Myeloid A disintegrin and metalloproteinase domain 10 deficiency modulates atherosclerotic plaque composition by shifting the balance from inflammation toward fibrosis.

A disintegrin and metalloproteinase domain 10 (ADAM10) is a metalloprotease involved in cleavage of various cell surface molecules, such as adhesion molecules, chemokines, and growth factor receptors. Although we have previously shown an association of ADAM10 expression with atherosclerotic plaque progression, a causal role of ADAM10 in atherosclerosis has not been investigated. Bone marrow from conditional knockout mice lacking Adam10 in the myeloid lineage or from littermate controls was transplanted into lethally irradiated low density lipoprotein receptor Ldlr(-/-) mice on an atherogenic diet. Myeloid Adam10 deficiency did not affect plaque size, but it increased plaque collagen content. Matrix metalloproteinase 9 and 13 expression and matrix metalloproteinase 2 gelatinase activity were significantly impaired in Adam10-deficient macrophages, whereas their capacity to stimulate collagen production was unchanged. Furthermore, relative macrophage content in advanced atherosclerotic lesions was decreased. In vitro, Adam10-deficient macrophages showed reduced migration toward monocyte chemoattractant protein-1 and transmigration through collagen. In addition, Adam10-deficient macrophages displayed increased anti-inflammatory phenotype with elevated IL-10, and reduced production of proinflammatory tumor necrosis factor, IL-12, and nitric oxide in response to lipopolysaccharide. These data suggest a critical role of Adam10 for leukocyte recruitment, inflammatory mediator production, and extracellular matrix degradation. Thereby, myeloid ADAM10 may play a causal role in modulating atherosclerotic plaque stability.

Reprogramming macrophages to an anti-inflammatory phenotype by helminth antigens reduces murine atherosclerosis.

Atherosclerosis is a lipid-driven inflammatory disease of the vessel wall, characterized by the chronic activation of macrophages. We investigated whether the helminth-derived antigens [soluble egg antigens (SEAs)] could modulate macrophage inflammatory responses and protect against atherosclerosis in mice. In bone marrow-derived macrophages, SEAs induce anti-inflammatory macrophages, typified by high levels of IL-10 and reduced secretion of proinflammatory mediators. In hyperlipidemic LDLR(-/-) mice, SEA treatment reduced plaque size by 44%, and plaques were less advanced compared with PBS-injected littermate controls. The atheroprotective effect of SEAs was found to be mainly independent of cholesterol lowering and T-lymphocyte responses but instead could be attributed to diminished myeloid cell activation. SEAs reduced circulating neutrophils and inflammatory Ly6C(high) monocytes, and macrophages showed high IL-10 production. In line with the observed systemic effects, atherosclerotic lesions of SEA-treated mice showed reduced intraplaque inflammation as inflammatory markers [TNF-α, monocyte chemotactic protein 1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and CD68], neutrophil content, and newly recruited macrophages were decreased. We show that SEA treatment protects against atherosclerosis development by dampening inflammatory responses. In the future, helminth-derived components may provide novel opportunities to treat chronic inflammatory diseases, as they diminish systemic inflammation and reduce the activation of immune cells.

A disintegrin and metalloprotease 10 is a novel mediator of vascular endothelial growth factor-induced endothelial cell function in angiogenesis and is associated with atherosclerosis.

OBJECTIVE: To elucidate the downstream mechanisms of vascular endothelial growth factor receptor 2 (VEGFR2), a key receptor in angiogenesis, which has been associated with atherosclerotic plaque growth and instability. METHODS AND RESULTS: By using a yeast-2-hybrid assay, we identified A Disintegrin And Metalloprotease 10 (ADAM10) as a novel binding partner of VEGFR2. ADAM10 is a metalloprotease with sheddase activity involved in cell migration; however, its exact function in endothelial cells (ECs), angiogenesis, and atherosclerosis is largely unknown. For the first time to our knowledge, we show ADAM10 expression in human atherosclerotic lesions, associated with plaque progression and neovascularization. We demonstrate ADAM10 expression and activity in ECs to be induced by VEGF; also, ADAM10 mediates the ectodomain shedding of VEGFR2. Furthermore, VEGF induces ADAM10-mediated cleavage of vascular endothelium (VE)-cadherin, which could increase vascular permeability and facilitate EC migration. Indeed, VEGF increases vascular permeability in an ADAM10- and ADAM17-dependent way; inhibition of ADAM10 reduces EC migration and chemotaxis. CONCLUSIONS: These data provide the first evidence of ADAM10 expression in atherosclerosis and neovascularization. ADAM10 plays a functional role in VEGF-induced EC function. These data open perspectives for novel therapeutic interventions in vascular diseases.

Whole body and hematopoietic ADAM8 deficiency does not influence advanced atherosclerotic lesion development, despite its association with human plaque progression.

