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a First Department of Pathology,  \$ i: l8 O0 l" X* n; f: u
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b Department of Ophthalmology,
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c Regeneration Research Center for Intractable Diseases,, i. g$ l4 p, }7 T* k

d Department of Anatomical Pathology,

e Second Department of Internal Medicine, and1 t7 J' }6 [/ u8 k7 G
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f Department of Neurology, Kansai Medical University, Moriguchi, Osaka, Japan8 L5 _$ p+ B# w: E1 z
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Key Words. Bone marrow cell ? G-CSF ? M-CSF ? Neovascularization
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Correspondence: Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, Moriguchi, Osaka, 570-8507, Japan. Telephone: 81-066-992-1001(ex. 2470); FAX 81-066-992-1219; e-mail: ikehara@takii.kmu.ac.jp2 V7 I# a: K' R+ E' c+ \' l: e& Z
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ABSTRACT/ L- l- }2 K" F4 O* ]2 G- ?
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It has been reported that endothelial progenitor cells (EPCs) exist in the peripheral blood, and that they can differentiate into the endothelial cells of blood vessels . Bone marrow cells (BMCs) have also been reported to differentiate into the endothelial cells of blood vessels .

Several cytokines, such as granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF) are known to augment the numbers and functions of granulocyte-monocyte lineage cells . G-CSF and GM-CSF have been reported to mobilize peripheral blood stem cells (PBSCs)  and EPCs . However, it remains unclear whether M-CSF contributes to neovascularization and mobilizes EPCs from the bone marrow. Here, we report that M-CSF mobilizes EPCs from the bone marrow and that both M-CSF as well as G-CSF augment neovascularization of ischemic limbs.
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MATERIALS AND METHODS/ g4 \9 L5 G. z0 z' i" d3 a5 k' T

Neovascularization Induced by G-CSF with or without M-CSF in Ischemic Limbs
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We first examined whether either G-CSF or M-CSF, or both, is involved in the formation of new blood vessels. Either G-CSF or M-CSF, or both, was administered to mice in which the right hindlimbs were induced with ischemia, as described in Materials and Methods. On day 3, the skeletal muscles of the lower limbs were collected. The specimens were stained with anit-CD31 Ab, and blood vessels were detected by CD31  cells and their shape (morphology).

As shown in Figure 1, nontreated limbs showed similar numbers of blood vessels in all groups. However, the ischemic hindlimbs of mice treated with either G-CSF or M-CSF, or both, showed a significant increase in the number of blood vessels in the skeletal muscles. We also obtained similar results using anti-CD146 Ab, which is also a specific marker for endothelial cells (data not shown).% u5 N5 x2 T$ V! B6 }/ S8 h
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Figure 1. Augmentation of neovascularization in ischemic hindlimbs by either granulocyte colony-stimulating factor (G-CSF) or macrophage colony-stimulating factor (M-CSF), or both. (A): Hindlimb-ischemic mice were prepared, and either G-SCF or M-CSF, or both, (each at 250 μg/kg) was administered to the mice for 3 consecutive days. One day after the last injection, the skeletal muscles of the ischemic hindlimbs and the nontreated hindlimbs were collected and snap-frozen in liquid nitrogen. The blood vessels in the muscles were visualized by staining with fluorescein isothiocyanate (FITC)–anti-CD31 Ab, which is expressed on the endothelial cells of blood vessels, and samples were observed by confocal laser microscopy (original magnification: x40, bars = 50 μm). (B): The number of blood vessels (per field) in which endothelial cells express CD31 molecules was calculated, and the mean ± SD is shown (n = 4). *p 9 a, g8 |4 W+ ^% `" W
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There are three possibilities: (1) Either G-CSF or M-CSF, or both, accelerate the extension of existing blood vessels, (2) Either G-CSF or M-CSF, or both, accelerate the generation of new blood vessels from EPCs in the peripheral blood, and (3) both (1) and (2).

