Jennifer Raftis

Increased platelet activation in patients with stable and acute exacerbation of COPD

Rationale Chronic obstructive pulmonary disease (COPD) is associated with systemic inflammation and cardiovascular disease. Interaction between inflammatory cells and activated platelets is important in the pathogenesis of atherothrombosis and may contribute to cardiovascular risk in patients with COPD. Objectives To assess platelet-monocyte aggregation in patients with COPD and matched controls, and in patients with an acute exacerbation of COPD. Methods 18 men with COPD and 16 male controls matched for age and cigarette smoke exposure were recruited. A further 12 patients were investigated during and at least 2 weeks after hospitalisation for an acute exacerbation. Platelet-monocyte aggregation and platelet P-selectin expression were determined using flow cytometry. Results Patients with COPD had increased circulating platelet-monocyte aggregates compared with controls (mean (SD) 25.3 (8.3)% vs 19.5 (4.0)%, p=0.01). Platelet-monocyte aggregation was further increased during an acute exacerbation compared with convalescence (32.0 (11.0)% vs 25.5 (6.4)%, p=0.03). Platelet P-selectin expression and soluble P-selectin did not differ between groups. Conclusions Patients with stable COPD have increased circulating platelet-monocyte aggregates compared with well-matched controls. Platelet activation is further increased in patients with COPD during an acute exacerbation. These findings identify a novel mechanism to explain the increased cardiovascular risk in COPD and suggest platelet inhibition as a plausible therapeutic target.

Nanoparticles and the cardiovascular system: a critical review

Nanoparticles (NPs) are tiny particles with a diameter of less than 100 nm. Traffic exhaust is a major source of combustion-derived NPs (CDNPs), which represent a significant component in urban air pollution. Epidemiological, panel and controlled human chamber studies clearly demonstrate that exposure to CDNPs is associated with multiple adverse cardiovascular effects in both healthy individuals and those with pre-existing cardiovascular disease. NPs are also manufactured from a large range of materials for industrial use in a vast array of products including for use as novel imaging agents for medical use. There is currently little information available on the impacts of manufactured NPs in humans, but experimental studies demonstrate similarities to the detrimental cardiovascular actions of CDNPs. This review describes the evidence for these cardiovascular effects and attempts to resolve the paradox between the adverse effects of the unintentional exposure of CDNPs and the intentional delivery of manufactured NPs for medical purposes.

Inhaled Nanoparticles Accumulate at Sites of Vascular Disease

The development of engineered nanomaterials is growing exponentially, despite concerns over their potential similarities to environmental nanoparticles that are associated with significant cardiorespiratory morbidity and mortality. The mechanisms through which inhalation of nanoparticles could trigger acute cardiovascular events are emerging, but a fundamental unanswered question remains: Do inhaled nanoparticles translocate from the lung in man and directly contribute to the pathogenesis of cardiovascular disease? In complementary clinical and experimental studies, we used gold nanoparticles to evaluate particle translocation, permitting detection by high-resolution inductively coupled mass spectrometry and Raman microscopy. Healthy volunteers were exposed to nanoparticles by acute inhalation, followed by repeated sampling of blood and urine. Gold was detected in the blood and urine within 15 min to 24 h after exposure, and was still present 3 months after exposure. Levels were greater following inhalation of 5 nm (primary diameter) particles compared to 30 nm particles. Studies in mice demonstrated the accumulation in the blood and liver following pulmonary exposure to a broader size range of gold nanoparticles (2-200 nm primary diameter), with translocation markedly greater for particles <10 nm diameter. Gold nanoparticles preferentially accumulated in inflammation-rich vascular lesions of fat-fed apolipoproteinE-deficient mice. Furthermore, following inhalation, gold particles could be detected in surgical specimens of carotid artery disease from patients at risk of stroke. Translocation of inhaled nanoparticles into the systemic circulation and accumulation at sites of vascular inflammation provides a direct mechanism that can explain the link between environmental nanoparticles and cardiovascular disease and has major implications for risk management in the use of engineered nanomaterials.

