Domenico Cucinotta

Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats

BACKGROUND We investigated the effect of genistein, a phytoestrogen derived from a soy diet with a flavonoid chemical structure, on endothelial dysfunction induced by estrogen deficiency in rats. METHODS Female mature Sprague-Dawley rats were subjected to a bilateral ovariectomy (OVX rats). Sham-operated animals (Sham OVX rats) were used as controls. Three weeks after surgery animals were randomized to the following treatments: genistein (0.2 mg/kg/day, s.c. for 4 weeks), 17 beta-estradiol (20 micrograms/kg/day, s.c. for 4 weeks) or their respective vehicles. Mean arterial blood pressure (MAP), heart rate (HR), total plasma cholesterol, plasma estradiol, plasma genistein levels and uterine weights were studied. Furthermore, we investigated acetylcholine (ACh 10 nM-10 microM) and sodium nitroprusside: (SN 15-30 nM) induced relaxation of aortic rings as well as NG-L-arginine (L-NMA: 10-100 microM) induced vasoconstriction in phenylephrine precontracted aortic segments and calcium-dependent nitric oxide synthase (cNOS) activity in homogenates of lungs taken from both sham OVX and OVX rats. RESULTS Untreated OVX rats had, compared with sham OVX animals, unchanged body weight, MAP, HR and plasma cholesterol. In contrast ovariectomy impaired endothelial responses, blunted L-NMA induced contraction (L-NMA 100 microM: Sham OVX = 2.1 +/- 0.2 g/mg tissue; OVX = 1.7 +/- 0.4 g/mg tissue) and reduced cNOS activity. Treatment with 17 beta-estradiol increased the hormone plasma levels, reverted the endothelial dysfunction and increased cNOS activity in lung homogenates. Genistein supplementation enhanced the circulating levels of the phytoestrogen and affected NOS activity and endothelial dysfunction to the same extent. CONCLUSIONS Our data suggest that genistein and 17 beta-estradiol show overlapping effects on experimental endothelial dysfunction.

Effect of genistein on endothelial function in postmenopausal women: a randomized, double-blind, controlled study

PURPOSE Genistein, a phytoestrogen found in soybeans, corrects endothelial dysfunction induced by oophorectomy in animals. Using a double-blind, controlled, randomized design, we evaluated its effects on endothelial function in women. METHODS We enrolled 79 healthy postmenopausal women (mean [+/- SD] age, 56 +/- 4 years) and randomly assigned them to receive continuous estrogen/progestin therapy (n = 26; 17beta-estradiol [1 mg/d] combined with norethisterone acetate [0.5 mg/d]), genistein (n = 27; 54 mg/d), or placebo (n = 26). Brachial artery flow-mediated, endothelium-dependent vasodilation and plasma levels of nitrites/nitrates (a marker of nitric oxide metabolism) and endothelin-1 were measured at baseline and after 1 year of therapy. RESULTS Treatment with genistein increased levels of nitrites/nitrates (mean increase, 21 micromol/L; 95% confidence interval [CI]: 15 to 26 micromol/L; P <0.001 vs. placebo); estrogen/progestin therapy caused similar changes (P <0.001 vs. placebo). Plasma endothelin-1 levels decreased following 12 months of genistein (mean decrease, 7 pg/mL; 95% CI: 3 to 10 pg/mL; P <0.001 vs. placebo) and after 12 months of estrogen/progestin (P <0.001 vs. placebo). When compared with placebo, brachial artery flow-mediated dilation was improved by genistein (mean increase, 5.5%; 95% CI: 3.9% to 7.0%; P <0.001) and by estrogen/progestin (P <0.001). There were no significant differences between estrogen and genistein for any of these parameters (all P >0.4). CONCLUSION One year of genistein therapy improves endothelium function in postmenopausal women to a similar extent as does an estrogen/progestin regimen.

The metabolic syndrome is a risk indicator of microvascular and macrovascular complications in diabetes: results from Metascreen, a multicenter diabetes clinic-based survey.

OBJECTIVE—We aimed at assessing the degree of association and the predictive power of the metabolic syndrome with regard to clinically detectable complications in patients with diabetes. RESEARCH DESIGN AND METHODS—Metascreen is a cross-sectional survey of metabolic syndrome and clinically detected diabetes complications performed in 8,497 patients (7,859 with type 2 diabetes and 638 with type 1 diabetes) randomly chosen in 176 diabetes outpatient clinics throughout Italy. The metabolic syndrome was defined according to either the American Heart Association/National Heart, Lung, and Blood Institute (AHA/NHLBI) or the International Diabetes Federation (IDF) diagnostic criteria. Multivariate analyses of the association(s) between either AHA/NHLBI or IDF metabolic syndrome and clinical complications were performed. Receiver-operator characteristic (ROC) curves were constructed to compare the predictive power of the two sets of diagnostic criteria of the metabolic syndrome. RESULTS—Either definition of the metabolic syndrome was an independent statistical indicator of the presence of nephropathy and neuropathy (P < 0.02–0.01) in type 1 diabetes and of all complications (P < 0.0001), including cardiovascular disease and retinopathy, in type 2 diabetes. For each complication, the ROC curves based on either AHA/NHLBI or IDF metabolic syndrome were similar to each other and to the ROC curves constructed with all continuous traits compounding the metabolic syndrome. CONCLUSIONS—The metabolic syndrome, defined according to AHA/NHLBI or IDF diagnostic criteria, is an independent clinical indicator and may be involved in the pathogenesis of both macro- and microvascular complications of diabetes.

