Spatiotemporal Coordination of RPE Cell Quality by Extracellular Vesicle miR-494-3p Via Competitive Interplays With SIRT3 or PTEN.

Purpose: To reveal the molecular mechanism underlying degeneration in human retinal pigment epithelial (hRPE) cells with dysfunctional mitochondrial homeostasis. Methods: The expression of recently identified miR-494-3p in extracellular vesicles (EV) released from induced-pluripotential-stem-cell-derived human RPE (iPS-hRPE), during coculture with macrophages (Mps) was investigated in iPS-hRPE and ARPE cells differentiated in the presence of nicotinamide (Nic-ARPE). The expression of phosphatase and tensin homolog (PTEN), sirtuin3 (SIRT3), and mitochondrial marker proteins before and after the transfection of miR-494-3p inhibitor and mimic, and the changes in mitochondrial metabolism, membrane potential, and oxidative phosphorylation (OXPHOS) were monitored. Results: Compared with senescent dedifferentiated ARPE19 cells, iPS-hRPE and Nic-ARPE cells expressed elevated levels of mitochondrial marker proteins but a repressed cellular miR-494-3p level. The expression of target proteins of miR-494-3p, PTEN, and SIRT3 was upregulated along with the differentiation disposition of these RPE cells. The ratio of PTEN/SIRT3 in de-differentiated ARPE19 cells was surprisingly elevated by around 20 times compared with that in iPS-hRPE and Nic-ARPE cells. The novel molecular interplay of EV miR-494-3p either with mitochondria selective SIRT3 or organelle nonselective PTEN was found to participate in the degeneration of hRPE cells by inducing mitochondrial dysfunctions and repressed OXPHOS, mitochondrial membrane potential, and ATP and NAD+ production. Conclusions: Our results demonstrate a clear causal link between miR-494-3p and hRPE cell degeneration via the regulation of mitochondrial integrity. EV miR-494-3p may play a pivotal role in pathogenic spreading of degenerated hRPE cells from the local perifovea throughout the macula.

The Interplay Between Metabolites and MicroRNAs in Aqueous Humor to Coordinate Corneal Endothelium Integrity.

Purpose: The purpose of the study was to clarify the interplay between metabolites and microRNAs (miRs) in the aqueous humor (AqH) of bullous keratopathy (BK) patients to retain human corneal endothelium (HCE) integrity. Design: Prospective, comparative, observational study. Participants: A total of 55 patients with BK and 31 patients with cataract (Cat) as control. Methods: A biostatic analysis of miRs and metabolites in the AqH, hierarchical clustering, and a least absolute shrinkage and selection operator (Lasso) analysis were employed. The miR levels in AqH of BK (n = 18) and Cat (n = 8) patients were determined using 3D-Gene human miR chips. Hierarchical clusters of metabolites detected by liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry in AqH specimens from 2 disease groups, BK (total n = 55) and Cat (total n = 31), were analyzed twice to confirm the reproducibility. The analytical procedure applied for investigating the association between metabolites and miRs in AqH was the exploratory data analysis of biostatistics to avoid any kind of prejudice. This research procedure includes a heat-map, cluster analysis, feature extraction techniques by principal component analysis, and a regression analysis method by Lasso. The cellular and released miR levels were validated using reverse transcription polymerase chain reaction and mitochondria membrane potential was assessed to determine the functional features of the released miRs. Main Outcome Measures: Identification of interacting metabolites and miRs in AqH attenuating HCE degeneration. Results: The metabolites that decreased in the AqH of BK patients revealed that 3-hydroxyisobutyric acid (HIB), 2-aminobutyric acid (AB) and branched-chain amino acids, and serine were categorized into the same cluster by hierarchical clustering of metabolites. The positive association of HIB with miR-34a-5p was confirmed (P = 0.018), and the Lasso analysis identified the interplay between miR-34a-5p and HIB, between miR-24-3p and AB, and between miR-34c-5p and serine (P = 0.041, 0.027, and 0.009, respectively). 3-hydroxyisobutyric acid upregulated the cellular miR-34a expression, mitochondrial membrane potential, and release of miR-184 in dedifferentiated cultured HCE cells. Conclusions: Metabolites and miRs in AqH may synchronize in ensuring the integrity of the HCE to maintain efficient dehydration from the stroma. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Novel Vertical Cup-to-Disc Classification to Identify Normal Eyes That Maintain Non-Glaucoma Status: A 10-Year Longitudinal Study.