The influence of the menstrual cycle, normal pregnancy and pre-eclampsia on platelet activation

Platelet activation has a key role in mediating thrombotic and inflammatory events. This study aimed to determine the influence of the menstrual cycle, pregnancy and pre-eclampsia on in vivo platelet activation. Twelve healthy nulliparous, non-smoking women with regular menses were studied over a single menstrual cycle. Twenty-one healthy primigravida pregnant women were studied longitudinally at 16, 24, 32 and 37 weeks gestation and seven weeks post-partum. Sixteen primigravida women with pre-eclampsia were studied at time of diagnosis and at seven weeks post-partum. Platelet-monocyte aggregates and platelet-surface P-selectin expression were assessed by flow-cytometry. Soluble P-selectin and CD40 ligand (CD40L) were measured by ELISA. Markers of platelet activation did not vary over the menstrual cycle. Platelet-monocyte aggregates were greater in the third trimester of pregnancy compared to non-pregnant women (p=0.003). Platelet surface and plasma soluble P-selectin concentrations increased with gestation (p<0.0001) and were raised by 24 weeks of pregnancy compared to non-pregnant women (p< or =0.02 for both) and together with platelet monocyte aggregates, decreased post-partum (p< or =0.02). Soluble CD40L concentrations fell in pregnancy, reaching a nadir at mid-gestation (p=0.002). There were no differences in markers of platelet activation between normal and pre-eclamptic pregnancies. In conclusion, platelet activation is increased in pregnancy and increases with gestation but is unaffected by pre-eclampsia. This suggests that systemic platelet activation is a feature of pregnancy but this is not affected by established pre-eclampsia.

Determination of Adsorption Affinity of Nanoparticles for Interleukin-8 Secreted From A549 Cells by In Vitro Cell-Free and Cell-Based Assays

The evaluation of the potential of nanoparticles (NP) for adsorbing biomolecules and use of control approaches are important for accurate presentation of in vitro analytical data. In this study, seven types of NP including carbon black (CB), cerium dioxide (CeO2), copper oxide (CuO), indium trioxide (In2O3), nickel oxide (NiO), silicon dioxide (SiO2), and titanium dioxide (TiO2) were used for determining the adsorption ability of interleukin-8 (IL-8) under either (1) a cell-free condition where NP were incubated with supernatant of A549 cells, or (2) a cell-based condition, where cells were treated with NP. Under the cell-free condition, CB and TiO2 NP showed a high adsorption affinity for IL-8 in supernatants of both lipopolysaccharide (LPS)-stimulated and unstimulated A549 cells. In contrast, SiO2 and In2O3 NP displayed a relatively low adsorption affinity. Further, IL-8 adsorption was markedly reduced when NP were predispersed in fetal bovine serum. The results obtained under cell-based conditions using both stimulated and unstimulated cells were consistent with those of the cell-free condition. Data indicate that adsorption of IL-8 onto NP surface is variable depending on type of NP, preparation method of NP, and cellular inflammatory state. Thus, the cell-free adsorption assay may be utilized for reliable interpretation of data produced by in vitro cell-based methodology.

The in vitro respiratory toxicity of cristobalite-bearing volcanic ash

Ash from dome-forming volcanoes poses a unique hazard to millions of people worldwide due to an abundance of respirable cristobalite, a crystalline silica polymorph. Crystalline silica is an established respiratory hazard in other mixed dusts, but its toxicity strongly depends on sample provenance. Previous studies suggest that cristobalite-bearing volcanic ash is not as bio-reactive as may be expected for a dust containing crystalline silica. We systematically address the hazard posed by volcanic cristobalite by analysing a range of dome-related ash samples, and interpret the crystalline silica hazard according to the mineralogical nature of volcanic cristobalite. Samples are sourced from five well-characterized dome-forming volcanoes that span a range of magmatic compositions, specifically selecting samples rich in cristobalite (up to 16wt%). Isolated respirable fractions are used to investigate the in vitro response of THP-1 macrophages and A549 type II epithelial cells in cytotoxicity, cellular stress, and pro-inflammatory assays associated with crystalline silica toxicity. Dome-related ash is minimally reactive in vitro for a range of source compositions and cristobalite contents. Cristobalite-based toxicity is not evident in the assays employed, supporting the notion that crystalline silica provenance influences reactivity. Macrophages experienced minimal ash-induced cytotoxicity and intracellular reduction of glutathione; however, production of IL-1β, IL-6 and IL-8 were sample-dependent. Lung epithelial cells experienced moderate apoptosis, sample-dependent reduction of glutathione, and minimal cytokine production. We suggest that protracted interaction between particles and epithelial cells may never arise due to effective clearance by macrophages. However, volcanic ash has the propensity to incite a low, but significant, and sample-dependent response; the effect of this response in vivo is unknown and prolonged exposure may yet pose a hazard.

