Novel bioactive lipids enhanced HDL-mediated cholesterol efflux from macrophages through the ABCA1 receptor pathway.

High-density lipoprotein (HDL) has traditionally been acknowledged as "good cholesterol" owing to its significant association with a decreased risk of atherosclerosis. This association is primarily attributed to HDL's direct involvement in cholesterol efflux capacity, which plays a pivotal role in reverse cholesterol transport. A novel active compound from Nannochloropsis microalgae termed lyso-DGTS, a lipid that contains EPA fatty acids, was previously isolated and found to increase paraoxonase 1 activity and enhance HDL-mediated cholesterol efflux and HDL-induced endothelial nitric oxide release. Here, the effect of different lyso-DGTS derivatives and analogs on HDL-mediated cholesterol efflux from macrophages was examined, and the mechanism was explored. Structure-activity relationships were established to characterize the essential lipid moieties responsible for HDL-mediated cholesterol efflux from macrophages. Lyso-DGTS, 1-carboxy-N-N-N-trimethyl-3-oleamidopropan-1-aminium, and lyso-platelet-activating factor increased HDL-mediated cholesterol efflux from macrophages dose-dependently, mainly via the ABCA1-mediated cholesterol efflux pathway. The effect of lyso-DGTS derivatives and analogs on the surface polarity of HDL was examined using the Laurdan generalized polarization (GP) assay. A reverse Pearson linear regression was obtained between Laurdan GP values and HDL-mediated cholesterol efflux. Because the incorporation of bioactive lipids into the surface phospholipid layer of HDL leads to a decrease in Laurdan GP, these bioactive lipids may induce lower phospholipid ordering and greater free space on the HDL particle surface, thereby enhancing apolipoprotein A1 binding to the ABCA1 receptor and improving ABCA1 cholesterol-mediated efflux. Our findings suggest a beneficial effect of lyso-DGTS and its bioactive lipid derivatives on increasing HDL-mediated cholesterol efflux activity from macrophages, which may impact atherosclerosis attenuation.

Lyso-DGTS Lipid Derivatives Enhance PON1 Activities and Prevent Oxidation of LDL: A Structure-Activity Relationship Study.

Paraoxonase 1 (PON1) plays a role in regulating reverse cholesterol transport and has antioxidative, anti-inflammatory, antiapoptotic, vasodilative, and antithrombotic activities. Scientists are currently focused on the modulation of PON1 expression using different pharmacological, nutritional, and lifestyle approaches. We previously isolated a novel active compound from Nannochloropsis microalgae-lyso-diacylglyceryltrimethylhomoserine (lyso-DGTS)-which increased PON1 activity, HDL-cholesterol efflux, and endothelial nitric oxide release. Here, to explore this important lipid moiety's effect on PON1 activities, we examined the effect of synthesized lipid derivatives and endogenous analogs of lyso-DGTS on PON1 lactonase and arylesterase activities and LDL oxidation using structure-activity relationship (SAR) methods. Six lipids significantly elevated recombinant PON1 (rePON1) lactonase activity in a dose-dependent manner, and four lipids significantly increased rePON1 arylesterase activity. Using tryptophan fluorescence-quenching assay and a molecular docking method, lipid-PON1 interactions were characterized. An inverse correlation was obtained between the lactonase activity of PON1 and the docking energy of the lipid-PON1 complex. Furthermore, five of the lipids increased the LDL oxidation lag time and inhibited its propagation. Our findings suggest a beneficial effect of lyso-DGTS or lyso-DGTS derivatives through increased PON1 activity and prevention of LDL oxidation.

Induction of glutathione biosynthesis by glycine-based treatment mitigates atherosclerosis.

