Cosimo Marzocca

Inducible Nitric Oxide Synthase Expression in Human Colorectal Cancer : Correlation with Tumor Angiogenesis

To investigate the potential involvement of the nitric oxide (NO) pathway in colorectal carcinogenesis, we correlated the expression and the activity of inducible nitric oxide synthase (iNOS) with the degree of tumor angiogenesis in human colorectal cancer. Tumor samples and adjacent normal mucosa were obtained from 46 surgical specimens. Immunohistochemical expression of iNOS, vascular endothelial growth factor (VEGF), and CD31 was analyzed on paraffin-embedded tissue sections. iNOS activity and cyclic GMP levels were assessed by specific biochemical assays. iNOS protein expression was determined by Western blot analysis. iNOS and VEGF mRNA levels were evaluated using Northern blot analysis. Both iNOS and VEGF expressions correlated significantly with intratumor microvessel density (r(s) = 0.31, P = 0.02 and r(s) = 0.67, P < 0.0001, respectively). A significant correlation was also found between iNOS and VEGF expression (P = 0.001). iNOS activity and cyclic GMP production were significantly higher in the cancer specimens than in the normal mucosa (P < 0.0001 and P < 0.0001, respectively), as well as in metastatic tumors than in nonmetastatic ones (P = 0.002 and P = 0.04, respectively). Western and Northern blot analyses confirmed the up-regulation of the iNOS protein and gene in the tumor specimens as compared with normal mucosa. NO seems to play a role in colorectal cancer growth by promoting tumor angiogenesis.

Protective effects of M40403, a selective superoxide dismutase mimetic, in myocardial ischaemia and reperfusion injury in vivo.

Myocardial injury caused by ischaemia and reperfusion comes from multiple pathogenic events, including endothelial damage, neutrophil extravasation into tissue, mast cell activation, and peroxidation of cell membrane lipids. These events are followed by myocardial cell alterations resulting eventually in cell necrosis. An enhanced formation of reactive oxygen species is widely accepted as a stimulus for tissue destruction and cardiac failure. In this study, we have investigated the cardioprotective effects of M40403 in myocardial ischaemia‐reperfusion injury. M40403 is a low molecular weight, synthetic manganese containing superoxide dismutase mimetic (SODm) that selectively removes superoxide anion. Ischaemia was induced in rat hearts in vivo by ligating the left anterior descending coronary artery. Thirty minutes after the induction of ischaemia, the ligature was removed and reperfusion allowed to occur for at least 60 min. M40403 (0.1–1 mg kg−1) was given intravenously 15 min before ischaemia. The results obtained in this study showed that M40403 significantly reduced the extent of myocardial damage, mast cell degranulation and the incidence of ventricular arrhythmias. Furthermore, M40403 significantly attenuated, in a dose‐dependent manner, neutrophil infiltration in the myocardium as well as the associated induction of lipid peroxidation. Calcium overload seen post‐reperfusion of the ischaemic myocardium was also reduced by M40403. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in cardiac tissue taken after reperfusion: this was attenuated by M40403. Moreover reperfused cardiac tissue sections showed positive staining for P‐selectin and for anti‐intercellular adhesion molecule (ICAM‐1) in the vascular endothelial cells. M40403 treatment markedly reduced the intensity and degree of P‐selectin and ICAM‐1 in these tissues. No staining for nitrotyrosine, P‐selectin or ICAM‐1 was found in cardiac tissue taken at the end of the ischaemic period. Overall, M40403 treatment reduced the morphological signs of myocardial cell injury and significantly improved survival. Taken together, these results clearly indicate that M40403 treatment exerts a protective effect against ischaemia‐reperfusion‐induced myocardial injury, supporting a key role for superoxide anion in reperfusion injuries. This suggests that synthetic enzymes of SOD such as M40403, offer a novel therapeutic approach for the treatment of ischaemic heart disease where superoxide anion plays a dominant role.

