Stefano Rizza

Ghrelin Improves Endothelial Function in Patients With Metabolic Syndrome

Background— Metabolic syndrome importantly accelerates the atherosclerotic process, the earliest event of which is endothelial dysfunction. Ghrelin, a gastric peptide with cardiovascular actions, has been shown to inhibit proatherogenic changes in experimental models. This study therefore investigated whether ghrelin administration might beneficially affect endothelial function in metabolic syndrome. Methods and Results— Endothelium-dependent and -independent vasodilator responses to intra-arterial infusion of increasing doses of acetylcholine and sodium nitroprusside (SNP), respectively, were assessed by strain-gauge plethysmography before and after local administration of human ghrelin (200 &mgr;g/min). During saline, the vasodilator response to acetylcholine was significantly blunted (P=0.008) in patients with metabolic syndrome (n=12, 5 female) compared with controls (n=12, 7 female), whereas the vasodilator response to SNP was not different between groups (P=0.68). In patients with metabolic syndrome, basal plasma ghrelin was significantly lower than in controls (P=0.02). In these patients, ghrelin infusion markedly increased intravascular concentrations of the peptide (P<0.001) and resulted in a potentiation of the vasodilator response to acetylcholine (P=0.001 versus saline) but not to SNP (P=0.22). This effect was likely related to enhanced nitric oxide bioavailability because, in a group of patients with metabolic syndrome (n=6, 2 female), ghrelin had no effect on the vasodilator response to acetylcholine (P=0.78 versus saline) after nitric oxide inhibition by NG-monomethyl-l-arginine. Conclusions— 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.

Citrus polyphenol hesperidin stimulates production of nitric oxide in endothelial cells while improving endothelial function and reducing inflammatory markers in patients with metabolic syndrome

CONTEXT Hesperidin, a citrus flavonoid, and its metabolite hesperetin may have vascular actions relevant to their health benefits. Molecular and physiological mechanisms of hesperetin actions are unknown. OBJECTIVE We tested whether hesperetin stimulates production of nitric oxide (NO) from vascular endothelium and evaluated endothelial function in subjects with metabolic syndrome on oral hesperidin therapy. DESIGN, SETTING, AND INTERVENTIONS: Cellular mechanisms of action of hesperetin were evaluated in bovine aortic endothelial cells (BAEC) in primary culture. A randomized, placebo-controlled, double-blind, crossover trial examined whether oral hesperidin administration (500 mg once daily for 3 wk) improves endothelial function in individuals with metabolic syndrome (n = 24). MAIN OUTCOME MEASURE We measured the difference in brachial artery flow-mediated dilation between placebo and hesperidin treatment periods. RESULTS Treatment of BAEC with hesperetin acutely stimulated phosphorylation of Src, Akt, AMP kinase, and endothelial NO synthase to produce NO; this required generation of H(2)O(2). Increased adhesion of monocytes to BAEC and expression of vascular cell adhesion molecule-1 in response to TNF-α treatment was reduced by pretreatment with hesperetin. In the clinical study, when compared with placebo, hesperidin treatment increased flow-mediated dilation (10.26 ± 1.19 vs. 7.78 ± 0.76%; P = 0.02) and reduced concentrations of circulating inflammatory biomarkers (high-sensitivity C-reactive protein, serum amyloid A protein, soluble E-selectin). CONCLUSIONS 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.

Timp3 deficiency in insulin receptor–haploinsufficient mice promotes diabetes and vascular inflammation via increased TNF-α

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-alpha-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-alpha, 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.

Benfotiamine counteracts glucose toxicity effects on endothelial progenitor cell differentiation via Akt/FoxO signaling

Dysfunction of mature endothelial cells is thought to play a major role in both micro- and macrovascular complications of diabetes. However, recent advances in biology of endothelial progenitor cells (EPCs) have highlighted their involvement in diabetes complications. To determine the effect of glucotoxicity on EPCs, human EPCs have been isolated from peripheral blood mononuclear cells of healthy donors and cultured in the presence or absence of high glucose (33 mmol/l) or high glucose plus benfotiamine to scavenge glucotoxicity. Morphological analysis revealed that high glucose significantly affected the number of endothelial cell colony forming units, uptake and binding of acLDL and Lectin-1, and the ability to differentiate into CD31- and vascular endothelial growth factor receptor 2–positive cells. Functional analysis outlined a reduced EPC involvement in de novo tube formation, when cocultured with mature endothelial cells (human umbilical vein endothelial cells) on matrigel. To explain the observed phenotypes, we have investigated the signal transduction pathways known to be involved in EPC growth and differentiation. Our results indicate that hyperglycemia impairs EPC differentiation and that the process can be restored by benfotiamine administration, via the modulation of Akt/FoxO1 activity.

TIMP3 Is Reduced in Atherosclerotic Plaques From Subjects With Type 2 Diabetes and Increased by SirT1

OBJECTIVE Atherosclerosis 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 METHODS We 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. RESULTS Here, 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. CONCLUSIONS In 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.

