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Neutrophils as a key cellular target for angiostatin: implications for regulation of angiogenesis and inflammation ¹

ROBERTO BENELLI^*,, MONICA MORINI, FABIO CARROZZINO, NICOLETTA FERRARI, SIMONA MINGHELLI^*, LEONARDO SANTI^*,, MARCO CASSATELLA, DOUGLAS M. NOONAN and ADRIANA ALBINI²

^* Centro di Biotecnologie Avanzate, 16132 Genova, Italy, the
Tumor Progression Section and the
Molecular Biology Laboratory, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genova, Italy;
Department of Oncology, Biology and Biotechnology, Università di Genova, Genova, Italy; and the
Department of Pathology, General Pathology Section, Università di Verona, 37134 Verona, Italy

²Correspondence: Molecular Biology Laboratory, Istituto Nazionale per la Ricerca sul Cancro, c/o Centro di Biotecnologie Avanzate, Largo Rosanna Benzi 10, 16132 Genova, Italy. E-mail: adriana.albini{at}istge.it

SPECIFIC AIMS

Angiostatin is an effective angiogenesis inhibitor whose mechanisms are poorly understood. Since leukocytes play an important role in angiogenesis, we investigated the ability of angiostatin to inhibit angiogenesis-associated leukocyte functions in vitro and leukocyte-mediated angiogenesis in vivo in response to CXCR2 ligands and inflammation-related stimuli.

PRINCIPAL FINDINGS

1. Angiostatin inhibits leukocyte migration in vitro
Angiostatin inhibition of angiogenesis has been observed in preclinical models, but its mechanism has not been fully elucidated. We have examined the effects of angiostatin on the migration of monocytes and neutrophils to specific chemotactic factors. Angiostatin (consisting of kringle repeats 1–4 of plasminogen) inhibited monocyte migration to chemoattractants such as MCP-1 (50 µg/ml) and fMLP (Fig. 1 ) by 25–50%, with inhibition reaching significance at high concentrations. An even stronger dose-dependent angiostatin inhibition of polymorphonuclear cell (PMN, >96% neutrophils) migration to the CXCR1 and CXCR2 agonist interleukin 8 (IL-8, 50 µg/ml) was observed (Fig. 1) , with significant inhibition at 1.25–2.5 µg/ml. Similar results were obtained using agonists specific for CXCR2, MIP-2 (200 µg/ml) (Fig. 1) , or Gro (200 µg/ml) (not shown) as chemoattractants. Since neutrophils appeared to have a higher sensitivity to angiostatin inhibition and are key mediators of CXCR2 agonist-induced angiogenesis in vivo, we concentrated our studies on this cell type.

View larger version (48K): [in this window] [in a new window]   Figure 1. Inhibition of monocyte or neutrophil migration by angiostatin (Ast) (in µg/ml). Means ± SE are shown; significant inhibition of migration relative to positive controls by ANOVA: ***P < 0.0001; **P < 0.01; *P < 0.05. Migration to the positive controls was always significantly higher than that to sfm alone (ANOVA, P<0.0001).

To test whether the effects on neutrophil migration were specific to angiostatin, the activity of whole plasminogen and an angiostatin-like plasminogen fragment (angiostatin K1–3, or Ang K1–3) was examined. Neutrophil migration to MIP-2 was significantly inhibited by 2.5 or 5 µg/ml angiostatin whereas equimolar concentrations of whole plasminogen did not significantly affect migration (Fig. 1) . Ang K1–3, which contains the first three kringle repeats known to have angiostatin-like activities, significantly inhibited neutrophil migration with a dose dependence similar to angiostatin (Fig. 1) . These data confirm that the inhibition of neutrophil migration was specific for angiostatin.

Other granulocyte chemoattractants that do not activate CXCR2 [fMLP and the protein kinase C (PKC) activator TPA] also induced neutrophil migration, which again was significantly inhibited by similar concentrations of angiostatin (Fig. 1) . The ability of angiostatin to inhibit TPA-induced granulocyte migration suggests that angiostatin treatment disturbs the MAP-kinase pathway downstream of PKC.

