Novel mechanisms for hesperetin action in endothelial cells inform effects of oral hesperidin treatment to improve endothelial dysfunction and reduce circulating markers of inflammation in our exploratory clinical trial. Hesperetin has vasculoprotective actions that may explain beneficial cardiovascular effects of citrus consumption.

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

Aging mitochondrial dysfunction evaluated by metabolomic profiling is associated with MACEs, independently of standard predictors.

These findings indicate that ghrelin reverses endothelial dysfunction in patients with metabolic syndrome by increasing nitric oxide bioactivity, thereby suggesting that decreased circulating levels of the peptide, such as those found in these patients, might play a role in the pathobiology of atherosclerosis.

OBJECTIVEAtherosclerosis is accelerated in subjects with type 2 diabetes by unknown mechanisms. We identified tissue inhibitor of metalloproteinase 3 (TIMP3), the endogenous inhibitor of A disintegrin and metalloprotease domain 17 (ADAM17) and other matrix metalloproteinases (MMPs), as a gene modifier for insulin resistance and vascular inflammation in mice. We tested its association with atherosclerosis in subjects with type 2 diabetes and identified Sirtuin 1 (SirT1) as a major regulator of TIMP3 expression.RESEARCH DESIGN AND METHODSWe investigated ADAM10, ADAM17, MMP9, TIMP1, TIMP2, TIMP3, and TIMP4 expression levels in human carotid atherosclerotic plaques (n = 60) from subjects with and without diabetes. Human vascular smooth muscle cells exposed to several metabolic stimuli were used to identify regulators of TIMP3 expression. SirT1 small interference RNA, cDNA, and TIMP3 promoter gene reporter were used to study SirT1-dependent regulation of TIMP3.RESULTSHere, we show that in human carotid atherosclerotic plaques, TIMP3 was significantly reduced in subjects with type 2 diabetes, leading to ADAM17 and MMP9 overactivity. Reduced expression of TIMP3 was associated in vivo with SirT1 levels. In smooth muscle cells, inhibition of SirT1 activity and levels reduced TIMP3 expression, whereas SirT1 overexpression increased TIMP3 promoter activity.CONCLUSIONSIn atherosclerotic plaques from subjects with type 2 diabetes, the deregulation of ADAM17 and MMP9 activities is related to inadequate expression of TIMP3 via SirT1. Studies in vascular cells confirmed the role of SirT1 in tuning TIMP3 expression.

Endothelial dysfunction and impaired autophagic activity have a crucial role in aging-related diseases such as cardiovascular dysfunction and atherosclerosis. We have identified miR-216a as a microRNA that is induced during endothelial aging and, according to the computational analysis, among its targets includes two autophagy-related genes, Beclin1 (BECN1) and ATG5. Therefore, we have evaluated the role of miR-216a as a molecular component involved in the loss of autophagic function during endothelial aging. The inverse correlation between miR-216a and autophagic genes was conserved during human umbilical vein endothelial cells (HUVECs) aging and in vivo models of human atherosclerosis and heart failure. Luciferase experiments indicated BECN1, but not ATG5 as a direct target of miR-216a. HUVECs were transfected in order to modulate miR-216a expression and stimulated with 100 μg/ml oxidized low-density lipoprotein (ox-LDL) to induce a stress repairing autophagic process. We found that in young HUVECs, miR-216a overexpression repressed BECN1 and ATG5 expression and the ox-LDL induced autophagy, as evaluated by microtubule-associated protein 1 light chain 3 (LC3B) analysis and cytofluorimetric assay. Moreover, miR-216a stimulated ox-LDL accumulation and monocyte adhesion in HUVECs. Conversely, inhibition of miR-216a in old HUVECs rescued the ability to induce a protective autophagy in response to ox-LDL stimulus. In conclusion, mir-216a controls ox-LDL induced autophagy in HUVECs by regulating intracellular levels of BECN1 and may have a relevant role in the pathogenesis of cardiovascular disorders and atherosclerosis.

BackgroundHigh serum resistin has been associated with increased risk of cardiovascular disease in the general population, Only sparse and conflicting results, limited to Asian individuals, have been reported, so far, in type 2 diabetes. We studied the role of serum resistin on coronary artery disease, major cardiovascular events and all-cause mortality in type 2 diabetes.MethodsWe tested the association of circulating resistin concentrations with coronary artery disease, major cardiovascular events (cardiovascular death, non-fatal myocardial infarction and non-fatal stroke) and all-cause mortality in 2,313 diabetic patients of European ancestry from two cross-sectional and two prospective studies. In addition, the expression of resistin gene (RETN) was measured in blood cells of 68 diabetic patients and correlated with their serum resistin levels.ResultsIn a model comprising age, sex, smoking habits, BMI, HbA1c, and insulin, antihypertensive and antidyslipidemic therapies, serum resistin was associated with coronary artery disease in both cross-sectional studies: OR (95%CI) per SD increment = 1.35 (1.10–1.64) and 1.99 (1.55–2.55). Additionally, serum resistin predicted incident major cardiovascular events (HR per SD increment = 1.31; 1.10–1.56) and all-cause mortality (HR per SD increment = 1.16; 1.06–1.26). Adjusting also for fibrinogen levels affected the association with coronary artery disease and incident cardiovascular events, but not that with all cause-mortality. Finally, serum resistin was positively correlated with RETN mRNA expression (rho = 0.343).ConclusionsThis is the first study showing that high serum resistin (a likely consequence, at least partly, of increased RETN expression) is a risk factor for cardiovascular disease and all-cause mortality in diabetic patients of European ancestry.