SpringerLink
Log in

Neutrophils promote inflammatory angiogenesis via release of preformed VEGF in an in vivo corneal model

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

We investigated the role of neutrophilic cells (neutrophils) in inflammatory angiogenesis and explored the possible mechanisms involved. Corneal angiogenesis was induced in vivo with a 75% silver nitrate applicator. Depletion of neutrophils was accomplished by the intraperitoneal administration of RB6-8C5, a neutrophil-depleting antibody. Angiogenesis, neutrophil infiltration, and the localization of vascular endothelial growth factor (VEGF) were evaluated by biomicroscopic observations, histology, and immunohistochemistry in control and RB6-8C5 treatment groups. Protein levels of VEGF, macrophage inflammatory protein-1alpha (MIP-1α), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor alpha in the cornea were determined by enzyme-linked immunosorbent assay. An in vitro model of neutrophil activation was also used to examine the ability of neutrophils to produce and release VEGF, MIP-1α, and MIP-2. At day 1 after injury, neutrophil infiltration in the cornea was highest, and VEGF was expressed in the infiltrating neutrophils. The enhanced protein levels of VEGF, MIP-1α, and MIP-2 correlated with the degree of neutrophil infiltration. Neutrophil depletion significantly inhibited corneal angiogenesis and reduced the protein levels of VEGF, MIP-1α, and MIP-2 in the cornea. Upon stimulation, isolated neutrophils released VEGF from preformed stores and MIP-1α and MIP-2 by de novo synthesis. Neutrophil depletion thus significantly impaired inflammatory angiogenesis, identifying neutrophils as an important player in inflammatory angiogenesis. Neutrophils may exercise their angiogenic function by releasing proangiogenic factors such as VEGF. Intervention measures targeting neutrophils may therefore help to deal with abnormal angiogenesis involved in chronic inflammatory diseases.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Germany)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Alon T, Hemo I, Itin A, Pe’er J, Stone J, Keshet E (1995) Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med 1:1024–1028

    Article  CAS  PubMed  Google Scholar 

  • Armstrong DA, Major JA, Chudyk A, Hamilton TA (2004) Neutrophil chemoattractant genes KC and MIP-2 are expressed in different cell populations at sites of surgical injury. J Leukoc Biol 75:641–648

    Article  CAS  PubMed  Google Scholar 

  • Azar DT (2006) Corneal angiogenic privilege: angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis). Trans Am Ophthalmol Soc 104:264–302

    PubMed  Google Scholar 

  • Ballara SC, Miotla JM, Paleolog EM (1999) New vessels, new approaches: angiogenesis as a therapeutic target in musculoskeletal disorders. Int J Exp Pathol 80:235–250

    Article  CAS  PubMed  Google Scholar 

  • Benelli R, Morini M, Carrozzino F, Ferrari N, Minghelli S, Santi L, Cassatella M, Noonan DM, Albini A (2002) Neutrophils as a key cellular target for angiostatin: implications for regulation of angiogenesis and inflammation. FASEB J 16:267–269

    CAS  PubMed  Google Scholar 

  • Binétruy-Tournaire R, Demangel C, Malavaud B, Vassy R, Rouyre S, Kraemer M, Plouët J, Derbin C, Perret G, Mazié JC (2000) Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis. EMBO J 19:1525–1533

    Article  PubMed  Google Scholar 

  • Clauss M, Gerlach M, Gerlach H, Brett J, Wang F, Familletti PC, Pan YC, Olander JV, Connolly DT, Stern D (1990) Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration. J Exp Med 172:1535–1545

    Article  CAS  PubMed  Google Scholar 

  • Daley JM, Thomay AA, Connolly MD, Reichner JS, Albina JE (2008) Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J Leukoc Biol 83:64–70

    Article  CAS  PubMed  Google Scholar 

  • Davatelis G, Tekamp-Olson P, Wolpe SD, Hermsen K, Luedke C, Gallegos C, Coit D, Merryweather J, Cerami A (1988) Cloning and characterization of a cDNA for murine macrophage inflammatory protein (MIP), a novel monokine with inflammatory and chemokinetic properties. J Exp Med 167:1939–1944

    Article  CAS  PubMed  Google Scholar 

  • Endlich B, Armstrong D, Brodsky J, Novotny M, Hamilton TA (2002) Distinct temporal patterns of macrophage-inflammatory protein-2 and KC chemokine gene expression in surgical injury. J Immunol 168:3586–3594

    CAS  PubMed  Google Scholar 

  • Fahey TJ 3rd, Sherry B, Tracey KJ, Deventer S van, Jones WG 2nd, Minei JP, Morgello S, Shires GT, Cerami A (1990) Cytokine production in a model of wound healing: the appearance of MIP-1, MIP-2, cachectin/TNF and IL-1. Cytokine 12:92–99

    Article  Google Scholar 

  • Fromer CH, Klintworth GK (1975) An evaluation of the role of leukocytes in the pathogenesis of experimentally induced corneal vascularization. Am J Pathol 79:537–554

