RESUMEN
Purpose: This study evaluated the dysregulation of TCF4 isoforms and differential exon usage (DEU) in corneal endothelial cells (CECs) of Fuchs endothelial corneal dystrophy (FECD) with or without trinucleotide repeat (TNR) expansion in the intron region of the TCF4 gene. Methods: Three RNA-Seq datasets of CECs (our own and two other previously published datasets) derived from non-FECD control and FECD subjects were analyzed to identify TCF4 isoforms and DEU events dysregulated in FECD by comparing control subjects to those with FECD with TNR expansion and FECD without TNR expansion. Results: Our RNA-Seq data demonstrated upregulation of three TCF4 isoforms and downregulation of two isoforms in FECD without TNR expansion compared to the controls. In FECD with TNR expansion, one isoform was upregulated and one isoform was downregulated compared to the control. Additional analysis using two other datasets identified that the TCF4-277 isoform was upregulated in common in all three datasets in FECD with TNR expansion, whereas no isoform was dysregulated in FECD without TNR expansion. DEU analysis showed that one exon (E174) upstream of the TNR, which only encompassed TCF4-277, was upregulated in common in all three datasets, whereas eight exons downstream of the TNR were downregulated in common in all three datasets in FECD with TNR expansion. Conclusions: This study identified TCF4-277 as a dysregulated isoform in FECD with TNR expansion, suggesting a potential contribution of TCF4-277 to FECD pathophysiology.
Asunto(s)
Endotelio Corneal , Distrofia Endotelial de Fuchs , Factor de Transcripción 4 , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Endotelio Corneal/metabolismo , Endotelio Corneal/patología , Exones/genética , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Regulación de la Expresión Génica , Isoformas de Proteínas/genética , Factor de Transcripción 4/genética , Factor de Transcripción 4/metabolismo , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Fuchs endothelial corneal dystrophy (FECD) is a complex corneal disease characterized by the progressive decline and morphological changes of corneal endothelial cells (CECs) that leads to corneal edema and vision loss. The most common mutation in FECD is an intronic CTG repeat expansion in transcription factor 4 (TCF4) that leads to its altered expression. Corneal endothelial wound healing occurs primarily through cell enlargement and migration, and FECD CECs have been shown to display increased migration speeds. In this study, we aim to determine whether TCF4 can promote cellular migration in FECD CECs. We generated stable CEC lines derived from FECD patients that overexpressed different TCF4 isoforms and investigated epithelial-to-mesenchymal (EMT) expression, morphological analysis and cellular migration speeds. We found that full length TCF4-B isoform overexpression promotes cellular migration in FECD CECs in an EMT-independent manner. RNA-sequencing identified several pathways including the negative regulation of microtubules, with TUBB4A (tubulin beta 4A class IVa) as the top upregulated gene. TUBB4A expression was increased in FECD ex vivo specimens, and there was altered expression of cytoskeleton proteins, tubulin and actin, compared to normal healthy donor ex vivo specimens. Additionally, there was increased acetylation and detyrosination of microtubules in FECD supporting that microtubule stability is altered in FECD and could promote cellular migration. Future studies could be aimed at investigating if targeting the cytoskeleton and microtubules would have therapeutic potential for FECD by promoting cellular migration and regeneration.
