Serine to proline mutation at position 341 of MYOC impairs trabecular meshwork function by causing autophagy deregulation.

Glaucoma is a highly heritable disease, and myocilin was the first identified causal and most common pathogenic gene in glaucoma. Serine-to-proline mutation at position 341 of myocilin (MYOCS341P) is associated with severe glaucoma phenotypes in a five-generation primary open-angle glaucoma family. However, the underlying mechanisms are underexplored. Herein, we established the MYOCS341P transgenic mouse model and characterized the glaucoma phenotypes. Further, we systematically explored the functional differences between wild-type and MYOCS341P through immunoprecipitation, mass spectrometry, and RNA-seq analyses. We found that MYOCS341P transgenic mice exhibit glaucoma phenotypes, characterized by reduced aqueous humor outflow, elevated intraocular pressure, decreased trabecular meshwork (TM) cell number, narrowed Schlemm's canal, retinal ganglion cell loss, and visual impairment. Mechanistically, the secretion of dysfunctional MYOCS341P accumulated in the endoplasmic reticulum (ER), inducing ER stress and dysregulation of autophagy, thereby promoting TM cell death. We describe an effective transgenic model for mechanistic studies and the screening of therapeutic targets. Our data generated from high-throughput analyses help elucidate the mechanism underlying mutant MYOC-related glaucoma.

The optic nerve head perfusion and its correlation with the macular blood perfusion in unilateral idiopathic macular hole: an optical coherence tomography angiography study.

AIM: To compare the optic nerve head (ONH) perfusion in both eyes of unilateral idiopathic macular hole (IMH) with normal control group by using optical coherence tomography angiography (OCTA) and investigate its correlationship with the macular blood perfusion. METHODS: We performed a prospective and cross-sectional study that included 19 patients with full-thickness unilateral IMH and 24 age- and sex-matched controls. All participants received OCTA test. The ONH perfusion was evaluated by the regions of peripapillary and whole en face (the sum of peripapillary and optic disc). The potential correlationship between ONH and parafovea were implied. All the data were performed using the nonparametric test. RESULTS: The mean values of ONH presented that normal control >IMH >unaffected eyes. A statistical variation was found between three groups in the region of temporal (P=0.007). Vessel density notablely decreased on the layers of superficial, deep and choroid of parafovea region in IMH group. The correlative coefficients showed that respectively whole en face and deep retina: r=0.528, peripapillary and deep retina: r=0.525, whole en face and choriocapillaries: r=0.569, peripapillary and choriocapillaries: r=0.504. CONCLUSION: Our study demonstrate a reduced ONH vessel density in both eyes of IMH patients and the vessel density of ONH in IMH eyes are positively correlated with both the retina capillary and choriocapillary in parafoveal. The reduction of vessel densities may indicate the hypoperfusion in IMH eyes.

MicroRNA regulation of MDM2-p53 loop in pterygium.

PURPOSE: The pathogenesis of pterygium has been linked to limbal stem cell damage, abnormal apoptosis and cellular proliferation. In this study, we investigated the epigenetic regulation through microRNA expression in the pathogenesis of pterygium. METHODS: Human full-length primary pterygia were microdissected into head and body regions. Specific microRNA and mRNA expression was assayed by TaqMan® real-time quantitative polymerase chain reaction (qPCR). Tissue localization of target microRNAs was performed by LNA-based in situ hybridization. MicroRNA-145 (miR-145) mimics were transfected to primary culture of human pterygial cells, followed by analyses of cell cycle changes, apoptosis, p53 and MDM2 expression using flow cytometry and qPCR. RESULTS: The expression of miR-145 was markedly higher in primary human pterygium than in limbus and conjunctiva. Both miR-143 and miR-145 were predominantly expressed in the basal pterygial epithelium. Oncogene MDM2 expression was abundant in pterygial epithelium and stroma, while the expression pattern was opposite to that of miR-145. Ectopic expression of miR-145 in pterygial cells induced G1 arrest, down-regulated MDM2 and elevated p53 expression. CONCLUSIONS: Our study showed that miR-145 suppressed MDM2 expression, which subsequently influenced the p53-related cell growth pattern in pterygial epithelium. The regulatory miR-145/MDM2-p53 loop can serve as a potential target for treatment of pterygium.

Quantitative Choriocapillaris Perfusion Before and After Vitrectomy in Idiopathic Epiretinal Membrane by Optical Coherence Tomography Angiography.

