Nitric oxide, prostanoids, cyclooxygenase, and angiogenesis in colon and breast cancer.

PURPOSE: Several studies have shown an overexpression of cyclooxygenase-2 (COX-2) and elevated levels of prostacyclin (PGI(2)) and thromboxane (TXA(2)) in colon cancer. In this report, we determined the distribution of inducible form of nitric oxide synthase (iNOS), PGI(2), and TXA(2) in cancerous and adjoining areas of specimens from human colon and breast cancer obtained during surgery. Additionally, we investigated differences in expression and histological localization of COX-2 in colon and breast cancer. EXPERIMENTAL DESIGN: Specimens were obtained during surgery, one centrally located, the second from an adjacent, cancer-free area. Activity of iNOS was determined, using the conversion of L-[(14)C]arginine to L-[(14)C]citrulline. PGI(2) and TXA(2) were measured as their stable metabolites, using enzyme immunoassay. A standard immunoperoxidase method was used for immunohistochemical expression of COX-2. RESULTS: Significant differences in iNOS, PGI(2), and TXA(2) expressions between colon and breast cancer were noted, with an enhanced expression of COX-2 in colon cancer, including the cancerous, adjoining, and stromatous fields. CONCLUSIONS: Increased expression of iNOS and production of prostanoids in colon cancer parallels the increase in COX-2, confirming the importance of this enzyme in colon cancer. The overexpression of COX-2, prostanoids, and nitric oxide in areas adjoining the tumor indicates increased metastatic potential for neoplastic cells in this area. Inflammatory changes in the tissue adjoining the cancer may play a role. COX-2 may result in the formation of new blood vessels and the spread of cancer.

Myocardial ischemia and infarction: growth of ideas.

This report reviews the author's involvement in the growth of ideas and basic concepts in myocardial ischemia resulting in the histological changes of myocardial infarction. Concepts arising from the study of myocardial substrate utilization, activation of the inducible form of nitric oxide synthase and production of prostacyclin and thromboxane in the infarcted heart are presented. New approaches are discussed dealing with the effects of nonsteroidal anti-inflammatory drugs on myocardial production of nitric oxide and prostanoids, and with the relevance of the inducible form of cyclooxygenase. The review also records a number of significant similarities between angiogenesis in the ischemic heart and some cancers. Angiogenesis in both instances originates from inflammatory reactions, illustrating how different tissues and organs such as ischemic heart muscle and cancer react to similar pathological stimuli in an identical manner. This multifocal approach opens new concepts on myocardial ischemia and cancer.

Effect of cyclooxygenase-2 inhibitor (celecoxib) on the infarcted heart in situ.

Several attempts have been made to replace aspirin with compounds without gastric toxicity; a cyclooxygenase-2 (COX-2) inhibitor, celecoxib, and a nitric oxide-aspirin, NCX-4016, have been developed for this purpose. This paper compares effects of celecoxib, NCX-4016 and aspirin on production of prostacyclin (PGI2) and thromboxane A2 (TXA2) and activation of the inducible form of nitric oxide synthase (iNOS) in infarcted heart in situ. Aspirin was most effective in reducing myocardial PGI2 synthesis and formation of TXA2. Myocardial effects of celecoxib resemble those of NCX-4016, although the two compounds have different modes of action.

Some aspects of biochemistry of myocardial infarction.

This review deals with biochemical changes in infarcted heart muscle. Two main topics are emphasized: changes in substrate metabolism and in myocardial nitric oxide (NO), and prostacyclin and thromboxane formation. Alterations in glucose metabolism in infarcted heart are discussed with special reference to its myocardial utilization in ischemia. The biochemical basis of the increase in NO and prostanoids and the relationship between the enzyme producing nitric oxide (NOS) and cyclooxygenases (COX) responsible for formation of prostanoids are described. The relevance of these findings to clinical conditions and to angiogenesis in heart muscle are stressed.

Nitric oxide donors.

Nitric oxide (NO) donors are pharmacologically active substances that release NO in vivo or in vitro. NO has a variety of functions such as the release of prostanoids, inhibition of platelet aggregation, effect on angiogenesis, and production of oxygen free radicals. This report discusses the chemical and pharmacological characteristics of NO donors, their effect on platelet function and cyclooxygenase, their cardiac action including myocardial infarction, and release of superoxide anions. This review stresses NO tolerance and the effect of NO donors on angiogenesis in myocardial infarction and in solid tumors.

