Circulating Endothelial Progenitor Cells Decreased in Patients with Sclerodermatous Chronic Graft-versus-Host Disease Kazuho Shimura, Eishi Ashihara,

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Circulating Endothelial Progenitor Cells Decreased in Patients with Sclerodermatous Chronic Graft-versus-Host Disease Kazuho Shimura, Eishi Ashihara, Chihiro Shimazaki, Shinsaku Matsunaga, Kyoko Taniguchi, Hitoji Uchiyama, Yosuke Matsumoto, Shinya Kimura, Hiroaki Matsubara, Masafumi Taniwaki, Taira Maekawa Biology of Blood and Marrow Transplantation Volume 14, Issue 4, Pages 426-437 (April 2008) DOI: 10.1016/j.bbmt.2008.02.001 Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

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. Biology of Blood and Marrow Transplantation 2008 14, 426-437DOI: (10.1016/j.bbmt.2008.02.001) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

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. Biology of Blood and Marrow Transplantation 2008 14, 426-437DOI: (10.1016/j.bbmt.2008.02.001) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

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. Biology of Blood and Marrow Transplantation 2008 14, 426-437DOI: (10.1016/j.bbmt.2008.02.001) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

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). Biology of Blood and Marrow Transplantation 2008 14, 426-437DOI: (10.1016/j.bbmt.2008.02.001) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

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. Biology of Blood and Marrow Transplantation 2008 14, 426-437DOI: (10.1016/j.bbmt.2008.02.001) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions