Use of Tracer Elements for Estimating Community Exposure to Marcellus Shale Development Operations.

Since 2009, unconventional natural gas development (UNGD) has significantly increased in Appalachia's Marcellus Shale formation. Elevations of fine particulate matter <2.5 µm (PM2.5), have been documented in areas surrounding drilling operations during well stimulation. Furthermore, many communities are experiencing increased industrial activities and probable UNGD air pollutant exposures. Recent studies have associated UNGD emissions with health effects based on distances from well pads. In this study, PM2.5 filter samples were collected on an active gas well pad in Morgantown, West Virginia, and three locations downwind during hydraulic stimulation. Fine particulate samples were analyzed for major and trace elements. An experimental source identification model was developed to determine which elements appeared to be traceable downwind of the UNGD site and whether these elements corresponded to PM2.5 measurements. Results suggest that 1) magnesium may be useful for detecting the reach of UNGD point source emissions, 2) complex surface topographic and meteorological conditions in the Marcellus Shale region could be modeled and confounding sources discounted, and 3) well pad emissions may be measurable at distances of at least 7 km. If shown to be more widely applicable, future tracer studies could enhance epidemiological studies showing health effects of UNGD-associated emissions at ≥15 km.

Cardiac and mitochondrial dysfunction following acute pulmonary exposure to mountaintop removal mining particulate matter.

Throughout the United States, air pollution correlates with adverse health outcomes, and cardiovascular disease incidence is commonly increased following environmental exposure. In areas surrounding active mountaintop removal mines (MTM), a further increase in cardiovascular morbidity is observed and may be attributed in part to particulate matter (PM) released from the mine. The mitochondrion has been shown to be central in the etiology of many cardiovascular diseases, yet its roles in PM-related cardiovascular effects are not realized. In this study, we sought to elucidate the cardiac processes that are disrupted following exposure to mountaintop removal mining particulate matter (PM MTM). To address this question, we exposed male Sprague-Dawley rats to PM MTM, collected within one mile of an active MTM site, using intratracheal instillation. Twenty-four hours following exposure, we evaluated cardiac function, apoptotic indices, and mitochondrial function. PM MTM exposure elicited a significant decrease in ejection fraction and fractional shortening compared with controls. Investigation into the cellular impacts of PM MTM exposure identified a significant increase in mitochondrial-induced apoptotic signaling, as reflected by an increase in TUNEL-positive nuclei and increased caspase-3 and -9 activities. Finally, a significant increase in mitochondrial transition pore opening leading to decreased mitochondrial function was identified following exposure. In conclusion, our data suggest that pulmonary exposure to PM MTM increases cardiac mitochondrial-associated apoptotic signaling and decreases mitochondrial function concomitant with decreased cardiac function. These results suggest that increased cardiovascular disease incidence in populations surrounding MTM mines may be associated with increased cardiac cell apoptotic signaling and decreased mitochondrial function.

Appalachian mountaintop mining particulate matter induces neoplastic transformation of human bronchial epithelial cells and promotes tumor formation.

Epidemiological studies suggest that living near mountaintop coal mining (MTM) activities is one of the contributing factors for high lung cancer incidence. The purpose of this study was to investigate the long-term carcinogenic potential of MTM particulate matter (PMMTM) exposure on human bronchial epithelial cells. Our results show that chronic exposure (3 months) to noncytotoxic, physiological relevant concentration (1 µg/mL) of PMMTM, but not control particle PMCON, induced neoplastic transformation, accelerated cell proliferation, and enhanced cell migration of the exposed lung cells. Xenograft transplantation of the PMMTM-exposed cells in mice caused no apparent tumor formation, but promoted tumor growth of human lung carcinoma H460 cells, suggesting the tumor-promoting effect of PMMTM. Chronic exposure to the main inorganic chemical constituent of PMMTM, molybdenum but not silica, similarly induced cell transformation and tumor promotion, suggesting the contribution of molybdenum, at least in part, in the PMMTM effects. These results provide new evidence for the carcinogenic potential of PMMTM and support further risk assessment and implementation of exposure control for PMMTM.

Biokinetically-based in vitro cardiotoxicity of residual oil fly ash: hazard identification and mechanisms of injury.

