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.

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.

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.

Geriatric nutritional risk index is associated with retinopathy in patients with type 2 diabetes.

The geriatric nutritional risk index (GNRI) is a nutrition-related risk assessment tool and has been used in various clinical settings. The relationship between body mass index (BMI) and the associated risk of diabetic retinopathy (DR) remains inconclusive. We aimed to evaluate the association between GNRI and DR in patients with type 2 diabetes. We included a total of 1359 patients with type 2 diabetes who followed up in our diabetes clinic and underwent fundus photographic examinations from August 2006 to February 2014. DR was assessed by retinal ophthalmologists using comprehensive ophthalmologic examinations. Patients were divided into tertiles according to their GNRI category. Patients in a lower GNRI tertile tended to have a higher proportion of nonproliferative DR (NPDR) and proliferative DR (PDR) compared with those in the other tertiles. The risk of PDR was higher in patients included in GNRI tertile 1 (Odds ratio (OR) 2.252, 95% Confidence Interval (CI) 1.080-4.823, P = 0.033) and GNRI tertile 2 (OR 2.602, 95% CI 1.323-5.336, P = 0.007) compared with those in GNRI tertile 3. In patients with lower GNRIs, the prevalence of DR was higher than in those with higher GNRIs. When GNRI was compared with BMI using the area under the curve, overall accuracy was high in GNRI. The risk of PDR was high in patients with low GNRI and there is an inverse association between GNRI scores and prevalence of DR. GNRI might be a useful tool to predict DR in patients with type 2 diabetes.

Low Contrast Visual Evoked Potentials for Early Detection of Optic Neuritis.

Optic neuritis (ON) detection is important for the early diagnosis and management of multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). However, the conventional high-contrast visual evoked potential (VEP) used for ON detection lacks sensitivity for identifying ON presenting as mild or unremarkable visual disturbance, which is common in first-episode ON. Therefore, this study aimed to investigate whether a change in contrast or check size improves the sensitivity of VEP to first-ever ON. In total, 60 patients with the demyelinating disease (29 MS and 31 idiopathic patients with ON) without ON or with first-ever ON at least 6 months prior and 32 healthy controls underwent neuro-ophthalmic evaluations. VEPs were induced using three pattern-reversal checkerboard stimuli having, respectively, 10% contrast with a check size of 32' (LC32 VEP), 100% contrast with a check size of 32' (HC32 VEP; conventional VEP), and 100% contrast with a check size of 16' (HC16 VEP). The receiver operating characteristic (ROC) curve analysis and area under the curve (AUC) were calculated to determine the most appropriate VEP method for detecting optic nerve involvement. The optimal cut-off point was determined using the Youden index (J-index). The McNemar test was used to determine whether dichotomous proportions were equivalent. In comparison with first-ever ON eyes (n = 39) and healthy eyes (n = 64), LC32 VEP showed the highest AUC for discriminating ON (0.750, p < 0.001; 0.730 for HC32 VEP, p < 0.001; 0.702 for HC16 VEP, p = 0.001). In the first-ever ON group, LC32 VEP and conventional HC32 VEP were abnormal in 76.9 and 43.6%, respectively (McNemar, p < 0.001), and combining these tests did not improve sensitivity. These indicate that LC32 VEP is the most sensitive method for detecting first-ever ON. Visual evoked potential with 10% contrast stimuli was superior to conventional VEP for detecting first-ever ON. Thus, adding these LC stimuli might be helpful in identifying optic nerve involvement in ON with mild or unremarkable visual impairment.

Inducible Expression of Several Drosophila melanogaster Genes Encoding Juvenile Hormone Binding Proteins by a Plant Diterpene Secondary Metabolite, Methyl Lucidone.

