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Kinetics of circulating endothelial progenitor cells in mice with type II collagen arthritis
Blood Cells, Molecules, and Diseases 35 (2005) 236 – 240 www.elsevier.com/locate/ybcmd
Kinetics of circulating endothelial progenitor cells in mice with type II collagen arthritis Daitaro Kurosaka*, Jun Yasuda, Ken Yoshida, Chiho Yasuda, Yasuhiko Toyokawa, Toru Yokoyama, Isamu Kingetsu, Akio Yamada Department of Internal Medicine, Division of Rheumatology, Jikei University School of Medicine, 3-25-8, Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan Submitted 7 June 2005 Available online 14 July 2005 (Communicated by M. Lichtman, M.D., 9 June 2005)
Abstract Objective: To examine the significance in arthritis of circulating endothelial progenitor cells (cEPCs) reportedly increasing in neovascularization. Methods: Arthritis was induced by immunizing DBA/1J mice with bovine type II collagen on day 0. Age-matched normal DBA/1J mice were used as controls. Blood was collected from these mice on days 7, 14, 21, 28, and 35. Peripheral blood CD45 , CD34+, Flk-1+, CD117+ cells were regarded as cEPCs (Flk-1 = vascular endothelial growth factor receptor 2). The number of cEPCs per 100 CD45+ cells was calculated by four-color flow cytometry, and compared with the arthritis score. Results: Arthritis developed about 3 days after booster immunization (day 21). On days 7, 14, and 21, no difference in cEPCs/100 CD45+ cells was noted between the arthritis and control groups. On days 28 and 35, cEPCs/100 CD45+ cells in the arthritis group were significantly greater in number than those in the control group. cEPCs/100 CD45+ cells on day 28 were greater in number than those on day 35. On day 28, a correlation was found between cEPCs/100 CD45+ cells and arthritis score. Conclusion: In mice with type II collagen-induced arthritis, an increase in cEPCs was associated with the onset of arthritis. The number of cEPCs was greater during the development and progression of arthritis than that at the time of its establishment, suggesting that cEPCs are involved in the pathogenesis of arthritis. D 2005 Elsevier Inc. All rights reserved. Keywords: Endothelial progenitor cell; Rheumatoid arthritis; Collagen induced arthritis; Neovascularization
Introduction Neovascularization occurring in mature individuals was previously thought to occur by angiogenesis, involving the proliferation and migration of endothelial cells of preexisting blood vessels. However, studies have discovered endothelial progenitor cells in the blood of mature individuals, showing that, in addition to angiogenesis, vasculogenesis in neovascularization in mature individuals exists, involving the mobilization of endothelial progenitor cells from the marrow, their incorporation into local
* Corresponding author. Fax: +81 3 3578 9078. E-mail address: [email protected] (D. Kurosaka). 1079-9796/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2005.06.001
tissues, proliferation, differentiation, and migration [1]. Vasculogenesis involving these endothelial progenitor cells has been reported to be important not only in physiological neovascularization, but also in various pathological states such as cancer and cardiovascular disease [2– 4]. For example, when tumors are transplanted into mice, endothelial progenitor cells are immobilized from the marrow, and participate in the formation of tumor blood vessels [5]. On the other hand, in rheumatoid arthritis, neovascularization is observed in the inflammatory synovial membrane, and is considered to be essential for the nourishment of proliferating synovial tissue. In this study, we examined the kinetics of circulating endothelial progenitor cells (cEPCs) during the development of arthritis in animal
D. Kurosaka et al. / Blood Cells, Molecules, and Diseases 35 (2005) 236 – 240
models of rheumatoid arthritis, that is, mice with type II collagen-induced arthritis.
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14, 21, 28, and 35 were n = 10, 9, 11, 11, and 11, respectively. As controls, 9, 9, 10, 10, and 10 age-matched normal DBA/1J mice were analyzed on days 7, 14, 21, 28, and 35, respectively.
