Persimmon Leaves (Diospyros kaki) Extract Enhances the Viability of Human Corneal Endothelial Cells by Improving Na+-K+-ATPase Activity

The Na+/K+-ATPase, present in the basolateral membrane of human corneal endothelial cells (HCECs), is known to play an important role for corneal transparency. Na+/K+-ATPase dysfunction is one of the major causes of corneal decompensation. The ethanol extract of Diospyros kaki (EEDK) has been reported to increase corneal cell viability. Thus, we treated HCECs with EEDK and studied its effects on HCECs survival and Na+/K+-ATPase against cytotoxic drugs like staurosporine (ST) and ouabain (OU). Firstly, survival assays, (MTT assay and live dead-imaging) showed that decreased HCECs viability by ST and OU was significantly recovered by EEDK co-treatment. Secondly, Na+/K+-ATPase activity assays revealed that EEDK enhanced Na+/K+-ATPase enzymatic activity (* p < 0.01) with/without ST and OU. Finally, Na+/K+-ATPase expression analysis (Western Blot and confocal microscopy) demonstrated that EEDK treatment with/without ST and OU facilitates Na+/K+-ATPase expression in HCECs. Taken together, our findings led us to the conclusion that EEDK might aid HCECs survival in vitro by increasing the activity and expression of Na+/K+-ATPase enzyme. Since Na+/K+-ATPase activity is important to maintain cellular function of HCECs, we suggest that EEDK can be a potential effective agent against corneal edema and related corneal disorders.

Investigating the Effects of Coenzyme Q10 on Human Corneal Endothelial Cells

Purpose. To investigate the effects of Coenzyme Q10 (CoQ10) treatment on immortalised human corneal endothelial cells (HCEC-12). Methods. HCEC-12 cells were cultured in different concentrations of CoQ10 (0.1%, 0.2%, 0.5%, and 1.0%) and analysed using live/dead staining assay to determine appropriate concentration for subsequent experiments. Cells were pretreated with CoQ10 before inducing apoptosis by ethanol (EtOH) treatment for 30 seconds which was followed by posttreatment with CoQ10. Viable, apoptotic, and dead cell proportions were analysed using Annexin V-FITC immunofluorescence staining. Mitochondrial intensity and respiratory functions were also investigated using MitoTracker staining and a Seahorse XFe24 analyser, respectively. Results were compared to a positive control for apoptosis. The experiments were carried out in triplicates. Graphpad prism software was used for statistical analysis where p < 0.05 was deemed statistically significant. Results. CoQ10 treatment at 0.5% and 1% showed 92% and 30% viable cells compared with 0.1% and 0.2% that showed 96% and 94% viable cells, respectively ( p = 0.0562 ). 0.1% and 0.2% concentrations were, thus, used for subsequent experiments. Annexin V-FITC apoptotic analysis showed 2% at 0.1% and 3% at 0.2% of apoptotic cells ( p = 0.0824 ). Mitochondrial respiratory function and mitochondrial intensity increased in apoptotic cells following 0.1% CoQ10 treatment. Conclusion. 0.1% CoQ10 was found optimal for reducing apoptosis and increasing metabolic activity on human corneal endothelial cell line. These results support the need for further ex vivo studies to investigate the safety profile of CoQ10 as an antiapoptotic agent for human corneal endothelium.

Extracellular Vesicles Derived From Human Corneal Endothelial Cells Inhibit Proliferation of Human Corneal Endothelial Cells

Corneal endothelial cells (CEnCs) are a monolayer of hexagonal cells that are responsible for maintaining the function and transparency of the cornea. Damage or dysfunction of CEnCs could lead to blindness. Human CEnCs (HCEnCs) have shown limited proliferative capacity in vivo hence, their maintenance is crucial. Extracellular vesicles (EVs), are responsible for inter- and intra-cellular communication, proliferation, cell-differentiation, migration, and many other complex biological processes. Therefore, we investigated the effect of EVs (derived from human corneal endothelial cell line – HCEC-12) on corneal endothelial cells. HCEC-12 cells were starved with serum-depleted media for 72 hours. The media was ultracentrifuged at 100,000xg to isolate the EVs. EV counting, characterization, internalization and localization were performed using NanoSight, flow cytometry, Dil labelling and confocal microscopy respectively. HCEC-12 and HCEnCs were cultured with media supplemented with EVs. Extracted EVs showed a homogeneous mixture of exosomes and microvesicles. Cells with EVs decreased the proliferation rate; increased apoptosis and cell size; showed poor wound healing response in vitro and on ex vivo human, porcine, and rabbit CECs. Thirteen miRNAs were found in the EV sample using next generation sequencing. We observed that increased cellular uptake of EVs by CECs limit the proliferative capacity of HCEnCs. These preliminary data may help in understanding the pathology of corneal endothelial dysfunction and provide further insights in the development of future therapeutic treatment options.

Proliferation Increasing Genetic Engineering in Human Corneal Endothelial Cells: A Literature Review

The corneal endothelium is the inner layer of the cornea. Despite comprising only a monolayer of cells, dysfunction of this layer renders millions of people visually impaired worldwide. Currently, corneal endothelial transplantation is the only viable means of restoring vision for these patients. However, because the supply of corneal endothelial grafts does not meet the demand, many patients remain on waiting lists, or are not treated at all. Possible alternative treatment strategies include intracameral injection of human corneal endothelial cells (HCEnCs), biomedical engineering of endothelial grafts and increasing the HCEnC density on grafts that would otherwise have been unsuitable for transplantation. Unfortunately, the limited proliferative capacity of HCEnCs proves to be a major bottleneck to make these alternatives beneficial. To tackle this constraint, proliferation enhancing genetic engineering is being investigated. This review presents the diverse array of genes that have been targeted by different genetic engineering strategies to increase the proliferative capacity of HCEnCs and their relevance for clinical and research applications. Together these proliferation-related genes form the basis to obtain a stable and safe supply of HCEnCs that can tackle the corneal endothelial donor shortage.

Human corneal endothelial cells grafts to replace cadaveric donor corneas

Human corneal endothelial cells (HCEC) grafts to replace cadaveric donor corneas have made tremendous progress in recent years, and this article highlights the most recent discoveries and important achievements in this area. Cell injection therapy with cultured HCEC has just finished its first clinical research, which showed promising results. This follows on the heels of the success of autologous limbal stem cell transplantation in bioengineered ocular tissue transplantation. Transplantation techniques like bioengineered endothelium sheet transplantation and full-thickness corneal transplantation are likely to become more prevalent in the future decade. This goal will be achieved by a combination of current advancements in HCEC propagation, high-quality culture media, and 3D or 4D tissue culture, which will be the trend in the future.