Allan M. 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.

The role of nitric oxide and cell adhesion molecules on the microcirculation in ischaemia-reperfusion

The microcirculation undergoes a profound degree of endothelial dysfunction within minutes (i.e., 2.5 to 5 min) following reperfusion of ischaemic vasculature. This has been documented in the coronary and mesenteric microcirculation. The endothelial dysfunction is characterized by a loss in basal and agonist-mediated nitric oxide (NO) produced by the vascular endothelium. The loss of NO results in upregulation of cell adhesion molecules (CAMs) particularly P-selectin 10-20 min following reperfusion. Thus, CAM upregulation renders the endothelium sticky, and a marked degree of leukocyte adherence (particularly neutrophils) occurs 20 min following reperfusion. This enhanced involvement of neutrophils leads to neutrophil infiltration into the underlying tissue (e.g., myocardium) within 2-3 h of reperfusion. The infiltration of neutrophils leads to reperfusion injury (i.e., necrosis) which is significant at 3 h but becomes profound at 4.5 h following reperfusion. Cardiac necrosis can be significantly attenuated by treatment with NO, an organic NO donor, L-arginine, or specific blockers of CAMs given just prior to reperfusion. This approach is a promising one for a variety of types of reperfusion injury.

The role of L-arginine in ameliorating reperfusion injury after myocardial ischemia in the cat.

BackgroundMyocardial ischemia followed by reperfusion results in endothelial dysfunction characterized by a reduced release of endothelium-derived relaxing factor (EDRF). Because EDRF has been characterized as nitric oxide, we examined the ability of L-arginine, the substrate for nitric oxide synthesis, to protect in a feline model of myocardial ischemia plus reperfusion. Methods and ResultsThe effects of L-arginine were investigated in a 6-hour model of myocardial ischemia and reperfusion in pentobarbital-anesthetized cats. A bolus administration (30 mg/kg) of L-arginine, or its enantiomer D-arginine, was given followed by a continuous infusion of 10 mg/kg/min for 1 hour starting 10 minutes before reperfusion. Myocardial ischemia plus reperfusion in cats receiving D-arginine resulted in severe myocardial injury and endothelial dysfunction characterized by marked myocardial necrosis, high cardiac myeloperoxidase activity in ischemic cardiac tissue, and loss of acetylcholine and A-23187–induced endothelium-dependent relaxation in coronary artery rings. In contrast, myocardial ischemia plus reperfusion cats treated with L-arginine exhibited a reduced area of cardiac necrosis (16±2% versus 41±5% of area at risk, p<0.01), lower myeloperoxidase activity in the ischemic region (03±0.08 versus 0.8±0.10 units/100 mg tissue, p<0.05), and significant preservation of acetylcholine- (p<0.01) and A-23187-(p<0.01) induced endothelial-dependent relaxation. ConclusionsThese results demonstrate the ability of L-arginine to reduce necrotic injury in a cat model of myocardial ischemia plus reperfusion, and this reduction in infarct size is associated with the preservation of endothelial function and attenuation of neutrophil accumulation in ischemic cardiac tissue.

Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia.

Endothelial dysfunction occurs after myocardial ischemia and reperfusion characterized by a marked reduction in endothelium‐dependent relaxation (EDR) due to reduced release or action of endothelium‐derived relaxing factor (EDRF). This reduced EDR occurs in coronary rings isolated from cats 2.5 min after reperfusion and in isolated perfused cat hearts 2.5 min after reperfusion. No decrease in EDR occurs before reperfusion in either preparation, suggesting that this impairment in EDR occurs during reperfusion. The decrease in EDR occurs soon after the generation of superoxide radicals by the reperfused coronary endothelium. Accumulation of neutrophils and myocardial cell injury does not occur until 3–4.5 h after reperfusion. Thus, endothelial generation of superoxide radicals acts as a trigger mechanism for endothelial dysfunction which is then amplified by neutrophil adherence and diapedesis into the ischemic region enhancing post‐reperfusion ischemic injury. Agents that preserve endothelial function or inhibit neutrophil activation (e.g., superoxide dismutase, prostacyclin analogs, TGF‐β, antibodies to adhesive proteins) can protect against endothelial dysfunction and myocardial injury, if administered before reperfusion.—Lefer, A. M.; Tsao, P. S.; Lefer, D. J.; Ma, X.‐1. Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia. FASEB J. 5: 2029–2034; 1991.

