ABSTRACT
Angiopoietin-like 8 (Angptl8) inhibits lipolysis in the circulation together with Angplt3 and controls post-prandial fat storage in white adipose tissue (WAT). It is strongly induced by insulin in vivo in WAT and in vitro in adipocytes. In this study we addressed the function of Angptl8 in adipocytes by its stable lentivirus-mediated knock-down in 3T3-L1 cells, followed by analyses of triglyceride (TG) storage, lipid droplet (LD) morphology, the cellular lipidome, lipolysis, and gene expression. Depletion of Angptl8 did not drastically affect the adipocyte differentiation of 3T3-L1 cells but resulted in a moderate (18-19%) reduction of stored TGs. The lipidome analysis revealed a reduction of alkyl- phosphatidylcholines (PCs) and phosphatidylethanolamine (PE) plasmalogens, as well as saturated PCs and PEs. Importantly, the Angptl8 depleted cells displayed enhanced lipolysis as measured by release of non-esterified fatty acids (NEFAs). Consistently, mRNAs encoding Angptl4 and Leptin, which facilitate lipolysis, as well as Cpt1a, Cpt1b, and Pgc-1α involved in FA oxidation, were elevated. The Angptl8 mRNA itself was suppressed by pharmacologic treatments inducing lipolysis: stimulation with the ß-adrenergic agonist isoproterenol or with the adenylate cyclase activator forskolin. To conclude, knock-down of Angptl8 in adipocytes suggests that the protein acts to inhibit intracellular lipolysis, analogous to its activity in the circulation. Depletion of Angptl8 results in an altered cellular phospholipid composition. The findings identify Angptl8 as a central insulin-regulated controller of adipocyte lipid metabolism.
Subject(s)
Adipocytes/metabolism , Angiopoietins/metabolism , Lipolysis , Phospholipids/metabolism , 3T3-L1 Cells , Angiopoietin-Like Protein 8 , Angiopoietin-like Proteins , Angiopoietins/deficiency , Animals , Cells, Cultured , MiceABSTRACT
BACKGROUND: Familial combined hypolipidemia, a Mendelian condition characterized by substantial reductions in all 3 major lipid fractions, is caused by mutations that inactivate the gene angiopoietin-like 3 (ANGPTL3). Whether ANGPTL3 deficiency reduces risk of coronary artery disease (CAD) is unknown. OBJECTIVES: The study goal was to leverage 3 distinct lines of evidence-a family that included individuals with complete (compound heterozygote) ANGPTL3 deficiency, a population based-study of humans with partial (heterozygote) ANGPTL3 deficiency, and biomarker levels in patients with myocardial infarction (MI)-to test whether ANGPTL3 deficiency is associated with lower risk for CAD. METHODS: We assessed coronary atherosclerotic burden in 3 individuals with complete ANGPTL3 deficiency and 3 wild-type first-degree relatives using computed tomography angiography. In the population, ANGPTL3 loss-of-function (LOF) mutations were ascertained in up to 21,980 people with CAD and 158,200 control subjects. LOF mutations were defined as nonsense, frameshift, and splice-site variants, along with missense variants resulting in <25% of wild-type ANGPTL3 activity in a mouse model. In a biomarker study, circulating ANGPTL3 concentration was measured in 1,493 people who presented with MI and 3,232 control subjects. RESULTS: The 3 individuals with complete ANGPTL3 deficiency showed no evidence of coronary atherosclerotic plaque. ANGPTL3 gene sequencing demonstrated that approximately 1 in 309 people was a heterozygous carrier for an LOF mutation. Compared with those without mutation, heterozygous carriers of ANGPTL3 LOF mutations demonstrated a 17% reduction in circulating triglycerides and a 12% reduction in low-density lipoprotein cholesterol. Carrier status was associated with a 34% reduction in odds of CAD (odds ratio: 0.66; 95% confidence interval: 0.44 to 0.98; p = 0.04). Individuals in the lowest tertile of circulating ANGPTL3 concentrations, compared with the highest, had reduced odds of MI (adjusted odds ratio: 0.65; 95% confidence interval: 0.55 to 0.77; p < 0.001). CONCLUSIONS: ANGPTL3 deficiency is associated with protection from CAD.
