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Granulocyte colony-stimulating factor promotes neovascularization by releasing vascular endothelial growth factor from neutrophils

Yuichi Ohki^*,, Beate Heissig^,, Yayoi Sato^*,, Haruyo Akiyama, Zhenping Zhu^||, Daniel J. Hicklin^||, Kazunori Shimada^*, Hideoki Ogawa, Hiroyuki Daida^*, Koichi Hattori^,,1 and Akimichi Ohsaka

^* Department of Cardiology,
Atopy (Allergy) Research Center,
Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan;
Center of Experimental Medicine, Institute of Medical Science at the University of Tokyo, Minato-ku, Tokyo, Japan; and
^|| ImClone Systems Incorporated, New York, New York, USA

¹Correspondence: 4-6-1, Shirokanedai, Minato-ku, Japan. E-mail: khattori{at}ims.u-tokyo.ac.jp

SPECIFIC AIMS

The granulocyte colony-stimulating factor (G-CSF), a growth factor used for stem and progenitor cell mobilization in malignant and nonmalignant disease, has been shown to have angiogenic potential.

We hypothesized that G-CSF promotes angiogenesis by 1) increasing the number of myelo-monocytic cells (neutrophils), a rich source of vascular endothelial growth factor (VEGF), a potent angiogenic factor, and 2) the ability of VEGF to co-mobilize VEGF receptor-2 (VEGFR2)⁺ endothelial progenitor cells (EPCs) and VEGF receptor-1 (VEGFR1)⁺ hematopoietic progenitor cells (HPCs) from bone marrow (BM). We tried to understand 3) whether G-CSF-mediated angiogenesis in the ischemic tissue is driven primarily by VEGF signaling through VEGFR1, VEGFR2, or both receptors.

PRINCIPAL FINDINGS

1. G-CSF improves EPC mobilization and angiogenesis by stimulating VEGF secretion from neutrophils
We investigated whether G-CSF promotes angiogenesis indirectly by promoting the secretion of angiogenic factors, like VEGF. Different FACS-isolated hematopoietic cell fractions were cultured in serum-free medium in the presence or absence of G-CSF for 48 h. VEGF concentrations were higher in supernatants of cultures of unstimulated monocytes/macrophages (Gr-1^–, CD11b⁺) than negative controls (Gr-1^–CD11b^– cells: 15.4±3.9 pg/mL vs. 7.6±0.3 pg/mL n=6, P<0.01). Similarly, culture supernatants of G-CSF-unstimulated neutrophils (Gr-1⁺, CD11b^–) contained high levels of VEGF compared with negative controls (15.0±3.6 pg/mL vs. 7.6±0.3 pg/mL, n=6, P<0.01). G-CSF significantly increased VEGF release into the supernatant, when neutrophils (26.3±11.1 pg/mL vs. 15.0±3.6 pg/mL, n=6, P<0.05), but not monocytes culture supernatants, were assayed (16.2±2.3 pg/mL vs. 15.4±3.9 pg/mL, n=6, N.S.). Taken together, these data indicate that G-CSF can increase VEGF release from myelo-monocytic cells, specifically neutrophils (Fig. 1 A).

View larger version (25K): [in this window] [in a new window]   Figure 1. G-CSF improves EPC mobilization and angiogenesis by stimulating VEGF secretion from neutrophils. A) FACS isolated Gr-1+CD11– cells from PBMCs were cultured in serum-free medium for 48 h in vitro. Cell-free supernatants were analyzed for VEGF by ELISA. B, C) G-CSF was administrated intramuscularly in nonischemic mice. B) Total neutrophils were counted using a Neubauer hematocytometer. C) Plasma VEGF levels in G-CSF-treated and untreated mice were determined by ELISA (2 independent experiments). D) G-CSF was administrated intramuscularly in mice with hindlimb ischemia. Plasma samples were collected and stored at –20°C. Plasma VEGF levels of 2 independent experiments were determined by ELISA. Values are mean ± SE (n=6 at each time point). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. controls.

We next confirmed in vivo that G-CSF resulted in the systemic increase in VEGF plasma levels. G-CSF-treated mice showed a 7-fold increase in neutrophil counts on day 5, returning to the control level by day 10 after injection (Fig. 1B ). Concurrent with augmented neutrophil counts, plasma VEGF levels increased in G-CSF treatment group, reaching peak level at day 5 (n=6) (Fig. 1C ). In another set of experiments, mice with hindlimb ischemia that had also received G-CSF injections further augmented plasma VEGF levels (n=6, P<0.001 on days 5 and 10 after start of G-CSF treatment) (Fig. 1D ).

