Local gene transfer of tissue factor pathway inhibitor regulates intimal hyperplasia in atherosclerotic arteries.

Tissue factor (TF), the initiator of blood coagulation and thrombosis, is up-regulated after vascular injury and in atherosclerotic states. Systemic administration of recombinant TF pathway inhibitor (TFPI) has been reported to decrease intimal hyperplasia after vascular injury and also to suppress systemic mechanisms of blood coagulation and thrombosis. Here we report that, in heritable hyperlipidemic Watanabe rabbits, adenoviral gene transfer of TFPI to balloon-injured atherosclerotic arteries reduced the extent of intimal hyperplasia by 43% (P < 0.05) compared with a control vector used at identical titer (1 x 10(10) plaque-forming units/ml). Platelet aggregation and coagulation studies performed 7 days after local gene transfer of TFPI failed to show any impairment in systemic hemostasis. At time of sacrifice, 4 weeks after vascular injury, the 10 Ad-TFPI treated carotid arteries were free of thrombi, whereas two control-treated arteries were occluded (P, not significant). These findings suggest that TFPI overexpressed in atherosclerotic arteries can regulate hyperplastic response to injury in the absence of changes in the hemostatic system, establishing a role for local TF regulation as target for gene transfer-based antirestenosis therapies.

Inhibition of coronary thrombosis and local inflammation by a noncarbohydrate selectin inhibitor.

We tested the hypothesis that selectin inhibition with blocking antibodies or a small-molecular-weight inhibitor of L-, P-, and E-selectin, methoxybenzoylpropionic acid (MBPA), prevents thrombus formation in a canine coronary Folts' model. Cyclic flow variations (CFVs) were induced by crush injury and constriction of the left anterior descending coronary artery in dogs. Systemic infusion of antibodies to P- and L-selectin abolished CFVs, respectively, in 50% and 17% of treated dogs [P = not significant (NS)]. The combination of P- and L-selectin antibodies suppressed CFVs in 60% of treated dogs (P = NS). In contrast, systemic selectin blockade by intravenous infusion or local adventitial application of MBPA markedly reduced CFVs and, in addition, reduced myocardial myeloperoxidase (MPO) activity. We conclude that inhibition of L-, P-, and E-selectin binding by a small-molecular-weight, noncarbohydrate compound markedly reduces arterial thrombosis, whereas systemic administration of antibodies to L- and P-selectin fail to reproduce this antithrombotic effect. These results underscore the role of selectins in the pathogenesis of arterial thrombosis under high shear stress and suggest that inhibition of P- and L- selectin may not suffice to prevent thrombus formation in this model. The role of E-selectin in thrombus formation in this model awaits further testing.

The emerging clinical potential of cardiovascular gene therapy.

Despite considerable progress, pharmacological therapies have not provided a complete solution for common cardiovascular problems, including recurrent thrombosis, restenosis, and vein graft deterioration. Optimal drug dosage, reproducing plasma concentrations achieved in animal studies establishing proof-of-principle, would often be too toxic to administer, especially when given over prolonged periods of time. Local gene therapy aims at overexpressing proteins that: (1) regulate the cell cycle of VSMC; (2) inhibit VSMC migration; (3) endow the endothelium with its vasoprotective properties; and (4) stimulate growth of endothelium and angiogenesis. Alternatively, some approaches tend to suppress gene expression of proteins believed to promote VSMC proliferation and migration. In sharp contrast to drug treatments, local gene therapy limits expression of the beneficial agent to the injured vascular site, and there, it can extend the presence of this agent to weeks and, with some gene vectors, to many months. The clinical potential of this approach has led to the initiation of trials that currently evaluate gene therapy approaches to the attenuation of peripheral and myocardial ischaemia and the prevention of vein graft disease.

