Mariarita Arlotta

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.

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.

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.

Adrenocorticotropin reverses vascular dysfunction and protects against splanchnic artery occlusion shock

Tumour necrosis factor (TNF‐α) is involved in the pathogenesis of splanchnic artery occlusion (SAO) shock. On the other hand, inhibition of TNF‐α is an important component of the mechanism of action of melanocortins in reversing haemorrhagic shock. We therefore investigated the effects of the melanocortin peptide ACTH‐(1–24) (adrenocorticotropin fragment 1–24) on the vascular failure induced by SAO shock. SAO‐shocked rats had a decreased survival rate (0% at 4 h of reperfusion, while sham‐shocked rats survived for more than 4 h), enhanced serum TNF‐α concentrations (755±81 U ml−1), decreased mean arterial blood pressure, leukopenia, and increased ileal leukocyte accumulation, as revealed by means of myeloperoxidase activity (MPO=9.4±1 U g−1 tissue). Moreover, aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE, 1 nM–10 μM) (Emax and ED50 in shocked rats=7.16 mN mg−1 tissue and 120 nM, respectively; Emax and ED50 in sham‐shocked rats=16.31 mN mg−1 tissue and 100 nM, respectively), reduced responsiveness to acetylcholine (ACh, 10 nM‐10 μM) (Emax and ED50 in shocked rats=30% relaxation and 520 nM, respectively; Emax and ED50 in sham‐shocked rats=82% relaxation and 510 nM, respectively) and increased staining for intercellular adhesion molecule‐1 (ICAM‐1). ACTH‐(1–24) [160 μg kg−1 intravenously (i.v.), 5 min after SAO] increased survival rate [SAO+ACTH‐(1–24)=80% at 4 h of reperfusion], reversed hypotension, reduced serum TNF‐α (55±13 U ml−1), ameliorated leukopenia, reduced ileal MPO (1.2±0.2 U g−1 tissue), restored the reactivity to PE, improved the responsiveness to ACh and blunted the enhanced immunostaining for ICAM‐1 in the aorta. Adrenalectomy only in part–but not significantly–reduced the ACTH‐induced shock reversal, the survival rate of SAO+ACTH‐(1–24) adrenalectomized rats being 60% at 4 h of reperfusion; and methylprednisolone (80 mg−1 i.v., 5 min after SAO) had a non‐significant effect (10% survival) at 4 h of reperfusion. The present data show that melanocortins are effective also in SAO shock, their effect being, at least in part, mediated by reduced production of TNF‐α. Furthermore, they demonstrate, for the first time, that this inhibition is responsible for the adrenocorticotropin‐induced reversal of vascular failure and leukocyte accumulation.

The effects of recombinant human granulocyte-colony stimulating factor on vascular dysfunction and splanchnic ischaemia-reperfusion injury.

The aim of our study was to investigate the effects of recombinant human granulocyte‐colony stimulating factor in a rat model of splanchnic ischaemia‐reperfusion injury. Male anaesthetized rats were subjected to clamping of the splanchnic arteries for 45 min. This surgical procedure resulted in an irreversible state of shock (splanchnic artery occlusion shock; SAO shock). Sham operated animals were used as controls. Survival rate, serum tumour necrosis factor‐α (TNF‐α), neutrophil count, bone marrow myeloid precursor cells, myeloperoxidase activity (MPO; studied as a quantitative means to assess leukocyte accumulation), mean arterial blood pressure and the responsiveness of aortic rings to phenylephrine (PE, 1 nm–10 μm) 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), increased serum levels of TNF‐α (201±10 u ml−1; sham shocked rats=undetectable), neutropenia, enhanced MPO activity in the ileum (0.11±0.06 u × 10−3 g−1 tissue; sham shocked rats=0.02±0.001 u × 10−3 g−1 tissue) and in the lung (1.5±0.2 u × 10−3 g−1 tissue; sham shocked rats=0.19±0.05 u × 10−3 g−1 tissue) and unchanged bone marrow myeloid precursor cells. Furthermore aortic rings from shocked rats showed a marked hyporeactivity to PE. Administration of recombinant human granulocyte colony stimulating factor (rh G‐CSF; 5, 10 and 20 μg kg−1 5 min following the release of occlusion) increased in a dose‐dependent manner survival rate (90% at 4 h of reperfusion with the dose of 20 u × 10−3 g kg−1), reduced serum TNF‐α (13±5 u ml−1) and MPO activity in the ileum (0.065±0.002 u × 10−3 g−1 tissue) and in the lung (0.7±0.03 μ g kg−1 tissue), improved neutropenia and mean arterial blood pressure but did not modify bone marrow myeloid progenitor cells. Furthermore rh G‐CSF, either in vivo or in vitro (200 nm for 1 h in the organ bath), restored to control values the hyporeactivity to PE. Finally rh G‐CSF potently inhibited the activity of inducible nitric oxide synthase in peritoneal macrophages activated with endotoxin. Our results suggest that rh G‐CSF protects against splanchnic ischaemia reperfusion injury by a mechanism(s) that does not depend upon its haematopoietic effects.