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Movimiento Celular , Endotelio Corneal , Distrofia Endotelial de Fuchs , Microtúbulos , Factor de Transcripción 4 , Humanos , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Distrofia Endotelial de Fuchs/patología , Movimiento Celular/genética , Microtúbulos/metabolismo , Factor de Transcripción 4/metabolismo , Factor de Transcripción 4/genética , Endotelio Corneal/metabolismo , Endotelio Corneal/patología , Masculino , Femenino , Transición Epitelial-Mesenquimal/genética , Anciano , Células Endoteliales/metabolismo , Células Endoteliales/patología , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Persona de Mediana Edad , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genéticaRESUMEN
Purpose: Fuchs endothelial corneal dystrophy (FECD) is characterized by Descemet's membrane (DM) abnormalities, namely an increased thickness and a progressive appearance of guttae and fibrillar membranes. The goal of this study was to identify abnormal extracellular matrix (ECM) proteins expressed in FECD DMs and to evaluate their impact on cell adhesion and migration. Methods: Gene expression profiles from in vitro (GSE112039) and ex vivo (GSE74123) healthy and FECD corneal endothelial cells were analyzed to identify deregulated matrisome genes. Healthy and end-stage FECD DMs were fixed and analyzed for guttae size and height. Immunostaining of fibronectin, tenascin-C, osteopontin, and type XIV collagen was performed on ex vivo specimens, as well as on tissue-engineered corneal endothelium reconstructed using healthy and FECD cells. An analysis of ECM protein expression according to guttae and fibrillar membrane was performed using immunofluorescent staining and phase contrast microscopy. Finally, cell adhesion was evaluated on fibronectin, tenascin-C, and osteopontin, and cell migration was studied on fibronectin and tenascin-C. Results: SPP1 (osteopontin), FN1 (fibronectin), and TNC (tenascin-C) genes were upregulated in FECD ex vivo cells, and SSP1 was upregulated in both in vitro and ex vivo FECD conditions. Osteopontin, fibronectin, tenascin-C, and type XIV collagen were expressed in FECD specimens, with differences in their location. Corneal endothelial cell adhesion was not significantly affected by fibronectin or tenascin-C but was decreased by osteopontin. The combination of fibronectin and tenascin-C significantly increased cell migration. Conclusions: This study highlights new abnormal ECM components in FECD, suggests a certain chronology in their deposition, and demonstrates their impact on cell behavior.
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Movimiento Celular , Endotelio Corneal , Fibronectinas , Distrofia Endotelial de Fuchs , Osteopontina , Tenascina , Humanos , Tenascina/metabolismo , Tenascina/genética , Fibronectinas/metabolismo , Fibronectinas/genética , Osteopontina/metabolismo , Osteopontina/genética , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Endotelio Corneal/metabolismo , Endotelio Corneal/patología , Anciano , Adhesión Celular , Células Cultivadas , Femenino , Masculino , Regulación de la Expresión Génica , Persona de Mediana Edad , Lámina Limitante Posterior/metabolismo , Lámina Limitante Posterior/patologíaRESUMEN
Fuchs endothelial corneal dystrophy (FECD) is a leading indication for corneal transplantation, but its molecular etiology remains poorly understood. We performed genome-wide association studies (GWAS) of FECD in the Million Veteran Program followed by multi-ancestry meta-analysis with the previous largest FECD GWAS, for a total of 3970 cases and 333,794 controls. We confirm the previous four loci, and identify eight novel loci: SSBP3, THSD7A, LAMB1, PIDD1, RORA, HS3ST3B1, LAMA5, and COL18A1. We further confirm the TCF4 locus in GWAS for admixed African and Hispanic/Latino ancestries and show an enrichment of European-ancestry haplotypes at TCF4 in FECD cases. Among the novel associations are low frequency missense variants in laminin genes LAMA5 and LAMB1 which, together with previously reported LAMC1, form laminin-511 (LM511). AlphaFold 2 protein modeling, validated through homology, suggests that mutations at LAMA5 and LAMB1 may destabilize LM511 by altering inter-domain interactions or extracellular matrix binding. Finally, phenome-wide association scans and colocalization analyses suggest that the TCF4 CTG18.1 trinucleotide repeat expansion leads to dysregulation of ion transport in the corneal endothelium and has pleiotropic effects on renal function.
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Distrofia Endotelial de Fuchs , Humanos , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Estudio de Asociación del Genoma Completo , Factor de Transcripción 4/genética , Colágeno , Laminina/genéticaRESUMEN
Fuchs endothelial corneal dystrophy (FECD) is the leading cause of endothelial keratoplasty without efficacious drug treatment. Recent studies have emphasized the involvement of epigenetic regulation in FECD development. Long non-coding RNAs (lncRNAs) are recognized as crucial epigenetic regulators in diverse cellular processes and ocular diseases. In this study, we revealed the expression patterns of lncRNAs using high-throughput sequencing technology in FECD mouse model, and identified 979 significantly dysregulated lncRNAs. By comparing the data from FECD human cell model, we obtained a series of homologous lncRNAs with similar expression patterns, and revealed that these homologous lncRNAs were enriched in FECD related biological functions, with apoptosis (mmu04210) showing the highest enrichment score. In addition, we investigated the role of lncRNA zinc finger antisense 1 (ZFAS1) in apoptotic process. This study would broaden our understanding of epigenetic regulation in FECD development, and provide potential anti-apoptotic targets for FECD therapy.
