RESUMO
Very-long-chain polyunsaturated fatty acids (VLCPUFAs; C24-38) constitute a unique class of PUFA that have important biological roles, but the lack of a suitable dietary source has limited research in this field. We produced an n-3 C24-28-rich VLCPUFA-oil concentrated from fish oil to study its bioavailability and physiological functions in C57BL/6J mice. The serum and retinal C24:5 levels increased significantly compared to control after a single-dose gavage, and VLCPUFAs were incorporated into the liver, brain, and eyes after 8-week supplementation. Dietary VLCPUFAs resulted in favorable cardiometabolic changes, and improved electroretinography responses and visual performance. VLCPUFA supplementation changed the expression of genes involved in PPAR signaling pathways. Further in vitro studies demonstrated that the VLCPUFA-oil and chemically synthesized C24:5 are potent agonists for PPARs. The multiple potential beneficial effects of fish oil-derived VLCPUFAs on cardiometabolic risk and eye health in mice support future efforts to develop VLCPUFA-oil into a supplemental therapy.
RESUMO
Introduction: Defects in lipolysis can lead to hypertriglyceridemia, which can trigger acute pancreatitis and is also associated with cardiovascular disease. Decreasing plasma triglycerides (TGs) by activating lipoprotein lipase (LPL) with ApoC2 mimetic peptides is a new treatment strategy for hypertriglyceridemia. We recently described a dual ApoC2 mimetic/ApoC3 antagonist peptide called D6PV that effectively lowered TG in several mouse models but has limitations in terms of drug development. The aim of this study was to create the next generation of ApoC2 mimetic peptides. Methods: We employed hydrocarbon staples, as well as select amino acid substitutions, to make short single helical mimetic peptides based on the last helix of ApoC2. Peptides were first tested for their ability to activate LPL and then in hypertriglyceridemia mouse models. All-atom simulations of peptides were performed in a lipid-trilayer model of TG-rich lipoproteins to discern their possible mechanism of action. Results: We designed a single stapled peptide called SP1 (21 residues), and a double stapled (stitched) peptide called SP2 (21 residues) and its N-terminal acylated analogue, SP2a. The hydrocarbon staples increased the amphipathicity of the peptides and their ability to bind lipids without interfering with LPL activation. Indeed, from all-atom simulations, the conformations of SP1 and SP2a are restrained by the staples and maintains the proper orientation of the LPL activation motif, while still allowing their deeper insertion into the lipid-trilayer model. Intraperitoneal injection of stapled peptides (1-5â umoles/kg) into ApoC2-hypomorphic mice or human ApoC3-transgenic resulted in an 80%-90% reduction in plasma TG within 3â h, similar to the much longer D6PV peptide (41 residues). Other modifications (replacement L-Glu20, L-Glu21 with their D-isomers, N-methylation of Gly19, Met2NorLeu and Ala1alpha-methylAla substitutions, N-terminal octanoylation) were introduced into the SP2a peptide. These changes made SP2a highly resistant to proteolysis against trypsin, pepsin, and Proteinase K, while maintaining similar efficacy in lowering plasma TG in mice. Conclusion: We describe a new generation of ApoC2 mimetic peptides based on hydron carbon stapling that are at least equally potent to earlier peptides but are much shorter and resistant to proteolysis and could be further developed into a new therapy for hypertriglyceridemia.
RESUMO
Both monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) play important roles in lipid metabolism, and diets enriched with either of these two fatty acids are associated with decreased cardiovascular risk. Conventional soybean oil (CSO), a common food ingredient, predominantly contains linoleic acid (LA; C18:2), a n-6 PUFA. Recently, a modified soybean oil (MSO) enriched in oleic acid (C18:1), a n-9 MUFA, has been developed, because of its improved chemical stability to oxidation. However, the effect of the different dietary soybean oils on cardiovascular disease remains unknown. To test whether diets rich in CSO versus MSO would attenuate atherosclerosis development, LDL receptor knock-out (LDLR-KO) mice were fed a Western diet enriched in saturated fatty acids (control), or a Western diet supplemented with 5% (w/w) LA-rich CSO or high-oleic MSO for 12 weeks. Both soybean oils contained a similar amount of linolenic acid (C18:3 n-3). The CSO diet decreased plasma lipid levels and the cholesterol content of VLDL and LDL by approximately 18% (p < 0.05), likely from increased hepatic levels of PUFA, which favorably regulated genes involved in cholesterol metabolism. The MSO diet, but not the CSO diet, suppressed atherosclerotic plaque size compared to the Western control diet (Control Western diet: 6.5 ± 0.9%; CSO diet: 6.4 ± 0.7%; MSO diet: 4.0 ± 0.5%) (p < 0.05), independent of plasma lipid level changes. The MSO diet also decreased the ratio of n-6/n-3 PUFA in the liver (Control Western diet: 4.5 ± 0.2; CSO diet: 6.1 ± 0.2; MSO diet: 2.9 ± 0.2) (p < 0.05), which correlated with favorable hepatic gene expression changes in lipid metabolism and markers of systemic inflammation. In conclusion, supplementation of the Western diet with MSO, but not CSO, reduced atherosclerosis development in LDLR-KO mice independent of changes in plasma lipids.