The effect of aspirin and two nitric oxide donors on the infarcted heart in situ.

Nitric oxide (NO) donors are heterogeneous substances which release NO, a biologically active compound. NO released by nitric oxide donors has important effects on the circulation by causing vasodilation, diminishing myocardial contractile force, inhibiting platelet aggregation, and counteracting the effects of thromboxane A2. In the infarcted heart, activation of the inducible form of nitric oxide synthase (iNOS) and the formation of prostacyclin and thromboxane A2 by cyclooxygenase (COX) were increased. Myocardial infarction also resulted in increased myocardial NO production. Aspirin (acetylsalicylic acid. ASA) at low concentration (35 mg/kg/day) fails to change iNOS production, in contrast to higher dose (150 mg/kg/day) which, as previously shown, inhibits iNOS activity. ASA at all doses also suppresses myocardial prostanoid formation because of inhibition of COX. Recently, two NO donors have been synthesized: NCX 4016 and Diethylenetriamine/NO (DETA/NO). NCX 4016 combines an NO-releasing moiety with a carboxylic residue via an esteric bond. We describe here that NCX 4016 (65 mg/kg/day) increased prostacyclin and thromboxane A2 production in the infarcted heart muscle, overcoming the inhibitory effects of ASA. As a result of nitric oxide release, oxidation products of NO (NO2- and NO3-; NOx) in arterial blood rose following administration of NCX 4016. On oral administration, NCX 4016 did not change systemic arterial pressure. The effects of a single NO donor, DETA/NO (1.0 mg/kg/day) on the infarcted heart were also investigated On intravenous administration, the compound increased NO concentration in arterial blood slightly but to a lesser degree than NCX 4016. Like NCX 4016, it raised myocardial production of prostacyclin and thromboxane A2 in the infarcted heart. However, it caused a severe fall in blood pressure. These findings demonstrate that newly-synthesized NO donors release nitric oxide in situ and increase myocardial production of prostanoids. NCX 4016 has therapeutic potential because it can be orally administered, lacks hypotensive effects, increases blood levels of nitric oxide and myocardial prostacyclin production.

The pharmacology of a new nitric oxide donor: B-NOD.

We describe here a new compound, B-NOD, which, in vitro and in situ, releases nitric oxide (NO). Its activity in situ persists for more than 7 h, it does not cause a fall in blood pressure or an increase in heart rate and can be orally administered. It increases cyclic guanosine monophosphate (cGMP) and prevents platelet aggregation. In vitro, its release of NO is augmented by the presence of living cells (blood platelets). B-NOD may be useful in a number of clinical conditions in which prolonged release of NO without hemodynamic effects are desirable. A combination of aspirin with B-NOD could be formulated in which the individual concentrations of aspirin and B-NOD may be useful in the long-term treatment of coronary artery disease and in clinical situations in which long-term release of NO may be beneficial.

New look at myocardial infarction: toward a better aspirin.

The evidence for the formation of NO and of its oxidation products, as well as of prostacyclin and thromboxane by the infarcted heart muscle is reviewed. The importance of inflammatory cells, primarily macrophages of cardiac origin is documented. Because of its side effects on gastric mucosa and kidney by aspirin, several modifications of aspirin are currently being developed. These are based on eliminating their inflammatory effect by selective inhibition of COX-2, or by attaching an NO-delivering side chain to the aspirin molecule (NO-aspirin), or by combining two preparations, an NO donor with aspirin. NO-aspirins and the combination of an NO-donor with aspirin promise to be beneficial in the early stages of myocardial infarction. Unfortunately, the main beneficial effect of aspirin, that of inhibition of thrombus formation, is also the cause for its most dreaded complication, hemorrhagic stroke. None of the new aspirins is able to prevent this complication.

Biochemistry of the infarcted heart.

I am honored by the invitation to contribute to a volume in Neuroscience, dedicated to Professor Galoyan, whose accomplishments in the field of neuroscience and circulation have been unique. In his book, Dr. Galoyan has summarized the results of his discovery of cardioactive neurohormones. His discovery of biosynthesis of cytokines in the neurosecretory cells of the hypothalamus have opened a new page in immunology.

