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    • Several culture techniques have been

    2018-11-08

    Several culture techniques have been developed for ex vivo expansion of limbal epithelial cells, including those that used intact or denuded AM as a supporting matrix; adopted an airlifting technique to promote epithelial differentiation; and prepared 3T3 fibroblasts as feeder layers (Sun et al., 2005). Previous studies have shown that limbal epithelial thyrotropin releasing hormone expanded on denuded AM grow faster than those on intact AM, and distinctly morphologic differences are also noted between these two culture conditions (Sun et al., 2005). Recent studies indicate that intact AM preserves the limbal epithelial phenotype, whereas denuded AM promotes corneal phenotype (Grueterich et al., 2002). Taken together, these facts reflect that the different status of microenvironments of the AM may modulate the outgrowth of limbal epithelial cells cultured on AM. The interaction between expanded limbal epithelial cells and underlying AM is complex yet interesting. Since the amniotic basement membrane contains several extracellular matrix (ECM) components such as type IV collagen and laminin-5 (Endo et al., 2004; Kurpakus-Wheater, 2001), the significance and involvement of matrix degradation enzymes in this in vitro culture model should deserve further investigation. To overcome ECM barriers (the basement membrane of AM), advancing limbal epithelial cells may produce proteases such as matrix metalloproteases (MMPs) (Endo et al., 2004; Li et al., 2006) or protease activators such as urokinase (Cheng et al., 2009) at their leading edge, where complex proteolysis can direct migration and preserve ECM attachment. MMPs, a family of Ca- and Zn-dependent endopeptidases, are able to degrade most of the ECM components and to regulate several physiological processes such as cell migration, proliferation and apoptosis through cleavage and release of ECM microenvironment molecules and modulation of gene expression (Nelson et al., 2000; Vu and Werb, 2000). Both MMP-2 and MMP-9 are important members of the MMP family which are involved in the cleavage of denatured collagens and native basement membrane components and the primary matrix-degrading enzymes produced by the corneal epithelium (Fini and Girard, 1990). Furthermore, MMPs have been shown to play a role in epithelial proliferation and wound healing (Fini et al., 1992). Several lines of evidence suggest that MMP-9 expression is induced in cells at the front of the migrating epithelial sheet as it begins to resurface the wounded area following injury (Legrand et al., 1999). Previously, we have demonstrated that both latent and active forms of MMP-9 are up-regulated during the outgrowth of human limbal explants cultured on intact AM (with devitalized amniotic epithelial cells preserved; H/Aintact group) and denuded AM. In contrast to MMP-9, MMP-2 is usually constitutively expressed throughout the culture period, but the activity is higher on denuded AM than that on intact AM (Sun et al., 2005; Li et al., 2006). We also used a specific MMP-2 antibody in the H/Aintact group and found that it was not as effective as MMP-9 antibody for inhibiting the outgrowth of limbal explants. In addition, the results from histologic staining showed that the devitalized amniotic epithelial cells under expanded limbal epithelial cells are barely discernible compared with those on naive intact AM (Sun et al., 2005), implying a key role for MMP-9 in limbal epithelial migration and ECM remodeling in the H/Aintact group. However, the molecular mechanisms by which MMP-9 is regulated in the context of cell-cell matrix interactions remain unclear. Post-translational modification of MMP has been reported to remove amino acids from the propeptide of MMP or to make structural changes. It has been reported that pro-MMP-9 can be modified and activated in vitro by many proteases such as plasmin, MMP-7, MMP-3, and MMP-13 (Cheng et al., 2009). However, plasmin was reported to be generated from plasminogen by the endogenous uPA and it was recognized as a direct activator of pro-MMP-9 (Saunders et al., 2005). In previous study, pro-MMP-9 has been shown to be post-translationally activated by uPA/plasmin, which is necessary for ECM remodeling and facilitating limbal epithelial cell migration in this culture model of human limbal explants on intact AM (Cheng et al., 2009). In addition, MMP activity is primarily controlled at the level of transcription (Fini et al., 1998). Transcription can be enhanced or inhibited through regulatory proteins that bind to response elements in the promoters of each MMP gene. The gene structure and promoter of MMP-9 have been characterized (Sato and Seiki, 1993) and binding sites for several transcriptional factors, including AP-1, SP-1, Ets and NF-κB are found in MMP-9 promoter region. Several studies have also indicated that NF-κB activation is required for maintaining cancer cell invasion and promoting epithelial wound healing (Liang et al., 2009; Egan et al., 2003). However, the role of NF-κB in the regulation of MMP-9 expression in the current model remains elusive.