Assuntos
Aterosclerose , Ácidos Graxos Ômega-3 , Animais , Colesterol/metabolismo , Suplementos Nutricionais , Ácidos Graxos/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Graxos Insaturados/metabolismo , Ácido Linoleico , Camundongos , Camundongos Knockout , Ácido Oleico , Receptores de LDL/genética , Óleo de SojaRESUMO
While serum-circulating complement destroys invading pathogens, intracellularly active complement, termed the "complosome," functions as a vital orchestrator of cell-metabolic events underlying T cell effector responses. Whether intracellular complement is also nonredundant for the activity of myeloid immune cells is currently unknown. Here, we show that monocytes and macrophages constitutively express complement component (C) 5 and generate autocrine C5a via formation of an intracellular C5 convertase. Cholesterol crystal sensing by macrophages induced C5aR1 signaling on mitochondrial membranes, which shifted ATP production via reverse electron chain flux toward reactive oxygen species generation and anaerobic glycolysis to favor IL-1ß production, both at the transcriptional level and processing of proIL-1ß. Consequently, atherosclerosis-prone mice lacking macrophage-specific C5ar1 had ameliorated cardiovascular disease on a high-cholesterol diet. Conversely, inflammatory gene signatures and IL-1ß produced by cells in unstable atherosclerotic plaques of patients were normalized by a specific cell-permeable C5aR1 antagonist. Deficiency of the macrophage cell-autonomous C5 system also protected mice from crystal nephropathy mediated by folic acid. These data demonstrate the unexpected intracellular formation of a C5 convertase and identify C5aR1 as a direct modulator of mitochondrial function and inflammatory output from myeloid cells. Together, these findings suggest that the complosome is a contributor to the biologic processes underlying sterile inflammation and indicate that targeting this system could be beneficial in macrophage-dependent diseases, such as atherosclerosis.
Assuntos
Inflamação/imunologia , Interleucina-1beta/biossíntese , Macrófagos/imunologia , Receptor da Anafilatoxina C5a/imunologia , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor da Anafilatoxina C5a/deficiênciaRESUMO
Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease characterized by low HDL-C levels, low plasma cholesterol esterification, and the formation of Lipoprotein-X (Lp-X), an abnormal cholesterol-rich lipoprotein particle. LCAT deficiency causes corneal opacities, normochromic normocytic anemia, and progressive renal disease due to Lp-X deposition in the glomeruli. Recombinant LCAT is being investigated as a potential therapy for this disorder. Several hepatic disorders, namely primary biliary cirrhosis, primary sclerosing cholangitis, cholestatic liver disease, and chronic alcoholism also develop Lp-X, which may contribute to the complications of these disorders. We aimed to test the hypothesis that an increase in plasma LCAT could prevent the formation of Lp-X in other diseases besides FLD. We generated a murine model of intrahepatic cholestasis in LCAT-deficient (KO), wild type (WT), and LCAT-transgenic (Tg) mice by gavaging mice with alpha-naphthylisothiocyanate (ANIT), a drug well known to induce intrahepatic cholestasis. Three days after the treatment, all mice developed hyperbilirubinemia and elevated liver function markers (ALT, AST, Alkaline Phosphatase). The presence of high levels of LCAT in the LCAT-Tg mice, however, prevented the formation of Lp-X and other plasma lipid abnormalities in WT and LCAT-KO mice. In addition, we demonstrated that multiple injections of recombinant human LCAT can prevent significant accumulation of Lp-X after ANIT treatment in WT mice. In summary, LCAT can protect against the formation of Lp-X in a murine model of cholestasis and thus recombinant LCAT could be a potential therapy to prevent the formation of Lp-X in other diseases besides FLD.
