Grantee Research Project Results
2009 Progress Report: Endothelial Cell Responses to PM—In Vitro and In Vivo
EPA Grant Number: R832414C002Subproject: this is subproject number 002 , established and managed by the Center Director under grant R832414
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: UC Davis Center for Children's Environmental Health and Disease Prevention
Center Director: Van de Water, Judith
Title: Endothelial Cell Responses to PM—In Vitro and In Vivo
Investigators: Wilson, Dennis , Rutledge, John
Current Investigators: Wilson, Dennis , Barakat, Abdul , Anastasio, Cort , Tablin, Fern , Rutledge, John
Institution: University of California - Davis
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2011)
Project Period Covered by this Report: July 1, 2008 through June 30,2009
RFA: Particulate Matter Research Centers (2004) RFA Text | Recipients Lists
Research Category: Human Health , Air
Objective:
Our previous work demonstrated that collected PM2.5 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 TGFb family signaling was elicited by PM as it is by several other endothelial cell stimuli. We determined that PM2.5 does not stimulate TGFb family second messenger responses. In the last 2 years, 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 winter rural 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 have completed evaluation of a variety of monocyte source cytokine activities by measuring gene expression in a human monocyte cell line by RT-PCR. We found that treatment with summer urban PM resulted in a mixture of upregulated and downregulated activities. Using fluorescent SiO2 particles, we found that particles were adherent but not phagocytosed during the time course of gene upregulation. 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 positive 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.
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
Progress Summary:
Specific aim 1: To characterize human endothelial cell culture responses to direct concentrated ambient PM2.5 exposure.
Microarray gene analysis in cultured human aortic endothelial cells (HAEC) exposed to PM2.5.
Characterize PAH receptor signaling in response to PM2.5
Evaluate ROS generation and associated Nrf2 signaling in PM2.5 treated HAEC
We have completed our comparison of the gene responses to collected samples of summer and winter urban PM in cell cultures of aortic endothelium and bronchial epithelial cells. The number of gene responses and their magnitude varied significantly between ambient particles collected in summer or winter. Samples collected in summer up-regulated significantly more genes in both target cells than did particles from winter. In general, the fold changes resulting from particles collected in summer were more robust than those from winter. The exception to this was the responses related to xenobiotic metabolism. Responses in endothelial cells included genes associated with inflammation and xenobiotic metabolism, specifically CYP 1A1, an aryl hydrocarbon response element related gene (Figure 1a and b). Associated xenobiotic response genes included aldehyde dehydrogenase and TCCD induced polyribosomal polymerase. Endothelial inflammatory responses included upregulation of E selectin and CCL2 (MCP-1). Endothelial cells also consistently responded by up-regulation of activating transcription factor 3, a transcription factor associated with the stress associated protein kinase/JNK subset of map kinase signaling.
Human bronchial epithelial cells also responded with a correlative mixture of pro-inflammatory genes and those involved with xenobiotic metabolism. In contrast to endothelium, HBEC upregulated CYP 1B1 and 1A1. Of particular importance relative to responses associated with asthma, HBEC had highest fold changes for the dendritic cell chemotactant, CCL20. Interleukin 8 and IL-1a were also prominent in the HBEC response (Figures 2A and 2B).
Given the unique signature of xenobiotic metabolism induction in response to PM, we further characterized activation of the AhR response element in assays demonstrating its nuclear translocation and transcriptional activation of a luciferase based AhR response element. Interestingly, both fold changes of CYP 1A1 and 1B1 as well as induction of the AhR luciferase response element (Figure 3) were higher in winter samples than those collected in summer. This is in contrast to genes more directly associated with inflammation which had more genes with significant responses and greater fold changes in cultures treated with summer source PM. This suggests that differing seasonal composition of PM alters the nature of cellular responses. Indeed, winter sample PM characterization demonstrates a higher concentration of PAH related compounds relative to summer samples (see Project 3 report for details).
