Bone Marrow Mesenchymal Stem Cells (BMSCs)-Derived Exosomes Promotes Proliferation and Differentiation of Retinal Neuron-Like Cells to Repair Corneal Epithelial Damage

Dry eye disease (DED) is a common ocular surface disease. Bone marrow mesenchymal stem cells (BMSCs) can differentiate into various cells, and BMSC-derived exosomes (BMSC-exo) is essential to maintaining BMSCs stemness. This study aimed to elucidate the mechanism underlying BMSCexo in DED. Sixty rats with corneal epithelial injury were treated with BMSCs or BMSC-exo and untreated (each group, n = 20) followed by analysis of the effect of BMSCs and BMSC-exo by evaluating the corneal epithelium damage via measuring the Basso-Beattie-Bresnahan (BBB) score on 1st, 3rd, 7th, 14th, 28th day after treatments. TUNEL staining assessed cell apoptosis, NF200 expression and the number of BrdU-positive cells. There was no significant difference in BBB scores among three groups on the 1st and 3rd day after treatment (p > 0.05) with significant difference on the 7th, 14th, and 28th day (p <0.05); compared with control group, BMSCs group and combination group had significantly higher BBB score (p < 0.05). The amount of apoptotic cells rose on 3rd and then gradually decreased since 7th day. Moreover, BMSCs and BMSC-exo decreased the apoptotic index and increased absorbance of NF200 and BrdU-positive rate (p < 0.05). BMSC-exo alleviates corneal epithelial damage in DED and facilitates wound healing possibly through reducing cell apoptosis and increasing retinal neuron-like cell proliferation protein.

Corneal epithelial defects following vitreoretinal surgery: incidence and outcomes from the DISCOVER study

AIM: To investigate the incidence, risk factors, clinical course, and outcomes of corneal epithelial defects (CED) following vitreoretinal surgery in a prospective study setting. METHODS: This was a post-hoc analysis of all participants in DISCOVER intraoperative optical coherence tomography study. Subjects with CED 1d after surgery without intraoperative corneal debridement was defined as the postoperative CED group. Subjects who underwent intraoperative debridement were defined as intraoperative debridement group. Eyes were matched 2:1 with controls (eyes without postoperative CED) for comparative assessment. The primary outcomes were the incidence of CED on postoperative day one and the incidence of required intraoperative debridement. Secondary outcomes included time to defect closure, delayed healing (&#x003E;2wk), visual acuity (VA) and presence of scarring at one year and cornea consult. RESULTS: This study included 856 eyes that underwent vitreoretinal surgery. Intraoperative corneal debridement was performed to 61 (7.1%) subjects and postoperative CED developed spontaneously in 94 (11.0%) subjects. Significant factors associated with postoperative CED included prolonged surgical duration (P=0.003), diabetes mellitus (P=0.04), postoperative ocular hypotension (P&#x003C;0.001). Prolonged surgical duration was associated with intraoperative debridement. Delayed defect closure time (&#x003E;2wk) was associated with corneal scar formation at the end of the 1y in all epithelial defect subjects (P&#x003C;0.001). The overall rate of corneal scarring for all eyes undergoing vitrectomy was 1.8%. CONCLUSION: Prolonged duration of surgery is the strongest factor associated with both intraoperative debridement and spontaneous postoperative CED. Delayed defect closure is associated with a greater risk of corneal scarring at one year. The overall rate of corneal scarring following vitrectomy is low at &#x003C;2%.

TLR4-Dependent DUOX2 Activation Triggered Oxidative Stress and Promoted HMGB1 Release in Dry Eye

Dry eye disease (DED) is one of the most common ocular surface diseases worldwide. DED has been characterized by excessive accumulation of reactive oxygen species (ROS), following significant corneal epithelial cell death and ocular surface inflammation. However, the key regulatory factor remains unclear. In this study, we tended to explore whether DUOX2 contributed to DED development and the underlying mechanism. Human corneal epithelial (HCE) cells were treated with hyperosmolarity, C57BL/6 mice were injected of subcutaneous scopolamine to imitate DED. Expression of mRNA was investigated by RNA sequencing (RNA-seq) and quantitative real-time PCR (qPCR). Protein changes and distribution of DUOX2, high mobility group box 1 (HMGB1), Toll-like receptor 4 (TLR4), and 4-hydroxynonenal (4-HNE) were evaluated by western blot assays and immunofluorescence. Cell death was assessed by Cell Counting Kit-8 (CCK8), lactate dehydrogenase (LDH) release, and propidium iodide (PI) staining. Cellular ROS levels and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. RNA-seq and western blot assay indicated a significant increase of DUOX2 dependent of TLR4 activation in DED both in vitro and in vivo. Immunofluorescence revealed significant translocation of HMGB1 within corneal epithelial cells under hyperosmolar stress. Interestingly, after ablated DUOX2 expression by siRNA, we found a remarkable decrease of ROS level and recovered MMP in HCE cells. Moreover, knockdown of DUOX2 greatly inhibited HMGB1 release, protected cell viability and abolished inflammatory activation. Taken together, our data here suggest that upregulation of DUOX2 plays a crucial role in ROS production, thereafter, induce HMGB1 release and cell death, which triggers ocular surface inflammation in DED.

