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.

The existence of senescent cells in conjunctival epithelium from elderly individuals.

PURPOSE: The ocular surface microenvironment changes with aging. However, it remains unclear if cellular senescence influences the ocular surface. We investigated the presence of p16INK4a-expressing senescent cells in healthy human conjunctiva. STUDY DESIGN: Clinical and experimental. METHODS: Healthy conjunctival tissue samples were obtained from middle-aged and elderly subjects. RT-qPCR was performed to assess the expression of senescence markers CDKN2A (p16INK4a) and CDKN1A (p21CIP1/WAF1) and immunostaining was performed to examine the expression of the senescence marker p16INK4a, stem cell markers Ki67 and p63, tight-junction marker ZO-1. RESULTS: Our study involved 19 conjunctival tissue samples (10 elderly and 9 middle-aged), mean age [elderly: 75.8 ± 3.7 years (72-81), middle-aged: 52.7 ± 7 years (38-59)], sex (elderly: 3 men, 7 women; middle-aged: 3 men, 6 women). The expression of p16INK4a was significantly increased at the RNA level in the elderly compared to middle-aged (p < 0.05). Positivity rate of p16INK4a was significantly elevated in the elderly (15.0 ± 7.8%) compared to middle-aged (0.2 ± 0.6%) (p < 0.05). Positivity rate of Ki67and p63 was significantly reduced in the elderly (1.7 ± 1.7% and 16.5 ± 9.5%) compared to middle-aged (3.9 ± 1.8% and 24.7 ± 5.7%) (p < 0.05). ZO-1 expression was reduced in tissue samples showing p16INK4a-positivity but retained in tissue samples in which p16INK4a was undetectable. CONCLUSIONS: Senescent cells accumulate with age in the conjunctival epithelium, accompanied by a decrease in Ki67, p63 and ZO-1 expressing cells.

Guttae Morphology After Cultured Corneal Endothelial Cell Transplant in Fuchs Endothelial Corneal Dystrophy.

Importance: Whether guttae in Fuchs endothelial corneal dystrophy (FECD) can be removed by polishing without Descemet stripping and whether postoperative maintenance of reduced guttae can be achieved through cultured corneal endothelial cell (CEC) transplant therapy are critical issues to be addressed. Objective: To investigate the decrease of guttae through polishing degenerated CECs and abnormal extracellular matrix (ECM) without Descemet stripping and to observe the behavior of guttae following cultured CEC transplant. Design, Setting, and Participants: This case series prospective observational study was conducted in a hospital outpatient clinic setting. Between December 2013 and January 2019, 22 eyes with corneal endothelial failure caused by FECD received cultured CEC transplant therapy at Kyoto Prefectural University Hospital. Of these, 15 eyes were consistently monitored at the same central corneal area during the preoperative phase, as well as in the early (within 1 year) and late (after 3 years) postoperative phases. The images from these phases were categorized into 3 groups: typical guttae, atypical guttae, and no guttae. Exposures: Cultured CEC transplant therapy. Main Outcomes: Proportion of guttae in the observable area was measured, comparing the early and late postoperative phases for each group. Results: The mean age of the patients at the time of surgery was 69 years (range, 49-79 years). All 15 eyes exhibited the presence of confluent guttae preoperatively (100%). Among these, 3 of 15 eyes belonged to male patients. The early postoperative phase of guttae morphologies was classified into 3 groups: 5 eyes with typical guttae, 7 with atypical guttae, and 3 with no guttae. The decrease in the number of these guttae was achieved by surgical procedures. The median percentage of guttae in the typical guttae, atypical guttae, and no guttae groups was 41.8%, 44.4%, and 16.2%, respectively, in the early phase, and 42.2%, 38.2%, and 18.8%, respectively, in the late phase. Conclusions and Relevance: The findings demonstrate that in some cases of FECD, guttae can be removed by scraping and polishing abnormal ECM and degenerated CECs, while preserving the Descemet membrane. Furthermore, cultured CEC transplant resulted in no increase in guttae for up to 3 years, providing insights into surgically eliminating guttae.

Senescent characteristics of human corneal endothelial cells upon ultraviolet-A exposure.

PURPOSE: The objective of this study was to investigate the senescent phenotypes of human corneal endothelial cells (hCEnCs) upon treatment with ultraviolet (UV)-A. METHODS: We assessed cell morphology, senescence-associated ß-galactosidase (SA-ß-gal) activity, cell proliferation and expression of senescence markers (p16 and p21) in hCEnCs exposed to UV-A radiation, and senescent hCEnCs induced by ionizing radiation (IR) were used as positive controls. We performed RNA sequencing and proteomics analyses to compare gene and protein expression profiles between UV-A- and IR-induced senescent hCEnCs, and we also compared the results to non-senescent hCEnCs. RESULTS: Cells exposed to 5 J/cm2 of UV-A or to IR exhibited typical senescent phenotypes, including enlargement, increased SA-ß-gal activity, decreased cell proliferation and elevated expression of p16 and p21. RNA-Seq analysis revealed that 83.9% of the genes significantly upregulated and 82.6% of the genes significantly downregulated in UV-A-induced senescent hCEnCs overlapped with the genes regulated in IR-induced senescent hCEnCs. Proteomics also revealed that 93.8% of the proteins significantly upregulated in UV-A-induced senescent hCEnCs overlapped with those induced by IR. In proteomics analyses, senescent hCEnCs induced by UV-A exhibited elevated expression levels of several factors part of the senescence-associated secretory phenotype. CONCLUSIONS: In this study, where senescence was induced by UV-A, a more physiological stress for hCEnCs compared to IR, we determined that UV-A modulated the expression of many genes and proteins typically altered upon IR treatment, a more conventional method of senescence induction, even though UV-A also modulated specific pathways unrelated to IR.

