2006 Progress Report: Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle CompositionEPA Grant Number: R832415C005
Subproject: this is subproject number 005 , established and managed by the Center Director under grant R832415
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Rochester PM Center
Center Director: Oberdörster, Günter
Title: Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle Composition
Investigators: Finkelstein, Jacob N.
Current Investigators: Finkelstein, Jacob N. , Prather, Kimberly A. , Rahman, Arshad , Oakes, David , Phipps, Richard
Institution: University of Rochester
Current Institution: University of Rochester , University of California - San Diego
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2012)
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
RFA: Particulate Matter Research Centers (2004) RFA Text | Recipients Lists
Research Category: Human Health , Air
The experiments proposed within this project are designed to address specific mechanistic hypotheses regarding the interactions between inhaled ultrafine particles and specific pulmonary cell populations. We will use cell lines and primary cells derived from rats and humans to test the hypothesis that increased morbidity and mortality in susceptible populations is due to the unique characteristics of ultrafine particles inducing oxidative stress and activation of target cells. It is further suggested that this ability is based on composition that is related to specific sources. The proposed in vitro experiments are intended to provide a link between the whole animal and controlled clinical (human) exposures, described in the 2006 individual Annual Reports of this Particle Center, by elucidating specific mechanisms that are triggered following particle cell contact and to test the specific hypothesis that many of the subsequent physiologic effects are the consequences of cellular oxidative stress, cell activation, and apoptosis.
Differential Responses to Particles by Endothelial Cells
During the current funding period our experiments have focused on a number of critical issues that relate to the overall goals of the Rochester PM Center. A key point of the proposed studies is the emphasis on real world particles in lieu of laboratory surrogates. The delay in the characterization of the HUCAPS concentrator in Rochester has delayed this effort somewhat. However, it has enabled us to continue mechanistic studies with defined composition particles that may ultimately be important in attributing effects seen in specific cell populations to unique sources.
The one source of real world particles available for in vitro studies was the material collected as part of the MAPS, multi-Center multisite particle collection effort begun during the previous grant period.
As shown in Figure 1a and b, vascular endothelial cells (HUVEC) exposed to concentrated fine and ultrafine particles respond through increased production of IL-6. This cytokine was chosen as a potential sentinel as a result of experiments from the animal and clinical studies core, which suggested a possible acute phase response following particle inhalation. Interestingly, using this marker and cell type we revealed a differential response from particles collected from certain sites. It is hypothesized that this relates to the abundance of vehicle emissions at these sites. More detailed analyses and source calculations are planned with the help of Core 1 as the compositional data are provided.
Figure 1. Response of Cultured Cells of Concentrated Ambient Particles
An additional important piece of data was revealed as a consequence of this study. When epithelial cells (A549 cells) were similarly exposed to these materials no differential response based on site selection was noted.
To verify this cell specific difference and to relate this potential mechanistic difference to studies from Cores 3 and 4, we carried out a direct comparison of the response of the epithelium and vascular endothelium with a series of well-characterized particles. We chose these carbon particles as they had previously been used for in vivo studies within the center (Figure 2). While these studies are interesting and important, they do not necessarily address the most relevant question, that of the response of the microvascular endothelium.
Figure 2. Production of IL-6 by HUVEC and A549 Cells Following Exposure to Various Types of Carbon Particles
Figure 3 begins to address this as we sought to establish a pulmonary microvascular endothelial cell culture system. The data shown compare culture of these primary cells on two different surfaces to determine how this would affect their cytokine production. We conclude that these microvascular cells are responsive to stimulation in vitro and thus present an appropriate model system to screen the activity of different particles.
Figure 3. Response of Microvascular Endothelial Cells to Varying Stimuli
Role of Oxidative Stress in the Response to Particles
A major hypothesis of this PM center is that cellular activation of cells following interaction with particles is a result of cellular oxidative stress. Studies in Core 4 show the direct ability of various types of particles to induce oxidative stress in an acellular milieu. This may be an important characteristic that differentiates the activity of various particle types but does not directly address the question of whether particles increase the oxidative stress response inside cells and whether particle uptake is a critical mechanism in cellular activation. We have addressed this in a number of ways. Using two fluorescent dyes that vary in their cellular permeability we have looked at both intracellular and extracellular dye oxidation after adding particles. In addition, we have assessed oxidative stress under conditions in which particle uptake is blocked. The result of these studies suggests that with ultrafine particles, uptake is required to induce cellular oxidative stress, regardless of composition.
Additional studies shown in Figure 4 suggest that cytokine production by cells following particle addition can be attenuated by prior incubation with both a soluble and lipophilic antioxidant treatment.
Figure 4. Antioxidants Reduce PM-Induced Cytokine Production
As part of our in vitro modeling effort in this core, we have begun to investigate various possible models of diabetic cells. It has recently been suggested that culture of cells in high glucose can mimic some of the altered responses seen in diabetes. In Figure 5, we examined the effect of high glucose on the expression of IL-6 by vascular endothelial cells. Using TNF as a test stimulus our data show a suppression of IL-6 when glucose is raised to 10 or 30 mM. As these studies continue, we will investigate the effect of glucose on particle-induced responses, including cytokines and oxidative stress.
Figure 5. Effect of Added Glucose on IL-6 Production by Vascular Endothelial Cells in Culture
In the coming year, we plan to continue to characterize the response of the microvascular endothelium to particles and begin to model the effects of diabetes on these responses. Recent studies suggest that hyperglycemia can significantly alter the oxidative stress response in the epithelium and we plan to assess the effect of this treatment on cytokine production following particles. Also, in collaboration with Core 4 we plan to determine the effect of serum isolated from diabetic animals, both PM exposed and controls, on the ability of vascular endothelial cells to be restimulated by concentrated PM. We also expect to extend these studies to cells of neuronal origin and possibly cardiac muscle. Measurement of prostaglandin production and COX-2 activation will be evaluated with respect to its usefulness as a marker.
Also, in support of the in vivo projects, we will evaluate in vitro effects of particles of differing composition. We will begin studies of concentrated real world particles and continue to examine the cytokine response to ultrafine and fine particles containing elemental carbon, metals, and organic carbon compounds.
Journal Articles:No journal articles submitted with this report: View all 13 publications for this subproject
Supplemental Keywords:endothelial cells, diabetic, glucose, oxidative stress,, RFA, Scientific Discipline, Health, PHYSICAL ASPECTS, Air, particulate matter, Genetics, Health Risk Assessment, Risk Assessments, Physical Processes, Biology, altered gene expression, atmospheric particulate matter, atmospheric particles, long term exposure, airway disease, exposure, human exposure, ambient particle health effects, atmospheric aerosol particles, PM, aersol particles
Progress and Final Reports:Original Abstract
Main Center Abstract and Reports:R832415 Rochester PM Center
Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R832415C001 Characterization and Source Apportionment
R832415C002 Epidemiological Studies on Extra Pulmonary Effects of Fresh and Aged Urban Aerosols from Different Sources
R832415C003 Human Clinical Studies of Concentrated Ambient Ultrafine and Fine Particles
R832415C004 Animal models: Cardiovascular Disease, CNS Injury and Ultrafine Particle Biokinetics
R832415C005 Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle Composition