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.

In Vivo Electroporation Improves Retinal Delivery of Intravitreally Injected Exosomes.

Purpose: Mesenchymal stem cell (MSC)-derived exosomes are promising therapeutic agents and natural nanoscale delivery platforms for treating degenerative retinal diseases. This study investigated the effect of electroporation on the retinal delivery of intravitreally administered MSC-derived exosomes in a murine model. Methods: Exosomes isolated from adipose tissue-derived MSCs were stained with ExoGlow exosome-specific dye and administered to the right eyes of 40 Sprague-Dawley rats. Electroporation was performed in 20 rats immediately after intravitreal injection (electroporation group); 5 square pulses of 40 V/cm for 50 ms each with 950-ms intervals were administered. The remaining 20 rats were assigned to the no-electroporation group. The eyeballs were harvested 24 h later for evaluation. The total number of fluorescent particles per hyperfield was counted from the retinal flat mounts to quantify the retinal delivery of exosomes. Tissue damage after electroporation was evaluated using retinal histological sections and a terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling (TUNEL) assay. Results: A significantly higher number of fluorescent particles per hyperfield were observed in the retinal flat mounts of the electroporation group compared with that in the no-electroporation group (599.0 ± 307.5 vs. 376.9 ± 175.4; P = 0.013). Retinal histological sections and TUNEL assays showed no signs of tissue damage after electroporation. Conclusions: In vivo electroporation can improve the retinal delivery of intravitreally injected exosomes.

A p-Tyr42 RhoA Inhibitor Promotes the Regeneration of Human Corneal Endothelial Cells by Ameliorating Cellular Senescence.

The development of treatment strategies for human corneal endothelial cells (hCECs) disease is necessary because hCECs do not regenerate in vivo due to the properties that are similar to senescence. This study is performed to investigate the role of a p-Tyr42 RhoA inhibitor (MH4, ELMED Inc., Chuncheon) in transforming growth factor-beta (TGF-ß)- or H2O2-induced cellular senescence of hCECs. Cultured hCECs were treated with MH4. The cell shape, proliferation rate, and cell cycle phases were analyzed. Moreover, cell adhesion assays and immunofluorescence staining for F-actin, Ki-67, and E-cadherin were performed. Additionally, the cells were treated with TGF-ß or H2O2 to induce senescence, and mitochondrial oxidative reactive oxygen species (ROS) levels, mitochondrial membrane potential, and NF-κB translocation were evaluated. LC3II/LC3I levels were determined using Western blotting to analyze autophagy. MH4 promotes hCEC proliferation, shifts the cell cycle, attenuates actin distribution, and increases E-cadherin expression. TGF-ß and H2O2 induce senescence by increasing mitochondrial ROS levels and NF-κB translocation into the nucleus; however, this effect is attenuated by MH4. Moreover, TGF-ß and H2O2 decrease the mitochondrial membrane potential and induce autophagy, while MH4 reverses these effects. In conclusion, MH4, a p-Tyr42 RhoA inhibitor, promotes the regeneration of hCECs and protects hCECs against TGF-ß- and H2O2-induced senescence via the ROS/NF-κB/mitochondrial pathway.

Role of NADPH Oxidase 4 in Corneal Endothelial Cells Is Mediated by Endoplasmic Reticulum Stress and Autophagy.

