F.A. Nicolini

Increased secretion of tumor necrosis factor-alpha and interferon-gamma by mononuclear leukocytes in patients with ischemic heart disease. Relevance in superoxide anion generation.

BACKGROUND There is growing evidence for a pathogenic role for cytokines in atherogenesis. The presence of certain cytokines has been documented in human atherosclerotic vessels. This study was designed to investigate cytokine production by mononuclear leukocytes from patients with ischemic heart disease. METHODS AND RESULTS We measured kinetics of secretion of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) by mononuclear leukocytes from 8 control subjects, 10 patients with stable angina pectoris, and 10 patients with unstable angina pectoris. Mononuclear leukocytes were isolated and incubated with or without the plant lectin mitogen concanavalin A for 48 hours. TNF-alpha and IFN-gamma secretion were measured by ELISA. The effect of TNF-alpha and IFN-gamma on superoxide radical generation by neutrophils was also examined. Secretion of both TNF-alpha and IFN-gamma by mononuclear leukocytes increased progressively over 48 hours, and it was consistently higher (P < .02) in patients compared with control subjects. A similar increase in cytokine secretion was observed in patients with stable or unstable angina pectoris. In addition, there was no relation between the severity of coronary artery disease by angiography and cytokine secretion. Basal neutrophil superoxide radical generation was increased in patients with ischemic heart disease, and incubation with cytokines failed to further stimulate superoxide generation in these patients. CONCLUSIONS Similar increases in cytokine secretion by mononuclear leukocytes in stable or unstable angina pectoris indicate that the increased cytokine release is not a nonspecific inflammatory response in acute myocardial ischemia. Increased cytokine secretion in ischemic heart disease may play a role in superoxide radical generation, endothelial injury, deposition and activation of cellular elements on the vessel wall, and possibly in the progression of atherosclerosis.

Sustained reflow in dogs with coronary thrombosis with K2P, a novel mutant of tissue-plasminogen activator.

Coronary artery reocclusion after thrombolysis with human recombinant tissue-type plasminogen activator (rt-PA) is related to the short half-life of this agent in plasma. K2P, a mutant of rt-PA lacking the fibronectin fingerlike, epidermal growth factor-like and first kringle domains (amino acids 6 to 173) and having the glycosylation site Asn184 mutagenized to Gln, has been produced in Chinese hamster ovary cells. In this study we compared the thrombolytic effect of K2P and rt-PA in dogs with electrically induced coronary artery thrombosis. Both agents were given intravenously in equimolar amounts over 20 min after the occlusive thrombus was stable for 30 min; dogs were monitored for 1 h after reperfusion if flow occurred. Coronary blood flow was restored by rt-PA in 6 (60%) of 10 dogs. The restored flow lasted for 49 +/- 12 min and mean flow at 60 min from the start of reperfusion was 7 +/- 3 ml/min. The reocclusion rate was 50% (three of six dogs). Flow was restored in five (100%) of five dogs by K2P. The restored blood flow lasted during the entire 1-h observation period in all but one dog and mean flow at 60 min was 49 +/- 16 ml/min (p less than 0.02 vs. flow in rt-PA-treated dogs). Restored coronary blood flow showed marked cyclic flow variations in rt-PA-treated but not in K2P-treated dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostacyclin analogue iloprost decreases thrombolytic potential of tissue-type plasminogen activator in canine coronary thrombosis.

