Patrizia Canale

Thrombolytic therapy with urokinase reduces increased circulating endothelial adhesion molecules in acute myocardial infarction.

The aim was to investigate circulating E-selectin and Intercellular Adhesion Molecule-1 (ICAM-1) in acute myocardial infarction. Our study was carried out in 80 patients, 40 hospitalized for acute myocardial infarction (AMI), 20 suffering from chronic stable angina and 20 healthy control subjects. Samples of venous blood were taken from all patients at the moment of hospitalization and after 2, 4, 6, 8, 10, 12 and 24 hours from the thrombolytic treatment (AMI+urokinase) or conventional therapy (AMI+nitroglycerin), for the dosage of creatinine kinase (CK) and adhesion molecules. The CK was determined by means of a Hitachi 901 automatic analyser using an enzymatic method (reagents Boheringer-Biochemia, Germany). Soluble E-selectin (sE-selectin) and soluble ICAM-1 (sICAM-1) were measured in the serum using a specific immunoassay (British Biotechnology Products). The serum levels of Tumor Necrosis Factor (TNF-α) were evaluated using an immunoenzymatic assay to quantitate the serum levels of the cytokine British Biotechnology Products). Patients with acute myocardial infarction (AMI) had increased serum levels of soluble E-selectin (sE-selectin; AMI+urokinase= 312±20 ng/ml; AMI+nitroglycerin=334±15 ng/ml) and soluble ICAM-1 (sICAM-1; AMI+urokinase= 629±30ng/ml; AMI+nitroglycerin=655±25 ng/ml) compared to both patients with chronic angina (sE-selectin =67±10 ng/ml; sICAM-1=230±20 ng/ml) and healthy control subjects (sE-selectin=53±15 ng/ml; sICAM-1 200±16 ng/ml). Furthermore patients with acute myocardial infarction also had increased serum levels of Tumor Necrosis Factor (TNF-α=309±10 pg/ml; control subjects=13±5 pg/ml). Thrombolytic therapy with urokinase (1,000,000 IU as an intravenous bolus for 5 minutes, followed by an infusion of an additional 1,000,000 IU for the following two hours) succeeded in producing reperfusion and reduced the serum levels of sE-selectin (52±13 ng/ml) and sICAM-1 (202±31 ng/ml). In contrast patients not eligible for thrombolytic therapy and therefore treated with conventional therapy (a continuous i.v. infusion of nitroglycerin at the dose of 50 mg/die) did not show any significant reduction in both sE-selectin and sICAM-1 throughout the study. Our results confirm previous experimental data and indicate that adhesion mechanisms supporting leukocyte-endothelium interaction may also be operative in human acute myocardial infarction.

Antibodies against intercellular adhesion molecule 1 protect against myocardial ischaemia-reperfusion injury in rat

In this study we have assayed the pathophysiological role of intercellular adhesion molecule (ICAM-1), a cytokine-inducible adhesion molecule, in a model of ischaemia reperfusion in the rat. Anaesthetized rats were subjected to occlusion (1 h) of the left main coronary artery followed by reperfusion (1 h). Sham myocardial ischaemia-reperfusion rats (Sham MI/R) were used as controls. Myocardial ischaemia plus reperfusion in untreated rats decreased survival rate, produced a marked myocardial necrosis, increased serum creatine phosphokinase activity, and cardiac myeloperoxidase activity (a marker enzyme commonly used to assess polymorphonuclear leukocyte accumulation). Furthermore, rats subjected to myocardial ischaemia-reperfusion showed an increased pressure rate index, studied as a quantitative means for assessing myocardial oxygen demand. Treatment with monoclonal anti-rat ICAM-1 (1 mg/kg i.v.), 3 h before occlusion of the left main coronary artery, significantly lowered serum creatine phosphokinase activity, blunted leukocyte accumulation and protected the myocardium from injury subsequent to ischaemia and reperfusion injury. These investigations have revealed that ICAM-1 is a critical adhesion molecule in the pathogenesis of ischaemia-reperfusion injury. In addition these results suggest that the use of monoclonal antibodies raised against ICAM-1 can represent a useful tool for the prevention of ischaemia-reperfusion damage.

E-selectin in the pathogenesis of experimental myocardial ischemia-reperfusion injury.

