David J. Lefer

Diminished basal nitric oxide release after myocardial ischemia and reperfusion promotes neutrophil adherence to coronary endothelium.

We measured changes in basal release of nitric oxide and its effect on polymorphonuclear leukocyte (PMN) adherence to endothelial cells (ECs) in a feline model of myocardial ischemia (90 minutes) and reperfusion. Basal release of nitric oxide from the left anterior descending coronary artery (LAD) after myocardial ischemia/reperfusion and from the control left circumflex coronary artery (LCX) was assessed by NG-nitro L-arginine methyl ester (L-NAME)-induced vasocontraction. L-NAME induced a significant EC-dependent vasocontraction in control LCX rings (0.28 +/- 0.04 g), which was fully reversed by L-arginine but not D-arginine. L-NAME-induced vasocontraction of LAD rings was not significantly changed after 90 minutes of myocardial ischemia without reperfusion. However, 10 minutes of reperfusion reduced the L-NAME-induced vasocontraction to 0.13 +/- 0.04 g (p < 0.05), and this was restored by addition of 3 mM L-arginine but not D-arginine. Longer periods of reperfusion progressively decreased L-NAME-induced vasocontraction. After 270 minutes of reperfusion, L-NAME-induced vasocontraction was virtually abolished. Myocardial ischemia without reperfusion did not increase PMN adherence to ECs. However, PMN adherence to LAD ECs was significantly increased after 20 minutes of reperfusion (39 +/- 6 to 105 +/- 9 PMNs/mm2, p < 0.01), and incubation of LAD segments with L-arginine significantly attenuated this increase in PMN adherence. After 270 minutes of reperfusion, PMN adherence to LAD ECs was further increased to 224 +/- 10 PMNs/mm2 (p < 0.001). This increase in PMN adherence was almost completely blocked by MAb R15.7, a monoclonal antibody against CD18 of PMNs, and was significantly attenuated by MAb RR1/1, a monoclonal antibody against intercellular adhesion molecule-1 of ECs (p < 0.01). These results indicate that decreased basal release of endothelium-derived relaxing factor after myocardial ischemia/reperfusion precedes enhanced PMN adherence to the coronary endothelium, which may lead to PMN-induced myocardial injury.

Time course of endothelial dysfunction and myocardial injury during myocardial ischemia and reperfusion in the cat.

Myocardial ischemia and reperfusion have been shown to impair coronary vasorelaxation to endothelium-dependent vasodilators. To examine the time course of this dysfunction, occlusion of the left anterior descending (LAD) coronary artery (90 minutes) was followed by reperfusion for 0, 2.5, 5, 20, 180, or 270 minutes. Coronary arterial rings from the ischemic LAD and control left circumflex (LCx) arteries were tested for responsiveness to the endothelium-dependent receptor-mediated vasodilator, acetylcholine (ACh), and the endothelium-dependent nonreceptor-mediated vasodilator, A23187, as well as the endothelium-independent vasodilator, NaNO2. ACh relaxation was not impaired after 90 minutes of ischemia without reperfusion. However, 2.5 minutes of reperfusion resulted in depressed ACh responses (36 +/- 10% of control) that was further reduced to 16 +/- 6% at 20 minutes, and remained comparably depressed at every time thereafter. A23187 vasodilator responses were also attenuated after reperfusion, although the reduced response occurred later (that is, at 20 minutes). There was no significant decrease in response to NaNO2 in the LAD at any time or to any vasodilator in LCx control rings. Treatment with recombinant human superoxide dismutase (hSOD, 5 mg/kg/hr, that is, 15,545 SOD units/kg/hr), starting 10 minutes before reperfusion, preserved the vasodilator response to ACh (82 +/- 6%) and A23187, but treatment with the hydroxyl ion scavenger N-(2-mercapto proprionyl)-glycine (MPG) (8 mg/kg/hr) only protected the A23187 response. No damage to the surface of the endothelium was observed by scanning electron microscopy at any time point. Myocardial cell damage increased with time of reperfusion as assessed by increasing plasma CK activities and amounts of necrotic tissue indexed to area at risk. Significant myocardial injury occurred at 3 hours after reperfusion. These findings suggest that endothelial dysfunction resulting in reduced endothelium-derived relaxing factor release occurs before the development of myocardial cell necrosis and may be due to oxygen-derived free radicals produced rapidly on reperfusion.

