High-cholesterol Diet Feeding Research Articles

A novel mouse model of familial combined hyperlipidemia and atherosclerosis.

Within the context of residual cardiovascular risk in post-statin era, emerging evidence from epidemiologic and human genetic studies have demonstratedthattriglyceride (TG)-rich lipoproteinsand their remnants are causally related to cardiovascular risk. While,carriers of loss-of-function mutations of ApoC3 havelow TG levelsand are protected from cardiovascular disease (CVD). Of translational significance,siRNAs/antisense oligonucleotide (ASO) targeting ApoC3 is beneficial for patients with atheroscleroticCVD. Therefore, animal models of atherosclerosiswith bothhypercholesterolemia and hypertriglyceridemia are important for the discovery of novel therapeutic strategies targeting TG-lowering on top of traditional cholesterol-lowering.In this study, we constructed a novel mouse model of familial combined hyperlipidemia through inserting a human ApoC3 transgene(hApoC3-Tg) into C57BL/6 J mice and injecting a gain-of-function variant of adeno-associated virus-proprotein convertase subtilisin/kexin type 9 (AAV-PCSK9)-D377Y concurrently with high cholesterol diet (HCD) feeding for 16 weeks. In the last 10 weeks, hApoC3-Tg mice were orally treated with a combination of atorvastatin (10 mg·kg-1·d-1) and fenofibrate (100 mg·kg-1·d-1). HCD-treated hApoC3-Tg mice demonstrated elevated levels of serum TG, total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C). Oral administration of atorvastatin and fenofibrate significantly decreased the plaque sizes of en face aorta, aortic sinus and innominate artery accompanied by improved lipid profileand distribution. In summary,this novel mouse model is of considerable clinical relevance for evaluation of anti-atherosclerotic drugsby targeting both hypercholesterolemia and hypertriglyceridemia.

Hepatic polypeptide nutrient solution improves high-cholesterol diet-induced rats with nonalcoholic fatty liver disease by activating AMP-activated protein kinase signaling pathway.

Hepatic polypeptide nutrient solution (HP) is a mixture of hepatoprotective peptides derived from fresh porcine liver with various effects. However, the role and mechanisms of HP in nonalcoholic fatty liver disease (NAFLD) are still not well understood. We investigated the effects of HP NAFLD rats induced by high-cholesterol diet (HCD) and its underlying mechanisms. Rats were provided with HCD for 4 weeks and then received HP or metformin after 2 weeks of HCD feeding. The study found that HP reduced cholesterol and triglyceride levels in rats with NAFLD (all p < .05). Histopathological examination also showed that HP improved the liver lesions induced by the HCD diet. Furthermore, the oxidative stress and inflammatory responses of NAFLD rats treated with HP were also improved. In addition, it was discovered that HP triggered the activation of AMPK and decreased the expression of SREBP-1c and FAS while enhancing the expression of PPAR α and CPT-1 in liver. These findings indicated that HP might have therapeutic potential for NAFLD, possibly via activating AMPK signaling pathway.

Abstract 17224: Macrophage Expressed CD36 Promotes Plaque Vulnerability in Atherosclerosis

Introduction: Atherosclerosis is characterised by a chronic lipid-driven inflammation that accounts for the majority of deaths worldwide. CD36 is an immunoregulatory scavenger receptor at the interface of lipid metabolism and inflammation that is critically involved in atherogenesis. However, due to the lack of cell-specific animal models the exact role of CD36 on macrophages in atherosclerosis remains unknown to date. Research Question: Here, we aimed to study the role of macrophage expressed CD36 in atherogenesis. Methods: To screen for a potential role of macrophage expressed CD36 in atherosclerosis we assessed CD36 gene expression across different leukocyte populations in scRNAseq data from atherosclerotic LDLR-/- mice. To evaluate the role of macrophage expressed CD36 in atherogenesis, we generated tamoxifen inducible macrophage (CX3CR1)-specific CD36-deficient mice on a LDLR-/- background. To interrogate a potential role of CD36 in human atherosclerosis, human atherosclerotic plaques were studied by scRNAseq. Results: Analysis of murine scRNAseq data identified macrophages as the main CD36 expressing cell type. In line, CD36 protein expression in aortic cell suspensions from LDLR-/- mice was significantly higher in macrophages compared to B and T cells as assessed by flow cytometry. Notably, macrophage-specific CD36 deficiency substantially reduced lesional necrotic core area after 16 weeks of high cholesterol diet feeding indicating ameliorated plaque vulnerability. In vitro, macrophage-specific CD36 deficiency reduced ROS production and oxLDL uptake. Similar to our observations in mice, cellular CD36 expression was highest in human macrophages/monocytes. Pathway analysis revealed that CD36-expressing macrophages exhibited a more pro-inflammatory gene signature than CD36-negative macrophages. Finally, unstable carotid plaques displayed a significantly higher CD36 expression compared to stable lesions. Conclusion: All in all, our data indicates a pro-inflammatory role of macrophage expressed CD36 in murine and human atherosclerosis. These findings suggest that targeting CD36 on macrophages may represent a potential target for anti-atherosclerotic therapy.