BACKGROUND AND OBJECTIVES: To compare choriocapillaris perfusion of the fovea between patients with idiopathic epiretinal membrane (iERM) and normal controls and determine whether surgery affects it. PATIENTS AND METHODS: Both eyes of 45 patients with iERM and 28 healthy subjects were scanned by optical coherence tomography angiography (OCTA) preoperatively, and 25 of the patients were scanned postoperatively. Central parameters measured included flow area and parafovea vessel density of the foveal choriocapillaris. RESULTS: Both parameters were significantly lower in eyes with iERM (both P < .001) compared with both the unaffected fellow eyes and the normal control eyes (both P < .001) preoperatively, and they increased as the result of surgery, although it did not reach statistical significance. However, the differences between both eyes of patients became insignificant after surgery (both P > .05). CONCLUSIONS: OCTA may provide quantitative information regarding choriocapillaris perfusion. iERM influences the foveal choriocapillaris perfusion, which is reversible by surgery. [Ophthalmic Surg Lasers Imaging Retina. 2017;48:906-915.].

OCT angiography quantifying choriocapillary circulation in idiopathic macular hole before and after surgery.

PURPOSE: To investigate the choriocapillary circulation in the macular area for eyes with unilateral idiopathic macular hole (IMH) before and after vitrectomy using optical coherence tomography angiography (OCTA). METHODS: A prospective study of 25 patients with unilateral IMH who underwent vitrectomy and 30 age- and sex-matched healthy controls were recruited. Choriocapillary circulation was measured by OCTA to obtain two measurements: flow area and parafovea vessel density. RESULTS: Flow area and parafovea vessel density of choriocapillaris in the macular area were significantly smaller and lower in IMH eyes than unaffected fellow eyes and healthy control eyes (p < 0.001), while no difference was found between unaffected fellow eyes and the healthy control eyes. One month after vitrectomy, the choriocapillary flow area and parafovea vessel density of IMH eyes significantly increased compared to the peroperative measurements (p < 0.001). Association analysis found that choriocapillary circulation measurements were negatively correlated with macular hole diameters in IMH eyes (p < 0.001), but was independent with best-corrected visual acuity (BCVA). CONCLUSIONS: The macular choriocapillary flow area and parafovea vessel density in IMH eyes were lower than those of normal controls. In addition, the choriocapillary circulation was negatively correlated with macular hole diameter. Our findings suggested that choroidal circulation in the macular area might be affected by the intact structure of the fovea.

Chitosan Promoted the Corneal Epithelial Wound Healing via Activation of ERK Pathway.

PURPOSE: To investigate the mechanism of chitosan promoting corneal wound healing though evaluating its effect on extracellular signal-regulated kinases (ERK) and p38 pathway activity in a rabbit animal model. METHODS: Cell proliferation and migration assay were performed 24 hours after chitosan treatment. The activity of ERK and p38 pathways was detected by using immunofluorescence and Western blotting in the presence of chitosan and an ERK inhibitor. In vivo study of epithelial debridement wounds was performed on 8 mm rabbit corneas in the presence of chitosan and an ERK pathway inhibitor. RESULTS: Immunostaining with Ki67 and migrating assay showed that chitosan could upregulate the cell proliferation and promote the cell migration. Chitosan activated the ERK pathway in 5 min to 30 min after treatment but did not affect the p38 pathway. ERK inhibitor PD98059 can inhibit the chitosan-stimulated ERK phosphorylation. Chitosan increased the corneal epithelial wound closure in organ culture and ERK inhibition with PD98059 blocked the effect of chitosan on wound healing. CONCLUSIONS: Chitosan promoted corneal epithelial proliferation and migration during the wound healing in rabbits' eye. Chitosan-stimulated epithelial wound healing is partially mediated through the activation of the ERK pathway but not the p38 pathway.

Signature microRNAs in human cornea limbal epithelium.

