Antonino Saitta

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.

17β-oestradiol reduces cardiac leukocyte accumulation in myocardial ischaemia reperfusion injury in rat

Abstract We investigated whether oestrogens modulate the phenomenon of leukocyte accumulation during ischaemia and reperfusion of the myocardium. Anaesthetized rats were subjected to total occlusion (1 h) of the left main coronary artery followed by 1 h reperfusion. Sham myocardial ischaemia–reperfusion rats (Sham) were used as controls. Myocardial necrosis, myocardial myeloperoxidase activity, serum creatinine phosphokinase activity, serum and macrophages tumour necrosis factor (TNF-α) and the myocardial staining of intercellular adhesion molecule-1 (ICAM-1) were evaluated. Myocardial ischaemia plus reperfusion in untreated rats produced marked myocardial necrosis, increased serum creatinine phosphokinase activity (348±38 U/ml) and cardiac myeloperoxidase activity, a marker of polymorphonuclear leukocyte accumulation, both in the area at risk and in the necrotic area (MPO; 9±1.1 U×10−3/g tissue and 8.2±1 U×10−3/g tissue, respectively), and induced a marked increase in the macrophage (156±14 U/ml at the end of reperfusion) and serum (344±12 U/ml, at the end of reperfusion) levels of TNF-α. Finally, myocardial ischaemia–reperfusion injury increased ICAM-1 staining in the myocardium. Administration of 17β-oestradiol (5, 10 and 20 μg/kg, i.m. 5 min after induction of myocardial ischaemia–reperfusion injury), lowered myocardial necrosis and myeloperoxidase activity in the area at risk and in the necrotic area, reduced serum and macrophages TNF-α (20±3 U/ml and 9±3 U/ml, respectively) and decreased serum creatinine phosphokinase activity (67±3 U/ml). Oestrogen treatment also blunted the increased staining of ICAM-1 in the injured myocardium. Finally, 17β-oestradiol added in vitro to peritoneal macrophages collected from untreated rats subjected to myocardial ischaemia–reperfusion injury, significantly reduced TNF-α production. Our results suggest that 17β-oestradiol, by inhibiting TNF-α production, limits the deleterious ICAM-1-mediated binding of leukocytes to injured mycardium and protects against myocardial ischaemia–reperfusion injury.

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.

Pulse wave velocity and augmentation index, but not intima-media thickness, are early indicators of vascular damage in hypercholesterolemic children

Eur J Clin Invest 2010; 40 (3): 250–257

IRFI 042, a novel dual vitamin E-like antioxidant, inhibits activation of nuclear factor-κB and reduces the inflammatory response in myocardial ischemia–reperfusion injury

BACKGROUND Nuclear factor-kappaB (NF-kappaB) is a ubiquitous rapid response transcription factor involved in inflammatory reactions which exerts its effect by expressing cytokines, chemokines, and cell adhesion molecules. Oxidative stress causes NF-kappaB activation. IRFI 042 is a novel dual vitamin E-like antioxidant and we, therefore, investigated its ability to protect the heart from oxidative stress and to halt the inflammatory response in a model of myocardial ischaemia-reperfusion injury. METHODS Anaesthetized rats were subjected to total occlusion (45 min) of the left main coronary artery followed by 5-h reperfusion (MI/R). Sham myocardial ischaemia rats (sham-operated rats) were used as controls. Myocardial necrosis, cardiac output, cardiac and plasma vitamin E levels, myocardial malondialdehyde (MAL), cardiac SOD activity and elastase content (an index of leukocyte infiltration), hydroxyl radical (OH&z.ccirf;) formation, cardiac amount of mRNA codifying for ICAM-1 (evaluated by the means of reverse transcriptase polymerase chain reaction) and ICAM-1 immunostaining in the at-risk myocardium were investigated. NF-kappaB activation and the inhibitory protein of NF-kappaB, I-kappaBalpha, were also studied in at-risk myocardium by using electrophoretic mobility shift assay (EMSA) and Western blot analysis, respectively. RESULTS The ischaemia-reperfusion model produced wide heart necrosis (area at risk-necrotic area=52+/-5%; necrotic area-left ventricle=28+/-3%), increased cardiac MAL, an index of lipid peroxidation (area at risk=62.5+/-3.9 nmol/g tissue; necrotic area=80.3+/-5.1 nmol/g tissue), induced tissue neutrophil infiltration, and caused a marked decrease in endogenous antioxidants. Furthermore, myocardial ischaemia plus reperfusion caused in the area at risk peak message for ICAM-1 at 3 h of reperfusion and increased cardiac ICAM-1 immunostaining at 5 h of reperfusion. NF-kappaB activation was also evident at 0.5 h of reperfusion and reached its maximum at 2 h of reperfusion. I-kappaBalpha was markedly decreased at 45 min of occlusion; peak reduction was observed at 1 h of reperfusion and thereafter it was gradually restored. Intraperitoneal administration of IRFI 042 (5, 10, 20 mg/kg, 5 min after reperfusion) reduced myocardial necrosis expressed as a percentage either of the area at risk (18+/-4%) or the total left ventricle (11+/-2%), and improved cardiac output. This treatment also limited membrane lipid peroxidation in the area at risk (MAL=14.3+/-2.5 nmol/g tissue) and in the necrotic area (MAL=26.5+/-3.7 nmol/g tissue), restored the endogenous antioxidants vitamin E and superoxide dismutase, and inhibited detrimental hydroxyl radical formation. Finally, IRFI 042 blocked the activation of NF-kappaB, reduced cardiac ICAM-1 expression, and blunted tissue elastase content, an index of leukocytes accumulation at the site of injury. CONCLUSIONS Our data suggest that IRFI 042 is cardioprotective during myocardial infarction by limiting reperfusion-induced oxidative stress and by halting the inflammatory response.

