Low-density Lipoprotein Receptor-deficient Mice Research Articles

Low‐dose oral sirolimus reduces atherogenesis, vascular inflammation and modulates plaque composition in mice lacking the LDL receptor

Chronic proliferative responses of different vascular cell types have been involved in the pathogenesis of atherosclerosis. However, their functional role remains to be established. Sirolimus reduces neointimal proliferation after balloon angioplasty and chronic graft vessel disease. These studies were undertaken to investigate the effects of this anti-proliferative drug on atherogenesis. Low-density lipoprotein receptor-deficient (LDL r-KO) mice on a cholesterol-rich diet were randomized to receive placebo or sirolimus (0.1; 0.3; or 1 mg.kg(-1)) in their diet for 8 or 16 weeks. In both studies, plasma levels of the drug increased in a dose-dependent fashion, animals gained weight normally and, among groups, plasma lipids levels did not differ significantly. Compared with placebo, plasma levels of interleukin-6, monocyte chemoattractant protein-1, interferon gamma, tumour necrosis factor alpha and CD40, and their mRNA levels in aortic tissue were significantly reduced in sirolimus-treated mice. This effect resulted in a significant and dose-dependent reduction in atherosclerotic lesions, in both the root and aortic tree. Also these lesions contained less monocyte/macrophages and smooth muscle cells, but more collagen. The present results demonstrated that at low doses, sirolimus was an effective and safe anti-atherogenic agent in the LDL r-KO mice. It attenuated the progression of atherosclerosis and modulated the plaque phenotype by reducing the pro-inflammatory vascular responses typical of the disease.

Antiatherosclerotic Effects of Artemisia princeps Pampanini cv. Sajabal in LDL Receptor Deficient Mice

Antiatherosclerotic effects of ethanolic extracts of Artemisia princeps Pampanini cv. Sajabal (ESJ) were investigated in low-density lipoprotein receptor deficient (LDLR(-/-)) mice. The Western diet-induced high levels of total cholesterol and triglyceride were similar in the ESJ and control groups. However, circulating oxidized LDL was significantly decreased in the ESJ group (p < 0.05). ESJ also markedly decreased aortic expression levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta), and reduced the aortic lesion formation and macrophage accumulation by 36.7% (p < 0.05) and 43% (p < 0.01) in the control group, respectively. Additionally, ESJ inhibited atherogenic properties with cytokine-induced surface expression of cell adhesion molecules, chemokines, and monocyte adhesion to the human umbilical vein endothelial cells (HUVECs), and simultaneously suppressed nuclear factor-kappaB (NF-kappaB) activation. These results suggest that ethanolic extracts of Artemisia princeps Pampanini cv. Sajabal contributes to the antiatherosclerotic and anti-inflammatory activities in LDLR(-/-) mice.

Developmental programming of sex-dependent alterations in lipid metabolism: a role for long-term, sex-specific alterations in LDL-receptor expression. Focus on “Developmental programming of lipid metabolism and aortic vascular function in C57BL/6 mice: a novel study suggesting an involvement of LDL-receptor”

the nobel prize in physiology or medicine was awarded to Brown and Goldstein in 1985 for their discovery of the low-density lipoprotein (LDL) receptor and its importance in the regulation of cholesterol metabolism ([2][1]). Cholesterol, an essential component of the mammalian cell membrane, is

RAGE mediates oxidized LDL-induced pro-inflammatory effects and atherosclerosis in non-diabetic LDL receptor-deficient mice

Receptor for advanced glycation end products (RAGE) plays a pivotal role in the genesis of diabetic vascular diseases. To further explore the mechanisms underlying atherosclerosis under non-diabetic conditions, we examined the effect of RAGE deficiency on atherosclerosis in hyperlipidaemic mice. RAGE-/- mice were crossed with low-density lipoprotein receptor-deficient (LDLr-/-) mice to generate the double knockout (DKO) mice. After feeding with high-fat diet for 12 weeks, aortic atherosclerotic lesions were analysed histologically in these mice. Although there were no differences in serum levels of glucose and known RAGE ligands between DKO and LDLr-/- mice, DKO mice exhibited a significant decrease in the size and macrophage content in atherosclerotic lesions compared with LDLr-/- mice. Expression of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the aorta was lower in DKO mice than in LDLr-/- mice. Fluorescence-based assays revealed that oxidative stress in the vessel wall was attenuated in DKO mice than in LDLr-/- mice. Cell culture experiments revealed that RAGE mediated oxidative LDL-induced activation of p42/44 mitogen-activated protein kinases and oxidative stress in macrophages. Oxidative LDL may be a ligand of RAGE in the hyperlipidaemic state. RAGE inactivation inhibits the atherosclerosis through reducing oxLDL-induced pro-inflammatory responses and oxidative stress in hyperlipidaemia.

