Circulating Endothelial Progenitor Cells Decreased in Patients with Sclerodermatous Chronic Graft-versus-Host Disease

Article Outline

• Abstract
• Introduction
• Materials and Methods
  • Patients
  • Quantification of Circulating CD34+ Cells and EPCs Frequencies
  • Evaluation of CFU-ECs in the Peripheral Blood
  • Evaluation of Angiogenic Cytokine Levels in the Sera
  • Statistical Analysis
• Results
  • Patients Characeristics
  • Time Course of Circulating CD34+ Cells and EPCs Frequencies after allo-SCT
  • Decreased Circulating CD34+ Cells and EPCs Frequencies Are Associated with s-cGVHD
  • CFU-EC Assays Confirm Reduction of EPCs in s-cGVHD Patients
  • CFU-ECs Are Derived from Donor Cells of allo-SCT
  • Allo-SCT Recipients Had Higher Levels of Angiogenic Cytokines Than Healthy Volunteers
• Discussion
• Acknowledgments
• References
• Copyright

Abstract 

Chronic graft-versus-host disease (cGVHD) is a common late complication of allogeneic stem cell transplantation (allo-SCT). Some cGVHD patients develop skin lesions, and the skin lesions in sclerodermatous cGVHD (s-cGVHD) patients resemble those in progressive systemic sclerosis (PSS), which is characterized by impaired production of circulating endothelial progenitor cells (EPCs). We investigated, retrospectively, whether low EPC production may promote the development of sclerodermatous lesions in cGVHD. Peripheral blood (PB) was obtained from 14 healthy volunteers and 27 allo-SCT patients. Five patients developed s-cGVHD. CD34⁺ cells were purified by using the magnetic cell-sorting separation system, and the CD34⁺/CD133⁺/vascular endothelial growth factor (VEGF) receptor-2⁺ EPCs were quantified. The endothelial cell colony-formation potential was evaluated. Serum VEGF and basic fibroblast growth factor (b-FGF) concentrations were measured by ELISA. The s-cGVHD patients had significantly lower median circulating EPCs frequencies than non-s-cGVHD patients or control (145 of 20 mL [interquartial range—IQR 107-193] versus 1083.5 [IQR 669.3-2151]; P = .0023, and versus 1530.5 [IQR 961.3-2158]; P = .0012, respectively). They also had impaired median endothelial-forming ability compared to non-s-cGVHD patients or controls (3.8 [IQR 1.0-4.3] versus 12.8 [IQR 8.8-28.8], and versus 26.4 [IQR 23.6-30.6], respectively; P = .0012). Their VEGF and b-FGF serum levels were also higher than in controls. In conclusion, s-cGVHD patients show findings consistent with those seen in PSS with impaired vasculogenesis that may limit blood perfusion and may contribute to the development of sclerodermatous lesions.

Key Words: Allogeneic stem cell transplantation, Endothelial progenitor cells, Sclerodermatous chronic GVHD, CD34, VEGR-2

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Introduction 

Allogeneic stem cell transplantation (allo-SCT) is an important therapy for hematologic diseasese. However, despite advances in the procedures and posttransplantation immunosuppressive therapy, over 50% of allo-SCT recipients develop acute or chronic graft-versus-host disease (aGHVD, cGVHD). Chronic GVHD is 1 of the most common late complications, as it occurs in 25%-80% of transplant recipients [1]. It affects the quality of life of long-term survivors after allo-SCT, and its clinical signs resemble those of autoimmune diseases. It has been suggested that fibrotic manifestations associated with cGVHD may be caused by microvessel loss because of cytotoxic T cells injury to microvessel endothelial cells in various organs [2]. As antibodies against host cells are detected in cGVHD showing autoimmune disease-like manifestations [3], the pathophysiology of sclerodermatous cGVHD (s-cGVHD) resembles to those observed in progressive systemic sclerosis (PSS). PSS is characterized by vasculopathy [4], and a recent study revealed that patients with PSS have low frequencies of circulating endothelial progenitor cells (EPCs), which impairs vasculogenesis [5]. On the basis of the similarities with PSS, we postulate that allo-SCT recipients with s-cGVHD may also have low circulating EPC frequencies. We retrospectively investigated the differences of the circulating EPC numbers in the peripheral blood (PB) and the ability of the PB mononuclear cells (PBMNCs) to form endothelial cell colonies by using the standard endothelial cell colony-forming unit (CFU-EC) assay between normal volunteers and 27 allo-SCT patients, of which 5 developed s-cGVHD. We showed that s-cGVHD patients had lower circulating EPC frequencies and impaired endothelial colony-forming potential, even though these patients had elevated angiogenic cytokine levels in their serum.