A Multicenter Study on the Prevalence of Diabetic Neuropathy in Italy

OBJECTIVE The prevalence of neuropathy, a common complication of diabetes, was determined in diabetic patients recruited from 109 outpatient diabetes clinics in Italy. RESEARCH DESIGN AND METHODS Neuropathy was diagnosed using the Diabetic Neuropathy Index (DNI), a standardized examination developed for use in the outpatient setting. A total of 8,757 diabetic patients were studied, 51.2% men and 48.8% women, with average and median ages of 56 and 58 years, respectively. RESULTS Of the 8,757 patients, 32.3% had neuropathy, defined as a positive score of > 2 points on the DNI. A total of 2,033 (49.6% men and 50.4% women) were administered the Diabetic Neuropathy Score (DNS), the second component of the screening program, by a neurologist. This component consists of a quantitative neurological examination and nerve conduction studies that together provide a summated score. A total of 335 patients (16.5%) were not neuropathic, and 395 (19.4%) had borderline, 453 (22.3%) mild, 592 (29.1%) moderate, and 258 (12.7%) severe neuropathy. The concordance between a positive score on the DNI and a DNS indicating neuropathy was 83.5%. The severity of neuropathy increased with both age and disease duration. Of patients with neuropathy, 64.1% had an average age between 58 and 59 years with a disease duration between 12.4 ± 8.4 years (mild neuropathy) and 15.6 ± 9.7 years (severe neuropathy). CONCLUSIONS Neuropathy is a common complication of diabetes and, in this study, was present in 32.3% of all patients. An increased awareness of the high prevalence of neuropathy can lead to early therapeutic intervention and possible prevention of later neuropathic complications, such as infection and foot ulcers.

Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse.

Aims/hypothesisWe studied the gene therapy efficacy of diabetes-associated wound healing disorder with an adeno-associated virus (AAV) vector expressing the 165-amino acid isoform of human vascular endothelial growth factor-A (VEGF-A) by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ db+/db+ mice and their normal littermates (db+/+m).MethodsAnimals were randomized to receive intradermally into the wound edges either rAAV-LacZ (a control gene), or rAAV-VEGF165. Animals were killed on different days (7 and 14 days after skin injury) and wounded skin tissues were used for gene marker studies, histological evaluation and immunohistochemistry, and wound breaking strength analysis. Furthermore we studied the VEGF mature protein in the wounds.ResultsWe found that AAV vectors are highly efficient for gene transfer to the mouse skin, displaying an exquisite tropism for the panniculus carnosus by using the beta-galactosidase activity assay. We confirmed the increased expression of the angiogenic factor at day 7 by measuring the wound content of the mature protein. Delivery of VEGF165 to incisional skin wounds of diabetic mice resulted in a remarkable induction of new vessel formation with consequent improvement in the wound healing process. The rAAV-VEGF165 gene improved wound healing in diabetic mice through the stimulation of angiogenesis, reepithelization, synthesis and maturation of extracellular matrix. Moreover the recombinant AAV encoding the human VEGF165 increased the breaking strenght of the wound and enhanced the wound content of VEGF.Conclusion/interpretationOur study suggests that VEGF gene transfer might represent a new approach to treat wound healing disorders associated with diabetes.

Inflammatory markers in women with a recent history of gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is a risk factor for both Type 2 diabetes (DM2) and insulin-resistance syndrome (IRS). C-reactive protein (CRP), fibrinogen and leukocyte count are increased in the IRS and predict DM2 and cardiovascular disease (CVD). The chemochine monocyte chemoattractant protein-1 (MCP-1/CCL2) is also elevated in DM2 and CVD. Recent evidence suggests a relation between chronic inflammation and GDM, but post-delivery information on inflammatory markers in these high-risk women is lacking. Serum levels of CRP, fibrinogen, MCP-1/CCL2, and leukocyte blood count have been assessed in 26 women with and 26 women without a recent history of GDM, matched for age, body mass index (BMI), post-partum duration and parity. DM2 was excluded in all the participants by an oral glucose tolerance test (OGTT). Women with previous GDM showed significantly higher CRP (p=0.007) and fibrinogen (p=0.02) serum concentrations, whereas MCP-1/CCL2 serum levels and leukocyte blood count were comparable in the two groups. Overall, CRP levels significantly correlated with BMI (r=0.40, p=0.03), waist-to-hip ratio (WHR) (r=0.44, p=0.001), fasting insulin (r=0.27, p=0.04), insulin-resistance assessed by means of the homeostatic model (HOMA) (r=0.28, p=0.04), and fibrinogen concentration (r=0.49, p=0.0001). At linear regression analysis, only WHR and fibrinogen were independently associated with CRP levels. In conclusion, the increase of inflammatory markers may be one of the first detectable disorders in healthy women at high risk of DM2 and IRS, like those with a GDM history.