Effects of dietary sodium on reactive oxygen species formation and endothelial dysfunction in low-density lipoprotein receptor-deficient mice on high-fat diet

Hypertension and high serum cholesterol level are important risk factors for atherosclerosis and coronary heart disease. In the present study we tested the hypothesis whether high sodium intake, when given in combination with Western type high-fat diet, induces endothelial dysfunction and promotes atherosclerosis. Furthermore, the role and enzyme sources of increased oxidative stress were examined. Low-density lipoprotein receptor-deficient mice (LDLR(-/-)) and control C57Bl/6 mice received either high-fat, normal-sodium diet (fat 18% and cholesterol 0.5%; NaCl 0.7%; w/w) or high-fat, high-sodium diet (7% NaCl w/w) for 12 weeks. Superoxide formation was assessed by lucigenin enhanced chemiluminescence, endothelial functions were examined ex vivo, and atherosclerotic lesions from the aorta were assessed by light microscopy. High-fat, high-sodium diet increased systolic blood pressure in LDLR(-/-) mice but not in C57Bl/6 mice, whereas it induced cardiac hypertrophy in both mouse strains. Dietary combination of fat and sodium induced endothelial dysfunction in LDLR(-/-) mice. Preincubation with a superoxide scavenger Tiron normalized endothelial dysfunction, whereas the hydrogen peroxide scavenger catalase did not alter endothelial function. High sodium intake induced superoxide formation in LDLR(-/-) mice on high-fat diet. Stimulation of muscarinic receptors in the endothelial cells by acetylcholine increased superoxide generation, whereas preincubation with the nitric oxide synthase (NOS) inhibitor L-arginine methyl ester or endothelium removal reduced superoxide production. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase by apocynin decreased vascular superoxide formation whereas the xanthine oxidase inhibitor oxypurinol did not significantly affect oxidative stress in LDLR(-/-) mice. In conclusion, the detrimental effects of dietary sodium on endothelial function and progression of atherosclerosis in LDLR(-/-) mice on high-fat diet are mediated by increased ROS formation mainly through uncoupled NOS and NADPH oxidase. The present study also underscores the importance of superoxide and endothelial NOS uncoupling in the pathogenesis of endothelial dysfunction.

Abstract 3693: Early Growth Response Gene-1 Deficiency In Bone-marrow-derived Cells Reduces Macrophage Accumulation And Atherosclerotic Lesion Formation In A Hyperlipidemic Mouse Model

Early growth response gene-1 (Egr-1), a prototype of a family of zinc-finger transcription factors, is a master regulator of many genes which play important roles in cardiovascular diseases. Within atherosclerotic lesions Egr-1 is expressed in several cell types, such as smooth muscle cells, endothelial cells and monocytes/macrophages. Since macrophages play a pivotal role in atherosclerotic lesion development, this study investigated the effects of Egr-1-deficiency within bone-marrow derived cells on the development of atherosclerosis in a hyperlipidemic mouse model. Therefore we transplanted bone-marrow from Egr-1 deficient mice and wild type controls into lethally irradiated low-density lipoprotein receptor deficient mice. After 20 weeks on a western diet atherosclerotic lesions within the aortic sinus and gene expression of inflammatory genes in the aortas of the recipients were evaluated. Mice receiving Egr-1 deficient bone-marrow had less atherosclerotic lesion development compared with mice receiving wild type bone-marrow (318 736 ± 98 910μm 2 vs. 404 539 ± 92 408μm 2 , p&lt;0.05). The size of the necrotic core within the lesions was also reduced. Immunohistochemistry revealed that mice receiving Egr-1 deficient bone-marrow had less macrophages in comparison to controls. Gene expression analysis in the aortas of the mice demonstrated reduced expression of Vascular Cell Adhesion Molecule (VCAM-1), an important adhesion molecule during the development of atherosclerosis. These results were validated with in vitro studies where Egr-1-deficient peritoneal macrophages revealed less VCAM-1 mRNA expression after stimulation with lipopolysaccharide in comparison to wildtype macrophages. This study demonstrates that bone-marrow derived Egr-1 promotes macrophage accumulation and atherosclerotic lesion development possible over an increased expression of VCAM-1.