Platelets play an important role in the formation of a coronary thrombus and reocclusion after thrombolysis. Therefore, we examined the thrombolytic potential of concomitant intravenous administration of potent platelet inhibitor iloprost, a prostacyclin analogue, with tissue-type plasminogen activator (t-PA; n = 8) and t-PA alone (n = 9) in dogs with an electrically induced occlusive coronary artery thrombus. t-PA (0.75 mg/kg) was given over 20 minutes, and iloprost (4 micrograms/kg) was given over 40 minutes. Reperfusion rate was 63% (five of eight dogs) in the t-PA plus iloprost group and 67% (six of nine dogs) in the t-PA alone group (p = NS). The time to thrombolysis (or reperfusion) in the t-PA plus iloprost group was almost twice as great as in the t-PA alone group (33.0 +/- 13.3 vs. 18.5 +/- 6.7 minutes, mean +/- SD, p less than 0.02), and the duration of reperfusion was much shorter (3.4 +/- 1.8 vs. 39.3 +/- 17.4 minutes, p less than 0.005). Peak coronary artery blood flow after reperfusion in the t-PA plus iloprost group was also less (20 +/- 17 ml/min) than in the t-PA alone group (58 +/- 21 ml/min, p less than 0.005). Reocclusion occurred in all dogs given t-PA with iloprost despite potent synergistic platelet inhibitory effects of t-PA and iloprost, whereas four of six dogs given t-PA alone reoccluded. Neither regimen exerted a significant beneficial effect on regional myocardial shortening during coronary reperfusion. Plasma levels of t-PA were lower when iloprost was given with t-PA (1,022 +/- 360 vs. 1,459 +/- 270 ng/ml in t-PA alone group, p less than 0.05). The detrimental effects of iloprost identified in this study may relate to the reduction in plasma t-PA concentrations by its degradation in the liver caused by the prostacyclin analogue iloprost.

Platelet-leukocyte-endothelial interactions in coronary artery disease

It is generally recognized that formation of a platelet-fibrin-rich thrombus in an atherosclerotic coronary artery is the basis of unstable angina and acute myocardial infarction. Platelet hyperactivity has been identified in coronary risk factors such as hyperlipidemia and diabetes mellitus. Persistent activation of these cells results in release of growth factors that may contribute to the progression of atherosclerosis. Several recent studies show that endothelium, by generating or metabolizing a host of vasoactive substances, plays a critical role in the modulation of vascular tone. Important among these substances are prostacyclin (PGI2) and endothelium-derived relaxing factor (EDRF). The endothelium-dependent modulation of coronary artery tone correlates with the severity of atherosclerosis and the number of coronary risk factors. Procedures such as angioplasty and coronary bypass surgery injure the endothelium. The loss of endothelial smooth muscle relaxant function may contribute to the vasoconstriction and thrombosis often observed soon after these procedures. Thrombolysis (and subsequent reperfusion of the coronary artery) is also associated with severe endothelial dysfunction, with a resulting vasoconstrictor influence on the coronary vascular bed. Activation of leukocytes and their presence in the reperfused myocardium contribute to progression of myocardial injury by release of oxygen free radicals and proteolytic enzymes. Thus, it seems that a perturbation in this delicate equilibrium in cellular interactions relates to genesis and progression of myocardial ischemia.

Leukocyte elastase inhibition and t-PA-induced coronary artery thrombolysis in dogs: Beneficial effects on myocardial histology

Leukocyte-derived elastase is released following coronary artery occlusion and reperfusion and may contribute to reperfusion-related myocardial injury. Leukocyte infiltration into the reperfused myocardium may also contribute to ischemic injury following reflow. In the present study, we examined the effects of tissue-plasminogen activator (t-PA, 1 mg/kg over 20 minutes) given intravenously with either saline or a leukocyte elastase inhibitor (ICI 200,880, 5 mg/kg) in dogs with electrically-induced coronary artery thrombosis. ICI 200,880 administration increased elastase inhibitory activity without affecting t-PA and plasminogen activator inhibitor (PAI-1) activities. Time to reflow, magnitude of peak coronary blood flow, and duration of reflow were not different in dogs given t-PA with saline or with the elastase inhibitor. However, administration of the elastase inhibitor decreased the histologically-determined leukocyte infiltration and severity of myocardial injury in dogs subjected to coronary artery thrombosis and subsequent thrombolysis. These early observations suggest that elastase release during reperfusion may be an important mediator of anoxia-reoxygenation-mediated tissue injury.

