Amniotic membrane transplantation combined with conjunctival flap covering surgery for the treatment of corneal perforations in fungal keratitis.

Purpose: To investigate the efficiency of amniotic membrane transplantation (AMT) combined with conjunctival flap covering surgery (CFCS) for patients with corneal perforations in fungal keratitis (FK). Methods: In this non-comparative, retrospective case series, 16 participants of corneal perforation in FK were successfully treated by a combination of multilayer AMT and bipedicle conjunctival flap with partial tenon's capsule. Corneal healing, recurrence of FK, visual acuity, and relevant complications were reported as outcome measures. Results: Sixteen patients (13 male, 3 female) had a mean age of 58.8 ± 10.3 (range 29-72) years. The mean diameter of corneal perforation was 1.9 ± 0.7 (range 0.5-2.8) mm. Corneal perforations healed and all the patients preserved their eyeballs. During the 11.0 ± 4.4 (range 6-18) months of follow-up, there was no recurrence of FK in any of these cases. Visual acuity improved in 15 eyes (93.8 %) and remained unchanged in 1 patient (6.3 %) who had no light perception when first admitted. All 6 patients who accepted secondary keratoplasty showed improved best corrected visual acuity of more than 4 lines. The most frequently found fungi were Aspergillus species (6 of 16, 37.5 %) and Fusarium species (4 of 16, 25.0 %), followed by 1 Scedosporium apiospermum (1 of 16, 6.3 %). Conclusions: Combination AMT with CFCS is a safe and effective surgery for patients with corneal perforations in FK, particularly where eye banks and fresh corneas are not available. This surgery could preserve the integrity of the eyeball and avoid the recurrence of FK. Besides, it provides a greater opportunity for further optical keratoplasty.

Roles of Optineurin and Extracellular Vesicles in Glaucomatous Retinal Cell Loss.

OBJECTIVE: To explore the role of extracellular vesicles (EVs) in the pathogenesis of glaucoma caused by E50K mutation. METHODS: A photoreceptor cell line, RGC-5, was transfected with empty plasmids and plasmids expressing wild-type (WT) optineurin (OPTN) or E50K OPTN to investigate the effects of OPTN glaucoma as well as to identify the role of EVs in glaucoma pathology. The RGC-5 cells were also stimulated with glutamate, and their viability was evaluated using flow cytometry or CCK-8 assay. EVs were extracted, labeled with PKH-26, and added into the medium for normal RGC-5 culture, and the status of the cells was observed thereafter. RESULTS: WT OPTN overexpression, E50K OPTN, and glutamate stimulation induced apoptosis of RGC-5 cells. However, when glutamate stimulation was used as an add-on treatment, the degree of apoptosis in WT OPTN-overexpressing RGC-5 cells was significantly lower than that in E50K OPTN-expressing and normal RGC-5 cells. The viability of normal RGC-5 cells was reduced when co-cultured with WT OPTN-overexpressing RGC-5 or E50K OPTN-overexpressing RGC-5. EVs released by the latter two transfected lines similarly reduced normal RGC-5 survival. CONCLUSION: Our results indicate that WT OPTN overexpression may lead to photoreceptor apoptosis. However, overexpression also confers a degree of protection against high concentrations of extracellular glutamate. Additionally, EVs released by transfected RGC-5 cells may regulate the cell state. These findings may improve our understanding of the mechanisms of cell-cell interactions in pathological conditions, providing a basis for the use of EVs as novel targets for early diagnosis and treatment of glaucoma.

A Novel Glycolysis-Related Signature for Predicting the Prognosis and Immune Infiltration of Uveal Melanoma.

