Suppression of the JNK pathway by induction of a metabolic stress response prevents vascular injury and dysfunction

Abstract

Background: Oxidative injury and dysfunction of the vascular endothelium are early and causal features of many vascular diseases. Single antioxidant strategies to prevent vascular injury have met with mixed results.

Methods and results: Here, we report that induction of a metabolic stress response with adenosine monophosphate kinase (AMPK) prevents oxidative endothelial cell injury. This response is characterized by stabilization of the mitochondrion and increased mitochondrial biogenesis, resulting in attenuation of oxidative c-Jun N-terminal kinase (JNK) activation. We report that peroxisome proliferator coactivator 1alpha is a key downstream target of AMPK that is both necessary and sufficient for the metabolic stress response and JNK attenuation. Moreover, induction of the metabolic stress response in vivo attenuates reactive oxygen species-mediated JNK activation and endothelial dysfunction in response to angiotensin II in wild-type mice but not in animals lacking either the endothelial isoform of AMPK or peroxisome proliferator coactivator 1alpha.

Conclusions: These data highlight AMPK and peroxisome proliferator coactivator 1alpha as potential therapeutic targets for the amelioration of endothelial dysfunction and, as a consequence, vascular disease.

Figure 1. Peroxide induces AMPK activation in endothelium

PAECs in 6-well plates were exposed to H₂O₂ as indicated, lysed, and the lysates probed for (A) phosphorylation of AMPK and ACC, (B) AMPK activity, and (C) ATP content as described in “Methods.” PAECs were then treated with H₂O₂ as indicated after treatment with either the AMPK inhibitor, compound C (25 uM; D) or dominant-negative AMPK adenovirus (E) and cell death or viability determined by LDH release and MTS assay, respectively as described in “Methods.” (F) PAECs were treated with dominant-negative AMPK adenovirus and AMPK activation assessed after H₂O₂ exposure by phosphorylation of the AMPK target, acetyl-CoA carboxylase (ACC).

Figure 2. Chronic AMPK activation induces stress adaptation in endothelium

PAECs in 6-well plates were exposed to AICAR as indicated, lysed, and the lysates probed for (A) phosphorylation of AMPK and ACC and (B) AMPK activity; *p<0.05 vs. 0uM by one-way ANOVA and Dunnett's test. PAECs were treated with either 20 uM compound C (C) or dominant-negative AMPK adenovirus (D) before a 20h exposure to 1mM AICAR; *p<0.05 vs. CTL, ^‡p<0.05 vs. AICAR by two-way ANOVA. Cells were then treated with H₂O₂ and either cell death or viability determined as indicated by LDH release or MTT assay, respectively. (E) PAECs were treated with Metformin as indicated and AMPK activity determined in cell lysates as ³²P incorporation into the SAMS peptide as described in “Methods,” *p<0.05 vs. 0mM by one-way ANOVA with a post hoc Dunnett's test. (F) PAECs were treated with 1mM metformin or AICAR prior to a 2h exposure to H₂O₂ as indicated and cell death determined by LDH release; *p<0.05 vs. CTL 50 uM H₂O₂, ^‡p<0.05 vs. CTL 100 uM H₂O₂ both by one-way ANOVA with a post hoc Dunnett's test.

Figure 3. AMPK-mediated adaptation involves the mitochondrion

(A) PAECs in 6-well plates were exposed to AICAR, metformin (MET), or buffer alone (CTL) as in Fig. 2 followed by assessment of JNK, Akt, and p38 MAP kinase activation as described. (B) PAECs treated as in (A) were lysed and the content of the indicated proteins determined by immunoblotting. (C) PAECs treated with AICAR as in (A) were washed and exposed to 100 μM H₂O₂ (60 min) before loading with 2.5 μg/ml JC-1 (final conc.) and then examined either qualitatively by microscopy (C) or quantitatively (D) in a plate reader for red (ex 550nm; em 600nm) and green (ex 485nm; em 535nm) fluorescence. Images are representative of 3 independent experiments and quantitative analysis represents mean ± S.E.M of 3 independent experiments; *p<0.05 vs. without H₂O₂ by two-way ANOVA and a Tukey's post hoc test. (E) PAEC were treated as in (A) and loaded with 10μM dihydrorhodamine before H₂O₂ treatment and fluorescence (ex 480nm, em 535nm) detection; n=5, , ^‡ p<0.05 vs. control H₂O₂ treated by two-way ANOVA and a Tukey test. PAEC were incubated with 1μM MitoQ as described before assessment of H₂O₂-induced mitochondrial ROS (F) or H₂O₂-induced JNK activation (G); images are representative of n=4, *p<0.05 vs. no H₂O₂.

