = 3). SMCs were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171711#pone.0171711.g001" target="_blank">Fig 1D</a> (G). <i>Eln</i> mRNA levels are expressed relative to the control-treated (no SAA or DRB) sample ± SD (n = 3). SMCs were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171711#pone.0171711.g001" target="_blank">Fig 1F</a> (H). <i>Eln</i> mRNA levels are expressed relative to the control-treated sample ± SD (n = 3).</p

Long-term treatment with SAA decreases insoluble elastin accumulation.

<p>SMCs were cultured for 2 weeks, then treated (or control-treated) with SAA for 2 weeks (A-C). <i>Mmp9</i> (A), <i>Eln</i> (B) and <i>Col1a1</i> (C) mRNA levels are expressed relative to the control-treated sample ± SD (n = 3). SMCs were cultured for 2 weeks, then either harvested for baseline measurements or treated (or control-treated) with SAA for 2 weeks (D, E). Insoluble elastin and total protein were determined by amino acid analysis. Data are expressed as insoluble elastin (μg/cm²) ± SD (n = 3–4) (D) or insoluble elastin (μg/cm²)/insoluble elastin (μg/cm²) + hot alkali-soluble protein (μg/cm²) ± SD (n = 3–4) (E). SMCs were treated as in Fig 4A (F, G). Cultures were visualized by phase contrast microscopy (F) or stained with Congo red and visualized by fluorescence microscopy (G).</p

SAA up-regulates <i>Mmp9</i> mRNA expression and down-regulates <i>Eln</i> mRNA expression via TLR2.

<p>SMCs pretreated with <i>siTlr2</i> or a control <i>(siCtl</i>) were treated (+) or control-treated (-) with SAA for 24 hours. <i>Tlr2</i> (A), <i>Mmp9</i> (B) and <i>Eln</i> (C) mRNA levels are expressed relative to the control-treated (<i>siCtl</i>, no SAA) sample ± SD (n = 3).</p

TLR2 activation increases MMP9 expression and decreases tropoelastin expression.

<p>SMCs were treated (or control-treated) with SAA, <i>Pg</i> LPS or LTA for 24 hours (A). <i>Tlr2</i> mRNA levels are expressed relative to the control-treated sample ± SD (n = 3). SMCs were co-transfected with NFκB-luc and a <i>Renilla</i> construct, the latter to normalize transfection efficiency (B). Cells were treated (or control-treated) with SAA or <i>Pg</i> LPS for 24 hours. Data are expressed as luciferase/<i>Renilla</i> ± SD (n = 5). SMCs were treated with SAA, <i>Pg</i> LPS, LTA, Pam or FSL as in Fig 6A (C, D). <i>Nos2</i> (C) and <i>Mmp9</i> (D) mRNA levels are expressed relative to the control-treated sample ± SD (n = 3). SMCs were treated as in Fig 6A and Western blot analysis performed with antibodies directed against MMP9 and tubulin (E). SMCs were treated as in Fig 6D (F). <i>Eln</i> mRNA levels are expressed relative to the control-treated sample ± SD (n = 3). SMCs were treated as in Fig 6A and Western blot analysis performed with antibodies directed against tropoelastin and tubulin (G). SMCs were transfected as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171711#pone.0171711.g002" target="_blank">Fig 2F</a> (H). Cells were treated (or control-treated) with SAA, <i>Pg</i> LPS or LTA for 24 hours. Data are expressed as luciferase/<i>Renilla</i> ± SD (n = 5).</p

SYBR qPCR rat primers.

<p>SYBR qPCR rat primers.</p

TaqMan qPCR primers.

<p>TaqMan qPCR primers.</p

Methyldopa blocks MHC class II binding to disease-specific antigens in autoimmune diabetes

Major histocompatibility (MHC) class II molecules are strongly associated with many autoimmune disorders. In type 1 diabetes, the DQ8 molecule is common, confers significant disease risk and is involved in disease pathogenesis. We hypothesized blocking DQ8 antigen presentation will provide a treatment by preventing recognition of self-peptides by pathogenic T-cells. We used the crystal structure of DQ8 to select drug-like small molecules predicted to bind structural pockets in the antigen binding cleft. A number of compounds inhibited DQ8 antigen presentation in vitro with one compound preventing insulin autoantibody production and delaying diabetes onset in an animal model of spontaneous autoimmune diabetes. We discovered an existing drug, methyldopa, blocked DQ8 and treated recent onset type 1 diabetes patients having the DQ8 allele. Methyldopa specifically inhibited DQ8 antigen presentation along with reducing inflammatory T-cell responses toward insulin, highlighting the relevance of blocking disease specific MHC II antigen presentation to treat autoimmunity