Expression Of CCR2, CXCR3 And Their Ligands In Atherosclerotic Mice

Atherosclerosis is a chronic inflammatory disease. Recruitment and migration of circulating leukocytes into the intima of the vascular wall is an important event in the initiation and progression of atherosclerosis. Recent studies have implicated that this process is mainly regulated by the production of chemokines of vascular cells. Chemokines are a superfamily of small proinflammatory cytokines involved in trafficking and activation of leukocytes and arterial cells. The effects of chemokines are mainly mediated by activating chemokine receptors expressed on the target cell.Among the most thoroughly characterized chemokines are the monocyte chemoattractant proteins (MCPs). The effects of MCPs are mediated mainly through activation of their cognate receptor, CC chemokine receptor 2 (CCR2), which is highly expressed on circulating monocytes, T lymphocytes, basophils, dendritic cells and natural killer cells. Monocyte chemoattractant protein-1 (MCP-1), the major ligand for CCR2, is one of the most important chemokines that regulates migration and infiltration of monocytes and is involved in the pathogenesis of atherosclerosis by promoting recruitment, migration of inflammatory cells and activation of these cells in atherosclerotic lesions. CXC chemokine receptor 3 (CXCR3), expressed on activated T helper type 1 cells, regulates the migration and infiltration of activated T helper type 1 cells and functions as a common receptor for CXCL9 (monokine-induced by IFN-γ, MIG), CXCL10 (IFN-γ-induced protein of 10 kDa, IP-10), and CXCL11 (IFN-γ-inducible T cellα-chemoattractant, I-TAC). Recent studies have provided evidence that CXCR3 crucially affects the recruitment and responses of T cells in atherosclerosis.Therefore, in order to better determine the implication of CCR2, CXCR3 and their ligands in atherosclerosis development as well as to investigate the possible transcriptional regulators involved in the early induction of MCP-1, we performed a time course study of lesion formation in apolipoprotein E-null mice (apoE-/- mice) fed a high fat and cholesterol diet and correlated lesion progression with the expression of CCR2, CCR2 chemokines, CXCR3, CXCR3 chemokines and the important MCP-1 transcription regulators AP-1 and NF-κB in vivo.Our results showed that:In parallel to hypercholesterolemia and the progression of atherosclerotic lesions in apoE-/- mice, MCP-1,CCR2 and CXCR3 mRNA expression increased significantly from the early stages of AS and peaked after 10 and 12 weeks of diet, respectively. The expression of IP-10 and I-TAC were also significantly induced during early atherogenesis, but peaked after 4 weeks of diet. Mig expression was induced after 6 weeks of diet and reached their highest value after 8 weeks of diet, wheres MCP-2 and MCP-3 mRNA expression were only elevated in the late phases of the lesion development. Immunostaining revealed that early MCP-1 expression was localized to vascular smooth muscle cells (VSMCs) and that in advanced lesions, both neointimal VSMCs and intimal macrophages expressed high levels of MCP-1. During the early (0 and 4 weeks of diet) induction of MCP-1 in VSMCs, the regulatory activator protein-1 (AP-1) proteins c-Jun and c-Fos were highly expressed and observed within the VSMCs nucleus, whereas the nuclear factor-κB (NF-κB) protein p65 was only observed within the nucleus of VSMCs after 4 weeks of diet. CCR2 was also indentified on intimal macrophages, endothelial cells and VSMCs in advanced lesions.In conclusion, our results provide fundamental information on the expression kinetics of CCR2, CXCR3 and their ligands during atherogenesis in apoE-/- mice and suggest that, medial VSMCs might be the primary source of MCP-1 for the initial recruitment of monocytes into the intima. The earliest induction of MCP-1 in VSMCs appears to correlate with AP-1 but not NF-κB regulatory pathways and both pathways might be involved in the regulation of MCP-1 expression as atherosclerotic lesions progress. The identification of CCR2 on endothelial cells and VSMCs of apoE-/- mice also suggest that CCR2 may not only mediate monocyte infiltration but may participate in endothelial cell and VSMC migration and proliferation during atherosclerosis development.