Human corneal-endothelial cells (hCEnCs) are located on the inner layer of the cornea. Injury to CEnCs leads to permanent corneal edema, requiring corneal transplantation. NADPH oxidase 4 (NOX4) has been reported to be implicated in the pathogenesis of CEnCs diseases. Thus, we investigated the role of NOX4 in CEnCs in this study. In an animal study, siRNA for NOX4 (siNOX4) or plasmid for NOX4 (pNOX4) was introduced into the corneal endothelium of rats by electroporation, using a square-wave electroporator (ECM830, Havard apparatus) to decrease or increase the expression of NOX4, respectively, and the rat corneas were cryoinjured through contact with a metal rod of 3 mm diameter frozen in liquid nitrogen for 10 min. The immunofluorescence staining of NOX4 and 8-OHdG showed that the levels of NOX4 and 8-OHdG were decreased in the siNOX4 group compared to the siControl, and increased in the pNOX4 group compared to the pControl at one week after treatment. Without cryoinjury, corneal opacity was more severe, and the density of CEnCs was lower, in pNOX4-treated rats compared to pControl. After cryoinjury, the corneas were more transparent, and the CEnC density was higher, in siNOX4-treated rats. The hCEnCs were cultured and transfected with siNOX4 and pNOX4. The silencing of NOX4 in hCEnCs resulted in a normal cell shape, higher viability, and higher proliferation rate than those transfected with the siControl, while NOX4 overexpression had the opposite effect. NOX4 overexpression increased the number of senescent cells and intracellular oxidative stress levels. NOX4 overexpression increased ATF4 and ATF6 levels, and nuclear translocation of XBP-1, which is the endoplasmic reticulum (ER) stress marker, while the silencing of NOX4 had the opposite effect. Additionally, the mitochondrial membrane potential was hyperpolarized by the silencing of NOX4, and depolarized by NOX4 overexpression. The LC3II levels, a marker of autophagy, were decreased by the silencing of NOX4, and increased by NOX4 overexpression. In conclusion, NOX4 plays a pivotal role in the wound-healing and senescence of hCEnCs, by modulating oxidative stress, ER stress, and autophagy. The regulation of NOX4 may be a potential therapeutic strategy for regulating the homeostasis of CEnCs, and treating corneal-endothelial diseases.

Effect of multivitamin drug on intractable dry eye symptoms.

Dry eye is a disorder of tear film and ocular surface characterized by ocular discomforts. It is associated with multiple causes and sometimes intractable. We investigated the effect of oral multivitamin supplementation (MVG) on dry eyes. Tear break-up time (TBUT), fluorescein ocular surface staining score, and tear secretion Schirmer test were measured in dry eye patients refractory to conventional topical treatment. The ocular surface disease index (OSDI), visual analog pain score (VAS), and modified standardized patient evaluation of eye dryness questionnaire were analyzed. In total, 42 eyes of 42 patients were included. TBUT increased at 1 and 3 months compared to baseline (p < 0.05). OSDI decreased at 1 and 3 months compared to baseline (p < 0.05). VAS score, impact on life, and frequency of total symptoms decreased at 3 months compared to baseline (p < 0.05). Oral administration of MVG, a vitamin complex formulation, was effective in stabilizing tear stability and alleviating symptoms in patients with intractable dry eye. Thus, it may be a viable treatment option for intractable dry eye.

Association of Dry Eye Diseases and Auditory Sensitivity.

This study aimed to evaluate the association between dry eye and inner ear diseases in a Korean population. Methods: Data from the Korean National Health and Nutrition Survey (KNHANES V, 2010−2012), a national cross-sectional health examination and survey, were collected by the Korea Centers for Disease Control and Prevention. The association between dry eye and inner ear disease was determined using the chi-square test and logistic regression analysis. The individuals were divided into two age groups (<60 and ≥60 years). Results: In total, 17,542 individuals (n = 11,932 in the <60 years group and n = 5610 in the ≥60 years group) were enrolled. After adjusting for confounding factors, the logistic regression model revealed that the associated factors were dizziness and loss of balance experience (OR, 1.315; 95% CI, 1.143−1.513), self-awareness of abnormal voice (OR, 1.372; 95% CI, 1.120−1.679), subjective hearing discomfort (OR, 1.278; CI, 1.084−1.506), and tinnitus (OR, 1.265; 95% CI, 1.101−1.453). The inversely associated factor for dry eye was bilateral hearing loss (OR, 0.497; 95% CI, 0.367−0.672). The hearing threshold was lower in the dry eye group than in the non-dry eye group (p < 0.05). Conclusions: Tinnitus was associated with dry eye and bilateral hearing loss was inversely associated with dry eye. These results suggest that hypersensitivity of the senses and nerves, which is neuropathic hyperesthesia, is one of the main mechanisms of dry eye. Treatment of neuropathy may help in treating dry eye associated with dizziness or tinnitus.

