2008 Progress Report: Endothelial Cell Responses to PM—In Vitro and In Vivo
EPA Grant Number:
Subproject: this is subproject number 002 , established and managed by the Center Director under
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
San Joaquin Valley Aerosol Health Effects Research Center (SAHERC)
Wexler, Anthony S.
Endothelial Cell Responses to PM—In Vitro
and In Vivo
, Rutledge, John
University of California - Davis
EPA Project Officer:
October 1, 2005 through
September 30, 2010
(Extended to September 30, 2011)
Project Period Covered by this Report:
October 1, 2007 through September 30,2008
Particulate Matter Research Centers (2004)
The overall goal of this project is to determine the relationship between vascular disease and systemic effects of particulate matter.
We will evaluate the transcriptional responses to environmentally derived particulate matter in human endothelial cell cultures in the context of functional models of inflammation and coagulation system activation. Microarray transcriptional screening of environmental samples collected in the SJV Inhalation Exposure Project will identify characteristic endothelial cell responses. Based on these data, key gene transcripts with potential influence on pro-inflammatory and hemostatic activities will be used to develop a panel of RT-PCR based transcriptional assays. We will study the functional significance of these activities by examining PM-induced alterations in the endothelial cell permeability barrier and inflammatory cell interactions with the endothelium of intact vessels. To determine the significance of endothelial cell reactions in vivo, we will extrapolate our understanding of the endothelial cell transcriptional response to determine whether and where similar responses occur in concentrated ambient particle (CAPs) exposed animals using histology, laser capture microdissection, and RT-PCR assays of potential target organs and vessels. We will extend these results to cardiovascular disease using mice as models. We will determine whether PM selectively accumulates in atherosclerotic plaque from isolated arteries of predisposed ApoE-/- mice and determine with similar in vivo laser capture studies whether this selectively upregulates proinflammatory reactions in plaque of CAPs-exposed atherosclerotic mice.
Our previous work demonstrated that collected PM 2.5 stimulates pro-inflammatory and PAH metabolizing genes in cultured human endothelial cells. We further examined PAH response elements and demonstrated that HAEC respond to PM2.5 by activating AHR signaling. Finally, we examined the hypothesis that TGFβ family signaling was elicited by PM as it is by several other endothelial cell stimuli. We determined that PM2.5 does not stimulate TGFβ family second messenger responses. In the current year progress, we extended the gene response studies to cultured human bronchiolar cells and determined that similar proinflammatory and PAH response genes were upregulated but that signaling activites were less evident in epithelial cells. Overall, both cell types had only modest gene responses compared with treatments with endothelial cell toxins or lipolysis products of blood lipids. Based on evidence that napthoquinone (NQ) is a photo-oxidation product of vehicular emissions, we evaluated its ROS generating capacity as free compound or bound to proteins. We found a modest ROS generation in cultured cells that was markedly enhanced by pre-binding NQ to a sulfydryl containing protein. These results suggest that protein binding by reactive intermediates of PAH metabolism are not necessarily detoxifying reactions and that bound intermediates can retain ROS generating activity. This also implies that binding of reactive intermediates in organic fractions of airborne particulates may exert a stabilizing effect that enhances their toxicity.
In collaboration with project 3, we asked whether systemic inflammatory and pro-coagulant responses occurred in CAPs exposed mice. We found increased platelet numbers and an increased proportion of activated platelets in mice exposed for 2 weeks to CAPs. These findings were correlated with a modest upregulation of circulating cytokines involved with immune response regulation and bone marrow stimulation. We next asked whether PM interaction with monocytes might contribute to systemic cytokine secretion. We found upregulation of genes for several cytokine activities in human blood monocytes treated with collected PM2.5. Finally, we asked whether pulmonary expression of pro-inflammatory adhesion molecules could be detected by immunohistochemistry in CAPs exposed mice. While we demonstrated enhanced staining in postive control mice from ETS experiments, no significant differences were found in CAPs exposed mice. These findings extend the understanding of potential mechanisms of cardiovascular injury to the concept that pro-inflammatory activation of endothelium and moncyte-platelet-endothelial interactions could initiate thrombotic events in susceptible regions of arteries such as atherosclerotic plaque.
Specific aim 1: To characterize human endothelial cell culture responses to direct concentrated ambient PM 2.5 exposure.
Microarray gene analysis in cultured human aortic endothelial cells (HAEC) exposed to PM 2.5.
