Low-density Lipoprotein Receptor Knockout Mice Research Articles

Red wine consumption mitigates the cognitive impairments in low-density lipoprotein receptor knockout (LDLr−/−) mice

ABSTRACT Although the benefits of moderate intake of red wine in decreasing incidence of cardiovascular diseases associated to hypercholesterolemia are well recognized, there are still widespread misconceptions about its effects on the hypercholesterolemia-related cognitive impairments. Herein we investigated the putative benefits of regular red wine consumption on cognitive performance of low-density lipoprotein receptor knockout (LDLr−/−) mice, an animal model of familial hypercholesterolemia, which display cognitive impairments since early ages. The red wine was diluted into the drinking water to a final concentration of 6% ethanol and was available for 60 days for LDLr−/− mice fed a normal or high-cholesterol diet. The results indicated that moderate red wine consumption did not alter locomotor parameters and liver toxicity. Across multiple cognitive tasks evaluating spatial learning/reference memory and recognition/identification memory, hypercholesterolemic mice drinking red wine performed significantly better than water group, regardless of diet. Additionally, immunofluorescence assays indicated a reduction of astrocyte activation and lectin stain in the hippocampus of LDLr−/− mice under consumption of red wine. These findings demonstrate that the moderate consumption of red wine attenuates short- and long-term memory decline associated with hypercholesterolemia in mice and suggest that it could be through a neurovascular action.

Aggravation of atherosclerosis by pulmonary exposure to indium oxide nanoparticles

The use of indium oxide (In2O3) and indium-metal hybrids for various applications, including the manufacture of batteries and liquid crystal displays, increases the chances of human exposure to In2O3 via inhalation, especially in occupational settings. However, there is little information available on the toxic effects of In2O3 nanoparticles (NPs) on secondary organs following pulmonary exposure. In this study, we evaluated the effect of In2O3 NPs on atherosclerotic plaque formation and the related mechanisms after pulmonary exposure in low-density lipoprotein receptor knockout (Ldlr−/−) mice. At 10 weeks after a single pharyngeal aspiration, In2O3 NPs caused chronic active inflammation, pulmonary alveolar proteinosis, and accumulation of inflammatory cells in the peribronchial and perivascular areas of the lungs. The expression of pro-inflammatory cytokines in the lung tissue, including TNF-α and MCP-1, was markedly increased by treatment with In2O3 NPs. In the In2O3 NP-treated groups, the levels of total cholesterol and low-density lipoprotein in the plasma were increased, whereas HDL cholesterol showed no significant changes compared to vehicle control. The formation of atherosclerotic lesions was increased by treatment with In2O3 NPs. Real-time PCR analysis of the aorta showed that IL-6 and MCP-1 expression was up-regulated upon treatment with In2O3 NPs. These results suggested that the pulmonary inflammation induced by In2O3 NPs aggravates the progression of atherosclerotic plaque formation, possibly by the alteration of the plasma lipid profile and enhancement of the aortic inflammatory processes.

Effect of Camellia sinensis teas on left ventricular hypertrophy and insulin resistance in dyslipidemic mice.

The control of dyslipidemia using plants is an important subject of studies since it has numerous benefits in cardiovascular protection. The objective of this study was to evaluate the effect of three Camellia sinensis L. teas (green, red, and white) on left ventricular hypertrophy and insulin resistance in low-density lipoprotein receptor knockout (LDLr-/-) mice fed a high-fat diet. The LDLr-/- mice were divided into four experimental groups: Group C: standard feed; Group CT: standard feed and three teas, Group HL: high-fat feed; HLT Group: high-fat feed and three teas. The three types of tea (green, red, and white) originated from different processing of the Camellia sinensis L. plant, and were administered associated once a day at a dose of 25 mg/kg by gavage for 60 days. The teas partially prevented hyperlipidemia, the decrease of the serum levels of high-density lipoproteins (HDL), insulin resistance, and increased C-reactive protein (CRP) levels, and completely prevented left ventricular hypertrophy in LDLr -/- mice of the HLT group. In conclusion, the three Camellia sinensis L. teas used to control genetic dyslipidemia associated with a high-fat diet can be used as an auxiliary treatment associated with the control of lipid intake, thus promoting cardiac protection against hyperlipidemia.