PRCIS: We propose a new classification model to serve as a control for future genomic studies of glaucoma by distinguishing normal subjects maintaining non-glaucoma status for 10 years using the vertical cup-to-disc ratio (VCDR). PURPOSE: This study aimed to develop a classification for distinguishing subjects maintaining non-glaucoma status for 10 years using the VCDR. PARTICIPANTS AND METHODS: Among 842 volunteers 40 years and older, 421 volunteers participated in the second ophthalmic examination 10 years after their first examination. Each volunteer was diagnosed either as healthy normal or glaucoma suspect (GS) in the first glaucoma screening examinations. The former was further classified into the 3 grades of N1, N2, and N3. Specifically, N1 represented (1) VCDR <0.3; (2) no notching or nerve fiber layer defect; and (3) no undermining, N2 indicated 0.3≤VCDR<0.6 and conditions (2) and (3) of N1; and N3 represented 0.3≤VCDR<0.6 with undermining and condition (2), or 0.6≤VCDR<0.7 and condition (2) of N1. Glaucoma transition rates (GTRs) were evaluated in 421 volunteers who returned to participate after a 10-year period. RESULTS: GTRs were calculated as 1.3% in both N1 and N2, 3.9% in N3, and 18.2% in GS. The ratio of volunteers in the same category maintenance rate increased from N1 to N3. CONCLUSION: GTRs were lower in N1 and N2 than in N3 or GS during the 10-year study period. This novel classification of healthy non-glaucoma subjects may help identify those, especially Japanese males, who maintain a non-glaucoma status for an extended period of 10 years.

Cellular Interplay Through Extracellular Vesicle miR-184 Alleviates Corneal Endothelium Degeneration.

Objective: The objective of the study was to reveal the presence of cellular interplay through extracellular vesicle (EV) microRNAs (miRs), to dampen the vicious cycle to degenerate human corneal endothelium (HCE) tissues. Design: Prospective, comparative, observational study. Methods: The miR levels in neonate-derived corneal tissues, in the aqueous humor (AqH) of bullous keratoplasty and cataract patients, as well as in the culture supernatant (CS) and EV of cultured human corneal endothelial cells (hCECs), were determined using 3D-Gene human miR chips and then validated using the real-time polymerase chain reaction. The extracellularly released miRs were profiled after the forced downregulation of cellular miR-34a, either by an miR-34a inhibitor or exposure to H2O2. The senescence-associated secretory phenotypes and mitochondrial membrane potential (MMP) were assessed to determine the functional features of the released miRs. Main Outcome Measures: Identification of functional miRs attenuating HCE degeneration. Results: The miRs in AqH were classified into 2 groups: expression in 1 group was significantly reduced in neonate-derived tissues, whereas that in the other group remained almost constant, independent of aging. The miR-34a and -29 families were typical in the former group, whereas miR-184 and -24-3p were typical in the latter. Additionally, a larger amount of the latter miRs was detected in AqH compared with those of the former miRs. There was also a greater abundance of miR-184 and -24-3p in hCECs, EV, and CS in fully mature CD44-/dull hCEC, leading to sufficient clinical tissue regenerative capacity in cell injection therapy. The repression of cellular miR-34a, either due to miR-34a inhibitors or exposure to oxidative stress, unexpectedly resulted in the elevated release of miR-184 and -24-3p. Secretions of VEGF, interleukin 6, monocyte chemotactic protein-1, and MMP were all repressed in both mature CD44-/dull and degenerated CD44+++ hCEC, transfected with an miR-184 mimic. Conclusions: The elevated release of miR-184 into AqH may constitute cellular interplay that prevents the aggravation of HCE degeneration induced by oxidative stress, thereby sustaining tissue homeostasis in HCE.

Quiescent innate and adaptive immune responses maintain the long-term integrity of corneal endothelium reconstituted through allogeneic cell injection therapy.

