Daniel Nyhan

Arginase Reciprocally Regulates Nitric Oxide Synthase Activity and Contributes to Endothelial Dysfunction in Aging Blood Vessels

Background—Although abnormal l-arginine NO signaling contributes to endothelial dysfunction in the aging cardiovascular system, the biochemical mechanisms remain controversial. l-arginine, the NO synthase (NOS) precursor, is also a substrate for arginase. We tested the hypotheses that arginase reciprocally regulates NOS by modulating l-arginine bioavailability and that arginase is upregulated in aging vasculature, contributing to depressed endothelial function. Methods and Results—Inhibition of arginase with (S)-(2-boronoethyl)-l-cysteine, HCl (BEC) produced vasodilation in aortic rings from young (Y) adult rats (maximum effect, 46.4±9.4% at 10−5 mol/L, P <0.01). Similar vasorelaxation was elicited with the additional arginase inhibitors N-hydroxy-nor-l-arginine (nor-NOHA) and difluoromethylornithine (DFMO). This effect required intact endothelium and was prevented by 1H-oxadiazole quinoxalin-1-one (P <0.05 and P <0.001, respectively), a soluble guanylyl cyclase inhibitor. DFMO-elicited vasodilation was greater in old (O) compared with Y rat aortic rings (60±6% versus 39±6%, P <0.05). In addition, BEC restored depressed l-arginine (10−4 mol/L)–dependent vasorelaxant responses in O rings to those of Y. Arginase activity and expression were increased in O rings, whereas NOS activity and cyclic GMP levels were decreased. BEC and DFMO suppressed arginase activity and restored NOS activity and cyclic GMP levels in O vessels to those of Y. Conclusions—These findings demonstrate that arginase modulates NOS activity, likely by regulating intracellular l-arginine availability. Arginase upregulation contributes to endothelial dysfunction of aging and may therefore be a therapeutic target.

Endothelial Arginase II A Novel Target for the Treatment of Atherosclerosis

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 atherogenic-prone apolipoprotein E–null (ApoE−/−) and wild-type mice fed a high cholesterol diet. In ApoE−/− mice, selective arginase II inhibition or deletion of the arginase II gene (Arg II−/− 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.

Arginase inhibition restores NOS coupling and reverses endothelial dysfunction and vascular stiffness in old rats

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 (O2(-)) production than young. Acute inhibition of both NOS, with N(G)-nitro-l-arginine methyl ester, and arginase, with 2S-amino- 6-boronohexanoic acid (ABH), significantly reduced O2(-) 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 O2(-) 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.

Knockdown of arginase I restores NO signaling in the vasculature of old rats

Arginase, expressed in endothelial cells and upregulated in aging blood vessels, competes with NO synthase (NOS) for l-arginine, thus modulating vasoreactivity and attenuating NO signaling. Moreover, arginase inhibition restores endothelial NOS signaling and l-arginine responsiveness in old rat aorta. The arginase isoform responsible for modulating NOS, however, remains unknown. Because isoform-specific arginase inhibitors are unavailable, we used an antisense (AS) oligonucleotide approach to knockdown arginase I (Arg I). Western blot and quantitative PCR confirmed that Arg I is the predominant isoform expressed in endothelialized aortic rings and is upregulated in old rats compared with young. Aortic rings from 22-month-old rats were incubated for 24 hours with sense (S), AS oligonucleotides, or medium alone (C). Immunohistochemistry, immunoblotting, and enzyme assay confirmed a significant knockdown of Arg I protein and arginase activity in AS but not S or C rings. Conversely, calcium-dependent NOS activity and vascular metabolites of NO was increased in AS versus S or C rings. Acetylcholine (endothelial-dependent) vasorelaxant responses were enhanced in AS versus S or C treated rings. In addition, 1H-oxadiazolo quinoxalin-1-one (10 &mgr;mol/L), a soluble guanylyl cyclase inhibitor, increased the phenylephrine response in AS compared with S and C rings suggesting increased NO bioavailability. Finally, l-arginine (0.1 mmol/L)-induced relaxation was increased in AS versus C rings. These data support our hypothesis that Arg I plays a critical role in the pathobiology of age-related endothelial dysfunction. AS oligonucleotides may, therefore, represent a novel therapeutic strategy against age-related vascular endothelial dysfunction.

Inducible NO Synthase–Dependent S-Nitrosylation and Activation of Arginase1 Contribute to Age-Related Endothelial Dysfunction

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 Km of arginase (>1 mmol/L) compared with NOS (2 to 20 &mgr;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 Km value 6-fold. Finally, arginase1 nitrosylation is increased (and thus its Km 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.

Oxidized Low-Density Lipoprotein–Dependent Endothelial Arginase II Activation Contributes to Impaired Nitric Oxide Signaling

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.

Arginase and vascular aging

Vascular and associated ventricular stiffness is one of the hallmarks of the aging cardiovascular system. Both an increase in reactive oxygen species production and a decrease in nitric oxide (NO) bioavailability contribute to the endothelial dysfunction that underlies this vascular stiffness, independent of other age-related vascular pathologies such as atherosclerosis. The activation/upregulation of arginase appears to be an important contributor to age-related endothelial dysfunction by a mechanism that involves substrate (L-arginine) limitation for NO synthase (NOS) 3 and therefore NO synthesis. Not only does this lead to impaired NO production but also it contributes to the enhanced production of reactive oxygen species by NOS. Although arginase abundance is increased in vascular aging models, it appears that posttranslational modification by S-nitrosylation of the enzyme enhances its activity as well. The S-nitrosylation is mediated by the induction of NOS2 in the endothelium. Furthermore, arginase activation contributes to aging-related vascular changes by mechanisms that are not directly related to changes in NO signaling, including polyamine-dependent vascular smooth muscle proliferation and collagen synthesis. Taken together, arginase may represent an as yet elusive target for the modification of age-related vascular and ventricular stiffness contributing to cardiovascular morbidity and mortality.

Decreased S-Nitrosylation of Tissue Transglutaminase Contributes to Age-Related Increases in Vascular Stiffness

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-NG-nitroarginine 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.

Vascular Stiffness and Increased Pulse Pressure in the Aging Cardiovascular System

Aging leads to a multitude of changes in the cardiovascular system, including systolic hypertension, increased central vascular stiffness, and increased pulse pressure. In this paper we will review the effects of age-associated increased vascular stiffness on systolic blood pressure, pulse pressure, augmentation index, and cardiac workload. Additionally we will describe pulse wave velocity as a method to measure vascular stiffness and review the impact of increased vascular stiffness as an index of vascular health and as a predictor of adverse cardiovascular outcomes. Furthermore, we will discuss the underlying mechanisms and how these may be modified in order to change the outcomes. A thorough understanding of these concepts is of paramount importance and has therapeutic implications for the increasingly elderly population.

Implications of Vascular Aging

Chronological age is a well-established risk factor for the development of cardiovascular diseases. The changes that accumulate in the vasculature with age, however, are highly variable. It is now increasingly recognized that indices of vascular health are more reliable than age per se in predicting adverse cardiovascular outcomes. The variation in the accrual of these age-related vascular changes is a function of multiple genetic and environmental factors. In this review, we highlight some of the pathophysiological mechanisms that characterize the vascular aging phenotype. Furthermore, we provide an overview of the key outcome studies that address the value of these vascular health indices in general and discuss potential effects on perioperative cardiovascular outcomes.