Lower circulating levels of glycine are consistently reported in association with cardiovascular disease (CVD), but the causative role and therapeutic potential of glycine in atherosclerosis, the underlying cause of most CVDs, remain to be established. Here, following the identification of reduced circulating glycine in patients with significant coronary artery disease (sCAD), we investigated a causative role of glycine in atherosclerosis by modulating glycine availability in atheroprone mice. We further evaluated the atheroprotective potential of DT-109, a recently identified glycine-based compound with dual lipid/glucose-lowering properties. Glycine deficiency enhanced, while glycine supplementation attenuated, atherosclerosis development in apolipoprotein E-deficient (Apoe-/-) mice. DT-109 treatment showed the most significant atheroprotective effects and lowered atherosclerosis in the whole aortic tree and aortic sinus concomitant with reduced superoxide. In Apoe-/- mice with established atherosclerosis, DT-109 treatment significantly reduced atherosclerosis and aortic superoxide independent of lipid-lowering effects. Targeted metabolomics and kinetics studies revealed that DT-109 induces glutathione formation in mononuclear cells. In bone marrow-derived macrophages (BMDMs), glycine and DT-109 attenuated superoxide formation induced by glycine deficiency. This was abolished in BMDMs from glutamate-cysteine ligase modifier subunit-deficient (Gclm-/-) mice in which glutathione biosynthesis is impaired. Metabolic flux and carbon tracing experiments revealed that glycine deficiency inhibits glutathione formation in BMDMs while glycine-based treatment induces de novo glutathione biosynthesis. Through a combination of studies in patients with CAD, in vivo studies using atherosclerotic mice and in vitro studies using macrophages, we demonstrated a causative role of glycine in atherosclerosis and identified glycine-based treatment as an approach to mitigate atherosclerosis through antioxidant effects mediated by induction of glutathione biosynthesis.

A Case of Severe COVID-19 Pneumonia Diagnosed in Bronchoscopy With Negative Repeated Nasopharyngeal Swabs.

Coronavirus disease 2019 (COVID-19) is a worldwide pandemic that had emerged in China since December 2019. The disease affects all age groups, with clinical manifestations in the spectrum from asymptomatic to rapidly lethal multi-organ failure, mainly involving the respiratory system. Diagnosis is confirmed mainly by a positive real-time polymerase chain reaction (PCR) nasopharyngeal swab. It is highly recommended to avoid performing invasive procedures in COVID-19 subjects to prevent the potential for dissemination of the pathogen. Treatment consists in particular of respiratory support and symptom relief. Dexamethasone is widely used with encouraging response. There were no cases in the literature that were diagnosed with positive reverse transcription-polymerase chain reaction (RT-PCR) testing only from fluid of involved organs, while repeated nasopharyngeal swabs returned negative for COVID-19. We here describe a case of COVID-19 that presented with moderate-severe pulmonary involvement, diagnosed by RT-PCR testing from broncho-alveolar lavage, while several nasopharyngeal swabs were consistently negative. The patient experienced no improvement under wide-spectrum antibiotics administered initially, and greatly improved after receiving systemic corticosteroids. One can realize from our case that COVID-19 could not be ruled out upon repeated negative RT-PCR nasopharyngeal swabs, and in subjects with highly suspected COVID-19, it is justified to perform invasive procedures, but still using maximal protective measures.

Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

Dysregulated glycine metabolism is emerging as a common denominator in cardiometabolic diseases, but its contribution to atherosclerosis remains unclear. In this study, we demonstrate impaired glycine-oxalate metabolism through alanine-glyoxylate aminotransferase (AGXT) in atherosclerosis. As found in patients with atherosclerosis, the glycine/oxalate ratio is decreased in atherosclerotic mice concomitant with suppression of AGXT. Agxt deletion in apolipoprotein E-deficient (Apoe-/-) mice decreases the glycine/oxalate ratio and increases atherosclerosis with induction of hepatic pro-atherogenic pathways, predominantly cytokine/chemokine signaling and dysregulated redox homeostasis. Consistently, circulating and aortic C-C motif chemokine ligand 5 (CCL5) and superoxide in lesional macrophages are increased. Similar findings are observed following dietary oxalate overload in Apoe-/- mice. In macrophages, oxalate induces mitochondrial dysfunction and superoxide accumulation, leading to increased CCL5. Conversely, AGXT overexpression in Apoe-/- mice increases the glycine/oxalate ratio and decreases aortic superoxide, CCL5, and atherosclerosis. Our findings uncover dysregulated oxalate metabolism via suppressed AGXT as a driver and therapeutic target in atherosclerosis.