Epidemiological studies have associated air pollution particulate matter (PM) exposure with adverse cardiovascular effects. Identification of causal PM sources is critically needed to support regulatory decisions to protect public health. This research examines the in vitro cardiotoxicity of bioavailable constituents of residual oil fly ash (ROFA) employing in vivo, biokinetically-based, concentrations determined from their pulmonary deposition. Pulmonary deposition of ROFA led to a rapid increase in plasma vanadium (V) levels that were prolonged in hypertensive animals without systemic inflammation. ROFA cardiotoxicity was evaluated using neonatal rat cardiomyocyte (RCM) cultures exposed to particle-free leachates of ROFA (ROFA-L) at levels present in exposed rat plasma. Cardiotoxicity was observed at low levels (3.13 µg/mL) of ROFA-L 24 h post-exposure. Dimethylthiourea (28 mM) inhibited ROFA-L-induced cytotoxicity at high (25-12.5 µg/mL) doses, suggesting that oxidative stress is responsible at high ROFA-L doses. Cardiotoxicity could not be reproduced using a V + Ni + Fe mixture or a ROFA-L depleted of these metals, suggesting that ROFA-L cardiotoxicity requires the full complement of bioavailable constituents. Susceptibility of RCMs to ROFA-L-induced cytotoxicity was increased following tyrosine phosphorylation inhibition, suggesting that phosphotyrosine signaling pathways play a critical role in regulating ROFA-L-induced cardiotoxicity. These data demonstrate that bioavailable constituents of ROFA are capable of direct adverse cardiac effects.

Whole-body nanoparticle aerosol inhalation exposures.

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle inhalation toxicology studies, the aerosols in a chamber housing the experimental animals must have: 1) a steady concentration maintained at a desired level for the entire exposure period; 2) a homogenous composition free of contaminants; and 3) a stable size distribution with a geometric mean diameter < 200 nm and a geometric standard deviation σg < 2.5 (5). The generation of aerosols containing nanoparticles is quite challenging because nanoparticles easily agglomerate. This is largely due to very strong inter-particle forces and the formation of large fractal structures in tens or hundreds of microns in size (6), which are difficult to be broken up. Several common aerosol generators, including nebulizers, fluidized beds, Venturi aspirators and the Wright dust feed, were tested; however, none were able to produce nanoparticle aerosols which satisfy all criteria (5). A whole-body nanoparticle aerosol inhalation exposure system was fabricated, validated and utilized for nano-TiO2 inhalation toxicology studies. Critical components: 1) novel nano-TiO2 aerosol generator; 2) 0.5 m(3) whole-body inhalation exposure chamber; and 3) monitor and control system. Nano-TiO2 aerosols generated from bulk dry nano-TiO2 powders (primary diameter of 21 nm, bulk density of 3.8 g/cm(3)) were delivered into the exposure chamber at a flow rate of 90 LPM (10.8 air changes/hr). Particle size distribution and mass concentration profiles were measured continuously with a scanning mobility particle sizer (SMPS), and an electric low pressure impactor (ELPI). The aerosol mass concentration (C) was verified gravimetrically (mg/m(3)). The mass (M) of the collected particles was determined as M = (Mpost-Mpre), where Mpre and Mpost are masses of the filter before and after sampling (mg). The mass concentration was calculated as C = M/(Q*t), where Q is sampling flowrate (m(3)/min), and t is the sampling time (minute). The chamber pressure, temperature, relative humidity (RH), O2 and CO2 concentrations were monitored and controlled continuously. Nano-TiO2 aerosols collected on Nuclepore filters were analyzed with a scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis. In summary, we report that the nano-particle aerosols generated and delivered to our exposure chamber have: 1) steady mass concentration; 2) homogenous composition free of contaminants; 3) stable particle size distributions with a count-median aerodynamic diameter of 157 nm during aerosol generation. This system reliably and repeatedly creates test atmospheres that simulate occupational, environmental or domestic ENM aerosol exposures.

Air pollution particulate matter collected from an Appalachian mountaintop mining site induces microvascular dysfunction.

