Mechanisms Underlying Acceleration of Blood Flow Recovery in Ischemic Limbs by M - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:06 |只看该作者 |倒序浏览 |打印

作者：Keiji Nakanoa,b, Yasushi Adachia,c, Keizo Minaminoa, Masayoshi Iwasakia, Akio Shigematsua, Naoko Kiriyamaa, Yasuhiro Suzukia, Yasushi Koikea, Hiromi Mukaidea, Shoichiro Taniuchib, Yohnosuke Kobayashia, Kazunari Kanekob,c, Susumu Ikeharaa,c





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      【摘要】
   Recently we reported that macrophage colony-stimulating factor (M-CSF) can mobilize endothelial progenitor cells (EPCs) from the bone marrow into the peripheral blood, resulting in an increase in the number of blood vessels and augmentation of blood flow in the ischemia-induced legs. M-CSF accelerates neovascularization of ischemic lesions resulting from the mobilization of EPCs. In the present paper, we analyze the mechanisms underling the mobilization of EPCs by M-CSF. M-CSF augments the production of vascular endothelial growth factor (VEGF) from the bone marrow cells, especially from myeloid lineage cells. In vivo administration of anti-VEGF antibody abrogates both the acceleration of the recovery of blood flow in the ischemia-induced limbs by M-CSF and the augmentation of the mobilization of EPCs induced by M-CSF. These results suggest that the M-CSF contributes to rapid recovery of blood flow in ischemic lesions by mobilization of EPCs from the bone marrow through augmentation of VEGF production in the bone marrow and that the VEGF is mainly produced by myeloid lineage cells. ) Y+ G) }6 ]9 t' P0 }
      【关键词】 Macrophage colony-stimulating factor Vascular endothelial growth factor Neovascularization Endothelial progenitor cell2 n* d7 n$ o& f3 V1 p
   INTRODUCTION, L& B" b$ b# o3 m

Asahara et al. have reported that endothelial progenitor cells (EPCs) exist in the bone marrow (BM) and the peripheral blood (PB) and that the EPCs can differentiate into the endothelial cells of the blood vessels . Thus, usage of EPCs in the BM is a novel and desirable therapy for ischemic diseases.

Three kinds of colony stimulating factors (CSF) are known at present: granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). It has been already reported that both G-CSF and GM-CSF can activate myeloid lineage cells and can mobilize hematopoietic precursor cells .: W) o) p4 s" p& [7 c% M0 z
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Recently, we have shown that M-CSF, as well as G-CSF, mobilizes EPCs from the BM into the PB, resulting in the acceleration of neovascularization in the ischemia-induced legs . However, the mechanisms underlying mobilization of EPCs by M-CSF are unclear. In this paper, we focus on clarifying the mechanisms underlying the mobilization of EPCs by M-CSF. We demonstrate that M-CSF can mobilize EPCs through vascular endothelial growth factor (VEGF) mainly produced by myeloid lineage cells in the BM, resulting in the augmentation of blood flow and the increased number of blood vessels in ischemia-induced legs.

MATERIALS AND METHODS# N* r) @6 H9 x

Mice# y) z4 o  o- }* E1 N  t$ N
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C57BL/6 mice (B6) were purchased from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan, http://web.kyoto-inet.or.jp/people/simizv/index.htm).2 c2 Q! E7 G- Z. h

Reagents

Anti-CD3, anti-CD19, anti-CD11b, anti-CD11c, anti-Ter119, anti-CD45, anti-Flk-1, and anti-NK1.1 antibodies (Abs) were purchased from BD Biosciences (San Diego, http://www.bdbiosciences.com). M-CSF was kindly donated by Morinaga (Zama, Japan, http://www.morinagamilk.co.jp/menu/english.html). G-CSF was kindly donated by Chugai Pharmaceutical Co. (Tokyo). Anti-CD115 (receptor for M-CSF) Ab was purchased from eBioscience (San Diego). Anti-VEGF Ab was purchased from Lab Vision (Fremont, CA, http://www.labvision.com).