A case of rapidly progressive upper limb ischemic necrosis in a patient with COVID-19.

BACKGROUND: For more than a year, health systems all over the world have been combating the global coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disease was first described in the city of Wuhan in China, presenting as an atypical infection of the lower respiratory tract. METHODS: COVID-19 is characterized by multisystemic involvement, and mortality is attributed mainly to the respiratory system involvement, which may lead to severe acute respiratory distress syndrome and respiratory failure. Several COVID-19-associated complications are being increasingly reported, including arterial and venous thromboembolic events that may lead to amputation of the affected limbs. So far, a large number of reports have described hypercoagulability crises leading to amputation of the lower limbs. However, a search of the National Library of Medicine (MEDLINE) revealed no cases of urgent upper limb amputation in COVID-19 patients. RESULTS: This article describes a novel case of upper limb ischemia in a COVID-19 patient, with rapid progression to hand necrosis, requiring urgent through-arm amputation of the upper limb. CONCLUSIONS: This case emphasizes the need for anticoagulant therapy in COVID-19 patients and to maintain a constant awareness of the possible thromboembolic COVID-19-related sequelae.

Glycine-based treatment ameliorates NAFLD by modulating fatty acid oxidation, glutathione synthesis, and the gut microbiome.

Nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH) has reached epidemic proportions with no pharmacological therapy approved. Lower circulating glycine is consistently reported in patients with NAFLD, but the causes for reduced glycine, its role as a causative factor, and its therapeutic potential remain unclear. We performed transcriptomics in livers from humans and mice with NAFLD and found suppression of glycine biosynthetic genes, primarily alanine-glyoxylate aminotransferase 1 (AGXT1). Genetic (Agxt1 -/- mice) and dietary approaches to limit glycine availability resulted in exacerbated diet-induced hyperlipidemia and steatohepatitis, with suppressed mitochondrial/peroxisomal fatty acid ß-oxidation (FAO) and enhanced inflammation as the underlying pathways. We explored glycine-based compounds with dual lipid/glucose-lowering properties as potential therapies for NAFLD and identified a tripeptide (Gly-Gly-L-Leu, DT-109) that improved body composition and lowered circulating glucose, lipids, transaminases, proinflammatory cytokines, and steatohepatitis in mice with established NASH induced by a high-fat, cholesterol, and fructose diet. We applied metagenomics, transcriptomics, and metabolomics to explore the underlying mechanisms. The bacterial genus Clostridium sensu stricto was markedly increased in mice with NASH and decreased after DT-109 treatment. DT-109 induced hepatic FAO pathways, lowered lipotoxicity, and stimulated de novo glutathione synthesis. In turn, inflammatory infiltration and hepatic fibrosis were attenuated via suppression of NF-κB target genes and TGFß/SMAD signaling. Unlike its effects on the gut microbiome, DT-109 stimulated FAO and glutathione synthesis independent of NASH. In conclusion, impaired glycine metabolism may play a causative role in NAFLD. Glycine-based treatment attenuates experimental NAFLD by stimulating hepatic FAO and glutathione synthesis, thus warranting clinical evaluation.

p38 MAPK in Glucose Metabolism of Skeletal Muscle: Beneficial or Harmful?

Skeletal muscles respond to environmental and physiological changes by varying their size, fiber type, and metabolic properties. P38 mitogen-activated protein kinase (MAPK) is one of several signaling pathways that drive the metabolic adaptation of skeletal muscle to exercise. p38 MAPK also participates in the development of pathological traits resulting from excessive caloric intake and obesity that cause metabolic syndrome and type 2 diabetes (T2D). Whereas p38 MAPK increases insulin-independent glucose uptake and oxidative metabolism in muscles during exercise, it contrastingly mediates insulin resistance and glucose intolerance during metabolic syndrome development. This article provides an overview of the apparent contradicting roles of p38 MAPK in the adaptation of skeletal muscles to exercise and to pathological conditions leading to glucose intolerance and T2D. Here, we focus on the involvement of p38 MAPK in glucose metabolism of skeletal muscle, and discuss the possibility of targeting this pathway to prevent the development of T2D.

