J. Vinten-Johansen

Antineutrophil and myocardial protecting actions of a novel nitric oxide donor after acute myocardial ischemia and reperfusion of dogs.

BackgroundIt has recently been demonstrated that myocardial ischemia and reperfusion results in a marked decrease in the release of nitric oxide (NO) by the coronary endothelium. NO may possess cardioprotective properties, possibly related to inhibition of neutrophil-related activities. We tested the hypothesis that a cysteine-containing nitric oxide donor compound, SPM-5185, would reduce infarct size and inhibit neutrophil-related activities (adherence to coronary vascular endothelium, accumulation). Methods and ResultsThe effects of intracoronary infusion of SPM-5185 were investigated in a 5.5-hour model of myocardial ischemia (1 hour) and reperfusion (4.5 hours) (MI-R) in anesthetized, open-chest dogs. SPM-5185 (500 nmol/L) or saline vehicle was infused for 4.5 hours into the left anterior descending coronary artery (LAD) at the time of reperfusion after 1 hour of LAD occlusion. MI-R in dogs receiving saline vehicle resulted in severe myocardial injury characterized by dyskinesis, a profound elevation of plasma creatine kinase, marked myocardial necrosis, and high cardiac myeloperoxidase (MPO) activity in the ischemic and necrotic zones. In contrast, treatment with SPM-5185 resulted in a modest restoration of regional function, a reduction of myocardial necrosis expressed as a percentage of the area at risk (12.5±3.2% versus 41.7±5.4%, P<.001), and significant reductions of MPO activity in the ischemic zone (0.8±0.1 versus 2.5±0.7 U/100 mg tissue, P<.05) and the necrotic zone (1.6±0.2 versus 3.3±0.6 U/100 mg tissue, P<.05). In additional studies, SPM-5185 (500 nmol/L) significantly (P<.001) attenuated the adherence of LTB4-stimulated canine neutrophils to autologous segments of coronary artery and attenuated the neutrophil-induced contraction of isolated coronary arterial rings. ConclusionsSPM-5185 reduces myocardial necrosis and neutrophil accumulation in an acute model of canine myocardial ischemia and reperfusion. This reduction in myocardial cell injury may be partially related to the inhibitory actions of this novel NO donor on neutrophil adherence to the coronary endothelium.

Myocardial protective effects of adenosine. Infarct size reduction with pretreatment and continued receptor stimulation during ischemia.

BackgroundWe hypothesized that 1) endogenous adenosine released during ischemia conferred an inherent cardioprotection, and 2) a pretreatment dose of adenosine before ischemia would provide additional protection independent of hemodynamic effects. Methods and ResultsThirty-six anesthetized New Zealand White rabbits underwent 30 minutes of regional ischemia produced by coronary occlusion followed by 2 hours of reperfusion. The adenosine group (ADO, n=9) received a 5-minute pretreatment infusion of 140 pg/kg/min of adenosine before ischemia. A control group (SAL, n=9) received saline before ischemia. To separate the effects of adenosine used as a pretreatment versus the effects during ischemia, a third group (ADO+SPT, n=9) received adenosine as pretreatment followed by 10 mg/kg 8-p-sulfophenyl theophylline (8-SPT), an A1/A2-receptor antagonist given before ischemia, thus allowing pretreatment with adenosine but antagonizing its effects during ischemia. To preclude any protection from endogenous adenosine released during ischemia, the fourth group (SAL+SPT, n=9) received saline as pretreatment and 8-SPT before ischemia. Area of necrosis within the area at risk (infarct size) was determined with tetrazolium and Evans blue stains, and transmural blood flow was measured using radioactive microspheres. Collateral blood flow in the area at risk was similar in all groups, as was the size of the area at risk. Infarct size was reduced by adenosine pretreatment (ADO, 8A±47.2%) in contrast to saline vehicle (SAL, 27.8±63%; p<0.05 versus ADO). α1/α2-Receptor blockade after adenosine pretreatment abolished the ischemic protection provided by pretreatment adenosine (ADO+SPT, 42.7±83%; p<0.05 versus ADO). Finally, receptor blockade of endogenously released adenosine without adenosine pretreatment increased infarct size by 24% over the nonpretreated saline group (SAL+SPT, 51.5±9.00%; p<0.05 versus SAL). ConclusionsWe conclude that 1) endogenous adenosine building up during ischemia is cardioprotective, and 2) pretreatment with adenosine confers cardioprotection independent of hemodynamic effects. Whether pretreatment effects of adenosine subsequently modulate the effects of endogenous adenosine (through alterations in receptor population or sensitivity) or endogenous and exogenous adenosine represent additive compartments is unclear.