Materials and methods Evaluation of arthritis Animal models An emulsion (100 Al) of 0.3% bovine type II collagen (Collagen Research Center, Tokyo, Japan) solution in acetic acid and an equal volume of 2 complete Freund’s adjuvant (Difco Lab, Detroit, MI) was injected intracutaneously into the tail root of 5-week-old male DBA/1J mice on day 0. On day 21, the mice were boost-immunized by an intracutaneous injection into the tail root of an emulsion of 0.3% bovine type II collagen solution in acetic acid and an equal volume of 1 incomplete Freund’s adjuvant (Difco Lab, Detroit, MI). The numbers of mice analyzed on days 7,
Arthritis was evaluated by arthritis score: 0 (no swelling), 1 (finger swelling or mild swelling), 2 (clear swelling), 3 (severe swelling), and 4 (arthrokleisis or joint deformity). The total arthritis score was obtained by adding the score of each of the four limbs. Leukocyte count Blood leukocytes in heparinized blood were counted using a Celltac-alpha (Nihon Kohden, Tokyo, Japan) on days 14, 21, 28, and 35.
Fig. 1. Analysis of cEPCs. (A) Analysis of cEPCs by four-color flow cytometry: (a) Analysis gate used to exclude platelets, dead cells, and cell debris. (b) Gate used to exclude CD45+ hematopoietic cells. (c) Separation of CD34+ cells. (d) Separation of Flk-1+ cells. (e) Measurement of CD117+ cells (CD45 , CD34+, Flk-1+, CD117+ cells). (B) Blood leukocyte counts in the control and arthritis groups. SSC, side light scatter; FSC, forward light scatter; FLK, Flk-1 = vascular endothelial growth factor receptor 2; CIA, type II collagen-induced arthritis model mouse group.
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D. Kurosaka et al. / Blood Cells, Molecules, and Diseases 35 (2005) 236 – 240
Measurement of endothelial progenitor cell counts
Statistical analysis
Mice were euthanized with diethyl ether on days 7, 14, 21, 28, and 35, and heparinized blood was obtained from the heart. Circulating endothelial progenitor cells (cEPCs) were counted according to the method of Capillo et al. [5] using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA). Heparinized whole blood (100 Al) was hemolyzed, and stained with peridinin chlorophyll-a protein-conjugated anti-mouse CD45 monoclonal antibody (BD Biosciences, San Jose, CA), phycoerythrin-conjugated anti-mouse Flk-1 antibody (eBioscience, San Diego, CA), fluorescein isothiocyanate-conjugated rat anti-mouse CD34 monoclonal antibody (BD Biosciences), and allophycocyanin-conjugated anti-mouse CD117 monoclonal antibody (eBioscience). First, platelets, dead cells, and cell debris were excluded by forward light scatter (FSC) and side light scatter (SSC) (Fig. 1A-a). Next, CD45+ cells were excluded by gating (Fig. 1A-b), and then CD34+, Flk-1+ cells among CD45 cells were separated (Figs. 1A-c,d). Among these cells, CD117+ cells were regarded as cEPCs (Fig. 1A-e). Finally, the number of CD45+ cells and that of CD45 , CD34+, Flk-1+, CD117+ cells were measured, and the number of CD45 , CD34+, Flk-1+, CD117+ cells per 100 CD45+ cells (cEPCs/100 CD45+ cells) was calculated. After acquisition of at least 100,000 cells/sample, analyses were considered as informative when adequate number of events (typically 50– 200) were collected in cEPCs enumeration gates. Percentages of stained cells were determined and compared with appropriate negative controls. Positive staining was defined as being greater than non-specific background staining.
The Mann – Whitney U test was used to compare cEPCs/ 100 CD45+ cells and peripheral leukocyte counts between the arthritis and control groups. The correlation between cEPCs/100 CD45+ cells and arthritis score was assessed by Spearman’s rank correlation coefficient. P < 0.05 was considered significant.
Results Although most mice developed arthritis about 3 days after booster immunization (day 24), some mice had already developed it on the day of booster immunization (day 21). The mean arthritis scores on days 28 and 35 were 4.1 T 1.0 (SE) and 6.3 T 0.7 (SE), respectively (Fig. 2A). We evaluated the kinetics of cEPCs as follows. First, we counted blood leukocytes in the control and arthritis groups on days 14, 21, 28, and 35. The mean leukocyte counts T SE/Al in the control group at these timepoints were 6057 T 720, 7771 T 708, 7510 T 744, and 7020 T 589, respectively, and those in the arthritis group were 5364 T 720, 7131 T 508, 6764 T 875, and 8560 T 1158, respectively. Comparison between the 2 groups by the Mann – Whitney U test revealed P = 0.255, P = 0.526, P = 0.379, and P = 0.295 for the respective timepoints, showing no significant differences (Fig. 1B). On the other hand, since CD45 can be used as a leukocyte marker, the finding of no differences in leukocyte counts leads to no differences in CD45+ cell counts. Thus, we judged that the number of cEPCs relative to CD45+ cells could be used to
Fig. 2. cEPCs and arthritis score. (A) The arthritis score at each timepoint. (B) The number of cEPCs per 100 CD45+ cells at each timepoint. cEPCs/100 CD45+ cells: the number of cEPCs per 100 CD45+ cells. *P < 0.05 (comparison between the control and arthritis groups on the same day). **P < 0.05 (comparison between arthritis score and cEPCs/100 CD45+ cells on days 28 and 35 in the arthritis group).