Evaluation on the Effectiveness of High Cholesterol Diet Feeding in Inducing Early and Established Atherosclerotic Lesions in New Zealand White Rabbits.

Various methods were used to induce atherosclerosis in rabbits. One of the most common methods used is high-cholesterol diet (HCD) feeding. However, the exact amount and duration of HCD feeding to induce early and established atherosclerosis in New Zealand white rabbits (NZWR) continue to be debated among researchers. Therefore, this study aims to evaluate the effectiveness of 1% HCD feeding in inducing early and established atherosclerosis lesions in NZWR. A total of 50 g/kg/day of 1% HCD was fed to three to four months old male rabbits weighing 1.8 to 2.0 kg for four and eight weeks to induce early and established atherosclerosis respectively. The body weight and lipid profile were measured at baseline and post-HCD intervention. Following euthanasia, the aorta was excised and prepared for histology and immunohistochemical analysis to confirm the stages of atherosclerosis. The mean body weight of the rabbits in early and established atherosclerosis groups increased significantly up to 17.5% (p = 0.026) and 19.75% (p = 0.019) respectively compared to baseline. The total cholesterol level dramatically elevated up to 13-fold (p = 0.005) and 38-fold (p = 0.013) compared to baseline, after four and eight weeks of 1% HCD feeding respectively. The low-density lipoprotein level significantly increased up to 42-fold (p = 0.006) and 128-fold (p = 0.011) compared to baseline, after four and eight weeks of 1% HCD feeding respectively. Rabbits fed with four and eight weeks 1% HCD significantly developed 5.79% (p = 0.008) and 21.52% (p = 0.008) aortic lesion areas compared to the control group. Histological evaluation in the aorta showed accumulation of foam cells in early atherosclerosis group and formation of fibrous plaque and lipid core in the established atherosclerosis group. Rabbits fed with eight weeks HCD showed higher tissue expressions of ICAM-1, VCAM-1, e-selectin, IL-6, IL-8, NF-κBp65, and MMP-12 compared to four weeks of HCD intervention. A total of 50 g/kg/day of 1% HCD for four and eight weeks is sufficient to induce early and established atherosclerosis in NZWR respectively. The consistent results through this method could facilitate researchers in inducing early and established atherosclerosis in NZWR.

PANDORA-Seq unveils the hidden small noncoding RNA landscape in atherosclerosis of LDL receptor-deficient mice

Small noncoding RNAs (sncRNAs) play diverse roles in numerous biological processes. While the widely used RNA sequencing (RNA-Seq) method has advanced sncRNA discovery, RNA modifications can interfere with the complementary DNA library construction process, preventing the discovery of highly modified sncRNAs including transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) that may have important functions in disease development. To address this technical obstacle, we recently developed a novel PANDORA-Seq (Panoramic RNA Display by Overcoming RNA Modification Aborted Sequencing) method to overcome RNA modification-elicited sequence interferences. To identify novel sncRNAs associated with atherosclerosis development, LDL receptor-deficient (LDLR−/−) mice were fed a low-cholesterol diet or high-cholesterol diet (HCD) for 9 weeks. Total RNAs isolated from the intima were subjected to PANDORA-Seq and traditional RNA-Seq. By overcoming RNA modification-elicited limitations, PANDORA-Seq unveiled an rsRNA/tsRNA-enriched sncRNA landscape in the atherosclerotic intima of LDLR−/− mice, which was strikingly different from that detected by traditional RNA-Seq. While microRNAs were the dominant sncRNAs detected by traditional RNA-Seq, PANDORA-Seq substantially increased the reads of rsRNAs and tsRNAs. PANDORA-Seq also detected 1,383 differentially expressed sncRNAs induced by HCD feeding, including 1,160 rsRNAs and 195 tsRNAs. One of HCD-induced intimal tsRNAs, tsRNA-Arg-CCG, may contribute to atherosclerosis development by regulating the proatherogenic gene expression in endothelial cells. Overall, PANDORA-Seq revealed a hidden rsRNA and tsRNA population associated with atherosclerosis development. These understudied tsRNAs and rsRNAs, which are much more abundant than microRNAs in the atherosclerotic intima of LDLR−/− mice, warrant further investigations.

