EPAS1 Attenuates Atherosclerosis Initiation at Disturbed Flow Sites Through Endothelial Fatty Acid Uptake.

BACKGROUND: Atherosclerotic plaques form unevenly due to disturbed blood flow, causing localized endothelial cell (EC) dysfunction. Obesity exacerbates this process, but the underlying molecular mechanisms are unclear. The transcription factor EPAS1 (HIF2A) has regulatory roles in endothelium, but its involvement in atherosclerosis remains unexplored. This study investigates the potential interplay between EPAS1, obesity, and atherosclerosis. METHODS: Responses to shear stress were analyzed using cultured porcine aortic EC exposed to flow in vitro coupled with metabolic and molecular analyses and by en face immunostaining of murine aortic EC exposed to disturbed flow in vivo. Obesity and dyslipidemia were induced in mice via exposure to a high-fat diet or through Leptin gene deletion. The role of Epas1 in atherosclerosis was evaluated by inducible endothelial Epas1 deletion, followed by hypercholesterolemia induction (adeno-associated virus-PCSK9 [proprotein convertase subtilisin/kexin type 9]; high-fat diet). RESULTS: En face staining revealed EPAS1 enrichment at sites of disturbed blood flow that are prone to atherosclerosis initiation. Obese mice exhibited substantial reduction in endothelial EPAS1 expression. Sulforaphane, a compound with known atheroprotective effects, restored EPAS1 expression and concurrently reduced plasma triglyceride levels in obese mice. Consistently, triglyceride derivatives (free fatty acids) suppressed EPAS1 in cultured EC by upregulating the negative regulator PHD2. Clinical observations revealed that reduced serum EPAS1 correlated with increased endothelial PHD2 and PHD3 in obese individuals. Functionally, endothelial EPAS1 deletion increased lesion formation in hypercholesterolemic mice, indicating an atheroprotective function. Mechanistic insights revealed that EPAS1 protects arteries by maintaining endothelial proliferation by positively regulating the expression of the fatty acid-handling molecules CD36 (cluster of differentiation 36) and LIPG (endothelial type lipase G) to increase fatty acid beta-oxidation. CONCLUSIONS: Endothelial EPAS1 attenuates atherosclerosis at sites of disturbed flow by maintaining EC proliferation via fatty acid uptake and metabolism. This endothelial repair pathway is inhibited in obesity, suggesting a novel triglyceride-PHD2 modulation pathway suppressing EPAS1 expression. These findings have implications for therapeutic strategies addressing vascular dysfunction in obesity.

Distinct Roles of DRP1 in Conventional and Alternative Mitophagy in Obesity Cardiomyopathy.

BACKGROUND: Obesity induces cardiomyopathy characterized by hypertrophy and diastolic dysfunction. Whereas mitophagy mediated through an Atg7 (autophagy related 7)-dependent mechanism serves as an essential mechanism to maintain mitochondrial quality during the initial development of obesity cardiomyopathy, Rab9 (Ras-related protein Rab-9A)-dependent alternative mitophagy takes over the role during the chronic phase. Although it has been postulated that DRP1 (dynamin-related protein 1)-mediated mitochondrial fission and consequent separation of the damaged portions of mitochondria are essential for mitophagy, the involvement of DRP1 in mitophagy remains controversial. We investigated whether endogenous DRP1 is essential in mediating the 2 forms of mitophagy during high-fat diet (HFD)-induced obesity cardiomyopathy and, if so, what the underlying mechanisms are. METHODS: Mice were fed either a normal diet or an HFD (60 kcal %fat). Mitophagy was evaluated using cardiac-specific Mito-Keima mice. The role of DRP1 was evaluated using tamoxifen-inducible cardiac-specific Drp1knockout (Drp1 MCM) mice. RESULTS: Mitophagy was increased after 3 weeks of HFD consumption. The induction of mitophagy by HFD consumption was completely abolished in Drp1 MCM mouse hearts, in which both diastolic and systolic dysfunction were exacerbated. The increase in LC3 (microtubule-associated protein 1 light chain 3)-dependent general autophagy and colocalization between LC3 and mitochondrial proteins was abolished in Drp1 MCM mice. Activation of alternative mitophagy was also completely abolished in Drp1 MCM mice during the chronic phase of HFD consumption. DRP1 was phosphorylated at Ser616, localized at the mitochondria-associated membranes, and associated with Rab9 and Fis1 (fission protein 1) only during the chronic, but not acute, phase of HFD consumption. CONCLUSIONS: DRP1 is an essential factor in mitochondrial quality control during obesity cardiomyopathy that controls multiple forms of mitophagy. Although DRP1 regulates conventional mitophagy through a mitochondria-associated membrane-independent mechanism during the acute phase, it acts as a component of the mitophagy machinery at the mitochondria-associated membranes in alternative mitophagy during the chronic phase of HFD consumption.