    CAS  PubMed  Google Scholar 

  • Fujita S, Saika S, Kao WW, Fujita K, Miyamoto T, Ikeda K, Nakajima Y, Ohnishi Y (2007) Endogenous TNFalpha suppression of neovascularization in corneal stroma in mice. Invest Ophthalmol Vis Sci 48:3051–3055

    Article  PubMed  Google Scholar 

  • Gaudry M, Bregerie O, Andrieu V, El Benna J, Pocidalo MA, Hakim J (1997) Intracellular pool of vascular endothelial growth factor in human neutrophils. Blood 90:4153–4161

    CAS  PubMed  Google Scholar 

  • Griffioen AW, Molema G (2000) Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation. Pharmacol Rev 52:237–268

    CAS  PubMed  Google Scholar 

  • Heidenreich R, Rocken M, Ghoreschi K (2008) Angiogenesis: the new potential target for the therapy of psoriasis? Drug News Perspect 21:97–105

    Article  CAS  PubMed  Google Scholar 

  • Hestdal K, Ruscetti FW, Ihle JN, Jacobsen SE, Dubois CM, Kopp WC, Longo DL, Keller JR (1991) Characterization and regulation of RB6-8C5 antigen expression on murine bone marrow cells. J Immunol 147:22–28

    CAS  PubMed  Google Scholar 

  • Jackson JR, Seed MP, Kircher CH, Willoughby DA, Winkler JD (1997) The codependence of angiogenesis and chronic inflammation. FASEB J 11:457–465

    CAS  PubMed  Google Scholar 

  • Kasama T, Miwa Y, Isozaki T, Odai T, Adachi M, Kunkel SL (2005) Neutrophil-derived cytokines: potential therapeutic targets in inflammation. Curr Drug Targets Inflamm Allergy 4:273–279

    Article  CAS  PubMed  Google Scholar 

  • Kasama T, Strieter RM, Standiford TJ, Burdick MD, Kunkel SL (1993) Expression and regulation of human neutrophil-derived macrophage inflammatory protein 1 alpha. J Exp Med 178:63–72

    Article  CAS  PubMed  Google Scholar 

  • Keane MP, Belperio JA, Moore TA, Moore BB, Arenberg DA, Smith RE, Burdick MD, Kunkel SL, Strieter RM (1999) Neutralization of the CXC chemokine, macrophage inflammatory protein-2, attenuates bleomycin-induced pulmonary fibrosis. J Immunol 162:5511–5518

    CAS  PubMed  Google Scholar 

  • Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, Connolly DT (1989) Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 1246:1309–1312

    Article  Google Scholar 

  • Kibbey MC, Corcoran ML, Wahl LM, Kleinman HK (1994) Laminin SIKVAV peptide-induced angiogenesis in vivo is potentiated by neutrophils. J Cell Physiol 160:185–193

    Article  CAS  PubMed  Google Scholar 

  • Kim KJ, Li B, Houck K, Winer J, Ferrara N (1992) The vascular endothelial growth factor proteins: identification of biologically relevant regions by neutralizing monoclonal antibodies. Growth Factors 7:53–64

    Article  CAS  PubMed  Google Scholar 

  • Kim SJ, Uehara H, Karashima T, Mccarty M, Shih N, Fidler IJ (2001) Expression of interleukin-8 correlates with angiogenesis, tumorigenicity, and metastasis of human prostate cancer cells implanted orthotopically in nude mice. Neoplasia 3:33–42

    Article  CAS  PubMed  Google Scholar 

  • Lagasse E, Weissman IL (1996) Flow cytometric identification of murine neutrophils and monocytes. J Immunol Methods 197:139–150

    Article  CAS  PubMed  Google Scholar 

  • Leek RD, Lewis CE, Whitehouse R, Greenall M, Clarke J, Harris AL (1996) Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer Res 56:4625–4629

    CAS  PubMed  Google Scholar 

  • Lewinsohn DM, Bargatze RF, Butcher EC (1987) Leukocyte-endothelial cell recognition: evidence of a common molecular mechanism shared by neutrophils, lymphocytes, and other leukocytes. J Immunol 138:4313–4321

    CAS  PubMed  Google Scholar 

  • Liekens S, De Clercq E, Neyts J (2002) Angiogenesis: regulators and clinical applications. Biochem Pharmacol 61:253–270

    Article  Google Scholar 

  • Lingen MW (2001) Role of leukocytes and endothelial cells in the development of angiogenesis in inflammation and wound healing. Arch Pathol Lab Med 125:67–71

    CAS  PubMed  Google Scholar 

  • McCourt M, Wang JH, Sookhai S, Redmond HP (1999) Proinflammatory mediators stimulate neutrophil-directed angiogenesis. Arch Surg 134:1325–1331

