Wnt/ß-catenin signaling stimulates the self-renewal of conjunctival stem cells and promotes corneal conjunctivalization.

Limbal stem cell deficiency causes conjunctivalization characterized by the covering of the corneal surface with conjunctival epithelium. However, the driving force for the encroachment of these conjunctival cells is unclear. Conjunctival stem cells are bipotent stem cells that can proliferate and differentiate into conjunctival epithelial cells and goblet cells to maintain regeneration of the conjunctival epithelium. Here, we show a robust proliferative response of conjunctival stem cells and upregulation of Wnt2b and Wnt3a gene expression in the conjunctivae of mice with induced limbal stem cell deficiency. Topical application of the Wnt/ß-catenin signaling activator CHIR resulted in increased proliferation of ΔNp63α-positive stem cells in the basal layers of the bulbar and forniceal conjunctivae and enhanced invasion of conjunctival epithelial and goblet cells into the corneal surface. We also found that in cultures of stem cells isolated from the human conjunctiva, Wnt/ß-catenin pathway activation improved the expansion of the ΔNp63α/ABCG2 double-positive cell population by promoting the proliferation and preventing the differentiation of these cells. These expanded stem cells formed a stratified epithelium containing goblet cells under airlift culture conditions. Our data reveal that Wnt/ß-catenin signaling contributes to the pathological process of limbal stem cell deficiency by promoting the self-renewal of conjunctival stem cells and suggest that these cells are a driving force in corneal conjunctivalization.

Expression of Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2) by human and rabbit meibomian glands and meibocytes.

PURPOSE: Previously, we showed that Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), an essential enzyme required for meibum wax ester synthesis, was not expressed by immortalized human meibomian gland epithelial cells (hMGEC) in culture. To begin to understand the mechanisms controlling AWAT2 expression, we have analyzed its expression in human and rabbit meibomian glands and cultured meibocytes. METHODS: Rabbit meibocyte progenitor cells (rMPC) were first grown in Cnt-BM.1 basal medium (Cellntec) supplemented with rhEGF, FGF10, and ROCK inhibitor (Y-27632 dihydrochloride), and then passed at 70-80% confluency with Accutase. Differentiation of rMPC to meibocytes (rMC) was induced by removal of Y-27632 and addition of 1 mM calcium with and without PPARγ agonists. RNA from the tissue, primary, passaged rMPC and differentiated rMC were obtained for AWAT2 qPCR analysis. Proteins and cells were evaluated for western blotting and neutral lipid synthesis, respectively. For comparison, human meibomian glands were separated for RNA and protein analysis. hMGEC was cultured to collect RNA and protein. RESULTS: Rabbit rMPCs were successfully grown, passaged, and differentiated, showing a significant increase in lipid droplet accumulation. AWAT2 RNA was highly expressed in tissue but showed a -16.9 log2 fold decrease in primary and passaged rMPCs and was not induced by differentiation to rMC. By comparison, human meibomian glands showed high expression of AWAT2, and hMGEC expressed non-detectable levels of AWAT2 transcripts or protein. CONCLUSIONS: AWAT2 expression is lost in cultured rMPC and rMC suggesting that cells in culture do not undergo complete meibocyte differentiation and require yet to be identified culture conditions.

Bilateral Trichosporon asahii keratitis after ptosis correction: A case report.

RATIONALE: Fungal keratitis (FK) is a severe vision-threatening disease that can lead to corneal perforation or endophthalmitis despite proper treatment. It is important to diagnose the disease promptly due to its indolent nature and disproportionate disease symptoms. Trichosporon asahii is reported rarely as the causative organism of FK. We report a case of highly unusual bilateral T asahii keratitis following ptosis surgery. PATIENT CONCERNS: An 86-year-old female underwent bilateral levator resection surgery for ptosis. Postoperatively, the patient complained of gradually worsening bilateral ocular pain and a decrease in visual acuity associated with a chronic non-healing epithelial defect. DIAGNOSES: Both eyes of the patient were evaluated using best-corrected visual acuity, intraocular pressure, slit-lamp examination, fundus examination, and corneal culture. Multifocal deep stromal infiltrates were found in both corneas. Cultures from both corneal ulcers revealed growth of T asahii. Optical coherence tomographic examination showed bilateral macular edema. INTERVENTIONS: The patient was treated with revisional ptosis surgery, an antifungal agent for the corneal ulcer, and intravitreal injection of steroid for macular edema. OUTCOMES: Both eyes recovered well. Her best-corrected visual acuity improved from 20/200 to 20/40 in the right eye and from 20/100 to 20/40 in the left eye. LESSONS: FK can develop in the cornea when certain risk factors are present, including recent lid surgery, chronic keratitis, and steroid eye drop use. Identification and correction of risk factors can be beneficial in the treatment of FK.

