Wnt signalling inhibits adipogenesis in orbital fibroblasts from patients with Graves' orbitopathy.

BACKGROUND/AIMS: To investigate the role of Wnt signalling in adipogenesis using an in vitro model of Graves' orbitopathy (GO). METHODS: Orbital fat was obtained from patients with GO and non-GO participants for primary orbital fibroblast (OF) culture. Expression levels of Wnt5a, Wnt10b, ß-catenin, phospho-ß-catenin and cyclin D1 were compared between GO and non-GO OFs. These expression levels were also determined during adipogenesis of GO and non-GO OFs. The effects of a stimulator and inhibitor of Wnt signalling on adipogenesis of GO and non-GO OFs were investigated. RESULTS: Western blotting analysis showed significant reductions in ß-catenin and cyclin D1 and significant enhancement of phospho-ß-catenin in OFs from patients with GO, compared with OFs from non-GO participants (p<0.05). Expression of Wnt5a, Wnt10b, ß-catenin and cyclin D1 in OFs was highest on day 0, and then gradually declined after induction of adipogenic differentiation. The expression levels of PPARγ, C/EBPα and C/EBPß were reduced in Wnt stimulator-treated OFs in a dose-dependent manner. Oil red O staining confirmed that a stimulator of Wnt inhibited adipogenesis in GO OFs. CONCLUSION: These results indicate that Wnt signalling inhibits adipogenesis in OFs from patients with GO and non-GO participants. Further studies are required to examine the potential of Wnt signalling as a target for therapeutic strategies.

Association between refractive errors and horizontal strabismus: the Korea National Health and Nutrition Examination Survey.

PURPOSE: To investigate the association between refractive error and horizontal strabismus based on a Korean population-based survey. METHODS: The study included 22,887 participants >5 years of age who participated in the Korea National Health and Nutrition Examination Survey (KNHANES) from 2008 to 2011. Refractive error was classified into myopia, hyperopia, astigmatism, and anisometropia. The association between refractive error and exodeviation or esodeviation was assessed using multivariate logistic regression analysis. RESULTS: After adjusting for potential confounders, the analyses revealed an increased odds ratio (OR) for exodeviation with an increasing trend according to the severity of myopia (mild myopia, OR = 1.36 [95% CI, 1.11-1.66]; moderate myopia, OR = 1.75 [95% CI, 1.36-2.26]; severe myopia, OR = 2.01 [95% CI, 1.50-2.69]; Ptrend < 0.001). Hyperopia was not associated with exodeviation (P = 0.273). There were also significant associations for exodeviation with astigmatism (OR = 1.36 [95% CI, 1.16-1.59; P < 0.001]) and anisometropia (OR = 1.78 [95% CI, 1.38-2.30; P < 0.001]) In comparison, esodeviation was significantly associated with hyperopia (OR, 10.24 [95% CI, 4.43-23.70; P < 0.001]) and anisometropia (OR, 5.16 [95% CI, 2.27-11.76; P < 0.001]), while no significant association was found with myopic degree (P = 0.170) or astigmatism (P = 0.816). CONCLUSIONS: Refractive error was closely associated with exodeviation and esodeviation. These risk associations should be considered when managing refractive errors.

Effects of Stuffer DNA on the Suppression of Choroidal Neovascularization by a rAAV Expressing a mTOR-Inhibiting shRNA.

Choroidal neovascularization (CNV) is the defining characteristic of the wet subtype of age-related macular degeneration (AMD), which is a rapidly growing global health problem. Previously, we had demonstrated the therapeutic potential of gene therapy against CNV using short hairpin RNA (shRNA) delivered via recombinant adeno-associated virus (rAAV), which abrogates mammalian-to-mechanistic (mTOR) activity in a novel manner by simultaneously inhibiting both mTOR complexes. Both the target and use of gene therapy represent a novel treatment modality against AMD. Here, the xenogeneic GFP gene used as a reporter in previous studies was removed from the virus vector to further develop the therapeutic for clinical trials. Instead, a stuffer DNA derived from the 3' UTR of the human UBE3A gene was used to ensure optimal viral genome size for efficient rAAV assembly. The virus vector containing the stuffer DNA, rAAV2-shmTOR-SD, positively compares to one encoding the shRNA and a GFP expression cassette in terms of reducing CNV in a laser-induced mouse model, as determined by fundus fluorescein angiography. These results were confirmed via immunohistochemistry using anti-CD31, while a TUNEL assay showed that rAAV2-shmTOR-SD possesses anti-apoptotic properties as well. The qualities exhibited by rAAV2-shmTOR-SD demonstrate its potential as a human gene therapeutic for the treatment of wet AMD.

mTORC1 and mTORC2 are differentially engaged in the development of laser-induced CNV.

