Abstract-Lipid accumulation in arteries induces vascular inflammation and atherosclerosis, the major cause of heart attack and stroke in humans. Extreme hyperlipidemia induced in mice and rabbits enables modeling many aspects of human atherosclerosis, but microscopic examination of plaques is possible only postmortem. Here we report that feeding adult zebrafish (Danio rerio) a high-cholesterol diet (HCD) resulted in hypercholesterolemia, remarkable lipoprotein oxidation, and fatty streak formation in the arteries. Feeding an HCD supplemented with a fluorescent cholesteryl ester to optically transparent fli1:EGFP zebrafish larvae in which endothelial cells express green fluorescent protein (GFP), and using confocal microscopy enabled monitoring vascular lipid accumulation and the endothelial cell layer disorganization and thickening in a live animal. The HCD feeding also increased leakage of a fluorescent dextran from the blood vessels. Administering ezetimibe significantly diminished the HCD-induced endothelial cell layer thickening and improved its barrier function. Feeding HCD to lyz:DsRed2 larvae in which macrophages and granulocytes express DsRed resulted in the accumulation of fluorescent myeloid cells in the vascular wall. Using a fluorogenic substrate for phospholipase A 2 (PLA 2 ), we observed an increased vascular PLA 2 activity in live HCD-fed larvae compared to control larvae. Furthermore, by transplanting genetically modified murine cells into HCD-fed larvae, we demonstrated that toll-like receptor-4 was required for efficient in vivo lipid uptake by macrophages. These results suggest that the novel zebrafish model is suitable for studying temporal characteristics of certain inflammatory processes of early atherogenesis and the in vivo function of vascular cells.

Abstract-Toll-like receptor (TLR)4 recognizes microbial pathogens, such as lipopolysaccharide, and mediates lipopolysaccharide-induced proinflammatory cytokine secretion, as well as microbial uptake by macrophages. In addition to exogenous pathogens, TLR4 recognizes modified self, such as minimally oxidized low-density lipoprotein (mmLDL). Here we report that mmLDL and its active components, cholesteryl ester hydroperoxides, induce TLR4-dependent fluid phase uptake typical of macropinocytosis. We show that mmLDL induced recruitment of spleen tyrosine kinase (Syk) to a TLR4 signaling complex, TLR4 phosphorylation, activation of a Vav1-Ras-Raf-MEK-ERK1/2 signaling cascade, phosphorylation of paxillin, and activation of Rac, Cdc42, and Rho. These mmLDL-induced and TLR4-and Syk-dependent signaling events and cytoskeletal rearrangements lead to enhanced uptake of small molecules, dextran, and, most importantly, both native and oxidized LDL, resulting in intracellular lipid accumulation. An intravenous injection of fluorescently labeled mmLDL in wild-type mice resulted in its rapid accumulation in circulating monocytes, which was significantly attenuated in TLR4-deficient mice. These data describe a novel mechanism leading to enhanced lipoprotein uptake in macrophages that would contribute to foam cell formation and atherosclerosis. These data also suggest that cholesteryl ester hydroperoxides are an endogenous ligand for TLR4. Because TLR4 is highly expressed on the surface of circulating monocytes in patients with chronic inflammatory conditions, and cholesteryl ester hydroperoxides are present in plasma, lipid uptake by monocytes in circulation may contribute to the pathological roles of monocytes in chronic inflammatory diseases. T oll-like receptors (TLRs) are pattern recognition receptors (PRRs) that sense the presence of numerous pathogen-associated molecular patterns (PAMPs). 1 Activation of PRRs has been widely implicated in signaling mechanisms that contribute to chronic inflammatory diseases, including atherosclerosis. Although PRRs were originally postulated to recognize only exogenous pathogens, they are now increasingly documented to respond to endogenous modified self, such as modified and/or oxidized low-density lipoprotein (LDL). We have postulated that such modified LDL could become endogenous PAMPs and initiate lowgrade, but sustained, PRR-mediated inflammation and other immune responses. 2 TLR4 and TLR2 deficiency, as well as a deficiency in MyD88, a signaling adaptor molecule for TLR4 and other TLRs, result in reduced atherosclerosis in apoE Ϫ/Ϫ or LDLR Ϫ/Ϫ mice fed a high-fat diet. [3][4][5] Some human studies, though not all, have shown that inactivating polymorphisms in TLR4 are associated with decreased cardiovascular disease risk. 6 Although these data support a general understanding that TLRs regulate inflammation in atherosclerosis, specific mechanisms are poorly understood.Excessive lipoprotein accumulation by macrophages and the formation of lipid-loaded foam cells is a rate-limiting pro...