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Distrofia Endotelial de Fuchs , ARN Largo no Codificante , Animales , Humanos , Ratones , Endotelio Corneal/metabolismo , Epigénesis Genética , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , ARN Largo no Codificante/genética , Zinc/metabolismoRESUMEN
Maintaining corneal endothelial function is required for vision, and corneal endothelial dysfunction is a major cause of visual deficits and blindness worldwide. To date there has been a dearth of innovation for therapeutics targeting the corneal endothelium. However, recent advances in understanding the role of oxidative stress and mitochondrial dysfunction have revealed potential avenues for the development of new therapies. This review summarizes recent developments in elucidating the role of the NRF2 pathway in corneal endothelial health and disease, focusing specifically on Fuchs' endothelial corneal dystrophy and the loss of corneal endothelial cells associated with cataract surgery. The pro-mitochondrial and antioxidant phenotype elicited by NRF2 activation offers a promising opportunity for new therapeutics for the diseased corneal endothelium.
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Distrofia Endotelial de Fuchs , Factor 2 Relacionado con NF-E2 , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , Células Endoteliales/metabolismo , Endotelio Corneal/metabolismo , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Estrés OxidativoRESUMEN
Fuchs endothelial corneal dystrophy (FECD) is the leading indication for corneal transplantation worldwide. Our aim was to investigate the role of transient receptor potential vanilloid subtype 1 (TRPV1) and the associated immune regulation contributing to this pathological condition. Significant upregulation of TRPV1 was detected in the H2O2-induced in vitro FECD model. Based on gene expression microarray dataset GSE142538 and in vitro results, a comprehensive immune landscape was studied and a negative correlation was found between TRPV1 with different immune cells, especially regulatory T cells (Tregs). Functional analyses of the 313 TRPV1-related differentially expressed genes (DEGs) revealed the involvement of TRP-regulated calcium transport, as well as inflammatory and immune pathways. Four TRPV1-related core genes (MAPK14, GNB1, GNAQ, and ARRB2) were screened, validated by microarray dataset GSE112039 and the combined validation dataset E-GEAD-399 & 564, and verified by in vitro experiments. Our study suggested a potential crosstalk between TRPV1 and immune regulation contributing to FECD pathogenesis. The identified pivotal biomarkers and immune-related pathways provide a novel framework for future mechanistic and therapeutic studies of FECD.
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Distrofia Endotelial de Fuchs , Humanos , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Distrofia Endotelial de Fuchs/patología , Endotelio Corneal/metabolismo , Endotelio Corneal/patología , Peróxido de Hidrógeno/metabolismo , Regulación hacia Arriba , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
Fuchs endothelial corneal dystrophy (FECD) is a slowly evolving, bilateral disease of the corneal endothelium, characterized by an abnormal accumulation of extracellular matrix (ECM) in the basement membrane (Descemet's membrane, DM). This results in the formation of small round excrescences, called guttae, and a progressive disappearance of endothelial cells. In the intermediate stage, the numerous guttae create significant optical aberrations, and in the late stage, the loss of endothelial function leads to permanent corneal edema. The molecular components of guttae have not been fully elucidated. In the current study, we conducted shotgun proteomics of the DMs, including guttae, obtained from patients with FECD and revealed that 32 proteins were expressed only in the FECD-DMs but not in the DMs of control subjects. Subsequent enrichment analyses identified associations with multiple ECM-related pathways. Immunostaining of flat-mounted DMs confirmed that 4 of the top 5 identified proteins (hemoglobin α, SRPX2, tenascin-C, and hemoglobin γδεß) were expressed in FECD-DMs but not in non-FECD-DMs. Fibrinogen α was strongly expressed in FECD-DMs, but weakly expressed in non-FECD-DMs. We also demonstrated that matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) can display the in situ spatial distribution of biomolecules expressed in the DM, including the guttae.
