ApoE regulates hematopoietic stem cell proliferation, monocytosis, and monocyte accumulation in atherosclerotic lesions in mice

Leukocytosis is associated with increased cardiovascular disease risk in humans and develops in hypercholesterolemic atherosclerotic animal models. Leukocytosis is associated with the proliferation of hematopoietic stem and multipotential progenitor cells (HSPCs) in mice with deficiencies of the cholesterol efflux–promoting ABC transporters ABCA1 and ABCG1 in BM cells. Here, we have determined the role of endogenous apolipoprotein-mediated cholesterol efflux pathways in these processes. In Apoe^–/– mice fed a chow or Western-type diet, monocytosis and neutrophilia developed in association with the proliferation and expansion of HSPCs in the BM. In contrast, Apoa1^–/– mice showed no monocytosis compared with controls. ApoE was found on the surface of HSPCs, in a proteoglycan-bound pool, where it acted in an ABCA1- and ABCG1-dependent fashion to decrease cell proliferation. Accordingly, competitive BM transplantation experiments showed that ApoE acted cell autonomously to control HSPC proliferation, monocytosis, neutrophilia, and monocyte accumulation in atherosclerotic lesions. Infusion of reconstituted HDL and LXR activator treatment each reduced HSPC proliferation and monocytosis in Apoe^–/– mice. These studies suggest a specific role for proteoglycan-bound ApoE at the surface of HSPCs to promote cholesterol efflux via ABCA1/ABCG1 and decrease cell proliferation, monocytosis, and atherosclerosis. Although endogenous apoA-I was ineffective, pharmacologic approaches to increasing cholesterol efflux suppressed stem cell proliferative responses.

Atherosclerosis is initiated by deposition of cholesterol-rich lipoproteins in the artery wall, leading to monocyte-macrophage recruitment and chronic inflammation. Prospective studies have documented a robust, risk-adjusted association between leukocytosis and cardiovascular disease (CVD) (1). While atherosclerosis-associated inflammation might contribute to this relationship, there is also considerable evidence that leukocytosis directly enhances atherosclerosis and thrombosis (1). Numerous CVD risk factors — including obesity, smoking, sedentary lifestyles and metabolic syndrome (including individual components of metabolic syndrome, such as high triglycerides and low HDL) — are all associated with leukocytosis (2–4). While these associations have usually been made for total wbcs or neutrophils, monocytosis has also been specifically associated with CVD and with atherosclerotic plaque burden in both prospective and cross-sectional studies (5–8). Monocytosis has also been linked to atherosclerosis in animal models: Gerrity first noted the relationship among dietary hypercholesterolemia, monocytosis, and atherosclerosis in swine and rabbit models (9, 10) and described an increase in BM colony forming units in hypercholesterolemic pigs (10). In the Apoe^–/– mouse model of atherosclerosis, dietary hypercholesterolemia is associated with progressive monocytosis and an increase in the Ly-6C^hi (CCR2⁺) subset of monocytes, which enter lesions more readily than do Ly-6C^lo monocytes (11, 12). Emerging evidence also suggests that neutrophilia can promote early lesion development (13, 14). Monocytosis in Apoe^–/– mice has previously been shown to involve both increased production and decreased clearance of cells (11), but the detailed mechanisms underlying monocytosis and neutrophilia are poorly understood.

The ATP-binding cassette transporters ABCA1 and ABCG1 play an important role in mediating cellular cholesterol efflux to apoA-I and HDL, respectively (15). The absence of ABCG1 and ABCA1 in BM-derived cells in combination with hypercholesterolemia results in markedly accelerated atherosclerosis (16). These mice display dramatic expansion of myeloid cells, monocytosis, and neutrophilia, with an underlying proliferative defect of hematopoietic stem and multipotential progenitor cells (HSPCs) (17). HSPC hyperproliferation was linked to increased plasma membrane lipid rafts and increased plasma membrane levels of the common βsubunit of the IL-3/GM-CSF receptor (CBS; also referred to as IL-3Rβ or CD131). Reversing the defect in cholesterol efflux via marked overexpression of transgenic Apoa1 resulted in reduced expression of the CBS on HSPCs and abolition of the myeloproliferative phenotype (17). However, these studies did not define the role of endogenous apolipoproteins in controlling HSPC proliferation. As noted above, ApoE has a prominent role in controlling leukocytosis, and ApoE and ApoE-containing HDL interact with ABCA1 and ABCG1, respectively, to promote cellular cholesterol efflux (16, 18, 19), but ApoE has not been linked to the control of HSPC proliferation. The goal of this study was to assess the role of endogenous apoA-I and ApoE in the control of HSPC proliferation and monocytosis.