Skate cartilage extracts containing chondroitin sulfate ameliorates hyperlipidemia-induced inflammation and oxidative stress in high cholesterol diet-fed LDL receptor knockout mice in comparison with shark chondroitin sulfate.

BACKGROUND/OBJECTIVESIn this study, we investigated the beneficial effects of skate cartilage extracts containing chondroitin sulfate (SCS) on hyperlipidemia-induced inflammation and oxidative stress in high cholesterol diet (HCD)-fed mice in comparison with the effects of shark cartilage-derived chondroitin sulfate (CS).MATERIALS/METHODSLow-density lipoprotein receptor knockout (LDLR-KO) mice were fed HCD with an oral administration of CS (50 and 100 mg/kg BW/day), SCS (100 and 200 mg/kg BW/day), or water, respectively, for ten weeks.RESULTSThe administration of CS or SCS reduced the levels of serum triglyceride (TG), total cholesterol (TC), and LDL cholesterol and elevated the levels of high-density lipoprotein cholesterol, compared with those of the control group (P < 0.05). Furthermore, CS or SCS significantly attenuated inflammation by reducing the serum levels of interleukin (IL)-1β and hepatic protein expression levels of nuclear factor kappa B, inducible nitric oxide synthase, cyclooxygenase-2, and IL-1beta (P < 0.05). In particular, the serum level of tumor necrosis factor-alpha was reduced only in the 100 mg/kg BW/day of SCS-fed group, whereas the IL-6 level was reduced in the 100 and 200 mg/kg BW/day of SCS-fed groups (P < 0.05). In addition, lipid peroxidation and nitric oxide production were attenuated in the livers of the CS and SCS groups mediated by the upregulation of hepatic proteins of antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase (P < 0.05).CONCLUSIONSThese results suggest that the biological effects of SCS, similar to those of CS, are attributed to improved lipid profiles as well as suppressed inflammation and oxidative stress induced by the intake of HCD.

Lack of mitochondrial NADP(H)-transhydrogenase expression in macrophages exacerbates atherosclerosis in hypercholesterolemic mice.

The atherosclerosis prone LDL receptor knockout mice (Ldlr-/-, C57BL/6J background) carry a deletion of the NADP(H)-transhydrogenase gene (Nnt) encoding the mitochondrial enzyme that catalyzes NADPH synthesis. Here we hypothesize that both increased NADPH consumption (due to increased steroidogenesis) and decreased NADPH generation (due to Nnt deficiency) in Ldlr-/- mice contribute to establish a macrophage oxidative stress and increase atherosclerosis development. Thus, we compared peritoneal macrophages and liver mitochondria from three C57BL/6J mice lines: Ldlr and Nnt double mutant, single Nnt mutant and wild-type. We found increased oxidants production in both mitochondria and macrophages according to a gradient: double mutant > single mutant > wild-type. We also observed a parallel up-regulation of mitochondrial biogenesis (PGC1a, TFAM and respiratory complexes levels) and inflammatory (iNOS, IL6 and IL1b) markers in single and double mutant macrophages. When exposed to modified LDL, the single and double mutant cells exhibited significant increases in lipid accumulation leading to foam cell formation, the hallmark of atherosclerosis. Nnt deficiency cells showed up-regulation of CD36 and down-regulation of ABCA1 transporters what may explain lipid accumulation in macrophages. Finally, Nnt wild-type bone marrow transplantation into LDLr-/- mice resulted in reduced diet-induced atherosclerosis. Therefore, Nnt plays a critical role in the maintenance of macrophage redox, inflammatory and cholesterol homeostasis, which is relevant for delaying the atherogenesis process.

