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PURPOSE: To assess the effectiveness of switching from the concomitant use of brinzolamide 1% (BZM) and brimonidine 0.1% (BMD) to a BZM/BMD fixed-dose combination (BBFC) for the reduction of corneal epithelial damage. STUDY DESIGN: Retrospective cohort study. METHODS: This study involved 52 eyes of 52 glaucoma patients (26 women, 26 men; mean age: 67.0 ± 14.0 years) followed for more than 3 months after being switched from concomitant BZM and BMD to BBFC. Superficial punctate keratitis (SPK) was assessed by fluorescein staining according to the National Eye Institute classification, with the cornea divided into 5 areas: center, superior, nasal, temporal, and inferior. SPK density was graded as 0 (no SPK), 1 (separate SPK), 2 (moderately dense SPK), and 3 (high SPK with overlapping lesions). SPK scores and intraocular pressure (IOP) at pre switching to BBFC (pre-BBFC) and at 3-months post switching to BBFC (post-BBFC) were then compared using the Wilcoxon signed-rank test. RESULTS: At pre-BBFC and post-BBFC, respectively, mean IOP was 12.4 ± 2.5 and 12.4 ± 2.7 mmHg, thus illustrating no significant difference in IOP between pre and post switch (p = 0.924), and the mean SPK score for center, superior, nasal, temporal, and inferior was 0.06 ± 0.24, 0.04 ± 0.19, 0.52 ± 0.67, 0.15 ± 0.36, and 0.92 ± 0.74, and 0.04 ± 0.19, 0.02 ± 0.14, 0.37 ± 0.56, 0.04 ± 0.19, and 0.75 ± 0.62, thus clearly showing a significant reduction in SPK scores for the nasal, temporal, and inferior areas at post-BBFC compared to those at pre-BBFC (p < 0.05). CONCLUSION: Our findings reveal that compared with the concomitant use of BZM and BMD, BBFC is effective in reducing corneal epithelial damage.
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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.
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PURPOSE: To examine corneal graft survival via corneal endothelial cell density (ECD) and corneal endothelial cell loss (ECL) at 5 years post-transplantation in the eyes of patients with and without a history of undergoing glaucoma surgery according to the maturity of the donor corneal endothelial cells. DESIGN: Prospective cohort study. METHODS: This prospective cohort study included 17 patients with glaucoma and 51 patients without glaucoma who underwent Descemet's stripping automated endothelial keratoplasty or penetrating keratoplasty at the Baptist Eye Institute, Kyoto, Japan, between October 2014 and October 2016. Human corneal endothelial cells were cultured from residual peripheral donor cornea tissue, and the maturity of the cells was evaluated by cell surface markers (ie, CD166+, CD44-/dull, CD24-, and CD105-) using fluorescence-activated cell sorting. Kaplan-Meier analysis or the chi-square test was used to assess the rate of successful corneal graft survival post-transplantation. RESULTS: At 36 months postoperatively, the mean ECD and ECL in the glaucoma-bleb eyes were 1197 ± 352 cells/mm2 and 55.5% ± 13.9% in the high-maturity group and 853 ± 430 cells/mm2 and 67.7% ± 18.1% in the low-maturity group, respectively. Kaplan-Meier analysis revealed that at 5 years postoperatively, the overall rate of survival was 45%, that is, 100% in the high-maturity group and 25% in the low-maturity group (P < .05). CONCLUSIONS: The findings in this prospective cohort study revealed that the use of donor corneal grafts containing mature-differentiated corneal endothelial cells could maintain the survival of the transplanted graft for a long-term period, even in patients with a history of undergoing glaucoma surgery.