Juvenile hormones prevent molting and metamorphosis in the juvenile stages of insects. There are multiple genes encoding a conserved juvenile hormone binding protein (JHBP) domain in a single insect species. Although some JHBPs have been reported to serve as carriers to release hormones to target tissues, the molecular functions of the other members of the diverse JHBP family of proteins remain unclear. We characterized 16 JHBP genes with conserved JHBP domains in Drosophila melanogaster. Among them, seven JHBP genes were induced by feeding the flies with methyl lucidone, a plant diterpene secondary metabolite (PDSM). Induction was also observed upon feeding the juvenile hormone (JH) analog methoprene. Considering that methyl lucidone and methoprene perform opposite functions in JH-mediated regulation, specifically the heterodimeric binding between a JH receptor (JHR) and steroid receptor coactivator (SRC), the induction of these seven JHBP genes is independent of JH-mediated regulation by the JHR/SRC heterodimer. Tissue-specific gene expression profiling through the FlyAtlas 2 database indicated that some JHBP genes are mainly enriched in insect guts and rectal pads, indicating their possible role during food uptake. Hence, we propose that JHBPs are induced by PDSMs and respond to toxic plant molecules ingested during feeding.

Comparison of a preservative-free nonsteroidal anti-inflammatory drug and preservative-free corticosteroid after uneventful cataract surgery: multicenter, randomized, evaluator-blinded clinical trial.

PURPOSE: To compare the efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) and steroidal eyedrops for inflammation management after cataract surgery using slitlamp indicators. SETTING: 11 eye centers in South Korea. DESIGN: Randomized prospective multicenter study with a blinded evaluator. METHOD: In 125 (250 eyes) patients who underwent cataract surgery, bromfenac sodium hydrate 0.1% (NSAID group) was applied twice a day in 1 eye, whereas the other eye was treated with fluorometholone 0.1% (steroid group), 4 times a day for 4 weeks postoperatively. The primary efficacy outcome was the presence of anterior chamber cells and flare at 1 week postoperatively. Anterior chamber cells and flare at 4 to 8 weeks, corrected distance visual acuity, central corneal thickness, conjunctival hyperemia, dry eye parameters, foveal thickness, and ocular and visual discomfort were evaluated as secondary outcomes. RESULTS: At week 1, residual anterior chamber inflammation was not statistically significantly different between the groups (-1.03 ± 1.27 vs -0.95 ± 1.24, P = .4850). However, the NSAID group recovered from conjunctival hyperemia more rapidly than the steroid group (0.30 ± 0.52 vs 0.44 ± 0.81, P = .0144 at week 1). The increase in central corneal thickness in the NSAID group was less than that in the steroid group 1 week postoperatively (7.87 ± 22.46 vs 29.47 ± 46.60 µm, P < .0001). The change in foveal thickness in the NSAID group was significantly less than that in the steroid group (18.11 ± 68.19 vs 22.25 ± 42.37 µm, P = .0002). Lower levels of postoperative ocular and visual discomfort were reported in the NSAID group than in the steroid group under treatment. CONCLUSIONS: Preservative-free bromfenac was as effective as preservative-free fluorometholone eyedrops in anterior chamber inflammation control and showed better signs and symptoms after cataract surgery.

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.

Development of clinical application program for radiotherapy induced cancer risk calculation using Monte Carlo engine in volumetric-modulated arc therapy.

