Longhou Fang

Oxidation-Specific Epitopes Are Danger-Associated Molecular Patterns Recognized by Pattern Recognition Receptors of Innate Immunity

Oxidation reactions are vital parts of metabolism and signal transduction. However, they also produce reactive oxygen species, which damage lipids, proteins and DNA, generating “oxidation-specific” epitopes. In this review, we discuss the hypothesis that such common oxidation-specific epitopes are a major target of innate immunity, recognized by a variety of “pattern recognition receptors” (PRRs). By analogy with microbial “pathogen-associated molecular patterns” (PAMPs), we postulate that host-derived, oxidation-specific epitopes can be considered to represent “danger (or damage)-associated molecular patterns” (DAMPs). We also argue that oxidation-specific epitopes present on apoptotic cells and their cellular debris provided the primary evolutionary pressure for the selection of such PRRs. Furthermore, because many PAMPs on microbes share molecular identity and/or mimicry with oxidation-specific epitopes, such PAMPs provide a strong secondary selecting pressure for the same set of oxidation-specific PRRs as well. Because lipid peroxidation is ubiquitous and a major component of the inflammatory state associated with atherosclerosis, the understanding that oxidation-specific epitopes are DAMPs, and thus the target of multiple arcs of innate immunity, provides novel insights into the pathogenesis of atherosclerosis. As examples, we show that both cellular and soluble PRRs, such as CD36, toll-like receptor-4, natural antibodies, and C-reactive protein recognize common oxidation-specific DAMPs, such as oxidized phospholipids and oxidized cholesteryl esters, and mediate a variety of immune responses, from expression of proinflammatory genes to excessive intracellular lipoprotein accumulation to atheroprotective humoral immunity. These insights may lead to improved understanding of inflammation and atherogenesis and suggest new approaches to diagnosis and therapy.

Vascular Lipid Accumulation, Lipoprotein Oxidation, and Macrophage Lipid Uptake in Hypercholesterolemic Zebrafish

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 A2 (PLA2), we observed an increased vascular PLA2 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.

Control of angiogenesis by AIBP-mediated cholesterol efflux

Cholesterol is a structural component of the cell and is indispensable for normal cellular function, although 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 the apoA-I-containing high-density lipoprotein (HDL). Maintaining efficient cholesterol efflux is essential for normal cellular function. 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 to HDL and thereby regulates angiogenesis. AIBP- and HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 (also known as KDR) dimerization and signalling and inhibits vascular endothelial growth factor-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Notably, Aibp, a zebrafish homologue of human AIBP, regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell-autonomous regulator of angiogenesis. aibp knockdown results in dysregulated sprouting/branching angiogenesis, whereas forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1 (also known as Abca1a) 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 endothelial cells and that effective cholesterol efflux is critical for proper angiogenesis.

Oxidative Stress Activates Endothelial Innate Immunity via Sterol Regulatory Element Binding Protein 2 (SREBP2) Transactivation of MicroRNA-92a

Background— Oxidative stress activates endothelial innate immunity and disrupts endothelial functions, including endothelial nitric oxide synthase–derived nitric oxide bioavailability. Here, we postulated that oxidative stress induces sterol regulatory element–binding protein 2 (SREBP2) and microRNA-92a (miR-92a), which in turn activate endothelial innate immune response, leading to dysfunctional endothelium. Methods and Results— Using cultured endothelial cells challenged by diverse oxidative stresses, hypercholesterolemic zebrafish, and angiotensin II–infused or aged mice, we demonstrated that SREBP2 transactivation of microRNA-92a (miR-92a) is oxidative stress inducible. The SREBP2-induced miR-92a targets key molecules in endothelial homeostasis, including sirtuin 1, Krüppel-like factor 2, and Krüppel-like factor 4, leading to NOD-like receptor family pyrin domain-containing 3 inflammasome activation and endothelial nitric oxide synthase inhibition. In endothelial cell–specific SREBP2 transgenic mice, locked nucleic acid–modified antisense miR-92a attenuates inflammasome, improves vasodilation, and ameliorates angiotensin II–induced and aging-related atherogenesis. In patients with coronary artery disease, the level of circulating miR-92a is inversely correlated with endothelial cell–dependent, flow-mediated vasodilation and is positively correlated with serum level of interleukin-1&bgr;. Conclusions— Our findings suggest that SREBP2–miR-92a–inflammasome exacerbates endothelial dysfunction during oxidative stress. Identification of this mechanism may help in the diagnosis or treatment of disorders associated with oxidative stress, innate immune activation, and endothelial dysfunction.

