Arginase blockade lessens endothelial dysfunction after thrombosis

Arginase blockade lessens endothelial dysfunction after thrombosis

Arginase blockade lessens endothelial dysfunction after thrombosis Chandani Lewis, MD,a,b Weifei Zhu, PhD,a,b Mircea L. Pavkov, MD,b Corttrell M. Kinn...

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Arginase blockade lessens endothelial dysfunction after thrombosis Chandani Lewis, MD,a,b Weifei Zhu, PhD,a,b Mircea L. Pavkov, MD,b Corttrell M. Kinney, BS,a Paul E. DiCorleto, PhD,a and Vikram S. Kashyap, MDa,b Cleveland, Ohio Introduction: Acute arterial thrombosis causes endothelial dysfunction due to decreased nitric oxide bioactivity. Increased arginase activity may modulate intracellular L-arginine levels, the substrate for nitric oxide. The purpose of this study was to identify the role of arginase in endothelial dysfunction in cell culture and in the vasomotor response of arteries exposed to thrombus. Methods: Rat aortic endothelial cells were exposed to thrombin at different time points. The cell extract was analyzed by immunoblotting and real-time polymerase chain reaction. Adult male rats underwent infrarenal aortic thrombosis by clip ligature for 1 hour. Infrarenal aortic ring segments were harvested and placed in physiologic buffer baths, and a force transducer was used to measure endothelial-dependent relaxation (EDR) and endothelial-independent relaxation (EIR). Arginase blockade was performed by incubating infrarenal aortic ring segments with arginase inhibitors for 1 hour before measuring EDR. Whole tissue extracts also underwent immunoblot analysis. The EDR and EIR curves were compared with analyses of variance. Results: A 6.76 ⴞ 1.4-fold induction in arginase I message levels (P ⴝ .001) was found in rat aortic endothelial cells exposed to thrombin (30 U/mL), and arginase I protein levels increased 2.1 times. The eight infrarenal aortic ring segments exposed to thrombosis for 1 hour had diminished EDR curves compared with 14 nonthrombosed normal segments (controls). The maximum (ⴞ SEM) EDR (acetylcholine 10ⴚ5M dose) in control infrarenal aortic ring segments was 108% ⴞ 4.3% compared with 63% ⴞ 6.2% for thrombosed infrarenal aortic ring segments (P < .001). Exposure to arterial thrombosis resulted in a 3.8-times increase in arginase I protein levels in infrarenal aortic ring segments. Preincubation of nine infrarenal aortic ring segments with the nonspecific (difluoromethylornithine) and six with specific ([S]-[2-boronoethyl]-L-Cysteine-HCl [BEC]) arginase inhibitor for 1 hour significantly increased the maximum EDR compared with untreated thrombosed segments (104 ⴞ 5.2, 108 ⴞ 7.6 vs 63% ⴞ 6.2, P < .001). EDR curves for difluoromethylornithine- and BEC-treated infrarenal aortic ring segments were superimposed on control EDR curves. The EIR and the vasoconstriction with norepinephrine for all groups were similar. Conclusion: Endothelial cells exposed to thrombin have increased arginase I messenger RNA and protein levels. Arterial thrombosis causes endothelial dysfunction without affecting smooth muscle responsiveness. Arginase blockade can lead to normalization of arterial vasomotor function. ( J Vasc Surg 2008;48:441-6.) Clinical relevance. Acute arterial thrombosis may lead to endothelial dysfunction, manifested clinically by vasospasm and rethrombosis after initially successful thrombectomy or thrombolysis. We have identified that the key enzyme, arginase, which modulates levels of the nitric oxide precursor, L-arginine, is increased in arterial thrombosis. Thrombin, a component of thrombus, increases arginase levels, which may lead to decreased nitric oxide bioavailability. These studies indicate that arginase blockade restores endothelial function in arteries exposed to thrombosis. This work may lead to therapeutic alternatives including L-arginine supplementation or arginase blockade for patients with acute ischemia.