Figure 1A. Gene responses in human aortic endothelial cells treated with ambient particulate matter collected from urban Fresno in summer 2006. Initial microarray results on pooled culture treatment samples (n=4) were verified by individual RT-PCR analysis.
Figure 1B. Gene responses in human aortic endothelial cells treated with ambient particulate matter collected from urban Fresno in winter 2007. Initial microarray results on pooled culture treatment samples (n=4) were verified by individual RT-PCR analysis.
Figure 2A. Gene responses in human bronchial epithelial cells treated with ambient particulate matter collected from urban Fresno in summer 2006. Initial microarray results on pooled culture treatment samples (n=4) were verified by individual RT-PCR analysis.
Figure 2B. Gene responses in human bronchial epithelial cells treated with ambient particulate matter collected from urban Fresno in winter 2007. Initial microarray results on pooled culture treatment samples (n=4) were verified by individual RT-PCR analysis.
Figure 3. Activation of AhR response element by ambient particulate matter collected from urban Fresno in summer 2006 or winter 2007. Recombinant mouse hepatoma (H1L1.1c2) containing a stably integrated DRE-driven firefly luciferase reporter gene plasmid whose transcriptional activation occurs in an AhR-dependent manner treated with APM or TCDD (positive control). Comparison with equivalent concentration of 30 nm SiO2 particles
Figure 4: Nuclear translocation assays for AhR. HAEC treated with either summer or winter urban source APM were stained with Immunofluorescent antibodies to AhR and the percent of cells with prominent nuclear staining determined by visual counts. TCDD (1nM in EtOH) was used as a positive control. A significant response in the vehicle control for TCDD was attributed to the reported EtOH activation of AhR. A) Representative images for cells treated with summer APM. B) Percent nuclear staining for summer APM treated cells 
In this reporting period we began to measure ROS generation by PM collected with high-volume samplers during the Project 3 animal exposures at Fresno and Westside. Our initial work has focused on measuring hydrogen peroxide (HOOH) from PM2.5 extracted in a cell-free, phosphate-buffered saline solution containing 50 μM ascorbate (a typical plasma level). As shown in Figure 5, both winter and summer fine particles from Fresno generate high levels of HOOH. This urban PM2.5 generates approximately 10 – 20 times more HOOH than does the rural PM2.5 collected from Westside. The results in Figure 5 are for samples representing the same volume of air sampled; normalizing the results to PM mass does not change the picture substantially, except for reducing the Fresno Summer 2008 (SU08) response because of high PM2.5 mass.
Additional experiments have shown that this generation of HOOH requires one or more transition metals: treatment of the PM samples with a strong metal chelator (desferrioxamine) reduces HOOH generation tremendously (e.g., by an average of 94% in the Fresno samples). Our current hypothesis is that soluble copper is responsible for HOOH generation in these samples, since our positive Cu control is very efficient at generating HOOH and the Fresno PM2.5 extracts have much greater levels of soluble Cu than do the Westside extracts.
We have also begun to quantify HOOH generation by Fresno and Westside particles in the presence of human aortic endothelial cells (HAECs). We have found a cell culture medium that does not destroy HOOH (surprisingly, our commonly used HAEC media destroys HOOH rapidly), but our initial experiments show that HAECs themselves quickly degrade HOOH. Experiments with a catalase-inhibitor indicate that this enzyme is not responsible for the HOOH destruction. We are working on examining other potential mechanisms, both so that we might inhibit them and measure HOOH generation, but also because these mechanisms might have implications for ROS-mediated cell toxicity.
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. Our initial study demonstrating activation of systemic cytokines and platelet priming is in final review for publication in Inhalation Toxicology. We have performed similar analyses for field exposures in urban Fresno in summer 2008 and winter 2009. While evidence of platelet priming was present in CAPs exposed mice from both these studies, the systemic cytokine response was not evident in either.