Evaluation of Central Corneal Epithelial Thickness With Anterior Segment OCT in Patients With Type 2 Diabetes Mellitus

Aim: The aim of this study was to evaluate the central corneal thickness (CCT) and central corneal epithelial thickness (CCET) in patients with Type 2 diabetes mellitus (DM), and the effect of the duration of diabetes, the degree of diabetic retinopathy (DR), and HbA1c level. Method: CCT and CCET values ​​of 72 patients diagnosed with type 2 DM and 72 healthy individuals were measured by anterior segment optical coherence tomography (AS-OCT). The eye tear function was evaluated with the Tear Break-up Time test (TBUT) and the Schirmer test. From the results of fundus examination, the diabetic patients were grouped as those without DR, with non-proliferative DR, and with proliferative DR. The disease duration and the HbA1c levels were recorded. Results: In the diabetic patients, the mean CCT was determined to be thicker (p=0.025), the CCET was thinner (p=0.003), and the TBUT and Schirmer values were lower (p<0.001, p<0.001, respectively). The duration of diabetes and the HbA1c level were not found to have any statistically significant effect on these parameters (p>0.05). The presence of retinopathy had no significant effect on CCT, TBUT and Schirmer values. The CCET was determined to be thinner in patients with retinopathy (p<0.001). Conclusion: As the corneal epithelial thickness is reduced in patients with advanced diabetic retinopathy, corneal epithelial pathologies can be seen more often. Therefore, early and effective treatment can be started taking into consideration the complications which may develop associated with the corneal epithelium following surgical procedures, especially those applied to the cornea.

Activity-Dependent Neuroprotective Protein (ADNP)-Derived Peptide (NAP) Counteracts UV-B Radiation-Induced ROS Formation in Corneal Epithelium

The corneal epithelium, the outermost layer of the cornea, acts as a dynamic barrier preventing access to harmful agents into the intraocular space. It is subjected daily to different insults, and ultraviolet B (UV-B) irradiation represents one of the main causes of injury. In our previous study, we demonstrated the beneficial effects of pituitary adenylate cyclase-activating polypeptide (PACAP) against UV-B radiation damage in the human corneal endothelium. Some of its effects are mediated through the activation of the intracellular factor, known as the activity-dependent protein (ADNP). In the present paper, we have investigated the role of ADNP and the small peptide derived from ADNP, known as NAP, in the corneal epithelium. Here, we have demonstrated, for the first time, ADNP expression in human and rabbit corneal epithelium as well as its protective effect by treating the corneal epithelial cells exposed to UV-B radiations with NAP. Our results showed that NAP treatment prevents ROS formation by reducing UV-B-irradiation-induced apoptotic cell death and JNK signalling pathway activation. Further investigations are needed to deeply investigate the possible therapeutic use of NAP to counteract corneal UV-B damage.

Unilateral zebrafish corneal injury induces bilateral cell plasticity supporting wound closure

The cornea, transparent and outermost structure of camera-type eyes, is prone to environmental challenges, but has remarkable wound healing capabilities which enables to preserve vision. The manner in which cell plasticity impacts wound healing remains to be determined. In this study, we report rapid wound closure after zebrafish corneal epithelium abrasion. Furthermore, by investigating the cellular and molecular events taking place during corneal epithelial closure, we show the induction of a bilateral response to a unilateral wound. Our transcriptomic results, together with our TGF-beta receptor inhibition experiments, demonstrate conclusively the crucial role of TGF-beta signaling in corneal wound healing. Finally, our results on Pax6 expression and bilateral wound healing, demonstrate the decisive impact of epithelial cell plasticity on the pace of healing. Altogether, our study describes terminally differentiated cell competencies in the healing of an injured cornea. These findings will enhance the translation of research on cell plasticity to organ regeneration.

Expression of immune response genes in human corneal epithelial cells interacting with Aspergillus flavus conidia

Background Aspergillus flavus, one of the causative agents of human fungal keratitis, can be phagocytosed by human corneal epithelial (HCE) cells and the conidia containing phagosomes mature into phagolysosomes. But the immunological responses of human corneal epithelial cells interacting with A. flavus are not clear. In this study, we report the expression of immune response related genes of HCE cells exposed to A. flavus spores using targeted transcriptomics. Methods Human corneal epithelial cell line and primary cultures were grown in a six-well plate and used for coculture experiments. Internalization of the conidia was confirmed by immunofluorescence microscopy of the colocalized endosomal markers CD71 and LAMP1. Total RNA was isolated, and the quantity and quality of the isolated RNA were assessed using Qubit and Bioanalyzer. NanoString nCounter platform was used for the analysis of mRNA abundance using the Human Immunology panel. R-package and nSolver software were used for data analysis. KEGG and FunRich 3.1.3 tools were used to analyze the differentially expressed genes. Results Different morphotypes of conidia were observed after 6 h of coculture with human corneal epithelial cells and found to be internalized by epithelial cells. NanoString profiling showed more than 20 differentially expressed genes in immortalized human corneal epithelial cell line and more than ten differentially expressed genes in primary corneal epithelial cells. Distinct set of genes were altered in their expression in cell line and primary corneal epithelial cells. KEGG pathway analysis revealed that genes associated with TNF signaling, NF-KB signaling, and Th17 signaling were up-regulated, and genes associated with chemokine signaling and B cell receptor signaling were down regulated. FunRich pathway analysis showed that pathways such as CDC42 signaling, PI3K signaling, and Arf6 trafficking events were activated by the clinical isolates CI1123 and CI1698 in both type of cells. Conclusions Combining the transcript analysis data from cell lines and primary cultures, we showed the up regulation of immune defense genes in A. flavus infected cells. At the same time, chemokine signaling and B cell signaling pathways are downregulated. The variability in the expression levels in the immortalized cell line and the primary cultures is likely due to the variable epigenetic reprogramming in the immortalized cells and primary cultures in the absence of any changes in the genome. It highlights the importance of using both cell types in host-pathogen interaction studies.

Detection of Acanthamoeba spp. using carboxylesterase antibody and its usage for diagnosing Acanthamoeba-keratitis

Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool.