Novel ROCK Inhibitors, Sovesudil and PHP-0961, Enhance Proliferation, Adhesion and Migration of Corneal Endothelial Cells.

The loss or dysfunction of human corneal endothelial cells (hCEnCs) is a leading cause of blindness due to corneal failure. Corneal transplantation with a healthy donor cornea has been the only available treatment for corneal endothelial disease. However, the need for way to regenerate the CEnCs has been increased due to the global shortage of donor corneas. The aim of the study is to investigate whether novel Rho-kinase (ROCK) inhibitors can induce the cultivation and regeneration of hCEnCs. Cultured hCEnCs were treated with Y-27632, sovesudil, or PHP-0961 for 24 h. Cellular responses, including cell viability, cytotoxicity, proliferation, and Ki67 expression with ROCK inhibitors were evaluated. We also evaluated wound healing and cell adhesion assays. Porcine corneas were used ex vivo to evaluate the effects of Y-27632, sovesudil, and PHP-0961 on wound healing and regeneration. We performed live/dead cell assays and immunofluorescence staining for SRY (sex determining region Y)-box 2 (SOX2), ß-catenin, and ZO-1 on porcine corneas after ROCK inhibitor treatments. Cell viability, cell proliferation rate, and the number of Ki67-positive cells were higher in Y-27632, sovesudil and PHP-0961 treated cells compared to the control. There was no difference in LDH cytotoxicity test between any groups. Cells treated with Y-27632, sovesudil and PHP-0961 showed faster migration, wound healing, and cell adhesion. In the porcine ex vivo experiments, wound healing, the number of live cells, and SOX2-positive cells were higher in Y-27632, sovesudil and PHP-0961 treated corneas. In all experiments, sovesudil and PHP-0961, the novel ROCK inhibitors, were equal or superior to the results of the ROCK inhibitor positive control, Y-27632. In conclusion, sovesudil and PHP-0961, novel ROCK inhibitors have the capacity to regenerate hCEnCs by enhancing cell proliferation and adhesion between cells.

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.

Physical activity status in relation to quality of life and dietary habits in breast cancer survivors: subset analyses of KROG 14-09 nationwide questionnaire study.

PURPOSE: We investigated the relationship of physical activity with dietary habits and quality of life (QoL) in breast cancer survivors in accordance with the recommendations of the American Cancer Society. METHODS: Data of 928 breast cancer survivors were obtained from the KROG 14-09 study to measure QoL in early phase after adjuvant radiotherapy. According to the extent of physical activity, survivors were divided into four groups: inactivity (0-149 min/week, N = 144), regular activity (150-450 min/week, N = 309), moderate activity (451-900 min/week, N = 229), and marked activity (901-1800 min/week, N = 164) excluding hyperactivity (> 1800 min/week, N = 82) as it is a difficult condition to recommend to survivors. Global physical activity questionnaire, 5-dimensional questionnaire by EuroQoL (EQ-5D-3L), QoL Questionnaire-breast cancer (QLQ-BR23) from EORTC, and dietary habits were surveyed. A linear-to-linear association test for EQ-5D-3L and Kruskal-Wallis analysis for QLQ-BR23 and dietary habit were conducted. RESULTS: Overall, 15.5% respondents (144/928) were classified as physically inactive. The trends of frequent intake of fruits (p = 0.001) and vegetable (p = 0.005) and reluctance toward fatty food (p < 0.001) were observed in physically active groups. Mobility (p = 0.021) and anxiety (p = 0.030) of EQ-5D-3L, and systemic therapy side effect (p = 0.027) and future perspective (p = 0.008) of QLQ-BR23 were better in physically active groups besides body image (p = 0.003) for the survivors with breast-conserving surgery. However, moderate and marked activities did not further improve QoL than regular activity. CONCLUSION: Physicians and care-givers have to pay attention to inactive survivors to boost their physical activity, thereby facilitating a better QoL and dietary habit.

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.

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.

Questionnaire study of the dietary habits of breast cancer survivors and their relationship to quality of life (KROG 14-09).