Fish oil supplementation improves endothelial function in normoglycemic offspring of patients with type 2 diabetes

OBJECTIVE Offspring of patients with type 2 diabetes (OPDs) exhibits endothelial dysfunction (ED) associated with a chronic inflammatory state. N-3 polyunsaturated fatty acids (n-3 PUFA) may have antioxidant and anti-inflammatory properties that are beneficial for cardiovascular and metabolic health. Therefore, in the present study, we tested the hypothesis that dietary supplementation with fish oil rich in n-3 PUFA may improve ED in otherwise healthy OPDs. METHODS AND DESIGN A double-blind, placebo-controlled trial was conducted with 50 OPDs. Participants were randomized to treatment with either placebo or n-3 PUFA (2g/day) for 12 weeks. Before and after treatment we evaluated endothelial function (using flow-mediated dilation (FMD) of the brachial artery), circulating inflammatory markers (adiponectin, TNF-alpha, and high sensitivity-CRP), and insulin resistance (QUICKI). RESULTS No significant changes were observed in study outcomes in subjects treated with placebo. By contrast, when compared with baseline values, subjects treated with n-3 PUFA had significant improvement in FMD (9.1+/-5.8% vs. 11.7+/-4.4%, p=0.02) that was accompanied by decreased plasma triglycerides (117+/-73mg/dl vs. 86+/-44mg/dl, p=0.001) and TNF-alpha levels (8.9+/-2.3pg/ml vs. 6.8+/-2.7pg/ml, p=0.001), and a trend towards increased plasma adiponectin levels (7.8+/-4.5microg/ml vs. 9.5+/-5.1microg/ml, p=0.09). When data were analyzed by multiple regression analysis, decreased TNF-alpha after treatment with n-3 PUFA predicted increased FMD. CONCLUSION Dietary supplementation with n-3 PUFA significantly improved endothelial function and reduced pro-inflammatory markers in OPDs. Thus, fish oil consumption may have beneficial cardiovascular and metabolic health effects in otherwise healthy subjects predisposed to diabetes and its vascular complications.

Plasma Ghrelin in Anorexia, Bulimia, and Binge-Eating Disorder: Relations with Eating Patterns and Circulating Concentrations of Cortisol and Thyroid Hormones

The present study was designed to investigate the relations between plasma ghrelin concentrations, eating patterns, and circulating concentrations of cortisol and thyroid hormones in women with anorexia nervosa, bulimia nervosa, and binge-eating disorder. The patterns of disordered eating behavior were assessed using the Eating Attitudes Test (EAT-26) and the Bulimia Test-Revised (BULIT-R). In women with eating disorders, but not in healthy control women, plasma ghrelin concentrations were negatively correlated with body mass index (BMI) and plasma concentrations of thyreotropin (TSH), free T3 and free T4, and positively correlated with plasma concentrations of cortisol. The ghrelin concentrations of women with binge-eating and purging behavior were significantly lower than those of women with anorexia nervosa, restricting type, and there was a negative relation between the frequency and severity of binge-eating and purging behavior, as measured by the BULIT-R total score, and ghrelin concentrations. In a multivariate regression model controlling for the confounding effects of body mass index (BMI) and age, higher ghrelin concentrations were correlated with lower BULIT-R total scores. The results of this study did not confirm the hypothesis advanced in previous studies that ghrelin concentrations are higher in patients with binge-eating/purging forms of eating disorders. Based on these data, we suggest that, in women with eating disorders, ghrelin concentrations best reflect nutritional status rather than specific patterns of disordered eating behavior.

Metabolomics signature improves the prediction of cardiovascular events in elderly subjects

AIMS Age is one of the most important determinants of cardiovascular health, therefore the management of cardiovascular diseases (CVD) in elderly people entails great challenge. A possible explanation of vascular senescence process is the mitochondrial damage and dysfunction. We hypothesized that metabolomic profiling would identify biomarkers predicting major cardiovascular events (MACEs) in elderly people, improving the clinical standard cardiovascular risk factors. METHODS AND RESULTS Targeted-mass-spectrometry-based profiling of 49 metabolites was performed in a group of very old participants (n = 67, mean age = 85 ± 3 years) with a high rate of previous CVD (68%). Principal Component Analysis, Random Survival Forest analysis and Cox proportional hazards regression modeling were used to evaluate the relation between the metabolite factors and recurring MACEs. We tested discrimination ability and reclassification of clinical and metabolomic models. At follow-up (median = 3.5 years), 17 MACEs occurred (5 cardiovascular deaths, 1 nonfatal myocardial infarction, 7 nonfatal strokes and 4 peripheral artery surgeries) (incidence = 7.3% person-years). Metabolite factor 1, composed by medium- and long-chain acylcarnitines, and factor 7 (alanine) were independently associated with MACEs, after adjustment for clinical CV covariates [HR = 1.77 (95%CI = 1.11-2.81, p = 0.016) and HR = 2.18 (95%CI = 1.17-4.07, p = 0.014), respectively]. However, only factor 1 significantly increases the prediction accuracy of the Framingham Recurring-Coronary-Heart-Disease-Score, with a significant improvement in discrimination (integrated discrimination improvement = 7%, p = 0.01) and correctly reclassifying 41% of events and 37% of non-events resulting in a cNRI = 0.79 (p = 0.005). CONCLUSIONS Aging mitochondrial dysfunction evaluated by metabolomic profiling is associated with MACEs, independently of standard predictors.

MiR-216a: a link between endothelial dysfunction and autophagy

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.

Serum Resistin, Cardiovascular Disease and All-Cause Mortality in Patients with Type 2 Diabetes

Background High 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. Methods We 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. Results In 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). Conclusions This 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.