D. Kurosaka et al. / Blood Cells, Molecules, and Diseases 35 (2005) 236 – 240
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Fig. 3. Relationships between the arthritis score and the number of cEPCs on days 28 (A) and 35 (B). cEPCs/100 CD45+ cells: the number of cEPCs per 100 CD45+ cells.
compare the number of cEPCs between the 2 groups. Fig. 2 shows the arthritis score and cEPCs/100 CD45+ cells in arthritis and control groups at each timepoint. The two groups did not differ in cEPCs/100 CD45+ cells on days 7, 14, and 21. On days 28 and 35, cEPCs/100 CD45+ cells in the control group were 0.11 T 0.02 (SE) and 0.07 T 0.01 (SE), respectively, whereas those in the arthritis group were 0.52 T 0.15 (SE) and 0.17 T 0.03 (SE), respectively, which were significantly greater than those in the control group. Next, comparison of leukocyte counts on days 28 and 35 in the arthritis group by the Mann – Whitney U test showed no significant difference (P = 0.193). However, the Mann – Whitney U test showed that cEPCs/100 CD45+ cells in the arthritis group were greater on day 28 than on day 35 (P = 0.024 < 0.05). In addition, a significant correlation was found between cEPCs/100 CD45+ cells and arthritis score on day 28 (r = 0.807) (Fig. 3A), but not on day 35 (Fig. 3B).
Discussion Enhanced mobilization of endothelial progenitor cells from the marrow into the blood is required for their increase in the peripheral circulation. This occurs during the mechanism for repair of injured endothelial cells or during neovascularization. This event has been reported to be promoted by vascular endothelial growth factor (VEGF) [6], which is known to play an important role in the pathogenesis of arthritis [7]. Lu et al. have reported the kinetics of expression of VEGF in the joint region in mice with the same type II collagen-induced arthritis as that analyzed in this study [7]. The expression of VEGF in the joint region began to increase on about day 24, after the administration of type II collagen, peaking 4 days after the onset of arthritis, and thereafter decreasing gradually to the original level, 12 days after onset. On the other hand, cEPCs/100 CD45+ cells on day 28 were greater in number than those on day 35, indicating that, to the best of our observation, the number of cEPCs on day 28 was the highest, tending to decease by day
35. The time-course of these changes was similar to that of changes in the expression of VEGF, suggesting that the number of cEPCs is also associated with the expression of VEGF in type II collagen arthritis mice. A correlation was found between cEPCs/100 CD45+ cells and arthritis score on day 28, but not on day 35. On the other hand, the arthritis score was higher on day 35 than on day 28. These findings suggest that the number of cEPCs is involved in the pathological state, during the development and progression of arthritis, rather than at the time of its establishment. In addition to VEGF, angiopoietin 1 [8], stromal cellderived growth factor-1 [9], granulocyte-macrophage colony stimulating factor [10], and matrix metalloprotease-9 [11] have been reported as factors increasing the number of cEPCs. Interestingly, many of these factors have been reported to be important in the pathogenesis of arthritis [12 –15]; therefore, the relationships between these factors and cEPCs require future studies. Much is unknown as to the degree to which these increased cEPCs are actually involved in the pathological state of arthritis. Thus, although many issues have yet to be addressed, the finding of a correlation between the number of cEPCs and the severity of arthritis suggests that cEPCs are closely