Macrophage Colony-Stimulating Factor (M-CSF), As Well As Granulocyte Colony-Stimulating Factor (G-CSF), Accelerates Neovascularization
http://www.100md.com
《干细胞学杂志》
a First Department of Pathology,
b Department of Ophthalmology,
c Regeneration Research Center for Intractable Diseases,
d Department of Anatomical Pathology,
e Second Department of Internal Medicine, and
f Department of Neurology, Kansai Medical University, Moriguchi, Osaka, Japan
Key Words. Bone marrow cell ? G-CSF ? M-CSF ? Neovascularization
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.jp
ABSTRACT
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.
MATERIALS AND METHODS
Neovascularization Induced by G-CSF with or without M-CSF in Ischemic Limbs
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).
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 < .005. Abbreviation: FITC, fluorescein isothiocyanate.
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 Vessels
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.
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 < .005. (C): The number of EGFP+ blood vessels (per field) was calculated, and the mean ± SD is shown (n = 4). *p <.005. Abbreviations: BMC, bone marrow cells; EGFP, enhanced green fluorescent protein; PE, phycoerythrin.
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.
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.
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.
Increases in Number of EPCs
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.
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 < .005. Abbreviation: EPC, endothelial progenitor cells.
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.
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 < .005. Abbreviation: LDPI, Laser Doppler perfusion image.
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 < .005.
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).
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.
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
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.
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b Department of Ophthalmology,
c Regeneration Research Center for Intractable Diseases,
d Department of Anatomical Pathology,
e Second Department of Internal Medicine, and
f Department of Neurology, Kansai Medical University, Moriguchi, Osaka, Japan
Key Words. Bone marrow cell ? G-CSF ? M-CSF ? Neovascularization
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.jp
ABSTRACT
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.
MATERIALS AND METHODS
Neovascularization Induced by G-CSF with or without M-CSF in Ischemic Limbs
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).
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 < .005. Abbreviation: FITC, fluorescein isothiocyanate.
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 Vessels
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.
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 < .005. (C): The number of EGFP+ blood vessels (per field) was calculated, and the mean ± SD is shown (n = 4). *p <.005. Abbreviations: BMC, bone marrow cells; EGFP, enhanced green fluorescent protein; PE, phycoerythrin.
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.
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.
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.
Increases in Number of EPCs
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.
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 < .005. Abbreviation: EPC, endothelial progenitor cells.
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.
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 < .005. Abbreviation: LDPI, Laser Doppler perfusion image.
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 < .005.
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).
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.
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
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.
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