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The FASEB Journal • Research Communication

Hypercholesterolemia-induced priming ofhematopoietic stem and progenitor cellsaggravates atherosclerosis

Tom Seijkens,* Marten A. Hoeksema,* Linda Beckers,* Esther Smeets,* Svenja Meiler,*Johannes Levels,† Marc Tjwa,‡ Menno P. J. de Winther,* and Esther Lutgens*,§,1

*Department of Medical Biochemistry, Experimental Vascular Biology, and †Department ofExperimental Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam,The Netherlands; ‡Laboratory of Vascular Hematology/Angiogenesis, Institute for TransfusionMedicine, Goethe University Frankfurt, Frankfurt, Germany; and §Institute for CardiovascularPrevention (IPEK), Ludwig Maximilians University, Munich, Germany

ABSTRACT Modulation of hematopoietic stem andprogenitor cells (HSPCs) determines immune cell func-tion. In this study, we investigated how hypercholester-olemia affects HSPC biology and atherosclerosis. Hy-percholesterolemia induced loss of HSPC quiescence,characterized by increased proliferation and expres-sion of cyclin B1, C1, and D1, and a decreased expres-sion of Rb, resulting in a 3.6- fold increase in thenumber of HSPCs in hypercholesterolemic Ldlr!/!

mice. Competitive bone marrow (BM) transplantationsshowed that a hypercholesterolemic BM microenviron-ment activates HSPCs and skews their developmenttoward myeloid lineages. Conversely, hypercholesterol-emia-primed HSPCs acquired an enhanced propensityto generate myeloid cells, especially granulocytes andLy6Chigh monocytes, even in a normocholesterolemicBM microenvironment. In conformity, macrophagesdifferentiated from hypercholesterolemia-primed HSPCsproduced 17.0% more TNF-", 21.3% more IL-6, and10.5% more MCP1 than did their normocholesterolemiccounterparts. Hypercholesterolemia-induced priming of

HSPCs generated leukocytes that more readily mi-grated into the artery, which resulted in a 2.1-foldincrease in atherosclerotic plaque size. In addition,these plaques had a more advanced phenotype andexhibited a 1.2-fold increase in macrophages and 1.8-fold increase in granulocytes. These results identifyhypercholesterolemia-induced activation and primingof HSPCs as a novel pathway in the development ofatherosclerosis. Inhibition of this proinflammatory dif-ferentiation pathway on the HSPC level has the poten-tial to reduce atherosclerosis.—Seijkens, T., Hoek-sema, M. A., Beckers, L., Smeets, E., Meiler, S., Levels,J., Tjwa, M., de Winther, M. P. J., Lutgens, E. Hyper-cholesterolemia-induced priming of hematopoieticstem and progenitor cells aggravates atherosclerosis.FASEB J. 28, 2202–2213 (2014). www.fasebj.org

Key Words: ! lipoproteins ! inflammation

During homeostasis, hematopoiesis is balanced be-tween the myeloid, lymphoid, and erythroid–mega-karyocytic lineages. Hematopoietic stressors disturb thisbalance and skew hematopoietic stem and progenitorcell (HSPC) development. For example, severe hemor-rhage or hemolytic anemia skews HSPC developmenttoward the erythroid lineage, and bacterial infectionrapidly induces granulopoiesis and increases the num-ber of immature granulocytes in the peripheral blood(1–4). Besides these acute hematopoietic stressors,chronic inflammatory conditions alter HSPC homeosta-sis. Animals subjected to autoimmune arthritis or sys-temic lupus erythematosus exhibit increased myeloidoutput of the bone marrow (BM; refs. 5, 6). These

1 Correspondence: Department of Medical Biochemistry,Academic Medical Center (AMC), University of Amsterdam,Meibergdreef 15, 1105 CZ Amsterdam, The Netherlands.E-mail: [email protected]

doi: 10.1096/fj.13-243105This article includes supplemental data. Please visit http://

www.fasebj.org to obtain this information.

Abbreviations: !SMA, !-smooth muscle actin; ApoE, apoli-poprotein E; BM, bone marrow; BMC, bone marrow cell;BMDM, bone marrow -derived macrophage; BrdU, bromode-oxyuridine; BMT, bone marrow transplantation; cBMT, com-petitive bone marrow transplantation; CFU, colony-formingunit; CFU-G, colony-forming unit granulocyte; CFU-M, colony-forming unit megakaryocyte; ChIP, chromatin immunopre-cipitation; CMP, common myeloid progenitor; CVD, cardio-vascular disease; EDTA, ethylenediaminetetraacetic acid; E-MPP, early multipotent progenitor; GMP, granulocyte–monocyte progenitor; H&E, hematoxylin and eosin; HDL,high-density lipoprotein; HFD, high-fat diet; HSPC, hemato-poietic stem and progenitor cell; IL, interleukin; LDL, low-density lipoprotein; Ldlr"/", low-density lipoprotein receptorknockout; Lin", lineage negative; L-MMP, late multipotentprogenitor; LSK, Lin"Sca1#cKit"; LT-HSC, long-term hema-topoietic stem cell; MCP1, monocyte chemotactic protein 1;PBS, phosphate-buffered saline; qPCR, quantitative PCR; Rb,retinoblastoma; SCF, stem cell factor; ST-HSC, short-termhematopoietic stem cell; TNF-!, tumor necrosis factor !;VLDL, very low density lipoprotein

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myeloid cells subsequently participate in the ongoinginflammatory response. Hence, inflammation-associ-ated alterations in HSPC biology may have a decisiverole in the persistence and/or progression of chronicinflammatory diseases.