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Distrofia Endotelial de Fuchs , Humanos , Distrofia Endotelial de Fuchs/metabolismo , Lámina Limitante Posterior , Proteómica , Células Endoteliales/metabolismo , Endotelio Corneal/metabolismoRESUMEN
Fuchs endothelial corneal dystrophy (FECD) is the most common inherited corneal disease. Fibrillar focal excrescences called guttae and corneal edema due to corneal endothelial cell death result in progressive vision loss. Multiple genetic variants have been reported, but the pathogenesis of FECD is not fully understood. In this study, we used RNA-Seq to analyze differential gene expression in the corneal endothelium obtained from patients with FECD. Differential expression analysis of transcriptomic profiles revealed that expression of 2366 genes (1092 upregulated and 1274 downregulated genes) was significantly altered in the corneal endothelium of patients with FECD compared to healthy subjects. Gene ontology analysis demonstrated an enrichment of genes involved in extracellular matrix (ECM) organization, response to oxidative stress, and apoptotic signaling. Several pathway analyses consistently indicated the dysregulation of ECM-associated pathways. Our differential gene expression findings support the previously proposed underlying mechanisms, including oxidative stress and apoptosis of endothelial cells, as well as the phenotypic clinical FECD hallmark of ECM deposits. Further investigation focusing on differentially expressed genes related to these pathways might be beneficial for elucidating mechanisms and developing novel therapies.
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Distrofia Endotelial de Fuchs , Humanos , Distrofia Endotelial de Fuchs/metabolismo , Células Endoteliales/metabolismo , RNA-Seq , Endotelio Corneal/patología , Córnea/patologíaRESUMEN
Fuchs Endothelial Corneal Dystrophy (FECD), a late-onset oxidative stress disorder, is the most common cause of corneal endothelial degeneration and is genetically associated with CTG repeat expansion in Transcription Factor 4 (TCF4). We previously reported accumulation of nuclear (nDNA) and mitochondrial (mtDNA) damage in FECD. Specifically, mtDNA damage was a prominent finding in development of disease in the ultraviolet-A (UVA) induced FECD mouse model. We hypothesize that an aberrant DNA repair may contribute to the increased DNA damage seen in FECD. We analyzed differential expression profiles of 84 DNA repair genes by real-time PCR arrays using Human DNA Repair RT-Profiler plates using cDNA extracted from Descemet's membrane-corneal endothelium (DM-CE) obtained from FECD patients with expanded (>40) or non-expanded (<40) intronic CTG repeats in TCF4 gene and from age-matched normal donors. Change in mRNA expression of <0.5- or >2.0-fold in FECD relative to normal was set as cutoff for down- or upregulation. Downregulated mitochondrial genes were further validated using the UVA-based mouse model of FECD. FECD specimens exhibited downregulation of 9 genes and upregulation of 8 genes belonging to the four major DNA repair pathways, namely, base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), and double strand break (DSB) repair, compared to normal donors. MMR gene MSH2 and BER gene POLB were preferentially upregulated in expanded FECD. BER genes LIG3 and NEIL2, DSB repair genes PARP3 and TOP3A, NER gene XPC, and unclassified pathway gene TREX1, were downregulated in both expanded and non-expanded FECD. MtDNA repair genes, Lig3, Neil2, and Top3a, were also downregulated in the UVA-based mouse model of FECD. Our findings identify impaired DNA repair pathways that may play an important role in DNA damage due to oxidative stress as well as genetic predisposition noted in FECD.
