Matthew J. Campen

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 ETA receptor antagonist BQ-123 (100 ng/kg/d) via osmotic minipumps, Tempol (≈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–ETA receptor pathways.

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.

Vascular and Cardiac Impairments in Rats Inhaling Ozone and Diesel Exhaust Particles

Background Mechanisms of cardiovascular injuries from exposure to gas and particulate air pollutants are unknown. Objective We sought to determine whether episodic exposure of rats to ozone or diesel exhaust particles (DEP) causes differential cardiovascular impairments that are exacerbated by ozone plus DEP. Methods and results Male Wistar Kyoto rats (10–12 weeks of age) were exposed to air, ozone (0.4 ppm), DEP (2.1 mg/m3), or ozone (0.38 ppm) + DEP (2.2 mg/m3) for 5 hr/day, 1 day/week for 16 weeks, or to air, ozone (0.51 or 1.0 ppm), or DEP (1.9 mg/m3) for 5 hr/day for 2 days. At the end of each exposure period, we examined pulmonary and cardiovascular biomarkers of injury. In the 16-week study, we observed mild pulmonary pathology in the ozone, DEP, and ozone + DEP exposure groups, a slight decrease in circulating lymphocytes in the ozone and DEP groups, and decreased platelets in the DEP group. After 16 weeks of exposure, mRNA biomarkers of oxidative stress (hemeoxygenase-1), thrombosis (tissue factor, plasminogen activator inhibitor-1, tissue plasminogen activator, and von Willebrand factor), vasoconstriction (endothelin-1, endothelin receptors A and B, endothelial NO synthase) and proteolysis [matrix metalloprotease (MMP)-2, MMP-3, and tissue inhibitor of matrix metalloprotease-2] were increased by DEP and/or ozone in the aorta, but not in the heart. Aortic LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) mRNA and protein increased after ozone exposure, and LOX-1 protein increased after exposure to ozone + DEP. RAGE (receptor for advanced glycation end products) mRNA increased in the ozone + DEP group. Exposure to ozone or DEP depleted cardiac mitochondrial phospholipid fatty acids (DEP > ozone). The combined effect of ozone and DEP exposure was less pronounced than exposure to either pollutant alone. Exposure to ozone or DEP for 2 days (acute) caused mild changes in the aorta. Conclusions In animals exposed to ozone or DEP alone for 16 weeks, we observed elevated biomarkers of vascular impairments in the aorta, with the loss of phospholipid fatty acids in myocardial mitochondria. We conclude that there is a possible role of oxidized lipids and protein through LOX-1 and/or RAGE signaling.

Cardiac and thermoregulatory effects of instilled particulate matter-associated transition metals in healthy and cardiopulmonary-compromised rats

Particulate matter air pollution has been associated with cardiopulmonary morbidity and mortality in many recent epidemiological studies. Previous toxicological research has demonstrated profound cardiac and thermoregulatory changes in rats following exposure to residual oil fly ash (ROFA), a combustion-derived particulate. The response to ROFA appeared biphasic, consisting of both immediate (0-6 h) and delayed (24-96 h) bradycardia and hypothermia. Other studies have demonstrated that much of the pulmonary toxicity of ROFA was caused by its constitutive transition metals, namely, Fe, Ni, and V. This study examined the contributions of these metals to the observed cardiac and thermoregulatory changes caused by ROFA in conscious, unrestrained rats. Prior to exposure, each animal was surgically implanted with a radiotelemetry device capable of continuously monitoring heart rate, electrocardiographic, and core temperature data. Individual metals were intratracheally instilled in healthy rats ( n = 4 per metal species) and in rats with monocrotaline (MCT; 60 mg/kg)-induced pulmonary hypertension ( n = 10 per metal species); combinations of metals were instilled in MCT-treated rats only ( n = 6 per combination of metal species). Metals were administered in doses equivalent to those found in the highest dose of ROFA used in previous studies, that is, 105 w g Fe 2 (SO 4 ) 3 , 263 w g NiSO 4 , and 245 w g VSO 4 . Healthy and MCT-treated rats demonstrated similar responses to metals. Fe caused little response, whereas V caused marked bradycardia, arrhythmogenesis, and hypothermia immediately following instillation and lasting ~6 h. Ni caused no immediate response, but induced a delayed bradycardia, arrhythmogenesis, and hypothermia that began ~24 h after instillation and lasted for several days. When instilled in combination, Ni appeared to exacerbate the immediate effects of V, whereas Fe attenuated them. These data suggest that the biphasic response to instilled ROFA may result from a summation of the temporally different effects of V and Ni.

Impairment of coronary endothelial cell ETB 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.

Cardiovascular effects of inhaled diesel exhaust in spontaneously hypertensive rats.

