Reduction of myocardial infarct size by fluvastatin

Statins have a variety of cardioprotective properties following chronic treatment. In contrast, little is known about the acute effects. Reperfusion acutely injures the heart by activation of neutrophils as well as endothelial cells. Because statins are known to influence the processes pathogenetically involved, we hypothesized that acute application of statins attenuates the sequelae of cardiac reperfusion. In rats, myocardial infarction (MI) was induced by ligature of the left coronary artery followed by reperfusion. Myocardial blood flow (MBF) was determined by H2 clearance and regional myocardial function (fractional thickening, FT) by pulsed Doppler. MI size was measured by triphenyltetrazolium chloride (TTC) staining, neutrophil extravasation by determination of myeloperoxidase (MPO) activity, and nitric oxide generation via measurement of cGMP. Treatment with fluvastatin, administered intravenously 20 min before the onset of ischemia, significantly attenuated the decline of FT and MBF at the end of the reperfusion period and significantly reduced MI size. Furthermore, fluvastatin induced a significant reduction of MPO activity and an increase of cGMP level compared with the control group. The effect of fluvastatin was completely abolished following pretreatment of NG-nitro-l-arginine methyl ester (l-NAME). These findings suggest that acute application of fluvastatin reduces MI size and attenuates reperfusion injury. We propose that the underlying mechanism is at least partially an inhibition of inflammation and endothelial dysfunction by preventing the activation and extravasation of neutrophils.     

Reduction of experimental myocardial infarct size with hyperosmolar mannitol.

Hypertonic mannitol previously has been shown to improve cardiac function, increase collateral flow, and decrease epicardial ST segment elevation following coronary occlusion in anesthetized or awake dogs. The present study quantitates by morphologic techniques, the effect of hypertonic mannitol on infarct size. Ischemic injury was produced by proximal occlusion of the circumflex artery for 40 min and necrosis was assessed after 48 hr of reflow. One group of dogs was given isotonic saline and the other hypertonic mannitol beginning the infusions just prior to, during, and for a short period after the release of the circumflex coronary artery occlusion. Serum osmolality increased by approximately 40 mOsm in the mannitol group. The administration of hypertonic mannitol was associated with a 40-50% reduction in infarct size ventricular fibrillation during occlusion and following release of the circumflex coronary artery occlusion was greater in mannitol-treated dogs although the difference was not statistically significant. Thus, the data obtained in this study extend previous observations and provide direct evidence that hypertonic mannitol can reduce infarct size in dogs with temporary circumflex artery occlusion and reflow.     

Reduction of myocardial infarct size by human mesenchymal stem cell conditioned medium

Although paracrine effects of mesenchymal stem cells (MSCs) have been suggested previously, cardioprotection by human MSC secretions has never been demonstrated. Human MSC-conditioned medium (CM) was collected by following a clinically compliant protocol. In a porcine model of ischemia and reperfusion injury, intravenous and intracoronary MSC-CM treatment significantly reduced myocardial nuclear oxidative stress as determined by immunostaining for 8-hydroxy-2′-deoxyguanosine. In addition, expression levels of phospho-SMAD2 and active caspase 3 were diminished following CM treatment, suggesting that TGF-β signaling and apoptosis were reduced. This was associated with a 60% reduction in infarct size and marked improvement of systolic and diastolic cardiac performance as assessed with echocardiography and pressure volume loops. Fractionation studies revealed that only the fraction of the CM containing products > 1000 kDa (100–220 nm) provided cardioprotection in a mouse model of ischemia and reperfusion injury. This indicates that the responsible paracrine factor of human MSCs is likely a large complex rather than a single small molecule. These data identify human MSC-CM as a promising therapeutic option to reduce myocardial infarct size in patients with acute MI and suggest that the use of stem cell secretions could extend the applicability of stem cells for therapeutic purposes.     

Coronary endothelial dysfunction from ischemia and reperfusion: effect of reactive oxygen metabolite scavengers

