The role of integrin α₅β₁ in the regulation of corneal neovascularization

Abstract

Integrins are transmembrane receptor proteins critical for growth and stabilization of vessels, but the mechanisms by which integrin activities are involved in neoangiogenesis of the eye remain unclear. Specific inhibitors to fibronectin receptor integrin α₅β₁ impeded pathological neovascularization in vivo. Our objective was to determine whether α₅β₁ plays a role in ocular angiogenesis, and whether a novel α₅β₁-inhibiting small molecule is able to reduce angiogenesis in a model of inflammatory corneal neovascularization. Corneal neovascularization was induced in C57Bl/6 mice by NaOH-application and debridement of the limbal epithelium. Mice were randomized into six groups receiving either no treatment, or intraperitoneal osmotic pumps delivering three different doses of integrin antagonist or control substance on day 10 after scraping. In order to quantify the neovascular response, flatmounts were stained with FITC-CD31. Integrin α₅ expression was determined by immunohistochemistry and quantified by semiquantitative western blot analysis. Influence of integrin antagonist treatment on the mRNA expression of VEGF, bFGF and integrin α₅ was quantified by real-time RT–PCR. Vascularized corneas demonstrated a strong up-regulation of integrin α₅ within affected areas. Animals treated systemically with α₅β₁-inhibiting small molecule showed a significant inhibition and regression of corneal neovascularization. PCR analysis evinced a significant up-regulation of VEGF and integrin α₅ mRNA levels in injured animals compared to controls, and a significant reduction of integrin α₅ mRNA in substance-treated animals compared to control substance, but no significant differences of bFGF levels in all groups. Western blot analysis of integrin α₅β₁ protein expression showed a trend towards up-regulation in injured animals, both control substance-treated and those treated with the α₅β₁-inhibiting small molecule. Systemic delivery of an α₅β₁-inhibiting small molecule inhibits and regresses corneal neovascularization induced by mechanical-alkali burn corneal injury. These results suggest an essential role for the integrin α₅β₁ in pathological neovascular processes of the cornea. Integrin α₅β₁ inhibitors could become a new approach for treatment of neovascularization in the eye.

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 α_vβ₃ and α_vβ₅, 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 α_vβ₃ 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.