Reactive oxygen metabolite scavengers decrease functional coronary microvascular injury due to ischemia-reperfusion.

The role of reactive oxygen metabolites in ischemia-reperfusion coronary microvascular injury is unclear. To investigate this problem, we tested the effects of the reactive oxygen metabolite scavengers superoxide dismutase (SOD) and dimethylthiourea (DMTU) on ischemia-reperfusion-induced coronary microvascular dysfunction. As an index of vascular function, we assessed microvascular permeability with a double radioisotope protein leak index (PLI) method. Anesthetized dogs underwent 60 min of ischemia via left anterior descending (LAD) occlusion followed by 60 min of reperfusion. Untreated animals (n = 7) received saline. SOD-treated animals (n = 6) received 140 U.kg-1.min-1 (6.6 mg.kg-1.min-1) bovine SOD throughout ischemia and reperfusion. DMTU-treated animals (n = 5) received a 500 mg/kg bolus 30 min before ischemia. At the beginning of reperfusion, radiolabeled autologous protein (113mIn transferrin) and red blood cells (99mTc) were given intravenously for the assessment of permeability. In untreated dogs, ischemia-reperfusion increased the PLI of ischemic (flow less than 20 ml.min-1.100 g-1) myocardium more than threefold compared with that of nonischemic (flow greater than 100 ml.min-1.100 g-1) myocardium (ischemic-to-nonischemic PLI ratio = 3.49 +/- 0.48). SOD reduced the PLI of ischemic myocardium by 45% and DMTU reduced it by 66% (PLI = 9.25 +/- 1.30, 5.04 +/- 1.18, and 3.16 +/- 0.94, untreated, SOD, and DMTU, respectively). The PLI was increased proportional to the regional severity of ischemic blood flow. Both SOD and DMTU reduced the increase in protein leak at all levels of regional ischemic blood flow. Neither SOD nor DMTU increased regional myocardial blood flow to the occluded LAD zone.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional coronary microvascular injury evident as increased permeability due to brief ischemia and reperfusion.

Although morphological studies suggest that coronary vascular injury is a result of prolonged ischemia and subsequent reperfusion, whether functional coronary microvascular injury develops during brief in vivo ischemia is unclear. In other organs, permeability is a sensitive indicator of functional vascular injury. Therefore, a new double-indicator method of assessing vascular protein permeability, a method that is both sensitive and specific for vascular injury, was used to investigate the effects of ischemia of graded duration followed by reperfusion on coronary microvascular function. To help confirm functional coronary vascular injury, endothelium-dependent vasodilation of isolated coronary vascular rings also was examined. Microvascular permeability was quantitatively assessed as a protein leak index by measuring the rate of extravascular accumulation of radiolabeled protein (indium 113m transferrin) normalized for vascular surface area (technetium 99m erythrocytes). Anesthetized dogs underwent 0 (control), 15, 30, or 60 minutes of left anterior descending coronary artery occlusion followed by 60 minutes of reperfusion. Even 15 minutes of ischemia increased the protein leak index by 50% (3.16 +/- 0.30 ischemic vs. 2.09 +/- 0.11 control). Longer periods of ischemia increased the protein leak index in proportion to the duration of ischemia. The protein leak index increased threefold (6.51 +/- 0.60) after 60 minutes of ischemia. At each duration of ischemia, there was significant regional variation in the protein leak index that correlated with the severity of ischemic blood flow to that region measured with microspheres. Endothelial injury also was evident after 15 and 30 minutes of ischemia as impaired vasodilation of isolated coronary rings in response to the endothelium-dependent vasodilators acetylcholine and the calcium ionophore A23187. Electron microscopy and in vitro direct immunofluorescence revealed evidence of vascular injury after 60 minutes but not after 15 minutes of ischemia. We conclude that even brief ischemia and reperfusion cause functional coronary vascular injury evident as increased microvascular permeability and impaired endothelium-dependent vasodilation and that regional differences in the degree of microvascular injury correlate with differences in the severity of ischemia.

Aggregating platelets increase intracellular calcium in endothelial cells through release of adenine nucleotides.