Addition of fish oil to atherogenic high fat diet inhibited atherogenesis while olive oil did not, in LDL receptor KO mice

Background and aimsMediterranean diet has been associated with decreased cardiovascular morbidity and mortality. Both fish and olive oil are key components of this diet. Therefore, we compared their effects on nonalcoholic fatty liver disease (NAFLD) and atherogenesis in a mouse model, fed a high fat diet. Methods and resultsForty nine, female LDL receptor knockout (LDLR KO) mice were allocated into 3 groups and fed an atherogenic high fat (HF) diet for 9 weeks. The HF group was fed a high fat diet alone. A HF + OO group was fed a HF diet with added olive oil (60 ml/kg feed), and the third group (HF + FO) was fed a HF diet with added fish oil (60 ml/kg feed).Both additions of fish and olive oil, significantly decreased plasma cholesterol elevation compared to HF diet. Nevertheless, only fish oil addition reduced significantly atherosclerotic lesion area by 51% compared to HF group. Liver levels of eicosapentenoic (EPA) and docosahexaenoic (DHA) acids were several folds higher in HF + FO group than in HF and HF + OO groups. Liver levels of oleic acid were higher in HF + OO compared to the other groups. Moreover, Fish oil addition significantly decreased NAFLD scores related to steatosis and inflammation and lowered the expression of the inflammatory genes interleukin 6 (IL6) and monocyte chemoattractant protein 1 (MCP1). ConclusionThese results suggest that fish oil addition on top of an atherogenic, HF diet, is beneficial, while olive oil is not, in its effect on plaque formation and NAFLD in LDLR KO mice.

Anti-Atherosclerotic Properties of Wild Rice in Low-Density Lipoprotein Receptor Knockout Mice: The Gut Microbiome, Cytokines, and Metabolomics Study.

Background and aim: We previously reported the anti-atherogenic properties of wild rice in low-density lipoprotein receptor knockout (LDL-r-KO) mice. The present study aimed to discover the mechanism of action for such effects. Materials: Fecal and plasma samples from the wild rice treated and control mice were used. Fecal bacterial population was estimated while using 16S rDNA technology. The plasma samples were used to estimate the levels of 35 inflammatory markers and metabolomics, while using Meso Scale multiplex assay and liquid chromatography-mass spectrometry (LC-MS/MS) techniques. Results: Many bacteria, particularly Anaeroplasma sp., Acetatifactor sp., and Prophyromonadaceae sp., were found in higher quantities in the feces of wild rice fed mice as compared to the controls. Cytokine profiles were significantly different between the plasma of treated and control mice. Among them, an increase in the level of IL-10 and erythropoietin (EPO) could explain the anti-atherogenic properties of wild rice. Among many metabolites tested in plasma of these animals, surprisingly, we found an approximately 60% increase in the levels of glucose in the wild rice fed mice as compared to that in the control mice. Conclusion: Additional studies warrant further investigation of the interplay among gut microbiome, inflammatory status, and macronutrient metabolism.

Carvedilol Ameliorates Experimental Atherosclerosis by Regulating Cholesterol Efflux and Exosome Functions.

Carvedilol (Cav), a nonselective β-blocker with α1 adrenoceptor blocking effect, has been used as a standard therapy for coronary artery disease. This study investigated the effects of Cav on exosome expression and function, ATP-binding cassette transporter A1 (ABCA1) expression, and cholesterol efflux that are relevant to the process of atherosclerosis. Human monocytic (THP-1) cell line and human hepatic (Huh-7) cells were treated with Cav, and cholesterol efflux was measured. Exosomes from cell culture medium or mice serum were isolated using glycan-coated recognition beads. Low-density lipoprotein receptor knockout (ldlr−/−) mice were fed with high-fat diet and treated with Cav. Cav accentuated cholesterol efflux and enhanced the expressions of ABCA1 protein and mRNA in both THP-1 and Huh-7 cells. In addition, Cav increased expression and function of exosomal ABCA1 in THP-1 macrophage exosomes. The mechanisms were associated with inhibition of nuclear factor-κB (NF-κB) and protein kinase B (Akt). In hypercholesterolemic ldlr−/− mice, Cav enhanced serum exosomal ABCA1 expression and suppressed atherosclerosis by inhibiting lipid deposition and macrophage accumulation. Cav halts atherosclerosis by enhancing cholesterol efflux and increasing ABCA1 expression in macrophages and in exosomes, possibly through NF-κB and Akt signaling, which provides mechanistic insights regarding the beneficial effects of Cav on atherosclerotic cardiovascular disease.