Soybean Oil Modulates the Gut Microbiota Associated with Atherogenic Biomarkers.

During the last few decades there has been a staggering rise in human consumption of soybean-oil (SO). The microbiome and specific taxa composing it are dramatically affected by diet; specifically, by high-fat diets. Increasing evidence indicates the association between dysbiosis and health or disease state, including cardiovascular diseases (CVD) and atherosclerosis pathogenesis in human and animal models. To investigate the effects of high SO intake, C57BL/6 mice were orally supplemented with SO-based emulsion (SOE) for one month, followed by analyses of atherosclerosis-related biomarkers and microbiota profiling by 16S rRNA gene sequencing of fecal DNA. SOE-supplementation caused compositional changes to 64 taxa, including enrichment in Bacteroidetes, Mucispirillum, Prevotella and Ruminococcus, and decreased Firmicutes. These changes were previously associated with atherosclerosis in numerous studies. Among the shifted taxa, 40 significantly correlated with at least one atherosclerosis-related biomarker (FDR < 0.05), while 13 taxa positively correlated with the average of all biomarkers. These microbial alterations also caused a microbial-derived metabolic-pathways shift, including enrichment in different amino-acid metabolic-pathways known to be implicated in CVD. In conclusion, our results demonstrate dysbiosis following SOE supplementation associated with atherosclerosis-related biomarkers. These findings point to the microbiome as a possible mediator to CVD, and it may be implemented into non-invasive diagnostic tools or as potential therapeutic strategies.

Prednisone and Its Active Metabolite Prednisolone Attenuate Lipid Accumulation in Macrophages.

BACKGROUND: Synthetic forms of glucocorticoids (GCs; eg, prednisone, prednisolone) are anti-inflammatory drugs that are widely used in clinical practice. The role of GCs in cardiovascular diseases, including atherosclerosis, is highly controversial, and their impact on macrophage foam cell formation is still unknown. We investigated the effects of prednisone and prednisolone on macrophage oxidative stress and lipid metabolism. METHODS AND RESULTS: C57BL/6 mice were intraperitoneally injected with prednisone or prednisolone (5 mg/kg) for 4 weeks, followed by lipid metabolism analyses in the aorta and peritoneal macrophages. We also analyzed the effect of serum samples obtained from 9 healthy human volunteers before and after oral administration of prednisone (20 mg for 5 days) on J774A.1 macrophage atherogenicity. Finally, J774A.1 macrophages, human monocyte-derived macrophages, and fibroblasts were incubated with increasing concentrations (0-200 ng/mL) of prednisone or prednisolone, followed by determination of cellular oxidative status, and triglyceride and cholesterol metabolism. Prednisone and prednisolone treatment resulted in a significant reduction in triglyceride and cholesterol accumulation in macrophages, as observed in vivo, ex vivo, and in vitro. These effects were associated with GCs' inhibitory effect on triglyceride- and cholesterol-biosynthesis rates, through downregulation of diacylglycerol acyltransferase 1 and HMG-CoA reductase expression. Glucocorticoid-induced reduction of cellular lipid accumulation was mediated by the GC receptors on the macrophages, because the GC-receptor antagonist (RU486) abolished these effects. In fibroblasts, unlike macrophages, GCs showed no effects. CONCLUSION: Prednisone and prednisolone exhibit antiatherogenic activity by protecting macrophages from lipid accumulation and foam cell formation.

A Comparative Study of the Reverse Sural Fascio-Cutaneous Flap Outcomes in the Management of Foot and Ankle Soft Tissue Defects in Diabetic and Trauma Patients.