We evaluated the dietary habits of breast cancer survivors and investigated the relationship with quality of life (QoL), with 1,156 survivors recruited from 17 institutions. We used the Questionnaire Survey of Dietary Habits of Korean Adults (Q-DH-KOR) comprising 25 questions. The following indices were derived as follows: (1) quality of healthy dietary habits (Q-HD)-eight questions on number of meals, regularity, quantity, duration, skipping breakfast, dinner with companion(s), overeating and late-night snacks; (2) habits of nutritional balance (H-NB)-questions on consuming five food categories (grains, fruits, proteins, vegetables and dairy products); and (3) habits of unhealthy foods (H-UF)-questions on consuming three food categories (fatty, instant and fast foods). The times and regularity of meals, frequency of skipping breakfast, dinner with companion(s) and overeating were better in groups with high symptomatic and functional QoL. Symptomatic QoL positively affected Q-HD and H-NB (p < 0.001 and p = 0.024 respectively) and negatively affected H-UF (p = 0.02). Breast cancer survivors more frequently ate from the fruit, protein and vegetable categories than did the control group, with lower H-UF and higher Q-HD values (p < 0.001 and p < 0.001 respectively). Our findings supported the relationship between QoL and dietary habit and showed healthier dietary habits of breast cancer survivors than controls.

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.

High tibial osteotomy accelerates lateral compartment osteoarthritis in discoid meniscus patients.

PURPOSE: The valgus high tibial osteotomy (HTO) in patients with medial osteoarthritis and discoid lateral meniscus can result in increased load on the lateral compartment and hence a higher chances of tear. This may accelerate the progression of osteoarthritis on lateral compartment. We, therefore, carried out the case control study with a hypothesis that an HTO would accelerate the progression of osteoarthritis (OA) on lateral compartment in patients with complete discoid meniscus. METHODS: The records of all patients with open wedge HTO from 2008 to 2012 were evaluated for complete lateral discoid meniscus. The patient who had a valgus HTO with or without partial meniscectomy for medial compartmental OA was included for this study. Cases to control were chosen to match age, body mass index (BMI), pre-operative osteoarthritis grade, and deformity angles in ratio 1:2. Patient's records were studied for demographic data, clinical examination records, and pre-operative knee functional scores and radiological scores and were compared with post-operative data. RESULTS: Thirty-six patients out of 674 patients, who underwent an HTO, consisted of discoid meniscus group. 72 patients were chosen as control group. Four patients showed progression of OA on the lateral compartment in discoid group compared to none in control group. Although control groups showed a little bit better functional outcomes, there were no statistical differences between two groups (n.s.). CONCLUSION: The high tibial osteotomy could result in accelerated lateral compartment osteoarthritis in patients with complete discoid meniscus, and the procedure should be used with caution in such patients. LEVEL OF EVIDENCE: IV.

An assessment of quality of life for early phase after adjuvant radiotherapy in breast cancer survivors: a Korean multicenter survey (KROG 14-09).

BACKGROUNDS: Quality of life (QoL) has become a major concern as the survival time of breast cancer increases. We investigated the changes in QoL through comprehensive categorical analysis, for the first three years after breast cancer treatment including radiotherapy. METHODS: A total of 1156 patients were enrolled from 17 institutions. All survivors were grouped according to a surveillance period of 9-15 months (first year), 21-27 months (second year), and 33-39 months (third year) from the end of radiotherapy. The 5-dimensional questionnaire by the EuroQol group (EQ-5D) and the EORTC Quality of Life Questionnaire; breast cancer specific module (QLQ-BR23) were checked by self-administrated method. RESULTS: First, second and third year groups comprised 51.0, 28.9, and 21.0%. In EQ-5D-3 L (3-Likert scale) analysis, pain/discomfort and anxiety/depression categories showed lower QoL. In multivariate analyses of EQ-5D-VAS (visual-analogue scale), categories of pain/discomfort and self-care were improved with time; axillary dissection was a significant clinical factor deteriorates pain/discomfort, self-care and usual activities. In QLQ-BR23 analysis, the lowest scored category was sexual activity, followed by sexual enjoyment, future perspective, and hair loss, and the best scored category was breast symptoms. In multivariate analyses, arm symptoms, breast symptoms and body image were improved with time. CONCLUSIONS: Categories of pain/discomfort and self-care in EQ-5D-VAS, arm/breast symptoms and body image in QLQ-BR23 were improved, while categories of anxiety/depression and future perspective BR23 were not, suggesting necessity of psychosocial support. This research provides comprehensive information on the categorical aspects of QoL and changes during early follow-up after breast cancer treatment.

Effects of chalazia on corneal astigmatism : Large-sized chalazia in middle upper eyelids compress the cornea and induce the corneal astigmatism.