Hypothalamic α7 nicotinic acetylcholine receptor (α7nAChR) is downregulated by TNFα-induced Let-7 overexpression driven by fatty acids.

The α7nAChR is crucial to the anti-inflammatory reflex, and to the expression of neuropeptides that control food intake, but its expression can be decreased by environmental factors. We aimed to investigate whether microRNA modulation could be an underlying mechanism in the α7nAchR downregulation in mouse hypothalamus following a short-term exposure to an obesogenic diet. Bioinformatic analysis revealed Let-7 microRNAs as candidates to regulate Chrna7, which was confirmed by the luciferase assay. Mice exposed to an obesogenic diet for 3 days had increased Let-7a and decreased α7nAChR levels, accompanied by hypothalamic fatty acids and TNFα content. Hypothalamic neuronal cells exposed to fatty acids presented higher Let-7a and TNFα levels and lower Chrna7 expression, but when the cells were pre-treated with TLR4 inhibitor, Let-7a, TNFα, and Chrna7 were rescued to normal levels. Thus, the fatty acids overload trigger TNFα-induced Let-7 overexpression in hypothalamic neuronal cells, which negatively regulates α7nAChR, an event that can be related to hyperphagia and obesity predisposition in mice.

Myocardial Rev-erb-Mediated Diurnal Metabolic Rhythm and Obesity Paradox.

BACKGROUND: The nuclear receptor Rev-erbα/ß, a key component of the circadian clock, emerges as a drug target for heart diseases, but the function of cardiac Rev-erb has not been studied in vivo. Circadian disruption is implicated in heart diseases, but it is unknown whether cardiac molecular clock dysfunction is associated with the progression of any naturally occurring human heart diseases. Obesity paradox refers to the seemingly protective role of obesity for heart failure, but the mechanism is unclear. METHODS: We generated mouse lines with cardiac-specific Rev-erbα/ß knockout (KO), characterized cardiac phenotype, conducted multi-omics (RNA-sequencing, chromatin immunoprecipitation sequencing, proteomics, and metabolomics) analyses, and performed dietary and pharmacological rescue experiments to assess the time-of-the-day effects. We compared the temporal pattern of cardiac clock gene expression with the cardiac dilation severity in failing human hearts. RESULTS: KO mice display progressive dilated cardiomyopathy and lethal heart failure. Inducible ablation of Rev-erbα/ß in adult hearts causes similar phenotypes. Impaired fatty acid oxidation in the KO myocardium, in particular, in the light cycle, precedes contractile dysfunctions with a reciprocal overreliance on carbohydrate utilization, in particular, in the dark cycle. Increasing dietary lipid or sugar supply in the dark cycle does not affect cardiac dysfunctions in KO mice. However, obesity coupled with systemic insulin resistance paradoxically ameliorates cardiac dysfunctions in KO mice, associated with rescued expression of lipid oxidation genes only in the light cycle in phase with increased fatty acid availability from adipose lipolysis. Inhibition of glycolysis in the light cycle and lipid oxidation in the dark cycle, but not vice versa, ameliorate cardiac dysfunctions in KO mice. Altered temporal patterns of cardiac Rev-erb gene expression correlate with the cardiac dilation severity in human hearts with dilated cardiomyopathy. CONCLUSIONS: The study delineates temporal coordination between clock-mediated anticipation and nutrient-induced response in myocardial metabolism at multi-omics levels. The obesity paradox is attributable to increased cardiac lipid supply from adipose lipolysis in the fasting cycle due to systemic insulin resistance and adiposity. Cardiac molecular chronotypes may be involved in human dilated cardiomyopathy. Myocardial bioenergetics downstream of Rev-erb may be a chronotherapy target in treating heart failure and dilated cardiomyopathy.

