Ldlr Mice Research Articles

Long‐term curcumin administration protects against atherosclerosis via hepatic regulation of lipoprotein cholesterol metabolism

Atherosclerosis is a major cause of cardiovascular disease caused by high cholesterol. Stains are widely prescribed to lower cholesterol levels, but natural dietary compounds may also be effective. Therefore, we studied the effect of the natural dietary compound curcumin on atherosclerosis and its underlying mechanisms based on plasma and hepatic lipid metabolism. LDLR(-/-) mice were fed a high-cholesterol diet and treated with curcumin, lovastatin or control (n=10 per group) for 18 wk. Aortic arch sections revealed curcumin ameliorated early atherosclerotic lesions, lipid infiltration, ICAM-1 and VCAM-1 localization, similar to lovastatin treatment. Furthermore, curcumin lowered plasma cholesterol, triglycerides, LDL cholesterol and Apo B levels as well as CETP activity, while curcumin increased plasma HDL cholesterol and liver Apo A-I expression, similar to lovastatin treatment. Curcumin caused transcriptional inhibition of HMG-CoA reductase, independent of ACAT1 and ACAT2 expression. Hepatic PPARα and LXRα expression was upregulated by curcumin treatment. Hepatic complement factor D (Cfd) and systemic CRP levels, markers of immune complement pathway activation, were significantly reduced by curcumin treatment. Long-term curcumin treatment lowers plasma and hepatic cholesterol and suppresses early atherosclerotic lesions comparable to the protective effects of lovastatin. The anti-atherogenic effect of curcumin is mediated via multiple mechanisms including altered lipid, cholesterol and immune gene expression.

POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1) inhibits endothelial cell senescence through a p53 dependent pathway

Vascular cell senescence, induced by the DNA damage response or inflammatory stress, contributes to age-associated vascular disease. Using complementary DNA microarray technology, we found that the level of POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1) is downregulated during endothelial cell (EC) senescence. PATZ1 may have an important role as a transcriptional repressor in chromatin remodeling and transcription regulation; however, the role of PATZ1 in EC senescence and vascular aging remains unidentified. Knockdown of PATZ1 in young cells accelerated premature EC senescence, which was confirmed by growth arrest, increased p53 protein level and senescence-associated β-galactosidase (SA-β-gal) activity, and repression of EC tube formation. In contrast, overexpression of PATZ1 in senescent cells reversed senescent phenotypes. Cellular senescence induced by PATZ1 knockdown in young cells was rescued by knockdown of p53, but not by knockdown of p16(INK4a). PATZ1 knockdown increased ROS levels, and pretreatment with N-acetylcysteine abolished EC senescence induced by PATZ1 knockdown. Notably, PATZ1 immunoreactivity was lower in ECs of atherosclerotic tissues than those of normal arteries in LDLR(-/-) mice, and immunoreactivity also decreased in ECs of old human arteries. These results suggest that PATZ1 may have an important role in the regulation of EC senescence through an ROS-mediated p53-dependent pathway and contribute to vascular diseases associated with aging.

Flt3 Signaling-Dependent Dendritic Cells Protect against Atherosclerosis

Early events in atherosclerosis occur in the aortic intima and involve monocytes that become macrophages. We looked for these cells in the steady state adult mouse aorta, and surprisingly, we found a dominance of dendritic cells (DCs) in the intima. In contrast to aortic adventitial macrophages, CD11c(+)MHC II(hi) DCs were poorly phagocytic but were immune stimulatory. DCs were of two types primarily: classical Flt3-Flt3L signaling-dependent, CD103(+)CD11b(-) DCs and macrophage-colony stimulating factor (M-CSF)-dependent, CD14(+)CD11b(+)DC-SIGN(+) monocyte-derived DCs. Both types expanded during atherosclerosis. By crossing Flt3(-/-) to Ldlr(-/-) atherosclerosis-prone mice, we developed a selective and marked deficiency of classical CD103(+) aortic DCs, and they were associated with exacerbated atherosclerosis without alterations in blood lipids. Concomitantly, the Flt3(-/-)Ldlr(-/-) mice had fewer Foxp3(+) Treg cells and increased inflammatory cytokine mRNAs in the aorta. Therefore, functional DCs are dominant in normal aortic intima and, in contrast to macrophages, CD103(+) classical DCs are associated with atherosclerosis protection.

