Jan Emmerechts

Air Pollution–Related Prothrombotic Changes in Persons with Diabetes

Background Population studies suggest that persons with diabetes are more sensitive to the effects of particulate matter (PM) air pollution. However, the biological mechanisms of a possible prothrombotic effect underlying this enhanced susceptibility remain largely unknown. Objective We hypothesized that exposure to PM causes prothrombotic changes in persons with diabetes, possibly via systemic inflammation. Methods Our study included 137 nonsmoking adults with diabetes who were outpatients at the University Hospital Leuven. Recent exposure (2 hr before examination) to ambient PM was measured at the entrance of the hospital. Individual chronic exposure to PM was assessed by measuring the area occupied by carbon in airway macrophages obtained by sputum induction. Platelet function was measured ex vivo with the PFA-100 platelet function analyzer, which simulates a damaged blood vessel; we analyzed the function of platelets in primary hemostasis under high shear conditions. Total and differential blood leukocytes were counted. Results Independent of antiplatelet medication, an interquartile range (IQR) increase of 39.2 μg/m3 in PM10 (PM with aerodynamic diameter ≤ 10 μm) concentration measured 2 hr before the clinical examination (recent exposure) was associated with a decrease of 21.1 sec [95% confidence interval (CI), − 35.3 to − 6.8] in the PFA-100 closure time (i.e., increased platelet activation) and an increase in blood leukocytes of 512 per microliter of blood (95% CI, 45.2–979). Each area increase of 0.25 μm2 (IQR) in carbon load of airway macrophages (chronic exposure) was associated with an increase of 687 leukocytes per microliter of blood (95% CI, 224–1,150). Conclusions A relevant increase in recent PM exposure was associated with a change in platelet function toward a greater prothrombotic tendency. The magnitude of the change was about two-thirds (in the opposite direction) of the average effect of antiplatelet medication. Diabetic patients showed evidence of proinflammatory response to both recent and chronic exposure to PM air pollution.

Traffic air pollution and oxidized LDL.

Background Epidemiologic studies indirectly suggest that air pollution accelerates atherosclerosis. We hypothesized that individual exposure to particulate matter (PM) derived from fossil fuel would correlate with plasma concentrations of oxidized low-density lipoprotein (LDL), taken as a marker of atherosclerosis. We tested this hypothesis in patients with diabetes, who are at high risk for atherosclerosis. Methodology/Principal Findings In a cross-sectional study of non-smoking adult outpatients with diabetes we assessed individual chronic exposure to PM by measuring the area occupied by carbon in airway macrophages, collected by sputum induction and by determining the distance from the patients residence to a major road, through geocoding. These exposure indices were regressed against plasma concentrations of oxidized LDL, von Willebrand factor and plasminogen activator inhibitor 1 (PAI-1). We could assess the carbon load of airway macrophages in 79 subjects (58 percent). Each doubling in the distance of residence from major roads was associated with a 0.027 µm2 decrease (95% confidence interval (CI): −0.048 to −0.0051) in the carbon load of airway macrophages. Independently from other covariates, we found that each increase of 0.25 µm2 [interquartile range (IQR)] in carbon load was associated with an increase of 7.3 U/L (95% CI: 1.3 to 13.3) in plasma oxidized LDL. Each doubling in distance of residence from major roads was associated with a decrease of −2.9 U/L (95% CI: −5.2 to −0.72) in oxidized LDL. Neither the carbon load of macrophages nor the distance from residence to major roads, were associated with plasma von Willebrand factor or PAI-1. Conclusions The observed positive association, in a susceptible group of the general population, between plasma oxidized LDL levels and either the carbon load of airway macrophages or the proximity of the subjects residence to busy roads suggests a proatherogenic effect of traffic air pollution.

Evaluation of the profibrinolytic properties of an anti-TAFI monoclonal antibody in a mouse thromboembolism model.

