The unfolded protein response in human corneal endothelial cells following hypothermic storage: Implications of a novel stress pathway

Abstract

Human corneal endothelial cells (HCEC) have become increasingly important for a range of eye disease treatment therapies. Accordingly, a more detailed understanding of the processing and preservation associated stresses experienced by corneal cells might contribute to improved therapeutic outcomes. To this end, the unfolded protein response (UPR) pathway was investigated as a potential mediator of corneal cell death in response to hypothermic storage. Once preservation-induced failure had begun in HCECs stored at 4°C, it was noted that necrosis accounted for the majority of cell death but with significant apoptotic involvement, peaking at several hours post-storage (4-8 hours). Western blot analysis demonstrated changes associated with apoptotic activation (caspase 9, caspase 3, and PARP cleavage). Further, the activation of the UPR pathway was observed through increased and sustained levels of ER folding and chaperone proteins (Bip, PDI, and ERO1-Lα) in samples experiencing significant cell death. Modulation of the UPR pathway using the specific inhibitor, salubrinal, resulted in a 2-fold increase in cell survival in samples experiencing profound cold-induced failure. Furthermore, this increased cell survival was associated with increased membrane integrity, cell attachment, and decreased necrotic cell death populations. Conversely, addition of the UPR inducer, tunicamycin, during cold exposure resulted in a significant decrease in HCEC survival during the recovery period. These data implicate for the first time that this novel cell stress pathway may be activated in HCEC as a result of the complex stresses associated with hypothermic exposure. The data suggest that the targeted control of the UPR pathway during both processing and preservation protocols may improve cell survival and function of HCEC thus improving the clinical utility of these cells as well as whole human corneas.

Characterization and modulation of human mesenchymal stem cell stress pathway response following hypothermic storage

“…Further, the chemical modulators resveratrol and salubrinal were investigated to assess their impact on hMSC tolerance to hypothermic storage. These inhibitors were selected based on previous reports that demonstrated their differential roles as both protective and detrimental to cell viability and function in other cell systems[12]. Further, other investigations have also revealed that a single molecular modulator could have both a positive or negative effect on outcome which is specific to the cell type, storage temperature and medium[14].…”

“…Numerous studies have now established that bioprocessing techniques are associated with the activation of molecular-based stress responses which contribute to cell loss during and following processing leading to failure[3,4,5,6,12,13,15,30,35]. These molecular responses can manifest as apoptosis or programmed cell death signaling.…”

“…However, if the problem proves to be too severe or prolonged, the UPR pathway then shifts signaling towards its secondary function which is to trigger apoptosis. Reports have demonstrated a profound positive effect on cell tolerance to hypothermic conditions when utilizing salubrinal in varous cell systems implicating the vital role of the UPR on storage outcome[12,14,29]. Given the UPR’s increasing level of importance in this and related areas of research[1,2,19,21,27,41], salubrinal was included in this study to determine if UPR signaling plays a role in biopreservation failure of hMSC.…”

The sensitivity of human mesenchymal stem cells to vibration and cold storage conditions representative of cold transportation

“…Although cell apoptosis induced by hypothermia/rewarming has been investigated extensively, especially for tissue preservation of major organs, such as liver or kidney tissues, there are still different opinions on whether cell death induced by hypothermia is through the mode of apoptosis [44] or necrosis [45]. Hence, the involvement of apoptosis in cold-induced cell death could not be excluded totally [43]; further characterization, perhaps using an apoptosis DNA fragment test, is needed to identify and confirm the mode of cell death. In addition, the final Vibration & cold storage effects on hMSC N. I.…”

“…However, annexin V expression is not exclusive to apoptotic cells as translocation of phosphatidylserine to the external cell surface also occurs during cell necrosis and oncosis [41,42]. Research into cold storage effects have shown that cold storage alone resulted in mainly necrosis [28,43]. Hence the observed increase of annexin Vþ and PIþ cell populations with prolonged cold storage duration could indicate more cells proceeding into cell death through oncotic necrosis (also referred to as swelling necrosis), a form of passive or accidental cell death.…”