Using anesthetized mongrel dogs exposed to 60 min of ligation of the left anterior descending coronary artery followed by 60 min of reperfusion, we examined the effect of superoxide dismutase (SOD) and dimethylthiourea (DMTU) on evidence of endothelial injury in coronary rings studied in vitro. In 13 dogs treated with saline rings from the normal left circumflex coronary artery (LCF) relaxed by 98 +/- 4% when exposed to 10(-5) M acetylcholine whereas rings from the left anterior descending coronary artery (LAD) relaxed by 79 +/- 7% (p less than 0.05). In the same rings maximum relaxation with the ionophore A23187 was 107 +/- 5% versus 87 +/- 8% (p less than 0.05) for the LCF and the LAD, respectively. Comparisons of concentration-response curves through a range of doses of both acetylcholine and A23187 revealed significant differences for both vasodilators between the LCF and the LAD (p less than 0.01 for each). Nine dogs were treated with bovine SOD infused in the left atrium the last 20 min of ligation and throughout reperfusion (140 units/kg/min) and six other dogs were treated with DMTU 500 mg/kg i.v. given the last 30 min of the ligation period. Neither SOD nor DMTU prevented endothelial injury in the LAD. Despite pretreatment with these agents, there were significant reductions in maximum relaxation and in total concentration-response curves in the LAD as compared with the results in rings from the LCF with both acetylcholine and A23187. There were normal responses to nitroprusside in both the LCF and LAD in all three experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of myocardial infarct size by doxycycline: A role for plasmin inhibition

Myocardial ischemia-reperfusion (I/R) is associated with the activation of matrix metalloproteinases (MMPs) and serine proteases. We hypothesized that activation of MMPs and the serine protease plasmin contribute to early cardiac myocyte death following I/R and that broad-spectrum protease inhibition with doxycycline (DOX) preserves myocyte viability. Rats treated daily with or without DOX beginning 48 h prior to experimentation were subjected to 30 min of coronary occlusion and 2 days of reperfusion. DOX pre-treatment reduced infarct size by 37%. DOX attenuated increases in MMP-9 and plasmin levels as determined by gelatin zymography and immunoblot, respectively. Neutrophil extravasation was unaltered by DOX as assessed by myeloperoxidase (MPO) activity. To examine the contribution of MMP-9 and plasmin to myocyte injury, cultures of neonatal rat ventricular myocytes (NRVMs) were treated for 48 h with 83 kDa MMP-9 or plasminogen in the presence or absence of DOX. MMP-9 treatment did not affect myocyte viability. Plasminogen treatment led to increased plasmin activity, resulting in loss of ₁-integrin, NRVM detachment and apoptosis. DOX co-treatment inhibited plasmin activity and preserved NRVM attachment, whereas co-treatment with the broad-spectrum MMP inhibitor GM6001 had no effect. These results indicate that plasmin causes disruption of myocyte attachment and viability independently of MMP activation in vitro and that inhibition of plasmin by DOX may reduce I/R-induced myocyte death in vivo through the inhibition of plasmin. (Mol Cell Biochem 270: 1–11, 2005)     

Reduction of infarct size by orally administered des-aspartate-angiotensin I in the ischemic reperfused rat heart

Occlusion of the left main coronary artery for 45 min caused sizable infarct scaring of the left ventricular wall in the rat heart at 14 days post-reperfusion. Daily oral administration of des-aspartate-angiotensin I (DAA-I) for 14 days attenuated the area of the infarct scar and transmurality. The attenuation was dose-dependent and biphasic; maximum effective dose was 1524 nmol/kg, and doses higher than this were progressively inactive. The exact mechanism of the biphasic attenuation is not known, and receptor down-regulation by internalization, which has been implicated in a similar biphasic nature for the anticardiac hypertrophic action of DAA-I, could be a likely cause. Indomethacin (101 μmol/kg, i.p.), administered sequentially after the daily oral dose of DAA-I (1524 nmol/kg), completely inhibited the attenuation at 14 days post-reperfusion, indicating that prostaglandins may be involved in transducing the attenuation. The present findings support earlier indications that DAA-I exerts protective actions in cardiovascular pathologies in which angiotensin II is implicated. It is suggested that DAA-I exerts the cardioprotective action by acting on the same indomethacin-sensitive angiotensin AT₁ receptor. Although similar array of protective actions are also seen with another endogenous angiotensin, angiotensin-(1–7), the present findings demonstrate for the first time the ability of an endogenous angiotensin to reduce the infarct size of an ischemic-reperfusion injured rat heart.     

Inhibition of production of monocyte/macrophage-derived angiogenic activity by oxygen free-radical scavengers.

We showed previously that thiol-containing compounds inhibited the production of macrophage-mediated angiogenic activity. Since thiol-containing compounds may act on macrophages by affecting activation and inhibiting the production of oxygen free-radicals, we studied the effects of oxygen free-radical scavengers on production of angiogenic activity by elicited mouse peritoneal macrophages and lipopolysaccharide stimulated normal human monocytes. Monocyte/macrophage conditioned media were potently angiogenic when assayed in rat corneas, while conditioned media, from oxygen free-radical scavenger-treated cells were not. The inhibitory effect of oxygen free-radical scavengers was due to a direct effect on monocyte/macrophage production of angiogenic activity but was not due solely to a decrease in the production of the macrophage-derived angiogenic cytokine tumor necrosis factor-alpha. We conclude that oxygen free-radical scavengers are potent inhibitors of the production of macrophage-mediated angiogenic activity.     