Atherosclerosis is a chronic, hypercholesterolemia-driven inflammatory disease that results in the forma-tion of plaques in the arterial wall (7). In early stages,immune cells, especially monocytes and granulocytes,are actively recruited to sites of vascular inflammationand promote plaque development by recruiting moreinflammatory cells (8, 9). Subsequently, these immunecells produce proinflammatory chemokines and cyto-kines, thereby decisively influencing the propensity of agiven atherosclerotic plaque to rupture and causeclinical manifestations (e.g., myocardial infarction andischemic stroke; refs. 7, 8). An increased number ofperipheral blood leukocytes, especially monocytes andgranulocytes, is associated with occurrence and out-come of cardiovascular disease (CVD; refs. 10, 11).Hypercholesterolemia, a common risk factor in pa-tients with CVD, promotes leukocytosis (12–14). Hyper-cholesterolemia is characterized by increased levels oflow-density lipoprotein (LDL) and very low densitylipoprotein (VLDL) and decreased levels of high-den-sity lipoprotein (HDL; refs. 7, 13–16). As shown byother laboratories, lipoproteins regulate HSPC biology(17–19). HDL and apolipoprotein E (ApoE) suppressHSPC proliferation by regulating cholesterol efflux viathe Abca1/Abcg1 transporters, whereas LDL promotesHSPC proliferation and skews hematopoiesis towardthe myeloid lineage (17–19).

In this study, we investigated how hypercholesterol-emia-induced alterations in HSPC homeostasis affectthe inflammatory response and subsequent develop-ment of atherosclerosis in LDL receptor-knockout(Ldlr"/") mice.

MATERIALS AND METHODS

Mice

Male CD45.2-Ldlr"/" and CD45.1-Ldlr"/" mice were bred atthe animal facilities of Maastricht University (Maastricht, TheNetherlands) and the University of Amsterdam. The micereceived chow or a high-fat diet (HFD) containing 16% fatand 0.15% cholesterol. The mice were given ad libitum accessto food and were housed according to institutional guide-lines. The animal experiment and care committees of Maas-tricht University and the University of Amsterdam approvedthe animal experiments.

Hematology and lipoproteins

Blood was obtained by venous or cardiac puncture andcollected into ethylenediaminetetraacetic acid (EDTA)-con-taining tubes. Hematologic analysis was performed on aScilVet abc Plus# (ScilVet, Oostelbeers, The Netherlands).Individual lipoprotein levels were determined by fast-perfor-mance liquid chromatography (FPLC). In brief, the systemcontained a PU-980 ternary pump with an LG-980-02 linear

degasser, an FP-920 fluorescence detector, and a UV 975UV/VIS detector (Jasco, Tokyo, Japan). An extra P-50 pump(Pharmacia Biotech, Uppsala, Sweden) was used for inlineaddition of cholesterol PAP enzymatic reagent (Biomerieux,Marcy l’Etoile, France) at 0.1 ml/min. EDTA plasma wasdiluted 1:1 with Tris-buffered saline, and 30 $l sample/buffermixture was loaded on a Superose 6 HR 10/30 column (GEHealthcare, Life Sciences Division, Diegem, Belgium) forlipoprotein separation at a flow rate of 0.31 ml/min.

Flow cytometry

BM from age- and sex-matched Ldlr"/" mice was harvested incold phosphate-buffered saline (PBS). BM cell (BMC) sus-pension was prepared and filtered through a 70-$m nylonmesh (Falcon; BD Biosciences, Breda, The Netherlands).Lineage depletion by magnetic bead isolation was performedaccording to the manufacturer’s instructions (Lineage CellDepletion Kit; Miltenyi Biotec, Teterow, Germany). Blood wastreated with red blood cell lysis buffer. Staining was per-formed with anti-mouse antibodies against the followingantigens: CD3, 7-4, NK1.1, Ly6G, CD11b, CD5, and Gr-1 (allfrom BD Pharmingen, Breda, The Netherlands); CD117,Ter-119, CD45.1, CD45.2, B220 CD127, CD34, and CD32/16(all from Ebioscience, Vienna, Austria); CD135, CD150, andSca-1 (all from Biolegend, San Diego, CA, USA); and Ly6C(Miltenyi Biotech). Nonspecific binding was prevented bypreincubating the cells with an Fc receptor-blocking anti-body. For cell-cycle analysis, lineage-negative (Lin") BMCswere stained with the indicated primary antibodies, fixed in70% ethanol for 24 h, and treated with propidium iodide/RNase buffer (PI/RNase; BD Biosciences; ref. 20). Stainingwas analyzed by flow cytometry (FACSCanto II; BD Biosci-ences).