The enhancement of fibrinolysis constitutes a promising approach to treat thrombotic diseases. Activated thrombin activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis and is an attractive target to develop profibrinolytic drugs. TAFI can be activated by thrombin, thrombin/thrombomodulin, or plasmin, but the in vivo physiologic TAFI activator(s) are unknown. Here, we generated and characterized MA-TCK26D6, a monoclonal antibody raised against human TAFI, and examined its profibrinolytic properties in vitro and in vivo. In vitro, MA-TCK26D6 showed a strong profibrinolytic effect caused by inhibition of the plasmin-mediated TAFI activation. In vivo, MA-TCK26D6 significantly decreased fibrin deposition in the lungs of thromboembolism-induced mice. Moreover, in the presence of MA-TCK26D6, plasmin-α(2)-antiplasmin complexes in plasma of thromboembolism-induced mice were significantly increased compared with a control antibody, indicative of an acceleration of fibrinolysis through MA-TCK26D6. In this study, we show that plasmin is an important TAFI activator that hampers in vitro clot lysis. Furthermore, this is the first report on an anti-TAFI monoclonal antibody that demonstrates a strong profibrinolytic effect in a mouse thromboembolism model.

Air pollution-associated procoagulant changes: the role of circulating microvesicles.

Summary.  Background: Epidemiological studies suggest an association between exposure to particulate matter (PM) in air pollution and the risk of venous thromboembolism (VTE). Objectives: To investigate the underlying pathophysiological pathways linking PM exposure and VTE. Patients and methods: We assessed potential associations between PM exposure and coagulation and inflammation parameters, including circulating microvesicles, in a group of 233 patients with diabetes. Results: The numbers of circulating blood platelet‐derived and annexin V‐binding microvesicles were inversely associated with the current levels of PM2.5 or PM10, measured on the day of sampling. Recent past exposure to PM10, up to 1 week prior to blood sampling, estimated at the patients’ residential addresses, was associated with elevated high‐sensitivity C‐reactive protein (CRP), leukocytes and fibrinogen, as well as with tissue factor (TF)‐dependent procoagulant changes in thrombin generation assays. When longer windows of past exposure were considered, up to 1 year preceding blood sampling, procoagulant changes were evident from the strongly increased numbers of red blood cell‐derived circulating microvesicles and annexin V‐binding microvesicles, but they no longer associated with TF. Past PM exposure was never associated with activated partial thromboplastin time (aPTT), prothrombin time (PT), or factor (F) VII, FVIII, FXII or D‐dimers. Residential distance to a major road was only marginally correlated with procoagulant changes in FVIII and thrombin generation. Conclusions: Increases in the number of microvesicles and in their procoagulant properties, rather than increases in coagulation factors per se, seem to contribute to the risk of VTE, developing during prolonged exposure to air pollutants.

Circulating apoptotic endothelial cells and apoptotic endothelial microparticles independently predict the presence of cardiac allograft vasculopathy.

OBJECTIVES Maintenance of endothelial homeostasis may prevent the development of cardiac allograft vasculopathy (CAV). This study investigated whether biomarkers related to endothelial injury and endothelial repair discriminate between CAV-negative and CAV-positive heart transplant recipients. BACKGROUND CAV is the most important determinant of cardiac allograft survival and a major cause of death after heart transplantation. METHODS Fifty-two patients undergoing coronary angiography between 5 and 15 years after heart transplantation were recruited in this study. Flow cytometry was applied to quantify endothelial progenitor cells (EPCs), circulating endothelial cells (CECs), and endothelial microparticles. Cell culture was used for quantification of circulating EPC number and hematopoietic progenitor cell number and for analysis of EPC function. RESULTS The EPC number and function did not differ between CAV-negative and CAV-positive patients. In univariable models, age, creatinine, steroid dose, granulocyte colony-forming units, apoptotic CECs, and apoptotic endothelial microparticles discriminated between CAV-positive and CAV-negative patients. The logistic regression model containing apoptotic CECs and apoptotic endothelial microparticles as independent predictors provided high discrimination between CAV-positive and CAV-negative patients (C-statistic 0.812; 95% confidence interval: 0.692 to 0.932). In a logistic regression model with age and creatinine as covariates, apoptotic CECs (p = 0.0112) and apoptotic endothelial microparticles (p = 0.0141) were independent predictors (C-statistic 0.855; 95% confidence interval: 0.756 to 0.953). These 2 biomarkers remained independent predictors when steroid dose was introduced in the model. CONCLUSIONS The high discriminative ability of apoptotic CECs and apoptotic endothelial microparticles is a solid foundation for the development of clinical prediction models of CAV.

