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Blood vessels are continually exposed to circulating lipids, and elevation of ApoB-containing lipoproteins causes atherosclerosis. Lipoprotein metabolism is highly regulated by lipolysis, largely at the level of the capillary endothelium lining metabolically active tissues. How large blood vessels, the site of atherosclerotic vascular disease, regulate the flux of fatty acids (FAs) into triglyceride-rich (TG-rich) lipid droplets (LDs) is not known. In this study, we showed that deletion of the enzyme adipose TG lipase (ATGL) in the endothelium led to neutral lipid accumulation in vessels and impaired endothelialdependent vascular tone and nitric oxide synthesis to promote endothelial dysfunction. Mechanistically, the loss of ATGL led to endoplasmic reticulum stress-induced inflammation in the endothelium. Consistent with this mechanism, deletion of endothelial ATGL markedly increased lesion size in a model of atherosclerosis. Together, these data demonstrate that the dynamics of FA flux through LD affects endothelial cell homeostasis and consequently large vessel function during normal physiology and in a chronic disease state., Introduction The vascular endothelium is essential in lipid partitioning (1). Specifically, in metabolically active tissues (e.g., heart, skeletal muscle, adipose), triglyceride-rich (TG-rich) lipoproteins (chylomicrons, VLDL) are metabolized by lipoprotein lipase [...]

microRNA-21 (miR-21) is the most commonly upregulated miRNA in solid tumors. This cancer-associated microRNA (oncomiR) regulates various downstream effectors associated with tumor pathogenesis during all stages of carcinogenesis. In this study, we analyzed the function of miR-21 in noncancer cells of the tumor microenvironment to further evaluate its contribution to tumor progression. We report that the expression of miR-21 in cells of the tumor immune infiltrate, and in particular in macrophages, was responsible for promoting tumor growth. Absence of miR-21 expression in tumor- associated macrophages (TAMs), caused a global rewiring of their transcriptional regulatory network that was skewed toward a proinflammatory angiostatic phenotype. This promoted an antitumoral immune response characterized by a macrophage-mediated improvement of cytotoxic T-cell responses through the induction of cytokines and chemokines, including IL-12 and C-X-C motif chemokine 10. These effects translated to a reduction in tumor neovascularization and an induction of tumor cell death that led to decreased tumor growth. Additionally, using the carrier peptide pH (low) insertion peptide, we were able to target miR-21 in TAMs, which decreased tumor growth even under conditions where miR-21 expression was deficient in cancer cells. Consequently, miR-21 inhibition in TAMs induced an angiostatic and immunostimulatory activation with potential therapeutic implications., Introduction miRNAs are small endogenous non-protein-coding RNAs that posttranscriptionally regulate the expression of multiple target genes in different cell types. In this manner, miRNAs control many physiological cellular processes, as [...]

Brown adipose tissue (BAT), a crucial heat-generating organ, regulates whole-body energy metabolism by mediating thermogenesis. BAT inflammation is implicated in the pathogenesis of mitochondrial dysfunction and impaired thermogenesis. However, the link between BAT inflammation and systematic metabolism remains unclear. Herein, we use mice with BAT deficiency of thioredoxin-2 (TRX2), a protein that scavenges mitochondrial reactive oxygen species (ROS), to evaluate the impact of BAT inflammation on metabolism and thermogenesis and its underlying mechanism. Our results show that BAT-specific TRX2 ablation improves systematic metabolic performance via enhancing lipid uptake, which protects mice from diet-induced obesity, hypertriglyceridemia, and insulin resistance. TRX2 deficiency impairs adaptive thermogenesis by suppressing fatty acid oxidation. Mechanistically, loss of TRX2 induces excessive mitochondrial ROS, mitochondrial integrity disruption, and cytosolic release of mitochondrial DNA, which in turn activate aberrant innate immune responses in BAT, indudingthe cGAS/STING and the NLRP3 inflammasome pathways. We identify NLRP3 as a key converging point, as its inhibition reverses both the thermogenesis defect and the metabolic benefits seen under nutrient overload in BAT-specific 7rx2-deficient mice. In conclusion, we identify TRX2 as a critical hub integrating oxidative stress, inflammation, and lipid metabolism in BAT, uncovering an adaptive mechanism underlyingthe link between BAT inflammation and systematic metabolism., Introduction Obesity related to overnutrition is driven by energy intake and energy expenditure (EE) imbalance. White adipose tissue (WAT), having small numbers of mitochondria, plays a major role in energy [...]

