Neuroprotective effects of idebenone on hydrogen peroxide-induced oxidative damage in retinal ganglion cells-5.

PURPOSE: To investigate the neuroprotective effect of idebenone against hydrogen peroxide (H2O2)-induced oxidative damage in retinal ganglion cells-5 (RGC-5 cells). METHODS: RGC-5 cells were pre-treated with various idebenone concentrations (5, 10, and 20 µM) for 12 h and were then subjected to 300 µM H2O2 for a further 12 h. Apoptosis in RGC-5 was measured by flow cytometry. The changes of mitochondrial membrane potential (MMP) were detected by JC-1 staining. Autophagy in RGC-5 cells was observed by transmission electron microscopy. Western blots were used to measure the expression of autophagy-related protein light chain 3 (LC3), Beclin-1, and the release of Cytochrome c (Cyt-c). RESULTS: Flow cytometry showed that the apoptosis rates in the normal control group, H2O2 group, and idebenone groups were 6.48 ± 0.55%, 27.3 ± 0.51%, 22.8 ± 0.52%, 15.45 ± 0.81%, and 12.59 ± 0.58%, respectively (F = 559.7, P < 0.0001). After incubation with H2O2, the number of autophagosomes increased significantly, whereas it was decreased in the idebenone groups. After incubation of RGC-5 cells with H2O2, MMP levels were significantly decreased, while idebenone could prevent the decrease in MMP levels. Compared with that in the normal control group, LC3 II/I, the expression levels of Beclin-1 and Cyt-c were increased significantly in the H2O2 group (P < 0.05). Compared with that in the H2O2 group, LC3 II/I, the expression of Beclin-1 and Cyt-c was significantly decreased in idebenone groups (P < 0.05). CONCLUSIONS: Idebenone protects RGC-5 cells against H2O2-induced oxidative damage by reducing mitochondrial damage and autophagic activity.

Silencing of YAP attenuates pericyte-myofibroblast transition and subretinal fibrosis in experimental model of choroidal neovascularization.

Age-related macular degeneration (AMD) is the main reason of irreversible vision loss in the elderly. The subretinal fibrosis subsequent to choroidal neovascularization (CNV) is an important feature in the late stage of wet AMD and is considered to be one reason for incomplete response to anti-VEGF drugs. Recent studies have shown that pericyte-myofibroblast transition (PMT) is an important pathological process involving fibrotic diseases of various organs. However, the specific role and mechanism of PMT in the subretinal fibrosis of CNV have not been clarified. It has been clear that the Hippo pathway along with its downstream effector Yes-associated protein (YAP) plays an important role in both epithelial and endothelial myofibroblast development. Therefore, we speculate whether YAP participates in PMT of pericytes and promotes fibrosis of CNV. In this study, experimental CNV was induced by laser photocoagulation in C57BL/6J (B6) mice, and aberrant YAP overexpression was detected in the retinal pigment epithelial/choroid/sclera tissues of the laser-injured eyes. YAP knockdown reduced the proliferation, migration, and differentiation of human retinal microvascular pericytes in vitro. It also reduced subretinal fibrosis of laser-induced CNV in vivo. Moreover, by proteomics-based analysis of pericyte conditioned medium (PC-CM) and bioinformatic analyses, we identified that the crosstalk between Hippo/YAP and MAPK/Erk was involved in expression of filamin A in hypoxic pericytes. These findings suggest that Hippo/YAP and MAPK/Erk are linked together to mediate pericyte proliferation, migration as well as differentiation, which may embody potential implications for treatment in diseases related to CNV.

Changes in retinal vascular bifurcation in eyes with myopia.

