IGF2BP2 Maintains Retinal Pigment Epithelium Homeostasis by Stabilizing PAX6 and OTX2.

Purpose: N6-methyladenosine (m6A) methylation is a chemical modification that occurs on RNA molecules, where the hydrogen atom of adenine (A) nucleotides is replaced by a methyl group, forming N6-methyladenosine. This modification is a dynamic and reversible process that plays a crucial role in regulating various biological processes, including RNA stability, transport, translation, and degradation. Currently, there is a lack of research on the role of m6A modifications in maintaining the characteristics of RPE cells. m6A readers play a crucial role in executing the functions of m6A modifications, which prompted our investigation into their regulatory roles in the RPE. Methods: Phagocytosis assays, immunofluorescence staining, flow cytometry experiments, ß-galactosidase staining, and RNA sequencing (RNA-seq) were conducted to assess the functional and cellular characteristics changes in retinal pigment epithelium (RPE) cells following short-hairpin RNA-mediated knockdown of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). RNA-seq and ultraviolet crosslinking immunoprecipitation with high-throughput sequencing (HITS-CLIP) were employed to identify the target genes regulated by IGF2BP2. adeno-associated virus (AAV) subretinal injection was performed in 6- to 8-week-old C57 mice to reduce IGF2BP2 expression in the RPE, and the impact of IGF2BP2 knockdown on mouse visual function was assessed using immunofluorescence, quantitative real-time PCR, optical coherence tomography, and electroretinography. Results: IGF2BP2 was found to have a pronounced effect on RPE phagocytosis. Subsequent in-depth exploration revealed that IGF2BP2 modulates the mRNA stability of PAX6 and OTX2, and the loss of IGF2BP2 induces inflammatory and aging phenotypes in RPE cells. IGF2BP2 knockdown impaired RPE function, leading to retinal dysfunction in vivo. Conclusions: Our data suggest a crucial role of IGF2BP2 as an m6A reader in maintaining RPE homeostasis by regulating the stability of PAX6 and OTX2, making it a potential target for preventing the occurrence of retinal diseases related to RPE malfunction.

Deficiency of SECTM1 impairs corneal wound healing in aging.

The corneal epithelium is the outermost transparent barrier of the eyeball and undergoes continuous self-renewal by limbal stem cells (LSCs) during its lifetime; however, the impact of aging on LSCs remains largely unknown. Here, we showed that the healing ability of the cornea in elderly macaques (Macaca fascicularis) was significantly decreased compared to that of younger macaques. This delayed wound closure accompanied a disordered cell arrangement and corneal opacity. A novel cytokine, Secreted and Transmembrane 1 (SECTM1), was found to facilitate corneal healing and was upregulated in young macaques upon wounding. Mechanistically, SECTM1 is essential for LSC migration and proliferation, and may partially function through Cell Division Cycle Associated 7 (CDCA7). Notably, the topical application of SECTM1 to aged wounded corneas dramatically promoted re-epithelialization and improved corneal transparency in both mice and macaques. Our work suggests that aging may impair the expression of healing response factors and injury repair in non-human primate corneas, and that SECTM1 application could potentially benefit corneal wound healing in clinical treatment.

The single-cell transcriptomic atlas and RORA-mediated 3D epigenomic remodeling in driving corneal epithelial differentiation.

Proper differentiation of corneal epithelial cells (CECs) from limbal stem/progenitor cells (LSCs) is required for maintenance of ocular homeostasis and clear vision. Here, using a single-cell transcriptomic atlas, we delineate the comprehensive and refined molecular regulatory dynamics during human CEC development and differentiation. We find that RORA is a CEC-specific molecular switch that initiates and drives LSCs to differentiate into mature CECs by activating PITX1. RORA dictates CEC differentiation by establishing CEC-specific enhancers and chromatin interactions between CEC gene promoters and distal regulatory elements. Conversely, RORA silences LSC-specific promoters and disrupts promoter-anchored chromatin loops to turn off LSC genes. Collectively, our work provides detailed and comprehensive insights into the transcriptional dynamics and RORA-mediated epigenetic remodeling underlying human corneal epithelial differentiation.

