PAR4 Antagonism in Patients With Coronary Artery Disease Receiving Antiplatelet Therapies.

BACKGROUND: BMS-986141 is a novel potent highly selective antagonist of PAR (protease-activated receptor) type 4. PAR4 antagonism has been demonstrated to reduce thrombus formation in isolation and in combination with factor Xa inhibition in high shear conditions in healthy people. We sought to determine whether PAR4 antagonism had additive antithrombotic effects in patients with coronary artery disease who were receiving antiplatelet therapy. METHODS: Forty-five patients with stable coronary heart disease and 10 healthy volunteers completed a phase 2a open-label 4-arm single-center study. Patients were allocated to 1 of 3 treatment arms for 7 days: (1) ticagrelor (90 mg BID), (2) aspirin (75 mg QD), or (3) the combination of ticagrelor and aspirin. Agonist-induced platelet aggregation, platelet activation, and ex vivo thrombus formation were measured before and 2 and 24 hours after a single oral 4-mg dose of BMS-986141 on the first study visit day in all participants. RESULTS: BMS-986141 demonstrated highly selective inhibition of PAR4-AP (agonist peptide)-induced platelet aggregation, P-selectin expression, and platelet-monocyte aggregate expression (P≤0.001 for all), which were unaffected by concomitant antiplatelet therapies. PAR4 antagonism reduced ex vivo thrombus area in high shear conditions in healthy volunteers (-21%; P=0.001) and in patients receiving ticagrelor alone (-28%; P=0.001), aspirin alone (-23%; P=0.018), or both in combination (-24%; P≤0.001). Plasma concentration of BMS-986141 correlated with PAR4-AP-induced platelet responses (P≤0.001 for all) and total thrombus area under high shear stress conditions (P≤0.01 for all). CONCLUSIONS: PAR4 antagonism has additive antithrombotic effects when used in addition to ticagrelor, aspirin, or their combination, in patients with stable coronary heart disease. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT05093790.

New insights into the association of air pollution and kidney diseases by tracing gold nanoparticles with inductively coupled plasma mass spectrometry.

Exposure to particles from air pollution has been associated with kidney disease; however, the underlying biological mechanisms are incompletely understood. Inhaled particles can gain access to the circulation and, depending on their size, pass into urine, raising the possibility that particles may also sequester in the kidney and directly alter renal function. This study optimised an inductively coupled plasma mass spectrometry (ICP-MS) method to investigate the size dependency of particle accumulation in the kidneys of mice following pulmonary instillation (0.8 mg in total over 4 weeks) to gold nanoparticles (2, 3-4, 7-8, 14 or 40 nm or saline control). Due to the smallest particle sizes being below the limit of detection in single particle mode, ICP-MS was operated in total quantification mode. Gold was detected in all matrices of interest (blood, urine and kidney) from animals treated with all sizes of gold nanoparticles, at orders of magnitude higher than the methodological limit of detection in biological matrices (0.013 ng/mL). A size-dependent effect was observed, with smaller particles leading to greater levels of accumulation in tissues. This study highlights the value of a robust and reliable method by ICP-MS to detect extremely low levels of gold in biological samples for indirect particle tracing. The finding that nano-sized particles translocate from the lung to the kidney may provide a biological explanation for the associations between air pollution and kidney disease.

Exposure to Elevated Nitrogen Dioxide Concentrations and Cardiac Remodeling in Patients With Dilated Cardiomyopathy.