Urotensin II Enhances Advanced Aortic Atherosclerosis Formation and Delays Plaque Regression in Hyperlipidemic Rabbits.

Accumulated evidence shows that elevated urotensin II (UII) levels are associated with cardiovascular diseases. However, the role of UII in the initiation, progression, and regression of atherosclerosis remains to be verified. Different stages of atherosclerosis were induced in rabbits by a 0.3% high cholesterol diet (HCD) feeding, and either UII (5.4 μg/kg/h) or saline was chronically infused via osmotic mini-pumps. UII promoted atherosclerotic fatty streak formation in ovariectomized female rabbits (34% increase in gross lesion and 93% increase in microscopic lesion), and in male rabbits (39% increase in gross lesion). UII infusion significantly increased the plaque size of the carotid and subclavian arteries (69% increase over the control). In addition, UII infusion significantly enhanced the development of coronary lesions by increasing plaque size and lumen stenosis. Histopathological analysis revealed that aortic lesions in the UII group were characterized by increasing lesional macrophages, lipid deposition, and intra-plaque neovessel formation. UII infusion also significantly delayed the regression of atherosclerosis in rabbits by increasing the intra-plaque macrophage ratio. Furthermore, UII treatment led to a significant increase in NOX2 and HIF-1α/VEGF-A expression accompanied by increased reactive oxygen species levels in cultured macrophages. Tubule formation assays showed that UII exerted a pro-angiogenic effect in cultured endothelial cell lines and this effect was partly inhibited by urantide, a UII receptor antagonist. These findings suggest that UII can accelerate aortic and coronary plaque formation and enhance aortic plaque vulnerability, but delay the regression of atherosclerosis. The role of UII on angiogenesis in the lesion may be involved in complex plaque development.

A Systems Analysis of Phenotype Heterogeneity in APOE*3Leiden.CETP Mice Induced by Long-Term High-Fat High-Cholesterol Diet Feeding.

Within the human population, considerable variability exists between individuals in their susceptibility to develop obesity and dyslipidemia. In humans, this is thought to be caused by both genetic and environmental variation. APOE*3-Leiden.CETP mice, as part of an inbred mouse model in which mice develop the metabolic syndrome upon being fed a high-fat high-cholesterol diet, show large inter-individual variation in the parameters of the metabolic syndrome, despite a lack of genetic and environmental variation. In the present study, we set out to resolve what mechanisms could underlie this variation. We used measurements of glucose and lipid metabolism from a six-month longitudinal study on the development of the metabolic syndrome. Mice were classified as mice with either high plasma triglyceride (responders) or low plasma triglyceride (non-responders) at the baseline. Subsequently, we fitted the data to a dynamic computational model of whole-body glucose and lipid metabolism (MINGLeD) by making use of a hybrid modelling method called Adaptations in Parameter Trajectories (ADAPT). ADAPT integrates longitudinal data, and predicts how the parameters of the model must change through time in order to comply with the data and model constraints. To explain the phenotypic variation in plasma triglycerides, the ADAPT analysis suggested a decreased cholesterol absorption, higher energy expenditure and increased fecal fatty acid excretion in non-responders. While decreased cholesterol absorption and higher energy expenditure could not be confirmed, the experimental validation demonstrated that the non-responders were indeed characterized by increased fecal fatty acid excretion. Furthermore, the amount of fatty acids excreted strongly correlated with bile acid excretion, in particular deoxycholate. Since bile acids play an important role in the solubilization of lipids in the intestine, these results suggest that variation in bile acid homeostasis may in part drive the phenotypic variation in the APOE*3-Leiden.CETP mice.

Long noncoding TSI attenuates aortic valve calcification by suppressing TGF-β1-induced osteoblastic differentiation of valve interstitial cells