This study aims to clarify the immunogenicity in acquired and innate immune responses of cultured human corneal endothelial cells (hCECs) applied for cell injection therapy, a newly established modality for corneal endothelium failures. Thirty-four patients with corneal endothelial failure received injection of allogeneic hCEC suspension into anterior chamber. No sign of immunological rejection was observed in all 34 patients during the 5-8 years postoperative follow-up period. Cell injection therapy was successful in 2 patients treated for endothelial failure after penetrating keratoplasty and one patient with Descemet membrane stripping automated endothelial keratoplasty failure. ELISPOT assays performed in allo-mixed lymphocyte reaction to the alloantigen identical to that on the injected hCECs, elicited sparse IFN-γ-specific spots in the peripheral blood mononuclear cells of patients who received hCEC injection. The therapy generated simple and smooth graft-host junctions without wound stress. The injection of C57BL/6 CECs into the anterior chamber of BALB/c mice, which rejected C57BL/6 corneas 6 weeks ago, induced no sign of inflammatory reactions after the second challenge of alloantigen. Collectively, injection of the hCEC cell suspension in the aqueous humor induces immune tolerance that contributes to the survival of the reconstituted endothelium.

Comprehensive Analysis Identified the Circadian Clock and Global Circadian Gene Expression in Human Corneal Endothelial Cells.

Purpose: To investigate circadian clock oscillation and circadian global gene expression in cultured human corneal endothelial cells (cHCECs) to elucidate and assess the potential function of circadian regulation in HCECs. Methods: In this study, we introduced a circadian bioluminescence reporter, Bmal1:luciferase (Bmal1:luc), into cHCECs and subsequently monitored real-time bioluminescence rhythms. RNA-sequencing data analysis was then performed using sequential time-course samples of the cHCECs to obtain a comprehensive understanding of the circadian gene expression rhythms. The potential relevance of rhythmically expressed genes was then assessed by systematic approaches using functional clustering and individual gene annotations. Results: Bmal1:luc bioluminescence exhibited clear circadian oscillation in the cHCECs. The core clock genes and clock-related genes showed high-amplitude robust circadian messenger RNA (mRNA) expression rhythms in cHCECs after treatment with dexamethasone, and 329 genes that exhibited circadian mRNA expression rhythms were identified (i.e., genes involved in various physiological processes including glycolysis, mitochondrial function, antioxidative systems, hypoxic responses, apoptosis, and extracellular matrix regulation, which represent the physiological functions of HCECs). Conclusions: Our findings revealed that cHCECs have a robust and functional circadian clock, and our discovery that a large number of genes exhibit circadian mRNA expression rhythms in cHCECs suggests a potential contribution of circadian regulation to fine-tune HCEC functions for daily changes in the environment.

Potential participation of CTRP6, a complement regulator, in the pathology of age related macular degeneration.

PURPOSE: To investigate the localized expression of C1q/tumor necrosis factor related protein (CTRP) 6 in human age-related macular degeneration (AMD) retinal tissues. EXPERIMENTAL STUDY DESIGN: 4 AMD and 3 non-AMD whole eyes of Caucasian donors were used. Eyecups were excised at Eye Bank CorneaGen, Inc. METHODS: To elucidate the effects of CTRP6, C3b was measured by an enzyme-linked immunosorbent-like assay. CFB versus CTRP6 competitive binding assay was applied to clarify the inhibition by CTRP6 of C3bBb complex formation. The cornea, iris, lens, and vitreous were removed and the eyes were cut into a posterior eye-cup including the retina, choroid, and sclera. Six-µm-thick serial sections of frozen samples underwent hematoxylin-eosin (HE) staining and indirect immunohistochemical staining using primary antibodies, anti-CTRP6, -CTRP5, -CTRP10, -Complement factor H (CFH) and -Clusterin (CLU). Results The two in vitro studies confirmed that CTRP6 has an inhibitory effect on alternative pathways of complement (APC) function and that the molecular target of CTRP6 is the inhibition of the formation of C3bBb. Localized expression for CTRP6 and CFH was found in the drusen of the AMD eyes, both associated with APC inhibition, CLU associated with membrane-attack complex (MAC) inhibition, and CTRP5 associated with retinal degeneration. CONCLUSION: The localized expression of CTRP6 in the drusen of AMD eyes may open a new insight into the possible involvement of APC regulatory factors in the pathogenesis of AMD, together with the known CFH so far analyzed solely as an APC inhibitor.