INTRODUCTION: As the most common aggressive intraocular cancer in adults, uveal melanoma (UVM) threatens the survival and vision of many people. Glycolysis is a novel hallmark of cancer, but the role of glycolysis-related genes in UVM prognosis remains unknown. The purpose of the study was to establish a glycolysis-related gene signature (GRGS) to predict UVM prognosis. METHODS: Raw data were obtained from TCGA-UVM and GSE22138 datasets. The GRGS was established by univariate, LASSO, and multivariate Cox regression analyses. Kaplan-Meier survival and time-dependent receiver operating characteristic curves were used to evaluate the predictive ability of the GRGS. The relationships of the GRGS with infiltrating immune cell levels and mutations were analyzed with CIBERSORT and maftools. RESULTS: A novel GRGS (risk score = 0.690861*ISG20 + 0.070991*MET - 0.227520*SDC2 + 0.690223*FBP1 + 0.048008*CLN6 - 0.128520*SDC3) was developed for predicting UVM prognosis. The GRGS had robust predictive stability in UVM. Enrichment annotation suggested that the high-risk group had stronger adaptive immune responses and that the low-risk group had more innate immune cell infiltration. Moreover, BAP1 mutation was related to high risk, and SF3B1 mutation was related to low risk. CONCLUSIONS: This study developed and validated a novel GRGS to predict UVM prognosis and immune infiltration. The signature revealed an association between glycolysis-related genes and the tumor microenvironment, providing new insights into the role of glycolysis in UVM.

Effects of Exosomal microRNAs on Oral Mucosal Epithelial Cells Cocultured with Limbal Niche Cells.

Autologous cultivated oral mucosal epithelial transplantation (COMET) is an important method for the treatment of limbal stem cell deficiency (LSCD), but the appearance of peripheral corneal neovascularization after COMET has prevented its widespread use in clinical practice. Using limbal niche cells (LNCs) as feeders in the process of coculturing could inhibit the postoperative corneal neovascularization. However, the specific mechanism is still unclear. In this study, LNCs were used as feeder cells to alter the phenotype of cultured oral mucosal epithelial cells (COMECs) by mimicking the primitive limbal microenvironment. The high-throughput sequencing of COMECs cocultured with LNCs or 3T3 cells (named LNCs group and 3T3 groups) was performed, and differential miRNA expression was analyzed. A total of 99 known and 1 newly predicted miRNAs were significantly upregulated in the LNCs group, while 101 known and 8 newly predicted miRNAs were significantly downregulated. A total of 3000 target genes with the 60 most significantly differentially expressed miRNAs were predicted, and 7 upregulated and 7 downregulated miRNAs were ultimately screened. The supernatants obtained from both cocultures were found to be rich in exosomes, indicating that the intercellular communication between COMECs and LNCs or 3T3 cells was highly active. Furthermore, the expression levels of rno-miR-200-5p, rno-miR-204-5p, rno-miR-126a-3p, rno-miR-192-5p, rno-miR-211-5p, rno-miR-143-3p, and rno-miR-184 were significantly higher in the LNCs group compared to the 3T3 group, and the expression levels had a similar trend in exosomes. Meanwhile, sequencing of the cell lines revealed 7 miRNAs that were significantly downregulated in the LNCs group. Interestingly, in that case, rno-miR-23a-3p, rno-miR-379-5p, and rno-miR-127-5p were also significantly downregulated in the exosomes. In summary, this study suggested that signal transduction between cells mediated by exosomal miRNAs may be an important factor for the inhibition of angiogenesis by LNCs nourished COMECs.

Quantitative Assessment of Three-Dimensional Choroidal Vascularity and Choriocapillaris Flow Signal Voids in Myopic Patients Using SS-OCTA.

PURPOSE: To compare the choroidal vascularity of large- and middle-sized choroidal vessels and choriocapillaris (CC) perfusion in patients with different degrees of myopia using swept-source optical coherence tomography angiography (SS-OCTA). METHODS: One hundred and thirteen people with myopia were enrolled. SS-OCTA was performed to analyze the choroidal vascularity and CC perfusion. Three-dimensional (3D) choroidal vascularity index (CVI) and choroidal luminal volumes (LV) were obtained by artificial intelligence segmentation of the choroidal lumen in Volume OCT images. CC perfusion was assessed by flow signal voids (FSVs). RESULTS: In the macular, multiple linear regression model showed that choroidal thickness (CT), total choroidal volume, LV, and choroidal stromal volume were negatively correlated with axis length (AL), respectively (all p < 0.001). Three dimensional CVI was negatively associated with AL (p < 0.05). FSV% was positively correlated with age only (p < 0.001). Additionally, after adjustment for age and AL, FSV% had a significant negative correlation with CT (p < 0.05). CONCLUSION: Choroidal vascularity decreases gradually with increasing severity of myopia. The decrease of CC blood perfusion was related to a higher severity of myopia and the thinning of choroid.