Cataract Surgery Practice in the Republic of Korea: A Survey of the Korean Society of Cataract and Refractive Surgery 2020.

PURPOSE: To describe current cataract surgery practice patterns and trends among Korean ophthalmologists. METHODS: A survey was conducted among members of the Korean Society of Cataract and Refractive Surgery in October 2020. Of the 998 questionnaires, 262 (26.3%) were received for analysis. Data were analyzed using descriptive statistics and compared with those of previous surveys. RESULTS: The largest percentage of respondents (39%) had <5 years of practical experience, and 40% had >11 years of practical experience. The average, median, and mode monthly volumes of cataract surgeries performed by the Korean Society of Cataract and Refractive Surgery members were 31, 20, and 10 cases, respectively. Topical anesthesia was administered by 85% of the respondents. For intraocular lens (IOL) calculations, 96% of the respondents used optical biometry. The proportion of surgeons providing femtosecond laser-assisted cataract surgery increased significantly from 5% in 2018 to 29% in 2020. This increase was accompanied by an increase in the multifocal IOLs. Those who implant multifocal IOL for >10% of their cases increased from 16% (2018) to 29% (2020). Topical nonsteroidal anti-inflammatory drugs were prescribed postoperatively by 76% of the respondents. Most respondents (70%) prescribed these anti-inflammatory drugs for 4 weeks. CONCLUSIONS: This survey provided a comprehensive update on current cataract surgery practice in the Republic of Korea. The results highlighted the increasing use of premium IOLs, femtosecond laser-assisted cataract surgery, optical biometry, and topical anesthesia to better meet the patients' needs.

Comparison of corneal thickness measurements using ultrasound pachymetry, noncontact tonopachy, Pentacam HR, and Fourier-domain OCT.

ABSTRACT: We retrospectively compared the central corneal thickness (CCT) obtained by ultrasound pachymetry (USP; SP-3000, Tomey Corp., Nagoya, Japan), non-contact tonopachy (TP) (NT-530P, Nidek Co., Ltd., Gamagori, Japan), Pentacam HR (OCULUS Inc., Wetzlar, Germany), and RTVue optical coherence tomography (OCT) (Optovue Inc., Fremont, CA, USA) in 78 eyes of 78 healthy subjects with myopia. Agreement between the measurement methods was evaluated using 95% confidence intervals for the limits of agreement (LoA). The mean CCT values were 546.9 ± 34.7, 548.1 ±â€Š33.5, 559.2 ±â€Š34.0, and 547.2 ±â€Š34.8 µm for USP, non-contact TP, Pentacam, and RTVue, respectively. The thickest and the thinnest mean CCT values corresponded to those obtained by Pentacam HR and USP, respectively. Plots of the differences against the means showed the best agreement between USP and RTVue (LoA, 10.14-10.70 µm), while the largest discrepancy was observed between RTVue and Pentacam systems (LoA, -25.47-1.44 µm). Our data showed that CCT measurements using these 4 instruments were well correlated. However, the results from Pentacam differed significantly from those of the other instruments.

A Randomized, Placebo-Controlled Phase II Clinical Trial of 0.01% or 0.02% Cyclosporin A with 3% Trehalose in Patients with Dry Eye Disease.