Endothelial dysfunction, which is observed in various vascular conditions,1-3 has been attributed to the decreased nitric oxide (NO) production by endothelial NO synthase (eNOS). In several of these conditions, supplementation of L-arginine, the precursor of NO, has been shown to improve NO production even though eNOS activity and concentrations have been reported to be normal.4-7 This From the Department of Cell Biology, the Lerner Research Institutea and the Department of Vascular Surgery,b Cleveland Clinic. This work is supported by the American College of Surgeons Faculty Research Fellowship and the National Institutes of Health (HL 080847), both to Dr Kashyap. Competition of interest: none. Presented in part at the Second Annual Academic Surgical Congress, Phoenix, Ariz, Feb 2007. Reprint requests: Vikram S. Kashyap, MD, Cleveland Clinic, 9500 Euclid Ave, S40, Cleveland, OH 44195 (e-mail: [email protected]). 0741-5214/$34.00 Copyright © 2008 by The Society for Vascular Surgery. doi:10.1016/j.jvs.2008.02.030

indicates that a functional lack of L-arginine contributes to decreased NO levels and endothelial dysfunction. These surprising findings have led to the recognition of an “arginine paradox,” because normal intracellular concentrations of L-arginine should saturate the eNOS enzyme.8 The plausible explanations for this complex phenomenon include compartmentalization of eNOS and its substrate, alterations in L-arginine transport, or production of an endogenous NOS inhibitor like asymmetric dimethylarginine, or both. Increased arginase activity as an etiology for endothelial dysfunction is another possibility. Competition between eNOS and arginase for the substrate Larginine could lead to decreased NO production if the balance shifted to L-arginine metabolism by arginase. Arginase, an important enzyme of the urea cycle, is a binuclear manganese metalloenzyme and hydrolyses Larginine to ornithine and urea. Of the two isomers, arginase I is predominately expressed in the liver as a cytosolic 441

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enzyme and is the predominant isoform found in rodent endothelial cells. Arginase II is found in mitochondria and expressed in extrahepatic tissues, including the kidney. Arginase II is the predominant human isoform. Although these two enzymes have diverse functions, both can modulate NO production.9 Arterial as well as venous thrombosis depresses the ability of the vasculature to relax to various endothelial dependent stimuli.6,7,10 In experimental models, this is evidenced by depressed endothelial-dependent relaxation (EDR), a measure of the endothelium’s ability to produce NO after appropriate stimulation. A clinically relevant analog is seen in patients with peripheral arterial thrombosis and vasospasm that can prevent the successful restoration of blood flow. From prior observations, we hypothesize that increased arginase activity may contribute to the endothelial dysfunction in arteries exposed to acute thrombus and its major component, thrombin. In this study, we tested this hypothesis using cell culture to explore the mechanisms involved and an animal model of arterial thrombosis. MATERIAL AND METHODS Cell culture. Rat aortic endothelial cells (RAEC) were isolated and initially plated onto T75 flasks in a 37°, humidified, 5% carbon dioxide incubator. They were maintained in Dulbecco Modified Eagle’s Medium (Sigma, St. Louis, Mo) containing 15% fetal bovine serum, 0.009% heparin, and 0.015% endothelial cell growth supplement. The cells were grown to confluence before initiation of experiments and were used between passages 3 and 5. RAECs were rinsed in heparin-free media before exposure to bovine thrombin. Real-time polymerase chain reaction. Arginase I messenger RNA (mRNA) expression was measured using real-time polymerase chain reaction (RT-PCR) from nontreated cells (control) and cells treated with thrombin (10 and 30 U/mL) for 1, 4, and 6 hours. Total RNA was extracted from 2 ⫻ 106 RAECs using standard phenolchloroform extraction (Rneasy Mini Kit, Qiagen, Valencia, Calif). The quantity of total RNA was determined by A260 absorbance. Complementary DNA production and amplification were synthesized from total RNA (1 ␮g) isolated from control and treatment cells using RT-PCR (Roche, Indianapolis, Ind) according to the manufacturer’s instructions. Volume of the reaction mixture was made up to 25 ␮L, and 5 ␮L of this reaction mixture was used to perform PCR with the following specific rat arginase I primer pairs: Forward: 5=-GCCAATGAACAGCTGGCTGCTGT-3=; Reverse: 5=-TACATCGGCTTGCGAGATGTGGA-3=. RT-PCR was performed using SYBR green PCR core reagent and a Perkin-Elmer ABI PRISM 7700 sequence detector (PE Applied Biosystems, Foster City, Calif), according to the manufacturer’s instructions. This quantification is expressed as fold induction compared with controls. Western blotting. Cellular lysates from RAECs and whole tissue lysates from control and thrombosed infrarenal segments and were electrophoresed on a 12% sodium

