Platelet inhibition reduces cyclic flow variations and neointimal proliferation in normal and hypercholesterolemic-atherosclerotic canine coronary arteries.

BACKGROUND: Platelet-derived growth factors help stimulate the neointimal proliferation of restenosis after coronary interventions. Reducing platelet accumulation at treated sites may attenuate restenosis. We tested this hypothesis by inducing repetitive platelet aggregation at coronary angioplasty sites in dogs and measuring subsequent neointima formation. METHODS AND RESULTS: Cholesterol-sensitive dogs (n=74) received either 4% cholesterol-enriched diets for >8 months (n=29), creating visible atheromas, or normal canine diets (n=45). A coronary balloon angioplasty cyclic flow variation (CFV) model was used. One group of control dogs (group 1, n=8) had angioplasty with no arterial constriction applied and no drug treatment. Three other groups had arterial constrictors applied to provoke CFVs: group 2 (n=28) received no drug therapy, group 3 (n=18) received oral aspirin alone, and group 4 (n=20) received 3 oral antiplatelet agents: ridogrel, ketanserin, and clopidogrel (R+K+C) to simultaneously inhibit the thromboxane A(2), serotonin, and ADP pathways of platelet aggregation, respectively. Bleeding times were moderately prolonged in the aspirin-treated group (124+/-9 seconds after 3 weeks versus 76+/-6 seconds at baseline, P<0.01) and greatly prolonged on R+K+C (>600 versus 104+/-5 seconds, P<0.001). The frequency and severity of CFVs were inversely related to the degree of platelet inhibition and prolongation of bleeding times, as was sudden death due to acute thrombotic coronary occlusion. Quantitative histology at 8 weeks revealed increased intima-to-media ratio with CFVs: 0.89+/-0.14 in the untreated group 2 versus 0.11+/-0.04 in the control group (P<0.001). Intima-to-media ratio was significantly reduced with antiplatelet treatment (0.27+/-0.05 with aspirin treatment and 0.20+/-0.05 with R+K+C treatment, respectively, P<0.001). Cholesterol feeding did not appear to influence results. CONCLUSIONS: Repetitive platelet accumulation at coronary angioplasty sites caused enhanced neointimal proliferation by 8 weeks. Oral inhibitors of platelet aggregation attenuated platelet function, prolonged bleeding times, reduced or prevented cyclic flows and abrupt thrombotic occlusions, and thereby inhibited neointimal proliferation. Platelet inhibition should continue to receive attention in efforts to reduce restenosis after coronary interventions.

Long-term mortality benefit with abciximab in patients undergoing percutaneous coronary intervention.

OBJECTIVES: The goal of this study was to test: 1) if platelet glycoprotein IIb/IIIa (GP IIb/IIIa) blockade with abciximab bolus plus 12-h infusion reduces mortality after percutaneous coronary intervention (PCI); 2) if prevention of early myocardial infarction (MI) after PCI is a mechanism for reducing mortality; and 3) for risk factors for mortality after PCI. BACKGROUND: Studies of PCI suggest that MI after intervention is predictive of mortality. Abciximab, a platelet GP IIb/IIIa receptor inhibitor, has consistently reduced the incidence of MI among PCI patients in several trials. The presumed mechanism is prevention of platelet thrombus associated with vessel wall injury and downstream embolization into the microcirculation. METHODS: In eight trials, 5,154 patients were randomized to a regimen comprising conventional therapy plus a bolus of abciximab within 1 h before PCI followed by a 12-h infusion; 4,136 controls were randomized to conventional therapy alone. Patient follow-up from six months to three years was available. Survival differences are examined using proportional hazards regression and survival curves. RESULTS: A hazard ratio of 0.71 (95% confidence interval 0.57 to 0.89; p = 0.003) suggests a mortality benefit with abciximab. The absolute reduction in mortality was estimated to be 0.5% through 30 days, 0.7% through six months, 0.9% through one year and 1.8% through three years. Early MI explained 18% of the observed mortality benefit at one year. Multivariate regression suggests that patients with advanced cardiovascular disease may derive the greatest mortality benefit from abciximab. CONCLUSIONS: The evidence from 9,290 randomized PCI patients shows a mortality benefit provided by abciximab bolus plus 12-h infusion.

Modulation of C-reactive protein-mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-atherosclerosis drugs.

