The Role of TLR2 and TLR4 in Atherosclerosis and Vascular Calcification

• Main Authors: Guan-Lin Lee, 李冠霖
• Other Authors: Cheng-Chin Kuo
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/9v349a

博士 === 國防醫學院 === 生命科學研究所 === 105 === Abnormal activation of components of the innate immune system, such as Toll-like receptor (TLR), has been implicated in the pathological progression of metabolic inflammatory diseases including atherosclerosis and vascular calcification. Functional phenotype of vascular smooth muscle cells (VSMCs) is considered to be a vital event in the pathophysiology of atherosclerosis. Despite substantial evidence supporting the pathogenic role of TLR2 and TLR4 in the progression of vascular diseases, their roles in the regulation of VSMC function remains unclear. The goal of the present study was to elucidate the mechanism by which TLR2 and TLR4 regulate VSMC function and phenotypic switching. Migration assay revealed that activation of TLR2 and TLR4 by their specific agonists induced VSMCs migration. Inhibition experiments indicated that IL-6 secretion and VSMC migration induced by agonists of TLR2 and TLR4 were mediated through the activation of p38 mitogen-associated protein kinase (p38 MAPK) and extracellular signal–regulated kinase (ERK) 1/2 signaling. Neutralizing anti-IL-6 antibodies abrogated TLR2 and TLR4-driven VSMC migration and F-actin polymerization. Blockade of p38 MAPK or ERK1/2 signaling cascade inhibited agonists of TLR2 and TLR4-mediated activation of cAMP response element binding protein (CREB). Moreover, siRNA-mediated suppression of CREB production repressed IL-6 production and VSMC migration-induced by TLR2 and TLR4. Rac-1 inhibitor suppressed TLR2 and 4-driven VSMC migration but not IL-6 production. Further experiments with inhibitors and siRNA found that TLR4 activates PI3K/ATK signaling to induce F-spondin expression, subsequently controls CREB-mediated IL-6 production to promote VSMC migration. These findings provide mechanismistic insights into the essential role of F-spondin in VSMC function and atherosclerosis. In addition to VSMC migration, agonists of TLR2 and TLR4 also promoted VSMC chondrogenesis and calcification as demonstrated by Alcian blue and Alizarin red S (ARS) staining. Neutralizing anti-IL-6 antibodies attenuated TLR2 and TLR4-mediated VSMC chondrogenesis, but did not affect VSMC calcification. Interestingly, TLR2 and TLR4 activated c-Jun N-terminal kinases (JNKs) - and ERK1/2 signaling to downregulate calcification inhibitor, osteoprotegerin (OPG) levels, which in turn not only promoted VSMC chondrogenesis but also trigged VSMCs calcification. Importantly, the lower OPG level and increased atherogenic calcification were clearly detected in high-fat diet (HFD)-fed ApoE-/- mice compared with HFD-fed ApoE-/-Tlr2-/- mice. Based on our findings, we concluded that, upon specific ligand binding, both TLR2 and TLR4 activate MAPKs to trigger VSMC migration and VSMC chondrogenesis, which in turn progressively triggers atherogenesis and atherogenic calcification.