Hypercholesterolemia-associated leukocytosis is associated with expansion of HSPCs in Apoe^–/– mice. Previous studies have documented neutrophilia and monocytosis in Apoe^–/– mice, especially after feeding a high-fat, high-cholesterol Western-type diet (WTD) (11, 12). To determine whether this reflects HSPC proliferation, we analyzed blood and BM myeloid populations in Apoe^–/– and Ldlr^–/– mice in response to feeding a WTD for 10 or 20 weeks (Figure 1). Apoe^–/– mice fed the WTD developed prominent monocytosis and neutrophilia (Figure 1, B and C; for absolute values, see Supplemental Figure 1, A and B; supplemental material available online with this article; doi: 10.1172/JCI57559DS1). This was paralleled by a marked 3-fold expansion of the HSPC population in the BM (Figure 1, D and E), as well as an increase in GM-CFUs (Figure 1F) and increased proliferation of BM myeloid cells (as measured by [³H]-thymidine incorporation into DNA; Figure 1G).

Figure 1

Feeding Ldlr^–/– and Apoe^–/– mice a WTD induces leukocytosis, expansion of HSPCs, increased GM-CFUs, and proliferation of BM myeloid cells. 8-week-old mice were fed a WTD for the indicated time periods. (A) Plasma cholesterol levels. (B and C) Monocytes and neutrophils were analyzed by flow cytometry and expressed as a percentage of CD45⁺ leukocytes. (D and E) The HSPC population was assessed by flow cytometry and is expressed as a percentage of total BM cells. (F) GM-CFU assays from isolated BM. (G) BM proliferation was evaluated by [³H]-thymidine incorporation into DNA. (A–G) *P < 0.05 vs. 0 weeks; ^{^}P < 0.05 vs. Ldlr^–/– at the respective time point. Data are mean ± SEM, n = 6–8. (H) WT, Apoa1^–/–, Apoe^–/–, and Apoa1^–/–Apoe^–/– mice were fed a chow diet until 20 weeks of age. The population of blood monocytes was identified by flow cytometry. *P < 0.05 vs. WT. Data are mean ± SEM, n = 5–8.

In contrast to the Apoe^–/– mice, Ldlr^–/– mice after 10 weeks on the WTD exhibited no significant monocytosis, slight neutrophilia (Supplemental Figure 1B), no expansion of HSPCs, and a 2-fold increase in BM myeloid proliferative response to exogenous growth factors (Figure 1G). After 20 weeks on the WTD, Ldlr^–/– mice displayed modest increases in all of these parameters. There were significant genotype-specific effects for all blood and BM parameters (i.e., greater responses in Apoe^–/– than in Ldlr^–/– mice) at both 10 and 20 weeks (P < 0.05, ANOVA), even though blood cholesterol levels were higher in WTD-fed Ldlr^–/– than Apoe^–/– mice (Figure 1A). These findings suggest that increased proliferation and expansion of HSPCs may be at least partly responsible for the monocytosis and neutrophilia that develops in Apoe^–/– and Ldlr^–/– mice after feeding the WTD. The effects were much more prominent in Apoe^–/– mice, suggestive of a specific role of ApoE in suppressing HSPC and BM myeloid proliferation.

Deficiency of Apoa1 in mice and in humans with Mendelian disorders causing low HDL is not associated with monocytosis. To directly compare the role of endogenous ApoE and apoA-I in control of HSPC proliferation and monocytosis, we assessed these phenotypes in chow-fed WT, Apoa1^–/–, Apoe^–/–, and Apoe^–/–Apoa1^–/– mice. Whereas Apoe^–/– mice showed significant monocytosis, there was no monocytosis in Apoa1^–/– mice, and no additional monocytosis was observed when Apoa1^–/–Apoe^–/– were compared with Apoe^–/– mice (Figure 1H and Supplemental Figure 1C). There was also no expansion of HSPCs in Apoa1^–/– compared with WT mice, and although ApoA1^–/–Apoe^–/– mice did have expansion of their HSPCs compared with WT mice, this was not further increased compared with Apoe^–/– mice (data not shown). Plasma cholesterol levels were lower in Apoa1^–/–Apoe^–/– than in Apoe^–/– mice (247 ± 14 mg/dl vs. 418 ± 38 mg/dl; mean ± SEM), consistent with previous reports (20), and this could have contributed to the lack of difference in monocytes between these groups. Nonetheless, these collective findings suggest that the role of endogenous ApoE is more important than that of apoA-I in controlling HSPC proliferation. We also determined whether there was significant monocytosis in humans with Mendelian disorders causing extremely low HDL cholesterol (HDL-C) and apoA-I levels. In a small study of humans with homozygous deficiencies of Abca1 (Tangier disease) or lecithin-cholesterol acyltransferase (Lcat), there was no increase in blood monocyte or neutrophil counts (Supplemental Table 1).

