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Objectives: Our goal was to demonstrate that microparticles (MPs) are the endogenous signal leading to neovessel formation through CD40 ligation in human atherosclerotic plaques., Background: Vulnerable atherosclerotic plaques prone to rupture are characterized by an increased number of vasa vasorum and frequent intraplaque hemorrhage. Although inflammatory cytokines, growth factors, or CD40/CD40 ligand (CD40L) are possible candidates, the mechanism of atherosclerotic plaque neovascularization remains unknown. Atherosclerotic plaques contain large amounts of membrane-shed submicron MPs released after cell activation or apoptosis., Methods: Microparticles were isolated from endarterectomy specimens surgically obtained from 26 patients and characterized by phosphatidylserine exposure and specific markers of cellular origin., Results: Plaque MPs increased both endothelial proliferation assessed by (3)H-thymidine incorporation and cell number and stimulated in vivo angiogenesis in Matrigel (BD Biosciences, San Diego, California) assays performed in wild-type and BalbC/Nude mice, whereas circulating MPs had no effect. Microparticles from symptomatic patients expressed more CD40L and were more potent in inducing endothelial proliferation, when compared with asymptomatic plaque MPs. Most of CD40L+ MPs (93%) isolated from human plaques were of macrophage origin. Microparticle-induced endothelial proliferation was impaired by CD40L or CD40-neutralizing antibodies and abolished after endothelial CD40-ribonucleic acid silencing. In addition, the proangiogenic effect of plaque MPs was abolished in Matrigel assays performed in the presence of CD40L-neutralizing antibodies or in CD40-deficient mice., Conclusions: These results demonstrate that MPs isolated from human atherosclerotic lesions express CD40L, stimulate endothelial cell proliferation after CD40 ligation, and promote in vivo angiogenesis. Therefore, MPs could represent a major determinant of intraplaque neovascularization and plaque vulnerability.

Objective: To study the role of Mer receptor tyrosine kinase (mertk) in atherosclerosis., Methods and Results: We irradiated and reconstituted atherosclerosis-susceptible C57Bl/6 low-density lipoprotein receptor-deficient female mice (ldlr(-/-)) with either a mertk(+/+) or mertk(-/-) (tyrosine kinase-defective mertk) bone marrow. The mice were put on high-fat diet for either 8 or 15 weeks. Mertk deficiency led to increased accumulation of apoptotic cells within the lesions, promoted a proinflammatory immune response, and accelerated lesion development., Conclusions: Mertk expression by bone marrow-derived cells is required for the disposal of apoptotic cells and controls lesion development and inflammation.

Human atherosclerotic plaques express the metalloprotease tumor necrosis factor (TNF)-alpha converting enzyme (TACE/ADAM-17), which cleaves several transmembrane proteins including TNF and its receptors (TNFR-1 and TNFR-2). Plaques also harbor submicron vesicles (microparticles, MPs) released from plasma membranes after cell activation or apoptosis. We sought to examine whether TACE/ADAM17 is present on human plaque MPs and whether these MPs would affect TNF and TNFR-1 cellular shedding. Flow cytometry analysis detected 12,867 +/- 2007 TACE/ADAM17(+) MPs/mg of plaques isolated from 25 patients undergoing endarterectomy but none in healthy human internal mammary arteries. Plaque MPs harbored mainly mature active TACE/ADAM17 and dose dependently cleaved a pro-TNF mimetic peptide, whereas a preferential TACE/ADAM17 inhibitor (TMI-2) and recombinant TIMP-3 prevented this cleavage. Plaque MPs increased TNF shedding from the human cell line ECV-304 overexpressing TNF (ECV-304(TNF)), as well as TNFR-1 shedding from activated human umbilical vein endothelial cells or ECV-304(TNF) cells, without affecting TNF or TNFR-1 synthesis. MPs also activated the shedding of the endothelial protein C receptor from human umbilical vein endothelial cells. All these effects were inhibited by TMI-2. The present study shows that human plaque MPs carry catalytically active TACE/ADAM17 and significantly enhance the cell surface processing of the TACE/ADAM17 substrates TNF, TNFR-1, and endothelial protein C receptor, suggesting that TACE/ADAM17(+) MPs could regulate the inflammatory balance in the culprit lesion.

Background: Atherosclerosis is an immunoinflammatory disease; however, the key factors responsible for the maintenance of immune regulation in a proinflammatory milieu are poorly understood., Methods and Results: Here, we show that milk fat globule-EGF factor 8 (Mfge8, also known as lactadherin) is expressed in normal and atherosclerotic human arteries and is involved in phagocytic clearance of apoptotic cells by peritoneal macrophages. Disruption of bone marrow-derived Mfge8 in a murine model of atherosclerosis leads to substantial accumulation of apoptotic debris both systemically and within the developing lipid lesions. The accumulation of apoptotic material is associated with a reduction in interleukin-10 in the spleen but an increase in interferon-gamma production in both the spleen and the atherosclerotic arteries. In addition, we report a dendritic cell-dependent alteration of natural regulatory T-cell function in the absence of Mfge8. These events are associated with a marked acceleration of atherosclerosis., Conclusions: Lack of Mfge8 in bone marrow-derived cells enhances the accumulation of apoptotic cell corpses in atherosclerosis and alters the protective immune response, which leads to an acceleration of plaque development.

Objectives: Inflammatory macrophage apoptosis is critical to atherosclerotic plaque formation, but its mechanisms remain enigmatic. We hypothesized that inhibitor of apoptosis protein (IAP) survivin regulates macrophage death in atherosclerosis., Methods and Results: Western blot analysis revealed discrete survivin expression in human aorta lipid streaks but virtually none in advanced atherosclerotic plaques, despite increased XIAP and cIAP2 levels. Survivin was detected in CD68-positive macrophages infiltrating human lipid streaks by immunohistochemistry. In advanced atherosclerotic plaques, only rare macrophages outside the necrotic core or occasional fibrous cap smooth muscle cells expressed survivin. In vitro, macrophage colony-stimulating factor-stimulated mouse macrophage survivin expression, proliferation and resistance to apoptosis. Conversely, prolonged oxidized low-density lipoprotein treatment abolished macrophage survivin expression and triggered apoptosis after 12 hours, despite enhanced XIAP and cIAP2 expression. Adenoviral overexpression of survivin conferred macrophages with sustained resistance to apoptosis after oxidized low-density lipoprotein, tumor necrosis factor-alpha, or staurosporine challenge., Conclusions: Survivin is a critical modulator of atherosclerotic macrophage apoptosis under dual control by growth factors and oxidized lipids accumulating in atheroma. In early lipid streaks, growth factor-stimulated survivin expression may contribute to macrophage accumulation and survival, but dysregulation of survivin expression caused by recurrent oxidized low-density lipoprotein exposure may favor apoptosis in advanced atherosclerotic plaques, despite upregulated cIAP2 and XIAP.