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BACKGROUND: Corneal endothelial cell (CEC) disorders, such as Fuchs's endothelial corneal dystrophy, induce abnormal corneal hydration and result in corneal haziness and vision loss known as bullous keratopathy. We investigated whether injection of cultured human CECs supplemented with a rho-associated protein kinase (ROCK) inhibitor into the anterior chamber could increase CEC density. METHODS: We performed an uncontrolled, single-group study involving 11 persons who had received a diagnosis of bullous keratopathy and had no detectable CECs. Human CECs were cultured from a donor cornea; a total of 1×106 passaged cells were supplemented with a ROCK inhibitor (final volume, 300 µl) and injected into the anterior chamber of the eye that was selected for treatment. After the procedure, patients were placed in a prone position for 3 hours. The primary outcome was restoration of corneal transparency, with a CEC density of more than 500 cells per square millimeter at the central cornea at 24 weeks after cell injection. Secondary outcomes were a corneal thickness of less than 630 µm and an improvement in best corrected visual acuity equivalent to two lines or more on a Landolt C eye chart at 24 weeks after cell injection. RESULTS: At 24 weeks after cell injection, we recorded a CEC density of more than 500 cells per square millimeter (range, 947 to 2833) in 11 of the 11 treated eyes (100%; 95% confidence interval [CI], 72 to 100), of which 10 had a CEC density exceeding 1000 cells per square millimeter. A corneal thickness of less than 630 µm (range, 489 to 640) was attained in 10 of the 11 treated eyes (91%; 95% CI, 59 to 100), and an improvement in best corrected visual acuity of two lines or more was recorded in 9 of the 11 treated eyes (82%; 95% CI, 48 to 98). CONCLUSIONS: Injection of human CECs supplemented with a ROCK inhibitor was followed by an increase in CEC density after 24 weeks in 11 persons with bullous keratopathy. (Funded by the Japan Agency for Medical Research and Development and others; UMIN number, UMIN000012534 .).
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Córnea/citologia , Doenças da Córnea/terapia , Transplante de Córnea , Células Endoteliais/transplante , Inibidores de Proteínas Quinases/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Terapia Combinada , Córnea/anatomia & histologia , Córnea/cirurgia , Doenças da Córnea/tratamento farmacológico , Doenças da Córnea/cirurgia , Células Endoteliais/metabolismo , Feminino , Humanos , Pressão Intraocular , Masculino , Pessoa de Meia-IdadeRESUMO
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.
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Sinergismo Farmacológico , Epigênese Genética , Transição Epitelial-Mesenquimal , Epitélio Pigmentado da Retina/patologia , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Humanos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismoRESUMO
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.
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Amidas/uso terapêutico , Edema da Córnea/terapia , Endotélio Corneano/transplante , Distrofia Endotelial de Fuchs/terapia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , Adulto , Idoso , Câmara Anterior , Contagem de Células , Células Cultivadas , Terapia Combinada , Edema da Córnea/diagnóstico , Edema da Córnea/fisiopatologia , Endotélio Corneano/citologia , Feminino , Seguimentos , Distrofia Endotelial de Fuchs/diagnóstico , Distrofia Endotelial de Fuchs/fisiopatologia , Rejeição de Enxerto/prevenção & controle , Humanos , Injeções Intraoculares , Pressão Intraocular/fisiologia , Masculino , Pessoa de Meia-Idade , Decúbito Ventral , Estudos Prospectivos , Medicina Regenerativa , Microscopia com Lâmpada de Fenda , Acuidade Visual/fisiologiaRESUMO
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.
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Comunicação Celular/fisiologia , Vesículas Extracelulares/metabolismo , Imunidade Inata/fisiologia , Macrófagos/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Tetraspanina 30/metabolismo , Animais , Western Blotting , Linhagem Celular , Quimiocina CCL2/metabolismo , Técnicas de Cocultura , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Exossomos/genética , Citometria de Fluxo , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
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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
BACKGROUND: Stevens-Johnson syndrome (SJS) and its severe form, toxic epidermal necrolysis (TEN), are acute inflammatory vesiculobullous reactions of the skin and mucous membranes, including the ocular surface, oral cavity, and genitals. These reactions are very rare but are often associated with inciting drugs, infectious agents, or both. OBJECTIVE: We sought to identify susceptibility loci for cold medicine-related SJS/TEN (CM-SJS/TEN) with severe mucosal involvement (SMI). METHODS: A genome-wide association study was performed in 808 Japanese subjects (117 patients with CM-SJS/TEN with SMI and 691 healthy control subjects), and subsequent replication studies were performed in 204 other Japanese subjects (16 cases and 188 control subjects), 117 Korean subjects (27 cases and 90 control subjects), 76 Indian subjects (20 cases and 56 control subjects), and 174 Brazilian subjects (39 cases and 135 control subjects). RESULTS: In addition to the most significant susceptibility region, HLA-A, we identified IKZF1, which encodes Ikaros, as a novel susceptibility gene (meta-analysis, rs4917014 [G vs. T]; odds ratio, 0.5; P = 8.5 × 10(-11)). Furthermore, quantitative ratios of the IKZF1 alternative splicing isoforms Ik1 and Ik2 were significantly associated with rs4917014 genotypes. CONCLUSION: We identified IKZF1 as a susceptibility gene for CM-SJS/TEN with SMI not only in Japanese subjects but also in Korean and Indian subjects and showed that the Ik2/Ik1 ratio might be influenced by IKZF1 single nucleotide polymorphisms, which were significantly associated with susceptibility to CM-SJS/TEN with SMI.