BACKGROUND: Empirical evidence suggests a strong link between exposure to air pollution and heart failure incidence, hospitalizations, and mortality, but the biological basis of this remains unclear. We sought to determine the relationship between differential air pollution levels and changes in cardiac structure and function in patients with dilated cardiomyopathy. METHODS AND RESULTS: We undertook a prospective longitudinal observational cohort study of patients in England with dilated cardiomyopathy (enrollment 2009-2015, n = 716, 66% male, 85% Caucasian) and conducted cross sectional analysis at the time of study enrollment. Annual average air pollution exposure estimates for nitrogen dioxide (NO2) and particulate matter with diameter of 2.5 µm or less (PM2.5) at enrolment were assigned to each residential postcode (on average 12 households). The relationship between air pollution and cardiac morphology was assessed using linear regression modelling. Greater ambient exposure to NO2 was associated with higher indexed left ventricular (LV) mass (4.3 g/m2 increase per interquartile range increase in NO2, 95% confidence interval 1.9-7.0 g/m2) and lower LV ejection fraction (-1.5% decrease per interquartile range increase in NO2, 95% confidence interval -2.7% to -0.2%), independent of age, sex, socioeconomic status, and clinical covariates. The associations were robust to adjustment for smoking status and geographical clustering by postcode area. The effect of air pollution on LV mass was greatest in women. These effects were specific to NO2 exposure. CONCLUSIONS: Exposure to air pollution is associated with raised LV mass and lower LV ejection fraction, with the strongest effect in women. Although epidemiological associations between air pollution and heart failure have been established and supported by preclinical studies, our findings provide novel empirical evidence of cardiac remodeling and exposure to air pollution with important clinical and public health implications.

A Review of Road Traffic-Derived Non-Exhaust Particles: Emissions, Physicochemical Characteristics, Health Risks, and Mitigation Measures.

Implementation of regulatory standards has reduced exhaust emissions of particulate matter from road traffic substantially in the developed world. However, nonexhaust particle emissions arising from the wear of brakes, tires, and the road surface, together with the resuspension of road dust, are unregulated and exceed exhaust emissions in many jurisdictions. While knowledge of the sources of nonexhaust particles is fairly good, source-specific measurements of airborne concentrations are few, and studies of the toxicology and epidemiology do not give a clear picture of the health risk posed. This paper reviews the current state of knowledge, with a strong focus on health-related research, highlighting areas where further research is an essential prerequisite for developing focused policy responses to nonexhaust particles.

Nitrate and nitrite contamination in drinking water and cancer risk: A systematic review with meta-analysis.

BACKGROUND: Pollution of water sources, largely from wide-scale agricultural fertilizer use has resulted in nitrate and nitrite contamination of drinking water. The effects on human health of raised nitrate and nitrite levels in drinking water are currently unclear. OBJECTIVES: We conducted a systematic review of peer-reviewed literature on the association of nitrate and nitrite in drinking water with human health with a specific focus on cancer. METHODS: We searched eight databases from 1 January 1990 until 28 February 2021. Meta-analyses were conducted when studies had the same exposure metric and outcome. RESULTS: Of 9835 studies identified in the literature search, we found 111 studies reporting health outcomes, 60 of which reported cancer outcomes (38 case-control studies; 12 cohort studies; 10 other study designs). Most studies were set in the USA (24), Europe (20) and Taiwan (14), with only 3 studies from low and middle-income countries. Nitrate exposure in water (59 studies) was more commonly investigated than nitrite exposure (4 studies). Colorectal (15 studies) and gastric (13 studies) cancers were the most reported. In meta-analyses (4 studies) we identified a positive association of nitrate exposure with gastric cancer, OR = 1.91 (95%CI = 1.09-3.33) per 10 mg/L increment in nitrate ion. We found no association of nitrate exposure with colorectal cancer (10 studies; OR = 1.02 [95%CI = 0.96-1.08]) or cancers at any other site. CONCLUSIONS: We identified an association of nitrate in drinking water with gastric cancer but with no other cancer site. There is currently a paucity of robust studies from settings with high levels nitrate pollution in drinking water. Research into this area will be valuable to ascertain the true health burden of nitrate contamination of water and the need for public policies to protect human health.

Acute cardiovascular effects of controlled exposure to dilute Petrodiesel and biodiesel exhaust in healthy volunteers: a crossover study.

