Andrew J. Lucking

Diesel exhaust inhalation increases thrombus formation in man

AIMS Although the mechanism is unclear, exposure to traffic-derived air pollution is a trigger for acute myocardial infarction (MI). The aim of this study is to investigate the effect of diesel exhaust inhalation on platelet activation and thrombus formation in men. METHODS AND RESULTS In a double-blind randomized crossover study, 20 healthy volunteers were exposed to dilute diesel exhaust (350 microg/m(3)) and filtered air. Thrombus formation, coagulation, platelet activation, and inflammatory markers were measured at 2 and 6 h following exposure. Thrombus formation was measured using the Badimon ex vivo perfusion chamber. Platelet activation was assessed by flow cytometry. Compared with filtered air, diesel exhaust inhalation increased thrombus formation under low- and high-shear conditions by 24% [change in thrombus area 2229 microm(2), 95% confidence interval (CI) 1143-3315 microm(2), P = 0.0002] and 19% (change in thrombus area 2451 microm(2), 95% CI 1190-3712 microm(2), P = 0.0005), respectively. This increased thrombogenicity was seen at 2 and 6 h, using two different diesel engines and fuels. Diesel exhaust also increased platelet-neutrophil and platelet-monocyte aggregates by 52% (absolute change 6%, 95% CI 2-10%, P = 0.01) and 30% (absolute change 3%, 95% CI 0.2-7%, P = 0.03), respectively, at 2 h following exposure compared with filtered air. CONCLUSION Inhalation of diesel exhaust increases ex vivo thrombus formation and causes in vivo platelet activation in man. These findings provide a potential mechanism linking exposure to combustion-derived air pollution with the triggering of acute MI.

Combustion-derived nanoparticulate induces the adverse vascular effects of diesel exhaust inhalation

Aim Exposure to road traffic and air pollution may be a trigger of acute myocardial infarction, but the individual pollutants responsible for this effect have not been established. We assess the role of combustion-derived-nanoparticles in mediating the adverse cardiovascular effects of air pollution. Methods and results To determine the in vivo effects of inhalation of diesel exhaust components, 16 healthy volunteers were exposed to (i) dilute diesel exhaust, (ii) pure carbon nanoparticulate, (iii) filtered diesel exhaust, or (iv) filtered air, in a randomized double blind cross-over study. Following each exposure, forearm blood flow was measured during intra-brachial bradykinin, acetylcholine, sodium nitroprusside, and verapamil infusions. Compared with filtered air, inhalation of diesel exhaust increased systolic blood pressure (145 ± 4 vs. 133 ± 3 mmHg, P< 0.05) and attenuated vasodilatation to bradykinin (P= 0.005), acetylcholine (P= 0.008), and sodium nitroprusside (P< 0.001). Exposure to pure carbon nanoparticulate or filtered exhaust had no effect on endothelium-dependent or -independent vasodilatation. To determine the direct vascular effects of nanoparticulate, isolated rat aortic rings (n= 6–9 per group) were assessed in vitro by wire myography and exposed to diesel exhaust particulate, pure carbon nanoparticulate and vehicle. Compared with vehicle, diesel exhaust particulate (but not pure carbon nanoparticulate) attenuated both acetylcholine (P< 0.001) and sodium-nitroprusside (P= 0.019)-induced vasorelaxation. These effects were partially attributable to both soluble and insoluble components of the particulate. Conclusion Combustion-derived nanoparticulate appears to predominately mediate the adverse vascular effects of diesel exhaust inhalation. This provides a rationale for testing environmental health interventions targeted at reducing traffic-derived particulate emissions.