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Materials and Methods 

Patients 

We recruited 27 allo-SCT patients and 14 healthy volunteers. Allo-SCT was performed at the Division of Hematology and Oncology, Hospital of Kyoto Prefectural University of Medicine, from November 1994 through to April 2006. All patients had maintained complete remission at the time of the study. Acute GVHD was diagnosed based on the standard criteria 6, 7, 8, and cGVHD was diagnosed based on the scoring system of National Institute of Health Consensus Development Project on Criteria for Clinical Trials in Chronic GVHD [9]. Acute GVHD occurred in 18 patients, whereas cGVHD occurred in 18 patients. Healthy controls were age-matched volunteer donors. PB samples were obtained from the 41 participants between June 2006 and April 2007. In accordance with the Declaration of Helsinki recommendations, all procedures were approved by the institutional review board at Kyoto Prefectural University of Medicine, and written informed consent was obtained from every participant. We collected PB from May through November of 2006. PB was obtained once from each patient at various time points after receiving the allo-SCT, ranging from 69 days to 4432 days after transplantation.

Quantification of Circulating CD34⁺ Cells and EPCs Frequencies 

To quantify the CD34⁺ cell frequencies in the PB, we designed a flow cytometric gating strategy on the basis of the International Society of Hematotherapy and Graft Engineering guidelines [10]. Thus, we stained whole blood collected in ethylenediaminetetraacetic acid (EDTA)-containing TruCOUNT tubes (BD Bioscience, San Jose, CA) according to the manufacturer's instructions. Briefly, 20 μL of fluorescein isothiocyanate (FITC)-conjugated anti-CD45/phycoerythrin (PE)-conjugated anti-CD34 combination (BD Bioscience) and 20 μL of 7-amino-actinomycin D (7-AAD) were mixed with 50 μL of PB, and the mixture was incubated at room temperature for 15 minutes. After red blood cell lysis, we used FACSCalibur (BD Bioscience) to acquire 100,000 CD45⁺ cells events. There are counting beads in TruCOUNT tubes and the frequency of CD34⁺ cells/μL of whole blood was calculated as follows: CD34⁺ cells/μL = (number of CD34⁺ cell events) × (number of beads per test tube)/{(number of counted beads) × 100}. The mean frequency of CD34⁺ cells in the PB was obtained from 2 or 3 replicates.

The frequency of the EPCs in the PB was determined by isolating the PBMNCs from 20 mL heparinized PB by Ficoll-Hypaque density centrifugation. The CD34⁺ cells were enriched from the PBMNCs by using the magnetic cell sorting (MACS) separation system (Miltenyi, Gladbach, Germany). The average purity of the enriched CD34⁺ cell populations was 86.5%. The sorted cells were then stained with FITC-conjugated anti-CD34 mAb, PE-conjugated antivascular endothelial growth factor receptor (VEGFR)-2 mAb (R&D Systems, Minneapolis, MN), and allophycocyanin-conjugated anti-CD133 mAb (Miltenyi). Appropriate isotype control Abs served as controls. Viable cells were identified by gating on their forward and side scatters, and the expression by the CD34⁺ cells of VEGFR-2 and CD133 was assessed. We defined EPCs as CD34⁺/VEGFR-2⁺/CD133+ cells 11, 12 and calculated the circulating EPC frequency as follows: EPCs /20 mL PB = (the absolute number of CD34⁺ cells/μL) × {(the number of events of VEGFR-2⁺/CD133⁺ cell events in the CD34⁺ cell gates)/(the number of events of CD34⁺ cell events)} × 20,000.

