Grantee Research Project Results

2013 Progress Report: Cardiovascular Consequences of Immune Modification by Traffic-Related Emissions

EPA Grant Number: R834796C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R834796
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: University of Washington Center for Clean Air Research
Center Director: Vedal, Sverre
Title: Cardiovascular Consequences of Immune Modification by Traffic-Related Emissions
Investigators: Campen, Matthew J. , McDonald, Jacob D. , Lund, Amie K. , Rosenfeld, Michael
Institution: University of New Mexico , University of Washington , Lovelace Biomedical & Environmental Research Institute
Current Institution: Lovelace Biomedical & Environmental Research Institute , University of New Mexico , University of Washington
EPA Project Officer: Callan, Richard
Project Period: December 1, 2010 through November 30, 2015 (Extended to November 30, 2017)
Project Period Covered by this Report: December 1, 2012 through November 30,2013
RFA: Clean Air Research Centers (2009) RFA Text | Recipients Lists
Research Category: Human Health , Air

Objective:

Traffic-related emissions are associated with the incidence and progression of acute and chronic cardiovascular sequelae in human population studies. Such phenomena of near-roadway health effects have yet to be characterized toxicologically.

Because of overlapping issues related to noise, socioeconomic status, ethnicity, etc., there is a need to better understand the biological plausibility that fresh mixtures of vehicular emissions have a more potent than expected impact on human health. We hypothesize that the complex mixtures produced by traffic are inherently more toxic due to the combined presence of both particulates and volatile organic emissions. Furthermore, we hypothesize that emissions-induced oxidation of certain endogenous phospholipids, presumably from the pulmonary surfactant, can stimulate the activity of immune cells through such receptors and in turn promote the invasion of existing vascular lesions.

Approach: This project will use complex roadway mixtures as generated and characterized in the laboratory. In Aim 1, we will ascertain (1) the potentiating effects of physical and photochemical aging on fresh emissions and (2) interactions of vehicular emissions with pertinent co-pollutants (ozone, road dust), both in terms of driving systemic vascular oxidative stress. In Aim 2, we will examine the effects of the emissions-induced oxidative modifications to endogenous phospholipids, in terms of activating immune-modulating receptors such as LOX-1, CD-36, TLR-2, and TLR-4. This Aim will utilize transgenic models to examine the roles of these receptors, as well as characterize the lipidomic alterations in various tissues. Lastly, in Aim 3, we will further explore the role of specific immune cell populations as participants in the innate and adaptive responses to emissions-induced phospholipid modifications. In this Aim, we will utilize mouse models of immunodeficiency, including SCID and B-Cell deficient models. Additionally, we will pursue bone-marrow transplants from mice lacking those receptors described in Aim 2 to mechanistically establish the involvement of the oxidatively modified phospholipids.

Owing to suggestions from the advisory committee, we have focused on the nature and bioactivity of circulating factors induced by pollutant exposures, as these appear to be ligands that interact with the scavenger receptors of interest in Aims 2 and 3. This has been an area of significant progress for the past year.

Progress Summary:

The primary accomplishment of this past year was an investigation into the role of bloodborne ligands and their interaction with the CD36 scavenger receptor in driving systemic vascular effects resulting from ozone (O₃) exposure. O₃ was used as a model pollutant that has no direct access to the circulation, due to its high reactivity, and also because O₃ is an important contributor to the photochemical smog mixtures being developed in Project 2.

In brief, wildtype (WT) or CD36-null mice were exposed for 4 h to 1 ppm O₃ and studies were conducted 24 h post-exposure. Aortas were harvested and tested for relaxation responses to acetylcholine (ACh), and endothelium-dependent vasodilator. While O₃-exposed WT mice exhibited a reduction in the vasorelaxation response to ACh, CD36-null mice were protected (Figure 1). Followup studies revealed that CD36-null mice were also protected from pulmonary inflammation, which required the development of a novel method to test the contribution of circulating ligands on the vascular effects.

To accomplish this, we used a dilute serum mixture in the aortic ring baths, taking serum from exposed mice and treating aortas from unexposed mice. When we used unexposed WT aortas, we found that serum obtained from O₃-exposed mice – both WT and CD36-null – could impair aortic responses to ACh (Figure 2). However, when vessels were obtained from unexposed CD36-null mice, the circulating components failed to elicit a response. Combined, we concluded that (1) CD36 is involved in the pulmonary inflammatory response to O₃; (2) neither inflammation nor CD36 are required for the generation of circulating vasoactive factors; and (3) those circulating factors inhibit vasorelaxation via a CD36-dependent pathway.

In attempting to ascertain the nature of chemical changes in the circulation following pollutant exposure, we have examined cytokine levels via multiplex assay (Millipore). O₃ induced no changes in serum levels of all cytokines examined (Figure 3). Additionally, we examined 4-hydroxynonenal adducts on proteins and found those to be significantly elevated on a number of as-yet-unidentified proteins (bands on immunoblots). Proteomics work with Andrew Ottens of Virginia Commonwealth University has revealed a great number of altered small peptides (<5kD) in the serum, and we are investing efforts to link such changes with bioactivity.

Working closely with Project 2, we also implemented two long-term (50 day) studies to mixed vehicular emissions (combined gasoline and diesel exhausts). In one study, male ApoE-/- mice were exposed to whole MVE, MVE with particulate matter (PM) removed by filtration (MVE–PM), and MVE with gases removed by denudation (MVE–G). Analysis of those tissues is ongoing, but the results indicate a likely interaction between gases and particulate fractions of the whole emissions. For instance, the effects of MVE on vascular 3-nitrotyrosine and MMP9 protein levels was largely ameliorated by either denudation of gases or filtration of particles. MVE-mediated inflammation appeared reduced by denudation, but not PM filtration, though the statistical conclusions are hampered by a low subject number.

