JNK inhibition reduces apoptosis and neovascularization in a murine model of age-related macular degeneration

Abstract

Age-related macular degeneration (AMD) is the leading cause of registered blindness among the elderly and affects over 30 million people worldwide. It is well established that oxidative stress, inflammation, and apoptosis play critical roles in pathogenesis of AMD. In advanced wet AMD, although, most of the severe vision loss is due to bleeding and exudation of choroidal neovascularization (CNV), and it is well known that vascular endothelial growth factor (VEGF) plays a pivotal role in the growth of the abnormal blood vessels. VEGF suppression therapy improves visual acuity in AMD patients. However, there are unresolved issues, including safety and cost. Here we show that mice lacking c-Jun N-terminal kinase 1 (JNK1) exhibit decreased inflammation, reduced CNV, lower levels of choroidal VEGF, and impaired choroidal macrophage recruitment in a murine model of wet AMD (laser-induced CNV). Interestingly, we also detected a substantial reduction in choroidal apoptosis of JNK1-deficient mice. Intravitreal injection of a pan-caspase inhibitor reduced neovascularization in the laser-induced CNV model, suggesting that apoptosis plays a role in laser-induced pathological angiogenesis. Intravitreal injection of a specific JNK inhibitor decreased choroidal VEGF expression and reduced pathological CNV. These results suggest that JNK1 plays a key role in linking oxidative stress, inflammation, macrophage recruitment apoptosis, and VEGF production in wet AMD and pharmacological JNK inhibition offers a unique and alternative avenue for prevention and treatment of AMD.

Fig. 1.

JNK is activated in a murine model of CNV. Choroids from WT mice, with no intervention (B) or subjected to laser-CNV (C and D, eyes were collected 1 or 3 d after laser treatment, respectively) were stained with a rabbit anti-phospho c-Jun antibody and examined by indirect immunofluorescence (arrows). A was a 3D laser CNV section blocked with nonspecific rabbit serum followed by the same anti-rabbit IgG secondary antibody as isotype control. Original magnification, 100×.

Fig. 2.

JNK1 regulates neovascularization and inflammation in a murine model of CNV. Ten days after laser treatment, mice (n = 20) were sacrificed and choroidal flat mounts were generated. AlexaFluor-conjugated isolectin was used for CNV immunolabeling. Representative images of computer generated 3D front and side views of CNV lesions in choroidal flat mounts from WT (A and C) and Jnk1^−/− mice (B and D) are shown. (E) CNV lesion volume was measured by constructed 3D image using Velocity Software (PerkinElmer, Waltham, MA) and expressed as mm³ (means ± SEM; *P < 0.01 vs. WT). (F and G) The laser-treated CNV flat mounted slides were stained with biotin-T15 antibody followed by FITC-streptavidin showing the reduction of oxPL in CNV lesions in Jnk1^−/− mice (G) compared to that in WT (F). Panel H shows the relative fluorescent intensity for oxPL epitope. The choroidal flat mounts were originally examined by a Zeiss LSM 510 confocal microscope (Carl Zeiss, Inc.) with 200× magnification. (Scale bars: A–D, 100 mm; F and G, 50 mm.)

Fig. 3.

JNK1 regulates VEGF secretion in a murine model of CNV. (A) Choroidal tissues were collected at 3 d after laser administration, placed in 100 μL of RPMI medium supplemented with 10% FCS in 96 wells/ plate, and then kept at 37 °C for 16 h. VEGF levels were analyzed in the supernatants of homogenized tissue by ELISA. Results are averages of two experiments using at least four mice per genotype. Results are expressed as means ± SEM, *P < 0.05 vs. WT. (B–G) Choroidal tissue from either WT (B–D) or Jnk1^−/− (E–G) mice, subjected to laser-CNV and collected 3 d after laser treatment, were stained by immunofluorescence with isolectin (B and E) and anti-VEGF antibody (C and F, arrows). D and G show VEGF, isolectin, and DAPI overlapping staining. Original magnification, 200×.

Fig. 4.

Reduced macrophage infiltration in Jnk1^−/− mice. (A–F) Choroidal tissue from either WT (A–C) or Jnk1^−/− (D–F) mice, subjected to laser-CNV and collected 3 d after laser treatment, were stained by immunofluorescence with isolectin (A and D) and anti-F4/80 antibody (B and E, arrows). C and F show F4/80, isolectin, and DAPI overlapping staining. Original magnification, 200×. (G) Choroidal RNA was extracted at day 3 after laser treatment and analyzed by qPCR in triplicates for expression of F4/80 mRNA. mRNA amounts were normalized to 18S rRNA. (H and I) qPCR showed that the MCP-1 level is elevated significantly in both retina and choroid after laser-CNV treatment in wild-type mice, however, only very moderate increase in MCP-1 level in both retina and choroid was seen in Jnk1^−/− mice. Results are expressed as means ± SEM, *P < 0.05 vs. WT.

Fig. 5.

JNK1 regulates apoptosis in a murine model of CNV. (A) WT mice (n = 10) were given an intravitreal injection of 1 μL of pan-caspase inhibitor Z-VAD-FMK (20 μM) right after laser treatment. An equal volume of PBS was given in the fellow eye as a control. Ten days after laser treatment, mice were killed and choroidal flat mounts generated. FITC-conjugated isolectin was used to immunolabel the CNV. Flat mounts were examined and CNV quantified. Results are expressed by CNV area (μm²) as means ± SEM, *P < 0.01 vs. PBS. (B) The retina of Jnk1^−/− and WT control mice were examined for apoptosis by an in situ TUNEL assay with retinal cross-sections obtained 6, 12, and 24 h after laser-CNV treatment. Results are expressed as number of apoptotic cells within the 20× viewing area (n = 3, means ± SEM, *P < 0.01 vs. WT). (C–H) Representative images of retinal cross-sections of WT control (C–E) and Jnk1^−/− (F–H) mice stained by an in situ TUNEL assay 6, 12, and 24 h after laser-CNV treatment (arrows indicate the positive TUNEL staining). Original magnification, 200×.

Fig. 6.

Intravitreal injection of a JNK inhibitor prevents CNV (A–C). WT mice (n = 10) were given an intravitreal injection of 1 μL of D-JNKi peptide (2 mM) right after laser treatment. An equal volume of PBS was given by intravitreal injection in the fellow eye as a control. Ten days after laser treatment, mice were killed and choroidal flat mounts generated. FITC-conjugated isolectin was used to immunolabel the CNV. (A and B) Representative CNV lesions in choroidal flat mounts from PBS and D-JNKi–treated mice. CNV lesion areas were larger in control (A) compared with D-JNKi–treated (B) mice. Original magnification, 200×. (C) Flat mounts were examined and CNV quantified. Results are expressed as means ± SEM, *P < 0.01 vs. PBS.