Preventing Cholesterol-Induced Perk (Protein Kinase RNA-Like Endoplasmic Reticulum Kinase) Signaling in Smooth Muscle Cells Blocks Atherosclerotic Plaque Formation.

BACKGROUND: Vascular smooth muscle cells (SMCs) undergo complex phenotypic modulation with atherosclerotic plaque formation in hyperlipidemic mice, which is characterized by de-differentiation and heterogeneous increases in the expression of macrophage, fibroblast, osteogenic, and stem cell markers. An increase of cellular cholesterol in SMCs triggers similar phenotypic changes in vitro with exposure to free cholesterol due to cholesterol entering the endoplasmic reticulum, triggering endoplasmic reticulum stress and activating Perk (protein kinase RNA-like endoplasmic reticulum kinase) signaling. METHODS: We generated an SMC-specific Perk knockout mouse model, induced hyperlipidemia in the mice by AAV-PCSK9DY injection, and subjected them to a high-fat diet. We then assessed atherosclerotic plaque formation and performed single-cell transcriptomic studies using aortic tissue from these mice. RESULTS: SMC-specific deletion of Perk reduces atherosclerotic plaque formation in male hyperlipidemic mice by 80%. Single-cell transcriptomic data identify 2 clusters of modulated SMCs in hyperlipidemic mice, one of which is absent when Perk is deleted in SMCs. The 2 modulated SMC clusters have significant overlap of transcriptional changes, but the Perk-dependent cluster uniquely shows a global decrease in the number of transcripts. SMC-specific Perk deletion also prevents migration of both contractile and modulated SMCs from the medial layer of the aorta. CONCLUSIONS: Our results indicate that hypercholesterolemia drives both Perk-dependent and Perk-independent SMC modulation and that deficiency of Perk significantly blocks atherosclerotic plaque formation.

Disruption of Phospholipid Transfer Protein-Mediated High-Density Lipoprotein Maturation Reduces Scavenger Receptor BI Deficiency-Driven Atherosclerosis Susceptibility Despite Unexpected Metabolic Complications.

OBJECTIVE: We tested the hypothesis that enlarged, dysfunctional HDL (high-density lipoprotein) particles contribute to the augmented atherosclerosis susceptibility associated with SR-BI (scavenger receptor BI) deficiency in mice. Approach and Results: We eliminated the ability of HDL particles to fully mature by targeting PLTP (phospholipid transfer protein) functionality. Particle size of the HDL population was almost fully normalized in male and female SR-BI×PLTP double knockout mice. In contrast, the plasma unesterified cholesterol to cholesteryl ester ratio remained elevated. The PLTP deficiency-induced reduction in HDL size in SR-BI knockout mice resulted in a normalized aortic tissue oxidative stress status on Western-type diet. Atherosclerosis susceptibility was-however-only partially reversed in double knockout mice, which can likely be attributed to the fact that they developed a metabolic syndrome-like phenotype characterized by obesity, hypertriglyceridemia, and a reduced glucose tolerance. Mechanistic studies in chow diet-fed mice revealed that the diminished glucose tolerance was probably secondary to the exaggerated postprandial triglyceride response. The absence of PLTP did not affect LPL (lipoprotein lipase)-mediated triglyceride lipolysis but rather modified the ability of VLDL (very low-density lipoprotein)/chylomicron remnants to be cleared from the circulation by the liver through receptors other than SR-BI. As a result, livers of double knockout mice only cleared 26% of the fractional dose of [14C]cholesteryl oleate after intravenous VLDL-like particle injection. CONCLUSIONS: We have shown that disruption of PLTP-mediated HDL maturation reduces SR-BI deficiency-driven atherosclerosis susceptibility in mice despite the induction of proatherogenic metabolic complications in the double knockout mice.