Purpose: To compare the efficacy and safety of HU00701 (0.01% cyclosporin A + 3% trehalose), HU007 (0.02% cyclosporin A + 3% trehalose) (all w/v), and placebo in patients with moderate to severe dry eye disease (DED). Methods: This was a multicenter, randomized, double-masked, parallel, placebo-controlled phase II study. In total, 114 patients were randomly assigned to the HU00701, HU007, placebo, or reference group. There was a 2-week run-in period before the 12-week intervention. Efficacy and safety were evaluated every 4 weeks. Results: The primary endpoint, change in corneal staining score from baseline to week 12, did not differ significantly among the control, HU00701, and HU007 groups in the full analysis. Of the secondary endpoints, only the tear film breakup time differed significantly at week 12 between the placebo and HU00701 groups. Twenty adverse events were reported by 15 patients, but the rate did not differ significantly among the 4 groups. The laboratory test, vital signs, and physical examination data showed no significant changes during the study. Conclusions: HU00701 and HU007 are safe, and HU007 effectively reduces the corneal staining score in patients with moderate-to-severe DED (NCT02917512).

Eicosapentaenoic acid (EPA) activates PPARγ signaling leading to cell cycle exit, lipid accumulation, and autophagy in human meibomian gland epithelial cells (hMGEC).

PURPOSE: The purpose of this study was to access the ability of the natural PPAR agonist, eicosapentaenoic acid (EPA), to activate PPAR gamma (γ) signaling leading to meibocyte differentiation in human meibomian gland epithelial cell (hMGEC). METHODS: HMGEC were exposed to EPA, alone and in combination with the specific PPARγ antagonist, T0070907, to selectively block PPARγ signaling. Expression of PPARγ response genes were evaluated by qPCR. Effect on cell cycle was evaluated using Ki-67 labelling and western blots. During differentiation, autophagy was monitored using the Autophagy Tandem Sensor (ATS) and LysoTracker. Lipid accumulation was characterized by Stimulated Raman Scattering microscopy (SRS) and neutral lipid staining in combination with ER-Tracker, LysoTracker, and ATS. Autophagy was also investigated using western blotting. Seahorse XF analysis was performed to monitor mitochondrial function. RESULTS: EPA specifically upregulated expression of genes related to lipid synthesis and induced cell cycle exit through reduced cyclin D1 expression and increased p21 and p27 expression. EPA also induced accumulation of lipid droplets in a time and dose dependent manner (P < 0.05) by specific PPARγ signaling. Lipid analysis identified both de novo synthesis and extracellular transport of lipid to form lipid droplets that were localized to the ER. PPARγ signaling also induced activation of AMPK-ULK1 signaling and autophagy, while inhibition of autophagy induced mitochondrial crisis with no effect on lipid accumulation. CONCLUSIONS: EPA induces meibocyte differentiation through PPARγ activation that is characterized by cell cycle exit, de novo and transported lipid accumulation in the ER, and autophagy.

Income and Education are Independently Associated with Visual Impairment: The Korean National Health and Nutrition Examination Survey 2010-2012.

Purpose: To investigate the association of visual impairment (VI) with socioeconomic status, including the highest educational level and household income when other confounding variables were adjusted. Methods: The Korean National Health and Nutrition Examination Survey 2010-2012 data were analyzed. The final analyses included a total of 16,905 subjects with their demographic, socioeconomic, and health data. The socioeconomic data included the household income and the highest educational level. Household income was categorized into quartiles. The educational attainment was categorized as ≤ elementary school, middle school, high school, and ≥ university. VI was defined when the distance-corrected VA was worse than 0.32 (20/63 Snellen) in the better-seeing eye according to the definition of the World Health Organization. Four multiple logistic regression models were used to determine the odds ratio (OR) and 95% confidence interval (CI) between VI and household income or between VI and the highest educational level, by adjusting for several confounding variables. Results: The prevalence of VI was 5.0% (844 subjects). Multiple logistic regression analyses indicated that the adjusted OR was 1.729 (95% CI: 1.108-2.697) for developing VI in subjects with ≤ elementary school as their highest education level compared to those with ≥ university education. Also, the adjusted OR of subjects in the first quartile of household income was 1.502 (95% CI: 1.061-2.127) for developing VI compared to those in the fourth quartile of household income. Conclusions: The present study showed that household income and education were independently associated with VI even after adjusting for significant confounding variables.

Herpes simplex virus linear endotheliitis in a post-keratoplasty patient: A case report.