Hematopoietic Npc1 mutation shifts gut microbiota composition in Ldlr\u2212/\u2212 mice on a high-fat, high-cholesterol diet

While the link between diet-induced changes in gut microbiota and lipid metabolism in metabolic syndrome (MetS) has been established, the contribution of host genetics is rather unexplored. As several findings suggested a role for the lysosomal lipid transporter Niemann-Pick type C1 (NPC1) in macrophages during MetS, we here explored whether a hematopoietic Npc1 mutation, induced via bone marrow transplantation, influences gut microbiota composition in low-density lipoprotein receptor knockout (Ldlr−/−) mice fed a high-fat, high-cholesterol (HFC) diet for 12 weeks. Ldlr−/− mice fed a HFC diet mimic a human plasma lipoprotein profile and show features of MetS, providing a model to explore the role of host genetics on gut microbiota under MetS conditions. Fecal samples were used to profile the microbial composition by 16 s ribosomal RNA gene sequencing. The hematopoietic Npc1 mutation shifted the gut microbiota composition and increased microbial richness and diversity. Variations in plasma lipid levels correlated with microbial diversity and richness as well as with several bacterial genera. This study suggests that host genetic influences on lipid metabolism affect the gut microbiome under MetS conditions. Future research investigating the role of host genetics on gut microbiota might therefore lead to identification of diagnostic and therapeutic targets for MetS.

Intermittent hypoxia and hypercapnia induces inhibitor of nuclear factor-κB kinase subunit β-dependent atherosclerosis in pulmonary arteries.

Clinical studies have shown that obstructive sleep apnea (OSA) increases atherosclerosis risk. The inflammation, especially mediated by the macrophages via nuclear factor-κB (NF-κB), has been speculated to contribute to atherogenicity in OSA patients. Inhibitor of NF-κB kinase-β (IKKβ) is an essential element of the NF-κB pathway and is linked to atherosclerosis. We previously reported that atherosclerosis was accelerated in pulmonary artery (PA) but not in aorta when low-density lipoprotein receptor knockout (Ldlr-/-) mice were exposed to intermittent hypoxia/hypercapnia (IHH), a surrogate for recurrent upper-airway obstruction. Therefore, we hypothesized that IKKβ-dependent NF-κB activation in monocytes and macrophages plays a role in IHH-induced PA atherosclerosis. To test this hypothesis, myeloid restricted IKKβ deletion (IkkβΔMye) or control (IkkβF/F) mice were crossed with Ldlr-/- mice to generate double-knockout mice. Then, the mice were exposed to IHH or room air (Air) on high-fat diet for 8 or 16 wk. Lesions of PA and aorta were examined in IkkβΔMye;Ldlr-/- and IkkβF/F;Ldlr-/- male mice under IHH vs. Air. The results revealed that IKKβ deletion abolished IHH-induced PA atherosclerosis after 8-wk exposure but not after 16-wk exposure (8 wk: IkkβF/F;Ldlr-/-, IHH 13.5 ± 1.4 vs. Air 5.7 ± 0.7%, P < 0.01; IkkβΔMye;Ldlr-/-, IHH 7.4 ± 1.9% vs. Air 4.6 ± 1.3%, P = 0.24). Both IKKβ deletion and IHH had no effects on atherosclerosis in the aorta. Our findings demonstrate that IKKβ-dependent NF-κB activity in myeloid-lineage cells plays a critical role in IHH-induced PA atherosclerosis at the early stage.

TLR4 (Toll-Like Receptor 4)-Dependent Signaling Drives Extracellular Catabolism of LDL (Low-Density Lipoprotein) Aggregates.

