Gene Profiling In Atherosclerosis Reveals A Key Role For Small Inducible Cytokines - Validation Using A Novel Monocyte Chemoattractant Protein Monoclonal Antibody

Background - Pathological aspects of atherosclerosis are well described, but gene profiles during atherosclerotic plaque progression are largely unidentified.Methods and Results - Microarray analysis was performed on mRNA of aortic arches of ApoE (-/-) mice fed normal chow ( NC group) or Western- type diet ( WD group) for 3, 4.5, and 6 months. Of 10 176 reporters, 387 were differentially (> 2X) expressed in at least 1 group compared with a common reference ( ApoE (-/-) , 3- month NC group). The number of differentially expressed genes increased during plaque progression. Time- related expression clustering and functional grouping of differentially expressed genes suggested important functions for genes involved in inflammation ( especially the small inducible cytokines monocyte chemoattractant protein [ MCP]- 1, MCP- 5, macrophage inflammatory protein [ MIP]- 1 alpha MIP- 1 beta , MIP- 2, and fractalkine) and matrix degradation ( cathepsin- S, matrix metalloproteinase- 2/ 12). Validation experiments focused on the gene cluster of small inducible cytokines. Real- time polymerase chain reaction revealed a plaque progression - dependent increase in mRNA levels of MCP- 1, MCP- 5, MIP- 1 alpha, and MIP- 1 beta. ELISA for MCP- 1 and MCP- 5 showed similar results. Immunohistochemistry for MCP- 1, MCP- 5, and MIP- 1 alpha located their expression to plaque macrophages. An inhibiting antibody for MCP- 1 and MCP- 5 ( 11K2) was designed and administered to ApoE (-/-) mice for 12 weeks starting at the age of 5 or 17 weeks. 11K2 treatment reduced plaque area and macrophage and CD45(+) cell content and increased collagen content, thereby inducing a stable plaque phenotype.Conclusions - Gene profiling of atherosclerotic plaque progression in ApoE (-/-) mice revealed upregulation of the gene cluster of small inducible cytokines. Further expression and in vivo validation studies showed that this gene cluster mediates plaque progression and stability.