Single-cell transcriptomics implicates the FEZ1-DKK1 axis in the regulation of corneal epithelial cell proliferation and senescence.

Limbal stem/progenitor cells (LSC) represent the source of corneal epithelium renewal. LSC proliferation and differentiation are essential for corneal homeostasis, however, the regulatory mechanism remains largely unexplored. Here, we performed single-cell RNA sequencing and discovered proliferation heterogeneity as well as spontaneously differentiated and senescent cell subgroups in multiply passaged primary LSC. Fasciculation and elongation protein zeta 1 (FEZ1) and Dickkopf-1 (DKK1) were identified as two significant regulators of LSC proliferation and senescence. These two factors were mainly expressed in undifferentiated corneal epithelial cells (CECs). Knocking down the expression of either FEZ1 or DKK1 reduced cell division and caused cell cycle arrest. We observed that DKK1 acted as a downstream target of FEZ1 in LSC and that exogenous DKK1 protein partially prevented growth arrest and senescence upon FEZ1 suppression in vitro. In a mouse model of corneal injury, DKK1 also rescued the corneal epithelium after recovery was inhibited by FEZ1 suppression. Hence, the FEZ1-DKK1 axis was required for CEC proliferation and the juvenile state and can potentially be targeted as a therapeutic strategy for promoting recovery after corneal injury.

Arrested Phase Separation Enables High-Performance Keratoprostheses.

Corneal transplantation is impeded by donor shortages, immune rejection, and ethical reservations. Pre-made cornea prostheses (keratoprostheses) offer a proven option to alleviate these issues. Ideal keratoprostheses must possess optical clarity and mechanical robustness, but also high permeability, processability, and recyclability. Here, it is shown that rationally controlling the extent of arrested phase separation can lead to optimized multiscale structure that reconciles permeability and transparency, a previously conflicting goal by common pore-forming strategies. The process is simply accomplished by hydrothermally treating a dense and transparent hydrophobic association hydrogel. The examination of multiscale structure evolution during hydrothermal treatment reveals that the phase separation with upper miscibility gap evolves to confer time-dependent pore growth due to slow dynamics of polymer-rich phase which is close to vitrification. Such a process can render a combination of multiple desired properties that equal or surpass those of the state-of-the-art keratoprostheses. In vivo tests confirm that the keratoprosthesis can effectively repair corneal perforation and restore a transparent cornea with treatment outcomes akin to that of allo-keratoplasty. The keratoprosthesis is easy to access and convenient to carry, and thus would be an effective temporary substitute for a corneal allograft in emergency conditions.

Cotransplantation of Limbal Epithelial and Stromal Cells for Ocular Surface Reconstruction.

Purpose: To propose an improved stem cell-based strategy for limbal stem cell deficiency (LSCD) treatment. Design: Experimental randomized or parallel-group animal study. Subjects: Fifty adult male New Zealand white rabbits. Methods: Human limbal stem/progenitor cells (LSCs) and limbal stromal stem/progenitor cells (LSSCs) were cultured in serum-free conditions and further differentiated into corneal epithelial cells and keratocytes, respectively. All cell types were characterized with lineage-specific markers. Gene expression analysis was performed to identify the potential function of LSSCs in corneal regeneration. Two LSCD models of rabbits for transplantations were used: transplantation performed at the time of limbal and corneal epithelial excision (LSCD model) and transplantation performed after clinical signs were induced in an LSCD model (pLSCD model). The pLSCD model better mimics the pathologic changes and symptoms of human LSCD. Rabbit models received LSC or LSC plus LSSC treatment. Corneal epithelial defects, neovascularization, and opacity were assessed every 3 weeks for 24 weeks. ZsGreen-labeled LSSCs were used for short-term tracking in vivo. Main Outcome Measures: Rates of corneal epithelial defect area, corneal neovascularization and opacity scores, graft survival rate, and immunofluorescence staining of specific markers. Results: Both LSC transplantation and LSC plus LSSC cotransplantation effectively repaired the corneal surface in the LSCD model. These 2 strategies showed no significant differences in terms of graft survival rate or epithelial repair. However, corneal opacity was observed in the LSC group (in 3 of 8 rabbits), but not in the LSC plus LSSC group. Notably, when treating LSCD rabbits with distinguishable stromal opacification and neovascularization, cotransplantation of LSCs and LSSCs exhibited significantly better therapeutic effects than transplantation of LSCs alone, with graft survival rates of 87.5% and 37.5%, respectively. The implanted LSSCs could differentiate into keratocytes during the wound-healing process. RNA sequencing analysis showed that the stromal cells produced not only a collagen-rich extracellular matrix to facilitate reconstruction of the lamellar structure, but also niche factors that accelerated epithelial cell growth and inhibited angiogenesis and inflammation. Conclusions: These findings highlight the support of stromal cells in niche homeostasis and tissue regeneration, providing LSC plus LSSC cotransplantation as a new treatment strategy for corneal blindness.