Increased production of thromboxane A2 by coronary arteries after thrombolysis

The coronary artery produces large amounts of prostacyclin (PGI2) and a small amount of thromboxane A2 (TXA2); this high PGI2/TXA2 ratio contributes to the antithrombotic properties of the coronary artery. This study was designed to determine whether this ratio changes after coronary artery thrombosis and thrombolysis and accounts for coronary artery reocclusion. Anesthetized dogs (N = 12) were subjected to electrically induced coronary artery thrombosis and tissue plasminogen activator-induced thrombolysis. Thrombolysis was achieved in 11 dogs, and the coronary artery reoccluded in five of these dogs after the initial reperfusion. Spontaneous and ionophore A23,187-stimulated PGI2 and TXA2 synthesis in normal circumflex and ischemic-reperfused left anterior descending coronary artery segments was measured by radioimmunoassay of thromboxane B2 and 6-keto-prostaglandin F1 alpha, respectively. Production of TXA2 was 413% to 656% greater in left anterior descending segments (from the region of thrombosis and sites proximal and distal to the thrombus) compared with normal circumflex segments (p < 0.001). Production of PGI2 was also increased but only by 46% to 80% in the left anterior descending segments compared with normal circumflex segments (p < 0.05). TXA2 production was greater in coronary artery segments that reocculded compared with segments that stayed open (p < 0.02). Scanning electron microscopy revealed platelet deposition in thrombosed left anterior descending segments but not in segments proximal or distal to the thrombus site, indicating that the vascular wall per se may be a source of increased production of TXA2.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effect of unstimulated neutrophils on platelet aggregation by release of a factor similar to Endothelium-Derived Relaxing Factor (EDRF)

Previous studies have indicated a possible role for polymorphonuclear leukocytes (PMNLs) in the maintenance of hemostasis and vascular tone. We now demonstrate that unstimulated isolated PMNLs maintained at 37 degrees inhibited human platelet aggregation in a concentration- and time-dependent fashion. In addition, PMNLs increased platelet cyclic GMP concentrations. The platelet aggregation inhibitory effect of PMNLs was potentiated by superoxide dismutase and attenuated by hemoglobin and methylene blue. This inhibitory effect of PMNLs was not observed in 48-hr-old killed cells and was not modulated by aspirin treatment or by adenosine deaminase. These observations suggest that human PMNLs maintained at 37 degrees produce a substance with biological characteristics similar to those of the endothelium-derived relaxing factor.

Verapamil and aspirin modulate platelet-mediated vasomotion in arterial segments with intact or disrupted endothelium☆

OBJECTIVES This study was designed to examine the effects of verapamil and aspirin, which decrease thromboxane A2 and serotonin release, on the modulation of vascular tone by platelets. BACKGROUND Aggregating platelets cause constriction of de-endothelialized arterial segments through thromboxane A2 and serotonin release. These cells cause relaxation of arterial segments with intact endothelium through release of the endothelium-derived relaxing factor. METHODS Healthy subjects were given either no drug, verapamil or aspirin for > or = 5 days before their platelets were obtained. The effects of platelets obtained from subjects before and after treatment with aspirin or verapamil on the tone of rat aortic rings were determined. RESULTS As expected, control platelets (before verapamil or aspirin treatment) induced concentration-dependent relaxation of rat aortic rings with intact endothelium and a concentration-dependent contraction of de-endothelialized rings. Verapamil treatment enhanced (p < 0.02) the platelet-mediated relaxation in rings with intact endothelium and abolished platelet-mediated constriction (p < 0.01) in the de-endothelialized rings. Aspirin treatment also abolished (p < 0.05) platelet-mediated constriction of the de-endothelialized rings. The de-endothelialized rings contracted normally in response to the synthetic thromboxane A2 analogue U46,619, as well as to serotonin, indicating that the vascular smooth muscle response to thromboxane A2 and serotonin was intact. CONCLUSIONS This study provides evidence for the modulation of platelet-mediated vasoconstriction of de-endothelialized arterial segments by prior treatment of subjects with verapamil or aspirin. In clinical syndromes characterized by endothelial dysfunction or disruption, treatment with verapamil or aspirin may modify platelet-vessel wall interactions.

