Inhibition of thrombus formation by endothelin-1 in canine models of arterial thrombosis.

The effect of endothelin-1 (ET-1) on thrombus formation in vivo was evaluated in two well-established canine models of coronary artery thrombosis. First, the possible antithrombotic effect of ET-1 was examined using the cyclic flow reduction (CFR) model of coronary artery stenosis, vascular endothelial cell and intimal smooth muscle cell injury, and periodic acute platelet thrombus formation. Using a rating system of 0 (no inhibition) to 3 (complete inhibition), ET-1 administration at 0.1, 0.5, and 1.0 microgram/kg, i.v. bolus, produced scores of 1.0 +/- 0.2 (n = 10), 1.8 +/- 0.4 (n = 8), and 2.1 +/- 0.3 (n = 7), respectively. ET-1 injection inhibited ex vivo platelet aggregation induced by ADP and U-46619 by 30-60%. When aspirin was administered at 5 mg/kg prior to ET-1 administration at 0.5 microgramoff, ET-1 produced a CFR rating of 2.7 +/- 0.2 (n = 6). However, higher dose aspirin (30 mg/kg, i.v.) significantly inhibited the antithrombotic effect of ET-1 (0.5 +/- 0.5, n = 4). The antithrombotic effect of ET-1 was also examined using an electrolytic injury model of arterial thrombosis. The time required to produce an occlusive thrombus during the experiments in which ET-1 was administered at 10 and 20 ng.kg-1.min-1 was 77 +/- 15 (p < 0.08) and 105 +/- 16 min (p < 0.05), respectively, compared to 44 +/- 5 min when vehicle was infused. Cardiovascular changes following occlusion were not significantly different between dogs given ET-1 and those given vehicle, suggesting that elevated plasma levels of ET-1 did not exacerbate the adverse effects of coronary occlusion. In addition, plasma ET-1 levels were elevated significantly after occlusion in the dogs given vehicle (from 7.4 to 12.4 pg/ml). Taken together, these date provide further evidence to support the notion that ET-1 release during ischemia may be involved in a protective mechanism that impeded thrombus formation in the stenosed coronary artery.

Protection of ischemic/reperfused canine myocardium by CL18/6, a monoclonal antibody to adhesion molecule ICAM-1.

OBJECTIVE: A blocking monoclonal antibody to intercellular adhesion molecule-1 (ICAM-1), CL18/6, previously has been demonstrated to inhibit neutrophil attachment to isolated vascular endothelium and cardiomyocytes. Due to the well known participation of ICAM-1 in the inflammatory responses associated with myocardial ischemia/reperfusion injury, we investigated if CL18/6 could attenuate myocardial ischemia/reperfusion injury in vivo. METHODS: Saline (3-5 ml, i.v., n = 6), non-blocking control MAb CL18/1D8 or CL18/6 (both 0.5 mg kg-1, i.v., n = 4) were administered prior to coronary occlusion (1 h) and subsequent reperfusion (5 h) produced by inflation of a coronary balloon angioplasty catheter in isoflurane-anesthetized, closed-chest dogs. Heart rate and arterial pressure were measured, and regional myocardial blood flow (rMBF), and myeloperoxidase activity (MPO) to index local neutrophil sequestration, were determined. Myocardial infarct size (IS) was evaluated using the tetrazolium staining technique and expressed as a percent of area at risk (AR). RESULTS: Changes in heart rate and arterial pressure were insignificant throughout the experiment. rMBF (mean +/- s.e.m.) in the ischemic subendocardium for each treatment group was: Saline (0.07 +/- 0.02 ml min-1 g-1); CL18/1D8 (0.04 +/- 0.02); CL18/6 (0.06 +/- 0.02). IS/AR% was: saline (37 +/- 3%); CL18/1D8 (39 +/- 9%); CL18/6 (15 +/- 4%*); * = significantly different from CL18/1D8 and saline, P < 0.05. MPO assayed from AR immediately adjacent to the infarct was significantly reduced below infarct MPO only in the CL18/6 treated group-36%). CONCLUSIONS: The results indicate that CL18/6 antagonism of ICAM-1 provided cardioprotection associated with reduced neutrophil activity in vulnerable myocardium, and suggest that ICAM-1 mediated neutrophil sequestration in endangered cardiac tissue is an important mechanism of myocardial ischemia/reperfusion injury.

Induction of neointimal hyperplasia by coronary angioplasty balloon overinflation: comparison of feeder pigs to Yucatan minipigs.

We evaluated the use of simple balloon overinflation to induce neointimal hyperplasia in a porcine model of coronary artery restenosis. By using standard percutaneous transluminal coronary angioplasty techniques, left anterior descending (LAD) and/or left circumflex (LCX) coronary arteries of either juvenile feeder pigs or adult Yucatan minipigs were intentionally overinflated. Four weeks later, resultant neointimal hyperplastic responses (neointima/media area; NI/M) were quantitated morphometrically. At all ballooned sites neointimal hyperplasia occurred only when the internal elastic lamina (IEL) was ruptured; the degree of hyperplasia correlated directly with the injury index, that is, the percentage of IEL circumference that fractured (r = 0.74; n = 25; p < 0.05). Despite similar injury indexes in the LAD bed, there was a trend (p = 0.07; analysis of variance) toward greater NI/M ratios in the Yucatan minipig versus the feeder pig group (1.14 +/- 0.21 vs 0.73 +/- 0.09, n = 7/group). We found no such trend in the LCX bed, where the injury index (25.7% +/- 3.5%) was significantly greater than that of the LAD (18.2% +/- 1.2%, p < 0.05). If variations in balloon-induced vascular injury are accounted for, the technique of balloon overinflation of coronary arteries should prove useful in testing potential antirestenotic agents in either adult or juvenile pigs.