MiR-302a Regenerates Human Corneal Endothelial Cells against IFN-γ-Induced Cell Death.

Damage to human corneal endothelial cells (hCECs) leads to bullous keratopathy because these cells cannot be regenerated in vivo. In this study, we investigated the protective role of microRNA (miR)-302a against interferon-γ (IFN-γ)-induced senescence and cell death of hCECs. Cultured hCECs were transfected with miR-302a and treated with IFN-γ (20 ng/mL) to evaluate the protective effect of miR-302a on IFN-γ-induced cell death. Senescence was evaluated by the senescence-associated ß-galactosidase (SA-ß-gal) assay, and the secretion of senescence-associated secretory phenotype (SASP) factors was analyzed. Mitochondrial function and endoplasmic reticulum (ER) stress were assessed. We revealed that miR-302a enhanced the cell viability and proliferation of hCECs and that IFN-γ increased the cell size, the number of SA-ß-gal-positive cells, and SASP factors, and arrested the cell cycle, which was eliminated by miR-302a. miR-302a ameliorated mitochondrial oxidative stress and ER stress levels which were induced by IFN-γ. IFN-γ decreased the mitochondrial membrane potential and promoted autophagy, which was eliminated by miR-302a. The in vivo study showed that regeneration of rat CECs was promoted in the miR-302a group by inhibiting IFN-γ and enhancing mitochondrial function. In conclusion, miR-302a eliminated IFN-γ-induced senescence and cellular damage by regulating the oxidative and ER stress, and promoting the proliferation of CECs. Therefore, miR-302a may be a therapeutic option to protect hCECs against IFN-γ-induced stress.

Role of Neutrophils on the Ocular Surface.

The ocular surface is a gateway that contacts the outside and receives stimulation from the outside. The corneal innate immune system is composed of many types of cells, including epithelial cells, fibroblasts, natural killer cells, macrophages, neutrophils, dendritic cells, mast cells, basophils, eosinophils, mucin, and lysozyme. Neutrophil infiltration and degranulation occur on the ocular surface. Degranulation, neutrophil extracellular traps formation, called NETosis, and autophagy in neutrophils are involved in the pathogenesis of ocular surface diseases. It is necessary to understand the role of neutrophils on the ocular surface. Furthermore, there is a need for research on therapeutic agents targeting neutrophils and neutrophil extracellular trap formation for ocular surface diseases.

Role of Choline in Ocular Diseases.

Choline is essential for maintaining the structure and function of cells in humans. Choline plays an important role in eye health and disease. It is a precursor of acetylcholine, a neurotransmitter of the parasympathetic nervous system, and it is involved in the production and secretion of tears by the lacrimal glands. It also contributes to the stability of the cells and tears on the ocular surface and is involved in retinal development and differentiation. Choline deficiency is associated with retinal hemorrhage, glaucoma, and dry eye syndrome. Choline supplementation may be effective for treating these diseases.

miR-30c-1 encourages human corneal endothelial cells to regenerate through ameliorating senescence.