Assuntos
1-Naftilisotiocianato/efeitos adversos , Colestase Intra-Hepática/tratamento farmacológico , Lipoproteína-X/sangue , Fosfatidilcolina-Esterol O-Aciltransferase/uso terapêutico , Animais , Colestase Intra-Hepática/induzido quimicamente , Colestase Intra-Hepática/metabolismo , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Humanos , Lipoproteína-X/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Fosfatidilcolina-Esterol O-Aciltransferase/farmacologiaRESUMO
Recent genetic studies have established that hypertriglyceridemia (HTG) is causally related to cardiovascular disease, making it an active area for drug development. We describe a strategy for lowering triglycerides (TGs) with an apolipoprotein C-II (apoC-II) mimetic peptide called D6PV that activates lipoprotein lipase (LPL), the main plasma TG-hydrolyzing enzyme, and antagonizes the TG-raising effect of apoC-III. The design of D6PV was motivated by a combination of all-atom molecular dynamics simulation of apoC-II on the Anton 2 supercomputer, structural prediction programs, and biophysical techniques. Efficacy of D6PV was assessed ex vivo in human HTG plasma and was found to be more potent than full-length apoC-II in activating LPL. D6PV markedly lowered TG by more than 80% within a few hours in both apoC-II-deficient mice and hAPOC3-transgenic (Tg) mice. In hAPOC3-Tg mice, D6PV treatment reduced plasma apoC-III by 80% and apoB by 65%. Furthermore, low-density lipoprotein (LDL) cholesterol did not accumulate but rather was decreased by 10% when hAPOC3-Tg mice lacking the LDL-receptor (hAPOC3-Tg × Ldlr-/- ) were treated with the peptide. D6PV lowered TG by 50% in whole-body inducible Lpl knockout (iLpl-/- ) mice, confirming that it can also act independently of LPL. D6PV displayed good subcutaneous bioavailability of about 80% in nonhuman primates. Because it binds to high-density lipoproteins, which serve as a long-term reservoir, it also has an extended terminal half-life (42 to 50 hours) in nonhuman primates. In summary, D6PV decreases plasma TG by acting as a dual apoC-II mimetic and apoC-III antagonist, thereby demonstrating its potential as a treatment for HTG.
Assuntos
Apolipoproteína C-III/antagonistas & inibidores , Apolipoproteína C-II/agonistas , Peptídeos/farmacologia , Triglicerídeos/sangue , Animais , Modelos Animais de Doenças , Feminino , Meia-Vida , Humanos , Hipertrigliceridemia/sangue , Hipertrigliceridemia/tratamento farmacológico , Lipólise , Lipase Lipoproteica/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/farmacocinética , Peptídeos/uso terapêutico , PrimatasRESUMO
OBJECTIVE: Highly elevated plasma levels of interleukin-10 (IL-10) are causally associated with "Disappearing HDL Syndrome" and low plasma LDL-cholesterol, but the underlying mechanism is poorly understood. Fluid-phase endocytosis, a process highly dependent on actin dynamics, enables cells to internalize relatively high amounts of extracellular fluids and solutes. We sought to investigate whether IL-10 induces lipoprotein uptake by fluid-phase endocytosis in macrophages. METHODS AND RESULTS: Macrophages (RAW264.7, Kupffer and human) were incubated with vehicle (PBS) or IL-10 (20â¯ng/ml) for 7â¯days. Uptake of HDL, LDL, and/or fluid-phase endocytosis probes (albumin-Alexa680®, 70â¯kDa FITC-Dextran and Lucifer Yellow, LY) was evaluated by FACS. Intracellular cofilin and phosphorylated cofilin (p-cofilin) levels were determined by immunoblotting. Macrophage uptake of lipoproteins and probes was non-saturable and increased after IL-10 incubation (pâ¯<â¯0.0001). Furthermore, pre-incubation with fluid-phase endocytosis inhibitors (LY294002, Latrunculin A, and Amiloride) significantly reduced uptake (pâ¯<â¯0.05). IL-10 increased the cofilin/p-cofilin ratio (pâ¯=â¯0.021), signifying increased cofilin activation and hence filamentous actin. Consistently, phalloidin staining revealed increased filamentous actin in macrophages after IL-10 treatment (pâ¯=â¯0.0018). Finally, RNA-seq analysis demonstrated enrichment of gene sets related to actin filament dynamics, membrane ruffle formation and endocytosis in IL-10-treated macrophages (pâ¯<â¯0.05). IL-10 did not alter mRNA levels of Ldlr, Vldlr, Scarb1, Cd36 or Lrp1. In primary human monocyte-derived macrophages and murine Kupffer cells, IL-10 incubation also increased uptake of lipoproteins, albumin and LY (pâ¯<â¯0.01). CONCLUSIONS: Interleukin-10 induces the uptake of HDL and LDL by fluid-phase endocytosis by increasing actin-filament rearrangement in macrophages, thus providing a plausible mechanism contributing to "Disappearing HDL Syndrome".