Synthesis and biological evaluation of antiplatelet 2-aminochromones.

The synthesis and biological evaluation of a series of antiplatelet 2-morpholinylchromones has been described. Modification of the C-7 phenylmethoxy group of 8-methyl-7-(phenylmethoxy)-2-(4-morpholinyl)-4H-1-benzopyran-4-one (2) has led to the discovery of a series of 7-[(amino-ethyl)oxy]-8-methyl derivatives which are potent inhibitors of ADP-induced platelet aggregation. Several members of this class proved active in preventing platelet-dependent thrombus formation in the dog, including 8-methyl-7-[2-(4-methyl-1-piperazinyl)ethoxy]-2-(4- morpholinyl)-4H-1-benzopyran-4-one (39) which was devoid of hemodynamic effects at the effective antithrombotic dose.

In vivo demonstration of enhanced arterial constrictor response to serotonin following focal endothelial cell loss.

Arterial sensitivity to vasospasm was assessed prior to and 2 weeks following a 15-min period of external compression of the superficial femoral artery in dogs. Compression was achieved by placing a plastic cuff around the artery to produce a 40-60% reduction in the artery cross-sectional area. An additional six dogs were used to assess morphologic changes produced in the artery immediately and at 2 weeks after compression. Angiography following intraarterial infusions of serotonin (10 and 30 micrograms/min), norepinephrine (0.1 microgram/min), ergonovine (20 micrograms/min), and the thromboxane mimic U-46,619 (0.1 microgram/min) demonstrated a specific sensitivity to serotonin 2 weeks after the 15-min application of external arterial compression. The serotonin response was antagonized by the specific serotonin (5-HT2) receptor antagonist, ketanserin. Scanning electron microscopy of the acutely injured luminal surface revealed loss of endothelium and deposition of platelets. Patchy areas with intact endothelium and migrating leukocytes were located within the denuded sites. Two weeks after constrictor placement, the compressed area appeared as a raised or semiraised lesion in which the orientation and shape of the luminal cells were distinctly delineated from the adjacent noninjured segments. However, the luminal cells appeared to be endothelium that had regrown over the previously denuded area. The results of this study demonstrate, in an in vivo model, an enhancement in serotonin-mediated vasoconstriction following intimal injury and repair and support the suggestion that endothelial damage or dysfunction may play a role in the pathophysiology of arterial spasm.

Effects of low doses of aspirin and dipyridamole on platelet aggregation in the dog coronary artery.

The circumflex coronary artery of pentobarbital-anesthetized dogs was partially obstructed with an externally applied rigid plastic band. Platelet aggregation at the site of stenosis caused a gradual decline in blood flow in the artery, which was monitored with an electromagnetic flow probe placed proximally to the obstructor. The effects of drugs on platelet aggregation were evaluated by monitoring changes in both the rate and the degree of decline in blood flow. In most dogs, aspirin inhibited intravascular platelet aggregation (ED50 = 1 mg/kg). Dipyridamole, even at doses that severely depressed blood pressure (1 mg/kg), had no effect on platelet aggregation. However, in dogs that had been pretreated with a low dose of dipyridamole (0.2 mg/kg), the antiaggregatory activity of aspirin was enhanced. This potentiation was evident only at low doses of aspirin (0.03 and 0.1 mg/kg), where the drug was 10 times more active; at high aspirin doses, which depressed vascular cyclooxygenase, no potentiation was seen. Further evidence that the mechanism of this synergism may depend on endogenous prostacyclin production at the site of the partial obstruction was seen when cyclooxygenase inhibitors applied topically on the exposed artery eliminated the antiaggregatory effect of low doses of aspirin. It is important to note that the protective effect of dipyridamole and low-dose aspirin was less than that seen at the high doses of aspirin alone, suggesting that the theoretical benefits of platelet-specific doses of aspirin may be overstated.

Effect of pulmonary and renal circulations on activity of atrial natriuretic factor.

The effect of passage through the pulmonary and renal circulation on the activity of extracted and synthetic atrial natriuretic factors was determined by monitoring the amount of the vascular relaxing activity surviving passage through the organ. When crude atrial extract was infused through isolated perfused lungs of the guinea pig, approximately 75% of its activity survived. The activity of a 23-amino acid synthetic atrial peptide was decreased 21% on passage through these lungs, but this loss of activity was not significantly different from the crude extract. Analogous experiments in vivo showed that passage through the pulmonary circulation of the dog did not alter the activity of either the crude atrial extract or of synthetic atrial peptide. In contrast to the lack of effect of the pulmonary circulation, approximately 80% of the activity of crude atrial extract and synthetic atrial peptide was removed by the isolated perfused kidney of the rabbit, and in vivo the dog kidney removed approximately 80% of the activity of both these atrial substances. By surviving transit through the pulmonary circulation, these materials exhibit a necessary property of a circulating hormone stored in the right atrium, and the fact that the renal circulation extracts most of these materials is consistent with the kidney being a target organ of this putative hormone.