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...

Rationale: Although an age-related decrease in NO bioavailability contributes to vascular stiffness, the underlying molecular mechanisms remain incompletely understood. We hypothesize that NO constrains the activity of the matrix crosslinking enzyme tissue transglutaminase (TG2) via S-nitrosylation in young vessels, a process that is reversed in aging. Objective: We sought to determine whether endothelium-dependent NO regulates TG2 activity by S-nitrosylation and whether this contributes to age-related vascular stiffness. Methods and Results: We first demonstrate that NO suppresses activity and increases S-nitrosylation of TG2 in cellular models. Next, we show that nitric oxide synthase (NOS) inhibition leads to increased surface and extracellular matrix-associated TG2. We then demonstrate that endothelium-derived bioactive NO primarily mediates its effects through TG2, using TG2 ؊/؊ mice chronically treated with the NOS inhibitor L-N Gnitroarginine methyl ester (L-NAME). We confirm that TG2 activity is modulated by endothelium-derived bioactive NO in young rat aorta. In aging rat aorta, although TG2 expression remains unaltered, its activity increases and S-nitrosylation decreases. Furthermore, TG2 inhibition decreases vascular stiffness in aging rats. Finally, TG2 activity and matrix crosslinks are augmented with age in human aorta, whereas abundance remains unchanged. Conclusions: Decreased S-nitrosylation of TG2 and increased TG activity lead to enhanced matrix crosslinking and contribute to vascular stiffening in aging. TG2 appears to be the member of the transglutaminase family primarily contributing to this phenotype. Inhibition of TG2 could thus represent a therapeutic target for age-associated vascular stiffness and isolated systolic hypertension. (Circ Res. 2010;107:117-125.)Key Words: tissue transglutaminase Ⅲ S-nitrosylation Ⅲ S-nitrosation Ⅲ aging Ⅲ vascular stiffness A ging is associated with alterations in the properties of all elements of the vascular wall including endothelium, vascular smooth muscle, and matrix. 1 These changes result in increased vascular stiffness and isolated systolic hypertension. In addition, increased vascular stiffness promotes atherosclerosis at various sites in the vascular tree, such as the carotid artery. 2,3 Both dynamic changes (alterations in endothelial function and effects on vascular smooth muscle contractility), as well as structural alterations (eg, fracturing of elastin, increased collagen content, and accumulation of advanced glycation end products) have been described in aging. Vessel structure can additionally be regulated by alterations in matrix crosslinking. 1 Transglutaminases (TGs) are enzymes that catalyze a transamidation reaction, leading to the crosslinking of proteins through the formation of the stable N--(␥-glutamyl)lysine isopeptide bonds. 4,5 At least 3 of the 9 members of the TG superfamily are expressed in vascular systems. Tissue transglutaminase (TG2) in particular is ubiquitously expressed in vasculature, including in endothelial ce...

Non-technical summary A high concentration of cholesterol in the blood, known as hypercholersterolaemia, in the absence of overt atherosclerotic disease induces changes throughout the circulation including an inability to fully respond to vasodilatory stimuli. Here we show that skin blood flow responses are reduced in hypercholersterolaemic men and women partly due to an upregulation of the arginase pathway. Arginase competes for the common substrate L-arginine for the synthesis of the vasoprotective molecule nitric oxide. After 3 months of oral atrovastatin (cholesterol lowering medication) intervention, arginase activity was decreased and skin blood flow responses resembled those of healthy men and women. This suggests that upregulated arginase contributes to decreased vasoreactivity in hyperocholesterolaemic humans and that atrovastatin therapy restores functional vasodilatory properties by decreasing arginase activity.Abstract Elevated low-density lipoproteins (LDLs) are associated with vascular dysfunction evident in the cutaneous microvasculature. We hypothesized that uncoupled endothelial nitric oxide synthase (NOS3) through upregulated arginase contributes to cutaneous microvascular dysfunction in hyperocholesterolaemic (HC) humans and that a statin intervention would decrease arginase activity. Five microdialysis fibres were placed in the skin of nine normocholesterolaemic (NC: LDL level 95 ± 4 mg dl −1 ) and nine hypercholesterolaemic (HC: LDL: 177 ± 6 mg dl −1 ) men and women before and after 3 months of systemic atrovastatin. Sites served as control, NOS inhibited, arginase inhibited, L-arginine supplemented and arginase inhibited plus L-arginine supplemented. Skin blood flow was measured while local skin heating (42• C) induced NO-dependent vasodilatation. L-NAME was infused after the established plateau in all sites to quantify NO-dependent vasodilatation. Data were normalized to maximum cutaneous vascular conductance (CVC max ). Skin samples were obtained to measure total arginase activity and arginase I and arginase II protein. Vasodilatation was reduced in hyperocholesterolaemic subjects (HC: 76 ± 2 vs. NC: 94 ± 3%CVC max , P < 0.001) as was NO-dependent vasodilatation (HC: 43 ± 5 vs. NC: 62 ± 4%CVC max , P < 0.001). The plateau and NO-dependent vasodilatation were augmented in HC with arginase inhibition (92 ± 2, 67 ± 2%CVC max , P < 0.001), L-arginine (93 ± 2, 71 ± 5%CVC max , P < 0.001) and combined treatments (94 ± 4, 65 ± 5%CVC max , P < 0.001) but not in NC. After statin intervention (LDL: 98 ± 5 mg dl −1 ) there was no longer a difference between control sites (88 ± 4, 61 ± 5%CVC max ) and localized microdialysis treatment sites (all P > 0.05). Arginase activity and protein were increased in HC skin (P < 0.05 vs. NC) and activity decreased with atrovastatin treatment (P < 0.05). Reduced NOS3 substrate availability through upregulated arginase contributes to cutaneous microvascular dysfunction in hyperocholesterolaemic humans, which is corrected with atorvastatin therapy.