Evaluation of CFU-ECs in the Peripheral Blood 

We also determined the ability of the PBMNCs from these patients to form endothelial cell colonies by using the EndoCult Liquid Medium Kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's protocol. Briefly, PBMNCs were resuspended in complete EndoCult medium and seeded at 5 × 10⁶ cells/well onto fibronectin-coated tissue culture plates (BD Bioscience). After 48 hours, the wells were washed with medium and the nonadherent cells were collected and plated in their existing medium at 10⁶ cells/well into 24-well fibronectin-coated tissue culture plates for 3 days. Colonies were defined as a central core of round cells surrounded by elongated sprouting cells at the colony periphery. The number of CFU-ECs was counted under an inverted microscope in duplicate or triplicate. To confirm that the colony cells are endothelial cells, fluorescent staining was used to detect the binding of FITC-conjugated Ulex europaeus lectin (Sigma, St. Louis, MO) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (acLDL) (Molecular Probes, Eugene, OR) [13]. We also stained the colony cells with mAbs against endothelial lineage surface markers. For this purpose, the colony cells were harvested after treatment with 0.05% trypsin/EDTA (Gibco, Tokyo, Japan) and were spun down onto slides by using Cytospin-3 (ThermoShandon, Cheshire, UK). The cells were fixed with 2% paraformaldehyde, blocked with goat immunoglobulin, and then incubated at 4°C overnight with mouse anti-CD31 mAb (Dako, Tokyo, Japan). Thereafter, the cells were incubated with FITC-conjugated mouse specific mAb (Sigma) for 1 hour at room temperature and then cells were incubated with propidium iodide for 30 minutes at room temperature. The stained cells were examined by FV1000 laser scanning confocal microscopy (Olympus, Tokyo, Japan).

To confirm that the CFU-ECs obtained from the recipients' PBMNCs were of donor origin, the CFU-ECs obtained from female patients who had received stem cells from a male donor were subjected to fluorescein in situ hybridization (FISH) analysis for the Y-chromosome as previously described [14]. Briefly, the CFU-EC cells were centrifuged onto the silane-coated glass slides described above, fixed in Carnoy's solution (methanol and acetic acid in a 3:1 ratio) for 10 minutes at ambient temperature, and then air dried. After denaturation at 70°C for 5 minutes in 70% formamide/2 × saline sodium citrate (SSC) (1× SSC consists of 0.15 M sodium chloride and 0.015 M sodium citrate), the slides were dehydrated in an ethanol series at room temperature and air dried. The probes mixture used to detect the X and Y chromosomes (X chromosome, CEP X (DXZ1); Y chromosome, CEP Y (DYZ1); Abbott Vysis, Chiba, Japan) was denatured at 73°C for 5 minutes, placed at 45°C, and applied onto the slide. After overnight hybridization in a humidified incubator at 37°C, the slides were washed in 0.4 × SSC/0.3% NP40 solution at 73°C for 30 minutes, then in 2 × SSC/0.1% NP40 solution at ambient temperature for 1 day and air dried. The slides were then counterstained with 4′, 6-diamidino-2-phenylindole (DAPI II, Abbott Vysis) and mounted in Vectashield (Vector Laboratories, Burlingame, CA). The FISH images were analyzed by using a fluorescence microscope BX40-RF (Olympus) and captured with a CCD camera (SenSys0400-G1; Photometrics, Tucson, AZ).

Evaluation of Angiogenic Cytokine Levels in the Sera 

We assessed the serum levels of VEGF and basic fibroblast growth factor (b-FGF) by using specific ELISA Kits (Quantikine, R&D Systems) according to the manufacturer's instructions.