Gradual Reperfusion Reduces Infarct Size and Endothelial Injury but Augments Neutrophil Accumulation

BACKGROUND Reperfusion causes injury to the coronary artery endothelium primarily by neutrophil-mediated mechanisms. However, factors other than neutrophils may govern the extent of myocardial necrosis. This study tests the hypothesis that gradual initiation of reflow will reduce reperfusion injury and preserve postischemic endothelial function. METHODS In 16 anesthetized dogs, the left anterior descending artery was ligated for 60 minutes. In one group, reperfusion was initiated abruptly (abrupt, n = 8), whereas in the gradual reperfusion group (ramp, n = 8), flow was slowly initiated during the first 30 minutes of reperfusion. After reperfusion, coronary artery segments were isolated to assess postischemic endothelial function. RESULTS Infarct size (area of necrosis/area at risk) was significantly reduced in the ramp group (28.2% +/- 2.0%) compared with abrupt (41.6% +/- 1.4%). Neutrophil accumulation (myeloperoxidase) in the area at risk was significantly greater in the ramp group compared with abrupt (8.0 +/- 1.3 versus 3.5 +/- 0.8 U/g tissue). In isolated postischemic left anterior descending arterial rings, the concentration of acetylcholine that elicited a response 50% of the maximum possible response was significantly greater in abrupt (-6.88 +/- 0.04 log [mol/L]) than ramp (-7.62 +/- 0.04 log [mol/L]) and control (-7.68 +/- 0.003 log [mol/L]), suggesting endothelial dysfunction. The concentration of A23187 that elicited a response 50% of the maximum possible response was similarly greater in abrupt (-7.24 +/- 0.03 log [mol/L]) versus ramp (-7.62 +/- 0.03 log [mol/L]) and control (-7.8 +/- 0.04 log [mol/L]). Smooth muscle dysfunction (response to sodium nitrite) also occurred in the abrupt rings. CONCLUSIONS Gradual reperfusion of an ischemic area reduces infarct size and preserves endothelial function but paradoxically increases neutrophil accumulation within the area at risk.

Endogenous nitric oxide (NO) protects against ischaemia-reperfusion injury in the rabbit

OBJECTIVES Recent studies suggest that nitric oxide (NO) is deleterious in models of shock and hypoxia-reoxygenation However, the role of endogenous NO in ischaemia-reperfusion injury in vivo remains controversial. We tested the hypothesis that blockade of endogenous NO produced during myocardial ischaemia-reperfusion or during reperfusion alone in vivo increases infarct size after coronary occlusion in the rabbit, and conversely, supplementation with L-arginine would reduce infarct size. METHODS Ketamine-xylazine anaesthetised New Zealand white rabbits were subjected to left coronary artery occlusion for 30 min and reperfusion for 120 min. The rabbits were divided into five groups: (1) saline (VEH); (2) L-nitro arginine (L-NA), a NO-synthase inhibitor, was infused intravenously (15 mg/kg bolus followed by 7.5 mg/kg h-1) before coronary occlusion to block NO synthase activity during ischaemia and reperfusion (IR); (3) L-NA was administered during reperfusion only (R) at the same dose as in the IR group; (4) D-arginine (D-ARG) (25 mg/kg bolus followed by 4 mg/kg min-1), the non-metabolised enantiomer of L-arginine was infused intravenously during reperfusion only; (5) L-arginine (L-ARG) (25 mg/kg bolus followed by 4 mg/kg min-1), the physiological precursor of NO, was infused intravenously during reperfusion only. RESULTS L-NA infusion in the IR and R groups caused an increase in mean arterial pressure and a decrease in heart rate; however, no significant change in pressure rate product (PRP) occurred immediately after drug infusion. PRP did not change significantly during the experiment across groups except at the end of reperfusion. The area at risk was comparable in all groups, averaging 29(1)%. The infarct size (triphenyltetrazolium chloride) expressed as a percent of area at risk was 27(2)% for the untreated vehicle group. In contrast, L-NA significantly (P < 0.05) increased infarct size in the IR group, 51(2)%; this augmented infarct size persisted when NO synthase activity was blocked during reperfusion only in the R group, 50(2)%. There was no significant (P < 0.05) difference in infarct size between the IR and the R groups. D-ARG-treated group showed a comparable increase in infarct size 48(2)% versus the IR and R groups. However, supplementation of NO with L-arginine (L-ARG) showed no reduction in infarct size, 24(3)%, over vehicle group (VEH). CONCLUSIONS We conclude that (1) blockade of NO synthase activity with L-NA increases infarct size, (2) this effect was expressed primarily during reperfusion, (3) D-arginine mimicked the infarct augmentation of L-NA, while (4) L-arginine supplementation did not reduce infarct size. These data imply that endogenous NO production exerts a tonic cardioprotective effect on myocardial infarct following coronary reperfusion.