A novel atherogenic epitope from Mycobacterium tuberculosis heat shock protein 65 enhances atherosclerosis in rabbit and LDL receptor-deficient mice

Previous studies have established that Mycobacterium tuberculosis heat shock protein 65 (mHSP65) plays an important role in immune-associated diseases as an autoimmune factor. Some overlapping epitopes of mHSP65 may serve as initiators of both atherosclerosis and other autoimmune-associated diseases. In the present study, atherosclerosis was significantly enhanced in high-cholesterol diet (HCD)-fed New Zealand white rabbits immunized with mHSP65(91-105) compared with PBS-immunized or BSA-immunized rabbits. Immunizing wild-type C57BL/6J mice with mHSP65(91-105) induced the aortic endothelial injury. Although western blot demonstrated that specific antibodies against mHSP65(91-105) can cross-react with recombinant human heat shock protein 60, specific antibodies against mHSP65(91-105) had no direct effects on HUVECs in vitro. Laser scanning confocal microscopy showed that mHSP65(91-105) localized in the cytoplasm of HUVECs, even when HUVECs were heat shocked at 42°C. mHSP65(91-105)-specific splenic cells secreted more IFN-γ than controls. Also, adoptive transfer of mHSP65(91-105)-specific splenic cells can accelerate atherosclerosis in ldlr( -/- ) mice. We can conclude that the (auto)immune response to mHSP65(91-105) accelerates atherosclerosis in animal models, and that the response of Th1 plays an important role in this progress.

Binding of CD40L to Mac-1's I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis--but does not affect immunity and thrombosis in mice.

CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the β-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr(-/-) mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.

Adverse effects of hyperlipidemia on bone regeneration and strength

Hyperlipidemia increases the risk for generation of lipid oxidation products, which accumulate in the subendothelial spaces of vasculature and bone. Atherogenic high-fat diets increase serum levels of oxidized lipids, which are known to attenuate osteogenesis in culture and to promote bone loss in mice. In this study, we investigated whether oxidized lipids affect bone regeneration and mechanical strength. Wild-type (WT) and hyperlipidemic (Ldlr(-/-)) mice were placed on a high-fat (HF) diet for 13 weeks. Bilateral cranial defects were introduced on each side of the sagittal suture, and 5 weeks postsurgery on the respective diets, the repair/regeneration of cranial bones and mechanical properties of femoral bones were assessed. MicroCT and histological analyses demonstrated that bone regeneration was significantly impaired by the HF diet in WT and Ldlr(-/-) mice. In femoral bone, cortical bone volume fraction (bone volume [BV]/tissue volume [TV]) was significantly reduced, whereas cortical porosity was increased by the HF diet in Ldlr(-/-) but not in WT mice. Femoral bone strength and stiffness, measured by three-point bending analysis, were significantly reduced by the HF diet in Ldlr(-/-), but not in WT mice. Serum analysis showed that the HF diet significantly increased levels of parathyroid hormone, tumor necrosis factor (TNF)-α, calcium, and phosphorus, whereas it reduced procollagen type I N-terminal propeptide, a serum marker of bone formation, in Ldlr(-/-), but not in WT mice. The serum level of carboxyl-terminal collagen crosslinks, a marker for bone resorption, was also 1.7-fold greater in Ldlr(-/-) mice. These findings suggest that hyperlipidemia induces secondary hyperparathyroidism and impairs bone regeneration and mechanical strength.