2. Neutrophils directly mediate CXCR2 ligand-induced angiogenesis in vivo
It has been shown that IL-8 binds CXCR1 and CXCR2, which is also a receptor for the chemokines MIP-2 and GRO. These CXCR2 ligands (also known as ELR chemokines due to the presence of this characteristic amino acid motif) are known to be potent angiogenic factors. We therefore examined the ability of these CXCR2 agonists to induce angiogenesis in vivo in neutropenic mice.

In the Matrigel implant model in vivo, IL-8, MIP-2, and GRO (all used the concentration of 50 µg/ml) induced potent angiogenic responses in normal control animals as indicated by the mean hemoglobin level as an indicator of the angiogenic response (Fig. 2 a, b). Intraperitoneal injections of the rat anti-mouse Ly-6G (Gr-1) monoclonal antibody induced a marked neutropenia. The strong angiogenic response to IL-8, MIP-2, or GRO in control animals was essentially absent in neutropenic animals, with significant differences with all three ligands (Fig. 2a ). Addition of angiostatin at 0.5 or 2.5 µg/ml in the Matrigel pellet significantly inhibited the angiogenesis induced by IL-8, MIP-2, or GRO in vivo (Fig. 2) . Consistent with the in vitro observations, equimolar concentrations of the Ang K1–3 plasminogen fragment gave significant inhibition of angiogenesis in vivo whereas inclusion of equimolar concentrations of whole plasminogen had little effect (Fig. 2b ). Twice-daily s.c. administration of Ang K1–3 also significantly inhibited MIP-2-induced angiogenesis (Fig. 2b ). Histological examination of the implants containing IL-8 demonstrated the formation of dilated hemorrhagic vessels and a massive infiltrate of leukocytes through the Matrigel. Angiostatin (2.5 µg/ml) decreased the infiltration of inflammatory cells and the formation of blood vessels. Myeloperoxidase staining indicated a strong infiltration of neutrophils and monocytes/macrophages into the gels in response to IL-8.

View larger version (32K): [in this window] [in a new window]   Figure 2. Vascularization of Matrigel implants after 4 days in vivo and reduction by neutropenia (a) or angiostatin (b) as assessed by hemoglobin content in neutropenic mice or after treatment with angiostatin. The data were pooled from independent experiments; means ± SE are shown. Significant inhibition of hemoglobin content relative to positive controls by ANOVA: ***P < 0.0001; **P < 0.01; *P < 0.05; by t test (Mann-Whitney): °°°P < 0.002; °°P < 0.02; °P < 0.05.

3. Angiostatin inhibits leukocyte recruitment and inflammation-related angiogenesis in vivo
LPS is a sign of bacteria intrusion recognized by phagocytes and a powerful proinflammatory stimulus. Inclusion of LPS into the Matrigel sponges resulted in an intense leukocyte infiltrate accompanied by a rapid and intense angiogenic response (Fig. 2b ), with poorly organized hemorrhagic vessels entering the gel. Addition of angiostatin along with the LPS dramatically reduced the leukocyte infiltrate and significantly inhibited the angiogenic response (Fig. 2b ), indicating that angiostatin inhibits inflammation in vivo. These data provide further evidence of the link between angiogenic potential and leukocyte recruitment in vivo and the ability of angiostatin to potently counteract leukocyte-induced angiogenesis.

4. Angiostatin receptor expression by neutrophils
The ability of angiostatin to inhibit neutrophil function in vitro and in vivo suggested that these cells may express receptors for angiostatin. One angiostatin receptor described is the cell surface ATP synthase, which is active on endothelial cells surfaces and inhibited by angiostatin; another is angiomotin, a recently described protein apparently directly involved in chemotaxis whose activity is inhibited by angiostatin. RT-PCR analysis demonstrated clear expression of the ATP synthase mRNA by PMNs and HL-60. The expression of ATP synthase mRNA correlated with the cell surface expression of ATP synthase protein on > 98% of neutrophils by flow cytometry analysis. Highly purified neutrophils clearly expressed mRNA for angiomotin. Thus, PMNs clearly express mRNAs and protein for known angiostatin receptors, though it is not clear whether these receptors mediate the effects of angiostatin on leukocytes.