Subject(s)
Angiopoietins/deficiency , Coronary Artery Disease/genetics , Adult , Angiopoietin-Like Protein 3 , Angiopoietin-like Proteins , Angiopoietins/blood , Angiopoietins/genetics , Animals , Atherosclerosis/genetics , Case-Control Studies , Female , Humans , Lipids/blood , Male , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Mutation, Missense , Myocardial Infarction/blood , Risk FactorsABSTRACT
The intestinal epithelium continually self-renews and can rapidly regenerate after damage. Dysregulation of intestinal epithelial homeostasis leads to severe inflammatory bowel disease. Additionally, aberrant signaling by the secreted protein angiopoietin-like protein 2 (ANGPTL2) causes chronic inflammation in a variety of diseases. However, little is known about the physiologic role of ANGPTL2 in normal tissue homeostasis and during wound repair following injury. Here, we assessed ANGPTL2 function in intestinal physiology and disease in vivo Although intestinal development proceeded normally in Angptl2-deficient mice, expression levels of the intestinal stem cell (ISC) marker gene Lgr5 decreased, which was associated with decreased transcriptional activity of ß-catenin in Angptl2-deficient mice. Epithelial regeneration after injury was significantly impaired in Angptl2-deficient relative to wild-type mice. ANGPTL2 was expressed and functioned within the mesenchymal compartment cells known as intestinal subepithelial myofibroblasts (ISEMFs). ANGPTL2 derived from ISEMFs maintained the intestinal stem cell niche by modulating levels of competing signaling between bone morphogenetic protein (BMP) and ß-catenin. These results support the importance of ANGPTL2 in the stem cell niche in regulating stemness and epithelial wound healing in the intestine.
Subject(s)
Angiopoietins/biosynthesis , Gene Expression Regulation , Homeostasis , Intestinal Mucosa/injuries , Intestinal Mucosa/physiology , Regeneration , Stem Cell Niche , Angiopoietin-Like Protein 2 , Angiopoietin-like Proteins , Angiopoietins/deficiency , Animals , Disease Models, Animal , Mice , Mice, Knockout , Receptors, G-Protein-Coupled/analysis , Wound Healing , beta Catenin/analysisABSTRACT
Dynamic control of endothelial cell junctions is essential for vascular homeostasis and angiogenesis. We recently provided genetic evidence that ANGPTL4 is a key regulator of vascular integrity both during developmental and in hypoxia-induced pathological conditions. The purpose of the present study was to decipher the molecular mechanisms through which ANGPTL4 regulates vascular integrity. Using surface plasmon resonance and proximity ligation assays, we show that ANGPTL4 binds integrin αvß3. In vitro and in vivo functional assays with Angptl4-deficient mice demonstrate that ANGPTL4-αvß3 interaction is necessary to mediate ANGPTL4 vasoprotective effects. Mechanistically, ANGPTL4-αvß3 interaction enhances Src recruitment to integrin αvß3 and inhibits Src signalling downstream of vascular endothelial growth factor receptor 2 (VEFGR2), thereby repressing hypoxia-induced breakdown of VEGFR2-VE-cadherin and VEGFR2-αvß3 complexes. We further demonstrate that intravitreal injection of recombinant human ANGPTL4 limits vascular permeability and leads to increased adherens junction and tight junction integrity. These findings identify a novel mechanism by which ANGPTL4 counteracts hypoxia-driven vascular permeability through integrin αvß3 binding, modulation of VEGFR2-Src kinase signalling, and endothelial junction stabilization. We further demonstrate that Angptl4-deficient mice show increased vascular leakage in vivo in a model of laser-induced choroidal neovascularization, indicating that this newly identified ANGPTL4-αvß3 axis might be a target for pharmaceutical intervention in pathological conditions. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Subject(s)
Angiopoietins/metabolism , Capillary Permeability/physiology , Integrin alphaVbeta3/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Angiopoietin-Like Protein 4 , Angiopoietins/deficiency , Animals , Cell Hypoxia/physiology , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/physiopathology , Humans , Mice, Knockout , Phosphorylation/physiology , Retina/metabolism , Signal Transduction/physiology , src-Family Kinases/metabolismABSTRACT
The consequences of angiopoietin-like protein 3 (ANGPTL3) deficiency on postprandial lipid and lipoprotein metabolism has not been investigated in humans. We studied 7 homozygous (undetectable circulating ANGPTL3 levels) and 31 heterozygous (50% of circulating ANGPTL3 levels) subjects with familial combined hypolipidemia (FHBL2) due to inactivating ANGPTL3 mutations in comparison with 35 controls. All subjects were evaluated at fasting and during 6 h after a high fat meal. Postprandial lipid and lipoprotein changes were quantified by calculating the areas under the curve (AUCs) using the 6 h concentration data. Plasma changes of ß-hydroxybutyric acid (ß-HBA) were measured as marker of hepatic oxidation of fatty acids. Compared with controls, homozygotes showed lower incremental AUCs (iAUCs) of total TG (-69%, P < 0.001), TG-rich lipoproteins (-90%, P < 0.001), apoB-48 (-78%, P = 0.032), and larger absolute increase of FFA (128%, P < 00.1). Also, heterozygotes displayed attenuated postprandial lipemia, but the difference was significant only for the iAUC of apoB-48 (-28%; P < 0.05). During the postprandial period, homozygotes, but not heterozygotes, showed a lower increase of ß-HBA. Our findings demonstrate that complete ANGPTL3 deficiency associates with highly reduced postprandial lipemia probably due to faster catabolism of intestinally derived lipoproteins, larger expansion of the postprandial FFA pool, and decreased influx of dietary-derived fatty acids into the liver. These results add information on mechanisms underlying hypolipidemia in FHBL2.
