Tissue and cellular characterisation of nucleolin in a murine model of corneal angiogenesis.

PURPOSE: Corneal neovascularisation (CNV), with consequent loss of transparency, is due to an imbalance of proangiogenic factors. Cell-surface nucleolin (NCL) has been associated with neo-angiogenesis. There are studies identifying NCL translocation from nucleus to the cell surface, which is essential for endothelial cell proliferation. To find the possible role of NCL in the generation of corneal neovessels, the aim of this study is to characterise the NCL presence and cell-localisation in non-injured corneas, as well as to describe the changes in NCL cell and tissue localisation in CNV, and to analyse the effect of bevacizumab on NCL cellular and tissular distribution. METHODS: Suture-induced CNV was performed in mice. The corneal tissues were obtained and the histological and co-immunofluorescence assays were performed using different proteins, such as CD31, cadherin and isolectin B4. To determine the possible role of VEGF in NCL presence and localisation in our CNV model, bevacizumab was concomitantly used. RESULTS: Nucleolin was principally observed in the nucleus of the basal epithelial cells of normal corneas. Interestingly, angiogenesis-induced changes were observed in the localisation of NCL, not only in tissue but also at the cellular level where NCL was extranuclear in epithelial cells, stromal cells and neovessels. In contrast, these changes were reverted when bevacizumab was used. Besides, NCL was able to stain only aberrant corneal neovessels in comparison with retinal vessels. CONCLUSIONS: NCL mobilisation outside the nucleus during angiogenesis could have a possible role as a proangiogenic molecule in the corneal tissue.

Anti-Inflammatory and Anti-Fibrotic Effects of Human Amniotic Membrane Mesenchymal Stem Cells and Their Potential in Corneal Repair.

Acute ocular chemical burns are ophthalmic emergencies requiring immediate diagnosis and treatment as they may lead to permanent impairment of vision. The clinical manifestations of such burns are produced by exacerbated innate immune response via the infiltration of inflammatory cells and activation of stromal fibroblasts. New therapies are emerging that are dedicated to repair mechanisms that improve the ocular surface after damage; for example, transplantation of stem cells (SC) has been successfully reported for this purpose. The pursuit of easily accessible, noninvasive procedures to obtain SC has led researchers to focus on human tissues such as amniotic membrane. Human amniotic mesenchymal SC (hAM-MSC) inhibits proinflammatory and fibrotic processes in different diseases. hAM-MSC expresses low levels of classical MHC-I and they do not express MHC-II, making them suitable for regenerative medicine. The aim of this study was to evaluate the effect of intracameral injection of hAM-MSC on the clinical manifestations, the infiltration of inflammatory cells, and the activation of stromal fibroblasts in a corneal alkali-burn model. We also determined the in vitro effect of hAM-MSC conditioned medium (CM) on α-SMA+ human limbal myofibroblast (HLM) frequency and on release of neutrophil extracellular traps (NETs). Our results show that intracameral hAM-MSC injection reduces neovascularization, opacity, stromal inflammatory cell infiltrate, and stromal α-SMA+ cells in our model. Moreover, in in vitro assays, CM from hAM-MSC decreased the quantity of α-SMA+ HLM and the release of NETs. These results suggest that intracameral hAM-MSC injection induces an anti-inflammatory and anti-fibrotic environment that promotes corneal wound healing. Stem Cells Translational Medicine 2018;7:906-917.