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

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 Apolipoprotein E-deficient (apoe(-/-)Arg2(+/+)) and Arg2-deficient apoe(-/-) (apoe(-/-)arg2(-/-)) 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 ARG2 but not by Arginase inhibitors. In line with the above observations, genetic ablation of Arg2 in apoe(-/-) 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.

AKT/PKB, v-akt murine thymoma viral oncogene homolog 1; ANOVA, analysis of variance; AR, aortic roots; ARG1, arginase 1; ARG2, arginase 2; ARGINASE; Atg, autophagy-related; BEC, S-12 bromoethyl-L-cystine-HCl; BECN1, Beclin 1, autophagy-related; Baf A1, bafilomycin A1; CMV, cytomegalovirus; EC, endothelial cell; H160F, inactive mutant of mouse ARG2; HAEC, human aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; LC3, microtubule-associated protein 1 light chain 3; LDL, low-density lipoprotein; MTOR; MTOR, mechanistic target of rapamycin; NOS3/eNOS, nitric oxide synthase 3 (endothelial cell); PE, phosphatidylethanolamine; PRKAA; PRKAA/AMPK, protein kinase, AMP-activated, α catalytic subunit; PtdIns3K, phosphatidylinositol 3-kinase; RPS6, ribosomal protein S6; RPS6KB1/S6K1, ribosomal protein S6 kinase, 70kDa, polypeptide 1; SA-ß-gal, senescence-associated-β-gal; SMC, smooth muscle cells; SQSTM1/p62, sequestosome 1; TP53/p53, tumor protein 53; Three-MA, 3-methyladenine; ULK1, unc-51 like autophagy activating kinase 1; VWF, von Willebrand factor; WT, wild type; apoe−/−, Apolipoprotein E-deficient; arg2−/−, arginase type II deficient; atherosclerosis; autophagy; endothelial cells; nor-NOHA, Nω-hydroxy-nor-Arginine; senescence; shRNA, short hairpin RNA.