Cis-regulatory chromatin loops analysis identifies GRHL3 as a master regulator of surface epithelium commitment.

Understanding the regulatory network of cell fate acquisition remains a major challenge. Using the induction of surface epithelium (SE) from human embryonic stem cells as a paradigm, we show that the dynamic changes in morphology-related genes (MRGs) closely correspond to SE fate transitions. The marked remodeling of cytoskeleton indicates the initiation of SE differentiation. By integrating promoter interactions, epigenomic features, and transcriptome, we delineate an SE-specific cis-regulatory network and identify grainyhead-like 3 (GRHL3) as an initiation factor sufficient to drive SE commitment. Mechanically, GRHL3 primes the SE chromatin accessibility landscape and activates SE-initiating gene expression. In addition, the evaluation of GRHL3-mediated promoter interactions unveils a positive feedback loop of GRHL3 and bone morphogenetic protein 4 on SE fate decisions. Our work proposes a concept that MRGs could be used to identify cell fate transitions and provides insights into regulatory principles of SE lineage development and stem cell-based regenerative medicine.

Healing Ability of Central Corneal Epithelium in Rabbit Ocular Surface Injury Models.

Purpose: Wound healing of the corneal epithelium mainly involves two types of cells: limbal stem/progenitor cells (LSCs) and differentiated central corneal epithelial cells (CECs). The healing ability of CECs is still debatable, and its correlated transcriptomic alterations during wound healing are yet to be elucidated. This study aimed to determine the healing ability and mechanisms underlying the actions of CECs using rabbit ocular surface injury models. Methods: A central corneal ring-like residual epithelium model was used to investigate the healing ability of CECs. Uninjured and injury-stimulated LSCs and CECs were collected for transcriptomic analysis. The analysis results were verified by quantitative reverse transcriptase polymerase chain reaction, immunofluorescence staining, and two types of rabbit corneal injury models. Results: During wound healing, the upregulated genes in LSCs were mostly enriched in the mitotic cell cycle-related processes, but those in CECs were mostly enriched in cell adhesion and migration. CECs could repair the epithelial defects successfully at one-time injuries. However, after repetitive injuries, the CECs repaired notably slower and failed to completely heal the defect, but the LSCs repaired even faster than the one-time injury. Conclusions: Our results indicated rabbit CECs repair the epithelial defect mainly depending on migration and its proliferative ability is limited, and LSCs are the main source of regenerative epithelial cells. Translational Relevance: This study provides information on gene expression in the corneal epithelium during wound healing, indicating that regulation of the cell cycle, cell adhesion, and migration may be the basis for future treatment strategies for corneal wound healing.

Comprehensive 3D epigenomic maps define limbal stem/progenitor cell function and identity.

The insights into how genome topology couples with epigenetic states to govern the function and identity of the corneal epithelium are poorly understood. Here, we generate a high-resolution Hi-C interaction map of human limbal stem/progenitor cells (LSCs) and show that chromatin multi-hierarchical organisation is coupled to gene expression. By integrating Hi-C, epigenome and transcriptome data, we characterize the comprehensive 3D epigenomic landscapes of LSCs. We find that super-silencers mediate gene repression associated with corneal development, differentiation and disease via chromatin looping and/or proximity. Super-enhancer (SE) interaction analysis identified a set of SE interactive hubs that contribute to LSC-specific gene activation. These active and inactive element-anchored loop networks occur within the cohesin-occupied CTCF-CTCF loops. We further reveal a coordinated regulatory network of core transcription factors based on SE-promoter interactions. Our results provide detailed insights into the genome organization principle for epigenetic regulation of gene expression in stratified epithelia.