Abstract 3614: Adiponectin Inhibits Vascular Inflammation and Rapid Progression of Atherosclerosis Induced by Angiotensin II

Adiponectin, an adipocytokine derived from adipose tissue, has anti-inflammatory and antiatherogenic properties and low plasma adiponectin levels are associated with the metabolic syndrome and cardiovascular disease. However, it is unknown whether elevated plasma adiponectin levels provide atheroprotection against angiotensin II (AngII)-induced vascular inflammation and accelerated atherosclerosis. We investigated whether adenoviral adiponectin expression provides protection against AngII-induced inflammation and atherosclerosis. Thirteen week-old low-density lipoprotein receptor-deficient (LDLR −/− ) mice were fed Western diet and infused with Ang-II. Mice were injected with 5 X 10 8 particles of adenovirus expressing either GFP (Ad-GFP, n=16/group) or mouse adiponectin (Ad-APN, n=16/group). Eight weeks after injection, plasma adiponectin levels in Ad-APN-injected mice were increased 12-fold compared to control Ad-GFP mice (306.4 mg/dL vs 25.3 mg/dL). Elevation of plasma adiponectin levels led to a significant increase in plasma HDL levels (23.6% increase vs. control), whereas the plasma cholesterol and triglyceride levels were unchanged. Quantification of atherosclerosis in the entire aortas by en face method, revealed a significant inhibition of atherosclerosis in Ad-APN mice (45% reduction, p&lt;0.01 vs. Ad-GFP mice). Furthermore, adiponectin expression substantially inhibited the mRNA expression of inflammatory and atherogenic genes (ICAM-1, osteopontin, MCP-1 and CCR2) in the vessel wall. Interestingly, adiponectin also increased the mRNA expression of critical reverse cholesterol transport genes, ABCA1 and ABCG1, in the aorta. These results demonstrate that increasing plasma adiponectin levels may be an effective therapeutic strategy to inhibit angiotensin-II induced vascular inflammation and rapid progression of atherosclerosis.

Leukocyte-derived hepatic lipase increases HDL and decreases en face aortic atherosclerosis in LDLr−/− mice expressing CETP

In addition to hepatic expression, cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) are expressed by human macrophages. The combined actions of these proteins have profound effects on HDL structure and function. It is not known how these HDL changes influence atherosclerosis. To elucidate the role of leukocyte-derived HL on atherosclerosis in a background of CETP expression, we studied low density lipoprotein receptor-deficient mice expressing human CETP (CETPtgLDLr -/-) with a leukocyte-derived HL deficiency (HL -/- BM). HL(-/-) bone marrow (BM), CETPtgLDLr(-/-) mice were generated via bone marrow transplantation. Wild-type bone marrow was transplanted into CETPtgLDLr(-/-) mice to generate HL +/+ BM, CETPtgLDLr(-/-) controls. The chimeras were fed a high-fat, high-cholesterol diet for 14 weeks to promote atherosclerosis. In female HL(-/-) BM, CETPtgLDLr(-/-) mice plasma HDL-cholesterol concentration during high-fat feeding was decreased 27% when compared with HL +/+ BM, CETPtgLDLr(-/-) mice (P < 0.05), and this was associated with a 96% increase in en face aortic atherosclerosis (P < 0.05). In male CETPtgLDLr(-/-) mice, leukocyte-derived HL deficiency was associated with a 16% decrease in plasma HDL-cholesterol concentration and a 25% increase in aortic atherosclerosis. Thus, leukocyte-derived HL in CETPtgLDLr(-/-) mice has an atheroprotective role that may involve increased HDL levels.

