The Regulation of Vascular Wall Cells by a TLR Ligand and Gp130 Cytokines

Abstract

<p>Atherosclerosis is a disease affecting the blood vessels that is inflammatory in nature, and plays an important role in cardiovascular disease (CVD), one of the leading causes of morbidity and mortality worldwide. Oncostatin M (OSM), a member of the IL-6/gp130 cytokine family, has been implicated in atherosclerosis both in mouse models and in humans. OSM synergizes with other stimuli in various systems to regulate cells. Infectious pathogens as well as danger associated host molecules stimulate members of the innate immune system, including Toll-like Receptors (TLRs), to respond in a pro-inflammatory manner to cause cell activation and cytokine release. Experiments were performed to determine whether OSM and LPS (a TLR-4 ligand) synergize in regulation of vascular wall cells <em>in vitro</em>.</p> <p>Upon stimulation of Aortic Adventitial Fibroblasts from mice (MAAFs) and humans (HAoAFs) as well as Human Aortic Smooth Muscle Cells (HAoSMCs) with LPS in combination with OSM, it was determined that there was a synergistic increase in IL-6 and VEGF levels in the cell supernatants as measured by ELISA compared to either treatment alone. MAAFs were also able to synergistically express KC upon stimulation with LPS and OSM, while in HAoAFs and HAoSMCs, LPS induced IL-8 levels were supressed by OSM. These effects were unique to OSM among gp130 cytokine members, as treatment of these cells with LPS in combination with LIF, IL-6, IL-31, or IL-11 had no marked effects compared to LPS alone. Furthermore, MCP-1 steady state mRNA levels were elevated 6 hours post stimulation with LPS and OSM compared to either treatment alone in HAoAFs and HAoSMCs.</p> <p>While OSM did not appear to modulate TLR-4 expression, OSM treatment resulted in an increased phosphorylation signal in STAT-1,-3, and -5, as well as Akt in MAAFs and HAoAFs. In addition, combined LPS and OSM stimulation resulted in an increased phosphorylation signal of the MAPK p38 compared to either treatment alone. Furthermore, a neutralizing antibody to the OSMr-β was able to inhibit HAoAF IL-6 responses to PBMC conditioned medium. Together, these findings indicate that OSM and LPS can synergize <em>in vitro </em>to induce the expression of inflammatory factors in vascular wall cells, emphasizing the potential role of OSM, TLR-4 ligands, and adventitial fibroblasts in vascular inflammation.</p>