A proteoglycan pool of ApoE on the surface of HSPCs controls cell proliferation. While Apoe^–/– mice have elevated cholesterol and reduced HDL levels, our experiments pointed to a more specific role of ApoE in controlling HSPC proliferation. To determine whether ApoE is produced in HSPCs, we assessed in vivo mRNA levels in different populations of BM myeloid cells from WT mice with and without LXR activator treatment. Apoe showed highest expression in HSPCs relative to lineage committed progenitor populations, and was induced in HSPCs, and to a lesser extent in granulocyte-macrophage progenitors (GMPs), by LXR activator treatment (Figure 2A). In contrast, Apoa1 mRNA was not detected in HSPCs. The HSPC population comprises long-term repopulating HSCs (LT-HSCs), short-term repopulating HSCs (ST-HSCs), and the multipotential progenitor (MPP) cell MPP1–MPP3 populations (21, 22). Analysis of different populations within the HSPC fraction showed that Apoe was most highly expressed in the stem cell–containing populations — in descending order, LT-HSCs and ST-HSCs/MPP1s, followed by MPP2s, and finally MPP3s — and was upregulated by LXR treatment in LT-HSC, ST-HSC/MPP1, and MPP2 subsets (Figure 2B). These findings are consistent with an earlier report based on microarrays, suggesting higher expression of Apoe mRNA in HSPCs relative to MPP cells (23). In contrast, Abca1 and Abcg1 were highly expressed and strongly induced by LXR activation in all HSPC subsets, with lower expression in myeloid progenitor populations (Supplemental Figure 2, A and B). Interestingly, flow cytometry revealed a pool of ApoE protein on the surface of WT HSPCs that was increased 2.8-fold after LXR activator treatment (Figure 2C). Cell surface ApoE was more prominent in the stem cell populations (i.e., LT-HSCs and ST-HSCs/MPP1s) than in MPP2s or MPP3s (Figure 2D). Combined treatment with heparinase to remove cell surface proteoglycans, and with 4-methylumbelliferyl β-D-xyloside (β-DX) to prevent their resynthesis (24), resulted in substantial loss of ApoE from the surface of HSPCs (Figure 2E), which suggests that ApoE was bound to proteoglycans on the surface of HSPCs.

Figure 2

Apoe is expressed on the surface of HSPCs and interacts with ABCA1 and ABCG1 to modulate HSPC proliferation. (A–D) WT mice were injected with saline or LXR activator (T0901317, 25 mg/kg body weight). (A and B) HSPC and progenitor populations were isolated from the BM, cDNA was prepared, and different mRNAs were quantified by real-time PCR. Data are mean ± SEM (n = 5). (A) Expression in stem and progenitor cell subsets. *P < 0.05 vs. vehicle. (B) BM was subjected to FACS to obtain LT-HSC (LT), ST-HSC (ST) and MPP1, MPP2, and MPP3 subsets. *P < 0.05 vs. vehicle; ^{^}P < 0.001 vs. LT; ^#P < 0.001 vs. ST. (C and D) ApoE protein was measured on the surface of (C) HSPCs and (D) HSPC subsets via flow cytometry. **P < 0.01 vs. vehicle. (E and F) BM was isolated, ApoE was cleaved from the surface using heparinase, and resynthesis of HSPGs was prevented using β-DX. BM cells were then incubated for 12 hours in IMDM with EdU (1 μM) with or without IL-3 and/or LXR activator (3 μM). (E) ApoE surface expression on HSPCs was measured via flow cytometry. (F) HSPC proliferation was determined by EdU incorporation. *P < 0.05, **P < 0.01, ***P < 0.001 vs. same treatment without heparinase; ^{^^}P < 0.01 vs. no treatment. Data are mean ± SEM.

To assess the hypothesis that the cell surface pool of ApoE acts to control proliferation of HSPCs, we measured proliferative responses after removal of heparan sulfate proteoglycans (HSPGs), including under conditions of LXR activation to increase cell surface ApoE and addition of exogenous growth factor (IL-3) to stimulate proliferation. Under all conditions, heparinase treatment led to significantly increased proliferation of WT, but not Apoe^–/–, HSPCs (Figure 2F). This indicates that proteoglycan-bound cell surface ApoE acts to suppress the proliferation of HSPCs. Heparinase treatment of Abca1^–/–Abcg1^–/– HSPCs, albeit relatively effective at removing ApoE, did not increase their proliferation under any condition. Even though HSPC proliferation was greatest in Abca1^–/–Abcg1^–/– compared with the other genotypes under basal conditions, this was still significantly increased in the presence of IL-3, which indicates that the cells’ proliferative responses could still be modulated. This suggests that proteoglycan-bound ApoE requires expression of ABCA1 and ABCG1 to exert an antiproliferative effect, most likely by stimulating cholesterol and/or phospholipid efflux via the transporters.