Aggregating platelets relax isolated coronary arteries through the release of endothelium-derived relaxing factor (EDRF). Since release of EDRF may be calcium dependent, we tested if and how aggregating platelets stimulated a calcium response in cultured endothelial cells. Aggregating platelets caused a transient increase in intracellular calcium in endothelial cells loaded with the fluorescent calcium indicator fura-2. The adenine nucleotides ADP and ATP, but not other platelet-derived mediators, mimicked the platelet-induced calcium response, and inhibition of adenine nucleotides impaired the response to aggregating platelets. Thus, aggregating platelets release adenine nucleotides and stimulate a rise in intracellular calcium in cultured endothelial cells. This calcium response may represent the intracellular transduction mechanism by which aggregating platelets induce endothelial release of EDRF and subsequent relaxation of coronary arteries.

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)

Lidocaine reduces canine infarct size and decreases release of a lipid peroxidation product.

Whether and how lidocaine reduced infarct size in a canine model of ischemia and reperfusion was investigated. Twenty dogs underwent a 90-min left anterior descending artery ligation and 300 min of reperfusion. Infarct size was measured by triphenyl tetrazolium chloride and the region at risk by 99Tc-labeled albumin microspheres injected during ischemia. In 10 dogs, lidocaine (70 micrograms/kg/min i.v.) was infused 90 min prior to and during ischemia and reperfusion, while 10 dogs were untreated. The ratio of infarct to risk area was 35.2 +/- 3.4% (SEM) in lidocaine dogs vs. 48.5 +/- 5.3% in untreated dogs (p less than 0.05). Lidocaine did not reduce neutrophil accumulation in ischemic and reperfused myocardium at 5 h of reperfusion, inhibit stimulated neutrophil superoxide production, or scavenge superoxide in vitro. However, during early reperfusion, lidocaine reduced coronary sinus levels of a lipid peroxidation product (conjugated dienes). Thus, clinically relevant lidocaine infusion rates reduced myocardial infarct size when given prior to and during ischemia and reperfusion. This protective effect may be due to lidocaine's membrane stabilizing effects, which could have protected the myocardial cell membrane from lipid peroxidation.

Dimethylthiourea, but not dimethylsulfoxide, reduces canine myocardial infarct size.

We studied the effect of treatment with two diffusible, low molecular weight scavengers of toxic oxygen metabolites, dimethylthiourea (DMTU) and dimethylsulfoxide (DMSO), on canine infarcts caused by 90 min of ischemia and 3 h of reperfusion. Infarct size was determined by incubating ventricular slices with triphenyl tetrazolium chloride. Areas at risk were determined by autoradiography of 99Tc microspheres injected in vivo during ischemia and were similar (p greater than 0.05) in DMTU, DMSO, and saline treated dogs. However, the ratio of infarct size to area at risk was reduced (p less than 0.05) in dogs treated 30 min before reperfusion with 500 mg/kg DMTU (31.1 +/- 4.6%, n = 9) compared with saline treated dogs (53.4 +/- 4.6% n = 9). In contrast, the ratio of infarct size to area at risk was not significantly different (p greater than 0.05) in dogs treated with 2000 mg/kg DMSO 30 min before reperfusion (43.7 +/- 4.3%) compared to saline treated dogs. The serum concentration of DMTU (4.5 mM) was one-tenth that of DMSO (48 mM) in early reperfusion. Therefore, DMTU but not DMSO protected against post-ischemic cardiac reperfusion injury.

Reperfusion after acute coronary occlusion in dogs impairs endothelium-dependent relaxation to acetylcholine and augments contractile reactivity in vitro.

Endothelial injury may contribute to the augmented coronary vascular tone seen in myocardial ischemia by impairing endothelial production or release of vasodilators. In vitro reactivity of arterial rings was studied after 60 min of coronary occlusion and 60 min of reperfusion in anesthetized dogs. Ischemia without reperfusion blunted contractile reactivity to potassium chloride (KCl), whereas ischemia plus reperfusion augmented contractile responses to both KCl and ergonovine. The response to acetylcholine, an endothelium-dependent vasodilator, was abolished in reperfused arteries, whereas the response to nitroprusside, an endothelium-independent vasodilator, was intact. Verapamil pretreatment restored KCl contractile responses to normal in reperfused coronary rings and partially restored endothelium-dependent relaxation. Electron microscopy revealed a nondenuding epicardial coronary endothelial injury in reperfused arteries. These data support the hypothesis that reperfusion of ischemic myocardium augments reactivity to vasoconstrictor agents by causing endothelial cell damage, excessive calcium influx, and loss of modulating vasodilator function.

Temporal delay of venous blood correlates with onset of exercise hyperpnea.