BACKGROUND: C-reactive protein (CRP) induces adhesion molecule expression by endothelial cells. However, the effects of CRP on chemokine expression by endothelial cells are not known. METHODS AND RESULTS: We tested the effects of CRP on the production of the chemokines monocyte chemoattractant protein-1 (MCP-1) and RANTES in cultured human umbilical vein endothelial cells. The secretion of chemokines was assessed by ELISA. Incubation with 100 microgram/mL recombinant human CRP induced a 7-fold increase in MCP-1 but no change in RANTES secretion. We showed that the effect of CRP on MCP-1 was present even at 5 microgram/mL CRP, with stepwise increases as the CRP concentration was increased to 10, 50, and 100 microgram/mL. The effect of CRP on MCP-1 induction was not influenced by aspirin (at concentrations up to 1 mmol/L), but it was significantly inhibited by 5 micromol/L simvastatin. The peroxisome proliferator-activated receptor-alpha activators fenofibrate (100 micromol/L) and Wy-14649 (100 micromol/L) almost completely abolished the induction of MCP-1, but the peroxisome proliferator-activated receptor-gamma activator ciglitazone had only a moderate effect. CONCLUSIONS: These results further strengthen the role of CRP in the pathogenesis of vascular inflammation and, likely, atherosclerosis and provide a crucial insight into a novel mechanism of action of anti-atherosclerosis drugs such as simvastatin and fenofibrate.

Development of atherosclerotic plaque with endothelial disruption in Watanabe heritable hyperlipidemic rabbit aortas.

To better understand the morphogenesis of atherosclerotic plaque, we evaluated temporal distribution of leukocytes, macrophages, foam cells, vascular smooth muscle cells, and subendothelial lipid in Watanabe heritable hyperlipedimic (WHHL) rabbit aortas. Aortas of WHHL (n=20) and New Zealand White (NZW, controls; n=8) rabbits were perfusion fixed at 1, 3, 6, and 12 months of age. At initial gross evaluation of lipid distribution, we identified aortic areas at high risk for lesion development. In WHHL rabbits, the lipid-positive portion of high-risk areas increased from 3% at 1 month to 50% at 12 months; during the same period, adherent cell count increased from <1 leukocyte and monocyte/mm(2) to 25 leukocytes, 44 monocytes, and 10 foam cells/mm(2). Controls showed no increase over time in lipid-positive areas or cellular adherence to the endothelium. One-month-old WHHL rabbit aortas had scattered lipid-positive cells in the intima (primarily branch points). Immunostaining of these areas did not show rabbit macrophages (RAM antibody) but were actin positive. Occasionally, platelets and monocytes adhered to the endothelial surface. By age 3 months, well-defined fatty streaks/atherosclerotic plaques had RAM-positive cells within foam cell core, along core margins, and in focal clusters in the fibrous cap and subendothelium. By age 12 months, isolated RAM-positive cells were on the endothelial surface, and surface morphology showed endothelial cell disruption foci containing clusters of macrophages and foam cells. Our results indicate that lipid accumulation (extra- and intracellular) is important in the early development of atherosclerotic lesions; a corresponding, slower accumulation of adherent cells on the lesion surface promotes lipid conversion from fatty streak to plaque.

Selective augmentation of prostacyclin production by combined prostacyclin synthase and cyclooxygenase-1 gene transfer.

BACKGROUND: We tested the hypothesis that combined cyclooxygenase-1 (COX-1) and prostacyclin synthase (PGIS) gene transfer selectively augments prostacyclin production without a concurrent overproduction of other prostanoids. METHODS AND RESULTS: ECV304 cells were transfected with bicistronic pCOX-1/PGIS versus pCOX-1 or pPGIS, and prostanoids were analyzed. Contrary to the high prostaglandin E2 synthesis in pCOX-1 transfected cells, selective prostacyclin formation was noted with bicistronic plasmid transfection. Next, we determined the optimal ratio of Ad-COX-1 to Ad-PGIS by transfecting human umbilical vein endothelial cells with various titers of these 2 adenoviral constructs and determined the level of protein expression and prostanoid synthesis. Our results show that optimal ratios of adenoviral titers to achieve a large prostacyclin augmentation without overproduction of prostaglandin E2 or F2alpha were 50 to 100 plaque forming units (pfu) of Ad-COX-1 to 50 pfu of Ad-PGIS per cell. A higher Ad-PGIS to Ad-COX-1 ratio caused a paradoxical decline in prostacyclin synthesis. CONCLUSIONS: Prostacyclin synthesis can be selectively augmented by cotransfecting endothelial cells with an optimal ratio of COX-1 to PGIS. Combined COX-1 and PGIS gene transfer has the potential for therapeutic augmentation of prostacyclin.

