Bone Marrow–derived Endothelial Progenitor Cells Promote Hematopoietic Reconstitution After Hematopoietic Stem Cell Transplantation

Bone Marrow– derived Endothelial Progenitor Cells Promote Hematopoietic Reconstitution After Hematopoietic Stem Cell Transplantation Z. Yan, L. Zeng, Z. Li, H. Zhang, W. Chen, L. Jia, C. Chen, H. Cheng, J. Cao, and K. Xu ABSTRACT Objective. A hematopoietic deficit is a serious complication after hematopoietic stem cell transplantation. It has been shown that fetal blood– derived endothelial progenitor cells (EPCs) can promote hematopoietic reconstitution after transplantation. This study investigated whether EPCs from bone marrow (BM) of adult mice could promote hematopoietic reconstitution. Methods. Lethally irradiated BALB/c mice were administered BM cells or BM cells plus EPCs. Results. The results showed that EPC-treated mice displayed accelerated recovery of peripheral blood white blood cells and reticulocytes. But the platelets were not significantly different with versus without EPCs. Accelerated recovery of BM sinusoidal vessels, promotion of stem cell implantation, and decreased adipocyte formation were associated with the mechanism. Systemic administration of anti-vascular endothelial cadherin antibody neutralized these effects significantly. Conclusion. These data showed that BM-derived EPC infusions augmented hematopoiesis suggesting a new approach to promote hematopoiesis. ELAYED reconstitution is a serious complication after hematopoietic stem cell (HSC) transplantation that not only increases the risk of infection and bleeding, but also represents one of the principal factors affecting a patient’s prognosis. Currently, the clinical solution to delayed hematopoietic reconstitution relies mainly on artificially synthesized cytokines, such as granulocyte colony stimulating factor, granulocytemonocyte colony stimulating factor, and thrombopoietin. However, these treatments are not effective for some patients. Current studies suggest that delayed hematopoietic reconstitution after transplantation has several main causes, including the quantity and quality of transplanted stem cells, an abnormal bone marrow (BM) microenvironment,1 complications, and the use of drugs (ie, Acyclovir and Imatinib). Moreover, an abnormal BM microenvironment appears to be the most important factor contributing to prolonged hematopoietic reconstitution.2 Previous studies have suggested that damage to the BM sinus endothelium (Fig 1) and adipocyte proliferation in the BM microenvironment after transplantation are the two most important factors affecting reconstitution.3 BM sinus endothelium is

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the main component of vascular niches, containing approximately 60% of HSCs.4 The vascular endothelia growth factor receptor-2 (VEGFR2) is a key signaling molecule contributing to hematopoietic reconstitution; blockade of the VEGFR2 affects stem cell implantation.5 Endothelial progenitor cells (EPCs) the precursors of endothelial cells6 are found mainly in peripheral blood (PB), BM, and cord blood.7 Numerous studies have shown that EPCs participate in the repair of injured blood vessels, contributing to the recovery of target organ function.8 –10 Salter et al observed that EPCs originating from fetal blood promote hematopoietic reconstitution after transplantaFrom the Department of Haematology (Z.Y., L.Z., Z.L., H.Z., W.C., L.J., C.C., H.C., J.C., K.X.), Affiliated Hospital of Xuzhou Medical College, and the Lab of Transplant Immunology (L.Z., K.X.), Xuzhou Medical College, Xuzhou, China. Supported by the Jiangsu and Jiangsu Nature Science foundation of college (10KJB320027) and national Nature Science foundation (30971281, 81070446). Address reprint requests to Kai-lin Xu, No. 99, West Road Huaihai, Xuzhou, China. E-mail: [email protected] 0041-1345/–see front matter http://dx.doi.org/10.1016/j.transproceed.2012.03.064 427

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Fig 1. Endothelial progenitor cell (EPC) infusions accelerate the recovery of mature blood counts. Panels A and B showed leukocyte counts in bone marrow (BM) and BME groups at days 10 and 14 posttransplantation. The difference between these two groups is statistically significant. Panels C and D showed reticulocyte counts at days 10 and 14 posttransplantation. The difference between these two groups is statistically significant. Panels E and F showed platelet counts at day 10 and 14 posttransplantation. There are no differences between the two groups. Therefore, leukocytes recovered to levels that met the standard for hematopoietic reconstitution 4 days earlier in the group that underwent BM and EPC transplantation.

tion. The mechanism underlying this effect seemed to mainly be related to repair of the BM sinus endothelium.11 However, fetal blood– originated EPCs are relatively difficult to obtain, limiting future clinical applications. In vitro induced EPCs derived from a wide variety of sources maintain the ability to repair injured endothelium. We confirmed that infusion of EPCs, induced from BM cells, into irradiated mice accelerated reconstitution of BM HSCs, PB counts, and BM sinusoidal vessels, namely hematopoietic reconstitution. MATERIAL AND METHODS Mice The recipients were 8- to 10-week-old BALB/c female mice (H-2Kd), and the donors were 8- to 10-weel-old C57BL/6 male mice (H-2kb) that were purchased from our animal center and maintained in laminar flow racks with sterile rodent chow and acidified water. All experiments and protocols were performed in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines.