Production of prostanoids and nitric oxide by infarcted heart in situ and the effect of aspirin.

The production of prostacyclin (PGI2) and thromboxane A2 (TXA2) in infarcted and noninfarcted portions of the rabbit heart was studied prior to and following administration of acetylsalicylic acid (aspirin). Aspirin was administered intravenously (iv) as water-soluble Aspisol, d-lysinmono (acetylsalicylate) (Bayer, Leverkusen, Germany) into an ear vein. A branch of the left circumflex coronary artery was ligated. The animals were divided into three groups. The first group received 150 mg/kg/day of aspirin (75 mg/kg of aspirin every 12 h, n = 10). The first administration of aspirin was 1 h after ligation of the coronary artery and the last injection was 1 h before euthanasia. The second group received 5 mg/kg/day of aspirin (every 24 h, n = 10). A separate group of rabbits not receiving aspirin served as controls (n = 12). Two days following onset of ischemia, inducible form of nitric oxide synthase (iNOS) was measured in heart muscle and the oxidation products of nitric oxide (nitrite, NO-2 plus nitrate, NO-3: their sum referred to as NOx) were determined in arterial and coronary venous blood. Concentrations of both PGI2 and TXA2 were elevated in the infarcted portions of the heart compared to the noninfarcted regions. Formation of prostanoids was accompanied by increased activation of iNOS. Both doses of aspirin diminished the concentrations of PGI2 and TXA2 in infarcted heart muscle; in contrast, small doses of aspirin failed to influence myocardial iNOS activity. Apparently small doses of aspirin changed the relationship of iNOS to cyclooxygenase (COX). Coronary arterial-venous difference of NOx and myocardial iNOS activity showed parallel increases. Diminution of prostacyclin by aspirin can damage gastric mucosa and interfere with vasodilatation. Since NO counters these deficiencies, a combination of aspirin with a nitric oxide donor may be advantageous.

Effect of acetylsalicylic acid on nitric oxide production in infarcted heart in situ.

The effect of Acetylsalicylic Acid (ASA, Aspirin) on the myocardial production of the inducible form of nitric oxide synthase (iNOS) and the oxidation products of nitric oxide (nitrite, NO-2 and nitrate, NO-3: NOx) were studied in the rabbit heart two days after ligation of a branch of the left circumflex coronary artery. ASA was administered intravenously as AspisolR, DL-Lysinmono(acetylsalicylate) which is soluble in water. Animals received a total dose of 250, 375, or 500 mg/kg of ASA in five divided doses intravenously. Significant inhibition of iNOS was noted in the infarcted portion of the myocardium at 375 and 500 mg/kg of ASA. The reduction in myocardial nitric oxide (NO) production was paralleled by a diminution in coronary arterial-venous difference of NOx, demonstrating that ASA inhibition extended also to the oxidation products of NO. ASA is an inhibitor of cyclooxygenase (COX). The inhibition of iNOS by ASA demonstrates the close relationship between COX and iNOS activity in the heart in situ. Whether activity of the infarcted heart is influenced by the diminution in the production of NO by ASA is not known.

Dexamethasone on inducible nitric oxide synthase and nitrite/nitrate production in myocardial infarction.

The effect of dexamethasone (DEX) on the activation of the inducible form of nitric oxide synthase (iNOS) and its relation to the formation of oxidation products of nitric oxide (NO2-+ NO3-, NOx) in infarcted heart muscle of rabbits was investigated. Myocardial infarction was produced by ligation of the first anterior branch of the left circumflex coronary artery. NOx was determined by chemiluminescence and iNOS by conversion of L-[14C]-arginine to L-[14C]-citrulline. The rise in myocardial iNOS production, which followed onset of myocardial ischemia, was suppressed by DEX. In addition, following coronary artery ligation, coronary arterial venous differences of NOx increased markedly; this effect was also partially abolished by DEX. Cardiac origin of NOx was confirmed by the linear relationship between coronary A-V NOx difference and myocardial formation of iNOS. This relationship was preserved after administration of DEX. Therefore glucocorticoids interfere with the myocardial NO production and thus diminish the concentration of its oxidative products in plasma.

Production of oxidative products of nitric oxide in infarcted human heart.

