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Manganese Supplementation Reduces High Glucose-induced Monocyte Adhesion to Endothelial Cells and Endothelial Dysfunction in Zucker Diabetic Fatty Rats

Open AccessPublished:January 17, 2013DOI:https://doi.org/10.1074/jbc.M112.447805
      Endothelial dysfunction is a hallmark of increased vascular inflammation, dyslipidemia, and the development of atherosclerosis in diabetes. Previous studies have reported lower levels of Mn2+ in the plasma and lymphocytes of diabetic patients and in the heart and aortic tissue of patients with atherosclerosis. This study examines the hypothesis that Mn2+ supplementation can reduce the markers/risk factors of endothelial dysfunction in type 2 diabetes. Human umbilical vein endothelial cells (HUVECs) were cultured with or without Mn2+ supplementation and then exposed to high glucose (HG, 25 mm) to mimic diabetic conditions. Mn2+ supplementation caused a reduction in monocyte adhesion to HUVECs treated with HG or MCP-1. Mn2+ also inhibited ROS levels, MCP-1 secretion, and ICAM-1 up-regulation in HUVECs treated with HG. Silencing studies using siRNA against MnSOD showed that similar results were observed in MnSOD knockdown HUVECs following Mn2+ supplementation, suggesting that the effect of manganese on monocyte adhesion to endothelial cells is mediated by ROS and ICAM-1, but not MnSOD. To validate the relevance of our findings in vivo, Zucker diabetic fatty rats were gavaged daily with water (placebo) or MnCl2 (16 mg/kg of body weight) for 7 weeks. When compared with placebo, Mn2+-supplemented rats showed lower blood levels of ICAM-1 (17%, p < 0.04), cholesterol (25%, p < 0.05), and MCP-1 (28%, p = 0.25). These in vitro and in vivo studies demonstrate that Mn2+ supplementation can down-regulate ICAM-1 expression and ROS independently of MnSOD, leading to a decrease in monocyte adhesion to endothelial cells, and therefore can lower the risk of endothelial dysfunction in diabetes.
      Background: Mn2+ levels are lower in blood of diabetic and atherosclerosis patients.
      Results: Mn2+ supplementation reduces monocyte adhesion in endothelial cells by down-regulating ROS, ICAM-1 expression, and MCP-1 secretion, and lowers blood levels of ICAM-1 and cholesterol in ZDF rats.
      Conclusion: Mn2+ supplementation is beneficial in lowering markers of endothelial dysfunction.
      Significance: Mn2+ supplementation can potentially prevent or delay progression of atherosclerosis.