The role of E-selectin in the pathogenesis of an experimental model of myocardial ischemia-reperfusion injury was investigated. Pentobarbital anesthetized rats underwent left main coronary artery ligation (1 h) followed by reperfusion (1 h; MI/R). Sham operated rats were used as controls (sham MI/R). Myocardial ischemia-reperfusion injury reduced survival rate (50%), caused severe myocardial damage (necrotic area/area-at-risk 69.8 +/- 5%; necrotic area/total area = 56 +/- 7.6%), increased serum creatine phosphokinase activity (sham MI/R = 33 +/- 3 U/ml; MI/R = 215 +/- 13 U/ml), and elevated myeloperoxidase activity (investigated as an index of leukocyte adhesion and accumulation; sham MI/R = 0.11 +/- 0.02 U x 10(-3)/g tissue) in the area-at-risk (7.5 +/- 1.7 U x 10(-3)/g tissue) and in the necrotic area (7.8 +/- 2.2 U x 10(-3)/g tissue). Furthermore, MI/R rats had an increased pressure rate index, studied as a quantitative means for assessing myocardial oxygen demand. Administration of a hyperimmune serum containing antibodies against E-selectin significantly improved survival rate (80%), reduced myocardial injury (necrotic area/area-at-risk = 26.4 +/- 7%, P < 0.005; necrotic area/total area 19.1 +/- 2.8%, P < 0.005), lowered serum creatine phospokinase activity (85 +/- 5 U/ml, P < 0.001) and decreased myeloperoxidase activity in the area at risk (3.7 +/- 1.3 U x 10(-3)/g tissue, P < 0.001) and in the necrotic area (3.0 +/- 0.7 U x 10(-3)/g tissue). Finally, the administration of anti E-selectin antibodies improved the PRI in MI/R rats. The present data suggest that E-selectin in vivo plays a key role in the pathogenesis of myocardial ischemia/reperfusion injury.

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.

Improved survival and reversal of endothelial dysfunction by the 21-aminosteroid, U-74389G in splanchnic ischaemia-reperfusion injury in the rat

1 Anaesthetized rats subjected to total occlusion of the superior mesenteric artery and the coeliac trunk for 45 min developed a severe shock state (splanchnic artery occlusion, SAO shock) resulting in death within 70–90 min after release of the occlusion. Sham‐operated animals were used as controls. 2 Survival rate, survival time, serum tumour necrosis factor (TNF‐α), white blood cell (WBC) count, mean arterial blood pressure (MAP), plasma malonylaldehyde (MAL), myeloperoxidase activity (MPO) and the responsiveness to acetylcholine (ACh 10 nM‐10 μM) of aortic rings were investigated. 3 SAO shocked rats had a decreased survival rate and survival time (74 ± 10 min, while sham‐shocked rats survived more than 4 h), reduced mean arterial blood pressure, increased serum levels of TNF‐α (267 ± 13 u ml−1) and plasma levels of MAL (57 ± 7 nmol ml−1), enhanced MPO activity in the ileum (0.23 ± 0.04 u × 10−3 g−1 tissue) and in the lung (2.2 ± 0.8 u × 10−3 g−1 tissue), leukopenia and reduced responsiveness to ACh of aortic rings. 4 The 21‐aminosteroid U‐74389G (30 mg kg−1, i.v.) increased survival (survival time = 232 ± 15 min), lowered the serum levels of TNF‐α and the plasma levels of MAL, reduced leukopenia and MPO activity both in the ileum (0.021 ± 0.004 u × 10−3 g−1 tissue) and in the lung (0.23 ± 0.03 u × 10−3 g−1 tissue), improved MAP and restored the responsiveness to ACh of aortic rings. 5 Our data suggest that U‐74389G is a potent lipid peroxidation inhibitor and that it has antishock and endothelial protective actions.

Contribution of intercellular adhesion molecule 1 (ICAM-1) to the pathogenesis of splanchnic artery occlusion shock in the rat

1 It has been suggested that leukocytes play a key role in the pathogenesis of splanchnic artery occlusion shock. Intercellular adhesion molecule 1 (ICAM‐1) is an adhesion molecule of crucial importance in the phenomenon of leukocyte accumulation. 2 We investigated the involvement of ICAM‐1 in the pathogenesis of splanchnic artery occlusion shock. Splanchnic artery occlusion (SAO) shock was induced in anaesthetized rats by clamping splanchnic arteries for 45 min. Sham‐operated animals were used as controls. Survival time, serum tumour necrosis factor‐α (TNF‐α), white blood cell (WBC) count, mean arterial blood pressure, myeloperoxidase activity (MPO; studied as a quantitative means to assess leukocyte accumulation) and the responsiveness to acetylcholine of aortic rings were investigated. SAO shocked rats had a decreased survival time (90 ± 9.5 min, while sham‐shocked rats survived more than 4 h), reduced mean arterial blood pressure, increased serum levels of TNF‐α (201 ± 10 u ml−1) and MPO activity in the ileum (0.15 ± 0.03 u × 10−3 per g tissue) and in the lung (1.9 ± 0.8 u × 10−3 per g tissue), leukopenia and reduced responsiveness to acetylcholine (ACh, 10nM‐10 μm) of aortic rings. 3 Administration of monoclonal antibody raised against rat ICAM‐1 significantly increased survival time (225 ± 9 min), reduced leukopenia and MPO activity both in the ileum (0.031 ± 0.003 u × 10−3 per g tissue) and in the lung 0.23 ± 0.03 u × 10−3 per g tissue), improved the cardiovascular changes and restored the responsiveness to ACh of aortic rings. 4 Our findings are consistent with an involvement of adhesion mechanisms in vivo in the pathogenesis of SAO shock and suggest that specific adhesion mechanisms, which support leukocyte accumulation, may represent potentially important therapeutic targets in circulatory shock.

