Inhibitors of corneal inflammation and angiogenesis : Prospectives and challenges

Pathologic angiogenesis is involved in cancer and several blinding conditions such as wet age-related macular degeneration, proliferative retinopathies and corneal neovascularization. In these dieseases, the angiogenic triggers are hypoxia and inflammation, and both involve the main angiogenic mediator, which is Vascular Endothelial Growth Factor (VEGF). Among available treatments, anti-VEGF often shows limited or temporary efficacy, while steroids are potentially responsible for many side-effects. This thesis presents a series of linked studies aimed at elucidating the early pathologic changes leading to inflammation and corneal neovascularization, and how various treatments affect this process. In this thesis, anti-inflammatory and anti-angiogenic treatments are applied in corneal neovascularization models, to identify VEGF-independent pathways and other novel factors as future therapy targets, as well as to investigate the endogenous modulation of angiogenesis. A model of experimental neovascularization in the rat cornea was used as main model, where the neovascular response is triggered by a surgical suture placed into the cornea. Investigational treatments (anti-Vegf, dexamethasone, IMD0354, Gap27, or control substances) were then given topically, with the exception of IMD0354, which was given systemically. The effects in the cornea were studied in vivo with slit lamp photography to assess and quantify macroscopic vessel growth and using in vivo confocal microscopy (IVCM) to study cell infiltration and limbal vessel dilation and detect microscopic vessel sprouts; these examinations were performed longitudinally. Genomic analysis with RNA microarray, selected gene expression with q-RT-PCR, and selected protein expression in tissue (immunohistochemistry, immunofluorescence, Western blot) were performed at different time-points. Moreover, other experiments on cell cultures (HUVEC and HCEC), organ cultures (human corneas), ex vivo models (aortic rings) and in vivo studies (zebrafish vasculogenesis) were performed. Dexamethasone suppressed limbal vasodilation and corneal neovascularization more than anti-Vegf, despite no difference in inflammatory cell infiltration into the cornea. Five-hundred eleven fewer genes were differentially expressed in dexamethasone-treated corneas relative to naïve corneas, compared to anti-Vegf. Among them, several major pro-angiogenic and pro-inflammatory factors and chemokines were suppressed only by dexamethasone and represent novel candidate factors to target in order to improve anti-VEGF treatment. On the other hand, selective inhibition of a single inflammatory pathway (NF-κB), despite showing similar early effects as dexamethasone in suppressing tissue inflammation, was not effective enough to suppress new vessel growth. The same factors suppressed by dexamethasone are also inhibited in endogenous modulation of angiogenesis. Surprisingly, dexamethasone activated several complement factors, which could possibly be beneficial in the anti-angiogenic response. In a different therapeutic approach, promoting cell migration to accelerate epithelial wound closure similarly was not sufficient to avoid inflammation and angiogenesis in the cornea. In conclusion, new and more effective treatments are needed for corneal inflammation and neovascularization with fewer side-effects. In this thesis, several novel factors and mechanisms related to inflammation are identified, factors that are not addressed by anti-Vegf therapy, and therefore represent interesting objects for further study, as they have the potential to be targets for adjuvant therapy. Specific anti-inflammatory treatment as well as therapeutic activation of endogenous regulatory pathways, and potentially complement modulation, might represent new strategies to improve anti-angiogenic therapy, but when used alone they do not seem to avoid corneal neovascularization