In the present study, we studied the role of microRNA-30c-1 (miR-30c-1) on transforming growth factor beta1 (TGF-ß1)-induced senescence of hCECs. hCECs were transfected by miR-30c-1 and treated with TGF-ß1 to assess the inhibitory effect of miR-30c-1 on TGF-ß1-induced senescence. Cell viability and proliferation rate in miR-30c-1-transfected cells was elevated compared with control. Cell cycle analysis revealed that cell abundance in S phase was elevated in miR-30c-1-treated cells compared with control. TGF-ß1 increased the senescence of hCECs; however, this was ameliorated by miR-30c-1. TGF-ß1 increased the size of hCECs, the ratio of senescence-associated beta-galactosidase-stained cells, secretion of senescence-associated secretory phenotype factors, the oxidative stress, and arrested the cell cycle, all of which were ameliorated by miR-30c-1 treatment. miR-30c-1 also suppressed a TGF-ß1-induced depolarization of mitochondrial membrane potential and a TGF-ß1 stimulated increase in levels of cleaved poly (ADP-ribose) polymerase (PARP), cleaved caspase 3, and microtubule-associated proteins 1A/1B light chain 3B II. In conclusion, miR-30c-1 promoted the proliferation of hCECs through ameliorating the TGF- ß1-induced senescence of hCECs and reducing cell death of hCECs. Thus, miR-30c-1 may be a therapeutic target for hCECs regeneration.

SIRT1 Activation Using CRISPR/dCas9 Promotes Regeneration of Human Corneal Endothelial Cells through Inhibiting Senescence.

Human corneal endothelial cells (hCECs) are restricted in proliferative capacity in vivo. Reduction in the number of hCEC leads to persistent corneal edema requiring corneal transplantation. This study demonstrates the functions of SIRT1 in hCECs and its potential for corneal endothelial regeneration. Cell morphology, cell growth rates and proliferation-associated proteins were compared in normal and senescent hCECs. SIRT1 was activated using the CRISPR/dCas9 activation system (SIRT1a). The plasmids were transfected into CECs of six-week-old Sprague-Dawley rats using electroporation and cryoinjury was performed. Senescent cells were larger, elongated and showed lower proliferation rates and lower SIRT1 levels. SIRT1 activation promoted the wound healing of CECs. In vivo transfection of SIRT1a promoted the regeneration of CECs. The proportion of the S-phase cells was lower in senescent cells and elevated upon SIRT1a activation. SIRT1 regulated cell proliferation, proliferation-associated proteins, mitochondrial membrane potential, and oxidative stress levels. In conclusion, corneal endothelial senescence is related with a decreased SIRT1 level. SIRT1a promotes the regeneration of CECs by inhibiting cytokine-induced cell death and senescence. Gene function activation therapy using SIRT1a may serve as a novel treatment strategy for hCEC diseases.

COL8A2 Regulates the Fate of Corneal Endothelial Cells.

Purpose: To investigate the effect of COL8A2 repression on corneal endothelial cells (CECs) in vitro and in vivo. Methods: Cultured human CECs (hCECs) were transfected with COL8A2 siRNA (siCOL8A2), and the cell viability and proliferation rate were measured. The expression of cell proliferation-associated molecules was evaluated by Western blotting and real-time reverse transcription PCR. Cell shape, Wingless-INT (WNT) signaling, and mitochondrial oxidative stress were also measured. For in vivo experiments, siCOL8A2 was transfected into rat CECs (rCECs), and corneal opacity and corneal endothelium were evaluated. Results: After transfection with siCOL8A2, COL8A2 expression was reduced (80%). Cell viability, cell proliferation rate, cyclin D1 expression, and the number of cells in the S-phase were reduced in siCOL8A2-treated cells. The cell attained a fibroblast-like shape, and SNAI1, pSMAD2, and ß-catenin expression, along with mitochondrial mass and oxidative stress levels, were altered. Corneal opacity increased, and the CECs were changed in rats in the siCOL8A2 group. Conclusions: COL8A2 is required to maintain normal wound healing and CEC function.

Effect of SOX2 Repression on Corneal Endothelial Cells.