BACKGROUND: The purpose of this study is to develop a clinical application program that automatically calculates the effect for secondary cancer risk (SCR) of individual patient. The program was designed based on accurate dose calculations using patient computed tomography (CT) data and Monte Carlo engine. Automated patient-specific evaluation program was configured to calculate SCR. METHODS: The application program is designed to re-calculate the beam sequence of treatment plan using the Monte Carlo engine and patient CT data, so it is possible to accurately calculate and evaluate scatter and leakage radiation, difficult to calculate in TPS. The Monte Carlo dose calculation system was performed through stoichiometric calibration using patient CT data. The automatic SCR evaluation program in application program created with a MATLAB was set to analyze the results to calculate SCR. The SCR for organ of patient was calculated based on Biological Effects of Ionizing Radiation (BEIR) VII models. The program is designed to sequentially calculate organ equivalent dose (OED), excess absolute risk (EAR), excess relative risk (ERR), and the lifetime attributable risk (LAR) in consideration of 3D dose distribution analysis. In order to confirm the usefulness of the developed clinical application program, the result values from clinical application program were compared with the manual calculation method used in the previous study. RESULTS: The OED values calculated in program were calculated to be at most approximately 13.3% higher than results in TPS. The SCR result calculated by the developed clinical application program showed a maximum difference of 1.24% compared to the result of the conventional manual calculation method. And it was confirmed that EAR, ERR and LAR values can be easily calculated by changing the biological parameters. CONCLUSIONS: We have developed a patient-specific SCR evaluation program that can be used conveniently in the clinic. The program consists of a Monte Carlo dose calculation system for accurate calculation of scatter and leakage radiation and a patient-specific automatic SCR evaluation program using 3D dose distribution. The clinical application program that improved the disadvantages of the existing process can be used as an index for evaluating a patient treatment plan.

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.

Serum Biomarkers for the Diagnosis of Glaucoma.

Despite the importance of the early detection of glaucoma, most patients with progressive glaucoma show minimal symptoms. We aimed to evaluate biomarkers for glaucoma diagnosis in Korea. Forty-two volunteers with/without open-angle glaucoma were enrolled from January through October 2015-divided into a control or open-angle glaucoma group, which was further divided into normal-tension glaucoma (NTG) and high-tension glaucoma (HTG) groups-and underwent assessments for myelin basic protein (MBP), heat shock protein 60, anti-Sjögren's-syndrome-related antigen A (SSA) and antigen B (SSB), anti-α-fodrin, and anti-nucleic acid. The glaucoma group showed a higher serum MBP level and lower serum anti-α-fodrin antibody level than the control group (p < 0.05). The NTG group showed higher serum anti-SSA and anti-SSB levels and lower anti-α-fodrin IgG/IgA levels than the HTG group. In the receiver operating characteristic curve analysis, the area under the curve (AUC) for serum MBP level was 0.917 in discriminating between controls and patients with glaucoma. Between the NTG and HTG groups, anti-SSA, anti-SSB, and anti-α-fodrin IgG/IgA levels showed an AUC above 0.8. Thus, these biomarkers were useful for diagnosing glaucoma and discriminating between controls and patients with glaucoma, and patients with NTG and HTG.

Comparison of Dose Distribution in Regional Lymph Nodes in Whole-Breast Radiotherapy vs. Whole-Breast Plus Regional Lymph Node Irradiation: An In Silico Planning Study in Participating Institutions of the Phase III Randomized Trial (KROG 1701).

The purpose of the current in silico planning study is to compare radiation doses of whole-breast irradiation (WBI) and whole-breast plus regional lymph node irradiation (WBI+RNI) administered to the regional lymph nodes (RLN) in pN1 breast cancer. Twenty-four participating institutions were asked to create plans of WBI and WBI+RNI for two dummy cases. To compare target coverage between the participants, an isodose line equal to 90% of the prescribed dose was converted to an isodose contour (contour90% iso). The relative nodal dose (RND) was obtained using the ratio of RLN dose to the target dose. The Fleiss's kappa values which represent inter-observer agreement of contour90% iso were over 0.68. For RNI, 6 institutions included axillary lymph node (ALN), supraclavicular lymph node (SCN), and internal mammary lymph node (IMN), while 18 hospitals included only ALN and SCN. The median RND between the WBI and WBI+RNI were as follows: 0.64 vs. 1.05 (ALN level I), 0.27 vs. 1.08 (ALN level II), 0.02 vs. 1.12 (ALN level III), 0.01 vs. 1.12 (SCN), and 0.54 vs. 0.82 (IMN). In all nodal regions, the RND was significantly lower in WBI than in WBI+RNI (p < 0.01). In this study, we could identify the nodal dose difference between WBI and WBI+RNI.

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.