Endothelial Progenitor Cell Culture EPCs were isolated from mononuclear cells (MNCs) of C57BL/6 mice BM using the previously described procedure.12 MNCs were seeded onto fibronectin (FN)– coated (1 ␮g/cm2, Chemicon International, Temecula, Calif, United States) plates using endothelialbasal medium (EBM-2), 10% fetal bovine serum (FBS; Gibco, Logan, Utah, United State), and 1⫻ glutamine-penicillin-streptomycin (GPS; Invitrogen, Carlsbad, Calif, United States). Unbound cells were removed at 48 hours; the coherent fraction was maintained in culture using EBM-2 supplemented with 10% FBS. SingleQuots (except for hydrocortisone), and 1⫻ GPS for 7 days growth before being used in the experiments.

Transplantation of HSCs Lethally irradiated (8.5 Gy) 8- to 10-week-old female BALB/c mice (H-2kd) were injected via the tail vein with one of the following inocula: (1) phosphate-buffered saline (PBS); (2) T cell– depleted BM (TCD-BM) cells (5 ⫻ 106/mouse; the BM group); (3) TCD-BM cells (5 ⫻ 106/mouse) plus EPCs (5 ⫻ 106/mouse; the BME group); (4) TCD-BM cells (5 ⫻ 106/mouse), and EPCs (5 ⫻

HEMATOPOIESIS 106/mouse) with subsequent treatment with vascular endothelial (VE) cadherin antibody (every 2 days); the BMEa group. All mice were administered cyclosporine (2.5 mg/kg · d⫺1) intraperitoneally beginning on the first day posttransplantation to prevent acute graft-versus-host disease (aGVHD). Survival was monitored daily, and PB counts, every 2 days.

Flow Cytometry Analysis To identify EPCs, unbound cells from MNC plates were removed after 48 hours. Adherent cells were maintained in culture using EBM-2 (phenol red free). After digestion using 2.5% trypsin, by filtration through a nylon screen (45 ␮m, Sefar America, Kansas City, Mo, United States, a single cell suspension was obtained. Cells were stained with Per-CP-conjugated anti-CD45 antibody (clone: 30-F11), fluorescein isothicocyanate (FITC)– conjugated anti-CD133 antibody (clone: 13A4), PE-conjugated anti-VEGF antibody (clone: Avas12a1), or APC-conjugated anti-CD144 antibody (clone: 16b1). For KLS cell analyses, BM-MNCs were stained with Per-CP-conjugated anti-CD45 antibody (clone: 30-F11), PEconjugated anti-c-kit antibody (clone: 2B8), FITC-conjugated antiLin antibody (including CD3, B220, Mac1/Gr1, Ter119), and APC-conjugated anti-Sca-1 antibody (clone: D7). All antibodies were purchased from BD Pharmingen (San Diego, Calif, United States). Chimerism in transplanted animals was measured by staining PB MNCs with FITC-anti-H-2Kb (clone: AF6-88.5) and PE-anti-H-2Kd (SF1-1.1). The cells were analyzed using an FACScan flow cytometer (BD Biosciences, San Jose, Calif, United States) equipped with CellQuest software.

Histology For histology, whole tibia and femora were fixed in 4% paraformaldehyde, decalcified in 5% formic acid, and embedded in paraffin. Sections of 4 ␮m were stained with hematoxylin and eosin for histological examination.

429 deparaffinized, blocked for 30 minuted in 5% horse serum, and incubated with an MECA-32 monoclonal antibody (1.6 ␮g/mL) overnight at 4°C. Horseradish peroxidase– conjugated secondary antibody and 3, 3-diaminobenzidine (DAB) were used for detection. The sections were counterstained with hematoxylin and mounted using Permount (Fisher Scientific, Hampton, NH, United States). Images were taken with an Olympus IX-71. Images were assembled into multipanel figures using Adobe Illustrator CS 7.0 (Adobe Systems, San Jose, Calif, United States).

Statistical Analyses The measured data were represented as mean values ⫾ SD. SPSS 13.0 software was used for statistical analyses. Changes in leukocytes, platelets, and KLS cells were analyzed with the Student t test. A P value of less than .05 was considered statistically significant.