Particle Traps Prevent Adverse Vascular and Prothrombotic Effects of Diesel Engine Exhaust Inhalation in Men

Background— In controlled human exposure studies, diesel engine exhaust inhalation impairs vascular function and enhances thrombus formation. The aim of the present study was to establish whether an exhaust particle trap could prevent these adverse cardiovascular effects in men. Methods and Results— Nineteen healthy volunteers (mean age, 25±3 years) were exposed to filtered air and diesel exhaust in the presence or absence of a particle trap for 1 hour in a randomized, double-blind, 3-way crossover trial. Bilateral forearm blood flow and plasma fibrinolytic factors were assessed with venous occlusion plethysmography and blood sampling during intra-arterial infusion of acetylcholine, bradykinin, sodium nitroprusside, and verapamil. Ex vivo thrombus formation was determined with the use of the Badimon chamber. Compared with filtered air, diesel exhaust inhalation was associated with reduced vasodilatation and increased ex vivo thrombus formation under both low- and high-shear conditions. The particle trap markedly reduced diesel exhaust particulate number (from 150 000 to 300 000/cm3 to 30 to 300/cm3; P<0.001) and mass (320±10 to 7.2±2.0 &mgr;g/m3; P<0.001), and was associated with increased vasodilatation, reduced thrombus formation, and an increase in tissue-type plasminogen activator release. Conclusions— Exhaust particle traps are a highly efficient method of reducing particle emissions from diesel engines. With a range of surrogate measures, the use of a particle trap prevents several adverse cardiovascular effects of exhaust inhalation in men. Given these beneficial effects on biomarkers of cardiovascular health, the widespread use of particle traps on diesel-powered vehicles may have substantial public health benefits and reduce the burden of cardiovascular disease. Clinical Trial Registration— http://www.clinicaltrials.gov. Unique identifier: NCT00745446.

Experimental exposure to diesel exhaust increases arterial stiffness in man

IntroductionExposure to air pollution is associated with increased cardiovascular morbidity, although the underlying mechanisms are unclear. Vascular dysfunction reduces arterial compliance and increases central arterial pressure and left ventricular after-load. We determined the effect of diesel exhaust exposure on arterial compliance using a validated non-invasive measure of arterial stiffness.MethodsIn a double-blind randomized fashion, 12 healthy volunteers were exposed to diesel exhaust (approximately 350 μg/m3) or filtered air for one hour during moderate exercise. Arterial stiffness was measured using applanation tonometry at the radial artery for pulse wave analysis (PWA), as well as at the femoral and carotid arteries for pulse wave velocity (PWV). PWA was performed 10, 20 and 30 min, and carotid-femoral PWV 40 min, post-exposure. Augmentation pressure (AP), augmentation index (AIx) and time to wave reflection (Tr) were calculated.ResultsBlood pressure, AP and AIx were generally low reflecting compliant arteries. In comparison to filtered air, diesel exhaust exposure induced an increase in AP of 2.5 mmHg (p = 0.02) and in AIx of 7.8% (p = 0.01), along with a 16 ms reduction in Tr (p = 0.03), 10 minutes post-exposure.ConclusionAcute exposure to diesel exhaust is associated with an immediate and transient increase in arterial stiffness. This may, in part, explain the increased risk for cardiovascular disease associated with air pollution exposure. If our findings are confirmed in larger cohorts of susceptible populations, this simple non-invasive method of assessing arterial stiffness may become a useful technique in measuring the impact of real world exposures to combustion derived-air pollution.

Cardiovascular effects of tumour necrosis factor α antagonism in patients with acute myocardial infarction: a first in human study

Objective The inflammatory cytokine, tumour necrosis factor α (TNF-α), exerts deleterious cardiovascular effects. We wished to determine the effects of TNF-α antagonism on endothelial function and platelet activation in patients with acute myocardial infarction. Design and setting and patients A double-blind, parallel group, randomised controlled trial performed in a tertiary referral cardiac centre. 26 patients presenting with acute myocardial infarction randomised to receive an intravenous infusion of etanercept (10 mg) or saline placebo. Main outcome measures Leucocyte count, plasma cytokine concentrations, flow cytometric measures of platelet activation and peripheral vasomotor and fibrinolytic function were determined before and 24 h after study intervention. Results Consistent with effective conjugation of circulating TNF-α, plasma TNF-α concentrations increased in all patients following etanercept (254±15 vs 0.12±0.02 pg/ml; p<0.0001), but not saline infusion. Etanercept treatment reduced neutrophil (7.4±0.6 vs 8.8±0.6×109 cells/l; p=0.03) and plasma interleukin-6 concentrations (5.8±2.0 vs 10.6±4.0 pg/ml; p=0.012) at 24 h but increased platelet–monocyte aggregation (30±5 vs 20±3%; p=0.02). Vasodilatation in response to substance P, acetylcholine and sodium nitroprusside, and acute tissue plasminogen activator release were unaffected by either treatment (p>0.1 for all). Conclusions Following acute myocardial infarction, etanercept reduces systemic inflammation but increases platelet activation without affecting peripheral vasomotor or fibrinolytic function. We conclude that TNF-α antagonism is unlikely to be a beneficial therapeutic strategy in patients with acute myocardial infarction.

