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(Circulation Research. 2008;102:923.)
© 2008 American Heart Association, Inc.

Molecular Medicine

Endothelial Arginase II

A Novel Target for the Treatment of Atherosclerosis

Sungwoo Ryoo^*, Gaurav Gupta^*, Alexandre Benjo, Hyun Kyo Lim, Andre Camara, Gautam Sikka, Hyun Kyung Lim, Jayson Sohi, Lakshmi Santhanam, Kevin Soucy, Eric Tuday, Ezra Baraban, Monica Ilies, Gary Gerstenblith, Daniel Nyhan, Artin Shoukas, David W. Christianson, Nicholas J. Alp, Hunter C. Champion, David Huso, Dan E. Berkowitz

From the Departments of Anesthesiology/Critical Care Medicine (S.R., A.B., H. Kyo Lim, A.C., G.S., H. Kyung Lim, E.B., D.N., D.E.B.), Biomedical Engineering (G. Gupta, J.S., L.S., K.S., E.T., A.S., D.E.B.), Medicine (G. Gerstenblith, H.C.C.), Division of Cardiology, Molecular and Comparative Pathobiology (D.H.), the Johns Hopkins Medical Institutions, Baltimore, Md; Department of Chemistry (M.I., D.W.C.), University of Pennsylvania, Philadelphia; Department of Cardiovascular Medicine (N.J.A.), John Radcliffe Hospital, University of Oxford, UK; and Institute of Long Life, Department of Anesthesiology and Pain Medicine (H. Kyung Lim), Yonsei University Wonju College of Medicine, Wonju, Korea.

Correspondence to Dan Berkowitz, MD, Associate Professor, Anesthesiology/CCM, Tower 711, The Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287. E-mail dberkowi{at}bme.jhu.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

Key Words: vascular stiffness • eNOS uncoupling • pulse wave velocity • nitric oxide • L-arginine

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In 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^12–14 and pulmonary hypertension,^15,16 aging,^5,17,18 diabetes,¹⁹ and erectile dysfunction²⁰ 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 OxLDL-dependent activation of arginase causes impaired vascular NO production, increased ROS production, and endothelial dysfunction in atherogenic (apolipoprotein E–null [ApoE^–/–]) mice; and (2) whether arginase inhibition prevents the development of endothelial dysfunction and vascular stiffness and attenuates plaque development in this model.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Arginase activity was measured by assaying for urea using a colorimetric method with -isonitrosopropiophenone. NO was determined by measuring NOx (Griess reaction), as well as the NO-sensitive fluorescent dye DAF-FM DA. Vascular and endothelial ROS was measured using lucigenin chemiluminescence, as well as the O₂^–-sensitive fluorescent dye dihydroethidine (DHE). Endothelial function was determined in mouse aortic rings in organ chambers, and vascular stiffness was measured using high frequency Doppler. eNOS dimer/monomer ratios were determined using nondenaturing, low-temperature SDS-PAGE and immunoblotting, and biopterin levels were determined by high-performance liquid chromatography. All experimental procedures were approved by the Institutional Animal Care and Use committee at The Johns Hopkins University School of Medicine. All methods have been described previously^17,26 or are provided in the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lipid Profiles
See Table I in the online data supplement.

Endothelial ArgII Is the Primary Isoform Upregulated/Activated in ApoE^–/– Mice
Arginase activity was increased in the aorta of wild-type (WT) mice fed a high-cholesterol (HC) diet (1.25% cholesterol, 0% cholate; Research Diets) for 6 to 8 weeks versus normal diet (ND) (WT+ND versus WT+HC, 685.0±22.1 versus 819.0±85.0 pmol urea/mg protein per minute; P<0.001, n=6) (Figure 1A). Arginase activity was further elevated in ApoE^–/– mice fed a HC diet (WT+ND versus ApoE^–/–+HC, 685.0±22.1 versus 895.7±23.7 pmol urea/mg protein per minute; P<0.001, n=6). ArgII^–/– mice had significantly reduced vascular arginase activity, indicating that ArgII is the predominant isoform present in the vascular endothelium. ArgII^–/– mice fed a HC diet did not exhibit an increase in vascular arginase activity, suggesting that OxLDL modulates endothelial cells ArgII activity. In addition, elevated ArgII activity in ApoE^–/– was inhibited with the arginase specific inhibitor S-(2-boronoethyl)-L-cysteine (BEC) (ApoE^–/–+HC versus ApoE^–/–+HC+BEC, 895.7±23.7 versus 592.7±39.3 pmol urea/mg protein per minute; P<0.001, n=6). In separate experiments, we confirmed the cellular source of arginase activity, ie, whether endothelial ArgII accounts for the inhibitable component of total vascular arginase activity. Removal of the endothelium in WT mice significantly reduced arginase activity such that it was not significantly different from endothelial-intact (E⁺) ArgII^–/– mouse aorta (Figure 1B). Furthermore, removal of the endothelium in ApoE^–/– mice significantly reduced its activity. In addition, inhibition of arginase in E⁺ ApoE^–/– aortas reduces arginase activity to that of the endothelial-denuded (E^–) vessels.

