ARG2 impairs endothelial autophagy through regulation of MTOR and PRKAA/AMPK signaling in advanced atherosclerosis

<p>Impaired autophagy function and enhanced ARG2 (arginase 2)-MTOR (mechanistic target of rapamycin) crosstalk are implicated in vascular aging and atherosclerosis. We are interested in the role of ARG2 and the potential underlying mechanism(s) in modulation of endothelial autophagy. Using human nonsenescent “young” and replicative senescent endothelial cells as well as <i>Apolipoprotein E</i>-deficient (<i>apoe</i>^−/−<i>Arg2</i>^+/+) and <i>Arg2</i>-deficient <i>apoe</i>^−/− (<i>apoe</i>^−/−<i>arg2</i>^−/−) mice fed a high-fat diet for 10 wk as the atherosclerotic animal model, we show here that overexpression of ARG2 in the young cells suppresses endothelial autophagy with concomitant enhanced expression of RICTOR, the essential component of the MTORC2 complex, leading to activation of the AKT-MTORC1-RPS6KB1/S6K1 (ribosomal protein S6 kinase, 70kDa, polypeptide 1) cascade and inhibition of PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit). Expression of an inactive ARG2 mutant (H160F) had the same effect. Moreover, silencing RPS6KB1 or expression of a constitutively active PRKAA prevented autophagy suppression by ARG2 or H160F. In senescent cells, enhanced ARG2-RICTOR-AKT-MTORC1-RPS6KB1 and decreased PRKAA signaling and autophagy were observed, which was reversed by silencing <i>ARG2</i> but not by arginase inhibitors. In line with the above observations, genetic ablation of <i>Arg2</i> in <i>apoe</i>^−/− mice reduced RPS6KB1, enhanced PRKAA signaling and endothelial autophagy in aortas, which was associated with reduced atherosclerosis lesion formation. Taken together, the results demonstrate that ARG2 impairs endothelial autophagy independently of the L-arginine ureahydrolase activity through activation of RPS6KB1 and inhibition of PRKAA, which is implicated in atherogenesis.</p

Increased S6K1 activity in senescent endothelial cells.

<p>(<b>A</b>) A representative SA-ß-gal staining of young and senescent HUVECs. (<b>B</b>) Immunoblotting analysis of S6-S235/S236 (p-S6), total S6, and tubulin protein levels in the young and senescent endothelial cells. Quantification of the signals is shown in the lower panel. n = 6 in each group. **p<0.01. Scale bar = 0.2 mm.</p

Resveratrol reduces superoxide generation and enhances NO production in senescent endothelial cells.

<p>Young and senescent HUVEC cells were treated with solvent as control (C), resveratrol (Resv, 10 µmol/L) or rapamycin (Rapa, 20 ng/ml) for one hour and then subjected to (<b>A</b>) MitoSox, (<b>B</b>) DHE, and (<b>C</b>) DAF-2DA staining. Quantification of the signals from six independent experiments is shown in the corresponding right panels. *p<0.05, **p<0.01 and ***p<0.001 between indicated groups. Scale bar = 0.2 mm.</p

Inhibition of S6K1 suppresses superoxide generation in the aortas of old rats.

<p>Confocal microscopic <i>en face</i> detection of MitoSox staining for mitochondrial superoxide anion production followed by counterstaining with DAPI for endothelial nuclei in the intact thoracic aorta segments from young and old rats, which were treated with resveratrol (Resv) or rapamycin (Rapa) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019237#pone-0019237-g010" target="_blank">Fig. 10</a>. The upper panels show representative images from a young and an old rat and lower panel shows quantification of the fluorescence intensity from three animals in each group. Scale bar = 50 µm. ***p<0.001 between indicated groups.</p

Increased oxidative stress in senescent endothelial cells.

