Stress erythropoiesis in atherogenic mice

Abstract

Bone marrow erythropoiesis is mainly homeostatic and a demand of oxygen in tissues activates stress erythropoiesis in the spleen. Here, we show an increase in the number of circulating erythrocytes in apolipoprotein E^-/- mice fed a Western high-fat diet, with similar number of circulating leukocytes and CD41⁺ events (platelets). Atherogenic conditions increase spleen erythropoiesis with no variations of this cell lineage in the bone marrow. Spleens from atherogenic mice show augmented number of late-stage erythroblasts and biased differentiation of progenitor cells towards the erythroid cell lineage, with an increase of CD71⁺CD41CD34^-CD117⁺Sca1^-Lin^- cells (erythroid-primed megakaryocyte-erythroid progenitors), which is consistent with the way in which atherogenesis modifies the expression of pro-erythroid and pro-megakaryocytic genes in megakaryocyte-erythroid progenitors. These data explain the transiently improved response to an acute severe hemolytic anemia insult found in atherogenic mice in comparison to control mice, as well as the higher burst-forming unit-erythroid and colony forming unit-erythroid capacity of splenocytes from atherogenic mice. In conclusion, our work demonstrates that, along with the well stablished enhancement of monocytosis during atherogenesis, stress erythropoiesis in apolipoprotein E^-/- mice fed a Western high fat diet results in increased numbers of circulating red blood cells.

Figure 1

Splenic Ter119⁺CD45⁻ and Ter119⁻CD45⁺ cells in ApoE^+/+ and ApoE^−/− mice fed with CD or with WD. (A) Number of Ter119⁺CD45⁻ and Ter119⁻CD45⁺ cells in spleen from ApoE^+/+ and ApoE^−/− mice fed either with a CD or with a WD for the last 13 weeks (n = 5, from 3 independent experiments). (B) Representative graphs showing the percentage of splenic Ter119⁺CD45⁻ and Ter119⁻CD45⁺ cells in ApoE^+/+ fed with a CD and ApoE^−/− mice fed with a WD, and percentage of Ter119⁺CD45⁻ and Ter119⁻CD45⁺ cells in spleens from mice described in A (n = 13–5, from 6–3 independent experiments). (C) Spleen weight from mice described in B. Two-tailed Student's t-tests were used for comparisons between the two groups (mean ± SEM; n = 13, from 6 independent experiments). **p < 0.01. (A,B) Data show the mean ± SEM. One-way ANOVA with Bonferroni correction was used to compare all pairs of columns between groups. ***p < 0.001.

Figure 2

Splenic and BM Ery. A, Ery. B, and Ery. C in ApoE^+/+ and ApoE^−/− mice fed with CD or with WD. (A) Number and percentage of Ery. A, Ery. B, and Ery. C in spleen from ApoE^+/+ mice and ApoE^−/− fed either with a CD or with a WD for the last 13 weeks. One-way ANOVA with Bonferroni correction was used to compare all pairs of columns between groups (mean ± SEM; n = 13–5, from 6–3 independent experiments), **p < 0.01, ***p < 0.001. (B) BM cellularity, number of Ter119⁺CD45⁻ cells and of Ery. A, Ery. B, and Ery. C in BM from ApoE^+/+ mice with a CD and ApoE^−/− fed with a WD for the last 13 weeks (mean ± SEM; n = 12–10, from 5 independent experiments).

Figure 3

Erythroid parameters and platelets in blood from atherogenic and control mice. (A) Number of circulating RBC from ApoE^+/+ and ApoE^−/− mice fed either with a CD or with a WD for the last 13 weeks (n = 13–6, from 6–2 independent experiments). One-way ANOVA with Bonferroni correction was used to compare all pairs of columns between groups. (B) Hematocrit, hemoglobin, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) determined in blood from ApoE^+/+ mice with a CD and ApoE^−/− fed with a WD for the last 13 weeks. Two-tailed Student's t-tests were used for comparisons between two groups (n = 7–7, from 3 independent experiments). (C) Epo mRNA levels in kidney and circulating Epo in mice described in B (n = 10–12, from 4–5 independent experiments). (D) Hepcidin mRNA levels in liver (n = 7–9, from 4 independent experiments) and circulating hepcidin in mice described in B (n = 7–10, from 4 independent experiments). (E,F) Number of circulating CD41⁺ events (E) and leukocytes (F) from mice described in B (n = 5–9, from 2–4 independent experiments). (A–F) Data show the mean ± SEM. (A,B) *p < 0.05, **p < 0.01.

Figure 4

Cell progenitors in spleen from atherogenic and control mice. (A) Graphs showing the number of live CD34⁺LSK (CD34⁺CD117⁺Sca⁺Lin⁻) cells, CMPs (CD34⁺CD117⁺Sca1⁻CD16/32⁻Lin⁻) GMPs (CD34⁺CD117⁺Sca1⁻CD16/32⁺Lin⁻), and MEPs (CD34⁻CD117⁺Sca1⁻CD16/32⁻Lin⁻) in spleens from ApoE^+/+ mice fed with a CD and ApoE^−/− mice fed with a WD for 13 weeks (n = 5, from 2 independent experiments). (B) Graphs showing the percentage of CD71⁻CD41⁻MEPs, CD71⁺CD41⁻MEPs, and CD71⁻CD41⁺MEPs respect to total splenic MEPs (n = 7, from 3 independent experiments), and a representative dot plot showing the staining of total splenic MEPs with CD71 and CD41. (C) Number of CD71⁺CD41⁻MEPs in spleens from mice described in A (n = 7, from 3 independent experiments). (D) Percentage of splenic CD41⁺ cells from mice described in A (n = 10–7, from 4 independent experiments). (A–D) Data show the mean ± SEM. Two-tailed Student's t-tests were used for comparisons between two groups.*p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Stress erythropoiesis in control and atherogenic mice. (A) Graphs showing the number of live CD34⁺CD133⁺CD117⁺Sca1⁺Lin⁻ cells in the spleen and BM from ApoE^+/+ mice fed with a CD and ApoE^−/− mice fed with a WD for 13 weeks (n = 5, from 3 independent experiments). (B) GDF15 and BMP4 mRNA levels in the spleen from mice described in A (n = 12, from 4–5 independent experiments). (C) Circulating RBC in mice described in A injected with 100 mg/kg mouse of PHZ (n = 7–8, from 2 independent experiments). Data show the significant differences between atherogenic and control mice within the same day. (A–C) Data show the mean ± SEM. (B,C) Two-tailed Student's t-tests were used for comparisons between two groups *p < 0.05, **p < 0.01.

Figure 6

Erythroid-colony forming assays of splenocytes and BM cells from control and atherogenic mice. Splenocytes and BM cells from ApoE^+/+ fed with a CD and ApoE^−/− mice fed a WD for the last 13 weeks were subjected to BFU-E and CFU-E analysis as described under “Materials and methods”. (A) BFU-E morphological characteristics (upper panel) and BFU-E counted after 9 days of incubation (lower panels). (B) CFU-E morphological characteristics (upper panel), CFU-E progenitors from mice described in (A) counted after 2 days of incubation (lower panels). (A,B) Data show the mean ± SEM, n = 4, triplicate in each assay. Two-tailed Student's t-tests were used for comparisons between two groups.*p < 0.05, **p < 0.01.