Induction of paraoxonase 1 and apolipoprotein A-I gene expression by aspirin

Low-dose aspirin therapy has become a standard in the treatment of cardiovascular diseases. Aspirin has been shown to inhibit atherosclerosis in mouse models. To determine the mechanisms by which aspirin might inhibit atherosclerosis, we incubated HEPG2 cells and rat primary hepatocytes with aspirin or salicylic acid and noted an increase in paraoxonase 1(PON1) activity in the medium, together with an induction of PON1 and apolipoprotein A-I (apoA-I) gene expression. Mice treated with aspirin also showed a 2-fold increase in plasma PON1 activity and a significant induction of both PON1 and apoA-I gene expression in the liver. The induction of the PON1 gene in cell culture was accompanied by an increase in arylhydrocarbon receptor (AhR) gene expression. Accordingly, aspirin treatment of AhR(-/-) animals failed to induce PON1 gene expression. We previously suggested that aspirin might be hydrolyzed by serum PON1, which could account for its short plasma half-life of 10 min. Taken together with the current studies, we suggest that the antiatherosclerotic effects of aspirin might be mediated by its hydrolytic product salicylate and that the induction of PON1 and apoA-I might be important in the cardioprotective effects of aspirin.

Phage display selection of peptides that home to atherosclerotic plaques: IL‐4 receptor as a candidate target in atherosclerosis

Imaging or drug delivery tools for atherosclerosis based on the plaque biology are still insufficient. Here, we attempted to identify peptides that selectively home to atherosclerotic plaques using phage display. A phage library containing random peptides was ex viv screened for binding to human atheroma tissues. After three to four rounds of selection, the DNA inserts of phage clones wer sequenced. A peptide sequence, CRKRLDRNC, was the most frequently occurring one. Intravenously injected phage displaying the CRKRLDRNC peptide was observed to home to atherosclerotic aortic tissues of low-density lipoprotein receptor-deficient (Ldlr−/–) mice at higher levels than to normal aortic tissues of wild-type mice. Moreover, a fluorescein- or radioisotope-conjugated synthetic CRKRLDRNC peptide, but not a control peptide, homed in vivo to atherosclerotic plaques in Ldlr−/– mice, while homing of the peptide to other organs such as brain was minimal. The homing peptide co-localized with endothelial cells, macrophages and smooth muscle cells a mouse and human atherosclerotic plaques. Homology search revealed that the CRKRLDRNC peptide shares a motif of interleukin-receptor (IL-4) that is critical for binding to its receptor. The peptide indeed co-localized with IL-4 receptor (IL-4R) at atherosclerotic plaques. Moreover, the peptide bound to cultured cells expressing IL-4R on the cell surface and the binding was inhibited by the knock-down of IL-4R. These results show that the CRKRLDRNC peptide homes to atherosclerotic plaques through binding to IL-4R as its target and may be a useful tool for selective drug delivery and molecular imaging of atherosclerosis.

Apolipoprotein E predisposes to obesity and related metabolic dysfunctions in mice

Obesity is a central feature of the metabolic syndrome and is associated with increased risk for insulin resistance and typeII diabetes. Here, we investigated the contribution of human apoliproteinE3 and mouse apoliproteinE to the development of diet-induced obesity in response to western-type diet. Our data show that apolipoproteinE contributes to the development of obesity and other related metabolic disorders, and that human apolipoproteinE3 is more potent than mouse apolipoproteinE in promoting obesity in response to western-type diet. Specifically, we found that apolipoproteinE3 knock-in mice fed western-type diet for 24 weeks became obese and developed hyperglycemia, hyperinsulinemia, hyperleptinemia, glucose intolerance and insulin resistance that were more severe than in C57BL/6 mice. In contrast, apolipoproteinE-deficient mice fed western-type diet for the same period were resistant to diet-induced obesity, had normal plasma glucose, leptin and insulin levels, and exhibited normal responses to glucose tolerance and insulin resistance tests. Furthermore, low-density lipoprotein receptor-deficient mice were more sensitive to the development of diet-induced obesity and insulin resistance than apolipoprotein E-deficient mice, but were still more resistant than C57BL/6 mice, raising the possibility that low-density lipoprotein receptor mediates, at least in part, the effects of apolipoproteinE on obesity. Taken together, our findings suggest that, in addition to other previously identified mechanisms of obesity, apolipoproteinE and possibly the chylomicron pathway are also important contributors to the development of obesity and related metabolic dysfunctions in mice.