Monoclonal antibody to ICAM-1 preserves postischemic blood flow and reduces infarct size after ischemia-reperfusion in rabbit

Neutrophils are pivotal in the pathogenesis of reperfusion injury leading to myocardial infarction. Firm adhesion of PMN to endothelium may be initiated by the interaction between constitutively expressed intercellular adhesion molecule‐1 (ICAM‐1) on endothelium and β2 integrin (CDllb/CD18) on neutrophils. We tested the hypothesis that a monoclonal antibody (mAb RR1/1) against ICAM‐1 would preserve postischemic myocardial blood flow and attenuate myocardial injury in an anesthetized rabbit model of coronary occlusion and reperfusion. Either mAb RR1/1 or isotypematched control mAb (R3.1) was injected 10 min before reperfusion. Postischemic myocardial blood flow in the area at risk (Ar) and necrotic area was significantly improved with mAb RR1/1 treatment compared with vehicle and mAb R3.1 during the reperfusion period. RR1/1 had no effect on nonischemic zone blood flow. The Ar as a percent of left ventricle was comparable between groups. Infarct size (TTC) as a percent of Ar was significantly reduced by mAb RR1/1 compared with saline vehicle and mAb R3.1. Plasma creatine kinase activity confirmed the reduction of infarct size in mAb RR1/1 group. In in vitro studies, 40 μg/mL mAb RR1/1, which approximates the plasma concentration of 2 mg/kg mAb RR1/1, markedly inhibited platelet‐activating factor‐stimulated neutrophil adherence to rabbit aortic endothelium. We conclude that blockade of ICAM‐1 during reperfusion reduces postischemic perfusion defects and attenuates the progression of myocardial injury leading to necrosis. This cardioprotection by mAb RR1/1 may be due to inhibition of neutrophil adhesion to the coronary endothelium. J. Leukoc. Biol. 62: 292–300; 1997.

Coronary artery endothelial dysfunction after global ischemia, blood cardioplegia, and reperfusion

This study tests the hypothesis that blood cardioplegia (BCP) attenuates endothelial dysfunction related to nitric oxide after global normothermic ischemia, cardioplegic arrest, and reperfusion in anesthetized open-chest dogs placed on cardiopulmonary bypass. The dogs were divided into five groups to identify the time when endothelial injury occurred: group 1 = control without ischemia; group 2 = 45 minutes of normothermic ischemia only; group 3 = 45 minutes of normothermic ischemia plus unmodified reperfusion; group 4 = 45 minutes of ischemia plus intermittent BCP without reperfusion; and group 5 = ischemia plus BCP and reperfusion. In vitro coronary vascular relaxation responses to the nitric oxide stimulator acetylcholine (endothelium-dependent, receptor-dependent), the calcium ionophore A23187 (endothelium-dependent, receptor-independent), and acidified NaNO2 (endothelium-independent) were measured at the end of the protocol. Maximum in vitro coronary vascular responses to acetylcholine were similar among groups 1, 2, and 4, indicating an absence of endothelial injury. In contrast, significantly impaired relaxations to acetylcholine were demonstrated in the two reperfused groups (groups 3 and 5). Relaxation responses to A23187 and NaNO2 were not altered markedly in any group. Electron microscopy showed intact endothelium in groups 1, 2, and 4. However, moderately severe endothelium damage was seen in groups 3 and 5. We conclude that morphologic and functional endothelial damage occurs after blood reperfusion with or without BCP, and 1-hour hypothermic BCP arrest after normothermic ischemia is not associated with extension of endothelial damage.