BACKGROUND: Air pollution derived from combustion is associated with considerable cardiorespiratory morbidity and mortality in addition to environmental effects. Replacing petrodiesel with biodiesel may have ecological benefits, but impacts on human health remain unquantified. The objective was to compare acute cardiovascular effects of blended and pure biodiesel exhaust exposure against known adverse effects of petrodiesel exhaust (PDE) exposure in human subjects. In two randomized controlled double-blind crossover studies, healthy volunteers were exposed to PDE or biodiesel exhaust for one hour. In study one, 16 subjects were exposed, on separate occasions, to PDE and 30% rapeseed methyl ester biodiesel blend (RME30) exhaust, aiming at PM10 300 µg/m3. In study two, 19 male subjects were separately exposed to PDE and exhaust from a 100% RME fuel (RME100) using similar engine load and exhaust dilution. Generated exhaust was analyzed for physicochemical composition and oxidative potential. Following exposure, vascular endothelial function was assessed using forearm venous occlusion plethysmography and ex vivo thrombus formation was assessed using a Badimon chamber model of acute arterial injury. Biomarkers of inflammation, platelet activation and fibrinolysis were measured in the blood. RESULTS: In study 1, PDE and RME30 exposures were at comparable PM levels (314 ± 27 µg/m3; (PM10 ± SD) and 309 ± 30 µg/m3 respectively), whereas in study 2, the PDE exposure concentrations remained similar (310 ± 34 µg/m3), but RME100 levels were lower in PM (165 ± 16 µg/m3) and PAHs, but higher in particle number concentration. Compared to PDE, PM from RME had less oxidative potential. Forearm infusion of the vasodilators acetylcholine, bradykinin, sodium nitroprusside and verapamil resulted in dose-dependent increases in blood flow after all exposures. Vasodilatation and ex vivo thrombus formation were similar following exposure to exhaust from petrodiesel and the two biodiesel formulations (RME30 and RME100). There were no significant differences in blood biomarkers or exhaled nitric oxide levels between exposures. CONCLUSIONS: Despite differences in PM composition and particle reactivity, controlled exposure to biodiesel exhaust was associated with similar cardiovascular effects to PDE. We suggest that the potential adverse health effects of biodiesel fuel emissions should be taken into account when evaluating future fuel policies. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01337882 /NCT01883466. Date of first enrollment March 11, 2011, registered April 19, 2011, i.e. retrospectively registered.

Nanotoxicology: The Need for a Human Touch?

With the ever-expanding number of manufactured nanomaterials (MNMs) under development there is a vital need for nanotoxicology studies that test the potential for MNMs to cause harm to health. An extensive body of work in cell cultures and animal models is vital to understanding the physicochemical characteristics of MNMs and the biological mechanisms that underlie any detrimental actions to cells and organs. In human subjects, exposure monitoring is combined with measurement of selected health parameters in small panel studies, especially in occupational settings. However, the availability of further in vivo human data would greatly assist the risk assessment of MNMs. Here, the potential for controlled inhalation exposures of MNMs in human subjects is discussed. Controlled exposures to carbon, gold, aluminum, and zinc nanoparticles in humans have already set a precedence to demonstrate the feasibility of this approach. These studies have provided considerable insight into the potential (or not) of nanoparticles to induce inflammation, alter lung function, affect the vasculature, reach the systemic circulation, and accumulate in other organs. The need for further controlled exposures of MNMs in human volunteers - to establish no-effect limits, biological mechanisms, and provide vital data for the risk assessment of MNMs - is advocated.

Identification of potential markers for internal exposure to ambient ozone in oral cavity of healthy adults.