We have performed experiments to determine the effects of PM 2.5 on endothelial cell barrier permeability. We examined the effect of rural winter source APM on the actin cytoskelton of HAEC and in related experiments evaluated the activation of the Nrf2 oxidant response element. We found that ambient PM had no effect on either system in vitro (Figure 6). Similarly, we asked whether treatment with ambient PM altered endothelial cell monolayer barrier permeability using a high throughput real time electrical resistance based permeability system. We compared treatment with rural winter source APM with the response to an equivalent concentration of 30nM SiO2. Surprisingly, the SiO2 treatment had a significant effect on barrier function while APM, even at a relatively high concentration of 50 ug/ml was without effect (Figure 7).
Figure 6: Effects of APM treatment on HAEC monolayer actin cytoskeleton and Nrf-2 nuclear translocation. Positive control for cytoskeltal rearrangement was treatment with Thrombin while Nrf-2 positive control treatment used dithiothreatol (D3T).
Figure 7: Transendothelial impedance measurements in HAEC treated with either 10 or 50 ug/ml summer urban source APM or 10 ug/ml 30 nm SiO2 particles.
Specific aim 3: To compare the nature and location of endothelial cell responses in pulmonary and cardiac tissue from CAPs exposed mice.
We have completed evaluation of standard histology and immunohistochemical analysis of lung and heart sections from mice in the 2007-9 field studies that included summer and winter rural and urban exposures. There are no evident inflammatory changes in H+E sections and little evidence of endothelial cell activation by immunohistochemistry. We also performed microarray studies of RNA isolated from these field studies and found inconsistent responses in whole lung gene arrays. PCR analysis showed no significant responses in selected genes between control and CAPs exposed animals and very little response in laser capture studies of gene responses in specific lung sub-compartments was evident (Figure 8). We are in the process of embedding the nasal cavities to determine whether there is an inflammatory response that can explain the consistent priming of platelets we have seen in the last 3 studies.
Figure 8. A) Gene expression by microarray and RT-PCR from whole lung of mice exposed to rural winter CAPs. B) Gene expression by RT-PCR of airways, vessels and alveolar parenchyma isolated from frozen sections of lungs from mice exposed to winter urban CAPs using laser capture microdissection. Upregulation of CYP 1A1 was evident in some but not all CAPs exposed animals; this was not statistically significant and no quantitative changes in other genes examined were found.
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.
Specific aim 4: To determine the effects of CAPs exposure on monocyte activation
Our previous studies demonstrated upregulation of cytokines in winter urban APM treated monocyte cell lines (THP-1). We have confirmed these studies with freshly isolated human monocytes and extended them to ask whether a similar upregulation of AhR related gene responses occurs (Table 1). We found upregulation of CYP 1A1 in monocyte cell lines and an even more robust response in freshly isolate human monocytes. While treatment with SiO2 increased some pro-inflammatory genes, this response was markedly less than in response to PM treatment, especially in fresh monocyte isolations.
Table 1. Summary of gene expression from THP-1 and fresh monocytes treated with APM and SiO2.