OBJECTIVE: Air pollution PM is associated with cardiovascular morbidity and mortality. In Appalachia, PM from mining may represent a health burden to this sensitive population that leads the nation in cardiovascular disease, among others. Cardiovascular consequences following inhalation of PM(MTM) are unclear, but must be identified to establish causal effects. METHODS: PM was collected within 1 mile of an active MTM site in southern WV. The PM was extracted and was primarily <10 µm in diameter (PM10), consisting largely of sulfur (38%) and silica (24%). Adult male rats were IT with 300 µg PM(MTM) . Twenty-four hours following exposure, rats were prepared for intravital microscopy, or isolated arteriole experiments. RESULTS: PM(MTM) exposure blunted endothelium-dependent dilation in mesenteric and coronary arterioles by 26%, and 25%, respectively, as well as endothelium-independent dilation. In vivo, PM(MTM) exposure inhibited endothelium-dependent arteriolar dilation (60% reduction). α-adrenergic receptor blockade inhibited PVNS-induced vasoconstriction in exposed animals compared with sham. CONCLUSIONS: These data suggest that PM(MTM) exposure impairs microvascular function in disparate microvascular beds, through alterations in NO-mediated dilation and sympathetic nerve influences. Microvascular dysfunction may contribute to cardiovascular disease in regions with MTM sites.

Xenobiotic particle exposure and microvascular endpoints: a call to arms.

Xenobiotic particles can be considered in two genres: air pollution particulate matter and engineered nanoparticles. Particle exposures can occur in the greater environment, the workplace, and our homes. The majority of research in this field has, justifiably, focused on pulmonary reactions and outcomes. More recent investigations indicate that cardiovascular effects are capable of correlating with established mortality and morbidity epidemiological data following particle exposures. While the preliminary and general cardiovascular toxicology has been defined, the mechanisms behind these effects, specifically within the microcirculation, are largely unexplored. Therefore, the purpose of this review is several fold: first, a historical background on toxicological aspects of particle research is presented. Second, essential definitions, terminology, and techniques that may be unfamiliar to the microvascular scientist will be discussed. Third, the most current concepts and hypotheses driving cardiovascular research in this field will be reviewed. Lastly, potential future directions for the microvascular scientist will be suggested. Collectively speaking, microvascular research in the particle exposure field represents far more than a "niche." The immediate demand for basic, translational, and clinical studies is high and diverse. Microvascular scientists at all career stages are strongly encouraged to expand their research interests to include investigations associated with particle exposures.

Nanoparticle inhalation alters systemic arteriolar vasoreactivity through sympathetic and cyclooxygenase-mediated pathways.

The widespread increase in the production and use of nanomaterials has increased the potential for nanoparticle exposure; however, the biological effects of nanoparticle inhalation are poorly understood. Rats were exposed to nanosized titanium dioxide aerosols (10 µg lung burden); at 24 h post-exposure, the spinotrapezius muscle was prepared for intravital microscopy. Nanoparticle exposure did not alter perivascular nerve stimulation (PVNS)-induced arteriolar constriction under normal conditions; however, adrenergic receptor inhibition revealed a more robust effect. Nanoparticle inhalation reduced arteriolar dilation in response to active hyperaemia (AH). In both PVNS and AH experiments, nitric oxide synthase (NOS) inhibition affected only controls. Whereas cyclooxygenase (COX) inhibition only attenuated AH-induced arteriolar dilation in nanoparticle-exposed animals. This group displayed an enhanced U46619 constriction and attenuated iloprost-induced dilation. Collectively, these studies indicate that nanoparticle exposure reduces microvascular NO bioavailability and alters COX-mediated vasoreactivity. Furthermore, the enhanced adrenergic receptor sensitivity suggests an augmented sympathetic responsiveness.

Formation of vascular S-nitrosothiols and plasma nitrates/nitrites following inhalation of diesel emissions.

