br Introduction Atherosclerosis is a chronic inflammatory di

Introduction Atherosclerosis is a chronic inflammatory disease characterized by lipid and leukocyte accumulation within the arterial wall. Homocysteine (Hcy) is a thiol-containing amino BAMB-4 sale derivative derived from the metabolism of dietary methionine. Epidemiological studies have shown that elevated plasma level of Hcy, known as hyperhomocysteinemia (HHcy), is an independent risk factor for atherosclerosis [1], [2], [3]. Our previous studies indicated that HHcy accelerates atherosclerotic development in apolipoprotein E-deficient (ApoE-/-) mice [4] and T-cell activation, with elevated reactive oxygen species (ROS) production and promoted proliferation, plays an important role in this process [5], [6], [7]. Recent studies suggest that HHcy promotes atherosclerosis in ApoE-/- mice by reducing S-nitrosylated protein (SNO-protein) levels in the aorta, and causes significant reduction of protein S-nitrosylation accompanied by increasing ROS in vascular endothelial cells [8], [9]. Substantial evidence indicates accumulation of ROS provides microenvironment within cells for altering the reversible nitrosative modification level of redox-sensitive residues in proteins [10]. S-nitrosylation, the covalent addition of an NO group to a reactive free thiol of proteins to form S-nitrosothiols (SNOs), is emerging as a critical reversible post-translational modification involved in the regulation of T-cell functions [11], [12]. The regulation of S-nitrosylation/denitrosylation, being as a redox switch, depends on several key enzymes, such as nitric oxide synthases (NOS) and denitrosylases [13]. S-nitrosoglutathione (GSNO) reductase (GSNOR) is the enzyme that metabolizes GSNO, a major physiological NO derivative, thus regulating the equilibrium between SNO-proteins and GSNO [14]. Genetic deletion of GSNOR in mice causes excessive protein S-nitrosylation, increased apoptosis and reduced number of T cells in the thymus [15], which indicates a protective role of GSNOR in T-cell development via regulation of S-nitrosylation. However, whether GSNOR-induced denitrosylation contributes to T cell activation and atherosclerotic development remains unknown. Over the years, multiple T-cell functions in response to S-nitrosylation have become increasingly recognized. SNO-proteins, such as the caspase family (−1, −3, −8), NF-κB and Bcl-2, are key regulators in T-cell apoptosis [16], [17], [18], [19]. During immunization, NO produced by inducible nitric oxide synthase (iNOS) suppresses the survival of T cells to control the persistence of CD4+ and CD8+ T-cell immune memory [20]. Moreover, accumulating evidence suggests a protective role of S-nitrosylation in various autoimmune diseases by modulating the differentiation of T helper (Th) cell subsets, including Th-1, − 2 and − 17 [11]. These previous studies indicated the direct or indirect regulatory effects of S-nitrosylation on T-cell apoptosis, survival, differentiation and development, but the regulatory effects of S-nitrosylation on Hcy-induced primary T-cell activation, including cytokine secretion and proliferation, remain to be fully elucidated. Our previous work showed that HHcy promotes Akt phosphorylation in T cells to accelerate atherosclerosis [6]. The phosphoinositide-3 kinase (PI3K)/Akt pathway is critical for regulating T-cell proliferation, metabolism, cytokine production and survival [21], [22], [23]. Upon activation, naïve T cells develop into Teff cells that enter the bloodstream and are recruited into atherosclerotic plaques, where they proliferate and produce proinflammatory cytokines [24]. Recent reports have shown that Akt can be S-nitrosylated in muscle cells and esophageal squamous cells, leading to its inhibited kinase activity in diabetic models, post-burn injury, and squamous cell differentiation [25], [26], [27]. Considering the crucial role of Akt-mediated T-cell activation in HHcy-accelerated atherosclerosis, whether and how S-nitrosylation of Akt regulates Hcy-induced T-cell activation and the mechanism underlying the intracellular pathway remain to be determined.