Human Oxidation-Specific Antibodies Reduce Foam Cell Formation and Atherosclerosis Progression

We sought to assess the in vivo importance of scavenger receptor (SR)-mediated uptake of oxidized low-density lipoprotein (OxLDL) in atherogenesis and to test the efficacy of human antibody IK17-Fab or IK17 single-chain Fv fragment (IK17-scFv), which lacks immunologic properties of intact antibodies other than the ability to inhibit uptake of OxLDL by macrophages, to inhibit atherosclerosis. The unregulated uptake of OxLDL by macrophage SR contributes to foam cell formation, but the importance of this pathway in vivo is uncertain. Cholesterol-fed low-density lipoprotein receptor knockout (LDLR(-/-)) mice were treated with intraperitoneal infusion of human IK17-Fab (2.5 mg/kg) 3 times per week for 14 weeks. Because anti-human antibodies developed in these mice, LDLR(-/-)/low-density lipoprotein receptor Rag 1 double-knockout mice (lacking the ability to make immunoglobulins due to loss of T- and B-cell function) were treated with an adenoviral vector encoding adenovirus expressed (Adv)-IK17-scFv or control adenovirus-enhanced green fluorescent protein vector intravenously every 2 weeks for 16 weeks. In LDLR(-/-) mice, infusion of IK17-Fab was able to sustain IK17 plasma levels for the first 8 weeks, but these diminished afterward due to increasing murine anti-IK17 antibody titers. Despite this, after 14 weeks, a 29% decrease in en face atherosclerosis was noted compared with phosphate-buffered saline-treated mice. In LDLR(-/-)/low-density lipoprotein receptor Rag 1 double-knockout mice, sustained levels of plasma IK17-scFv was achieved by Adv-IK17-scFv-mediated hepatic expression, which led to a 46% reduction (p < 0.001) in en face atherosclerosis compared with adenovirus-enhanced green fluorescent protein vector. Importantly, peritoneal macrophages isolated from Adv-IK17-scFv treated mice had decreased lipid accumulation compared with adenovirus-enhanced green fluorescent protein-treated mice. These data support an important role for SR-mediated uptake of OxLDL in the pathogenesis of atherosclerosis and demonstrate that oxidation-specific antibodies reduce the progression of atherosclerosis, suggesting their potential in treating cardiovascular disease in humans.

26 The role of properdin in murine atherosclerosis

BackgroundAtherosclerosis in humans and mice has inflammatory, immune and metabolic components. Mice lacking low-density lipoprotein receptor (LDLR(−/−)) may be protected from atherogenesis by some components of the complement system. The...

AAV8-Mediated Long-Term Expression of Human LCAT Significantly Improves Lipid Profiles in hCETP;Ldlr+/− Mice

Lecithin:cholesterol acyltransferase (LCAT) is the key circulating enzyme responsible for high-density lipoprotein (HDL) cholesterol esterification, HDL maturation, and potentially reverse cholesterol transport. To further explore LCAT's mechanism of action on lipoprotein metabolism, we employed adeno-associated viral vector (AAV) serotype 8 to achieve long-term (32-week) high level expression of human LCAT in hCETP;Ldlr(+/-) mice, and characterized the lipid profiles in detail. The mice had a marked increase in HDL cholesterol, HDL particle size, and significant reduction in low-density lipoprotein (LDL) cholesterol, plasma triglycerides, and plasma apoB. Plasma LCAT activity significantly increased with humanized substrate specificity. HDL cholesteryl esters increased in a fashion that fits human LCAT specificity. HDL phosphatidylcholines trended toward decrease, with no change observed for HDL lysophosphatidylcholines. Triglycerides reduction appeared to reside in all lipoprotein particles (very low-density lipoprotein (VLDL), LDL, and HDL), with HDL triglycerides composition highly reflective of VLDL, suggesting that changes in HDL triglycerides were primarily driven by the altered triglycerides metabolism in VLDL. In summary, in this human-like model for lipoprotein metabolism, AAV8-mediated overexpression of human LCAT resulted in profound changes in plasma lipid profiles. Detailed lipid analyses in the lipoprotein particles suggest that LCAT's beneficial effect on lipid metabolism includes not only enhanced HDL cholesterol esterification but also improved metabolism of apoB-containing particles and triglycerides. Our findings thus shed new light on LCAT's mechanism of action and lend support to its therapeutic potential in treating dyslipidemia.