Intracellular pH affects mitochondrial homeostasis in cultured human corneal endothelial cells prepared for cell injection therapy.

This study aimed to uncover the mechanism responsible for the clinical efficacy of cell injection therapy with fully differentiated cultured cells. Analysis of polarized expression of ion transporters on cultured human corneal endothelial cells (CECs) subpopulations (SPs) was performed. The intracellular pH (pHi) between two CEC SPs, distinct in the proportion of differentiated cells, was measured, and the association with mitochondrial respiration homeostasis was investigated. The effects of the ion transporter inhibition by their selective inhibitors or siRNA transfection were also explored. Na+/K+-ATPase, Aquaporin 1, SLC4A11, NBCe1, NHE1 as transporters, and ZO-1, were all selectively expressed in differentiated SPs, but were almost null in the cell-state-transitioned SPs. We also confirmed that the pHi of CEC SPs affected their mitochondrial respiration by modulating the expression of these ion transporters via inhibitors or siRNA transfection. Ion and water transporters might participate in the maintenance of pHi and mitochondria homeostasis in differentiated SPs, which may contribute, combined with integral barrier functions, to efficient water efflux. The differences in intracellular pH between the two SPs is attributed to variations in the expression profile of specific ion transporters and mitochondrial functions, which may associate with the efficacy of the SPs in cell injection therapy.

Repressed miR-34a Expression Dictates the Cell Fate to Corneal Endothelium Failure.

Purpose: To reveal the mechanism triggering the functional disparity between degenerated and non-degenerated corneal endothelium cells in the water efflux from corneal stroma to the anterior chamber. Methods: The varied levels of the microRNA (miR)-34, miR-378, and miR-146 family in human corneal endothelium and cultured cells thereof were investigated using 3D-Gene Human miRNA Oligo Chips. Concomitantly, CD44, p53, c-Myc, matrix metalloprotease (MMP)-2 expression, and Ras homolog gene family member A (Rho A) activity was correlated to the expression intensities of these microRNAs, partly complemented with their altered expression levels with the transfection of the corresponding mimics and inhibitors. The levels of miRs were further associated with intracellular pH (pHi) and mitochondrial energy homeostasis. Results: P53-inducible miR-34a/b repressed CD44 expression, and CD44 was repressed with the elevated c-Myc. The repressed miR-34a activated the CD44 downstream factors Rho A and MMP-2. MiR-34a mimics downregulated pHi, inducing the skewing of mitochondrial respiration to oxidative phosphorylation. The oxidative stress (H2O2) induced on human corneal endothelial cells, which repressed miR-34a/b expression, may account for the impaired signaling cascade to mitochondrial metabolic homeostasis necessary for an efficient water efflux from the corneal stroma. Conclusions: The upregulated expression of CD44, through repressed miR-34a/b by reactive oxygen species and elevated c-Myc by oxidative stress, may impair mitochondrial metabolic homeostasis, leading to human corneal endothelial failure.

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

PURPOSE: To investigate the safety and efficacy of cultured human corneal endothelial cell (hCEC) injection therapy with mature differentiated (mature) cell subpopulations (SPs) for corneal endothelial failure (CEF). DESIGN: Comparative, interventional case series. METHODS: This study involved 18 eyes with CEF that underwent cultured hCEC injection therapy, categorized into 2 groups: (1) 11 eyes administered a relatively lower proportion (0.1 to 76.3%) of mature cell SPs (group 1 [Gr1]), and (2) 7 eyes administered a relatively higher proportion (>90%) of mature cell SPs (group 2 [Gr2]). From 1 week to 3 years postoperation, corneal endothelial cell (CEC) density (CECD), central corneal thickness (CCT), and best-corrected visual acuity (BCVA) were recorded, and the CEC parameter's "spring constant" was calculated. The proportion of mature SPs was evaluated by fluorescence-activated cell sorting analysis based on cell-surface markers. RESULTS: At 3 years postoperation, corneal restoration with improved BCVA was attained in 10 of the 11 Gr1 eyes and all Gr2 eyes, the median CECD in Gr2 (3083 cells/mm2; range, 2182-4417 cells/mm2) was higher than that in Gr1 (1349 cells/mm2; range, 746-2104 cells/mm2) (P < .001), and the spring constant verified the superiority of the mature cultured hCECs. From 24 weeks through 3 years postoperation, the median percentage of CECD decrease was 3.2% in Gr2 and 23.6% in Gr1 (P < .005). CCT recovery was prompt and constant in Gr2, while diverse in Gr1. No adverse events were observed. CONCLUSION: Our findings showed that mature cell SPs for hCEC injection therapy provide rapid recovery of CCT, better CECD, and low CECD attrition over 3 years postsurgery.