      Introduction

      Manganese is an essential micronutrient that serves as a cofactor for many enzyme systems. Metalloenzymes, or manganese-containing enzymes, such as arginase, pyruvate carboxylase, and manganese superoxide dismutase (MnSOD),
      • Anetor J.I.
      • Asiribo O.A.
      • Adedapo K.S.
      • Akingbola T.S.
      • Olorunnisola O.S.
      • Adeniyi F.A.
      Increased plasma manganese, partially reduced ascorbate,1 and absence of mitochondrial oxidative stress in type 2 diabetes mellitus: implications for the superoxide uncoupling protein 2 (UCP-2) pathway.
      require Mn2+ to function. MnSOD is the major mitochondrial antioxidant and is responsible for protecting the cell from reactive oxygen species (ROS) by scavenging mitochondrial superoxide (
      • Fridovich I.
      Superoxide anion radical (O), superoxide dismutases, and related matters.
      ). MnSOD acts by catalyzing the conversion of superoxide radicals (such as O2) to hydrogen peroxide, which is further metabolized to water by other antioxidant enzymes such as catalase and glutathione peroxidase (
      • Anetor J.I.
      • Asiribo O.A.
      • Adedapo K.S.
      • Akingbola T.S.
      • Olorunnisola O.S.
      • Adeniyi F.A.
      Increased plasma manganese, partially reduced ascorbate,1 and absence of mitochondrial oxidative stress in type 2 diabetes mellitus: implications for the superoxide uncoupling protein 2 (UCP-2) pathway.
      ). At low concentrations, Mn2+ ions have been shown to have antioxidant properties with the ability to scavenge superoxide and hydroxyl radicals (
      • Hussain S.
      • Ali S.F.
      Manganese scavenges superoxide and hydroxyl radicals: an in vitro study in rats.
      ). Several studies have reported that changes in dietary Mn2+ induced changes in MnSOD activity and that MnSOD activity was reduced in heart and livers of Mn2+-deficient animals (
      • Paynter D.I.
      Changes in activity of the manganese superoxide dismutase enzyme in tissues of the rat with changes in dietary manganese.
      ,
      • Davis C.D.
      • Ney D.M.
      • Greger J.L.
      Manganese, iron, and lipid interactions in rats.
      • Zidenberg-Cherr S.
      • Keen C.L.
      • Lönnerdal B.
      • Hurley L.S.
      Superoxide dismutase activity and lipid peroxidation in the rat: developmental correlations affected by manganese deficiency.
      ). Previous studies report lower levels of Mn2+ in the plasma and lymphocytes of type 2 diabetic patients (
      • Ekmekcioglu C.
      • Prohaska C.
      • Pomazal K.
      • Steffan I.
      • Schernthaner G.
      • Marktl W.
      Concentrations of seven trace elements in different hematological matrices in patients with type 2 diabetes as compared to healthy controls.
      ,
      • Kazi T.G.
      • Afridi H.I.
      • Kazi N.
      • Jamali M.K.
      • Arain M.B.
      • Jalbani N.
      • Kandhro G.A.
      Copper, chromium, manganese, iron, nickel, and zinc levels in biological samples of diabetes mellitus patients.
      ) and in the heart and aortic tissue of patients with atherosclerosis when compared with those of healthy controls (
      • Volkov N.F.
      Cobalt, manganese, and zinc content in the blood of atherosclerosis patients.
      ). Other studies have also shown beneficial effect of Mn2+ on lipid metabolism and a decrease in total serum cholesterol, aorta cholesterol, and regression of atherosclerosis following manganese supplementation in cholesterol-fed rabbits (
      • Bomb B.S.
      • Kumawat D.C.
      • Bomb P.
      • Taly A.B.
      • Bedi T.
      • Bedi H.K.
      Effect of manganese on regression of atherosclerosis in cholesterol fed rabbits.
      ). The mechanisms by which Mn2+ can reduce cholesterol, however, are unknown. Also, a possible beneficial role of Mn2+ supplementation alone (without MnSOD) on vascular inflammation has never been investigated. Endothelial dysfunction and vascular inflammation, characterized by monocyte adhesion to endothelial cells and increased levels of MCP-1, ROS, and ICAM-1, are known to play a significant role in the development of atherosclerosis (
      • Peluso I.
      • Morabito G.
      • Urban L.
      • Ioannone F.
      • Serafini M.
      Oxidative stress in atherosclerosis development: the central role of LDL and oxidative burst.
      ,
      • Panee J.
      Monocyte chemoattractant protein 1 (MCP-1) in obesity and diabetes.
      • Rains J.L.
      • Kanikarla-Marie P.
      • Jain S.K.
      Hyperketonemia induces upregulation of LFA-1 in monocytes, which is mediated by ROS and p38 MAPK activation.
      ). This study examines the hypothesis that Mn2+ supplementation can prevent vascular inflammation and endothelial dysfunction in type 2 diabetes and that Mn2+ can have a beneficial role independently of MnSOD. Our results demonstrate that Mn2+ supplementation reduces ROS levels, MCP-1 secretion, ICAM-1 expression, and the adhesion of monocytes to endothelial cells. Furthermore, similar results were obtained in MnSOD knockdown human umbilical vein endothelial cells (HUVECs). Further studies in vivo showed that Mn2+ supplementation lowers blood levels of ICAM-1 and cholesterol in Zucker diabetic fatty rats. These in vitro and in vivo studies demonstrate that Mn2+ supplementation can lower markers of oxidative stress and endothelial dysfunction, such as monocyte adhesion to endothelial cells, ICAM-1, ROS, MCP-1, and cholesterol, thereby lowering the risk of endothelial dysfunction in diabetes. We also show for the first time that Mn2+ supplementation can have beneficial effects on endothelial cells independently of MnSOD.