Recombinant human erythropoietin inhibits iNOS activity and reverts vascular dysfunction in splanchnic artery occlusion shock

We investigated the effects of recombinant human erythropoietin (rh‐EPO) in splanchnic artery occlusion (SAO) shock. Sham operated animals were used as controls. Survival rate, mean arterial blood pressure (MAP), serum Tumor Necrosis Factor (TNF‐α), plasma nitrite/nitrate concentrations, red blood cell (RBC) count, blood haemoglobin (Hb), the responsiveness of aortic rings to phenylephrine (PE, 1 nM–10 μM) and the activity of inducible nitric oxide synthase (iNOS) were studied. SAO shocked rats had a decreased survival rate (0% at 4 h of reperfusion, while sham shocked rats survived more than 4 h), enhanced serum TNF‐α concentrations, increased plasma nitrite/nitrate levels (60±9.5 μM; sham shocked rats=2±0.4 μM), decreased MAP, unchanged RBC count and blood Hb and enhanced iNOS activity in the aorta. Moreover aortic rings from shocked rats showed a marked hyporeactivity to PE. Rh‐EPO (25, 50 and 100 U 100 g−1, 5 min following the onset of reperfusion) increased survival rate (70% at 4 h of reperfusion with the highest dose), reduced plasma nitrite/nitrate concentrations (10.3±3.3 μM), increased MAP, did not change RBC count and blood Hb, and inhibited iNOS activity in thoracic aortae. Furthermore rh‐EPO, either in vivo or in vitro (10 U for 1 h in the organ bath), restored to control values the hyporeactivity to PE. Finally rh‐EPO inhibited the activity of iNOS in peritoneal macrophages activated with endotoxin. Our data suggest that rh‐EPO protects against SAO shock by inhibiting iNOS activity.

Oxidative stress causes nuclear factor-κB activation in acute hypovolemic hemorrhagic shock