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

To explore new molecular targets for therapy in human model systems by discerning the role of extracellular vesicle (EV) microRNAs (miRs) secreted by human retinal pigment epithelium (hRPE) cells and their cellular interplay with macrophages (Mps). Human Mps differentiated from THP-1 cells stimulated by phorbol myristate acetate were co-cultured with induced pluripotent stem cell-derived differentiated hRPE (iPS-hRPE) cells in Transwell® system separated by 0.40 µm or 0.03 µm filters. EV-associated CD63+ proteins (CD63+ EV) were detected by western blotting, and secreted EVs were analyzed by Nanosight tracking. The miR profiles of the secreted EVs were determined using 3D-gene human microRNA chips (Toray Industries, Inc.). Levels of CD63+ EV were increased in co-cultures concomitantly with the increased production of EV particles (50-150 nm). The increased production of EVs was associated with higher production of MCP-1, IL-6, IL-8 from hRPE cells, and VEGF and repressed production of TNF-α from Mps and pigment epithelium-derived factor (PEDF) from RPE cells. Ultracentrifugation of semi-purified EVs increased the secretion of these pro-inflammatory cytokines and EV particles from hRPE cells, but this effect was eliminated in transwells equipped with 0.03 µm filters, whereas no repression of PEDF and TNF-α secretion occurred. 3D-gene miR analysis revealed a selective increase in secretion of miR494-3p in EVs from iPS-hRPE cells during the interplay with Mps. The miRs in EVs secreted by hRPE cells may have a critical role in the vicious inflammatory cycle, whereas repression of TNF-α and PEDF require cell-to-cell contact that is independent of EVs or exosomes. MiR494-3p may be a candidate molecular target of diagnosis and therapy for age-related macular degeneration.

CD63+ extracellular vesicles from retinal pigment epithelial cells participate in crosstalk with macrophages in the innate inflammatory axis.

The aim of the study is to clarify the participation of extracellular vesicles (EV) secreted by murine primary retinal pigment epithelial (mpRPE) cells in the cell to cell communication with macrophages (Mps), firstly described by the authors in 2016. In ocular inflammation, Mps act as sources of tumor necrosis factor-α (TNF-α), an activator of RPE cells. TNF-α stimulates the production of monocyte chemotactic protein (MCP-1) by RPE cells, thereby causing greater recruitment of Mps to the sub-RPE space. Murine RAW 264.7 Mps cells were co-cultured with C57BL/6 mouse mpRPE cells, either together or separated in transwells, vertically or horizontally connectable, with 0.40 or 0.03 µm membrane filters. The association of EV with mpRPE or RAW 264.7 was quantified by fluorescence cell sorting (FACS) using Qdot655 streptavidin-conjugated biotinylated EV. Increased levels of CD63+ EV were detected in co-cultures by western blotting or FACS analysis, in accordance with the increased production of nanoparticles (50-150 nm) detected by Nanosight tracking analysis. The gene expressions of cytokines, MCP-1, IL-6, IL-8, and VEGF in mpRPE cells and the corresponding proteins were increased in co-cultures even in transwells, vertically connected with 0.40 µm membrane filters, while the repressed TNF-α protein production was not affected. Most of the CD63+ EVs produced by mpRPE cells in co-cultures were associated with Raw264.7, but not with mpRPE cells. Semi-purified CD63+ EV secreted from mpRPE cells, increased the secretion of MCP-1, IL-6, and VEGF in co-cultures with RAW 264.7. Culture chamber separation horizontally connected with 0.03 µm membrane filters reduced this increased secretion. Collectively, mpRPE derived CD63+ EV partly participate in the sub-retinal innate inflammation. To evaluate the functional damage of RPE cells upon chronic exposure to here defined EVs will be the critical issue to uncover their roles in chronic retinal degenerative diseases.