Loss of FOXC1 contributes to the corneal epithelial fate switch and pathogenesis.

Forkhead box C1 (FOXC1) is required for neural crest and ocular development, and mutations in FOXC1 lead to inherited Axenfeld-Rieger syndrome. Here, we find that FOXC1 and paired box 6 (PAX6) are co-expressed in the human limbus and central corneal epithelium. Deficiency of FOXC1 and alternation in epithelial features occur in patients with corneal ulcers. FOXC1 governs the fate of the corneal epithelium by directly binding to lineage-specific open promoters or enhancers marked by H3K4me2. FOXC1 depletion not only activates the keratinization pathway and reprograms corneal epithelial cells into skin-like epithelial cells, but also disrupts the collagen metabolic process and interferon signaling pathways. Loss of interferon regulatory factor 1 and PAX6 induced by FOXC1 dysfunction is linked to the corneal ulcer. Collectively, our results reveal a FOXC1-mediated regulatory network responsible for corneal epithelial homeostasis and provide a potential therapeutic target for corneal ulcer.

In vivo post-contrast 1H-MRS evaluation of malignant and benign breast lesions: a meta-analysis.

The aim of this study is to perform a meta-analysis to evaluate the diagnostic performance of the in vivo post-contrast proton magnetic resonance spectroscopy (MRS) for benign/malignant discrimination of focal breast lesions. Sixteen studies with a total of 661 malignant breast lesions and 388 benign breast lesions were included. The pooled sensitivity and specificity of post-contrast 1H-MRS were 74 % (95 % confidence interval (CI) 70-77 %) and 78 % (95 % CI 73-82 %), respectively. The positive likelihood ratio (PLR) and the negative likelihood ratio (NLR) were 4.00 (95 % CI 2.74-5.84) and 0.25 (95 % CI 0.17-0.37), respectively. From the fitted summary receiver operating characteristics curve (SROC), the AUC and Q* index were 0.89 and 0.83. Publication bias was present (t = 2.43, P = 0.029). Meta-regression analysis suggested that neither threshold effect nor evaluated covariates including method of choline analysis, strength of field, pulse sequence, repetition time (TR), and time interval were sources of heterogeneity (all P values >0.05). In vivo post-contrast 1H-MRS was useful for differentiation between malignant and benign focal breast lesions. However, pooled diagnostic measures might be overestimated. The standardization of the acquisition protocol as well as the post-processing method for post-contrast proton MRS need to be established for the future study.

Differential diagnosis between hepatic metastases and benign focal lesions using DWI with parallel acquisition technique: a meta-analysis.

We aim to investigate the diagnostic capability of diffusion-weighted imaging using parallel acquisition technique for the differentiation between hepatic metastases and benign focal lesions with a meta-analysis. The meta-analysis included a total of 858 hepatic metastases and 440 benign liver lesions from nine studies. The pooled sensitivity and specificity of diffusion-weighted imaging (DWI) were 0.87 (95% CI, 0.84-0.89) and 0.90 (95% CI, 0.87-0.93), respectively. The positive likelihood ratio (PLR) and negative likelihood ratio (NLR) were 8.50 (95% CI, 4.97-14.52) and 0.17 (95% CI, 0.11-0.26), respectively. The P value for χ (2) heterogeneity for all pooled estimates was <0.05. From the fitted summary receiver operating characteristics (SROC), the area under the curve (AUC) and Q* index were 0.95 and 0.88, respectively. Publication bias is not present (t = -0.76, P = 0.471). The meta-regression analysis indicated that evaluated covariates included patient number, patient population, mean age, maximum of b factor, number of cysts, number of hemangiomas, and field were not sources of heterogeneity (all P value >0.05). Diffusion-weighted imaging was useful for differentiation between hepatic metastases and benign focal lesions. The diffusion characteristics of the benign hepatocellular lesions, including cases of focal nodular hyperplasia (FNH) and adenoma, have rarely been reported and need further studies. The diagnostic capability of DWI with parallel acquisition technique for differentiation between metastases and benign hepatic focal lesions might be overestimated.