View larger version (46K): [in this window] [in a new window]   Figure 1. Endothelial arginase is activated in atherogenic ApoE–/– mice and WT mice fed a HC diet. A, Inhibition of arginase with BEC or KO of the ArgII gene (ArgII–/–) prevents atherogenic diet-induced increase in arginase activity in WT and ApoE–/– mice. WT (C57Bl6J) and ArgII–/– mice were fed a ND or HC diet. Atherosclerosis-prone ApoE–/– mice fed a HC diet were implanted with osmotic infusion pumps containing the arginase inhibitor BEC or water vehicle placebo (PL). Arginase activity was measured as described in Materials and Methods. Arginase activity is increased in WT (HC) and ApoE–/– mice. BEC resulted in a marked decrease in arginase activity such that the activity was no different from ArgII–/– mice. *P<0.001, WT (ND) vs WT (HC) and ApoE–/– (HC); #P<0.001, ApoE–/– vs (BEC); P<0.05, WT (HC) vs ArgII–/– (HC). The primary source of the inhibitable component of arginase activity resides in the endothelium (B) because removal of the endothelium in ApoE–/– and WT reduced activity to that of aorta from endothelial-intact BEC-treated or ArgII–/– mice, respectively. *P<0.01, WT (E+) vs WT (E–) and ApoE–/– (E+) vs ApoE–/– (E–); P=NS, WT (E–) vs ArgII–/– (E+); P=NS, ApoE–/– (E–) vs ApoE–/– (BEC). Increase in arginase activity is not associated with an increase in protein abundance (C).

We next examined the abundance of arginase protein in the aorta of the mice strains. ArgII was not expressed in ArgII^–/– mice (Figure 1C). Although arginase activity was increased in aortic tissue of ApoE^–/– mice, this was not associated with an increase in protein abundance (Figure 1C). This is consistent with our observations in endothelial cells and the observation of others.^22,27

Arginase Inhibition Restores NO Production in OxLDL-treated Human Aortic Endothelial Cells and Endothelium of ApoE^–/– Mice
A HC diet decreased NO production in aorta of WT mice, which was further decreased in ApoE^–/– mice. Moreover, ApoE^–/– mice treated with BEC were protected from HC mediated decrease in vascular NOx (ApoE^–/–+HC+BEC versus ApoE^–/–+HC, 13.6±1.9 versus 9.8±1.2 µmol/mg protein; P<0.001, n=10) such that the levels were no different from WT mice fed a ND (ApoE^–/–+HC+BEC versus WT+ND, 13.6±1.9 versus 14.0±1.9 µmol/mg protein; P=NS, n=10). This is consistent with our results in ArgII^–/– mice fed a HC diet: vascular NO production was unchanged. Interestingly, ArgII^–/– mice had a higher basal level of vascular NOx production than their WT background controls (n=10 from 5 mice).

Because arginase inhibition increases NOx in ApoE^–/– mice, we tested whether arginase inhibition attenuated OxLDL, induced decreases in NO production in human aortic endothelial cells (HAECs) using an NO-sensitive fluorescent dye, DAF-FM DA acetate (see Materials and Methods).^17,26 Incubation of HAECs with OxLDLs decreased the DAF fluorescence slope (Figure 2B; 1.56±0.33 versus 0.53±0.02, baseline versus OxLDL; slope±SEM, P<0.01, n=8). Remarkably, arginase inhibition (BEC 10 µmol/L) restored the DAF fluorescence rate to baseline (1.56±0.33 versus 1.411±0.34, baseline versus OxLDL+BEC; slope±SEM, P=NS, n=8), suggesting that OxLDL-induced decreases endothelial NO production is arginase-dependent (Figure 2C). N^G-Nitro-L-arginine methyl ester (L-NAME) resulted in an almost complete reduction in slope of the DAF fluorescence to 0 (slope=0.05±0.03, n=8).