PURPOSE: Human corneal endothelial cells (hCECs) pump out water from the stroma and maintain the clarity of the cornea. The sex-determining region Y-box 2 (SOX2) participates in differentiation during the development of the anterior segment of the eye and is found in the periphery of wounded corneas. This study was performed to investigate the effect of SOX2 repression on hCECs. METHODS: Cultured hCECs were transfected by siRNA for SOX2. The wound healing rate and cell viability were measured. The cell proliferation-associated protein level was evaluated by Western blotting and RT-PCR. The energy production and mitochondrial function were measured, and cell shape and WNT signaling were assessed. RESULTS: Upon transfecting the cultured cells with siRNA for SOX2, the SOX2 level was reduced by 80%. The wound healing rate and viability were also reduced. Additionally, CDK1, cyclin D1, SIRT1, and ATP5B levels were reduced, and CDKN2A and pAMPK levels were increased. Mitochondrial oxidative stress and mitochondrial viability decreased, and the cell shape became elongated. Furthermore, SMAD1, SNAI1, WNT3A, and ß-catenin levels were increased. CONCLUSION: SOX2 repression disrupts the normal metabolism of hCECs through modulating WNT signaling and mitochondrial functions.

Effect of Oral Choline Alfoscerate on Patients with Keratoconjunctivitis Sicca.

Keratoconjunctivitis sicca (KCS) or dry eye is a disease characterized by ocular surface symptoms. This study aimed to investigate the effectiveness of oral choline alfoscerate (CA) administration as a treatment for KCS. The medical records of dry eye patients who were refractory to topical eyedrops and then took oral CA were reviewed. Results of tear break-up time (TBUT), fluorescein ocular surface staining score (FSS), and tear secretion by the Schirmer test (STT) were analyzed. The results of the ocular surface disease index (OSDI), visual analog pain score (VAS), reporting of the severity and frequency of symptoms, and the modified Standardized Patient Evaluation of Eye Dryness (SPEED) questionnaire were also analyzed. The records of 47 patients were analyzed for this study. The mean age was 62.8 ± 9.3 years, and the patients included 9 males and 38 females. TBUT, OSDI, and VAS significantly improved after CA administration compared to before (p < 0.05, paired t-test). After CA administration, symptom frequency and impact on life improved (p < 0.05, paired t-test). No significant change in photophobia or FSS was identified. In conclusion, oral CA administration was effective in improving tear stability and alleviating symptoms of KCS.

Transcription Factor 4 Regulates the Regeneration of Corneal Endothelial Cells.

Purpose: Human corneal endothelial cells (hCECs) have limited regenerative capacity in vivo. Reduced hCEC density results in bullous keratopathy requiring corneal transplantation. This study reveals the role of transcription factor 4 (TCF4) in hCEC diseases and suggests that TCF4 may be a molecular target for hCEC regeneration. Methods: Cell shape, cell proliferation rates, and proliferation-associated proteins were evaluated in normal or senescent hCECs. TCF4 was blocked by siRNA (si-TCF4) or activated using clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 activation systems (pl-TCF4). The corneal endothelium of six-week-old Sprague-Dawley (SD) rats was transfected by electroporation followed by cryoinjury. Results: Cell proliferation rates and TCF4 levels were reduced in senescent cells. TCF4 CRISPR activation enhanced corneal endothelial wound healing. TCF4 regulated mitochondrial functions including mitochondrial membrane potential, mitochondrial superoxide levels, and energy production. The percentage of cells in the S-phase was reduced with si-TCF4 and increased with pl-TCF4. Cell proliferation and cell cycle-associated proteins were regulated by TCF4. Autophagy was induced by si-TCF4. In vivo transfection of CRISPR/dCas9 activation systems (a-TCF4) induced regeneration of corneal endothelium. Conclusions: Corneal endothelial diseases are associated with TCF4 reduction; TCF4 may be a potential target for hCEC diseases. Gene therapy using TCF4 CRISPR/dCas9 may be an effective treatment for hCEC diseases.

O-GlcNAc Signaling Augmentation Protects Human Corneal Endothelial Cells from Oxidative Stress via AKT Pathway Activation.