BACKGROUND: Ozone is a highly oxidative gaseous pollutant associated with adverse health outcomes, but markers for internal exposure to ambient ozone are not well-established. METHODS: We aimed to evaluate the feasibility and suitability of the markers in oral microbiome for ambient ozone exposure. Between March and May in 2018, 97 healthy adults were examined on 2 or 3 occasions for oral swab sampling. Hourly concentrations of ambient ozone 1-7 days preceding sampling were collected. Mixed-effect models were fitted to examine the associations between ambient ozone and the diversity and taxon abundances of oral microbiome. Receiver operating characteristic (ROC) curves estimated the accuracies of markers to delineate between samples exposed to different concentrations of ambient ozone. The associations between the makers and lung function were further examined by linear mixed effect models. RESULTS: The averages of daily mean concentrations of ambient ozone (O3-daily), maximum 8-h means (O3-8hmax) and 1-h maximums (O3-1hmax) were respectively 72 µg/m³, 123 µg/m³ and 144 µg/m³. O3-daily was positively associated with α-diversity of oral microbiome, but the exposure-response curves only yielded positive associations in the range of O3-daily from 60 µg/m³ to 75 µg/m³. Results of O3-8hmax and O3-1hmax were consistent with these of O3-daily. With an interquartile range increase in O3-daily at lag04, the abundance of Proteobacteria decreased by 3.1% (95% CI: -4.0%, -2.2%) and Firmicutes increased by 3.3% (95% CI: 2.3%, 4.3%), whilst the Proteobacteria:Firmicutes ratio (P/F) decreased by 0.9 (95% CI: -1.5, -0.4). The areas under ROC curves for Proteobacteria, Firmicutes and P/F were 0.8535, 0.7569 and 0.8929, respectively. Proteobacteria and P/F were associated with forced expiratory volume in the first second and fractional exhaled nitric oxide significantly. CONCLUSION: Ambient ozone disturbs oral microbial homeostasis. Proteobacteria, Firmicutes and their ratio may be potential markers for short-term ambient ozone exposure, and indicators of airway inflammation or lung function decline.

Silica nanoparticles trigger the vascular endothelial dysfunction and prethrombotic state via miR-451 directly regulating the IL6R signaling pathway.

BACKGROUND: Safety evaluation is a prerequisite for nanomaterials in a wide range of fields, including chemical industries, medicine or food sciences. Previously, we had demonstrated that SiNPs could trigger the thrombotic effects in vivo, but the underlying mechanisms remain unknown. This study was aimed to explore and verify the role of miR-451a on SiNPs-induced vascular endothelial dysfunction and pre-thrombotic state. RESULTS: The color doppler ultrasound results showed that SiNPs had the inhibitory effects on aorta velocity and cardiac output. The histological and ultrastructural analysis manifested that SiNPs could induce the vascular endothelial damage. In addition, the expression level of MDA was elevated while the activity of SOD and GSH-Px were decreased in aortic arch triggered by SiNPs, accompanied with the release of iNOS and decline of eNOS in blood serum. The immunohistochemistry results showed that the positive staining of TF and PECAM-1 were elevated in a dose-dependent manner induced by SiNPs. The activation of coagulation function occurred via shortened TT, PT and APTT while the FIB was elevated markedly induced by SiNPs. Coagulant factors (TF, FXa and vWF) and PLT numbers were increased whereas the levels of anticoagulant factors (ATIII, TFPI and t-PA) were decreased. Microarray analysis showed that the down-regulated miR-451a could target the gene expression of IL6R, which further activated the JAK/STAT signaling pathway triggered by SiNPs. Dual-luciferase reporter gene assay confirmed the directly target relationship between miR-451a and IL6R. Additionally, the chemical mimics of miR-451a led to attenuate the expression of IL6R/STAT/TF signaling pathway in vitro and in vivo induced by SiNPs, while the inhibitor of miR-451a enhanced the activation of IL6R/STAT/TF signaling pathway. CONCLUSIONS: In summary, SiNPs could accelerate the vascular endothelial dysfunction and prethrombotic state via miR-451a negative regulating the IL6R/STAT/TF signaling pathway.

Role of chemical composition and redox modification of poorly soluble nanomaterials on their ability to enhance allergic airway sensitisation in mice.

