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. 2017 Apr 3;214(4):1029-1047.
doi: 10.1084/jem.20161802. Epub 2017 Mar 22.

Secretogranin III as a Disease-Associated Ligand for Antiangiogenic Therapy of Diabetic Retinopathy

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Free PMC article

Secretogranin III as a Disease-Associated Ligand for Antiangiogenic Therapy of Diabetic Retinopathy

Michelle E LeBlanc et al. J Exp Med. .
Free PMC article

Abstract

Diabetic retinopathy (DR) is a leading cause of vision loss with retinal vascular leakage and/or neovascularization. Current antiangiogenic therapy against vascular endothelial growth factor (VEGF) has limited efficacy. In this study, we applied a new technology of comparative ligandomics to diabetic and control mice for the differential mapping of disease-related endothelial ligands. Secretogranin III (Scg3) was discovered as a novel disease-associated ligand with selective binding and angiogenic activity in diabetic but not healthy vessels. In contrast, VEGF bound to and induced angiogenesis in both diabetic and normal vasculature. Scg3 and VEGF signal through distinct receptor pathways. Importantly, Scg3-neutralizing antibodies alleviated retinal vascular leakage in diabetic mice with high efficacy. Furthermore, anti-Scg3 prevented retinal neovascularization in oxygen-induced retinopathy mice, a surrogate model for retinopathy of prematurity (ROP). ROP is the most common cause of vision impairment in children, with no approved drug therapy. These results suggest that Scg3 is a promising target for novel antiangiogenic therapy of DR and ROP.

