The role of integrin α5β1 in the regulation of corneal neovascularization
Introduction
The angiogenic process called neovascularization constitutes the final common path of multiple ophthalmological diseases resulting in visual decay. Corneal neovascularization is a complication of many entities, including trauma, chemical injury, and following keratoplasty. A broad selection of substances has been tested for their potential to inhibit angiogenesis, however, regression, the more important therapeutic feature, has rarely been described (Ambati et al., 2002, Zhang et al., 2005).
Angiogenesis is a highly regulated process which occurs in response to various proangiogenic stimuli like growth factors, cytokines and other physiologic molecules as well as other factors like hypoxia and low pH (Folkman and Shing, 1992). The angiogenic cascade for development of new blood vessels requires the cooperation of a variety of molecules that regulate necessary cellular processes such as extracellular matrix remodeling, invasion, migration, proliferation, differentiation and tube formation of endothelial cells (Brooks, 1996). Activation, survival, targeting, migration and endothelial cell adhesion to the extracellular matrix are fundamental steps in the process of angiogenesis, and most of these interactions are mediated by integrins.
Integrins are a family of transmembrane glycoproteins, consisting of an alpha and beta chain, mediating cell-cell and cell-matrix interactions (Hynes, 1992). Several of them have been identified as receptors involved in angiogenic pathways and are up-regulated on activated endothelial cells (Brooks et al., 1994, Collo and Pepper, 1999, Drake et al., 1995, Friedlander et al., 1995, Kim et al., 2000, Stromblad and Cheresh, 1996a, Stromblad and Cheresh, 1996b).
Therefore the integrin receptor family, in particular the integrins αvβ3 and αvβ5, has undergone intense analysis on their physiological function and potential in manipulating vasculo- and angiogenesis (Drake et al., 1995, Friedlander et al., 1995, Friedlander et al., 1996, Stromblad and Cheresh, 1996a).
However, knockout studies in mice have shown that deletion of αvβ3 and/or β5 fails to block developmental angiogenesis and in some cases may enhance vasculogenesis and angiogenesis (Bader et al., 1998, Hodivala-Dilke et al., 1999, Huang et al., 2000, Hynes, 1992, Hynes, 2002). In contrast, α5β1-integrin seems to have a definite impact on blood vessel formation and maturation (Francis et al., 2002, Yang et al., 1993). Knockout mice for α5-integrin, which pairs only with β1, show embryonic lethality with major vascular defects such as marked decrease in the complexity of the vasculature and not appropriately formed vessels in mouse embryos and embryoid bodies (Francis et al., 2002, Yang et al., 1993).
The integrin α5β1 receptor negotiates endothelial cell adhesion and migration along the extracellular matrix by recognition of fibronectin, an extracellular matrix protein, and provides proliferative signals to vascular cells (Kim et al., 2000). Although several integrins bind to fibronectin (Hynes, 1992), integrin α5β1 is generally selective for fibronectin (Pytela et al., 1985). Moreover, integrin α5β1 seems to be only minimally expressed in quiescent vascular cells, whereas newly growing blood vessels and areas of pathological neovascularization show strong up-regulation of α5β1 (Kim et al., 2000, Magnussen et al., 2005, Parsons-Wingerter et al., 2005, Zhang et al., 2002). Pharmacological studies with integrin inhibitors confirmed the important role of α5β1 in vascularization processes. Integrin α5 receptor inhibition as well as monoclonal antibodies to the cell-binding domain of fibronectin reduced pathological neovascularization and tumor growth in tumor animal models, as well as in the chick CAM-model (Kim et al., 2000). Alterations of aortic vasculogenesis with antibodies to β1-integrins have been elucidated by Drake et al. (1992b).
In this study, we evaluated the efficacy of a novel anti-integrin α5β1 small molecule JSM5562 in a model of corneal injury and neovascularization. The changes in integrin expression and mRNA-expression of different growth factors and cytokines relevant for the regulation of neovascularization were investigated.
Section snippets
Animals
Female C57 Bl/6 mice, weighting 20–25 g, were purchased from Jackson Laboratories (Bar Harbor, MA). All animal experiments followed the guidelines of the Association for Research in Vision and Ophthalmology and were approved by the Animal Care and Use Committee of Cologne (Regierungspraesidium Koeln), Germany. All surgical procedures were performed under general anesthesia with 10 mg/kg xylazine hydrochloride 2% (Xylazin; Riemser Arzneimittel AG, Riems, Germany) and 50 mg/kg ketamine hydrochloride
Qualitative integrin-α5 expression
In order to visualize α5β1 integrin expression in physiologic corneas as well as injured corneas, with or without JSM5562-treatment, integrin-α5 staining was performed on corneal flatmounts collected on day 10 after injury. Co-labeling for CD31 was conducted and merged images were taken to identify all vessels and to localize the integrin expression. Representative images are shown in Fig. 1. Integrin-α5 expression was visible in the limbal areas and definite areas of neovascularization.
Discussion
We examined the efficacy of the anti-integrin α5β1 small molecule JSM5562 for inhibition and regression of corneal neovascularization induced by corneal injury due to mechanical and alkali burn trauma. After onset of corneal neovascularization, animals were either treated with JSM5562 or control substance administered with i.p. implanted osmotic mini-pumps. Digital quantification of corneal neovascularization on whole corneal flatmounts demonstrated that integrin α5β1-inhibitor JSM5562 is
Acknowledgements
This work was funded by Deutsche Forschungsgemeinschaft DFG Jo 324/4-1, DFG Jo 324/6-2 (Emmy Noether), Kämpgen Stiftung, and the ZMMK Köln (TV 76). The authors thank Claudia Gavranic, Martina Becker and Frank Lacina for expert technical assistance. These data were presented at the ARVO meeting, 2005 in Fort Lauderdale, FL, USA.
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These authors contributed equally to this work.