RESULTS Implantation of EPCs Promotes the Early Recovery of PB Cells

EPCs were obtained from BM using a procedure established previously in our laboratory. Cell counts greater than 0.5 ⫻ 109/L for granulocytes and greater than 1 ⫻ 109/L for leukocytes were believed to be the minimum standard for hematopoietic reconstitution after transplantation. Mice receiving transplantation of BM plus EPCs granulocytes greater than 1 ⫻ 109/L at day 10, 4 days ahead of hosts treated with BM only (Fig 1A, 1B). Reticulocytes recovered significantly earlier as well (Fig 1C,D). But the platelets showed no significant difference with versus or without EPC infusion (Fig 1E, F). Hence, implantation of EPCs promoted the early recovery of WBCs and reticulocytes.

Immunohistochemistry

Transplantation of EPCs Promotes Early Homing and Localization of HSCs

We used MECA-32 (BioLegend) monoclonal antibody to identify endothelium. For immunohistochemistry, 7-␮m-thick sections were

The process of homing and localization of HSCs in the BM microenvironment are essential for hematopoietic reconsti-

Fig 2. Change of KLS cells in the bone marrow (BM) of recipient mice at days 7, 14, and 21 posttransplantation. Panel A represents the proportion of KLS cells of BM (1.68 ⫾ 0.06)% and BME (2.80 ⫾ 0.38)% at day 7. Panel B represents the proportion of KLS cells of BM (2.80 ⫾ 0.40)% and BME (4.15 ⫾ 0.37)% at day 14. Panel C represents the proportion of KLS cells of BM (0.35 ⫾ 0.25)% and BME (0.27 ⫾ 0.028)% at day 21. Therefore, the transplantation of hematopoietic stem cells combined with endothelial progenitor cells increases the proportion of KSL cells.

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Fig 3. Endothelial progenitor cell infusions accelerate the recovery of bone marrow (BM) sinusoidal vessels at day 14 posttransplantation. (A) Sinusoidal expression of MECA-32 in the control group (brown), (B) Sinusoidal expression of MECA-32 in the irradiation group, (C) There are few expressions of MECA-32 in BM group, (D) More expressions of MECA-32 in the BME group.

tution. KLS (Lin⫺/c-Kit⫹/Sca-1⫹) cells and KL (Lin⫺1/ c-Kit⫹/Sca-1⫺) cells represent specific maturation states of stem and progenitor cells. Their numbers correlate closely with the degree of hematopoietic reconstitution after transplantation. We observed that the number of KLS cells among the BM elements at days 7 and 14 after BM plus EPC cell transplantation were 2.80 ⫾ 0.38% and 4.15 ⫾ 0.37%, respectively. In contrast, they were 1.68 ⫾ 0.06% and 2.80 ⫾ 0.40% respectively, among mice treated with BM only (P ⬍ .05). However, at 21 days after transplantation, the numbers of KSL cells in the BM were 0.27 ⫾ 0.028% and 0.35 ⫾ 0.25%, respectively (P ⬎ .05); (Figs. 2 and 3). Implantation of EPCs Promotes BM Sinus Repair

The integrity of the BM sinus is vital to hematopoietic reconstitution after transplantation. MECA-32 (BioLegend) is a specific endothelial marker. We noticed that markedly few MECA-32 are expressed in mice after irradiation. Surprisingly, there was greater expression of MECA-32 in mice receiving BM cells plus EPCs than BM only, suggesting that these EPCs promote BM sinus repair (Fig 3). Infusion of EPCs Reduces Adipocytes Formation

Increased adipocytes in the BM microenvironment after HSC transplantation inhibit normal hematopoietic activity. We observed adipocytes to arise in the BM microenvironment beginning at day 7 after transplantation and gradually increasing thereafter. However, enhanced hematopoietic

recovery after EPCs showed markedly fewer adipocytes in the marrow cavity, suggesting that infusion of EPCs reduced adipocyte formation, a mechanism that needs further exploration (Fig 4). Administration of VE-cadherin Antibody Abrogates the Effect of EPC Infusions on Hematopoietic Recovery

VE-cadherin, an endothelial-specific adhesion molecule located at endothelial cell junctions, is of vital important for the maintenance and control of endothelial cell contacts, to control blood vessel formation and to regulate cellular signaling processes. The observation that EPC-treated displayed concordant early recovery of BM sinusoidal vessels and hematopoiesis a causal relationship between BM vascular recovery and hematopoietic reconstitution in mice that received EPC transplantation. Administration of antiVE-cadherin antibody to mice that received BM cells and EPCs abrogated the EPC effect on hematopoietic reconstitution. The numbers of leukocytes, reticulocytes, and platelets were significantly different between the two groups at days 10 and 14 after transplantation (Fig 5A). Meanwhile, we observed greater expression of MECA-32 in mice receiving EPCs than those with BM only (Fig 5B). These data suggested a relationship between BM microvascular recovery and hematopoietic reconstitution. DISCUSSION