View larger version (39K): [in this window] [in a new window]   Figure 2. Arginase inhibition reciprocally decreases NO production in ApoE–/– mice and endothelial cells stimulated with OxLDL. A, Vascular NO was measured in mouse aorta using the Griess method. *P<0.001, WT (ND) vs WT (HC) and ApoE–/– (HC); #P<0.001, ApoE–/– vs ApoE–/–(BEC); P<0.05, WT (HC) vs Arg II–/– (HC); **P<0.01, WT (ND) vs ArgII–/– (ND). HAECS were loaded with DAF (5 µmol/L), and fluorescence was measured. Cells were then stimulated with OxLDL (50 µg/mL, 10 minutes), and the change on the slope of the fluorescence was monitored. The effect of BEC (10 µmol/L) on the slope of the DAF fluorescence was then determined. Cumulative data are shown in B, and a representative trace is shown in C. *P<0.001, control vs OXLDL; #P<0.001, OxLDL vs OxLDL (BEC); P<0.001, control vs L-NAME. Slope of DAF fluorescence was also measured in DAF-loaded mouse aorta (en face, endothelial side up) (D). *P<0.001, ApoE–/– vs WT; #P<0.001, ApoE–/– vs ApoE–/– (BEC); P<0.01, ArgII–/– vs WT; **P<0.001, WT vs WT (L-NAME). Endothelial cells isolated from aorta of WT and ArgII–/– mice were confirmed using the endothelial cell–specific protein eNOS (E). Following stimulation of the cells with OxLDL (50 µg/mL), mouse endothelial cells were stained with the NO-sensitive fluorescent dye DAF (F). eNOS expression in ArgII–/– and ApoE–/– mice demonstrates a decrease in abundance in ArgII–/– and a increase in abundance in ApoE–/– (G). *P<0.01, WT (ND) vs WT (HC); #P<0.05, WT (ND) vs ApoE–/– (HC); P<0.01, WT (ND) vs ArgII–/– (ND).

Using a similar DAF bioassay, we examined mouse aorta en face (endothelial side up) from WT, ArgII^–/–, and ApoE^–/– mice treated with and without BEC (Figure 2D). The rate of DAF fluorescence accumulation was markedly depressed in ApoE^–/– mice. ApoE^–/– mice acutely treated with BEC had an increased DAF fluorescence slope (ApoE^–/– versus ApoE^–/–+BEC, 0.74±0.16 versus 6.36±2.99; P<0.01, n=4 aorta). In Arg II^–/–, the DAF fluorescence slope was higher than that of WT mice. Predictably, eNOS inhibition with L-NAME decreased fluorescence intensity in both WT and ArgII^–/– mice (WT versus WT+L-NAME, 4.68±0.88 versus 0.99±0.14, P<0.05; ArgII^–/– versus ArgII^–/–+L-NAME, 6.43±0.56 versus 1.2±0.18, P<0.05; n=4). We next isolated primary endothelial cells from WT and Arg II^–/– mouse aorta respectively. The endothelial cell phenotype was confirmed by the presence of eNOS expression (Figure 2E). OxLDL significantly decreased DAF fluorescence in WT cells. In endothelial cells from ArgII^–/– mice, basal DAF fluorescence was increased (Figure 2F). Furthermore, in endothelial cells from ArgII^–/– mice, OxLDL failed to decrease NO production (Figure 2F).