Statistical Analysis 

Comparison between any 2 groups of participants was done by using the nonparametric Mann-Whitney U test. In the case of cytokine levels under the lowest detectable values by an ELISA kit, the median values between 0 and the lowest detectable values were used for the statistical analyses. The significance of differences between 2 groups analyzed by the Mann-Whitney U test with the Bonferroni correction (3 comparisons in 3 groups) was considered to be significant at P < .0167. The correlations between the number of circulating CD34⁺ cells and EPCs, and the examined days after transplantation were assessed using Spearman's rank correlation test. P values <0.05 were considered to be statistically significant.

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Results 

Patients Characeristics 

The characteristics of the 27 allo-SCT patients are shown in Table 1. The stem cell sources used for allo-SCT were bone marrow (14 patients), PB (11 patients), and umbilical cord blood (2 patients). PB was obtained once from each patient at various time points after receiving the allo-SCT, ranging from 69 days to 4432 days after transplantation. Five patients developed s-cGVHD around the 500th days after allo-SCT, 2 of which had received PB and 3 of which received BM. These patients were tested 593, 1250, 2163, 2257, and 3708 days after transplantation, respectively. We evaluated sclerodermatous lesions in s-cGVHD using a modified Rodnan skin score, which is used for the evaluations of skin lesions in PSS [15]. To assess the extent of skin involvement, 17 body areas are palpated and scored on the following scale: 0 = normal, 1 = thickened, 2 = thickened, unable to move, and 3 = thickened, unable to pinch (maximum score = 51). This scoring scale is used for the evaluations of s-cGVHD [16]. The mean modified Rodnan skin score of the s-cGVHD patients was 26.2.

Table 1. Patients' Characteristics

Pt. indicates patient; SCT, stem cell transplantation; GVHD, graft-versus-host disease; F, female; M, male; MDS, myelodysplastic syndrome; AML, acute myelogenous leukemia;

CML, chronic myelogenous leukemia; NHL, non-Hodgkin's lymphoma; CMML, chronic myelomonocytic leukemia; ALL, acute lymphoblastic leukemia; MM, multiple myeloma; AA, aplastic anemia; LCH, Langerhans cell histiocytosis; CY, cyclophosphamide; TBL, total-body irradiation;, Flu, fludarabine; l-PAM, melphalan; 2CdA, 2-chrolodeoxyadenosine; BU, busulfan; ATG, antithymocyte globulin; ETP, etoposide; Msib, matched sibling; MUD, matched unrelated donor; MMUD, mismatched unrelated donor; MMRD, mismatched related donor; PB, peripheral blood; BM, bone marrow; UCB, umbilical cord blood; PS, performance score; S, skin; M, mouth; E, eyes; GI, gastrointestinal tract; Li, liver; Lu, lungs; J, joints and fascia; CsA, cyslosporine A; FK, tacrolimus; PSL, predonisolone.

Time Course of Circulating CD34⁺ Cells and EPCs Frequencies after allo-SCT 

The PBs of the 27 patients and 14 healthy controls were subjected to flow cytometric analysis to determine the circulating EPC frequencies. Circulating EPCs were identified as CD34⁺/CD133⁺/VEGFR-2⁺ cells. Representative flow cytometric data are shown in Figure 1. The data of each patient was shown in Table 2. The frequencies of circulating CD34⁺ cells and EPCs in the allo-SCT patients are shown in Figure 2A and B, respectively. The correlations between the CD34⁺ cell numbers and the days after transplantation, and the EPC numbers and the days in non-s-cGVHD patients are statistically significant (Rs = 0.508; P = .02, and Rs = 0.614; P = .005, respectively), whereas the correlations between the CD34⁺ cell numbers and the days after transplantation, and the EPC numbers and the days are not significantly in all patients including the s-cGVHD patients (Rs = 0.254; P = .19, and Rs = 0.306; P = .12, respectively). These observations suggests that there could be a time-dependent change in the circulating CD34⁺ cell and EPC frequencies in the allo-SCT patients without s-cGVHD and that the frequencies were very low up until 500 days after transplantation, at which point they started to recover and fall into the normal donor range of frequencies. However, when s-cGVHD was present, the frequencies of these cell types remained low, which suggests that these patients have impaired endothelial differentiation.