Heme Oxygenase-1 and the Ischemia-Reperfusion Injury in the Rat Heart

Carbon monoxide (CO) is a signaling gas produced intracellularly by heme oxygenase (HO) enzymes using heme as a substrate. During heme breakdown, HO-1 and HO-2 release CO, biliverdin, and Fe2+. In this study, we investigated the effects of manipulation of the HO-1 system in an in vivo model of focal ischemia–reperfusion (FIR) in the rat heart. Male Wistar albino rats, under general anesthesia and artificial ventilation, underwent thoracotomy, the pericardium was opened, and a silk suture was placed around the left descending coronary artery; ischemia was induced by tightening the suture and was monitored for 30 min. Subsequently, the ligature was released to allow reperfusion lasting for 60 min. The first group of rats was sham operated and injected intraperitoneally (ip) with saline. The second group underwent FIR. The third group was treated ip 18 hr before FIR with hemin (4 mg/kg). The fourth group was pretreated ip 24 hr before FIR and 6 hr before hemin with zinc protoporphyrin IX (ZnPP-IX, 50 μg/kg). Specimens of the left ventricle were taken for determination of HO expression and activity, infarct size, malonyldialdehyde (MDA) production, and tissue calcium content. FIR led to a significant increase in the generation of MDA and notably raised tissue calcium levels. Induction of HO-1 by hemin significantly decreased infarct size, incidence of reperfusion arrhythmias, MDA generation, and calcium overload induced by FIR. These effects were prevented by the HO-1 inhibitor ZnPP-IX. The present experiments show that the concerted actions of CO, iron, and biliverdin/bilirubin modulate the FIR-induced myocardial injury.

Haeme oxygenase‐1 and cardiac anaphylaxis

Haeme oxygenase (HO) is an enzyme mainly localized in the smooth endoplasmic reticulum and involved in haeme degradation and in the generation of carbon monoxide (CO). Here we investigate (1) whether the inducible isoform of HO (HO‐1) is expressed in the isolated heart of the guinea‐pig and (2) the functional significance of HO‐1 on the response to antigen in isolated hearts taken from actively sensitized guinea‐pigs. Both the HO‐1 expression and activity are consistently increased in hearts from guinea‐pigs pretreated with hemin, an HO‐1 inducer (4 mg kg−1 i.p., 18 h before antigen challenge). The administration of the HO‐1 inhibitor zinc‐protoporphyrin IX (ZnPP‐IX, 50 μmol kg−1, i.p., 6 h before hemin) abolished the increase of both the HO‐1 expression and activity. In vitro challenge with the specific antigen of hearts from actively sensitized animals evokes a positive inotropic and chronotropic effect, a coronary constriction followed by dilation and an increase in the amount of histamine in the perfusates. In hearts from hemin‐pretreated animals, antigen challenge did not modify the heart rate and the force of contraction; the coronary outflow was significantly increased and a diminution of the release of histamine was observed. The patterns of cardiac anaphylaxis were fully restored in hearts from animals treated with ZnPP‐IX 6 h before hemin. In isolated hearts perfused with a Tyrode solution gassed with 100% CO for 5 min and successively reoxygenated, the response to antigen was similar to that observed in hearts from hemin‐pretreated animals. Pretreatment with hemin or the exposure to exogenous CO were linked to an increase in cardiac cyclic GMP levels and to a decrease of tissue Ca2+ levels. The study demonstrates that overexpression of HO‐1 inhibits cardiac anaphylaxis through the generation of CO which, in turn, decreases the release of histamine through a cyclic GMP‐ and Ca2+‐dependent mechanism.

Acute Cyanide Intoxication Treated with a Combination of Hydroxycobalamin, Sodium Nitrite, and Sodium Thiosulfate

An 80-year-old diabetic patient was admitted to the hospital because of sudden unconsciousness and severe metabolic acidosis. His son reported the possibility of cyanide poisoning. Clinical data and the detection of cyanide in blood and gastric material confirmed this possibility. Supportive therapy and the following antidotes—sodium nitrite two doses 300 mg IV, sodium thiosulfate 3 g IV, and hydroxocobalamin 4 g in 24 hours—were administered immediately and the patient completely recovered in 48 hours. Our observations suggest that timely and appropriate use of antidotes for cyanide intoxication may prevent death, even in aged diabetic patients.

A plant histaminase modulates cardiac anaphylactic response in guinea pig.

The effect of a copper amine oxidase (histaminase) purified from the pea seedling as free or immobilized enzyme on the response to specific antigen was studied in isolated hearts from actively sensitized guinea pigs. In vitro challenge with the specific antigen of hearts from actively sensitized animals evokes a positive inotropic and chronotropic effect, a coronary constriction, followed by dilation and an increase in the amount of histamine and nitrites, the oxidation product of nitric oxide, in the perfusates. In the presence of both forms of histaminases, the positive inotropic and chronotropic responses as well as the coronary constriction and the release of histamine were fully blocked. The amount of nitrites, appearing in the perfusates when anaphylaxis is elicited in the presence of both forms of histaminases, is significantly increased, as well as nitric oxide synthase activity and cyclic GMP content in cardiac tissue, while cardiac calcium overload was significantly prevented. These observations demonstrate that the decrease in the anaphylactic release of histamine and the subsequent abatement of the cardiac response to antigen can be accounted for by the inactivation by histaminase of the released histamine and by a stimulation of endogenous nitric oxide production.