Assuntos
Interleucina-10/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Aterosclerose/sangue , Aterosclerose/metabolismo , Células Cultivadas , Cofilina 1/metabolismo , Endocitose , Humanos , Camundongos , Cultura Primária de Células , Proteínas Recombinantes/metabolismoRESUMO
We describe simple, sensitive and robust methods to monitor lipoprotein remodeling and cholesterol and apolipoprotein exchange, using fluorescent Lissamine Rhodamine B head-group tagged phosphatidylethanolamine (*PE) as a lipoprotein reference marker. Fluorescent Bodipy cholesterol (*Chol) and *PE directly incorporated into whole plasma lipoproteins in proportion to lipoprotein cholesterol and phospholipid mass, respectively. *Chol, but not *PE, passively exchanged between isolated plasma lipoproteins. Fluorescent apoA-I (*apoA-I) specifically bound to high-density lipoprotein (HDL) and remodeled *PE- and *Chol-labeled synthetic lipoprotein-X multilamellar vesicles (MLV) into a pre-ß HDL-like particle containing *PE, *Chol, and *apoA-I. Fluorescent MLV-derived *PE specifically incorporated into plasma HDL, whereas MLV-derived *Chol incorporation into plasma lipoproteins was similar to direct *Chol incorporation, consistent with apoA-I-mediated remodeling of fluorescent MLV to HDL with concomitant exchange of *Chol between lipoproteins. Based on these findings, we developed a model system to study lipid transfer by depositing fluorescent *PE and *Chol-labeled on calcium silicate hydrate crystals, forming dense lipid-coated donor particles that are readily separated from acceptor lipoprotein particles by low-speed centrifugation. Transfer of *PE from donor particles to mouse plasma lipoproteins was shown to be HDL-specific and apoA-I-dependent. Transfer of donor particle *PE and *Chol to HDL in whole human plasma was highly correlated. Taken together, these studies suggest that cell-free *PE efflux monitors apoA-I functionality.
RESUMO
SCOPE: Palmitoleic acid (palmitoleate; C16:1 n-7), an omega-7 monounsaturated fatty acid (MUFA) found in plants and marine sources, has been shown to favorably modulate lipid and glucose metabolism. However, its impact on atherosclerosis has not been examined in detail. METHODS AND RESULTS: LDL receptor knock out (LDLR-KO) mice are fed a Western diet supplemented with 5% (w/w) palmitoleate concentrate, oleic-rich olive oil, or none (control) for 12 weeks. Dietary palmitoleate increases hepatic C16:1 levels, improves plasma and hepatic lipid/lipoprotein profiles (≈40% decrease in triglycerides), and reduces the atherosclerotic plaque area by ≈45% compared with control or olive oil group (p < 0.05). These favorable changes are accompanied by the downregulation of key genes, such as Srebp1c, Scd1, Il-1ß, and Tnfα. ApoB-depleted plasma from mice fed palmitoleate has increased cholesterol efflux capacity by 20% from ABCA1-expressing cells (p < 0.05). A beneficial effect of palmitoleate on glucose metabolism (54% decreased in HOMA-IR, p < 0.05) is also observed. CONCLUSIONS: Dietary-supplemented palmitoleate reduces atherosclerosis development in LDLR-KO mice, and is associated with improvement of lipid and glucose metabolism and favorable changes in regulatory genes involved in lipogenesis and inflammation. These findings imply the potential role of dietary palmitoleate in the prevention of cardiovascular disease and diet-induced metabolic disorders.
Assuntos
Aterosclerose/prevenção & controle , Ácidos Graxos Monoinsaturados/administração & dosagem , Hiperlipidemias/prevenção & controle , Receptores de LDL/fisiologia , Animais , Composição Corporal/efeitos dos fármacos , Colesterol/metabolismo , Feminino , Glucose/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Regulation of lipid absorption by enterocytes can influence metabolic status in humans and contribute to obesity and related complications. The intracellular steps of chylomicron biogenesis and transport from the Endoplasmic Reticulum (ER) to the Golgi complex have been described, but the mechanisms for post-Golgi transport and secretion of chylomicrons have not been identified. Using a newly generated Dennd5b-/- mouse, we demonstrate an essential role for this gene in Golgi to plasma membrane transport of chylomicron secretory vesicles. In mice, loss of Dennd5b results in resistance to western diet induced obesity, changes in plasma lipids, and reduced aortic atherosclerosis. In humans, two independent exome sequencing studies reveal that a common DENND5B variant, p.(R52K), is correlated with body mass index. These studies establish an important role for DENND5B in post-Golgi chylomicron secretion and a subsequent influence on body composition and peripheral lipoprotein metabolism.
