[Abstract unavailable

Background-Aging is a major risk factor for the development of atherosclerosis and coronary artery disease. Through a microarray approach, we have identified a microRNA (miR-217) that is progressively expressed in endothelial cells with aging. miR-217 regulates the expression of silent information regulator 1 (SirT1), a major regulator of longevity and metabolic disorders that is progressively reduced in multiple tissues during aging. Methods and Results-miR-217 inhibits SirT1 expression through a miR-217-

Background-Hyperglycemia impairs functional properties of cytosolic and nuclear proteins via O-linked glycosylation modification (O-GlcNAcylation). We studied the effects of O-GlcNAcylation on insulin signaling in human coronary artery endothelial cells. Methods and Results-O-GlcNAcylation impaired the metabolic branch of insulin signaling, ie, insulin receptor (IR) activation of the IR substrate (IRS)/phosphatidylinositol 3-kinase (PI3-K)/Akt, whereas it enhanced the mitogenic branch, ie, ERK-1/2 and p38 (mitogen-activated protein kinase). Both in vivo and in vitro phosphorylation of endothelial nitric oxide synthase (eNOS) by Akt were reduced by hyperglycemia and hexosamine activation. Insulin-induced eNOS activity in vivo was reduced by hyperglycemia and hexosamine activation, which was coupled to increased activation and expression of matrix metalloproteinase-2 and -9; these phenomena were reversed by inhibition of the hexosamine pathway. Finally, carotid plaques from type 2 diabetic patients showed increased endothelial O-GlcNAcylation with respect to nondiabetics. Conclusions-Our

STAT1 is an essential transcription factor for macrophage activation by IFN-gamma and requires phosphorylation of the C-terminal Ser727 for transcriptional activity. In macrophages, Ser727 phosphorylation in response to bacterial lipopolysaccharide (LPS), UV irradiation, or TNF-alpha occurred through a signaling path sensitive to the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 whereas IFN-gamma-mediated Ser727 phosphorylation was not inhibited by the drug. Consistently, SB203580 did not affect IFN-gamma-mediated, Stat1-dependent transcription but inhibited its enhancement by LPS. Furthermore, LPS, UV irradiation, and TNF-alpha caused activation of p38 MAPK whereas IFN-gamma did not. An essential role for p38 MAPK activity in STAT1 Ser727 phosphorylation was confirmed by using cells expressing an SB203580-resistant p38 MAPK. In such cells, STAT1 Ser727 phosphorylation in response to UV irradiation was found to be SB203580 insensitive. Targeted disruption of the mapkap-k2 gene, encoding a kinase downstream of p38 MAPK with a key role in LPS-stimulated TNF-alpha production and stress-induced heat shock protein 25 phosphorylation, was without a significant effect on UV-mediated Ser727 phosphorylation. The recombinant Stat1 C terminus was phosphorylated in vitro by p38MAPKalpha and beta but not by MAPK-activated protein kinase 2. Janus kinase 2 activity, previously reported to be required for IFN-gamma-mediated Ser727 phosphorylation, was not needed for LPS-mediated Ser727 phosphorylation, and activation of Janus kinase 2 did not cause the appearance of STAT1 Ser727 kinase activity. Our data suggest that STAT1 is phosphorylated at Ser727 by a stress-activated signaling pathway either through p38 MAPK directly or through an unidentified kinase downstream of p38MAPK.

Activation of inflammatory pathways may contribute to the beginning and the progression of both atherosclerosis and type 2 diabetes. Here we report a novel interaction between insulin action and control of inflammation, resulting in glucose intolerance and vascular inflammation and amenable to therapeutic modulation. In insulin receptor heterozygous (Insr +/-) mice, we identified the deficiency of tissue inhibitor of metalloproteinase 3 (Timp3, an inhibitor of both TNF-α-converting enzyme [TACE] and MMPs) as a common bond between glucose intolerance and vascular inflammation. Among Insr +/-mice, those that develop diabetes have reduced Timp3 and increased TACE activity. Unchecked TACE activity causes an increase in levels of soluble TNF-α, which subsequently promotes diabetes and vascular inflammation. Double heterozygous Insr +/-Timp3 +/-mice develop mild hyperglycemia and hyperinsulinemia at 3 months and overt glucose intolerance and hyperinsulinemia at 6 months. A therapeutic role for Timp3/TACE modulation is supported by the observation that pharmacological inhibition of TACE led to marked reduction of hyperglycemia and vascular inflammation in Insr +/-diabetic mice, as well as by the observation of increased insulin sensitivity in Tace +/-mice compared with WT mice. Our results suggest that an interplay between reduced insulin action and unchecked TACE activity promotes diabetes and vascular inflammation.

However, recent studies have demonstrated that circulating bone marrow-derived endothelial progenitor cells (EPCs) tightly contribute to adult blood vessel formation (4,5). The EPCs promote in vivo re-endothelization and are able to be incorporated into new vessels in animal models of hind limb ischemia (6,7). EPCs are involved in processes like myocardial ischemia and infarction, wound healing, and endogenous endothelial repair (8 -11). Furthermore, in vivo studies in animal models and in vitro studies using EPCs from type 1 diabetic patients revealed a potential role for glucotoxicity in impairing EPC function (7,(12)(13)(14).High glucose induces pathological alterations through increased formation of advanced glycosylation end product, activation of aldose reductase and protein kinase C, and increased flux through the hexosamine pathway. All of these mechanisms seem to reflect a single hyperglycemiainduced process of overproduction of superoxide anion by the mitochondrial electron transport chain (15). Superoxide inhibits the glycolytic enzyme glyceraldehyde phosphate dehydrogenase, diverting upstream metabolites from glycolysis toward the glucose-driven signaling pathways that cause hyperglycemic damage (16). These processes may be in part reduced by transketolase activation through its cofactor thiamine. In fact, both thiamine and benfotiamine have been shown to correct microvascular and macrovascular complications of diabetes, although at a different extent, blocking three major pathways of hyperglycemic damage (16 -19).Interestingly, the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway is crucial for both endothelial cell function and EPC differentiation (20 -22). The PI 3-kinase/ Akt pathway is known to direct cellular processes like differentiation and stress resistance through a tight regulation of the forkhead family of transcription factors (FoxO1/3a/4). FoxO1 and FoxO3a were recently found to play a role in angiogenesis and vasculogenesis (23-26). We and others have recently shown that both genetic and metabolic factors impair activation of the PI 3-kinase/Akt/ FoxO pathway in mature endothelial cells (27)(28)(29). In this study, we investigated the impact of glucose toxicity on the ability of EPCs to differentiate into mature endothelial cells, and we also tested benfotiamine capacity to bypass the negative effects of high glucose concentrations. RESEARCH DESIGN AND METHODSEPC isolation and culture. EPCs were obtained by isolating peripheral mononuclear cells from human blood buffy coats using Ficoll density centrifugation. Recovered cells were washed twice with PBS. Unselected mononuclear cells were plated on fibronectin-coated culture dishes (Biocoat; Becton Dickinson Labware) at a density of 10 6 cells/ml in Medium 199 (Invitrogen), supplemented with 20% fetal bovine serum, 100 units/ml penicillin/streptomycin (Invitrogen), and 0.05 mg/ml bovine pituitary extract (Invitrogen) and in From the

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