Cardiovascular effects of tumour necrosis factor α antagonism in patients with acute myocardial infarction

Objective The inflammatory cytokine, tumour necrosis factor α (TNF-α), exerts deleterious cardiovascular effects. We wished to determine the effects of TNF-α antagonism on endothelial function and platelet activation in patients with acute myocardial infarction. Design and setting and patients A double-blind, parallel group, randomised controlled trial performed in a tertiary referral cardiac centre. 26 patients presenting with acute myocardial infarction randomised to receive an intravenous infusion of etanercept (10 mg) or saline placebo. Main outcome measures Leucocyte count, plasma cytokine concentrations, flow cytometric measures of platelet activation and peripheral vasomotor and fibrinolytic function were determined before and 24 h after study intervention. Results Consistent with effective conjugation of circulating TNF-α, plasma TNF-α concentrations increased in all patients following etanercept (254±15 vs 0.12±0.02 pg/ml; p<0.0001), but not saline infusion. Etanercept treatment reduced neutrophil (7.4±0.6 vs 8.8±0.6×109 cells/l; p=0.03) and plasma interleukin-6 concentrations (5.8±2.0 vs 10.6±4.0 pg/ml; p=0.012) at 24 h but increased platelet–monocyte aggregation (30±5 vs 20±3%; p=0.02). Vasodilatation in response to substance P, acetylcholine and sodium nitroprusside, and acute tissue plasminogen activator release were unaffected by either treatment (p>0.1 for all). Conclusions Following acute myocardial infarction, etanercept reduces systemic inflammation but increases platelet activation without affecting peripheral vasomotor or fibrinolytic function. We conclude that TNF-α antagonism is unlikely to be a beneficial therapeutic strategy in patients with acute myocardial infarction.

P-selectin antagonism reduces thrombus formation in humans

Summary.  Background: Interaction of P‐selectin with its glycoprotein ligand (P‐selectin glycoprotein ligand type 1) mediates inflammatory processes that may also include vascular thrombosis. Platelet P‐selectin expression is increased in patients with coronary heart disease, and its antagonism represents a potential future therapeutic target for the prevention and treatment of atherothrombosis. Aim: To investigate the effects of the novel small molecule P‐selectin antagonist PSI‐697 on thrombus formation in humans. Methods and Results: In a double‐blind randomized crossover study, thrombus formation was measured in 12 healthy volunteers, using the Badimon ex vivo perfusion chamber under conditions of low and high shear stress. Saline placebo, low‐dose (2 m) and high‐dose (20 m) PSI‐697 and the glycoprotein IIb–IIIa receptor antagonist tirofiban (50 ng mL−1) were administered into the extracorporeal circuit prior to the perfusion chamber. As compared with saline placebo, blockade of platelet glycoprotein IIb–IIIa receptor with tirofiban produced 28% and 56% reductions in thrombus formation in the low‐shear and high‐shear chambers, respectively. PSI‐697 caused a dose‐dependent, but more modest, reduction in thrombus formation. Low‐dose PSI‐796 (2 m) reduced total thrombus area by 14% (P = 0.04) and 30% (P = 0.0002) in the low‐shear and high‐shear chambers, respectively. At the high dose (20 m), PSI‐697 reduced total thrombus area by 18% (P = 0.0094) and 41% (P = 0.0008) in the low‐shear and high‐shear chambers, respectively. Conclusions: P‐selectin antagonism with PSI‐697 reduces ex vivo thrombus formation in humans. These findings provide further evidence that P‐selectin antagonism may be a potential target for the prevention and treatment of cardiovascular disease.