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dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) gel. The gel was soaked in protein transfer buffer (39mM glycine, 48mM Tris base, 0.037% SDS, 20% methanol) for 20 minutes at room temperature and then transferred to nitrocellulose using a Trans-Blot semi-dry transfer cell (Bio-Rad, Hercules, Calif). The blot was blocked with 5% bovine serum albumin (BSA). Next, the blot was incubated with polyclonal antiarginase I (sc-18351) and II (sc-20151) antibody (Santa Cruz Biotechnology, Santa Cruz, Calif) in 5% BSA overnight at 4°C. The blot was washed three times with Tris-buffered saline Tween-20 and incubated for 1 hour with goat antirabbit horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch, West Grove, Pa) at room temperature. Protein bands were detected using chemiluminescence. Animals. Adult, male Sprague-Dawley rats weighing 350 to 500 mg were used for all experiments. Animals were handled and cared for under Cleveland Clinic guidelines and in compliance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy Press, Washington, DC, 1996) and the American Association for the Accreditation of Laboratory Animal Care (AAALAC). The experimental protocols were approved by the Cleveland Clinic Institutional Animal Care and Use Committee and were performed in accordance with the recommendations of Good Laboratory Practices. Surgical procedures. Intraperitoneal injections of ketamine (80 mg/kg) and xylazine (8 mg/kg) were used for anesthesia. The aorta was ligated with small surgical clips just distal to the renal arteries and proximal to the bifurcation to induce arterial thrombosis, as previously described.6 Aortic segments were harvested before any intervention (controls), or after a prescribed time interval of thrombosis. The presence of intraluminal thrombus was confirmed in all animals at the time of tissue harvest. Studies of endothelial and smooth muscle cell function. The infrarenal aorta was carefully harvested, placed in Krebs-Henseleit physiologic solution (118mM NaCl, 25mM NaHCO3, 5.6mM glucose, 1.2mM KH2PO4, 4.7mM KCL, 0.6mM MgSO4, 2.5mM CaCl2), and sectioned into 4-mm segments. The segments were mounted on standard tungsten wire triangles (A-M Systems, Everett, Wash), attached to isometric force displacement transducers (Radnoti Systems Inc, Monrovia, Calif), and placed into tissue baths. The tissue baths were temperature controlled with a heated water jacket at 37°C. Standard Krebs-Henseleit solution was used with a gas mixture of 95% oxygen and 5% carbon dioxide bubbled into the baths. Preload (2 g) was applied to the arterial rings, and the vessels were allowed to equilibrate for 1 hour. After equilibration, the arteries were constricted using a high potassium solution (120mM KCl) and allowed to re-equilibrate. This represented the maximal contractile force for the artery (F-max). The baths were then emptied, and segments were rinsed three times with the Krebs-Henseleit solution and again allowed to equilibrate.

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Fig 2. Effect of thrombosis on endothelial-dependent relaxation (EDR) of rat infrarenal aortic (IRA) ring segments. EDR is plotted as percentage relaxation of baseline on the y axis, with log increases in the dose of acetylcholine (Ach, 10⫺9M to 10⫺4M) on the x axis. EDR of control (nonthrombosed) rat IRA (diamonds) is compared with IRAs after 1 hour of thrombosis (squares). EDR is depressed in the thrombosis group for all acetylcholine doses (n ⫽ 5 in control and n ⫽ 4 thrombosed group). *P ⬍ .05, **P ⬍ .001 by two-way analysis of variance. Data are presented with the standard error.