Oxidized cholesteryl esters and phospholipids in zebrafish larvae fed a high cholesterol diet: macrophage binding and activation.

A novel hypercholesterolemic zebrafish model has been developed to study early events of atherogenesis. This model utilizes optically transparent zebrafish larvae, fed a high cholesterol diet (HCD), to monitor processes of vascular inflammation in live animals. Because lipoprotein oxidation is an important factor in the development of atherosclerosis, in this study, we characterized the oxidized lipid milieu in HCD-fed zebrafish larvae. Using liquid chromatography-mass spectrometry, we show that feeding an HCD for only 2 weeks resulted in up to 70-fold increases in specific oxidized cholesteryl esters, identical to those present in human minimally oxidized LDL and in murine atherosclerotic lesions. The levels of oxidized phospholipids, such as 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine, and of various lysophosphatidylcholines were also significantly elevated. Moreover, lipoproteins isolated from homogenates of HCD-fed larvae induced cell spreading as well as ERK1/2, Akt, and JNK phosphorylation in murine macrophages. Removal of apoB-containing lipoproteins from the zebrafish homogenates with an anti-human LDL antibody, as well as reducing lipid hydroperoxides with ebselen, resulted in inhibition of macrophage activation. The TLR4 deficiency in murine macrophages prevented their activation with zebrafish lipoproteins. Using biotinylated homogenates of HCD-fed larvae, we demonstrated that their components bound to murine macrophages, and this binding was effectively competed by minimally oxidized LDL but not by native LDL. These data provide evidence that molecular lipid determinants of proatherogenic macrophage phenotypes are present in large quantities in hypercholesterolemic zebrafish larvae and support the use of the HCD-fed zebrafish as a valuable model to study early events of atherogenesis.

Toll-Like Receptor-4 and Lipoprotein Accumulation in Macrophages

Excessive lipid accumulation in macrophages, also known as foam cell formation, is a key process during the development of atherosclerosis, leading to vascular inflammation and plaque growth. Recent studies have identified a new mechanism of macrophage lipid accumulation in which minimally oxidized low-density lipoprotein (mmLDL) and its active components, polyoxygenated cholesteryl ester hydroperoxides, are involved in endogenous activation of toll-like receptor-4 (TLR4), leading to recruitment of spleen tyrosine kinase (Syk), robust cytoskeletal rearrangements and macropinocytosis. In hyperlipidemic environments, mmLDL-induced, TLR4- and Syk-dependent macropinocytosis leads to substantial lipid accumulation in macrophages and monocytes, which may constitute an important mechanism of foam cell formation in atherosclerosis. A novel hypercholesterolemic zebrafish model of early stages of atherosclerosis was used to demonstrate that the TLR4 deficiency significantly reduces the in vivo rate of macrophage lipid accumulation in vascular lesions.

In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish

Oxidative modification of LDL is an early pathological event in the development of atherosclerosis. Oxidation events such as malondialdehyde (MDA) formation may produce specific, immunogenic epitopes. Indeed, antibodies to MDA-derived epitopes are widely used in atherosclerosis research and have been demonstrated to enable cardiovascular imaging. In this study, we engineered a transgenic zebrafish with temperature-inducible expression of an EGFP-labeled single-chain human monoclonal antibody, IK17, which binds to MDA-LDL, and used optically transparent zebrafish larvae for imaging studies. Feeding a high-cholesterol diet (HCD) supplemented with a red fluorescent lipid marker to the transgenic zebrafish resulted in vascular lipid accumulation, quantified in live animals using confocal microscopy. After heat shock-induced expression of IK17-EGFP, we measured the time course of vascular accumulation of IK17-specific MDA epitopes. Treatment with either an antioxidant or a regression diet resulted in reduced IK17 binding to vascular lesions. Interestingly, homogenates of IK17-EGFP-expressing larvae bound to MDA-LDL and inhibited MDA-LDL binding to macrophages. Moreover, sustained expression of IK17-EGFP effectively prevented HCD-induced lipid accumulation in the vascular wall, suggesting that the antibody itself may have therapeutic effects. Thus, we conclude that HCD-fed zebrafish larvae with conditional expression of EGFP-labeled oxidation-specific antibodies afford an efficient method of testing dietary and/or other therapeutic antioxidant strategies that may ultimately be applied to humans.