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• Figure 1 

  Detection of EPCs in the PB. Representative flow cytometric data showing the detection of EPCs in the PB of a healthy volunteer (A), a patient without s-cGVHD (B), and a patient with s-cGVHD (C). The upper right quadrant of the individual dot-plot images contains the CD34⁺/CD133⁺/VEGFR-2⁺ EPCs. The frequency of EPCs in the gated CD34⁺ cells and the absolute number of EPCs detected in 20 mL of peripheral blood are shown in each panel. The graph (D) shows negative control data obtained by using isotype control antibodies.

Table 2. Absolute Numbers of CD34, EPCs, CFU-EC, and Serum Cytokine Levels

EPC indicates endothelial progenitor cell; CFU-ECs, endothelial cell colony-forming unit; VEGF, vascular endothelial growth factor; b-FGF, basic fibroblast growth factor.

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• Figure 2 

  Scattergrams showing the frequencies of CD34⁺ cells (A) and EPCs (B) in the PB from normal donors, s-cGVHD patients, and non-s-cGVHD patients (with and without cGVHD) relative to when the PBs were obtained after allo-SCT. There was a clear time-dependent change in the circulating CD34⁺ cell (A) and EPC (B) frequencies in the allo-SCT patients, as the frequencies were very low up until 500 days after transplantation, at which point they started to recover and fall into the normal donor range of frequencies. However, when s-cGVHD was present, the frequencies of these cell types remained low. Black triangles, white triangles, and red squares represent the absolute cell numbers in the PB of non-s-cGVHD patients without cGVHD, non-s-cGVHD with cGVHD, and s-cGVHD patients, respectively. Black circles represent the absolute cell numbers in the PB of healthy volunteers.

Decreased Circulating CD34⁺ Cells and EPCs Frequencies Are Associated with s-cGVHD 

To confirm that the s-cGVHD patients had lower EPCs frequencies and had impaired endothelial differentiation, we statistically compared the data of the 5 s-cGHVD with those of the 14 non-s-cGVHD patients who were tested >500 days after allo-SCT. This is because the s-cGVHD in our cases occurred about 500 days after allo-SCT, and it was around 500 days that the CD34⁺ cell and EPC frequencies started to recover. The median CD34⁺ cell frequencies of the healthy volunteers, the 14 non-s-cGVHD, and the s-cGVHD patients were 1.74/μL PB (interquartile range [IQR] 1.34-2.01), 0.85 (0.73-1.62), and 0.41 (0.17-0.42), respectively (Figure 3A). The non-s-cGVHD patients had significantly lower frequencies than control (P = .018), whereas the s-cGVHD patients had significantly lower frequencies when compared to either the healthy volunteers (P = .0012) or non-s-cGVHD (P = .014) groups by the Mann-Whitney U test with the Bonferroni correction. Thus, although hematopoiesis generally remains slightly decreased after allo-SCT, patients with s-cGVHD have very significantly impaired hematopoiesis as shown in the previous report [17]. A similar analysis of the EPC frequencies revealed median frequencies in the healthy volunteer, non-s-cGVHD, and s-cGVHD groups of 1530.5 cells/20 mL (IQR 961.3-2158), 1083.5 (669.3-2151), and 145 (107-193), respectively (Figure 3B). The difference between the healthy volunteer and non-s-cGVHD groups was not significant, as determined by the Mann-Whitney U test with the Bonferroni correction. In contrast, the s-cGVHD group had significantly lower EPC frequencies than both the healthy volunteer and non-s-cGVHD groups (P = .0012 and .0023, respectively). We investigated the frequencies of circulating EPCs of non-s-cGVHD patients by divided into those with and without c-GVHD. Those of the patients without cGVHD and with cGVHD (without s-cGVHD) were 1460.5, and 89.5/20mL of PB, respectively. The frequencies of EPCs in s-cGVHD patients were significantly lower than those in patients without c-GVHD (P = .006) and with c-GVHD (P = .0084). However, there was no significant difference between those in patients without c-GVHD and with c-GVHD (P = .197). We also evaluated the impacts of immunosuppressants on the frequencies of circulating EPCs in 14 patients who were tested >500 days after allo-SCT. The medians of the EPC frequencies in patients treated with and without the immunosuppressants are 990, and 1177 cells/20mL of PB, respectively. There were no significant differences (P = .549). Immunosuppression had no influences on the circulating EPC frequencies. Taken together, the sclerodermatous changes in cGVHD have impacts on the circulating EPC frequencies after allo-SCT.