BACKGROUND: A chalazion is a common eyelid disease that causes eye morbidity due to inflammation and cosmetic disfigurement. Corneal topographic changes are important factors in corneal refractive surgery, intraocular lens power calculations for cataract surgery, and visual acuity assessments. However, the effects of chalazia on corneal astigmatism have not been thoroughly investigated. The changes in corneal astigmatism according to chalazion size and location is necessary for better outcome of ocular surgery. The aim of this study is to evaluate changes in corneal astigmatism according to chalazion size and location. METHODS: In this cross-sectional study, a total of 44 eyes from 33 patients were included in the chalazion group and 70 eyes from 46 patients comprised the control group. Chalazia were classified according to location and size. An autokeratorefractometer (KR8100, Topcon; Japan) and a Galilei™ dual-Scheimpflug analyzer (Ziemer Group; Port, Switzerland) were utilized to evaluate corneal changes. RESULT: Oblique astigmatism was greater in the chalazion group compared with the control group (p < 0.05). Astigmatism by simulated keratometry (simK), steep K by simK, total root mean square, second order aberration, oblique astigmatism, and vertical astigmatism were significantly greater in the upper eyelid group (p < 0.05). Astigmatism by simK, second order aberration, oblique astigmatism, and vertical astigmatism were significantly greater in the large-sized chalazion group (p < 0.05). Corneal wavefront aberration was the greatest in the upper eyelid chalazion group, whole area group, and large-sized chalazion group (p < 0.05). CONCLUSIONS: Large-sized chalazia in the whole upper eyelid should be treated in the early phase because they induced the greatest change in corneal topography. Chalazion should be treated before corneal topography is performed preoperatively and before the diagnosis of corneal diseases.

Stemness characteristics of human corneal endothelial cells cultured in various media.

PURPOSE: To investigate stemness characteristics of human corneal endothelial cells (HCECs) cultured in various media. METHODS: Human corneal endothelial cells were isolated using a sphere-forming assay. Cells were allowed to attach to the bottom of culture plates and were cultured in different media designated as medium A (Opti-MEM I with 8% fetal bovine serum), medium B (DMEM/F12 with B27 supplement), medium E (DMEM/F12 with epidermal growth factor [EGF]), and medium BE (DMEM/F12 with B27 supplement and EGF), respectively. Cell morphology was evaluated with an phase-contrast inverted microscope. Immunofluorescence staining and western blotting of nestin, octamer-binding transcription factor (OCT3/4), glial fibrillary acidic protein (GFAP), zonula occludens-1 (ZO-1), collagen VIII alpha2, and Na-K ATPase was performed. Cell proliferation was assessed with a cell counting kit-8 assay. RESULTS: A few cultured cells stained with nestin. The cells cultured in medium A expressed high levels of GFAP, OCT3/4, and nestin, and higher levels of ZO-1 were expressed in the cells cultured in medium A and medium B compared with cells cultured in the other media. The cells cultured in medium A assumed a fibroblast-like shape, whereas the cells cultured in medium B and medium BE appeared as mosaics. Cell proliferation was highest in medium A compared with those cultured in the other media. CONCLUSIONS: Cultured HCECs expressed stem cell markers, including nestin, OCT3/4, and GFAP. The expression of stem cell markers differed according to the culture media and associated proliferation rate.

A case of fungal keratitis and onychomycosis simultaneously infected by Trichophyton species.

BACKGROUND: Fungal keratitis is difficult to treat that can result in corneal blindness requiring penetrating keratoplasty and in fungal endothalmitis. We report a case of fungal keratitis and onychomycosis simultaneously infected by Trichophyton. CASE PRESENTATION: A 77-year old male presented with ocular pain, conjunctival injection, and severe loss of vision in his left eye. His best corrected visual acuity was hand movements in the left eye, and slit-lamp examination showed a corneal ulcer with feathery margin and hypopyon. Bacterial and fungal smear/culture showed no organism, and there was no improvement in spite of treatment with topical fortified 5% cefazolin and 2% tobramycin. Trichophyton species was identified by repeated cultures. We found onychomycosis on the patient's foot, where the same fungal species were identified. Regimen was changed to topical itraconazole and systemic intravenous itraconazole. No clinical improvement was observed, so therapeutic penetrating keratoplasty and cryotherapy was done with continuation of antifungal therapy. The graft was clear at postoperative 1 month and no evidence of recurrence was found. CONCLUSION: It is important to identify the pathogen of keratitis because early identification of pathogen causing keratitis provides the appropriate treatment in early phase of keratitis. It is necessary to search for other fungal skin infections such as onychomycosis and athelete's foot considering the fungal keratitis following skin infection. In addition, fungal skin infection including onychomycosis should be treated for prevention of fungal keratitis as soon as possible.

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.

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.