Assuntos
Índice de Massa Corporal , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/fisiologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Absorção Intestinal , Obesidade/prevenção & controle , Triglicerídeos/metabolismo , Animais , Transporte Biológico , Dieta Hiperlipídica/efeitos adversos , Feminino , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Knockout , Obesidade/etiologia , Obesidade/metabolismoRESUMO
AIM: Plasma apolipoprotein C-II (apoC-II) activates lipoprotein lipase (LPL) and thus lowers plasma triglycerides (TG). We previously reported that a human apoC-II mimetic peptide (C-II-a) decreased plasma TG in apoC-II mutant mice, as well as in apoE-knockout mice. Because it is unknown what tissues take up free fatty acids (FFAs) released from TG after C-II-a peptide administration, we investigated in mice TG plasma clearance and tissue incorporation, using 3H-triolein as a tracer, with and without C-II-a treatment. METHODS AND RESULTS: Intralipid® fat emulsion was labeled with 3H-triolein and then mixed with or without C-II-a. Addition of the peptide did not alter mean particle size of the lipid emulsion particles (298 nm) but accelerated their plasma clearance. After intravenous injection into C57BL/6N mice, the plasma half-life of the 3H-triolein for control and C-II-a treated emulsions was 18.3 ± 2.2 min and 14.8 ± 0.1 min, respectively. In apoC-II mutant mice, the plasma half-life of 3H-triolein for injected control and C-II-a treated emulsions was 30.1 ± 0.1 min and 14.8 ± 0.1 min, respectively. C57BL/6N and apoC-II mutant mice at 120 minutes after the injection showed increased tissue incorporation of radioactivity in white adipose tissue when C-II-a treated emulsion was used. Higher radiolabeled uptake of lipids from C-II-a treated emulsion was also observed in the skeletal muscle of C57BL/6N mice only. In case of apoC-II mutant mice, decreased uptake of radioactive lipids was observed in the liver and kidney after addition of C-II-a to the lipid emulsion. CONCLUSIONS: C-II-a peptide promotes the plasma clearance of TG-rich lipid emulsions in wild type and apoC-II mutant mice and promotes the incorporation of fatty acids from TG in the lipid emulsions into specific peripheral tissues.
RESUMO
Familial LCAT deficiency (FLD) is due to mutations in lecithin:cholesterol acyltransferase (LCAT), a plasma enzyme that esterifies cholesterol on lipoproteins. FLD is associated with markedly reduced levels of plasma high-density lipoprotein and cholesteryl ester and the formation of a nephrotoxic lipoprotein called LpX. We used a mouse model in which the LCAT gene is deleted and a truncated version of the SREBP1a gene is expressed in the liver under the control of a protein-rich/carbohydrate-low (PRCL) diet-regulated PEPCK promoter. This mouse was found to form abundant amounts of LpX in the plasma and was used to determine whether treatment with recombinant human LCAT (rhLCAT) could prevent LpX formation and renal injury. After 9 days on the PRCL diet, plasma total and free cholesterol, as well as phospholipids, increased 6.1 ± 0.6-, 9.6 ± 0.9-, and 6.7 ± 0.7-fold, respectively, and liver cholesterol and triglyceride concentrations increased 1.7 ± 0.4- and 2.8 ±0.9-fold, respectively, compared with chow-fed animals. Transmission electron microscopy revealed robust accumulation of lipid droplets in hepatocytes and the appearance of multilamellar LpX particles in liver sinusoids and bile canaliculi. In the kidney, LpX was found in glomerular endothelial cells, podocytes, the glomerular basement membrane, and the mesangium. The urine albumin/creatinine ratio increased 30-fold on the PRCL diet compared with chow-fed controls. Treatment of these mice with intravenous rhLCAT restored the normal lipoprotein profile, eliminated LpX in plasma and kidneys, and markedly decreased proteinuria. The combined results suggest that rhLCAT infusion could be an effective therapy for the prevention of renal disease in patients with FLD.
