Grantee Research Project Results
Final Report: Novel Markers of Air Pollution-induced Vascular Toxicity
EPA Grant Number: R833990Title: Novel Markers of Air Pollution-induced Vascular Toxicity
Investigators: Campen, Matthew J. , Lund, Amie K.
Institution: Lovelace Biomedical & Environmental Research Institute , New Mexico State University - Main Campus
EPA Project Officer: Hahn, Intaek
Project Period: November 1, 2008 through October 31, 2011 (Extended to October 31, 2012)
Project Amount: $500,000
RFA: Development of Environmental Health Outcome Indicators (2007) RFA Text | Recipients Lists
Research Category: Air Quality and Air Toxics , Air Toxics
Objective:
Over the past decade, it has become clear that air pollution has an important impact on cardiovascular disease, especially on those outcomes where atherosclerosis, an inflammatory disease of arteries, plays a significant role. We have been developing a model over the past few years focused on a molecular pathway that may well link both chronic growth and development of atherosclerotic plaques, as well as the eventual destabilization and rupture, which is the precipitating event in infarction, stroke, and aneurysm. This pathway relates to the regulation of vascular gelatinases via induction by oxidatively modified lipids.
Several markers of atherosclerosis have been investigated in humans exposed under controlled conditions to particulate matter (PM), ozone (03), or diesel exhaust (DE). These include IL-6, C-reactive protein (CRP), and tumor necrosis factor-alpha (TNF-a), among others. Many of these markers have good specificity with chronic arterial inflammatory disease but are otherwise uncharacterized for acute exposure to airborne pollutants. That is, there is no a priori reason to assume that pollutants act through these markers, especially acutely.
This project began with the concept that specific biomarkers in the plasma or serum could provide better information regarding the nature of the exposure and the potential impact on cardiovascular health. However, the concept evolved to the recognition that biased approaches to identify specific markers, even when candidates were derived from unbiased screens such as microarray, would limit our ability to make inferences regarding the health impacts of such biomarkers. Thus, in the course of this grant we developed an endothelial cell biosensor approach that utilizes whole plasma or serum from subjects (human, rat, mouse, etc) to derive a more holistic understanding of the relative impacts of environmental exposures on cardiovascular health.
Initially, we assayed a number of candidate biomarkers in serum from murine studies of air pollution exposures. These included osteopontin, soluble receptor for oxidized LDL (LOX-1 ), and matrix metalloproteinase-9 (MMP-9). We followed up these studies with mechanistic work to determine whether select multiligand endothelial cell receptors, namely LOX- I and CD36, played a role in mediating vascular effects of inhaled pollutants. These studies provided compelling information that, in fact, both multiligand receptors may play a role, and that information changed our perspective on the likelihood that single mediators in the serum or plasma may be facilitating the transfer of toxicity from the lung to the systemic blood vessels.
The results of this project included the observation of previously unreported markers altered by air pollutants, but more importantly included the development of a novel biosensor assay that has great potential for I) identification of susceptible individuals, 2) comparative assessment of causal components of the complex air pollution mixture, and 3) improved understanding of the biological mechanisms involved in air pollution-induced cardiovascular toxicity.
Summary/Accomplishments (Outputs/Outcomes):
Specific Aim l involved the investigation of candidate biomarkers in banked plasma from a number of studies conducted within the National Environmental Respiratory Center (NERC) Core studies. The details of these studies are previously published in great detail, including both characterization of pollutant atmospheres (McDonald et al., 2004; McDonald et al., 2006; McDonald et al., 2012) and health outcomes (Mauderly et al., 2011; Reed et al., 2004; Reed et al., 2006). Because we had observed increased plasma MMP9 activity in human plasma following exposure to diesel emissions (Lund et al., 2009), we assayed for several related biomarkers in mice, including MMP-9, soluble LOX- I and osteopontin. Plasma samples were banked from the previous exposures to a simulated downwind coal combustion atmosphere, hardwood smoke combustion, and diesel emissions. ln all three atmospheres, a filtered air control atmosphere was compared against 3 concentrations of v-.rhole emissions plus an additional permutation wherein the whole emissions were filtered to remove particulate matter, thereby exposing mice to just gases (model described in Campen et al., 2010).
