Published Online
on June 20, 2005

A more recent version of this article appeared on June 28, 2005

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Submitted on September 30, 2004
Revised on February 7, 2005
Accepted on March 29, 2005

Gene Profiling in Atherosclerosis Reveals a Key Role for Small Inducible Cytokines. Validation Using a Novel Monocyte Chemoattractant Protein Monoclonal Antibody

Esther Lutgens MD, PhD^*, Birgit Faber PhD, Kitty Schapira BSc, Chris T.A. Evelo PhD, Rachel van Haaften PhD, Sylvia Heeneman PhD, Kitty B.J.M. Cleutjens PhD, Ann Pascale Bijnens PhD, Linda Beckers BSc, J. Gordon Porter PhD, Charles R. Mackay PhD, Paul Rennert PhD, Veronique Bailly PhD, Matthew Jarpe PhD, Brian Dolinski PhD, Victor Koteliansky MD, PhD, Tony de Fougerolles PhD, and Mat J.A.P. Daemen MD, PhD

From the Departments of Pathology (E.L., B.F., K.S., S.H., K.B.J.M.C., A.P.B., L.B., M.J.A.P.D.) and Bioinformatics (C.T.A.E., R.v.H.), Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, the Netherlands; Incyte Corporation, Palo Alto, Calif (J.G.P.); Arthritis and Inflammation Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia (C.R.M.); and Biogen-Idec Inc, Cambridge, Mass (P.R., V.B., M.J., B.D., V.K., T.d.F.). Drs de Fougerolles and Koteliansky are currently affiliated with Alnylam Pharmaceuticals, Cambridge, Mass.

^* To whom correspondence should be addressed. E-mail: e.lutgens{at}path.unimaas.nl.

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, MIP-1, 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, and MIP-1. ELISA for MCP-1 and MCP-5 showed similar results. Immunohistochemistry for MCP-1, MCP-5, and MIP-1 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.

Key words: atherosclerosis • genes • inflammation

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