Louisville PM2.5 and VACES characterization.
was concentrated using a VACES, and the CAP concentration varied between experiments as a function of ambient PM2.5
levels and meteorological conditions [e.g., temperature and relative humidity; see Supplemental Material, Table 1
)]. We observed little difference between ambient PM2.5
and chemical composition and physical properties of CAP (organic carbon, elemental carbon, sulfate, nitrate, ammonium, elements, particle size distribution; ). For example, ambient PM2.5
and CAP showed no significant difference of mass fraction (as a percentage) in chemical or elemental composition (; see also Supplemental Material, Table 2
). The number median diameter of ambient PM2.5
(0.33 µm for August 2009; 0.36 µm for June 2010) was similar to CAP number median diameter (0.53 µm, August 2009; 0.38 µm, June 2010) (). These data indicate the VACES concentrated ambient PM2.5
but did not alter its specific properties.
Figure 1 Chemical and physical characterization of ambient PM2.5 and CAP in Louisville, Kentucky. (A) Overall chemical composition of PM2.5 and CAP. (B) Overall particle size distributions as analyzed on the basis of equivalent number diameter. (C,D) Elemental (more ...) CAP exposure decreases the levels of circulating EPC.
Our previous study (O’Toole et al. 2010
) showed that 9-day CAP exposure decreases circulating levels of EPCs in mice; however, we did not examine either the nature or the mechanism of this response. Hence, to understand the effects of CAP on EPCs, we first examined the time course and reversibility of the response. EPC levels in the blood were measured by counting events positive for endothelial (Flk-1) and stem cell (Sca-1) antigens using flow cytometry (). Flk-1+
cells were gated in the lymphocytic population as indicated in the representative flow cytometry plots of side scatter and forward scatter (, top). The number of cells positive for both PE-Sca-1 and APC-Flk-1 (PE-Sca-1+
) was counted from plots, and the percentage of total gated cells isolated from mice exposed for 9 days to either HEPA-filtered air or CAP showed fewer EPCs following CAP exposure (, bottom). For additional characterization, blood cells were cultured for 4 days () and uptake of DiI-acLDL and binding to FITC-UE-lectin were analyzed. Microscopic images showed the outgrowth of DiI-acLDL+
spindle-shaped cells, as indicated by arrow in the merged images. Most of these cells were positive for both endothelial cell–specific markers (76 ± 7%; , merged images). Previous analysis shows that these cells are 3–5 µm in diameter and are positive for Id-1, CXCR4, and several other progenitor and lymphocytic cell markers (Wheat et al. 2011
Figure 2 CAP exposure decreases circulating levels of Flk-1+/Sca-1+ cells. (A) Representative flow cytometry plots of side scatter (SSC) and forward scatter (FSC) or PE-Sca-1+/APC-Flk-1+ cells isolated from mice exposed for 9 days (more ...)
To examine whether PM2.5
exposure leads to a progressive decrease in EPC levels, we exposed mice to CAP (6 hr/day) for 4, 9, or 30 days. As shown in , CAP exposure led to a progressive decrease in the number of circulating Flk-1+
cells; however, we observed no significant changes in the levels of Sca-1+
cells (). Complete blood count (CBC) analyses showed no effect of CAP exposure on the numbers of leukocytes or red blood cells [see Supplemental Material, Table 3
)]. Time-course analysis revealed a nonlinear relationship between dose and EPC level. A steep decline in EPC levels was observed within 4 days of CAP exposure (33% decrease; p
< 0.05); whereas longer exposures were associated with a shallower response. The half-maximal response duration (ED50
) calculated from a first-order exponential equation was approximately 12 days (). The dose–response relationship was nonlinear with an ED50
value of < 1 µg cumulative pulmonary CAP load (). Suppression of circulating EPC levels by CAP was reversible. After 9 days of exposure, a 7-day exposure-free period led to complete recovery of circulating Flk-1+
cells (). These observations indicate that exposure to CAP results in a rapid, specific, and reversible decrease in circulating EPC levels at low cumulative doses of CAP exposure.