Novel insights into the development of atherosclerosis in hemophilia A mouse models

Cardiovascular diseases in aging people with hemophilia (PWH) represent a growing concern. The underlying hypocoagulability probably provides a protective effect against acute thrombus formation, but the limited data available show no preventive effect against the development of atherogenesis in PWH. Atherosclerosis-prone mice are attractive tools for the study of atherosclerosis development, and may provide insights into disease progression in PWH. Severe hemophilia A (factor VIII-deficient [FVIII(o)]) mice were crossed with mice lacking apolipoprotein E (ApoE(-/-)) or mice lacking the LDL receptor (LDLR(-/-)), and then compared to hemostatically normal littermate controls. After mice had received atherogenic diets for 8, 22 or 37 weeks, atherosclerotic lesion size and phenotypic characterization were analyzed in the aortic sinus and whole aortas. ApoE(-/-)/FVIII(o) mice showed a time-dependent protective effect against the development of atherosclerosis, beginning after 22 diet-weeks and persisting to 37 diet-weeks in both the aorta sinus and whole aorta as compared with ApoE(-/-) mice. Notably, the FVIII deficiency did not influence the progression of atherosclerosis in the FVIII(o)/LDLR(-/-) model as compared with controls at early or late time points. Hypocoagulability ameliorates vascular disease in the ApoE-deficient model in a lipid-independent manner. Interestingly, FVIII deficiency did not affect the development of atherosclerosis in LDLR(-/-) mice. In contrast to the ApoE model, the LDLR model resembles the lipid profile that is commonly observed in humans with atherosclerosis. These findings, to a certain extent, support the notion of atherosclerosis development in the complete absence of FVIII.

RNAi-mediated knockdown of HMG CoA reductase enhances gene expression from physiologically regulated low-density lipoprotein receptor therapeutic vectors in vivo

The development of novel strategies to enhance gene expression from therapeutic vectors may prove advantageous for complementation gene therapy. This applies to therapeutic expression of the low-density lipoprotein receptor (LDLR) gene to treat familial hypercholesterolaemia (FH), where appropriate gene regulation could enhance therapeutic effect. We have previously reported that LDLR genomic DNA expression vectors can be regulated in vivo by pravastatin. In the current study, we investigated whether targeted knockdown of the mevalonate pathway in conjunction with LDLR delivery would lead to enhanced LDLR transgene expression and improved phenotype recovery. We demonstrated here that knockdown of HMG CoA reductase (HMGCR) by up to 70% using small interfering RNAs (siRNAs) led to a significant increase in binding and internalisation of LDL particles in vitro in mouse and human cells. In vivo co-injection of LDLR promoter luciferase expression plasmids with siRNAs or microRNA (miRNA) expression vectors targeting mouse Hmgcr led to at least a 10-fold increase in luciferase expression. Injection of Ldlr(-/-) mice with pLDLR-LDLR expression plasmids led to a significant reduction in plasmid LDL cholesterol, which was further enhanced by co-injection with miRNA expression vectors targeted to mouse Hmgcr. Our data suggest that targeted knockdown of HMGCR may enhance gene therapy outcomes for FH.

Unexpected roles for PCSK9 in lipid metabolism

To consider the evidence that PCSK9 has effects on lipoprotein metabolism that are in addition to its role in promoting the degradation of the LDL receptor. Transgenic mice expressing human PCSK9 under physiological control have recently been described. As well as the expected effects on LDL-receptor protein levels in the liver, mice expressing the gain-of-function mutant D374Y secrete more triglyceride than control mice or mice expressing wild-type PCSK9, supporting earlier suggestions that apoB synthesis is increased in hepatocytes expressing D374Y PCSK9 and that patients heterozygous for PCSK9 mutations have increased apoB synthesis. No increase in triglyceride secretion was observed in LDLR mice, suggesting that the effect of PCSK9 on triglyceride secretion is to some extent independent of the LDL receptor. Other recent studies have shown an association between serum PCSK9 concentration and serum triglyceride, but care has to be taken in interpretation of these results as it has also been shown that the level of PCSK9 in human serum shows strong diurnal variation. Understanding the physiology of PCSK9 is important because this protein has become a major new target for lipid lowering therapy.