ApoE acts in a cell-autonomous fashion to regulate myelopoiesis. The ability of ApoE to suppress HSPC proliferation in vivo could be mediated either by exogenous ApoE derived from the circulation or by ApoE made in HSPCs acting to control their own proliferation (Figure 2, E and F). To test the hypothesis that ApoE acts in vivo in an autocrine or cell-autonomous fashion, we performed competitive BM transplantation experiments. Equal portions of BM from WT CD45.1 and WT CD45.2 mice or from WT CD45.1 and Apoe^–/– CD45.2 mice were transplanted into WT or Ldlr^–/– recipients and, after BM reconstitution, were fed WTD for 10 weeks (Figure 3A). We chose Ldlr^–/– rather than Apoe^–/– recipients since introduction of WT BM into Apoe^–/– mice results in marked lowering of plasma cholesterol levels (25, 26) that would act as a confounding variable affecting myeloid proliferation (i.e., mixture 1 would contain more WT BM and thus produce lower cholesterol levels than mixture 2). We confirmed that plasma cholesterol levels were indeed similar for recipients of WT+WT and WT+Apoe^–/– BM (WT recipients, 138 ± 6 and 147 ± 4 mg/dl, respectively; Ldlr^–/– recipients, 1,287 ± 97 and 1,552 ± 80 mg/dl, respectively; n = 6, mean ± SEM; P = NS for BM donor genotype effect). In WT recipients with very mild hypercholesterolemia, there was a greater contribution of Apoe^–/– BM to blood monocytes and neutrophils, but only after prolonged WTD feeding (6–10 weeks; Figure 3, B and D). In hypercholesterolemic Ldlr^–/– recipients, a competitive advantage of the Apoe^–/– BM–derived blood monocytes and neutrophils was observed in moderately hypercholesterolemic conditions when mice were on a chow diet (i.e., 0 weeks), and this was markedly enhanced when the mice were fed WTD (Figure 3, C and E). Analysis of HSPCs and myeloid progenitors revealed increased numbers of Apoe^–/– BM–derived (i.e., CD45.2) HSPCs, myeloid progenitors, monocytes, and neutrophils in both BM and spleen (Figure 3, F and G, and Supplemental Figure 4, A–F). Consistent with the findings on blood leukocytes, the cell-autonomous expansion of HSPCs, common myeloid progenitors (CMPs), and granulocyte-macrophage progenitors (GMPs) was much more prominent in hypercholesterolemic BM recipients than in chow-fed recipients. These findings revealed a cell-autonomous proliferative advantage of Apoe^–/– HSPCs and myeloid progenitors in the BM and spleen, especially in a hypercholesterolemic environment, as an underlying cause of leukocytosis in hypercholesterolemic Apoe^–/– mice.

Figure 3

Leukocytosis and expansion of BM stem cells in Apoe^–/– mice is cell autonomous and enhanced in a hypercholesterolemic environment, resulting in preferential accumulation of BM-derived cells in atherosclerotic lesions. (A) Experimental overview. Equally mixed portions of BM from the respective genotypes were transplanted into either WT or Ldlr^–/– mice, which were fed a WTD for 10 weeks. (B–G) Ratio of CD45.2/CD45.1 monocytes over time (B and C), CD45.2/CD45.1 neutrophils over time (D and E), and CD45.2/CD45.1 BM stem cell populations (F and G) in WT (B, D, and F) and Ldlr^–/– (C, E, and G) recipients. (H and I) Accumulation of CD45.1 or CD45.2 BM-derived cells in atherosclerotic lesions in Ldlr^–/– recipients. (H) Representative stacked images of lesions from serial sections — stained for CD45.1 or CD45.2 (green); nuclei stained with TO-PRO-3 (blue) — presented as a combined overlay. Transmitted light images were all obtained by confocal microscopy (original magnification, ×20). (I) Accumulation of BM-derived cells (CD45.1- or CD45.2-positive cells per lesion) presented as a ratio of CD45.2⁺ to CD45.1⁺ cells in the lesion. Analysis was performed on 2 serial sections for either CD45.1 or CD45.2 cells for each mouse. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT/WT. Data are mean ± SEM (n = 6).

Cell-autonomous expansion of Apoe^–/– HSPCs and leukocytosis results in increased leukocyte accumulation in atherosclerotic lesions. Previous studies have shown that hypercholesterolemia-induced monocytosis in Apoe^–/– mice is associated with increased monocyte entry into atherosclerotic plaques (11, 12). To determine whether the cell-autonomous expansion of monocytes also leads to increased accumulation of the Apoe^–/– population of monocytes in plaques, we analyzed lesions from Ldlr^–/– recipients. There was no preferential accumulation of WT CD45.1– versus WT CD45.2–specific BM–derived cells in the lesions of Ldlr^–/– recipients (assessed as the percentage of nuclear stained cells that also stained for CD45.1 or CD45.2 in serial sections). However, there was a clear preponderance of Apoe^–/– CD45.2–positive over WT CD45.1–positive cells (Figure 3, H and I), indicative of preferential accumulation of Apoe^–/– BM–derived cells in plaques of Ldlr^–/– mice that had been transplanted with mixed WT and Apoe^–/– BM. Lesions showed positive staining with CD68, but not Ly6-G (data not shown), which indicated that accumulating cells were predominantly monocytes/macrophages, not neutrophils. As expected, the lesions in Ldlr^–/– recipients transplanted with Apoe^–/– CD45.2 BM were significantly larger than those in mice transplanted with WT CD45.2 BM (Supplemental Figure 5). These findings suggest that the increased cell-autonomous proliferation of Apoe^–/– HSPCs led to monocytosis and increased entry of monocytes into lesions, contributing to the formation of larger lesions.