Differentiation from BMCs into Blood Vessels9 Y- J8 i, Q" P

It has been reported that the EPCs in the peripheral blood and BMCs differentiate into endothelial cells of blood vessels . We therefore examined whether BMCs could differentiate into endothelial cells of blood vessels. One month after the BMCs of EGFP-transgenic mice were transplanted into the lethally irradiated B6 mice, unilateral hindlimb ischemia was induced. Either G-CSF or M-CSF, or both, was then injected i.p. for 3 days (day 0 to day 2). On day 3, the mice were sacrificed, and frozen sections of the gastrocnemius muscles were prepared to examine vascularization using a confocal microscopy. In the ischemic limbs of either G-CSF or M-CSF, or both, treated mice, we found cells expressing both EGFP and CD31 (Fig. 2). The numbers of blood vessels in each group were similar to those in the experiment using B6 mice (Fig. 1 and Fig. 2B), suggesting that BMT had no effect on the neovascularization by either G-CSF or M-CSF, or both.& [; @, E6 _, p. A
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Figure 2. Contribution of BMCs to neovascularization of ischemic limbs. BMCs of EGFP–transgenic mice were transplanted into lethally irradiated B6 mice. One month after bone marrow transplantation, hindlimb ischemia was induced in the mice. Either granulocyte colony-stimulating factor (G-CSF) or macrophage colony-stimulating factor (M-CSF), or both (each at 250 μg/kg) was administered to the mice for 3 consecutive days. One day after the last injection, the skeletal muscles of the ischemic hindlimbs and nontreated hindlimbs were collected and snap-frozen in liquid nitrogen. The blood vessels in the muscles were visualized by staining with PE–anti-CD31 Ab, followed by counting EGFP-positive or -negative intramuscular capillaries using a confocal microscopy. (A): Representative data of the staining. Arrows show EGFP  blood vessels. (B): The number of blood vessels (per field) was calculated, and the mean ± SD is shown (n = 4). *p
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Next, we counted EGFP /CD31  capillaries (Fig. 2C), since EGFP  cells are thought to be derived from transplanted BMCs. Bone marrow–derived endothelial cells, the number of which increased in ischemic limbs of cytokine-treated mice, accounted for close to half of the augmented blood vessels in the cytokine-treated groups (Fig. 2B, 2C). These results suggest that G-CSF and M-CSF induce not only the proliferation of existing endothelial cells but also the differentiation from BMCs into the endothelial cells.* r& {( E) X9 W
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Since there are several reports suggesting that cell fusion instead of regeneration should be considered , we performed further experiments to assess whether some new blood vessels are truly derived from BMCs. BMT from C3H mice to B6 mice were performed, as described in Materials and Methods. One month after BMT, we confirmed that more than 90% of the peripheral blood showed donor-type MHC (H-2k) molecules, suggesting that host BMCs (H-2b) were replaced by donor BMCs (H-2k). We induced unilateral hindlimb ischemia in the mice, followed by cytokine injection for 3 days, as described above. As shown in Figure 3, the endothelium cells, which wereH-2k /H-2b–/CD31 , existed in the capillary walls of ischemia-induced muscle of cytokine-treated mice. Moreover, the numbers of H-2k /H-2b–/CD31  capillary were similar to those of EGFP  capillaries (Fig. 2 and data not shown). These results suggest that the expression of donor MHC molecules by endothelial cells was not due to cell fusion but to real differentiation from donor BMCs.- Z# t1 g+ F: `/ H
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Figure 3. Contribution of bone marrow–derived cells to neovascularization due to differentiation into endothelium, but not due to cell-to-cell fusion. To discriminate real differentiation from cell-to-cell fusion, we performed further BMT experiments. One month after BMT (from C3H mice into lethally irradiated B6 mice), hindlimb ischemia was induced in the mice. Either granulocyte colony-stimulating factor (G-CSF) or macrophage colony-stimulating factor (M-CSF), or both (each at 250 μg/kg), was administered to the mice for 3 consecutive days. One day after the last injection, the skeletal muscles of the ischemic hindlimbs and the nontreated hindlimbs were collected and snap-frozen in liquid nitrogen. The blood vessels in the muscles were visualized by staining with PE–anti-CD31 Ab (red), FITC–labeled anti-H-2b Ab (green), and biotin-labeled H-2k Ab (orange), followed by staining with Alexa-labeled avidin (blue). Representative data of H-2k /H-2b– () or H-2b /H-2k– () blood vessels are shown (original magnification: x60, bars = 5 μm). Abbreviations: BMT, bone marrow transplant; FITC, fluorescein isothiocyanate; PE, phycoerythrin.
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Increases in Number of EPCs& L' ~& o( e1 R% ?
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We next examined the number of EPC-enriched populations present in the circulating blood. Several surface markers of EPCs have been reported: Sca-1 /CD3–/CD19– cells are thought of as an EPC-enriched population , and both Flk-1 /CD45– cells and Sca-1 /c-kit /CD45– cells have been reported to be an EPC-enriched fraction . The cell numbers in these populations increased in the mice treated with cytokines, particularly G-CSF plus M-CSF (Fig. 4). These results suggest that G-CSF and M-CSF accelerate the mobilization of EPCs from the bone marrow.
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Figure 4. Augmentation of EPCs in the peripheral blood of mice treated with either granulocyte colony-stimulating factor (G-CSF) or macrophage colony-stimulating factor (M-CSF), or both. G-CSF and M-CSF (each at 250 μg/kg) were injected i.p. to B6 mice for a continuous 3 days. One day after the last injection, white blood cells of the mice were collected and analyzed, followed by calculation of the number of Sca-1 /CD3–/CD19– cells, Flk-1 /CD45– cells, or Sca-1 /c-kit /CD45– cells in the peripheral blood. *p 1 b! ?/ a, b; d, e" r; q9 ?; M- j
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Functional Analyses of Increased Blood Vessels by G-CSF and M-CSF