RATIONALE: To report a case of herpes simplex virus (HSV) linear endotheliitis in a 57-year-old male who had underwent keratoplasty 10 years ago. The characteristic linear keratic precipitates (KPs) resembled the Khodadoust line in graft rejection. The differential diagnosis is essential, because the treatment regimen is different between HSV linear endotheliitis and graft rejection. PATIENT CONCERNS: The patient developed a sudden onset of ocular pain and a decrease in visual acuity in his right eye. The patient had received penetrating keratoplasty in the eye 10 years ago. DIAGNOSES: The ocular disease was evaluated using several ocular examinations, including best-corrected visual acuity (BCVA), intraocular pressure, slit lamp examination, fundus examination, and aqueous humor tap. Characteristic linear endothelial KPs were found both in the host cornea and graft cornea. Stromal edema was evident in both the donor and recipient corneas. The aqueous humor was sampled for viral polymerase chain reaction (PCR) analysis. The sample was investigated for the possible presence of HSV I, HSV II, cytomegalovirus, and varicella zoster virus. The PCR was positive for HSV I and negative for HSV II, cytomegalovirus, and varicella zoster virus. INTERVENTIONS: The patient was treated with both antiviral and steroid treatments for 1 month. Thereafter, prophylactic antiviral treatment was continued. OUTCOMES: The subjective symptoms had improved and the cornea edema and the linear endothelial KPs had disappeared. The BCVA improved from 20/200 to 20/80. LESSONS: HSV linear endotheliitis is the most severe form of HSV endotheliitis. This case showed characteristic endothelial KPs, which were different from the Khodadoust line of graft rejection.

Retrospective observational study of micro-monovision small incision lenticule extraction (SMILE) for the correction of presbyopia and myopia.

This study was aimed to evaluate refractive and visual outcomes after micro-monovision small incision lenticule extraction (SMILE) in patients with presbyopia and myopia. In total, 72 patients (144 eyes) with a mean age of 46.0 ±â€Š4.9 years were included in this study. The dominant eye was treated for distance vision and the nondominant eye for near vision by targeting between -0.50 and -1.75 diopters (D). Treatment efficacy, safety, and refractive stability were calculated from postoperative data including refraction, binocular uncorrected distance visual acuity (UDVA), binocular uncorrected near visual acuity, monocular uncorrected distance visual acuity, and monocular corrected distance visual acuity (CDVA). Six months post-surgery, binocular UDVA was better than or equal to 20/20 in 88% of patients. No loss in 2 or more lines was observed in the Snellen lines of corrected distance visual acuity. Mean spherical equivalent (SE) for the distance eye was -0.18 ±â€Š0.37 D, whereas the attempted and achieved SE in the near eye were -0.90 ±â€Š0.44 and -0.99 ±â€Š0.54 D, respectively. In total, 79% of eyes were within ±0.50 D, and 98% within ±1.00 D, of the intended refraction. A UDVA of 0.0 logMAR (20/20) or better, and an uncorrected near visual acuity of Jaeger (J) of 3 (20/32) or better, were observed in 83% of patients. Micromonovision refractive surgery using SMILE enhanced functional near vision in presbyopic patients.

Effects of Ranibizumab, Bevacizumab, and Aflibercept on Senescent Retinal Pigment Epithelial Cells.

PURPOSE: Anti-vascular endothelial growth factor (VEGF) agents have been used for the last 10 years, but their safety profile, including cytotoxicity against various ocular cells such as retinal pigment epithelial (RPE) cells, remains a serious concern. Safety studies of VEGF agents conducted to date have primarily relied on healthy RPE cells. In this study, we assessed the safety of three anti-VEGF agents, namely, ranibizumab, bevacizumab, and aflibercept, on senescent RPE cells. METHODS: Senescent human induced pluripotent stem cell-derived RPE cells were generated by continuous replication and confirmed with senescence biomarkers. The viability, proliferation, protein expression, and phagocytosis of the senescent RPE cells were characterized 3 days after anti-VEGF treatment with clinical doses of ranibizumab, bevacizumab, or aflibercept. RESULTS: Clinical doses of ranibizumab, bevacizumab, or aflibercept did not decrease the viability or alter proliferation of senescent RPE cells. In addition, the anti-VEGF agents did not induce additional senescence, impair the protein expression of zonula occludens-1 and RPE65, or reduce the phagocytosis capacity of senescent RPE cells. CONCLUSIONS: Clinical dosages of ranibizumab, bevacizumab, or aflibercept do not induce significant cytotoxicity in senescent RPE cells.