OBJECTIVE: To evaluate the effect of myopia on retinal vascular bifurcation. METHODS: A cross-sectional study that retrospectively analyzed the fundus photographs and clinical data of 493 people who participated in routine physical examinations in Huadong Sanatorium. One eye of each subject was included in the analysis. Retinal vascular bifurcation measurements were extracted by using a validated computer program. One-way ANOVA and analysis of covariance were performed to compare the measurements across high myopia, low to moderate myopia, and non-myopia groups. RESULTS: The mean age was 41.83 ± 10.43 years and 63.49% were women. The mean spherical equivalent refraction (SER) was - 4.59 ± 3.07 D. Ninety-nine (20.08%) eyes met the definition of high myopia (SER ≤ -6.0 D), along with 234 (47.46%) low to moderate myopia (-6.0 D < SER <-0.5 D), and 160 (32.45%) non-myopia (SER ≥ -0.5 D). The differences in the arteriolar branching angle, venular branching coefficient, venular asymmetry ratio, venular angular asymmetry, and venular junctional exponent among the three groups remained significant (p < 0.05) after multivariate adjustment. Pairwise comparisons showed arteriolar branching angle and venular angular asymmetry in high myopia were significantly lower than low to moderate myopia (p < 0.001, p = 0.014 respectively) and non-myopia (p = 0.007, p = 0.048 respectively). Venular asymmetry ratio and venular branching coefficient in high myopia were significantly higher than low to moderate myopia (p = 0.029, p = 0.001 respectively) and non-myopia (p = 0.041, p = 0.043 respectively). There was a significant difference in venular junctional exponent between high myopia and low to moderate myopia (p = 0.031). CONCLUSION: The vascular bifurcation differs in dependence on the myopic refractive error and a significant increase in the difference can be observed in high myopic eyes.

YAP promotes ocular neovascularization by modifying PFKFB3-driven endothelial glycolysis.

Ocular neovascularization is the leading cause of vision impairment in a variety of ocular diseases, such as age-related macular degeneration and retinopathy of prematurity. Emerging studies have suggested that the yes-associated protein (YAP), a downstream effector of the Hippo pathway, is involved in the pathological angiogenesis, but the mechanism are largely unknown. Here, we demonstrated that hypoxic treatment triggered YAP expression and nuclear translocation in human umbilical vein endothelial cells (HUVECs). YAP acted as a transcriptional co-activator working together with transcriptional enhancer activator domain 1 (TEAD1) to binds the promoter of the key glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3 (PFKFB3), and thereby increases PFKFB3 expression. Moreover, silencing of YAP inhibited glycolysis as well as proliferation, migration, sprouting and tube formation of HUVECs under hypoxia, all of which could be reversed by enforced expression of PFKFB3. Finally, our animal study also showed that intravitreal injection of small interfering RNA of YAP or PFKFB3 dramatically suppressed the neovascular growth in mouse models of choroidal neovascularization and oxygen-induced retinopathy. These findings provide new insights into a previously unrecognized effect of YAP on endothelial glycolysis and highlight the potential of targeting YAP/PFKFB3 axis in the treatment of ocular neovascularization.

YAP nuclear-cytoplasmic translocation is regulated by mechanical signaling, protein modification, and metabolism.

Nuclear-cytoplasmic transport is necessary for the biological function of nuclear proteins. The mechanism underlying this process is very complex and has been a subject of intense research. Yes-associated protein (YAP), a Hippo signaling pathway effector, localizes to both the cytoplasm and the nucleus and can influence cell proliferation, stem cell status, and tissue homeostasis. Recent studies have focused on the significance of YAP distribution between the nucleus and the cytoplasm in disease, but it remains unclear how this dynamic process is regulated. In this review, we discuss YAP nuclear-cytoplasmic transport under different physiological and pathological conditions in terms of mechanical signaling, protein modification, and metabolism. Understanding the mechanisms underlying nuclear-cytoplasmic YAP transport mechanism under different physiological and pathological conditions may help identify important targets for disease treatment.

Ocular biometric parameters are associated with non-contact tonometry measured intraocular pressure in non-pathologic myopic patients.