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Endotélio Corneano , Glaucoma , Sobrevivência de Enxerto , Pressão Intraocular , Doadores de Tecidos , Humanos , Sobrevivência de Enxerto/fisiologia , Estudos Prospectivos , Masculino , Feminino , Endotélio Corneano/patologia , Idoso , Contagem de Células , Pessoa de Meia-Idade , Pressão Intraocular/fisiologia , Glaucoma/cirurgia , Glaucoma/fisiopatologia , Perda de Células Endoteliais da Córnea/diagnóstico , Ceratoplastia Penetrante , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior , Seguimentos , Citometria de Fluxo , Idoso de 80 Anos ou mais , Acuidade Visual/fisiologiaRESUMO
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.
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Vesículas Extracelulares , MicroRNAs , Sirtuína 3 , Humanos , MicroRNAs/genética , Sirtuína 3/genética , Diferenciação Celular , PTEN Fosfo-Hidrolase/genéticaRESUMO
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.
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Purpose: To report the safety, efficacy, and long-term outcome in a case of Fuchs endothelial corneal dystrophy (FECD) treated by Rho-associated protein kinase (ROCK)-inhibitor eye drops in combination with removal of degenerated corneal endothelial cells (CECs) subsequent to transcorneal freezing. Observations: A 52-year-old Japanese man diagnosed with early-stage FECD developed central corneal edema with decreased visual acuity (VA) in his left eye and was treated by ROCK inhibitor eye drops (Y-27632 10mM) q.i.d. for 1 week starting immediately subsequent to the removal of the damaged CECs via 2-mm-diameter transcorneal freezing in May 18, 2010. Before treatment, the best-corrected VA (BCVA) was 20/20 OD and 20/63 OS, and the central corneal thickness in the left eye was 643 µm and specular microscopy image at the central cornea was not detected due to edema. Corneal transparency recovered, and the BCVA improved to 20/20 within two weeks. At 12 years post treatment, the cornea in left eye remained transparent without corneal edema, and the CEC density at the central cornea was 1294 cells/mm2 and the central corneal thickness was 581 µm. The annual decrease of CECs at the central cornea was 1.1%, and VA was maintained at 20/25. Multiple guttae were observed in the peripheral region, but few in the central region were removed by transcorneal freezing treatment, and relatively normal and healthy CECs were observed. Conclusions and importance: The findings in this case suggest the potential long-term safety and efficacy of the medical therapy by ROCK-inhibitor eye drop for early-stage FECD.
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Skeletal muscle stem cells (MuSCs) have been proposed as suitable candidates for cell therapy to muscular disorders since they exhibit a good capacity for myogenic regeneration. However, for better therapeutic outcomes, it is necessary to isolate human MuSCs from a suitable tissue source that possess high myogenic differentiation. In this context, isolated CD56+CD82+ cells from extra eyelid tissues were tested in vitro myogenic differentiation potential. Primary human myogenic cells derived from extra eyelids including orbicularis oculi, might be good candidates for human muscle stem cell-based research.
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Pálpebras , Fibras Musculares Esqueléticas , Humanos , Diferenciação Celular , Células-Tronco , Músculo Esquelético/fisiologiaRESUMO
Purpose: Corneal endothelial cell density (ECD) gradually decreases after corneal transplantation by unknown biologic, biophysical, or immunologic mechanism. Our purpose was to assess the association between donor corneal endothelial cell (CEC) maturity in culture and postoperative endothelial cell loss (ECL) after successful corneal transplantation. Design: Prospective cohort study. Participants: This cohort study was conducted at Baptist Eye Institute, Kyoto, Japan, between October 2014 and October 2016. It included 68 patients with a 36-month follow-up period who had undergone successful Descemet stripping automated endothelial keratoplasty (DSAEK) or penetrating keratoplasty. Methods: Human CECs (HCECs) from remaining peripheral donor corneas were cultured and evaluated for maturity by surface markers (CD166+, CD44-/dull, CD24-, and CD105-) using fluorescence-activated cell sorting. Postoperative ECD was assessed according to the mature-differentiated HCEC contents: high-maturity group: > 70%, middle-maturity group: 10% to 70%, low-maturity group: < 10%. The successful rate of ECD maintained at 1500 cells/mm2 at 36 months postoperative was analyzed using the log-rank test. Main Outcome Measures: Endothelial cell density and ECL at 36 months postoperative. Results: The 68 included patients (mean [standard deviation] age 68.1 [13.6] years, 47.1% women, 52.9% DSAEK). The high, middle, and low-maturity groups included 17, 32, and 19 eyes, respectively. At 36 months postoperative, the mean (standard deviation) ECD significantly decreased to 911 (388) cells/mm2 by 66% in the low-maturity group, compared with 1604 (436) by 40% and 1424 (613) cells/mm2 by 50% in the high and middle-maturity groups (P < 0.001 and P = 0.007, respectively) and the low-maturity group significantly failed to maintain ECD at 1500 cells/mm2 at 36 months postoperative (P < 0.001). Additional ECD analysis for patients who underwent DSAEK alone displayed a significant failure to maintain ECD at 1500 cells/mm2 at 36 months postoperative (P < 0.001). Conclusions: The high content of mature-differentiated HCECs expressed in culture by the donor peripheral cornea was coincident with low ECL, suggesting that a high-maturity CEC content predicts long-term graft survival. Understanding the molecular mechanism for maintaining HCEC maturity could elucidate the mechanism of ECL after corneal transplantation and aid in developing effective interventions. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.
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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.
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Glaucoma , Hipertensão Ocular , Disco Óptico , Masculino , Humanos , Estudos Longitudinais , Pressão Intraocular , Glaucoma/diagnóstico , Hipertensão Ocular/diagnósticoRESUMO
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.
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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.
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Doenças da Córnea , Endotélio Corneano , Camundongos , Animais , Humanos , Células Alógenas , Células Endoteliais , Leucócitos Mononucleares , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos BALB C , Isoantígenos , Imunidade , Doenças da Córnea/cirurgiaRESUMO
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.
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Relógios Circadianos , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Células Endoteliais/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
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.
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Degeneração Macular , Corioide/patologia , Colágeno , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Humanos , Fatores Imunológicos , Degeneração Macular/diagnóstico , Retina/patologiaRESUMO
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.
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Células Endoteliais , Mitocôndrias , Proteínas de Transporte de Ânions , Antiporters , Células Cultivadas , Homeostase , Humanos , Concentração de Íons de Hidrogênio , RNA Interferente Pequeno/genética , ÁguaRESUMO
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.
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Endotélio Corneano , MicroRNAs , Células Endoteliais/metabolismo , Endotélio Corneano/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Água/metabolismoRESUMO
PURPOSE: Carriers of functionally deficient mutations in the CYP39A1 gene have been recently reported to have a 2-fold increased risk of exfoliation syndrome (XFS). The aim of this study was to evaluate the risk of blindness and related clinical phenotypes of XFS patients carrying the loss-of-function CYP39A1 G204E mutation in comparison with XFS patients without any CYP39A1 mutation. DESIGN: Retrospective case study. PARTICIPANTS: A total of 35 patients diagnosed with XFS carrying the CYP39A1 G204E mutation and 150 XFS patients without any CYP39A1 mutation who were randomly selected from the Japanese XFS cohort. METHODS: Two-sided Fisher exact test with an alpha level < 0.05 was used to estimate the significance of the calculated odds ratio (OR) for all categorical measures. Comparisons between groups of subjects were performed using linear mixed effect models with group as random effect and taking possible dependence between eyes within a subject into account. MAIN OUTCOME MEASURES: Primary analysis compared the incidence of blindness (defined as visual acuity [VA] < 0.05 decimal), prevalence of exfoliation glaucoma (XFG), history of glaucoma surgery, and indices of glaucoma severity such as visual field (VF) mean deviation (MD), intraocular pressure (IOP), and vertical cup-disc ratio (CDR) between CYP39A1 G204E carriers and those without any CYP39A1 mutation. RESULTS: The overall risk for blindness was significantly higher in XFS patients carrying the CYP39A1 G204E variant (10/35 [28.6%]) compared with XFS patients without any CYP39A1 mutations (8/150 [5.4%]; odds ratio [OR], 7.1; 95% confidence interval [CI], 2.7-20.2]; P < 0.001). A higher proportion of XFS patients with the CYP39A1 G204E mutation (23/35 [65.7%]) had evidence of XFG in at least 1 eye compared with the comparison group (41/150 [27.3%]; OR, 5.1; 95% CI, 2.4-11.4]; P < 0.0001). Significantly higher peak IOP, larger vertical CDR, and worse VF MD were also found in CYP39A1 G204E variant carriers (P < 0.001). Additionally, patients with the CYP39A1 G204E mutation (18/35 [51.4%]) required more laser or glaucoma surgical interventions compared with those without any CYP39A1 mutation (32/150 [21.3%], P < 0.001). CONCLUSIONS: Patients with XFS carrying the CYP39A1 G204E mutation had significantly increased risk of blindness, higher occurrence of XFG, and more severe glaucoma compared with patients with XFS without any CYP39A1 mutation.