Subject(s)
Angiopoietins/genetics , Fatty Acids, Nonesterified/blood , Hypobetalipoproteinemias/blood , Lipids/blood , Angiopoietin-Like Protein 3 , Angiopoietin-like Proteins , Angiopoietins/blood , Angiopoietins/deficiency , Apolipoprotein B-48/blood , Female , Heterozygote , Homozygote , Humans , Hypobetalipoproteinemias/genetics , Hypobetalipoproteinemias/pathology , Lipoproteins/blood , Male , Middle Aged , Mutation , Postprandial Period , Triglycerides/bloodABSTRACT
Humans and mice lacking angiopoietin-like protein 3 (ANGPTL3) have pan-hypolipidemia. ANGPTL3 inhibits two intravascular lipases, LPL and endothelial lipase, and the low plasma TG and HDL-cholesterol levels in ANGPTL3 deficiency reflect increased activity of these enzymes. The mechanism responsible for the low LDL-cholesterol levels associated with ANGPTL3 deficiency is not known. Here we used an anti-ANGPTL3 monoclonal antibody (REGN1500) to inactivate ANGPTL3 in mice with genetic deficiencies in key proteins involved in clearance of ApoB-containing lipoproteins. REGN1500 treatment consistently reduced plasma cholesterol levels in mice in which Apoe, Ldlr, Lrp1, and Sdc1 were inactivated singly or in combination, but did not alter clearance of rabbit (125)I-ßVLDL or mouse (125)I-LDL. Despite a 61% reduction in VLDL-TG production, VLDL-ApoB-100 production was unchanged in REGN1500-treated animals. Hepatic TG content, fatty acid synthesis, and fatty acid oxidation were similar in REGN1500 and control antibody-treated animals. Taken together, our findings indicate that inactivation of ANGPTL3 does not affect the number of ApoB-containing lipoproteins secreted by the liver but alters the particles that are made such that they are cleared more rapidly from the circulation via a noncanonical pathway(s). The increased clearance of lipolytic remnants results in decreased production of LDL in ANGPTL3-deficient animals.
Subject(s)
Angiopoietins/genetics , Gene Silencing , Lipoproteins, VLDL/metabolism , Liver/metabolism , Triglycerides/metabolism , Angiopoietin-Like Protein 3 , Angiopoietin-like Proteins , Angiopoietins/deficiency , Angiopoietins/immunology , Animals , Antibodies, Monoclonal/immunology , Apolipoproteins E/deficiency , Cholesterol/blood , Humans , Low Density Lipoprotein Receptor-Related Protein-1 , Male , Mice , Rabbits , Receptors, LDL/deficiency , Syndecan-1/deficiency , Tumor Suppressor Proteins/deficiencySubject(s)
Angiopoietins/metabolism , Cell Differentiation , Epithelial Cells/metabolism , Lipogenesis , Sebaceous Glands/metabolism , Angiopoietin-Like Protein 4 , Angiopoietins/deficiency , Angiopoietins/genetics , Animals , Cell Differentiation/drug effects , Cell Line , Epithelial Cells/drug effects , Humans , Linoleic Acid/pharmacology , Lipogenesis/drug effects , Mice, Knockout , RNA Interference , RNA, Messenger/metabolism , Sebaceous Glands/cytology , Sebaceous Glands/drug effects , Thiazolidinediones/pharmacology , Time Factors , Transfection , Up-RegulationABSTRACT
OBJECTIVE: Cardiovascular disease (CVD), the most common morbidity resulting from atherosclerosis, remains a frequent cause of death. Efforts to develop effective therapeutic strategies have focused on vascular inflammation as a critical pathology driving atherosclerosis progression. Nonetheless, molecular mechanisms underlying this activity remain unclear. Here, we ask whether angiopoietin-like protein 2 (Angptl2), a proinflammatory protein, contributes to vascular inflammation that promotes atherosclerosis progression. APPROACH AND RESULTS: Histological analysis revealed abundant Angptl2 expression in endothelial cells and macrophages infiltrating atheromatous plaques in patients with cardiovascular disease. Angptl2 knockout in apolipoprotein E-deficient mice (ApoE(-/-)/Angptl2(-/-)) attenuated atherosclerosis progression by decreasing the number of macrophages infiltrating atheromatous plaques, reducing vascular inflammation. Bone marrow transplantation experiments showed that Angptl2 deficiency in endothelial cells attenuated atherosclerosis development. Conversely, ApoE(-/-) mice crossed with transgenic mice expressing Angptl2 driven by the Tie2 promoter (ApoE(-/-)/Tie2-Angptl2 Tg), which drives Angptl2 expression in endothelial cells but not monocytes/macrophages, showed accelerated plaque formation and vascular inflammation because of increased numbers of infiltrated macrophages in atheromatous plaques. Tie2-Angptl2 Tg mice alone did not develop plaques but exhibited endothelium-dependent vasodilatory dysfunction, likely because of decreased production of endothelial cell-derived nitric oxide. Conversely, Angptl2(-/-) mice exhibited less severe endothelial dysfunction than did wild-type mice when fed a high-fat diet. In vitro, Angptl2 activated proinflammatory nuclear factor-κB signaling in endothelial cells and increased monocyte/macrophage chemotaxis. CONCLUSIONS: Endothelial cell-derived Angptl2 accelerates vascular inflammation by activating proinflammatory signaling in endothelial cells and increasing macrophage infiltration, leading to endothelial dysfunction and atherosclerosis progression.