In vivo neutralization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury.

The cardioprotective effects of an mAb to P-selectin designated mAb PB1.3 was examined in a feline model of myocardial ischemia (MI) and reperfusion. PB1.3 (1 mg/kg), administered after 80 min of ischemia (i.e., 10 min before reperfusion), significantly attenuated myocardial necrosis compared to a non-blocking mAb (NBP1.6) for P-selectin (15 +/- 3 vs 35 +/- 3% of area at risk, P < 0.01). Moreover, endothelial release of endothelium derived relaxing factor, as assessed by relaxation to acetylcholine, was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with mAb PB1.3 compared to mAb NBP1.6 (67 +/- 6 vs 11 +/- 3, P < 0.01). This endothelial preservation was directly related to reduced endothelial adherence of PMNs in ischemic-reperfused coronary arteries. Immunohistochemical localization of P-selectin was significantly upregulated in the cytoplasm of endothelial cells that lined coronary arteries and veins after 90 min of ischemia and 20 min of reperfusion. The principal site of intracytoplasmic expression was in venous vessels. mAb PB1.3 significantly decreased (P < 0.01) adherence of unstimulated PMNs to thrombin and histamine stimulated endothelial cells in a concentration-dependent manner in vitro. These results demonstrate that PMN adherence to endothelium by P-selectin is an important early consequence of reperfusion injury, and a specific monoclonal antibody to P-selectin exerts significant endothelial preservation and cardioprotection in myocardial ischemia and reperfusion.

Coronary endothelial and cardiac protective effects of a monoclonal antibody to intercellular adhesion molecule-1 in myocardial ischemia and reperfusion.

BackgroundIntercellular adhesion molecule-1 (ICAM-1) is a major ligand on endothelial cells for adherence of activated polymorphonuclear leukocytes (PMNs). The major purpose of this study was to study the effects of RR1/1, a monoclonal antibody against ICAM-1 (i.e., MAb RR1/1), on myocardial injury and endothelial dysfunction associated with myocardial ischemia and reperfusion. Methods and ResultsEither MAb RR1/1 (2 mg/kg, n=7), an antibody that was found to bind selectively to endothelial cells in the cat, or MAb R3.1 (2 mg/kg, n=7), a nonbinding control antibody, was given as an intravenous bolus 10 minutes before reperfusion. Two hundred eighty minutes later, hearts were excised. The left ventricle area-at-risk (AAR) was similar in MAb RR1/1 (297±2%) and MAb R3.1 (30±3%) groups. In MAb R3.1-treated cats, 90 minutes of myocardial ischemia plus 4.5 hours of reperfusion induced a significant myocardial injury (necrotic tissue/AAR, 28±2%), high myeloperoxidase activity (0.65±0.16 units/100 mg ischemic tissue), and a marked decrease in endothelium-dependent vasorelaxation in isolated left anterior descending coronary arteries (vasorelaxation to acetylcholine, 29±3%) with no change in endothelium-independent vasorelaxation (relaxation to NaNO2, 91±3%). However, cats treated with MAb RR1/1 developed significantly less myocardial necrosis (10±2% of the AAR, p<0.0l), lower myeloperoxidase activity in ischemic myocardial tissue (0.2±0.03 units/100 mg ischemic tissue, p<0.01), and enhanced vasorelaxant responses to endothelial-dependent relaxation to acetylcholine (53±5%) compared with ischemic/reperfused cats treated with MAb R3.1. Furthermore, addition of MAb RR1/1 in vitro significantly inhibited unstimulated PMN adherence to ischemicreperfused coronary artery endothelium. ConclusionsThese results suggest that ICAM-1-dependent PMN adherence plays an important role in reperfusion injury, and that PMN adherence and infiltration contribute significantly to coronary endothelial dysfunction.

Monoclonal antibody to L-selectin attenuates neutrophil accumulation and protects ischemic reperfused cat myocardium.