Epigenetic regulation of the epithelial mesenchymal transition induced by synergistic action of TNF-α and TGF-ß in retinal pigment epithelial cells.

To clarify the influence of tumor necrosis factor (TNF)-α on fibrotic phenotypes induced by transforming growth factor (TGF)-ß in retinal pigment epithelial cells (RPECs) by epigenetic regulation. Human primary retinal pigment epithelial cells (RPECs including ARPE19) were used in cultures in the presence or absence of TNF-α and/or TGF-ß2. RT2 Profiler™ (Qiagen) was used for PCR Array for fibrosis and epithelial mesenchymal transition (EMT). Microarray analysis by 3D gene DNA chip was outsourced to Toray Industries Inc. Quantification of histone acetyl transferase (HAT)-related and histone deacetylase (HDAC) related gene expression were also analyzed. HDAC and HAT activity was measured using an EpiQuik HDAC and HAT Activity/Inhibition Assay Kit (Epigentek). CD44, MMP-9, HAT, and HDAC in RPECs were analyzed by western blotting. Analysis of expression of the EMT/fibrosis related CD44 and MMP-9 phenotypes induced by TNF-α+TGF-ß2 revealed four alterations in RPECs: 1) abolition of TGF-ß2-induced α-SMA by TNF-α; 2) synergy between TNF-α+TGF-ß2 for induction of CD44 and MMP-9 phenotypes 3) no inhibition of HDAC activity by either TNF-α or TGF-ß2; and 4) significant inhibition of HAT activity by either TNF-α or TGF-ß2, but no synergy. The HDAC activation through HAT inhibition by TNF-α+TGF-ß was counteracted by HDAC inhibitors, leading to the inhibition of EMT/fibrosis. EMT/fibrotic CD44 and MMP-9 phenotypes were epigenetically upregulated by concerted action of TNF-α and TGF-ß in RPECs. The intervention in epigenetic regulation may hold potential in preventing intraocular proliferative diseases.

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

PURPOSE: To report the safety and efficacy of a novel cell injection therapy using cultured human corneal endothelial cells (hCECs) for endothelial failure conditions via the report of the long-term 5-year postoperative clinical data from a first-in-humans clinical trial group. DESIGN: Prospective observational study. PARTICIPANTS: This study involved 11 eyes of 11 patients with pseudophakic endothelial failure conditions who underwent hCEC injection therapy between December 2013 and December 2014. METHODS: All patients underwent follow-up examinations at 1 week, 4 weeks, 12 weeks, and 24 weeks and 1 year, 2 years, 3 years, 4 years, and 5 years after surgery. Specific corneal endothelial cell parameters (i.e., corneal endothelial cell density [ECD], coefficient of variation of area, and percentage of hexagonal cells) and central corneal thickness, best-corrected visual acuity (BCVA) on a Landolt C eye chart, and intraocular pressure (IOP) were recorded. MAIN OUTCOME MEASURES: The primary outcome was the change in central ECD after cell injection therapy, and the secondary outcome was corneal thickness, BCVA, and IOP during the 5-year-postoperative follow-up period. RESULTS: At 5 years after surgery, normal corneal endothelial function was restored in 10 of the 11 eyes, the mean ± standard deviation central corneal ECD was 1257 ± 467 cells/mm2 (range, 601-2067 cells/mm2), BCVA improved significantly in 10 treated eyes, the mean visual acuity changed from 0.876 logarithm of the minimum angle of resolution before surgery to 0.046 logarithm of the minimum angle of resolution after surgery, and no major adverse reactions directly related to the hCEC injection therapy were observed. CONCLUSIONS: The findings in this study confirmed the safety and efficacy of cultured hCEC injection therapy for up to 5 years after surgery.