Subject(s)
Angiopoietins/metabolism , Atherosclerosis/metabolism , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Inflammation Mediators/metabolism , Vasculitis/metabolism , Aged, 80 and over , Angiopoietin-Like Protein 2 , Angiopoietin-like Proteins , Angiopoietins/deficiency , Angiopoietins/genetics , Animals , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Atherosclerosis/genetics , Atherosclerosis/immunology , Atherosclerosis/pathology , Atherosclerosis/prevention & control , Bone Marrow Transplantation , Cells, Cultured , Chemotaxis, Leukocyte , Diet, High-Fat , Disease Models, Animal , Disease Progression , Dyslipidemias/metabolism , Dyslipidemias/physiopathology , Endothelial Cells/immunology , Endothelial Cells/pathology , Endothelium, Vascular/immunology , Endothelium, Vascular/pathology , Endothelium, Vascular/physiopathology , Female , Humans , Integrin alpha5beta1/metabolism , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Monocytes/immunology , Monocytes/metabolism , NF-kappa B/metabolism , NFATC Transcription Factors/metabolism , Nitric Oxide/metabolism , Obesity/metabolism , Obesity/physiopathology , Plaque, Atherosclerotic , Signal Transduction , Time Factors , Vasculitis/genetics , Vasculitis/immunology , Vasculitis/pathology , Vasculitis/prevention & control , VasodilationABSTRACT
AIMS: Given the impact of vascular injuries and oedema on brain damage caused during stroke, vascular protection represents a major medical need. We hypothesized that angiopoietin-like 4 (ANGPTL4), a regulator of endothelial barrier integrity, might exert a protective effect during ischaemic stroke. METHODS AND RESULTS: Using a murine transient ischaemic stroke model, treatment with recombinant ANGPTL4 led to significantly decreased infarct size and improved behaviour. Quantitative characteristics of the vascular network (density and branchpoints) were preserved in ANGPTL4-treated mice. Integrity of tight and adherens junctions was also quantified and ANGPTL4-treated mice displayed increased VE-cadherin and claudin-5-positive areas. Brain oedema was thus significantly decreased in ANGPTL4-treated mice. In accordance, vascular damage and infarct severity were increased in angptl4-deficient mice thus providing genetic evidence that ANGPTL4 preserves brain tissue from ischaemia-induced alterations. Altogether, these data show that ANGPTL4 protects not only the global vascular network, but also interendothelial junctions and controls both deleterious inflammatory response and oedema. Mechanistically, ANGPTL4 counteracted VEGF signalling and thereby diminished Src-signalling downstream from VEGFR2. This led to decreased VEGFR2-VE-cadherin complex disruption, increased stability of junctions and thus increased endothelial cell barrier integrity of the cerebral microcirculation. In addition, ANGPTL4 prevented neuronal loss in the ischaemic area. CONCLUSION: These results, therefore, show ANGPTL4 counteracts the loss of vascular integrity in ischaemic stroke, by restricting Src kinase signalling downstream from VEGFR2. ANGPTL4 treatment thus reduces oedema, infarct size, neuronal loss, and improves mice behaviour. These results suggest that ANGPTL4 constitutes a relevant target for vasculoprotection and cerebral protection during stroke.
