BackgroundMacrophage‐mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg‐II) in macrophage function remains elusive. This study characterizes the role of Arg‐II in macrophage inflammatory responses and its impact on obesity‐linked type II diabetes mellitus and atherosclerosis.Methods and ResultsIn human monocytes, silencing Arg‐II decreases the monocytes’ adhesion to endothelial cells and their production of proinflammatory mediators stimulated by oxidized low‐density lipoprotein or lipopolysaccharides, as evaluated by real‐time quantitative reverse transcription‐polymerase chain reaction and enzyme‐linked immunosorbent assay. Macrophages differentiated from bone marrow cells of Arg‐II–deficient (Arg‐II−/−) mice express lower levels of lipopolysaccharide‐induced proinflammatory mediators than do macrophages of wild‐type mice. Importantly, reintroducing Arg‐II cDNA into Arg‐II−/− macrophages restores the inflammatory responses, with concomitant enhancement of mitochondrial reactive oxygen species. Scavenging of reactive oxygen species by N‐acetylcysteine prevents the Arg‐II–mediated inflammatory responses. Moreover, high‐fat diet–induced infiltration of macrophages in various organs and expression of proinflammatory cytokines in adipose tissue are blunted in Arg‐II−/− mice. Accordingly, Arg‐II−/− mice reveal lower fasting blood glucose and improved glucose tolerance and insulin sensitivity. Furthermore, apolipoprotein E (ApoE)–deficient mice with Arg‐II deficiency (ApoE−/−Arg‐II−/−) display reduced lesion size with characteristics of stable plaques, such as decreased macrophage inflammation and necrotic core. In vivo adoptive transfer experiments reveal that fewer donor ApoE−/−Arg‐II−/− than ApoE−/−Arg‐II+/+ monocytes infiltrate into the plaque of ApoE−/−Arg‐II+/+ mice. Conversely, recipient ApoE−/−Arg‐II−/− mice accumulate fewer donor monocytes than do recipient ApoE−/−Arg‐II+/+ animals.ConclusionsArg‐II promotes macrophage proinflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis. Targeting Arg‐II represents a potential therapeutic strategy in type II diabetes mellitus and atherosclerosis. (J Am Heart Assoc. 2012;1:e000992 doi: 10.1161/JAHA.112.000992.)
SummaryAugmented activities of both arginase and S6K1 are involved in endothelial dysfunction in aging. This study was to investigate whether or not there is a crosstalk between arginase and S6K1 in endothelial inflammation and aging in senescent human umbilical vein endothelial cells and in aging mouse models. We show increased arginase-II (Arg-II) expression ⁄ activity in senescent endothelial cells. Silencing Arg-II in senescent cells suppresses eNOSuncoupling, several senescence markers such as senescence-associated-b-galactosidase activity, p53-S15, p21, and expression of vascular adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1). Conversely, overexpressing Arg-II in nonsenescent cells promotes eNOS-uncoupling, endothelial senescence, and enhances VCAM1 ⁄ ICAM1 levels and monocyte adhesion, which are inhibited by co-expressing superoxide dismutase-1. Moreover, overexpressing S6K1 in nonsenescent cells increases, whereas silencing S6K1 in senescent cells decreases Arg-II gene expression ⁄ activity through regulation of Arg-II mRNA stability. Furthermore, S6K1 overexpression exerts the same effects as Arg-II on endothelial senescence and inflammation responses, which are prevented by silencing Arg-II, demonstrating a role of Arg-II as the mediator of S6K1-induced endothelial aging. Interestingly, mice that are deficient in Arg-II gene (Arg-II ) ⁄ ) ) are not only protected from age-associated increase in Arg-II, VCAM1 ⁄ ICAM1, aging markers, and eNOS-uncoupling in the aortas but also reveal a decrease in S6K1 activity. Similarly, silencing Arg-II in senescent cells decreases S6K1 activity, demonstrating that Arg-II also stimulates S6K1 in aging. Our study reveals a novel mechanism of mutual positive regulation between S6K1 and Arg-II in endothelial inflammation and aging. Targeting S6K1 and ⁄ or Arg-II may decelerate vascular aging and age-associated cardiovascular disease development.