CAP exposure does not induce EPC death or tissue deposition.
CAP-induced depletion of circulating EPCs could be due to an increase in cell death (apoptosis/necrosis), enhanced recruitment/homing to sites of injury, or a decrease in mobilization from bone marrow. Hence, we tested each of these possibilities. To determine whether CAP-induced EPC depletion was due to an increase in EPC death, we examined apoptosis and necrosis in the circulating Flk-1+
cell population by flow cytometry. Exposure to CAP depleted circulating Flk-1+
cells; however, we did not observe an increase in markers of apoptosis (Annexin-V+
) or necrosis (7AAD+
) compared with exposure to filtered air [see Supplemental Material, Figure 1A,B
)]. These results suggest that EPC depletion in CAP-exposed mice is not due to an increase in cell death.
To test the possibility that EPCs are depleted due to increased recruitment to sites of injury (e.g., lung) or other stem cell niches (e.g., spleen), we measured the abundance of the EPC-specific protein Id-1 (Mellick et al. 2010
) by Western blot analysis. Immunoblots of lysates prepared from lung, aorta, heart, and spleen showed no differences in the abundance of Id-1 protein in these organs after 9 days of exposure to air or CAP [see Supplemental Material, Figure 1C
)]. Thus, EPC depletion in CAP-exposed mice could not be attributed to an increase in EPC recruitment to sites of injury or other stem cell niches. Hence, we studied the effects of CAP exposure on EPC mobilization.
CAP exposure increases resident EPCs in bone marrow. We examined PM2.5-induced changes in bone marrow EPCs by culturing cells from the bone marrow on fibronectin-coated plates for 7 days. Nearly 90% of outgrowing spindle-shaped cells were positive for acLDL uptake and UE-lectin binding (). After 10 days in culture, these cells formed tube-like structures () when seeded on Matrigel, indicating that they had differentiated into endothelial cells. To determine the effects of PM2.5 on this cell population, we exposed mice to CAP for 9 days and measured bone marrow Flk-1+/Sca-1+ cells by flow cytometry (). We found that in contrast to decreased EPC levels in blood, CAP-exposure led to a significant increase in the number of resident EPCs in bone marrow ().
Figure 3 CAP exposure increases bone marrow resident Flk-1+/Sca-1+ cells. (A) Fluorescence images (top, 10×; bottom, 40×) of bone marrow-derived DiI-acLDL+/FITC-UE-lectin+ cells isolated from unexposed animals and cultured for 7 (more ...)
To confirm this finding, we isolated bone marrow from mice exposed for 9 days to CAP or filtered air and cultured the cells in fibronectin-coated dishes. Clusters of cells (cluster forming unit; CFU) appeared within 24 hr after seeding and were counted after 2, 4, and 7 days in culture (; typically 5–20 CFU/well). Cultures of bone marrow cells from CAP-exposed mice developed 50% more CFUs than those from mice breathing filtered air (). In both groups, the number of clusters dissipated within 7 days of culture, giving rise to spindle-shaped cells (), which stained positive for Sca-1 and Flk-1, as well as for acLDL and UE-lectin (). The number of Flk-1+/Sca-1+ cells or acLDL+/UE-lectin+ cells was greater in the bone marrow from CAP-exposed mice than in those from mice exposed to filtered air (). These data confirmed flow cytometry results (), showing that CAP exposure increases EPC levels in the bone marrow. Thus, suppression of circulating EPCs in CAP-exposed mice does not appear to be a consequence of EPC depletion in the bone marrow but perhaps is due to a defect in the release of EPCs from the bone marrow.
Figure 4 CAP exposure enhances the outgrowth of BMDCs isolated from mice exposed to air or CAP for 9 days. Phase contrast images (A) and quantification (B) of counted CFUs (fold change) after 2, 4, and 7 days of culture of BMDCs. (C) Fluorescence images of BMDCs (more ...) CAP exposure suppresses VEGF signaling.