Subject(s)
Angiopoietins/pharmacology , Brain Ischemia/prevention & control , Nootropic Agents/pharmacology , Stroke/prevention & control , Angiopoietin-Like Protein 4 , Angiopoietins/deficiency , Animals , Blood-Brain Barrier/physiology , Brain/blood supply , Brain Edema/prevention & control , Brain Ischemia/physiopathology , Cadherins/physiology , Cell Membrane Permeability/drug effects , Encephalitis/physiopathology , Endothelial Cells/physiology , Endothelium, Vascular/physiology , Humans , Male , Mice , Mice, Inbred C57BL , Stroke/physiopathology , Vascular Endothelial Growth Factor A/pharmacology , Vascular Endothelial Growth Factor Receptor-2/physiology , src-Family Kinases/physiologyABSTRACT
OBJECTIVE: Angiopoietin-like 3 (Angptl3) is a regulator of lipoprotein metabolism at least by inhibiting lipoprotein lipase activity. Loss-of-function mutations in ANGPTL3 cause familial combined hypolipidemia through an unknown mechanism. APPROACH AND RESULTS: We compared lipolytic activities, lipoprotein composition, and other lipid-related enzyme/lipid transfer proteins in carriers of the S17X loss-of-function mutation in ANGPTL3 and in age- and sex-matched noncarrier controls. Gel filtration analysis revealed a severely disturbed lipoprotein profile and a reduction in size and triglyceride content of very low density lipoprotein in homozygotes as compared with heterozygotes and noncarriers. S17X homozygotes had significantly higher lipoprotein lipase activity and mass in postheparin plasma, whereas heterozygotes showed no difference in these parameters when compared with noncarriers. No changes in hepatic lipase, endothelial lipase, paraoxonase 1, phospholipid transfer protein, and cholesterol ester transfer protein activities were associated with the S17X mutation. Plasma free fatty acid, insulin, glucose, and homeostatic model assessment of insulin resistance were significantly lower in homozygous subjects compared with heterozygotes and noncarriers subjects. CONCLUSIONS: These results indicate that, although partial Angptl3 deficiency did not affect the activities of lipolytic enzymes, the complete absence of Angptl3 results in an increased lipoprotein lipase activity and mass and low circulating free fatty acid levels. This latter effect is probably because of decreased mobilization of free fatty acid from fat stores in human adipose tissue and may result in reduced hepatic very low density lipoprotein synthesis and secretion via attenuated hepatic free fatty acid supply. Altogether, Angptl3 may affect insulin sensitivity and play a role in modulating both lipid and glucose metabolism.
Subject(s)
Angiopoietins/deficiency , Fatty Acids, Nonesterified/blood , Hypobetalipoproteinemias/enzymology , Insulin Resistance , Lipoprotein Lipase/blood , Adult , Aged , Analysis of Variance , Angiopoietin-Like Protein 3 , Angiopoietin-like Proteins , Angiopoietins/genetics , Biomarkers/blood , Blood Glucose/analysis , Case-Control Studies , Chi-Square Distribution , Down-Regulation , Female , Heterozygote , Homozygote , Humans , Hypobetalipoproteinemias/blood , Hypobetalipoproteinemias/genetics , Hypobetalipoproteinemias/physiopathology , Insulin/blood , Italy , Linear Models , Lipase/blood , Lipoproteins, LDL/blood , Male , Middle Aged , Mutation , Triglycerides/blood , Up-RegulationABSTRACT
BACKGROUND: Lipoprotein lipase (LPL) hydrolyzes triglycerides in lipoproteins and makes fatty acids available for tissue metabolism. The activity of the enzyme is modulated in a tissue specific manner by interaction with other proteins. We have studied how feeding/fasting and some related perturbations affect the expression, in rat adipose tissue, of three such proteins, LMF1, an ER protein necessary for folding of LPL into its active dimeric form, the endogenous LPL inhibitor ANGPTL4, and GPIHBP1, that transfers LPL across the endothelium. RESULTS: The system underwent moderate circadian oscillations, for LPL in phase with food intake, for ANGPTL4 and GPIHBP1 in the opposite direction. Studies with cycloheximide showed that whereas LPL protein turns over rapidly, ANGPTL4 protein turns over more slowly. Studies with the transcription blocker Actinomycin D showed that transcripts for ANGPTL4 and GPIHBP1, but not LMF1 or LPL, turn over rapidly. When food was withdrawn the expression of ANGPTL4 and GPIHBP1 increased rapidly, and LPL activity decreased. On re-feeding and after injection of insulin the expression of ANGPTL4 and GPIHBP1 decreased rapidly, and LPL activity increased. In ANGPTL4(-/-) mice adipose tissue LPL activity did not show these responses. In old, obese rats that showed signs of insulin resistance, the responses of ANGPTL4 and GPIHBP1 mRNA and of LPL activity were severely blunted (at 26 weeks of age) or almost abolished (at 52 weeks of age). CONCLUSIONS: This study demonstrates directly that ANGPTL4 is necessary for rapid modulation of LPL activity in adipose tissue. ANGPTL4 message levels responded very rapidly to changes in the nutritional state. LPL activity always changed in the opposite direction. This did not happen in Angptl4(-/-) mice. GPIHBP1 message levels also changed rapidly and in the same direction as ANGPTL4, i.e. increased on fasting when LPL activity decreased. This was unexpected because GPIHBP1 is known to stabilize LPL. The plasticity of the LPL system is severely blunted or completely lost in insulin resistant rats.