Short-term exposure to particulate matter induces arterial but not venous thrombosis in healthy mice

Summary.  Background:  Epidemiological findings suggest an association between exposure to particulate matter (PM) and venous thrombo‐embolism. Objectives:  To investigate arterial vs. venous thrombosis, inflammation and coagulation in mice, (sub)acutely exposed to two types of PM. Methods:  Various doses (25, 100 and 200 μg per animal) of urban particulate matter (UPM) or diesel exhaust particles (DEP) were intratracheally (i.t.) instilled in C57Bl6/n mice and several endpoints measured at 4, 10 and 24 h. Mice were also repeatedly exposed to 100 μg per animal on three consecutive days with endpoints measured 24 h after the last instillation. Results:  Exposure to 200 μg per mouse UPM enhanced arterial thrombosis, but neither UPM nor DEP significantly enhanced venous thrombosis. Both types of PM induced dose‐dependent increases in broncho‐alveolar lavage fluid (BALF) total cell numbers (mainly neutrophils) and cytokines (IL‐6, KC, MCP‐1, RANTES, MIP‐1α), with peaks at 4 h and overall higher values for UPM than for DEP. Systemic inflammation was limited to increased serum IL‐6 levels, 4 h after UPM. Both types of PM induced similar and dose‐dependent but modest increases in factor (F)VII, FVIII and fibrinogen. Three repeated instillations did not or only modestly enhance the proinflammatory and procoagulant status. Conclusions:  Compared with DEP, UPM induced more pronounced pulmonary inflammation, but both particle types triggered similar and mild short‐term systemic effects. Hence, acute exposure to PM triggers activation of primary hemostasis in the mouse, but no substantial secondary hemostasis activation, resulting in arterial but not venous thrombogenicity.

Changed gene expression in brains of mice exposed to traffic in a highway tunnel.

Context: Air pollution has been suggested to have an impact on the brain. Objective: The objective was to assess the expression of inflammation-related genes in the brains of mice that had been exposed for 5 days to a well-characterized traffic-polluted environment, i.e. a highway tunnel. Materials and methods: Twenty C57BL6 mice were randomly allocated to four groups of five animals. Two groups were placed in the tunnel for 5 days (mean PM 2.5, 55.1 μg/m3, mean elemental carbon, EC 13.9 μg/m3) in cages with or without filter, two control groups were housed outside the tunnel. Animals were assessed within 24 hours after the last exposure day. Lung injury and inflammation were assessed by bronchoalveolar lavage (BAL) and histology. Blood leukocytosis and coagulation parameters were determined in peripheral blood. The olfactory bulb and hippocampus were analyzed for changes in expression of inflammatory genes and brain-derived neurotrophic factor (BDNF). Results and discussion: Although carbon particles were abundant in alveolar macrophages of exposed mice and absent in non-exposed mice, there was no evidence of pulmonary or systemic inflammation. There was an increased expression of genes involved in inflammatory response (COX2, NOS2, NOS3, and NFE2L2) in the hippocampus of the exposed mice. In the olfactory bulb, a downregulation was found for IL1α, COX2, NFE2L2, IL6, and BDNF. Conclusion: Although this short-term exposure to traffic-related pollution did not induce pulmonary or systemic inflammation, the expression of inflammatory genes was affected in different brain areas. The decreased BDNF expression in the olfactory bulb suggests lower brain neurotrophic support in response to traffic-related air pollution.