|
|
mRNA Expression (fold change from water control)
|
|||
|
|
PM (50 µg/mL)
|
SiO2 (50 µg/mL)
|
||
|
|
THP-1 monocytes
|
Fresh monocytes
|
THP-1 monocytes
|
Fresh monocytes
|
|
TNFα
|
2.67*
|
3.6*
|
4.3*
|
1.24
|
|
IL-1β
|
8.92*
|
4.82*
|
2.68*
|
1.57*
|
|
IL-6
|
34.92*
|
1.23
|
7.61*
|
1.97*
|
|
IL-8
|
8.73*
|
10.54*
|
2.46*
|
2.54*
|
|
COX-2
|
2.77*
|
3.53*
|
2.48
|
1.81*
|
|
GM-CSF
|
16.47*
|
5.79*
|
6.21
|
1.63
|
|
NFκB
|
1.89*
|
2.25*
|
0.98
|
0.94
|
|
IL-10
|
0.58
|
1.07
|
0.70
|
1.23
|
|
IL-12
|
0.43**
|
1.15
|
1.21
|
4.89*
|
|
CCL3 (MIP-1α)
|
0.61
|
1.71
|
1.58
|
1.14
|
|
CCL5 (Rantes)
|
0.99
|
1.12
|
2.15
|
1.61*
|
|
CYP1a1
|
3.21*
|
26.54*
|
|
3.24
|
|
Aldh3a1
|
0.74
|
1.08
|
|
2.52
|
*Significantly higher than water control
**Significantly lower than water control
This work continues to evolve. Recent experiments with polymixin B demonstrate partial abrogation of inflammatory responses suggesting a component of endotoxin mediated signaling through induction of NFkb. Other components of the response, including CYP 1A1 induction, are unaffected.
Monocyte endothelial interactions in response to PM:
We asked whether treatment of either HAEC or monocytes with PM would alter monocyte adhesion to endothelial cell monolayers. We found that treating HAEC monolayers resulted in a modest increase in monocyte adhesion but that treatment of monocytes markedly increased their adhesion to untreated endothelial cell monolayers (Figure 9). We further asked whether PM exposed HAEC elaborated factors that activated monocytes and found a similar activation pattern to that seen with APM treatment of monocytes alone (Figure 10).
Figure 9. Effect of APM treatment on monocyte adhesion to HAEC monolayers. HAEC or Monocytes were pretreated with either 10 or 50 ug/ml winter urban APM
Figure 10. Effect of supernatant from winter urban APM treated HAEC cultures on monocyte activation. Gene responses evaluated by RT-PCR.
Future Activities:
Ongoing Questions:
Are the limited responses in field studies a consequence of dose, composition or nasal filtration?
Histopathologic evaluation of nasal cavities of mice from field studies
Intratracheal instillations of calculated equivalent cumulative doses
Comparison of IT responses to collected particulates vs. low and high PAH synthetic soot preparations. Supported also by CARB (Tablin & Wilson)
Comparison of field exposures with equivalent concentrations of low and high PAH synthetic soot
What mediator secreted by PM treated endothelium activates monocytes?
Multiplex analysis of culture supernatants
Selective deletion by antibody binding, specific inhibitors or siRNA downregulation
Does a similar endothelial response to PM activate platelets in vitro?
PM induced platelet – endothelial cell or monocyte adhesion in vitro
Measure activation of platelets co-cultured with PM treated EC or supernatants by flow cytometry.
What is the interface between ROS generation, CYP induction and AhR responses and inflammation?
Measure exogeneous hydroxyl radical (OH) generation by the Fresno and Westside particles in the presence and absence of HAECs
Determine intracellular ROS generation in response to PM treatment in vitro using flow cytometry
Characterize activation of endogenous ROS generating systems in response to varying source PM
Determine relative sensitivity of CYP induction and AhR activation in field source and synthetic low and high PAH content PM
Determine whether antioxidants, inhibitors of AhR and Stress response pathways alter upregulation of pro-inflammatory activities in epithelium, monocytes and endothelium.
Journal Articles:
No journal articles submitted with this report: View all 15 publications for this subprojectSupplemental Keywords:
Health, RFA, Scientific Discipline, Air, Health Risk Assessment, Risk Assessments, particulate matter, Environmental Chemistry, Epidemiology, ambient aerosol, lung disease, human exposure, long term exposure, airborne particulate matter, concentrated air particles, ambient particle health effects, epidemiological studies, PM, inhalation toxicology, toxicology, vascular dysfunction, acute cardiovascular effects, cardiovascular disease, human health risk, lung injury, microarray studies, airway diseaseProgress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R832414 UC Davis Center for Children's Environmental Health and Disease Prevention 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
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.