To determine a possible role of other mechanisms contributing to the preferential accumulation of Apoe^–/– CD45.2 cells in lesions, we assessed some of the key steps of monocyte recruitment and entry into the lesion. This is a potentially important process in atherosclerotic lesion development, as ApoE appears to have little effect on the ability of macrophages to leave lesions (27). We found that Apoe^–/– Ly6-C^hi monocytes had slightly increased expression of VLA-4 (17%), but not of other cell surface activation markers (Supplemental Figure 6, A–D). This was associated with an approximately 35% increase in Apoe^–/– Ly6-C^hi monocyte adhesion to endothelial cells (Supplemental Figure 6, E and F). Ly6-C^lo monocytes were less adhesive than Ly6-C^hi cells, and there was no difference between the genotypes. Importantly, there was no difference in the migration of WT versus Apoe^–/– Ly6-C^hi (CCR2⁺) monocytes in response to MCP-1 (CCL2) in a Transwell migration assay (Supplemental Figure 6, G and H). These findings suggest that the increase in Apoe^–/– CD45.2 cells in lesions may mainly reflect increased circulating monocyte numbers.

Increased membrane cholera toxin B staining, and higher levels of the CBS on the surface of Apoe^–/– HSPCs, with enhanced signaling through ERK1/2 and STAT5. To determine whether deletion of Apoe in HSPCs causes reorganization of membrane lipids, we performed cholera toxin-B (CTx-B) staining. There was an increase in CTx-B staining of HSPCs from Apoe^–/– mice (Figure 4A), consistent with an association between increased lipid raft formation and increased membrane cholesterol content (28–30). There also appeared to be an increase in neutral lipid in the HSPCs of Apoe^–/– mice, as shown by increased staining with BODIPY 493/503 (Supplemental Figure 7 and refs. 31, 32). Increased incorporation of 5-ethynyl-2′deoxyuridine (EdU) into HSPCs (Figure 4B) indicated enhanced proliferation, consistent with data in Figure 1, and increased numbers of cells in the HSPC fraction expressed the CBS on their surface (Figure 4C). As the ligands for the CBS include IL-3 and GM-CSF, we next sought to determine whether related signaling pathways are activated in Apoe^–/– HSPCs. Indeed, we observed an increase in p-ERK1/2 and p-STAT5 (Figure 4D), classical pathways involved in cell survival and proliferation (33–36). To link these findings with increased cholesterol content in the cell membrane (lipid rafts) and enhanced IL-3/GM-CSF signaling, we performed an in vitro proliferation assay, in which cholesterol levels in WT and Apoe^–/– BM progenitor cells were modulated using cyclodextrin (CyD) (37, 38). Apoe^–/– progenitors displayed increased proliferation compared with WT progenitors in response to IL-3/GM-CSF (Figure 4E). In both genotypes, increasing cellular cholesterol levels enhanced proliferation, and removal of cellular cholesterol by CyD or reconstituted HDL (rHDL; cholesterol-poor phospholipid/apoA-I complexes) decreased proliferation. Incubation of Apoe^–/– BM cells with rHDL also showed a dose-dependent decrease in GM-CFUs (Figure 4F), indicative of a reduced capacity to produce leukocytes. These findings suggest that membrane cholesterol enrichment in Apoe^–/– HSPCs leads to increased cell surface expression of the CBS and signaling via ERK and STAT5 pathways, leading to proliferation that can be reversed by cholesterol depletion using rHDL or CyD.

Figure 4

Cellular cholesterol levels modulate HSPC proliferation through the CBS. (A) WT and Apoe^–/– mice were fed a WTD for 4 weeks and infused with saline or rHDL (80 mg/kg). 96 hours after infusion, HSPCS were isolated via flow cytometry and stained with CTx-B for lipid rafts. HSPCs were viewed on a confocal microscope, and lipid raft abundance was quantified. Scale bars: 5 μM. (B–D) WT and Apoe^–/– mice were fed a WTD for 4 weeks, and BM HSPCs were analyzed by flow cytometry. (B) In vivo HSPC proliferation, determined by injecting mice with EdU 18 hours prior to sacrifice and assessing EdU incorporation into DNA. (C) Percent HSPCs expressing the CBS. (D) Phospho-flow for p-ERK1/2 and p-STAT5 in HSPCs. *P < 0.05, **P < 0.01 vs. WT; ^{^^}P < 0.01 vs. Apoe^–/– plus saline. (E) BM was isolated from WT and Apoe^–/– mice and incubated with IL-3 and GM-CSF in the presence or absence of cholesterol-loaded CyD (Chol-CyD; 6.6 mg/ml), CyD (6.6 mg/ml), or rHDL (1 mg/ml), and proliferation was assessed by [³H]-thymidine incorporation. *P < 0.05 versus respective control; ^{^}P < 0.05 vs. WT. (F) GM-CFU assay from BM isolated from Apoe^–/– mice and incubated with increasing concentrations of rHDL. *P < 0.05, ***P < 0.001 vs. control. Data are mean ± SEM.