Anorectic activity of NG-nitro-L-arginine, an inhibitor of brain nitric oxide synthase, in obese Zucker rats

We investigated the effects of NG-nitro-L-arginine (L-NO Arg) administration (12.5, 25 and 50 mg/kg i.p.) on food consumption and body weight of male obese Zucker rats (fa/fa) and in their lean age-matched controls (FA/?; FA/FA), both groups aged 14 weeks. Acute or repeated administration of L-NO Arg reduced food intake and body weight in both obese and lean rats. However the lean rats showed tolerance to the L-NO Arg effects after 5 days of treatment. L-NO Arg anorexia was suppressed by pretreatment with metergoline. These results suggest that L-NO Arg may represent a new anorectic drug.

Skin lesions in diabetes mellitus: prevalence and clinical correlations.

With the aim to assess the prevalence and the main clinical correlations of skin lesions in diabetes mellitus, 457 diabetic subjects consecutively attending an outpatient clinic underwent a dermatological examination. Neurovascular foot lesions were excluded. Thirty-five of 64 IDDM patients (54%) had skin alterations mainly consisting of vitiligo (9% of all patients), psoriasis (9%) and eczema (8%). The most frequent skin lesions observed in 240/393 NIDDM subjects (61%) were represented by infections (20% of all patients) and diabetic dermopathy (12.5%), while other lesions were not common. NIDDM patients with skin infections had a worse metabolic control, and those with diabetic dermopathy had a greater prevalence of neuropathy and large vessel disease than patients without skin lesions. These data show that the prevalence of skin diseases in a large, unselected diabetic population is higher than expected and indicate that, in most cases, a careful dermatological examination and a better metabolic control are needed in order to improve quality of life in these patients.

Food deprivation increases brain nitric oxide synthase and depresses brain serotonin levels in rats

We studied nitric oxide (NO) synthase activity and serotonin content in the diencephalon of 24 hr food deprived rats. NO synthase activity was significantly increased whereas serotonin levels together with those of tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) were reduced in food deprived rats when compared to control rats. NG-Nitro-L-arginine (L-NO Arg), an inhibitor of NO synthase, was used as a tool to study the role of NO in food deprivation. Twenty-four hr food deprived male Sprague-Dawley rats were intraperitoneally (i.p.) administered L-NO Arg (12.5, 25 and 50 mg/kg) before food presentation. Control rats received a NaCl (0.9%) solution. Food consumption was monitored 1 and 2 hr after food presentation. L-NO Arg administration produced a dose-dependent reduction in food intake. Pretreatment with metergoline (2 mg/kg) but not with ritanserin (1 mg/kg) antagonized the anorectic effect of L-NO Arg. Moreover, in the diencephalon L-NO Arg significantly reduced NO synthase activity whereas it increased serotonin levels. Our data indicate that NO might have a physiological role in the regulation of food intake and suggest that brain NO may modulate the central serotoninergic system.

Cardioprotection by the phytoestrogen genistein in experimental myocardial ischaemia-reperfusion injury.

Soybean phytoestrogens have no oestrogen agonist effects on the reproductive system and therefore it is reasonable to explore the potential of these naturally occurring plant oestrogens in the cardiovascular pathology. We therefore investigated the effects of genistein in a rat model of myocardial ischaemia‐reperfusion injury. Anaesthetized rats were subjected to total occlusion (45 min) of the left main coronary artery followed by 5 h reperfusion (MI/R). Sham operated rats were used as controls. Myocardial necrosis, myocardial myeloperoxidase activity (MPO), serum creatinine phosphokinase activity (CPK), serum and macrophage Tumour Necrosis Factor‐α (TNF‐α), cardiac intercellular adhesion molecule‐1 (ICAM‐1) immunostaining, cardiac mRNA for ICAM‐1 evaluated by the means of reverse transcriptase polymerase chain reaction (RT–PCR), ventricular arrhythmias and myocardial contractility (left ventricle dP/dtmax) were evaluated. Myocardial ischaemia and reperfusion in untreated rats produced marked myocardial necrosis, increased serum CPK activity and MPO activity both in the area‐at‐risk and in the necrotic area, reduced myocardial contractility, caused ventricular arrhythmias and induced a marked increase in serum and macrophage TNF‐α. Furthermore myocardial ischaemia‐reperfusion injury increased ICAM‐1 expression in the myocardium. Administration of genistein (1 mg kg−1, i.v., 5 min after coronary artery occlusion) lowered myocardial necrosis and MPO activity in the area‐at‐risk and in the necrotic area, decreased serum CPK activity, increased myocardial contractility, decreased the occurrence of ventricular arrhythmias, reduced serum and macrophages levels of TNF‐α and blunted ICAM‐1 expression in the injured myocardium. Finally genistein added in vitro to peritoneal macrophages collected from untreated rats subjected to myocardial ischaemia‐reperfusion injury significantly reduced TNF‐α production. Our data suggest that genistein limits the inflammatory response and protects against myocardial ischaemia‐reperfusion injury.