Zebrafish models of dyslipidemia: Relevance to atherosclerosis and angiogenesis

Lipid and lipoprotein metabolism in zebrafish and in humans are remarkably similar. Zebrafish express all major nuclear receptors, lipid transporters, apolipoproteins and enzymes involved in lipoprotein metabolism. Unlike mice, zebrafish express cetp and the Cetp activity is detected in zebrafish plasma. Feeding zebrafish a high cholesterol diet, without any genetic intervention, results in significant hypercholesterolemia and robust lipoprotein oxidation, making zebrafish an attractive animal model to study mechanisms relevant to early development of human atherosclerosis. These studies are facilitated by the optical transparency of zebrafish larvae and the availability of transgenic zebrafish expressing fluorescent proteins in endothelial cells and macrophages. Thus, vascular processes can be monitored in live animals. In this review article, we discuss recent advances in using dyslipidemic zebrafish in atherosclerosis-related studies. We also summarize recent work connecting lipid metabolism with regulation of angiogenesis, the work that considerably benefited from using the zebrafish model. These studies uncovered the role of aibp, abca1, abcg1, mtp, apoB, and apoC2 in regulation of angiogenesis in zebrafish and paved the way for future studies in mammals, which may suggest new therapeutic approaches to modulation of excessive or diminished angiogenesis contributing to the pathogenesis of human disease.

Emerging applications for zebrafish as a model organism to study oxidative mechanisms and their roles in inflammation and vascular accumulation of oxidized lipids

With the advent of genetic engineering, zebrafish (Danio rerio) were recognized as an attractive model organism to study many biological processes. Remarkably, the small size and optical transparency of zebrafish larvae enable high-resolution imaging of live animals. Zebrafish respond to various environmental and pathological factors with robust oxidative stress. In this article, we provide an overview of the molecular mechanisms involved in oxidative stress and antioxidant response in zebrafish. Existing applications of genetically encoded fluorescent sensors allow imaging, in real time, of the production of H(2)O(2) and studying its involvement in inflammatory responses, as well as activation of the oxidation-sensitive transcription factors HIF and NRF2. Oxidative stress, combined with hyperlipidemia, leads to oxidation of lipoproteins, the process that contributes significantly to the development of atherosclerosis in humans. Recent work found that feeding zebrafish a high-cholesterol diet results in hypercholesterolemia, vascular lipid accumulation, and extreme lipoprotein oxidation. Generation of a transgenic zebrafish expressing a green fluorescent protein-tagged human antibody to malondialdehyde (MDA)-modified LDL makes possible the in vivo visualization of MDA epitopes in the vascular wall and testing of the efficacy of antioxidants and dietary interventions. Thus, using zebrafish as a model organism provides important advantages in studying the roles of reactive oxygen species and lipid oxidation in basic biologic and pathologic processes.

Ezetimibe and Simvastatin Reduce Cholesterol Levels in Zebrafish Larvae Fed a High-Cholesterol Diet

Cholesterol-fed zebrafish is an emerging animal model to study metabolic, oxidative, and inflammatory vascular processes relevant to pathogenesis of human atherosclerosis. Zebrafish fed a high-cholesterol diet (HCD) develop hypercholesterolemia and are characterized by profound lipoprotein oxidation and vascular lipid accumulation. Using optically translucent zebrafish larvae has the advantage of monitoring vascular pathology and assessing the efficacy of drug candidates in live animals. Thus, we investigated whether simvastatin and ezetimibe, the principal drugs used in management of hypercholesterolemia in humans, would also reduce cholesterol levels in HCD-fed zebrafish larvae. We found that ezetimibe was well tolerated by zebrafish and effectively reduced cholesterol levels in HCD-fed larvae. In contrast, simvastatin added to water was poorly tolerated by zebrafish larvae and, when added to food, had little effect on cholesterol levels in HCD-fed larvae. Combination of low doses of ezetimibe and simvastatin had an additive effect in reducing cholesterol levels in zebrafish. These results suggest that ezetimibe exerts in zebrafish a therapeutic effect similar to that in humans and that the hypercholesterolemic zebrafish can be used as a low-cost and informative model for testing new drug candidates and for investigating mechanisms of action for existing drugs targeting dyslipidemia.