Short-Term Ultraviolet A Irradiation Leads to Dysfunction of the Limbal Niche Cells and an Antilymphangiogenic and Anti-inflammatory Micromilieu

Abstract

PURPOSE. We analyzed the effects of short-term ultraviolet A (UVA) irradiation on the putative limbal stem cell phenotype, limbal fibroblasts, corneal inflammation, and corneal (lymph)angiogenic privilege.METHODS. Primary human limbal epithelial cells and fibroblasts were irradiated with 5.2 J/cm 2 of UVA. The limbal epithelial cell phenotype was assessed using P63a, cytokeratin 15, integrin b1 (marking stem and transient amplifying cells), and cytokeratin 3 (a differentiation marker) as well as by a colony-forming efficiency (CFE) assay. An epithelial-fibroblast coculture model was used to compare the ability of irradiated and nonirradiated fibroblasts to support the putative limbal stem cell phenotype. The effects of the conditioned media of irradiated and nonirradiated cells on proliferation and tube formation of human lymphatic and blood endothelial cells also were tested. The levels of factors related to angiogenesis and inflammation were assessed in a protein array and using ELISA.RESULTS. Ultraviolet A induced phenotypical changes of limbal epithelial cells, as their CFE and putative stem cell/transient amplifying marker expression decreased. Limbal epithelial cells cocultured with UVA-irradiated limbal fibroblasts also exhibited differentiation and CFE decrease. Conditioned media from irradiated limbal epithelial cells and fibroblasts inhibited lymphatic endothelial cell proliferation and tube network complexity. Levels of monocyte chemoattractant protein 1 (MCP1) were reduced following UVA irradiation of both cell populations, while levels of IFN-c increased in irradiated limbal epithelial cells.CONCLUSIONS. These data imply a key role of cellular components of the limbal niche following short-term UVA irradiation. Overall, UVA irradiation leads to dysfunction of these cells and a anti(lymph)angiogenic and anti-inflammatory micromilieu.

The Role of Limbal Epithelial Stem Cells in Regulating Corneal (Lymph)angiogenic Privilege and the Micromilieu of the Limbal Niche following UV Exposure

“…The resident epithelial stem cells in the limbus constantly replenish the cornea with epithelial cells which are lost due to normal wear and tear or injury [ 15 ]. Other studies demonstrated that corneal fibroblast signalling permits vessel invasion under pathological conditions [ 78 – 80 ] while our own data illustrated the proangiogenic paracrine activity of human limbal fibroblasts on blood and lymphatic endothelial cells by promoting migration, proliferation, and tube formation compared to human limbal epithelial cells and controls [ 81 ].…”

“…Our group has recently used an in vitro approach to study UVA- and UVB-induced changes in human limbal epithelial cell and fibroblast phenotype and functionality and their paracrine signalling regulating (lymph)angiogenesis and inflammation [ 81 , 122 ].…”

“…Notably, limbal epithelial cells which were cocultured with UV-irradiated limbal fibroblasts also lost their putative LESC characteristics. For the first time, we showed that UVA and UVB disrupted the function of limbal fibroblasts in maintaining the limbal epithelial cell phenotype in a coculture [ 81 , 122 ]. We have previously referred to the significance of the spatial proximity and cell-cell contact between the limbal fibroblasts and putative LESCs for the stem cell maintenance [ 14 ].…”

“…Specifically, conditioned media produced by both irradiated cell types hindered tube formation and proliferation of lymphatic endothelial cells. MCP1 was downregulated as a result of UVA irradiation of both cell types, while IFN- γ was upregulated in limbal epithelial cells [ 81 ].…”

“…In this light, long-term UVA insult may contribute to neovascularisation. Possible damage due to long-term UVA exposure is the subject of future studies and not reported in this review, (lymph)angiogenesis via downregulation of macrophage-recruiting cytokines [ 81 ]. These data put forward the UVA-induced damage to the limbal niche phenotype and function while showing an antilymphangiogenic and anti-inflammatory effect.…”