Assuntos
Modelos Animais de Doenças , Rim/metabolismo , Deficiência da Lecitina Colesterol Aciltransferase/tratamento farmacológico , Deficiência da Lecitina Colesterol Aciltransferase/metabolismo , Lipoproteína-X/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/administração & dosagem , Animais , Dieta com Restrição de Carboidratos/efeitos adversos , Proteínas Alimentares/efeitos adversos , Feminino , Rim/efeitos dos fármacos , Rim/patologia , Deficiência da Lecitina Colesterol Aciltransferase/patologia , Lipoproteína-X/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
Increasing evidence suggests that neutrophil extracellular traps (NETs) may play a role in promoting atherosclerotic plaque lesions in humans and in murine models. The exact pathways involved in NET-driven atherogenesis remain to be systematically characterized. To assess the extent to which myeloid-specific peptidylarginine deiminase 4 (PAD4) and PAD4-dependent NET formation contribute to atherosclerosis, mice with myeloid-specific deletion of PAD4 were generated and backcrossed to Apoe-/- mice. The kinetics of atherosclerosis development were determined. NETs, but not macrophage extracellular traps, were present in atherosclerotic lesions as early as 3 weeks after initiating high-fat chow. The presence of NETs was associated with the development of atherosclerosis and with inflammatory responses in the aorta. Specific deletion of PAD4 in the myeloid lineage significantly reduced atherosclerosis burden in association with diminished NET formation and reduced inflammatory responses in the aorta. NETs stimulated macrophages to synthesize inflammatory mediators, including IL-1ß, CCL2, CXCL1, and CXCL2. Our data support the notion that NETs promote atherosclerosis and that the use of specific PAD4 inhibitors may have therapeutic benefits in this potentially devastating condition.
Assuntos
Aterosclerose/etiologia , Aterosclerose/metabolismo , Deleção de Genes , Hidrolases/genética , Células Mieloides/imunologia , Células Mieloides/metabolismo , Animais , Aorta/imunologia , Aorta/metabolismo , Aorta/patologia , Aterosclerose/patologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Citocinas/metabolismo , Desoxirribonuclease I/metabolismo , Modelos Animais de Doenças , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Proteína-Arginina Desiminase do Tipo 4RESUMO
BACKGROUND AND AIMS: Concentrated fish oils, containing a mixture of long-chain monounsaturated fatty acids (LCMUFA) with aliphatic chains longer than 18 C atoms (i.e., C20:1 and C22:1), have been shown to attenuate atherosclerosis development in mouse models. It is not clear, however, how individual LCMUFA isomers may act on atherosclerosis. METHODS: In the present study, we used saury fish oil-derived concentrates enriched in either C20:1 or C22:1 isomer fractions to investigate their individual effect on atherosclerosis and lipoprotein metabolism. LDLR-deficient (LDLr-/-) mice were fed a Western diet supplemented with 5% (w/w) of either C20:1 or C22:1 concentrate for 12 wk. RESULTS: Compared to the control Western diet with no supplement, both LCMUFA isomers increased hepatic levels of LCMUFA by 2â¼3-fold (p < 0.05), and decreased atherosclerotic lesion areas by more than 40% (p < 0.05), although there were no major differences in plasma lipoproteins or hepatic lipid content. Both LCMUFA isomers significantly decreased plasma CRP levels, improved Abca1-dependent cholesterol efflux capacity of apoB-depleted plasma, and enhanced Ppar transcriptional activities in HepG2 cells. LC-MS/MS proteomic analysis of lipoproteins (HDL, LDL and VLDL) revealed that both LCMUFA isomer diets resulted in similar potentially beneficial alterations in proteins involved in complement activation, blood coagulation, and lipid metabolism. Several lipoprotein proteome changes were significantly correlated with atherosclerotic plaque reduction. CONCLUSIONS: Dietary supplementation with the LCMUFA isomers C20:1 or C22:1 was equally effective in reducing atherosclerosis in LDLr-/-mice and this may partly occur through activation of the Ppar signaling pathways and favorable alterations in the proteome of lipoproteins.