We next examined the abundance of eNOS in aorta. eNOS expression was significantly increased and NO production decreased in ApoE^–/– mice, whereas eNOS expression was decreased and NO production increased in ArgII^–/– mice (Figure 2G). This is consistent with the idea of eNOS uncoupling.²⁴

Arginase Inhibition Prevents OxLDL-Dependent eNOS Uncoupling
To determine whether arginase activation (associated with L-arginine depletion) contributes to eNOS uncoupling, we measured O₂^– using the O₂^–-sensitive dye DHE in isolated aorta en face from WT, ArgII^–/–, and ApoE^–/– mice treated with L-NAME (10 µmol/L), BEC (10 µmol/L), or both (see Materials and Methods). There was a significant increase in ROS production in the aorta of ApoE^–/– versus WT mice (n=4 aorta) (Figure 3A).²⁸ Supporting our hypothesis, BEC treatment significantly decreased ROS production, particularly in ApoE^–/– mice (5.99±0.67 versus 1.15±0.19 versus ApoE^–/–+BEC, 80% reduction) (Figure 3A). Interestingly, treatment with L-NAME (10 µmol/L) in ApoE^–/– mice led to a significant reduction in ROS (65% reduction) while increasing the slope of DHE response in WT mice. Furthermore, basal ROS signal was significantly decreased in the aorta of ArgII^–/– mice. Finally, in HAECs, OxLDL increased DHE fluorescence, which was markedly attenuated in the presence of the arginase inhibitor BEC (Figure 3B).

View larger version (33K): [in this window] [in a new window]   Figure 3. OxLDL-dependent arginase activation results in eNOS uncoupling and ROS production. ROS production was measured in mouse aorta (cumulative data and representative traces inset) (A) and HAECs (B) using the O2–-specific fluorescent dye DHE. Slope of DHE fluorescence over time was determined in aorta from WT, ArgII–/–, and ApoE–/– (HC) mice both before and following incubation with L-NAME (10 µmol/L) or BEC (10 µmol/L) or a combination of both. *P<0.01, ApoE–/– vs WT; #P<0.01, ApoE–/– (or WT)+BEC vs ApoE–/– (or WT); P<0.05, WT (L-NAME) vs WT; **P<0.01, ApoE–/– vs ApoE–/– (L-NAME); ##P<0.05, ArgII–/– vs WT; P<0.01, WT vs WT (BEC+L-NAME) and ApoE–/– (HC) vs ApoE–/– (BEC+L-NAME). Positive control, H2O2 (1 µmol/L) treatment of aorta and pretreatment with the O2– scavenger MnTBAP (10 µmol/L). BEC decreased ROS production in OxLDL-stimulated HAECs (B). Lucigenin chemiluminescence (5 µmol/L) was used for quantitative assessment of ROS production in WT and ArgII–/– endothelial cells treated with OxLDL (C). *P<0.01 WT vs WT (OxLDL); ¶P<0.001 WT (OxLDL) vs WT (OxLDL+BEC); P<0.001, WT vs Arg II–/–; #P<0.001, WT (OxLDL) vs ArgII–/– (OxLDL). On the other hand, the amount of BH4, total biopterin, or the BH4:BH2 ratio was not significantly changed in ApoE–/– mice treated with BEC (D). E, Native 4°C polyacrylamide gel electrophoresis and Western blot for eNOS to determine eNOS dimerization in the presence of OxLDL alone or following pretreatment of the cells with L-arginine (50 µmol/L), L-NAME (100 µmol/L), or BEC (10 µmol/L). *P<0.001 vs C; #P<0.05 vs OxLDL (E). BEC did not have any effect on xanthine oxidase–derived O2– production (F).

We next used lucigenin chemiluminescence (5 µmol/L lucigenin) to quantify ROS in primary aortic endothelial cells derived from the experimental mice. Stimulation of WT endothelial cells with OxLDL resulted in a significant increase in ROS (WT versus WT+OxLDL, 100±5.6 versus 173±6.0; P<0.001) (Figure 3C). Preincubation with BEC markedly attenuated an OxLDL-dependent increase in ROS. Consistent with our DHE fluorescence results (Figure 3A), endothelial cells from ArgII^–/– mice had a significantly reduced basal ROS production (WT versus ArgII^–/–, 100±5.6 versus 61.2±6.1; P<0.001). Furthermore, OxLDL-stimulated ROS production was almost completely prevented in ArgII^–/– endothelial cells (Figure 3C).