<p>Fluorescent signals of (<b>A</b>) MitoSox, (<b>B</b>) DHE, and (<b>C</b>) DAF-2DA staining for detection of mitochondrial, cytoplasmic superoxide generation (red) and NO production (green) in young and senescent HUVECs, respectively. Quantification of the signals from four independent experiments of each group is shown in the corresponding lower panels. *p<0.05, **p<0.01 vs. young cells. Scale bar = 0.2 mm. (<b>D</b>) Immunoblotting showing increased eNOS protein level in senescent cells. Quantification of eNOS protein level is shown in the lower panel. n = 9 in each group. **p<0.01 vs. young cells.</p

eNOS uncoupling in senescent endothelial cells.

<p>(<b>A</b>) Immunoblotting analysis of eNOS-dimers and -monomers in the young and senescent HUVEC endothelial cells. Ponseau S staining served as loading control. Quantification of the eNOS-dimer/monomer is shown in the lower panel. n = 4 in each group. *p<0.05. (<b>B</b>) DHE staining for detection of cytoplasmic superoxide generation in young and senescent endothelial cells with or without pre-treatment of the cells with L-NAME (1 mmol/L) for 1 hour as indicated. Quantification of the signals from six independent experiments of each group is shown in the corresponding lower panels. ***p<0.001 between indicated groups. Scale bar = 0.2 mm.</p

Over-expression of an active S6K1 mutant causes eNOS uncoupling in endothelial cells.

<p>Young HUVEC cells were transduced either with an empty rAd vector as control or with rAd/CMV-HA-S6K1ca (a constitutively active S6K1 mutant). Two days post transduction, cells were subjected to (<b>A</b>) Immunoblotting analysis of expression of eNOS-dimers, -monomers with anti-eNOS antibody, HA-S6K1-ca with anti-HA antibody. Ponseau S staining served as loading control. Quantification of the eNOS-dimer/monomer ratio is shown in the lower panel. n = 8 in each group. (<b>B</b>) DHE staining was performed 1 hour after L-NAME (1 mmol/L) treatment. Quantification of the signals is shown in the corresponding lower panels. n = 8. **p<0.01 and ***p<0.001 between indicated groups. Scale bar = 0.2 mm.</p

Silencing S6K1 reduces superoxide and enhances NO production in senescent endothelial cells.

<p>Senescent HUVECs were transduced either with rAd/U6-LacZ^shRNA as control or with rAd/U6-S6K1^shRNA. Four days post transduction, cells were subjected to (<b>A</b>) immunoblotting analysis to reveal the effective silencing of S6K1 and (<b>B</b>) MitoSox, DHE, and DAF-2DA staining. Quantification of the signals from four independent experiments of each group is shown in the corresponding lower panels. ***p<0.001 vs. rAd/U6-LacZ^shRNA control cells. Scale bar = 0.2 mm.</p

Over-expression of an active S6K1 mutant enhances superoxide, decreases NO production and induces premature senescence in young endothelial cells.

<p>Young HUVECs were transduced either with an empty rAd vector as control or with rAd/CMV-HA-S6K1-ca (a constitutively active S6K1 mutant). (<b>A</b>) Immunoblotting analysis of expression of HA-S6K1-ca with anti-HA antibody or antibodies against S6-S235/S236 (p-S6), total S6, and tubulin on day 2 of post transduction. (<b>B</b>) Four days post transduction, cells were subjected to SA-ß-gal staining. (<b>C</b>) MitoSox, DHE, and DAF-2DA staining were performed on day 2 of post transduction. Quantification of the signals is shown in the corresponding lower panels. n = 6. **p<0.01 and ***p<0.001 vs. control cells. Scale bar = 0.2 mm.</p

Resveratrol inhibits Akt and S6K1 signalling in senescent endothelial cells.

<p>Young or senescent HUVECs were treated with either solvent or resveratrol (Resv, 10 µmol/L) for one hour. The cell lysates were then prepared and subjected to immunoblotting analysis of Akt-S473 (p-Akt), total Akt, S6-S235/S236 (p-S6), and total S6. Quantification of the signals is shown in the lower panels. n = 6, ***p<0.001 between indicated groups.</p