LRG1 Expression Is Elevated in the Eyes of Patients with Neovascular Age-Related Macular Degeneration

Figures

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  Figure A1. LRG1 is not expressed in normal choroidal and retinal vessels (from patient with retinoblastoma). Serial paraffin-embedded sections from human normal choroids were analyzed by immunohistochemical staining using anti-CD34 and anti-LRG1 antibodies. In the right panels, arrowheads indicate choroidal and retinal blood vessels. Left panels show entire eye sections, and blue rectangles indicate the magnification areas shown in the right panels. Scale bars: 250 μm.

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  Figure A2. Example panels showing the scores used for vessel density, LRG1 expression, and α-SMA positivity evaluation. Paraffin-embedded sections from CNVMs were analyzed by sIHC staining using anti-CD34, anti-LRG1, or anti- α-SMA antibodies, respectively. Scale bars: 100 μm.

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  Figure 1. LRG1 is not expressed in normal choroidal and retinal vessels. The eye from a patient with a lacrimal sac tumor was resected, formalin-fixed, and paraffin-embedded. Serial sections were analyzed by immunohistochemical staining using anti-CD34 and anti-LRG1 antibodies. In the right panels, arrows indicate choroidal and retinal blood vessels. Left panels show entire eye sections, and blue rectangles indicate the magnification areas shown in the right panels. Scale bars: 250 μm.

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  Figure 2. LRG1 is expressed in pathological vessels of CNVM. Serial CNV membrane sections were analyzed by immunohistochemistry using anti-LRG1, anti-CD34, and anti-α-SMA antibodies. High magnifications of stained sections displayed in the blue rectangles are shown on the right. Arrows indicate blood vessels positive for LRG1. (a) CNV membrane from an anti-VEGFA-naïve eye with LRG1 expression in vessels and myofibroblasts. (b) CNV membrane from an anti-VEGFA-treated eye with LRG1 expression in vessels. Scale bars: 250 μm; 50 μm for magnifications.

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  Figure 3. LRG1 is expressed in pathological vessels and myofibroblasts of CNVM. CNV membrane sections were analyzed by multiplex immunohistochemistry using anti-LRG1 (brown staining) and anti-CD34 (red staining), or anti-LRG1 (brown staining) and anti-α-SMA (red staining) antibody combinations. Results of three representative patients are shown. Red arrows indicate blood vessels negative for LRG1; red/brown arrows indicate blood vessels or myofibroblasts positive for both LRG1 and CD34, or LRG1 and α-SMA. Scale bar: 50 μm.

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  Figure 4. Analysis of the humor aqueous levels of LRG1. (a) LRG1 protein concentrations in the aqueous humor of 58 naïve nvAMD patients (nvAMD) and 51 controls (Cntr.) (described in Table 2) were determined by ELISA. Each sample was measured twice. Data are presented as box and whisker plots displaying the median, lower, and upper quartiles (boxes) and minimum–maximum (whiskers). The P value was calculated by a two-tailed unpaired Student’s t-test. (b) Western blot analysis of LRG1 levels in the aqueous humor of 18 randomly selected nvAMD patients and controls. Loading control is shown as Ponceau S staining. (c) Quantitative analysis of panel (b) shows a significant increase in LRG1 expression in nvAMD patients. LRG1 intensity was normalized by the intensity of Ponceau-stained proteins, and protein levels were presented as fold changes. The P value was calculated by a Mann–Whitney nonparametric U test.