The role of integrin alpha5beta1 in the regulation of corneal neovascularization.

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 alpha(5)beta(1) impeded pathological neovascularization in vivo. Our objective was to determine whether alpha(5)beta(1) plays a role in ocular angiogenesis, and whether a novel alpha(5)beta(1)-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 alpha(5) 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 alpha(5) was quantified by real-time RT-PCR. Vascularized corneas demonstrated a strong up-regulation of integrin alpha(5) within affected areas. Animals treated systemically with alpha(5)beta(1)-inhibiting small molecule showed a significant inhibition and regression of corneal neovascularization. PCR analysis evinced a significant up-regulation of VEGF and integrin alpha(5) mRNA levels in injured animals compared to controls, and a significant reduction of integrin alpha(5) mRNA in substance-treated animals compared to control substance, but no significant differences of bFGF levels in all groups. Western blot analysis of integrin alpha(5)beta(1) protein expression showed a trend towards up-regulation in injured animals, both control substance-treated and those treated with the alpha(5)beta(1)-inhibiting small molecule. Systemic delivery of an alpha(5)beta(1)-inhibiting small molecule inhibits and regresses corneal neovascularization induced by mechanical-alkali burn corneal injury. These results suggest an essential role for the integrin alpha(5)beta(1) in pathological neovascular processes of the cornea. Integrin alpha(5)beta(1) inhibitors could become a new approach for treatment of neovascularization in the eye.

Inhibition of TNF-alpha reduces laser-induced choroidal neovascularization.

To investigate the role of the TNF-alpha in the development of laser-induced choroidal neovascularization (CNV) in a mouse model. Four separate laser burns were applied to induce ruptures of Bruch's membrane and subsequent choroidal neovascularization in C57BL/6J mice. TNF-alpha protein expression was semiquantitatively assessed by Western blot analysis of the choroidal and RPE layer from mice with or without laser treatment. To investigate the effect of TNF-alpha inhibition on CNV formation, animals were treated for 7 days via intraperitonealy implanted osmotic pumps either 3 days before or after laser injury with recombinant TNF receptor P75 (etanercept), a chimeric monoclonal antibody (infliximab), or a purified rat anti-mouse/rat TNF monoclonal antibody (TNF-mAb), respectively. Fluorescein angiography, flat-mount preparations, and histopathology were performed at day 7, 10, or 14 after laser treatment. Western blotting demonstrated that TNF-alpha expression was 4.57-fold higher in the choroid and RPE one week after laser injury compared to control mice without laser. When evaluated one and two weeks after laser injury, etanercept and infliximab given from the 3rd day before laser-damage significantly reduced CNV size and pathological fluorescein leakage compared to the control group after laser treatment only. The inhibitory effect of the monoclonal TNF-alpha antibody on CNV formation was evident two weeks after photocoagulation but not after one week. Only etanercept administered 3 days after laser injury still reduced significantly the development of CNV lesions. Histopathology confirmed that CNV lesions in treated mice were smaller in size compared to the control animals without TNF inhibitor treatment. In conclusion, anti-TNF-alpha treatment with different inhibitors reduces both the size and the leakage of laser-induced CNV. These results suggest the involvement of TNF-alpha in the development of laser-induced CNV and its potential use as a therapeutic agent in the age-related macular degeneration.

The role of PDGF receptor inhibitors and PI3-kinase signaling in the pathogenesis of corneal neovascularization.

PURPOSE: Corneal neovascularization remains an unsolved therapeutic problem. Platelet-derived growth factor (PDGF) is directly linked to vessel formation and stabilization. This study was undertaken to elucidate the mechanisms by which PDGF exerts its effects on corneal angiogenesis. METHODS: Corneal neovascularization was induced in C57 mice by removal of the limbal epithelium. When mature vessels appeared after 7 days, mice were treated with the PDGF receptor-beta inhibitor AG 1296 or the phosphatidylinositol 3-kinase (PI3-K)-inhibitors wortmannin and LY294002, respectively, using an intraperitoneally implanted miniosmotic pump. At day 14 after scraping, corneas of treated and untreated (control) mice were dissected and immunostained with FITC-CD31 antibody for endothelial cells and with Cy3-SMA (smooth muscle actin) for pericytes. VEGF (vascular endothelial growth factor), ang1/2 (angiopoietin 1 and 2), and PDGF mRNA levels of treated and untreated corneas were determined by real-time RT-PCR. RESULTS: Mice treated with the PDGF inhibitor AG 1296 showed an inhibition of corneal neovascularization of 21.1% and a reduction of pericytes of 52% in the newly formed vessels compared with untreated animals. VEGF, ang1, ang2, and PDGF mRNA expression was reduced in the corneas of AG 1296-treated mice compared with the respective control. Treatment with the PI3-K inhibitors wortmannin and LY29002 had similar effects, inducing a decrease in corneal neovascularization and a reduction of VEGF, ang1, ang2, and PDGF mRNA levels. CONCLUSIONS: Inhibition of the PDGF signal pathway results in loss of pericytes and a reduction in vessel density in the neovascularized cornea that correlates with reduced expression of PDGF, ang1/2, and VEGF mRNA. Furthermore, PI3-K was shown to be involved in the regulation of VEGF, ang1, and PDGF, as the PI3-K inhibitors wortmannin or LY294002 had similar effects. Because PDGF is a known stimulus for PI3-K activation, it can be postulated that the observed decrease in VEGF, ang1/2, and PDGF mRNA levels on administration of the PDGF inhibitor is caused by the decreased activation of the PI3-K signaling cascade.