Fire Simulation and Cardiovascular Health in Firefighters

Background: Rates of myocardial infarction in firefighters are increased during fire suppression duties, and are likely to reflect a combination of factors including extreme physical exertion and heat exposure. We assessed the effects of simulated fire suppression on measures of cardiovascular health in healthy firefighters. Methods: In an open-label randomized crossover study, 19 healthy firefighters (age, 41±7 years; 16 males) performed a standardized training exercise in a fire simulation facility or light duties for 20 minutes. After each exposure, ex vivo thrombus formation, fibrinolysis, platelet activation, and forearm blood flow in response to intra-arterial infusions of endothelial-dependent and -independent vasodilators were measured. Results: After fire simulation training, core temperature increased (1.0±0.1°C) and weight reduced (0.46±0.14 kg, P<0.001 for both). In comparison with control, exposure to fire simulation increased thrombus formation under low-shear (73±14%) and high-shear (66±14%) conditions (P<0.001 for both) and increased platelet-monocyte binding (7±10%, P=0.03). There was a dose-dependent increase in forearm blood flow with all vasodilators (P<0.001), which was attenuated by fire simulation in response to acetylcholine (P=0.01) and sodium nitroprusside (P=0.004). This was associated with a rise in fibrinolytic capacity, asymptomatic myocardial ischemia, and an increase in plasma cardiac troponin I concentrations (1.4 [0.8–2.5] versus 3.0 [1.7–6.4] ng/L, P=0.010). Conclusions: Exposure to extreme heat and physical exertion during fire suppression activates platelets, increases thrombus formation, impairs vascular function, and promotes myocardial ischemia and injury in healthy firefighters. Our findings provide pathogenic mechanisms to explain the association between fire suppression activity and acute myocardial infarction in firefighters. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01812317.

PAR4 (Protease-Activated Receptor 4) Antagonism With BMS-986120 Inhibits Human Ex Vivo Thrombus Formation

Objective— BMS-986120 is a novel first-in-class oral PAR4 (protease-activated receptor 4) antagonist with potent and selective antiplatelet effects. We sought to determine for the first time, the effect of BMS-986120 on human ex vivo thrombus formation. Approach and Results— Forty healthy volunteers completed a phase 1 parallel-group PROBE trial (Prospective Randomized Open-Label Blinded End Point). Ex vivo platelet activation, platelet aggregation, and thrombus formation were measured at 0, 2, and 24 hours after (1) oral BMS-986120 (60 mg) or (2) oral aspirin (600 mg) followed at 18 hours with oral aspirin (600 mg) and oral clopidogrel (600 mg). BMS-986120 demonstrated highly selective and reversible inhibition of PAR4 agonist peptide (100 &mgr;M)-stimulated P-selectin expression, platelet-monocyte aggregates, and platelet aggregation (P<0.001 for all). Compared with pretreatment, total thrombus area (&mgr;m2/mm) at high shear was reduced by 29.2% (95% confidence interval, 18.3%–38.7%; P<0.001) at 2 hours and by 21.4% (9.3%–32.0%; P=0.002) at 24 hours. Reductions in thrombus formation were driven by a decrease in platelet-rich thrombus deposition: 34.8% (19.3%–47.3%; P<0.001) at 2 hours and 23.3% (5.1%–38.0%; P=0.016) at 24 hours. In contrast to aspirin alone, or in combination with clopidogrel, BMS-986120 had no effect on thrombus formation at low shear (P=nonsignificant). BMS-986120 administration was not associated with an increase in coagulation times or serious adverse events. Conclusions— BMS-986120 is a highly selective and reversible oral PAR4 antagonist that substantially reduces platelet-rich thrombus formation under conditions of high shear stress. Our results suggest PAR4 antagonism has major potential as a therapeutic antiplatelet strategy. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT02439190.