Infusion of rHDL suppresses leukocytosis and HSPC proliferation in Apoe^–/– mice. To determine whether rHDL could reverse leukocytosis and HSPC proliferation in vivo, we gave a single infusion of rHDL (CSL-111) to 4-week WTD-fed Apoe^–/– mice (39). Although there was a transient increase in HDL-C and apoA-I levels, total cholesterol (saline, 1,157 ± 143 mg/dl; rHDL, 1,080 ± 61 mg/dl; n = 10, P = NS) and HDL-C (saline, 33 ± 0.8 mg/dl, rHDL, 36 ± 3.2 mg/dl; n = 6, P = NS) remained unchanged 96 hours after infusion. There was a significant dose-related suppression of monocytosis and neutrophilia, with a nadir at about 1 week after infusion and a maximum effect at the 80-mg/kg rHDL dose, followed by a return to control levels after 14 days (Figure 5A and Supplemental Figure 8, A and B). A transient decrease in leukocytes seen at 24 hours was also observed in the saline group, likely representative of a nonspecific response. Both monocyte subsets, Ly6-C^hi and Ly6-C^lo, were suppressed (Figure 5A and Supplemental Figure 8, C and D). The reduction in blood monocyte numbers paralleled a decrease in EdU incorporation (Figure 5B), consistent with diminished proliferation of monocytes or their precursors.

Figure 5

Infusion of rHDL attenuates monocytosis and neutrophilia by decreasing cellular cholesterol, CBS expression, and HSPC cell cycling in the BM of 4-week WTD-fed Apoe^–/– mice. After WTD feeding for 4 weeks, Apoe^–/– mice were infused with either saline or rHDL (CSL-111; 80 mg/kg), and analysis was performed after 7 days. (A and B) Blood experiments. Data are mean ± SEM (n = 8). (A) Monocytes were assessed via flow cytometry and converted to cells/μl using counts from the complete blood cell analyses. (B) Monocyte proliferation, determined by i.v. injection of EdU 18 hours prior to sacrifice. Proliferation was measured by EdU incorporation into blood monocytes, as determined by flow cytometry. (C–F) BM experiments. Data are mean ± SEM (n = 6–8). (C) HSPCs and downstream progenitors and (D) HSPC subsets, quantified by flow cytometry. (E) HSPCs expressing the CBS. (F) Percent cells in the G₂M phase of the cell cycle. (A–F) *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT plus saline; ^{^}P < 0.05, ^{^^}P < 0.01, ^{^^^}P < 0.001 vs. Apoe^–/– plus saline.

To assess the effects of rHDL on BM HSPCs and myeloid populations, we sacrificed Apoe^–/– or WT mice at 7 days after infusion of 80 mg/kg rHDL. This confirmed significant expansion of HSPC and myeloid precursor (CMP and GMP) populations, but not megakaryocyte-erythroid progenitor (MEP) or common lymphoid progenitor (CLP) populations, in Apoe^–/– versus WT mice. There was a complete reversal of HSPC and myeloid progenitor expansion by rHDL infusion (Figure 5C). Analysis of HSPC subpopulations showed that LT-HSC, ST-HSC/MPP1, MPP2, and MPP3 populations were expanded in Apoe^–/– mice, but only the ST/MPP1 and MPP2 populations were suppressed by rHDL (Figure 5D). Consistent with our proposed mechanism, rHDL infusion caused a reduction in lipid rafts and a decrease in the number of HSPCs expressing the CBS (Figure 5E). This led to a dampening of downstream signaling pathways (p-ERK1/2 and p-STAT5), which resulted in a decrease in HSPC proliferation, as determined by EdU incorporation (Supplemental Figure 9, A and B). Decreased proliferation was confirmed by a reduction in cell cycling of BM HSPCs, CMPs, and GMPs after rHDL infusion (Figure 5F). The spleen is a known reservoir for monocytes (40) and is home to a small population of HSPCs that actively produce leukocytes, especially under conditions of extramedullary hematopoiesis (41, 42). Therefore, we also examined the effect of rHDL infusion on splenic myeloid cells. There was a marked expansion of HSPCs, CMPs, and GMPs in the spleen of Apoe^–/– mice, with increased EdU incorporation by CMPs and GMPs, but not HSPCs (Supplemental Figure 9, C and D). As in the BM, these changes were partly reversed by rHDL infusion.