Cholesterol is a structural component of the cell, indispensable for normal cellular function, but its excess often leads to abnormal proliferation, migration, inflammatory responses and/or cell death. To prevent cholesterol overload, ATP-binding cassette (ABC) transporters mediate cholesterol efflux from the cells to apolipoprotein A-I (ApoA-I) and to the ApoA-I-containing high-density lipoprotein (HDL)1-3. Maintaining efficient cholesterol efflux is essential for normal cellular function4-6. However, the role of cholesterol efflux in angiogenesis and the identity of its local regulators are poorly understood. Here we show that ApoA-I binding protein (AIBP) accelerates cholesterol efflux from endothelial cells (EC) to HDL and thereby regulates angiogenesis. AIBP/HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 dimerization and signaling, and inhibits VEGF-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Remarkably, Aibp regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell autonomous regulator of zebrafish angiogenesis. Aibp knockdown results in dysregulated sprouting/branching angiogenesis, while forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1/Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1/Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from EC and that effective cholesterol efflux is critical for proper angiogenesis.

Abstract-Oxidative modification of low-density lipoprotein (LDL) plays a causative role in the development of atherosclerosis. In this study, we demonstrate that minimally oxidized LDL (mmLDL) stimulates intracellular reactive oxygen species (ROS) generation in macrophages through NADPH oxidase 2 (gp91phox/Nox2), which, in turn, induces production of RANTES and migration of smooth muscle cells. Peritoneal macrophages from gp91phox/Nox2 Ϫ/Ϫ mice or J774 macrophages in which Nox2 was knocked down by small interfering RNA failed to generate ROS in response to mmLDL. Because mmLDL-induced cytoskeletal changes were dependent on Toll-like receptor (TLR)4, we analyzed ROS generation in peritoneal macrophages from wild-type, TLR4 Ϫ/Ϫ , or MyD88 Ϫ/Ϫ mice and found that mmLDLmediated ROS was generated in a TLR4-dependent, but MyD88-independent, manner. Furthermore, we found that ROS generation required the recruitment and activation of spleen tyrosine kinase (Syk) and that mmLDL also induced phospholipase PLC␥1 phosphorylation and protein kinase C membrane translocation. Importantly, the phospholipase C␥1 phosphorylation was reduced in J774 cells expressing Syk-specific short hairpin RNA. Nox2 modulated mmLDL activation of macrophages by regulating the expression of proinflammatory cytokines interleukin-1␤, interleukin-6, and RANTES. We showed that purified RANTES was able to stimulate migration of mouse aortic smooth muscle cells and addition of neutralizing antibody against RANTES abolished the migration of mouse aortic smooth muscle cells stimulated by mmLDL-stimulated macrophages. These results suggest that mmLDL induces generation of ROS through sequential activation of TLR4, Syk, phospholipase C␥1, protein kinase C, and gp91phox/Nox2 and thereby stimulates expression of proinflammatory cytokines. These data help explain mechanisms by which endogenous ligands, such as mmLDL, can induce TLR4-dependent, proatherogenic activation of macrophages. (Circ Res. 2009;104:210-218.) Key Words: minimally oxidized LDL Ⅲ reactive oxygen species Ⅲ NADPH oxidase 2 Ⅲ RANTES Ⅲ atherosclerosis R eactive oxygen species (ROS), including hydrogen peroxide and superoxide anion, are generally considered cytotoxic. 1 However, in recent years, many reports have demonstrated that intracellular ROS, produced in mammalian cells in response to the activation of various receptors, also serve as important second messengers in cell signaling. 1 The major source of ROS in phagocytes is an NADPH oxidase complex. 2 It is composed of 6 protein components: 2 transmembrane flavocytochrome b proteins (gp91phox and p22phox) and 4 cytosolic proteins (p47phox, p67phox, p40phox, and Rac). The catalytic protein in this complex is gp91phox, recently renamed as Nox2. The NH 3 -terminal region of gp91phox/Nox2 consists of transmembrane domains that bind 2 heme groups, and the COOH-terminal region contains NADPH/FAD binding sites. The cytosolic component of the complex, p47phox, plays a critical role in an assembly of the whole NADPH oxidase complex on activation o...