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.

Peroxynitrite inhibits leukocyte-endothelial cell interactions and protects against ischemia-reperfusion injury in rats.

Peroxynitrite (ONOO-) anion, formed by the interaction of superoxide with nitric oxide (NO), has previously been implicated as a cytotoxic agent. However, the effects of this free radical species on neutrophil (PMN)-endothelial cell interactions is largely unknown. We investigated the direct actions of ONOO- on PMN adhesion to endothelial cells in vitro and in vivo, as well as the effects of ONOO- on PMN-mediated myocardial ischemia-reperfusion injury. In vitro, peroxynitrite (100-1,000 nM) inhibited the adhesion of rat PMNs to the endothelium of isolated thrombin- or H2O2-stimulated rat mesenteric artery (P < 0.01 vs. thrombin or H2O2 alone). In vivo, in the rat mesentery, thrombin (0.5 U/ml) or N(G)-nitro-L-arginine-methyl ester (50 microM) significantly increased venular leukocyte rolling and adherence, which were also significantly (P < 0.01) attenuated by ONOO (800 nM) accompanied by reduced P-selectin expression on the endothelial cell surface. Isolated perfused rat hearts were subjected to global ischemia and reperfusion with rat PMNs (10(8) cells), which resulted in profound cardiac depression (i.e., a marked reduction in left ventricular developed pressure and maximal rate of development of left ventricular pressure). Infusion of ONOO- reversed the myocardial contractile dysfunction of ischemic-reperfused rat hearts to near baseline levels, and markedly attenuated the accumulation of PMNs in the postischemic heart. The present study provides strong evidence that nanomolar concentrations of ONOO- both inhibit leukocyte-endothelial cell interactions and exert cytoprotective effects in myocardial ischemia-reperfusion injury. Furthermore, our results suggest that the inhibition of P-selectin expression by peroxynitrite is a key mechanism of the modulatory actions of ONOO- on leukocyte-endothelial cell interactions.

Vascular effects of HMG CoA-reductase inhibitors (statins) unrelated to cholesterol lowering : new concepts for cardiovascular disease

Time for primary review 18 days. In 1976, Endo et al. [1] reported on the discovery of a specific competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl (HMG) CoA reductase. This compound, isolated from fungi, was called mevastatin. In 1988, Alberts [2] described a more potent HMG CoA reductase inhibitor, lovastatin, isolated from Aspergillus terreus . These two compounds started the development of a series of cholesterol lowering agents, now commonly called ‘statins’. Statins block the conversion of HMG CoA to mevalonic acid and thus dramatically attenuate the biosynthesis of cholesterol. These effects occur largely in the liver, where the statins primarily distribute [2]. The major effect of these pharmacologic agents is a marked reduction in LDL-cholesterol levels in the blood. On the basis of these and other findings, the statins have been widely prescribed in humans with elevated serum cholesterol levels. These statins are well tolerated, are very safe, and effectively lower serum LDL-cholesterol levels and maintain this effect over periods of many years [3]. In one clinical trial, 4444 patients were carefully studied and not only was simvastatin shown to be safe and effective in lowering circulating cholesterol, it also significantly improved survival in patients with coronary heart disease (CHD) [4], and carried a 37% reduction in the risk of treated patients undergoing myocardial vascularization procedures [4]. These findings speak eloquently to the clinical usefulness of the statins. For many years, all the beneficial effects of the statins were attributed to their cholesterol lowering effect. Nevertheless, quite recently, the statins were found to exert direct cardiovascular effects which clearly are independent of their cholesterol lowering effects, and are not directly attributable to a reduction in serum cholesterol levels. The purpose of this review is to describe, evaluate, and discuss the major mechanisms underlying these non-lipid-lowering effects … * Corresponding author. Tel.: +1-215-503-7760; fax: +1-215-503-2073 allan.m.lefer{at}mail.tju.edu

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.