Reduction in surgical ischemic-reperfusion injury with adenosine and nitric oxide therapy

Ischemia and reperfusion impair the inherent capacity of the heart to protect itself from related pathophysiologic events by reducing endogenous oxygen radical scavengers and inhibitors. However, other endogenously produced agents, notably adenosine and nitric oxide, are produced during ischemia, reperfusion, or both. These autacoids have several cardioprotection actions in common, particularly antineutrophil effects and inhibition of endothelial-neutrophil interactions, which are key initial steps in ischemic-reperfusion injury. Studies have shown that nitric oxide exerts cardioprotection primarily during reperfusion. Adenosine, on the other hand, protects the myocardium to some extent during both ischemia and reperfusion, thereby covering both periods during which myocardial injury may be sustained during a cardiac operation. Native adenosine or active analogues, or donors of nitric oxide, may be given before or in conjunction with cardioplegia solutions. However, these endogenous agents can also be pharmacologically recruited to provide a new potent therapeutic approach against surgical ischemic-reperfusion injury. This article reviews the cardioprotective effects of primarily endogenous nitric oxide and adenosine in both nonsurgical and surgical models of ischemia-reperfusion injury. Both adenosine and nitric oxide provide potent cardioprotection in surgical and nonsurgical models of ischemia-reperfusion. An important mechanism in this cardioprotection is attenuation of neutrophil-mediated damage.

Blood cardioplegia enhanced with nitric oxide donor SPM-5185 counteracts postischemic endothelial and ventricular dysfunction

This study tested the hypothesis that enhancement of blood cardioplegia with the nitric oxide donor agent SPM-5185 inhibits postischemic left ventricular and coronary endothelial dysfunction. Eighteen anesthetized dogs supported by total vented bypass were subjected to 30 minutes of normothermic ischemia followed by 4 degrees C multidose blood cardioplegia. Hearts received either standard blood cardioplegia (vehicle group; n = 6), blood cardioplegia with 1 mumol/L SPM-5185 (low-dose group; n = 6), or 10 mumol/L SPM-5185 (high-dose group; n = 6). After 60 minutes of cardioplegic arrest, the heart was reperfused for a total of 60 minutes, first in the beating empty state for 30 minutes and then after discontinuation of bypass for 30 minutes. Baseline and postischemic left ventricular function was assessed by the slope of the end-systolic pressure-volume (impedance catheter) relation. Postischemic end-systolic pressure-volume relation was depressed by 53.7% of preischemic values in the vehicle group (from 8.2 +/- 1.0 to 3.8 +/- 0.3 mm Hg/ml) and by 33.7% (from 9.2 +/- 1.1 to 6.1 +/- 0.5 mm Hg/ml) in the low-dose group. In contrast, there was complete postischemic functional recovery in the high-dose group (from 7.6 +/- 1.1 to 7.2 +/- 1.2 mm Hg/ml). In coronary arteries isolated from these hearts, endothelium-dependent maximal relaxation to acetylcholine was impaired by 27% in the vehicle group and by 18% in the low-dose group, whereas the high-dose group showed complete endothelium-dependent relaxation. Myeloperoxidase activity, an index of neutrophil accumulation in postischemic myocardium, was elevated in the vehicle and low-dose groups (3.36 +/- 0.58 and 2.56 +/- 0.68 U/100 mg tissue) but was significantly reduced in the high-dose group to 1.27 +/- 0.45 U/100 mg tissue. We conclude that inclusion of 10 mumol/L nitric oxide donor SPM-5185 in blood cardioplegia improves postischemic ventricular performance and endothelial function in ischemically injured hearts, possibly via inhibition of neutrophil-mediated damage.