Detection of CD115  Cells in BM

To examine the expression of CD115 in various lineages of BMCs, BMCs were stained with anti-CD115 Ab lineage-specific Abs. To detect the expression of CD115 on erythroid lineage cells, myeloid lineage cells, and B lymphocytes, BMCs were incubated with biotin-labeled anti-Ter119 PE-labeled anti-CD115 Ab followed by staining with fluorescein isothio-cyanate (FITC)-labeled avidin, FITC-labeled anti-Gr-1 Ab PE-labeled anti-CD115 Ab, FITC-labeled anti-CD11b Ab PE-labeled anti-CD115 Ab, and FITC-labeled anti-CD19 Ab PE-labeled anti-CD115 Ab. The stained cells were analyzed with a FACScan (Becton, Dickinson and Company, San Diego, http://www.bd.com). Each lineage of cells was gated, and the expressions of CD115 in the cells were analyzed. To analyze the expression of CD115 in lineage-negative (Lin¨C) cells, BMCs were stained with biotin-labeled anti-Ter119 Ab biotin-labeled anti-Gr-1 Ab biotin-labeled anti-Mac-1 Ab biotin-labeled anti-CD19 Ab biotin-labeled anti-CD3 Ab biotin-labeled anti-NK1.1 Ab biotin-labeled anti-CD11c Ab PE-labeled anti-CD115 Ab followed by staining with PE Cy5-labeled avidin. Lin¨C cells were gated, and the expression of CD115 in the cells was analyzed. To examine the expression of CD115 of EPCs, BMCs were stained with FITC-labeled anti-CD45 Ab, PE-labeled anti-Flk-1 Ab, and biotin-labeled anti-CD115 Ab followed by staining with PE Cy5-labeled avidin. CD45¨C /Flk-1  cells were gated, and the expression of CD115 of the cells was analyzed.' W( _3 k. U2 H' T& A/ F

Preparation of Hind Limb Ischemia/ D) j0 a2 r- A, @
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On day 0, unilateral hind limb ischemia was induced by ligation of the right femoral arteries and veins of B6 mice, as previously described .
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Administration of G-CSF, M-CSF, and anti-VEGF Ab

Human recombinant M-CSF (250 µg/kg) or human recombinant G-CSF (250 µg/kg) was administered to the mice intraperitoneally for 3 days (day 0, day 1, and day 2). The dosage of M-CSF and G-CSF was determined according to our previous experiments . Anti-VEGF Ab (250 µg/mouse) was intraperitoneally administered on day 0, simultaneously with injection of M-CSF or G-CSF.
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Laser Doppler Perfusion Image5 h9 I5 G, I0 z6 u

The hind limbs of mice were shaved using a razor. The mice were anesthetized with 160 mg/kg pentobarbital and fixed supine on a cork plate. We next measured the blood flow of limbs using a laser Doppler perfusion image (LDPI) analyzer (Moor Instruments, Millway Devon, U.K., http://www.moor.co.uk), as described previously . LDPI indexes were shown as the ratio of the blood flow of the ischemic (right) to normal (left) limb.3 c) H$ \/ R5 V) v. y4 h
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Cell Culture

BMCs and spleen cells were adjusted to 1 x 106/ml in RPMI 1,640 containing 10% of fetal calf serum with or without M-CSF (10 ng/ml).
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For purification of Terr119  cells, Gr-1  cells, Mac-1  cells, or CD19  cells, BMCs were incubated with PE-labeled anti-Terr119, Gr-1  cells, Mac-1  cells, or CD19  Abs, and then positive cells for each Ab were sorted using an EPICS ALTRA cell sorter (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). For preparation of Lin¨C cells, BMCs were cultured with PE-labeled anti-Terr119, anti-Gr-1, anti-Mac-1, anti-CD19, anti-CD11c, and anti-NK1.1 Abs, and cells negative to these Abs were collected using the EPICS ALTRA. These cells were cultured with or without M-CSF (10 ng/ml) for 3 days.  b, |! }" _, h9 A6 x8 F
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Enzyme-Linked Immunosorbent Assay (ELISA) for VEGF
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Supernatants of cultured cells were collected, and ELISAs for VEGF were performed using the Quantikine Mouse VEGF immunoassay kit (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) following the manufacturer¡¯s instructions.
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Estimation of Number of EPCs in Peripheral Blood; D7 t. z6 F" H) t

B6 mice were injected with M-CSF (250 µg/kg) for 3 days. Some of the mice were intraperitoneally injected with anti-VEGF Ab (250 µg/mouse) simultaneously with the injection of cytokines on day 0. Four days after the first injection of M-CSF (day 3), PBs of the mice were collected, and numbers of nuclear cells were analyzed using an SF-3000 autoanalyzer for the peripheral blood (Sysmex, Kobe, Japan). The cells were then stained with PE-labeled anti-Flk-1 Ab, biotin-labeled anti-CD45 Ab, and biotin-labeled anti-Terr119 Ab, followed by staining with PE Cy5-labeled avidin. The cells were analyzed with a FACScan. The nuclear cells were gated using development of forward scatter and side scatter, and the percentages of EPCs that show Flk-1 /CD45¨C/Terr119¨C were analyzed, followed by calculation of absolute number of EPCs in PB from the numbers of nuclear cells and percentages of EPCs .- K$ k9 G7 b4 ?0 [+ [