involved in the pathogenesis of arthritis. References [1] T. Asahara, T. Murohara, A. Sullivan, M. Silver, R. van der Zee, T. Li, B. Witzenbichler, G. Schatteman, J.M. Isner, Isolation of putative progenitor endothelial cells for angiogenesis, Science 275 (1997) 964 – 967. [2] T. Asahara, H. Masuda, T. Takahashi, C. Kalka, C. Pastore, M. Silver, M. Kearne, M. Magner, J.M. Isner, Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization, Circ. Res. 85 (1999) 221 – 228. [3] W. Hilbe, S. Dirnhofer, F. Oberwasserlechner, T. Schmid, E. Gunsilius, G. Hilbe, E. Woll, C.M. Kahler, CD133 positive endothelial progenitor cells contribute to the tumour vasculature in non-small cell lung cancer, J. Clin. Pathol. 57 (2004) 965 – 969. [4] J.M. Hill, G. Zalos, J.P. Halcox, W.H. Schenke, M.A. Waclawiw,
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D. Kurosaka et al. / Blood Cells, Molecules, and Diseases 35 (2005) 236 – 240 A.A. Quyyumi, T. Finkel, Circulating endothelial progenitor cells, vascular function, and cardiovascular risk, N. Engl. J. Med. 348 (2003) 593 – 600. M. Capillo, P. Mancuso, A. Gobbi, S. Monestiroli, G. Pruneri, C. Dell’Agnola, L. Shultz, F. Bertolini, Continuous infusion of endostatin inhibits differentiation, mobilization, and clonogenic potential of endothelial cell progenitors, Clin. Cancer Res. 9 (2003) 377 – 382. T. Asahara, T. Takahashi, H. Masuda, C. Kalka, D. Chen, H. Iwaguro, Y. Inai, M. Silver, J.M. Isner, VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells, EMBO J. 18 (1999) 3964 – 3972. J. Lu, T. Kasama, K. Kobayashi, Y. Yoda, F. Shiozawa, M. Hanyuda, M. Negishi, H. Ide, M. Adachi, Vascular endothelial growth factor expression and regulation of murine collagen-induced arthritis, J. Immunol. 164 (2000) 5922 – 5927. K. Hattori, S. Dias, B. Heissig, N.R. Hackett, D. Lyden, M. Tateno, D.J. Hicklin, Z. Zhu, L. Witte, R.G. Crystal, M.A. Moore, S. Rafii, Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells, J. Exp. Med. 193 (2001) 1005 – 1014. J. Yamaguchi, K.F. Kusano, O. Masuo, A. Kawamoto, M. Silver, S. Murasawa, M. Bosch-Marce, H. Masuda, D.W. Losordo, J.M. Isner, T. Asahara, Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization, Circulation 107 (2003) 1322 – 1328.
[10] T. Takahashi, C. Kalka, H. Masuda, D. Chen, M. Silver, M. Kearney, M. Magner, J.M. Isner, T. Asahara, Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization, Nat. Med. 5 (1999) 434 – 438. [11] B. Heissig, K. Hattori, S. Dias, M. Friedrich, B. Ferris, N.R. Hackett, R.G. Crystal, P. Besmer, D. Lyden, M.A. Moore, Z. Werb, S. Rafii, Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand, Cell 109 (2002) 625 – 637. [12] E.M. Gravallese, A.R. Pettit, R. Lee, R. Madore, C. Manning, A. Tsay, J. Gaspar, M.B. Goldring, S.R. Goldring, P. Oettgen, Angiopoietin-1 is expressed in the synovium of patients with rheumatoid arthritis and is induced by tumour necrosis factor alpha, Ann. Rheum. Dis. 62 (2003) 100 – 107. [13] M.C. Blades, F. Ingegnoli, S.K. Wheller, A. Manzo, S. Wahid, G.S. Panayi, M. Perretti, C. Pitzalis, Stromal cell-derived factor 1 (CXCL12) induces monocyte migration into human synovium transplanted onto SCID Mice, Arthritis Rheum. 46 (2002) 824 – 836. [14] I.K. Campbell, M.J. Rich, R.J. Bischof, A.R. Dunn, D. Grail, J.A. Hamilton, Protection from collagen-induced arthritis in granulocytemacrophage colony-stimulating factor-deficient mice, J. Immunol. 161 (1998) 3639 – 3644. [15] T. Itoh, H. Matsuda, M. Tanioka, K. Kuwabara, S. Itohara, R. Suzuki, The role of matrix metalloproteinase-2 and matrix metalloproteinase-9 in antibody-induced arthritis, J. Immunol. 169 (2002) 2643 – 2647.
