Asymmetric dimethylarginine triggers macrophage apoptosis via the endoplasmic reticulum stress pathway

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is emerging as a key contributing factor in atherogenesis, a process in turn known to involve macrophage apoptosis. The aim of this study was to determine the effect of ADMA on macrophage apoptosis, with specific reference to the endoplasmic reticulum (ER) stress pathway. Macrophage apoptosis was evaluated by Annexin V- Propidium iodide (PI) and Hoechst 33258 staining assays. Levels of the ER stress marker glucose regulated protein 78 (GRP78) were characterized by western blot. Levels of the proapoptotic C/EBP-homologous protein (CHOP) were evaluated by western blot and reverse transcription polymerase chain reaction (RT-PCR), and caspase-4 activity was measured using a colorimetric protease assay kit. We observed ADMA dose- and time-dependent increases in macrophage levels of GRP78. Similar ADMA dose- and time-dependent increases were detected in intracellular caspase-4 activity and macrophage apoptosis, all of which were sensitive to treatment with siRNAs for protein kinase RNA-like ER kinase and inositol-requiring protein-1 (IRE1), the ADMA antagonist L-arginine, as well as inhibitors of eukaryotic translation initiation factor-2 (salubrinal), IRE1 (irestatin 9389), and c-Jun N-terminal kinase (SP600125). Our results indicate that ADMA triggers macrophage apoptosis via the ER stress pathway.