Biomarkers were measured with commercially available kits according to manufacturers' recommendations. While the results revealed significant changes for all markers in certain exposure groups, the overalI patterns of response were inconsistent with what had been previously shown in humans acutely exposed (Figure 1).
Figure I. Specific biomarkers hypothesized to be altered in plasma following pollutant exposure. Levels of osteopontin, soluble LOX-Land MMP-9 were measured in the plasma of ApoE-/- mice exposed to increasing concentrations of coal combustion emissions. woodsmoke emissions (WS) or diesel emissions. Asterisks indicate significant difference from filtered air control (P<0.05) by ANOVA.
Additionally, we observed elevated LOX-I in rat aortas exposed to ozone and/or diesel exhaust particulate matter (Kodavanti et al., 201'1). These results were consistent with our findings of fresh vehicular exhaust emissions, described in Aim 2 (Lund et al., 2011)
In Specific Aim 2, we tested the roles ofLOX-1 and CD36 scavenger receptors in mediating the vascular effects of inhaled pollutants. We hypothesized that a number of non-specific factors formed in the lung would be released into the circulation and scavenging receptors such as CD36 and LOX-1 would be likely receptors for these ligands.
To test this hypothesis, we used two different models. In the first, ApoE-null hypercholesteremic mice were exposed to filtered air (control) or a mixture of gasoline and diesel emissions 6 h/d for 7 days (Lund et al, 20 I l ). Every other day a select cohort of mice were injected intraperitoneally with a neutralizing antibody against LOX-I, with remaining animals injected with a non-immune IgG for control purposes. After the exposure, we assessed vascular lipid peroxidation and inflammation in the aorta, among other endpoints. What we observed was a near-complete abrogation of the oxidative and inflammatory effects of the vehicular exhaust in mice with the LOX-I antibody treatment (Figure 2). We additionally observed several parallel markers in humans exposed to diesel emissions. These studies established a potential requirement for pattern recognition receptors, such as LOX-1, as mediators of the downstream cardiovascular health effects of inhaled pollutants.
In the second model, we used a short-term exposure to ozone (03; I ppm x 4 h) to study a common criteria pollutant that does NOT cross directly into the circulation. 03 is well characterized in terms of its reactivity, and know to penetrate no further than 0.1 micrometers into the airway epithelial lining fluid, where it reacts out with phospholipid and protein components, having a considerable affinity for glutathione, ascorbate, and urate. Thus, while the vehicular emissions certainly contain some components in the solid and gas phases that can achieve the circulation in varying concentrations, 03 specifically interacts with the lung surfactant to form secondary/tertiary/etc reaction products.
Figure 2 Mixed vehicular emissions (ME) induced aortic lipid peroxidation (measured by TBA RS) and circulating levels of oxidized LDL in ApoE-/ mice compared to filtered air-exposed controls. Co-treatment with an antibody against LOX- I abolished this effect (from Lund et al. 2011)
For the 03 model, we used wildtype (WT) and CD36-deficient mouse mice and assessed aortic vasorelaxation, as an index of endothelial function. In WT mice exposed whole-body to 03, the aortic response to acetylcholine (ACh) was greatly diminished compared to filtered air-exposed control mice. However, CD36-deficient mice were completely protected. Pulmonary inflammation, as measured by macrophage and neutrophil content in bronchoalveolar lavage fluid, also noted a'protection afforded by deficiency of CD36. Thus, it was not clear if the vascular effects were secondary to the ozone interactions with surfactant or tertiary and downstream of the inflammatory responses. To test this, we obtained serum from both WT and CD36-deficient mice exposed to filtered air or ozone and used that serum to treat naive aortas. With this homologous serum ex vivo study design, we found that 03 induces circulating factors in both WT and CD36-deficient mice, and these factors could impair vasorelaxation in WT but not CD36-deficient aortas. Thus, CD36 is essential for the detection of the ozonated by-products in the lung and in the circulation, but the generation-of the vasoactive substances is independent of this scavenger receptor (Robertson et al., 2013).