The lazaroid, U-74389G, inhibits inducible nitric oxide synthase activity, reverses vascular failure and protects against endotoxin shock

The aim of our study was to investigate the effect of the 21-aminosteroid U-74389G [21- < 4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl-pregna-1,4,9,(11) triene-3,20-dione(z)-2-butenedionate] on the l-arginine-nitric oxide (NO) pathway in a rat model of endotoxin shock. Endotoxin shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg/kg of Salmonella Enteritidis lipopolysaccharide (LPS). Rats were treated with U-74389G (7.5, 15 and 30 mg/kg i.v.) or vehicle (1 ml/kg i.v.) 5 min after endotoxin challenge. Lipopolysaccharide administration reduced survival rate (0%, 72 h after endotoxin administration) decreased mean arterial blood pressure, enhanced plasma concentration of bilirubin and alanine aminotransferase and increased plasma nitrite concentrations. Lipopolysaccharide injection also increased the activity of inducible NO synthase in the liver and in the aorta. Furthermore aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (1 nM-10 microM). In addition lipopolysaccharide (50 microg/ml for 4 h) in vitro stimulation significantly increased nitrite production in peritoneal macrophages harvested from normal rats. Treatment with U-74389G (15 and 30 mg/kg i.v., 5 min after endotoxin challenge) significantly protected against lipopolysaccharide-induced lethality (90% survival rate 24 h and 80% 72 h after lipopolysaccharide injection, respectively, following the highest dose of the drug), reduced hypotension, ameliorated liver function, decreased plasma nitrite levels, restored the hyporeactivity of aortic rings to their control values and inhibited the activity of inducible NO synthase in the liver and in the aorta. Finally, U-74389G in vitro (12.5, 25 and 50 microM) significantly inhibited nitrite production in endotoxin stimulated peritoneal macrophages. The data suggest that U-74389G may exert beneficial effects in an experimental model of septic shock by inhibiting the activity of the inducible NO synthase.

Inhibition of tumour necrosis factor and reversal of endotoxin-induced shock by U-83836E, a ‘second generation’ lazaroid in rats

1 Antioxidants can exert protective effects in endotoxic shock by either a reduction of the oxidant damage or attenuation of Tumour Necrosis Factor (TNF‐α) production. 2 Lazaroids are a family of compounds that inhibit lipid peroxidation. Besides, they can also reduce TNF‐α. U‐83836E is a new lazaroid lacking the glucocorticoid ring. 3 Aim of our study was to investigate the effect of U‐83836E on TNF‐α production either in vivo or in vitro. Endotoxic shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg kg−1 of S. enteritidis lipopolysaccharide (LPS). LPS administration reduced survival rate (0% survival, 72 h after endotoxin administration), decreased mean arterial blood pressure, increased serum and macrophage TNF‐α and enhanced plasma malonylaldehyde (MAL) levels. Furthermore aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE 1 nm–10 μm). 4 Treatment with U‐83836E (7.5, 15 and 30 mg kg−1, i.v.) 5 min after endotoxin challenge significantly protected against LPS induced lethality (90% survival rate and 80% survival rate 24 h and 72 h after LPS injection respectively, following the highest dose of the drug), reduced hypotension, blunted plasma MAL, decreased serum and macrophage TNF‐α and restored the hyporeactivity of aortic rings to control values. In vitro LPS stimulation (50 μg ml−1 for 4 h) significantly increased cytokine production in macrophages (MΦ) harvested from untreated normal rats. Pretreatment with pertussis toxin (PT; 0.1, 1 and 10 ng ml−1 4 h before LPS) significantly increased TNF‐α production. PT effects on these LPS responses were correlated with a PT mediated ADP ribosylation of a 41 kDa protein. U‐83836E (50 μm) reduced, in a dose dependent manner, LPS induced TNF‐α production and inhibited the PT effects on cytokine production and on ADP ribosylation of the protein. 5 Our data suggest that lazaroids may affect the early events associated with LPS receptor mediated activation of a G protein in LPS induced TNF‐α production. These molecular events may explain, at least in part, the in vivo inhibition of cytokine production and reversal of endotoxic shock.

The involvement of tumour necrosis factor-α in the protective effects of 17β oestradiol in splanchnic ischaemia-reperfusion injury

1 Tumour necrosis factor‐α (TNF‐α) is a cytokine that is implicated in the pathogenesis of ischaemic states and atherosclerosis. We tested the hypothesis that the vasoprotective effects of the oestrogens may be mediated in vivo by inhibition of the formation of TNF‐α. 2 Anaesthetized rats, subjected to total occlusion of the superior mesenteric artery and the coeliac trunk for 45 min developed a severe shock state resulting in a fatal outcome within 75–90 min after the release of occlusion. Sham‐operated animals were used as controls. 3 Splanchnic artery occlusion (SAO) shocked rats had a marked hypotension, enhanced levels of TNF‐α in serum and macrophages, leukopenia and increased ileal leukocyte accumulation, studied by means of myeloperoxidase activity (MPO). Furthermore, aortae from SAO rats showed a marked hyporeactivity to phenylephrine (PE, 1 nM–10 μM), reduced responsiveness to acetylcholine (ACh, 10 nM–10 μM) and an increased staining for intercellular adhesion molecule‐1 (ICAM‐1). 4 In vivo administration of 17β oestradiol (500 μg kg−1, i.m., three hours before the induction of SAO) increased survival rate (100%, 4 h after SAO), enhanced mean arterial blood pressure; reduced serum TNF‐α (25±5 u ml−1 vs 379±16 u ml−1), ameliorated leukopaenia and reduced ileal MPO (0.7±0.02 u 10−3 g−1 tissue vs 4.2±0.4 u 10−3 g−1 tissue). Furthermore aortae from SAO rats treated with 17β oestradiol exhibited a greater contractile response to phenylephrine, improved responsiveness to ACh and a blunted staining of ICAM‐1. Finally 17β oestradiol, added in vitro to peritoneal macrophages collected from untreated SAO rats, significantly reduced TNF‐α production. 5 Our results suggest that inhibition of TNF‐αin vivo may explain, at least in part, the vasoprotective effects of oestrogens.