Gene expression signatures of human senescent corneal and conjunctival epithelial cells.

PURPOSE: This study aimed to investigate the senescent phenotypes of human corneal and conjunctival epithelial cells. METHODS: We examined cell morphology, senescence-associated ß-galactosidase (SA-ß-gal) activity, cell proliferation, and expression of senescence markers (p16 and p21). RNA sequencing analysis was conducted to compare gene expression profiles between senescent and non-senescent cells. Finally, the potential involvement of senescent cells in the pathogenesis of ocular surface diseases was investigated. RESULTS: X-irradiated corneal and conjunctival epithelial cells exhibited typical senescence phenotypes, i.e., flattened morphologies, increased SA-ß-gal activity, decreased cell proliferation, and increased expression of senescence markers, p16 and p21. RNA-seq analysis revealed substantial differences in gene expression profiles between senescent corneal (SCo) and conjunctival epithelial cells (SCj). Moreover, SCj were detected in pathological conjunctival tissues associated with limbal stem cell deficiency (LSCD) due to Stevens-Johnson syndrome or chemical burns, potentially being involved in abnormal differentiation. CONCLUSION: This study highlights the cellular and molecular characteristics of senescent ocular surface cells, particularly in SCj that show abnormal keratin expression, and their potential roles in severe ocular surface diseases and pathology.

Dyslipidemia Exacerbates Meibomian Gland Dysfunction: A Systematic Review and Meta-Analysis.

Dry eye is a multifactorial and common age-related ocular surface disease. Dyslipidemia has been reported to be involved in meibomian gland dysfunction (MGD). However, it has not been clearly identified which lipid abnormality is responsible for MGD. In this systematic review and meta-analysis, we discuss how lipid profile changes with aging is responsible for MGD development. METHODS: An article search was performed in PubMed, EMBASE, and Web of Science. Eleven studies involving dyslipidemia in patients with MGD were identified. Five out of eleven studies were further analyzed with meta-analysis. The preferred reporting items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines were followed. Study-specific estimates (prevalence of dyslipidemia in MGD patients) were combined using one-group meta-analysis in a random-effects model. RESULTS: Meta-analysis revealed that high total cholesterol (TC) and high triglycerides (TG) were significantly associated with MGD prevalence, with odds ratios of 5.245 (95% confidence interval [CI]: 1.582-17.389; p < 0.001) and 3.264 (95% CI: 1.047-10.181; p < 0.001), respectively, but high low-density lipoprotein (LDL) and low high-density lipoprotein (HDL) were not identified. Systematic review found that the percentage of MGD patients with TC ≥ 200 mg/dL ranged from 20.0-77.6%, TG ≥ 150 mg/dL ranged from 8.3-89.7%, whereas, in the aged-match-adjusted controls, TC range of 200 mg/dL or higher and TG range of 150 mg/dL was 6.1-45.1% and 1.1-47.8%, respectively. The severity of MGD was higher with dyslipidemia. CONCLUSION: Dyslipidemia and higher TC and TG are significant risk factors for MGD.

Cellular senescence exacerbates features of aging in the eyes.

Aging is a process often associated with various age-related diseases. Senescence is one of the hallmarks of aging, and senescent cells acquire a complex, often pro-inflammatory, secretory phenotype termed the senescence-associated secretory phenotype (SASP). Here we show that ocular surface cells from human cornea become senescent upon X-irradiation, characterized by increased SA-ß-gal activity, decreased cell proliferation, increased expression of p16, and disruption of epithelial barrier. Comprehensive transcriptomic and proteomic analysis revealed that human senescent ocular cells acquire a SASP that disrupts epithelial barrier function. During aging in mice, senescent ocular cells accumulate, resulting in decreased epithelial barrier and chronic inflammation. Lacrimal gland excision, which leads to symptoms of dry eye (DE), resulted in corneal opacity associated with severe angiogenesis only in aged mice but not in young mice, and early senolytic treatment protected old DE mice from corneal opacity. In conclusion, senescent cells alter the ocular microenvironment through their SASP and eliminating these cells could represent a potential approach to alleviate symptoms associated with aged ocular surface.

Impact of aging on the pathophysiology of dry eye disease: A systematic review and meta-analysis.

PURPOSE: Dry eye disease (DED) is a common age-related ocular surface disease. However, it is unknown how aging influences the ocular surface microenvironment. This systematic review aims to investigate how the aging process changes the ocular surface microenvironment and impacts the development of DED. METHODS: An article search was performed in PubMed, EMBASE, and Web of Science. 44 studies reporting on age-related ocular changes and 14 large epidemiological studies involving the prevalence of DED were identified. 8 out of 14 epidemiological studies were further analyzed with meta-analysis. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines were followed. Study-specific estimates (impact of aging on the prevalence of DED) were combined using one-group meta-analysis in a random-effects model. RESULTS: Meta-analysis revealed the prevalence of DED in the elderly aged 60 years old or older was 5519 of 60107 (9.2%) and the odds ratio of aging compared to younger age was 1.313 (95% confidence interval [CI]; 1.107, 1.557). With increasing age, the integrity of the ocular surface and tear film stability decreased. Various inflammatory cells, including senescent-associated T-cells, infiltrated the ocular surface epithelium, lacrimal gland, and meibomian gland, accompanied by senescence-related changes, including accumulation of 8-OHdG and lipofuscin-like inclusions, increased expression of p53 and apoptosis-related genes, and decreased Ki67 positive cells. CONCLUSIONS: The aging process greatly impacts the ocular surface microenvironment, consequently leading to DED.

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.

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.

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.