Controlled coronary hydrodynamics at the time of reperfusion reduces postischemic injury

Background: In a canine preparation of 2 hours of coronary occlusion and 2 hours of reperfusion, we tested the hypothesis that controlled hydrodynamic conditions (coronary pressure and flow) of reperfusion would decrease infarct size and improve regional functional recovery. Methods: In 28 anesthetized, open-chest dogs, segmental contractile work and diastolic stiffness (sonomicrometry), and myocardial blood flow (15 μm microspheres) were measured in the segment per fused by the left anterior descending (LAD) and circumflex coronary arteries. After 2 hours of LAD occlusion, reperfusion was established using an extracorporeal perfusion circuit to control intracoronary pressure and blood flow. Twelve dogs did not complete the protocol or meet exclusion criteria. In eight dogs (abrupt group), blood flow was abruptly restored and continued for 2 hours of reperfusion. In eight dogs (ramp group), blood flow was adjusted so that LAD perfusion pressure was linearly incremented over the first 30 minutes of reflow, and equalized to mean arterial pressure thereafter. Results: Two hours of LAD occlusion reduced myocardial blood flow in the ischemic zone from 67.7 ± 11.7 to 11.7 ± 4.7 mL/min/100g (P Conclusions: Slowly increasing coronary pressure and blood flow during the early phase of reperfusion 1) increases postischemic systolic and diastolic functional recovery, 2) improves postischemic blood flow, and 3) reduces infarct size by 67%. These data suggest that the mode of reperfusion may be a factor in determining myocardial injury after reversible occlusion.

l-Arginine inhibits neutrophil adherence and coronary artery dysfunction

BACKGROUND Nitric oxide (NO) attenuates neutrophil (PMN)-mediated damage, partly by inhibiting superoxide anion (O2-) generation and adherence to the coronary artery endothelium. L-Arginine is the endogenous substrate for production of NO via the NO synthase pathway. This study tested the hypothesis that the endogenous NO precursor L-arginine (L-Arg) would reduce PMN-induced coronary artery dysfunction by attenuating O2- production and neutrophil adherence. METHODS Neutrophils and left anterior descending (LAD) coronary artery segments were isolated from normal, anesthetised (30 mg/kg i.v. pentobarbitone) dogs. LAD segments were either cut into 2-3 mm rings and mounted in organ chambers to measure vascular tone responses to acetylcholine (endothelium-receptor-dependent) and acidified NaNO2 (smooth muscle), or cut into segments to measure adherence of fluorescently labeled neutrophils by epifluorescence microscopy. RESULTS L-Arg had no direct inhibitory effect on O2- production (cytochrome c reduction) by PMN activated with platelet activating factor (PAF) (34.6 +/- 4.8 nmol vs. 34.2 +/- 4.1 nmol). L-Arg (10 mmol) reduced adherence of fluorescently labeled PMN to isolated canine coronary artery endothelium activated by 100 nM PAF from 187 +/- 11 to 41 +/- 6 PMN/mm2, P < 0.05. This inhibition of adherence was reversed by N-var pi-nitro-L-arginine (L-NA, 1 mmol) (175 +/- 20 PMN/mm2) and by the NO scavenger, carboxy-PTIO (600 mu M, 157 +/- 23 PMN/mm2). D-arginine, the nonmetabolised enantiomer of L-arginine, (D-Arg, 10 mmol) did not reduce adherence (162 +/- 20 PMN/mm2). To determine the effect of PMN on coronary artery endothelial function, canine coronary artery rings were transiently incubated with activated PMNs in organ chambers to induce dysfunction. After washout of PMN, the EC50 (- log M) derived from post-injury concentration-relaxation responses to acetylcholine was significantly less in 10 mmol L-Arg (6.94 +/- 0.08) than untreated rings (6.47 +/- 0.06). In contrast, 10 mmol D-Arg could not reverse this dysfunction (6.48 +/- 0.11). CONCLUSIONS L-Arg reduces PMN-induced coronary endothelial dysfunction by inhibition of adherence via the L-arginine-NO pathway.