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ADN Glicosilasas , Distrofia Endotelial de Fuchs , Animales , Ratones , Humanos , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Endotelio Corneal/metabolismo , Predisposición Genética a la Enfermedad , Reparación del ADN/genética , ADN Mitocondrial/genética , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismoRESUMEN
Purpose: In the United States, 70% of Fuchs' endothelial corneal dystrophy (FECD) cases are caused by an intronic trinucleotide repeat expansion in the TCF4 gene. CUG repeat RNA transcripts from this expansion accumulate as nuclear foci in the corneal endothelium. In this study, we sought to detect foci in other anterior segment cell types and assess their molecular impact. Methods: We examined CUG repeat RNA foci appearance, expression of downstream affected genes, gene splicing, and TCF4 RNA expression in corneal endothelium, corneal stromal keratocytes, corneal epithelium, trabecular meshwork cells, and lens epithelium. Results: CUG repeat RNA foci, the hallmark of FECD in corneal endothelium (found in 84% of endothelial cells), are less detectable in trabecular meshwork cells (41%), much less prevalent in stromal keratocytes (11%) or corneal epithelium (4%), and absent in lens epithelium. With few exceptions including mis-splicing in the trabecular meshwork, differential gene expression and splicing changes associated with the expanded repeat in corneal endothelial cells are not observed in other cell types. Expression of the TCF4 transcripts including full-length isoforms containing the repeat sequence at the 5' end is much higher in the corneal endothelium or trabecular meshwork than in the corneal stroma or corneal epithelium. Conclusions: Expression of the CUG repeat containing TCF4 transcripts is higher in the corneal endothelium, likely contributing to foci formation and the large molecular and pathologic impact on those cells. Further studies are warranted to examine any glaucoma risk and impact of the observed foci in the trabecular meshwork of these patients.
Asunto(s)
Distrofia Endotelial de Fuchs , Expansión de Repetición de Trinucleótido , Humanos , Expansión de Repetición de Trinucleótido/genética , Células Endoteliales/metabolismo , Factor de Transcripción 4/genética , Distrofia Endotelial de Fuchs/metabolismo , Endotelio Corneal/metabolismo , ARN/genética , ARN/metabolismoRESUMEN
Late-onset Fuchs endothelial corneal dystrophy (FECD) is a disease affecting the corneal endothelium (CE), associated with a cytosine-thymine-guanine repeat expansion at the CTG18.1 locus in the transcription factor 4 (TCF4) gene. It is unknown whether CTG18.1 expansions affect global methylation including TCF4 gene in CE or whether global CE methylation changes at advanced age. Using genome-wide DNA methylation array, we investigated methylation in CE from FECD patients with CTG18.1 expansions and studied the methylation in healthy CE at different ages. The most revealing DNA methylation findings were analyzed by gene expression and protein analysis. 3488 CpGs had significantly altered methylation pattern in FECD though no substantial changes were found in TCF4. The most hypermethylated site was in a predicted promoter of aquaporin 1 (AQP1) gene, and the most hypomethylated site was in a predicted promoter of coagulation factor V (F5 for gene, FV for protein). In FECD, AQP1 mRNA expression was variable, while F5 gene expression showed a ~ 23-fold increase. FV protein was present in both healthy and affected CE. Further gene expression analysis of coagulation factors interacting with FV revealed a ~ 34-fold increase of thrombomodulin (THBD). THBD protein was detected only in CE from FECD patients. Additionally, we observed an age-dependent hypomethylation in elderly healthy CE.Thus, tissue-specific genome-wide and gene-specific methylation changes associated with altered gene expression were discovered in FECD. TCF4 pathological methylation in FECD because of CTG18.1 expansion was ruled out.
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Distrofia Endotelial de Fuchs , Humanos , Anciano , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Distrofia Endotelial de Fuchs/patología , Factor V/genética , Factor V/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Trombomodulina/genética , Trombomodulina/metabolismo , Metilación de ADN/genética , Factor de Transcripción 4/genética , Factor de Transcripción 4/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Expansión de Repetición de TrinucleótidoRESUMEN
PURPOSE: Fuchs endothelial corneal dystrophy (FECD) is a progressive corneal disease that impacts the structure and stiffness of the Descemet's membrane (DM), the substratum for corneal endothelial cells (CECs). These structural alterations of the DM could contribute to the loss of the CECs resulting in corneal edema and blindness. Oxidative stress and transforming growth factor-ß (TGF-ß) pathways have been implicated in endothelial cell loss and endothelial to mesenchymal transition of CECs in FECD. Ascorbic acid (AA) is found at high concentrations in FECD and its impact on CEC survival has been investigated. However, how TGF-ß and AA effect the composition and rigidity of the CEC's matrix remains unknown. METHODS: In this study, we investigated the effect of AA, TGF-ß1 and TGF-ß3 on the deposition, ultrastructure, stiffness, and composition of the extracellular matrix (ECM) secreted by primary bovine corneal endothelial cells (BCECs). RESULTS: Immunofluorescence and electron microscopy post-decellularization demonstrated a robust deposition and distinct structure of ECM in response to treatments. AFM measurements showed that the modulus of the matrix in BCECs treated with TGF-ß1 and TGF-ß3 was significantly lower than the controls. There was no difference in the stiffness of the matrix between the AA-treated cell and controls. Gene Ontology analysis of the proteomics results revealed that AA modulates the oxidative stress pathway in the matrix while TGF-ß induces the expression of matrix proteins collagen IV, laminin, and lysyl oxidase homolog 1. CONCLUSIONS: Molecular pathways identified in this study demonstrate the differential role of soluble factors in the pathogenesis of FECD.