Mood disorders are associated with the reduction of brain derived neurotrophic factor in the hypocampus in rats submitted to the hipercaloric diet.

Adipose tissue accumulation, resulting from the consumption of hypercaloric foods, can cause a dysfunction of the endocrine system. Such endocrine changes can influence the expression of various neurochemicals including brain-derived neurotrophic factor (BDNF) - associated with cognitive and emotional problems. Here, we investigated the effects of a hypercaloric diet on depression- and anxiety-like behaviors in young rats along with concomitant changes in BDNF expression levels in the hippocampus. Eight week-old Wistar rats (n = 20) were divided in: control diet (CD) group which received industrial food (n = 8) and hypercaloric diet (HD) group which received animal fat and soybean oil (n = 12). After 45 days on the diet, the animals were evaluated: body weight and blood biochemical analisys. Changes in mood disposition were evaluated using forced swim test and the elevated plus-maze, whereas hippocampal BDNF expression levels were quantified by ELISA. After 45 weeks, the CD group showed a significant increase in body weight relative to the HD group. However, the HD rats had a body fat percentage and exhibited increased level of the biochemical markers. Furthermore, the animals in the HD group presented increased immobility time in the forced swimming test, as well as reduced response to plus-maze test suggesting a depression- and anxiety-like emotional state. In addition, the HD group also showed lower BDNF expression levels in the hippocampus. This study demonstrates that a hypercaloric diet induced increase in adipose tissue concentration in young rats was associated with reduced hippocampal BDNF expression and resulted in an increase in depression- and anxiety-like behaviors. Graphical abstract.

[Establishment of high-fat diet-induced obesity and insulin resistance model in rats].

OBJECTIVE: To investigate the appropriate conditions and duration for establishing a high-fat diet-induced obesity and insulin resistance model in rats. METHODS: Forty-five 6-week-old male Sprague-Dawley (SD) rats were randomly assigned into 2 groups: (1) control group (CON), (2) high-fat diet group (HFD). HFD was fed with a high-fat diet (45% kcal from fat) while CON with chow diet. After four-weeks of high-fat diet feeding, the rats of obesity resistance (OR) were eliminated according to body weight sorting, whereas obese (OB) rats were continued feeding a high-fat diet until 12 weeks. Body weight and food intake were recorded weekly. Glucose tolerance was evaluated by oral glucose tolerance test (OGTT) in 4 weeks, 8 weeks and 12 weeks. At the end of 12 weeks, insulin releasing test and visceral fat mass were measured and HE staining of the liver, adipose tissue and pancreatic tissue were conducted. RESULTS: After 4 weeks of a high-fat diet, the body weight of HFD was 17.8% higher than that of CON (P=0.001), and the rate of obesity was 67.6%-78.4%. Glucose tolerance of OB rats was impaired with a higher blood glucose concentration at 120 min (P<0.001) and a higher area under the curve (AUC, P=0.037) in OGTT compared with CON. The rate of obesity and insulin-resistance rats was 79.3%. After 8 weeks of feeding, the body weight in OB was 30.4% higher than CON (P<0.001). In OGTT, blood glucose levels at 60 min and 120 min were 35.6% and 36.4% higher than those in CON (both P<0.001), and AUC was 21.7% (P<0.001) higher than that of CON. The rate of obesity and insulin-resistance rats was 100.0%. After 12 weeks of feeding, the body weight in OB was 36.9% higher than that in CON (P<0.001). In OGTT, the blood glucose levels at 60 min and 120 min were 24.8% (P=0.001) and 34.6% (P<0.001) higher than those in CON, and AUC was 16.1% (P=0.019) higher than that of CON. The rate of obesity and insulin-resistance rats was 93.3%. The insulin releasing test showed that serum insulin concentration at each time point (0, 30, 60, 120 min) was higher than that in CON, with a 6.3-times higher than that in CON at 120 min (P=0.008). Pathological changes were observed in islets and liver in the OB rats. CONCLUSION: After 4 weeks of a high-fat diet (45% kcal from fat) feeding in six-weeks SD rats, the rats of OR were eliminated. Impaired glucose tolerance was found in OB rats after 4 weeks of feeding, and the rate was higher after 8-12 weeks of high-fat diet feeding.