The intermediary metabolite pyruvate attenuates stunning and reduces infarct size in in vivo porcine myocardium

The intermediary metabolite pyruvate has been shown to exert significant beneficial effects in in vitro models of myocardial oxidative stress and ischemia-reperfusion injury. However, there have been few reports of the ability of pyruvate to attenuate myocardial stunning or reduce infarct size in vivo. This study tested whether supraphysiological levels of pyruvate protect against reversible and irreversible in vivo myocardial ischemia-reperfusion injury. Anesthetized, open-chest pigs (n = 7/group) underwent 15 min of left anterior descending coronary artery (LAD) occlusion and 3 h of reperfusion to induce stunning. Load-insensitive contractility measurements of regional preload recruitable stroke work (PRSW) and PRSW area (PRSWA) were generated. Vehicle or pyruvate (100 mg/kg i.v. bolus + 10 mg x kg(-1) x min(-1) intra-atrial infusion) was administered during ischemia and for the first hour of reperfusion. In infarct studies, pigs (n = 6/group) underwent 1 h of LAD ischemia and 3 h of reperfusion. Group I pigs received vehicle or pyruvate for 30 min before and throughout ischemia. In group II, the infusion was extended through 1 h of reperfusion. In the stunning protocol, pyruvate significantly improved the recovery of PRSWA at 1 h (50 +/- 4% vs. 23 +/- 3% in controls) and 3 h (69 +/- 5% vs. 39 +/- 3% in controls) reperfusion. Control pigs exhibited infarct sizes of 66 +/- 1% of the area at risk. The pyruvate I protocol was associated with an infarct size of 49 +/- 3% (P < 0.05), whereas the pyruvate II protocol was associated with an infarct size of 30 +/- 2% (P < 0.05 vs. control and pyruvate I). These findings suggest that pyruvate attenuates stunning and decreases myocardial infarction in vivo in part by reduction of reperfusion injury. Metabolic interventions such as pyruvate should be considered when designing the optimal therapeutic strategies for limiting myocardial ischemia-reperfusion injury.     

Inhibition by adenosine of reactive oxygen metabolite production by human polymorphonuclear leucocytes.

The stimulation of reactive oxygen metabolite production from human polymorphonuclear leucocytes by chemotactic peptide (fMet-Leu-Phe) was inhibited by adenosine with a K0.5 of 0.6 microM. Dipyridamole (0.1 microM), an inhibitor of adenosine uptake, did not prevent the effect of adenosine. Non-metabolizable analogues could substitute for adenosine in the potency order N-ethoxycarboxamideadenosine greater than 2-chloroadenosine greater than adenosine greater than L-N6-(phenylisopropyl)adenosine = D-N6-(phenylisopropyl)adenosine, which is characteristic of an A2 adenosine receptor. The effects of adenosine, 2-chloroadenosine and N-ethoxycarboxamideadenosine were reversed by 8-phenyltheophylline. When endocytosis was inhibited with cytochalasin B, cells were still susceptible to adenosine receptor agonists. 2-Chloroadenosine (10 microM) reduced the activation of respiration in response to chemotactic peptide from 3.3-fold to 1.4-fold. Activation of reactive oxygen metabolite production in response to latex beads was not reversed by adenosine or its analogues. It was concluded that adenosine acts at an A2 adenosine receptor to antagonize the activation of polymorphonuclear leucocytes by those stimuli, such as chemotactic peptide, which cause an increase in the intracellular free Ca2+ concentration.     

Effects of scavengers for active oxygen species on cell death by cryptogein

The hypersensitive reaction is a type of programmed cell death in plants. Cryptogein is a proteinaceous elicitor secreted from Phythophthora cryptogea. In one current model, active oxygen species (AOS) trigger programmed cell death in plants. In this study, we examined a variety of AOS scavengers to elucidate the function of AOS in the death program. Most of these AOS scavengers, including tiron, a scavenger for superoxide radical, catalase for hydrogen peroxide, and hydroquinone, sodium ascorbate and propyl gallate for free radicals, almost completely removed extracellular AOS. However, none of the reagents completely blocked the cell death process. Other reagents, such as histidine and dimethylfuran, scavengers for singlet oxygen, and diphenyleneiodonium chloride, an inhibitor of NADPH oxidase, showed significant toxicity in BY-2 cells. These results indicate that AOS produced in the extracellular space do not play a role in hypersensitive cell death.     