We tested the hypothesis that humoral factors contribute to the onset of exercise hyperpnea in an electrically induced model of isocapnic exercise in alpha-chloralose-anesthetized dogs. A cannula placed in the inferior vena cava (IVC) permitted hindlimb venous blood to flow either directly to the lungs or through a variable-length extracorporeal circuit. Mean transit times (MTT) of blood from exercising hindlimbs were measured from the arrival at the pulmonary artery of green dye injected into the saphenous vein. Onset of hyperpnea was determined by the half time of the ventilatory response (T 1/2), the time required to reach 50% of the steady-state ventilation. In seven dogs, T 1/2 was directly related to MTT (P less than 0.001), suggesting that blood-borne substances released at the onset of exercise contribute to the hyperpneic response. The T 1/2-MTT relation persisted following L2 cord transection (n = 4), suggesting that intraspinal afferents are not required for this response. Chemoreceptor denervation (n = 4) slowed the onset of exercise hyperpnea but did not alter the T 1/2-MTT relation. In this model of electrically induced "exercise" in which neurogenic influences have been minimized, humoral factors alone may stimulate ventilation sufficiently to produce arterial isocapnia.

Oxygen-radical-mediated permeability edema and vasoconstriction in isolated perfused rabbit lungs.

Oxygen radicals have been implicated in the pathogenesis of permeability pulmonary edema. To determine directly if O2 radicals can cause increased alveolar-capillary membrane (ACM) permeability and low-pressure permeability edema, we chemically produced O2 radicals in the sale perfusates of isolated rabbit lungs. The O2 radicals generated by xanthine oxidase caused protein-rich edema and increases in lung perfusion pressures that were inhibitable by catalase (hydrogen peroxide scavenger) or dimethylthiourea (hydroxyl radical scavenger) but not by superoxide dismutase. To determine the effect of O2 radicals on ACM permeability without interference from increased perfusion pressures, we used papaverine to maintain baseline perfusion pressures during O2 radical exposure and then assessed ACM integrity by evaluating the response of isolated lungs to elevated outflow pressures (10 mmHg for 10 min). Under these conditions, increased ACM permeability manifested by weight gains and lavage albumin accumulations occurred in lungs treated with xanthine oxidase but not in control lungs. We conclude that O2 radicals can cause increased ACM permeability and vasoconstriction in isolated lungs.

Granulocytes mediate acute edematous lung injury in rabbits and in isolated rabbit lungs perfused with phorbol myristate acetate: role of oxygen radicals.

Acute edematous lung injury is associated with a marked increase in the number of granulocytes in the alveoli and microvasculature of the lung. Phorbol myristate acetate (PMA) causes granulocytes to adhere, aggregate, and release oxygen radicals and granular enzymes. We found that intravenously injected PMA caused a protein-rich edema in lungs of control rabbits but not in granulocytopenic rabbits pretreated with nitrogen mustard. Specifically, control rabbits treated with PMA had higher lung weight to body weight ratios (6.4 +/- 1.0 X 10(-3)) and lung lavage albumin concentrations (190 +/- 44 mg/dl) than granulocytopenic rabbits pretreated with nitrogen mustard and then given PMA (4.74 +/- 0.23 X 10(-3) and 9.9 +/- 3.8 mg/dl, respectively). To further clarify the role of granulocytes in the production of edema, additional experiments were conducted in an isolated perfused rabbit lung. Addition of purified granulocytes and PMA to the balanced salt perfusate caused lung edema, whereas neither granulocytes nor PMA alone caused edema. Specifically, increases in lung weights (42 +/- 9.2 g) and albumin concentrations (1,182 +/- mg/dl) in lung lavages from isolated lungs exposed to granulocytes and PMA were greater than increases in lung weights or albumin concentrations in lung lavages from isolated lungs exposed to granulocytes alone (2.0 +/- 0.4 g and 15 +/- 0.6 mg/dl), or to PMA alone (6.0 +/- 0.6 g and 81 +/- 34 mg/dl). To determine the contribution of oxygen radicals to the pathogenesis of the edema, chronic granulomatous disease granulocytes, which are deficient in oxygen radical production, were added to the isolated lung perfusate. Chronic granulomatous disease granulocytes and PMA did not cause edema in isolated lungs (delta lung weight 1.0 +/- 0.2 g and lavage albumin 12 +/- 5.0 mg/dl) whereas granulocytes from normal human subjects and PMA did (delta lung weight 43 +/- 5.2 g and lavage albumin 1,120 +/- 54 mg/dl). These data suggest that oxygen radicals released from stimulated granulocytes contribute to the pathogenesis of acute edematous lung injury.