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 lipoprotein (OxLDL) impairs NO signaling and endothelial function, and contributes to the pathogenesis of atherosclerosis. Arginase reciprocally regulates NO levels in endothelial cells by competing with NO synthase for the substrate L-arginine. In human aortic endothelial cells, OxLDL stimulation increased arginase enzyme activity in a time-and dose-dependent manner. Arginase activity reached its maximum as early as 5 minutes, was maintained for a period of more than 48 hours, and was associated with a reciprocal decrease in NO metabolite (NOx [nitrite and nitrate]) production. Furthermore, OxLDL induced arginase II mRNA expression after 4 hours. Small interfering RNA targeted to arginase II decreased both the quantity and the activity of arginase from baseline, prevented OxLDL-dependent increases in arginase activity, and induced an increase in NOx production. Immunofluorescence analysis revealed an association of arginase II with the microtubule cytoskeleton. Microtubule disruption with nocodazole caused a dramatic redistribution of arginase II to a diffuse cytosolic pattern, increased arginase activity, and decreased NOx production, which was restored in the presence of the specific arginase inhibitor (S)-(2-boronoethyl)-L-cysteine (BEC). On the other hand, epothilone B prevented microtubule disruption and inhibited OxLDL-dependent increases in arginase activity and attenuated OxLDL-dependent decreases in NOx. Preincubation of rat aortic rings with OxLDL resulted in an increase in arginase activity and a decrease in NOx production. This was reversed by arginase inhibition with the BEC. Thus, OxLDLs increase arginase activity by a sequence of regulatory events that involve early activation through decreased association with microtubules and a later increase in transcription. Furthermore, increased arginase activity contributes to OxLDL-dependent impairment of NOx production. Arginase, therefore, represents a novel target for therapy in atherosclerosis.

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,...

Endothelial dysfunction and increased arterial stiffness contribute to multiple vascular diseases and are hallmarks of cardiovascular aging. To investigate the effects of aging on shear stress-induced endothelial nitric oxide (NO) signaling and aortic stiffness, we studied young (3-4 mo) and old (22-24 mo) rats in vivo and in vitro. Old rat aorta demonstrated impaired vasorelaxation to acetylcholine and sphingosine 1-phosphate, while responses to sodium nitroprusside were similar to those in young aorta. In a customized flow chamber, aortic sections preincubated with the NO-sensitive dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, were subjected to steady-state flow with shear stress increase from 0.4 to 6.4 dyn/cm(2). In young aorta, this shear step amplified 4-amino-5-methylamino-2',7'-difluorofluorescein fluorescence rate by 70.6 +/- 13.9%, while the old aorta response was significantly attenuated (23.6 +/- 11.3%, P < 0.05). Endothelial NO synthase (eNOS) inhibition, by N(G)-monomethyl-l-arginine, abolished any fluorescence rate increase. Furthermore, impaired NO production was associated with a significant reduction of the phosphorylated-Akt-to-total-Akt ratio in aged aorta (P < 0.05). Correspondingly, the phosphorylated-to-total-eNOS ratio in aged aortic endothelium was markedly lower than in young endothelium (P < 0.001). Lastly, pulse wave velocity, an in vivo measure of vascular stiffness, in old rats (5.99 +/- 0.191 m/s) and in N(omega)-nitro-l-arginine methyl ester-treated rats (4.96 +/- 0.118 m/s) was significantly greater than that in young rats (3.64 +/- 0.068 m/s, P < 0.001). Similarly, eNOS-knockout mice demonstrated higher pulse wave velocity than wild-type mice (P < 0.001). Thus impaired Akt-dependent NO synthase activation is a potential mechanism for decreased NO bioavailability and endothelial dysfunction, which likely contributes to age-associated vascular stiffness.