We examined the functional effects of G-CSF and M-CSF on improvement in the blood flow in the ischemic limbs using LDPI and thermography. As shown in Figure 5, blood flow recovered rapidly under the condition with either G-CSF or M-CSF (or both). Particularly, G-CSF plus M-CSF showed remarkable recovery, an example of which was the LPDI of ischemic limbs in the mice treated with G-CSF and M-CSF, reaching around 78% on day 7, while that of ischemic limbs in the untreated mice was only around 38%. These results were confirmed using thermography (Fig. 6). All three CSF-treated mice showed better skin temperature of ischemic limbs than did limbs of saline-treated mice.( Q: B) }5 r6 x5 s' d( C! S" X6 J

Figure 5. Recovery of blood flow using laser Doppler perfusion image (LDPI). We measured the blood flow of the lower limbs, using an LDPI analyzer. Before ischemia induction, we confirmed that bilateral legs showed similar blood flow. After ischemia induction, blood flow of the legs was measured (day 0). We administered either granulocyte colony-stimulating factor (G-CSF) or macrophage colony-stimulating factor (M-CSF), or both (each at 250 μg/kg), to the mice for a continuous 3 days (day 0–2). At 3 and 7 days after surgery, we measured blood flow using an LPDI analyzer, followed by calculation of the ratio of the ischemic (right) to normal (left) limb blood flow. (A): Representative figures of LDPIs showing the time course of the ratio of the ischemic to normal limb blood flow (mean ± SD) (n = 4). *p
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Figure 6. Recovery of blood flow using a thermography. After either granulocyte colony-stimulating factor (G-CSF) or macrophage colony-stimulating factor (M-CSF), or both (each at 250 μg/kg) was administered to ischemic hindlimb mice for 3 days, the skin temperature was measured using a thermograph. Differences of skin thermographs of right (treated) and left (nontreated) legs are shown. (A): Representative data of thermography on day 3. (B): Mean ± SD of the differences of the skin temperatures on day 3 (n = 4). *p
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Increase in VEGF Production in Bone Marrow by G-CSF and M-CSF

To clarify the mechanisms underlying the mobilization of EPCs in the peripheral blood by G-CSF and M-CSF, we performed ELISA for VEGF, since it has been reported that M-CSF stimulates monocytes to produce VEGF . At first, we performed ELISA using the sera of mice treated with either G-CSF or M-CSF, or both (each at 250 μg/kg) for 3 days, as described in Material and Methods. However, we did not detect VEGF in the sera using the ELISA kit (data not shown).* ~, J5 M) p$ e) E9 C$ J