To confirm that neutrophils (Gr-1-positive cells) are the major source for VEGF after G-CSF treatment in vivo, we costained ischemic muscle tissue of vehicle- or G-CSF-treated mice with Gr-1 and VEGF antibodies. Especially in the muscle tissue of G-CSF-treated mice, the absolute number of Gr-1⁺VEGF⁺ double positive cells increased significantly.

2. G-CSF promotes mobilization of BM-derived EPCs
To study the effect of G-CSF on endothelial progenitor cell (EPC) mobilization, BALB/C mice were treated with an i.p. injection of G-CSF (200 µg/kg/day) for 5 days. Negative control mice received vehicle. VEGF (300 ng/body) was used as positive control. Murine peripheral blood mononuclear cells (PBMCs) were collected on days 0, 3, 5, 7, and 10. To determine the number of mobilized EPCs within the PBMCs, a culture assay was used. EPCs could be distinguished from mature endothelial cells by their capacity to form colonies (CFU-ECs) in endothelial growth medium. CFU-ECs showed DiI-acetylated-LDL (DiI-Ac LDL) uptake and stained positive for VEGF receptor-2 (VEGFR2) in vitro by immunohistochemistry. Concomitant with the peak value of white blood cells, we detected a significant increase in circulating CFU-ECs in PBMCs of G-CSF-treated mice, which reached a peak on day 5. Whereas the administration of recombinant VEGF resulted in CFU-EC mobilization on day 3, mobilization of CFU-ECs by G-CSF was slightly delayed (peak day 5) in vivo.

3. G-CSF administration improves revascularization in a hindlimb ischemia model and involves VEGFR1 signalings
We used a murine model of hindlimb ischemia to determine whether local administration of G-CSF enhances neovascularization of ischemic tissues. After operative excision of the right femoral artery, G-CSF was injected intramuscularly daily for 5 days. 21 days later, mice were killed and the gastrocnemius muscles of these mice were isolated and H&E staining and CD31 staining was performed. The capillary density in CD31-stained slides, as an index of angiogenesis, was significantly higher in the G-CSF-treated group (365±43.6/mm²) compared with the BSA group (246±38.9/mm², P<0.01, n=7).

Functional assessment of subcutaneous blood perfusion in animals after femoral ligation treated with and without G-CSF was performed using Laser Doppler Perfusion Image (LDPI) analysis. Blood flow measured at baseline was similar in both G-CSF and control groups. The blood flow in the ischemic limb recovered faster in the G-CSF-treated group especially on days 7 and 14, compared with the BSA-treated group. Furthermore, a higher perfusion was noted on day 21 in the ischemic limb of G-CSF group (P<0.01, n=7) compared with BSA controls.

Finally we asked whether G-CSF mediated angiogenesis was primarily driven by VEGF signaling through VEGFR1, VEGFR2 or both receptors. Using a murine ischemic hindlimb model G-CSF-treated mice were coinjected with neutralizing antibodies against either anti-mouse VEGFR1 with monoclonal antibodies (anti-VEGFR1 mAb: 800 µg/body), anti-mouse VEGFR2 with monoclonal antibodies (anti-VEGFR2 mAb: 800 µg/body), or a combination of both. In treated mice, blood perfusion was analyzed on day 7 using LDPI analysis (Fig. 2A ). A significant decreased blood perfusion was found in ischemic limb mice that had received anti-VEGFR1 mAb or a combination of the two antibodies (anti-VEGFR1 and anti-VEGFR2) compared with the control group and anti-VEGFR2 mAb-treated group (P<0.01, n=4) (Fig. 2B ). Histological examination revealed that in mice treated with anti-VEGFR1 mAb (152±11.3/mm², n=4) there was a significant decrease in capillary density compared with control group (248±14.5/mm², n=4, P<0.01) and mice treated with anti-VEGFR2 mAb (219±9.8/mm², n=4, P<0.01). In contrast, in mice treated with anti-VEGFR2 mAb, capillary density was reduced much less than in mice treated with anti-VEGFR1 mAb. But a significant reduction of capillary density was found in anti-VEGFR2-treated mice vs. control (P<0.05). A combination therapy using both antibodies further decreased the capillary density vs. treatment with anti-VEGFR1 mAb alone (P<0.05) (Fig. 2C-G ). These data set forth the concept that the G-CSF mediated angiogenesis is driven by the release of VEGF from G-CSF-responsive myelo-monocytic cells, especially neutrophils, which promote the corecruitment of VEGFR1⁺ cells contributing to neo-angiogenesis.