Colony-forming unit in culture (CFU-C) assays

BM was isolated from normo- and hypercholesterolemicmice, and a single-cell suspension was prepared (20). BMCs(1%104) were cultured in 2 ml semisolid methylcellulosemedium supplemented with growth factors (MethoCult; StemCell Technologies, Grenoble, France) at 37°C in 98% humid-ity and 5% CO2 for 7 d. CFU-granulocyte, erythrocyte,monocyte, megakaryocyte (CFU-GEMM), CFU-granulocyte-macrophage (CFU-GM), CFU-megakaryocyte (CFU-M), andCFU-granulocyte (CFU-G) colonies were scored after 7 d withan inverted microscope by T.S., in a blinded protocol.

Competitive BM transplantation (cBMT)

C57Bl6CD45.2-recipient mice were housed in filter-top cagesand received drinking water containing antibiotics (poly-myxin B sulfate, 6000 U/ml, and neomycin, 100 $g/ml) for 5wk, starting 1 wk before the BMT. At 1 d before BMT, themice were lethally irradiated (9.5 Gy, 0.5 Gy/min; MU15F/225 kV; Philips, Eindhoven, The Netherlands). The next day,the donor mice (CD45.2-Ldlr"/" and normocholesterolemicand hypercholesterolemic CD45.1-Ldlr"/" mice) were eutha-nized, and BMCs were obtained as just described. The donormice were fed chow or an HFD for 4 wk. BM mononuclearcells were isolated via Lympholiter-M (Cedarlane-Sanbio,Uden, The Netherlands) and density centrifugation (20).Test cells (5%105) and competitor cells (1%106) were trans-planted into the recipient mice. Reconstitution of peripheralblood leukocytes was analyzed by flow cytometry, as describedabove, at 4, 8, 12, 16, and 20 wk after BMT.

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Bromodeoxyuridine (BrdU) incorporation

BrdU was injected intraperitoneally at 0.2 mg/g. BM wascollected 16 h after injection, and Lin" cells were isolated asdescribed earlier. BrdU incorporation was determined byintracellular staining with anti-BrdU antibodies, using theFITC BrdU Flow Kit (BD Biosciences).

Atherosclerosis

Male CD45.2-Ldlr"/" mice received HFD, starting at the ageof 6 wk. At 2 wk after the start of the HFD, the miceunderwent a cBMT, as just described. The donor mice werefed chow or an HFD for 4 wk. Test cells (5%105) andcompetitor cells (1%106) were transplanted into the recipientmice. The mice were euthanized at the age of 18 wk, and thearterial tree was perfused with PBS containing nitroprusside.The aortic arch and its main branch points were excised,fixed overnight in 1% paraformaldehyde in PBS, and embed-ded in paraffin. Twenty consecutive, longitudinal sections of

the aortic arch were selected for histologic analysis. Forplaque area and morphology, 4 sections (20 $m apart) werestained with hematoxylin and eosin (H&E), as describedpreviously (21, 22). For phenotypic analysis, immunohisto-chemistry was performed for CD3 (Dako, Heverlee, Bel-gium), CD45 (BD Biosciences), Mac-3 (BD Biosciences), and!-smooth muscle actin (!SMA; Sigma-Aldrich, St. Louis, MO,USA). Sirius red staining was performed as described else-where (23). Morphometric analyses were performed withLas4.0 software (Leica, Wetzlar, Germany). Organs wereanalyzed by H&E staining, no abnormalities were observed.There were no differences between the experimental groupsin body weight and cholesterol levels.

In vitro macrophage culture

BMCs were isolated from normo- and hypercholesterolemicmice and cultured in RPMI supplemented with 15% L929-conditioned medium to generate BM-derived macrophages

Figure 1. Hypercholesterolemia induces leukocytosis by disrupting HSPC quiescence. A) Hypercholesterolemia increasedperipheral blood leukocyte levels. B) Peripheral blood leukocyte counts correlated with total plasma cholesterol levels. C)Number of LSK cells in the BM was increased in hypercholesterolemic mice. D) CFU assays revealed that hypercholesterolemicBM produced more colonies. E) BrdU incorporation was increased in hypercholesterolemic LSK cells. F) Propidium iodide flowcytometry showed that the number of quiescent cells decreased in hypercholesterolemic BM. G) Expression of cyclins increasedin hypercholesterolemic LSK cells. n & 4–6/group. *P ' 0.05; **P ' 0.01. ***P ' 0.001.

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(BMDMs; ref. 24). BMDMs were activated by lipopolysaccha-ride (LPS; 10 ng/ml; Sigma-Aldrich) for 6 h.

Quantitative PCR (qPCR) and ELISA

RNA was isolated from sorted Lin"Sca1#cKit" (LSK) cellsand BMDMs and reverse transcribed with an iScript cDNAsynthesis kit (Bio-Rad, Veenendaal, The Netherlands). qPCR

was performed with a SYBR Green PCR kit (Applied Biosys-tems, Leusden, The Netherlands) on a ViiA7 real-time PCRsystem (Applied Biosystems). Primer sequences are shown inSupplemental Table S1. Enzyme-linked immunosorbent as-says (ELISAs) were used to determine monocyte chemotacticprotein 1 (MCP1), tumor necrosis factor ! (TNF-!), interleu-kin 6 (IL-6), and IL-10 levels (Invitrogen, Leusden, TheNetherlands).