Mechanisms of Urokinase Plasminogen Activator (uPA)-mediated Atherosclerosis: ROLE OF THE uPA RECEPTOR AND S100A8/A9 PROTEINS

Data from clinical studies, cell culture, and animal models implicate the urokinase plasminogen activator (uPA)/uPA receptor (uPAR)/plasminogen system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/uPAR/plasminogen stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression and mice genetically deficient in uPAR to elucidate mechanisms of uPA/uPAR/plasminogen-accelerated atherosclerosis and aneurysm formation. We found that macrophage-specific uPA overexpression accelerates atherosclerosis and causes aortic root dilation in fat-fed Ldlr(-/-) mice (as we previously reported in Apoe(-/-) mice). Macrophage-expressed uPA accelerates atherosclerosis by stimulation of lesion progression rather than initiation and causes disproportionate lipid accumulation in early lesions. uPA-accelerated atherosclerosis and aortic dilation are largely, if not completely, independent of uPAR. In the absence of uPA overexpression, however, uPAR contributes modestly to both atherosclerosis and aortic dilation. Microarray studies identified S100A8 and S100A9 mRNA as the most highly up-regulated transcripts in uPA-overexpressing macrophages; up-regulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also up-regulated in the aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is up-regulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Macrophage microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm in a second animal model that macrophage-expressed uPA stimulates atherosclerosis and aortic dilation. They also reveal uPAR independence of these actions and implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease.

Hematopoietic Fas Deficiency Does Not Affect Experimental Atherosclerotic Lesion Formation despite Inducing a Proatherogenic State

The Fas death receptor (CD95) is expressed on macrophages, smooth muscle cells, and T cells within atherosclerotic lesions. Given the dual roles of Fas in both apoptotic and nonapoptotic signaling, the aim of the present study was to test the effect of hematopoietic Fas deficiency on experimental atherosclerosis in low-density lipoprotein receptor-null mice (Ldlr(-/-)). Bone marrow from Fas(-/-) mice was used to reconstitute irradiated Ldlr(-/-) mice as a model for atherosclerosis. After 16 weeks on an 0.5% cholesterol diet, no differences were noted in brachiocephalic artery lesion size, cellularity, or vessel wall apoptosis. However, Ldlr(-/-) mice reconstituted with Fas(-/-) hematopoietic cells had elevated hyperlipidemia [80% increase, relative to wild-type (WT) controls; P < 0.001] and showed marked elevation of plasma levels of CXCL1/KC, CCL2/MCP-1, IL-6, IL-10, IL-12 subunit p70, and soluble Fas ligand (P < 0.01), as well as systemic microvascular inflammation. It was not possible to assess later stages of atherosclerosis because of increased mortality in Fas(-/-) bone marrow recipients. Our data indicate that hematopoietic Fas deficiency does not affect early atherosclerotic lesion development in Ldlr(-/-) mice.

Antisense oligonucleotide reduction of apoB-ameliorated atherosclerosis in LDL receptor-deficient mice

Chronic elevations of plasma apolipoprotein B (apoB) are strongly associated with cardiovascular disease. We have previously demonstrated that inhibition of hepatic apoB mRNA using antisense oligonucleotides (ASO) results in reductions of apoB, VLDL, and LDL in several preclinical animal models and humans. In this study, we evaluated the anti-atherogenic effects of a murine-specific apoB ASO (ISIS 147764) in hypercholesterolemic LDLr deficient (LDLr(-/-)) mice. ISIS 147764 was administered weekly at 25-100 mg/kg for 10-12 weeks and produced dose-dependent reductions of hepatic apoB mRNA and plasma LDL by 60-90%. No effects on these parameters were seen in mice receiving control ASOs. ApoB ASO treatment also produced dose-dependent reductions of aortic en face and sinus atherosclerosis from 50-90%, with high-dose treatment displaying less disease than the saline-treated, chow-fed LDLr(-/-) mice. No changes in intestinal cholesterol absorption were seen with apoB ASO treatment, suggesting that the cholesterol-lowering pharmacology of 147764 was primarily due to inhibition of hepatic apoB synthesis and secretion. In summary, ASO-mediated suppression of apoB mRNA expression profoundly reduced plasma lipids and atherogenesis in LDLr(-/-) mice, leading to the hypothesis that apoB inhibition in humans with impaired LDLr activity may produce similar effects.

