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.

Image enhancement of color fundus photographs for age-related macular degeneration: the Shanghai Changfeng Study.

AIM: To develop and evaluate a new fundus image optimization software based on red, green, blue channels (RGB) for the evaluation of age-related macular degeneration (AMD) in the Chinese population. METHODS: Fundus images that were diagnosed as AMD from the Shanghai Changfeng Study database were analyzed to develop a standardized optimization procedure. Image brightness, contrast, and color balance were measured. Differences between central lesion area and normal retinal area under different image brightness, contrast, and color balance were observed. The optimal optimization parameters were determined based on the visual system to avoid image distortion. A paired-sample diagnostic test was used to evaluate the enhancement software. Fundus optical coherence tomography (OCT) was used as the gold standard. Diagnostic performances were compared between original images and optimized images using McNemar's test. RESULTS: A fundus image optimization procedure was developed using 86 fundus images of 74 subjects diagnosed with AMD. By observing gray-scale images, choroid can be best displayed in red channel and retina in green channel was found. There was limited information in blue channel. Totally 104 participants were included in the paired sample diagnostic test to assess the performance of the optimization software. After the image enhancement, sensitivity increased from 74% to 88% (P=0.008), specificity decreased slightly from 88% to 84% (P=0.500), and Youden index increased by 0.11. CONCLUSION: The standardized image optimization software increases diagnostic sensitivity and may help ophthalmologists in AMD diagnosis and screening.

Lipopolysaccharide Promotes Choroidal Neovascularization by Up-Regulation of CXCR4 and CXCR7 Expression in Choroid Endothelial Cell.

Stromal cell-derived factor-1 (SDF-1) has been confirmed to participate in the formation of choroidal neovascularization (CNV) via its two receptors: CXC chemokine receptors 4 (CXCR4) and CXCR7. Previous studies have indicated that the activation of Toll-like receptors (TLRs) by lipopolysaccharide (LPS) might elevate CXCR4 and/or CXCR7 expression in tumor cells, enhancing the response to SDF-1 to promote invasion and cell dissemination. However, the impact of LPS on the CXCR4 and CXCR7 expression in endothelial cells and subsequent pathological angiogenesis formation remains to be elucidated. The present study shows that LPS enhanced the CXCR4 and CXCR7 expression via activation of the TLR4 pathway in choroid-retinal endothelial (RF/6A) cells. In addition, the transcriptional regulation of CXCR4 and CXCR7 by LPS was found to be mediated by phosphorylation of the extracellular signal-related kinase (ERK) 1/2 and activation of nuclear factor kappa B (NF-κB) signaling pathways, which were blocked by ERK- or NF-κB-specific inhibitors. Furthermore, the increased CXCR4 and CXCR7 expression resulted in increased SDF-1-induced RF/6A cells proliferation, migration and tube formation. In vivo, LPS-treated rat had significantly higher mRNA levels of CXCR4 and CXCR7 expression and lager laser-induced CNV area than vehicle-treated rat. SDF-1 blockade with a neutralizing antibody attenuated the progression of CNV in LPS-treated rat after a single intravitreal injection. Altogether, these results demonstrated that LPS might influence CNV formation by enhancing CXCR7 and CXCR7 expression in endothelial cells, possibly providing a new perspective for the treatment of CNV-associated diseases.

The Relationship between Visual Field Global Indices and Retinal Nerve Fiber Layer Thickness in Healthy Myopes.

The aim of the current study was to investigate the association between the thickness of the retinal nerve fiber layer (RNFL) and central visual field indices in otherwise healthy myopes. In total, 57 otherwise healthy subjects were cross-sectionally studied. General ophthalmic examinations, refractive measurements, RNFL thickness by spectral domain optical coherence tomography (OCT), and central visual fields were examined. Linear models were used to assess the associations. In this young and mid-aged population, the mean spherical equivalent was -4.79 (SD 1.66) and -4.59 (SD 1.88) diopters in the right and left eyes, respectively. Approximately 7% to 14% of the eyes showed the average RNFL thickness out of the normal range. The temporal RNFL was remarkably thicker, whereas the nasal RNFL was thinner. The higher the refractive error, the thinner the RNFL thickness. A thicker overall RNFL was significantly associated with decreased mean sensitivity and increased mean defect, and further adjustments for age, sex, refractive error, optic disk area, or ocular magnification did not change the association. Although nonpathologic myopia does not significantly affect central visual field global indices, its effects on the RNFL may be linked with performance on the central visual field test.