Purpose: To investigate the effect of inhibitor of O-glycosylation on human corneal endothelial cells (HCECs) under oxidative stress.Methods: HCECs were cultured and treated with 10 mM tert-butyl hydroperoxide (tBHP) with or without PUGNAc, a known inhibitor of OGA. Cell viability was assessed. Mitochondrial membrane potential (ΔΨm) was measured. Intracellular Ca2+ levels and mitochondrial Ca2+ levels were measured. Intracellular reactive oxygen species formation was measured. Levels of O-linked ß-N-acetylglucosamine (O-GlcNAc), AKT, and pAKT were evaluated by Western blotting.Results: O-GlcNAc augmentation by PUGNAc increased cell viability, attenuated the loss of ΔΨm, and intracellular ROS against tBHP-induced oxidative stress (p < .05). O-GlcNAc augmentation reduced tBHP-induced mitochondrial calcium overload (p < .05) while it did not have any effect on intracellular calcium overload with tBHP. Furthermore, AKT signaling was activated in the cells with O-GlcNAc augmentation.Conclusions: O-GlcNAc signaling augmentation protects HCECs from oxidative stress via activation of AKT pathways.

Rapamycin Rescues Endoplasmic Reticulum Stress-Induced Dry Eye Syndrome in Mice.

Purpose: To investigate whether rapamycin protects tear production and the ocular surface during endoplasmic reticulum (ER) stress-induced dry eye syndrome in mice. Methods: Tunicamycin was injected intraperitoneally in BALB/c mice without or with rapamycin (TM or RM5 group). Peritoneal injection of PBS performed in vehicle group. Group without injection served as control. Blinking rate, fluorescein staining score (FSS), and phenol red thread tear production test were measured at 4 days, 1 week, and 2 weeks after treatment. Levels of inflammatory and angiogenic cytokines were measured by ELISA. Results: Blinking rate and FSS were elevated, and tear production was decreased in TM group compared with controls (P < 0.05 for all), which was ameliorated by rapamycin at 1 and 2 weeks. Levels of inflammatory and angiogenic cytokines in the cornea and lacrimal glands were higher in the TM group than controls, and lower in the RM5 group than the TM group at 1 and 2 weeks (P < 0.05 for all). Conclusion: Rapamycin protected tear production and the ocular surface against this dry eye syndrome by ameliorating ER stress-induced vascular damage and inflammation of lacrimal glands and the ocular surface.

Vitamin D Enhances the Efficacy of Topical Artificial Tears in Patients With Dry Eye Disease.

PURPOSE: To investigate the efficacy of topical carbomer-based lipid-containing artificial tears (CLAT) and hyaluronate (HU) in patients with dry eye disease (DED) based on serum 25-hydroxyvitamin D (25HD) levels and cholecalciferol (vitamin D) supplementation. METHODS: A total of 116 patients with DED from June 2015 to June 2016 were included. The participants were divided into the vitamin D deficiency (VDD) group and the non-VDD group according to their serum 25HD levels. The patients determined the ways of cholecalciferol supplementation. Ocular Surface Disease Index (OSDI) score, visual analog pain scale score, lid hyperemia, tear breakup time (TBUT), corneal fluorescein staining score, and Schirmer test were compared between baseline and 2 weeks posttreatment after topical applications and between before and after cholecalciferol supplementation. RESULTS: The OSDI and visual analog pain scale scores of both VDD and non-VDD groups decreased after application of topical CLAT and HU compared with baseline values (P < 0.05 for all, paired t test). TBUT, corneal fluorescein staining score, and lid hyperemia in the VDD group remained unaffected by topical CLAT and HU, whereas those in the non-VDD group were improved (3.2 ± 1.7 vs. 4.1 ± 2.2, 0.5 ± 0.7 vs. 0.4 ± 0.6, and 2.2 ± 0.8 vs. 1.9 ± 0.7 in the non-VDD group, P = 0.001, 0.030, and 0.012, respectively). OSDI score, TBUT, and lid margin hyperemia were improved in the intramuscular group after cholecalciferol supplementation compared with pretreatment (33.2 ± 23.2 vs. 28.5 ± 21.9, 3.5 ± 1.9 vs. 6.0 ± 2.5, and 2.2 ± 0.7 vs. 1.2 ± 0.8, P < 0.05, Wilcoxon rank test). CONCLUSIONS: The effect of topical CLAT and HU was dependent on serum 25HD levels. Cholecalciferol supplementation enhanced the efficacy of topical treatment and may be a useful adjuvant therapy for patients with DED refractory to topical lubricants.