Antiatherosclerotic Effects of a Novel Synthetic Tissue-Selective Steroidal Liver X Receptor Agonist in Low-Density Lipoprotein Receptor-Deficient Mice

Liver X receptor (LXR) agonists have the potential to treat atherosclerosis based on their ability to enhance reverse cholesterol transport. However, their side effects, such as induction of liver lipogenesis and triglyceridemia, may limit their pharmaceutical development. In contrast to the nonsteroidal LXR agonist N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide (T0901317), 3alpha, 6alpha, 24-trihydroxy-24, 24-di(trifluoromethyl)-5beta-cholane (ATI-829), a novel potent synthetic steroidal LXR agonist, was a poor inducer of sterol regulatory element-binding protein 1c expression in hepatoma HepG2 cells, whereas both compounds increased ABCA1 expression in macrophage THP-1 cells. In male low-density lipoprotein receptor-deficient mice, ATI-829 selectively activated LXR target gene expression in mouse intestines and macrophages but not in the liver. A significant increase in liver triglyceride and plasma triglyceriderich small very low-density lipoprotein (VLDL) was observed in T0901317 but not ATI-829-treated mice. Compared with vehicle-treated mice, atherosclerosis development was significantly inhibited in the innominate artery after treatment with either compound. However, in the aortic root, inhibition of atherosclerosis was only observed in the right (right coronary artery-associated sinus) but not the left coronary-related sinus (left coronary artery-associated sinus; LC) of mice treated with either compound. Lesions in the innominate artery were less complex after treatment with either compound and contained mostly macrophage foam cells. In contrast, LC lesions were more complex and had a large collagen-positive fibrous cap and less macrophage foam cell area after treatment with either compound. The T0901317-induced hypertriglyceridemia was accompanied by an increase in small triglyceride-rich VLDL that may influence LXR agonist-mediated antiatherosclerotic effects at certain vascular sites. ATI-829, by selectively activating LXR in certain tissues without inducing hypertriglyceridemia, is a good candidate for drug development.

Identification of a peptide ligand recognizing dysfunctional endothelial cells for targeting atherosclerosis

Targeting ligands to dysfunctional or activated endothelial cells overlying atherosclerotic plaques could provide tools for selective drug delivery to atherosclerotic lesions. To identify peptides selectively targeting dysfunctional endothelial cells, a phage library was screened against tumor necrosis factor-alpha (TNF-alpha) activated bovine aortic endothelial cells (BAECs). Five rounds of biopanning were carried out and the phage clones selected were examined for their DNA inserts. A phage clone displaying the CLWTVGGGC sequence occurred most frequently and was found to bind specifically to TNF-alpha activated BAECs over untreated cells. On the other hand, bindings of the phage clone to human umbilical vein endothelial cells, lymphatic endothelial cells, and epithelial cells were minimal. Flow cytometric and fluorescence microscopic studies showed the preferential binding of the CLWTVGGGC peptide to TNF-alpha activated BAECs compared to untreated cells. In vivo studies demonstrated selective homing and co-localization of the CLWTVGGGC peptide to dysfunctional endothelial cells overlying atherosclerotic plaques in low-density lipoprotein receptor-deficient mice. These results demonstrate that the CLWTVGGGC peptide could specifically recognize dysfunctional endothelial cells at atherosclerotic plaques and be used as a targeting ligand for drug delivery and imaging of atherosclerosis.

Brief Report: Accelerated Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice Lacking the Membrane-Bound Complement Regulator CD59

Whereas studies in humans and animal models have suggested a role for complement activation in atherosclerosis, there has been little analysis of the importance of complement regulators. We tested the hypothesis that the terminal pathway inhibitor CD59 plays an essential role in limiting the proinflammatory effects of complement activation. CD59 gene targeted mice (CD59a(-/-)) mice were crossed with low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. CD59-deficient Ldlr(-/-) mice had significantly more extensive en face Sudan IV staining of thoracoabdominal aorta than Ldlr(-/-) single knock-outs, both after a low-fat diet (6.51+/-0.36% versus 2.63+/-0.56%, P<0.001) or a high-fat diet (17.05+/-2.15% versus 7.69+/-1.17%, P<0.004). Accelerated lesion formation in CD59a(-/-)/Ldlr(-/-) mice on a high-fat diet was associated with increased lesional vascular smooth muscle cell (VSMC) number and fibrous cap formation. Our data show that CD59 deficiency accelerates the development of lesions and increases plaque VSMC composition. Assuming that the main function of CD59 is to prevent the development of C5b-9 membrane attack complexes, our observations are consistent with the terminal complement pathway having proatherogenic potential in the Ldlr(-/-) mouse model, and highlight the importance of complement regulation.