Figure 4. AMPK-activation in endothelial cells induces PGC-1α-dependent mitochondrial biogenesis

(A) PAECs were exposed to AICAR as indicated, lysed, and subjected to immunoblotting for assessment of AMPK activation (p-ACC) and the levels of PGC-1α and mitochondrial transcription factor A (Mt-TFA). (A) BAECs were transfected with the Mt-TFA-promoter linked to a luciferase reporter prior to incubation with AICAR ± compound C followed by assessment of luciferase activity. Adenoviral transfection of human PGC-1α served as positive control. (C) PAECs were incubated with AICAR or metformin as indicated and mitochondrial mass determined fluorometrically with Mitotracker Green or nonyl-acridine orange (NAO) as indicated (*p<0.05 vs. CTL by two-way ANOVA and Dunnet's test) HUVECs were transfected with adenoviral vectors expressing either β-galactosidase (LacZ) or human PGC-1α. Cells were then lysed and assessed for the indicated proteins (D) or mitochondrial mass using Mitotracker (E; *p<0.05 vs. CTL by one-way ANOVA and a Dunnett's test). (F) HUVECs were treated with the indicated siRNA or buffer control for 72hr before a 24 hour incubation with AICAR. Mitochondrial mass was then determined using Mitotracker Green, *p<0.05 vs. no AICAR exposure. All experiments are N=5 – 7.

Figure 5. Mitochondrial biogenesis protects endothelial cells from H₂O₂-mediated toxicity

(A) PAECs were incubated with AICAR or metformin (MET) for 24h before exposure to TNF-α as indicated. Cells were lysed and immunoblotted for c-Jun or its phosphorylated form. (B and C) HUVECs were treated with 24h of AICAR (1mM) or metformin (MET; 5mM) or transfected with control (LacZ) or PGC-1α adenovirus. Cells were then treated with H₂O₂ as indicated for 2h and cell death or viability determined by LDH release or MTT assay, respectively (*p<0.05 vs. CTL, ^‡P<0.05 vs. H₂O₂ alone by one-way ANOVA with Tukey test, n=5). (D and E) HUVECs were incubated (72h) in media alone or media with siRNA against α₁-AMPK, PGC-1α, or scrambled control (Scr) before incubation with or without AICAR for 24h. After incubation, cells were treated with H₂O₂ and LDH release or cell survival assessed as in B and C, respectively (*p<0.05 vs. no additions by one-way ANOVA with Dunnett's test). (F) HUVECs were incubated with control (Ad-LacZ) or PGC-1α adenovirus (Ad-PGC-1α) for 48h before exposure to H₂O₂ or TNF-α (10ng/mL) as indicated. Cells were lysed and immunoblotted for phosphorylated c-Jun as well as total PGC-1α, and actin.

Figure 6. Metabolic stress protects the endothelium from angiotensin II-mediated dysfunction

Mice (C57Bl6) were infused with angiotensin II (ATII; 1.0 mg/kg/d) or vehicle (CTL) via osmotic minipumps for 7d and each group was also treated with AICAR (200 mg/kg/d) or vehicle by sc injection once daily. Aortae were harvested and assessed for (A) endothelium-dependent relaxation to acetylcholine, (B) superoxide by dihydroethidium staining (E=endothelium; A=adventitia), and (D) JNK activation as c-Jun phosphorylation; (*p<0.05 vs. vehicle alone; ‡P<0.05 vs. angiotensin II alone both by two-way ANOVA interaction term). Hearts were also harvested for (C) NADPH oxidase activity using NADPH-driven lucigenin chemiluminescence as described. Mice lacking α1-AMPK (E) or PGC-1α (F) and littermate controls were infused with angiotensin II or vehicle and each group was also treated with either AICAR or vehicle as in (A; *p<0.05 vs vehicle infusion).

Figure 7. Metabolic stress protects the endothelium from LPS-induced dysfunction

Wild-type (C57Bl6) or α1-AMPK-null mice were injected with intravenous LPS (10 mg/kg/d) or vehicle (CTL) via tail vein as indicated. The indicated groups were also pre-treated (7d) with AICAR (200 mg/kg/d) by sc injection once daily. Aortae were harvested and assessed for (A) endothelium-dependent relaxation to acetylcholine or (B) superoxide by dihydroethidium staining as in Fig. 6. (*p<0.05 vs. vehicle alone by two-way ANOVA interaction term).