IntroductionCalcific aortic valve disease (CAVD) is an active and cellular-driven fibrocalcific process characterised by differentiation of valve interstitial cells (VICs) towards an osteogenic-like phenotype. A recently identified lncRNA, lncTSI, has been reported to inhibit fibrogenesis through transforming growth factor (TGF)-β/Smad3 pathway. Here, the present study aimed to investigate the role of lncTSI in CAVD. MethodsThe effect of TGF-β1 on lncTSI of VICs was measured. TGF-β1, RUNX2 and collagen I expression between calcified aortic valve tissue and normal samples by immunohistochemistry and western blotting. Human VICs were cultured and treated with TGF-β1. SiRNA and pcDNA3.1-lncTSI plasmid transfection were used to silence and overexpress lncTSI in VICs for 48 h, Smads phosphorylation, RUNX2 and collagen I expression were then verified by western blotting. In ApoE−/− mice fed with 0.25 % high-cholesterol diet, AAV2-lncTSI were injected intravenously to observe their effect on the formation of aortic valve calcification. ResultslncTSI was highly expressed in VICs treated with TGF-β1. lncTSI was transcriptionally regulated by Smad3 and reversely inhibited TGF-β1-induced Smad3 phosphorylation and downregulated profibrotic gene expression. Silencing lncTSI increased TGF-β1–induced Smad3 phosphorylation, and subsequently, upregulated RUNX2 and collagen I expressions in VICs. While overexpression of lncTSI reversed the production of RUNX2 and collagen I in VICs. In a mouse CAVD model of 24 week 0.25 % high-cholesterol diet feeding, overexpression of lncTSI significantly reduced calcium deposition, RUNX2, pSmad3, and collagen I expression in aortic valve leaflets, with less aortic valve stenosis. ConclusionsThe novel findings of present study suggested that lncTSI alleviated aortic valve calcification through negative regulation of the TGF-β/Smad3 pathway. The results may help elucidate new diagnostic and therapeutic targets to prevent CAVD progression.

Abstract 153: The Differential Effects Of Weight Loss And Regain On Atherosclerosis

Heightened inflammation in obesity is a major contributor to many diseases, including atherosclerosis and other cardiovascular diseases (CVD). Although weight loss is extremely beneficial in improving many obesity-related conditions, weight regain worsens disease outcomes, including CVD, compared to maintenance of an obese state. Nonetheless, how weight loss promotes inflammation resolution and why weight regain worsens inflammation is poorly understood and was investigated here. Using high-fat high-cholesterol diet feeding of atherogenic mice (Ldlr deficient), we promoted weight loss after the establishment of atherosclerosis and obesity by using dietary restriction, e.g. reducing daily food consumption by 30% for 2 weeks, while maintaining hyperlipidemia. For weight regain, some mice were then given free access to food for 6 weeks. Strikingly, after 2 weeks of dietary restriction we observed atherosclerosis resolution, while weight regain accelerated disease re-progression. Single-cell analysis of visceral adipose and plaque leukocytes revealed enrichment in both tissues of a novel macrophage population with dietary restriction, which is distinguished by high expression of the antibody receptor Fcgr4, a known phagocytosis mediator. Conversely, Fcgr4 + macrophages disappeared from plaques and adipose following weight regain. Mechanistically, Fcgr4 + macrophages from the adipose tissue induced clearance of plaque necrotic core via enhanced efferocytosis. Moreover, we discovered that weight loss and regain differentially influence immune progenitors. Bone marrow (BM) transplantation showed that while weight loss and obese BM recipients had similar plaque inflammation, weight regain BM recipients had larger, more inflammatory plaques. In conclusion, our data are the first to directly show that initial weight loss promotes resolution of atherosclerosis, independently of plasma cholesterol changes and through the enrichment of specialized pro-resolving macrophages in adipose tissue that communicate with plaques. Conversely, weight regain accelerates disease progression via inflammatory reprogramming of immune progenitors.

Albino mice with the point mutation at the tyrosinase locus show high cholesterol diet-induced NASH susceptibility

Non-alcoholic fatty liver disease (NAFLD) constitutes a metabolic disorder with high worldwide prevalence and increasing incidence. The inflammatory progressive state, non-alcoholic steatohepatitis (NASH), leads to liver fibrosis and carcinogenesis. Here, we evaluated whether tyrosinase mutation underlies NASH pathophysiology. Tyrosinase point-mutated B6 (Cg)-Tyrc-2J/J mice (B6 albino) and C57BL/6J black mice (B6 black) were fed with high cholesterol diet (HCD) for 10 weeks. Normal diet-fed mice served as controls. HCD-fed B6 albino exhibited high NASH susceptibility compared to B6 black, a phenotype not previously reported. Liver injury occurred in approximately 50% of B6 albino from one post HCD feeding, with elevated serum alanine aminotransferase and aspartate aminotransferase levels. NASH was induced following 2 weeks in severe-phenotypic B6 albino (sB6), but B6 black exhibited no symptoms, even after 10 weeks. HCD-fed sB6 albino showed significantly higher mortality rate. Histological analysis of the liver revealed significant inflammatory cell and lipid infiltration and severe fibrosis. Serum lipoprotein analysis revealed significantly higher chylomicron and very low-density lipoprotein levels in sB6 albino. Moreover, significantly higher small intestinal lipid absorption and lower fecal lipid excretion occurred together with elevated intestinal NPC1L1 expression. As the tyrosinase point mutation represents the only genetic difference between B6 albino and B6 black, our work will facilitate the identification of susceptible genetic factors for NASH development and expand the understanding of NASH pathophysiology.

