JNK1 regulates VEGF expression and neovascularization in a murine model of retinopathy. Litters of Jnk1^+/+ and Jnk1^−/− pups with at least two nursing dams per group were placed under hyperoxia (75% oxygen) or ambient air for 5 days on P7. After 5 days, on P12, mice were returned to ambient air until P17, when they were killed; their eyes were enucleated, and retinas were isolated. (A) Whole-mounted retinas from mice exposed to hyperoxia followed by normoxia were stained with Alexa Fluor 594-conjugated B4 isolectin from G. simplicifolia and viewed by fluorescent microscopy. (B) Areas of vascular obliteration and neovascular tufts were quantified by using at least 6 mice per genotype. Results are expressed as means ± SEM. *, P < 0.05 vs. WT mice. (C) Retinal proteins were extracted at P17, and VEGF was quantified by ELISA. Results are averages of two experiments using at least 6 mice per genotype. Results are expressed as means ± SEM. *, P < 0.05 vs. control; #, P < 0.05 vs. WT mice. (D) Retinal RNA was extracted at P15, and VEGF mRNA was analyzed by Q-RT-PCR (n = 4 per genotype). Results are expressed as means ± SEM. *, P < 0.05 vs. control; #, P < 0.05 vs. WT mice. (E) BMDM from either Jnk1^+/+ or Jnk1^−/− mice were exposed to normoxia (Po₂ = 21%) or hypoxia (Po₂ = 0.5%) for 4 h. RNA was extracted, and VEGF mRNA was quantified by Q-RT-PCR. Results are expressed as means ± SEM. *, P < 0.05 vs. normoxic Jnk1^+/+ BMDM; #, P < 0.05 vs. hypoxic Jnk1^+/+ BMDM. (F) BMDM were treated as above, and VEGF protein expression was analyzed by ELISA. Results are expressed as means ± SEM. *, P < 0.05 vs. normoxic Jnk1^+/+ BMDM; #, P < 0.05 vs. hypoxic Jnk1^+/+ BMDM. (G) U87 cells were preincubated (1 h) with D-JNKi (1 μM) and cultured under normoxia (O₂ = 21%) or hypoxia (O₂ = 0.5%) for 4 h. RNA was extracted, and VEGF expression was analyzed by Q-RT-PCR. Results are expressed as means ± SEM. *, P < 0.05 vs. normoxic control cells; #, P < 0.05 vs. hypoxic control cells.

Hypoxia induces JNK activation. RAW264.7 macrophages were cultured either normoxia (Po₂ = 21%) or hypoxia (Po₂ = 0.5%). (A) Cell lysates were obtained after 1 h under normoxia or hypoxia, and JNK activity was measured by immunocomplex kinase assay using GST-c-Jun (1–79) as a substrate. (B) Amounts of phosphorylated (P) JNK and c-Jun were analyzed by immunobloting at different times after initiation of hypoxia. Numbers below P-JNK and P-c-Jun blots represent relative values normalized to total JNK and c-Jun amounts. (C) Cells were preincubated without or with BHA (10 μM) for 1 h, cultured under normoxia or hypoxia for 1 h, and analyzed as in B. (D) Retinas from P15 mice either under normoxia or subjected to OIR were stained with antibodies to phospho-c-Jun (a and b) or VEGF (c and d). g, ganglionar cell layer; inl, inner nuclear layer.

JNK1 does not regulate HIF-1α expression. BMDM from either WT or Jnk1^−/− mice were cultured under normoxia (Po₂ = 21%) or hypoxia (Po₂ = 0.5%) for 4 h. (A) Cells were harvested, and cytosolic and nuclear extracts were prepared. Protein expression of JNK1/2 (cytosolic) and HIF-1α (nuclear) was examined by immunoblotting. (B) RNA was extracted and quantified by Q-RT-PCR. Results of three separate experiments done in triplicate are expressed as means ± SEM.

JNK1 regulates VEGF transcriptional activity. (A) RAW264.7 macrophages were transfected with pVEGF-Luc, pVEGF^ΔAP−1-Luc, or pVEGF^ΔHRE-Luc reporters. After 24 h, cells were treated with the hypoxia mimetic CoCl₂ (200 μM). Some of the cells were pretreated with D-JNKi (1 μM) for 1 h. Results represent mean ± SEM luciferase activity relative to the internal control pRL-TK, in three separate experiments performed in triplicate. (B) Jnk1^+/+ and Jnk1^−/− BMDM were cultured under normoxia (Po₂ = 21%) or hypoxia (Po₂ = 0.5%). At different time points, cell lysates were prepared and analyzed for phosphorylated (P) or total amounts of the indicated proteins by immunoblotting. Numbers below P-c-Jun blot represent relative values normalized to total c-Jun.(C) ChIP was performed with anti-phospho-S63-c-Jun or a control antibody by using fixed and sheared chromatin isolated from RAW264.7 mouse macrophages cultured under normoxia or hypoxia for 1 h. The VEGF promoter fragment, which contains an AP-1 site at −1093/−1086 bp, was detected by PCR. The actin promoter served as a control.

Intravitreal injection of a JNK inhibitor prevents retinal neovascularization. Mice subjected to OIR were injected intravitreally once with either PBS or D-JNKi at P14. (A) c-Jun phosphorylation was examined by immunohistochemistry at P15. (B) On P17, mice were killed, and their eyes were enucleated, and retinas were isolated and stained as in Fig. 1. (C) Areas of vascular obliteration and neovascular tufts were quantified as in Fig. 1A (n = 10 per genotype). Results are expressed as means ± SEM. *, P < 0.05 vs. WT mice. (D) Protein was extracted from retinas at P17, and VEGF concentration was measured by ELISA (n = 8 per genotype). Results are expressed as means ± SEM. *, P < 0.05 vs. control; #, P < 0.05 vs. WT mice. (E) Mice injected at P18 with PBS or D-JNKi were assessed at P28 by staining retinal cross-sections with hematoxylin and eosin under normoxic conditions.