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• Figure 3 

  CD34⁺ cell (A) and EPC (B) frequencies in the PB from normal donors, s-cGVHD patients, and non-s-cGVHD patients. The CD34⁺ cell and EPC frequencies are expressed as cells per microliters and per 20 mL of PB, respectively. The non-s-cGVHD patients consist only of the 14 patients who gave PB >500 days after transplantation. In non-s-cGVHD patients, white circles represent patients with cGVHD. The P values were determined by using the Mann-Whitney U test with the Bonferroni correction.

CFU-EC Assays Confirm Reduction of EPCs in s-cGVHD Patients 

We next assessed the ability of endothelial colony in PBMNCs of each group. The median CFU-EC numbers per 10⁶ nonadherent cells in the healthy volunteer, non-s-cGVHD, and s-cGVHD groups were 26.4 (IQR 23.6-30.6), 12.8 (IQR 8.8-28.8), and 3.8 (IQR 1.0-4.3), respectively (Figure 4A). The s-cGVHD patients had significantly lower CFU-EC forming ability than the other 2 groups (P = .0012 and .0012, respectively), as determined by the Mann-Whitney U test with the Bonferroni correction. We also assessed the phenotype of the cells making up the colonies by fluorescent immunostaining. The cells in the colonies from all 3 groups showed lectin-binding activity and uptake of acLDL (Figure 4B), and expressed the endothelial marker CD31 (Figure 4C).

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• Figure 4 

  Endothelial cell differentiation potential of nonadherent PBMNCs from the normal donors, s-cGVHD patients, and non-s-cGVHD patients. The number of CFU-ECs per 10⁶ nonadherent PBMNCs from each participant is shown in (A). The non-s-cGVHD patients consist only of the 14 patients who gave PB >500 days after transplantation. In non-s-cGVHD patients, white circles represent patients with cGVHD. The P values were determined by using the Mann-Whitney U test with the Bonferroni correction. The colony cells had lectin-binding activity and took up of acLDL (B), and expressed CD31 (C). FISH analysis revealed the Y-chromosome signal in the colony cells from female patients who had received male donor stem cells (D). The white bars represent 100 μm (B), 5 μm (C), and 10 μm (D).

CFU-ECs Are Derived from Donor Cells of allo-SCT 

We next subjected the colonies from the patients to FISH analysis to confirm that these cells were of donor origin. There were too few colonies from the s-cGVHD patients to permit FISH analysis. However, when the colonies from non-s-cGVHD female patients who had received male donor stem cells were subjected to FISH, Y-FISH signal was detected in over 95% of the 500 colony cells that were tested (Figure 4D). Thus, it is likely that the circulating CFU-ECs of all the allo-SCT patients were of donor origin.

Allo-SCT Recipients Had Higher Levels of Angiogenic Cytokines Than Healthy Volunteers 

Last, we measured the serum levels of VEGF and b-FGF (Figure 5). The non-s-cGVHD patients and s-cGVHD had higher median VEGF levels (37.5 [IQR 24-135.3]) and 53 (33-107), respectively) than the healthy volunteers (<20 [<20-24.8], P = .0015 and P = .0012, respectively), as determined by the Mann-Whitney U test with the Bonferroni correction. However, the VEGF levels of the 2 patient groups did not differ significantly. With regard to the serum levels of b-FGF, the non-s-cGVHD patients had significantly higher median levels (23.5 IQR [12.5-28.5]) than the healthy volunteers (<10 [<10-11.75], P = .0012), whereas the s-cGVHD patients tended to have higher levels (16 [12-69]) than the normal donors (P = .04). A significant difference in b-FGF levels was not observed between the 2 patient groups.