Influence of across-pupil sutures on the optical quality of intraocular lenses.

PURPOSE: In cases of pupillary capture after previous transscleral fixation of an intraocular lens (IOL), an across-pupil suture technique is sometimes used to stabilize the IOL. We investigated the optical effects of 10-0 polypropylene sutures placed across the pupil. METHODS: Optical performance was evaluated using the optical bench metrology system, and a single-piece IOL was used in an optical bench system consisting of a model eye, a high-resolution Hartmann-Shack wave front sensor, and an image capturing device with 2.0, 3.0, 4.0, and 4.5 mm apertures with distance focus. To simulate across-pupil sutures, two 10-0 polypropylene sutures were placed 2 mm apart across the pupil. The focus image, spherical aberration, and image quality were measured and compared with and without sutures. RESULTS: When pupil size increased, spherical aberration increased, irrespective of sutures. No difference was found in spherical aberration with and without sutures, and image qualities at 17.6 and 35.2 cycles per degree were not affected by the across-pupil sutures. CONCLUSIONS: The differences in optical quality using across-pupil 10-0 polypropylene sutures were clinically negligible.

Tyrosine Kinase Inhibitor, Vatalanib, Inhibits Proliferation and Migration of Human Pterygial Fibroblasts.

PURPOSE: Vatalanib is a small-molecule tyrosine kinase inhibitor. We investigated the effects of vatalanib on the proliferation and migration of cultured human pterygial fibroblasts (HPFs). METHODS: Pterygium tissues were obtained after pterygium excision surgery and subjected to primary culture. HPFs were treated with vatalanib at various concentrations. Mitomycin C (MMC) was used as a positive control. Cell proliferation and migration assays were used to investigate the effects of vatalanib. Cell death was measured using flow cytometry analysis. Western blot analysis was performed to identify signaling molecules associated with the response to vatalanib. RESULTS: Vatalanib inhibited both proliferation and migration of HPFs in a dose-dependent manner. Cell proliferation was significantly suppressed by vatalanib (10 and 100 µM) and MMC (0.004% and 0.04%) treatments. Migration assays revealed significant HPF delay when treated with vatalanib (1, 10, and 100 µM) and MMC (0.004% and 0.04%) compared with that in a negative control. Cell death analysis showed that high concentrations of vatalanib (100 µM) and MMC (0.004% and 0.04%) decreased cell numbers. Western blot analysis of vatalanib-treated cells showed vascular endothelial growth factor and transforming growth factor-ß significantly reduced, but there was no alteration in p53 protein levels in HPFs. CONCLUSIONS: These results indicate that vatalanib significantly suppressed the proliferation and migration of HPFs by decreasing vascular endothelial growth factor and transforming growth factor-ß. Vatalanib showed less toxicity than that of MMC. Based on these results, vatalanib may potentially serve as a new adjuvant treatment after pterygium excision surgery.

The Antiangiogenic Effects of Gold Nanoparticles on Experimental Choroidal Neovascularization in Mice.

Purpose: The purpose of this study was to evaluate the antiangiogenic effect of gold nanoparticles (AuNPs) on experimental choroidal neovascularization (CNV) in mice. Methods: Choroidal neovascularization was induced by rupturing the Bruch's membrane using laser photocoagulation in C57BL/6 mice. The following day, intravitreal injections of AuNPs were administered. The control group received PBS injection of the same volume. Two weeks after laser injury, CNV lesions were evaluated by examination of choroidal flat-mounts using fluorescein-labeled dextran and immunofluorescence staining with isolectin B4. The effects of AuNPs on endothelial cell tube formation, proliferation, and cytotoxicity were evaluated using human umbilical vein endothelial cells (HUVECs) or human RPE cells. The activity of extracellular signal-regulated kinase (ERK)1/2, protein kinase B (Akt), and focal adhesion kinase (FAK) signaling pathways was also analyzed. Results: The AuNPs reduced the extent of CNV. Mice treated with intravitreal AuNPs injections exhibited a 67.9% reduction in the extent of CNV lesions compared with the control group (P < 0.001). The size of the isolectin B4-labeled area was also significantly smaller in AuNP-treated groups compared with the control group (P < 0.001). Gold nanoparticles decreased vascular endothelial growth factor-induced HUVEC tube formation and proliferation but showed no RPE cell toxicity with the treatment doses administered. The phosphorylation of ERK1/2, Akt, and FAK in HUVECs was suppressed by AuNPs. Conclusions: Gold nanoparticles can inhibit laser-induced CNV in mice and may have an indication for the treatment of CNV.