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Distrofia Endotelial de Fuchs , Factor de Crecimiento Transformador beta1 , Animales , Bovinos , Factor de Crecimiento Transformador beta1/metabolismo , Células Endoteliales/metabolismo , Factor de Crecimiento Transformador beta3/metabolismo , Distrofia Endotelial de Fuchs/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Endotelio Corneal/metabolismoRESUMEN
AIM: a review of miRNA expression connected to epithelial mesenchymal transition studies in Fuchs' endothelial corneal dystrophy (FECD). METHODS: literature search strategy-PubMed central database, using "miRNA" or "microRNA" and "epithelial mesenchymal transition" or "EMT" and "Fuchs' endothelial corneal dystrophy" or "FECD" as keywords. Experimental or clinical studies on humans published in English regarding miRNA profiles of epithelial mesenchymal transition in Fuchs' endothelial corneal dystrophy published between 2009 and 2022 were included. CONCLUSION: The publications regarding the miRNA profiles of epithelial mesenchymal transition in Fuchs' endothelial corneal dystrophy are scarce but provide some valuable information about the potential biomarkers differentiating aging changes from early disease stages characterized by epithelial mesenchymal transition. In the corneal tissue of FECD patients, miRNA-184 seed-region mutation as well as unidirectional downregulation of total miRNA expression led by the miRNA-29 were demonstrated. For early diagnostics the miRNA of epithelial mesenchymal transition in aqueous humor should be analyzed and used as biomarkers.
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Distrofia Endotelial de Fuchs , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Transición Epitelial-Mesenquimal/genética , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , CórneaRESUMEN
BACKGROUND: To investigate and compare the metabolic profiles in the aqueous humour of Han Chinese patients with Fuchs' syndrome and presumed viral-induced anterior uveitis (PVIAU). METHODS: The metabolites in the aqueous humour of 20 Fuchs' syndrome patients, 20 PVIAU patients and 20 senile cataract control patients were detected by liquid chromatography with mass spectrometry. Differential metabolites were analysed by Student's t test, multivariate analysis, cluster analysis and correlation analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to explore the potential disrupted metabolic pathways in Fuchs' syndrome and PVIAU. RESULTS: Comparisons of metabolic profiles identified 29 differential metabolites between Fuchs' syndrome patients and controls, 36 differential metabolites between PVIAU patients and controls, and 30 differential metabolites between Fuchs' syndrome patients and PVIAU patients. DL-serine was markedly elevated in Fuchs' syndrome, and 1-palmitoyl-sn-glycero-3-phosphocholine in PVIAU. KEGG pathway analysis suggested that the differential metabolites in Fuchs' syndrome compared with control were mostly enriched in central carbon metabolism in cancer, adenosine triphosphate-binding cassette (ABC) transporters and mineral absorption, while those in PVIAU compared with control were mostly enriched in protein digestion and absorption, biosynthesis of unsaturated fatty acids, and ABC transporters. The metabolic pathways differentially affected in Fuchs' syndrome compared to PVIAU included central carbon metabolism in cancer, protein digestion and absorption and ascorbate and aldarate metabolism. CONCLUSIONS: In Fuchs' syndrome and PVIAU patients, the aqueous humour exhibited specific metabolic profiles and enriched metabolic pathways, which provides a better understanding of the pathogenesis of Fuchs' syndrome and PVIAU in Han Chinese patients.