PURPOSES: This research aimed to investigate effects and risk factors on non-contact tonometer (NCT) readings in healthy myopic subjects by employing cross-sectional study design. METHODS: Totally, sixty otherwise healthy myopic volunteers (mean 28.4 years old) with 90% female were recruited in ophthalmic clinic. The routine ophthalmic tests, refractive evaluation, examination central corneal thickness (CCT), depth of anterior chamber, axial length, corneal curvature, white-to-white and NCT were assessed at baseline. The linear-mixed model was utilized to evaluate correlation between the readings and ocular biometric parameters. RESULTS: For population in this study, mean spherical equivalents were - 4.85 ± 1.79 diopters in right eyes and - 4.63 ± 1.95 diopters in left eyes. Meanwhile, 28.3% of the eyes had a refractive error exceeding - 6.0 diopters. The mean NCT reading was 15.02 ± 3.02 mmHg in left eyes and 15.33 ± 2.96 mmHg in right eyes. Among the factors analyzed, CCT was the most significant parameter associated with NCT readings. After adjusting for the other factors, per one standard deviation increase of central corneal thickness (36.11 µm) was associated a 1.14 (95% confidence interval 0.53-1.77) mmHg elevated NCT reading. The average central corneal curvature, age and spherical equivalence were also significantly and independently associated with NCT readings. CONCLUSIONS: Central corneal thickness, age, corneal curvature and degree of myopia were independently associated with NCT measured intraocular pressure. Central corneal thickness is one of the most influential factors.

miR-539-5p inhibits experimental choroidal neovascularization by targeting CXCR7.

Stromal cell-derived factor-1 (SDF-1) has been previously confirmed to participate in the formation of choroidal neovascularization (CNV) via its receptor, CXC chemokine receptor (CXCR) 4; CXCR7 is a recently identified receptor for SDF-1. The molecular mechanisms and therapeutic value of CXCR7 in CNV remain undefined. In this study, experimental CNV was induced by laser photocoagulation in Brown-Norway pigmented rats, and aberrant CXCR7 overexpression was detected in the retinal pigment epithelial/choroid/sclera tissues of laser-injured eyes. Blockade of CXCR7 activation via CXCR7 knockdown or neutralizing Ab administration inhibited SDF-1-induced cell survival and the tubular formation of human retinal microvascular endothelial cells (HRMECs) in vitro and reduced CNV leakage and lesion size in vivo. By using microRNA array screening and bioinformatic analyses, we identified miR-539-5p as a regulator of CXCR7. Transfection of HRMECs and choroid-retinal endothelial (RF/6A) cells with the miR-539-5p mimic inhibited their survival and tube formation, whereas CXCR7 overexpression rescued the suppressive effect of miR-539-5p. The antiangiogenic activities of the miR-539-5p mimic were additionally demonstrated in vivo by intravitreal injection. ERK1/2 and AKT signaling downstream of CXCR7 is involved in the miR-539-5p regulation of endothelial cell behaviors. These findings suggest that the manipulation of miR-539-5p/CXCR7 levels may have important therapeutic implications in CNV-associated diseases.-Feng, Y., Wang, J., Yuan, Y., Zhang, X., Shen, M., Yuan, F. miR-539-5p inhibits experimental choroidal neovascularization by targeting CXCR7.

Are blood lipids associated with microvascular complications among type 2 diabetes mellitus patients? A cross-sectional study in Shanghai, China.

BACKGROUND: Although there are several studies to investigate the association between blood lipids and microvascular complications, these studies reported conflicting results. The aim of the current study was to explore the association between blood lipid parameters and the risk of microvascular complications, especially the dose-response association between them, among community patients with type 2 diabetes mellitus (T2DM) in Shanghai, China. METHODS: The cross-sectional study was conducted in 6 community health service centers in Shanghai between December 2014 and December 2016.The associations between blood lipids and diabetic kidney disease (DKD) or diabetic retinopathy (DR) were assessed using multiple logistic regression. Restricted cubic spline (RCS) was employed to estimate the dose-response relation of blood lipids and the risk of microvascular complications. RESULTS: A total of 3698 participants were included in the final analysis to study the association between blood lipids and DKD, wherein 33.2% of participants had DKD and 1374 were included for the analysis of the association between blood lipids and DR, wherein 23.2% of participants had DR. DKD odds ratio was increased by 1.16(95%CI,1.08-1.25), 1.21(95%CI,1.13-1.30), 1.18(95%CI,1.10-1.26) for comparing fourth to first quartiles of triglycerides (TG), TG/high-density lipoprotein cholesterol (HDL-C), non-HDL-C/HDL-C, respectively, and decreased by 0.83(95%CI,0.78-0.89) for comparing fourth to first quartiles of HDL-C. Furthermore, the dose-response association between TG, HDL-C, TG/HDL-C, non-HDL-C/HDL-C and the risk of DKD demonstrated turning points in TG of 1.90 mmol/L, HDL-C of 1.62 mmol/L, TG/HDL-C of 2.00, non-HDL-C/HDL-C of 3.09, respectively. However, no significant association was found between blood lipid parameters and DR. CONCLUSIONS: This community-based study indicated that TG, HDL-C, TG/HDL-C, non-HDL-C/HDL-C were independently associated with DKD but not DR.