OBJECTIVES: We sought to assess whether oxidation products of nitric oxide (NO), nitrite (NO2-) and nitrate (NO3-), referred to as NOx, are released by the heart of patients after acute myocardial infarction (AMI) and whether NOx can be determined in peripheral blood of these patients. BACKGROUND: Previously we reported that in experimental myocardial infarction (rabbits) NOx is released mainly by inflammatory cells (macrophages) in the myocardium 3 days after onset of ischemia. NOx is formed in heart muscle from NO; NO originates through the activity of the inducible form of nitric oxide synthase (iNOS). METHODS: Eight patients with acute anterior MI and an equal number of controls were studied. Coronary venous blood was obtained by coronary sinus catheterization; NOx concentrations in coronary sinus, in arterial and peripheral venous plasma were measured. Left ventricular end-diastolic pressure was determined. Measurements were carried out 24, 48 and 72 h after onset of symptoms. The type and location of coronary arterial lesions were determined by coronary angiography. Plasma NO3- was reduced to NO2- by nitrate reductase before determination of NO2- concentration by chemiluminescence. RESULTS: The results provided evidence that in patients with acute anterior MI, the myocardial production of nitrite and nitrate (NOx) was increased, as well as the coronary arterial-venous difference. Increased NOx production by the infarcted heart accounted for the increase of NOx concentration in arterial and the peripheral venous plasma. The peak elevation of NOx occurred on days 2 and 3 after onset of the symptoms, suggesting that NOx production was at least in part the result of production of NO by inflammatory cells (macrophages) in the heart. CONCLUSIONS: The appearance of oxidative products of NO (NO2- and NO3-) in peripheral blood of patients with acute MI is the result of their increased release from infarcted heart during the inflammatory phase of myocardial ischemia. Further studies are needed to define the clinical value of these observations.

Myocardial infarction and regulatory myosin light chain.

Myosin from cardiac muscle consists of two heavy chains and two pairs of light chain. Regulatory myosin light chain (RMLC) is phosphorylated by a Ca2+ and calmodulin dependent myosin light chain kinase. The impact of experimental myocardial infarction on cardiac RMLC was studied. The left anterior descending coronary artery of rabbits was ligated. Three, 7 and 14 days later the animals were euthanized, sections of the heart were frozen in liquid nitrogen and later subjected to 2-dimensional electrophoresis. Isoelectric focusing was carried out at a pH range of 4.5-5.4. Reproducible patterns of protein separation showed four spots with proteins of phosphorylatable regulatory light chains shifted to a more negative pH as compared to essential light chain. We investigated changes in phosphorylation of RMLC in infarcted heart muscle. As compared to sham operated animals, a decline in phosphorylation of RMLC was present in both infarcted and non-infarcted portions of the left ventricle; the latter was significant 7 days following the onset of ischemia. In contrast, the decline in percent phosphorylation in the infarcted area was not significant. The amount of RMLC decreased significantly in the infarcted portion. A highly significant reduction in the percent of viable cardiomyocytes accompanied the decline in phosphorylation. There was a significant correlation of RMLC following administration of isoproterenol, 7 and 14 days following onset of ischemia. Only faint traces of essential atrial myosin light chain (ALC-1) were present in the non-infarcted portion of the left ventricle. No correlation was found between percent phosphorylation and the amount of RMLC (density) following infusion of saline or isoproterenol. Isoproterenol significantly increased percent phosphorylation without altering the amount of RMLC protein. We conclude that myocardial infarction profoundly affects regulatory myosin light chain phosphorylation in the infarcted and non-infarcted areas of the myocardium and that RMLC plays a significant part in myocardial contractility.

Oxidation products of nitric oxide, NO2 and NO3, in plasma after experimental myocardial infarction.