Statistical Analyses

Statistical analyses were performed with Student¡¯s t test. Values of p
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RESULTS/ q9 b* Z* ~( F7 y

Anti-VEGF Ab Abrogates Acceleration of Blood Flow by M-CSF but Not Acceleration by G-CSF1 A6 \+ c2 X& c$ w) f4 j3 N

We have reported that M-CSF and G-CSF can accelerate neovascularization of ischemia-induced legs and that both cytokines can induce the production of VEGF in the bone marrow cells but not spleen cells . These results also suggest that M-CSF effects acceleration of blood flow through VEGF.

Figure 1. Anti-VEGF Ab abrogates the effects of M-CSF on blood flow recovery but not the effects of G-CSF. Hind limb model was prepared by ligation of femoral artery and vein. Just after ischemia induction, blood flow in the ischemic limbs was measured using a laser Doppler perfusion image (LPDI) analyzer (day 0), as described in Materials and Methods. The blood flow was shown as an LPDI index. The mice were then injected with G-CSF, M-CSF, G-CSF anti-VEGF Ab, or M-CSF anti-VEGF Ab, followed by observation of recovery of the blood flow. G-CSF or M-CSF was injected on three consecutive days. The time course of the blood flow of the ischemic limbs is shown. Representative data are shown from four independent experiments (A). Means and SDs of four independent experiments are shown (B). *, p - p) ^) S% s. V1 {
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Expression of Receptor for M-CSF in BMCs
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Next, we analyzed the expression of receptor for M-CSF (CD115) in various lineage cells in the BM. As shown in Figure 2, mainly Gr-1  cells and Mac-1  cells express CD115, whereas only a very low number of EPCs (less than 5%) express CD115, suggesting that M-CSF does not work directly on the EPCs but, rather, works on the EPCs via another factor. These results are compatible with the abrogation of the effects of M-CSF by the administration of anti-VEGF Ab.
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Figure 2. Expression of CD115 (receptor for M-CSF) in BMCs. BMCs were stained with anti-CD115 and various lineage cells, as described in Materials and Methods. The indicated lineage cells or lineage-negative cells were gated, and the expressions of CD115 of the cells were analyzed. Thin lines show isotype-matched control, and bold lines show the expressions of CD115. Representative data are shown for three independent experiments. Abbreviation: BMC, bone marrow cell; EPC, endothelial progenitor cell.

Myeloid-Lineage Cells in the BM Produce VEGF by Stimulation of M-CSF

We measured serum VEGF levels using ELISA, but we detected VEGF neither in the serum of nontreated mice nor in the serum of M-CSF-administered mice (data not shown; . In the present study, we measured the concentrations of VEGF on an absolute scale and with the time course. As shown in Figure 3A, BMCs produced VEGF, and the production of VEGF was enhanced by M-CSF, whereas spleen cells produced only a small quantity of VEGF even when cultured with M-CSF.

Figure 3. Production of VEGF by stimulation of M-CSF. (A): Production of VEGF from spleen cells or BMCs under the stimulation of M-CSF was measured with ELISA. BMCs or spleen cells (1 x 106 cells per ml) were cultured with or without M-CSF (10 ng/ml). Supernatants of the medium were collected, and concentrations of VEGF were measured. Means and SDs of the concentration of VEGF over the time course are shown (n = 4). (B): Production of VEGF from each population in the BM under the stimulation of M-CSF. Ter119  cells, Gr-1  cells, Mac-1  cells, CD19  cells, and Lin¨C (Ter119¨C, Gr-1¨C, Mac-1¨C, CD19¨C, CD11c¨C, CD3¨C, and NK1.1¨C) cells were obtained using an EPICS ALTRA cell sorter, as described in Materials and Methods. After the cells (5 x 105 cells per ml) were cultured with or without M-CSF (10 ng/ml) for 3 days, the supernatants were collected, and the concentrations of VEGF were examined using enzyme-linked immunosorbent assay. Means of duplicated examinations are shown. Representative data are shown for three independent experiments. *, p 2 R  N+ `" z: L& S+ m* {
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Next, we examined the production VEGF in various populations of BMCs in the presence of M-CSF. As shown in Figure 3B, Mac-1  cells, Gr-1  cells, and Lin¨C cells, especially Mac-1  cells and Gr-1  cells, produced VE
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