Prolongation of alloskin graft survival by catalytic scavengers of reactive oxygen species

We tested the effects of salen manganese (Salen-Mn) complexes, which are scavengers of reactive oxygen species exhibiting superoxide dismutase and catalase activities on the rejection of and alloresponse to fully allogeneic skin grafts in mice. We showed that pre-transplant treatment of C57Bl/6 donor skin or of BALB/c recipients with Salen-Mn complexes significantly delayed allograft rejection. ELISPOT analysis of alloimmune response of treated mice revealed a significant reduction of the frequency of type 1 cytokine (pro-inflammatory) producing T-cells, while the number of activated T-cells producing type 2 cytokines was elevated. In addition, anti-oxidative treatment of graft recipients resulted in a profound inhibition of their donor-specific cytotoxic T-cell response. Our results indicate that salen manganese complexes mediate their effect on graft rejection both by reducing the susceptibility of graft tissue to ROS-mediated injury and by exerting an anti-inflammatory effect in recipients.     

Oxygen radical-mediated mutagenic effect of asbestos on human lymphocytes: suppression by oxygen radical scavengers.

The mutagenic effect of chrysotile asbestos fibers and zeolite and latex particles on human lymphocytes in whole blood has been studied. It was concluded that their mutagenic activities were mediated by oxygen radicals because they were inhibited by antioxidant enzymes (SOD and catalase) and oxygen radical scavengers (rutin, ascorbic acid, and bemitil). It was proposed that oxygen radicals were released by phagocytes activated upon exposure to mineral dusts and fibers. The study of lucigenin- and luminol-amplified chemiluminescence of peritoneal macrophages stimulated by chrysotile fibers and zeolite and latex particles has shown that their mutagenic action is probably mediated by different oxygen species, namely, by the iron-oxygen complexes (perferryl ions) plus hydrogen peroxide, hydrogen peroxide, and superoxide ion, respectively. From the oxygen radical scavengers studied, rutin was the most effective inhibitor of the mutagenic effect of mineral fibers and dusts.     

Effects of iron and oxygen species scavengers on Listeria spp. chemiluminescence

Listeria monocytogenes and Listeria innocua are able, under certain conditions, to produce chemiluminescence (CL), which is amplified by luminol. Kinetic studies of CL by L. monocytogenes and L. innocua show a close parallelism between CL and growth curves during the exponential phase, with a maximum of CL reached just before entrance of bacteria into the stationary phase. CL is tightly correlated with the release of oxygen compounds. The reactive oxygen species scavengers tryptophan, mannitol, and tiron, as well as cellobiose and high temperature, were assessed with regard to CL in the two Listeria species. Only tiron strongly reduced the CL emitted by L. monocytogenes and L. innocua. On the other hand, charcoal pretreatment of the growth medium inhibited the CL, whereas ferric citrate strongly increased the CL of L. monocytogenes and L. innocua. These data suggest that iron and superoxide radical are implicated in the CL produced by these bacteria, but this phenomenon is not correlated to virulence.     

Changes in oxygen consumption induced by t-butyl hydroperoxide in perfused rat liver. Effect of free-radical scavengers.

The addition of t-butyl hydroperoxide to perfused rat liver elicited a biphasic effect on hepatic respiration. A rapid fall in liver oxygen consumption was initially observed, followed by a recovery phase leading to respiratory rates higher than the initial steady-state values of oxygen uptake. This overshoot in hepatic oxygen uptake was abolished by free-radical scavengers such as (+)-cyanidanol-3 or butylated hydroxyanisole at concentrations that did not alter mitochondrial respiration. (+)-Cyanidanol-3 was also able to facilitate the recovery of respiration, the diminution in the calculated rate of hydroperoxide utilization and the decrease in liver GSH content produced by two consecutive pulses of t-butyl hydroperoxide. It is suggested that the t-butyl hydroperoxide-induced overshoot in liver respiration is related to increased utilization of oxygen for lipid peroxidation as a consequence of free radicals produced in the scission of the hydroperoxide by cellular haemoproteins.     