Figure 3. While WT mice showed a significant impairment in endothelial function following acute 03 exposure, CD36 mice were protected from this effect (from Roberison et al. in press).
Thus, in Aim 2, we were successful in demonstrating that vascular scavenger receptors or pattern recognition receptors are vital to the pathological responses to inhaled substances. We are still uncertain as to the biochemical nature of the vasoactive substances, but are continuing work to clarify this important facet of the mechanism underlying cardiovascular health effects of inhaled pollutants.
In Specific Aim 3, we proposed to use information obtained in Specific Aims 1 and 2 to develop hypotheses for protein or biochemical markers to test in human plasma. While we did measure a few specific targets, such as soluble LOX-1 and MMP-9, we ultimately felt that an unbiased approach to capture the impact of complete circulatory changes on bioactivity would provide a more informative outcome. Thus, we developed a straightforward biosensor assay utilizing primary human coronary artery endothelial cells to assess the holistic inflammatory potential of serum or plasma obtained from exposed individuals. This assay, now utilized in several toxicology and efficacy studies, has also been adapted to rodent studies and has profound impact on our appreciation of the mechanisms underlying the cardiovascular health effects of air pollution.
In the initial phases of this Aim, we examined several key biomarkers in the serum of humans exposed to diesel emissions in a controlled setting. We found small, but consistent increases in plasma MMP-9 concentration and activity, endothelin-1 concentration, and nitrate/nitrite (NOx) levels (Lund et al., 2009). Using the same samples, we subsequently assayed for the concentration of soluble LOX- I receptor, and again found this to be increased (Figure 4).
Figure 4. Plasma from healthy human subjects obtained after exposure to diesel (left) or nitrogen dioxide (NO2; right) showed higher levels of soluble LOX-1 compared to both baseline levels and air exposure trends (from Lund et al., 2011 and Channell et al., 2012).
While these outcomes were significant and conducted with a relevant human exposure study design, the overall changes in the measured serum factors were modest (20- 40% above baseline) and certainly not to the extent needed to induce changes in vascular function or pathology. Moreover, substantial intra-subject variability meant that such biomarkers may have limited value for a cross-sectional study design. Because of these issues, we were motivated to develop a functional assay that addressed all circulating components simultaneously. Figure 5 illustrates the assay strategy in a straightforward manner.
Figure 5. General schematic for the endothelial inflammatory potential assay developed to assess functional outcomes of holistic changes in plasma composition following exposure to air pollutants. Briefly. primary coronary artery endothelial cells are incubated with I0% plasma or serum in media for a period of time and endothelial responses are measured with standard molecular techniques (adapted from Channell et al., 2012)
Figure 6. Plasma from healthy human subjects obtained after exposure to diesel (grey) or nitrogen dioxide (NO2: red) induced significant upregulation of vascular cell adhesion molecule- I (VCAM) in primary human coronary artery endothelial cells (adapted from Channell et al., 2012). We speculate that the prolonged plasma inflammatory potential increase seen in diesel emissions relates to the retention of particulate matter, which leads to c continuing interaction with lung surfactant components.