This study was aimed to identify the signature microRNAs, which regulate the biological processes of corneal epithelial progenitor cell (CEPC) homeostasis and regulation through characterizing the differential expression profile of microRNAs in human limbal epithelium containing adult CEPC versus central corneal epithelium without CEPC. MicroRNA microarray had identified 37 microRNAs enriched in human corneal epithelium. Among them, nine were significantly upregulated in limbal epithelium and one in central corneal epithelium after validation by TaqMan® real-time polymerase chain reaction. In addition to our previous finding of miR-143 and 145, the expression of miR-10b, 126, and 155 was localized in limbal epithelium (LE) (predominantly basal layers) by using locked nucleic acid-based in situ hybridization. Potential target genes were predicted by TargetScan Human v6.0 and compared to the reported human cornea epithelial gene profile GSE5543. Analyzed by web-based Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and DAVID Functional Annotation Bioinformatics Resources v6.7, the downregulated genes were involved in pathways of immune response and cellular protection, apoptosis, and cell movement whereas upregulated genes with cell survival, cell-matrix interaction, and cell-cell adhesion. We found a constant occurrence of miR-143, 145, and 155 in all KEGG pathways regulating limbal epithelial events. By Ingenuity Systems (IPA®) analysis, these microRNAs could cooperatively regulate cell growth and apoptosis via tumor necrosis factor activation and MYC repression. Our findings thus suggest a unique microRNA signature existing in human limbal epithelium and participating in CEPC homeostasis.

Evaluation of crowded optic nerve head and small scleral canal in intrapapillary hemorrhage with adjacent peripapillary subretinal hemorrhage.

BACKGROUND: Intrapapillary hemorrhage with adjacent peripapillary subretinal hemorrhage (IHAPSH) is a clinical syndrome most commonly affecting myopic eyes with tilted discs that usually resolves spontaneously without treatment. Subretinal hemorrhage usually occurs peripapillary on the nasally adjacent side near the optic disc. The etiology of this condition is still unknown. The purpose of this study was to determine if a crowded optic nerve head and small scleral canal are involved in the pathogenetic mechanisms of IHAPSH. METHODS: Twelve subjects with IHAPSH diagnosed at the Affiliated Ophthalmology Hospital of the First Clinical College of Harbin Medical University and 24 control subjects were examined. The size of the inner aspect of the scleral canal and level of nerve fiber crowding of the optic nerve head were analyzed with optic nerve head analysis software packet of the Stratus Optical Coherence Tomography software and manual segmentation software. The Mann-Whitney U test and multiple comparisons (with the Bonferroni correction method) were performed. p values less than 0.002 (two-sided) were considered statistically significant. The area, perimeter, and the perimeter/area ratio of the optic disc, vertical and horizontal diameter of the inner aspect of the scleral canal, vertical integrated rim area (VIRA), and the rim area were calculated. RESULTS: The area and perimeter of the optic disc and the horizontal diameter of the inner aspect of the scleral canal were significantly lower in the affected and contralateral eyes of the subjects with IHAPSH than in the eyes of the controls. Conversely, the IHAPSH-affected and contralateral eyes had significantly higher perimeter/area ratio of the optic disc, VIRA, and rim area values than the control eyes. The VIRA and rim area were greater in the IHAPSH-affected eyes than in the contralateral eyes. CONCLUSIONS: Patients with IHAPSH have smaller optic discs and scleral canals than control subjects, with a higher level of nerve fiber crowding.

[Clinical characteristics and pathogenesis of intracapillary hemorrhage with adjacent peripapillary subretinal hemorrhage].

OBJECTIVE: To study the clinical characteristics and pathogenesis of intracapillary hemorrhage with adjacent peripapillary subretinal hemorrhage (IHAPSH). METHODS: A retrospective review of 8 patients with IHAPSH. The demographical data of the patients, the symptoms, initial and final visual acuities, biomicroscopic findings, fundus photographs, and the results of various special examinations, including fluoresce in angiography, B-scan ultrasonography, optical coherence tomography (OCT) and CT were analyzed. RESULTS: Fundus examination of 8 patients showed various changes of optic disc, including: a small and tilted optic disc with elevation and blurring of the nasal or superonasal edge. Hemorrhage of the optic disc was present in the cup and/or on the nasal edge, extended to the surface of the optic disc, the superficial layer of the retina and the subretinal space. Subretinal hemorrhage was located on the nasal, superior and inferior side of the optic disc. OCT examination showed an elevated optic nerve head with an enlarged nasal peripapillary subretinal space. CONCLUSIONS: In the IHAPSH patient, bleeding may originate from the posterior ciliary artery that traverse the sclera window of the disc, passes through the space between the optic nerve and the scleral canal, reaches the edge of the optic disc and the subretinal space, penetrates into the optic nerve tissues anterior to the lamina cribrosa, reaches the optic cup and extends to the surface of the optic disc, the retina or the vitreous.