To determine the importance of vascular tetrahydrobiopterin levels on eNOS uncoupling in the setting of arginase activation, we measured aortic biopterins in experimental mice. Importantly, we found no significant difference in the levels of total biopterins, tetrahydrobiopterin (BH₄), or the BH₄:BH₂ ratios between WT and ApoE^–/– mice with or without BEC treatment (Figure 3D). These data are consistent with the idea that arginase activation by OxLDL leads to eNOS uncoupling, in this setting, independently of BH₄ levels or oxidation, and thus most likely by a specific effect of L-arginine substrate depletion.

We next tested the hypothesis that inhibition of arginase would protect the dimer form of eNOS. HAECs were incubated with OxLDL (50 µg/mL). Cell homogenates were separated on nondenaturing gels at 4°C, and the ratio of eNOS dimers to monomeric eNOS was determined. As can be seen in Figure 3E, Western blots for eNOS reveal a significant dimer band in unstimulated HAECs. Stimulation of endothelial cells with OxLDL results in a decrease in the intensity of the dimer band (untreated control versus OxLDL, ratio: 0.95±0.03 versus 0.40±0.03; P<0.001, n=3). Interestingly, inhibition of arginase with BEC prevented the decrease in eNOS dimer bands in OxLDL-stimulated endothelial cells (0.69±0.06, P<0.05, n=3). In addition, L-NAME also prevented OxLDL-dependent decreases in dimer formation, and treatment of cells with L-arginine partially preserved eNOS dimer stability. This suggests that ROS produced by eNOS may contribute to destabilization of the eNOS dimer by disruption of the Zn²⁺ thiolate complex.

Given the effect of arginase inhibition on ROS production, we wished to confirm that BEC was not acting simply as a ROS scavenger. Using lucigenin chemiluminescence, xanthine oxidase was stimulated in vitro to produce O₂^– with the addition of the substrate xanthine. As can be seen in Figure 3F, BEC had no effect on the chemiluminescence signal. In contrast, the signal was completely quenched with MnTBAP (10 µmol/L). These data confirm that BEC is not acting as a free radical scavenger.

Arginase Inhibition Restores Endothelial Function in ApoE^–/– Mice
Because both inhibition of arginase and deletion of the ArgII gene prevent cholesterol-induced decreases in vascular NO production, we determined whether endothelial function was altered in our mouse models. Vascular relaxation in response to the endothelial-dependent vasodilator acetylcholine (ACh) was measured in prostaglandin F2 (10 nmol/L) or phenylephrine (1 µmol/L) (data not shown) preconstricted mouse thoracic aortic rings (Figure 4). ACh caused a dose-dependent relaxation in aortic rings from WT mice. The response to ACh in ApoE^–/– mice fed a HC diet was significantly attenuated E_max, maximal vasorelaxant response (ApoE^–/–+ HC versus WT+ND, 31.1±3.3% (n=8) versus 66.6±2.5% (n=8); P<0.001). Interestingly, ApoE^–/– mice treated with BEC had a markedly enhanced response to ACh, such that it tended to be greater than WT mice fed a ND (ApoE^–/–+BEC versus WT+ND, 80.8.5±3.6% (n=8) versus 66.6±2.5% (n=8), P<0.001). The response to ACh in ArgII^–/– mice fed a ND were significantly greater than WT (71±3.0% versus 66.5±2.5%, P<0.05, n=8), consistent with our findings in the mouse carotid,²⁹ whereas responses in aorta from in ArgII^–/– mice fed a HC diet were not significantly different from WT (59.7±3.6 versus 66.5±2.5; n=8; P=NS). The responses to the endothelial-independent vasodilator (NO donor) sodium nitroprusside were similar in all aortic rings studied.

View larger version (13K): [in this window] [in a new window]   Figure 4. Arginase inhibition improves endothelial function in ApoE–/– mice and WT mice fed an atherogenic diet. Vasodilator dose responses to the endothelial-dependent, NO-coupled mediator ACh (1 nmol/L to 10 µmol/L) and sodium nitroprusside (SNP) (1 nmol/L to 10 µmol/L) (inset) in prostaglandin F2 (PGF2) (10 nmol/L) preconstricted thoracic aortic rings from WT and ApoE–/– treated with vehicle or the arginase inhibitor BEC: cumulative data (A) and representative trace (B). *P<0.01, WT vs ApoE–/–; #P<0.01, ApoE–/– vs ApoE–/– (BEC); P<0.05, ApoE–/– (BEC) vs WT. Endothelial function in WT mice and ArgII–/– mice fed a ND or HC diet (C). *P<0.05, ArgII–/– vs WT.