View larger version (49K): [in this window] [in a new window]   Figure 2. G-CSF promotes angiogenesis by releasing VEGF and signaling through VEGFR1. Starting from the day of right femoral artery ligation, G-CSF was injected into ischemic tissue for 5 days with anti-VEGFR1 Ab (800 µg), anti-VEGFR2 Ab (800 µg), a combination of both antibodies and control antibody (IgG Ab) on day 1, 3 and 5 by i.p. injection. LDPI analysis was performed on day 7. Mice were killed and the gastrocnemius muscle was isolated for histological examination. A) Representative LDPIs: blood perfusion (red to yellow) was augmented in control and anti-VEGFR2 Ab-treated groups compared with anti-VEGFR1 Ab and combination with antibodies-treated groups (green to blue). B) The ischemic normal blood flow ratio using LDPI analysis is shown. The blood flow in the ischemic limb of anti-VEGFR1 Ab group and combination with antibodies-treated group were significantly lower than in the control group and anti-VEGFR2 Ab-treated group. C–F) H&E staining of ischemic muscle tissue from G-CSF-treated HL-ischemic mice coinjected with C) vehicle + IgG Ab, D) G-CSF + anti-VEGFR1 Ab, E) G-CSF + anti-VEGFR2 Ab, F) G-CSF + anti-VEGFR1 Ab + anti-VEGFR2 Ab. D) x100. G) Quantification of the capillary density after CD31 staining. Values are mean ± SE (n=4). *P < 0.05 and **P < 0.01 vs. controls.

CONCLUSIONS AND SIGNIFICANCE

As the main stem cell mobilizing agent, G-CSF has been shown to have angiogenic potential. We demonstrate for the first time that the angiogenesis-promoting effect of G-CSF is partly mediated by enhanced mobilization of mature hematopoietic cells, specifically neutrophils and immature or mature endothelial cells. We demonstrated that G-CSF stimulation significantly augmented VEGF secretion from neutrophils in vitro and promoted angiogenesis in vivo using a hindlimb model.

Even though monocytes/macrophages have a functional G-CSF receptor, we showed that VEGF production by monocytes/macrophages was independent of G-CSF stimulation. We demonstrated that G-CSF can amplify VEGF secretion from neutrophils by two distinct pathways: G-CSF can increase the absolute number of neutrophils and, on the other hand, can improve the VEGF production per neutrophil above the level seen in steady state neutrophils. The importance of this finding is underscored by a recent study demonstrating that VEGF-A can induce neutrophil migration through extravascular tissue in vivo after binding to VEGFR1 on neutrophils, resulting in further VEGF release in human endometrium. We showed that G-CSF induced angiogenesis was due to activation of the VEGF/VEGFR1 signaling pathway, because administration anti-VEGFR1 mAb in vivo significantly suppressed angiogenesis/vasculogenesis in G-CSF-treated mice with hindlimb ischemia, whereas the use of anti-VEGFR2 mAb had a less profound effect. These data are in accordance with other groups showing that endogenous VEGF from inflammatory tissue can increase neutrophil migration into inflammatory site via VEGFR1 pathway.

Several other groups have reported that both G-CSF and granulocyte/macrophage colony-stimulating factor (GM-CSF) induced the proliferation and migration of endothelial cells and that these cytokine-induced EPCs can contribute to neovascularization of ischemic tissue.

In conclusion, we provide evidence that G-CSF promotes mobilization of EPCs and increases neutrophil counts in vivo. G-CSF-activated neutrophils release VEGF setting an "angiogenic environment" to further promote EPC mobilization and local recruitment of angiogenic cells, which finally contribute to vessel formation. Local administration of G-CSF into ischemic tissues in adults might be a treatment strategy to promote collateral vessel formation by modulating the cellular and angiogenic growth factors in the muscle niche. Studies are needed to determine the possible risk associated with induction of neo-angiogenesis in patients with malignant tumors and thrombosis by administration of cytokines during the course of chemotherapy.

View larger version (40K): [in this window] [in a new window]   Figure 3. Schematic diagram. G-CSF can increase the absolute number of circulating neutrophils and their release of VEGF. Endogenous VEGF from inflammatory tissue can induce neutrophils migration into inflammatory site via VEGFR1 pathway, resulting in further VEGF release. On the other hand, further VEGF release can enhance comobilization of VEGFR1-expressing HPCs and VEGFR2-expressing EPCs, which are not able to incorporate into vascular structure but can release angiogenic factors and induce inflammatory neovascularization.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3496fje;

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