Marcella Ferlito

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.

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.

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.

Effect of cross-tolerance between endotoxin and TNF-α or IL-1β on cellular signaling and mediator production

Abstract: Endotoxin [lipopolysaccharide (LPS)] tolerance suppresses macrophage/monocyte proinflammatory‐mediator production. This phenomenon also confers cross‐tolerance to other stimuli including tumor necrosis factor (TNF) α and interleukin (IL)‐1β. Post‐receptor convergence of signal transduction pathways might occur after LPS, IL‐1β, and TNF‐α stimulation. Therefore, it was hypothesized that down‐regulation of common signaling molecules induces cross‐tolerance among these stimuli. LPS tolerance and cross‐tolerance were examined in THP‐1 cells. Phosphorylation of MAP kinases and degradation of inhibitor κBα (IκBα) DNA binding of nuclear factor‐κB (NF‐κB), and mediator production were examined. In naive cells, LPS, TNF‐α, and IL‐1β induced IκBα degradation, kinase phosphorylation, and NF‐κB DNA binding. LPS stimulation induced production of TNF‐α or TxB2 and degradation of IRAK. However, neither TNF‐α nor IL‐1β induced IRAK degradation or stimulated TNF‐α or TxB2 production in naive cells. Pretreatment with each stimulus induced homologous tolerance to restimulation with the same agonist. LPS tolerance also suppressed LPS‐induced TxB2 and TNF‐α production. LPS pretreatment induced cross‐tolerance to TNF‐α or IL‐1β stimulation. Pretreatment with TNF‐α induced cross‐tolerance to LPS‐induced signaling events and TxB2 production. Although pretreatment with IL‐1β did not induce cross‐tolerance to LPS‐induced signaling events, it strongly inhibited LPS TNF‐α and TxB2 production. These data demonstrate that IL‐1β induces cross‐tolerance to LPS‐induced mediator production without suppressing LPS‐induced signaling to MAP kinases or NF‐κB activation.

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.

Signal transduction events in Chinese hamster ovary cells expressing human CD14; effect of endotoxin desensitization.

Previous studies suggest that endotoxin (LPS) stimulation of CD14 receptors may be coupled to heterotrimeric G proteins. However, characterization of the G protein-coupled signaling pathways is incomplete. Also, specific changes in the transduction pathways occur in a phenomenon known as LPS tolerance or desensitization induced by prior exposure to LPS. In the present study, we examined potential CD14-dependent G protein-coupled signaling events in response to LPS, and changes in signaling in these pathways during LPS desensitization in Chinese Hamster Ovary (CHO) cells. LPS stimulated inhibitory kappa B alpha (IkappaB alpha) degradation and p38 phosphorylation in CHO cells transfected with human CD14 receptor (CHO-CD14), but not in CHO cells transfected with vector only. However, activation of these signaling events diverged early in the signal transduction pathways. Pretreatment with pertussis toxin, which inactivates inhibitor G protein (G alpha i) function, significantly inhibited LPS-induced p38 phosphorylation, but not LPS-induced IkappaB alpha degradation. Mastoparan, a putative G alpha i agonist, synergized with LPS to induce p38 phosphorylation. Thus, LPS stimulation of p38 phosphorylation is, in part, G alpha i coupled, whereas IkappaB alpha degradation is not. In subsequent studies, CHO-CD14 cells were desensitized by prior LPS exposure. LPS-desensitized cells exhibited augmented IkappaB alpha content and were refractory to LPS-induced IkappaB alpha degradation and p38 phosphorylation. Pretreatment with cycloheximide, a protein synthesis inhibitor, prevented the effect of LPS desensitization on augmenting cellular IkappaB alpha content and its refractoriness to LPS-induced degradation. However, cycloheximide pretreatment did not prevent impaired p38 phosphorylation in desensitized cells. IkappaB alpha upregulation in LPS tolerance may occur through increased synthesis and/or induction of protein that suppress IkappaB alpha degradation. The latter protein synthesis-dependent mechanisms may be distinct from mechanismis inhibiting p38 phosphorylation in tolerance. These findings suggest that LPS tolerance induces CD14-dependent signaling alterations in G alpha i-coupled pathways leading to mitogen-activated (MAP) kinase activation as well as G alpha i-independent pathways inducing IkappaB alpha degradation.

Participation of tumour necrosis factor and nitric oxide in the mediation of vascular dysfunction in splanchnic artery occlusion shock.

1 Splanchnic artery occlusion (SAO) shock is characterized by irreversible circulatory failure. Tumour necrosis factor (TNF‐α) may affect the 1‐arginine/nitric oxide (NO) pathway, thus contributing to the cardiovascular derangements of circulatory shock. 2 We investigated the contribution of both TNF‐α and the 1‐arginine/nitric oxide pathway to the vascular dysfunction of SAO shock. Anaesthetized rats, subjected to total occlusion of the superior mesenteric artery and the coeliac trunk for 45 min developed a severe shock state (SAO shock) resulting in a fatal outcome within 75–90 min after the release of occlusion. Sham operated animals were used as controls. SAO shocked rats had also a marked hypotension and enhanced macrophage and serum levels of TNF‐α. Furthermore, aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE 1 nm‐10 μm) and reduced responsiveness to acetylcholine (ACh lOnM‐lOμm). Endothelium‐denuded aortic rings had also a marked hyporeactivity to phenylephrine, which was restored to control values by in vitro administration of N° nitro‐1‐arginine‐methyl ester (1‐NAME 10 μm). 3 In vivo administration of cloricromene (2 mg kg−1, i.v.), an inhibitor of TNF‐α biosynthesis, increased survival, enhanced mean arterial blood pressure and reduced macrophage and serum levels of TNF‐α. Furthermore, aortic rings from shocked rats treated with cloricromene exhibited a greater contractile response to phenylephrine and improved responsiveness to ACh when compared to aortic rings from vehicle‐treated SAO shocked rats. 4 Our results suggest that TNF‐α alters both endothelial and muscular L‐arginine/nitric oxide pathways which in turn produce vascular dysfunction in SAO shock.