BACKGROUND: Engineered nanoparticles (NPs) have been shown to enhance allergic airways disease in mice. However, the influence of the different physicochemical properties of these particles on their adjuvant properties is largely unknown. Here we investigate the effects of chemical composition and redox activity of poorly soluble NPs on their adjuvant potency in a mouse model of airway hypersensitivity. RESULTS: NPs of roughly similar sizes with different chemical composition and redox activity, including CeO2, Zr-doped CeO2, Co3O4, Fe-doped Co3O4(using Fe2O3 or Fe3O4) and TiO2 NPs, all showed adjuvant activity. OVA induced immune responses following intranasal exposure of BALB/c mice to 0.02% OVA in combination with 200 µg NPs during sensitization (on day 1, 3, 6 and 8) and 0.5% OVA only during challenge (day 22, 23 and 24) were more pronounced compared to the same OVA treatment regime without NPs. Changes in OVA-specific IgE and IgG1 plasma levels, differential cell count and cytokines in bronchoalveolar lavage fluid (BALF), and histopathological detection of mucosa cell metaplasia and eosinophil density in the conducting airways were observed. Adjuvant activity of the CeO2 NPs was primarily mediated via the Th2 response, while that of the Co3O4 NPs was characterised by no or less marked increases in IgE plasma levels, BALF IL-4 and IL-5 concentrations and percentages of eosinophils in BALF and more pronounced increases in BALF IL-6 concentrations and percentages of lymphocytes in BALF. Co-exposure to Co3O4 NPs with OVA and subsequent OVA challenge also induced perivascular and peribronchiolar lymphoid cell accumulation and formation of ectopic lymphoid tissue in lungs. Responses to OVA combined with various NPs were not affected by the amount of doping or redox activity of the NPs. CONCLUSIONS: The findings indicate that chemical composition of NPs influences both the relative potency of NPs to exacerbate allergic airway sensitization and the type of immune response. However, no relation between the acellular redox activity and the observed adjuvant activity of the different NPs was found. Further research is needed to pinpoint the precise physiological properties of NPs and biological mechanisms determining adjuvant activity in order to facilitate a safe-by-design approach to NP development.

Pravastatin ameliorates placental vascular defects, fetal growth, and cardiac function in a model of glucocorticoid excess.

Fetoplacental glucocorticoid overexposure is a significant mechanism underlying fetal growth restriction and the programming of adverse health outcomes in the adult. Placental glucocorticoid inactivation by 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) plays a key role. We previously discovered that Hsd11b2(-/-) mice, lacking 11ß-HSD2, show marked underdevelopment of the placental vasculature. We now explore the consequences for fetal cardiovascular development and whether this is reversible. We studied Hsd11b2(+/+), Hsd11b2(+/-), and Hsd11b2(-/-) littermates from heterozygous (Hsd11b(+/-)) matings at embryonic day (E)14.5 and E17.5, where all three genotypes were present to control for maternal effects. Using high-resolution ultrasound, we found that umbilical vein blood velocity in Hsd11b2(-/-) fetuses did not undergo the normal gestational increase seen in Hsd11b2(+/+) littermates. Similarly, the resistance index in the umbilical artery did not show the normal gestational decline. Surprisingly, given that 11ß-HSD2 absence is predicted to initiate early maturation, the E/A wave ratio was reduced at E17.5 in Hsd11b2(-/-) fetuses, suggesting impaired cardiac function. Pravastatin administration from E6.5, which increases placental vascular endothelial growth factor A and, thus, vascularization, increased placental fetal capillary volume, ameliorated the aberrant umbilical cord velocity, normalized fetal weight, and improved the cardiac function of Hsd11b2(-/-) fetuses. This improved cardiac function occurred despite persisting indications of increased glucocorticoid exposure in the Hsd11b2(-/-) fetal heart. Thus, the pravastatin-induced enhancement of fetal capillaries within the placenta and the resultant hemodynamic changes correspond with restored fetal cardiac function. Statins may represent a useful therapeutic approach to intrauterine growth retardation due to placental vascular hypofunction.

Ambient air pollution and thrombosis.

