Episodic Exposure to Fine Particulate Air Pollution Decreases Circulating Levels of Endothelial Progenitor Cells
Rationale: Acute and chronic exposures to airborne particulate matter (PM) have been linked in epidemiological studies to a wide spectrum of cardiovascular disorders that are characterized by a dysfunctional endothelium. The pathophysiological mechanisms underlying these associations are unclear.
Objective: To examine whether exposure to fine PM with an aerodynamic diameter of <2.5 μm (PM2.5) affects the circulating levels of endothelial progenitor cell (EPC) populations, systemic inflammation and coagulation.
Methods and Results: Phenotypically distinct EPC populations were quantified by flow cytometry in young (18 to 25 years) adult humans exposed to episodic increases in PM2.5 along the Wasatch Mountain Front in Utah. In addition, Sca-1+/Flk-1+ cells were measured in the peripheral blood of mice exposed to concentrated particles from ambient air in Louisville, Ky. In both studies, PM exposure was negatively correlated with circulating EPC levels. In humans, statistically significant associations between PM2.5 exposure and the plasma levels of platelet–monocyte aggregates, high-density lipoprotein, and nonalbumin protein were also observed. Episodic increases in PM2.5 did not change plasma levels of C-reactive protein, interleukin-1β, interleukin-6, fibrinogen, or serum amyloid A.
Conclusions: Episodic exposure to PM2.5 induces reversible vascular injury, reflected in part by depletion of circulating EPC levels, and increases in platelet activation and the plasma level of high-density lipoprotein. These changes were also accompanied by an increase in nonalbumin protein and may be related to mechanisms by which exposure to particulate air pollution increases the risk of cardiovascular disease and adverse cardiovascular events.
Acute and chronic exposure to elevated levels of fine airborne particulate matter (PM) is associated with an increase in the incidence of adverse cardiovascular events,1,2 atherogenesis, cardiovascular disease (CVD) risk, and cardiovascular mortality. In urban environments, fine PM (PM with aerodynamic diameter of <2.5 μm [PM2.5]) is generated mostly by fossil fuel combustion in automobiles or by industrial processes. Although several mechanisms have been proposed to account for the link between PM exposure and CVD risk, endothelial dysfunction has emerged as a key feature of PM toxicity. Inhalation of concentrated PM2.5 induces acute conduit artery vasoconstriction in humans and chronic deficits in endothelium-mediated vasodilation in mice.1,2
The adult endothelium is a differentiated cell layer that provides a nonthrombotic interface between parenchymal cells and peripheral blood. Defects in its function arise because of the upregulated expression of proinflammatory and prothrombotic molecules or from defective, endogenous repair capacity. Evidence from multiple studies suggests that the endothelium is continually repaired by progenitor cells mobilized from specific niches such as the bone marrow. These cells express both endothelial and stem cell markers, and their circulating levels in blood are reflective of CVD risk and burden.3,4 The present study was designed to examine how exposure to PM2.5 affects endothelial progenitor cell (EPC) populations and whether this was associated with changes in systemic inflammation, coagulation, or plasma lipids.
For the human study, 16 (8 male and 8 female), young (18 to 25 years of age), nonsmoking, healthy (no existing acute or chronic disease) adults of normal weight (body mass index, 19 to 25) with no reported exposure to second-hand smoke were recruited in Provo, Utah. In the Utah Valley of the Wasatch Front, winter temperature inversion episodes elevate PM levels as emissions become trapped in a stagnant air mass near the valley floor. These episodes occur under somewhat predictable conditions that include a combination of snow cover, high barometric pressure, and low or falling clearing index.5 Arrangements were made with the research participants to have their blood drawn 4 times between January and early March of 2009 during a period of high pollution (PM2.5 >40 μg/m3), a period of moderate pollution (PM2.5, ≈20 to 40 μg/m3), and 2 periods of low pollution (PM2.5 <10 μg/m3) (Figure 1A). In the murine study, 28 C57BL/6 mice were exposed to either filtered air or PM2.5, concentrated ≈8-fold from ambient downtown Louisville air for 6 hours per day for 9 consecutive days (see the Online Data Supplement, available at http://circres.ahajournals.org) at 2 different times.