Combined low-dose rHDL and LXR activator treatment reduces myeloid proliferation. We previously showed that LXR activators could suppress proliferation of BM myeloid cells in an ABCA1/ABCG1-dependent fashion (17). Since Apoe^–/– mice have residual apoA-I–containing HDL, we reasoned that LXR activators would induce Abca1/Abcg1 expression (Supplemental Figure 2), promote cholesterol efflux to endogenous or infused apoA-I–containing HDL, and suppress myeloid proliferation. Thus, we assessed the effects of LXR activator treatment and/or rHDL infusion on monocyte counts and HSPC proliferation in 4-week WTD-fed WT or Apoe^–/– mice. We used a submaximal dose of 40 mg/kg, to bring out possible additive effects of LXR and rHDL treatments. The combined treatments significantly reduced monocyte and neutrophil counts, whereas single treatments did not have significant effects (Supplemental Figure 10, A and B). Unlike treatment with rHDL, LXR activator treatment did reduce monocyte counts in WT mice (Supplemental Figure 10, A and B), suggestive of an effect on additional pathways compared with rHDL. Single and combined treatments significantly decreased EdU incorporation into the monocytes in blood of Apoe^–/– mice (Supplemental Figure 10C). Analysis of BM cells revealed that LXR activator or combined treatment significantly reduced the number and proliferation of HSPCs while suppressing expression of the CBS. These effects were only observed in WTD-fed Apoe^–/– mice (Supplemental Figure 10, D–F). Together with data shown in Supplemental Figure 2, these findings suggest that in Apoe^–/– mice, induction of Abca1/Abcg1 in HSPCs by LXR activator treatments leads to cholesterol efflux to endogenous HDL or infused rHDL, leading to suppression of HSPC proliferation and reduced numbers of monocytes in blood.

This study suggests a key role of cell surface proteoglycan-bound ApoE in controlling HSPC proliferation, myeloid cell expansion, monocytosis, and accumulation of monocytes in atherosclerotic lesions. ApoE appears to interact with ABCA1/ABCG1 in HSPCs, promoting cholesterol efflux and decreasing the cell surface expression and downstream signaling of the IL-3 receptor. Although infusion of rHDL into Apoe^–/– mice reversed these defects, circulating endogenous apoA-I and ApoE appeared to have relatively minor roles in controlling HSPC proliferation.

Even though ApoE is relatively abundant in the circulation, our studies revealed a major cell-autonomous role of ApoE in controlling proliferation of HSPCs and subsequent expansion of myeloid lineage cells. Apoe mRNA and cell surface protein were relatively abundant in the LT-HSC and ST-HSC populations, suggestive of a role in controlling stem cell proliferation and emergence from quiescence. Although Abca1 and Abcg1 were also highly expressed in HSPCs relative to other myeloid lineage cells, they were equally expressed in multipotential progenitor and stem cells, pointing to a key regulatory role of ApoE on stem cells. We documented an LXR-inducible pool of ApoE on the surface of HSPCs and showed that cell surface ApoE was bound to proteoglycans. Heparinase treatments indicated that proteoglycan-bound cell surface ApoE acts to suppress HSPC proliferation. In contrast, heparinase treatment did not affect proliferation of Abca1^–/–Abcg1^–/– HSPCs, even though it resulted in release of ApoE from the surface of these cells. This genetic evidence is consistent with a model in which ApoE concentration at the cell surface by HSPGs facilitates its interaction with ABCA1/ABCG1, promoting cholesterol and/or phospholipid efflux (Figure 6). This conclusion is also supported by the similar downstream effects of ApoE and ABCA/ABCG1 deficiencies: cholesterol accumulation, increased cell surface levels, and signaling of the CBS via ERK and STAT5 signaling pathways (Figure 6). These findings are reminiscent of the binding of ApoE to the surface of hepatocytes or macrophages by interaction with HSPGs (24) and could explain the observation that ApoE expressed within macrophages is more effective at promoting cholesterol efflux than ApoE added to media (43).

Figure 6

Endogenous cholesterol pathways regulate HSPC proliferation. HSPCs possess an active cholesterol efflux system expressing Abca1, Abcg1, and Apoe, which are responsive to LXRs. (i) ApoE protein is secreted and bound to cell surface HSPGs, inducing cholesterol efflux by interacting with ABCA1 and/or ABCG1. Importantly, circulating ApoE or ApoE from neighboring cells appears to be an inefficient acceptor of cholesterol (C) from HSPCs. (ii) In Apoe-deficient HSPCs, or when ApoE is removed from the cell surface by cleavage of the HSPGs, cholesterol efflux pathways are disrupted, and cholesterol begins to accumulate in the cells. (iii) This results in an increased population expressing the CBS, and the cells become hypersensitive to IL-3/GM-CSF signaling, inducing phosphorylation of EKR1/2 and STAT5. (iv) The net result is increased proliferation and expansion of HSPCs, leukocytosis, and accelerated atherosclerosis. Black and gray arrows denote antiproliferative and proliferative pathways, respectively.

The competitive BM transplantation experiments strongly suggest a causal link between the cell-autonomous effects of ApoE on the proliferation of HSPCs and myeloid cells and the number of leukocytes in atherosclerotic lesions (Figure 3). This is consistent with prior studies showing that monocytosis in WTD-fed Apoe^–/– mice is associated with increased entry of monocytes into lesions (11, 12) and the strong genetic evidence indicating that the blood monocyte count is rate-limiting for the extent of atherosclerotic lesion development in both Ldlr^–/– and Apoe^–/– mice (44–47). There may be additional mechanisms involved in accumulation of Apoe^–/– leukocytes, such as increased monocyte activation, endothelial transmigration, or retention. Examination of these processes showed that Apoe^–/– Ly6-C^hi monocytes were slightly activated and had a moderate increase in adhesion to endothelial cells. However, Apoe^–/– monocytes may be primed to respond to in situ activation signals, and this could also play a significant role in atherogenesis in vivo. We did not determine a potential effect of ApoE on monocyte or macrophage egress from lesions; however, a recent study indicates that ApoE does not likely affect this process (27). Given the magnitude of the 3- to 4-fold cell-autonomous increases in neutrophil and monocyte counts arising from Apoe deficiency (Figure 3, C and E), this is likely to be an important mechanism underlying monocyte accumulation in lesions in Apoe^–/– mice.