Protective effects of L-659,989, a platelet-activating factor receptor antagonist, in myocardial ischemia and reperfusion in rats.

Summary The cardioprotective effects of L-659,989, a specific platelet-activating factor (PAF) receptor antagonist, were investigated in an ischemia/reperfusion model in rats. Pentobarbital-anesthetized rats were subjected to left main coronary artery occlusion (1 h) followed by reperfusion (1 h) (MI/R); Sham-operated rats were used as controls (Sham MI/R). Rats receiving vehicle showed reduced survival rate (60%), marked myocardial injury (necrotic area/total area = 54.5 ± 6%; necrotic area/area at risk 76.6 ± 6.7%), high serum creatine phosphokinase (CPK) activity (150 ± 10 U/ml), and increased myocardial myeloperoxidase (MPO) activity in the area at risk (AR, 6.2 ± 0.5 U x 10-3/g protein) and in the necrotic area (6.6 ± 0.7 U x 10-3/g protein). PAF plasma levels increased significantly during reperfusion and peaked at 15 min of reperfusion. Administration of L-659,989 enhanced survival rate (80%), reduced myocardial damage (necrotic area/total area 25.6 ± 3.5%; necrotic area/AR 34.6 ± 5.4%), attenuated the increase in serum CPK (50 ± 6 U/ml) and decreased MPO activity both in the AR (2.8 ± 0.3 U x 10-3/g tissue) and in the necrotic area (2.3 ± 0.5 U x 10-3/g tissue). Our results suggest that PAF-inducing adhesion and activation of polymorphonuclear leukocytes (PMN) plays a significant role in the injury associated with ischemia/reperfusion

Effects of S‐ethylisothiourea, a potent inhibitor of nitric oxide synthase, alone or in combination with a nitric oxide donor in splanchnic artery occlusion shock

1 The aim of this study was to compare the effects of an intravenous infusion of a potent and non selective nitric oxide synthase inhibitor S‐ethylisothiourea (Ethyl‐TU) with that of a nitric oxide (NO) donor on the pathological sequelae associated with splanchnic artery occlusion (SAO) shock. In addition the effects of the combination of these two treatments were also investigated. 2 SAO shock was induced in anaesthetized rats by clamping splanchnic arteries for 45 min. Sham operated animals were used as controls. Survival time, white blood cell (WBC) count, mean arterial blood pressure, myeloperoxidase activity (MPO; studied as a quantitative means to evaluate neutrophil accumulation) and the responsiveness of aortic rings to acetylcholine (ACh, 10 nM‐10 μm) and to phenylephrine (PE, 1 nM‐10 μm) were studied. 3 SAO shocked rats had a decreased survival rate (0% survival 2 h after the release of occlusion) and survival time (76 ± 10 min), increased MPO activity in the ileum (3.39 ± 0.8 u × 10−3 g−1 tissue), a marked leukopenia and a profound hypotension. In addition aortic rings from shocked rats showed a marked hyporeactivity to PE and reduced responsiveness to ACh. Endothelium denuded aortic rings had also a marked hyporeactivity to PE. 4 In vivo administration of Ethyl‐TU (0.1 mg kg−1 h−1, beginning 1 min after the onset of reperfusion) significantly increased survival time and rate, improved mean arterial blood pressure, restored the responsiveness to PE, but did not change MPO activity, leukopenia or the impairment in the responsiveness of aortic rings to ACh. Addition of Ethyl‐TU (2 μm) to endothelium denuded aortic rings in vitro, restored the marked hyporeactivity to PE. Administration of the NO donor C87‐3754 (0.75 mg kg−1 h−1, beginning 1 min after the onset of reperfusion) slightly increased survival time and reduced MPO activity and leukopenia, but did not change survival rate and mean arterial blood pressure. In addition C87‐3754 restored the responsiveness of aortic rings to ACh to control values, but did not modify the hyporeactivity to PE. The combination of these two interventions produced a higher degree of protection than either Ethyl‐TU or C87‐3754 alone. In fact, co‐administration of Ethyl‐TU plus C87‐3754 completely prevented mortality, reduced MPO activity, attenuated leukopenia and the profound hypotension and restored the impaired responsiveness of aortic rings to PE and ACh. 5 Our study suggests that treatment with a nitric oxide synthase inhibitor combined with an NO donor may be a new therapeutic approach to the treatment of splanchnic artery occlusion shock.