Thus, in the second phase of Aim 3, obtained primary coronary artery endothelial cells and treated them with plasma from healthy human subjects exposed to diesel emissions (100 µg PM/m3) or nitrogen dioxide (500 ppb) for 2 h. Plasma was obtained immediately before the exposures, immediately after, then 24 h after, and each subject received an exposure to one pollutant plus a filtered air control exposure on a separate occasion. Endothelial cells were grown to confluence and incubated with a 10% or 30% mixture of plasma in media for 24h. Endothelial cell responses, measure by quantitative polymerase chain reaction assessment of mRNA, including modest but significant upregulation of vascular cell adhesion molecule- I (VCAM-1), intracellular adhesion molecule-I (ICAM-1), and p selectin, all essential adhesion molecules required for initiation of vascular inflammation. Additionally, interleukin-8 was measured in the media of the endothelial cell after incubation and was found to be significantly elevated relative to filtered air exposures.
Conclusions:
While results from the initial Aims lacked the clarity of outcome that we anticipated, these results led us to consider truly novel approaches for assessing cardiovascular effects and risk, rather than just novel biomarkers. The development of the endothelial cell assays, which assesses the response to the complete serum or plasma, has a great potential to better capture the total vascular inflammatory impact of acute and chronic environmental exposures. Beyond the valuable application to environmental health, we have already explored the value of this approach in an efficacy study with a month -long clinical trial with resveratrol/grape seed extract versus placebo. While plasma cytokines were unresponsive to treatment, healthy subjects taking the resveratrol supplement showed a ver'y consistent reduction in the inflammatory potential of their plasma, while subjects taking placebo showed no effect. Thus, this novel inflammatory potential assay holds tremendous potential for both safety/toxicity as well as clinical diagnostic and therapeutic efficacy research.
References:
1. Agarwal B*, Campen MJ*, Channel MM, WhetTy SJ, Varamini B, Davis JG, Baur JA, Smoliga JM. Resveratrol for primary prevention of atherosclerosis: clinical trial evidence for improved gene expression in vascular endothelium. Int .I Cardiol. In Press, 2012. *Co-1st authorship.
2. Campen MJ, Lund AK, Knuckles TL, Conklin DJ, Bishop B, Young D, Seilkop S, Seagrave J, Reed MD, McDonald JO. Inhaled diesel emissions alter atherosclerotic plaque composition in ApoE(-/-) mice. Toxicol Appl Pharmacol. 2010 Feb 1;242(3):310-7.
3. Channell MC, Aragon M, Paffett ML, Devlin R, Campen MJ. Circulating factors induce coronary endothelial cell activation following exposure to inhaled diesel exhaust and nitrogen dioxide in humans: Evidence from a novel translational in vitro model. Toxicol Sci. 127: I 79-186, 2012.
4. Kodavanti UP, Thomas R, Ledbettef AD, Schladweiler MC, Shannahan JH, Wallenbom JO, Lund AK, Campen MJ, Butler EO, Gottipolu RR, Nyska A, Richards JE, Andrews D, Jaskot RH, McKee .I, Kotha SR, Patel RB, Parinandi NL. Vascular and Cardiac Impairments in Rats Inhaling Ozone and Diesel Exhaust Particles. Environ Health Perspect, 119:312-318, 2011.
5. Lund AK, Lucero J, Lucas S, Madden MC, McDonald JD, Seagrave JC, Knuckles TL, Campen M.I. Vehicular emissions induce vascular MMP-9 expression and activity associated with endothelin-1-mediated pathways. Arterioscler Thromb Vase Biol. 29(4):511-7, 2009.
6. Lund AK, Lucero J, Harman M, Mathews N, Madden M, McDonald JD, Seagrave J, and Campen MJ. The Oxidized Low Density Lipoprotein Receptor Mediates Vascular Effects of Inhaled Vehicle Emissions. Am J Resp Crit Care Med, 184:82-91, 2011. •
7. Mauderly JL, Barrett EG, Gigliotti AP, McDonald JD, Reed MD, Seagrave J, Mitchell LA, Seilkop SK. Health effects of subchronic inhalation exposure to simulated downwind coal combustion emissions. lnhal Toxicol. 2011 May;23(6):349-62.