Arginase Inhibition Restores Arterial Compliance to Normal in ApoE^–/– Mice
We next investigated the effect of arginase inhibition on vascular stiffness, a marker of vascular health in vivo, by measuring aortic pulse wave velocity (PWV) using high-frequency Doppler before and at the end of the intervention (treatment or no treatment; see Materials and Methods for details). As illustrated in Figure 5A and supplemental Table II, WT mice fed a HC diet developed a significant increase in vascular stiffness, as measured by their PWV, consistent with a significant alteration in vascular properties (WT+ND versus WT+HC, 3.77±0.07 versus 4.59±0.12; n=9, P<0.001). ApoE^–/– mice have a significantly increased PWV compared with WT controls fed a ND (ApoE^–/–+HC versus WT+ND, 4.85±0.08 m/sec (n=7) versus 3.88±0.06 m/sec (n=9); P<0.001). Interestingly, ApoE^–/– mice treated with BEC showed a dramatic and consistent reduction in PWV (ApoE^–/–+HC versus ApoE^–/–+HC+BEC, 4.73±0.08 versus 3.99±0.06 m/sec; P<0.001, n=9) such that the PWV was not significantly different from WT (ApoE^–/–+HC+BEC versus WT+ND, 3.99±0.06 versus 3.88±0.06 m/sec; P=NS). Intriguingly, ArgII^–/– mice demonstrated a reduced baseline PWV compared with WT controls (ArgII^–/–+ND versus WT+ND, 3.13±0.04 versus 3.88±0.06 m/sec; n=8, P<0.05). Furthermore, although ArgII^–/– mice demonstrated a small increase in PWV after being fed a HC diet, the percentage increase was markedly reduced compared to WT mice fed a HC diet (ArgII^–/–+HC versus WT+HC, 8.5±1.7% versus 21.8±2.1%; P<0.001) (Figure 5B).

View larger version (17K): [in this window] [in a new window]   Figure 5. Arginase inhibition restores vascular compliance to normal in ApoE–/– and WT mice fed an atherogenic diet. Paired measurements of vascular stiffness as measured by PWV before and after treatment in all groups of mice (A). PWV velocity is shown in supplemental Table II. *P<0.001, WT (ND) vs WT (HC); P<0.001, WT (ND) vs ApoE–/–; #P<0.001, ApoE–/– vs ApoE–/– (BEC); ¶P<0.01, WT vs ArgII–/–. The percentage change increase in PWV in WT fed a high-fat diet compared with ArgII (high-fat diet) mice (C). *P<0.01, WT (HC) vs ArgII–/– (HC). Effect of a second arginase-specific inhibitor, ABH, on PWV, arginase activity, and NO production. #P<0.0001, ApoE–/– (placebo [PL]) vs ApoE–/– (ABH); *P<0.05, ApoE–/– (placebo) vs ApoE–/– (ABH).

2(S)-Amino-6-Boronohexanoic Acid, Another Potent Inhibitor of Arginase, Increases Vascular NO and Decreases Vascular Stiffness in ApoE^–/– Mice
In a select number of assays, we wished to determine whether a second boronic acid inhibitor of arginase has the same effect as BEC on vascular NO production and vascular stiffness. Four ApoE^–/– mice were randomized to receive either 2(S)-amino-6-boronohexanoic acid (ABH)⁸ (200 µg/d) in their drinking water for 2 weeks or placebo. As demonstrated in Figure 6, ABH treatment resulted in a significant reduction in vascular arginase activity (900.00±62.57 versus 712±17.27, ApoE^–/– versus ApoE^–/–+ABH; P<0.01, n=6 from 3 mice). This was associated with a reciprocal increase in vascular NOx production (8.50±0.60 versus 10.20±0.20, ApoE^–/– versus ApoE^–/–+ABH; P<0.01, n=6 from 3 mice). Furthermore, mice treated with ABH demonstrated a dramatic decrease in PWV (4.37±0.02 versus 3.72±0.0, n=4, P<0.0001).