Epidemiological studies have associated traffic-related airborne pollution with adverse cardiovascular outcomes. Nitric oxide (NO) is a common component of fresh diesel and gasoline engine emissions that rapidly transforms both in the atmosphere and once inhaled. Because of this rapid transformation, limited information is available in terms of potential human exposures and adverse health effects. Young rats were exposed to whole diesel emissions (DE) adjusted to 300 µg/m(3) of particulate matter (containing 3.5 ppm NO) or 0, 3, or 10 ppm NO as a positive control. Animals were also pre-injected (ip) with either saline or N-acetylcysteine (NAC), a precursor of glutathione. Predictably, pure NO exposures led to a concentration-dependent increase in plasma nitrates compared to controls, which lasted for roughly 4 h postexposure. Whole DE exposure for 1 h also led to a doubling of plasma NOx. NAC injection increased the levels of plasma nitrates and nitrites (NOx) in the DE exposure group. Inhibition of nitric oxide symthase (NOS) by N(G)-nitro-L-arginine (L-NNA) did not block the rise in plasma NOx, demonstrating that the increase was entirely due to exogenous sources. Both DE and pure NO exposures paradoxically led to elevated eNOS expression in aortic tissue. Furthermore, coronary arterioles from NO-exposed animals exhibited greater constriction to endothelin-1 compared to controls, consistent with a derangement of the NOS system. Thus, NO may be an important contributor to traffic-related cardiovascular morbidity, although further research is necessary for proper hazard identification.

A comparison of vascular effects from complex and individual air pollutants indicates a role for monoxide gases and volatile hydrocarbons.

BACKGROUND: Emerging evidence suggests that the systemic vasculature may be a target of inhaled pollutants of vehicular origin. We have identified several murine markers of vascular toxicity that appear sensitive to inhalation exposures to combustion emissions. OBJECTIVE: We sought to examine the relative impact of various pollutant atmospheres and specific individual components on these markers of altered vascular transcription and lipid peroxidation. METHODS: Apolipoprotein E knockout (ApoE(-/-)) mice were exposed to whole combustion emissions (gasoline, diesel, coal, hardwood), biogenically derived secondary organic aerosols (SOAs), or prominent combustion-source gases [nitric oxide (NO), NO(2), carbon monoxide (CO)] for 6 hr/day for 7 days. Aortas were assayed for transcriptional alterations of endothelin-1 (ET-1), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-2 (TIMP-2), and heme oxygenase-1 (HO-1), along with measures of vascular lipid peroxides (LPOs) and gelatinase activity. RESULTS: We noted transcriptional alterations with exposures to gasoline and diesel emissions. Interestingly, ET-1 and MMP-9 transcriptional effects could be recreated by exposure to CO and NO, but not NO(2) or SOAs. Gelatinase activity aligned with levels of volatile hydrocarbons and also monoxide gases. Neither gases nor particles induced vascular LPO despite potent effects from whole vehicular emissions. CONCLUSIONS: In this head-to-head comparison of the effects of several pollutants and pollutant mixtures, we found an important contribution to vascular toxicity from readily bioavailable monoxide gases and possibly from volatile hydrocarbons. These data support a role for traffic-related pollutants in driving cardiopulmonary morbidity and mortality.

Inhaled diesel emissions alter atherosclerotic plaque composition in ApoE(-/-) mice.

Recent epidemiological studies suggest that traffic-related air pollution may have detrimental effects on cardiovascular health. Previous studies reveal that gasoline emissions can induce several enzyme pathways involved in the formation and development of atherosclerotic plaques. As a direct comparison, the present study examined the impact of diesel engine emissions on these pathways, and further examined the effects on vascular lesion pathology. Apolipoprotein E-null mice were simultaneously placed on a high-fat chow diet and exposed to four concentrations, plus a high concentration exposure with particulates (PM) removed by filtration, of diesel emissions for 6 h/day for 50 days. Aortas were subsequently assayed for alterations in matrix metalloproteinase-9, endothelin-1, and several other biomarkers. Diesel induced dose-related alterations in gene markers of vascular remodeling and aortic lipid peroxidation; filtration of PM did not significantly alter these vascular responses, indicating that the gaseous portion of the exhaust was a principal driver. Immunohistochemical analysis of aortic leaflet sections revealed no net increase in lesion area, but a significant decrease in lipid-rich regions and increasing trends in macrophage accumulation and collagen content, suggesting that plaques were advanced to a more fragile, potentially more vulnerable state by diesel exhaust exposure. Combined with previous studies, these results indicate that whole emissions from mobile sources may have a significant role in promoting chronic vascular disease.

Impairment of coronary endothelial cell ET(B) receptor function after short-term inhalation exposure to whole diesel emissions.