The effect of TC14012 on alkali burn-induced corneal neovascularization in mice.

AIMS: To observe the effect of TC14012 (a CXCR4 antagonist and CXCR7 agonist) on alkali burn-induced corneal neovascularization (CNV) in a mouse model. METHODS: CNV was induced in vivo by alkali burns on the corneas of BALB/c mice. A total of 54 mice treated with alkali burns were randomly divided into 3 groups, each of which received one of the following treatments: bilateral subconjunctival injections of TC14012 for 3 consecutive days, bilateral subconjunctival injections of balanced saline (BS) for 3 consecutive days or no treatment (blank control). The areas of CNV were measured on days 3, 7 and 14 after the alkali burns. CXCR4, CXCR7, vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP) mRNAs were detected and quantified by real-time reverse transcription PCR on days 7 and 14. Additionally, the expression of the proteins CXCR4, CXCR7, VEGF, ß-arrestin 2, total ERK1/2 and phospho-ERK1/2 was determined by Western blotting. RESULTS: On day 7 after the alkali burns, the CNV area, VEGF, MMP-2 and MMP-9 mRNA levels, and VEGF, ß-arrestin 2 and phospho-ERK1/2 protein levels were increased in the TC14012 group compared with the nontreatment and BS groups. However, on day 14, the CNV area, CXCR4, CXCR7, VEGF, MMP-2 and MMP-9 mRNA levels, and the CXCR4, CXCR7, VEGF and ß-arrestin 2 protein levels were significantly decreased in the TC14012 group. CONCLUSIONS: TC14012 initially enhanced alkali burn-induced CNV but reduced CNV in later stages. In addition to CXCR4, CXCR7 is involved in the pathogenesis of CNV.

5-Aza-2'-Deoxycytidine Ameliorates Choroidal Neovascularization by Inhibiting the Wnt/ß-Catenin Signaling Pathway.

Purpose: Choroidal neovascularization (CNV) can constitute the final pathology of many ocular diseases and result in severe vision loss. Studies have demonstrated that DNA methylation is critical in retinal development, aging, and disorders. The current work investigated the effects and underlying mechanism of 5-Aza-2'-deoxycytidine (5-aza-dC), a suppressor of DNA methylation, in the pathological progression of CNV. Methods: The DNA methylation profiles of retinal pigment epithelial (RPE)/choroidal complexes in normal and laser-induced CNV mice were assessed by Arraystar Mouse RefSeq Promoter Arrays. The CNV area and blood flow density and intensity were observed by optical coherence tomography angiography, and fluorescence leakage was examined by fundus fluorescein angiography in CNV mice with systemic administration of 5-aza-dC. The effects of 5-aza-dC on the biological functions of bEnd.3 cells were estimated by related assays. Notum gene promoter methylation was measured using bisulfite sequencing PCR. Methyltransferases and Wnt signaling-related genes were detected in animal and cell culture experiments by real-time PCR and immunoblot. Results: Methyltransferases were upregulated, but Notum (a secretion inhibitor of Wnt signaling) was downregulated in the RPE/choroidal complexes of mice with experimental CNV. Intraperitoneal injection of 5-aza-dC inactivated the Wnt pathway and ameliorated the lesion area and the intensity and density of blood flow, as well as the degree of leakage in CNV. In vitro, vascular endothelial growth factor A (VEGFA) stimulation promoted methyltransferases expression and suppressed Notum expression, consequently activating Wnt signaling, whereas exogenous 5-aza-dC reversed VEGFA-induced hyperpermeability, proliferation, migration, and tube formation in bEnd.3 cells via demethylation of Notum promoter. Conclusions: We observed that 5-aza-dC attenuates the growth of CNV by inhibiting the Wnt signaling pathway via promoter demethylation of the Wnt antagonist Notum. These findings provide a theoretical basis for methylation-based treatment with the Notum gene as a potential target for CNV treatment.