High Fat Diet and High Cholesterol Diet Reduce Hepatic Vitamin D-25-Hydroxylase Expression and Serum 25-Hydroxyvitamin D3 Level through Elevating Circulating Cholesterol, Glucose, and Insulin Levels.

Low circulating 25-hydroxyvitamin D (25(OH)D) levels associate with obesity, diabetes, and hyperlipidemia, but the underlying mechanisms remain uncertain. As energy-dense diet contributes to these disorders, this study investigates whether diet could impair vitamin D metabolism. Compared with control chow-fed mice, high fat diet (HFD)-fed mice show lower serum 25(OH)D3 and 1,25(OH)2 D3 levels, lower hepatic vitamin D 25-hydroxylase Cyp2r1 expression but comparable renal vitamin D metabolic enzymes expression. Time course studies show that after HFD feeding, the serum concentrations of cholesterol, triglycerides, fatty acids, glucose, and insulin elevate sequentially and before the reduction of hepatic Cyp2r1 expression and serum 25(OH)D3 levels. Hepatic Cyp2r1 expression also reduces after consuming high fat and high sucrose diet. After high cholesterol diet feeding, serum total cholesterol rises and hepatic Cyp2r1 expression decreases ahead of the reduction of serum 25(OH)D3 . In vitro studies demonstrate that high concentrations of cholesterol, glucose, and insulin significantly inhibit Cyp2r1expression in primary murine hepatocytes. Further studies show that dietary restriction in HFD-fed mice ameliorates hypercholesterolemia, hyperglycemia, and hypertriglyceridemia, and elevates hepatic Cyp2r1 expression and serum 25(OH)D3 level. These findings suggest that diet-induced elevation of circulating cholesterol, glucose, and insulin reduces serum 25(OH)D3 level through suppressing hepatic Cyp2r1 expression.

Smooth Muscle Overexpression of PGC1α Attenuates Atherosclerosis in Rabbits.

[Figure: see text].

Efficacy of nobiletin in improving hypercholesterolemia and nonalcoholic fatty liver disease in high-cholesterol diet-fed mice

BACKGROUND/OBJECTIVESNobiletin (NOB), a citrus flavonoid, is reported to have beneficial effects on cardiovascular and metabolic health. However, there is limited research investigating the effect of long-term supplementation with low-dose NOB on high-cholesterol diet (HCD)-induced hypercholesterolemia and non-obese nonalcoholic fatty liver disease (NAFLD). Therefore, we investigated the influence of NOB on hypercholesterolemia and NAFLD in HCD-fed mice.SUBJECTS/METHODSC57BL/6J mice were fed a normal diet (ND) or HCD (35 kcal% fat, 1.25% cholesterol, 0.5% cholic acid) with or without NOB (0.02%) for 20 weeks.RESULTSHCD feeding markedly reduced the final body weight compared to ND feeding, with no apparent energy intake differences. NOB supplementation suppressed HCD-induced weight loss without altering energy intake. Moreover, NOB significantly decreased the total cholesterol (TC) levels and the low-density lipoprotein (LDL)/very-LDL-cholesterol to TC ratio, and increased the high-density lipoprotein-cholesterol/TC ratio in plasma, compared to those for HCD feeding alone. The plasma levels of inflammatory and atherosclerosis markers (C-reactive protein, oxidized LDL, interleukin [IL]-1β, IL-6, and plasminogen activator inhibitor-1) were significantly lower, whereas those of anti-atherogenic adiponectin and paraoxonase were higher in the NOB-supplemented group than in the HCD control group. Furthermore, NOB significantly decreased liver weight, hepatic cholesterol and triglyceride contents, and lipid droplet accumulation by inhibiting messenger RNA expression of hepatic genes and activity levels of cholesterol synthesis-, esterification-, and fatty acid synthesis-associated enzymes, concomitantly enhancing fatty acid oxidation-related gene expression and enzyme activities. Dietary NOB supplementation may protect against hypercholesterolemia and NAFLD via regulation of hepatic lipid metabolism in HCD-fed mice; these effects are associated with the amelioration of inflammation and reductions in the levels of atherosclerosis-associated cardiovascular markers.CONCLUSIONSThe present study suggests that NOB may serve as a potential therapeutic agent for the treatment of HCD-induced hypercholesterolemia and NAFLD.

