The Rappaport Family Institute for Research in the Medical science

Haifa, Israel

The Rappaport Family Institute for Research in the Medical science

Haifa, Israel
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Meilin E.,The Rappaport Family Institute for Research in the Medical Science | Aviram M.,The Rappaport Family Institute for Research in the Medical Science | Hayek T.,The Rappaport Family Institute for Research in the Medical Science
BioFactors | Year: 2011

Diabetes mellitus (DM) is a major risk factor for the development of atherosclerosis, and high-serum levels of insulin are strongly associated with type 2 DM. Atherosclerosis is characterized by lipid-laden macrophage foam cell formations, which contain substantial amount of cholesterol and triglycerides (TG). This study analyzed for the first time, the effects of insulin on TG metabolism in macrophages under normal and diabetic conditions. Mouse peritoneal macrophages from C57BL6 mice were cultured under normal (5 mM) or high (diabetic condition, 25 mM) glucose concentration, with or without insulin, followed by the assessment of TGs metabolism in these cells. Under diabetic condition, insulin increased TG accumulation in macrophages by 100%, decreased cellular TG degradation by 21%, and increased C-reactive protein levels in macrophages by 83%. Insulin decreased hormone-sensitive lipase mRNA and protein expression by 28 and 60%, respectively, and adipose TG lipase (ATGL) protein expression by 36%, with no significant reduction in ATGL mRNA levels. The inhibition of insulin-mediated phosphorylation, and the addition of cyclic adenosine 3'5'-monoposphate, abolished the insulin-mediated inhibition of TGs degradation in cells. Insulin increases macrophage TGs accumulation only under diabetic conditions, suggesting that impaired glycemic control in diabetic patients treated with insulin may contribute to foam cell formations and enhanced inflammation in macrophages. © 2011 International Union of Biochemistry and Molecular Biology, Inc.


Chulsky S.,The Rappaport Family Institute for Research in the Medical science | Paland N.,The Rappaport Family Institute for Research in the Medical science | Lazarovich A.,The Rappaport Family Institute for Research in the Medical science | Fuhrman B.,The Rappaport Family Institute for Research in the Medical science
Atherosclerosis | Year: 2014

Objectives: The aim of the present study was to investigate the effect of urokinase-type plasminogen activator (uPA) on the expression of the scavenger receptor class B type I (SR-BI) in hepatocytes, and its impact on the removal of HDL-cholesteryl ester (CE) in the liver. Methods and results: Huh7 hepatoma cell lines were incubated with increasing concentrations of uPA. uPA dose-dependently decreased SR-BI protein expression, as determined by flow cytometry (FACS) and by Western blot assays, and down-regulated SR-BI gene expression. Functionally, uPA decreased both the cellular binding of HDL to Huh7 hepatocytes, and the selective uptake of CE from HDL, as determined by several methods including BODIPY staining, cellular cholesterol determination and chasing radio-labeled CE transfer from HDL to the cells. These results were further confirmed using primary rat hepatocyes. The effect of uPA on hepatic SR-BI expression was mediated via binding to the uPA receptor (uPAR). Invivo, SR-BI protein and gene expressions were found to be increased in hepatocytes derived from the uPAR-KO mice compared to C57Bl/6 mice, and in parallel HDL-cholesterol levels in plasma derived from uPAR-KO mice were decreased. Moreover, deficiency of uPAR significantly accelerated the plasma decay of injected HDL-[3H]CE. Conclusions: The results of this study suggest that uPA decreases the removal of HDL-CE in the liver via suppression of the hepatic SR-BI expression. Impaired reverse cholesterol transport (RCT) may result in atherogenic dysfunctional HDL metabolism and may contribute to atherosclerosis development. © 2013 Elsevier Ireland Ltd.


Rosenblat M.,The Rappaport Family Institute for Research in the Medical science | Volkova N.,The Rappaport Family Institute for Research in the Medical science | Aviram M.,The Rappaport Family Institute for Research in the Medical science
Atherosclerosis | Year: 2010

