Abstract
Purpose :
Previous studies have shown that storage of donor corneal endothelial cells (dCEnCs) at 4°C and subsequent rewarming prior to transplantation can damage mitochondria and decrease cell viability. The Insulin-like growth factor binding protein-3 (IGFBP-3) is a pleiotropic protein with known roles in cell growth and survival. This study investigated the effect of IGFBP-3 on mitochondrial fitness in dCEnCs subjected to hypothermic storage.
Methods :
Human donor corneas were obtained from Tissue Transplant Services at UT Southwestern Medical Center. dCEnCs were harvested by removal of Descemet’s followed by trypsinization. Cells were plated on collagen IV coated plates and cultured in medium containing 4% fetal bovine serum. Cells were serum starved for 24 hours prior to cold exposure (4°C) for 15 minutes, 2 or 6 hours with and without recombinant human (rh)IGFBP-3. Mitochondrial respiration and glycolysis were measured in real time using a Seahorse assay. Mitochondrial fragmentation and polarization were further assessed by double-labeling with MitoTracker and TMRE, respectively. Amplex red was used to quantify levels of reactive oxygen species (ROS). Western blotting was used to determine the expression of mitochondrial proteins.
Results :
Short term cold exposure induced cell migration that was visible at 2 hours. By 6 hours, dCEnCs formed distinct morphological clusters that were not evident in cells cultured at 37°C or rhIGFBP-3 treated cells. At the 6-hour time point, dCEnCs also demonstrated a reduction in cell-substrate adhesion that was recovered by co-treatment with rhIGFBP-3, while cell-cell adhesion appeared unchanged. Despite a measurable increase in mitochondrial oxygen consumption, cold exposure induced mitochondrial hyperpolarization and fragmentation. Co-treatment with rhIGFBP-3 significantly increased oxygen consumption. This was associated with an improvement in mitochondrial morphology. Expression levels of Superoxide dismutase (SOD) 1 and SOD2 were decreased following 6 hours of cold exposure but returned to near normal levels when treated with rhIGFBP-3. There were no detectable differences in ROS levels between groups.
Conclusions :
IGFBP-3 promotes mitochondrial fitness and cell-substrate adhesion in dCEnCs in cell culture in vitro. Further studies are needed to determine the effects of IGFBP-3 on dCEnCs during transplantation in vivo.
This is a 2021 ARVO Annual Meeting abstract.