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.

Potential Factors of Corneal Endothelial Cells for Progression in Children with Uveitis

Objective. To observe the morphological changes and abnormal structure of corneal endothelial cells in children with uveitis, to analyze the related factors affecting the morphological changes of corneal endothelial cells, and to explore the clinical application of a corneal endothelial microscope in children with uveitis. Methods. The corneal endothelial cells of 70 patients with uveitis were photographed with the Topcon SP-3000 noncontact corneal endothelial microscope, and the corneal endothelial cell density (CD), average cell area (AVE), coefficient of variation of the cell area (CV), and percentage of hexagonal cells (PHC) were measured with the IMAGEnet system. Twenty-eight patients (56 eyes) with monocular uveitis were selected, with the affected eyes (28 eyes) as the experimental group and the contralateral healthy eyes (28 eyes) as the control group. The corneal endothelial cell parameters between the two groups were statistically analyzed. The parameters of corneal endothelial cells in 70 children with uveitis were compared, and the effects of the course of the disease, inflammatory cells in the anterior chamber, and posterior corneal deposition (KP) on the parameters of corneal endothelial cells were analyzed. Results. There are four abnormal forms of the corneal endothelium in children with uveitis: enlarged cell area gap, irregular cell shape, blurred intercellular space, and cell loss. KP showed irregular high reflective white spots in the corneal endothelial microscope images, surrounded by dark areas, and existed in all the eyes with dusty KP found in slit lamp examination and a small number of eyes without obvious KP. Comparing the corneal endothelial cell parameters between the experimental group and the control group, it was found that the corneal endothelial CD and PHC of the former were lower than those of the latter, and the difference was statistically significant ( P < 0.001 and P = 0.018 , respectively). The AVE and CA of the former were higher than those of the latter ( P = 0.013 and P = 0.046 , respectively). The corneal endothelial cell density of the eyes with a course of the disease of more than 1 year was lower than that of the eyes with a course of the disease less than 1 year, the coefficient of variation of the corneal endothelial cell area of the eyes with KP was higher than that of the eyes without KP, and the difference was statistically significant ( P = 0.003 and P = 0.030 , respectively). Conclusion. Corneal endothelial microscopy is one of the important methods for the detection of uveitis with high sensitivity. The change of morphological parameters of corneal endothelial cells is one of the important indexes to assist in the diagnosis of uveitis and can be further promoted in ophthalmological examination.

Lower Fractions of TCF4 Transcripts Spanning over the CTG18.1 Trinucleotide Repeat in Human Corneal Endothelium

Fuchs’ endothelial corneal dystrophy (FECD) is a bilateral disease of the cornea caused by gradual loss of corneal endothelial cells. Late-onset FECD is strongly associated with the CTG18.1 trinucleotide repeat expansion in the Transcription Factor 4 gene (TCF4), which forms RNA nuclear foci in corneal endothelial cells. To date, 46 RefSeq transcripts of TCF4 are annotated by the National Center of Biotechnology information (NCBI), however the effect of the CTG18.1 expansion on expression of alternative TCF4 transcripts is not completely understood. To investigate this, we used droplet digital PCR for quantification of TCF4 transcripts spanning over the CTG18.1 and transcripts with transcription start sites immediately downstream of the CTG18.1. TCF4 expression was analysed in corneal endothelium and in whole blood of FECD patients with and without CTG18.1 expansion, in non-FECD controls without CTG18.1 expansion, and in five additional control tissues. Subtle changes in transcription levels in groups of TCF4 transcripts were detected. In corneal endothelium, we found a lower fraction of transcripts spanning over the CTG18.1 tract compared to all other tissues investigated.