Protective effects of BAY U 3405, a thromboxane A2 receptor antagonist, in endotoxin shock.

The present study was designed to investigate the effects of BAY U 3405, a new thromboxane A2 (TxA2) receptor antagonist, in endotoxin shock. Endotoxin shock (ES) was induced in male rats by an i.v. injection of Salmonella enteritidis lipopolysaccharide (LPS; 20 mg kg-1). LPS administration caused animal death (survival = 0%, 48 h after endotoxin challenge), systemic hypotension, depressed phagocytosis and increased blood levels of TNF-alpha, TxB2 and 6-keto-PGF1 alpha, reduced white blood cell (WBC) count (ES = 5.9 +/- 1 x 10(3) mm-3; CTRL = 13.4 +/- 5 x 10(3) mm-3) and enhanced myeloperoxidase (MPO) activity, studied as a quantitative means for assessing leukocyte accumulation, in the ileum (ES = 0.24 +/- 0.7 U g-1 fresh tissue; CTRL = 0.13 +/- 0.04 U g-1 fresh tissue), in the heart (ES = 0.41 +/- 0.1 U g-1 fresh tissue; CTRL = 0.16 +/- 0.08 U g-1 fresh tissue) and in the lung (ES = 0.68 +/- 0.11 U g-1 fresh tissue; CTRL = 0.19 +/- 0.05 U g-1 fresh tissue). Furthermore, endotoxin administration produced characteristic damage of the gastric mucosa consisting of haemmorrhagic infiltrates. BAY U 3405 (30 mg kg-1 i.v., 30 min before endotoxin challenge) increased survival rate (45% survival rate 48 h after endotoxin challenge), reduced hypotension, decreased TNF-alpha levels in serum, enhanced phagocytic activity (ES = 25.6 +/- 1.9%, BAY U 3405 = 45.9 +/- 0.4%, P < 0.001) and lowered MPO activity in the ileum (0.14 +/- 0.05 U g-1 fresh tissue), in the heart (0.18 +/- 0.08 U g-1 fresh tissue) and in the lung (0.44 +/- 0.09 U g-1 fresh tissue). Finally, the gastric alterations were significantly reduced in rats pretreated with BAY U 3405. These data suggest that this thromboxane receptor antagonist might be a useful drug in shock conditions.

TCV-309, a novel platelet activating factor antagonist, inhibits leukocyte accumulation and protects against splanchnic artery occlusion shock

The aim of this study was to evaluate: (1) the accumulation of leukocytes in the ileum and the lung during splanchnic artery occlusion (SAO) shock; (2) the role of platelet-activating factor (PAF) and tumor necrosis factor (TNF-α) in this phenomenon. Untreated anesthetized rats subjected to total occlusion of the celiac, superior and inferior mesenteric arteries for 45 min, followed by reperfusion, uniformly died within 90 min after reperfusion. The mean survival time was 93±7 min. The neutrophilic infiltrate was quantitated in the ileum and in the lung using a myeloperoxidase (MPO) assay. MPO activity in the ileum and in the lung averaged 0.05±0.03 and 0.4±0.02 U×10−3/g protein in animals killed before occlusion. MPO activity did not change in rats killed immediately before reperfusion and was significantly elevated (0.11±0.02 and 1.7±0.6 U×10−3/g protein in the ileum and the lung, respectively) in those killed 80 min after the beginning of the reperfusion. The histological examination confirmed the accumulation of leukocytes in the mucosa of the ileum and the lung over the 80 min. SAO shocked rats exhibited leukopenia and increased serum levels of TNF-α. In order to evaluate the role of PAF and TNF-α in SAO shock, a powerful PAF receptor antagonist, TCV-309 (5 μg/kg i.v.), was injected 5 min after reperfusion. TCV-309 increased survival time, lowered serum TNF-α, reduced MPO activity in both the ileum and the lung and ameliorated leukopenia induced by SAO shock. In addition, the drug significantly reduced ileal necrosis and pulmonary morphological alterations induced by shock. These results suggest an important role for PAF in the adhesion of leukocytes in SAO shock.