Vascular Semaphorin 7A Upregulation by Disturbed Flow Promotes Atherosclerosis Through Endothelial ß1 Integrin.

OBJECTIVE: Accumulating evidence suggests a role of semaphorins in vascular homeostasis. Here, we investigate the role of Sema7A (semaphorin 7A) in atherosclerosis and its underlying mechanism. APPROACH AND RESULTS: Using genetically engineered Sema7A-/-ApoE-/- mice, we showed that deletion of Sema7A attenuates atherosclerotic plaque formation primarily in the aorta of ApoE-/- mice on a high-fat diet. A higher level of Sema7A in the atheroprone lesser curvature suggests a correlation of Sema7A with disturbed flow. This notion is supported by elevated Sema7A expression in human umbilical venous endothelial cells either subjected to oscillatory shear stress or treated with the PKA (protein kinase A)/CREB (cAMP response element-binding protein) inhibitor H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl hydrate). Further studies using the partial carotid artery ligation model showed that disturbed flow in the left carotid artery of Sema7A+/+ApoE-/- mice promoted the expression of endothelial Sema7A and cell adhesion molecules, leukocyte adhesion, and plaque formation, whereas such changes were attenuated in Sema7A-/-ApoE-/- mice. Further studies showed that blockage of ß1 integrin, a known Sema7A receptor, or inhibition of FAK (focal adhesion kinase), MEK1/2 (mitogen-activated protein kinase kinase 1/2), or NF-κB (nuclear factor-κB) significantly reduced the expression of cell adhesion molecules and THP-1 (human acute monocytic leukemia cell line) monocyte adhesion in Sema7A-overexpressing human umbilical venous endothelial cells. Studies using chimeric mice suggest that vascular, most likely endothelial, Sema7A plays a major role in atherogenesis. CONCLUSIONS: Our findings indicate a significant role of Sema7A in atherosclerosis by mediating endothelial dysfunction in a ß1 integrin-dependent manner.

Effect of a high-fat diet on the rat bladder wall and bioactive action of Brazil nut oil.

High-fat diet-induced obesity is associated with metabolic disorders. The Brazil nut has bioactive substances and has been used to control the damage caused by obesity in several organs. The work intended to show the damage caused by high-fat diet in the bladder wall and if the Brazil nut oil added to the diet could ameliorate or reverse this effect. Sixty-day-old rats were divided into two groups: C (control, n = 30) and HF (high-fat, n = 30) diets. At 90 days, 10 animals of each group were sacrificed. The others were divided into 4 groups: C and HF (animals that maintained their previous diet, n = 10 for each group) and C / Bno and HF / Bno (animals whose control or high-fat diet was supplemented by Brazil nut oil, n = 10 for each group). Sacrifice occurred at 120 days, and the bladders were removed and analyzed. Epithelial height was increased in the HF compared to the C group. In contrast, the C / Bno had a lower epithelial height compared to the others. The percentage of collagen between the detrusor muscle fibers was significantly greater in C / Bno, HF and HF / Bno than in control group. The HF had a larger muscle fiber diameter than the C group, while the C / Bno presented lower values than the HF and HF / Bno groups. HF diets induced bladder wall damage. These changes in the rat's bladder wall were partially reversed by the Bno.

STING-IRF3 Triggers Endothelial Inflammation in Response to Free Fatty Acid-Induced Mitochondrial Damage in Diet-Induced Obesity.