Objective: To examine whether the beneficial effects of PJ consumption by mice on their macrophages are mediated via PJ-induced increment in serum paraoxonase 1 (PON1) activity and/or in macrophage PON2 expression. Methods and results: We performed studies in peritoneal macrophages (MPM) from C57BL/6 control mice, or from PON1KO mice, or from PON2KO mice that consumed PJ (200 μg of gallic acid equivalents/mouse/day, for 1 month period).PJ consumption by C57BL/6 mice resulted in a significant increment, by 36% in serum PON1 catalytic activities, and upregulated MPM PON2 expression.In MPM from C57BL/6 or from PON1KO mice that consumed PJ, the extent of cell-mediated LDL oxidation was decreased by 22%, and that of cellular superoxide release by 20-26%. In contrast, PJ consumption by PON2KO mice resulted in a minimal inhibitory effect on macrophage oxidative stress by only 4-9%. Unlike PJ antioxidative effects in MPM, PJ anti-atherogenic effects on MPM cholesterol and triglyceride metabolism were similar in all mice groups that consumed PJ. After PJ consumption, cellular cholesterol content was decreased by 14-19%, and this could be attributed to a significant inhibition in MPM cholesterol biosynthesis rate by 20-32%, and/or to stimulation of HDL-mediated cholesterol efflux from the cells by 22-37%. Similarly, MPM triglyceride content and triglyceride biosynthesis rate were both significantly decreased after PJ consumption, by 16-27% and by 22-28%, respectively. Conclusion: PJ consumption antioxidative properties on mouse macrophages, but not PJ beneficial effects on macrophage cholesterol and triglyceride metabolism, are mediated via PJ-induced stimulation of macrophage PON2 expression. Serum PON1 stimulation by PJ consumption, however, was not involved in PJ-induced effects on macrophages. © 2010 Elsevier Ireland Ltd.


Khateeb J.,The Rappaport Family Institute for Research in the Medical science | Gantman A.,The Rappaport Family Institute for Research in the Medical science | Kreitenberg A.J.,The Rappaport Family Institute for Research in the Medical science | Aviram M.,The Rappaport Family Institute for Research in the Medical science | Fuhrman B.,The Rappaport Family Institute for Research in the Medical science
Atherosclerosis | Year: 2010

Objective: Serum paraoxonase-1 (PON1) expression is regulated by polyphenols, shown to activate the peroxisome proliferator-activated receptor γ (PPARγ). Pomegranate juice (PJ) is a polyphenol-rich fruit. Because promoter sequence of PON1 gene indicates that it could be regulated by nuclear receptors, we investigated the effect of PJ polyphenols on PON1 gene expression in HuH7 hepatocytes. Methods and results: PON1 protein or mRNA expression, determined by immunocytochemistry, or quantitative PCR, respectively, as well as PON1 gene promoter activation, was significantly increased in hepatocytes incubated with PJ or with its major polyphenols punicalagin, or gallic acid (GA). Ellagic acid (EA) elicited only modest stimulatory effect. Accordingly, PJ, punicalagin, GA, and less so EA, dose-dependently increased cell-associated and hepatocyte-secreted PON1 arylesterase activity. Functionally, the secreted PON1 exhibited biological activity by protecting LDL and HDL from oxidation. Finally, PJ polyphenols upregulated the hepatocyte PON1 expression, at least in part, via the intracellular signaling cascade PPARγ-PKA-cAMP. Conclusions: This study shows for the first time that PJ polyphenols have a specific transcriptional role in hepatocyte PON1 expression upregulation, and its secretion to the medium. We conclude that the anti-atherogenic characteristics of PJ polyphenols are modulated, at least in part, via hepatocyte PON1 upregulation and its subsequent release to the medium. © 2009 Elsevier Ireland Ltd. All rights reserved.


Gantman A.,The Rappaport Family Institute for Research in the Medical science | Fuhrman B.,The Rappaport Family Institute for Research in the Medical science | Aviram M.,The Rappaport Family Institute for Research in the Medical science | Hayek T.,The Rappaport Family Institute for Research in the Medical science
Biochemical and Biophysical Research Communications | Year: 2010

Aims: Diabetes is associated with atherogenesis and macrophage-foam cell formation, due in part to a decrease in HDL-mediated cholesterol efflux from macrophages. This study examined the expression of proteins involved in cholesterol transport, i.e. ABCA1 and SR-BI, under diabetic conditions. Methods and results: ABCA1 expression was similar, whereas SR-BI expression (mRNA and protein) was significantly increased in mouse peritoneal macrophages (MPM) harvested from C57Bl/6 diabetic mice, compared to MPM from control non-diabetic mice. Similar results were obtained in vitro in J-774A.1 macrophage-like cell line incubated with high (30 mM) vs. low (5 mM) glucose concentrations. Accordingly, association and internalization of HDL to MPM from diabetic mice, or to J-774A.1 macrophages grown under diabetic conditions was significantly higher compared to control cells. Unexpectedly, however, increased macrophage SR-BI expression was associated with a substantial reduction in HDL-mediated cholesterol efflux from the macrophages. Moreover, total cellular cholesterol content was increased by 28% in macrophages incubated with HDL under high glucose concentrations, compared to low glucose concentrations. This effect was abolished by a rabbit polyclonal anti-SR-BI, which blocks binding to the receptor, or alternatively by using BLT1, a specific inhibitor of lipid transport via the SR-BI. Conclusions: Diabetes stimulates the expression of SR-BI in macrophages and leads to a shift in its activity from HDL-mediated cholesterol efflux to HDL-mediated cholesterol influx. These effects may lead to increased foam cell formation and atherosclerosis development. © 2009 Elsevier Inc. All rights reserved.