Pioneering studies have shown that transplantation of WT BM into Apoe^–/– mice leads to a marked decrease in atherosclerosis, associated with reductions in plasma cholesterol levels (48), which illustrates the central role of ApoE in the clearance of chylomicron and VLDL remnants (49). Our findings are consistent with earlier reports indicating that ApoE in BM-derived cells also exerts antiatherogenic effects independent of its ability to lower cholesterol levels or to alter the lipoprotein profile (26, 50). Although these effects were likely mediated in part through expression of ApoE in lesional macrophages, our present results suggest that effects on HSPC and myeloid cell proliferation may also have been involved.

Even though endogenous circulating apoA-I and HDL were relatively ineffective at controlling HSPC proliferation, infusion of pharmacological doses of cholesterol-poor phospholipid/apoA-I complexes (i.e., rHDL) could reverse these defects. Increasing HDL levels in Apoe^–/– mice and other atherosclerosis models, either by infusions of HDL or transgenic overexpression of Apoa1, has consistently been shown to reduce atherosclerosis (51). In the present studies, we used a preparation of rHDL (CSL-111) previously shown to cause regression of coronary atherosclerosis, stabilization of peripheral vascular lesions, and decreased inflammation in humans at doses similar to those used here (80 mg/kg) (39, 52–54). rHDL suppressed HSPC and GMP proliferation in BM and spleen and reduced blood monocyte and neutrophil counts. Although we showed that the intrinsic cholesterol efflux pathway mediated by ApoE in HSPCs was more important than circulating HDL in controlling their proliferation, infusions of rHDL (cholesterol free particles with a high cholesterol efflux potential) also altered membrane lipid rafts of HSPCs, suppressing proliferation and monocytosis. This pharmacologic effect likely reflects the large dose of rHDL — the effect was seen at 80 but not 40 mg/kg (Supplemental Figure 8) — with a high cholesterol efflux capacity. Moreover, the acute infusion will result in rapid removal of cellular cholesterol, while the longstanding depletion of Apoa1 in the knockout model may be compensated by changes in cholesterol homeostasis (such as increased synthesis) as well as other efflux pathways, notably ApoE. We also showed that LXR activation, alone or in combination with a low dose of rHDL, reduced HSPC proliferation and monocytosis. LXR activators induced Abca1, Abcg1, and Apoe in HSPCs, and LXR activators appeared to be acting via a mechanism similar to that of rHDL, i.e., by decreasing the cell surface expression of the CBS on HSPCs. However, LXRs are widely expressed with many target genes, and additional mechanisms may be involved.

In conclusion, we describe a mechanism that we believe to be novel, in which ApoE acts in a cell-intrinsic fashion to regulate HSPC proliferation, monocytosis, and neutrophilia, likely by promoting cholesterol efflux from HSPCs via ABCA1/ABCG1. Importantly, we have also shown that treatment with pharmacological doses of the potent cholesterol acceptor rHDL bypassed this cell-intrinsic mechanism and restored normal HSPC proliferative responses and blood leukocyte levels in the setting of atherosclerosis. This could represent a novel antiatherogenic mechanism of rHDL infusions.

View Supplemental data

This work was supported by NIH grants HL107653 and 54591 and by the Foundation Leducq. M. Akhtari was a research fellow supported by the Sarnoff Cardiovascular Research Foundation. M. Sanson is supported by NIH grant HL-084312. L. Yvan-Charvet is a recipient of American Heart Association grant SDG2160053. The authors thank the flow cytometry unit at Columbia University for HSPC sorting, George Kuriakose for atherosclerotic lesion sectioning, and CSL Australia for providing the rHDL used in this study.

Address correspondence to: Andrew J. Murphy, Division of Molecular Medicine, 630 W 168th St., Room 8-401, Columbia University, New York, New York 10032, USA. Phone: 212.305.5789; Fax: 212.305.5052; E-mail: am3440@columbia.edu.

Conflict of interest: Alan R. Tall serves on scientific advisory boards for Merck and Arisaph Pharmaceuticals and provides paid consulting services to Roche, Merck, Arisaph Pharmaceuticals, and CSL Limited related to the development of drugs that would increase HDL levels.

Reference information: J Clin Invest. 2011;121(10):4138–4149. doi:10.1172/JCI57559.

See the related article at ApoE controls the interface linking lipids and inflammation in atherosclerosis.

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