Introduction. Soft tissue defects in the distal leg and foot are challenging conditions for reconstruction. The widely used reverse sural fascio-cutaneous flap (RSFCF) has been reported with large variation in complication frequency. Some authors reported higher complications in the diabetic population when compared with trauma patients. We compared the reliability of the RSFCF in treating such defects among both populations. Methods. This is a retrospective series with a prospective data collection of 24 patients (11 with type 2 diabetes and 13 in trauma settings) treated with an ipsilateral RSFCF for soft tissue defects of the distal leg and the rear foot. Healing events and complications were recorded and compared for both groups. The mean follow-up was 32 months. Results. Diabetic group versus trauma group showed the following results; mean flap healing time of 24 versus 22 days, donor site healing time of 14 versus 16 days, 1 total flap necrosis in both groups, 3 versus 2 cases of skin edge necrosis, 2 cases of temporary venous congestion in both groups, and 8 versus 10 cases of transient hypoesthesia of the lateral border of the foot. No infection was encountered in both groups and no recurrence of infection in the primary infected diabetic patients. Conclusions. We found the RSFCF to be useful, reproducible, and reliable in treating soft tissue defects with a very low frequency of serious complications. Diabetic patients were found to have similar outcomes when compared with trauma patients. Therefore, diabetes might not be a major factor of flap failure. Levels of Evidence: Level III: Therapeutic.

Leucine supplementation attenuates macrophage foam-cell formation: Studies in humans, mice, and cultured macrophages.

Whereas atherogenicity of dietary lipids has been largely studied, relatively little is known about the possible contribution of dietary amino acids to macrophage foam-cell formation, a hallmark of early atherogenesis. Recently, we showed that leucine has antiatherogenic properties in the macrophage model system. In this study, an in-depth investigation of the role of leucine in macrophage lipid metabolism was conducted by supplementing humans, mice, or cultured macrophages with leucine. Macrophage incubation with serum obtained from healthy adults supplemented with leucine (5 g/d, 3 weeks) significantly decreased cellular cholesterol mass by inhibiting the rate of cholesterol biosynthesis and increasing cholesterol efflux from macrophages. Similarly, leucine supplementation to C57BL/6 mice (8 weeks) resulted in decreased cholesterol content in their harvested peritoneal macrophages (MPM) in relation with reduced cholesterol biosynthesis rate. Studies in J774A.1 murine macrophages revealed that leucine dose-dependently decreased cellular cholesterol and triglyceride mass. Macrophages treated with leucine (0.2 mM) showed attenuated uptake of very low-density lipoproteins and triglyceride biosynthesis rate, with a concurrent down-regulation of diacylglycerol acyltransferase-1, a key enzyme catalyzing triglyceride biosynthesis in macrophages. Similar effects were observed when macrophages were treated with α-ketoisocaproate, a key leucine metabolite. Finally, both in vivo and in vitro leucine supplementation significantly improved macrophage mitochondrial respiration and ATP production. The above studies, conducted in human, mice, and cultured macrophages, highlight a protective role for leucine attenuating macrophage foam-cell formation by mechanisms related to the metabolism of cholesterol, triglycerides, and energy production. © 2018 BioFactors, 44(3):245-262, 2018.

Heparanase Inhibition Reduces Glucose Levels, Blood Pressure, and Oxidative Stress in Apolipoprotein E Knockout Mice.

BACKGROUND: Atherosclerosis is a multifactorial process. Emerging evidence highlights a role of the enzyme heparanase in various disease states, including atherosclerosis formation and progression. OBJECTIVE: The aim of the study was to investigate the effect of heparanase inhibition on blood pressure, blood glucose levels, and oxidative stress in apoE-/- mice. METHODS: Male apoE-/- mice were divided into two groups: one treated by the heparanase inhibitor PG545, administered intraperitoneally weekly for seven weeks, and the other serving as control group (injected with saline). Blood pressure was measured a day before sacrificing the animals. Serum glucose levels and lipid profile were measured. Assessment of oxidative stress was performed as well. RESULTS: PG545 significantly lowered blood pressure and serum glucose levels in treated mice. It also caused significant reduction of the serum oxidative stress. For safety concerns, liver enzymes were assessed, and PG545 caused significant elevation only of alanine aminotransferase, but not of the other hepatic enzymes. CONCLUSION: Heparanase inhibition by PG545 caused marked reduction of blood pressure, serum glucose levels, and oxidative stress in apolipoprotein E deficient mice, possibly via direct favorable metabolic and hemodynamic changes caused by the inhibitor. Possible hepatotoxic and weight wasting effects are subject for future investigation.