Measurement of platelet-activating factor in a canine model of coronary thrombosis and in endarterectomy samples from patients with advanced coronary artery disease.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid mediator of numerous inflammatory and thrombotic responses. The purpose of this study was to determine if PAF synthesis is elevated in damaged coronary arteries after a sustained period of cyclic flow variation (CFV), a phenomenon caused by alternating periods of thrombosis and reperfusion at sites of endothelial injury. Cyclic flow was established and maintained in the left anterior descending coronary arteries (LADs) of 10 dogs. After 8 hours of CFV, the section of damaged LAD containing the thrombus and control sections of the circumflex artery, carotid artery, and saphenous vein was excised, and the total lipids were extracted. The PAF was then purified by silica column chromatography and high-performance liquid chromatography and assayed by both a rabbit platelet bioassay and a PAF radioimmunoassay. With the platelet bioassay, PAF levels of 8.9 +/- 4.0 (range, 4.8 to 15.5) pg/mg wet wt were found in the damaged LADs from the 10 dogs. This PAF bioactivity was completely inhibited by a PAF receptor antagonist. When the radioimmunoassay was used, slightly higher PAF levels of 16.3 +/- 12.9 (range, 4.5 to 41.8) pg/mg wet wt were observed in the LADs. Overall, these PAF levels were 3- to 64-fold higher than in the control vessels when either assay method was used. Although increases in PAF were observed in the damaged LADs, measurements of PAF in blood samples taken from the LAD and the aorta (control) failed to demonstrate any site-specific increase of PAF in the blood. In related experiments, PAF was also measured in 23 endarterectomy samples taken from the coronary arteries of 16 patients with severe atherosclerosis. The PAF levels in these samples were highly variable (2.9 +/- 2.2 [range, 0.3 to 8.5] pg/mg wet wt) and showed no correlation with tissue mass, suggesting that PAF is affected by factors other than the simple presence of atherosclerotic tissue in the vessel. These findings provide direct evidence that PAF is synthesized locally at the site of endothelial injury during thrombosis and that PAF accumulates in the atherosclerotic plaque of some patients with advanced coronary artery disease.

Serotonin S2 and thromboxane A2-prostaglandin H2 receptor blockade provide protection against epinephrine-induced cyclic flow variations in severely narrowed canine coronary arteries.

The object of this study was to test the hypothesis that administration of both serotonin S2 and thromboxane A2-prostaglandin H2 (PGH2) receptor antagonists provides significant protection against epinephrine-induced cyclic coronary artery flow variations in open chest, anesthetized dogs with severe proximal coronary artery stenosis and endothelial injury. Three groups of dogs were studied. In Group 1 (n = 7) and Group 2 (n = 6), cyclic coronary flow variations were initiated after placement of a concentric constrictor around the left anterior descending coronary artery and were abolished by administration of either a thromboxane A2-prostaglandin H2 receptor antagonist, SQ29,548 (SQ) (Group 1), or a serotonin S2 receptor antagonist, LY53,857 (LY) (Group 2). Cyclic flow variations were restored with an epinephrine infusion and the second antagonist (LY for Group 1; SQ for Group 2) was administered to abolish epinephrine-induced cyclic flow variations. The rate of epinephrine infusion was increased until cyclic coronary flow variations returned (n = 8) or significant hemodynamic changes occurred. Plasma epinephrine concentrations were determined during a control period of cyclic coronary flow variations, after epinephrine restored cyclic flow variations in the presence of either SQ or LY, and again after epinephrine restored cyclic flow variations in the presence of both SQ and LY. A third group of dogs (Group 3, n = 9) required both SQ and LY to eliminate the initial cyclic coronary flow variations and infused epinephrine restored cyclic flow variations (n = 8). Plasma epinephrine concentrations were determined during a control period and after cyclic coronary flow variation restoration with epinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cooperative mediation by serotonin S2 and thromboxane A2/prostaglandin H2 receptor activation of cyclic flow variations in dogs with severe coronary artery stenoses.

We have reported previously that thromboxane A2/prostaglandin (PG)H2 and serotonin independently mediate the occurrence of cyclic flow variations (CFVs) in a canine preparation of severe coronary artery narrowing. This may be due to an effect of these substances on platelets and/or the vascular wall. We tested the hypothesis that there is a cooperative effect between thromboxane A2/PGH2 and serotonin receptor stimulation in the development of CFVs in this animal preparation. After placement of a hard plastic cylindrical constrictor around the left anterior descending coronary artery, CFVs develop and are characterized by repetitive cycles of declines in coronary blood flow and abrupt increases in flow. In a control group of dogs, CFV frequency (cycles/hour) and severity (lowest coronary blood flow just before its restoration) did not change significantly over a 3 hr interval. In a second group of dogs, CFVs were established after constrictor placement, abolished with the serotonin (5HT2) receptor antagonist ketanserin, and reestablished by the continuous infusion of serotonin into the left atrium. Serotonin-induced CFVs were then abolished with a thromboxane A2/PGH2 receptor antagonist, SQ29,548, or a thromboxane synthetase inhibitor, dazoxiben (UK37,248). The relative specificity of the respective antagonists, SQ29,548 and ketanserin, was determined in canine platelets and rat aortic vascular strips. No significant cross-reactivity between ketanserin and SQ29,548 was found. Thus, the data obtained in these studies demonstrate a cooperative interaction between thromboxane A2/PGH2 and serotonin S2 receptors that contributes to the development of CFVs in this experimental preparation.