Ambient levels of PM2.5 recorded in January to early March of 2009 by 2 Utah Valley monitoring sites are shown in Figure 1A. A detailed characterization of changes in PM composition during winter inversion has been recently published.6 A substantial air pollution episode with peak PM2.5 concentrations occurred around January 22nd, with a more moderate episode approximately 10 to 14 days later and a return to baseline levels thereafter. Blood samples were obtained 4 times from each study participant to provide measurements at both high and low PM levels for each individual. EPC populations were identified by a 7-color cytometry procedure. The data were regressed on PM2.5 (average of 24 hours before blood draw) controlling for subject-specific fixed effects, using heteroskedasticity-consistent covariance matrix estimators.
The most abundant progenitor cell population (CD31+/CD34+) was negatively correlated with ambient PM2.5 levels (Table 1). In addition, CD45±CD133 cell populations also demonstrated negative associations with varying statistical strength. The strongest statistical correlation was observed for CD34+/CD31+/CD45+/CD133+ cells (Figure 1B). Some data sets showed differences in variability. For example, in Figure 1B, at PM2.5 concentrations of ≈35 μg/m3, there is much less variability in some data. These results suggest the need to estimate standard errors and probability values based on heteroskedasticity-consistent covariance matrix estimators. In most cases, these estimators resulted in slightly larger standard errors and corresponding probability values. Nevertheless, similar regression results were observed when these specific measurements were deleted from the regression analysis.
In addition to EPC levels, changes in systemic inflammation, coagulation, and plasma lipids were measured. As listed in Table 1, acute exposure to PM2.5 was significantly correlated with an increase in platelet (CD41a+)–monocyte (CD45+) aggregates and high-density lipoprotein (HDL) cholesterol levels. No associations with serum amyloid A and C-reactive protein (Table 1) or low-density lipoprotein cholesterol, triglycerides, fibrinogen, stromal cell-derived factor-1, interleukin-6, interleukin-1β, vascular endothelial growth factor, platelet factor-4 (data not shown) were observed. PM2.5 levels were, however, most strongly associated with nonalbumin plasma protein (NAP) levels (Table 1). Absolute levels, or levels of these proteins expressed as a percentage of total plasma protein, were both highly associated with PM2.5 (Table 1). Unlike associations of PM with platelet–monocyte aggregates and HDL, which were of marginal significance, there was no anomalous heteroskedasticity observed in the NAP data. Given the incredible strength of the statistical association (P<0.0001) and given the remarkable consistency across all subjects (Table 1, Figure 1C), the association between NAP and PM2.5 is unlikely to be an artifact of multiple hypothesis testing. Finally, there were no changes in markers of liver or skeletal muscle injury (data not shown), indicating that the effects observed are not symptoms of general tissue injury.
Because several confounding factors can influence the levels of circulating EPCs in humans, we examined PM-induced changes in mice. These mice were exposed to filtered air or concentrated ambient particles (CAPs) from downtown Louisville air for 9 days, and blood levels of Sca-1+/Flk-1+ cells were measured. As shown in Figure 2, CAPs exposure resulted in a ≈30–50% decrease in Sca-1+/Flk-1+ cells during the 2 exposure periods. CAPs exposure was also associated with significant increases in total cholesterol and HDL but no changes in NAP (Table 2). No significant changes in the number of bone marrow–derived EPCs were observed after 1 week in culture (Online Figure VI). Collectively, these findings suggest that exposure to particulate air pollution in mice decreases EPC levels.
The major finding of this study is that exposure to high PM2.5 levels induces reversible vascular injury, as evidenced by a suppression of circulating EPC levels in both humans and mice. In humans, this was also accompanied by an increase in platelet activation and elevated levels of plasma HDL and NAP. No significant changes in markers of systemic inflammation or tissue injury were observed. These results support the notion that suppression of EPC levels in peripheral blood may be an important feature and, perhaps, a significant mechanism of PM-induced cardiovascular injury. Moreover, EPC level may be a sensitive, albeit nonspecific, biomarker of endothelial injury caused by PM exposure.
Although the mechanisms by which PM exposure decreases the circulating EPC levels remain unclear, concurrent increases in thrombosis and HDL indicate that the loss of EPCs from peripheral blood may be attributable to endothelial injury. Consistent with this idea, the increase in NAP is likely reflective of an increase in globulin levels. After albumin, globulins are the second-most abundant proteins in the plasma, and an increase in their levels may be reflective of a mild systemic immune response.