Figure 2. Hypercholesterolemic BM microenvironment activates HSPCs andpromotes myelopoiesis. A) cBMTs were performed, in which normocholes-terolemic CD45.1-Ldlr"/" BMCs were transplanted into chow- or HFD-fedCD45.2-Ldlr"/" recipients to analyze the effects of the hypercholesterolemicBM microenvironment on the development of HSPCs. B) Hypercholester-olemic BM microenvironment increased the number of LSK cells, CMPs, andGMPs. C) Hypercholesterolemic BM microenvironment activates HSPCs, as reflected by

increased CD45.1# leukocyte reconstitution of normocholesterolemic HSPCs in a hypercholesterolemic microenviroment. D, E) Nodifferences in the reconstitution of T cells (D) and B cells (E) were observed. F–I) Transplantation of normocholesterolemic BM intoa hypercholesterolemic microenvironment increased the reconstitution of myeloid cells, especially proinflammatory Ly6Chigh

monocytes. F) CD45.1#CD11b#Ly6G" cells. G) CD45.1#CD11b#Ly6G" cells. H) CD45.1# Ly6Ghigh monocytes. I) CD45.1#

Ly6Clow/" monocytes. n & 12–15/group. *P ' 0.05; **P ' 0.01.



Chromatin immunoprecipitation (ChIP)

BMCs were isolated as described earlier. For ChIP, 1.5–2.0 %107 BMCs were cross-linked with 1% formaldehyde.AcH3(K9/K14) ChIP was performed with 5 $g antibody (CellSignaling Technology, Danvers, MA), as described previously(25). ChIP-qPCR was performed on an ABI ViiA 7 PCR systemusing SYBR Green Fast (Applied Biosystems). Relative enrich-ments are presented as the percentage of input. Primersequences are shown in Supplemental Table S1.

Statistical analysis

Data are presented as means ( sem and were analyzed byStudent’s t test, or, when appropriate, by linear regression.Calculations were performed with GraphPad Prism 5.0 soft-ware (GraphPad Software, Inc., La Jolla, CA, USA). Values ofP ' 0.05 were considered statistically significant.

RESULTS

Hypercholesterolemic mice have an expanded HSPCpopulation

To investigate the effects of hypercholesterolemia onperipheral blood leukocyte numbers and HSPC re-

sponses, we fed Ldlr"/" mice chow or an HFD, contain-ing 16% fat and 0.15% cholesterol, for 10 wk andanalyzed peripheral blood, spleen, and BM. The HFDinduced a 3.5-fold increase in total plasma cholesterollevels, a 3.6-fold increase in LDL levels, and a 72-foldincrease in VLDL levels (Supplemental Fig. S1A, B). Nochanges in HDL levels were detected (SupplementalFig. S1A, B). Hypercholesterolemia reduced the HDL/LDL ratio from 0.73 to 0.30, reflecting a detrimentallipoprotein profile, comparable to human hypercholes-terolemia (26).

No differences in platelet and erythrocyte numbers,spleen weights, and body weights were observed (datanot shown). In addition, no differences in splenicleukocyte and HSPC populations were observed (datanot shown). The absolute number of leukocytes showeda 2.2-fold increase in hypercholesterolemic mice (Fig. 1A).Leukocyte subset analysis revealed that these miceexhibited a relative monocytosis, whereas relative lym-phocyte counts were decreased (Supplemental Fig.S1C). Hypercholesterolemia increased the absolutenumber of monocytes by 6.3-fold, granulocytes by 2.7-fold, and lymphocytes by 1.7-fold (Supplemental Fig.

Figure 3. The hypercholesterolemic BM microenvironment expresses increased levels of proinflammatory cytokines. A–D)Hypercholesterolemia did not affect the expression of GM-CSF (A), M-CSF (B), G-CSF (C), or SCF (D) in the BMmicroenvironment. E–I) The expression of the cytokines TNF-! (E), IL-1) (F), and IL-6 (G), but not of IL-12 (H) and IL-10 (I),was increased in hypercholesterolemic BM. n & 4–6/group. *P ' 0.05; **P ' 0.01.


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Figure 4. Hypercholesterolemia induces acell-intrinsic priming of HSPCs that per-sists during long-term normocholesterolemiaand promotes myelopoiesis. A) cBMTs wereperformed, in which hypercholesterolemicCD45.1-Ldlr"/" BMCs were transplantedinto chow-fed CD45.2-Ldlr"/" recipients,to analyze the development of hypercho-lesterolemia-primed HSPCs. B) Hypercho-lesterolemia-primed BM had an increased reconstitution of CMPs and GMPs, compared with nonprimed HSPCs. C)Hypercholesterolemia-primed BM had an increased total leukocyte reconstitution potential. D, E) T-cell (D) and B-cell (E)reconstitution was not affected. F–I) Hypercholesterolemia-primed HSPCs produced more granulocytes and monocytes,

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S1D). A strong correlation between total cholesterollevels and peripheral blood leukocyte count was ob-served, reflected by a Pearson’s correlation coefficientof 0.81 (Fig. 1B).