Mutations in Dyrk1b are associated with metabolic syndrome and nonalcoholic fatty liver disease in humans. Our investigations showed that DYRK1B levels are increased in the liver of patients with nonalcoholic steatohepatitis (NASH) and in mice fed with a high-fat, high-sucrose diet. Increasing Dyrk1b levels in the mouse liver enhanced de novo lipogenesis (DNL), fatty acid uptake, and triacylglycerol secretion and caused NASH and hyperlipidemia. Conversely, knockdown of Dyrk1b was protective against high-calorie-induced hepatic steatosis and fibrosis and hyperlipidemia. Mechanistically, Dyrk1b increased DNL by activating mTORC2 in a kinase-independent fashion. Accordingly, the Dyrk1b-induced NASH was fully rescued when mTORC2 was genetically disrupted. The elevated DNL was associated with increased plasma membrane sn-1,2-diacylglyerol levels and increased PKC[epsilon]-mediated IRK T1150 phosphorylation, which resulted in impaired activation of hepatic insulin signaling and reduced hepatic glycogen storage. These findings provide insights into the mechanisms that underlie Dyrk1binduced hepatic lipogenesis and hepatic insulin resistance and identify Dyrk1b as a therapeutic target for NASH and insulin resistance in the liver., Introduction Nonalcoholic fatty liver disease (NAFLD) is a rapidly growing disorder affecting nearly 25% of the adult population worldwide and is a major risk factor for nonalcoholic steatohepatitis (NASH), atherosclerosis, [...]

Hyperlipidemia is a risk factor for various cardiovascular and metabolic disorders. Overproduction of lipoproteins, a process that is dependent on microsomal triglyceride transfer protein (MTP), can contribute to hyperlipidemia. We [...]

Cardiovascular disease remains the leading cause of mortality in westernized countries, despite optimum medical therapy to reduce the levels of low-density lipoprotein (LDL)-associated cholesterol. The pursuit of novel therapies to target the residual risk has focused on raising the levels of high-density lipoprotein (HDL)-associated cholesterol in order to exploit its atheroprotective effects (1). MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of lipid metabolism and are thus a new class of target for therapeutic intervention (2). MicroRNA-33a and microRNA-33b (miR-33a/b) are intronic miRNAs whose encoding regions are embedded in the sterol-response-element-binding protein genes SREBF2 and SREBF1 (refs 3-5), respectively. These miRNAs repress expression of the cholesterol transporter ABCA1, which is a key regulator of HDL biogenesis. Recent studies in mice suggest that antagonizing miR-33a may be an effective strategy for raising plasma HDL levels (3-5) and providing protection against atherosclerosis (6); however, extrapolating these findings to humans is complicated by the fact that mice lack miR-33b, which is present only in the SREBF1 gene of medium and large mammals. Here we show in African green monkeys that systemic delivery of an anti-miRNA oligonucleotide that targets both miR-33a and miR-33b increased hepatic expression of ABCA1 and induced a sustained increase in plasma HDL levels over 12 weeks. Notably, miR-33 antagonism in this non-human primate model also increased the expression of miR-33 target genes involved in fatty acid oxidation (CROT, CPT1A, HADHB and PRKAA1) and reduced the expression of genes involved in fatty acid synthesis (SREBF1, FASN, ACLY and ACACA), resulting in a marked suppression of the plasma levels of very-low-density lipoprotein (VLDL)-associated triglycerides, a finding that has not previously been observed in mice. These data establish, in a model that is highly relevant to humans, that pharmacological inhibition of miR-33a and miR33b is a promising therapeutic strategy to raise plasma HDL and lower VLDL triglyceride levels for the treatment of dyslipidaemias that increase cardiovascular disease risk., Recent advances in the understanding of lipid metabolism have revealed that the genetic loci encoding the transcription factors SREBP1 and SREBP2 (known as SREBF1 and SREBF2) also encode the miRNAs [...]

Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL levels have remained elusive. Recent advances in the understanding of lipid metabolism have revealed that [...]

Blood vessel formation during ischemia and wound healing requires coordination of the inflammatory response with genes that regulate blood vessel assembly. Here we show that the reticulon family member 4B, aka Nogo-B, is upregulated in response to ischemia and is necessary for blood flow recovery secondary to ischemia and wound healing. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. Bone marrow-derived macrophages isolated from Nogo knock-out mice have reduced spreading and chemotaxis due to impaired Rac activation. Bone marrow reconstitution experiments show that Nogo in myeloid cells is necessary to promote macrophage homing and functional recovery after limb ischemia. Thus, endogenous Nogo coordinates macrophage-mediated inflammation with arteriogenesis, wound healing, and blood flow control. inflammation | ischemia | vascular doi/ 10.1073/pnas.0907359106

Akt1 is implicated in cell metabolism, survival migration, and gene expression; however, little is known about the role of specific Akt isoforms during inflammation in vivo. Thus, we directly explored the roles of the isoforms Akt1 and Akt2 in acute inflammation models by using mice deficient in either Akt1 or Akt2. [Akt1.sup.-/-] mice showed a markedly reduced edema versus [Akt2.sup.-/-] and WT controls, and the reduced inflammation was associated with a dramatic decrease in neutrophil and monocyte infiltration. The loss of Akt1 did not affect leukocyte functions in vitro, and bone marrow transplant experiments suggest that host Akt1 regulates leukocyte emigration into inflamed tissues. Moreover, carrageenan-induced edema and the direct propermeability actions of bradykinin and histamine were reduced dramatically in [Akt1.sup.-/-] versus WT mice. These findings are supported by in vitro experiments showing that Akt1 deficiency or blockade of nitric oxide synthase markedly reduces histamine-stimulated changes in transendothelial electrical resistance of microvascular endothelial cells. Collectively, these results suggest that Akt1 is necessary for acute inflammation and exerts its actions primarily via regulation of vascular permeability, leading to edema and leukocyte extravasation. endothelium | nitric oxide

