Sleep deprivation induces corneal endothelial dysfunction by downregulating Bmal1.

BACKGROUND: Sleep deprivation (SD) is a common public health problem that contributes to various physiological disorders and increases the risk of ocular diseases. However, whether sleep loss can damage corneal endothelial function remains unclear. This study aimed to determine the effect and possible mechanism of SD on the corneal endothelium. METHODS: Male C57BL/6J mice were subjected to establish SD models. After 10 days, quantitative RT-PCR (qRT-PCR) and western blot or immunostaining for the expression levels of zonula occludens-1 (ZO-1), ATPase Na+/K + transporting subunit alpha 1 (Atp1a1), and core clock genes in the corneal endothelium were evaluated. Reactive oxygen species staining and mitochondrial abundance characterized the mitochondrial function. The regulatory role of Bmal1 was confirmed by specifically knocking down or overexpressing basic helix-loop-helix ARNT like 1 protein (Bmal1) in vivo. In vitro, a mitochondrial stress test was conducted on cultured human corneal endothelial cells upon Bmal1 knockdown. RESULTS: SD damaged the barrier and pump functions of mouse corneal endothelium, accompanied by mitochondrial dysfunction. Interestingly, SD dramatically downregulated the core clock gene Bmal1 expression level. Bmal1 knockdown disrupted corneal endothelial function, while overexpression of Bmal1 ameliorated the dysfunction induced by SD. Mitochondrial bioenergetic deficiency mediated by Bmal1 was an underlying mechanism for SD induced corneal endothelial dysfunction. CONCLUSION: The downregulation of Bmal1 expression caused by SD led to corneal endothelial dysfunction via impairing mitochondrial bioenergetics. Our findings offered insight into how SD impairs the physiological function of the corneal endothelium and expanded the understanding of sleep loss leading to ocular diseases.

Protective effects of curcumin on corneal endothelial cell PANoptosis and monocyte adhesion induced by tumor necrosis factor-alpha and interferon-gamma in rats.

Decrease of human corneal endothelial cell (CEC) density leads to corneal edema, progressive corneal opacity, and reduced visual acuity. A reduction in CEC density may be related to elevated levels of inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interferon (INF)-γ. PANoptosis, characterized by the activation of apoptosis, necroptosis, and pyroptosis, could be a factor in the loss of CECs driven by TNF-α and INF-γ. Cytokines also stimulate monocytes adhesion to endothelium. It has been shown in previous research that curcumin plays protective roles against numerous corneal inflammatory diseases. However, it is not determined whether curcumin acts as an anti-PANoptotic agent or if it mitigates monocyte adhesion to CECs. Therefore, this research aimed to explor the potential therapeutic effects of curcumin and its underlying mechanisms in the loss of CECs. CEC injury models were established, and curcumin was injected subconjunctivally. Clinical evaluation of the corneas was conducted using a scoring system and anterior segment photography. Corneal observation was performed with hematoxylin and eosin staining and immunostaining of zona occludens-1(ZO-1). Apoptotic cells within the corneal endothelium were observed using TUNEL staining. The detection of primary proteins expression was accomplished through Western blot analysis. Interleukin (IL)-1ß and macrophage chemotactic protein 1 (MCP-1) levels were determined via ELISA, while the expression of cleaved caspase-3, gasdermin-D (GSDMD), phosphor-mixed lineage kinase domain-like protein (p-MLKL) and intercellular cell adhesion molecule-1 were confirmed by immunofluorescence. Myeloperoxidase (MPO) activity was measured in aqueous humors. Curcumin treatment attenuated the loss of CECs and corneal edema caused by TNF-α and IFN-γ. Besides, it decreased the count of TUNEL-positive cells, and inhibited the upregulation of cleaved caspase-3, cleaved caspase-6, cleaved caspase-7, and cleaved poly (ADP-ribose) polymerase. Moreover, both the expression and phosphorylation of MLKL and receptor-interacting protein 3 were decreased in curcumin-treated rats. Furthermore, curcumin also lowered the expression of cleaved caspase-1, diminished the levels of IL1ß and MCP-1, and inhibited the activity of MPO. Besides, the expression of intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, as well as the number of CD11b-positive cells adhered to the CECs decreased for the administration of curcumin.

Dysregulation of the TCF4 Isoform in Corneal Endothelial Cells of Patients With Fuchs Endothelial Corneal Dystrophy.

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.