SOX2 Activation Using CRISPR/dCas9 Promotes Wound Healing in Corneal Endothelial Cells.

There are no effective treatments for corneal endothelial diseases, except for corneal transplantation, as human corneal endothelial cells (hCECs) do not regenerate. The regeneration of hCECs could be induced through regulation of the expression of specific genes. In this study, we investigated whether the overexpression of sex-determining region Y-box 2 (SOX2) can regenerate hCECs in vivo and in vitro. SOX2 was activated using the clustered regularly interspaced short palindromic repeats (CRISPR)/deactivated CRISPR-associated protein 9 (dCas9) activation system. Genes were transfected into the corneal endothelium of Sprague-Dawley rats. Central corneal thickness and opacity were measured, and alizarin red S staining was performed. Corneal opacity and central corneal thickness were reduced in the SOX2 group compared with the control group. The density of CECs was higher in the SOX2 group compared with the control group. Additionally, hCECs were cultured and analyzed after overexpressing SOX2. Cell viability, proliferation rate, and the number of cells in S-phase were increased after SOX2 overexpression (p < .05). Cyclin-dependent kinase 1 and cyclin D1 were found to be overexpressed (p < .05). WNT signaling was repressed, and the AKT pathway was activated by SOX2 overexpression. Mitochondrial oxidative stress and energy production were increased by SOX2 overexpression (p < .05). In conclusion, SOX2 activation promotes wound healing and regeneration in CECs. SOX2 activation using the CRISPR/dCas9 system may thus be useful for the treatment of hCEC diseases. Stem Cells 2018;36:1851-12.

Utility of the optical quality analysis system for decision-making in cataract surgery.

BACKGROUND: A cataract is a common cause of vision impairment that requires surgery in older subjects. The Optical Quality Analysis System (OQAS, Visiometrics SL, Terrassa, Spain) assesses the optical quality of the eye in cataract patients. This study shows the role of the optical quality evaluation system for decision-making in cataract surgery. We investigated the clinical utility of the OQAS for decision-making in cataract surgery. METHODS: Sixty-seven eyes from 67 patients undergoing cataract surgery and 109 eyes from 109 control subjects were compared. The best corrected visual acuity (BCVA) was measured. The objective scatter index (OSI), modulation transfer function (MTF), Strehl ratio, predicted visual acuity (PVA) 100%, PVA 20%, and PVA 10% were measured using the OQAS. The sensitivity and specificity of the different parameters were analyzed using the receiver operating characteristic (ROC) curve. The main parameters measured were sensitivity and specificity. RESULTS: The BCVA, OSI, PVA 100%, PVA 20%, and PVA 10% were higher in the cataract group compared to those in the control group, while the MTF and Strehl ratios were lower (p <  0.001 for all). ROC analysis showed that the OSI had the largest area under the curve and that the sensitivity and specificity of the OSI were 83.9 and 84.6%, respectively, at the optimal cut-off point of 2.35. CONCLUSION: The MTF, OSI, Strehl ratio, PVA 100%, PVA 20% and PVA 10% may be useful parameters for preoperative decision-making in cataract surgery. The OSI appears to be the most effective parameter for this purpose.