BackgroundInteraction of CDii/CD18 located on neutrophil membranes with its endothelial counterreceptor, intercellular adhesion molecule-1, plays a major role in polymorphonuclear leukocyte (PMN)- mediated endothelial dysfunction and myocardial injury associated with ischemia and reperfusion. However, PMN-derived L-selectin, which is thought to play an early role in PMN rolling along the vascular endothelium, has not been studied in a setting of myocardial ischemia and reperfusion. Methods and ResultsIn this study, we evaluated the effects of a monoclonal antibody against L-selectin, DREG-200, in a feline model of myocardial ischemia (1.5 hours) and reperfusion (4.5 hours). DREG-200 (1 mg/kg) or an isotype-matched IgGl antibody, MAb R3.1, which does not cross-react in cats, was administered as a bolus 10 minutes before reperfusion. In MAb R3.1-treated cats, myocardial ischemia followed by reperfusion resulted in significant coronary vascular endothelial dysfunction, elevated cardiac myeloperoxidase activity indicative of neutrophil accumulation in the ischemic myocardium, and severe myocardial injury. In contrast, administration of DREG-200 at 1 mg/kg significantly attenuated myocardial necrosis (14 ± 4 versus 32 ± 3 expressed as percentage of area at risk, p<.001) and attenuated coronary endothelial dysfunction (P<.01) associated with ischemia/reperfusion. Moreover, myeloperoxidase activity in the ischemic myocardium was significantly lower than MAb R3.1-treated cats (0.4 ± 0.1 versus 0.9 ± 0.2 U/100 mg tissue, p<.05). ConclusionThese results demonstrate that blocking L-selectin with DREG-200 exerts a significant cardioprotective effect in a feline model of myocardial ischemia and reperfusion, indicating that L-selectin plays a significant role in mediating PMN accumulation and PMN-induced endothelial and myocardial injury after ischemia and reperfusion.

Synergism Between Platelets and Neutrophils in Provoking Cardiac Dysfunction After Ischemia and Reperfusion Role of Selectins

BACKGROUNDnNeutrophils (PMNs) are known to contribute to both cardiac dysfunction and myocardial necrosis after reperfusion of an ischemic heart. Moreover, platelets are also important blood cells that can aggravate myocardial ischemic injury. This study was designed to test the effects of PMNs and platelets separately and together in provoking cardiac dysfunction in isolated perfused rat hearts after ischemia and reperfusion.nnnMETHODS AND RESULTSnControl rat hearts not subjected to ischemia were perfused without blood cells for 80 minutes. Additional control rat hearts were perfused with 75x106 PMNs, with 100x106 platelets, or with 75x106 PMNs+100x106 platelets over a 5-minute perfusion followed by a 75-minute observation period. No significant reduction in coronary flow, left ventricular developed pressure (LVDP), or the first derivative of LVDP (dP/dtmax) was observed at the end of the observation period in any nonischemic group. Similarly, global ischemia (I) for 20 minutes followed by 45 minutes of reperfusion (R) produced no sustained effects on the final recovery of any of these parameters in any group of hearts perfused in the absence of blood cells. However, I/R hearts perfused with either PMNs or platelets alone exhibited decreases in these variables of 10% to 12% (P<0.05 from control). Furthermore, I/R hearts perfused with both PMNs and platelets exhibited decreases of 50% to 60% in all measurements of cardiac function (P<0.001). These dual-cell-perfused I/R hearts also exhibited marked increases in cardiac myeloperoxidase (MPO) activity, indicating a significant PMN infiltration, and enhanced P-selectin expression on the coronary microvascular endothelium. All cardiodynamic effects as well as MPO accumulation and PMN infiltration were markedly attenuated by a sialyl LewisX-oligosaccharide or a recombinant soluble P-selectin ligand, which inhibits selectin-mediated cell adhesion.nnnCONCLUSIONSnThese results provide evidence that platelets and neutrophils act synergistically in provoking postreperfusion cardiac dysfunction and that this may be largely due to cell-to-cell interactions mediated by P-selectin. These findings may help explain the reperfusion injury phenomenon.