Transcription factor 4 promotes increased corneal endothelial cellular migration by altering microtubules in Fuchs endothelial corneal dystrophy.

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.

Deciphering novel TCF4-driven mechanisms underlying a common triplet repeat expansion-mediated disease.

Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.

Alginate Hydrogel Integrated with a Human Fibroblast-Derived Extracellular Matrix Supports Corneal Endothelial Cell Functionality and Suppresses Endothelial-Mesenchymal Transition.

Human corneal transplantation is still the only option to restore the function of corneal endothelial cells (CECs). Therefore, there is an urgent need for hCEC delivery systems to replace the human donor cornea. Here, we propose an alginate hydrogel (AH)-based delivery system, where a human fibroblast-derived, decellularized extracellular matrix (ECM) was physically integrated with AH. This AH securely combined with the ECM (ECM-AH) was approximately 50 µm thick, transparent, and permeable. The surface roughness and surface potential provided ECM-AH with a favorable microenvironment for CEC adhesion and growth in vitro. More importantly, ECM-AH could support the structural (ZO-1) and functional (Na+/K+-ATPase) markers of hCECs, as assessed via western blotting and quantitative polymerase chain reaction, which were comparable with those of a ferritic nitrocarburizing (FNC)-coated substrate (a positive control). The cell density per unit area was also significantly better with ECM-AH than the FNC substrate at day 7. A simulation test of cell engraftment in vitro showed that hCECs were successfully transferred into the decellularized porcine corneal tissue, where they were mostly alive. Furthermore, we found out that the endothelial-mesenchymal transition (EnMT)-inductive factors (Smad2 and vimentin) were largely declined with the hCECs grown on ECM-AH, whereas the EnMT inhibitory factor (Smad7) was significantly elevated. The difference was statistically significant compared to that of the FNC substrate. Moreover, we also observed that TGF-ß1-treated hCECs showed faster recovery of cell phenotype on the ECM. Taken together, our study demonstrates that ECM-AH is a very promising material for hCEC culture and delivery, which endows an excellent microenvironment for cell function and phenotype maintenance.

Nicotinamide promotes the differentiation of functional corneal endothelial cells from human embryonic stem cells.

Corneal transplantation represents the primary therapeutic approach for managing corneal endothelial dysfunction, but corneal donors remain scarce. Anterior chamber cell injection emerges as a highly promising alternative strategy for corneal transplantation, with pluripotent stem cells (PSC) demonstrating considerable potential as an optimal cell source. Nevertheless, only a few studies have explored the differentiation of functional corneal endothelial-like cells originating from PSC. In this investigation, a chemical-defined protocol was successfully developed for the differentiation of functional corneal endothelial-like cells derived from human embryonic stem cells (hESC). The application of nicotinamide (NAM) exhibited a remarkable capability in suppressing the fibrotic phenotype, leading to the generation of more homogeneous and well-distinctive differentiated cells. Furthermore, NAM effectively suppressed the expression of genes implicated in endothelial cell migration and extracellular matrix synthesis. Notably, NAM also facilitated the upregulation of surface marker genes specific to functional corneal endothelial cells (CEC), including CD26 (-) CD44 (-∼+-) CD105 (-) CD133 (-) CD166 (+) CD200 (-). Moreover, in vitro functional assays were performed, revealing intact barrier properties and Na+/K+-ATP pump functionality in the differentiated cells treated with NAM. Consequently, our findings provide robust evidence supporting the capacity of NAM to enhance the differentiation of functional CEC originating from hESC, offering potential seed cells for therapeutic interventions of corneal endothelial dysfunction.

Expression and Impact of Fibronectin, Tenascin-C, Osteopontin, and Type XIV Collagen in Fuchs Endothelial Corneal Dystrophy.

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.

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

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

Long noncoding RNA ZFAS1: A novel anti-apoptotic target in Fuchs endothelial corneal dystrophy.

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.

Dynamic changes of endothelial and stromal cilia are required for the maintenance of corneal homeostasis.