Long-term Observation After Transplantation of Cultured Human Corneal Endothelial Cells for Corneal Endothelial Dysfunction

Background At present, corneal transplantation is still the only way to treat serious corneal diseases caused by corneal endothelial dysfunction. However, the shortage of donor cornea tissues and human corneal endothelial cells (HCECs) remains a worldwide challenge. We cultivated HCECs by the use of a conditioned medium from orbital adipose-derived stem cells (OASC-CM) in vitro. Then the HCECs were used to treat animal corneal endothelial dysfunction models via cell transplantation. The initial effect was gratifying. The purpose of this study was to conduct a long-term observation and evaluation after cell transplantation. Methods First, orbital adipose-derived stem cells (OASCs) were isolated to prepare conditioned medium (CM). Then HCECs were cultivated and expanded by the usage of CM (CM-HCECs). Related CEC markers were analyzed by immunofluorescence. Cells proliferation ability was also tested. CM-HCECs were then transplanted into monkey corneal endothelial dysfunction models by cell injection. We carried out a 24-month postoperative preclinical observation and verified the long-term effect by histological examination and transcriptome sequencing. Results CM-HCECs expressed HCEC related markers and maintained polygonal cell morphology after several passages. During 24 months of cell transplantation into the monkey's anterior chamber, the cornea thickness and transparency kept healthy status, and the corneal endothelial cell density remained in the normal range. Gene sequencing showed that the gene expression pattern of CM-HCECs was similar to that of transplanted cells and HCECs. Conclusions The proliferation and repair ability of HCECs were significantly improved due to the effect of OASC-CM. The result of this study confirmed long-term therapeutic efficacy of CM-HCECs in vivo. Our research provided an extensive cell source and a promising prospect for regenerative medicine and cell-based therapy.

The Response of Corneal Endothelial Cells to Shear Stress in an In Vitro Flow Model

Purpose. Corneal endothelial cells are usually exposed to shear stress caused by the aqueous humour, which is similar to the exposure of vascular endothelial cells to shear stress caused by blood flow. However, the effect of fluid shear stress on corneal endothelial cells is still poorly understood. The purpose of this study was to explore whether the shear stress that results from the aqueous humour influences corneal endothelial cells. Methods. An in vitro model was established to generate fluid flow on cells, and the effect of fluid flow on corneal endothelial cells after exposure to two levels of shear stress for different durations was investigated. The mRNA and protein expression of corneal endothelium-related markers in rabbit corneal endothelial cells was evaluated by real-time PCR and western blotting. Results. The expression of the corneal endothelium-related markers ZO-1, N-cadherin, and Na+-K+-ATPase in rabbit corneal endothelial cells (RCECs) was upregulated at both the mRNA and protein levels after exposure to shear stress. Conclusion. This study demonstrates that RCECs respond favourably to fluid shear stress, which may contribute to the maintenance of corneal endothelial cell function. Furthermore, this study also provides a theoretical foundation for further investigating the response of human corneal endothelial cells to the shear stress caused by the aqueous humour.

Receptor interacting protein 3 kinase, not 1 kinase, through MLKL-mediated necroptosis is involved in UVA-induced corneal endothelium cell death

Ultraviolet (UV) is one of the most energetic radiations in the solar spectrum that can result in various tissue injury disorders. Previous studies demonstrated that UVA, which represents 95% of incident photovoltaic radiation, induces corneal endothelial cells (CECs) death. Programmed cell death (PCD) has been implicated in numerous ophthalmologic diseases. Here, we investigated receptor-interacting protein 3 kinase (RIPK3), a key signaling molecule of PCD, in UVA-induced injury using a short-term corneal endothelium (CE) culture model. UVA irradiation activated RIPK3 and mediated necroptosis both in mouse CE and primary human CECs (pHCECs). UVA irradiation was associated with upregulation of key necroptotic molecules (DAI, TRIF, and MLKL) that lie downstream of RIPK3. Moreover, RIPK3 inhibition or silencing in primary corneal endothelial cells suppresses UVA-induced cell death, along with downregulation of MLKL in pHCECs. In addition, genetic inhibition or knockout of RIPK3 in mice (RIPK3K51A and RIPK3−/− mice) similarly attenuates cell death and the levels of necroptosis in ex vivo UVA irradiation experiments. In conclusion, these results identify RIPK3, not RIPK1, as a critical regulator of UVA-induced cell death in CE and indicate its potential as a future protective target.