Vascular endothelial growth factor regulates primate choroid-retinal endothelial cell proliferation and tube formation through PI3K/Akt and MEK/ERK dependent signaling.

Vascular endothelial growth factor (VEGF) is a hypoxia-induced angiogenic protein that exhibits a broad range of biological and pathological effects in wet age-related macular degeneration and proliferative diabetic retinopathy. However, its specific mechanism is still not fully understood. Here, we examined the effects of VEGF on choroid-retinal endothelial cells (RF/6A) proliferation and tube formation, and the underlying signal pathways responsible in this process. RF/6A cells were pretreated with MEK inhibitor or PI3K inhibitor, and then incubated in a hypoxia chamber. Real-time PCR and Western blot analysis were carried out to explore VEGF expression on mRNA and protein levels. Hypoxia inducible factor-1α (HIF-1α) and VEGFR2 expression levels were also investigated in the presence and absence of hypoxic conditions. CCK-8 analysis and tube formation assay were tested under hypoxia, exogenous recombinant VEGF, and different signal pathway inhibitors, respectively. Mean while, the PI3K/Akt and MEK/ERK pathways in this process were also investigated. Our results showed that VEGF, HIF-1α, VEGFR2, p-ERK, and p-Akt were up-regulated in RF/6A cells under hypoxic conditions. MEK inhibitor (PD98059) and PI3K inhibitor (LY294002) decreased ERK and Akt activity, respectively, and reduced VEGF expression. VEGF-induced RF/6A proliferation and tube formation requires MEK/ERK and PI3K/Akt signaling, and both of the two pathways were needed in regulating VEGF expression. These suggest that VEGF plays an important role in RF/6A proliferation and tube formation, and MEK/ERK and PI3K/Akt pathway may be responsible for this process.

[Visual field defects in 169 cases of pituitary adenomas].

OBJECTIVE: To compare static visual field perimetry with Octopus 101 and Goldmann perimetry in patients with pituitary adenomas; To identify the factors of visual field defects and visual field prognosis. METHODS: Retrospective nested case-control study. Visual acuity, fundus, computed tomography (CT) or magnetic resonance imaging (MRI) and visual field were pre-operated assessed in 169 patients (338 eyes) diagnosed pituitary adenoma at the Department of Neurosurgery in Huashan Hospital between Feb. 2006 and Feb. 2007. 334 eyes were underwent Goldmann perimetry, while 323 eyes were underwent static visual field perimetry with Octopus 101 perimeter. In the 169 patients, 28 patients (56 eyes) were followed up post-operatively for 3 to 6 months. Statistical analysis was performed using SPSS 15.0 software. T test and rank sum test were used for continuous variables, and chi-square test and Fisher's exact test were used for categorical variables. We also carry out Logistic regression. RESULTS: Of the 169 patients, unilateral or bilateral visual field defects were observed in 133 cases. In these 323 eyes, 231 eyes showed visual field defects with static visual field perimetry, while 196 eyes in 334 eyes showed defects with Goldmann perimetry. Earlier age of onset, larger tumors and poor preoperative corrected visual acuity were more frequent in patients with visual field defects than in patients with normal visual fields (t = 4.802, 7.930; chi(2) = 28.210, P < 0.01). In the 28 followed-up patients (55 eyes), visual acuity was improved in 69.05% eyes and visual field was improved in 85.71% eyes after operation. There were significant differences in age of onset and preoperative visual field defects in upper temporal quadrants between patients with visual field returned to normal after the operation and patients with visual field improvement only (t = 2.525, P = 0.023; chi(2) = 6.218, P = 0.013). CONCLUSIONS: Static visual field perimetry with Octopus 101 perimeter is more sensitive than Goldmann perimetry in the early diagnosis of pituitary adenomas. Patients with visual field abnormalities are significantly with earlier onset age, larger tumors and worse preoperative visual acuity than patients free of visual field defects. The onset age and the degree of impairment in the preoperative visual field in the upper temporal quadrant may be significant factors for visual field prognosis.