View larger version (30K): [in this window] [in a new window]   Figure 6. Arginase inhibition in ApoE–/– mice reduces plaque load. ApoE–/– were randomized to receive 4 or 8 weeks of BEC treatment following 6 weeks of a HC diet. A representative descending thoracic aorta stained with Sudan IV to determine plaque area (A) and cumulative quantitative assessment of plaque area determined by pixel count of red-stained area (B). Histological sections of proximal ascending aorta from each group (C) were used to determine wall/plaque thickness (D). The quality of the plaque in the aorta of BEC-treated mice is significantly different with a marked reduction in foam cells. *P<0.01.

Arginase Inhibition Decreases Plaque Burden in ApoE^–/– Mice
BEC treatment reduced the area of the aorta with plaque lesions (Figure 6), as quantified by staining with the lipid dye Sudan IV (BEC 4 weeks versus placebo, 2.8±0.4 versus 1.4±0.1%; P<0.001) (Figure 6B). Treatment for 8 weeks further reduced plaque area (BEC 8 weeks versus BEC 4 weeks, 1.4±0.1 versus 1.0±0.1%; P=0.03). Histological assessment of plaque in the ascending aorta demonstrated that the average wall thickness in mice treated for 4 weeks or 8 weeks was significantly reduced (BEC 4 weeks versus placebo, 183±8 versus 240±15 µm; P<0.005) (Figure 6C and 6D). There is further trend toward a reduction in plaque thickness at 8 weeks.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have demonstrated that increased arginase activity in atherogenic mice contributes to impaired NO signaling and an increase in ROS production as a result of eNOS uncoupling. This leads to endothelial dysfunction and increased vascular stiffness. ArgII inhibition restores endothelial nitroso–redox balance and endothelial function and reduces vascular plaque burden, suggesting that ArgII could be a critically important target in atherosclerosis therapy.

Arginase activation, rather than an increase in protein abundance, appears to be responsible for the increased enzyme activity. This is supported by the results of this study in the aorta of ApoE^–/– mice and by the study of Ming et al.²⁷ Our previous results demonstrated that the increase in arginase activity was attributable to its rapid disassociation from the microtubular cytoskeletal structure.²²

Increased arginase activity and impaired NO signaling are now well recognized as contributing to several pathophysiological conditions. In contrast, the interaction between arginase and NOS isoforms under physiological, baseline conditions, is less clear. However, our previous studies in cardiomyocytes indicated that ArgII constrains NOS-1–dependent myocardial contractibility at baseline.⁶ Our present observations in the vasculature also support a physiological interaction because Arg^–/– mice demonstrated increased basal endothelial NO production, enhanced endothelial-dependent relaxation, and attenuated basal vascular stiffness.

Paradoxically, eNOS expression/abundance is actually increased in most animal models of atherosclerosis.³⁰ This is consistent with observations in eNOS-deficient and eNOS-overexpressing mice in which a HC diet results in decreased and increased measures of atherosclerosis, respectively.^31,32 Interestingly, NO production in ArgII^–/– mice is increased despite a decrease in eNOS abundance, suggesting that coupling rather than protein abundance is critical.

Several mechanisms could explain eNOS uncoupling in pathophysiologic conditions including: (1) substrate (L-arginine) depletion; (2) cofactor (BH4) depletion; (3) loss of dimerization; and (4) altered eNOS phosphorylation. These are interrelated and depend on spatial confinement of NO signaling and the nitroso-redox milieu. NOS uncoupling in the setting of cofactor (BH₄) or substrate (L-arginine) limitations could be amplified by an overabundance of the enzyme itself and is consistent with our results. Our data suggest that upregulation of arginase results in NOS uncoupling, and arginase inhibition results in recoupling, with restoration of nitroso–redox balance of endothelium function. In our experiments, the effect of arginase on NOS uncoupling was independent of vascular BH₄ concentration, suggesting that the mechanism underlying uncoupling was L-arginine limitation, rather than BH₄. eNOS uncoupling resulting from decreased BH₄ bioavailability has been demonstrated in experimental vascular disease states including diabetes,³³ hypertension,³⁴ and hypercholesterolemia.²⁸ Other studies have found that reduced L-arginine availability alone is sufficient to induce NOS uncoupling.³⁵ This is consistent with our findings in which the effects of arginase inhibition are independent of BH₄ levels. Peroxynitrite (ONOO^–)-induced direct oxidation of BH₄ is among the proposed mechanisms underlying eNOS uncoupling.³⁴ However, concentrations of ONOO^–, one-tenth to one-hundredth required to induce BH₄ oxidation can result in inhibition of BH₄ binding to the Cys99-thiolate center of the eNOS dimmer and to eNOS dimeric disruption.³⁶ Arginase inhibition alters the nitroso–redox milieu and, thus potentially, BH₄ oxidation but, more likely, BH₄ binding and eNOS dimer ratios. Further studies will be required to determine the relative contributions of these mechanisms. Our data suggest that the atherogenic process is, at least in part, attributable to arginase activation.