Assuntos
Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Suplementos Nutricionais , Ácidos Graxos Monoinsaturados/farmacologia , Óleos de Peixe/farmacologia , Hiperlipidemias/tratamento farmacológico , Lipoproteínas/sangue , Proteoma , Receptores de LDL/deficiência , Animais , Doenças da Aorta/sangue , Doenças da Aorta/genética , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/patologia , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Cromatografia Líquida , Dieta Ocidental , Modelos Animais de Doenças , Predisposição Genética para Doença , Células Hep G2 , Humanos , Hiperlipidemias/sangue , Hiperlipidemias/genética , Hiperlipidemias/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos Knockout , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Fenótipo , Placa Aterosclerótica , Proteômica/métodos , Receptores de LDL/genética , Espectrometria de Massas em TandemRESUMO
SCOPE: α-Cyclodextrin (α-CD), a cyclic polymer of glucose, has been shown to lower plasma cholesterol in animals and humans; however, its effect on atherosclerosis has not been previously described. METHODS AND RESULTS: apoE-knockout mice were fed either low-fat diet (LFD; 5.2% fat, w/w), or Western high fat diet (21.2% fat) containing either no additions (WD), 1.5% α-CD (WDA); 1.5% ß-CD (WDB); or 1.5% oligofructose-enriched inulin (WDI). Although plasma lipids were similar after 11 weeks on the WD vs. WDA diets, aortic atherosclerotic lesions were 65% less in mice on WDA compared to WD (P < 0.05), and similar to mice fed the LFD. No effect on atherosclerosis was observed for the other WD supplemented diets. By RNA-seq analysis of 16S rRNA, addition of α-CD to the WD resulted in significantly decreased cecal bacterial counts in genera Clostridium and Turicibacterium, and significantly increased Dehalobacteriaceae. At family level, Comamonadaceae significantly increased and Peptostreptococcaceae showed a negative trend. Several of these bacterial count changes correlated negatively with % atherosclerotic lesion and were associated with increased cecum weight and decreased plasma cholesterol levels. CONCLUSION: Addition of α-CD to the diet of apoE-knockout mice decreases atherosclerosis and is associated with changes in the gut flora.
Assuntos
Aterosclerose/dietoterapia , Microbioma Gastrointestinal/efeitos dos fármacos , Lipídeos/sangue , alfa-Ciclodextrinas/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Aterosclerose/microbiologia , Aterosclerose/patologia , Peso Corporal/efeitos dos fármacos , Ceco/efeitos dos fármacos , Ceco/microbiologia , Dieta com Restrição de Gorduras , Dieta Hiperlipídica/efeitos adversos , Suplementos Nutricionais , Feminino , Microbioma Gastrointestinal/genética , Absorção Intestinal , Lipídeos/farmacocinética , Camundongos Knockout para ApoE , alfa-Ciclodextrinas/metabolismo , beta-Ciclodextrinas/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
SCOPE: Fish oil-derived long-chain monounsaturated fatty acids (LCMUFA) containing chain lengths longer than 18 were previously shown to improve cardiovascular disease risk factors in mice. However, it is not known if LCMUFA also exerts anti-atherogenic effects. The main objective of the present study was to investigate the effect of LCMUFA on the development of atherosclerosis in mouse models. METHODS AND RESULTS: LDLR-KO mice were fed Western diet supplemented with 2% (w/w) of either LCMUFA concentrate, olive oil, or not (control) for 12 wk. LCMUFA, but not olive oil, significantly suppressed the development of atherosclerotic lesions and several plasma inflammatory cytokine levels, although there were no major differences in plasma lipids between the three groups. At higher doses 5% (w/w) LCMUFA supplementation was observed to reduce pro-atherogenic plasma lipoproteins and to also reduce atherosclerosis in ApoE-KO mice fed a Western diet. RNA sequencing and subsequent qPCR analyses revealed that LCMUFA upregulated PPAR signaling pathways in liver. In cell culture studies, apoB-depleted plasma from LDLR-K mice fed LCMUFA showed greater cholesterol efflux from macrophage-like THP-1 cells and ABCA1-overexpressing BHK cells. CONCLUSION: Our research showed for the first time that LCMUFA consumption protects against diet-induced atherosclerosis, possibly by upregulating the PPAR signaling pathway.