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Anti-Inflamatórios não Esteroides/efeitos adversos , Antígenos HLA-A/genética , Fator de Transcrição Ikaros/genética , Mucosa Bucal/efeitos dos fármacos , Medicamentos Compostos contra Resfriado, Influenza e Alergia/efeitos adversos , Síndrome de Stevens-Johnson/genética , Adolescente , Adulto , Idoso , Processamento Alternativo , Povo Asiático , Estudos de Casos e Controles , Feminino , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Antígenos HLA-A/imunologia , Humanos , Fator de Transcrição Ikaros/imunologia , Masculino , Pessoa de Meia-Idade , Mucosa Bucal/patologia , Razão de Chances , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Síndrome de Stevens-Johnson/etnologia , Síndrome de Stevens-Johnson/etiologia , Síndrome de Stevens-Johnson/patologia , População BrancaRESUMO
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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Corneal endothelial dysfunction accompanied by visual disturbance is a primary indication for corneal transplantation. We previously reported that the adhesion of corneal endothelial cells (CECs) to a substrate was enhanced by the selective ROCK inhibitor Y-27632. It is hypothesized that the inhibition of ROCK signaling may manipulate cell adhesion properties, thus enabling the transplantation of cultivated CECs as a form of regenerative medicine. In the present study, using a rabbit corneal endothelial dysfunction model, the transplantation of CECs in combination with Y-27632 successfully achieved the recovery of corneal transparency. Complications related to cell injection therapy, such as the abnormal deposition of the injected cells as well as the elevation of intraocular pressure, were not observed. Reconstructed corneal endothelium with Y-27632 exhibited a monolayer hexagonal cell shape with a normal expression of function-related markers, such as ZO-1, and Na(+)/K(+)-ATPase, whereas reconstruction without Y-27632 exhibited a stratified fibroblastic phenotype without the expression of markers. Moreover, transplantation of CECs in primates in the presence of the ROCK inhibitor also achieved the recovery of long-term corneal transparency with a monolayer hexagonal cell phenotype at a high cell density. Taken together, these results suggest that the selective ROCK inhibitor Y-27632 enables cultivated CEC-based therapy and that the modulation of Rho-ROCK signaling activity serves to enhance cell engraftment for cell-based regenerative medicine.
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Amidas/farmacologia , Endotélio Corneano/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Piridinas/farmacologia , Regeneração/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Amidas/uso terapêutico , Animais , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Terapia Combinada , Lesões da Córnea , Transplante de Córnea/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Células Endoteliais/transplante , Endotélio Corneano/lesões , Endotélio Corneano/fisiologia , Endotélio Corneano/transplante , Inibidores Enzimáticos/uso terapêutico , Macaca fascicularis , Fenótipo , Piridinas/uso terapêutico , Coelhos , Regeneração/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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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
Interleukin (IL)-15 is expressed in a variety of inflammatory diseases. However, the contribution of dendritic cell (DC)-derived IL-15 to the development of diseases is uncertain. Using established models of Propionibacterium acnes (P. acnes)- and zymosan-induced liver inflammation, we observed granuloma formation in the livers of wild-type (WT) and RAG-2(-/-) mice but not in those of IL-15(-/-) mice. We demonstrate that this is likely caused by an impaired sequential induction of IL-12, IFN-gamma, and chemokines necessary for monocyte migration. Likewise, lethal endotoxin shock was not induced in P. acnes- and zymosan-primed IL-15(-/-) mice or in WT mice treated with a new IL-15-neutralizing antibody. In both systems, proinflammatory cytokine production was impaired. Surprisingly, neither granuloma formation, lethal endotoxin shock, nor IL-15 production was induced in mice deficient for DCs, and adoptive transfer of WT but not IL-15(-/-) DCs restored the disease development in IL-15(-/-) mice. Collectively, these data indicate the importance of DC-derived IL-15 as a mediator of inflammatory responses in vivo.
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Células Dendríticas/metabolismo , Granuloma/imunologia , Hepatite/imunologia , Interleucina-15/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Hepatite/microbiologia , Imuno-Histoquímica , Interleucina-15/metabolismo , Camundongos , Camundongos Knockout , Propionibacterium acnes , ZimosanRESUMO
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 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
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 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.
Assuntos
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 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 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.
Assuntos
Endotélio Corneano , MicroRNAs , Células Endoteliais/metabolismo , Endotélio Corneano/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Água/metabolismoRESUMO
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.
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Córnea , Endotélio Corneano , Contagem de Células , Diferenciação Celular , Células Cultivadas , Células Endoteliais , HumanosRESUMO
Gelatinous drop-like dystrophy (GDLD) is a rare autosomal recessive form of corneal dystrophy characterized by subepithelial amyloid depositions on the cornea. Previous clinical and laboratory observations have strongly suggested that epithelial barrier function is significantly decreased in GDLD. Despite the decade-old identification of the tumor-associated calcium signal transducer 2 (TACSTD2) gene as a causative gene for GDLD, the mechanism by which the loss of function of this causative gene leads to the pathological consequence of this disease remains unknown. In this study, we investigated the functional relationship between the TACSTD2 gene and epithelial barrier function. Through the use of immunoprecipitation and a proximity ligation assay, we obtained evidence that the TACSTD2 protein directly binds to claudin 1 and 7 proteins. In addition, the loss of function of the TACSTD2 gene leads to decreased expression and change in the subcellular localization of tight junction-related proteins, including claudin 1, 4, 7, and ZO1 and occludin, both in diseased cornea and cultured corneal epithelial cells. These results indicate that loss of function of the TACSTD2 gene impairs epithelial barrier function through decreased expression and altered subcellular localization of tight junction-related proteins in GDLD corneas.