Subject(s)
Adipose Tissue/enzymology , Angiopoietins/physiology , Appetite Regulation/physiology , Lipoprotein Lipase/metabolism , Membrane Proteins/physiology , Receptors, Lipoprotein/physiology , Angiopoietin-Like Protein 4 , Angiopoietins/deficiency , Animals , Circadian Rhythm/physiology , Enzyme Activation/physiology , Insulin Resistance/genetics , Insulin Resistance/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Rats , Rats, Sprague-DawleyABSTRACT
OBJECTIVE: Recently, we reported that angiopoietin-like protein 2 (Angptl2) functions in various chronic inflammatory diseases. In the present study, we asked whether Angptl2 and its associated chronic inflammation contribute to abdominal aortic aneurysm (AAA). METHODS AND RESULTS: Immunohistochemistry revealed that Angptl2 is abundantly expressed in infiltrating macrophages within the vessel wall of patients with AAA and in a CaCl(2)-induced AAA mouse model. When Angptl2-deficient mice were used in the mouse model, they showed decreased AAA development compared with wild-type mice, as evidenced by reduction in aneurysmal size, less severe destruction of vessel structure, and lower expression of proinflammatory cytokines and matrix metalloproteinase-9. However, no difference in the number of infiltrating macrophages within the aortic aneurysmal vessel wall was observed between genotypes. AAA development was also significantly suppressed in wild-type mice that underwent Angptl2-deficient bone marrow transplantation. Expression levels of proinflammatory cytokines and metalloproteinase-9 in Angptl2-deficient macrophages were significantly decreased, and those decreases were rescued by treatment of Angptl2 deficient macrophages with exogenous Angptl2. CONCLUSIONS: Macrophage-derived Angptl2 contributes to AAA development by inducing inflammation and degradation of extracellular matrix in the vessel wall, suggesting that targeting the Angptl2-induced inflammatory axis in macrophages could represent a new strategy for AAA therapy.
Subject(s)
Angiopoietins/metabolism , Aorta, Abdominal/metabolism , Aortic Aneurysm, Abdominal/metabolism , Macrophages/metabolism , Angiopoietin-Like Protein 2 , Angiopoietin-like Proteins , Angiopoietins/deficiency , Angiopoietins/genetics , Animals , Aorta, Abdominal/immunology , Aorta, Abdominal/pathology , Aortic Aneurysm, Abdominal/chemically induced , Aortic Aneurysm, Abdominal/genetics , Aortic Aneurysm, Abdominal/immunology , Aortic Aneurysm, Abdominal/pathology , Aortic Aneurysm, Abdominal/prevention & control , Bone Marrow Transplantation , Calcium Chloride , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Extracellular Matrix/metabolism , Gene Expression Regulation , Genotype , Humans , Immunohistochemistry , Inflammation Mediators/metabolism , Macrophages/immunology , Macrophages/pathology , Male , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , PhenotypeABSTRACT
BACKGROUND: Increased permeability, predominantly controlled by endothelial junction stability, is an early event in the deterioration of vascular integrity in ischemic disorders. Hemorrhage, edema, and inflammation are the main features of reperfusion injuries, as observed in acute myocardial infarction (AMI). Thus, preservation of vascular integrity is fundamental in ischemic heart disease. Angiopoietins are pivotal modulators of cell-cell junctions and vascular integrity. We hypothesized that hypoxic induction of angiopoietin-like protein 4 (ANGPTL4) might modulate vascular damage, infarct size, and no-reflow during AMI. METHODS AND RESULTS: We showed that vascular permeability, hemorrhage, edema, inflammation, and infarct severity were increased in angptl4-deficient mice. We determined that decrease in vascular endothelial growth factor receptor 2 (VEGFR2) and VE-cadherin expression and increase in Src kinase phosphorylation downstream of VEGFR2 were accentuated after ischemia-reperfusion in the coronary microcirculation of angptl4-deficient mice. Both events led to altered VEGFR2/VE-cadherin complexes and to disrupted adherens junctions in the endothelial cells of angptl4-deficient mice that correlated with increased no-reflow. In vivo injection of recombinant human ANGPTL4 protected VEGF-driven dissociation of the VEGFR2/VE-cadherin complex, reduced myocardial infarct size, and the extent of no-reflow in mice and rabbits. CONCLUSIONS: These data showed that ANGPTL4 might constitute a relevant target for therapeutic vasculoprotection aimed at counteracting the effects of VEGF, thus being crucial for preventing no-reflow and conferring secondary cardioprotection during AMI.
