Abstract 295: Macrophages Induce Apoptosis of Arterial Smooth Muscle Cells via a Monocyte Chemoattractant Protein-1 (MCP-1) Mediated Cytotoxicity

Abstract

Objectives Excessive apoptosis of smooth muscle cells (SMCs) has been observed in human and experimental abdominal aortic aneurysm (AAA). We have previously demonstrated in a murine model of AAA that apoptotic SMCs attract monocytes and other leukocytes by producing MCP-1. In this study, we hypothesized that MCP-1 may contribute to aortic destruction also by enhancing pro-apoptotic capability of infiltrating macrophages. Methods and Results In a co-culture system MCP-1 primed RAW264.7 macrophages caused significantly more apoptosis of SMCs compared to control macrophages (1.53±0.11 fold increase in % of Annexin V PE+/7AAD-/CD11b- cells, n=5, p<0.05). To understand how MCP-1 may modulate cytotoxicity, we treated RAW cells with MCP-1 and analyzed Fas Ligand (FasL) expression by real-time PCR and flow cytometry. MCP-1 enhanced FasL mRNA level by 1.60±0.15 fold (p<0.05, n=4) and membrane FasL expression by 1.62±0.20 fold (p<0.05, n=3). Neutralizing FasL blocked SMC apoptosis. Consistently, we found MCP-1 elevated levels of cleaved caspase 8 and 3 in SMCs exposed to MCP-1 primed macrophages. We hypothesize that MCP-1 primed macrophages kill SMCs through a FasL-Fas-Caspase8- receptor interacting protein-1 (RIP1) mediated mechanism. Silencing RIP1 conferred apoptosis resistant to SMCs, protected them from being killed by macrophages. In situ proximity ligation assay showed that the exposed SMCs contained higher levels of RIP1-Caspase 8 complexes. In the mouse elastase injury model of aneurysm, immuno co-staining showed that TUNEL-positive SMCs were frequently surrounded by CD68+ macrophages, although little engulfing activity was observed. In parallel, aneurysm induction increased the level of RIP1-Caspase 8 complexes in medial SMCs, indirectly proved the occurrence of FasL mediated-kill of SMCs during aneurysm development. Conclusions Taken together, our data suggest that MCP-1 not only attract monocytes/macrophages but also can modulate their function. Mechanistically, MCP-1 primes macrophages by increasing levels of membrane bound FasL. Since apoptotic SMCs release MCP-1 in large quantity, this newly described role of MCP-1 may indicate a positive feedback loop between residential SMCs and infiltrating macrophages.