Development of The Chinese Version of Ultra-Low Vision Visual Functioning Questionnaire-150.

Purpose: The purpose of this study was to develop the Chinese version of Ultra-Low Vision Visual Functioning Questionnaire-150 (ULV-VFQ-150) and evaluate its psychometric function. Methods: A standardized procedure for the translation of ULV-VFQ-150 was carried out, including the forward translation, consistency check, back translation, back review, and coordination. Participants with ultra-low vision (ULV) were recruited for the questionnaire survey. Psychometric characteristics were evaluated using Rasch analysis based on Item Response Theory (IRT), and some items were revised and proofread accordingly. Results: In total, 70 out of 74 responders completed the Chinese ULV-VFQ-150, of which 10 were excluded because their vision did not meet the criterion of ULV. Therefore, 60 valid questionnaires were analyzed (valid response rate = 81.1%). The average age of eligible responders was 49.0 years (standard deviation = 16.0), with 35% female subjects (21/60). The person measures (ability) ranged from -1.7 to +4.9 logits, and the item measures (difficulty) ranged from -1.6 to +1.2 logits. The mean value of item difficulty and personnel ability were 0.00 and 0.62 logits, respectively. The reliability index was 0.87 for items and 0.99 for persons, and the overall fit is good. The items conform to unidimensionality as indicated by principal component analysis of the residuals. Conclusions: The Chinese version of ULV-VFQ-150 is a reliable questionnaire for evaluating both visual function and functional vision in people with ULV in China. Translational Relevance: The Chinese version of ULV-VFQ-150 is a new assessment of the visual function of people with ULV in China.

MicroRNA-376b-3p Suppresses Choroidal Neovascularization by Regulating Glutaminolysis in Endothelial Cells.

Purpose: Choroidal neovascularization (CNV) is a common pathological change of various ocular diseases that causes serious damage to central vision. Accumulated evidence shows that microRNAs (miRNAs) are closely related with the regulation of endothelial metabolism, which plays crucial roles in angiogenesis. Here, we investigate the molecular mechanism underlying the regulation of endothelial glutamine metabolism by miR-376b-3p in the progression of CNV. Methods: Human retinal microvascular endothelial cells (HRMECs) were transfected with control or miR-376b-3p mimics, and the expression of glutaminase 1 (GLS1), a rate-limiting enzyme in glutaminolysis, was detected by real-time PCR or Western blotting. The biological function and glutamine metabolism of transfected HRMECs were measured by related kits. Luciferase reporter assays were used to validate the CCAAT/enhancer-binding protein beta (CEBPB) was a target of miR-376b-3p. Chromatin immunoprecipitation and RNA immunoprecipitation assays were performed to verify the binding of CEBPB on the promoter region of GLS1. Fundus fluorescein angiography and immunofluorescence detected the effect of miR-376b-3p agomir on rat laser-induced CNV. Results: The expression of miR-376b-3p was decreased, whereas GLS1 expression was increased in the retinal pigment epithelial-choroidal complexes of rats with CNV. HRMECs transfected with miR-376b-3p mimic showed inhibition of CEBPB, resulting in the inactivation of GLS1 transcription and glutaminolysis. Moreover, the miR-376b-3p mimic inhibited proliferation, migration and tube formation but promoted apoptosis in HRMECs, whereas these effects counteracted by α-ketoglutarate supplementation or transfection with CEBPB overexpression plasmid. Finally, the intravitreal administration of the miR-376b-3p agomir restrained CNV formation. Conclusions: Collectively, miR-376b-3p is a suppressor of glutamine metabolism in endothelial cells that could be expected to become a therapeutic target for the treatment of CNV-related diseases.

