There is increasing evidence that upregulation of arginase contributes to impaired endothelial function in aging. In this study, we demonstrate that arginase upregulation leads to endothelial nitric oxide synthase (eNOS) uncoupling and that in vivo chronic inhibition of arginase restores nitroso-redox balance, improves endothelial function, and increases vascular compliance in old rats. Arginase activity in old rats was significantly increased compared with that shown in young rats. Old rats had significantly lower nitric oxide (NO) and higher superoxide (O 2 Ϫ ) production than young. Acute inhibition of both NOS, with N G -nitro-L-arginine methyl ester, and arginase, with 2(S)-amino-6-boronohexanoic acid (ABH), significantly reduced O 2 Ϫ production in old rats but not in young. In addition, the ratio of eNOS dimer to monomer in old rats was significantly decreased compared with that shown in young rats. These results suggest that eNOS was uncoupled in old rats. Although the expression of arginase 1 and eNOS was similar in young and old rats, inducible NOS (iNOS) was significantly upregulated. Furthermore, S-nitrosylation of arginase 1 was significantly elevated in old rats. These findings support our previously published finding that iNOS nitrosylates and activates arginase 1 (Santhanam et al., Circ Res 101: 692-702, 2007). Chronic arginase inhibition in old rats preserved eNOS dimer-to-monomer ratio and significantly reduced O 2 Ϫ production and enhanced endothelial-dependent vasorelaxation to ACh. In addition, ABH significantly reduced vascular stiffness in old rats. These data indicate that iNOS-dependent S-nitrosylation of arginase 1 and the increase in arginase activity lead to eNOS uncoupling, contributing to the nitroso-redox imbalance, endothelial dysfunction, and vascular stiffness observed in vascular aging. We suggest that arginase is a viable target for therapy in age-dependent vascular stiffness. aging; nitric oxide; S-nitrosylation; NOS uncoupling VASCULAR STIFFNESS (33) and decreased nitric oxide (NO) bioavailability (3, 19) are hallmarks of the aging cardiovascular system. Reactive oxygen species (ROS) production is also enhanced in aged blood vessels (12,23 Ϫ to levels potentially detrimental to vascular cell function and viability (22). This nitroso-redox imbalance contributes to aging-related endothelial dysfunction and vascular stiffness (6).Under normal physiological conditions, nitric oxide synthase (NOS) produces the potent vasodilator NO by catalyzing L-arginine to L-citrulline. This normal function of endothelial NOS (eNOS, NOS3) requires dimerization of the enzyme, the substrate L-arginine, and the essential cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) (24). However, the enzyme arginase uses L-arginine as a substrate (4) and reciprocally regulates NOS by substrate depletion (6,15,55). There is increasing evidence that upregulation of arginase functionally inhibits NOS activity and contributes to the pathophysiology of agerelated vascular dysfunction (6,45,55). Furthermore, phar...

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,...

Alternatively activated macrophages prevent lethal intestinal pathology caused by worm ova in mice infected with the human parasite Schistosoma mansoni through mechanisms that are currently unclear. This study demonstrates that arginase I (Arg I), a major product of IL-4– and IL-13–induced alternatively activated macrophages, prevents cachexia, neutrophilia, and endotoxemia during acute schistosomiasis. Specifically, Arg I-positive macrophages promote TGF-β production and Foxp3 expression, suppress Ag-specific T cell proliferation, and limit Th17 differentiation. S. mansoni-infected Arg I-deficient bone marrow chimeras develop a marked accumulation of worm ova within the ileum but impaired fecal egg excretion compared with infected wild-type bone marrow chimeras. Worm ova accumulation in the intestines of Arg I-deficient bone marrow chimeras was associated with intestinal hemorrhage and production of molecules associated with classical macrophage activation (increased production of IL-6, NO, and IL-12/IL-23p40), but whereas inhibition of NO synthase-2 has marginal effects, IL-12/IL-23p40 neutralization abrogates both cachexia and intestinal inflammation and reduces the number of ova within the gut. Thus, macrophage-derived Arg I protects hosts against excessive tissue injury caused by worm eggs during acute schistosomiasis by suppressing IL-12/IL-23p40 production and maintaining the Treg/Th17 balance within the intestinal mucosa.

Two series of halogenated sulfonamides have been prepared. The first consists of mono/dihalogenated sulfanilamides, whereas the second one consists of the mono/dihalogenated aminobenzolamides, incorporating equal or different halogens (F, Cl, Br, and I). These sulfonamides have been synthesized from the corresponding anilines by acetylation (protection of the amino group), chlorosulfonylation, followed either by amidation, or reaction with 5-amino-1,3,4-thiadiazole-2-sulfonamide (and eventually deacetylation). All these compounds, together with the six clinically used sulfonamide inhibitors (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, and brinzolamide) were investigated as inhibitors of the transmembrane, tumor-associated isozyme carbonic anhydrase (CA) IX. Inhibition data against the classical, physiologically relevant isozymes I, II, and IV were also obtained. CA IX shows an inhibition profile which is generally completely different from those of isozymes I, II, and IV, with potent inhibitors (inhibition constants in the range of 12-40 nM) among both simple aromatic (such as 3-fluoro-5-chloro-4-aminobenzenesulfonamide) as well as heterocyclic compounds (such as acetazolamide, methazolamide, 5-amino-1,3,4-thiadiazole-2-sulfonamide, aminobenzolamide, and dihalogenated aminobenzolamides). This first detailed CA IX inhibition study revealed many interesting leads, suggesting the possibility to design even more potent and eventually CA IX-selective inhibitors, with putative applications as antitumor agents.