Air pollution is a growing public health concern of global significance. Acute and chronic exposure is known to impair cardiovascular function, exacerbate disease and increase cardiovascular mortality. Several plausible biological mechanisms have been proposed for these associations, however, at present, the pathways are incomplete. A seminal review by the American Heart Association (2010) concluded that the thrombotic effects of particulate air pollution likely contributed to their effects on cardiovascular mortality and morbidity. The aim of the current review is to appraise the newly accumulated scientific evidence (2009-2016) on contribution of haemostasis and thrombosis towards cardiovascular disease induced by exposure to both particulate and gaseous pollutants.Seventy four publications were reviewed in-depth. The weight of evidence suggests that acute exposure to fine particulate matter (PM2.5) induces a shift in the haemostatic balance towards a pro-thrombotic/pro-coagulative state. Insufficient data was available to ascertain if a similar relationship exists for gaseous pollutants, and very few studies have addressed long-term exposure to ambient air pollution. Platelet activation, oxidative stress, interplay between interleukin-6 and tissue factor, all appear to be potentially important mechanisms in pollution-mediated thrombosis, together with an emerging role for circulating microvesicles and epigenetic changes.Overall, the recent literature supports, and arguably strengthens, the contention that air pollution contributes to cardiovascular morbidity by promoting haemostasis. The volume and diversity of the evidence highlights the complexity of the pathophysiologic mechanisms by which air pollution promotes thrombosis; multiple pathways are plausible and it is most likely they act in concert. Future research should address the role gaseous pollutants play in the cardiovascular effects of air pollution mixture and direct comparison of potentially susceptible groups to healthy individuals.

Assessing the bioactivity of crystalline silica in heated high-temperature insulation wools.

High-Temperature Insulation Wools (HTIW), such as alumino silicate wools (Refractory Ceramic Fibers) and Alkaline Earth Silicate wools, are used in high-temperature industries for thermal insulation. These materials have an amorphous glass-like structure. In some applications, exposure to high temperatures causes devitrification resulting in the formation of crystalline species including crystalline silica. The formation of this potentially carcinogenic material raises safety concerns regarding after-use handling and disposal. This study aims to determine whether cristobalite formed in HTIW is bioactive in vitro. Mouse macrophage (J774A.1) and human alveolar epithelial (A549) cell lines were exposed to pristine HTIW of different compositions, and corresponding heat-treated samples. Cell death, cytokine release, and reactive oxygen species (ROS) formation were assessed in both cell types. Cell responses to aluminum lactate-coated fibers were assessed to determine if responses were caused by crystalline silica. DQ12 α-quartz was used as positive control, and TiO2 as negative control. HTIW did not induce cell death or intracellular ROS, and their ability to induce pro-inflammatory mediator release was low. In contrast, DQ12 induced cytotoxicity, a strong pro-inflammatory response and ROS generation. The modest pro-inflammatory mediator responses of HTIW did not always coincide with the formation of cristobalite in heated fibers; therefore, we cannot confirm that devitrification of HTIW results in bioactive cristobalite in vitro. In conclusion, the biological responses to HTIW observed were not attributable to a single physicochemical characteristic; instead, a combination of physicochemical characteristics (cristobalite content, fiber chemistry, dimensions and material solubility) appear to contribute to induction of cellular responses.

Differences in the toxicity of cerium dioxide nanomaterials after inhalation can be explained by lung deposition, animal species and nanoforms.

Considerable differences in pulmonary responses have been observed in animals exposed to cerium dioxide nanoparticles via inhalation. These differences in pulmonary toxicity might be explained by differences in lung deposition, species susceptibility or physicochemical characteristics of the tested cerium dioxide nanoforms (i.e. same chemical substance, different size, shape, surface area or surface chemistry). In order to distinguish the relative importance of these different influencing factors, we performed a detailed analysis of the data from several inhalation studies with different exposure durations, species and nanoforms, namely published data on NM211 and NM212 (JRC repository), NanoAmor (commercially available) and our published and unpublished data on PROM (industry provided). Data were analyzed by comparing the observed pulmonary responses at similar external and internal dose levels. Our analyses confirm that rats are more sensitive to developing pulmonary inflammation compared to mice. The observed differences in responses do not result purely from differences in the delivered and retained doses (expressed in particle mass as well as surface area). In addition, the different nanoforms assessed showed differences in toxic potency likely due to differences in their physicochemical parameters. Primary particle and aggregate/agglomerate size distributions have a substantial impact on the deposited dose and consequently on the pulmonary response. However, in our evaluation size could not fully explain the difference observed in the analyzed studies indicating that the pulmonary response also depends on other physicochemical characteristics of the particles. It remains to be determined to what extent these findings can be generalized to other poorly soluble nanomaterials.