To investigate whether hypercholesterolemia-in-duced leukocytosis results from changes in HSPC ho-meostasis, we analyzed the BM. Flow cytometry revealeda 3.6-fold increase in the number of hematopoieticstem cells, defined as LSK cells, in hypercholester-olemic mice (Fig. 1C). Next, we analyzed the differentsubsets within the LSK population, including long-termhematopoietic stem cells (LT-HSCs; Lin"cKit#Sca1#

CD150#CD34"CD135" cells), short-term hematopoi-etic stem cells (ST-HSCs; Lin"cKit#Sca1#CD150#CD34#

CD135" cells), early multipotent progenitors (E-MPPs;Lin"cKit#Sca1#CD150"CD34#CD135" cells), and latemultipotent progenitors (L-MPPs; Lin"cKit#Sca1#

CD150"CD34#CD135# cells) (Supplemental Fig. S2A).No differences were observed, indicating that hyper-cholesterolemia did not affect the relative compositionof the LSK population. However, because the LSKpopulation was increased by 3.6-fold, the absolute num-ber of LT-HSCs, ST-HSCs, E-MPPs, and L-MMPs wasincreased in the hypercholesterolemic Ldlr"/" mice.The number of common myeloid progenitors (CMPs;Lin"cKit#Sca1"CD34#CD16/32low), granulocyte–mono-cyte progenitors (GMPs; Lin"cKit#Sca1"CD34#CD16/32# cells), and mature BM granulocytes and monocyteswere increased as well in hypercholesterolemic BM(Supplemental Fig. S2B–D). CFU assays showed that themyeloid colony-forming potential of hypercholester-olemic BM increased by 1.3-fold (Fig. 1D) Especially theCFU-G colonies and CFU-M colonies were increased(Supplemental Fig. S2E).

Together, these results show that hypercholesterol-emia induces quantitative and functional alterations inthe BM, characterized by an expanded HSPC popula-tion with an increased differentiation potential towardthe myeloid lineages.

Hypercholesterolemia disrupts HSPC quiescence

To assess whether the expansion of the HSPC popula-tion is a result of increased proliferation, we analyzedBrdU uptake in LSK cells in normo- and hypercholes-terolemic mice. BrdU incorporation was increased by1.8-fold in hypercholesterolemic LSK cells comparedwith their normocholesterolemic counterparts (Fig.1E). Flow cytometry using propidium iodide revealedthat fewer hypercholesterolemic Lin" cells were in theG0/G1 phase, indicating that these cells were lessquiescent than the normocholesterolemic Lin" cells(Fig. 1F). qPCR showed increased expression of cyclin

B1, D1, and E1 in hypercholesterolemic LSK cells,reflecting a proliferative cyclin profile (Fig. 1G).

Since Abca1, Abcg1, and increased expression ofgrowth factors have been reported to be responsible forthe hyperproliferative phenotype of HSPCs in ApoE"/"

mice, we determined the expression of these genes onLSK cells of our hypercholesterolemic Ldlr"/" mice.However, we did not observe any differences in theexpression of the Abca1 and Abcg transporters, or in theexpression of the GM-colony-stimulating factor (GM-CSF) receptor, M-CSF receptor, or G-CSF receptor (Supple-mental Fig. S3A–E). As the retinoblastoma (Rb) tumorsuppressor family has a critical role in the regulation ofboth cell proliferation and differentiation of HSPCs(27), we analyzed the expression of Rb family membersin normo- and hypercholesterolemic LSK cells. Hyper-cholesterolemic LSK cells showed reduced Rb expres-sion, compared to normocholesterolemic LSK cells(Supplemental Fig. S3F). The expression of the Rb-gene family members p107 and p130 was not affected(Supplemental Fig. S3G, H). ChIP revealed decreasedacetylation of histone H3 on lysine 9/14 within thepromoter of Rb, suggesting that epigenetic mecha-nisms regulate hypercholesterolemia-induced pheno-type of HSPCs (Supplemental Fig. S3I).

Overall, these results show that hypercholesterolemicHSPCs are less quiescent than normocholesterolemicHSPCs and have a hyperproliferative phenotype, possi-bly mediated via epigenetic mechanisms.

Naive HSPCs are activated by thehypercholesterolemic microenvironment

HSPC homeostasis is tightly regulated by the BM mi-croenvironment. To elucidate whether the BM mi-croenvironment is involved in the hypercholesterol-emia-induced activation of HSPCs, we performed cBMTs.To investigate the effect of a hypercholesterolemicmicroenvironment on HSPC differentiation, normo-cholesterolemic CD45.1-Ldlr"/" BMCs were trans-planted into chow- or HFD-fed CD45.2-Ldlr"/" recipi-ents (Fig. 2A).

