DOUBLE-STRANDED RNA AND BACTERIAL LPS-INDUCED ACTIVATION OF VASCULAR SMOOTH MUSCLE CELLS: ROLE OF TOLL-LIKE RECEPTOR 3 AND TOLL-LIKE RECEPTOR 4

Abstract

A growing body of evidence supports a central role of inflammation in the pathogenesis of atherosclerosis, however the triggers that initiate the inflammatory process have not been definitively identified. Infectious agents, including both bacteria and viruses, are among the proposed initiators of inflammation, yet the molecular mechanisms by which microbes activate cells in the vessel wall are unknown. Recent evidence supports a central role of transmembrane Toll-like receptors (TLRs) in cellular activation by microbial products. Ten different TLRs are activated by distinct microbial ligands, and in turn elicit differential cellular responses. To test the possible role of TLRs in vascular smooth muscle cell (VSMC) activation, we characterized the expression of TLR mRNAs in VSMC, and characterized the activation of VSMC by their respective microbial ligands. VSMC isolated from either human pulmonary artery or human aorta were found to express mRNA encoding TLR3, TLR4 and TLR6. TLR3 has recently emerged as an important receptor for poly ( IC), a mimic of double-stranded RNA that is produced by many viruses, whereas TLR4 is the receptor for bacterial lipopolysaccharide (LPS). Poly ( IC) was a potent stimulator of human VSMC, markedly stimulating the cellular release of monocyte-chemoattractant protein-1 (MCP-1), and the levels of cell-associated interleukin-1alpha ( IL-1alpha ), a potent SMC mitogen. LPS likewise stimulated MCP-1 release and IL-1alpha synthesis. Both poly ( IC) and LPS markedly induced NFkappaB activation in human VSMC, and in aortic SMC derived from wild-type mice that express functional TLR4. Poly ( IC) also activated NFkappaB in VSMC derived from mice that express a non-functional form of TLR4, whereas LPS was ineffective. Expression of a dominant negative form of TLR3 attenuated poly ( IC)-, but not LPSor IL-1alpha-induced NFkappaB activation in mouse aortic SMC. Poly ( IC ) also markedly stimulated proliferation in human VSMC and in mouse aortic VSMC derived from either wild-type or TLR4 mutant mice. In contrast, bacterial LPS did not affect proliferation either when added alone or in the presence of the LPS transfer factor CD14. These studies support the hypothesis that double-stranded RNA activates VSMC via TLR3, whereas bacterial LPS activates VSMC via TLR4, and that TLR3 and TLR4 appear to elicit differential effects on VSMC proliferation.