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• Figure 5 

  Serum VEGF (A) and b-FGF (B) levels in the normal donors, the non-s-cGVHD patients consist only of the 14 patients who gave PB >500 days after transplantation. In non-s-cGVHD patients, white circles represent patients with cGVHD. The P values were determined by using the Mann-Whitney U test with the Bonferroni correction.

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Discussion 

We showed here that within the first 500 days after allo-SCT, the circulating CD34⁺ cell and EPC frequencies in patient PBs were extremely low but that these frequencies then recovered to normal levels. However, if the patients developed s-cGVHD, which our 5 patients did around the 500th day after transplantation, their CD34⁺ cell and EPC frequencies remained low. Compared to healthy volunteers or non-s-cGVHD patients who were tested >500 days after allo-SCT, the s-cGVHD patients also formed endothelial cell colonies significantly less. The previous report demonstrated that the number of circulating EPCs is decreased in patients with PSS [5]. According to this report that the estimation of the circulating EPC numbers was performed by the similar flow cytometric procedures, the number of circulating CD34⁺/CD133⁺/VEGFR-2⁺ EPCs from healthy volunteers (the median number = 1074/20 mL of PB) is as high as that in our study (the median number = 1530.5/20 mL of PB). Because the frequencies of circulating EPCs in patients with PSS (the median number = 274/20 mL of PB) is as low as that in patients with s-cGVHD (the median number = 145/20 mL of PB), we concluded that the circulating EPCs frequencies are decreased in the patients with s-cGVHD similar to those in the patients with PSS.

Total-body irradiation (TBI) and/or chemotherapy are used as a conditioning regimen in allo-SCT. To evaluate the role of TBI as a potential cause for decreased CD34⁺ cells and EPCs, we compared these frequencies in patients who received TBI-containing regimen and in those who received non-TBI regimen. There were no significant differences between them (data not shown). Regardless of TBI, the conditioning regimen not only injures endothelial cells [18] but also damages the bone marrow (BM) stromal function [17], which decreases the number of progenitors in the BM. This probably explains why the allo-SCT recipients had lower circulating CD34⁺ cell frequencies shortly after transplantation compared to healthy volunteers. Because EPCs are derived from CD34⁺ hematopoietic cells [19], the conditioning regimen is probably also responsible for low EPC frequencies observed after allo-SCT. Supporting this is our FISH analysis, which showed that the circulating EPCs in the recipients are of donor origin. Thus, the conditioning regimen damages hematopoiesis and impairs the production of EPCs after allo-SCT. Subsequently, hematopoiesis from the donor stem cells then begins occurring. An elevation in angiogenic cytokines production then stimulates the EPCs to mobilize into the PB. These events lead to a recovery in circulating EPC numbers and endothelial cell colony formation potential.

It has been suggested previously that patients with cGVHD have a profound and sustained impairment of hematopoiesis [17]. This may suppress the production of EPCs in the bone marrow, and reduce CD34⁺ cell and EPC frequencies in the PB of the severe form of cGVHD such as s-cGVHD. In this study, s-cGVHD patients show findings consistent with those seen in PSS with impaired vasculogenesis [5]. Two mechanisms contribute to postnatal neovascularization. In the first, new blood vessels are produced from preexisting vessels by the sprouting of differentiated ECs (angiogenesis). In the second mechanism, EPCs are mobilized from the bone marrow and construct de novo blood vessels (vasculogenesis). Circulating EPCs are important to the process of vascular repair. The EPC numbers reflect vascular disease and impairment of vasculogenesis 20, 21, 22 and vasculogenesis contributes to vascular healing in response to vascular injuries or ischemia 23, 24. It is speculated that the decrease of EPCs frequencies may limit blood perfusion and may contribute to the development of sclerodermatous lesions in s-cGVHD patients as seen in patients with PSS.