The second study examined the impact of MVE on older (18 months) versus younger (2 months) mice. These assays are still being conducted, but several important conclusions regarding vulnerability are arising. Among the more interesting findings was that serum-induced vasorelaxation impairments due to MVE exposure were only observed with serum from young mice (Figure 4), but mostly due to the fact that serum from old mice exposed to filtered air conditions also induced vasorelaxation impairments. That is, aging leads to the generation of vasoactive factors independent from environmental exposures. Other biological assays are pending, including cardiac function by SPECT/CT, cardiac gene changes by qPCR, and lavage endpoints.

Future Activities:

Aim 1: Compare potency of mixed emissions and photochemically transformed emissions in terms of serum inflammatory potential.
Aim 2: Explore roles of TLR4 and LOX-1 with O₃ responses, as per CD36. Consider all KO models with MVE exposures.
Aim 3: Develop assays to measure monocyte response to serum factors, as a parallel to the endothelial assays.

Expected Results: Findings will (1) indicate the most potent combinations of urban roadway and background co-pollutants in terms of vascular toxicity and (2) detail the role of the immune system in mechanistically driving the systemic effects of inhaled pollutants.

Journal Articles on this Report : 4 Displayed | Download in RIS Format

Publications Views
Other subproject views:	All 26 publications	16 publications in selected types	All 16 journal articles
Other center views:	All 197 publications	94 publications in selected types	All 93 journal articles

Publications
Type	Citation	Sub Project	Document Sources
Journal Article	Campen MJ, Lund A, Rosenfeld M. Mechanisms linking traffic-related air pollution and atherosclerosis. Current Opinion in Pulmonary Medicine 2012;18(2):155-160.	R834796 (2012) R834796 (2013) R834796 (2015) R834796 (Final) R834796C003 (2012) R834796C003 (2013) R834796C003 (Final) R833990 (2010) R833990 (2011)	Full-text from PubMed Abstract from PubMed Associated PubMed link Abstract: Lippincott Williams & Wilkins-Abstract Exit
Journal Article	Lund AK, Doyle-Eisele M, Lin Y-H, Arashiro M, Surratt JD, Holmes T, Schilling KA, Seinfeld JH, Rohr AC, Knipping EM, McDonald, JD. The effects of α-pinene versus toluene-derived secondary organic aerosol exposure on the expression of markers associated with vascular disease. Inhalation Toxicology 2013;25(6):309-324.	R834796 (2013) R834796 (2014) R834796 (2015) R834796 (Final) R834796C002 (2015) R834796C002 (Final) R834796C003 (2013) R833990 (2011)	Abstract from PubMed Abstract: Taylor & Francis-Abstract Exit Other: UNC at Chapel Hill-Abstract Exit
Journal Article	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.	R834796 (2013) R834796 (2014) R834796 (2015) R834796 (Final) R834796C003 (2013) R834796C003 (2016) R834796C003 (Final) R833990 (2011) R833990 (Final)	Full-text from PubMed Abstract from PubMed Associated PubMed link Full-text: Oxford Journals-Full Text HTML Exit Abstract: Oxford Journals-Abstract Exit Other: Oxford Journals-Full Text PDF Exit
Journal Article	Yin F, Lawal A, Ricks J, Fox JR, Larson T, Navab M, Fogelman AM, Rosenfeld ME, Araujo JA. Diesel exhaust induces systemic lipid peroxidation and development of dysfunctional pro-oxidant and pro-inflammatory high-density lipoprotein. Arteriosclerosis, Thrombosis, and Vascular Biology 2013;33(6):1153-1161.	R834796 (2013) R834796 (2014) R834796 (2015) R834796 (Final) R834796C003 (2013) R834796C003 (2016) R834796C003 (Final)	Abstract from PubMed Full-text: American Heart Association-Full Text HTML Exit Abstract: American Heart Association-Abstract Exit Other: American Heart Association-Full Text PDF Exit

Supplemental Keywords:

Coronary artery disease, oxidized phospholipids, atherosclerosis, particulate matter, volatile organic compounds, carbon monoxide, ozone, Scientific Discipline, Health, Air, ENVIRONMENTAL MANAGEMENT, Air Quality, air toxics, Health Risk Assessment, Risk Assessments, mobile sources, Environmental Monitoring, Biochemistry, Risk Assessment, ambient air quality, atmospheric particulate matter, particulate matter, aerosol particles, air pollutants, motor vehicle emissions, vehicle emissions, air quality models, motor vehicle exhaust, airway disease, bioavailability, air pollution, particle exposure, atmospheric aerosols, ambient particle health effects, vascular dysfunction, cardiotoxicity, atmospheric chemistry, exposure assessment

Relevant Websites:

Center for Clear Air Research

Progress and Final Reports:

Original Abstract

Main Center Abstract and Reports:

R834796 University of Washington Center for Clean Air Research

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R834796C001 Exposure Mapping – Characterization of Gases and Particles for ExposureAssessment in Health Effects and Laboratory Studies
R834796C002 Simulated Roadway Exposure Atmospheres for Laboratory Animal and Human Studies
R834796C003 Cardiovascular Consequences of Immune Modification by Traffic-Related Emissions
R834796C004 Vascular Response to Traffic-Derived Inhalation in Humans
R834796C005 Effects of Long-Term Exposure to Traffic-Derived Particles and Gases on Subclinical Measures of Cardiovascular Disease in a Multi-Ethnic Cohort

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The 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.