Aggregation and modification of LDLs (low-density lipoproteins) promote their retention and accumulation in the arteries. This is a critical initiating factor during atherosclerosis. Macrophage catabolism of agLDL (aggregated LDL) occurs using a specialized extracellular, hydrolytic compartment, the lysosomal synapse. Compartment formation by local actin polymerization and delivery of lysosomal contents by exocytosis promotes acidification of the compartment and degradation of agLDL. Internalization of metabolites, such as cholesterol, promotes foam cell formation, a process that drives atherogenesis. Furthermore, there is accumulating evidence for the involvement of TLR4 (Toll-like receptor 4) and its adaptor protein MyD88 (myeloid differentiation primary response 88) in atherosclerosis. Here, we investigated the role of TLR4 in catabolism of agLDL using the lysosomal synapse and foam cell formation. Approach and Results: Using bone marrow-derived macrophages from knockout mice, we find that TLR4 and MyD88 regulate compartment formation, lysosome exocytosis, acidification of the compartment, and foam cell formation. Using siRNA (small interfering RNA), pharmacological inhibition and knockout bone marrow-derived macrophages, we implicate SYK (spleen tyrosine kinase), PI3K (phosphoinositide 3-kinase), and Akt in agLDL catabolism using the lysosomal synapse. Using bone marrow transplantation of LDL receptor knockout mice with TLR4 knockout bone marrow, we show that deficiency of TLR4 protects macrophages from lipid accumulation during atherosclerosis. Finally, we demonstrate that macrophages in vivo form an extracellular compartment and exocytose lysosome contents similar to that observed in vitro for degradation of agLDL. We present a mechanism in which interaction of macrophages with agLDL initiates a TLR4 signaling pathway, resulting in formation of the lysosomal synapse, catabolism of agLDL, and lipid accumulation in vitro and in vivo.

Genital Chlamydia infection in hyperlipidemic mouse models exacerbates atherosclerosis

Background and aimsAtherosclerosis is a chronic inflammatory disease, and recent studies have shown that infection at remote sites can contribute to the progression of atherosclerosis in hyperlipidemic mouse models. In this report, we tested the hypothesis that genital Chlamydia infection could accelerate the onset and progression of atherosclerosis. MethodsApolipoprotein E (Apoe−/−) and LDL receptor knockout (Ldlr−/−) mice on a high-fat diet were infected intra-vaginally with Chlamydia muridarum. Atherosclerotic lesions on the aortic sinuses and in the descending aorta were assessed at 8-weeks post-infection. Systemic, macrophage, and vascular site inflammatory responses were assessed and quantified. ResultsCompared to the uninfected groups, infected Apoe−/− and Ldlr−/− mice developed significantly more atherosclerotic lesions in the aortic sinus and in the descending aorta. Increased lesions were associated with higher circulating levels of serum amyloid A-1, IL-1β, TNF-α, and increased VCAM-1 expression in the aortic sinus, suggesting an association with inflammatory responses observed during C. muridarum infection. Genital infection courses were similar in Apoe−/−, Ldlr−/−, and wild type mice. Further, Apoe−/− mice developed severe uterine pathology with increased dilatations. Apoe-deficiency also augmented cytokine/chemokine response in C. muridarum infected macrophages, suggesting that the difference in macrophage response could have contributed to the genital pathology in Apoe−/− mice. ConclusionsOverall, these studies demonstrate that genital Chlamydia infection exacerbates atherosclerotic lesions in hyperlipidemic mouse and suggest a novel role for Apoe in full recovery of uterine anatomy after chlamydial infection.

Protease Activity in Vascular Disease.

Protease Activity in Vascular Disease.

Canonical Wnt Signaling Promotes Neovascularization Through Determination of Endothelial Progenitor Cell Fate via Metabolic Profile Regulation.

Endothelial progenitor cells (EPCs) contribute to blood vessel formation. Canonical Wnt signaling plays an important role in physiological and pathological angiogenesis and EPC fate regulation. However, the mechanism for Wnt signaling to regulate EPC fate in neovascularization (NV) has not been clearly defined. Here, we showed that very low‐density lipoprotein receptor knockout (Vldlr −/−) mice, a model of ocular NV induced by Wnt signaling overactivation, have increased EPC numbers in the bone marrow, blood, and retina, as well as an elevated mitochondrial membrane potential indicating higher mitochondrial function of EPCs in the circulation. Isolated EPCs from Vldlr −/− mice showed overactivated Wnt signaling, correlating with increased mitochondrial function, mass, and DNA copy numbers, compared with WT EPCs. Our results also demonstrated that Wnt signaling upregulated mitochondrial biogenesis and function, while inhibiting glycolysis in EPCs, which further decreased EPC stemness and promoted EPCs to a more active state toward differentiation, which may contribute to pathologic vascular formation. Fenofibric acid, an active metabolite of fenofibrate, inhibited Wnt signaling and mitochondrial function in EPCs and decreased EPC numbers in Vldlr −/−mice. It also decreased mitochondrial biogenesis and reactive oxygen species production in Vldlr −/− EPCs, which may be responsible for its therapeutic effect on diabetic retinopathy. These findings demonstrated that Wnt signaling regulates EPC fate through metabolism, suggesting potential application of the EPC metabolic profile as predictor and therapeutic target for neovascular diseases. stem cells 2019;37:1331–1343

Genetic Deficiency of Flavin-Containing Monooxygenase 3 ( Fmo3) Protects Against Thrombosis but Has Only a Minor Effect on Plasma Lipid Levels-Brief Report.