New developments in the detection of vulnerable plaque.

Failure of coronary angiography (luminography) in prediction of future acute coronary syndromes has cast a shadow of doubt over the value of this old gold-standard technique. The fact that angiographically invisible or nonsignificant lesions cause the majority of acute coronary syndromes has driven scientists to develop new diagnostic methods. In this article, we review the ongoing worldwide research on both invasive techniques (such as intravascular angioscopy and colorimetry, ultrasound, thermography, optical coherence tomography, near infrared spectroscopy, Raman spectroscopy, fluorescence emission spectroscopy, elastography, magnetic resonance imaging and spectroscopy, nuclear immunoscintigraphy, electrical impedance imaging, vascular tissue doppler, and shear stress imaging) and noninvasive techniques (such as MRI, contrast-enhanced MRI with and without immunolabeled agents, electron beam computed tomography, multi-slice spiral / helical computed tomography, and nuclear imaging, including positron emission tomography). Each of these techniques and their potential combination holds promise for characterization of plaques responsible for acute coronary syndromes, namely vulnerable plaque.

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.

Prospects for cardiovascular research.

Cardiovascular disease is the greatest threat to human life and health. The past decade has seen remarkable progress in clinical and basic cardiovascular research, and many areas of opportunity are promising. The pace of current progress in clinical and basic research is such that remarkable improvement in the quality and length of life for those at risk for cardiovascular disease is likely.

Growth suppression of human coronary vascular smooth muscle cells by gene transfer of the transcription factor E2F-1.

BACKGROUND: The transcription factor E2F-1 promotes S-phase entry and death in transformed cells and primary cardiomyocytes. We tested the hypothesis that overexpression of E2F-1 forces growth-arrested human coronary vascular smooth muscle cells (VSMCs) to enter the S phase, undergo apoptosis, and thereby regulate VSMC growth. METHODS AND RESULTS: Early-passage (8 times. CONCLUSIONS: Overexpression of the transcription factor E2F-1 regulates growth of human coronary VSMCs by forcing the cells to enter the S phase and then to die. Cell death appears to involve caspase 3-like activity, which, in the VSMCs, is markedly increased by overexpression of E2F-1.

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.

Direct proinflammatory effect of C-reactive protein on human endothelial cells.

BACKGROUND: The acute-phase reactant C-reactive protein (CRP) is an important risk factor for coronary heart disease. However, the possible effects of CRP on vascular cells are not known. METHODS AND RESULTS: We tested the effects of CRP on expression of adhesion molecules in both human umbilical vein and coronary artery endothelial cells. Expression of vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM-1), and E-selectin was assessed by flow cytometry. Incubation with recombinant human CRP (10 microg/mL) for 24 hours induced an approximately 10-fold increase in expression of ICAM-1 and a significant expression of VCAM-1, whereas a 6-hour incubation induced significant E-selectin expression. Adhesion molecule induction was similar to that observed in endothelial cells activated with interleukin-1beta. In coronary artery endothelial cells, induction of ICAM-1 and VCAM-1 was already present at 5 microg/mL and reached a maximum at 50 microg/mL, at which point a substantial increase in expression of E-selectin was also evident. The CRP effect was dependent on presence of human serum in the culture medium, because no effect was seen in cells cultured with serum-free medium. In contrast, interleukin-1beta was able to induce adhesion molecule expression in the absence of human serum. CONCLUSIONS: CRP induces adhesion molecule expression in human endothelial cells in the presence of serum. These findings support the hypothesis that CRP may play a direct role in promoting the inflammatory component of atherosclerosis and present a potential target for the treatment of atherosclerosis.

Redefining medical treatment in the management of unstable angina.

In 1994, the Agency for Health Care Policy and Research sponsored the development of guidelines for diagnosing and managing patients with unstable angina. Since their publication, several important developments have occurred. The prognostic value of biochemical assays for cardiac-specific troponins T and I have been shown in many studies. The possible role for C-reactive protein in determining prognosis deserves further investigation. Substantial clinical benefits have been obtained with intravenous inhibitors of the platelet glycoprotein (GP) IIb-IIIa receptor (abciximab, eptifibatide, tirofiban) and with one of the low-molecular-weight heparins (enoxaparin). The therapeutic potential of other low-molecular-weight heparins, direct thrombin inhibitors, and oral GP IIb-IIIa inhibitors remains to be clarified. On the basis of this evidence, consideration should be given to measuring serum levels of a cardiac troponin (either T or I) and using intravenous GP IIb-IIIa inhibitors and low-molecular-weight heparin in the standard management of patients with unstable angina.