In the present study, there were no differences of serum levels of angiogenic factors between the patients with and without s-cGVHD. The reasons why EPCs frequencies did not increase in the s-cGVHD patients desptie and increase of angiogenic factors could be that the EPCs themselves are damaged in s-cGVHD patients. One possibility is that EPCs may be exhausted by repairing the endothelial injuries of s-cGVHD. Del Papa et al. [25] described that the circulating EPCs are decreased despite the elevated serum VEGF levels in advanced PSS patients. They showed the decrease of CD133⁺ EPCs in the BM of advanced PSS patients and speculated that the decrease of the circulating EPCs is caused by the exhaustion of EPCs in the BM. We did not estimate the number of EPCs in the BM. However, the impairment of the hematopoiesis because of cGVHD could induce the decrease of EPCs in the BM. In addition to this, the mobilization of EPCs into the PB for the repairing endothelial injuries might have decreased further. The second possibility is that the EPCs in s-cGVHD patients may be functionally altered such that they become intrinsically hyporesponsive to angiogenic stimuli. A similar EPC dysfunction is observed in patients with diabetes mellitus type II [26]. As a result of this functional alteration of EPCs, endothelial colony formation and the mobilization of EPCs into the PB is impaired even though high levels of angiogenic cytokines such as VEGF and b-FGF are being produced. A third possibility to explain the low EPC frequencies in s-cGVHD patients is that EPC apoptosis may be increased in these patients. Supporting this is the fact that patients with coronary artery disease show continuous endothelial injury that may deplete the circulating EPC frequencies [20]. Moreover, EPCs have been shown to be sensitive to apoptotic induction [27]. Thus, it is possible that the transplanted stem cells or EPCs in s-cGVHD patients may be induced to apoptosis because of the chronic endothelial injury caused by GVHD [2]. Taken together, we speculated that the EPC frequencies are decreased in the s-cGHHD patients despite the lack of differences in the levels of the measeured angiogenic factors between s-cGVHD and non-s-cGVHD patients. Although the low EPC frequencies in s-cGVHD patients may be because of an imbalance between the angiogenic and angiostatic cytokines, we did not measure the serum levels of angiostatic cytokines like endostatin. It remains possible that s-cGVHD patients may be producing high levels of angiostatic factors that could suppress the differentiation or maturation of EPCs.

Recently it was reported that true EPCs are not CD34⁺/CD133⁺/VEGFR-2⁺ cells, but CD34⁺/CD45⁻ cells [28]. However, the hematopoietic origin CD34⁺/CD133⁺/VEGFR-2⁺ EPCs can differentiate into endothelial cells. These cells may be mainly responsible for maintaining the level of circulating EPCs and angiogenic capacity of wound or ischemic tissue [23]. Therefore, our results indicate that the poor recovery of circulating EPCs after allo-SCT is predictive for s-cGVHD characterized by poor vascularity in the skin lesions [2].

In conclusion, we demonstrated that the circulating EPCs frequencies and the ability of endothelial colony formation of PBMNCs are decreased in s-cGVHD patients after allo-SCT. It remains unclear how to treat cGVHD, in particular s-cGVHD, although several promising therapies are available 29, 30. It is possible that circulating EPC frequencies and endothelial cell colony formation ability may be good markers that indicate responsiveness to therapy. Moreover, our observations suggest that s-cGVHD patients develop sclerodermatous lesions because they suffer blood vessel loss because of chronic endothelial injury and low EPC frequencies; this, in turn, may impair blood perfusion and fibrosis of the skin, resulting in the development of sclerodermatous lesions in the patients with cGVHD. Given that relatively few s-cGVHD patients were examined in the present study, additional, more extensive, studies are needed to confirm these theories.

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Acknowledgments 

The authors disclose no commercial affiliations. We thank N. Urao and M. Goto for their excellent technical assistance. This work was partly supported by Grant-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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References 

1. Farag SS. Chronic graft-versus-host disease: where do we go from here?. Bone Marrow Transplant. 2004;33:569–577
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