Objective- FMO (flavin-containing monooxygenase) 3 converts bacterial-derived trimethylamine to trimethylamine N-oxide (TMAO), an independent risk factor for cardiovascular disease. We generated FMO3 knockout (FMO3KO) mouse to study its effects on plasma TMAO, lipids, glucose/insulin metabolism, thrombosis, and atherosclerosis. Approach and Results- Previous studies with an antisense oligonucleotide (ASO) knockdown strategy targeting FMO3 in LDLRKO (low-density lipoprotein receptor knockout) mice resulted in major reductions in TMAO levels and atherosclerosis, but also showed effects on plasma lipids, insulin, and glucose. Although FMO3KO mice generated via CRISPR/Cas9 technology bred onto the LDLRKO background did exhibit similar effects on TMAO levels, the effects on lipid metabolism were not as pronounced as with the ASO knockdown model. These differences could result from either off-target effects of the ASO or from a developmental adaptation to the FMO3 deficiency. To distinguish these possibilities, we treated wild-type and FMO3KO mice with control or FMO3 ASOs. FMO3-ASO treatment led to the same extent of lipid-lowering effects in the FMO3KO mice as the wild-type mice, indicating off-target effects. The levels of TMAO in LDLRKO mice fed an atherogenic diet are very low in both wild-type and FMO3KO mice, and no significant effect was observed on atherosclerosis. When FMO3KO and wild-type mice were maintained on a 0.5% choline diet, FMO3KO showed a marked reduction in both TMAO and in vivo thrombosis potential. Conclusions- FMO3KO markedly reduces systemic TMAO levels and thrombosis potential. However, the previously observed large effects of an FMO3 ASO on plasma lipid levels appear to be due partly to off-target effects.

Dietary Fruit and Vegetable Supplementation Suppresses Diet-induced Atherosclerosis in LDL Receptor Knockout Mice (OR24-07-19)

ObjectivesEpidemiological studies have shown that consumption of fruits and vegetables (F&V) is inversely associated with incidence of cardiovascular disease (CVD). However, the evidence for causality and underlying mechanisms is lacking. Our objective was to determine if increased consumption of F&V could prevent atherosclerosis and its underlying mechanisms. MethodsA unique blend of the most commonly consumed 24 F&V was freeze-dried into a powder and mixed into diets. Thirty six 4-week old male LDL receptor knockout mice were randomly assigned to one of 3 diet groups (12/group): low fat (LF, 10 kcal% fat), high-fat (27 kcal% fat) with 0% F&V (HF), and HF plus 15% F&V diet (HF + FV, equivalent to 8–9 servings for humans). After 20 weeks, mice were euthanized and blood, aorta, and liver tissue were collected. Aortic atherosclerotic lesion, hepatic steatosis, plasma lipid profile and pro-inflammatory cytokine levels were measured. ResultsNo significant differences were found in body weight among the 3 groups. Mice fed HF diet had larger aortic atherosclerotic lesion and hepatic steatosis area than mice fed LF diet by 6.5 and 1.9 fold, respectively (p < 0.001). HF + FV group had 80% less aortic lesion and hepatic steatosis than HF group (p < 0.001). Mice fed HF diet had significantly higher plasma TG and LDL and lower HDL levels than mice fed LF diet, and this dyslipidemia was prevented by F&V supplementation. Further, HF + FV group had lower plasma TNFα levels compared to HF0 group (p < 0.05). Spearman correlation analysis showed that aortic atherosclerotic lesion and hepatic steatosis area were negatively correlated with plasma HDL (p < 0.001) and significantly and positively correlated with TNFα, and the ratios of LDL/HDL, TG/HDL, and non HDL/HDL. ConclusionsOur results demonstrate a causal role of high intake of F&V in preventing HF-induced atherosclerosis and hepatic steatosis, which may be mediated through improved dyslipidemia and reduced inflammation. Funding SourcesU.S. Department of Agriculture – Agricultural Research Service.