Subconjunctival Injection of Transdifferentiated Oral Mucosal Epithelial Cells for Limbal Stem Cell Deficiency in Rats.

Rat limbal niche cells (LNCs) have been proven to induce transdifferentiation of oral mucosal epithelial cells (OMECs) into corneal epithelial-like cells termed transdifferentiated oral mucosal epithelial cells (T-OMECs). This investigation aimed to evaluate the effect of subconjunctival T-OMEC injections on alkali-induced limbal stem cell deficiency (LSCD) in rats. LNCs were cocultured with OMECs in the Transwell system to obtain T-OMECs, with NIH-3T3 cells serving as a control. Subconjunctival injection of single T-OMEC or OMEC suspension was performed immediately after corneal alkali injury. T-OMECs were prelabeled with the fluorescent dye CM-DiI in vitro and tracked in vivo. Corneal epithelial defect, opacity, and neovascularization were quantitatively analyzed. The degree of corneal epithelial defect (from day 1 onward), opacity (from day 5 onward), and neovascularization (from day 2 onward) was significantly less in the T-OMEC group than in the OMEC group. Cytokeratin 12 (CK12), pigment epithelium-derived factor, and soluble fms-like tyrosine kinase-1 were expressed at a higher rate following T-OMEC injection. Some CM-DiI-labeled cells were found to be coexpressed with CK12, Pax6, and ΔNp63α in the corneal epithelium after subconjunctival injection. Subconjunctival injection of T-OMECs prevents conjunctival invasion and maintains a normal corneal phenotype, which might be a novel strategy in the treatment of LSCD.

Characterization of a novel mutation in the MYOC gene in a Chinese family with primary open­angle glaucoma.

Although primary open­angle glaucoma (POAG)­related mutations in the myocilin (MYOC) gene have been reported, the underlying associations remain poorly understood. In the present study, the relationship between a MYOC mutation and POAG was investigated using ophthalmic examination and total exon gene sequencing in a Chinese family comprised of 5 individuals with POAG and 15 unaffected individuals. Pathogenic mutations underlying POAG were identified by whole­exome sequencing and subsequently validated by Sanger sequencing. Of the family members, nine (45%) harbored heterozygous p.D208Y mutations; among these, five had POAG and four were unaffected. The mean age at diagnosis was 26.2±4.12 years and the mean intraocular pressure (IOP) was 39.7±16.58 mmHg; all affected members complained of vision loss, headaches and eye swelling. Among the five cases of POAG, two presented with blindness. Among 10 members of the family who underwent comprehensive ophthalmologic examination, 3 individuals exhibited severe visual field defects. The mean age at the time of operation was 27.2±3.54 years. In the present study, a novel MYOC mutation (c.G622T: p.D208Y) was identified that was associated with severe visual impairment, high IOP and the need for frequent surgical interventions. Some carriers of the mutation were young and did not show signs of glaucoma. These individuals should be followed­up to firmly establish whether the mutated gene is pathogenic for POAG.

Limbal niche cells can reduce the angiogenic potential of cultivated oral mucosal epithelial cells.