[REDUCTION OF ANTIBIOTIC CONSUMPTION IN RAMBAM HEALTH CARE CAMPUS - THE ROLE OF AN ANTIBIOTIC STEWARDSHIP PROGRAM].

INTRODUCTION: Antibiotic stewardship programs (ASP) are designed to optimize antibiotic use in hospitals. Antibiotic consumption is one of the measures assessing the effects of ASPs. AIMS: To evaluate the effect of an ASP on antibiotic consumption in our hospital and compare it to hospitals in Israel and worldwide. METHODS: Between October 2012 and March 2013 an ASP was implemented in Rambam Hospital. The program included educational activities, publication of local guidelines for empirical antibiotic treatment, structured infectious diseases consultations, pre-authorization antibiotic restrictions and stop orders. We compared antibacterial antibiotic consumption in defined daily doses (DDD)/100 hospital days (HD) between the periods before (1/2010-3/2013) and after (4/2013-9/2014) implementing the ASP. The study was conducted in the medical departments, hematology, the intensive care unit (ICU) and all pediatric wards. RESULTS: Total antibiotic consumption before implementing the ASP was 96±11.2 DDD/100 HD in medical departments, 186.4±42.8 in the ICU and 185.5±59 in hematology; all values were higher than the worldwide-reported averages for these departments. Following the ASP, total antibiotic consumption decreased by 12% (p=0.008) in the medical departments and by 26% (p=0.002) in hematology, mostly due to reductions in non-restricted antibiotics. No significant changes were observed overall in the ICU and in pediatric wards. There was a significant reduction in consumption of vancomycin and carbapenems in all settings, the latter was reduced to nearly half. Amikacin use quadrupled in the medical departments. CONCLUSIONS: Implementation of an ASP lead to a reduction in non-restricted and restricted antibiotic consumption, especially carbapenems.

Atherogenicity of amino acids in the lipid-laden macrophage model system in vitro and in atherosclerotic mice: a key role for triglyceride metabolism.

Atherosclerosis-related research has focused mainly on the effects of lipids on macrophage foam cell formation and atherogenesis, whereas the role of amino acids (AAs) was understudied. The current study aimed to identify anti- or pro-atherogenic AA in the macrophage model system and to elucidate the underlying metabolic and molecular mechanisms. J774A.1 cultured macrophages were treated with increasing concentrations of each 1 of the 20 AAs. Macrophage atherogenicity was assessed in terms of cellular toxicity, generation of reactive oxygen species (ROS) and cellular cholesterol or triglyceride content. At nontoxic concentrations (up to 1 mM), modest effects on ROS generation or cholesterol content were noted, but six specific AAs significantly affected macrophage triglyceride content. Glycine, cysteine, alanine and leucine significantly decreased macrophage triglyceride content (by 24%-38%), through attenuated uptake of triglyceride-rich very low-density lipoprotein (VLDL) by macrophages. In contrast, glutamate and glutamine caused a marked triglyceride accumulation in macrophages (by 107% and 129%, respectively), via a diacylglycerol acyltransferase-1 (DGAT1)-dependent increase in triglyceride biosynthesis rate with a concurrent maturation of the sterol regulatory element-binding protein-1 (SREBP1). Supplementation of apolipoprotein E-deficient (apoE-/-) mice with glycine for 40 days significantly decreased the triglyceride levels in serum and in peritoneal macrophages (MPMs) isolated from the mice (by 19%). In contrast, glutamine supplementation significantly increased MPM ROS generation and the accumulation of cholesterol and that of triglycerides (by 48%), via enhanced uptake of LDL and VLDL. Altogether, the present findings reveal some novel roles for specific AA in macrophage atherogenicity, mainly through modulation of cellular triglyceride metabolism.