Figures

Figure 1.
Figure 1.
Schematics of comparative ligandomics. (A) Multi-round in vivo binding selection by OPD to enrich for retinal endothelial ligands in diabetic and healthy mice (see F). Three rounds of in vivo selection (three mice/round/group) were performed. Phage enrichment quantification is shown in G. (B) Global identification of all enriched ligands. After three rounds of selection, the cDNA inserts of enriched ligands were amplified by PCR and identified by NGS with simultaneous binding activity quantification for all identified ligands. (C) Quantitative comparison of entire ligandome profiles for diabetic versus control retina to systematically identify DR-associated endothelial ligands. (D) Enrichment of DR-high Scg3 and DR-low HRP-3. VEGF and GFP are internal positive and negative controls, respectively. ***, P < 0.001; diabetic versus healthy; χ2 test. (E) Binding activity plot for diabetic versus healthy retina. DR-associated ligands with increased or decreased binding to diabetic endothelium are classified as DR high or DR low, respectively. Non–DR-associated ligands and background binding showed similar binding activities in both conditions. DR-high Scg3, DR-low HRP-3, non–DR-associated VEGF, and background GFP are indicated. Pearson correlation coefficient: r = 0.489. (F) Diabetic mice develop retinal vascular leakage. C57BL/6 mice were treated with STZ to induce diabetes. Hyperglycemic mice were aged for 4 mo to develop DR. Retinal vascular leakage was analyzed by EB assay. Data are normalized against healthy mice treated with mock buffer. n = 12 mice. Data are ±SEM. One-way ANOVA was used. The experiment was repeated three times. (G) Phage enrichment by in vivo binding selection. The selection scheme is showed in A. Total phage bound to retinal endothelium was quantified by plaque assay after each round of selection. (A–E and G) Ligandomics was performed once.
Figure 2.
Figure 2.
Ligandomic profiling. (A and B) Ligandome profiles. A total of 844 and 1,548 putative ligands were identified by ligandomics for control (A) and diabetic (B) retina. VEGF-Phage and GFP-Phage were internal positive and negative controls, indicated by red columns. DR-high Scg3 and DR-low HRP-3 are indicated by yellow columns. Scg3 was not detected in healthy retina. GFP is used as the baseline of nonspecific binding to distinguish 417 and 817 ligands in control and diabetic retina with binding activity higher than the background. (C) Protein classification based on their roles in biological processes. (D) Activity ratio analyses to summarize the distribution patterns of binding activity (left Y axis for vertical bars) and activity ratios (right Y axis for the orange curve) for all 1,772 nonredundant ligands identified in the two conditions. Scg3 was identified with the highest binding activity ratio. (E) χ2 value distribution. The reliability of χ2 test is analyzed by plotting χ2 value against binding activity ratio. The results indicate that many ligands within the red triangle area have minimal binding activity changes but high χ2 values, suggesting that χ2 test alone may result in a large number of false positives. Based on this analysis, stringent criteria described in Results were used to identify 353 DR-high ligands and 105 DR-low ligands.
Figure 3.
Figure 3.
In vitro characterizations of Scg3 as a novel vascular permeability and angiogenic factor. (A–C) Scg3 promotes endothelial permeability. Permeability assay with Transwell inserts is illustrated in A. 1 µg/ml Scg3, 100 ng/ml VEGF, or PBS was added to the bottom (B) or upper (C) chamber. After 24 h, media were collected from the top chamber and quantified for leaked FITC-dextran. n = 3 wells. (D) Endothelial proliferation assay with HUVECs in 48-well plates. Cell number in each well was quantified at 48 h. Scg3, 1 µg/ml; VEGF, 50 ng/ml. n = 4 wells. (E and F) Scg3 induces endothelial proliferation only at a high concentration. HUVECs (n = 4 wells; E) or HRMVECs (n = 6; F) were incubated with increasing concentrations of Scg3 in 96-well plates for 48 h. Cells in each well were quantified. (G–J) Tube formation assay with HUVECs. (G) Representative images of the tube formation. Scg3, 300 ng/ml; VEGF, 50 ng/ml. Bar, 20 µm. (H) Total tube length per viewing field. (I) Number of tubes per viewing field. (J) Number of branching points per viewing field. n = 4 fields. (K and L) Scg3 stimulates the migration of HRMVECs. (K) Representative images of cell migration. Bar, 100 µm. (L) The percentage of the denuded area covered by migrated cells within the original scratch was quantified. n = 3. Experiments were independently repeated three times. One representative experiment is presented. Data are ±SEM. One-way ANOVA was used.
Figure 4.
Figure 4.
