Abstract-Oxidized low-density lipoproteins increase arginase activity and reciprocally decrease endothelial NO in human aortic endothelial cells. Here, we demonstrate that vascular endothelial arginase activity is increased in atherogenicprone apolipoprotein E-null (ApoE Ϫ/Ϫ ) and wild-type mice fed a high cholesterol diet. In ApoE Ϫ/Ϫ mice, selective arginase II inhibition or deletion of the arginase II gene (Arg II Ϫ/Ϫ mice) prevents high-cholesterol diet-dependent decreases in vascular NO production, decreases endothelial reactive oxygen species production, restores endothelial function, and prevents oxidized low-density lipoprotein-dependent increases in vascular stiffness. Furthermore, arginase inhibition significantly decreases plaque burden. These data indicate that arginase II plays a critical role in the pathophysiology of cholesterol-mediated endothelial dysfunction and represents a novel target for therapy in atherosclerosis. (Circ Res. 2008;102:923-932.) Key Words: vascular stiffness Ⅲ eNOS uncoupling Ⅲ pulse wave velocity Ⅲ nitric oxide Ⅲ L-arginine I n atherosclerosis, 1 oxidized low-density lipoprotein (OxLDL) is known to impair endothelial NO production by mechanisms that involve altered endothelial NO synthase (eNOS) expression, increased reactive oxygen species (ROS) production, 2 and alterations in proteins that regulate eNOS function (eg, caveolin and heat shock protein-90). 3 The concept has emerged that arginase, which shares the substrate L-arginine with NO synthase (NOS), reciprocally regulates NOS activity by competing for arginine and can inhibit NO-dependent processes by depleting the substrate pool for NO biosynthesis. This is dependent on L-arginine concentrations in microdomains in which NOS isoforms and/or arginase are located. 4 Reciprocal regulation of NOS by arginase has been demonstrated in cells/organs in which NO is an important signaling molecule including the endothelium, 5 cardiac myocyte, 6 penis, 7,8 airway, 9 skin, 10 and inflammatory cells. 11 Upregulation of arginase activity contributes to vasoregulatory dysfunction in systemic [12][13][14] and pulmonary hypertension, 15,16 aging, 5,17,18 diabetes, 19 and erectile dysfunction 20 and to bronchodilatory dysfunction in asthma. 21 In cultured endothelial cells, we have demonstrated that OxLDL-dependent activation and upregulation of arginase impairs NO production and endothelial function. 22 This novel mechanism may be pivotal in the pathogenesis of atherosclerosis. 23 We have demonstrated that OxLDL facilitates arginase II (ArgII) release from the endothelial microtubular structure, 22 and the resulting increased arginase activity contributes to impaired endothelial cell NO production. Finally, L-arginine depletion secondary to arginase activation and upregulation may result in eNOS uncoupling, 24,25 with increased endothelial ROS production and nitroso-redox imbalance.Our objectives were to determine: (1) whether OxLDLdependent activation of arginase causes impaired vascular NO production, increased ROS production,...

Abstract-Endothelial function is impaired in aging because of a decrease in NO bioavailability. This may be, in part, attributable to increased arginase activity, which reciprocally regulates NO synthase (NOS) by competing for the common substrate, L-arginine. However, the high K m of arginase (Ͼ1 mmol/L) compared with NOS (2 to 20 mol/L) seemingly makes direct competition for substrate unlikely. One of the mechanisms by which NO exerts its effects is by posttranslational modification through S-nitrosylation of protein cysteines. We tested the hypothesis that arginase1 activity is modulated by this mechanism, which serves to alter its substrate affinity, allowing competition with NOS for L-arginine. We demonstrate that arginase1 activity is altered by S-nitrosylation, both in vitro and ex vivo. Furthermore, using site-directed mutagenesis we demonstrate that 2 cysteine residues (C168 and C303) are able to undergo nitrosylation. S-Nitrosylation of C303 stabilizes the arginase1 trimer and reduces its K m value 6-fold. Finally, arginase1 nitrosylation is increased (and thus its K m decreased) in blood vessels from aging rats, likely contributing to impaired NO bioavailability and endothelial dysfunction. This is mediated by inducible NOS, which is expressed in the aging endothelium. These findings suggest that S-nitrosylated arginase1 can compete with NOS for L-arginine and contribute to endothelial dysfunction in the aging cardiovascular system. (Circ Res. 2007;101:692-702.)Key Words: arginase Ⅲ NO synthase Ⅲ S-nitrosylation Ⅲ aging A ging is accompanied by impaired endothelial function caused by reduced NO bioavailability. Arginase, the final enzyme of the urea cycle, uses L-arginine as a substrate 1-4 and reciprocally regulates NO synthase (NOS) by substrate depletion. [5][6][7][8] Increased arginase activity, therefore, leads to diminished production of NO. 6,[9][10][11][12] In vascular tissue, this further affects 2 important functions of NO: (1) cGMPdependent signaling and (2) modulation of protein function through S-nitrosylation. Several studies have demonstrated reciprocal regulation of arginase and NOS, where inhibition of arginase leads to increased NO activity. 7,8,13,14 Conversely, upregulation of arginase functionally inhibits NOS activity and contributes to the pathophysiology of several disease processes, 8,[15][16][17][18] including age-related vascular dysfunction. 5,6 The high K m value of human arginase for L-arginine (Ͼ1 mmol/L 19 ) is significantly higher than that of NOS (2 to 20 mol/L 20 ) and suggests that there should not be a direct competition for L-arginine. However, the role of arginase in regulating NOS activity through substrate depletion is now well detailed, as discussed above.Given the interaction between NOS and arginase signaling, we hypothesized that S-nitrosylation of arginase1 might be an important posttranslational modification mechanism that regulates its activity. The protein sequence for human arginase1 contains 3 cysteines, C45, C168, and C303, with C303 being very close...