Aqueous humor concentrations of vascular endothelial growth factor and pigment epithelium-derived factor in high myopic patients.

PURPOSE: To compare the aqueous humor levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in high myopic eyes and control eyes. METHODS: Aqueous humor samples were collected from 21 highly myopic eyes of 20 patients (high myopia group) and from 30 cataract eyes of 30 patients with no choroidal neovascularization (CNV) or other ocular or systemic diseases (control group). Of the 21 high myopic eyes, 13 had no complications secondary to high myopia (high myopia with no complications group), 3 had posterior staphyloma (high myopia with staphyloma group), and 5 had chorioretinal atrophy (high myopia with chorioretinal atrophy group). The aqueous humor levels of VEGF and PEDF were determined by using commercially available enzyme-linked immunosorbent assay kits. RESULTS: Aqueous humor levels of VEGF were significantly lower in the high myopia group compared to that in the control group (p<0.001). VEGF levels decreased with an increase in the axial length (p<0.001). PEDF levels tended to be higher in the high myopia group compared to that in the control group; however, the difference was not significant. Three high myopia groups had significantly lower VEGF/PEDF ratios than the control group (p=0.000, 0.002, and 0.005). CONCLUSIONS: Aqueous humor levels of VEGF in the high myopia group were significantly lower than those in the control group. The differing levels of VEGF and PEDF in the high myopia and control groups suggest that high myopia disrupts the VEGF/PEDF balance in retinal pigment epithelium (RPE) cells.

Oral alcohol administration disturbs tear film and ocular surface.

PURPOSE: To investigate whether ethanol administration disturbs the tear film and ocular surface. DESIGN: Case-control study. PARTICIPANTS: Twenty healthy male subjects were recruited. Ethanol was administered to 10 subjects and another 10 subjects served as controls. METHODS: Twenty healthy male subjects with no ocular disease were recruited. Ethanol (0.75 g/kg) was administered orally at 8 pm for 2 hours to 10 subjects. MAIN OUTCOME MEASURES: The tear film and ocular surface were evaluated at 6 pm before drinking, at midnight, and immediately (6 am) and 2 hours (8 am) after waking the next morning. Tear osmolarity, ethanol concentration in tears and serum, Schirmer's test results, tear film break-up time (TBUT), corneal punctuate erosion, and corneal sensitivity were measured. RESULTS: Ethanol was detected in tears and serum at midnight, but it was not detected the next morning. The mean tear osmolarity level increased in the alcohol group at midnight compared with that in the control group (P<0.001). The alcohol group showed a significantly shorter TBUT compared with the control group after drinking alcohol (P<0.001 at 12 am, P<0.001 at 6 am, and P = 0.002 at 8 am). There were significantly higher fluorescein staining scores in the alcohol group compared with those in the control group at 6 am and 8 am (P = 0.001 and P<0.001, respectively). No significant change was shown in corneal sensitivity or Schirmer's test results in either group. CONCLUSIONS: Orally administered ethanol was secreted into the tears. Ethanol in tears induced tear hyperosmolarity and shortened TBUT and triggered the development of ocular surface diseases. Similar changes could exacerbate signs and symptoms in patients with ocular surface disease.

Chemically injured keratocytes induce cytokine release by human peripheral mononuclear cells.

PURPOSE: To establish an in vitro model to study the role of keratocytes in corneal chemical burns and to investigate the interaction between chemically injured keratocytes and human peripheral blood mononuclear cells (PBMCs). METHODS: Human keratocytes, epithelial cells, and PBMCs were cultured. The PBMC stimulation assay was then performed using cultured human keratocytes, epithelial cells, and NaOH-treated keratocytes. Matrix metalloprotease-9 (MMP-9), transforming growth factor-beta 1 (TGF-ß1), and macrophage migration inhibitory factor (MIF) secretion profiles of activated PBMCs stimulated by NaOH-treated keratocytes were determined by ELISA. RESULTS: Human keratocytes stimulated PBMC proliferation (p=0.016), and keratocytes treated with various concentrations of NaOH further stimulated PBMC proliferation compared to control cells in a dose-dependent manner (p=0.028 and 0.009). MMP-9 and MIF levels were higher than in the negative controls, while TGF-ß1 levels did not differ from those of the negative controls. CONCLUSION: Our results suggest that PBMCs are stimulated by chemically injured keratocytes, and produce inflammatory cytokines in response. This may be a major mechanism underlying the process causing corneal chemical burn injuries. This model can be used as an in vitro model for further studies on corneal chemical burns.