Physiological concentrations of nitric oxide do not elicit an acute negative inotropic effect in unstimulated cardiac muscle

We examined the effect of several nitric oxide (NO) donors, authentic NO gas, and L-arginine in isolated cat and rat papillary muscles. We did not observe significant inotropic effects in response to any NO donor (ie, SPM-5185, C87-3754, and S-nitroso-N-acetylpenicillamine [SNAP]) from 1 nmol/L to 100 mumol/L. Similarly, authentic NO, at concentrations far in excess of those that maximally dilate the coronary vasculature (ie, 500 nmol/L), also failed to exert a detectable inotropic effect in these preparations. However, in the presence of 5 mumol/L norepinephrine, 500 nmol/L NO exerted a 12 +/- 3% decrease in isolated rat papillary muscle contractility (P < .05). Addition of L-arginine up to 25 mmol/L exerted no inotropic effects in isolated rat papillary muscles. However, at 50 mmol/L, L-arginine decreased contractile force by 21 +/- 4% (P < .01). On further examination, the negative inotropic effect of 50 mmol/L L-arginine appeared to be nonspecific, since the inactive stereoisomer, D-arginine, at 50 mmol/L exerted the same effect. Further studies in isolated adult rat cardiac myocytes elicited similar results, in that 50 mmol/L of L- and D-arginine equally decreased contraction amplitude and the underlying cytosolic calcium transient. Moreover, 500 nmol/L of the NO donor SPM-5185 only modestly decreased contraction amplitude or intracellular calcium in isolated rat cardiac myocytes. These results indicate that administration of physiological concentrations of exogenous NO does not acutely depress the inotropic state of the rat or cat heart to a physiologically significant extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardioprotective actions of a monoclonal antibody against CD-18 in myocardial ischemia-reperfusion injury.

BackgroundPrevious studies have demonstrated that polymorphonuclear leukocytes (PMNs) are locally activated in reperfused myocardium and contribute to the myocardial cell injury associated with reperfusion. It has been suggested that the adhesion of activated PMNs in reperfused myocardium is mediated by the PMN adhesion molecule CD-18. In the present study, we performed experiments to determine if the specific anti-CD-18 monoclonal antibody (MAb) R15.7 can prevent PMN adhesion and PMN-mediated reperfusion injury in the heart. Methods and ResultsStudies were performed with isolated, Langendorff-perfused rat hearts (nine per group) in which the hearts were subjected to 20 minutes of global ischemia followed by 45 minutes of reperfusion. Human PMNs (50 million) and rat plasma (HNRP) were infused directly into the coronary circulation of nonischemic and postischemic hearts. When HNRP was administered to nonischemic hearts, no significant alterations in coronary flow, left ventricular developed pressure, or left ventricular end-diastolic pressure were observed. When hearts were reperfused in the presence of HNRP, however, marked impairment of contractile function was observed with more than 90%o reduction in coronary flow throughout the reperfusion period (P<.001 versus baseline). In addition, left ventricular developed pressure was significantly depressed (P<.001 versus baseline) throughout the reperfusion period in the HNRP group and recovered to only 13.0±3.0%o at 45 minutes of reperfusion. Moreover, left ventricular end-diastolic pressure was significantly elevated (P<.001) in the HNRP group throughout the reperfusion period. Treatment with the anti-CD-18 monoclonal antibody MAb R15.7 (20 pug/mL) at the time of reperfusion resulted in a 92.9±4.9%o recovery of coronary flow (P<.001 versus HNRP) as well as a 71.0±10.1% recovery of left ventricular developed pressure (P<.001 versus HNRP). Administration of MAb R15.7 also very significantly attenuated the elevation in left ventricular end-diastolic pressure that was observed in the untreated HNRP (30.2±7.8 versus 110.3±10.3 mm Hg, p<.001) at 45 minutes of reperfusion. Cardiac myeloperoxidase activity, an index of PMN accumulation, was markedly reduced in the MAb R15.7 group at 45 minutes of reperfusion compared with the HNRP group (0.03±0.01 versus 0.3±0.05, p<.001). To determine that the protective effect ofMAb R15.7 was based on functional blocking of CD-18, additional experiments were performed with identical concentrations of MAb 3.1, which binds to the a-subunit of LFA-1. This PMN-binding but non-CD-18-blocking antibody had little effect on the recovery of postischemic function or coronary flow and did not reduce tissue myeloperoxidase activity ConclusionsThe administration of a specific anti-CD-18 monoclonal antibody, MAb R15.7, attenuates much of the PMN-mediated contractile dysfunction associated with this in vitro model of myocardial ischemia-reperfusion injury by limiting PMN accumulation. We conclude that CD-18-mediated adhesion may play a critical role in the pathogenesis of PMN-induced myocardial injury.