A biomimetic approach for enhancing the in vivo half-life of peptides.

The tremendous therapeutic potential of peptides has not yet been realized, mainly owing to their short in vivo half-life. Although conjugation to macromolecules has been a mainstay approach for enhancing protein half-life, the steric hindrance of macromolecules often harms the binding of peptides to target receptors, compromising the in vivo efficacy. Here we report a new strategy for enhancing the in vivo half-life of peptides without compromising their potency. Our approach involves endowing peptides with a small molecule that binds reversibly to the serum protein transthyretin. Although there are a few molecules that bind albumin reversibly, we are unaware of designed small molecules that reversibly bind other serum proteins and are used for half-life extension in vivo. We show here that our strategy was effective in enhancing the half-life of an agonist for GnRH receptor while maintaining its binding affinity, which was translated into superior in vivo efficacy.

Platelet activation independent of pulmonary inflammation contributes to diesel exhaust particulate-induced promotion of arterial thrombosis.

BACKGROUND: Accelerated thrombus formation induced by exposure to combustion-derived air pollution has been linked to alterations in endogenous fibrinolysis and platelet activation in response to pulmonary and systemic inflammation. We hypothesised that mechanisms independent of inflammation contribute to accelerated thrombus formation following exposure to diesel exhaust particles (DEP). METHODS: Thrombosis in rats was assessed 2, 6 and 24 h after administration of DEP, carbon black (CB; control carbon nanoparticle), DQ12 quartz microparticles (to induce pulmonary inflammation) or saline (vehicle) by either intra-tracheal instillation (0.5 mg, except Quartz; 0.125 mg) or intravenous injection (0.5 mg/kg). Thrombogenicity was assessed by carotid artery occlusion, fibrinolytic variables and platelet-monocyte aggregates. Measures of inflammation were determined in plasma and bronchoalveolar lavage fluid. Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI)-1 were measured following direct in vitro exposure of human umbilical vein endothelial cells (HUVECs) to DEP (10-150 µg/mL). RESULTS: Instillation of DEP reduced the time to thrombotic occlusion in vivo, coinciding with the peak of DEP-induced pulmonary inflammation (6 h). CB and DQ12 produced greater inflammation than DEP but did not alter time to thrombotic occlusion. Intravenous DEP produced an earlier (2 h) acceleration of thrombosis (as did CB) without pulmonary or systemic inflammation. DEP inhibited t-PA and PAI-1 release from HUVECs, and reduced the t-PA/PAI-1 ratio in vivo; similar effects in vivo were seen with CB and DQ12. DEP, but not CB or DQ12, increased platelet-monocyte aggregates. CONCLUSION: DEP accelerates arterial thrombus formation through increased platelet activation. This effect is dissociated from pulmonary and systemic inflammation and from impaired fibrinolytic function.

Regulation of angiogenesis through the efficient delivery of microRNAs into endothelial cells using polyamine-coated carbon nanotubes.

MicroRNAs (miRNAs) directly regulate gene expression at a post-transcriptional level and represent an attractive therapeutic target for a wide range of diseases. Here, we report a novel strategy for delivering miRNAs to endothelial cells (ECs) to regulate angiogenesis, using polymer functionalized carbon nanotubes (CNTs). CNTs were coated with two different polymers, polyethyleneimine (PEI) or polyamidoamine dendrimer (PAMAM), followed by conjugation of miR-503 oligonucleotides as recognized regulators of angiogenesis. We demonstrated a reduced toxicity for both polymer-coated CNTs, compared with pristine CNTs or polymers alone. Moreover, polymer-coated CNT stabilized miR-503 oligonucleotides and allowed their efficient delivery to ECs. The functionality of PAMAM-CNT-miR-503 complexes was further demonstrated in ECs through regulation of target genes, cell proliferation and angiogenic sprouting and in a mouse model of angiogenesis. This comprehensive series of experiments demonstrates that the use of polyamine-functionalized CNTs to deliver miRNAs is a novel and effective means to regulate angiogenesis.