Fibrin clot structure remains unaffected in young, healthy individuals after transient exposure to diesel exhaust

Exposure to urban particulate matter has been associated with an increased risk of cardiovascular disease and thrombosis. We studied the effects of transient exposure to diesel particles on fibrin clot structure of 16 healthy individuals (age 21- 44). The subjects were randomly exposed to diesel exhaust and filtered air on two separate occasions. Blood samples were collected before exposure, and 2 and 6 hours after exposure. There were no significant changes on clot permeability, maximum turbidity, lag time, fibre diameter, fibre density and fibrinogen level between samples taken after diesel exhaust exposure and samples taken after filtered air exposure. These data show that there are no prothrombotic changes in fibrin clot structure in young, healthy individuals exposed to diesel exhaust.

Effect of the small molecule plasminogen activator inhibitor-1 (PAI-1) inhibitor, PAI-749, in clinical models of fibrinolysis.

Summary.  Background: The principal inhibitor of fibrinolysis in vivo is plasminogen activator inhibitor‐1 (PAI‐1). PAI‐749 is a small molecule inhibitor of PAI‐1 with proven antithrombotic efficacy in several preclinical models. Objective: To assess the effect of PAI‐749, by using an established ex vivo clinical model of thrombosis and a range of complementary in vitro human plasma‐based and whole blood‐based models of fibrinolysis. Methods: In a double‐blind, randomized, crossover study, ex vivo thrombus formation was assessed using the Badimon chamber in 12 healthy volunteers during extracorporeal administration of tissue‐type plasminogen activator (t‐PA) in the presence of PAI‐749 or control. t‐PA‐mediated lysis of plasma clots and of whole blood model thrombi were assessed in vitro. The role of vitronectin was examined by assessing lysis of fibrin clots generated from purified plasma proteins. Results: There was a dose‐dependent reduction in ex vivo thrombus formation by t‐PA (P < 0.0001). PAI‐749 had no effect on in vitro or ex vivo thrombus formation or fibrinolysis in the presence or absence of t‐PA. Inhibition of PAI‐1 with a blocking antibody enhanced fibrinolysis in vitro (P < 0.05). Conclusions: Despite its efficacy in a purified human system and in preclinical models of thrombosis, the current study suggests that PAI‐749 does not affect thrombus formation or fibrinolysis in a range of established human plasma and whole blood‐based systems.

Characterisation and reproducibility of a human ex vivo model of thrombosis

BACKGROUND The Badimon chamber is a clinical ex vivo model of thrombosis that mimics flow conditions within the coronary circulation of man. The aims of this study were to characterise thrombus formation in the chamber and evaluate its reproducibility. METHODS Using blood from 24 healthy human volunteers, thrombus formation was assessed at low and high shear rates with porcine aortic tunica media as the thrombogenic substrate. Thrombus area was measured histomorphometrically. Reproducibility was assessed by paired measurements made both within and between days. Platelet activation was assessed before and at selected points within the extracorporeal circuit using flow cytometry, and fibrin content and distribution within the thrombus were assessed by immunohistochemistry. RESULTS Total thrombus area was highly reproducible within and between days in the low shear ([mean thrombus area, mean difference ± SEM] 8,018μm(2), 58±204μm(2) and 8,177μm(2), -154±168μm(2) respectively) and high shear chambers (11,802μm(2), -52±175μm(2) and 11,877μm(2), 220±181μm(2) respectively). Total thrombus area was greater in the high compared to the low shear chamber (11,970±285μm(2)versus 7,892±298μm(2); P<0.0001). Transit through the extracorporeal circuit did not result in platelet activation which was only detected after blood passed across the perfusion chambers (P=0.02 for platelet-monocyte aggregate formation and P=0.05 for P-selectin expression). Thrombus in the low shear chamber contained a greater proportion of fibrin (25.0±6.0% versus 8.3±1.6%, P<0.001). CONCLUSIONS The Badimon chamber provides a highly reproducible technique for the assessment of ex vivo platelet-rich thrombus formation in man.