Mammalian target of rapamycin (mTOR)/S6K1 signalling emerges as a critical regulator of aging. Yet, a role of mTOR/S6K1 in aging-associated vascular endothelial dysfunction remains unknown. In this study, we investigated the role of S6K1 in aging-associated endothelial dysfunction and effects of the polyphenol resveratrol on S6K1 in aging endothelial cells. We show here that senescent endothelial cells displayed higher S6K1 activity, increased superoxide production and decreased bioactive nitric oxide (NO) levels than young endothelial cells, which is contributed by eNOS uncoupling. Silencing S6K1 in senescent cells reduced superoxide generation and enhanced NO production. Conversely, over-expression of a constitutively active S6K1 mutant in young endothelial cells mimicked endothelial dysfunction of the senescent cells through eNOS uncoupling and induced premature cellular senescence. Like the mTOR/S6K1 inhibitor rapamycin, resveratrol inhibited S6K1 signalling, resulting in decreased superoxide generation and enhanced NO levels in the senescent cells. Consistent with the data from cultured cells, an enhanced S6K1 activity, increased superoxide generation, and decreased bioactive NO levels associated with eNOS uncoupling were also detected in aortas of old WKY rats (aged 20–24 months) as compared to the young animals (1–3 months). Treatment of aortas of old rats with resveratrol or rapamycin inhibited S6K1 activity, oxidative stress, and improved endothelial NO production. Our data demonstrate a causal role of the hyperactive S6K1 in eNOS uncoupling leading to endothelial dysfunction and vascular aging. Resveratrol improves endothelial function in aging, at least in part, through inhibition of S6K1. Targeting S6K1 may thus represent a novel therapeutic approach for aging-associated vascular disease.
Rajapakse AG, Ming XF, Carvas JM, Yang Z. The hexosamine biosynthesis inhibitor azaserine prevents endothelial inflammation and dysfunction under hyperglycemic condition through antioxidant effects. Am J Physiol Heart Circ Physiol 296: H815-H822, 2009. First published January 9, 2009 doi:10.1152/ajpheart.00756.2008.-Hexosamine biosynthetic pathway (HBP) accounts for some cardiovascular adverse effects of hyperglycemia. We investigated whether the HBP inhibitor azaserine protects against hyperglycemia-induced endothelial damage dependently of HBP. Human endothelial cells isolated from umbilical veins were exposed either to a high (30.5 mmol/l) or low concentration of glucose (5.5 mmol/l) for 4 days, followed by a stimulation with TNF-␣ (1 ng/ml, 24 h). The blockade of the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase inhibited HBP flux and oxidative stress (generation of superoxide and peroxynitrite) under the hyperglycemic condition and prevented the synergistic stimulation of VCAM-1 and ICAM-1 expression by hyperglycemia and TNF-␣. In the cells cultured under a low-glucose condition when no increased HBP flux occurred, azaserine enhanced the manganese-superoxide dismutase (MnSOD) protein level and also inhibited the oxidative stress and the expression of VCAM-1 and ICAM-1 in response to TNF-␣. Moreover, the polyphenol resveratrol inhibited the oxidative stress and adhesion molecule expression and did not decrease the HBP flux under the hyperglycemia condition. In addition, in isolated rat aortas exposed to hyperglycemic buffer for 5 h when no significant HBP flux occurred, azaserine upregulated the MnSOD protein level and prevented decreased endothelium-dependent relaxations to acetylcholine. In conclusion, hyperglycemia independently increases oxidative stress and HBP flux, amplifies endothelial inflammation, and impairs endothelial function mainly through oxidative stress and not the HBP pathway. Azaserine protects against hyperglycemic endothelial damage through its antioxidant effect independently of inhibiting HBP pathway. adhesion molecules; endothelium; glucose; O-linked acetylglusamine; superoxide DIABETES MELLITUS is highly associated with an increased risk of cardiovascular morbidity and mortality (10, 34). Chronic hyperglycemia causes glucotoxicity to vascular cells, particularly the endothelial cells, resulting in an increased incidence of cardiovascular events (7). Clinical and experimental studies provide compelling evidence showing that hyperglycemia is associated with impaired endothelium-dependent relaxations (7) and increased endothelial expression of adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) (20, 31). The decreased endothelial nitric oxide (NO) synthase (eNOS) function and upregulation of the adhesion molecules enhance the adhesion of inflammatory cells onto the endothelium followed by a transmigration of the cells into the subendothelial space, a key early process in atherogenesis (15). Numerous proi...
Background: Pharmacological inhibition of endothelial arginase-II has been shown to improve endothelial nitric oxide synthase (eNOS) function and reduce atherogenesis in animal models. We investigated whether the endothelial arginase II is involved in inflammatory responses in endothelial cells.