BACKGROUND: The mechanistic target of rapamycin (mTOR) pathway is a potential target to inhibit pathologic processes in choroidal neovascularization. However, the exact role of mTOR signaling in the development of CNV remains obscure. In this study, we assessed the role of mTORC1 and mTORC2 as well as the effect of rapamycin (sirolimus) on choroidal neovascularization (CNV) in a laser-induced mouse model. METHODS: In experiment A, we observed the natural course of CNV development and the dynamics of mTOR-related proteins during the 12 days after the laser injury. The expression of mTOR-related proteins was evaluated using Western blot (WB). Cryosections of CNV-induced mice were immunostained for the visualization of the vascular and extravascular components of the CNV. Experiment B was performed to confirm the critical period of mTOR signaling in the development of laser-induced CNV, we administered rapamycin before and/or during the active period of mTOR complexes. WB and immunofluorescence staining was performed to evaluate the mode of action and the effect of mTOR inhibition on CNV development. RESULTS: In experiment A, we detected high levels of p-mTOR S2448 and p-mTOR S2481 from the 5th to 12th day of laser injury. Immunofluorescence imaging of cryosections of mice sacrificed on day 7 revealed greater co-immunoreactivity of p-mTOR S2448 positive cells with CD11b and F4/80, while p-mTOR S2481 positive cells showed colocalization with CD31, α-SMA, and cytokeratin. In experiment B, rapamycin injection during the active period of mTOR signaling demonstrated near-complete inhibition of CNV lesion as well as significant induction of autophagy. CONCLUSION: Our study suggests the mTOR as a critical player during CNV development in laser-induced mouse model through differentially acting with the mTORC1 and mTORC2. mTORC1 activity was high predominantly in inflammatory cells in CNV lesion, while mTORC2 activity was higher in vascular components and the RPE.

Association between Three Heavy Metals and Dry Eye Disease in Korean Adults: Results of the Korean National Health and Nutrition Examination Survey.

PURPOSE: To investigate the associations between blood heavy metal concentrations and dry eye disease using a Korean population-based survey. METHODS: This study included 23,376 participants >40 years of age who participated in the Korean National Health and Nutrition Examination Survey from 2010 to 2012. Blood concentrations of lead, cadmium, and mercury were measured in all participants. The associations between blood heavy metal concentrations and dry eye disease were assessed using multivariate logistic regression analyses. RESULTS: After adjusting for potential confounders, including age, sex, lifestyle behaviors and sociodemographic factors, the analyses revealed an increased odds ratio (OR) for dry eye disease with higher blood mercury concentrations (tertile 2: OR, 1.22; 95% confidence interval [CI], 0.91 to 1.64; tertile 3: OR, 1.39; 95% CI, 1.02 to 1.89; p = 0.039). The prevalence of dry eye disease was not associated with blood lead (tertile 2: OR, 1.15; 95% CI, 0.87 to 1.51; tertile 3: OR, 0.83; 95% CI, 0.59 to 1.16; p = 0.283) or cadmium (tertile 2: OR, 1.05; 95% CI, 0.77 to 1.44; tertile 3: OR, 1.15; 95% CI, 0.84 to 1.58; p = 0.389) concentrations. There were no significant associations between any of the three heavy metals and dry eye disease in males after adjusting for potential confounding factors, but blood mercury concentrations in females were associated with dry eye disease (tertile 2: OR, 1.18; 95% CI, 0.83 to 1.69; tertile 3: OR, 1.58; 95% CI, 1.12 to 2.24; p = 0.009). CONCLUSIONS: Mercury concentrations in blood were associated with dry eye disease. Our results suggested that controlling environmental exposure to mercury may be necessary to reduce the incidence of dry eye disease.

En face optical coherence tomography findings in a case of Alport syndrome.

Alport syndrome is a rare hereditary disease that is associated with retinal abnormalities such as dot-and-fleck retinopathy and temporal macular thinning. The main pathophysiological process of Alport syndrome is loss of the collagen network in the basement membrane. However, the alterations in each retinal layer have not been fully evaluated. In the case presented here, we evaluated the retina of a patient with Alport syndrome using en face optical coherence tomography (OCT). The findings suggested that the primary alterations occur in the internal limiting membrane and the retinal pigment epithelium basement membrane which is a part of the Bruch's membrane. The adjacent retinal layers are damaged subsequently. In conclusion, en face OCT could be useful in evaluating retinal abnormalities and understanding their underlying pathophysiology in Alport syndrome.