Experimental studies of corneal neovascularisation

Abstract: The cornea contains no vessels, which is a prerequisite for the maintenance of its transparency. Vessel ingrowth leads to opacification resulting in visual impairment, sometimes complete blindness. This important clinical problem affects patients of all ages and may be due to chronic inflammation, hypoxia, chemical injury, stem cell deficiency, infection, dry eyes, a loss of sensation or transplant rejection. Corneal neovascularisation (NV) may be inhibited by anti-inflammatory treatment but not completely reversed or prevented. Although angiogenic research has translated into the development of new promising drugs, a deeper knowledge of angiogesis is needed to define optimal treatment options and save vision. The present studies aimed to broaden the understanding of corneal angiogenesis on a molecular, cellular and tissue level. All three studies were based on an experimental mouse model were a transcorneal silk suture causes inflammation and NV. Genetically modified mice lacking functional genes (knockout mice) for matrix metalloproteinase-2 (MMP-2) or interleukin-10 (IL-10) were used in the first two studies. In the third study the cornea model was an in vivo-part of an experiment cluster including the chick chorioallantoic membrane model (CAM), cell cultures, in-vitro gel-contraction and peptide-inhibitor experiments. The vessel visualization was performed with dye/ink perfusions and immunohystochemical stainings. NV was studied and quantified with a digital microscope with image analysis software, assessing spatiotemporal aspects. Studies of the inflammatory reaction were performed with immunohistochemistry for cellular markers while in-situ hybridization and real time-Polymerase Chain Reaction (rt-PCR) were used for identification of mRNA for relevant target molecules. MMP-2, a member of an enzyme family essential for degradation of extracellular matrix (ECM) and the vascular basement membrane, acted as an angiogenic regulator in this model. NV correlated with increased expression of MMP-2 mRNA and protein, both mainly found in activated keratocytes. In the second study, the anti-inflammatory and angio-regulatory cytokine IL-10 was found to act pro-angiogenically which could be explained neither by its anti-inflammatory effects nor by an apparent cross-talk with other potent angiogenic and immuno-modulatory factors. The third study revealed that tissue tension generated by activated myofibroblasts during wound contraction was able to mediate and direct the initial vascular invasion of the cornea and CAM-matrix gels through mainly vessel enlargement and elongation, independently of endothelial sprouting or proliferation. Neutralizing antibodies to vascular endothelial growth factor receptor -2 (VEGFR-2) could not prevent vessel ingrowth, while inhibition of gel contractibility did so. The results from this study may contribute to the already established theories on angiogenesis and this novel explanation model has been tentatively named looping angiogenesis . Taken together, these studies show that corneal neovascularisation is dependent on both chemical signals and mechanical factors. Further investigations of the interaction between these mechanisms will hopefully broaden the understanding of the complex and vital biological process that angiongesis represents.