Characterize PAH receptor signaling in response to PM 2.5
Evaluate ROS generation and associated Nrf2 signaling in PM 2.5 treated HAEC
We extended our previous microarray gene response studies to cultured human bronchiolar cells and determined that similar pro-inflammatory and PAH response genes were upregulated to that previously found with endothelial cells but that signaling activites were less evident in epithelial cells (Figure 1). Overall, both cell types had only modest gene responses compared with treatments with endothelial cell toxins or lipolysis products of blood lipids.
Figure 1; Comparison of gene responses to collected ambient PM 2.5 (Winter Urban 2007). HAEC or HBEC were treated with 10 ul/ml for 4 hours.
Our studies with PAH receptor signaling are complete and currently in manuscript preparation. We are currently performing ROS assays and evaluating Nrf2 nuclear translocation in cultured aortic endothelial cells exposed to collected ambient PM 2.5. A related project evaluated the effects of napthoquinone, an apparent product of photo-oxidation of vehicular napthalene release, on ROS generation and Nrf2 signaling in cultured human pulmonary endothelial cells (HPAEC). This study examined the potential that protein binding of reactive intermediates such as NQ was a detoxifying event. We found instead that while NQ alone elicited a modest ROS response and stimulated Nrf2 translocation to the nucleus, tranfection of cells with NQ bound to a glutathione residue rich protein elicited a markedly increased ROS response (Figure 2).
Figure 2: ROS generation based on CM-H2DCFDA fluorescence in HPAEC treated with NQ, Galectin adducted with NQ or Monocrotaline (M).
Specific aim 2: To determine the effects of direct PM exposure on permeability and pro-coagulant activity in endothelium
Monolayer permeability in endothelial cell cultures
Platelet activation and Systemic markers of coagulation and inflammation
We have accomplished studies of platelet activation and systemic markers of coagulation and inflammation in CAPs exposures to mice. We performed preliminary studies with Summer Rural exposures in 2007 and found evidence of platelet activation. We did a more thorough study with the Winter Rural study in 2008 that included both platelet studies and multiplex immunoassays for systemic cytokines. Using a panel of 32 mouse specific bead associated antibodies for cytokines, we found 7 upregulated in exposed mice (Figure 3). Of these, three are general markers of inflammation, two markers of TH-2 responses, one TH-1 associated and one associated with bone marrow stimulation. Mice exposed to CAPs for 2 weeks had increases in blood platelet counts. Platelets from exposed mice had significantly more aggregates than control mice. More platelets bound fibrinogen in response to thrombin and a significantly increased percentage expressed a marker of platelet lysosomal secretion, LAMP-1 (Figure 4).
We have yet to fully evaluate effects of PM 2.5 on endothelial cell barrier permeability. We presently are performing experiments evaluating effects of PM on HAEC actin cytoskeleton and have developed preliminary data with a high throughput real time electrical resistance based permeability system that will allow us to compare effects of multiple synthetic and environmental source PM samples.
Figure 3: Significantly altered cytokine responses in blood of mice exposed to Winter Rural source CAPs for 2 weeks.
Figure 4: Platelet activation in blood of mice exposed to Winter Rural source CAPs for 2 weeks. Platelet from exposued mice had an aggregated population that was absent in control mice and significant increases in fibrinogen binding activity as well as lysosomal secretion (degranulation) as determined by surface expression of Lysosomal Associated Membrane protein 1 (LAMP-1).
Specific aim 3: To compare the nature and location of endothelial cell responses in pulmonary and cardiac tissue from CAPs exposed mice.
Cytokine secretion in BAL (Collaboration with project 3)
We have developed immunofluorescent probes for VCAM-1 and E-selectin and verified their use in lungs of mice exposed to ETS. Figure 5 demonstrates positive responses for E-selectin expression in arterioles of mice exposed for 2 weeks to ETS. Figure 6 shows a relative lack of responses in the lungs of mice exposed for 2 weeks to CAPs. An apparent upreguation of VCAM-1in airway epithelium of urban CAPs exposed mice from Winter 2007 (Figure 7) was not replicable in exposures from rural exposures in Winter 2008 (data not shown).
Figure 5 and 6: Expression of E-Selectin in lungs of mice exposed to either ETS or CAPs for 2 weeks.
Figure 7: Expression of VCAM-1 in airway epithelium in response to a 2 week CAPs exposure: A) Immunohistochemistry, B) Subjective evaluation of VCAM and E-Selectin Expression.