Nitrite and nitrate (NO2 and NO3), the oxidative products of nitric oxide (NO), were elevated in the plasma of rabbits on the third day following ligation of a coronary artery. This elevation coincided with increased activity of the inducible form of nitric oxide synthase (iNOS) in infarcted heart muscle. Data are reported which relate the elevated plasma concentrations of NO2+NO3 (NO(x)) to the increased induction of iNOS in an infarcted heart. NO2 and NO3 in plasma were measured by chemiluminescence. Nitrate was converted to nitrite by nitrate reductase. Plasma from the ear vein, right and left ventricle, and coronary sinus were analyzed for NO(x), and iNOS activity was enzymatically determined in infarcted, risk, and normal areas of the heart. The production equivalent of NO(x) by the heart and lung was also calculated. In addition, the effect of a specific inhibitor of iNOS, S-methylisothiourea sulfate (SMT) on plasma concentration and myocardial production of NO(x) was determined. It was concluded that the elevation of plasma NO(x) following onset of myocardial ischemia was directly related to increased induction of iNOS in the heart. This conclusion was based on a proportional and simultaneous increase in NO(x) plasma concentration with myocardial iNOS activation. The inhibitory effect of SMT furnished additional confirmation of the relationship between myocardial iNOS activation and NO(x) plasma levels in experimental myocardial infarction.

Apoptosis in experimental myocardial infarction in situ and in the perfused heart in vitro.

We report the appearance of apoptotic cells in experimental myocardial infarction (rabbit heart) in in situ and in vitro preparations. Apoptosis was recognized by intravital staining with Hoechst 33342 (Ho342), by nick-end labeling (TUNEL) and by DNA laddering. A steady rise in the relative number of apoptotic cardiomyocytes (apoptotic index) was noted in in situ preparations. Apoptosis was first noted 6 h after the onset of ischemia with its highest value occurring after 72 h. Apoptotic nuclei were absent in remote areas of the left and right ventricles. Apoptotic nuclei within the infarcted area showed diminished intensity of Ho342 fluorescence. Three days after ischemia, a border zone adjacent to the infarcted area consisting of apoptotic macrophages was recognized. A novel finding was the appearance of apoptotic cardiomyocytes in the isolated perfused ischemic heart. Occurring as early as 50 min after the onset of ischemia, a high apoptotic index was present adjacent to the ligature placed around the coronary artery. This observation provides the opportunity to selectively examine factors leading to apoptosis in the ischemic heart under controlled experimental conditions.

Myocardial infarction and nitric oxide.

The report deals with the induction of the inducible form of nitric oxide synthase (iNOS) in infarcted heart muscle of rabbit and man. In the rabbit, nitric oxide synthase was significantly increased in the infarcted area beginning on the third day following ligation of a coronary artery. iNOS induction occurred primarily in macrophages. In man, iNOS immunoreactivity was also primarily localized in macrophages on the seventh day following death from myocardial infarction. Of the specific inhibitors of iNOS in infarcted heart muscle, S-methylisothiourea (SMT) was the most potent. Its greatest effect occurred in the normal non-affected area of the heart. Dexamethasone and cyclosporin A failed to inhibit iNOS. Apoptosis of macrophages commenced two days following ligation of a coronary artery.

Effect of inhibitors of inducible form of nitric oxide synthase in infarcted heart muscle.

Nitric oxide (NO), an unstable radical, is synthesized from L-arginine by the constitutive (cNOS) and inducible (iNOS) forms of NOS. cNOS is present mainly in endothelial cells and plays a role in the regulation of blood flow. iNOS, the dominant enzyme in heart muscle during myocardial infarction, allograft rejection, and cardiomyopathy, is activated in macrophages. We recently described a significant increase of iNOS activity in macrophages of infarcted rabbit myocardium 24 hours after coronary occlusion, with peak activity occurring 3 days following coronary artery ligation. Inhibitors of NOS are L-arginine derivatives that inhibit both cNOS and iNOS; S-methylisothiourea (SMT) and aminoguanidine (AMG) are specific inhibitors of iNOS. Cyclosporin A and dexamethasone inhibit by interfering with protein synthesis. iNOS inhibition by SMT, NG-nitro-L-arginine (L-NNA), AMG, cyclosporin A and dexamethasone was examined in homogenates of normal, risk and infarcted myocardium. Three days after coronary artery ligation, the heart was excised and divided into normal, risk and infarcted regions. The inhibitory effect was calculated as IC50. Results shows that SMT was the most potent inhibitor with the lowest IC50; its effect, as well as the effects of L-NNA and AMG, depended on the location in the myocardium. Inhibition for SMT and AMG was greater in the normal area than in the risk and infarcted regions. AMG induced an initial rise of iNOS followed by gradual decline in the area of risk and infarction. No inhibitory effects in cyclosporin A and dexamethasone were noted.