Kinetics of circulating endothelial progenitor cells in mice with type II collagen arthritis Daitaro Kurosaka*, Jun Yasuda, Ken Yoshida, Chiho Yasuda, Yasuhiko Toyokawa, Toru Yokoyama, Isamu Kingetsu, Akio Yamada Department of Internal Medicine, Division of Rheumatology, Jikei University School of Medicine, 3-25-8, Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan Submitted 7 June 2005 Available online 14 July 2005 (Communicated by M. Lichtman, M.D., 9 June 2005)
Abstract Objective: To examine the significance in arthritis of circulating endothelial progenitor cells (cEPCs) reportedly increasing in neovascularization. Methods: Arthritis was induced by immunizing DBA/1J mice with bovine type II collagen on day 0. Age-matched normal DBA/1J mice were used as controls. Blood was collected from these mice on days 7, 14, 21, 28, and 35. Peripheral blood CD45 , CD34+, Flk-1+, CD117+ cells were regarded as cEPCs (Flk-1 = vascular endothelial growth factor receptor 2). The number of cEPCs per 100 CD45+ cells was calculated by four-color flow cytometry, and compared with the arthritis score. Results: Arthritis developed about 3 days after booster immunization (day 21). On days 7, 14, and 21, no difference in cEPCs/100 CD45+ cells was noted between the arthritis and control groups. On days 28 and 35, cEPCs/100 CD45+ cells in the arthritis group were significantly greater in number than those in the control group. cEPCs/100 CD45+ cells on day 28 were greater in number than those on day 35. On day 28, a correlation was found between cEPCs/100 CD45+ cells and arthritis score. Conclusion: In mice with type II collagen-induced arthritis, an increase in cEPCs was associated with the onset of arthritis. The number of cEPCs was greater during the development and progression of arthritis than that at the time of its establishment, suggesting that cEPCs are involved in the pathogenesis of arthritis. D 2005 Elsevier Inc. All rights reserved. Keywords: Endothelial progenitor cell; Rheumatoid arthritis; Collagen induced arthritis; Neovascularization
Introduction Neovascularization occurring in mature individuals was previously thought to occur by angiogenesis, involving the proliferation and migration of endothelial cells of preexisting blood vessels. However, studies have discovered endothelial progenitor cells in the blood of mature individuals, showing that, in addition to angiogenesis, vasculogenesis in neovascularization in mature individuals exists, involving the mobilization of endothelial progenitor cells from the marrow, their incorporation into local
* Corresponding author. Fax: +81 3 3578 9078. E-mail address: [email protected] (D. Kurosaka). 1079-9796/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2005.06.001
tissues, proliferation, differentiation, and migration [1]. Vasculogenesis involving these endothelial progenitor cells has been reported to be important not only in physiological neovascularization, but also in various pathological states such as cancer and cardiovascular disease [2– 4]. For example, when tumors are transplanted into mice, endothelial progenitor cells are immobilized from the marrow, and participate in the formation of tumor blood vessels [5]. On the other hand, in rheumatoid arthritis, neovascularization is observed in the inflammatory synovial membrane, and is considered to be essential for the nourishment of proliferating synovial tissue. In this study, we examined the kinetics of circulating endothelial progenitor cells (cEPCs) during the development of arthritis in animal
D. Kurosaka et al. / Blood Cells, Molecules, and Diseases 35 (2005) 236 – 240
models of rheumatoid arthritis, that is, mice with type II collagen-induced arthritis.
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14, 21, 28, and 35 were n = 10, 9, 11, 11, and 11, respectively. As controls, 9, 9, 10, 10, and 10 age-matched normal DBA/1J mice were analyzed on days 7, 14, 21, 28, and 35, respectively.
Materials and methods Evaluation of arthritis Animal models An emulsion (100 Al) of 0.3% bovine type II collagen (Collagen Research Center, Tokyo, Japan) solution in acetic acid and an equal volume of 2 complete Freund’s adjuvant (Difco Lab, Detroit, MI) was injected intracutaneously into the tail root of 5-week-old male DBA/1J mice on day 0. On day 21, the mice were boost-immunized by an intracutaneous injection into the tail root of an emulsion of 0.3% bovine type II collagen solution in acetic acid and an equal volume of 1 incomplete Freund’s adjuvant (Difco Lab, Detroit, MI). The numbers of mice analyzed on days 7,
Arthritis was evaluated by arthritis score: 0 (no swelling), 1 (finger swelling or mild swelling), 2 (clear swelling), 3 (severe swelling), and 4 (arthrokleisis or joint deformity). The total arthritis score was obtained by adding the score of each of the four limbs. Leukocyte count Blood leukocytes in heparinized blood were counted using a Celltac-alpha (Nihon Kohden, Tokyo, Japan) on days 14, 21, 28, and 35.