Carboxylesterase 1d (Ces1d) does not contribute to cholesteryl ester hydrolysis in the liver

The liver is the central organ regulating cholesterol synthesis, storage, transport, and elimination. Mouse carboxylesterase 1d (Ces1d) and its human ortholog CES1 have been described to possess lipase activity and play roles in hepatic triacylglycerol metabolism and VLDL assembly. It has been proposed that Ces1d/CES1 might also catalyze cholesteryl ester (CE) hydrolysis in the liver and thus be responsible for the hydrolysis of HDL-derived CE; this could contribute to the final step in the reverse cholesterol transport (RCT) pathway, wherein cholesterol is secreted from the liver into bile and feces, either directly or after conversion to water-soluble bile salts. However, the proposed function of Ces1d/CES1 as a CE hydrolase is controversial. In this study, we interrogated the role hepatic Ces1d plays in cholesterol homeostasis using liver-specific Ces1d-deficient mice. We rationalized that if Ces1d is a major hepatic CE hydrolase, its absence would (1) reduce in vivo RCT flux and (2) provoke liver CE accumulation after a high-cholesterol diet challenge. We found that liver-specific Ces1d-deficient mice did not show any difference in the flux of in vivo HDL-to-feces RCT nor did it cause additional liver CE accumulation after high-fat, high-cholesterol Western-type diet feeding. These findings challenge the importance of Ces1d as a major hepatic CE hydrolase.

Cilostazol improves hyperglycemia-induced impaired vasculo-angiogenesis through stimulating adiponectin/adiponectin receptor 1/Sirtuin 1 signaling pathway

Abstract Background Cilostazol is an antiplatelet agent with vasodilating effect working through increasing intracellular concentration of cyclic adenosine monophosphate (AMP). We and others have previously found that cilostazol has a favorable effect on vasculo-angiogenesis. However, there is no study to evaluate the effect of cilostazol on adiponectin and its receptors. Purpose This study investigated the effects of cilostazol on adiponectin/adiponectin receptors and Sirtuin 1 (Sirt1)/AMP-activated protein kinase (AMPK) signaling pathway for preventing high glucose (HG)-induced impaired vasculo-angiogenesis in vitro and in vivo. Methods and results Human umbilical vein endothelial cells (HUVECs), seeded onto Transwell insert, and human aortic smooth muscle cells (HASMCs), seeded onto 6-well plate at lower level, were co-cultured in HG condition (25 mM). Adiponectin concentrations in the supernatant of 6-welll-plate and Transwell insert were significantly higher when HASMCs were treated with cilostazol in a dose-response manner but not significantly changed when only HUVECs were treated with cilostazol. HG downregulated protein expression of adiponectin receptor-1 (adipoR1), adipoR2, and Sirt1 and phosphorylation of AMPKα1, whereas cilostazol treatment restored expression of adipoR1 and Sirt1 proteins and upregulated phosphorylation of AMPKα1 in HUVECs treated with HG but not adipoR2. The stimulating effect of cilostazol on AMPKα1 or Sirt1 was attenuated with Sirt1 or AMPKα1 gene knockdown, respectively. By using gene knockdown of adiponectin receptors or AMPKα1, or treatment of the Sirt1 inhibitor, our data showed that cilostazol prevented apoptosis, and stimulated proliferation, chemotactic motility and capillary-like tube formation in HG-treated HUVECs through adipoR1, AMPK, and Sirt1 signaling pathway but not adipoR2. Fifteen-week-old male ICR hyperglycemic mice, induced by streptozosin injection and high cholesterol diet feeding, were treated intraperitoneally with cilostazol (10 mg/kg) 2 times per day since day 1 to day 7 after hindlimb ischemia. Recovery of blood flow ratio (ipsilateral/contralateral) in the ischemic hindlimb 14–21 days after surgery and circulating CD34+CD45dim cells were significantly attenuated by adipoR1 knockdown but not adipoR2. Capillary density in the ischemic muscles was significantly lower in both adipoR1- and adipoR2-knockdown mice. Expression of Sirt1 as well as phosphorylation of AMPKα1/acetyl-CoA carboxylase and Akt/endothelial nitric oxide synthase in ischemic muscles were significantly attenuated by gene knockdown of adipoR1 or adipoR2. Conclusions Our data suggest that cilostazol prevents high glucose-induced endothelial dysfunction in vascular endothelial cells as well as enhances vasculo-angiogenesis in hyperglycemic mice by upregulation of adiponectin/adipoR2 and its downstream signaling molecules, Sirt1/AMPK. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): The Ministry of Health and Welfare, Executive Yuan, Taiwan; The Ministry of Science and Technology, Executive Yuan, Taiwan

Liver-specific microRNA-185 knockout promotes cholesterol dysregulation in mice

BackgroundThe liver plays a key role in regulating whole body cholesterol homeostasis. Hepatic cholesterol accumulation causes liver injury in fatty liver disease and hypercholesterolemia increases the risk of cardiovascular disease. MicroRNAs (miRNAs, miRs) have been shown to regulate various pathways in cholesterol metabolism. Recently, miR-185 has been shown to regulate sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR) to modulate cholesterol synthesis and uptake. Materials and methodsThe role of miR-185 in regulating diet-induced metabolic disorders were studied in liver-specific miRNA-185 knockout (L-miR-185 KO) mice. ResultsL-miR-185 KO mice developed worsened hepatic steatosis upon high-fat high-cholesterol Western diet feeding with accumulation of triglyceride and cholesterol in the liver. In addition, L-miR-185 KO mice developed hypercholesterolemia upon Western diet feeding. Gene expression analysis showed that L-miR-185 KO mice did not show increased hepatic mRNA expression of SREBP2 or its targets LDLR and HMG-CoA reductase (HMGCR). Although expression of miR-185 mimic inhibited the mRNA of SREBP2, HMGCR and LDLR in HepG2 cells, miR-185 inhibitor did not increase the mRNA of SREBP2, HMGCR or LDLR in HepG2 cells. ConclusionsIn conclusion, we reported that L-miR-185 KO mice were more sensitive to Western diet induced hepatic steatosis and hypercholesterolemia. The molecular mechanisms underlying these metabolic changes remain to be investigated in future studies.

Epigenetic silencing of microRNA-125b-5p promotes liver fibrosis in nonalcoholic fatty liver disease via integrin α8-mediated activation of RhoA signaling pathway

BackgroundNonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases that may progress to liver fibrosis or cancer. The present study aimed to investigate the role of microRNA-125b-5p (miR-125b-5p) in NAFLD and to further explore underlying molecular mechanisms. MethodsA mouse model of NAFLD was constructed by high cholesterol diet feeding and a cell-model was developed by treating the mouse liver cell line NCTC1469 with palmitic acid. Gain- and loss-of-function experiments were performed to determine the effects of miR-125b-5p, integrin α8 (ITGA8), and the RhoA signaling pathway on liver fibrosis in NAFLD. After the expression levels of miR-125b-5p, ITGA8, and RhoA were determined, liver fibrosis was evaluated in vivo and in vitro. The binding relationship of miR-125b-5p and ITGA8 was then validated. Finally, miR-125b-5p promoter methylation in NAFLD liver tissues and cells was determined. ResultsIn NAFLD clinical samples, mouse model, and cell-model, miR-125b-5p expression was reduced, while ITGA8 expression was increased. Moreover, miR-125b-5p targeted and downregulated ITGA8, leading to inhibition of the RhoA signaling pathway. In NAFLD liver tissues and cells, the CpG island in the miR-125b-5p promoter was methylated, causing epigenetic silencing of miR-125b-5p. Both miR-125b-5p silencing and ITGA8 overexpression promoted in vitro and in vivo liver fibrosis in NAFLD via activation of the RhoA signaling pathway. ConclusionsCollectively, epigenetic silencing of miR-125b-5p upregulates ITGA8 expression to activate the RhoA signaling pathway, leading to liver fibrosis in NAFLD.

Trimethylamine N-oxide attenuates high-fat high-cholesterol diet-induced steatohepatitis by reducing hepatic cholesterol overload in rats.

BACKGROUNDTrimethylamine N-oxide (TMAO) has been shown to be involved in cardiovascular disease (CVD). However, its role in nonalcoholic steatohepatitis (NASH) is unknown.AIMTo determine the effect of TMAO on the progression of NASH.METHODSA rat model was induced by 16-wk high-fat high-cholesterol (HFHC) diet feeding and TMAO was administrated by daily oral gavage for 8 wk.RESULTSOral TMAO intervention attenuated HFHC diet-induced steatohepatitis in rats. Histological evaluation showed that TMAO treatment significantly alleviated lobular inflammation and hepatocyte ballooning in the livers of rats fed a HFHC diet. Serum levels of alanine aminotransferase and aspartate aminotransferase were also decreased by TMAO treatment. Moreover, hepatic endoplasmic reticulum (ER) stress and cell death were mitigated in HFHC diet-fed TMAO-treated rats. Hepatic and serum levels of cholesterol were both decreased by TMAO treatment in rats fed a HFHC diet. Furthermore, the expression levels of intestinal cholesterol transporters were detected. Interestingly, cholesterol influx-related Niemann-Pick C1-like 1 was downregulated and cholesterol efflux-related ABCG5/8 were upregulated by TMAO treatment in the small intestine. Gut microbiota analysis showed that TMAO could alter the gut microbial profile and restore the diversity of gut flora.CONCLUSIONThese data suggest that TMAO may modulate the gut microbiota, inhibit intestinal cholesterol absorption, and ameliorate hepatic ER stress and cell death under cholesterol overload, thereby attenuating HFHC diet-induced steatohepatitis in rats. Further studies are needed to evaluate the influence on CVD and define the safe does of TMAO treatment.