Subject(s)
Angiopoietins/therapeutic use , Endothelium, Vascular/metabolism , Myocardial Infarction/metabolism , Myocardial Infarction/prevention & control , No-Reflow Phenomenon/metabolism , No-Reflow Phenomenon/prevention & control , Angiopoietin-Like Protein 4 , Angiopoietins/deficiency , Animals , Cardiotonic Agents/metabolism , Cardiotonic Agents/therapeutic use , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/pathology , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Myocardial Infarction/pathology , Myocytes, Cardiac/metabolism , Rabbits , Random AllocationABSTRACT
Angiopoietin-like protein family 4 (Angptl4) has been shown to regulate lipoprotein metabolism through the inhibition of lipoprotein lipase (LPL). In familial hypercholesterolemia (FH), individuals lacking low-density lipoprotein receptor (LDLR) present not only hypercholesterolemia, but also increased plasma triglyceride (TG)-rich lipoprotein remnants, and develop atherosclerosis. In addition, the most common type of dyslipidemia in individuals with diabetes is increased TG levels. We first generated LDLR(-/-)Angptl4(-/-) mice to study the effect of Angptl4 deficiency on the lipid metabolism. Fasting total cholesterol, VLDL-C, LDL-C, HDL-C and TG levels were decreased in LDLR(-/-)Angptl4(-/-) mice compared with LDLR(-/-)Angptl4(+/+) mice. In particular, post olive oil-loaded TG excursion were largely attenuated in LDLR(-/-)Angptl4(-/-) mice compared with LDLR(-/-)Angptl4(+/+) mice. We next introduced diabetes by streptozotocin (STZ) treatment in Angptl4(-/-) mice and examined the impacts of Angptl4 deficiency. Compared with diabetic Angptl4(+/+) mice, diabetic Angptl4(-/-) mice showed the improvement of fasting and postprandial hypertriglyceridemia as well. Thus, targeted silencing of Angptl4 offers a potential therapeutic strategy for the treatment of dyslipidemia in individuals with FH and insulin deficient diabetes.
Subject(s)
Angiopoietins/deficiency , Diabetes Mellitus, Experimental/blood , Hyperlipoproteinemia Type II/blood , Triglycerides/blood , Angiopoietins/genetics , Animals , Cholesterol/blood , Fasting/blood , Lipoproteins, HDL/blood , Lipoproteins, LDL/blood , Male , Mice , Mice, Knockout , Receptors, LDL/geneticsABSTRACT
OBJECTIVE: The angiopoietin-like protein 4 (Angptl4)/fasting-induced adipose factor (Fiaf) is known as a regulator of peripheral lipid and glucose metabolism. In the present study, we investigated the physiological role of Angptl4 in central regulation of body weight homeostasis. RESEARCH DESIGN AND METHODS: Hypothalamic Angptl4 expression levels were measured using immunoblot assay during feeding manipulation or after administration of leptin, insulin, and nutrients. The effects of Angptl4 on food intake, body weight, and energy expenditure were determined following intracerebroventricular (ICV) administration of Angptl4 in C57BL/6 mice. Food intake, energy metabolism, and feeding responses to leptin, insulin, and nutrients were compared between Angptl4-null mice and their wild littermates. Finally, the relationship of hypothalamic AMP-activated protein kinase (AMPK) and Angptl4 was studied. RESULTS: Hypothalamic Angptl4 expression levels were increased upon food intake or administration of leptin, insulin, and nutrients. Furthermore, central administration of Angptl4 suppressed food intake and body weight gain but enhanced energy expenditure. These effects were mediated via suppression of hypothalamic AMPK activities. Consistently, Angptl4-null mice displayed increased body weight and hypothalamic AMPK activity but reduced energy expenditure. Food intake following a fast was significantly greater in Angptl4-null mice, which was normalized by centrally administered Angptl4. Moreover, anorectic responses to leptin, insulin, and glucose were diminished in Angptl4-null mice. In contrast, Angptl4-null mice were resistant to diet-induced obesity, indicating obesity-promoting effects of Angptl4 under the condition of fat-enriched diet. CONCLUSIONS: We have demonstrated that hypothalamic Angptl4 is regulated by physiological appetite regulators and mediates their anorexigenic effects via inhibition of hypothalamic AMPK activity. Therefore, Angptl4 appears to have an important role in central regulation of energy metabolism.
Subject(s)
Angiopoietins/physiology , Eating/physiology , Energy Intake , Hypothalamus/physiology , Angiopoietin-Like Protein 4 , Angiopoietins/deficiency , Angiopoietins/metabolism , Animals , Body Weight , Dietary Fats/metabolism , Energy Metabolism , Homeostasis , Insulin/pharmacology , Leptin/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Obese/genetics , Motor Activity , Obesity/etiologyABSTRACT
RATIONALE: Although dietary fatty acids are a major fuel for the heart, little is known about the direct effects of dietary fatty acids on gene regulation in the intact heart. OBJECTIVE: To study the effect of dietary fatty acids on cardiac gene expression and explore the functional consequences. METHODS AND RESULTS: Oral administration of synthetic triglycerides composed of one single fatty acid altered cardiac expression of numerous genes, many of which are involved in the oxidative stress response. The gene most significantly and consistently upregulated by dietary fatty acids encoded Angiopoietin-like protein (Angptl)4, a circulating inhibitor of lipoprotein lipase expressed by cardiomyocytes. Induction of Angptl4 by the fatty acid linolenic acid was specifically abolished in peroxisome proliferator-activated receptor (PPAR)beta/delta(-/-) and not PPARalpha(-/-) mice and was blunted on siRNA-mediated PPARbeta/delta knockdown in cultured cardiomyocytes. Consistent with these data, linolenic acid stimulated binding of PPARbeta/delta but not PPARalpha to the Angptl4 gene. Upregulation of Angptl4 resulted in decreased cardiac uptake of plasma triglyceride-derived fatty acids and decreased fatty acid-induced oxidative stress and lipid peroxidation. In contrast, Angptl4 deletion led to enhanced oxidative stress in the heart, both after an acute oral fat load and after prolonged high fat feeding. CONCLUSIONS: Stimulation of cardiac Angptl4 gene expression by dietary fatty acids and via PPARbeta/delta is part of a feedback mechanism aimed at protecting the heart against lipid overload and consequently fatty acid-induced oxidative stress.