Enhanced expression of vascular cell adhesion molecule-1 by corticotrophin-releasing hormone contributes to progression of atherosclerosis in LDL receptor-deficient mice

Peripherally produced corticotrophin-releasing hormone (CRH) is a strong proinflammatory factor involved in many inflammatory diseases. However, to date, there is no evidence about the action of CRH on atherosclerosis, a chronic disease characterized by inflammatory reactions. In this study we observed the effect of CRH on atherosclerosis in low-density lipoprotein receptor-deficient (LDLr−/−) mice. Twelve-week-old, male LDLr−/− mice were subcutaneously injected with CRH (10 μg/kg) or vehicle once a day for 8 weeks. The results indicated aortic atherosclerotic lesions were larger ( P < 0.01) in CRH-treated mice than those in untreated mice. CRH significantly up-regulated the expression of both protein and mRNA for vascular cell adhesion molecule-1 (VCAM-1), together with a markedly increased activation of nuclear factor kappa B (NF-κB) in aortas. In addition, the blood lipid levels were not influenced by CRH subcutaneous injection. The significant proatherogenic effect of CRH in LDLr−/− mice was largely attenuated by selective CRH receptor 1 (CRHR1) antagonist NBI27914 but not by specific CRH receptor 2 (CRHR2) antagonist antisauvagine-30 (anti-Svg-30). Meanwhile, both the enhanced expression of VCAM-1 and increased activation of NF-κB induced by CRH in aortas of LDLr−/− mice were also largely suppressed by NBI27914, whereas these inhibitory effects were not observed in anti-Svg-30 group. Taken together, these findings indicated that CRH may accelerate atherosclerosis progression in LDLr−/− mice via CRHR1. The enhanced VCAM-1 expression which probably resulted from increased activation of NF-κB induced by CRH, may be one of the important molecular mechanisms by which CRH accelerates atherosclerosis. This study provides a new insight into the effect of CRH on atherosclerosis and suggests a potential target for the prevention and treatment of atherosclerosis.

Defective Mer Receptor Tyrosine Kinase Signaling in Bone Marrow Cells Promotes Apoptotic Cell Accumulation and Accelerates Atherosclerosis

To study the role of Mer receptor tyrosine kinase (mertk) in atherosclerosis. 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. Mertk expression by bone marrow-derived cells is required for the disposal of apoptotic cells and controls lesion development and inflammation.

Antiatherogenic Effects of Cilostazol and Probucol Alone, and in Combination in Low Density Lipoprotein Receptor-deficient Mice Fed with a High Fat Diet

Cilostazol, an antiplatelet drug, and probucol, a cholesterol-lowering drug, are reported to ameliorate atherosclerosis in animal models. However, their combined effect on atherosclerosis is unclear. We therefore evaluated their combined effect on atherosclerotic lesions in LDL receptor-deficient mice. Male LDL receptor-deficient mice were fed a high fat diet with or without cilostazol alone, probucol alone, or with cilostazol and probucol in combination, for 8 weeks. Body weight and plasma lipid levels were measured before and during treatment. At the end of treatment, the size distribution of plasma lipoproteins was analyzed by HPLC and then plasma HDL cholesterol levels and en face aortic atherosclerotic lesion areas were measured. Probucol alone significantly decreased both total cholesterol and HDL cholesterol, while cilostazol alone did not decrease total cholesterol, but significantly increased HDL cholesterol. Both cilostazol alone and probucol alone significantly decreased atherosclerotic lesion areas, and their combined administration showed more significant decreases than when each drug was administered singly. The combination of cilostazol and probucol was more effective in preventing atherosclerotic lesion formation than the administration of each drug alone; this may provide us with a new strategy for treating atherosclerosis.