Aharoni S.,The Rappaport Family Institute for Research in the Medical science | Lati Y.,The Rappaport Family Institute for Research in the Medical science | Aviram M.,The Rappaport Family Institute for Research in the Medical science | Fuhrman B.,The Rappaport Family Institute for Research in the Medical science
BioFactors | Year: 2015

It was documented that pomegranate has anti-inflammatory effects. In this study, we investigated a direct effect of pomegranate juice (PJ) and its polyphenols on macrophage inflammatory phenotype. In vitro, PJ and its major polyphenols dose-dependently attenuated macrophage response to M1 proinflammatory activation in J774.A1 macrophage-like cell line. This was evidenced by a significant decrease in TNFα and IL-6 secretion in response to stimulation by IFNγ and Lipopolysaccharide. In addition, PJ and punicalagin dose-dependently promoted the macrophages toward a M2 anti-inflammatory phenotype, as determined by a significant increase in the spontaneous secretion of IL-10. In mice, supplementation with dietary PJ substantially inhibited the M2 to M1macrophage phenotypic shift associated with age, toward a favorable anti-inflammatory M2 phenotype. This effect was also reflected in the mice atherosclerotic plaques, as evaluated by the distinct expression of arginase isoforms. PJ consumption inhibited the increment of arginase II (Arg II, M1) mRNA expression during aging, and maintained the levels of Arg I (M2) expression similar to those in young mice aorta. This study demonstrates, for the first time, that pomegranate polyphenols directly suppress macrophage inflammatory responses and promote M1 to M2 switch in macrophage phenotype. Furthermore, this study indicates that PJ consumption may inhibit the progressive proinflammatory state in the aorta along atherosclerosis development with aging, due to a switch in macrophage phenotype from proinflammatory M1to anti-inflammatory M2. © 2015 International Union of Biochemistry and Molecular Biology.


Koren-Gluzer M.,The Rappaport Family Institute for Research in the Medical science | Aviram M.,The Rappaport Family Institute for Research in the Medical science | Hayek T.,The Rappaport Family Institute for Research in the Medical science
Biochemical and Biophysical Research Communications | Year: 2013

The aim of the present study was to analyze the metformin (MF) effect on two cellular atherogenic activities: cholesterol biosynthesis and oxidative-stress (OS) as studied in J774A.1 macrophage cell line. MF (2-5. mM) significantly and dose-dependently reduced macrophage cholesterol content and cholesterol biosynthesis rate from acetate, but not from mevalonate, by up to 68% and 71%, respectively. MF inhibitory effect on cholesterol biosynthesis was similar to that of simvastatin. In contrast to the above anti-atherogenic MF effect, MF significantly increased cellular OS as shown by enhancement of reactive oxygen species (ROS) production by up to 70%, and decrement in cellular reduced glutathione (GSH) levels by up to 67%. Macrophage paraoxonase2 (PON2) expression however, increased by MF, by up to 1.5 folds. To overcome the MF oxidation stimulation, macrophages were incubated with MF together with potent dietary antioxidants, i.e. -5. μg GAE/ml of pomegranate juice (PJ) or 30. μM of vitamin E (VE). Both of these potent antioxidants substantially reduced MF-induced OS, and in parallel, abolished MF inhibitory effect on cholesterol biosynthesis rate. We thus conclude that the inhibition of macrophage cholesterol biosynthesis by MF is related, at least in part, to MF-induced OS. © 2013 Elsevier Inc.


Koren-Gluzer M.,The Rappaport Family Institute for Research in the Medical science | Aviram M.,The Rappaport Family Institute for Research in the Medical science | Meilin E.,The Rappaport Family Institute for Research in the Medical science | Hayek T.,The Rappaport Family Institute for Research in the Medical science
Atherosclerosis | Year: 2011