MicroRNA-145 regulates human corneal epithelial differentiation.

BACKGROUND: Epigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium. METHODOLOGY/PRINCIPAL FINDINGS: Human LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin ß8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs. CONCLUSION/SIGNIFICANCE: We found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.

[Prevalence of diabetic retinopathy among the elderly in rural southern Shuangcheng city, Heilongjiang province].

OBJECTIVE: The purpose of this study was to understand the awareness, prevalence of diabetic retinopathy and treatment status of people aged over 50 and living in the rural areas of Shuangcheng city, Heilongjiang province, China. METHODS: Cluster sampling was used in randomly selected 5504 survey for ophthalmic clinical examination, in patients with diabetic retinopathy. A questionnaire in the state of knowledge about prevention and treatment was developed. RESULTS: Among the 5504 persons entering in the project, 5053 were examined on their eyes (91.8%). In this selected population, 56 persons (112 eyes) were diagnosed as diabetic retinopathy (1.108%), with 95% confidence interval (CI) as: 0.819% to 1.397%. Of 56 patients, 49 cases were non-proliferative diabetic retinopathy, accounting for 87.50% of the total number of patients with diabetic retinopathy; proliferative diabetic retinopathy 7 cases, accounting for 12.50% of the 112 eyes, 6.25% (7/112) having vitreous hemorrhage, 8.04% (9/112) having macular edema. For diabetic retinopathy prevalence rates, there was no significant difference in males and females. Between the per differential 10-year-old division, the difference was significant. Among the 60 to 69 group, a significantly higher prevalence rate was seen. Of the 112 eyes with diabetic retinopathy, 34 eyes (30.4%) were low vision [visual acuity < 20/60 (0.3) to ≥ 20/400 (0.05)]; 6 eyes (5.4%) were blind [visual acuity < 20/400 (0.05) to NLP]. The rate in the patients with PDR and fasting blood glucose was above 11.1 mmol/L was higher than having NPDR and fasting blood glucose below 11.1 mmol/L. Having fasting blood glucose 11.1 mmol/L and above with the course over five years among patients with PDR, the proportion of fasting blood glucose was higher than those with less than 11.1 mmol/L and diabetic retinopathy duration of less than five years. Of 56 patients with diabetic retinopathy, 38 cases (67.9%) did not receive any treatment. Among 18 cases (32.1%) with insulin or oral drug therapy, regularly using insulin or other medication (14.3%), only 1 (1.8%) case was given the treatment for diabetic retinopathy. Results from our survey showed that patients with diabetic retinopathy had a poor understanding about prevention and treatment of the disease. CONCLUSION: Long duration and high blood glucose in patients with diabetic retinopathy seemed to be the important risk factor. Early systematic drug use for prevention and blood glucose control was the key to prevent diabetic retinopathy. Patients with diabetic retinopathy in China had poor understanding about the prevention measures of the disease and lack of knowledge.

Proliferative and migratory aptitude in pterygium.

Pterygium is a chronic fibrovascular overgrowth on the corneal surface and is often associated with inflammation, astigmatism and obstructed vision. The common treatment is surgical removal but post-operative recurrences with more aggressive behavior are common. However, there is a controversy in the pathogenesis of primary pterygium between limbal stem cell failure versus proliferation. In this study, we explore the proliferative and migratory aptitude in pterygium by characterizing the growth and migration pattern of pterygial cells in the head (on the cornea), the neck (over the focal limbus), and the body (on the conjunctiva) epithelia of 12 full-length primary pterygia. Immunofluorescence and quantification analyses showed a spatial expression pattern of markers for stem cells, cell growth, and matrix metalloproteinases. Beside the basal epithelia in all three regions, p63α(strong) cells were located in suprabasal layers in head, weak in the body and absent in neck. Pertinent cell proliferation in head than body epithelia was revealed by its higher colony-forming efficiency. ATP-binding cassette transporter glycoprotein family member-2 and cytokeratin-15 were found mainly in the body basal epithelia, similar to that in normal conjunctiva. Much fewer proliferating stem-like cells in the neck region supported the limbal failure as a cause of pterygium formation. Pax6, matrix metalloproteinase-2 and -9 were more expressed in the head than in the other two regions. Our results indicate the importance of pterygium head in tissue growth and invasion and its likely involvement in post-operation recurrence.