An anti-CD18 antibody limits infarct size and preserves left ventricular function in dogs with ischemia and 48-hour reperfusion

OBJECTIVESnThis study investigated whether an antibody against neutrophil adhesion protein CD18 could limit myocardial infarct size and preserve left ventricular function after prolonged reperfusion in a canine model.nnnBACKGROUNDnMyocardial reperfusion injury is mediated in part by accumulation of activated neutrophils. Although antibodies against CD18 have been shown to reduce neutrophil influx and infarct size after ischemia and 3 to 4 h of reperfusion, it is unknown whether protection is sustained beyond this time or whether there is meaningful preservation of ventricular function.nnnMETHODSnDogs undergoing 90-min circumflex coronary artery occlusion and 48-h reperfusion were randomized to receive 1 mg/kg bodyweight of R15.7 (an anti-CD18 antibody, n = 12) or saline (control, n =12) 10 min before reperfusion. Contrast left ventriculography was used to measure left ventricular ejection fraction and regional chord shortening at baseline, during occlusion and at 48 h. Microspheres injected during occlusion were used to measure collateral flow and risk region size. Postmortem infarct size was measured with triphenyltetrazolium chloride.nnnRESULTSnIn the dose administered, R15.7 bound to neutrophils in vivo, with >85% saturation of CD18 for >24 h, with sustained antibody excess in the plasma. R15.7 significantly reduced infarct size after adjusting for the effect of collateral flow (p = 0.0002, analysis of covariance). In a subgroup of dogs with collateral flow <30% of nonischemic flow, infarct size was reduced from 34.6 +/- 3.9% (mean +/- SE) of the region at risk in the control group to 19.5 +/- 3.3% in the antibody group (p = 0.008). Ejection fraction and regional chord shortening did not differ between the two groups at baseline or during occlusion, but after 48-h reperfusion, ejection fraction and inferior wall regional cord shortening (representing the infarct zone) were both higher in the R15.7 group than the control group (43.6 +/- 2.9% vs. 28.5 +/- 1.8%, p < 0.01; 2.55 +/- 0.29% vs. 1.06 +/- 0.18%, p < 0.05).nnnCONCLUSIONSnA single injection of an anti-CD18 antibody given before reperfusion can limit myocardial infarct size by nearly 50% and preserve global and regional left ventricular function after 48 h of reperfusion.

Potentiation of leukotriene formation in pulmonary and vascular tissue

SummaryLeukotriene (LT) release from vascular and pulmonary tissue was assessed by a radioimmunoassay for peptide leukotrienes (i.e., LTC4, LTD4 and LTE4). The calcium ionophore A-23187 at 1–3 μg/ml and platelet activating factor (PAF) at 10 μg/ml produced marked formation of peptide leukotrienes in minced cat pulmonary tissue. This was also confirmed by bioassay of the incubates in isolated perfused cat coronary arteries. Rat pulmonary tissue was comparable to cat with regard to LT production, but guineapig lung produced about 30–50% less on a weight basis. In addition, aortic and coronary artery vessel walls produced significant amounts of LTs. The time course for maximal leukotriene production occurred at 45–60 min of incubation at 37°C in both the radioimmunoassay and the bioassay. Cat coronary artery constricted markedly to LTC4 or LTD4 (30–40 mm Hg) and to the lung or blood vessel incubate. This constriction was virtually totally blocked by the leukotriene antagonist FPL-55712, but not by the thromboxane receptor antagonist, pinane thromboxane A2, the α-adrenergic receptor antagonist, phenoxybenzamine, or the angiotensin receptor antagonist, saralasin. Thus, pulmonary and vascular tissue produce leukotrienes that appear to exert coronary constrictor effects on specific leukotriene receptors. These results indicate that the ischemia of shock and anaphylaxis may be accentuated by the release of peptide leukotrienes.