Assessment of visual function in blind mice and monkeys with subretinally implanted nanowire arrays as artificial photoreceptors.

Retinal prostheses could restore image-forming vision in conditions of photoreceptor degeneration. However, contrast sensitivity and visual acuity are often insufficient. Here we report the performance, in mice and monkeys with induced photoreceptor degeneration, of subretinally implanted gold-nanoparticle-coated titania nanowire arrays providing a spatial resolution of 77.5 µm and a temporal resolution of 3.92 Hz in ex vivo retinas (as determined by patch-clamp recording of retinal ganglion cells). In blind mice, the arrays allowed for the detection of drifting gratings and flashing objects at light-intensity thresholds of 15.70-18.09 µW mm-2, and offered visual acuities of 0.3-0.4 cycles per degree, as determined by recordings of visually evoked potentials and optomotor-response tests. In monkeys, the arrays were stable for 54 weeks, allowed for the detection of a 10-µW mm-2 beam of light (0.5° in beam angle) in visually guided saccade experiments, and induced plastic changes in the primary visual cortex, as indicated by long-term in vivo calcium imaging. Nanomaterials as artificial photoreceptors may ameliorate visual deficits in patients with photoreceptor degeneration.

[Anti-angiogenesis effect of AMD3100 in oxygen-induced retinopathy mice].

OBJECTIVE: To observe the inhibition of neovascularisation in the oxygen-induced retinopathy (OIR) mice by a stromal cell-derived factor 1 (SDF-1) antagonist. METHODS: Experimental study. Fifty-eight 7-day-old C57BL/6 mice were divided into 3 groups randomly, the control group (n = 17), the test group (n = 17) and the medication group (n = 24). According to the dosage of AMD3100, the medication group (n = 24) were divided into low dose group, high dose group, low dose control group, and high dose control group (each group n = 6). Each group (19-day-old) was sacrificed to perform ADPase staining, paraffin sections and immunohistochemical staining (anti-VEGF and anti-SDF-1). The average positive staining area percentage (APSAP) was measured as the outcomes and processed with the Students' t-test. RESULTS: Real-time PCR showed expression of both VEGF mRNA (0.080 ± 0.022 vs. 0.123 ± 0.032) and SDF-1 mRNA (0.731 ± 0.099 vs.0.544 ± 0.108) in retinas from the control group and test group, respectively. The expression of these factors in the test group was significantly higher (t = 2.488, P = 0.038;t = 2.864, P = 0.021). The number of neovascular endothelial nuclear that broke through the retinal internal limiting membrane in the paraffin section in the high dose group and the low dose group was significantly less than that in the self-control group (t = -9.507, P = 0.000; t = -10.761, P = 0.000). The appearance of ADPase staining sections in the medication group was more similar to the simple control group than that of the test group. Immunohistochemical staining sections showed that VEGF and SDF-1 expressed in neuroepithelial cells in each group. APSAP in the high dose group and the low dose group was significantly lower than that in the self-control group (VEGF: t = -7.249, P = 0.000; t = -9.02, P = 0.000; SDF-1: t = -5.246, P = 0.000; t = -5.216, P = 0.000). CONCLUSION: These results indicate that AMD3100 block the SDF-1 receptor to reduce the effect of SDF-1, decrease the production of VEGF protein and inhibite neovascularization.

Case Report: A Case of Cystoid Macular Edema in Retinitis Pigmentosa With Central Retinal Vein Occlusion.