The release of progenitor cells from a stem cell niche is a complex multistep process (Heissig et al. 2002
; Rafii and Lyden 2003
). EPCs are maintained in a quiescent state by contact with the stromal cells of the bone marrow and are mobilized after tissue injury in response to VEGF or stromal-derived factor-1α (SDF-1α). In the bone marrow, VEGF stimulates Akt, and this increases NO production by phosphorylating eNOS (Urbich and Dimmeler 2004
). We found that CAP exposure for 9 days did not affect a
) plasma levels of VEGF or SDF-1α [see Supplemental Material, Table 4
) acetylcholine-induced relaxation of the aorta isolated from these mice (see Supplemental Material, Table 5
); or c
) levels of VEGFR2 in the aorta (). VEGF (20 ng/mL for 15 min) stimulated significant increases in both Akt and eNOS phosphorylation in aortas isolated from mice breathing filtered air; however, these responses were abolished in VEGF-stimulated aortas of CAP-exposed mice (). From these data, we conclude that CAP exposure impairs vascular VEGF signaling.
Figure 5 CAP exposure impairs aortic VEGF-signaling. Representative Western blots and densitometric analysis of VEGFR-2 expression (A) and VEGF-induced phosphorylation of Akt (B) and eNOS (C). Western blots were developed from lysates of aortas from mice exposed (more ...)
We examined whether VEGF-induced mobilization of EPCs in mice was also affected by 9 days of CAP exposure. On days 6–9 of CAP exposure, mice received daily injections of VEGF; on day 9, 1 hr before euthanasia, mice received a single injection of AMD3100. Treatment with VEGF and AMD3100 (VEGF/AMD3100) led to a 2-fold increase in EPC levels in the blood of saline-injected mice exposed to filtered air; however, no significant increase in circulating levels of EPCs was observed in CAP-exposed mice (). Treatment with VEGF/AMD3100 did not affect circulating Sca-1+
cells or the CAP-induced retention of EPCs in the bone marrow [see Supplemental Material, Figure 2A,B
)]. These results indicate that CAP exposure prevents VEGF-induced EPC mobilization from bone marrow to peripheral blood.
Figure 6 CAP exposure impairs VEGF-induced but not SCF-mediated mobilization of bone marrow Flk-1+/Sca-1+ cells. (A) Representative flow cytometry plots of PE-Sca-1/APC-Flk-1 immunolabeling of mononuclear blood cells from mice exposed (more ...)
Stimulation of VEGFR2 in bone marrow results in MMP-9 activation, which in turn cleaves membrane-bound KitL to generate the soluble KitL (SCF or steel factor) (Heissig et al. 2002
). The binding of SCF to c-kit [chemokine receptor on stem cells (CD117)] on stem cells stimulates their mobilization (Thum et al. 2006
). We reasoned that if VEGF-induced release of membrane-bound SCF is inhibited but the events downstream of c-kit activation are not affected, then treatment with SCF should rescue CAP-induced EPC suppression. To test this hypothesis, we exposed mice to CAP for 9 days and then treated them with SCF and AMD3100 on the last day of exposure. Flow cytometry revealed that injection of SCF and AMD3100 led to a significant increase in the circulating level of Flk-1+
cells () in mice breathing filtered air, and moreover, SCF/AMD3100 treatment led to a significantly greater increase in the number of Flk-1+
cells in the blood of CAP-exposed mice. We observed no changes in the circulating levels of Sca-1+
cells () or other blood cells [see Supplemental Material, Table 6
)]; however, SCF/AMD3100 treatment also reversed the CAP-induced retention of EPCs in the bone marrow (see Supplemental Material, Figure 2C,D
). From these data we conclude that CAP exposure does not affect c-kit signaling and that the CAP-induced decrease in circulating EPC levels could be attributed, in part, to the inhibition of signaling events that are triggered by VEGF/VEGFR2 upstream of c-kit activation.