Particulate matter air pollution is associated with increased cardiovascular mortality. The present study examined the cardiac effects of diesel exhaust exposure in spontaneously hypertensive rats. These rats (4 mo old, n=6 males and 4–6 females/concentration) were exposed to one of five diesel exhaust levels (0, 30, 100, 300, and 1000 μg particles/m3) for 6 h per day for 7 d. Electrocardiographic measurements were obtained by radiotelemetry beginning 3 d prior to exposure and ending 4d after exposure cessation. Control rats displayed a reduced daytime heart rate from the beginning of the protocol, whereas exposed rats maintained a significantly elevated heart rate throughout the exposure. Daytime heart rate values for male control rats averaged 265±5 beats/min (mean±standard error [SE]), whereas values for exposed rats averaged 290±7 beats/min. This difference persisted during the evenings of the exposure period but was not observed at any time during the preex-posure or postexposure periods. The PQ interval, an index of atrioventricular node sensitivity, was significantly prolonged among exposed animals in a concentration-dependent manner. Increased heart rate with prolongation of the PQ interval may represent a substrate for ventricular arrhythmias. These results concur with previous reports suggesting that realistic exposure concentrations of air pollution affect the pacemaking system of rats.

Nicotine Primarily Suppresses Lung Th2 but Not Goblet Cell and Muscle Cell Responses to Allergens

Allergic asthma, an inflammatory disease characterized by the infiltration and activation of various leukocytes, the production of Th2 cytokines and leukotrienes, and atopy, also affects the function of other cell types, causing goblet cell hyperplasia/hypertrophy, increased mucus production/secretion, and airway hyperreactivity. Eosinophilic inflammation is a characteristic feature of human asthma, and recent evidence suggests that eosinophils also play a critical role in T cell trafficking in animal models of asthma. Nicotine is an anti-inflammatory, but the association between smoking and asthma is highly contentious and some report that smoking cessation increases the risk of asthma in ex-smokers. To ascertain the effects of nicotine on allergy/asthma, Brown Norway rats were treated with nicotine and sensitized and challenged with allergens. The results unequivocally show that, even after multiple allergen sensitizations, nicotine dramatically suppresses inflammatory/allergic parameters in the lung including the following: eosinophilic/lymphocytic emigration; mRNA and/or protein expression of the Th2 cytokines/chemokines IL-4, IL-5, IL-13, IL-25, and eotaxin; leukotriene C4; and total as well as allergen-specific IgE. Although nicotine did not significantly affect hexosaminidase release, IgG, or methacholine-induced airway resistance, it significantly decreased mucus content in bronchoalveolar lavage; interestingly, however, despite the strong suppression of IL-4/IL-13, nicotine significantly increased the intraepithelial-stored mucosubstances and Muc5ac mRNA expression. These results suggest that nicotine modulates allergy/asthma primarily by suppressing eosinophil trafficking and suppressing Th2 cytokine/chemokine responses without reducing goblet cell metaplasia or mucous production and may explain the lower risk of allergic diseases in smokers. To our knowledge this is the first direct evidence that nicotine modulates allergic responses.

Mechanisms of diesel-induced endothelial nitric oxide synthase dysfunction in coronary arterioles.

Background and objective Increased air pollutants correlate with increased incidence of cardiovascular disease potentially due to vascular dysfunction. We have reported that acute diesel engine exhaust (DE) exposure enhances vasoconstriction and diminishes acetylcholine (ACh)-induced dilation in coronary arteries in a nitric oxide synthase (NOS)-dependent manner. We hypothesize that acute DE inhalation leads to endothelial dysfunction by uncoupling NOS. Methods Rats inhaled fresh DE (300 μg particulate matter/m3) or filtered air for 5 hr. After off-gassing, intraseptal coronary arteries were isolated and dilation to ACh recorded using videomicroscopy. Results Arteries from DE-exposed animals dilated less to ACh than arteries from air-exposed animals. NOS inhibition did not affect ACh dilation in control arteries but increased dilation in the DE group, suggesting NOS does not normally contribute to ACh-induced dilation in coronary arteries but does contribute to endothelial dysfunction after DE inhalation. Cyclooxygenase (COX) inhibition did not affect ACh dilation in the DE group, but combined inhibition of NOS and COX diminished dilation in both groups and eliminated intergroup differences, suggesting that the two pathways interact. Superoxide scavenging increased ACh dilation in DE arteries, eliminating differences between groups. Tetrahydrobiopterin (BH4) supplementation with sepiapterin restored ACh-mediated dilation in the DE group in a NOS-dependent manner. Superoxide generation (dihydroethidium staining) was greater in DE arteries, and superoxide scavenging, BH4 supplementation, or NOS inhibition reduced the signal in DE but not air arteries. Conclusion Acute DE exposure appears to uncouple NOS, increasing reactive oxygen species generation and causing endothelial dysfunction, potentially because of depletion of BH4 limiting its bioavailability.

Heart rate variability in rodents: uses and caveats in toxicological studies

Heart rate variability (HRV) is a measure of cardiac pacing dynamics that has recently garnered a great deal of interest in environmental health studies. While the use of these measures has become popular, much uncertainty remains in the interpretation of results, both in terms of human and animal research. In humans, HRV endpoints, specifically chronic alterations in baseline HRV patterns, have been reasonably well characterized as prognostic indicators of adverse outcomes for a variety of diseases. However, such information is lacking for reversible HRV changes that may be induced by short-term exposures to environmental toxicants. Furthermore, there are minimal substantive data, either acute or chronic, regarding the pathological interpretation or prognostic value of toxicant-induced changes in HRV in rodents. The present report summarizes the physiological and clinical aspects of HRV, the methodological processes for obtaining these endpoints, and previous human and animal studies in the field of environmental health. Furthermore, we include a discussion of important caveats and recommendations for the interpretation of HRV data in animal research.

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), NO2, 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 NO2 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.