Vascular dysfunction and endothelial injury are well-described effects of PM exposure.1,2 Our observation that PM decreased EPC levels in humans and mice exposed to similar doses (25 μg/kg for per 24 hours per day and 54 μg/kg for 6 hours per day, respectively), and despite potential compositional differences between Provo and Louisville air, suggests that suppression of EPC levels is a robust response to PM exposure. Previous studies show that chronic exposure to tobacco smoke, which contains high levels of PM2.5 and other pollutants, is also associated with low EPC levels.7 Thus, exposure to air particulates and/or their copollutants from several sources may have the general property of reducing circulating EPC levels. Further studies are required to identify specific PM components that might be related to EPC suppression.
The CD34+ cell population, which was significantly correlated with PM2.5 levels, has been shown previously to be associated with CVD risk.4 Moreover, stronger association with CD133+ than CD133− cells suggests that PM exposure affects the immature, early EPC population. This population shows a 5-fold reduction in patients with coronary artery disease and is predictive of adverse cardiovascular events in patients with preexisting CVD.4 Significantly, the levels of nonmonocytic EPC population (CD45−) cells were also suppressed on PM exposure. Therefore, depletion of multiple EPC populations could contribute to CVD risk imposed by PM2.5 exposure by inducing deficits in endothelial repair and angiogenesis. However, we could not study functional changes because the effects of PM on EPC levels in humans were reversible. Moreover, ex vivo assays to assess EPC function or proliferation require prolonged (7- to 21-day) culture, during which time the PM-induced changes are likely to be lost. Nevertheless, reversible suppression of EPC levels suggests that exposure to PM induces a transient mismatch between EPC utilization and recruitment. Given that this gap is robustly associated with several cardiovascular diseases,3,4 it appears likely that depletion of circulating EPCs, along with changes in blood coagulation, lipids, and nonalbumin proteins is reflective of vascular injury induced by PM, even in the absence of overt cardiovascular disease.
We thank Dr David Ingram (Indiana University School of Medicine) and members of his laboratory for their help and advice in EPC analysis.
Sources of Funding
This work was supported, in part, by grants from the Environmental Protection Agency (833336701), National Center for Research Resources (RR024489), National Institute of Environmental Health Sciences (ES11860), and contract W81XWH-10-1-0398 from U.S. Army Medical Research and Material Command (USAMRMC) and TATRC. C.A.P. is partially supported by the Mary Lou Fulton Professorship.
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Novelty and Significance
What Is Known?
Several epidemiological studies show that acute exposure to elevated levels of fine airborne particulate matter is associated with an increase in the risk of adverse cardiovascular events.
Controlled laboratory exposure to particulate matter has been reported to induce acute conduit artery vasoconstriction in humans and chronic deficits in endothelium-mediated vasodilation in mice.
Endothelial progenitor cells (EPCs) in peripheral blood contribute to postembryonic endothelial repair and regeneration and a decrease in circulating EPC levels is reflective of cardiovascular disease risk and burden.
What New Information Does This Article Contribute?
An increase in airborne particulate matter attributable to winter temperature inversion-episode in Utah Valley of the Wasatch Front was associated with a reversible decrease in circulating levels of EPC in a cohort of young (18 to 25 years) healthy adults.
The increase in particulate matter was also accompanied by an increase in plasma levels of platelet–monocyte aggregates, high-density lipoprotein, and nonalbumin protein. No changes in C-reactive protein, interleukin-1β, interleukin-6, fibrinogen, or serum amyloid A were observed.
Circulating levels of EPC were also decreased in mice exposed to concentrated airborne particles from downtown Louisville, Ky.
Particulate air pollution contributes to cardiovascular dysfunction and mortality, but the mechanisms for this remain unclear. Here, we show that episodic exposure to high levels of particulate matter decreased circulating EPCs in young adults and that this effect was reversible. These effects were accompanied by an increase in markers of thrombosis but no change in systemic inflammation. Exposure to concentrated PM also decreased circulating EPCs in mice. Consistent data between humans and mice at 2 locales suggests that depletion of circulating EPCs is a characteristic feature of PM exposure and may be one mechanism by which PM contributes to cardiovascular disease.
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Original received April 21, 2010; revision received June 17, 2010; accepted June 17, 2010.