Significance: Cystoid macular edema (CME) is a common complication of retinitis pigmentosa (RP). However, CME in RP with central retinal vein occlusion (CRVO) is rare. Prompt administration of anti-vascular endothelial growth factor (anti-VEGF) medication can achieve a satisfactory prognosis. Purpose: This report describes a case of using anti-VEGF medication to treat CME secondary to RP with impending or mild CRVO. Case Report: A 26-year-old female presented for blurred vision in both eyes. Best-corrected visual acuity (BCVA) was 20/50 in the right eye and finger-counting in the left eye. According to ophthalmic examinations, CME secondary to RP in the right eye and CME secondary to RP with impending or mild CRVO in her left eye can be diagnosed. Central macular thickness (CMT) was 554 µ m in the right eye and 831 µm in the left eye. Only the left eye was treated with a single intravitreal injection of anti-VEGF medication. One month later, BCVA increased to 20/200 and CMT decreased to 162 µm in the left eye. Interestingly, BCVA in the right eye also had an improvement (20/40) and intraretinal fluid decreased significantly. However, 3 months after injection, these improvements of both eyes were not maintained. Conclusion: This is the second case of RP with CRVO. Intravitreal injection of anti-VEGF medication for addressing CME secondary to RP with CRVO is an effective treatment, but it needs to be reinjected.

Yes-associated protein promotes endothelial-to-mesenchymal transition of endothelial cells in choroidal neovascularization fibrosis.

AIM: To reveal whether and how Yes-associated protein (YAP) promotes the occurrence of subretinal fibrosis in age-related macular degeneration (AMD). METHODS: Cobalt chloride (CoCl2) was used in primary human umbilical vein endothelial cells (HUVECs) to induce hypoxia in vitro. Eight-week-old male C57BL/6J mice weighing 19-25 g were used for a choroidal neovascularization (CNV) model induced by laser photocoagulation in vivo. Expression levels of YAP, phosphorylated YAP, mesenchymal markers [α smooth muscle actin (α-SMA), vimentin, and Snail], and endothelial cell markers (CD31 and zonula occludens 1) were measured by Western blotting, quantitative real-time PCR, and immunofluorescence microscopy. Small molecules YC-1 (Lificiguat, a specific inhibitor of hypoxia-inducible factor 1α), CA3 (CIL56, an inhibitor of YAP), and XMU-MP-1 (an inhibitor of Hippo kinase MST1/2, which activates YAP) were used to explore the underlying mechanism. RESULTS: CoCl2 increased expression of mesenchymal markers, decreased expression of endothelial cell markers, and enhanced the ability of primary HUVECs to proliferate and migrate. YC-1 suppressed hypoxia-induced endothelial-to-mesenchymal transition (EndMT). Moreover, hypoxia promoted total expression, inhibited phosphorylation, and enhanced the transcriptional activity of YAP. XMU-MP-1 enhanced hypoxia-induced EndMT, whereas CA3 elicited the opposite effect. Expression of YAP, α-SMA, and vimentin were upregulated in the laser-induced CNV model. However, silencing of YAP by vitreous injection of small interfering RNA targeting YAP could reverse these changes. CONCLUSION: The findings reveal a critical role of the hypoxia-inducible factor-1α (HIF-1α)/YAP signaling axis in EndMT and provide a new therapeutic target for treatment of subretinal fibrosis in AMD.

Measurement of the Lid Margin Thickness in Meibomian Gland Dysfunction with Vernier Micrometer.

INTRODUCTION: To investigate the lid margin thickness (LMT) from the posterior lash line to the mucocutaneous junction at the middle position in adults with and without meibomian gland dysfunction (MGD) by vernier micrometer (VM). METHODS: This is a cross-sectional, observational study. A hundred eyes from 100 volunteers aged 20 to 79, including 56 normal participants and 44 participants with MGD, were recruited. Measurements of the LMT by VM were performed by the same person. RESULTS: The mean age of 56 normal subjects (24 males and 32 females) and 44 MGD subjects (16 males and 28 females) was 40.0 ± 13.2 years and 42.7 ± 17.1 years, respectively. There was a significant difference in the upper LMT between normal and MGD subjects (1.36 ± 0.25 vs. 1.60 ± 0.27 mm, P < 0.001), but not in the lower LMT (1.0 ± 0.23 vs. 1.10 ± 0.28 mm, P = 0.07). In both normal and MGD subjects, the upper or lower LMT was significantly positively correlated with age (P < 0.05), and the upper LMT was greater than the lower LMT (P < 0.001). In addition, the lower LMT in MGD subjects was significantly positively correlated with meibum expressibility (rs = 0.35, P = 0.02). CONCLUSIONS: The LMT was closely related to age and could be an important indicator for detecting MGD. Furthermore, we found that the upper LMT was greater than the lower LMT, and the lower LMT in MGD subjects seemed to be related to meibum expressibility.