Primary cilia are distributed extensively within the corneal epithelium and endothelium. However, the presence of cilia in the corneal stroma and the dynamic changes and roles of endothelial and stromal cilia in corneal homeostasis remain largely unknown. Here, we present compelling evidence for the presence of primary cilia in the corneal stroma, both in vivo and in vitro. We also demonstrate dynamic changes of both endothelial and stromal cilia during corneal development. In addition, our data show that cryoinjury triggers dramatic cilium formation in the corneal endothelium and stroma. Furthermore, depletion of cilia in mutant mice lacking intraflagellar transport protein 88 compromises the corneal endothelial capacity to establish the effective tissue barrier, leading to an upregulation of α-smooth muscle actin within the corneal stroma in response to cryoinjury. These observations underscore the essential involvement of corneal endothelial and stromal cilia in maintaining corneal homeostasis and provide an innovative strategy for the treatment of corneal injuries and diseases.

Impact of culture media on primary human corneal endothelial cells derived from old donors.

Corneal endothelial dysfunction is a major indication for corneal transplantation. However, a global shortage of donor corneal tissues and risks associated with corneal surgeries have prompted exploration of alternative options, including tissue-engineered grafts or cell injection therapy. Nonetheless, these approaches require a controlled culture of primary human corneal endothelial cells (HCEnCs). Although HCEnCs established from young donors are generally more proliferative and maintain a better phenotype, corneas from old donors are more frequently accessible from eye banks due to a lower corneal endothelial cell count than the necessary threshold required for transplantation. In this study, we investigated various culture media to evaluate which one is the most appropriate for stimulating the proliferation while maintaining cell morphology and function of HCEnCs derived from old donors (age >65 years). All experiments were performed on paired research-grade donor corneas, divided for the conditions under investigation in order to minimize the inter-donor variability. Cell morphology as well as expression of specific markers were assessed at both mRNA (CD166, SLC4A11, ATP1A1, COL8A1, α-SMA, CD44, COL1A1, CDKN2A, LAP2A and LAP2B) and protein (ZO-1, α-SMA, Ki67 and LAP2) levels. Results obtained showed how the Dual Media formulation maintained the hexagonal phenotype more efficiently than Single Medium, but cell size gradually increased with passages. In contrast, the Single Medium provided a higher proliferation rate and a prolonged in vitro expansion but acquired an elongated morphology. To summarize, Single medium and Dual media preserve morphology and functional phenotype of HCEnCs from old donor corneas at low passages while maintenance of the same cell features at high passages remains an active area of research. The new insights revealed within this work become particularly relevant considering that the elderly population a) is the main target of corneal endothelial therapy, b) represents the majority of corneal donors. Therefore, the proper expansion of HCEnCs from old donors is essential to develop novel personalised therapeutic strategies and reduce requirement of human corneal tissues globally.

Evaluation of biophysical alterations in the epithelial and endothelial layer of patients with Bullous Keratopathy.

The cornea is a fundamental ocular tissue for the sense of sight. Thanks to it, the refraction of two-thirds of light manages to participate in the visual process and protect against mechanical damage. Because it is transparent, avascular, and innervated, the cornea comprises five main layers: Epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Each layer plays a key role in the functionality and maintenance of ocular tissue, providing unique ultrastructural and biomechanical properties. Bullous Keratopathy (BK) is an endothelial dysfunction that leads to corneal edema, loss of visual acuity, epithelial blisters, and severe pain, among other symptoms. The corneal layers are subject to changes in their biophysical properties promoted by Keratopathy. In this context, the Atomic Force Microscopy (AFM) technique in air was used to investigate the anterior epithelial surface and the posterior endothelial surface, healthy and with BK, using a triangular silicone tip with a nominal spring constant of 0.4 N/m. Six human corneas (n = 6) samples were used for each analyzed group. Roughness data, calculated by third-order polynomial adjustment, adhesion, and Young's modulus, were obtained to serve as a comparison and identification of morphological and biomechanical changes possibly associated with the pathology, such as craters and in the epithelial layer and exposure of a fibrotic layer due to loss of the endothelial cell wall. Endothelial cell membrane area and volume data were calculated, obtaining a relevant comparison between the control and patient. Such results may provide new data on the physical properties of the ocular tissue to understand the physiology of the cornea when it has pathology.

Targeting the NRF2 pathway: A promising approach for corneal endothelial dysfunction.

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.

Transcriptomic meta-analysis reveals ERRα-mediated oxidative phosphorylation is downregulated in Fuchs' endothelial corneal dystrophy.