Carbon monoxide modulates the response of human basophils to FcεRI stimulation through the heme oxygenase pathway

We report the effects of exogenous and endogenous carbon monoxide (CO) on the immunological activation of human basophils. Hemin (1-100 microM), a heme oxygenase substrate analogue, significantly increased the formation of bilirubin from partially purified human basophils, thus indicating that these cells express heme oxygenase. This effect was reversed by preincubating the cells for 30 min with Zn-protoporphyrin IX (100 microM), a heme oxygenase inhibitor. Hemin (100 microM) also decreased immunoglobulin G anti-Fcepsilon (anti-IgE)-induced activation of basophils, measured by the expression of a membrane granule-associated protein, identified as cluster differentiation protein 63 (CD63), and by histamine release. These effects were reversed by Zn-protoporphyrin IX (100 microM), by oxyhemoglobin (HbO(2)), a CO scavenger (100 microM), and by 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ), an inhibitor of the soluble guanylyl cyclase (100 microM). Exposure of basophils to exogenous CO (10 microM for 30 min) also decreased their activation, while nitrogen (N(2)) was ineffective. HbO(2) and ODQ reversed the inhibition, reversing both membrane protein CD63 expression and histamine release to basal values. Both hemin and exogenous CO significantly raised cGMP levels in basophils and blunted the rise of calcium levels caused by immunological activation. This study suggests that CO increases cGMP formation, which in turn induces a fall in intracellular Ca(2+) concentration, thereby resulting in the inhibition of human basophil activation.

Evaluation of the effects of a novel carbon monoxide releasing molecule (CORM-3) in an in vitro model of cardiovascular inflammation.

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Histaminergic receptors modulate the coronary vascular response in isolated guinea pig hearts. Role of nitric oxide.

AbstractObjective: The effects of histamine and of histamine receptor agonists and antagonists on the coronary outflow and on the generation of nitric oxide (NO) were evaluated on isolated guinea pig hearts. Methods: Isolated guinea pig hearts were perfused for 50 min in a Langendorff apparatus with histamine (10–7– 10–8 M), in the absence or in the presence of NG-monome-thyl-L-arginine (L-NMMA, 10–4 M), a NO synthase inhibitor and of triprolidine (3⋅10–8 M) and cimetidine (10–7 M), H1 receptor and H2 receptor antagonists, and with trifluoromethyl-phenylhistamine (TFMPH, 10–7 M) and dimaprit (10–7 M), H1 and H2 receptor agonists. The effects of (R)-α-methylhistamine (10–7 M), a H3 receptor agonist and of FUB 181 (10–7 M), a H3 receptor antagonist, were studied in the presence of bradykinin (10–7 M). Results: Histamine increases the coronary outflow and the generation of NO in a concentration-dependent fashion. The effects were completely abolished by blocking NO-synthase (NOS) with L-NMMA (10 –4 M). The effects were also abolished by cimetidine (10 –7 M), H 2 receptor antagonist, and only scarcely affected by triprolidine (3⋅10 –8 M), H 1 receptor antagonist. The effects were reproduced by dimaprit (10 –7 M), H 2 receptor agonist, and only scarcely by TFMPH (10 –7 M), a selective H 1 receptor agonist. Bradykinin (10 –7 M) produces a sustained coronary dilation paralleled by a marked increase in the generation of NO; the effects were significantly reduced by L-NMMA. The stimulation of H 3 receptors by (R)-α-methylhistamine (10 –7 M) significantly reduced both effects, which reverted to normal with FUB 181 (10 –7 M), an H 3 receptor antagonist. Conclusion: These results suggest that, in isolated guinea pig hearts, histamine produces coronary dilation through an H 2 /H 3 -dependent mechanism involving the generation of nitric oxide.

Pea Seedling Histaminase as a Novel Therapeutic Approach to Anaphylactic and Inflammatory Disorders

Amine oxidases (AOs) are ubiquitous enzymes involved in the metabolism of biogenic amines. Copper AOs (Cu-AOs) catalyze the oxidative deamination of primary amine groups of several biogenic amines, such as putrescine, cadaverine, and histamine. In the present review, the effects of a plant amine oxidase (Cu-AO, histaminase, EC1.4.3.6) purified from pea seedlings in the modulation of IgE-mediated allergic reactions, and in the prevention of cardiac and splachnic postischemic reperfusion damage are reported.