OBJECTIVE: Metabolic stress in obesity induces endothelial inflammation and activation, which initiates adipose tissue inflammation, insulin resistance, and cardiovascular diseases. However, the mechanisms underlying endothelial inflammation induction are not completely understood. Stimulator of interferon genes (STING) is an important molecule in immunity and inflammation. In the present study, we sought to determine the role of STING in palmitic acid-induced endothelial activation/inflammation. APPROACH AND RESULTS: In cultured endothelial cells, palmitic acid treatment activated STING, as indicated by its perinuclear translocation and binding to interferon regulatory factor 3 (IRF3), leading to IRF3 phosphorylation and nuclear translocation. The activated IRF3 bound to the promoter of ICAM-1 (intercellular adhesion molecule 1) and induced ICAM-1 expression and monocyte-endothelial cell adhesion. When analyzing the upstream signaling, we found that palmitic acid activated STING by inducing mitochondrial damage. Palmitic acid treatment caused mitochondrial damage and leakage of mitochondrial DNA into the cytosol. Through the cytosolic DNA sensor cGAS (cyclic GMP-AMP synthase), the mitochondrial damage and leaked cytosolic mitochondrial DNA activated the STING-IRF3 pathway and increased ICAM-1 expression. In mice with diet-induced obesity, the STING-IRF3 pathway was activated in adipose tissue. However, STING deficiency (Stinggt/gt ) partially prevented diet-induced adipose tissue inflammation, obesity, insulin resistance, and glucose intolerance. CONCLUSIONS: The mitochondrial damage-cGAS-STING-IRF3 pathway is critically involved in metabolic stress-induced endothelial inflammation. STING may be a potential therapeutic target for preventing cardiovascular diseases and insulin resistance in obese individuals.

MicroRNA-206 prevents hepatosteatosis and hyperglycemia by facilitating insulin signaling and impairing lipogenesis.

BACKGROUND & AIMS: The paradox of selective hepatic insulin resistance, wherein the insulin-resistant liver fails to suppress glucose production but continues to produce lipids, has been central to the pathophysiology of hepatosteatosis and hyperglycemia. Our study was designed to investigate the mechanism(s) by which microRNA-206 alleviates the pathogenesis of hepatosteatosis and hyperglycemia. METHODS: Dietary obese mice induced by a high fat diet were used to study the role of microRNA-206 in the pathogenesis of hepatosteatosis and hyperglycemia. A mini-circle vector was used to deliver microRNA-206 into the livers of mice. RESULTS: Lipid accumulation impaired biogenesis of microRNA-206 in fatty livers of dietary obese mice and human hepatocytes (p<0.01). Delivery of microRNA-206 into the livers of dietary obese mice resulted in the strong therapeutic effects on hepatosteatosis and hyperglycemia. Mechanistically, miR-206 interacted with the 3' untranslated region of PTPN1 (protein tyrosine phosphatase, non-receptor type 1) and induced its degradation. By inhibiting PTPN1 expression, microRNA-206 facilitated insulin signaling by promoting phosphorylation of INSR (insulin receptor) and impaired hepatic lipogenesis by inhibiting Srebp1c transcription. By simultaneously modulating lipogenesis and insulin signaling, microRNA-206 reduced lipid (p=0.006) and glucose (p=0.018) production in human hepatocytes and livers of dietary obese mice (p<0.001 and p<0.01 respectively). Re-introduction of Ptpn1 into livers offset the inhibitory effects of microRNA-206, indicating that PTPN1 mediates the inhibitory effects of microRNA-206 on both hepatosteatosis and hyperglycemia. CONCLUSIONS: MicroRNA-206 is a potent inhibitor of lipid and glucose production by simultaneously facilitating insulin signaling and impairing hepatic lipogenesis. Our findings potentially provide a novel therapeutic agent for both hepatosteatosis and hyperglycemia. LAY SUMMARY: The epidemic of obesity is causing a sharp rise in the incidence of insulin resistance and its major complications, type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). However, there are no effective treatments because the mechanisms underlying both disorders are not well described. We identified microRNA-206 as a novel and effective inhibitor for both glucose and lipid production in liver and potentially provide a unique therapeutic drug for both hepatosteatosis and hyperglycemia.