      DISCUSSION

      Complications of atherosclerosis cause most morbidity and mortality in patients with diabetes (
      • Beckman J.A.
      • Creager M.A.
      • Libby P.
      Diabetes and atherosclerosis: epidemiology, pathophysiology, and management.
      ). More than 25 million persons in the United States have at least one clinical manifestation of atherosclerosis (
      • Faxon D.P.
      • Creager M.A.
      • Smith Jr., S.C.
      • Pasternak R.C.
      • Olin J.W.
      • Bettmann M.A.
      • Criqui M.H.
      • Milani R.V.
      • Loscalzo J.
      • Kaufman J.A.
      • Jones D.W.
      • Pearce W.H.
      Atherosclerotic Vascular Disease Conference: Executive Summary: Atherosclerotic Vascular Disease Conference proceeding for healthcare professionals from a special writing group of the American Heart Association.
      ). The key early event in the development of atherosclerosis is dysfunction of the endothelium, which is characterized by increased expression of cellular adhesion molecules, such as ICAM-1, and secretion of chemokines such as MCP-1. These events lead to the recruitment of monocytes to the arterial wall where they become macrophages and initiate chronic inflammation, leading to hyperlipidemia and atherosclerotic lesion development.
      Various studies report blood concentrations of manganese between 0.15 and 7 μm (
      • Ekmekcioglu C.
      • Prohaska C.
      • Pomazal K.
      • Steffan I.
      • Schernthaner G.
      • Marktl W.
      Concentrations of seven trace elements in different hematological matrices in patients with type 2 diabetes as compared to healthy controls.
      ,
      • Kazi T.G.
      • Afridi H.I.
      • Kazi N.
      • Jamali M.K.
      • Arain M.B.
      • Jalbani N.
      • Kandhro G.A.
      Copper, chromium, manganese, iron, nickel, and zinc levels in biological samples of diabetes mellitus patients.
      ,
      • Walter Jr., R.M.
      • Uriu-Hare J.Y.
      • Olin K.L.
      • Oster M.H.
      • Anawalt B.D.
      • Critchfield J.W.
      • Keen C.L.
      Copper, zinc, manganese, and magnesium status and complications of diabetes mellitus.
      ,
      • Nahar Z.
      • Azad M.A.
      • Rahman M.A.
      • Rahman M.A.
      • Bari W.
      • Islam S.N.
      • Islam M.S.
      • Hasnat A.
      Comparative analysis of serum manganese, zinc, calcium, copper, and magnesium level in panic disorder patients.
      ). Some studies have reported lower Mn2+ levels in lymphocytes (
      • Ekmekcioglu C.
      • Prohaska C.
      • Pomazal K.
      • Steffan I.
      • Schernthaner G.
      • Marktl W.
      Concentrations of seven trace elements in different hematological matrices in patients with type 2 diabetes as compared to healthy controls.
      ), plasma, and hair samples (
      • Kazi T.G.
      • Afridi H.I.
      • Kazi N.
      • Jamali M.K.
      • Arain M.B.
      • Jalbani N.
      • Kandhro G.A.
      Copper, chromium, manganese, iron, nickel, and zinc levels in biological samples of diabetes mellitus patients.
      ) of type 2 diabetic patients and in the heart and aorta tissue of patients with atherosclerosis when compared with those of healthy controls (
      • Volkov N.F.
      Cobalt, manganese, and zinc content in the blood of atherosclerosis patients.
      ). Manganese supplementation has been shown to cause a decrease in total serum cholesterol and aorta cholesterol and regression of atherosclerosis in cholesterol-fed rabbits (
      • Bomb B.S.
      • Kumawat D.C.
      • Bomb P.
      • Taly A.B.
      • Bedi T.
      • Bedi H.K.
      Effect of manganese on regression of atherosclerosis in cholesterol fed rabbits.
      ). However, there is no evidence showing a direct beneficial role of Mn2+ supplementation on endothelial function and vascular inflammation, and the mechanisms by which Mn2+ can reduce cholesterol levels are unknown. MCP-1 is a chemokine that promotes the recruitment of monocytes and macrophages to the subendothelial cell layer. Deposition of lipids within these monocytes and macrophages then leads to development of atherosclerotic lesions. MCP-1 is also produced after induction of oxidative stress or growth factors by a variety of cell types, including monocytes, smooth muscle cells, and endothelial cells, and plays an important role in vascular inflammation and atherosclerotic lesion formation (
      • Rollins B.J.
      Chemokines.
      • Xing L.
      • Remick D.G.
      Promoter elements responsible for antioxidant regulation of MCP-1 gene expression.
      ,
      • Lefer D.J.
      • Granger D.N.
      Monocyte rolling in early atherogenesis: Vital role in lesion development.
      • Egashira K.
      Molecular mechanisms mediating inflammation in vascular disease: Special reference to monocyte chemoattractant protein-1.
      ). Mn2+ is a potent antioxidant; it is the cofactor of the enzyme MnSOD, the main antioxidant enzyme in the mitochondria, and can also scavenge oxygen radicals itself. Various studies using Mn2+ link its effects with the function and role of MnSOD (