Next, we cultured spleen cells and BMCs (2 x 106/ml) with, or both G-CSF or M-CSF (each at 10 ng/ml), then we measured the VEGF using the ELISA kit. We did not detect VEGF in the supernatants of spleen cells even when cytokines had been added, but we did in the supernatants of BMCs (Fig. 7). The amount of VEGF in the supernatants of BMCs with either G-CSF or M-CSF, or both, increased. These results suggest that G-CSF and M-CSF stimulate BMCs, probably monocyte-lineage cells, to produce VEGF, followed by an increase in the number of EPCs and augmentation of their mobilization.
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Figure 7. Augmentation of vascular endothelial cell growth factor (VEGF) production by bone marrow cells (BMCs) in the presence of either granulocyte colony-stimulating factor (G-CSF) or macrophage colony-stimulating factor (M-CSF), or both, in vitro. Spleen cells or BMCs (each at 2 x 106/ml) were cultured with or without G-CSF or M-CSF, or both, for 1 day or 3 days. The concentrations of VEGF in the supernatants were measured by enzyme-linked immunoassay (ELISA).

DISCUSSION* `; X( p! O' E' {" r) ~4 f) h
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We thank professor H. Okabe (Osaka University, Osaka, Japan) for the donation of EGFP-transgenic mice. We also thank Ms. Sachiko Miura, Ms. Mari Murakami-Shinkawa, and Ms. Yoko Tokuyama for their expert technical assistance, and also Mr. Hilary Eastwick-Field and Ms. Keiko Ando for the preparation of this manuscript. Keizo Minamino and Yasushi Adachi contributed equally to this work.. `. p+ S2 N  V) z* @

REFERENCES: r1 ^( H% C4 B1 l

Asahara T, Murohara T, Sullivan A et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–967.
) O( L/ A, ~% x8 {6 e, z) J
Asahara T, Masuda H, Takahashi T et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Cir Res 1999;85:221–228.( u' U! O' H' e  R

Andreeff M, Welte K. Hematopoietic colony-stimulating factors. Semin Oncol 1989;16:211–229.

Tsuchiya M, Nomura H, Asano S et al. Characterization of recombination human granulocyte-colony stimulating factor produced in mouse cells. EMBO J 1987;6:611–616.
7 I; D5 T8 w9 f0 V. X+ j
Yanai N, Yamada M, Watanabe Y et al. The granulopoietic effect of human urinary colony stimulating factor on normal and cyclophosphasmide treated mice. Exp Hematol 1983;11:1027–1036.! z5 }1 Y# X4 N) y

Kawasaki ES, Ladner MB, Wang AM et al. Molecular cloning of a complementary DNA encoding human macrophage specific colony-stimulating factor (CSF-1). Science 1985;230:291–296.$ J0 e" t7 \, M9 ]0 e* z0 I; j! K
' j- k8 F: F$ Y& W
Yamamoto Y, Yasumizu R, Amoh Y et al. Characterization of peripheral blood stem cells in mice. Blood 1996;88:445–454.! T1 G0 D6 ~! {
2 N5 \7 `6 }, d7 Y+ U: R
Kocher AA, Schuster MD, Szabolcs MJ et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001;7:430–436./ q* o1 b0 k, W/ f5 z  F
- E$ q: i1 o. S3 K4 s
Takahasi T, Kalka C, Masuda H et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 1999;5:434–438.6 Y+ T" I5 {5 x! Q

Okabe M, Ikawa M, Kominami K et al. "Green mice" as a source of ubiquitous green cells. FEBS Lett 1997;407:313–319.1 ~6 Y# D. s8 V9 M3 b# z3 |
& [/ m( C9 P7 I
Kushida T, Inaba M, Hisha H et al. Intra-bone marrow injection of allogeneic bone marrow cells: a powerful new strategy for treatment of intractable autoimmune disease in MRL/lpr mice. Blood 2001;97:3292–3299.
% R2 A# k' n3 L. @1 M! d% K
Tomita M, Adachi Y, Yamada H et al. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. STEM CELLS 2002;20:279–283.; s+ w( b" ?; M$ t' `