OR33-2 Blocking FSH Inhibits Hepatic Cholesterol Biosynthesis and Reduces Serum Cholesterol

Menopause is associated with dyslipidemia and an increased risk of cardio-cerebrovascular disease. The classic view assumes that the underlying mechanism of dyslipidemia is attributed to an insufficiency of estrogen. In addition to a decrease in estrogen, circulating follicle-stimulating hormone (FSH) levels become elevated at menopause. In this study, we found that blocking FSH reduced serum cholesterol via inhibiting hepatic cholesterol biosynthesis. First, the epidemiological results showed that the serum FSH levels were positively correlated with the serum total cholesterol levels, even after excluding serum estrogen effects. In addition, the prevalence of hypercholesterolemia was significantly higher in peri-menopausal women than that in pre-menopausal women. Furthermore, we generated an FSH-elevated mouse model by intraperitoneal injection of exogenous FSH in ovariectomized (OVX) mice, in which a normal level of estrogen (E2) was maintained by exogenous supplementation. Consistently, the results indicated that FSH, independent of estrogen, increased the serum cholesterol level in the FSH-elevated mouse model. Moreover, blocking FSH by FSHβ-antibody or ablating the FSH receptor (FSHR) gene could effectively prevent hypercholesterolemia induced by FSH injection or high-cholesterol diet feeding. Mechanistically, FSH, binding with hepatic FSHRs, activated the Gi2a/β-arrestin-2/Akt pathway and subsequently inhibited the binding of FoxO1 with the SREBP-2 promoter, thus preventing FoxO1 from repressing SREBP-2 gene transcription. This effect, in turn, resulted in the upregulation of SREBP-2, which drove HMGCR nascent transcription and de novo cholesterol biosynthesis, leading to the increase of cholesterol accumulation. This study uncovers a new opportunity for treating hypercholesterolemia during menopause by blocking FSH signaling, particularly for women in peri-menopause characterized by only FSH elevation.

Blocking FSH inhibits hepatic cholesterol biosynthesis and reduces serum cholesterol.

Menopause is associated with dyslipidemia and an increased risk of cardio-cerebrovascular disease. The classic view assumes that the underlying mechanism of dyslipidemia is attributed to an insufficiency of estrogen. In addition to a decrease in estrogen, circulating follicle-stimulating hormone (FSH) levels become elevated at menopause. In this study, we find that blocking FSH reduces serum cholesterol via inhibiting hepatic cholesterol biosynthesis. First, epidemiological results show that the serum FSH levels are positively correlated with the serum total cholesterol levels, even afteradjustment by considering the effects of serum estrogen. In addition, the prevalence of hypercholesterolemia is significantly higher in peri-menopausal women than that in pre-menopausal women. Furthermore, we generated a mouse model of FSH elevation by intraperitoneally injecting exogenous FSH into ovariectomized (OVX) mice, in which a normal level of estrogen (E2) was maintained by exogenous supplementation. Consistently, the results indicate that FSH, independent of estrogen, increases the serum cholesterol level in this mouse model. Moreover, blocking FSH signaling by anti-FSHβ antibody or ablating the FSH receptor (FSHR) gene could effectively prevent hypercholesterolemia induced by FSH injection or high-cholesterol diet feeding. Mechanistically, FSH, via binding to hepatic FSHRs, activates the Gi2α/β-arrestin-2/Akt pathway and subsequently inhibits the binding of FoxO1 with the SREBP-2 promoter, thus preventing FoxO1 from repressing SREBP-2 gene transcription. This effect, in turn, results in the upregulation of SREBP-2, which drives HMGCR nascent transcription and de novo cholesterol biosynthesis, leading to the increase of cholesterol accumulation. This study uncovers that blocking FSH signaling might be a new strategy for treating hypercholesterolemia during menopause, particularly for women in peri-menopause characterized by FSH elevation only.