BACKGROUND: Autologous cultivated oral mucosal epithelial transplantation (COMET) is an important treatment for limbal stem cell deficiency. However, peripheral corneal neovascularization after surgery hinders its application. This study aims to employ a culture system using allogenic limbal niche cells (LNCs) instead of mouse-derived 3T3 cells as a feeder layer that could relieve postoperative neovascularization. METHODS: Rat oral mucosal epithelial cells (OMECs) were co-cultured with rat LNCs or 3T3 cells. Cultivated oral mucosal epithelial cells (COMECs) of different culture systems were identified by hematoxylin and eosin staining and immunocytochemistry. The expression levels of the angiogenesis-related factors were analyzed by RT-qPCR and western blotting/ELISA. Angiogenic potential was reconfirmed by cell viability and tube formation assays with human umbilical vein endothelial cells (HUVECs). RESULTS: COMECs were obtained from both culture systems successfully. Immunocytochemistry showed approximately equal percentages of positive staining cells for p63α (p = 0.9177), ABCG2 (p = 0.526), Ki67 (p = 0.0987), and CK3 (p = 0.4000) in COMECs of different groups. RT-qPCR and western blotting/ELISA showed that COMECs of the LNC group expressed a significantly lower amount of basic fibroblast growth factor (bFGF) (p = 0.0038 for RT-qPCR, p = 0.0026 for western blotting) but more pigment epithelium-derived factor (PEDF) (p = 0.0172 for RT-qPCR, p = 0.0253 for western blotting) and soluble fms-like tyrosine kinase-1 (sFlt-1) (p < 0.0001 for RT-qPCR, p = 0.0064 for ELISA) than the COMECs of the 3T3 group. Furthermore, compared with COMECs of the 3T3 group, COMECs of the LNC group could reduce the viability (p = 0.0002) and tube formation (p = 0.0002) of HUVECs. CONCLUSIONS: LNCs could substitute 3T3 cells for expanding OMECs in vitro, and the COMECs obtained in this system are less likely to induce postsurgical neovascularization, which provides an alternative option for an ex vivo culture system and promotes the application of COMET.

Limbal niche cells: a novel feeder cell for autologous cultivated oral mucosal epithelial transplantation.

AIM: To establish a culture system using conspecific limbal niche cells (LNCs) as feeders for autologous cultivated oral mucosal epithelial transplantation (COMET). MATERIALS & METHODS: Rabbit oral epithelial sheets, harvested from culture systems containing LNCs or 3T3 cells, were transplanted onto limbal stem cell-deficient rabbit eyes (COMET-3T3 or COMET-LNCs). RESULTS: After COMET, corneas were relatively restored, with the exception of mild neovascularization in one cornea of the COMET-3T3 group. CD34 was detected in COMET-3T3 group corneas. Corneas of the COMET-LNCs group expressed high levels of PEDF and sFlt-1, but low levels of bFGF, compared with expression in COMET-3T3 corneas. CONCLUSION: The culture system containing conspecific LNC feeders could substitute for the 3T3 cell system and decrease the risk of neovascularization after COMET.

Rat limbal niche cells can induce transdifferentiation of oral mucosal epithelial cells into corneal epithelial-like cells in vitro.

BACKGROUND: Cultivated oral mucosal epithelial cells (OMECs) are widely used in the treatment of limbal stem cell deficiency (LSCD) for their ocular reconstruction capability. As the most important component of the limbal microenvironment, limbal niche cells (LNCs) play a key role in the direction of stem cell differentiation. In this study, we investigated whether LNCs can induce the transdifferentiation of rat OMECs to corneal epithelial-like cells. METHODS: We isolated OMECs and LNCs from rats by dispase and collagenase, respectively, to establish a three-dimensional or Transwell coculturing system. NIH-3T3 cells and renewed LNCs were also used as feeder layers in the Transwell system to compare their ability to support the OMECs. The airlift method was used for the culture of OMECs to obtain a stratified epithelial sheet. Cocultured OMECs were characterized by reverse-transcription polymerase chain reaction, Western blotting, hematoxylin and eosin staining, and immunohistochemistry. RESULTS: The cocultured OMECs showed corneal epithelial-like morphology and expressed the corneal epithelial markers CK12 and Pax6 in most cocultured systems. Furthermore, we found that the expression level of CK12, Pax6, and proliferation marker Ki67 was upregulated when compared with that of other groups by renewing the LNCs in the Transwell system (p < 0.05, n = 3), suggesting that this might be a potential method for improving the efficiency of transdifferentiation. The obtained stratified epithelial sheet expressed CK3 and CK12. CONCLUSION: Through coculturing OMECs and LNCs in vitro, we successfully cultivated corneal epithelial-like OMECs. This investigation is of great significance for the treatment of LSCD and ocular surface reconstruction.