Although A Disintegrin And Metalloproteinase 8 (ADAM8) is not crucial for tissue development and homeostasis, it has been implicated in various inflammatory diseases by regulating processes like immune cell recruitment and activation. ADAM8 expression has been associated with human atherosclerosis development and myocardial infarction, however a causal role of ADAM8 in atherosclerosis has not been investigated thus far. In this study, we examined the expression of ADAM8 in early and progressed human atherosclerotic lesions, in which ADAM8 was significantly upregulated in vulnerable lesions. In addition, ADAM8 expression was most prominent in the shoulder region of human atherosclerotic lesions, characterized by the abundance of foam cells. In mice, Adam8 was highly expressed in circulating neutrophils and in macrophages. Moreover, ADAM8 deficient mouse macrophages displayed reduced secretion of inflammatory mediators. Remarkably, however, neither hematopoietic nor whole-body ADAM8 deficiency in mice affected atherosclerotic lesion size. Additionally, except for an increase in granulocyte content in plaques of ADAM8 deficient mice, lesion morphology was unaffected. Taken together, whole body and hematopoietic ADAM8 does not contribute to advanced atherosclerotic plaque development, at least in female mice, although its expression might still be valuable as a diagnostic/prognostic biomarker to distinguish between stable and unstable lesions.

High-Density Lipoproteins Exert Pro-inflammatory Effects on Macrophages via Passive Cholesterol Depletion and PKC-NF-κB/STAT1-IRF1 Signaling.

Membrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain elusive. Here we show overt pro-inflammatory effects of HDL-mediated passive cholesterol depletion and lipid raft disruption in murine and human primary macrophages in vitro. These pro-inflammatory effects were confirmed in vivo in peritoneal macrophages from apoA-I transgenic mice, which have elevated HDL levels. In line with these findings, the innate immune responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Expression analysis, ChIP-PCR, and combinatorial pharmacological and genetic intervention studies unveiled that both native and reconstituted HDL enhance Toll-like-receptor-induced signaling by activating a PKC-NF-κB/STAT1-IRF1 axis, leading to increased inflammatory cytokine expression. HDL's pro-inflammatory activity supports proper functioning of macrophage immune responses.

Deleting myeloid IL-10 receptor signalling attenuates atherosclerosis in LDLR-/- mice by altering intestinal cholesterol fluxes.

Inflammatory responses and cholesterol homeostasis are interconnected in atherogenesis. Interleukin (IL)-10 is an important anti-inflammatory cytokine, known to suppress atherosclerosis development. However, the specific cell types responsible for the atheroprotective effects of IL-10 remain to be defined and knowledge on the actions of IL-10 in cholesterol homeostasis is scarce. Here we investigated the functional involvement of myeloid IL-10-mediated atheroprotection. To do so, bone marrow from IL-10 receptor 1 (IL-10R1) wild-type and myeloid IL-10R1-deficient mice was transplanted to lethally irradiated female LDLR-/- mice. Hereafter, mice were given a high cholesterol diet for 10 weeks after which atherosclerosis development and cholesterol metabolism were investigated. In vitro, myeloid IL-10R1 deficiency resulted in a pro-inflammatory macrophage phenotype. However, in vivo significantly reduced lesion size and severity was observed. This phenotype was associated with lower myeloid cell accumulation and more apoptosis in the lesions. Additionally, a profound reduction in plasma and liver cholesterol was observed upon myeloid IL-10R1 deficiency, which was reflected in plaque lipid content. This decreased hypercholesterolaemia was associated with lowered very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) levels, likely as a response to decreased intestinal cholesterol absorption. In addition, IL-10R1 deficient mice demonstrated substantially higher faecal sterol loss caused by increased non-biliary cholesterol efflux. The induction of this process was linked to impaired ACAT2-mediated esterification of liver and plasma cholesterol. Overall, myeloid cells do not contribute to IL-10-mediated atheroprotection. In addition, this study demonstrates a novel connection between IL-10-mediated inflammation and cholesterol homeostasis in atherosclerosis. These findings make us reconsider IL-10 as a beneficial influence on atherosclerosis.

Hematopoietic miR155 deficiency enhances atherosclerosis and decreases plaque stability in hyperlipidemic mice.

microRNA-155 (miR155) is a central regulator of immune responses that is induced by inflammatory mediators. Although miR155 is considered to be a pro-inflammatory microRNA, in vitro reports show anti-inflammatory effects in lipid-loaded cells. In this study we examined the role of miR155 in atherosclerosis in vivo using bone marrow transplantation from miR155 deficient or wildtype mice to hyperlipidemic mice. Hematopoietic deficiency of miR155 enhanced atherosclerotic plaque development and decreased plaque stability, as evidenced by increased myeloid inflammatory cell recruitment to the plaque. The increased inflammatory state was mirrored by a decrease in circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells, and an increase in granulocytes (CD11b(+)Ly6G(+)) in blood of miR155(-/-) transplanted mice. Moreover, we show for the first time a crucial role of miR155 in monocyte subset differentiation, since hematopoietic deficiency of miR155 increases the 'inflammatory' monocyte subset (CD11b(+)Ly6G(-)Ly6C(hi)) and reduces 'resident' monocytes (CD11b(+)Ly6G(-)Ly6C(low)) in the circulation. Furthermore, cytokine production by resident peritoneal macrophages of miR155(-/-) transplanted hyperlipidemic mice was skewed towards a more pro-inflammatory state since anti-inflammatory IL-10 production was reduced. In conclusion, in this hyperlipidemic mouse model miR155 acts as an anti-inflammatory, atheroprotective microRNA. Additionally, besides a known role in lymphoid cell development, we show a crucial role of miR155 in myeloid lineage differentiation.