Cannabinoid Receptor 2 Signaling Does Not Modulate Atherogenesis in Mice

BackgroundStrong evidence supports a protective role of the cannabinoid receptor 2 (CB2) in inflammation and atherosclerosis. However, direct proof of its involvement in lesion formation is lacking. Therefore, the present study aimed to characterize the role of the CB2 receptor in Murine atherogenesis.Methods and FindingsLow density lipoprotein receptor-deficient (LDLR−/−) mice subjected to intraperitoneal injections of the selective CB2 receptor agonist JWH-133 or vehicle three times per week consumed high cholesterol diet (HCD) for 16 weeks. Surprisingly, intimal lesion size did not differ between both groups in sections of the aortic roots and arches, suggesting that CB2 activation does not modulate atherogenesis in vivo. Plaque content of lipids, macrophages, smooth muscle cells, T cells, and collagen were also similar between both groups. Moreover, CB2 −/−/LDLR−/− mice developed lesions of similar size containing more macrophages and lipids but similar amounts of smooth muscle cells and collagen fibers compared with CB2 +/+/LDLR−/− controls. While JWH-133 treatment reduced intraperitoneal macrophage accumulation in thioglycollate-illicited peritonitis, neither genetic deficiency nor pharmacologic activation of the CB2 receptor altered inflammatory cytokine expression in vivo or inflammatory cell adhesion in the flow chamber in vitro.ConclusionOur study demonstrates that both activation and deletion of the CB2 receptor do not relevantly modulate atherogenesis in mice. Our data do not challenge the multiple reports involving CB2 in other inflammatory processes. However, in the context of atherosclerosis, CB2 does not appear to be a suitable therapeutic target for reduction of the atherosclerotic plaque.

Hepatic PUFA content impacts fatty liver in mouse models of obesity &amp; diabetes

Non-alcoholic fatty liver disease (NAFLD) has increased in parallel with the incidence of obesity. NAFLD is characterized by abnormal accumulation of hepatic triglycerides, induction of inflammatory markers and reduced levels of hepatic C20-22 PUFA. Two approaches were used to determine if hepatic PUFA content impacts fatty liver & inflammation. LDL-R (−/−) mice fed a high fat-high cholesterol (HFHC) diet become obese and develop fatty liver; hepatic C20-22 PUFA are low while inflammatory markers (nuclear NFkB-p65 and MCP1 & CD68 expression) are induced. Supplementing the HFHC diet with omega-3 C20-22 PUFA prevented fatty liver and inflammation in these obese mice. The second approach used a high fat (lard) diet to induce obesity and diabetes in C57BL/6J mice. These mice develop fatty livers with low levels of C20-22 PUFA and fatty acid elongase-5 (Elovl5). Elovl5 is a key enzyme involved in PUFA synthesis. A recombinant adenovirus approach was used to restore hepatic Elovl5 activity to normal and elevate hepatic C20-22 PUFA content. This treatment restored hepatic triglycerides & nuclear NFkB-p65 content to normal. These studies establish that mechanisms controlling hepatic C20-22 PUFA content play an important role in regulating hepatic triglycerides and inflammation in mouse models of diet-induced fatty liver disease. Research support: NIFA (2009-65200-05846) and NIDDK (DK43220).

A novel potent synthetic steroidal liver X receptor agonist lowers plasma cholesterol and triglycerides and reduces atherosclerosis in LDLR–/– mice