BACKGROUND: Late-onset Fuchs' endothelial corneal dystrophy (FECD) is a degenerative disease of cornea and the leading indication for corneal transplantation. Genetically, FECD patients can be categorized as with (RE+) or without (RE-) the CTG trinucleotide repeat expansion in the transcription factor 4 gene. The molecular mechanisms underlying FECD remain unclear, though there are plausible pathogenic models proposed for RE+ FECD. METHOD: In this study, we performed a meta-analysis on RNA sequencing datasets of FECD corneal endothelium including 3 RE+ datasets and 2 RE- datasets, aiming to compare the transcriptomic profiles of RE+ and RE- FECD. Gene differential expression analysis, co-expression networks analysis, and pathway analysis were conducted. RESULTS: There was a striking similarity between RE+ and RE- transcriptomes. There were 1,184 genes significantly upregulated and 1,018 genes significantly downregulated in both RE+ and RE- cases. Pathway analysis identified multiple biological processes significantly enriched in both-mitochondrial functions, energy-related processes, ER-nucleus signaling pathway, demethylation, and RNA splicing were negatively enriched, whereas small GTPase mediated signaling, actin-filament processes, extracellular matrix organization, stem cell differentiation, and neutrophil mediated immunity were positively enriched. The translational initiation process was downregulated in the RE+ transcriptomes. Gene co-expression analysis identified modules with relatively distinct biological processes enriched including downregulation of mitochondrial respiratory chain complex assembly. The majority of oxidative phosphorylation (OXPHOS) subunit genes, as well as their upstream regulator gene estrogen-related receptor alpha (ESRRA), encoding ERRα, were downregulated in both RE+ and RE- cases, and the expression level of ESRRA was correlated with that of OXPHOS subunit genes. CONCLUSION: Meta-analysis increased the power of detecting differentially expressed genes. Integrating differential expression analysis with co-expression analysis helped understand the underlying molecular mechanisms. FECD RE+ and RE- transcriptomic profiles are much alike with the hallmark of downregulation of genes in pathways related to ERRα-mediated OXPHOS.

Integrative analysis of gene expression datasets in corneal endothelium samples of Fuchs endothelial corneal dystrophy.

FECD is an age-related progressive ocular disorder characterized by the gradual loss of corneal endothelial cells. Although the exact pathogenesis of FECD remains incompletely understood, differentially expressed genes in the corneal endothelium of FECD patients compared to controls have been reported in several studies. However, a consensus regarding consistently affected genes in FECD has not been established. To address this issue, we conducted a comprehensive meta-analysis incorporating five studies with data that met our predefined inclusion criteria. The combined dataset included 41 FECD patients and 26 controls. We conducted study-level analyses, followed by a meta-analysis, and subsequent functional enrichment analysis targeting the topmost DEGs. Our findings revealed a total of 1537 consistently dysregulated genes in the corneal endothelium of FECD patients. Notably, only 14.6% (224/1537) of these DEGs had been previously identified as statistically significant in individual datasets. Functional enrichment analysis revealed that the upregulated DEGs were significantly enriched in immune-related signaling pathways, with a particularly high enrichment in "The NLRP3 inflammasome" and "Inflammasomes" pathways. In conclusion, we successfully identify a set of consistently dysregulated genes in FECD, which are associated with both established and novel biological pathways. This study highlights the importance of further investigating the role of inflammasomes in FECD pathogenesis and exploring strategies to modulate inflammasome activation for the management of this debilitating condition.

Adipose Mesenchymal Stem Cell-Derived Exosomes Promote the Regeneration of Corneal Endothelium Through Ameliorating Senescence.

Purpose: Human corneal endothelial cells (hCECs) have been considered unable to regenerate in vivo, resulting in corneal decompensation after significant loss of hCECs. adipose-derived mesenchymal stem cell (ASC)-derived exosomes can regenerate tissues and organs. In this study, we investigated whether ASC-derived exosomes could protect and regenerate CECs. Methods: We performed cell viability and cell-cycle analyses to evaluate the effect of ASC-derived exosomes on the regeneration capacity of cultured hCECs. Transforming growth factor-ß (TGF-ß) and hydrogen peroxide (H2O2) were used to induce biological stress in CECs. The effect of ASC-derived exosomes on CECs was investigated in vivo. ASC-derived exosomes were introduced into rat CECs using electroporation, and rat corneas were injured using cryoinjury. Next-generation sequencing analysis was performed to compare the differentially expressed microRNAs (miRNAs) between ASC-derived and hCEC-derived exosomes. Results: ASC-derived exosomes induced CEC proliferation and suppressed TGF-ß- or H2O2-induced oxidative stress and senescence. ASC-derived exosomes protect hCECs against TGF-ß- or H2O2-induced endothelial-mesenchymal transition and mitophagy. In an in vivo study, ASC-derived exosomes promoted wound healing of rat CECs and protected the corneal endothelium against cryoinjury-induced corneal endothelium damage. Next-generation sequencing analysis revealed differentially expressed miRNAs for ASC-derived and hCEC-derived exosomes. They are involved in lysine degradation, adherens junction, the TGF-ß signaling pathway, the p53 signaling pathway, the Hippo signaling pathway, the forkhead box O (FoxO) signaling pathway, regulation of actin cytoskeleton, and RNA degradation based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Conclusions: ASC-derived exosomes promoted wound healing and regeneration of endothelial cells by inducing a shift in the cell cycle and suppressing senescence and autophagy.