1889-P: Inhibition of Acid Sphingomyelinase Attenuates Nonalcoholic Steatohepatitis and Atherosclerosis Induced by High-Fat Diet and LPS in LDL Receptor-Deficient Mice

Macrophage-mediated inflammation plays an essential role in diabetes-associated nonalcoholic steatohepatitis (NASH) and atherosclerosis. Our previous study has shown that acid sphingomyelinase (ASMase) plays a key role in the synergistic stimulation of inflammatory signaling by saturated fatty acid palmitate and lipopolysaccharide (LPS) in macrophages. In the current study, we assessed the effect of ASMase inhibition on NASH and atherosclerosis induced by lard-based, high palmitate-containing high-fat diet (HP-HFD) in combination with LPS in LDL receptor-knockout (LDLR-/-) mice. LDLR-/- mice were fed HP-HFD for 20 weeks and treated with or without a low dose of LPS and amitriptyline, an inhibitor of ASMase. Metabolic assays showed that amitriptyline reduced bodyweight, lipids and insulin resistance induced by HP-HFD or HP-HFD plus LPS. Histological study and Oil Red O staining of livers showed that while mice fed HP-HFD without LPS treatment had hepatic steatosis and hepatocellular ballooning, LPS further increased steatosis and ballooning by 70%. However, amitriptyline attenuated the effect of HP-HFD or HP-HFD plus LPS by 66%. Immunohistochemistry study showed that while mice fed HP-HFD without LPS treatment had a marked immunostaining of F4/80, a biomarker for macrophages, LPS further enhanced F4/80 immunostaining. However, amitriptyline significantly reduced F4/80 immunostaining. Furthermore, results showed that amitriptyline also attenuated atherosclerosis induced by HP-HFD plus LPS. To elucidate the underlying mechanisms, we found that amitriptyline downregulated proinflammatory cytokines by potently reducing sphingosine 1 phosphate, a bioactive sphingolipid. Taken together, this study showed that ASMase inhibition attenuated both NASH and atherogenesis induced by HP-HFD plus LPS and one of the mechanisms potentially involved in the attenuation is inhibition of S1P production. Disclosure Z. Lu: None. Y. Li: None. M.F. Lopes-Virella: None. Y. Huang: None. Funding U.S. Department of Veterans Affairs (1I01CX001825-01); National Institutes of Health

Sortilin promotes macrophage cholesterol accumulation and aortic atherosclerosis through lysosomal degradation of ATP-binding cassette transporter A1 protein.

Sortilin is closely associated with hyperlipidemia and the risk of atherosclerosis (AS). The role of sortilin and the underlying mechanism in peripheral macrophage are not fully understood. In this study, we investigated the effect of macrophage sortilin on ATP-binding cassette transporter A1 (ABCA1) expression, ABCA1-mediated cholesterol efflux, and aortic AS. Macrophage sortilin expression was upregulated by oxidized low-density lipoproteins (ox-LDLs) in both concentration- and time-dependent manners. Its expression reached the peak level when cells were incubated with 50μg/ml ox-LDL for 24h. Overexpression of sortilin in macrophage reduced cholesterol efflux, leading to an increase in intracellular total cholesterol, free cholesterol, and cholesterol ester. Sortilin was found to bind with ABCA1 protein and suppress macrophage ABCA1 expression, resulting in a decrease in cholesterol efflux from macrophages. The inhibitory effect of sortilin in cholesterol efflux was partially reversed by treatment with chloroquine, a lysosomal inhibitor. On the contrary, the ABCA1 protein level and ABCA1-mediated cholesterol efflux is increased by sortilin short hairpin RNA transfection. The fecal and biliary cholesterol 3H-sterol from cholesterol-laden mouse peritoneal macrophage was reduced by sortilin overexpression through lentivirus vector (LV)-sortilin in low-density lipoprotein receptor knockout mice, which was prevented by co-treatment with chloroquine. Treatment with LV-sortilin reduced plasma high-density lipoprotein and increased plasma ox-LDL levels. Accordingly, aortic lipid deposition and plaque area were exacerbated, and ABCA1 expression was reduced in mice in response to infection with LV-sortilin alone. These effects of LV-sortilin were partially reversed by chloroquine. Sortilin enhances lysosomal degradation of ABCA1 protein and suppresses ABCA1-mediated cholesterol efflux from macrophages, leading to foam cell formation and AS development.