Attenuation of coronary flow reserve and myocardial function after temporary subtotal coronary artery occlusion and increased myocardial oxygen demand in dogs

OBJECTIVES We examined whether subtotal coronary artery occlusion and reperfusion alter coronary flow reserve and regional myocardial function. BACKGROUND Total coronary artery occlusion followed by reperfusion results in decreased coronary flow reserve and regional myocardial dysfunction. METHODS Thirteen anesthetized dogs were subjected to subtotal occlusion of the left anterior descending coronary artery for 1 h, followed by reperfusion for 1 h. During subtotal left anterior descending occlusion, heart rate was increased by atrial pacing. After reperfusion, coronary flow reserve, indicated by reactive hyperemia, as well as coronary flow responses to acetylcholine and nitroglycerin, regional myocardial function and myocardial leukocyte accumulation were measured. RESULTS After reperfusion, coronary flow reserve was decreased in the ischemic left anterior descending but not the nonischemic circumflex coronary artery region. Myocardial function was also depressed in the left anterior descending coronary region and did not improve on reperfusion. Histologic study showed no leukocyte infiltration in the ischemic left anterior descending coronary region. Myeloperoxidase, an index of myocardial leukocyte accumulation, was similar in the left anterior descending and circumflex coronary regions. Sensitivity of epicardial left anterior descending coronary artery rings to the thromboxane A2 analog U46,619 was enhanced, and relaxation of these rings in response to endothelium-dependent relaxants was decreased. CONCLUSIONS Coronary flow reserve is reduced and regional myocardial function depressed after subtotal coronary artery occlusion and increased heart rate. A decreased synthesis or increased breakdown of endothelium-derived relaxing factor may be related to a decrease in coronary flow reserve. However, the reduction in coronary flow reserve appears to be unrelated to leukocyte accumulation in the reperfused region.

Adenosine protects against attenuation of flow reserve and myocardial function after coronary occlusion and reperfusion

Total coronary artery occlusion and reperfusion result in attenuation of coronary blood flow reserve, regional myocardial dysfunction, and myocardial leukocyte infiltration. To examine the effects of intracoronary adenosine on these occlusion and reperfusion-induced perturbations, we subjected 14 dogs to total left anterior descending (LAD) coronary artery occlusion (1 hour) and reperfusion (1 hour). Seven dogs received adenosine (3.75 mg/min into the LAD distal to the occlusion) over a 1-hour period starting 5 minutes before reperfusion, and the remaining seven dogs received saline solution. One dog in each group died of ventricular fibrillation during coronary artery occlusion. Coronary flow reserve, measured as peak reactive hyperemia (10 and 20 seconds of total coronary artery occlusion) and peak coronary blood flow response to acetylcholine (0.01 to 1.0 micrograms) and nitroglycerin (5 to 25 micrograms), was impaired in the LAD region after LAD occlusion and reperfusion in the saline-treated dogs (all p < 0.01 vs before occlusion and reperfusion); LAD regional myocardial shortening fraction measured by ultrasonic crystals was also diminished after occlusion and reperfusion in saline-treated dogs (-5% +/- 1% vs 12% +/- 2%; p < 0.02). The adenosine-treated dogs showed total protection against loss of coronary flow reserve (peak reactive hyperemia and blood flow increase in response to acetylcholine and nitroglycerin; all p values not significant vs before LAD occlusion and reperfusion). LAD regional myocardial shortening fraction was also preserved in adenosine-treated dogs (9% +/- 2% vs 14% +/- 2%; p not significant). Myocardial myeloperoxidase activity, measured as an index of myocardial leukocyte infiltration, was greater (p < 0.02) in the LAD ischemic-reperfused regions than in nonischemic circumflex regions in the saline-treated dogs. A similar difference in myeloperoxidase activities in the reperfused and control regions was not observed in the adenosine-treated dogs. Thus adenosine protects against loss of coronary flow reserve and regional myocardial function in dogs subjected to coronary artery occlusion and reperfusion.