Potent synthetic nonsteroidal liver X receptor (LXR) agonists like T0901317 induce triglyceridaemia and fatty liver, effects not observed with some natural and synthetic steroidal, relatively weak agonists of LXR. To determine if potency is responsible for the lack of side effects with some steroidal agonists, we investigated the in vivo effects of a novel steroidal LXR agonist, ATI-111, that is more potent than T0901317. Eight week old male LDLR(-/-) mice fed an atherogenic diet were orally treated with vehicle or ATI-111 at 3 and 5 mg·kg(-1) ·day(-1) for 8 weeks, and effects on plasma and liver lipid levels, expression of genes involved in lipid metabolism and on atherogenesis were analysed. ATI-111 increased the expression of genes involved in lipid transport, such as ABCA1, ABCG1 and ABCG5/G8, in intestine and macrophages; decreased ABCG1, apoE; and slightly increased ABCA1 and ABCG5/G8 expression in liver. ATI-111 markedly increased sterol regulatory element-binding protein (SREBP)-1c mRNA in some tissues, whereas acetyl-coenzyme A carboxylase and fatty acid synthase expression was unaffected or only slightly increased in intestine and liver. ATI-111 inhibited the conversion of SREBP-1c precursor form to its active form. Compared with vehicle-treated mice, the levels of hepatic lipids and liver-secreted nascent lipoproteins were not altered, while a significant decrease in plasma cholesterol and triglyceride levels was observed in ATI-111-treated mice. ATI-111 significantly inhibited atherogenesis in three separate vascular sites. ATI-111 is a promising candidate for further development as a treatment of certain vascular diseases as it lacks the significant side effects associated with nonsteroidal LXR agonists, the induction of fatty liver and hypertriglyceridaemia.

Macrophage LXRα gene therapy ameliorates atherosclerosis as well as hypertriglyceridemia in LDLR−/− mice

Liver X receptors (LXRs) are implicated in the regulation of cholesterol homeostasis, inflammatory response and atherogenesis. Administration of LXR agonists inhibits the progress of atherosclerosis, and also increases plasma triglyceride levels, representing an obstacle to their use in treating this disease. The objective of this study was to develop an alternative approach that could overcome this obstacle. Eight-week-old low-density lipoprotein receptor-deficient (LDLR(-/-)) mice were transplanted with hematopoietic stem cell (HSC)-enriched bone marrow cells transduced with lentivectors expressing either green fluorescent protein (GFP) (Lenti-SP-GFP, control) or LXRα (Lenti-SP-LXRα) driven by a synthetic macrophage promoter. At 4 weeks post-transplant, the mice were fed with a Western diet for 8 weeks and then killed. Compared with Lenti-SP-GFP mice, the Lenti-SP-LXRα mice had a 30% reduction in atherosclerotic lesions, which was accompanied by increases in levels of macrophage expression of cholesterol efflux genes apolipoprotein E and ATP-binding cassette A1, as well as decreases in plasma inflammatory cytokines interleukin-6 and tumor necrosis factor-α. Intriguingly, a 50% reduction of plasma triglyceride level was also observed. We conclude that HSC-based macrophage LXRα gene therapy ameliorates the development of atherosclerosis along with an unexpected concomitant reduction of plasma triglyceride levels in LDLR(-/-) mice. These findings highlight the potential value of macrophage LXR expression as an avenue for therapeutic intervention against atherosclerosis.

No Reduction of Atherosclerosis in C-reactive Protein (CRP)-deficient Mice

C-reactive protein (CRP), a phylogenetically highly conserved plasma protein, is the classical acute phase reactant in humans. Upon infection, inflammation, or tissue damage, its plasma level can rise within hours >1000-fold, providing an early, nonspecific disease indicator of prime clinical importance. In recent years, another aspect of CRP expression has attracted much scientific and public attention. Apart from transient, acute phase-associated spikes in plasma concentration, highly sensitive measurements have revealed stable interindividual differences of baseline CRP values in healthy persons. Strikingly, even modest elevations in stable baseline CRP plasma levels have been found to correlate with a significantly increased risk of future cardiovascular disease. These observations have triggered intense controversies about potential atherosclerosis-promoting properties of CRP. To directly assess potential effects of CRP on atherogenesis, we have generated CRP-deficient mice via gene targeting and introduced the inactivated allele into atherosclerosis-susceptible ApoE−/− and LDLR−/− mice, two well established mouse models of atherogenesis. Morphometric analyses of atherosclerotic plaques in CRP-deficient animals revealed equivalent or increased atherosclerotic lesions compared with controls, an experimental result, which does not support a proatherogenic role of CRP. In fact, our data suggest that mouse CRP may even mediate atheroprotective effects, adding a cautionary note to the idea of targeting CRP as therapeutic intervention against progressive cardiovascular disease.