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

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.

Pluripotent epigenetic regulator OBP-801 maintains filtering blebs in glaucoma filtration surgery model.

Inhibition of fibrosis is indispensable for maintaining filtering blebs after glaucoma filtration surgery (GFS). The purpose of this study was to investigate the ability of a pluripotent epigenetic regulator OBP-801 (OBP) to ameliorate extracellular matrix formation in a rabbit model of GFS. Rabbits that underwent GFS were treated with OBP. The gene expression profiles and intraocular pressure (IOP) were monitored until 30 postoperative days. The bleb tissues were evaluated for tissue fibrosis at 30 postoperative days. In in vitro models, OBP interfered the functions of diverse genes during the wound-healing process. In in vivo GFS models, the expressions of TGF-ß3, MMP-2, TIMP-2 and 3, LOX, COL1A and SERPINH1 were significantly inhibited at 30 postoperative days in the OBP group compared with those in the vehicle control group. OBP treatment involving subconjunctival injection or eye drops showed no adverse effects, and reduced levels of α-SMA and collagen deposition at the surgical wound site. OBP maintained the long-lived bleb without scar formation, and IOP was lower at 30 postoperative days compared with the vehicle control group. These findings suggest that OBP is an effective and useful candidate low-molecular-weight agent for improving wound healing and surgical outcomes in a rabbit model of GFS.

Polarized Expression of Ion Channels and Solute Carrier Family Transporters on Heterogeneous 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.

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

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-ßß 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.

In Vivo Fluorescence Visualization of Anterior Chamber Injected Human Corneal Endothelial Cells Labeled With Quantum Dots.

Purpose: The injection of cultured human corneal endothelial cells (cHCECs) into the anterior chamber (AC) is a newly developed modality for the successful treatment of corneal endothelium dysfunction. Here, we investigated whether or not cHCECs could be labeled using quantum dots (QDs) composed of semiconductor nanoparticle octa-arginine (R8) to trace injected cHCECs and examined the utility of in vivo fluorescence imaging to analyze the dynamics and accumulation of QD-labeled injected cHCECs in a corneal endothelial dysfunction mouse model. Methods: The cHCECs, either of high quality or with cell-state transition, were labeled by adding a mixture of QDs655 and R8. The labeling efficiency and the unchanging of the cell phenotypes by the labeling was confirmed by flow cytometry. The labeled cHCECs were injected into the AC of either healthy mice or mice with corneal endothelium damaged by cryogenic treatment. The kinetics of the injected cHCECs was traced quantitatively via multiphoton confocal laser microscopy. Results: QD labeling induced no morphologic change in the cHCECs or in the expression of the functional markers of cHCECs (i.e., Na+/K+-ATPase and zonula occludens-1). The injected cHCECs-QDs were quantitatively detected, and the retention of cHCECs-QDs was evident, from 3 to 48 hours post cell injection on the posterior surface in the cryogenically injured corneal endothelium mouse model eyes, yet not in the noninjured healthy control eyes. Conclusions: The findings of this study show that in the field of regenerative medicine, QD labeling of cells presents a convenient and sensitive method of finely monitoring the fate of injected cells in vivo.

A physical biomarker of the quality of cultured corneal endothelial cells and of the long-term prognosis of corneal restoration in patients.

Dysfunction of the corneal endothelium reduces the transparency of the cornea and can cause blindness. Because corneal endothelial cells have an extremely limited proliferative ability in vivo, treatment for corneal endothelial dysfunction involves the transplantation of donor corneal tissue. Corneal endothelium can also be restored via intraocular injection of endothelial cells in suspension after their expansion in vitro. Yet, because quality assessment during the expansion of the cells is a destructive process, a substantial number of the cultured cells are lost. Here, we show that the 'spring constant' of the effective interaction potential between endothelial cells in a confluent monolayer serves as a biomarker of the quality of corneal endothelial cells in vitro and of the long-term prognosis of corneal restoration in patients treated with culture-expanded endothelial cells or with transplanted corneas. The biomarker can be measured from phase contrast imaging in vitro and from specular microscopy in vivo, and may enable a shift from passive monitoring to pre-emptive intervention in patients with severe corneal disorders.