Lyophilised reconstituted human platelets increase thrombus formation in a clinical ex vivo model of deep arterial injury

Platelets are the principal component of the innate haemostatic system that protect from traumatic bleeding. We investigated whether lyophilised human platelets (LHPs) could enhance clot formation within platelet-free and whole blood environments using an ex vivo model of deep arterial injury. Lyophilised human platelets were produced from stored human platelets and characterised using conventional, fluorescent and electron microscopic techniques. LHPs were resuspended in platelet-free plasma (PFP) obtained from citrated whole human blood to form final concentrations of 0, 20 and 200 x 10⁹ LHPs/L. LHPs with recalcified PFP or whole blood were perfused through the chamber at low (212 s⁻¹) and high (1,690 s⁻¹) shear rates with porcine aortic tunica media as thrombogenic substrate. LHPs shared morphological characteristics with native human platelets and were incorporated into clot generated from PFP or whole blood. Histomorphometrically measured mean thrombus area increased in a dose-dependent manner following the addition of LHPs to PFP under conditions of high shear [704 μm² ± 186 μm² (mean ± SEM), 1,511 μm² ± 320 μm² and 2,378 μm² ± 315 μm², for LHPs at 0, 20 and 200 x 10⁹ /l, respectively (p= 0.012)]. Lyophilised human platelets retain haemostatic properties when reconstituted in both PFP and whole blood, and enhance thrombus formation in a model of deep arterial injury. These data suggest that LHPs have the potential to serve as a therapeutic intervention during haemorrhage under circumstances of trauma, and platelet depletion or dysfunction.

Ferumoxytol-enhanced magnetic resonance imaging assessing inflammation after myocardial infarction

Objectives Macrophages play a central role in the cellular inflammatory response to myocardial infarction (MI) and predict subsequent clinical outcomes. We aimed to assess temporal changes in cellular inflammation and tissue oedema in patients with acute MI using ultrasmallsuperparamagnetic particles of iron oxide (USPIO)-enhanced MRI. Methods Thirty-one patients were recruited following acute MI and followed up for 3 months with repeated T2 and USPIO-enhanced T2*-mapping MRI. Regions of interest were categorised into infarct, peri-infarct and remote myocardial zones, and compared with control tissues. Results Following a single dose, USPIO enhancement was detected in the myocardium until 24 hours (p<0.0001). Histology confirmed colocalisation of iron and macrophages within the infarcted, but not the non-infarcted, myocardium. Following repeated doses, USPIO uptake in the infarct zone peaked at days 2–3, and greater USPIO uptake was detected in the infarct zone compared with remote myocardium until days 10–16 (p<0.05). In contrast, T2-defined myocardial oedema peaked at days 3–9 and remained increased in the infarct zone throughout the 3-month follow-up period (p<0.01). Conclusion Myocardial macrophage activity can be detected using USPIO-enhanced MRI in the first 2 weeks following acute MI. This observed pattern of cellular inflammation is distinct, and provides complementary information to the more prolonged myocardial oedema detectable using T2 mapping. This imaging technique holds promise as a non-invasive method of assessing and monitoring myocardial cellular inflammation with potential application to diagnosis, risk stratification and assessment of novel anti-inflammatory therapeutic interventions. Trial registration number Trial registration number: 14663. Registered on UK Clinical Research Network (http://public.ukcrn.org.uk) and also ClinicalTrials.gov (https://clinicaltrials.gov/ct2/show/NCT02319278?term=DECIFER&rank=2).