Air pollutant levels positively correlate with increases in both acute and chronic cardiovascular disease. The pollutant diesel exhaust (DE) increases endothelin (ET) levels, suggesting that this peptide may contribute to DE-induced cardiovascular disease. We hypothesized that acute exposure to DE also enhances ET-1-mediated coronary artery constrictor sensitivity. Constrictor responses to KCl, U-46619, and ET-1 were recorded by videomicroscopy in pressurized intraseptal coronary arteries from rats exposed for 5 h to DE (300 microg/m(3)) or filtered air (Air). ET-1 constriction was augmented in arteries from DE-exposed rats. Nitric oxide synthase (NOS) inhibition [N(omega)-nitro-L-arginine (L-NNA), 100 microM] and endothelium inactivation augmented ET-1 responses in arteries from Air but not DE rats so that after either treatment responses were not different between groups. DE exposure did not affect KCl and U-46619 constrictor responses, while NOS inhibition augmented KCl constriction equally in both groups. Thus basal NOS activity does not appear to be affected by DE exposure. The endothelin type B (ET(B)) receptor antagonist BQ-788 (10 microM) inhibited ET-1 constriction in DE but not Air arteries, and constriction in the presence of the antagonist was not different between groups. Cytokine levels were not different in plasma from DE and AIR rats, suggesting that acute exposure to DE does not cause an immediate inflammatory response. In summary, a 5-h DE exposure selectively increases constrictor sensitivity to ET-1. This augmentation is endothelium-, NOS-, and ET(B) receptor dependent. These data suggest that DE exposure diminishes ET(B) receptor activation of endothelial NOS and augments ET(B)-dependent vasoconstriction. This augmented coronary vasoreactivity to ET-1 after DE, coupled with previous reports that DE induces production of ET-1, suggests that ET-1 may contribute to the increased incidence of cardiac events during acute increases in air pollution levels.

Vehicular emissions induce vascular MMP-9 expression and activity associated with endothelin-1-mediated pathways.

OBJECTIVE: Mechanisms of air pollution-induced exacerbation of cardiovascular disease are currently unknown, thus we examined the roles of vascular endothelin-1 (ET-1) and reactive oxygen species (ROS) in regulating mediators of vascular remodeling, namely matrix metalloproteinases (MMPs), after exposure to vehicle engine emissions. METHODS AND RESULTS: ApoE(-/-) mice were exposed by inhalation to filtered air or gasoline engine exhaust (GEE, 1:12 dilution) 6 hours per day for 1 or 7 days. Concurrently, mice were treated with either ET(A) receptor antagonist BQ-123 (100 ng/kg/d) via osmotic minipumps, Tempol (approximately 41 mg/kg/d, orally), or vehicle. GEE-exposure increased vascular MMP-2 and -9, endothelin-1 (ET-1), tissue inhibitor of metalloproteinases (TIMP)-2 mRNA and ROS levels. Aortic MMP protein and plasma MMP-9 were similarly upregulated. GEE-mediated increases in vascular ROS were attenuated by Tempol-treatment, as were MMP-2 and TIMP-2; whereas BQ-123 ameliorated GEE-induced vascular expression of MMP-9, MMP-2, ROS, and ET-1. In a parallel study, diesel exhaust exposure in volunteer human subjects induced significant increases in plasma ET-1 and MMP-9 expression and activity. CONCLUSIONS: These findings demonstrate that acute exposure to vehicular source air pollutants results in upregulation of circulating and vascular factors associated with progression of atherosclerosis, mediated in part through activation of ET-1-ET(A) receptor pathways.

Diesel exhaust exposure enhances venoconstriction via uncoupling of eNOS.

Environmental air pollution is associated with adverse cardiovascular events, including increased hospital admissions due to heart failure and myocardial infarction. The exact mechanism(s) by which air pollution affects the heart and vasculature is currently unknown. Recent studies have found that exposure to air pollution enhances arterial vasoconstriction in humans and animal models. Work in our laboratory has shown that diesel emissions (DE) enhance vasoconstriction of mouse coronary arteries. Thus, we hypothesized that DE could enhance vasoconstriction in arteries and veins through uncoupling of endothelial nitric oxide synthase (eNOS). To test this hypothesis, we first bubbled DE through a physiological saline solution and exposed isolated mesenteric veins. Second, we exposed animals, whole body, to DE at 350 microg/m(3) for 4 h, after which mesenteric arteries and veins were isolated. Results from these experiments show that saline bubbled with DE as well as inhaled DE enhances vasoconstriction in veins but not arteries. Exposure to several representative volatile organic compounds found in the DE-exposed saline did not enhance arterial constriction. L-nitro-arginine-methyl-ester (L-NAME), an eNOS inhibitor, normalized the control vessels to the DE-exposed vessels implicating an uncoupling of eNOS as a mechanism for enhanced vasoconstriction. The principal conclusions of this research are 1) veins exhibit endothelial dysfunction following in vivo and ex vivo exposures to DE, 2) veins appear to be more sensitive to DE effects than arteries, and 3) DE components most likely induce endothelial dysfunction through the uncoupling of eNOS.