2, 3, 4', 5-tetrahydroxystilbene-2-0-β-d Glycoside Attenuates Age- and Diet-Associated Non-Alcoholic Steatohepatitis and Atherosclerosis in LDL Receptor Knockout Mice and Its Possible Mechanisms.

The compound, 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (TSG), a primary bioactive polyphenolic component of Polygonum multiflorum exerts numerous pharmacological activities. However, its protective effect against non-alcoholic steatohepatitis (NASH), in the context of metabolic syndrome, remains poorly understood. The aim of the present study is to evaluate the effects of TSG treatment on middle-aged (12-mo-old) male LDLr−/− mice, which were fed a high fat diet for 12 weeks to induce metabolic syndrome and NASH. At the end of the experiment, the blood samples of mice were collected for determination of metabolic parameters. Liver and aorta tissues were collected for analysis, such as histology, immunofluorescence, hepatic lipid content, real-time PCR, and western blot. Our data show that TSG treatment improved the different aspects of NASH (steatosis, inflammation, and fibrosis) and atherosclerosis, as well as some of the metabolic basal characteristics. These modulatory effects of TSG are mediated, at least in part, through regulating key regulators of lipid metabolism (SREBP1c, PPARα and their target genes, ABCG5 and CYP7A1), inflammation (CD68, TNF-α, IL-6 and ICAM), fibrosis (α-SMA and TNFβ) and oxidative stress (NADPH-oxidase 2/4, CYP2E1 and antioxidant enzymes). These results suggest that TSG may be a promising candidate for preventing and treating the progression of NASH.

Fenofibrate-Loaded Biodegradable Nanoparticles for the Treatment of Experimental Diabetic Retinopathy and Neovascular Age-Related Macular Degeneration.

Fenofibrate is a peroxisome proliferator-activated receptor α (PPARα) agonist and has been shown to have therapeutic effects on diabetic retinopathy (DR). However, the effects of fenofibrate through systemic administration are not as potent as desired due to inefficient drug delivery to the retina. The present study aimed to explore the sustained therapeutic effects of fenofibrate-loaded biodegradable nanoparticles (NP) on both DR and neovascular age-related macular degeneration (AMD). Fenofibrate was successfully encapsulated into poly(lactic- co-glycolic acid) (PLGA) NP (Feno-NP), and Feno-NP were optimized by varying polymer composition to achieve high drug loading and prolonged drug release. The Feno-NP made of PLGA 34 kDa demonstrated a drug content of 6% w/w and a sustained drug release up to 60 days in vitro. Feno-NP (PLGA 34 kDa) was selected for following in vivo studies, and one single intravitreal (IVT) injection of Feno-NP into rat eyes with a 30G fine needle maintained sustained fenofibric acid drug level in the eye for more than 60 days. The efficacy of Feno-NP in DR and neovascular AMD was investigated using streptozotocin (STZ)-induced diabetic rats, laser-induced choroidal neovascularization (CNV) rats, and very low-density lipoprotein receptor knockout ( Vldlr -/-) mice. Therapeutic effects of Feno-NP were evaluated by measuring electroretinogram (ERG), retinal vascular leakage, leukostasis, CNV size, and retinal levels of vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1). In diabetic rats, Feno-NP ameliorated retinal dysfunctions, reduced retinal vascular leakage, inhibited retinal leukostasis, and downregulated the overexpression of VEGF and ICAM-1 at 8 weeks after one IVT injection. In addition, Feno-NP reduced retinal vascular leakage and CNV formation in both CNV rats and Vldlr -/- mice. Moreover, no toxicity of Feno-NP or Blank-NP to retinal structure and function was detected. Feno-NP exhibited good physiochemical characteristics and controlled drug release profile, conferring prolonged beneficial effects on DR and neovascular AMD.