8. McDonald JD, Barr EB and White RK. Design, Characterization, and Evaluation of a Small Scale Diesel Exhaust Exposure System. Aerosol Sci Technol 38: 62-78, 2004.
9. McDonald, J. D.; White, R. K.; Barr, E. B.; Zielinska, B.; Chow, J.C.; Grosjean,
£.Generation and Characterization of Hardwood Smoke Inhalation Exposure Atmospheres. Aerosol Sci. Technol. 40: 573-584, 2006.
10. McDonald JD, White RK, Holmes T, Mauderly J, Zielinska B, Chow JC. Simulated downwind coal combustion emissions for laboratory inhalation exposure atmospheres. lnhal Toxicol. 2012 Apr;24(5):3 10-9.
11. Reed MD, Gigliotti AP, McDonald JD, Seagrave JC, Seilkop SK, Mauderly JL. Health effects of subchronic exposure to environmental levels of diesel exhaust. Inhal Toxicol. 2004 Apr;16(4):177-93.
12. Reed MD, Campen MJ, Gigliotti AP, Harrod KS, McDonald JD, Seagrave JC, Mauderly JL, Seilkop SK. Health effects of subchronic exposure to environmental levels of hardwood smoke. Inhal Toxicol. 2006 Jul;I8(8):523-39.
13. Robertson S, Colombo ES, Lucas SN, Hall PR, Febbraio M, Paffett ML, Campen M.J. CD36 mediates endothelial dysfunction downstream of circulating factors induced by 03 exposure. Toxico/ Sci, in press, 2013.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 9 publications | 6 publications in selected types | All 6 journal articles |
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Channell MM, Paffett ML, Devlin RB, Madden MC, Campen MJ. Circulating factors induce coronary endothelial cell activation following exposure to inhaled diesel exhaust and nitrogen dioxide in humans: evidence from a novel translational in vitro model. Toxicological Sciences 2012;127(1):179-186. |
R833990 (2010) R833990 (2011) R833990 (Final) |
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Kodavanti UP, Thomas R, Ledbetter AD, Schladweiler MC, Shannahan JH, Wallenborn JG, Lund AK, Campen MJ, Butler EO, Gottipolu RR, Nyska A, Richards JE, Andrews D, Jaskot RH, McKee J, Kotha SR, Patel RB, Parinandi NL. Vascular and cardiac Impairments in rats inhaling ozone and diesel exhaust particles. Environmental Health Perspectives 2011;119(3):312-318. |
R833990 (2009) R833990 (2010) R833990 (2011) R833990 (Final) |
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Lund AK, Lucero J, Harman M, Madden MC, McDonald JD, Seagrave JC, Campen MJ. The oxidized low-density lipoprotein receptor mediates vascular effects of inhaled vehicle emissions. American Journal of Respiratory and Critical Care Medicine 2011;184(1):82-91. |
R833990 (2009) R833990 (2010) R833990 (2011) R833990 (Final) |
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Robertson S, Colombo ES, Lucas SN, Hall PR, Febbraio M, Paffett ML, Campen MJ. CD36 mediates endothelial dysfunction downstream of circulating factors induced by O3 exposure. Toxicological Sciences 2013;134(2):304-311. |
R833990 (2011) R833990 (Final) R834796 (2013) R834796 (2014) R834796 (2015) R834796 (Final) R834796C003 (2013) R834796C003 (2016) R834796C003 (Final) |
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Supplemental Keywords:
Particulate matter, ozone, diesel, gasoline, coal, woodsmoke, cardiovascular, vascular, endothelium, endothelial function, vasodilation, pattern recognition receptor, scavenger receptor, CD36, LOX-1, RFA, Health, Scientific Discipline, Air, particulate matter, Health Risk Assessment, Risk Assessments, ambient air quality, atmospheric particulate matter, chemical characteristics, human health effects, cardiovascular vulnerability, biological mechanisms, chemical composition, biological mechanism , human exposure, ambient particle health effects, autonomic dysfunction, oxidative stressProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.