Target size analysis by radiation inactivation: the use of free radical scavengers

Several model systems were employed to assess indirect effects that occur in the process of using radiation inactivation analysis to determine protein target sizes. In the absence of free radical scavengers, such as mannitol and benzoic acid, protein functional unit sizes can be drastically overestimated. In the case of glutamate dehydrogenase, inclusion of free radical scavengers reduced the apparent target size from that of a hexamer to that of a trimer based on enzyme activity determinations. For glucose-6-phosphate dehydrogenase, the apparent target size was reduced from a dimer to a monomer. The target sizes for both glutamate dehydrogenase and glucose-6-phosphate dehydrogenase in the presence of free radical scavengers corresponded to subunit sizes when determinations of protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or immunoblotting were done rather than enzyme activity. The free radical scavengers appear to compete with proteins for damage by secondary radiation products, since irradiation of these compounds can result in production of inhibitory species. Addition of benzoic acid/mannitol to samples undergoing irradiation was more effective in eliminating secondary damage than were 11 other potential free radical scavenging systems. Addition of a free radical scavenging system enables more accurate functional unit size determinations to be made using radiation inactivation analysis.     

Failure of oxygen radical scavengers to modify fatigue in electrically stimulated muscle.

We used in situ gastrocnemius muscle of anaesthetized dogs to test the hypothesis that O2 radical production during muscle contraction contributes to fatigue. Muscle tension was measured with a force transducer and blood flow was monitored with an electromagnetic flow probe. Muscle contractions were produced by stimulating the nerve for 15 min at 20 Hz, 12 trains/min, and a duty cycle of 0.25. Three groups of seven animals were given an infusion of 0.2 mL.min-1 of either saline, low-dose oxygen radical scavengers (250 IU.mL-1 superoxide dismutase, 640 IU.mL-1 polyethylene glycol (PEG)-catalase, 0.25 mg.mL-1 deferoxamine, and 0.1 mg.mL-1 oxypurinol), or high-dose oxygen radical scavengers (3300 IU.mL-1 superoxide dismutase, 6600 IU.mL-1 PEG-catalase, 2.5 mg.mL-1 deferoxamine, and 0.1 mg.mL-1 oxypurinol). Blood flow and vascular resistance of the gastrocnemius muscle during stimulation did not differ among groups. After 15 min of stimulation, the developed tension (represented as a percentage of initial tension developed) was 66 +/- 7% in the saline treated group, 70 +/- 6% in the low-dose group, and 70 +/- 4% in the high-dose group. The change in tension during recovery was not significant in the control or low-dose groups. However, there was partial recovery in the high-dose group. In conclusion, in this preparation, oxygen radical scavengers did not delay the development of decreased muscle tension.     

Ischemic preconditioning enhances scavenging activity of reactive oxygen species and diminishes transmural difference of infarct size

Reactive oxygen species (ROS) enhance myocardial ischemia-reperfusion (I/R) injury. Ischemic preconditioning (PC) provides potent cardioprotective effects in I/R. However, it has not been elucidated whether PC diminishes ROS stress in I/R and whether PC protects the myocardium from ROS stress transmurally and homogeneously. Isolated rabbit hearts perfused with Krebs-Henseleit buffer underwent 30 min of ischemia and 60 min of reperfusion. Hemodynamic changes and myocardial damage extent were analyzed in four groups. The control group underwent I/R alone. The H2O2 group underwent I/R with H2O2 infusion (50 microM) in the first minute of reperfusion to enhance oxidative stress. The PC and H2O2+PC groups underwent 5 min of PC before control and H2O2 protocols, respectively. Extracted myocardial DNA was analyzed for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative DNA damage, with the use of the HPLC-electrochemical detection method. Glutathione peroxidase (GPX) activity and the reduced form of GSH were measured by spectrophotometric assays. The myocardial infarct size was significantly reduced in the PC group (19 +/- 2%) compared with the control group (37 +/- 4%; P < 0.05), particularly in the subendocardium. H2O2 transmurally increased the infarct size by 59 +/- 4% (P < 0.05), which was significantly diminished in the H2O2+PC group (31 +/- 4%; P < 0.01). The GSH levels, but not GPX activity, were well preserved transmurally in protocols with PC. The 8-OHdG levels were significantly decreased in PC and were significantly enhanced in H2O2 (P < 0.01). These changes in oxidative DNA damage were effectively diminished by PC. In conclusion, PC enhanced the scavenging activity of GSH against ROS transmurally, reduced myocardial damage, particularly in the subendocardium, and diminished the transmural difference in myocardial infarct size.     

Inhibitory effect of active oxygen scavengers on the endocytosis of concanavalin A by lymphocytes

It has been shown that of active oxygen species regulate the activity of the processes of Con A endocytosis in rat thymus cells.