Fig 1: 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.
Fig 2: 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.
Fig 3: LRG1 is expressed in pathological vessels of CNVM. Serial CNV membrane sections were analyzed by immunohistochemistry using anti-LRG1, anti-CD34, and anti-a-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.
Fig 4: LRG1 and LPHN2 mediate microvessel sprouting and neurite outgrowth in the absence of TGF-ß1 under hyperglycemic conditions.a, b Ex vivo mouse aortic ring assay (a) and mouse cavernosum endothelial cell (MCEC) sprouting assay (b) under normal-glucose (NG) or high-glucose (HG) conditions with treatment of PBS (negative control) or LRG1 (1 µg/ml) in the presence of lentivirus (shCon or shLPHN2; 5 × 104 TU/ml) or anti-TGF-ß1 antibody (10 µg/ml) for 5 days. Sprouted microvessels were immunostained with the endothelial cell marker platelet/endothelial cell adhesion molecule-1 (PECAM-1) (red, a). The dotted line indicates the sprouting endothelial cell range (b). Scale bars, 100 µm. The intensity of the area of microvessel sprouting from aortic rings (means ± SEM (n = 4) (a) and endothelial cell sprouting from cavernous tissue (means ± SEM (n = 6) (b) were quantified (bottom). c and d ßIII-Tubulin immunostaining in mouse MPG tissue (c, top) and DRG tissue (d, top) under normal glucose (NG) or high glucose (HG) conditions with treatment of PBS (negative control) or LRG1 (1 µg/ml) in the presence of lentivirus (shCon or shLPHN2; 5 × 104 TU/ml) or anti-TGF-ß1 antibody (10 µg/ml) for 1 week. Scale bars, 100 µm. Quantification of ßIII-tubulin–immunopositive neurite length in MPG (c) and DRG (d) tissue and (bottom, means ± SEM (n = 4)). *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t test). N.S., not significant. The relative ratio of the NG group was defined as 1.
Fig 5: Identification of the LRG1-LPHN2 signaling pathway.a HUVECs were stimulated with LRG1 (1 µg/ml) for the indicated times under high-glucose conditions. Immunoprecipitants with an anti-LPHN2 antibody were analyzed by Western blotting using antibodies specific for pY-LPHN2 and LPHN2. b Cignal Finder GPCR signaling 10-pathway reporter array analysis of HUVECs after treatment with LRG1 (1 µg/ml). The results of dual-luciferase assays are presented as normalized relative luminescence signals (means ± SEM, n = 3). The relative ratio of the untreated group was defined as 1. **P < 0.01 (Student’s t test). c Network model showing interactions between the proteins with increased phosphorylation in response to LRG1. Node color indicates the increase (red) in the phosphorylation level. The color bar indicates the gradient of the log2-fold-change of phosphorylation levels by LRG1 with respect to those in untreated control conditions. Circled P on a node indicates phosphorylation of the corresponding protein. Arrows, activation; inhibition symbols, inhibition; solid arrows, direct activation; dotted arrows, indirect activation; gray lines, protein–protein interactions; and green lines, plasma membrane. d Western blot analysis of HUVECs stimulated with LRG1 (5 µg/ml) using the indicated antibodies. e Western blot analysis of HUVECs stimulated with 1 µg/ml LRG1 with or without pre-treatment with PP2 (10 µM) or LY294002 (10 µM). f and g HUVECs (f) and mouse DRG explants (g) were treated with LRG1 (1 µg/ml), LY (10 µM), PP2 (10 µM), LRG1 (1 µg/ml) + LY (10 µM), or LRG1 (1 µg/ml) + PP2 (10 µM). Top: Representative images of HUVEC tube formation (f) and ßIII-tubulin staining in mouse DRG explants (g). The relative ratio of the untreated group was defined as 1. Scale bars, 100 µm. Bottom: Master junctions (n = 4) (f) and ßIII-tubulin–immunopositive axon lengths in DRGs (g) were quantified using ImageJ, and the results are presented as the means ± SEM (n = 4). h Western blot analysis of HUVECs stimulated with 1 µg/ml LRG1 for 30 min or 7 hr using antibodies specific for angiogenic factors (VEGFA, angiopoietin-1, FGF2).
Supplier Page from MilliporeSigma for Anti-LRG1 antibody produced in rabbit