Fig. 1. Analysis of cEPCs. (A) Analysis of cEPCs by four-color flow cytometry: (a) Analysis gate used to exclude platelets, dead cells, and cell debris. (b) Gate used to exclude CD45+ hematopoietic cells. (c) Separation of CD34+ cells. (d) Separation of Flk-1+ cells. (e) Measurement of CD117+ cells (CD45 , CD34+, Flk-1+, CD117+ cells). (B) Blood leukocyte counts in the control and arthritis groups. SSC, side light scatter; FSC, forward light scatter; FLK, Flk-1 = vascular endothelial growth factor receptor 2; CIA, type II collagen-induced arthritis model mouse group.
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Measurement of endothelial progenitor cell counts
Statistical analysis
Mice were euthanized with diethyl ether on days 7, 14, 21, 28, and 35, and heparinized blood was obtained from the heart. Circulating endothelial progenitor cells (cEPCs) were counted according to the method of Capillo et al. [5] using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA). Heparinized whole blood (100 Al) was hemolyzed, and stained with peridinin chlorophyll-a protein-conjugated anti-mouse CD45 monoclonal antibody (BD Biosciences, San Jose, CA), phycoerythrin-conjugated anti-mouse Flk-1 antibody (eBioscience, San Diego, CA), fluorescein isothiocyanate-conjugated rat anti-mouse CD34 monoclonal antibody (BD Biosciences), and allophycocyanin-conjugated anti-mouse CD117 monoclonal antibody (eBioscience). First, platelets, dead cells, and cell debris were excluded by forward light scatter (FSC) and side light scatter (SSC) (Fig. 1A-a). Next, CD45+ cells were excluded by gating (Fig. 1A-b), and then CD34+, Flk-1+ cells among CD45 cells were separated (Figs. 1A-c,d). Among these cells, CD117+ cells were regarded as cEPCs (Fig. 1A-e). Finally, the number of CD45+ cells and that of CD45 , CD34+, Flk-1+, CD117+ cells were measured, and the number of CD45 , CD34+, Flk-1+, CD117+ cells per 100 CD45+ cells (cEPCs/100 CD45+ cells) was calculated. After acquisition of at least 100,000 cells/sample, analyses were considered as informative when adequate number of events (typically 50– 200) were collected in cEPCs enumeration gates. Percentages of stained cells were determined and compared with appropriate negative controls. Positive staining was defined as being greater than non-specific background staining.
The Mann – Whitney U test was used to compare cEPCs/ 100 CD45+ cells and peripheral leukocyte counts between the arthritis and control groups. The correlation between cEPCs/100 CD45+ cells and arthritis score was assessed by Spearman’s rank correlation coefficient. P < 0.05 was considered significant.
Results Although most mice developed arthritis about 3 days after booster immunization (day 24), some mice had already developed it on the day of booster immunization (day 21). The mean arthritis scores on days 28 and 35 were 4.1 T 1.0 (SE) and 6.3 T 0.7 (SE), respectively (Fig. 2A). We evaluated the kinetics of cEPCs as follows. First, we counted blood leukocytes in the control and arthritis groups on days 14, 21, 28, and 35. The mean leukocyte counts T SE/Al in the control group at these timepoints were 6057 T 720, 7771 T 708, 7510 T 744, and 7020 T 589, respectively, and those in the arthritis group were 5364 T 720, 7131 T 508, 6764 T 875, and 8560 T 1158, respectively. Comparison between the 2 groups by the Mann – Whitney U test revealed P = 0.255, P = 0.526, P = 0.379, and P = 0.295 for the respective timepoints, showing no significant differences (Fig. 1B). On the other hand, since CD45 can be used as a leukocyte marker, the finding of no differences in leukocyte counts leads to no differences in CD45+ cell counts. Thus, we judged that the number of cEPCs relative to CD45+ cells could be used to
Fig. 2. cEPCs and arthritis score. (A) The arthritis score at each timepoint. (B) The number of cEPCs per 100 CD45+ cells at each timepoint. cEPCs/100 CD45+ cells: the number of cEPCs per 100 CD45+ cells. *P < 0.05 (comparison between the control and arthritis groups on the same day). **P < 0.05 (comparison between arthritis score and cEPCs/100 CD45+ cells on days 28 and 35 in the arthritis group).