      • Paynter D.I.
      Changes in activity of the manganese superoxide dismutase enzyme in tissues of the rat with changes in dietary manganese.
      ,
      • Zidenberg-Cherr S.
      • Keen C.L.
      • Lönnerdal B.
      • Hurley L.S.
      Superoxide dismutase activity and lipid peroxidation in the rat: developmental correlations affected by manganese deficiency.
      ).
      This study demonstrates for the first time that Mn2+ supplementation down-regulates ICAM-1 expression, reduces ROS production, MCP-1 secretion, and monocyte adhesion in endothelial cells exposed to high glucose, and lowers blood levels of ICAM-1 and cholesterol in ZDF rats. In addition, our study provides a molecular mechanism for the beneficial effects of Mn2+ supplementation on lowering vascular inflammation markers in HUVECs and ZDF rats. We found that Mn2+ supplementation inhibits secretion of MCP-1 in endothelial cells and adipocyte cells and that the MCP-1-induced up-regulation of ICAM-1 expression and monocyte adhesion to endothelial cells can be inhibited with Mn2+ supplementation. Interestingly, Mn2+ supplementation also inhibited both the ICAM-1 down-regulation and the monocyte adhesion induced by exogenous MCP-1 treatment in endothelial cells. This demonstrates that the inhibition of MCP-1 secretion caused by Mn2+ supplementation may mediate the down-regulation of ICAM-1 expression and monocyte adhesion in endothelial cells.
      Hyperglycemia is known to increase oxidative stress and glycation of protein (
      • Jain S.K.
      • McVie R.
      • Duett J.
      • Herbst J.J.
      Erythrocyte membrane lipid peroxidation and glycosylated hemoglobin in diabetes.
      ,
      • Jain S.K.
      Hyperglycemia can cause membrane lipid peroxidation and osmotic fragility in human red blood cells.
      • Jain S.K.
      • Palmer M.
      The effect of oxygen radicals metabolites and vitamin E on glycosylation of proteins.
      ). In this study, we observed that ROS, which was inhibited by Mn2+ supplementation, was increased in HG-treated HUVEC cells. Also, the decrease in MnSOD activity observed could be due to increase in oxidative stress or glycation of MnSOD. This study reports that Mn2+ supplementation increases MnSOD activity in HG-treated HUVECs. However, further investigation shows that the effect of Mn2+ on monocyte adhesion to endothelial cells is independent of MnSOD. Although activity of MnSOD increases in cells supplemented with Mn2+, similar effects of Mn2+ supplementation on monocyte adhesion to endothelial cells, inhibition of ICAM-1 and ROS levels, were observed in MnSOD knockdown cells, suggesting that MnSOD does not play a role in the decreased monocyte-endothelial cell adhesion in Mn2+-supplemented endothelial cells.
      This study suggests that the effects of Mn2+ supplementation on monocyte adhesion to endothelial cells are mediated by the inhibition of ROS and ICAM-1 expression. This study also demonstrates that Mn2+ supplementation significantly lowers blood levels of ICAM-1 and cholesterol in ZDF rats. Thus, this study provides a novel molecular mechanism by which Mn2+ supplementation can prevent or delay endothelial dysfunction and atherosclerosis development using both cell culture and in vivo studies.
      The findings reported in this study could have broader significance. In addition to atherosclerosis, adhesion molecules such as ICAM-1 are also implicated in the progression of infection (
      • Bonazzi M.
      • Cossart P.
      Impenetrable barriers or entry portals? The role of cell-cell adhesion during infection.
      ,
      • Golias C.
      • Batistatou A.
      • Bablekos G.
      • Charalabopoulos A.
      • Peschos D.
      • Mitsopoulos P.
      • Charalabopoulos K.
      Physiology and pathophysiology of selectins, integrins, and IgSF cell adhesion molecules focusing on inflammation. A paradigm model on infectious endocarditis.
      • Khan A.G.
      • Pickl-Herk A.
      • Gajdzik L.
      • Marlovits T.C.
      • Fuchs R.
      • Blaas D.
      Human rhinovirus 14 enters rhabdomyosarcoma cells expressing ICAM-1 by a clathrin-, caveolin-, and flotillin-independent pathway.
      ). A recent study reported that manganese blocks intracellular trafficking of Shiga toxin and that manganese-supplemented mice were completely resistant to a lethal Shiga toxin challenge (
      • Mukhopadhyay S.
      • Linstedt A.D.
      Manganese blocks intracellular trafficking of shiga toxin and protects against Shiga toxicosis.
      ). Whether manganese inhibition of ICAM-1 expression occurs in other cell types and can play a beneficial role in preventing the progression of infection is not known and warrants investigation.

      Acknowledgments

      We thank Georgia Morgan for excellent editing of this manuscript.

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