Transplantation of CD45.1-Ldlr"/" normocholester-olemic BM into a hypercholesterolemic BM microenvi-ronment increased the reconstitution of CD45.1-Ldlr"/" LSK cells by 1.6-fold (Fig. 2B). In addition, thehypercholesterolemic niche induced a 2.3- and 2.8-foldincrease in the reconstitution of CD45.1-Ldlr"/" CMPand GMP cells, respectively (Fig. 2B). Accordingly, thehypercholesterolemic niche increased the reconstitu-tion of peripheral blood leukocytes at 2, 4, 6, and 10 wkafter cBMT, compared with transplantation into anormocholesterolemic BM microenvironment (Fig.

especially proinflammatory Ly6Chigh monocytes, even under long-term normocholesterolemic conditions. F) CD45.1#CD11b#

Ly6G" cells. G) CD45.1#CD11b#Ly6G" cells. H) CD45.1# Ly6Ghigh monocytes. I) CD45.1# Ly6Clow/" monocytes. J) BMDMsdifferentiated from hypercholesterolemic-primed BM, but cultured under normocholesterolemic conditions, producedincreased levels of the proinflammatory cytokines TNF-! and IL-6 and the chemokine MCP1, whereas the production of IL-10was not affected. n & 12–15/group. *P ' 0.05; **P ' 0.01; ***P ' 0.001.


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Figure 5. Hypercholesterolemia-induced activation of HSPCs aggravates atherosclerosis. A) cBMTs were performed in whichnormo- or hypercholesterolemic CD45.1-Ldlr"/" BMCs were transplanted into HFD-fed CD45.2-Ldlr"/" recipients, to analyzethe effects of hypercholesterolemia-induced HSPC activation on the development of atherosclerosis. B) NormocholesterolemicHSPCs, which were transplanted into an HFD-fed recipient, showed increased leukocyte reconstitution. C) Total atheroscleroticplaque area in the aortic arches of 18-wk-old Ldlr"/" mice was increased in those that received normocholesterolemic BM. D)Representative H&E-stained sections of the aortic arch that show the branch point of the left common carotid artery. Mice that

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2C). No differences in the reconstitution of T and Bcells were observed (Fig. 2D, E). However, the reconsti-tution of granulocytes and monocytes, especially in-flammatory Ly6Chigh monocytes was increased, whereaspatrolling Ly6Clow/" monocytes were not affected (Fig.2F–I). Thus, the hypercholesterolemic BM microenvi-ronment activated HSPCs and skewed hematopoiesisspecifically toward the proinflammatory myeloid lin-eages.

Next, we assessed the expression of myeloid growthfactors in the normo- and hypercholesterolemic BMmicroenvironment. We observed a trend toward de-creased expression of M-CSF and G-CSF under hyper-cholesterolemic conditions; no differences in the ex-pression of GM-CSF and stem cell factor (SCF) wereobserved (Fig. 3A–D). It is known that hypercholester-olemia increases the expression of proinflammatorycytokines in the BM microenvironment (28). Thesecytokines are known to alter HSPC homeostasis, (e.g.,increase proliferation and induce a myeloid lineagebias; refs. 29–31). Therefore, we analyzed the expres-sion of these cytokines in the normo- and hypercholes-terolemic BM microenvironments and found that hy-percholesterolemia increased the expression of TNF-!,IL-1), and IL-6 (Fig. 3E–G). No differences were ob-served in the expression of IL-12 and the anti-inflam-matory cytokine IL-10 (Fig. 3H, I).

Together, these data demonstrate that the hypercho-lesterolemic BM microenvironment activates HSPCsand induces a myeloid lineage bias, possibly mediatedby alterations in TNF-!, IL-1), and IL-6 expression inthe microenvironment.

Hypercholesterolemia induces a long-term, cellintrinsic priming of HSPCs

To investigate whether hypercholesterolemia results incell intrinsic alterations in HSPC development, weperformed a cBMT in which we transplanted normo- orhypercholesterolemic CD45.1-Ldlr"/" BM into chow-fed CD45.2-Ldlr"/" recipients (Fig. 4A).

At 10 wk after the cBMT, no differences were ob-served in the reconstitution of normo- or hypercholes-terolemic CD45.1-Ldlr"/" LSK cells (Fig. 4B). However,hypercholesterolemic LSK cells gave rise to more my-eloid progenitors, as indicated by a 1.9-fold increase inCMP and 2.2-fold increase in GMP (Fig. 4B). Thereconstitution of CD45.1-Ldlr"/" leukocytes was in-creased by 1.3-fold in mice that received BM fromhypercholesterolemic donors (Fig. 4C). We observedno differences in the reconstitution of T and B cells(Fig. 4D, E). However, hypercholesterolemic HSPCsexhibited an increased granulocyte and monocyte

reconstitution 10 wk after transplantation into a nor-mocholesterolemic microenvironment (Fig. 4F, G).Monocyte subset analysis revealed that especially theproinflammatory Ly6Chigh monocytes were increased,whereas the reconstitution of patrolling Ly6Clow/"

monocytes was not affected (Fig. 4H, I).To determine the functional effects of hypercholes-

terolemia-induced HSPC priming, BMCs were isolatedfrom chow- or HFD-fed Ldlr"/" mice and culturedunder normocholesterolemic conditions in L929-con-ditioned medium to generate BMDMs. Macrophagesdifferentiated from hypercholesterolemic HSPCs pro-duced more TNF-!, IL-6, and MCP1 on activation (Fig.4J). The production of the anti-inflammatory cytokineIL-10 was not affected.