Analysis of phenotype and outcome in essential thrombocythemia with CALR or JAK2 mutations

The JAK2 V617F mutation, the thrombopoietin receptor MPL W515K/L mutation and calreticulin (CALR) mutations are mutually exclusive in essential thrombocythemia and support a novel molecular categorization of essential thrombocythemia. CALR mutations account for approximately 30% of cases of essential thrombocythemia. In a retrospective study, we examined the frequency of MPL and CALR mutations in JAK2 V617F-negative cases of essential thrombocythemia (n=103). In addition, we compared the clinical phenotype and outcome of CALR mutant cases of essential thrombocythemia with a cohort of JAK2 V617F-positive essential thrombocythemia (n=57). CALR-positive cases represented 63.7% of double-negative cases of essential thrombocythemia, and most carried CALR type 1 or type 2 indels. However, we also identified one patient who was positive for both the JAK2 V617F and the CALR mutations. This study revealed that CALR mutant essential thrombocythemia is associated with younger age, higher platelet counts, lower erythrocyte counts, leukocyte counts, hemoglobin, and hematocrit, and increased risk of progression to myelofibrosis in comparison with JAK2 V617F-positive essential thrombocythemia. Analysis of the CALR mutant group according to indel type showed that CALR type 1 deletion is strongly associated with male gender. CALR mutant patients had a better overall survival than JAK2 V617F-positive patients, in particular patients of age 60 years or younger. In conclusion, this study in a Belgian cohort of patients supports and extends the growing body of evidence that CALR mutant cases of essential thrombocythemia are phenotypically distinct from JAK2 V617F-positive cases, with regards to clinical and hematologic presentation as well as overall survival.

The effect of air pollution on haemostasis

Ambient environmental air pollutants include gaseous and particulate components. In polluted air, especially particulate matter seems responsible for cardiovascular complications: It consists of a heterogeneous mixture of solid and liquid particles with different diameters ranging from large thoracic to ultrafine particles, with a diameter <100 nm. Ultrafines can penetrate deeply into the lung to deposit in the alveoli. Cardiovascular manifestations result both from short-term and long-term exposure and have been linked to interference with the autonomic nervous system, direct translocation into the systemic circulation, pulmonary inflammation and oxidative stress. Thrombotic complications associated with air pollution comprise arterial and probably venous thrombogenicity. This review describes the existing epidemiological and experimental evidence to explain the rapid induction of myocardial infarction within 1-2 hours after exposure to polluted air and advances several explanations as to why more chronic exposure will lead to enhanced venous thrombogenicity. Mechanisms such as platelet activation, endothelial dysfunction, coagulation factor changes and microvesicle production are discussed.

Progression of the Prothrombotic State in Aging Bmal1-Deficient Mice

Objective—The goal of this study was to examine the functional relationship between aging endothelium and thrombogenicity in a mouse model of premature aging. Methods and Results—Coagulation tests and factors, blood cell counts, aorta endothelial function, aorta gene expression, and FeCl3-induced thrombosis in mesenteric blood vessels were analyzed in 10- to 30-week-old brain and muscle ARNT-like protein-1 (Bmal1)–deficient (knockout [KO]) mice and wild-type littermates. Ten-week-old KO mice manifested shortened prothrombin times (9.7 versus 11.3 seconds in wild-type) and elevated plasma fibrinogen (264 versus 172 mg/dL). At 30 weeks, factor VII (198% versus 149%), and platelet counts (2049 versus 1354 K/&mgr;L) were increased in KO mice. Gene deficiency reduced the vasoactive nitric oxide production at 10 and 30 weeks and tended to reduce and increase the protein expression of thrombomodulin and von Willebrand factor, respectively, with aging. Shortened venular and arteriolar occlusion times on FeCl3-induced injury in 10-week-old KO mice confirmed higher thrombogenicity, culminating in priapism, observed in 60% of 25- to 30-week-old KO males. Conclusion—Endothelial dysfunction and a hypercoagulable state cause early arterial and venous thrombogenicity in Bmal1 KO mice. With aging, progressive endothelial dysfunction, rising platelet counts, and high factor VII further enhance thrombogenicity, provoking priapism.