Supplementation with linoleic acid-rich soybean oil stimulates macrophage foam cell formation via increased oxidative stress and diacylglycerol acyltransferase1-mediated triglyceride biosynthesis.

During the last decades there has been a staggering rise in human consumption of soybean oil (SO) and its major polyunsaturated fatty acid linoleic acid (LA). The role of SO or LA in cardiovascular diseases is highly controversial, and their impact on macrophage foam cell formation, the hallmark of early atherogenesis, is unclear. To investigate the effects of high SO or LA intake on macrophage lipid metabolism and the related mechanisms of action, C57BL/6 mice were orally supplemented with increasing levels of SO-based emulsion or equivalent levels of purified LA for 1 month, followed by analyses of lipid accumulation and peroxidation in aortas, serum and in peritoneal macrophages (MPM) of the mice. Lipid peroxidation and triglyceride mass in aortas from SO or LA supplemented mice were dose-dependently and significantly increased. In MPM from SO or LA supplemented mice, lipid peroxides were significantly increased and a marked accumulation of cellular triglycerides was found in accordance with enhanced triglyceride biosynthesis rate and overexpression of diacylglycerol acyltransferase1 (DGAT1), the key enzyme in triglyceride biosynthesis. In cultured J774A.1 macrophages treated with SO or LA, triglyceride accumulated via increased oxidative stress and a p38 mitogen-activated protein kinase (MAPK)-mediated overexpression of DGAT1. Accordingly, anti-oxidants (pomegranate polyphenols), inhibition of p38 MAPK (by SB202190) or DGAT1 (by oleanolic acid), all significantly attenuated SO or LA-induced macrophage triglyceride accumulation. These findings reveal novel mechanisms by which supplementation with SO or LA stimulate macrophage foam cell formation, suggesting a pro-atherogenic role for overconsumption of SO or LA. © 2016 BioFactors, 43(1):100-116, 2017.

Acrolein increases macrophage atherogenicity in association with gut microbiota remodeling in atherosclerotic mice: protective role for the polyphenol-rich pomegranate juice.

The unsaturated aldehyde acrolein is pro-atherogenic, and the polyphenol-rich pomegranate juice (PJ), known for its anti-oxidative/anti-atherogenic properties, inhibits macrophage foam cell formation, the hallmark feature of early atherosclerosis. This study aimed to investigate two unexplored areas of acrolein atherogenicity: macrophage lipid metabolism and the gut microbiota composition. The protective effects of PJ against acrolein atherogenicity were also evaluated. Atherosclerotic apolipoprotein E-deficient (apoE-/-) mice that were fed acrolein (3 mg/kg/day) for 1 month showed significant increases in serum and aortic cholesterol, triglycerides, and lipid peroxides. In peritoneal macrophages isolated from the mice and in J774A.1 cultured macrophages, acrolein exposure increased intracellular oxidative stress and stimulated cholesterol and triglyceride accumulation via enhanced rates of their biosynthesis and over-expression of key regulators of cellular lipid biosynthesis: sterol regulatory element-binding proteins (SREBPs), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), and diacylglycerol acyltransferase1 (DGAT1). Acrolein-fed mice demonstrated a major shift in the gut microbiota composition, including a significant phylum-level change in increased Firmicutes and decreased Bacteroidetes. At the family level, acrolein significantly increased the prevalence of Ruminococcaceae and Lachnospiraceae of which the Coprococcus genus was significantly and positively correlated with serum, aortic and macrophage lipid levels and peroxidation. The pro-atherogenic effects of acrolein on serum, aortas, macrophages, and the gut microbiota were substantially abolished by PJ. In conclusion, these findings provide novel mechanisms by which acrolein increases macrophage lipid accumulation and alters the gut microbiota composition in association with enhanced atherogenesis. Moreover, PJ was found as an effective strategy against acrolein atherogenicity.

Aqueous or lipid components of atherosclerotic lesion increase macrophage oxidation and lipid accumulation.