Amano K, Matsubara H, Iba O et al. Enhancement of ischemic-induced angiogenesis by eNOS overexpression. Hypertension 2003;41:156–162.7 x8 C8 I. T0 v* x- `* |
1 I" V5 X( v4 q# f2 h1 A
Shahin R, David L. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med 2003;9:702–712.  W+ k' t# f6 }, E9 ]

Wang X, Willenbring H, Akkari Y et al. Cell fusion is the principal source of bone marrow-derived hepatocytes. Nature 2003;422:897–901.; F( O% d6 k; P9 j% y
" M: n: n9 `1 \. Z& ^* b1 L! D
Eubank TD, Galloway M, Montague CM et al. M-CSF induces vascular endothelial growth factor production and angiogenic activity from human monocytes. J Immunol 2003;171:2637–2643.
, R" v, Q; e: Q' z- u. F
Risau W. Mechanisms of angiogenesis. Nature 1997;386:671–674.% s" J" m$ G2 `, Z  i
+ O, K# r+ t0 M
Kalka C, Masuda H, Takahashi T et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A 2000;97:3422–3427.
0 t2 i/ v* o: d( g5 d0 g( ~6 t
Tateishi-Yuyama E, Matsubara H, Murohara T et al. Therapeutic angiogenesis for patients with limb ischemia by autologous transplantation of bone-marrow cells: a pilot study and a randomized control trial. Lancet 2002;360:427–435.! m, Q1 v& I/ R+ @3 j3 t
. V9 _4 `! e& w) D
Suzu S, Yanai N, Saito M et al. Enhancement of the antibody-dependent tumoricidal activity of human monocytes. Jpn J Cancer Res 1990;81:79–84.

Scholz D, Cai WJ, Schaper W. Arteriogenesis, a new concept of vascular adaptation in occlusive disease. Angiogenesis 2001;4:247–257.

Carlo-Stella C, Di Nicola M, Magni M et al. Defibrotide in combination with granulocyte colony-stimulating factor significantly enhances the mobilization of primitive and committed peripheral blood progenitor cells in mice. Cancer Res 2002;62:6152–6157.
7 x2 a3 f' R5 w+ B+ ~+ M' S* }
LiuF, Poursine-Laurent J, Link DC. Expression of the G-CSF receptor on hematopoietic progenitor cells is not required for their mobilization by G-CSF. Blood 2000;95:3025–3031.
' i1 m2 o, z. M
Geppert A, Huber K. Inflammation and cardiovascular diseases: lessons that can be learned for the patient with cardiogenic shock in the intensive care unit. Curr Opin Crit Care 2004;10:347–353.: R- k0 V# i! a. O" {+ J
- m( w! m8 r- z$ \% f
Kang H-J, Kim H-S, Zhang S-Y et al. Effects of intracoronary infusion of peripheral blood stem-cells mobilized with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction: the MAGIC cell randomized clinical trial. Lancet 2004;363:751–756.( N4 s0 n, {" C( ]
0 O, y$ e+ A* \* C
Rajavashisth T, Qiao J-H, Tripathi S et al. Heterozygous osteopetrotic (op) mutation reduces atherosclerosis in LDL receptor-deficient mice. J Clin Invest 1998;101:2702–2710.3 @8 X1 K. }# U0 A% b* m: n. v, U5 t
- @% ^" I1 u( w
Bensinger W, Singer J, Appelbaum F et al. Autologous transplantation with peripheral blood mononuclear cells collected after administration of recombinant granulocyte stimulating factor. Blood 1993;81:3158–3163.
' ?  N9 i5 I) A  [( q+ L% ?
Nishikawa S-I, Nishikawa S, Hirashima M et al. Progressive lineage analysis by cell sorting and culture identified FLK1 VE-cadherin  cells at a diverging point of endothelial and hemopoietic lineages. Development 1998;125:1747–1756.8 Z/ y0 c% h& Y$ n" b( u; Y
$ \% A: K2 m* r! r& J0 _
Azoulay E, Attalah H, Yang K et al. Exacerbation by granulocyte colony-stimulating factor of prior acute lung injury: implication of neutrophils. Crit Care Med 2002;30:2115–2122.(Keizo Minaminoa,b, Yasush)

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