Fine oil combustion particle bioavailable constituents induce molecular profiles of oxidative stress, altered function, and cellular injury in cardiomyocytes.

Epidemiological studies have shown a positive association between exposure to air particulate matter (PM) pollution and adverse cardiovascular health effects in susceptible subpopulations such as those with pre-existing cardiovascular disease. The mechanism(s) through which pulmonary deposited PM, particularly fine PM2.5, PM with mass median aerodynamic diameter <2.5 microm, affects the cardiovascular system is currently not known and remains a major focus of investigation. In the present study, the transcriptosome and transcription factor proteome were examined in rat neonatal cardiomyocyte (RCM) cultures, following an acute exposure to bioavailable constituents of PM2.5 oil combustion particles designated residual oil fly ash leachate (ROFA-L). Out of 3924 genes examined, 38 genes were suppressed and 44 genes were induced following a 1-h exposure to 3.5 microg/ml of a particle-free leachate of ROFA (ROFA-L). Genomic alterations in pathways related to IGF-1, VEGF, IL-2, PI3/AKT, cardiovascular disease, and free radical scavenging, among others, were detected 1 h postexposure to ROFA-L. Global gene expression was altered in a manner consistent with cardiac myocyte electrophysiological remodeling, cellular oxidative stress, and apoptosis. ROFA-L altered the transcription factor proteome by suppressing activity of 24 and activating 40 transcription factors out of a total of 149. Genomic alterations were found to correlate with changes in transcription factor proteome. These acute changes indicate pathological molecular alterations, which may lead to possible chronic alterations to the cardiac myocyte. These data also potentially relate underlying cardiovascular effects from occupational exposure to ROFA and identify how particles from specific emission sources may mediate ambient PM cardiac effects.

Gasoline exhaust emissions induce vascular remodeling pathways involved in atherosclerosis.

Epidemiological evidence indicates that environmental air pollutants are positively associated with the development of chronic vascular disease; however, the mechanisms involved have not been fully elucidated. In the present study we examined molecular pathways associated with chronic vascular disease in atherosclerosis-prone apolipoprotein E-deficient (ApoE(-/-)) mice, including markers of vascular remodeling and oxidative stress, in response to exposure to the ubiquitous environmental pollutant, gasoline engine emissions. ApoE(-/-) mice, on a high-fat diet, were exposed by inhalation to either filtered air; 8, 40, or 60 mug/m(3) particulate matter whole exhaust; or filtered exhaust with gases matching the 60-mug/m(3) concentration, for 7 weeks. Aortas and plasma were collected and assayed for changes in histochemical markers, real-time reverse transcriptase-polymerase chain reaction, and indicators of oxidative damage. Inhalational exposure to gasoline engine emissions resulted in increased aortic mRNA expression of matrix metalloproteinase-3 (MMP-3), MMP-7, and MMP-9, tissue inhibitor of metalloproteinases-2, endothelin-1 and heme oxygenase-1 in ApoE(-/-) mice; increased aortic MMP-9 protein levels were confirmed through immunohistochemistry. Elevated reactive oxygen species were also observed in arteries from exposed animals, despite absence of plasma markers. Similar findings were also observed in the aortas of ApoE(-/-) mice exposed to particle-filtered atmosphere, implicating the gaseous components of the whole exhaust in mediating the expression of markers associated with the vasculopathy. These findings demonstrate that exposure to gasoline engine emissions results in the transcriptional upregulation of factors associated with vascular remodeling, as well as increased markers of vascular oxidative stress, which may contribute to the progression of atherosclerosis and reduced stability of vulnerable plaques.