INTRODUCTION AND OBJECTIVE: Understanding the interactions among atherosclerotic plaque components and arterial macrophages, is essential for elucidating the mechanisms involved in the development of atherosclerosis. We assessed the effects of lesion extracts on macrophages. METHODS: Mouse peritoneal macrophages from atherosclerotic normoglycemic or hyperglycemic apoE(-/-) mice were incubated with aortic aqueous or with aortic lipidic extracts (mAAE or mALE) derived from these mice. In parallel, J774A.1 cultured macrophages were incubated with increasing concentrations of extracts prepared from human carotid lesions: polar lesion aqueous extract (hLAE), nonpolar lesion lipid extract (hLLE), or with their combination. In all the above systems we performed analyses of macrophage oxidative status, cholesterol, and triglyceride metabolism. RESULTS: Aqueous or lipid extracts from either mice aorta or from human carotid lesions significantly increased macrophage oxidative stress as determined by reactive oxygen species (ROS) analysis. In parallel, a compensatory increase in the cellular antioxidant paraoxonase2 (PON2) activity and in macrophage glutathione content were observed following incubation with all extracts. Macrophage triglyceride mass and triglyceride biosynthesis rate were both significantly increased following treatment with the lipid extracts, secondary to upregulation of DGAT1. All extracts decreased cholesterol biosynthesis rate, through downregulation of HMGCR, the rate limiting enzyme in cholesterol biosynthesis. The combination of the human lesion extracts had the most significant effects. CONCLUSION: The present study demonstrates that atherosclerotic plaque constituents enhance macrophage cellular oxidative stress, and accumulation of cholesterol and triglycerides, as shown in both in vivo and in vitro model systems.

Paraoxonase 1 (PON1) and pomegranate influence circadian gene expression and period length.

The circadian timing system regulates key aspects of mammalian physiology. Here, we analyzed the effect of the endogenous antioxidant paraoxonase 1 (PON1), a high-density lipoprotein-associated lipolactonase that hydrolyses lipid peroxides and attenuates atherogenesis, on circadian gene expression in C57BL/6J and PON1KO mice fed a normal chow diet or a high-fat diet (HFD). Expression levels of core-clock transcripts Nr1d1, Per2, Cry2 and Bmal1 were altered in skeletal muscle in PON1-deficient mice in response to HFD. These findings were supported by circadian bioluminescence reporter assessments in mouse C2C12 and human primary myotubes, synchronized in vitro, where administration of PON1 or pomegranate juice modulated circadian period length.

Paraoxonase 2 Induces a Phenotypic Switch in Macrophage Polarization Favoring an M2 Anti-Inflammatory State.

Inflammatory processes are involved in atherosclerosis development. Macrophages play a major role in the early atherogenesis, and they are present in the atherosclerotic lesion in two phenotypes: proinflammatory (M1) or anti-inflammatory (M2). Paraoxonase 2 (PON2) is expressed in macrophages, and it was shown to protect against atherosclerosis. Thus, the aim of our study was to analyze the direct effect of PON2 on macrophage inflammatory phenotypes. Ex vivo studies were performed with murine peritoneal macrophages (MPM) harvested from control C57BL/6 and PON2-deficient (PON2KO) mice. PON2KO MPM showed an enhanced proinflammatory phenotype compared to the control, both in the basal state and following M1 activation by IFNγ and lipopolysaccharide (LPS). In parallel, PON2KO MPM also showed reduced anti-inflammatory responses in the basal state and also following M2 activation by IL-4. Moreover, the PON2-null MPM demonstrated enhanced phagocytosis and reactive oxygen species (ROS) production in the basal state and following M1 activation. The direct effect of PON2 was shown by transfecting human PON2 (hPON2) into PON2KO MPM. PON2 transfection attenuated the macrophages' response to M1 activation and enhanced M2 response. These PON2 effects were associated with attenuation of macrophages' abilities to phagocyte and to generate ROS. We conclude that PON2 promotes an M1 to M2 switch in macrophage phenotypes.