Objective: To analyze the direct effects of paraoxonase-1 (PON1) on diabetes development and on β-cell insulin release. Methods and results: Injection of rePON1 to mice, prior to STZ-induced diabetes, resulted in reduced incidence of diabetes, as well as, in higher serum insulin levels. Incubation of β-cells with PON1 also dose-dependently increased insulin secretion and its cellular content. PON1 increased cell survival under high glucose levels, but not under high STZ concentrations. The addition of the PON1 carrier in the circulation - HDL, to βTC3 cell line, had an additive effect on PON1-induced insulin secretion. PON1 administration to mice or incubation with β-cells was associated with a substantial decreased oxidative stress. Just like PON1, the dietary anti-oxidants, pomegranate juice, punicalagin (major polyphenol in pomegranate) or vitamin E, also increased insulin release from βTC3, but unlike PON1, failed to increase insulin cellular content, suggesting a possible role for PON1 in insulin biosynthesis, separately from PON1 antioxidative effect. Both, PON1 catalytic activity and PON1 association to HDL, were not required for PON1 stimulation of insulin release from β-cells. However, the PON1 free sulfhydryl group was shown to be essential for insulin release by PON1, as blocking the PON1 SH group, abolished PON1 stimulatory effect on insulin secretion. Conclusion: PON1 is a potent anti-diabetic enzyme that exerts this protection against diabetes through its antioxidative, as well as via its insulin stimulation properties on β-cells. © 2011 Elsevier Ireland Ltd.


Rosenblat M.,The Rappaport Family Institute for Research in the Medical science | Volkova N.,The Rappaport Family Institute for Research in the Medical science | Aviram M.,The Rappaport Family Institute for Research in the Medical science
BioFactors | Year: 2011

We analyzed, for the first time, the effects of recombinant PON1 (rePON1) intraperitoneal injection to C57BL/6 mice on their HDL and macrophage antiatherogenic properties. Thioglycolate-treated mice were injected with either saline (Control), or rePON1 (50 μg/mouse), and 20 H post injection, their blood samples and peritoneal macrophages (MPM) were collected. A significant increase in serum and HDL-PON1 arylesterase and lactonase activities was noted. Similarly, a significant increment, by 3.8 and 2.8 fold, in MPM-PON1 arylesterase and lactonase activities, respectively, as compared to the activities in control MPM was observed. The HDL from rePON1-injected mice was resistant to oxidation by copper ions as compared to control HDL. Furthermore, enrichment of the mouse HDL with rePON1 increased its ability to induce cholesterol efflux from J774A.1 macrophage cell line, and to inhibit macrophage-mediated LDL oxidation. In MPM from rePON1-injected mice vs. control MPM, there was a significant reduction in cholesterol mass, by 42%, in association with inhibition in cellular cholesterol biosynthesis rate, by 33%, and with significant stimulation, by 65%, of human HDL-mediated cholesterol efflux from the cells. We conclude that rePON1 injection to mice improved the mice HDL and MPM antiatherogenic properties, and these effects could probably lead to attenuation of atherosclerosis development. © 2011 International Union of Biochemistry and Molecular Biology, Inc.


Rosenblat M.,The Rappaport Family Institute for Research in the Medical science | Volkova N.,The Rappaport Family Institute for Research in the Medical science | Ward J.,The Rappaport Family Institute for Research in the Medical science | Aviram M.,The Rappaport Family Institute for Research in the Medical science
Atherosclerosis | Year: 2011

Objective: To analyze paraoxonase 1 (PON1) effect on monocyte-to-macrophage differentiation. Methods and results: THP-1 monocytic cell-line and mouse peritoneal macrophages (MPM) were studied. Markers for monocytes differentiation included: morphological changes, CD11b and CD36 expression, and cellular oxidative stress. PON1KO MPM were more differentiated than control C57BL/6 MPM. Intraperitoneal injection of recombinant PON1 (rePON1) to C57BL/6 or to PON1KO mice significantly increased serum, MPM, and tissues PON1 activities. These effects were associated with a significant decrease in CD11b in C57BL/6 and PON1KO MPM (by 21% and 35%, respectively), in CD36 (by 35% and 38%, respectively), and in cellular total peroxides content (by 18% and 20%, respectively). rePON1 also significantly inhibited CD11b and CD36 expression, and cellular total peroxides during PMA-induced THP-1 monocytes differentiation, by 68%, 56% and 53%, respectively. Similar effects were observed upon using reconstituted HDL (rHDL) +rePON1, or human HDL +rePON1, in comparison to rHDL or to human HDL, as well as, HDL from C57BL/6 vs. PON1KO mice. Inhibition of monocyte-to-macrophage differentiation was demonstrated also by several dietary antioxidants such as vitamin E, gallic acid, or punicalagin (the major polyphenol in pomegranate). Whereas NADPH oxidase was not involved in PON1 anti-differentiation effect, mitochondrial complex I could be involved, as rotenone (complex I inhibitor) significantly decreased (by 77%) the expression of CD11b during THP-1 differentiation. Finally, blocking PON1 sulfhydryl group with N-ethylmalemide significantly attenuated PON1 inhibitory effect on THP-I monocyte-to-macrophage differentiation. Conclusion: HDL-associated PON1 inhibits monocyte-to-macrophage differentiation, and this effect could be related to PON1 peroxidase-like activity which involves its free sulfhydryl group. © 2011 Elsevier Ireland Ltd.

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