Scg3 expression in the retina. (A) Scg3 is expressed in the retinal ganglion cell layer, inner and outer plexiform layer, photoreceptor inner segments, and retinal pigment epithelial cells. Few Scg3 signals were detected in the inner and outer nuclear layer and photoreceptor outer segments. Bar, 50 µm. (B) Western blotting to detect Scg3 expression in the retina of diabetic and healthy mice. (C) Quantification of Scg3 signal in B. n = 3 mice. (D) Scg3 expression in the vitreous fluid of diabetic and healthy mice. (E) Quantification of Scg3 signal in D. n = 4 mice. (F) Scg3 expression in the retina of OIR and healthy mice at P17. (G) Quantification of Scg3 signal in F. (H) Scg3 expression in the vitreous fluid of OIR and healthy mice at P17. (I) Quantification of Scg3 signal in H. n = 6 mice. Experiments were repeated three times with similar results. Data are ±SEM. n/s, not significant. One-way ANOVA was used.
Figure 5.
Figure 5.
Scg3 is a DR-high angiogenic factor. (A–E) DR-high Scg3 selectively stimulated corneal angiogenesis in diabetic but not control mice. In contrast, DR-low HRP-3 induced angiogenesis in healthy but not diabetic mice. VEGF promoted angiogenesis in both healthy and diabetic mice. (A) Representative photographic images of corneal angiogenesis in diabetic and healthy mice. (B) DiI staining of corneal blood vessels. (A and B) Asterisks indicate the position of corneal implant. Bars, 500 µm. (C–E) Quantification of corneal angiogenesis in a blinded manner. (C) Total number of corneal vessels. (D) Number of branching points. (E) Total angiogenesis score. Sample sizes (number of cornea) are indicated at the bottom of the graphs. Data from different mice within the same groups are pooled and analyzed using one-way ANOVA. Data are ±SEM.
Figure 6.
Figure 6.
Scg3 and VEGF have distinct receptor signaling pathways. (A–F) Scg3 does not bind to VEGFR1 or VEGFR2. Protein pulldown (A, C, and E) and ELISA (B, D, and F) assays failed to detect Scg3 interaction with aflibercept (A and B), VEGFR1-Fc (C and D), or VEGFR2-Fc (E and F). n = 3 wells. Data are ±SEM. One-way ANOVA was used. (G) Scg3 does not activate VEGFR2 in HRMVECs. (H–J) Scg3 and VEGF activate Src (H), ERK1/2 (I), and MEK (J) in HUVECs. (K and L) VEGF, but not Scg3, induces the phosphorylation of Akt and Stat3. Experiments were independently repeated three times.
Figure 7.
Figure 7.
Anti-Scg3 therapy of DR. (A) Affinity-purified anti-Scg3 pAb blocks Scg3-induced proliferation of HRMVECs. VEGF, 100 ng/ml; Scg3, 1 µg/ml; anti-Scg3 pAb, 2 µg/ml. n = 8 wells. (B) Anti-Scg3 pAb inhibits Scg3-induced spheroid sprouting of HRMVECs. VEGF, 2.5 ng/ml; Scg3, 15 ng/ml; anti-Scg3 pAb, 30 ng/ml. n = 8 spheroids. (C) Anti-Scg3 ML49.3 mAb inhibits Scg3-induced HRMVEC proliferation. Concentrations are as in A. n = 3 wells. (D) Anti-Scg3 mAb cannot neutralize VEGF-induced proliferation of HRMVECs. n = 3 wells. (E) ML49.3 mAb binds to both human Scg3 (hScg3) and mouse Scg3 (mScg3) as detected by ELISA assay. n = 3 wells. (F) Anti-Scg3 therapy of DR in STZ-induced diabetic mice. Anti-Scg3 pAb, mock affinity-purified pAb against an irrelevant antigen, control rabbit IgG, ML49.3 mAb (0.36 µg/1 µl/eye), aflibercept (2 µg/1 µl/eye), or PBS was intravitreally injected. Retinal vascular leakage was quantified by EB assay. Data are normalized to PBS. n = 5 mice (except n = 3 for mock pAb). (G) Anti-Scg3 therapy of DR in Ins2Akita diabetic mice. Doses are as in F. n = 3 mice (4 for anti-Scg3). Experiments were independently repeated three times (A–E) or were repeated twice in a blinded manner (F and G). One representative experiment is shown. Data are ±SEM. One-way ANOVA was used.
Figure 8.
Figure 8.
Anti-Scg3 therapy of OIR. (A) Representative images of OIR. Arrowheads indicate neovascularization (NV) and neovascularization tufts. Doses are as in Fig. 7 F. Bar, 500 µm. (B) Quantification of neovascularization. (C) Quantification of neovascularization tuft number. (D) Quantification of branching points. n = 13 (PBS), 11 (control IgG), 12 (aflibercept), 11 (anti-Scg3 pAb), and 13 (anti-Scg3 mAb) eyes. This experiment was repeated twice in a blinded manner. Data are ±SEM. One-way ANOVA was used.
Figure 9.
Figure 9.
Schematic models of comparative ligandomics, anti-Scg3 therapy, and Scg3 molecular mechanisms. (A) From comparative ligandomics to ligand-based therapy. This study encompasses the initial ligand screening, functional characterization, pathogenic analysis, target validation, and ligand-based therapy to demonstrate the validity and utility of comparative ligandomics. (B) Anti-Scg3 therapy. Scg3 is a unique angiogenic factor that minimally regulates normal ECs. The marked up-regulation of its receptors on diabetic ECs coupled with moderate Scg3 up-regulation in diabetic vitreous exacerbates retinal vascular leakage in DR. Scg3-neutralizing antibody alleviates the leakage by blocking Scg3 binding to its receptor. (C) Scg3 molecular mechanisms. Scg3 activates ERK and Src pathways, but not Akt and Stat3, through unknown receptors. ERK activated by Scg3 and VEGF may regulate different metabolic events (see the Scg3 as a disease-associated angiogenic ligand section of Discussion). FAK, focal adhesion kinase.

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