Corneal endothelial dysfunction: Evolving understanding and treatment options.

Five-Year Follow-up of First 11 Patients Undergoing Injection of Cultured Corneal Endothelial Cells for Corneal Endothelial Failure

Purpose Aiming to clarify the role of mitochondria in cell fate decision of cultured human corneal endothelial cell (cHCEC) subpopulations. Methods The mitochondrial respiratory ability were examined with Mito stress and Mito fuel flex test assays using an extracellular flux analyzer (XFe24; Agilent Technologies; Santa Clara, CA) for human corneal endothelium tissues, mature cHCECs and a variety of cell state transitioned cHCECs. Tricarboxylic acid cycle and acetyl-coenzyme A-related enzymes was analyzed by proteomics for cell lysates using liquid chromatography-tandem mass spectrometry for cHCEC subpopulations. Results The maximum oxygen consumption rate was found to become stable depending on the maturation of cHCECs. In the Mito stress tests, culture supplements, epidermal growth factor, SB203580, and SB431543 significantly repressed oxygen consumption rate, whereas a Rho-associated protein kinase inhibitor Y-27632 increased. Tricarboxylic acid cycle and mitochondria acetyl-coenzyme A-related enzymes were selectively upregulated in mature cHCECs, but not in cell state transitioned cHCECs. The maximum oxygen consumption rate was found to be higher in healthy human corneal endothelium tissues than those with deeply reduced cell density. An upregulated tricarboxylic acid cycle was linked with metabolic rewiring converting cHCECs to acquire the mitochondria-dependent oxidative phenotype. Conclusions Mitochondrial metabolic intermediates and energy metabolism are tightly linked to the endothelial cell fate and function. These findings will help us to standardize a protocol for endothelial cell injection.

Mitochondria as a Platform for Dictating the Cell Fate of Cultured Human Corneal Endothelial Cells.

Purpose: To provide a consistent implementation of a retinal ganglion cell (RGC) displacement model proposed by Drasdo et al. for macular structure-function analysis, customizable by axial length (AL).

Methods: The effect of axial length on the shape of the inner retina was measured on 235 optical coherence tomography (OCT) scans from healthy eyes, to provide evidence for geometric scaling of structures with eye size. Following this assumption, we applied the Drasdo model to map perimetric stimuli on the radially displaced RGCs using two different methods: Method 1 only displaced the center of the stimuli; Method 2 applied the displacement to every point on the edge of the stimuli. We compared the accuracy of the two methods by calculating, for each stimulus, the number of expected RGC receptive fields and the number RGCs calculated from the histology map, expected to be equivalent. The same calculation was repeated on RGC density maps derived from 28 OCT scans from 28 young healthy subjects (age < 40 years) to confirm our results on clinically available measurements.

Results: The size of the retinal structures significantly increased with AL (P < 0.001) and was well predicted by geometric scaling. Method 1 systematically underestimated the RGC counts by as much as 60%. No bias was observed with Method 2.

Conclusions: The Drasdo model can effectively account for AL assuming geometric scaling. Method 2 should be used for structure-function analyses.

Translational Relevance: We developed a free web App in Shiny R to make our results available for researchers.

/pdf/revisiting-the-drasdo-model-implications-for-structure-20r2gsqsih.pdf

Revisiting the Drasdo Model: Implications for Structure-Function Analysis of the Macular Region.

http://www.ajo.com/article/S0002939421005948/pdf

Superiority of Mature Differentiated Cultured Human Corneal Endothelial Cell Injection Therapy for Corneal Endothelial Failure

Purpose Aiming to clarify the metabolic interrogation in cell fate decision of cultured human corneal endothelial cells (cHCECs). Methods To analyze the metabolites in the culture supernatants (CS), 34 metabolome measurements were carried out for mature differentiated and a variety of cHCECs with cell state transition through a facility service. Integrated proteomics research for cell lysates by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed for 3 aliquots of each high-quality or low-quality cHCEC subpopulations (SP). The investigations for the focused genes involved in cHCEC metabolism were performed by using DAVID and its options "KEGG_PATHWAY." Results The clusters of metabolites coincided well with the distinct content of CD44-/+ SPs. Both secreted pyruvic acid and lactic acid in the CS were negatively correlated with the content of high-quality SPs. Lactic acid and pyruvic acid in the CS exhibited the positive correlation with that of Ile, Leu, and Ser, whereas the negative correlation was with glutamine. Platelet-derived growth factor-ββ in the CS negatively correlated with lactic acid in CS, indicating indirectly the positive correlation with the content of CD44-/+ SPs. Upregulated glycolytic enzymes and influx of glutamine to the tricarboxylic acid cycle may be linked with a metabolic rewiring converting oxidative metabolism in mature differentiated CD44-/+SPs into a glycolytic flux-dependent state in immature SPs with cell state transition. Conclusions The findings suggest that the cell fate decision of cHCECs may be dictated at least partly through metabolic rewiring.

Metabolites Interrogation in Cell Fate Decision of Cultured Human Corneal Endothelial Cells.

Purpose To clarify the expression profiles of ion channels and transporters of metabolic substrates among heterogeneous cultured human corneal endothelial cells (cHCECs) distinct in their effectiveness in reconstituting the corneal endothelium. Methods Integrated proteomics for cell lysates by liquid chromatography-tandem mass spectrometry was carried out from three aliquots of cHCECs enriched in either cluster of definition (CD)44-/+ (mature) cHCECs or CD44++/+++ cell-state transition (CST) cHCECs. The expression profiles of cations/anions, monocarboxylic acid transporters (MCTs), and solute carrier (SLC) family proteins, as well as carbonic anhydrases (CAs), were investigated. Results The polarized expression of cations/anions, MCTs, and SLC family proteins, as well as CAs, was clarified for mature and CST cHCECs. Most SLC4 family members, including SLC4A11 and SLC4A4 (NBCe1), were upregulated in the CST cHCECs, whereas SLC9A1 (Na+/H+ exchanger isoform one [NHE1]) and CA5B were detected only in the mature cHCECs. In addition, SLC25A42, catalyzing the entry of coenzyme A into the mitochondria, and SLC25A18, functioning as a mitochondrial glutamate carrier 2 (both relevant for providing the substrates for mitochondrial bioenergetics), were selectively expressed in the mature cHCECs. Conclusions Our findings may suggest the relevance of qualifying the polarized expression of these ion channels and transporter-like proteins to ensure not only the suitability but also the in vivo biological functionality of cHCECs selected for use in a cell-injection therapy.

Polarized Expression of Ion Channels and Solute Carrier Family Transporters on Heterogeneous Cultured Human Corneal Endothelial Cells.

Mitochondrial miRNA494-3p in extracellular vesicles participates in cellular interplay of iPS-Derived human retinal pigment epithelium with macrophages.

Tomoko Fujita

Papers

Metabolites Interrogation in Cell Fate Decision of Cultured Human Corneal Endothelial Cells.

Mitochondria as a Platform for Dictating the Cell Fate of Cultured Human Corneal Endothelial Cells.

Polarized Expression of Ion Channels and Solute Carrier Family Transporters on Heterogeneous Cultured Human Corneal Endothelial Cells.

Mitochondrial miRNA494-3p in extracellular vesicles participates in cellular interplay of iPS-Derived human retinal pigment epithelium with macrophages.