Fn14-Fc fusion protein regulates atherosclerosis in ApoE-/- mice and inhibits macrophage lipid uptake in vitro.

OBJECTIVE: TWEAK is a multifunctional cytokine belonging to the tumor necrosis factor superfamily and binds to the receptor Fn14. TWEAK and Fn14 are expressed in atherosclerotic plaques in areas rich in macrophages and foam cells. We investigated the role of TWEAK/Fn14 interactions in ApoE(-/-) mice and bone marrow-derived macrophages in vitro. METHODS AND RESULTS: ApoE(-/-) mice were treated with TWEAK-inhibiting fusion protein, Fn14-Fc, in an early (5 to 17 weeks of age) or delayed (17 to 29 weeks of age) setting. In the aortic arch, Fn14-Fc as compared to control treatment resulted in advanced plaques which were smaller (early treatment), fewer (delayed treatment), lower in fibrotic content (early and delayed treatment), and exhibited an increased macrophage content and smaller macrophage size (delayed treatment). There were no differences in apoptosis in atherosclerotic plaques after Fn14-Fc versus control Ab treatment. However, blocking TWEAK resulted in less macrophage uptake of modified lipids in vitro. CONCLUSIONS: Fn14-Fc fusion protein treatment did not prevent lesion initiation but inhibited some features of plaque progression and induced a unique advanced plaque phenotype with increased macrophage content and smaller macrophage size, which may be attributable to reduced lipid uptake. These findings indicate that TWEAK/Fn14 interactions regulate atherosclerosis and mediate lipid uptake in macrophages.

Control of atherosclerotic plaque vulnerability: insights from transgenic mice.

Atherosclerosis is a complex, progressive disease of the large systemic arteries. This multi-factorial disease is characterized by accumulation of lipids, cells and extracellular matrix in the vessel wall. The quest to unravel the molecular mechanisms leading to progression of human atherosclerotic plaques has lead to the development of a variety of animal models. Mice are easily amendable to transgenesis and multiple mutant and inbred strains have been generated in which potential regulators are manipulated and subsequently studied for effects on the development and progression of atherosclerosis. The scope of this review is to discuss the relevance, advantages and disadvantages of genetically-engineered mice to investigate mechanisms of plaque vulnerability. Features of human vulnerable lesions, such as large lipid-rich necrotic cores, active inflammation, matrix remodeling and signs of intraplaque hemorrhage are represented in mouse lesions. Here, we will discuss how atherosclerosis is modified by manipulations in apoptosis, lesional lipid metabolism, inflammatory pathways, matrix remodeling and thrombotic pathways in genetically-engineered mice, emphasizing the insights that have been gained from these studies for the control of plaque vulnerability.

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

Genetic deletion or antibody blockade of alpha1beta1 integrin induces a stable plaque phenotype in ApoE-/- mice.

OBJECTIVE: Adhesive interactions between cells and the extracellular matrix play an important role in inflammatory diseases like atherosclerosis. We investigated the role of the collagen-binding integrin alpha1beta1 in atherosclerosis. METHODS AND RESULTS: ApoE-/- mice were alpha1-deficient or received early or delayed anti-alpha1 antibody treatment. Deficiency in alpha1 integrin reduced the area of atherosclerotic plaques and altered plaque composition by reducing inflammation and increasing extracellular matrix. In advanced plaques, alpha1-deficient mice had a reduced macrophage and CD3+ cell content, collagen and smooth muscle cell content increased, lipid core sizes decreased, and cartilaginous metaplasia occurred. Anti-alpha1 antibody treatment reduced the macrophage content in initial plaques after early and delayed treatment, decreased the CD3+ cell content in advanced plaques after delayed treatment, and increased the collagen content in initial and advanced plaques after delayed treatment. Migration assays performed on alpha1-deficient macrophages on collagen I and IV substrata revealed that alpha1-deficient cells can migrate on collagen I, but not IV. Anti-alpha1 antibody treatment of ApoE-/- macrophages also inhibited migration of cells on collagen IV. CONCLUSIONS: Our results suggest that alpha1beta1 integrin is involved in atherosclerosis by mediating the migration of leukocytes to lesions by adhesion to collagen IV. Blocking this integrin reduces atherosclerosis and induces a stable plaque phenotype.