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Phos-tag Acrylamide HLE cells which were not UVB irradiated
HLE Phos-tag Acrylamide which were not UVB irradiated displayed the characteristic phenotype of small cobblestone-like, tightly packed colonies which co-expressed the putative LESC markers including P63a (Di Iorio et al., 2005) and Cytokeratin15 (Meyer-Blazejewska et al., 2010). The expression of β1 integrin by HLE indicates a basal cell phenotype as its presence in corneal epithelial cells has been found to be stronger in the basal layers in vivo and in the smaller tightly packed cells in vitro (Li et al., 2005). A partial loss of these markers is observed in BHLE cells, coinciding with enlarging cell morphology, an increase in K3-positive cell clusters and a significant drop in %CFE including the %CFE of colonies with a diameter larger than 2mm. These changes indicate loss of the putative stem cell phenotype following UVB irradiation. Remarkably, similar changes in marker localization, colony forming efficiency values and morphology are observed in the non-UV treated HLE which are co-cultured with the irradiated limbal fibroblasts (BHLF). The serum-free co-culture model used here, was previously shown to promote expansion of HLE displaying an enhanced putative stem cell like phenotype with an increased colony forming efficiency (Notara et al., 2010b) compared to the routine method of culture on growth arrested 3T3 fibroblast feeders (Rheinwald & Green, 1975; Detmar et al., 1993). For the first time, it is shown that UVB disrupts the ability of limbal fibroblasts to facilitate expansion of limbal epithelial cells in a co-culture whilst successfully maintaining their putative stem cell phenotype. Increasing evidence suggests that spatial proximity including physical contact between subsets of the limbal fibroblasts and putative stem cells within the niche is essential (Dziasko et al., 2014). The epithelial-fibroblast crosstalk in the limbal niche is fundamental as the latter contribute in mechanisms which prevent stem cell differentiation, such as the BMP/Wnt (Han et al., 2014), TGFβ/BMP (Joyce & Zieske, 1997; Nakatsu et al., 2013)and Notch (Tsai et al., 2014; Kulkarni et al., 2010) pathways. A disruption in the function of HLF, a key cellular component of the limbal microenvironment will be detrimental for the niche function. After investigating the direct impact of UVB on limbal epithelial cells and limbal fibroblasts, secondary effects on their pro(lymph)angiogenic activity were assessed. Therefore, conditioned media from the cells with and without UVB irradiation was used in functional assays of lymphatic and blood endothelial cells and for protein analysis. The aim was to investigate the synergistic effect of one or more soluble factors produced by the cells on (lymph)angiogenesis. Conditioned medium from HLF induced higher levels of LEC proliferation, wound closure and all parameters of tube network complexity in a tube formation assay. This effect was less pronounced for blood vascular endothelium. These data suggest a previously unreported stronger stimulatory effect of limbal fibroblasts in favor of lymphangiogenesis compared to hemangiogenesis. Earlier studies using a co-culture of an immortalized vascular endothelial cell line with limbal fibroblasts also indicated that the later had a stimulatory effect in tube formation; the opposite was observed in an older study using rabbit epithelial and fibroblast cells to investigate vascular endothelial cell proliferation (Eliason & Elliott, 1987). A proteomic approach in the form of an angiogenesis array as well as individual ELISA analyses were employed to elucidate the mechanisms involved in this response. The protein analyses data revealed a profile where both pro-angiogenic and anti-angiogenic signals were expressed in different levels by HLE and HLF. Specifically, the pro-angiogenic proteins endothelin 1 and amphiregulin, were strongly down-regulated. Endothelin 1 is both an angiogenesis (Wu et al., 2014; Zhang et al., 2014) and lymphangiogenesis (Spinella et al., 2009; Garrafa et al., 2012) promoter while amphiregulin may enhance lymphangiogenesis in skin (Marino et al., 2013) and hemangiogenesis in normal gut tissue (Shao et al., 2006)as well as in some cancer models (Fontanini et al., 1998; Ma et al., 1999). At the same time, MCP1, angiogenin, IGBP-3, VEGFA and VEGF C were significantly increased in HLF cells. MCP1 is both a pro-angiogenic chemokine (Hong et al., 2005) and a major inducer of monocyte recruitment (Palframan et al., 2001) while angiogenin is a pro-angiogenic growth factor found to stimulate both lymph-angiogenesis and hem-angiogenesis in cancer (Park et al., 2002) and in cornea (Shin et al., 2000; Kim et al., 1999). IGFBP3 is strongly expressed in superficial layers of the corneal epithelium (Robertson et al., 2007) and is upregulated in corneal fibroblasts during myofibroblast differentiation in corneal wound healing (Izumi et al., 2006). VEGFA and VEGFC are also up-regulated in HLF compared to HLE cells. VEGFC is the master regulator in cornea lymphangiogenesis; it is normally expressed in corneal epithelium but the ectopically expressed VEGFR3 acts as a ‘sink’ preventing VEGFC action and thus maintaining avascularity (Cursiefen et al., 2006; Cursiefen et al., 2005; Bock et al., 2008). VEGFA on the other hand is promoting cornea hem-angiogenesis via VEGFR-2 while also stimulating lymphangiogenesis (Cursiefen et al., 2004a). Given these data, with an emphasis to the VEGFA and VEGFC assessment, it is possible that this protein expression profile is tipping the balance towards a pro-lymphangiogenic and a weaker pro-hem-angiogenic effect of HLF compared to HLE. This also correlated with the outcome of the LEC and BEC functional assays. In vivo, since fibroblasts populate the cornea in low numbers compared to epithelial cells, the HLF pro-angiogenic effect observed in vitro is not evident. However, in a situation where the fibroblasts in the stroma become hyper-proliferative, like in pterygium (Su et al., 2011; Kim et al., 2013; Almeida Junior et al., 2008), this pro-angiogenic effect from the HLF may contribute to the neovascularization and recurrence of the condition.