Carnosine Supplementation Mitigates the Deleterious Effects of Particulate Matter Exposure in Mice

Abstract

Background Exposure to fine airborne particulate matter ( PM _2.5) induces quantitative and qualitative defects in bone marrow-derived endothelial progenitor cells of mice, and similar outcomes in humans may contribute to vascular dysfunction and the cardiovascular morbidity and mortality associated with PM _2.5 exposure. Nevertheless, mechanisms underlying the pervasive effects of PM _2.5 are unclear and effective interventional strategies to mitigate against PM _2.5 toxicity are lacking. Furthermore, whether PM _2.5 exposure affects other types of bone marrow stem cells leading to additional hematological or immunological dysfunction is not clear. Methods and Results Mice given normal drinking water or that supplemented with carnosine, a naturally occurring, nucleophilic di-peptide that binds reactive aldehydes, were exposed to filtered air or concentrated ambient particles. Mice drinking normal water and exposed to concentrated ambient particles demonstrated a depletion of bone marrow hematopoietic stem cells but no change in mesenchymal stem cells. However, HSC depletion was significantly attenuated when the mice were placed on drinking water containing carnosine. Carnosine supplementation also increased the levels of carnosine-propanal conjugates in the urine of CAPs-exposed mice and prevented the concentrated ambient particles-induced dysfunction of endothelial progenitor cells as assessed by in vitro and in vivo assays. Conclusions These results suggest that exposure to PM _2.5 has pervasive effects on different bone marrow stem cell populations and that PM _2.5-induced hematopoietic stem cells depletion, endothelial progenitor cell dysfunction, and defects in vascular repair can be mitigated by excess carnosine. Carnosine supplementation may be a viable approach for preventing PM _2.5-induced immune dysfunction and cardiovascular injury in humans.

Keywords: air pollution; endothelial progenitor cells; hematopoietic stem cells; ischemia.

Figure 1

Quantitative impact of CAPs exposure on mesenchymal stem cells and hematopoietic stem cell colony formation. Mice exposed to filtered air or CAPs for 9 days were euthanized and bone marrow cells were collected. MSCs in these isolates were quantified after culture in Mesencult and flow cytometry (A). Illustrated are the cumulative results (B: n=22 mice) and actual MSC numbers in 3 separate exposures of different CAPs levels (C; β: −0.0030; R2: 0.041; P: 0.180). From 16 exposures of different CAPs levels, HSCs were quantified in a colony‐forming assay (D). Illustrated is a representative colony (E) and the cumulative results (F; *P<0.05). Also illustrated (G; β: −0.0827; R2: 0.177; P<0.001) are actual colony counts in the filtered air (n=70 mice over 16 exposures) and CAPS‐exposed groups (n=4–7 mice per exposure). Levels of MSCs (C) and HSC colonies (G) in control mice inhaling filtered air are depicted at the 0 μg/m³ concentration. CAPs indicates concentrated ambient particles; HSCs, hematopoietic cells; MSCs, mesenchymal stem cells.

Figure 2

CAPs exposure increases urinary levels of carnosine‐propanal. Illustrated is the experimental outline (A). Mice were placed on normal drinking water or water supplemented with 1 mg/mL carnosine for 1 week before exposure to filtered air or CAPs. Urine was then collected for 12 hours before exposure and for 12 hours after a single‐day, 6‐hour exposure. Levels of carnosine‐propanal in the urine were measured as described in Methods and normalized to urinary creatinine. Illustrated are representative LC‐MS/MS ionograms obtained from pre‐exposure urine (B) and postexposure urine (C), where carnosine propanal elutes at ≈2.3 minutes. Also illustrated are the cumulative results (D). n=5; *P<0.05. C indicates carnosine‐containing water; N, normal water; LC‐MS/MS, liquid chromatography‐massspectrometry.

Figure 3

Carnosine supplementation limits HSC depletion. Illustrated is the experimental outline (A). Mice were placed on normal drinking water or that supplemented with 1 mg/mL carnosine for 1 week before exposure and then exposed to filtered air or CAPs for 9 days. Bone marrow cells were then isolated and HSC CFUs were quantified after culture in Colony Gel. Illustrated are the cumulative results (B; n=7–9 mice) and the average CFUs in 3 independent exposures (C). The average number of HSC colonies in control mice inhaling filtered air is depicted at the 0 μg/mL concentration (C). CAPs indicates concentrated ambient particles; CFUs, colony‐forming units; HSCs, hematopoietic cells.

Figure 4

Carnosine supplementation attenuates CAPs‐induced EPC dysfunction ex vivo. Illustrated is the experimental outline (A). Mice were placed on normal drinking water or that supplemented with 1 mg/mL carnosine for 1 week before exposure and then exposed to filtered air or CAPs for 9 days. Bone marrow cells were isolated and EPCs were cultured for 10 to 12 days and then used in functional assays. B, Illustrated are the relative doubling times of these cells normalized to filtered air controls. n=4 to 5; *P≤0.05. The cultured EPCs were also used in a tube formation assay. Illustrated are representative images (C) and quantitative data (D). n=5 to 7; *P<0.05. CAPs indicates concentrated ambient particles; EPCs, endothelial progenitor cells.

Figure 5

Carnosine supplementation attenuates CAPs‐induced defects in EPC‐mediated vascular reperfusion and tissue repair. A, Illustrated is the experimental outline. Mice were placed on normal drinking water or that supplemented with 1 mg/mL carnosine for 1 week before exposure and then exposed to filtered air or CAPs for 9 days. Bone marrow cells were isolated and EPCs cultured for 10 to 12 days and then injected into naïve mice that were subjected to HLI. Recovery of vascular perfusion in the injured limb was examined after 3 weeks by LDPI. Illustrated are representative LDPI images (B). The percent recoveries of vascular perfusion in HLI mice receiving cells from the various donors is also illustrated (C). n=7 to 24; *P<0.05. Isolated muscle sections from HLI mice injected with cells from the CAPs‐exposed donors were also stained with Sirius Red. Illustrated are representative images (D) and the relative staining intensity (E); n=4 to 5; *P<0.05. CAPs indicates concentrated ambient particles; EPCs, endothelial progenitor cells; HLI, hind limb ischemia; LDPI, Laser Doppler Perfusion Imaging.