CalDAG-GEFI Deficiency Reduces Atherosclerotic Lesion Development in Mice.

OBJECTIVE: Platelets are important for the development and progression of atherosclerotic lesions. However, relatively little is known about the contribution of platelet signaling to this pathological process. Our recent work identified 2 independent, yet synergistic, signaling pathways that lead to the activation of the small GTPase Rap1; one mediated by the guanine nucleotide exchange factor, CalDAG-GEFI (CDGI), the other by P2Y12, a platelet receptor for adenosine diphosphate and the target of antiplatelet drugs. In this study, we evaluated lesion formation in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr(-/-)) mice lacking CDGI or P2Y12 in hematopoietic cells. APPROACH AND RESULTS: Lethally irradiated Ldlr(-/-) mice were reconstituted with bone marrow from wild-type (WT), Caldaggef1(-/-) (cdgI(-/-)), p2y12(-/-), or cdgI(-/-)p2y12(-/-) (double knockout [DKO]) mice and fed a high-fat diet for 12 weeks. Ldlr(-/-) chimeras deficient for CDGI or P2Y12 developed significantly smaller atherosclerotic lesions in the aortic sinus and in aortas when compared with the Ldlr(-/-)/WT controls. We also observed a significant reduction in platelet-leukocyte aggregates in blood from hypercholesterolemic Ldlr(-/-)/cdgI(-/-) and Ldlr(-/-)/p2y12(-/-) chimeras. Consistently, fewer macrophages and neutrophils were detected in the aortic sinus of Ldlr(-/-)/cdgI(-/-) and Ldlr(-/-)/ p2y12(-/-) chimeras. Compared with controls, the plaque collagen content was significantly higher in Ldlr(-/-) chimeras lacking CDGI. Interestingly, no statistically significant additive effects were seen in Ldlr(-/-)/DKO chimeras when compared with chimeras lacking only CDGI. CONCLUSIONS: Our findings suggest that CDGI is critical for atherosclerotic plaque development in hypercholesterolemic Ldlr(-/-) mice because of its contribution to platelet-leukocyte aggregate formation and leukocyte recruitment to the lesion area.

Therapeutic Inhibition of miR-33 Promotes Fatty Acid Oxidation but Does Not Ameliorate Metabolic Dysfunction in Diet-Induced Obesity.

OBJECTIVE: miR-33 has emerged as an important regulator of lipid homeostasis. Inhibition of miR-33 has been demonstrated as protective against atherosclerosis; however, recent studies in mice suggest that miR-33 inhibition may have adverse effects on lipid and insulin metabolism. Given the therapeutic interest in miR-33 inhibitors for treating atherosclerosis, we sought to test whether pharmacologically inhibiting miR-33 at atheroprotective doses affected metabolic parameters in a mouse model of diet-induced obesity. APPROACH AND RESULTS: High-fat diet (HFD) feeding in conjunction with treatment of male mice with 10 mg/kg control anti-miR or anti-miR33 inhibitors for 20 weeks promoted equivalent weight gain in all groups. miR-33 inhibitors increased plasma total cholesterol and decreased serum triglycerides compared with control anti-miR, but not compared with PBS-treated mice. Metrics of insulin resistance were not altered in anti-miR33-treated mice compared with controls; however, respiratory exchange ratio was decreased in anti-miR33-treated mice. Hepatic expression of miR-33 targets Abca1 and Hadhb were derepressed on miR-33 inhibition. In contrast, protein levels of putative miR-33 target gene SREBP-1 or its downstream targets genes Fasn and Acc were not altered in anti-miR33-treated mice, and hepatic lipid accumulation did not differ between groups. In the adipose tissue, anti-miR33 treatment increased Ampk gene expression and markers of M2 macrophage polarization. CONCLUSIONS: We demonstrate in a mouse model of diet-induced obesity that therapeutic silencing of miR-33 may promote whole-body oxidative metabolism but does not affect metabolic dysregulation. This suggests that pharmacological inhibition of miR-33 at doses known to reduce atherosclerosis may be a safe future therapeutic.