A new atherosclerotic lesion probe based on hydrophobically modified chitosan nanoparticles functionalized by the atherosclerotic plaque targeted peptides

We developed a new imaging probe for atherosclerotic lesion imaging by chemically conjugating an atherosclerotic plaque-homing peptide (termed the AP peptide) to hydrophobically modified glycol chitosan (HGC) nanoparticles. The AP peptide was previously discovered by using an in vivo phage display screening method. HGC nanoparticles were labeled with the near-infrared (NIR) fluorophore Cy5.5, yielding nanoparticles 314 nm in diameter. The binding characteristics of nanoparticles to cytokine (TNF-α)-activated bovine aortic endothelial cells (BAECs) were studied in vitro under static conditions and in a dynamic flow environment. AP-tagged HGC-Cy5.5 nanoparticles (100 µg/ml, 2 h incubation) bound more avidly to TNF-α-activated BAECs than to unactivated BAECs. Nanoparticles were mostly located in the membranes of BAECs, although some were taken up by the cells and were visible in the cytoplasm, suggesting that the AP peptides in HGC nanoparticles retained target selectivity for activated BAECs. Binding selectivity of AP-tagged HGC-Cy5.5 nanoparticles was also studied in vivo. NIR fluorescence imaging demonstrated that AP-tagged HGC-Cy5.5 nanoparticles bound better to atherosclerotic lesions in a low-density lipoprotein receptor-deficient ( Ldlr -/- ) atherosclerotic mouse than to such lesions in a normal mouse. These results suggest that the newly designed AP-tagged HGC-Cy5.5 nanoparticles may be useful for atherosclerotic lesion imaging, and may also be employed to elucidate pathophysiological changes, at the molecular level, on atherosclerotic endothelium.

Hierarchical contributions of IgM and complement C1q in protection against murine atherosclerosis

Natural IgM antibodies and complement C1q have been implicated in atherogenesis through their ability to bind tissue debris such as denatured low‐density lipoprotein (LDL) and apoptotic cells. To investigate their relative contributions, mice deficient in serum IgM (sIgM), C1q or both C1q and sIgM were crossed with LDL receptor deficient mice (Ldlr−/−) and studied at 22 weeks. sIgM.Ldlr−/− mice showed increased lesion size in comparison to Ldlr−/− mice, both en face (~7‐fold, p&lt;0.0001) and at the aortic root (~5‐fold, p&lt;0.0001). Similar findings were noted on a high fat diet with a 98% increase en face (p=0.0009) and a 23% increase in aortic root lesion area (p=0.006) in sIgM.Ldlr−/− mice, and increased smooth muscle cell infiltration (p=0.007). En face and aortic root lesions were substantially larger in sIgM.Ldlr−/− than C1q.Ldlr−/− mice on either diet (all p&lt;0.005), and the effects of combined C1q and serum IgM deficiency were no different to those of sIgM deficiency alone. Thus although IgM antibodies and C1q are both significantly atheroprotective, IgM makes a more prominent contribution. The major mechanism of protection by IgM therefore appears to be independent of classical pathway activation.Funded by the Wellcome Trust and British Heart Foundation

Genetic and Pharmacological Targeting of Phosphoinositide 3-Kinase-γ Reduces Atherosclerosis and Favors Plaque Stability by Modulating Inflammatory Processes

The role of inflammation at all stages of the atherosclerotic process has become an active area of investigation, and there is a notable quest for novel and innovative drugs for the treatment of atherosclerosis. The lipid kinase phosphoinositide 3-kinase-gamma (PI3Kgamma) is thought to be a key player in various inflammatory, autoimmune, and allergic processes. These properties and the expression of PI3Kgamma in the cardiovascular system suggest that PI3Kgamma plays a role in atherosclerosis. Here, we demonstrate that a specific PI3Kgamma inhibitor (AS605240) is effective in murine models of established atherosclerosis. Intraperitoneal administration of AS605240 (10 mg/kg daily) significantly decreased early atherosclerotic lesions in apolipoprotein E-deficient mice and attenuated advanced atherosclerosis in low-density lipoprotein receptor-deficient mice. Furthermore, PI3Kgamma levels were elevated in both human and murine atherosclerotic lesions. Comparison of low-density lipoprotein receptor-deficient mice transplanted with wild-type or PI3Kgamma-deficient bone marrow demonstrated that functional PI3Kgamma in the hematopoietic lineage is required for atherosclerotic progression. Alleviation of atherosclerosis by targeting of PI3Kgamma activity was accompanied by decreased macrophage and T-cell infiltration, as well as increased plaque stabilization. These data identify PI3Kgamma as a new target in atherosclerosis with the potential to modulate multiple stages of atherosclerotic lesion formation, such as fatty streak constitution, cellular composition, and final fibrous cap establishment.