Differential Effects of Bevacizumab, Ranibizumab, and Aflibercept on the Viability and Wound Healing of Corneal Epithelial Cells.

PURPOSE: This study compared the effects of 3 antivascular endothelial growth factor (VEGF) agents (bevacizumab, ranibizumab, and aflibercept) on corneal epithelial cell viability and wound healing using human corneal epithelial cells (HCECs). METHODS: To determine the cytotoxic effects of anti-VEGF agents on HCECs, HCEC viability was determined at various concentrations of these agents. An in vitro migration assay was used to investigate the migration of HCECs treated with 3 anti-VEGF agents. The protein level of extracellular signal-regulated kinase was used to evaluate the effect of anti-VEGF treatment on cell proliferation. The protein levels of p38 mitogen-activated protein kinase (MAPK) were analyzed by Western blotting to investigate cell migration. RESULTS: After 24 or 48 h of exposure, aflibercept treatment showed no apparent effect on cell viability; however, bevacizumab and ranibizumab treatment decreased cell viability at high concentrations (1 and 2 mg/mL). A migration assay showed that HCEC migration was different among the 3 anti-VEGF treatment groups. Bevacizumab significantly delayed HCEC migration. Western blotting showed that bevacizumab treatment decreased the expression levels of phosphorylated p38 MAPK. CONCLUSIONS: Bevacizumab, the most widely used and investigated anti-VEGF agent, decreased epithelial cell migration and viability. Anti-VEGF agents other than bevacizumab might therefore be better for treating corneal neovascularization complicated with epithelial defects.

Polar Value Analysis of Corneal Astigmatism in Intrastromal Corneal Ring Segment Implantation.

Purpose. To evaluate surgically induced astigmatism (SIA) and the average corneal power change in symmetric intrastromal corneal ring segment (ICRS) implantation. Methods. The study included 34 eyes of 34 keratoconus patients who underwent symmetric Intacs SK ICRS implantation. The corneal pocket incision meridian was the preoperative steep meridian. Corneal power data were obtained before and 3 months after Intacs SK ICRS implantation using scanning-slit topography. Polar value analysis was used to evaluate the SIA. Hotelling's trace test was used to compare intraindividual changes. Results. Three months postoperatively, the combined mean polar value for SIA changed significantly (Hotelling's T2 = 0.375; P = 0.006). The SIA was 1.54 D at 99° and the average corneal power decreased significantly by 3.8 D. Conclusion. Intacs SK ICRS placement decreased the average corneal power and corneal astigmatism compared to the preoperative corneal power and astigmatism when the corneal pocket incision was made at the preoperative steep meridian.

Melissa Officinalis L. Extracts Protect Human Retinal Pigment Epithelial Cells against Oxidative Stress-Induced Apoptosis.

BACKGROUND: We evaluated the protective effect of ALS-L1023, an extract of Melissa officinalis L. (Labiatae; lemon balm) against oxidative stress-induced apoptosis in human retinal pigment epithelial cells (ARPE-19 cells). METHODS: ARPE-19 cells were incubated with ALS-L1023 for 24 h and then treated with hydrogen peroxide (H2O2). Oxidative stress-induced apoptosis and intracellular generation of reactive oxygen species (ROS) were assessed by flow cytometry. Caspase-3/7 activation and cleaved poly ADP-ribose polymerase (PARP) were measured to investigate the protective role of ALS-L1023 against apoptosis. The protective effect of ALS-L1023 against oxidative stress through activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) was evaluated by Western blot analysis. RESULTS: ALS-L1023 clearly reduced H2O2-induced cell apoptosis and intracellular production of ROS. H2O2-induced oxidative stress increased caspase-3/7 activity and apoptotic PARP cleavage, which were significantly inhibited by ALS-L1023. Activation of the PI3K/Akt pathway was associated with the protective effect of ALS-L1023 on ARPE-19 cells. CONCLUSIONS: ALS-L1023 protected human RPE cells against oxidative damage. This suggests that ALS-L1023 has therapeutic potential for the prevention of dry age-related macular degeneration.