CONCLUSIONS AND SIGNIFICANCE

Chemokines that are CXCR2 agonists are recognized to play a key role in angiogenesis in several tumor types. Although previous studies have suggested that the angiogenic effects of CXCR2 agonists may be due to functional CXCR2 expression on some endothelial cells, we show here that neutrophil depletion abrogates their angiogenic potential in vivo, demonstrating an essential role for these cells. Leukocyte infiltrates are often observed in the in vivo responses to a variety of angiogenic factors, particularly those that strongly attract leukocytes. Previous studies of the time course of Matrigel implant invasion in vivo in response to the angiogenic factor HIV-Tat by electron microscopy indicated that the initial cells invading the gels were PMNs, which appear to be actively degrading the matrix and creating clefts in the Matrigel. These clefts often contained red blood cells, suggesting they represent the beginning of a branching vessel. Macrophages were also observed in the clefts, some of which engulf red blood cells. The PMN and macrophage invasion was followed by endothelial cell entry into the clefts, which over time lined the channels and formed functional blood vessels. A similar cascade appears to occur in response to CXCR2 ligands (Fig. 3 );F3>: in the absence of neutrophils (neutropenic mice), there was no angiogenic response to the chemokines IL-8, MIP-2, or GRO. Angiostatin blocked IL-8-, MIP-2-, and GRO-induced angiogenesis in vivo, which was related to strong inhibition of PMN migration and metabolic activation in vitro. Inflammatory stimuli seem to induce a similar leukocyte-mediated angiogenesis in vivo that again was blocked by angiostatin, which also inhibited monocyte migration in vivo. That PMNs and macrophages can be induced to release significant quantities of angiogenic factors and enzymes linked to the angiogenic switch such as MMP-9 (gelatinase B) suggests that, when activated, these cells attract endothelial cells to form new vessels. This cascade appears to be critical for the angiogenesis induced by the CXCR2 ligand chemokines and inflammatory stimuli.

View larger version (96K): [in this window] [in a new window]   Figure 3. A schematic model of the leukocyte-mediated angiogenic cascade in vivo and the direct effects of angiostatin. Angiogenic chemotactic factors (ACF). The first cells that enter are PMNs, which appear to degrade the matrix and lead formation of a cleft (step 1). In response to some stimuli, macrophages also invade at intermediate times (step 2). Both activated PMNs (steps 2, 3) and macrophages (step 3) are able to release additional angiogenic factors (arrows), such as VEGF, that can subsequently induce endothelial cell entry. Endothelial cell recruitment and proliferation continues until the cleft originally formed by the PMNs is completely lined with endothelial cells and joins neighboring clefts to become a functional vessel (steps 4, 5). In neutropenic mice, the lack of neutrophils eliminates the critical first step of PMN recruitment (solid line), and there is no subsequent angiogenesis. Besides its ability to inhibit endothelial cells in the later stages (solid line), the presence of angiostatin also strongly inhibits PMNs (solid line) and, to a lesser degree, monocytes/macrophages (dashed line) at the initial phases of the cascade.

Angiostatin inhibition of leukocyte recruitment in angiogenesis, precociously interrupting an important cascade, may represent a key mechanism for angiostatin action. Since leukocytes release enzymes that could potentially generate angiostatin, angiostatin inhibition of leukocyte recruitment might represent a physiological feedback mechanism for control of inflammatory angiogenesis. The angiostatin inhibition of neutrophil recruitment and angiogenesis in response to fMLP in vitro and LPS in vivo supports this hypothesis. Our data indicate that angiostatin or angiostatin mimics may be effective anti-inflammatory agents as well, broadening its potential clinical applications.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0651fje; to cite this article, use FASEB J. (December 28, 2001) 10.1096/fj.01-0651fje

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