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Síndrome de Exfoliação , Glaucoma , Esteroide Hidroxilases , Cegueira/genética , Síndrome de Exfoliação/complicações , Síndrome de Exfoliação/genética , Glaucoma/complicações , Glaucoma/genética , Humanos , Estudos Retrospectivos , Esteroide Hidroxilases/genética , Campos VisuaisRESUMO
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.
Assuntos
Córnea , Endotélio Corneano , Contagem de Células , Diferenciação Celular , Células Cultivadas , Células Endoteliais , HumanosRESUMO
PURPOSE: To investigate the trend of seasonal variation of intraocular pressure (IOP) in patients with normal-tension glaucoma over a 20-year period by retrospectively analyzing the Kyoto Prefectural University of Medicine Glaucoma Registry database as real-world data. DESIGN: Retrospective cohort study. METHODS: Data points (n = 49,007) were extracted retrospectively from the medical records of 1774 patients with normal-tension glaucoma (665 male patients and 1109 female patients; mean ± SD age was 59.8 ± 14.4 years; and mean ± SD observation period was 5.6 ± 4.4 years) seen over the 20-year period. We first calculated the mean IOP from all available data of each month from January 1997 through December 2016. The data were then categorized into 5 groups of 4 consecutive years each (1997-2000, 2001-2004, 2005-2008, 2009-2012, and 2013-2016) and the mean IOP of each month within the group was calculated. Seasonal variations of IOP over the 20-year study period and in the 5 consecutive groups were then investigated via nonlinear multiple regression analysis. RESULTS: A continuous decrease of IOP was detected throughout the 20-year period (P < .001), with distinct seasonal variation. The annual mean ± SD IOP was highest (13.9 ± 2.7 mm Hg) in the oldest group (1997-2000), with a gradual decrease in each subsequent group, finally becoming lowest (12.3 ± 2.7 mm Hg) in the most recent group (2013-2016) (P < .001), and all of them were accompanied by distinct seasonal variation (P < .001). CONCLUSIONS: Based on the Kyoto Prefectural University of Medicine Glaucoma Registry real-world longitudinal data, our findings revealed a continuous decrease and distinct seasonal variation of IOP in patients with normal-tension glaucoma throughout the 20-year study period.
Assuntos
Glaucoma , Pressão Intraocular , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Estações do Ano , Tonometria OcularRESUMO
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.
Assuntos
Vesículas Extracelulares/metabolismo , Regulação da Expressão Gênica , Macrófagos/metabolismo , Degeneração Macular/genética , MicroRNAs/genética , Epitélio Pigmentado da Retina/metabolismo , Western Blotting , Comunicação Celular , Células Cultivadas , Vesículas Extracelulares/patologia , Humanos , Macrófagos/patologia , Degeneração Macular/metabolismo , Degeneração Macular/patologia , MicroRNAs/biossíntese , Epitélio Pigmentado da Retina/patologiaRESUMO
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.