Pathogenic role and therapeutic potential of pleiotrophin in mouse models of ocular vascular disease

Abstract

Angiogenic factors play an important role in the pathogenesis of diabetic retinopathy (DR), neovascular age-related macular degeneration (nAMD) and retinopathy of prematurity (ROP). Pleiotrophin, a well-known angiogenic factor, was recently reported to be upregulated in the vitreous fluid of patients with proliferative DR (PDR). However, its pathogenic role and therapeutic potential in ocular vascular diseases have not been defined in vivo. Here using corneal pocket assays, we demonstrated that pleiotrophin induced angiogenesis in vivo. To investigate the pathological role of pleiotrophin we used neutralizing antibody to block its function in multiple in vivo models of ocular vascular diseases. In a mouse model of DR, intravitreal injection of pleiotrophin-neutralizing antibody alleviated diabetic retinal vascular leakage. In a mouse model of oxygen-induced retinopathy (OIR), which is a surrogate model of ROP and PDR, we demonstrated that intravitreal injection of anti-pleiotrophin antibody prevented OIR-induced pathological retinal neovascularization and aberrant vessel tufts. Finally, pleiotrophin-neutralizing antibody ameliorated laser-induced choroidal neovascularization, a mouse model of nAMD, suggesting that pleiotrophin is involved in choroidal vascular disease. These findings suggest that pleiotrophin plays an important role in the pathogenesis of DR with retinal vascular leakage, ROP with retinal neovascularization and nAMD with choroidal neovascularization. The results also support pleiotrophin as a promising target for anti-angiogenic therapy.

Keywords: Anti-angiogenic therapy; Choroidal neovascularization; Diabetic retinopathy; Neovascular age-related macular degeneration; Oxygen-induced retinopathy; Pleiotrophin; Retinopathy of prematurity.

Fig. 1

In vitro functional analyses of Ptn. a Ptn induces endothelial proliferation. HRMVECs were incubated with Ptn, VEGF or PBS control for 48 h in 96-well plates and quantified. n = 8 wells. b, c Ptn promotes endothelial migration by wound healing assay. b Representative images of endothelial migration. HRMVECs were incubated with Ptn (200 ng/ml), VEGF (50 ng/ml) or PBS for 20 h. Bar 50 μm. c The percentage of the denuded area covered by migrated cells within the original scratch was quantified. n = 3 wells. d, e Ptn enhances HRMVEC migration in transwell assay. d Representative images of transwell migration assay with HRMVECs (DAPI signal). Ptn (200 ng/ml), VEGF (50 ng/ml) or PBS was in the bottom chamber for 8 h. Bar 50 μm. e, Quantification of migrated cells/well. n = 4 well. These assays were independently performed three times with similar outcomes. One representative experiment is shown. ±SEM, *p < 0.05, **p < 0.01, ***p < 0.001, versus control, t test.

Fig. 2

Ptn stimulates spheroid sprouting of HRMVECs. a Representative images of spheroid sprouting. Ptn, 20 ng/ml; VEGF, 5 ng/ml. Bar 100 μl. b Quantification of endothelial sprouts. The average length of sprouts per spheroid was quantified. The assay was independently performed three times with similar outcomes. One representative experiment is shown. n = 7 wells. ±SEM, ***p < 0.001, ****p < 0.0001, versus control, t test.

Fig. 3

Ptn activates ERK1/2 and Akt. HRMVECs were incubated with Ptn (200 ng/ml), VEGF (100 ng/ml) or PBS for 10 or 30 min. Phosphorylated and total ERK1/2 or Akt were detected by Western blot. These results were validated three times with similar outcomes.

Fig. 4

Ptn stimulates corneal angiogenesis. a Representative images of corneal angiogenesis by slit-lamp photography. Small pieces of filter papers presoaked in Ptn (250 ng/ml), VEGF (100 ng/ml) or PBS were implanted in corneal pockets to induce vascular growth for 6 days. Asterisk indicates filter paper position. b Representative images of corneal blood vessels labeled with fluorescent DiI dye. c–e Quantification of corneal angiogenesis. The numbers of new sprouting vessels into the cornea, branching points of corneal vessels and total new vessel score were quantified in c–e. n = 8 corneas for Ptn, 5 for VEGF and 8 for PBS. ±SEM, one-way ANOVA test.

Fig. 5

Anti-Ptn Ab neutralizes Ptn-induced endothelial proliferation. a Affinity-purified anti-Ptn Ab specifically recognizes Ptn in mouse retinal homogenate. ~200 μg/lane. b Proliferation assay with HRMVECs was performed as described in Fig. 1a. Ptn, 200 ng/ml; VEGF, 100 ng/ml; anti-Ptn Ab, 400 ng/ml. The assay was independently performed three times with similar outcomes. One representative experiment is shown. n = 8 wells. ±SEM, one-way ANOVA test.

Fig. 6

Anti-Ptn Ab alleviates retinal vascular leakage in diabetic mice. a Retinal vascular leakage in 4-month-diabetic mice. The leakage was quantified by Evan blue assay. n = 6 eyes. b Anti-Ptn therapy of DR. Intravitreal injection of anti-Ptn Ab reduced diabetic retinal vascular leakage. Data were normalized against PBS in fellow eyes. Anti-Ptn Ab, 1.28 μg/1 μl/eye; aflibercept, 2 μg/1 μl/eye; control IgG, 1.28 μg/1 μl/eye. n = 6 eyes for anti-Ptn and 5 for aflibercept or control IgG. ±SEM, one-way ANOVA test.

Fig. 7

Anti-Ptn Ab prevents OIR-induced pathological neovascularization. A Representative images of OIR. Anti-Ptn (0.1 μg/1 μl/eye), aflibercept (2 μg/1 μl/eye) or control IgG (0.1 μg/1 μl/eye) was intravitreally injected at P12. RNV was analyzed at P17. Arrowheads indicate neovascularization (NV) and NV tufts. Bar 500 μm. b Quantification of NV. c Quantification of NV tufts. d Quantification of branching points. e Quantification of avascular area in central retina. n = 11 eyes for anti-Ptn, 4 for aflibercept, 6 for control IgG, 4 for PBS and 8 for healthy control. ±SEM, one-way ANOVA test

Fig. 8

Anti-Ptn Ab ameliorates laser-induced CNV. Mice were treated intravitreally with anti-Ptn (0.36 μg/1 μl/eye), aflibercept (2 μg/1 μl/eye) or control IgG (0.36 μg/1 μl/eye) 3 days after laser photocoagulation. Fluorescein angiography was performed on Day 7. Eyecups without the retina were isolated from euthanized mice on Day 8, labeled with Alexa Fluor 488-isolectin B4, flat mounted and analyzed by confocal microscopy to detect CNV. a Representative images of fluorescein angiography. Bar 800 μm. b Quantification of fluorescence intensity in a. c Representative images of Alexa Fluor 488-isolectin B4-labeled CNV. Bar 125 μm. d Quantification of CNV 3D volume in c. e Quantification of CNV vessel density (i.e., fluorescence intensity) in c. f Quantification of CNV area size in c. n = 16 laser spots for anti-Ptn, 22 for aflibercept and 22 for control IgG. ±SEM, *p < 0.05, **p < 0.01, versus control IgG, one-way ANOVA test.