Thus, hypercholesterolemia induces a cell-intrinsicpriming of HSPCs, which persists under long-termnormocholesterolemic conditions, is characterized by ahyperproliferative phenotype, and specifically pro-motes the development myeloid cells with an increasedinflammatory propensity.

Hypercholesterolemia-induced activation of HSPCsaggravates atherosclerosis

To determine the effects of hypercholesterolemia-in-duced activation and priming of HSPCs on the devel-opment of atherosclerosis, we performed a cBMT inwhich HFD-fed CD45.2Ldlr"/" recipients receivednormo- or hypercholesterolemic CD45.1Ldlr"/" BMCs(Fig. 5A). Normocholesterolemic HSPCs showed anincreased leukocyte reconstitution (Fig. 5B), whichreflects the activation of normocholesterolemicHSPCs by the hypercholesterolemic microenviron-ment (Fig. 3A).

The aortic arch of the recipients was harvested 10 wkafter the cBMT, and the extent of atherosclerosis wasdetermined. A total of 56 lesions of mice that receivedhypercholesterolemic HSPCs and 63 lesions of micethat received normocholesterolemic HSPCs were ana-lyzed by histology. Hypercholesterolemia-induced acti-vation by the BM niche of naive HSPCs resulted in a2.1-fold increase in atherosclerotic plaque size (Fig.5C). Morphologic analysis revealed that these plaqueswere characterized by a more advanced phenotype(pathologic intimal thickening), whereas plaques inmice that received hypercholesterolemic HSPCs had amore initial phenotype (intimal xanthoma; Fig. 5D andSupplemental Fig. S4A). Hypercholesterolemia-in-duced activation of HSPCs increased the number ofCD45.1# plaque leukocytes by 1.6-fold, indicating thatthe progeny of the hypercholesterolemia-primedHSPCs promoted the inflammatory response underly-

received normocholesterolemic HSPCs (left) developed larger and more advanced atherosclerotic plaques than did mice thatreceived hypercholesterolemic HSPCs (right) Scale bars & 100 $m. E) Hypercholesterolemia-induced activation of HSPCsresulted in increased CD45.1# cell accumulation in atherosclerotic plaques. F–H) Hypercholesterolemia-induced activation ofHSPCs resulted in a more inflammatory plaque phenotype, characterized by increased granulocyte (Ly6G#; F), macrophage(MAC3#; G), and T-cell (CD3#; H) counts. n & 12–15/group. *P ' 0.05; **P ' 0.01.


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ing atherogenesis (Fig. 5E). In accordance, theseplaques had a more inflammatory phenotype, charac-terized by more macrophages (Mac3#), more granulo-cytes (Ly6G#), and more T cells (CD3# cells) (Fig.5F–H) and contained 1.9-fold more collagen, reflectingthe progressed plaque phenotype (Supplemental Fig.S4B). No differences in smooth muscle cell (!SMA#)content were observed (Supplemental Fig. S4C). To-gether, these data show that hypercholesterolemia-induced priming by the niche of naive HSPCs aggra-vates the development of atherosclerosis, possibly byincreasing the accumulation of proinflammatory my-eloid progeny in the plaques.

DISCUSSION

Over the past decades, numerous studies have reportedthe association between peripheral blood leukocytecounts, especially myeloid cells, and the occurrence ofCVD (10, 13, 31–34). For example, patients with anincreased granulocyte/lymphocyte ratio (*3) havemore advanced coronary artery disease [odds ratio(OR)&2.45; 95% confidence interval (CI), 1.76–3.42;P'0.001] and a higher risk of major cardiovascularevents in the next 3 yr [hazard ratio (HR)&1.55; 95%CI, 1.09–2.2; P&0.01] compared with patients with alower ratio ('2) (11). The mechanisms that link leu-kocyte counts to the development of atherosclerosis arepoorly understood (11). Hypercholesterolemia, a ma-jor risk factor for atherosclerosis, may promote leuko-cytosis by altering HSPC homeostasis (16–19). How-ever, the role of hypercholesterolemia-associatedalterations in HSPC homeostasis in the development ofatherosclerosis is unknown.

In this study, hypercholesterolemia induced a patho-logic HSPC phenotype, which is characterized by re-duced HSPC quiescence and a biased developmenttoward the myeloid lineages. Increased LDL and VLDLlevels and decreased HDL levels characterize hypercho-lesterolemia. Recent studies have demonstrated a rolefor lipoproteins in the regulation of HSPC biology.HDL and ApoE suppress HSPC proliferation inApoE"/" mice by promoting ABCa1- and ABCg1-medi-ated cholesterol efflux, thereby decreasing the expres-sion of growth factor receptors on the cell surface,including the IL-3 and GM-CSF receptors (17, 18). Inour study, in which we used Ldlr"/" mice, we observedthat the hypercholesterolemia-induced hyperprolifera-tive HSPC phenotype was independent of ApoE. More-over, we found that hypercholesterolemia did not affectthe gene expression of growth factor receptors such asGM-CSF, G-CSF, and M-CSF and ABC transporters, suchas ABCA1 and ABCG1 on HSPCs, or the expression ofhematopoietic growth factor in the BM microenviron-ment, indicating that other mechanisms are involved.As the Rb tumor-suppressor family is known for its rolein the integration of multiple cellular signals thatcontrol cell proliferation and differentiation, especiallyunder stress conditions (27, 35, 36), we analyzed the

expression of Rb family members in normo- and hyper-cholesterolemic HSPCs. We observed decreased geneexpression of Rb in hypercholesterolemic HSPCs,whereas p107 and p130 were not affected. Our resultsindicate that hypercholesterolemia-induced epigeneticmechanisms may be involved in the down-regulation ofRb, which may explain why hypercholesterolemia-primed HSPCs maintain their pathologic phenotypeunder long-term normocholesterolemic conditions.