Subject(s)
Angiopoietins/metabolism , Cardiomyopathies/prevention & control , Dietary Fats/metabolism , Fatty Acids, Unsaturated/metabolism , Myocardium/metabolism , Oxidative Stress , PPAR delta/metabolism , PPAR-beta/metabolism , Angiopoietin-Like Protein 4 , Angiopoietins/deficiency , Angiopoietins/genetics , Animals , Animals, Newborn , Cardiomyopathies/chemically induced , Cardiomyopathies/genetics , Cardiomyopathies/metabolism , Cells, Cultured , Cytoprotection , Dietary Fats/administration & dosage , Dietary Fats/blood , Dietary Fats/toxicity , Fatty Acids, Unsaturated/administration & dosage , Fatty Acids, Unsaturated/blood , Fatty Acids, Unsaturated/toxicity , Feedback, Physiological , Linoleic Acid/metabolism , Lipid Peroxidation , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Oleic Acid/metabolism , Oxidative Stress/genetics , PPAR delta/deficiency , PPAR delta/genetics , PPAR-beta/deficiency , PPAR-beta/genetics , RNA Interference , Time Factors , Up-Regulation , alpha-Linolenic Acid/metabolismABSTRACT
Recent studies of obesity have provided new insights into the mechanisms underlying insulin resistance and metabolic dysregulation. Numerous efforts have been made to identify key regulators of obesity-linked adipose tissue inflammation and insulin resistance. We found that angiopoietin-like protein 2 (Angptl2) was secreted by adipose tissue and that its circulating level was closely related to adiposity, systemic insulin resistance, and inflammation in both mice and humans. Angptl2 activated an inflammatory cascade in endothelial cells via integrin signaling and induced chemotaxis of monocytes/macrophages. Constitutive Angptl2 activation in vivo induced inflammation of the vasculature characterized by abundant attachment of leukocytes to the vessel walls and increased permeability. Angptl2 deletion ameliorated adipose tissue inflammation and systemic insulin resistance in diet-induced obese mice. Conversely, Angptl2 overexpression in adipose tissue caused local inflammation and systemic insulin resistance in nonobese mice. Thus, Angptl2 is a key adipocyte-derived inflammatory mediator that links obesity to systemic insulin resistance.
Subject(s)
Adipose Tissue, White/metabolism , Angiopoietins/metabolism , Inflammation/metabolism , Insulin Resistance/physiology , Obesity/metabolism , 3T3-L1 Cells , Adiposity/physiology , Angiopoietin-Like Protein 2 , Angiopoietin-like Proteins , Angiopoietins/deficiency , Angiopoietins/genetics , Animals , Cell Line , Endothelial Cells/metabolism , Humans , Integrins/metabolism , Leukocytes/immunology , Leukocytes/metabolism , Mice , Mice, Knockout , Mice, ObeseABSTRACT
Glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) binds both LPL and chylomicrons, suggesting that GPIHBP1 is a platform for LPL-dependent processing of triglyceride (TG)-rich lipoproteins. Here, we investigated whether GPIHBP1 affects LPL activity in the absence and presence of LPL inhibitors angiopoietin-like (ANGPTL)3 and ANGPTL4. Like heparin, GPIHBP1 stabilized but did not activate LPL. ANGPTL4 potently inhibited nonstabilized LPL as well as heparin-stabilized LPL but not GPIHBP1-stabilized LPL. Like ANGPTL4, ANGPTL3 inhibited nonstabilized LPL but not GPIHBP1-stabilized LPL. ANGPTL3 also inhibited heparin-stabilized LPL but with less potency than nonstabilized LPL. Consistent with these in vitro findings, fasting serum TGs of Angptl4(-/-)/Gpihbp1(-/-) mice were lower than those of Gpihbp1(-/-) mice and approached those of wild-type littermates. In contrast, serum TGs of Angptl3(-/-)/Gpihbp1(-/-) mice were only slightly lower than those of Gpihbp1(-/-) mice. Treating Gpihbp1(-/-) mice with ANGPTL4- or ANGPTL3-neutralizing antibodies recapitulated the double knockout phenotypes. These data suggest that GPIHBP1 functions as an LPL stabilizer. Moreover, therapeutic agents that prevent LPL inhibition by ANGPTL4 or, to a lesser extent, ANGPTL3, may benefit individuals with hyperlipidemia caused by gene mutations associated with decreased LPL stability.