Nuclear Factor kappaB (NFkappaB) is an ubiquitous rapid response transcription factor involved in inflammatory reactions and exerts its action by expressing cytokines, chemokines, and cell adhesion molecules. We investigated the role of NF-kappaB in acute hypovolemic hemorrhagic (Hem) shock. Hem shock was induced in male anesthetized rats by intermittently withdrawing blood from an iliac catheter over a period of 20 min (bleeding period) until mean arterial blood pressure (MAP) fell and stabilized within the range of 20-30 mmHg. Hemorrhagic shocked rats died in 26.3 +/- 2.1 min following the discontinuance of bleeding, experienced a marked hypotension (mean arterial blood pressure = 20-30 mmHg) and had enhanced plasma levels of Tumor Necrosis Factor-alpha (200 +/- 15 pg/ml, 20 min after the end of bleeding). Furthermore, aortas taken 20 min after bleeding from hemorrhagic shocked rats showed a marked hypo-reactivity to phenylephrine (PE; 1nM to 10 microM) compared with aortas harvested from sham shocked rats. Hem shocked rats also had increased levels of TNF-alpha mRNA in the liver (15-20 min after the end of bleeding) and enhanced plasma levels of 2,5-dihydroxybenzoic acid (2,5-DHBA; 6 +/- 2.2 microm), 2,3-dihydroxybenzoic acid (2,3-DHBA; 13 +/- 2.1 microm), both studied to evaluate OH(*) production. Electrophoretic mobility shift assay showed that liver NF-kappaB binding activity increased in the nucleus 10 min after the end of hemorrhage and remained elevated until the death of animals. Western blot analysis suggested that the levels of inhibitory IkappaBalpha protein in the cytoplasm became decreased at 5 min after the end of bleeding. IRFI-042, a vitamin E analogue (20 mg/kg intraperitoneally 2 min after the end of bleeding), inhibited the loss of IkappaBalpha protein from the cytoplasm and blunted the increase in NF-kappaB binding activity. Furthermore IRFI-042 increased survival time (117.8 +/- 6.51 min; p <.01) and survival rate (vehicle = 0% and IRFI-042 = 80%, at 120 min after the end of bleeding), reverted the marked hypotension, decreased liver mRNA for TNF-alpha, reduced plasma TNF-alpha (21 +/- 4.3 pg/ml), and restored to control values the hypo-reactivity to PE. Our results suggest that acute blood loss (50% of the estimated total blood volume over a period of 20 min) causes early activation of NF-kappaB, likely through an increased production of reactive oxygen species. This experiment indicates that NF-kappaB-triggered inflammatory cascade becomes early activated during acute hemorrhage even in the absence of resuscitation procedures.

Evidence for a Role of Nitric Oxide in Hypovolemic Hemorrhagic Shock

Summary: Hypovolemic hemorrhagic shock was induced in rats by intermittently withdrawing blood from an iliac catheter for 20 min until mean arterial blood pressure (MAP) decreased to 30 mm Hg. Survival rate, survival time, plasma myocardial depressant factor (MDF) activity, MAP, and microscopic gastric alterations were then evaluated. NG-nitro-L-arginine methyl-ester (L-NAME), a selective inhibitor of nitric oxide (NO) production from L-arginine, was injected intravenously (i.v.) after the bleeding was discontinued. Untreated hemorrhagic shocked rats died in 27 ± 3.3 min, had enhanced plasma activity of MDF, and exhibited hemorrhagic infiltrates in gastric fundus mucosa. L-NAME (5 and 10 mg/kg) significantly increased survival rate and time, blunted the increase in plasma MDF activity, and protected against the gastric lesions induced by hemorrhagic hypovolemic shock. All these protective effects were reversed by a bolus of L-arginine (30 mg/kg/i.v.), given 2 min after administration of L-NAME. Our findings suggest that NO production plays an important role in the pathophysiology of hemorrhagic shock.

Cyclosporin-A reduces leukocyte accumulation and protects against myocardial ischaemia reperfusion injury in rats.

The present study was designed to evaluate the effect of cyclosporin A in a rat model of myocardial ischaemia reperfusion injury (MI/R). Anaesthetized rats were subjected to total occlusion (20 min) of the left main coronary artery followed by 5 h reperfusion (MI/R). Sham myocardial ischaemia-reperfusion rats (Sham MI/R) were used as controls. Myocardial necrosis, myocardial myeloperoxidase activity (MPO), serum creatinine phosphokinase activity (CPK), serum tumor necrosis factor (TNF-alpha), cardiac mRNA for TNF-alpha, cardiac intercellular adhesion molecule-1 (ICAM-1) immunostaining and myocardial contractility (left ventricle dP/dtmax) were evaluated. Myocardial ischaemia plus reperfusion in untreated rats produced marked myocardial necrosis, increased serum CPK activity and myeloperoxidase activity (a marker of leukocyte accumulation) both in the area-at-risk and in the necrotic area, reduced myocardial contractility and induced a marked increase in the serum levels of the TNF-alpha. Furthermore increased cardiac mRNA for TNF-alpha was measurable within 10 to 20 min of left main coronary artery occlusion in the area-at-risk and increased levels were generally sustained for 0.5 h. Finally, myocardial ischaemia-reperfusion injury increased ICAM-1 staining in the myocardium. Administration of cyclosporin A (0.25, 0.5 and 1 mg/kg as an i.v. infusion 5 min after coronary artery occlusion) lowered myocardial necrosis and myeloperoxidase activity in the area-at-risk and in the necrotic area, decreased serum CPK activity, increased myocardial contractility, reduced serum levels of TNF-alpha and the cardiac cytokine mRNA levels, and blunted ICAM-1 immunostaining in the injured myocardium. The data suggest that cyclosporin A suppresses leukocyte accumulation and protects against myocardial ischaemia-reperfusion injury.