Besides the effects of direct priming of HSPCs byhypercholesterolemia, we also showed a prominent rolefor the hypercholesterolemic niche in altering thebiology of HSPCs. Using cBMTs, we showed that thehypercholesterolemic BM environment promotedHSPC proliferation and myeloid differentiation. Weobserved elevated expression of IL-1) and TNF-!, cyto-kines known to promote HSPC proliferation and my-eloid differentiation (3, 4, 28–30, 37, 38), suggestingthat these cytokines are responsible for the hypercho-lesterolemia-induced activation and priming of HSPCs.Interestingly, TNF-! is also known to increase cellproliferation by promoting RAF-1-mediated inactiva-tion of the Rb protein in vascular smooth muscle cells(39). A similar mechanism may also be true of myeloidcells, and may be an alternative explanation of thereduced Rb expression that we observed in hypercho-lesterolemic BM.

Our data show that there is a difference in dynamicresponses of HSPC priming in terms of proliferationand differentiation over time. The effects of niche-mediated alterations on mature leukocyte reconstitu-tion occur sooner after the BM transplantation, and areeven more prominent than the effects of hypercholes-terolemia-primed HSPCs. This suggests more a contin-uous role of the niche in the regulation of HSPCproliferation and shows that both direct and niche-induced hypercholesterolemic priming of HSPCs areimportant in HSPC proliferation and myeloid differen-tiation.

In early atherogenesis, myeloid cells are activelyrecruited to sites of vascular inflammation and criticallycontribute to the initiation of plaque development (8,9). We observed that hypercholesterolemia-inducedactivation of HSPCs not only increased the develop-ment of proinflammatory myeloid cells, but also in-creased leukocyte accumulation in the atheroscleroticplaques. After homing to the inflamed endothelium,these cells produce proinflammatory chemokines andcytokines, which further propagate atherogenesis. Weobserved increased T-cell accumulation in plaques ofmice that received normocholesterolemic BM com-pared with mice that received hypercholesterolemicBM. As shown by Kidani et al. and others (40, 41),hypercholesterolemia may directly affect T-cell progen-itors and effector T cells, however, we observed that thelipid background of the transplanted BM did not affectcirculating T-cell reconstitution or thymocyte reconsti-tution (data not shown). This suggests that the in-creased plaque T-cell content resulted from increasedrecruitment of these cells by proinflammatory plaque



macrophages and not from alterations in T-cell devel-opment. As macrophages are the most abundant leu-kocyte subset in atherosclerotic plaques, we analyzedwhether hypercholesterolemia-induced priming ofHSPCs affects the inflammatory propensity of thesecells. Compared to macrophages differentiated fromnormocholesterolemia-primed HSPCs, those derivedfrom hypercholesterolemia-primed HSPCs producedincreased levels of TNF-!, IL-6, and MCP1, therebyincreasing plaque inflammation and disease progres-sion.

In summary, we have shown that hypercholesterol-emia induces a pathologic HSPC phenotype state thatpersists under long-term normocholesterolemic condi-tions and favors the development of proinflammatorymyeloid cells that aggravate the development of athero-sclerosis. Inhibition of this novel proinflammatorymechanism on the HSPC level harbors the potential toreduce atherosclerosis.

This study was supported by the Dutch Heart Foundation(Dr. E. Dekker grant to T.S.; Dr. E. Dekker EstablishedInvestigator grant to E.L. and M.P.J.D.W.), the NetherlandsOrganization for Scientific Research (NOW; Vidi grant toE.L.; Vici grant to E.L.), the Academisch Medisch Centrum(AMC; fellowship to E.L. and M.P.J.D.W.), the HumboldtFoundation (Sofja Kovalevskaja grant to E.L.), and theDeutsche Forschungsgemeinschaft (DFG809 and SFB 1054 toE.L.). The authors acknowledge the support from the Neth-erlands CardioVascular Research Initiative (participants: theDutch Heart Foundation, Dutch Federation of UniversityMedical Centres, the Netherlands Organisation for HealthResearch and Development, and the Royal Netherlands Acad-emy of Sciences) for the GENIUS project Generating the BestEvidence-Based Pharmaceutical Targets for Atherosclerosis(CVON2011-19).

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Received for publication September 25, 2013.Accepted for publication January 13, 2014.



10.1096/fj.13-243105Access the most recent version at doi:2014 28: 2202-2213 originally published online January 30, 2014FASEB J

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