    Article  CAS  PubMed  Google Scholar 

  • Ogawa S, Yoshida S, Ono M, Onoue H, Ito Y, Ishibashi T, Inomata H, Kuwano M (1999) Induction of macrophage inflammatory protein-1alpha and vascular endothelial growth factor during inflammatory neovascularization in the mouse cornea. Angiogenesis 3:327–334

    Article  CAS  PubMed  Google Scholar 

  • Philipp W, Speicher L, Humpel C (2000) Expression of vascular endothelial growth factor and its receptors in inflamed and vascularized human corneas. Invest Ophthalmol Vis Sci 41:2514–2522

    CAS  PubMed  Google Scholar 

  • Scapini P, Lapinet-Vera JA, Gasperini S, Calzetti F, Bazzoni F, Cassatella MA (2000) The neutrophil as a cellular source of chemokines. Immunol Rev 177:195–203

    Article  CAS  PubMed  Google Scholar 

  • Scapini P, Morini M, Tecchio C, Minghelli S, Di Carlo E, Tanghetti E, Albini A, Lowell C, Berton G, Noonan DM, Cassatella MA (2004) CXCL1/macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth factor-A. J Immunol 172:5034–5040

    CAS  PubMed  Google Scholar 

  • Schanzlin DJ, Cyr RJ, Friedlaender MH (1983) Histopathology of corneal neovascularization. Arch Ophthalmol 101:472–474

    CAS  PubMed  Google Scholar 

  • Schlaeppi JM, Siemeister G, Weindel K, Schnell C, Wood J (1999) Characterization of a new potent, in vivo neutralizing monoclonal antibody to human vascular endothelial growth factor. J Cancer Res Clin Oncol 125:336–342

    Article  CAS  PubMed  Google Scholar 

  • Sunderkötter C, Roth J, Sorg C (1990) Immunohistochemical detection of bFGF and TNF-alpha in the course of inflammatory angiogenesis in the mouse cornea. Am J Pathol 137:511–515

    PubMed  Google Scholar 

  • Sunderkötter C, Goebeler M, Schulze-Osthoff K, Bhardwa R, Sorg C (1991) Macrophage-derived angiogenesis factors. Pharmacol Ther 51:195–216

    Article  PubMed  Google Scholar 

  • Sunderkötter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C (1994) Macrophages and angiogenesis. J Leukoc Biol 55:410–422

    PubMed  Google Scholar 

  • Szekanecz Z, Koch AE (2001) Chemokines and angiogenesis. Curr Opin Rheumatol 13:202–208

    Article  CAS  PubMed  Google Scholar 

  • Tanaka D, Kagari T, Doi H, Shimozato T (2006) Essential role of neutrophils in anti-type II collagen antibody and lipopolysaccharide-induced arthritis. Immunology 119:195–202

    Article  CAS  PubMed  Google Scholar 

  • Walsh DA, Haywood L (2001) Angiogenesis: a therapeutic target in arthritis. Curr Opin Invest Drugs 2:1054–1063

    CAS  Google Scholar 

  • Wang H, Keisar JA (1998) Vascular endothelial growth factor upregulates the expression of matrix metalloproteinases in vascular smooth muscle cells: role of flt-1. Circ Res 83:832–840

    CAS  PubMed  Google Scholar 

  • Wolpe SD, Cerami A (1989) Macrophage inflammatory proteins 1 and 2: members of a novel superfamily of cytokines. FASEB J 3:2565–2573

    CAS  PubMed  Google Scholar 

  • Wolpe SD, Davatelis G, Sherry B, Beutler B, Hesse DG, Nguyen HT, Moldawer LL, Nathan CF, Lowry SF, Cerami A (1988) Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties. J Exp Med 167:570–581

    Article  CAS  PubMed  Google Scholar 

  • Xue ML, Thakur A, Willcox M (2002) Macrophage inflammatory protein-2 and vascular endothelial growth factor regulate corneal neovascularization induced by infection with Pseudomonas aeruginosa in mice. Immunol Cell Biol 80:323–327

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to Professor Roger Beuerman of the Singapore Eye Research Institute for his helpful discussions and to Professor Teh Ming from the Department of Pathology, National University Hospital and Dr. Thai Tran from the Department of Physiology, National University of Singapore, Singapore for their kind support.

Author information

Authors and Affiliations

  1. Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Blk MD9, 2 Medical Drive, Singapore, 117597, Singapore

    Yue Gong & Dow-Rhoon Koh

Authors
  1. Yue Gong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dow-Rhoon Koh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dow-Rhoon Koh.

Additional information

This work was supported by the Yong Loo Lin School of Medicine, National University of Singapore, Singapore (grant no. R-185-000-170-101).

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gong, Y., Koh, DR. Neutrophils promote inflammatory angiogenesis via release of preformed VEGF in an in vivo corneal model. Cell Tissue Res 339, 437–448 (2010). https://doi.org/10.1007/s00441-009-0908-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-009-0908-5

Keywords

Search

Navigation