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Catarata , Distrofia Endotelial de Fuchs , Uveítis Anterior , Uveítis , Humanos , Humor Acuoso/metabolismo , Uveítis Anterior/complicaciones , Catarata/etiología , Síndrome , Metaboloma , Carbono/metabolismo , Distrofia Endotelial de Fuchs/metabolismo , Uveítis/complicacionesRESUMEN
Corneal endothelial cell (CEC) dysfunction causes corneal edema and severe visual impairment that require transplantation to restore vision. To address the unmet need of organ shortage, descemetorhexis without endothelial keratoplasty has been specifically employed to treat early stage Fuchs endothelial corneal dystrophy, which is pathophysiologically related to oxidative stress and exhibits centrally located corneal guttae. After stripping off central Descemet's membrane, rho-associated protein kinase (ROCK) inhibitor has been found to facilitate CEC migration, an energy-demanding task, thereby achieving wound closure. However, the correlation between ROCK inhibition and the change in bioenergetic status of CECs remained to be elucidated. Through transcriptomic profiling, we found that the inhibition of ROCK activity by the selective inhibitor, ripasudil or Y27632, promoted enrichment of oxidative phosphorylation (OXPHOS) gene set in bovine CECs (BCECs). Functional analysis revealed that ripasudil, a clinically approved anti-glaucoma agent, enhanced mitochondrial respiration, increased spare respiratory capacity, and induced overexpression of electron transport chain components through upregulation of AMP-activated protein kinase (AMPK) pathway. Accelerated BCEC migration and in vitro wound healing by ripasudil were diminished by OXPHOS and AMPK inhibition, but not by glycolysis inhibition. Correspondingly, lamellipodial protrusion and actin assembly that were augmented by ripasudil became reduced with additional OXPHOS or AMPK inhibition. These results indicate that ROCK inhibition induces metabolic reprogramming toward OXPHOS to support migration of CECs.
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Endotelio Corneal , Distrofia Endotelial de Fuchs , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Bovinos , Células Endoteliales/metabolismo , Endotelio Corneal/metabolismo , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Distrofia Endotelial de Fuchs/cirugía , Fosforilación Oxidativa , Quinasas Asociadas a rho/metabolismoRESUMEN
AIMS: To investigate the genetic and functional association of an intronic variant of LAMC1, rs3768617 with Fuchs endothelial corneal dystrophy (FECD) in the Indian population. METHODS: Blood samples were collected from age and sex matched 356 controls and 120 FECD patients after a detailed assessment via specular microscopy. Genomic DNA was extracted and genotyping was done by fluorescence based capillary electrophoresis. The genetic association of rs3768617 polymorphisms was computed by the chi-square (χ2) test. Bioinformatics studies were performed to find the allele specific binding of different transcription factors in the region of rs3768617 and functional evaluation assessed by luciferase assay followed by Electrophoretic Mobility Shift Assay (EMSA) and Chromatin Immunoprecipitation assay (ChIP). Immunofluorescence assay was carried out to check for any differential expression of GFI1B between control and FECD endothelium samples. RESULTS: SNP rs3768617 {chr1:183123365 (GRCh38.p13)} was found to be genetically associated with FECD in Indian population (p = 2.646 × 10-8). Luciferase assay suggested that the rs3768617 locus has a regulatory role. In silico analysis showed that the transcription factor, GFI1B binds to the risk allele 'G' of rs3768617, but not to the protective allele 'A' which was also experimentally validated by EMSA. High enrichment of DNA flanking the surrounding region of rs3768617 was also found in presence of GFI1B specific antibody in ChIP assay. There was a 0.63 fold decrease in GFI1B expression in FECD affected corneal endothelium compared to control endothelium. CONCLUSIONS: The genetic association of rs3768617 in LAMC1 with FECD pathogenesis is mediated by GFI1B, thus finding the functional role of LAMC1 in FECD pathogenesis.