Implication of G i proteins and Src tyrosine kinases in endotoxin-induced signal transduction events and mediator production

Previous studies have suggested that heterotrimeric G proteins and tyrosine kinases may be involved in lipopolysaccharide (LPS) signaling events. Signal transduction pathways activated by LPS were examined in human promonocytic THP-1 cells. We hypothesized that Gi proteins and Src tyrosine kinase differentially affect mitogen-activated protein (MAP) kinases (MAPK) and nuclear factor kappa (NF- B) activation. Post-receptor coupling to G i proteins were examined using pertussis toxin (PTx), which inhibits G i receptor-coupling. The involvement of the Src family of tyrosine kinases was examined using the selective Src tyrosine kinase inhibitor pyrazolopyrimidine-2 (PP2). Pretreatment of THP-1 cells with PTx attenuated LPS-induced activation of c-Jun-N-terminal kinase (JNK) and p38 kinase, and production of tumor necrosis factor-alpha (TNF-) and thromboxane B2 (TxB2). Pretreatment with PP2 inhibited TNF- and TxB2 production, but had no effect on p38 kinase or JNK signaling. Therefore, the G i-coupled signaling pathways and Src tyrosine kinase-coupled signaling pathways are necessary for LPS-induced TNF- and TxB2 production, but differ in their effects on MAPK activation. Neither PTx nor PP2 inhibited LPS-induced activation of interleukin receptor activated kinase (IRAK) or inhibitedtranslocation of NF- B. However, PP2 inhibitedLPS-inducedNF-B transactivation of a luciferase reporter gene construct in a concentration-dependent manner. Thus, LPS induction of Src tyrosine kinases may be essential in downstream NF- B transactivation of genes following DNA binding. PTx had no effect on NF- B activation of the reporter construct. These data suggest upstream divergence in signaling through G i pathways leading to MAPK activation and other signaling events leading to I B degradation and NF- B DNA binding.

Tacrolimus suppresses tumour necrosis factor-α and protects against splanchnic artery occlusion shock

Tumour necrosis factor (TNF‐α) is a pleiotropic cytokine which is deeply involved in the pathogenesis of splanchnic artery occlusion (SAO) shock. Tacrolimus, formerly known as FK506, is a macrolide antibiotic, that blocks the transcription of several proinflammatory cytokines including TNF‐α. Male anaesthetized rats were subjected to clamping of the splanchnic arteries for 45 min. This surgical procedure resulted in an irreversible state of shock (SAO shock). Sham operated animals were used as controls. 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 (415±12 U ml−1), decreased mean arterial blood pressure (MAP), leukopenia and increased ileal leukocyte accumulation studied by means of myeloperoxidase activity (MPO=7.5±0.3 U g−1 tissue). Moreover aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE, 1 nM–10 μM), reduced responsiveness to acetylcholine (ACh, 10 nM–10 μM) and increased staining for intercellular adhesion molecule‐1 (ICAM‐1). Furthermore increased mRNA for TNF‐α was observed in peritoneal macrophages of SAO shocked rats. Tacrolimus (100 μg kg−1, 5 min after splanchnic arteries occlusion) increased survival rate (SAO+Tacrolimus=100% at 4 h of reperfusion), reverted the marked hypotension, reduced serum TNF‐α (15±3 U ml−1), ameliorated leukopenia, reduced ileal MPO (0.9±0.01 U g−1 tissue), restored to control values the hyporeactivity to PE, improved the reduced responsiveness to ACh and blunted the enhanced immunostaining for ICAM‐1 in the aorta. Finally tacrolimus suppressed cytokine mRNA levels in peritoneal macrophages. The data suggest that tacrolimus may represent a new therapeutic approach in circulatory shock.

The severity of cholestatic injury is modulated by the genetic background.

Common bile duct ligation (CBDL) compromises the hepatic reticuloendothelial system by impairing the clearing of endotoxin and triggering an overwhelming inflammatory response. The response to endotoxin at the level of cytokine release and subsequent mortality depends on the genetic background in experimental mouse models. We hypothesized that the genetic make-up modulates the inflammatory responses after CBDL. The CBD was ligated in male A/J and B6 mice (8 weeks old). At 7 days post-CBDL, the presence of ascites was observed in 80% of B6 mice but in none of the A/J mice (P < 0.001). B6 mice showed higher mortality than A/J mice (P < 0.05). Both strains had marked cholestatic injury documented histologically. Liver chemistries were markedly elevated in both strains after injury. Plasma levels of the anti-inflammatory cytokine IL-10 were significantly higher in A/J than B6 mice at the 4- and 12-h time points (P < 0.05), whereas proinflammatory cytokine TNF-α levels were significantly higher in B6 than A/J mice at 2 h (P < 0.05). Both strains displayed activation of NF-κB after CBDL. In conclusion, the contrasting response observed after CBDL between A/J and B6 mice is largely attributable to genetic differences. Survival after CBDL was correlated with an increase in anti-inflammatory cytokines.