Nonalcoholic fatty liver disease (NAFLD) associates with obesity and type 2 diabetes. Hypoactive AMP-activated protein kinase (AMPK), hyperactive mammalian target of rapamycin (mTOR) signaling, and macrophage-mediated inflammation are mechanistically linked to NAFLD. Studies investigating roles of arginase particularly the extrahepatic isoform arginase-II (Arg-II) in obesity-associated NAFLD showed contradictory results. Here we demonstrate that Arg-II−/− mice reveal decreased hepatic steatosis, macrophage infiltration, TNF-α and IL-6 as compared to the wild type (WT) littermates fed high fat diet (HFD). A higher AMPK activation (no difference in mTOR signaling), lower levels of lipogenic transcription factor SREBP-1c and activity/expression of lipogenic enzymes were observed in the Arg-II−/− mice liver. Moreover, release of TNF-α and IL-6 from bone marrow-derived macrophages (BMM) of Arg-II−/− mice is decreased as compared to WT-BMM. Conditioned medium from Arg-II−/−-BMM exhibits weaker activity to facilitate triglyceride synthesis paralleled with lower expression of SREBP-1c and SCD-1 and higher AMPK activation in hepatocytes as compared to that from WT-BMM. These effects of BMM conditioned medium can be neutralized by neutralizing antibodies against TNF-α and IL-6. Thus, Arg-II-expressing macrophages facilitate diet-induced NAFLD through TNF-α and IL-6 in obesity.
Background: Endothelial nitric oxide synthase (eNOS)-uncoupling links obesity-associated insulin resistance and type-II diabetes to the increased incidence of cardiovascular disease. Studies have indicated that increased arginase is involved in eNOS-uncoupling through competing with the substrate L-arginine. Given that arginase-II (Arg-II) exerts some of its biological functions through crosstalk with signal transduction pathways, and that p38 mitogen-activated protein kinase (p38mapk) is involved in eNOS-uncoupling, we investigated here whether p38mapk is involved in Arg-II-mediated eNOS-uncoupling in a high fat diet (HFD)-induced obesity mouse model. Methods: Obesity was induced in wild type (WT) and Arg-II-deficient (Arg-II
BackgroundEndothelial nitric oxide synthase (eNOS)-uncoupling links obesity-associated insulin resistance and type-II diabetes to the increased incidence of cardiovascular disease. Studies have indicated that increased arginase is involved in eNOS-uncoupling through competing with the substrate L-arginine. Given that arginase-II (Arg-II) exerts some of its biological functions through crosstalk with signal transduction pathways, and that p38 mitogen-activated protein kinase (p38mapk) is involved in eNOS-uncoupling, we investigated here whether p38mapk is involved in Arg-II-mediated eNOS-uncoupling in a high fat diet (HFD)-induced obesity mouse model.MethodsObesity was induced in wild type (WT) and Arg-II-deficient (Arg-II-/-) mice on C57BL/6 J background by high-fat diet (HFD, 55% fat) for 14 weeks starting from age of 7 weeks. The entire aortas were isolated and subjected to 1) immunoblotting analysis of the protein level of eNOS, Arg-II and p38mapk activation; 2) arginase activity assay; 3) endothelium-dependent and independent vasomotor responses; 4) en face staining of superoxide anion and NO production with Dihydroethidium and 4,5-Diaminofluorescein Diacetate, respectively, to assess eNOS-uncoupling. To evaluate the role of p38mapk, isolated aortas were treated with p38mapk inhibitor SB203580 (10 μmol/L, 1 h) prior to the analysis. In addition, the role of p38mapk in Arg-II-induced eNOS-uncoupling was investigated in cultured human endothelial cells overexpressing Arg-II in the absence or presence of shRNA against p38mapk.ResultsHFD enhanced Arg-II expression/activity and p38mapk activity, which was associated with eNOS-uncoupling as revealed by decreased NO and enhanced L-NAME-inhibitable superoxide in aortas of WT obese mice. In accordance, WT obese mice revealed decreased endothelium-dependent relaxations to acetylcholine despite of higher eNOS protein level, whereas Arg-II-/- obese mice were protected from HFD-induced eNOS-uncoupling and endothelial dysfunction, which was associated with reduced p38mapk activation in aortas of the Arg-II-/- obese mice. Moreover, overexpression of Arg-II in human endothelial cells caused eNOS-uncoupling and augmented p38mapk activation. The Arg-II-induced eNOS-uncoupling was prevented by silencing p38mapk. Furthermore, pharmacological inhibition of p38mapk recouples eNOS in isolated aortas from WT obese mice.ConclusionsTaking together, we demonstrate here for the first time that Arg-II causes eNOS-uncoupling through activation of p38 mapk in HFD-induced obesity.
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