ROCK Inhibitors as an Alternative Therapy for Corneal Grafting: A Systematic Review.

Currently, corneal blindness is affecting >10 million individuals worldwide, and there is a significant unmet medical need because only 1.5% of transplantation needs are met globally due to a lack of high-quality grafts. In light of this global health disaster, researchers are developing corneal substitutes that can resemble the human cornea in vivo and replace human donor tissue. Thus, this review examines ROCK (Rho-associated coiled-coil containing protein kinases) inhibitors as a potential corneal wound-healing (CWH) therapy by reviewing the existing clinical and nonclinical findings. The systematic review was done from PubMed, Scopus, Web of Science, and Google Scholar for CWH, corneal injury, corneal endothelial wound healing, ROCK inhibitors, Fasudil, Netarsudil, Ripasudil, Y-27632, clinical trial, clinical study, case series, case reports, preclinical study, in vivo, and in vitro studies. After removing duplicates, all downloaded articles were examined. The literature search included the data till January 2023. This review summarized the results of ROCK inhibitors in clinical and preclinical trials. In a clinical trial, various ROCK inhibitors improved CWH in individuals with open-angle glaucoma, cataract, iris cyst, ocular hypertension, and other ocular diseases. ROCK inhibitors also improved ocular wound healing by increasing cell adhesion, migration, and proliferation in vitro and in vivo. ROCK inhibitors have antifibrotic, antiangiogenic, anti-inflammatory, and antiapoptotic characteristics in CWH, according to the existing research. ROCK inhibitors were effective topical treatments for corneal infections. Ripasudil, Y-27632, H-1152, Y-39983, and AMA0526 are a few new ROCK inhibitors that may help CWH and replace human donor tissue.

Crosstalk between TRPV1 and immune regulation in Fuchs endothelial corneal dystrophy.

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.

Manufacturing of human corneal endothelial grafts.

PURPOSE: Human corneal endothelial cells (HCECs) play a significant role in maintaining visual function. However, these cells are notorious for their limited proliferative capacity in vivo. Current treatment of corneal endothelial dysfunction resorts to corneal transplantation. Herein we describe an ex vivo engineering method to manufacture HCEC grafts suitable for transplantation through reprogramming into neural crest progenitors. METHODS: HCECs were isolated by collagenase A from stripped Descemet membrane of cadaveric corneoscleral rims, and induced reprogramming via knockdown with p120 and Kaiso siRNAs on collagen IV-coated atelocollagen. Engineered HCEC grafts were released after assessing their identity, potency, viability, purity and sterility. Phase contrast was used for monitoring cell shape, graft size, and cell density. Immunostaining was used to determine the normal HCEC phenotype with expression of N-cadherin, ZO-1, ATPase, acetyl-α-tubulin, γ-tubulin, p75NTR, α-catenin, ß-catenin, and F-actin. Stability of manufactured HCEC graft was evaluated after transit and storage for up to 3 weeks. The pump function of HCEC grafts was measured by lactate efflux. RESULTS: One HCEC graft suitable for corneal transplantation was generated from 1/8th of the donor corneoscleral rim with normal hexagonal cell shape, density, and phenotype. The manufactured grafts were stable for up to 3 weeks at 37 °C or up to 1 week at 22 °C in MESCM medium and after transcontinental shipping at room temperature by retaining normal morphology (hexagonal, >2000 cells/mm2, >8 mm diameter), phenotype, and pump function. CONCLUSIONS: This regenerative strategy through knockdown with p120 and Kaiso siRNAs can be used to manufacture HCEC grafts with normal phenotype, morphology and pump function following prolonged storage and shipping.