Anti-angiogenic effect of ALS-L1023, an extract of Melissa officinalis L., on experimental choroidal neovascularization in mice.

BACKGROUND: The effect of ALS-L1023, an extract of Melissa officinalis L. (Labiatae; lemon balm) leaves, on experimental choroidal neovascularization (CNV) in mice was evaluated. METHODS: C57BL/6 mice were given either vehicle or ALS-L1023 daily via oral gavage for 3 weeks (days 0-21). CNV was induced by rupturing Bruch's membrane using laser photocoagulation (day 7). Two weeks after laser injury (day 21), the CNV lesions were evaluated by an examination of choroidal flat mounts using fluorescein-labelled dextran, immunofluorescence staining with isolectin B4 and fluorescence angiography. The effects of ALS-L1023 on endothelial cell tube formation and the expression of phosphorylated extracellular signal-regulated kinase 1/2 were evaluated using human umbilical vein endothelial cells. RESULTS: The extent of CNV was reduced by ALS-L1023. Mice treated with 100 and 200 mg/kg/day of the material exhibited 44.3 and 68.1% reductions in the extent of CNV lesions, respectively, compared to the vehicle group (P < 0.001). The size of the isolectin B4-labelled area was also significantly decreased in the ALS-L1023-treated groups (P < 0.001). On fluorescein angiography, ALS-L1023-treated mice exhibited significantly less leakage of fluorescent material than did vehicle-treated mice. ALS-L1023 decreased vascular endothelial growth factor-induced human umbilical vein endothelial cell tube formation in a dose-dependent manner. The expression of phosphorylated extracellular signal-regulated kinase 1/2 was suppressed by ALS-L1023. CONCLUSIONS: The laser-induced CNV in mice can be inhibited by ALS-L1023. Therefore, it may have therapeutic potential for the treatment of diseases involving CNV.

Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF) Factor on Corneal Epithelial Cells in Corneal Wound Healing Model.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs). We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF). An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 µg/ml). MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 µg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.

Inhibition of Lymphangiogenesis and Hemangiogenesis in Corneal Inflammation by Subconjunctival Prox1 siRNA Injection in Rats.

PURPOSE: Prospero homeobox 1 (Prox1) siRNA is a small interfering RNA that is designed to specifically bind Prox1 mRNA. We determined whether Prox1 siRNA inhibits lymphangiogenesis and hemangiogenesis after acute corneal inflammation. METHODS: Three Prox1 siRNAs were synthesized and investigated for their effects on Prox1 mRNA expression and tube formation in human dermal lymphatic endothelial cells (HDLECs) in vitro. The in vivo effects of Prox1 siRNA were assessed in alkali burn-induced inflammatory corneal neovascularization in rats. Prox1 siRNA was administered via subconjunctival injection. Corneal flat mounts were stained for lymphatic vessel endothelial hyaluronan receptor (LYVE)-1 to show lymphatic vessels. Lymphangiogenesis and hemangiogenesis were analyzed morphometrically using Image J software. Corneal inflammatory cell infiltration was evaluated by immunostaining for F4/80 and CD45. Protein levels of LYVE-1, podoplanin, VEGF receptor 2 (VEGFR2), and VEGFR3 were analyzed by Western blotting. RESULTS: Prox1 siRNA treatment decreased Prox1 mRNA expression and tube formation in cultured HDLECs. Subconjunctival injection of Prox1 siRNA significantly inhibited alkali burn-induced lymphangiogenesis and hemangiogenesis in the cornea compared with those of scrambled siRNA (negative control). This inhibition was comparable to that induced by bevacizumab (positive control). Prospero homeobox 1 knockdown by Prox1 siRNA also inhibited macrophage and leukocyte infiltration into the cornea. Prox1 siRNA downregulated the expression of all four proteins. CONCLUSIONS: Prox1 siRNA is a strong inhibitor of inflammatory corneal lymphangiogenesis and hemangiogenesis in vivo. Prox1 siRNA may be useful in preventing immune rejection after penetrating keratoplasty by suppressing lymphangiogenesis.