D. Kurosaka et al. / Blood Cells, Molecules, and Diseases 35 (2005) 236 – 240
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Fig. 3. Relationships between the arthritis score and the number of cEPCs on days 28 (A) and 35 (B). cEPCs/100 CD45+ cells: the number of cEPCs per 100 CD45+ cells.
compare the number of cEPCs between the 2 groups. Fig. 2 shows the arthritis score and cEPCs/100 CD45+ cells in arthritis and control groups at each timepoint. The two groups did not differ in cEPCs/100 CD45+ cells on days 7, 14, and 21. On days 28 and 35, cEPCs/100 CD45+ cells in the control group were 0.11 T 0.02 (SE) and 0.07 T 0.01 (SE), respectively, whereas those in the arthritis group were 0.52 T 0.15 (SE) and 0.17 T 0.03 (SE), respectively, which were significantly greater than those in the control group. Next, comparison of leukocyte counts on days 28 and 35 in the arthritis group by the Mann – Whitney U test showed no significant difference (P = 0.193). However, the Mann – Whitney U test showed that cEPCs/100 CD45+ cells in the arthritis group were greater on day 28 than on day 35 (P = 0.024 < 0.05). In addition, a significant correlation was found between cEPCs/100 CD45+ cells and arthritis score on day 28 (r = 0.807) (Fig. 3A), but not on day 35 (Fig. 3B).
Discussion Enhanced mobilization of endothelial progenitor cells from the marrow into the blood is required for their increase in the peripheral circulation. This occurs during the mechanism for repair of injured endothelial cells or during neovascularization. This event has been reported to be promoted by vascular endothelial growth factor (VEGF) [6], which is known to play an important role in the pathogenesis of arthritis [7]. Lu et al. have reported the kinetics of expression of VEGF in the joint region in mice with the same type II collagen-induced arthritis as that analyzed in this study [7]. The expression of VEGF in the joint region began to increase on about day 24, after the administration of type II collagen, peaking 4 days after the onset of arthritis, and thereafter decreasing gradually to the original level, 12 days after onset. On the other hand, cEPCs/100 CD45+ cells on day 28 were greater in number than those on day 35, indicating that, to the best of our observation, the number of cEPCs on day 28 was the highest, tending to decease by day
35. The time-course of these changes was similar to that of changes in the expression of VEGF, suggesting that the number of cEPCs is also associated with the expression of VEGF in type II collagen arthritis mice. A correlation was found between cEPCs/100 CD45+ cells and arthritis score on day 28, but not on day 35. On the other hand, the arthritis score was higher on day 35 than on day 28. These findings suggest that the number of cEPCs is involved in the pathological state, during the development and progression of arthritis, rather than at the time of its establishment. In addition to VEGF, angiopoietin 1 [8], stromal cellderived growth factor-1 [9], granulocyte-macrophage colony stimulating factor [10], and matrix metalloprotease-9 [11] have been reported as factors increasing the number of cEPCs. Interestingly, many of these factors have been reported to be important in the pathogenesis of arthritis [12 –15]; therefore, the relationships between these factors and cEPCs require future studies. Much is unknown as to the degree to which these increased cEPCs are actually involved in the pathological state of arthritis. Thus, although many issues have yet to be addressed, the finding of a correlation between the number of cEPCs and the severity of arthritis suggests that cEPCs are closely involved in the pathogenesis of arthritis. References [1] T. Asahara, T. Murohara, A. Sullivan, M. Silver, R. van der Zee, T. Li, B. Witzenbichler, G. Schatteman, J.M. Isner, Isolation of putative progenitor endothelial cells for angiogenesis, Science 275 (1997) 964 – 967. [2] T. Asahara, H. Masuda, T. Takahashi, C. Kalka, C. Pastore, M. Silver, M. Kearne, M. Magner, J.M. Isner, Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization, Circ. Res. 85 (1999) 221 – 228. [3] W. Hilbe, S. Dirnhofer, F. Oberwasserlechner, T. Schmid, E. Gunsilius, G. Hilbe, E. Woll, C.M. Kahler, CD133 positive endothelial progenitor cells contribute to the tumour vasculature in non-small cell lung cancer, J. Clin. Pathol. 57 (2004) 965 – 969. [4] J.M. Hill, G. Zalos, J.P. Halcox, W.H. Schenke, M.A. Waclawiw,
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[5]
[6]
[7]
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