Atherosclerosis is an inflammatory disease that is associated with monocyte recruitment and subsequent differentiation into lipid-laden macrophages at sites of arterial lesions, leading to the development of atherosclerotic plaques. PLC is a key member of signaling pathways initiated by G protein--coupled ligands in macrophages. However, the role of this enzyme in the regulation of macrophage function is not known. Here, we studied macrophages from mice lacking PLC [beta]2, PLC [beta]3, or both PLC isoforms and found that PLC [beta]3 is the major functional PLC [beta] isoform in murine macrophages. Although PLC [beta]3 deficiency did not affect macrophage migration, adhesion, or phagocytosis, it resulted in macrophage hypersensitivity to multiple inducers of apoptosis. PLC [beta]3 appeared to regulate this sensitivity via PKC-dependent upregulation of Bcl-XL. The significance of PLC [beta] signaling in vivo was examined using the apoE-deficient mouse model of atherosclerosis. Mice lacking both PLC [beta]3 and apoE exhibited fewer total macrophages and increased macrophage apoptosis in atherosclerotic lesions, as well as reduced atherosclerotic lesion size when compared with mice lacking only apoE. These results demonstrate what we believe to be a novel role for PLC activity in promoting macrophage survival in atherosclerotic plaques and identify PLC [beta]3 as a potential target for treatment of atherosclerosis., Introduction PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate 2 second messengers, inositol 1,3,4-triphosphate and diacylgl-ycerol. While inositol 1,3,4-triphosphate regulates [Ca.sup.2+] efflux, it and diacylglycerol activate PKC (1). The [beta] family of [...]

Lipid modifications mediate the subcellular localization and biological activity of many proteins, including endothelial nitric oxide synthase (eNOS). This enzyme resides on the cytoplasmic aspect of the Golgi apparatus and in caveolae and is dually acylated by both N-myristoylation and S-palmitoylation. Palmitoylation-deficient mutants of eNOS release less nitric oxide (NO). We identify enzymes that palmitoylate eNOS in vivo. Transfection of human embryonic kidney 293 cells with the complementary DNA (cDNA) for eNOS and 23 cDNA clones encoding the Asp-His-His-Cys motif (DHHC) palmitoyl transferase family members showed that five clones (2, 3, 7, 8, and 21) enhanced incorporation of [[sup.3]H]palmitate into eNOS. Human endothelial cells express all five of these enzymes, which colocalize with eNOS in the Golgi and plasma membrane and interact with eNOS. Importantly, inhibition of DHHC-21 palmitoyl transferase, but not DHHC-3, in human endothelial cells reduces eNOS palmitoylation, eNOS targeting, and stimulated NO production. Collectively, our data describe five new Golgi-targeted DHHC enzymes in human endothelial cells and suggest a regulatory role of DHHC-21 in governing eNOS localization and function.

Red grape juice (RGJ) polyphenols have been shown to reduce circulating levels of LDL cholesterol and to increase LDL receptor activity. To explore the effect of RGJ-derived polyphenols on intracellular cholesterol homeostasis, human hepatocarcinoma HepG2 and promyelocytic HL-60 cell lines were incubated in serum-free medium, with or without LDL, in the presence or absence of RGJ. In the presence of LDL, RGJ increased both the activity and cell surface expression of the LDL receptor, and increased the cell total cholesterol content. In cells exposed to LDL, RGJ also increased levels of the active form of sterol regulatory element-binding protein-1 and mRNA expression of the LDL receptor and hydroxymethylglutaryI-CoA reductase. In contrast, RGJ caused a marked reduction in the expression of CYP7A1, apolipoprotein B, ABCA1, and ABCG5. Experiments using the acyl-CoA cholesterol acyltransferase inhibitor S-58035 indicated that no measurable free cholesterol from endocytosed LDL reaches the endoplasmic reticulum in cells treated with RGJ. Finally, fluorescence microscopy revealed that in RGJ-treated cells, Dil-labeled LDL did not colocalize with CD63, a protein localized at steady state in the internal vesicles of late endosomes. These results indicate that RGJ polyphenols disrupt or delay LDL trafficking through the endocytic pathway, thus preventing LDL cholesterol from exerting regulatory effects on intracellular lipid homeostasis. KEY WORDS: * LDL receptor * red grape juice polyphenols * cholesterol trafficking * LDL cholesterol