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Distrofia Endotelial de Fuchs/genética , Laminina/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Anciano , Alelos , Femenino , Distrofia Endotelial de Fuchs/metabolismo , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , India , Intrones , Laminina/metabolismo , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos , Factores de RiesgoRESUMEN
Fuchs' endothelial corneal dystrophy (FECD) is a bilateral disease of the cornea caused by gradual loss of corneal endothelial cells. Late-onset FECD is strongly associated with the CTG18.1 trinucleotide repeat expansion in the Transcription Factor 4 gene (TCF4), which forms RNA nuclear foci in corneal endothelial cells. To date, 46 RefSeq transcripts of TCF4 are annotated by the National Center of Biotechnology information (NCBI), however the effect of the CTG18.1 expansion on expression of alternative TCF4 transcripts is not completely understood. To investigate this, we used droplet digital PCR for quantification of TCF4 transcripts spanning over the CTG18.1 and transcripts with transcription start sites immediately downstream of the CTG18.1. TCF4 expression was analysed in corneal endothelium and in whole blood of FECD patients with and without CTG18.1 expansion, in non-FECD controls without CTG18.1 expansion, and in five additional control tissues. Subtle changes in transcription levels in groups of TCF4 transcripts were detected. In corneal endothelium, we found a lower fraction of transcripts spanning over the CTG18.1 tract compared to all other tissues investigated.
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Distrofia Endotelial de Fuchs/genética , Factor de Transcripción 4/genética , Expansión de Repetición de Trinucleótido/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Córnea , Células Endoteliales/metabolismo , Endotelio Corneal/metabolismo , Femenino , Distrofia Endotelial de Fuchs/metabolismo , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Factores de Transcripción/genéticaRESUMEN
Fuchs endothelial corneal dystrophy (FECD) is a genetically complex, heterogenous, age-related degenerative disease of corneal endothelial cells (CEnCs), occurring in the fifth decade of life with a higher incidence in females. It is characterized by extracellular matrix (ECM) protein deposition called corneal guttae, causing light glare and visual complaints in patients. Corneal transplantation is the only treatment option for FECD patients, which imposes a substantial socioeconomic burden. In FECD, CEnCs exhibit stress-induced senescence, oxidative stress, DNA damage, heightened reactive oxygen species (ROS) production, mitochondrial damage, and dysfunction as well as sustained endoplasmic reticulum (ER) stress. Among all of these, mitochondrial dysfunction involving altered mitochondrial bioenergetics and dynamics plays a critical role in FECD pathogenesis. Extreme stress initiates mitochondrial damage, leading to activation of autophagy, which involves clearance of damaged mitochondria called auto(mito)phagy. In this review, we discuss the role of mitochondrial dysfunction and mitophagy in FECD. This will provide insights into a novel mechanism of mitophagy in post-mitotic ocular cell loss and help us explore the potential treatment options for FECD.
Asunto(s)
Células Endoteliales/patología , Endotelio Corneal/patología , Distrofia Endotelial de Fuchs/patología , Mitocondrias/patología , Mitofagia , Animales , Daño del ADN , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Células Endoteliales/metabolismo , Endotelio Corneal/metabolismo , Metabolismo Energético , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Transducción de SeñalRESUMEN
PURPOSE: To investigate the association of body mass index (BMI) with Fuchs endothelial corneal dystrophy (FECD) severity and TCF4 CTG18.1 expansion. METHODS: A total of 343 patients with FECD were enrolled from the Mayo Clinic. FECD severity was graded by slit-lamp biomicroscopy. BMI values were obtained from the electronic medical records. DNA extracted from leukocytes was analyzed for CTG18.1 expansion length, with ≥40 repeats considered expanded. Wilcoxon signed-rank tests were used to compare FECD grade and CTG18.1 expansion length in patients by BMI (<25, ≥25 to <30, and ≥30 kg/m2). FECD grade was regressed on age, sex, BMI, and CTG18.1 expansion and, separately, BMI on CTG18.1 expansion. Models were investigated for effect modification by age and sex with an interaction term of P < 0.05 considered statistically significant. RESULTS: When examining the association between BMI and FECD, there was a significant interaction between BMI and sex (P for interaction = 0.004). When controlling for age and CTG18.1 expansion, a positive association was observed between BMI and FECD grade in women, but not in men. In addition, BMI was not associated with CTG18.1 expansion when controlling for age and sex. CONCLUSIONS: BMI was positively associated with FECD severity among women but not men. There was no significant association between BMI and CTG18.1 expansion. These findings suggest that increased BMI is potentially a modifiable risk factor for FECD disease progression among women.