Design of the blockade of the glycoprotein IIb/IIIa receptor to avoid vascular occlusion (BRAVO) trial.

BACKGROUND: Platelets play a key role in the pathogenesis of atherosclerosis, thrombosis, and acute coronary and cerebrovascular syndromes. Inhibition of platelet function by acetylsalicylic acid (aspirin) has been shown to reduce the incidence atherothrombotic events in patients with coronary, cerebrovascular, or peripheral vascular disease. Thienopyridine agents, however, including ticlopidine and clopidogrel, inhibit the adenosine diphosphate receptor and have modestly superior effects compared with aspirin on reduction of death, myocardial infarction, and stroke among a broad group of patients with vascular disease. More effective antithrombotic agents are still required to treat patients at high risk for recurrent vascular events. METHODS: Lotrafiban, a selective, nonpeptide antagonist of the human platelet fibrinogen receptor (glycoprotein [GP] IIb/IIIa [alphaIIb/beta3 integrin]), blocks the binding of fibrinogen to the GP IIb/IIIa receptor, which is the final common pathway of platelet aggregation. Lotrafiban at doses of up to 50 mg twice daily was well-tolerated in a 12-week, double-blind, placebo-controlled, dose-ranging study in patients with recent myocardial infarction, unstable angina, transient ischemic attack, or stroke when added to aspirin therapy. On the basis of these results, a dosing regimen was selected for the phase III Blockage of the Glycoprotein IIb/IIIa Receptor to Avoid Vascular Occlusion (BRAVO) trial based on pharmacodynamics and drug tolerability. In the pivotal BRAVO study, lotrafiban therapy is being evaluated in patients who have had a recent myocardial infarction, unstable angina, transient ischemic attack, or ischemic stroke, or who present at any time after a diagnosis of peripheral vascular disease combined with either cardiovascular or cerebrovascular disease. RESULTS: The efficacy evaluation will be based on a composite end point of clinical events (death by any cause, myocardial infarction, stroke, recurrent ischemia requiring hospitalization, or urgent ischemia-driven revascularization). The target enrollment is 9200 patients worldwide. Approximately 700 centers will participate and will be distributed within 30 countries across North America, Europe, Australia, and Asia.

Inhibition of the sodium-hydrogen exchanger with cariporide to prevent myocardial infarction in high-risk ischemic situations. Main results of the GUARDIAN trial. Guard during ischemia against necrosis (GUARDIAN) Investigators.

BACKGROUND: The transmembrane sodium/hydrogen exchanger maintains myocardial cell pH integrity during myocardial ischemia but paradoxically may precipitate cell necrosis. The development of cariporide, a potent and specific inhibitor of the exchanger, prompted this investigation of the potential of the drug to prevent myocardial cell necrosis. METHODS AND RESULTS: A total of 11 590 patients with unstable angina or non-ST-elevation myocardial infarction (MI) or undergoing high-risk percutaneous or surgical revascularization were randomized to receive placebo or 1 of 3 doses of cariporide for the period of risk. The trial failed to document benefit of cariporide over placebo on the primary end point of death or MI assessed after 36 days. Doses of 20 and 80 mg every 8 hours had no effect, whereas a dose of 120 mg was associated with a 10% risk reduction (98% CI 5.5% to 23.4%, P=0.12). With this dose, benefit was limited to patients undergoing bypass surgery (risk reduction 25%, 95% CI 3.1% to 41.5%, P=0.03) and was maintained after 6 months. No effect was seen on mortality. The rate of Q-wave MI was reduced by 32% across all entry diagnostic groups (2.6% versus 1.8%, P=0.03), but the rate of non-Q-wave MI was reduced only in patients undergoing surgery (7.1% versus 3.8%, P=0.005). There were no increases in clinically serious adverse events. CONCLUSIONS: No significant benefit of cariporide could be demonstrated across a wide range of clinical situations of risk. The trial documented safety of the drug and suggested that a high degree of inhibition of the exchanger could prevent cell necrosis in settings of ischemia-reperfusion.