Increased vascular stiffness is now a recognized cardiovascular risk factor³⁷ and correlates with indices of atherosclerosis in animal models.³⁸ Mechanistically, NO bioavailability clearly modulates arterial stiffness.^26,39 Our data demonstrate that the endothelial dysfunction observed in ApoE^–/– mice is associated with a marked increase in vascular aortic stiffness as measured by aortic PWV. This is consistent with the findings of others who demonstrated a similar phenomenon.^40,41 Moreover, we have demonstrated that inhibition of arginase restores PWV to WT levels. This is associated with a marked improvement in endothelial-dependent vasodilatory function in vitro.

Advanced atherosclerotic plaque development is the result of activation/inhibition of multiple complex interacting mechanisms. Overall, the atherosclerotic process and plaque evolution are inhibited by NO and enhanced by ROS. Arginase inhibition, thereby increasing NO bioavailability and decreasing ROS production, represents a relatively proximal target among the complex, interacting mechanisms that culminate in plaque formation. Despite advance plaque development in the ApoE^–/–, short-term arginase inhibition significantly decreased plaque burden. Early and long-term inhibition may afford further benefit.

Given the inflammatory nature of the atherogenic process, we cannot exclude conclusively the possibility that the inhibition of arginase in activated macrophages in atherosclerotic plaques may have contributed to the effects observed. Arginase I is coactivated with inducible NOS in macrophages in response to inflammation.^11,17 However, in a genomic study of foam versus nonfoam macrophages, it was observed that arginase I was downregulated,⁴² suggesting that its activation may not be an important contributor to the phenotype observed. Indeed, in BEC-treated ApoE^–/– mice the arginase activity was not significantly different from the ArgII^–/– mice. Because BEC is nonselective arginase inhibition, this suggests that arginase I inhibition, either in the vasculature or macrophages, does not confound the interpretation of our results.

We also cannot exclude the possibility that the effects of arginase inhibition may not, in part, be attributable to modulating of vascular smooth muscle arginase I. Arginase I overexpression enhances smooth muscle cell proliferation and migration, and arginase-specific inhibition attenuates this process.⁴³ It is possible that the attenuating effect of BEC on atherosclerosis development may, in part, be a function of inhibition of smooth muscle migration.

In summary, we have demonstrated that ArgII activity is upregulated in atherosclerosis-prone mice and is associated with impaired endothelial NO production, endothelial dysfunction, vascular stiffness and ultimately, aortic plaque development. Inhibition of endothelial arginase or deletion of the ArgII gene enhances NO production, restores endothelial function and aortic compliance, and reduces plaque burden. Because arginase is downstream of OxLDL, it represents a novel risk factor–independent target for the prevention and treatment of atherosclerotic vascular disease.

	Acknowledgments

 
We thank Dr O’Brien (Baylor College of Medicine, Houston, Tex) for the generous gift of the ArgII^–/– knockout (KO) mice.

Sources of Funding

This work was supported by NIH grants R01 AG021523 (to D.E.B.) and R01 GM49758 (D.W.C.), National Space Biomedical Research Institute grant CA00405 through the National Aeronautics and Space Administration (to A.S.), and National Aeronautics and Space Administration grant NNH04ZUU005N (to D.E.B.), and American Heart Association, Mid-Atlantic Affiliate Fellowship Award (to S.R.).

Disclosures

None.

	Footnotes

 
*Both authors contributed equally to this work.

This manuscript was sent to Donald D. Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Original received April 28, 2007; resubmission received December 12, 2007; revised resubmission received January 31, 2008; accepted February 20, 2008.

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