In this study, we analyzed different apoptosis-related proteins in corneal endothelial and epithelial cells, as well as in keratocytes both in vitro and ex vivo in corneal tissue. In addition to the detection of different apoptosis-related proteins as potential readouts, the other important aspect of this study was to quantify the presence of these proteins in cells and whole tissue as direct indicators of the extent of apoptosis in different corneal cell types. We focused our analysis on caspase-dependent mitochondrial cell death pathway proteins. We studied the most relevant apoptosis pathways from initiator caspases down to lamin cleavage, marking the very end of apoptosis (
Fig. 1). The caspases are central to the apoptosis pathways as the caspase cascade is responsible for the biochemical and morphologic changes associated with both intrinsic and extrinsic apoptosis pathways.
3,22 An important step prior to caspase activation is formation of the apoptosome. After apoptosis induction, cytochrome c is released from the mitochondria, which binds to Apaf-1 and forms the apoptosome, which in turn leads to activation of Caspase-9.
2 In our study, we analyzed the proteins constituting the apoptosome, Apaf-1, and Caspase-9. Apaf-1 was observed in all the three cell types; however, the levels of Apaf-1 were found to be decreased in the staurosporine-treated samples. Interestingly, these reduced levels of Apaf-1 also corresponded with the presence of cleaved Apaf-1 product in the treated cell lysates. This cleaved Apaf-1 protein has been reported in some of the previous studies.
16 The activated form of Caspase-9 was detected in the cell lysates. Following the activation of initiator caspases, the effector caspases are activated, and this results in the degradation of further downstream targets. Three effector caspases (3, 6, and 7) were analyzed in our study; these executioner caspases have been shown to have specific and nonredundant functions in the apoptosis process using Jurkat cell lysates.
23,24 As a result of this analysis, some of the findings from former studies could be confirmed and expanded in our work. The study on human corneal endothelial cells showing the presence of activated Caspase-3 and cleaved PARP in staurosporine-treated cells is in line with our results; the authors focused on the clarification whether endothelial cell death is indeed an apoptotic event.
9 Similarly Caspase-3 activation after apoptosis induction with staurosporine has been investigated in the corneal epithelial cell line by fluorometric assay. In other study concerning failure of corneal allografts in a mouse model active Caspase-3 was analyzed in cultured keratocytes and in mouse corneal tissue by Western blotting.
6,8 Our results showed the increased presence of activated Caspase-3 in the staurosporine-treated cell lysates of all the three cell types. The presence of activated Caspase-3 was highest in HCEp cells, and quantification of cleaved Caspase-3 bands showed that the results were significant across cell types (
Fig. 3A, right). The role of Caspases-3 and -9 has been widely studied with respect to apoptosis execution in corneal cells and tissue.
6,8,9 However, the other effector caspases -6 and -7 have not been investigated in corneal cells, so we analyzed these caspases, which play an important role in the execution phase of apoptosis.
24 Caspases-6 and -7 have been shown to have specific downstream targets. Lamin A has been reported to be a highly specific substrate for Caspase-6. In experiments with Hela and Jurkat nuclei, it was conclusively shown that when lamin A is present, it must be cleaved by Caspase-6 for the efficient culmination of the nuclear apoptosis process.
25 Our results clearly showed the presence of cleaved Caspase-6 in all studied cell types, and it was significantly higher for HCEp cells compared with the other two cell types (
Fig. 3B, left and right). The downstream nuclear targets of activated Caspase-6, which are lamins, were evidently detected in the HCEC-12, HCEp, and HCK cell lysates. The presence of cleaved lamin A/C products was significantly higher for HCEp cells compared with HCEC-12 and HCK cells, indicating that there is a direct relation between the presence of activated caspase-6 and its downstream nuclear substrate lamin, pointing toward the specificity of lamin for caspase-6. The cleavage product of another nuclear target, PARP, which is a substrate for Caspase-3 and -7,
3,9 was clearly detected in the staurosporine-treated cell lysates; however, on quantification, these results were not statistically significant. Comparing the obtained results between different cell types, it was observed that the presence of apoptosis marker proteins such as cleaved Caspase-3, cleaved Caspase-6, and cleaved lamin A/C was significantly higher in epithelial cell lysates compared with the other cells types, pointing toward the fact that epithelial cells reacted in a strong manner to staurosporine treatment. This might be attributed to their higher proliferation rate and metabolic activity.
8 To close the gap between basic research and tissue storage, all data were obtained not only in cell lines but also in full-thickness research corneas, representing the standard in corneal graft storage. Lamin A/C cleavage turned out to be the only end point apoptosis marker that could be quantified by Western blotting and also be analyzed by fluorescence microscopy in the corneal tissue. This is in line with studies in cell lines of different origins and is presented here in a comprehensive manner for corneal cells and tissue for the first time.
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