BAL cytokine assays were performed with material from mice exposed to Rural CAPs in Winter 2008. These assays did not detect significant levels of any of the cytokines in the panel in either control or treated mice. Future studies will require concentration methods to increase sensitivity.
Ongoing studies in specific aim 3 will compare immunohistochemical responses with HO-1 based on results with synthetic PM exposures in Project 1. Similarly, VCAM and E Selectin expression will be evaluated in cardiac vasculature of CAPs exposed mice.
Specific aim 4: To determine the effects of CAPs exposure on the progression of preexisting vascular disease in ApoE -/- mice.
We propose to significantly alter this aim with the intention to focus more on interaction of blood lipids, platelets and endothelium relative to PM and metabolic syndrome. In our revised aim, we will evaluate:
Monocyte upregulation of cytokines in response to collected PM 2.5
Phagocytosis and ROS production in monocytes exposed to collected PM 2.5
PM induced platelet-endothelial interaction in vitro.
We have completed evaluation of a variety of monocyte source cytokine activities by measuring gene expression in isolated human blood monocytes by RT-PCR. We find a mixture of upregulated and downregulated activities (Figure 8a and b). Ongoing studies are evaluating whether these responses are associated with PM phagocytosis using synthetic fluorescently labeled PM and Raman spectroscopic evaluation of monocytes exposed to collected PM 2.5. Preliminary fluorescence studies suggest synthetic silica particles are adherent but not phagocytosed during the time course of gene upregulation.
Figure 8a and b: cytokine gene responses to collected PM 2.5 (Winter Rural 2007) in isolated human blood monocytes treated with 50 ug/ml for 1 hour. A) Upregulated Activities. B) Downregulated activities
Proposed Studies Year 4:
Systemic Coagulation markers after intratracheal instillation of collected ambient PM 2.5: Supported also by CARB (Tablin & Wilson).
Evaluation of Monocyte Platelet and Endothelial cell interface in systemic coagulation in vitro.
Comparison of ROS generating capacity of differing source collected ambient PM 2.5 (Anastasio) vs. ROS generation and Nrf2signaling in cultured cells.
More focused evaluation of pro-coagulant mechanisms and activities in plasma in field exposure studies IL-11
Evaluate cytokines in BAL in collaboration with Project 3 and inflammatory mediator expression in airway epithelium and vasculature. Extend these studies to markers of ROS response elements.
Multiplex assay of endothelial cell barrier functional responses to synthetic and collected PM.
Microarray studies should provide significant insight into the specificity of responses to particulate matter. Signal transduction and endothelial cell functional studies will answer similar questions about specificity. These should help distinguish between non-specific oxidant injury and potential interference with specific endothelial cell activation pathways. The combination of gene and protein responses assayed in CAPs-exposed animals should provide significant evidence of PM-induced vascular proinflammatory and coagulant responses in normal tissues. Experiments with atheromatous mice exposed to CAPs should determine whether specific accumulation of PM in altered regions of vascular wall could contribute to the initiation of plaque rupture. They also will address the possibility that atheromatous plaque might be selectively susceptible to the induction of inflammatory responses to circulating PM.
No journal articles submitted with this report: View all 15 publications for this subproject
aerosol, ozone, exposure, health effects, human health, metabolism, sensitive populations, infants, children, PAH, metals, oxidants, agriculture, transportation,
, RFA, Health, Scientific Discipline, Air, particulate matter, Environmental Chemistry, Health Risk Assessment, Epidemiology, Risk Assessments, ambient aerosol, lung injury, air toxics, toxicology, long term exposure, lung disease, acute cardiovascular effects, airway disease, airborne particulate matter, human exposure, ambient particle health effects, epidemiological studies, PM, inhalation toxicology, vascular dysfunction, concentrated air particles, microarray studies, cardiovascular disease
Progress and Final Reports:
2006 Progress Report
2007 Progress Report
2009 Progress Report
2010 Progress Report
Main Center Abstract and Reports:
San Joaquin Valley Aerosol Health Effects Research Center (SAHERC)
Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R832414C001 Project 1 -- Pulmonary Metabolic Response
R832414C002 Endothelial Cell Responses to PM—In Vitro and In Vivo
R832414C003 Project 3 -- Inhalation Exposure Assessment of San Joaquin Valley Aerosol
R832414C004 Project 4 -- Transport and Fate Particles
R832414C005 Project 5 -- Architecture Development and Particle Deposition