Induced EPCs have been extensively studied in cardiovascular and ischemic diseases. They not only improve the

Fig 4. Endothelial progenitor cells decrease adipocyte formation in the bone marrow (BM) microenvironment. Panel A was the control. Panel B shows the changes in the BM microenvironment at day 14 posttransplantation of BM mononuclear cells only. Numerous lipid droplets are aggregated in the hematopoietic tissue. Panel C shows an abundance of hematopoietic tissue but only a few lipid droplets in the BM microenvironment of the group that underwent BM cell and endothelial progenitor cell transplantation.

HEMATOPOIESIS

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Fig 5. Administration of vascular endothelial– cadherin antibody abrogates the effects of endothelial progenitor cells (EPC) infusions on hematopoietic recovery. (A) Mice that were treated with EPCs plus VE-cadherin antibody showed significantly delayed recovery of white blood cell reticulocytes and platelets compared with mice treated with EPCs alone. (B) Few MECA-32 expressed in BM group after administration of VE-cadherin antibody than it in BME group.

blood supply to ischemic tissue, but also promote function recovery of host organs.13–15 In this study, we concluded that mice treated with BM cells plus EPCs facilitated the recovery of leukocytes and reticulocytes suggesting that EPCs promoted hematopoietic reconstitution. This effect is particularly important in terms of granulocyte recovery, which helps to reduce the chance of concurrent infections during the period of BM suppression after transplantation. Induced EPCs can be obtained directly from the donor, which avoids the limitations of other sources.

Promoting rapid implantation, proliferation, and differentiation of donor HSCs are the primary means to achieve hematopoietic reconstitution after transplantation. At present, enhancing the proliferation and differentiation of HSCs relies mainly on artificially synthesized cytokines. Our study found that the mice administered EPC showed significantly increased numbers of BM KLS cells compared hosts receiving BM only, suggesting that the administered EPCs supported the expansion of BM KSL cells after irradiation, thereby contributing to expedited hematopoietic reconsti-

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tution. However, how the EPCs regulate the proliferation and differentiation of KSL cells requires further investigation. Under physiological conditions, HSCs remain in a state of rapid proliferation and differentiation during the process of hematopoietic reconstitution, which requires the support of a favorable BM microenvironment. Previous studies from our team and others have reported that conditioning before HSC transplantation can cause severe injury to the BM microenvironment.16 –18 An inhospitable BM microenvironment is not favorable for the proliferation and differentiation of donor HSCs. Particularly, the endothelial sinus plays a critical role in hematopoietic reconstitution.19 After EPCs introduction, the BM sinus endothelium was repaired, providing a favorable niche for HSC implantation, proliferation, and differentiation. These findings suggest that repair of the BM sinus endothelium by EPCs is one of the factors that promote hematopoietic reconstitution. Adipocytes are another important component of the BM microenvironment. They secrete various cytokines, such as adiponectin, interleukin-6, interleukin-8, and prostaglandins, which may promote or inhibit hematopoiesis depending on the conditions.20 Using a transplantation model, we showed BM adipocytes increase by 7 days after transplantation.3 The mechanism underlying this effect may be related to adipocyte-mediated inflammation.21 By inhibiting the MAPK signaling pathway, more mesenchymal stem cells are induced to differentiate to adipocytes from osteoblasts.22 These adipocytes secrete significant amounts of negative regulatory factors that inhibit normal hematopoietic activity.3 Therefore, an optimal BM microenvironent is critical for the success of hematopoietic reconstitution. Our results showed that enhances hematopoietic recovery after BM plus EPCs was associated with markedly fewer adipocytes in the marrow, suggesting an association with promotion of reconstitution. However, how does inflammation affect adipocyte proliferation? How do adipocytes regulate hematopoietic reconstitution after transplantation? What is the relation between EPCs and adipocytes? All of these questions remain unclear. In conclusion, this study showed that in vitro induced BM EPCs can promote hematopoietic reconstitution after HSC transplantation. The underlying mechanisms contributing to the beneficial effects of EPCs include their ability to promote repair of the BM microenvironment, reduced adipocyte formation, and increased proliferation and differentiation of HSCs. However, our study showed that implantation of EPCs mainly promoted the early phases of hematopoietic reconstitution after transplantation and provided no significant advantages for later stages.

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