Assuntos
Aterosclerose/prevenção & controle , Ácidos Graxos Monoinsaturados/farmacologia , Óleos de Peixe/farmacologia , Animais , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Linhagem Celular , Colesterol/metabolismo , Citocinas/sangue , Modelos Animais de Doenças , Ácidos Graxos/análise , Ácidos Graxos Monoinsaturados/química , Óleos de Peixe/química , Humanos , Lipídeos/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Knockout , Receptores de LDL/genéticaRESUMO
Large population studies have shown that living at higher altitudes, which lowers ambient oxygen exposure, is associated with reduced cardiovascular disease mortality. However, hypoxia has also been reported to promote atherosclerosis by worsening lipid metabolism and inflammation. We sought to address these disparate reports by reducing the ambient oxygen exposure of ApoE-/- mice. We observed that long-term adaptation to 10% O2 (equivalent to oxygen content at â¼5000 m), compared to 21% O2 (room air at sea level), resulted in a marked decrease in aortic atherosclerosis in ApoE-/- mice. This effect was associated with increased expression of the anti-inflammatory cytokine interleukin-10 (IL-10), known to be anti-atherogenic and regulated by hypoxia-inducible transcription factor-1α (HIF-1α). Supporting these observations, ApoE-/- mice that were deficient in IL-10 (IL10-/- ApoE-/- double knockout) failed to show reduced atherosclerosis in 10% oxygen. Our study reveals a specific mechanism that can help explain the decreased prevalence of ischemic heart disease in populations living at high altitudes and identifies ambient oxygen exposure as a potential factor that could be modulated to alter pathogenesis. Key messages: Chronic low ambient oxygen exposure decreases atherosclerosis in mice. Anti-inflammatory cytokine IL-10 levels are increased by low ambient O2. This is consistent with the established role of HIF-1α in IL10 transactivation. Absence of IL-10 results in the loss of the anti-atherosclerosis effect of low O2. This mechanism may contribute to decreased atherosclerosis at high altitudes.
Assuntos
Doença da Altitude/epidemiologia , Aterosclerose/epidemiologia , Oxigênio/imunologia , Doença da Altitude/genética , Doença da Altitude/imunologia , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/imunologia , Aterosclerose/genética , Aterosclerose/imunologia , Linhagem Celular , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Inflamação/epidemiologia , Inflamação/genética , Inflamação/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Proteção , Regulação para CimaRESUMO
Human familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, chemical and biologic characteristics, to wild-type and Lcat-/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in Lcat-/- mice, which have low HDL, but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat-/- mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal Cxl10 expression in Lcat-/- mice. In conclusion, LpX is a nephrotoxic particle that in the absence of Lcat induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.
Assuntos
Glomérulos Renais/efeitos dos fármacos , Deficiência da Lecitina Colesterol Aciltransferase/metabolismo , Lipoproteína-X/toxicidade , Proteinúria/etiologia , Animais , Apolipoproteína A-I/metabolismo , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Membrana Basal Glomerular/efeitos dos fármacos , Membrana Basal Glomerular/patologia , Mesângio Glomerular/citologia , Mesângio Glomerular/metabolismo , Mesângio Glomerular/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Interleucina-6/metabolismo , Glomérulos Renais/patologia , Deficiência da Lecitina Colesterol Aciltransferase/patologia , Lipoproteína-X/metabolismo , Lipoproteína-X/farmacocinética , Lipoproteínas HDL/metabolismo , Lisossomos/metabolismo , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Fosfolipases A2/metabolismo , Pinocitose , Podócitos/metabolismo , Podócitos/patologia , Proteinúria/induzido quimicamente , Proteinúria/genética , Proteinúria/patologiaRESUMO
Apolipoprotein C-II (apoC-II) is a cofactor for lipoprotein lipase, a plasma enzyme that hydrolyzes triglycerides (TGs). ApoC-II deficiency in humans results in hypertriglyceridemia. We used zinc finger nucleases to create Apoc2 mutant mice to investigate the use of C-II-a, a short apoC-II mimetic peptide, as a therapy for apoC-II deficiency. Mutant mice produced a form of apoC-II with an uncleaved signal peptide that preferentially binds high-density lipoproteins (HDLs) due to a 3-amino acid deletion at the signal peptide cleavage site. Homozygous Apoc2 mutant mice had increased plasma TG (757.5 ± 281.2 mg/dl) and low HDL cholesterol (31.4 ± 14.7 mg/dl) compared with wild-type mice (TG, 55.9 ± 13.3 mg/dl; HDL cholesterol, 55.9 ± 14.3 mg/dl). TGs were found in light (density < 1.063 g/ml) lipoproteins in the size range of very-low-density lipoprotein and chylomicron remnants (40-200 nm). Intravenous injection of C-II-a (0.2, 1, and 5 µmol/kg) reduced plasma TG in a dose-dependent manner, with a maximum decrease of 90% occurring 30 minutes after the high dose. Plasma TG did not return to baseline until 48 hours later. Similar results were found with subcutaneous or intramuscular injections. Plasma half-life of C-II-a is 1.33 ± 0.72 hours, indicating that C-II-a only acutely activates lipolysis, and the sustained TG reduction is due to the relatively slow rate of new TG-rich lipoprotein synthesis. In summary, we describe a novel mouse model of apoC-II deficiency and show that an apoC-II mimetic peptide can reverse the hypertriglyceridemia in these mice, and thus could be a potential new therapy for apoC-II deficiency.