A Randomized, Controlled Trial of Treat-and-Extend vs. Pro Re Nata Regimen for Neovascular Age-Related Macular Degeneration.

Purpose: To compare the efficacy and safety of conbercept using a treat-and-extend (T&E) regimen vs. a pro re nata (PRN) regimen in Chinese patients with neovascular age-related macular degeneration (nAMD). Methods: This was a randomized, multicenter, non-inferiority study. After an initial loading phase of three consecutive monthly intravitreal injections of 0.5 mg Conbercept, the patients were treated to PRN or T&E regimen. The prespecified retreatment criteria was defined as a more than 5-letter decrease in BCVA from the previous visit or any evidence of new retinal hemorrhages, or the presence of any IRF and any SRF of more than 200 µm in height at the sub-foveal center. The primary outcome was the mean change in best-corrected visual acuity (BCVA) from baseline to 24 months, with a prespecified non-inferiority limit of -5 letters. Results: From July 2016 through August 2018, 141 participants were allocated and treated (T&E, n = 69; PRN, n = 72). About one fifth of the overall participants were dropped out during the 12-month follow-up (28/141, 19.9%), and about one thirds of the overall participants were lost during the 24-month follow-up (51/141, 36%). At 2 years, mean BCVA letter improvement was + 4.0 in the T&E group vs. + 5.1 in the PRN group, and T&E regimen was not non-inferior to PRN regimen [difference, -1.169 letters; 95% confidence interval (CI): -6.864 ∼ 4.526]. Subgroup analyses also demonstrate the similar results in PCV patients, naive patients and no-naive patients. The mean decrease in central subfield thickness were 180 ± 165 µm in the T&E group and 247 ± 230 µm in the PRN group, respectively. The patients in the PRN group had required significantly fewer injections than those in the T&E group (12.4 vs. 14.6 injections, P = 0.041). The types and rates of adverse events were comparable in the two treatment groups. Conclusion: These findings suggest that the T&E regimen was not non-inferior to the PRN regimen in patients with nAMD in terms of BCVA outcomes through 24 months. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT02802657.

Pharmacological Inhibition of Glutaminase 1 Attenuates Alkali-Induced Corneal Neovascularization by Modulating Macrophages.

Corneal neovascularization (CoNV) in response to chemical burns is a leading cause of vision impairment. Although glutamine metabolism plays a crucial role in macrophage polarization, its regulatory effect on macrophages involved in chemical burn-induced corneal injury is not known. Here, we elucidated the connection between the reprogramming of glutamine metabolism in macrophages and the development of alkali burn-induced CoNV. Glutaminase 1 (GLS1) expression was upregulated in the mouse corneas damaged with alkali burns and was primarily located in F4/80-positive macrophages. Treatment with a selective oral GLS1 inhibitor, CB-839 (telaglenastat), significantly decreased the distribution of polarized M2 macrophages in the alkali-injured corneas and suppressed the development of CoNV. In vitro studies further demonstrated that glutamine deprivation or CB-839 treatment inhibited the proliferation, adhesion, and M2 polarization of bone marrow-derived macrophages (BMDMs) from C57BL/6J mice. CB-839 treatment markedly attenuated the secretion of proangiogenic factors, including vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) from interleukin-4- (IL-4-) regulated M2 macrophages. Our findings revealed that GLS1 inhibition or glutamine deprivation prevented alkali-induced CoNV by inhibiting the infiltration and M2 polarization of macrophages. This work suggests that pharmacological GLS1 inhibition is a feasible and effective treatment strategy for chemical burn-related CoNV in humans.