Fig 1. A, Thrombin-induced arginase I messenger RNA (mRNA) expression in five separate experiments. B, Thrombin-induced arginine I protein expression in three separate experiments. *P ⫽ .001.

After re-equilibration, the arteries were constricted to 75% of F-max with 10⫺6M norepinephrine (Sigma). Acetylcholine (Sigma) was then added in incremental log concentrations from 10⫺9M to 10⫺4M for determination of EDR. Endothelial-independent relaxation (EIR) was measured in a similar fashion using sodium nitroprusside (Sigma) from 10⫺9M to 10⫺7M. Baths were rinsed and arterial segments brought to 75% F-max with norepinephrine between each reagent. Nonspecific arginase blockade was achieved by incubating the aortic segments in difluoromethylornithine (DFMO). Specific arginase blockade was accomplished using (S)-(2boronoethyl)-L-Cysteine-HCl (BEC). Incubation with BEC or DFMO (Alexis Biochemicals, San Diego, Calif) occurred for 1 hour during the equilibration period. Control vessels underwent equilibration with Krebs-Henseleit physiologic solution for 1 hour. For these experiments, a fixed norepinephrine dose of 10⫺6M was used to achieve contraction. EDR and EIR were measured as above. Experimental design. Normal, nonthrombosed aortas (n ⫽ 5) served as controls and were compared with aortas (n ⫽ 4) that underwent 1 hour of thrombosis. In experiments using arginase inhibitors, nonthrombosed control aortas (n ⫽ 3) were compared with aortas after 1 hour of thrombosis (n ⫽ 3) and thrombosed aortas treated with the nonspecific arginase inhibitor DFMO (n ⫽ 7) or the specific arginase inhibitor BEC (n ⫽ 6).

Statistical analysis. Data reported are mean ⫾ standard error of the mean. Single comparisons were made with a two-tailed Student paired and unpaired t tests. Multiple comparisons were made with two-way analysis of variance (ANOVA), and the Bonferroni t test was used for post hoc tests. Statistical significance was set at P ⬍ .05. Statistical analysis was performed using Prism software (GraphPad, San Diego, Calif). RESULTS Thrombin increases arginase I expression. Isolated RAECs were exposed to thrombin, a major component of arterial thrombus. Arginase I mRNA and protein expression were assayed after RAEC exposure to thrombin (30 U/mL) for 6 hours. RT-PCR results revealed a sevenfold increase in arginase I message levels (6.76 ⫾ 1.4-fold, P ⫽ .001, n ⫽ 5) compared with controls (Fig 1, A) Immunoblot analyses was performed to study the effect of thrombin on arginase I protein expression. Endothelial arginase I protein levels increased 2.1 times with exposure to thrombin, confirmed on three separate Western blots (Fig 1, B). Tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels both remained unchanged. Arginase I protein levels also increased (1.5 times) with 1 hour and 4 hour exposure times to thrombin 30 U/mL. Lower levels of thrombin exposure (10 U/mL) only showed minor changes in arginase I protein levels. Arginase II was not detectable in RAECs. Endothelial-dependent relaxation is diminished by arterial thrombosis. The effect of arterial thrombosis on endothelial function was measured. After 60 minutes of infrarenal aortic thrombosis, the aorta was harvested and EDR and EIR were measured in an organ chamber system. The nonthrombosed control rat aortas responded normally (Fig 2). EDR increased with increasing acetylcholine doses

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Fig 4. Effect of nonspecific arginase inhibition on endothelialdependent relaxation (EDR) of thrombosed rat infrarenal aortic (IRA) ring segments is shown. EDR is plotted as percentage relaxation of baseline, with log increases in the dose of acetylcholine (Ach, 10⫺9M to 10⫺4M). EDR of nonthrombosed (control IRA, diamonds; n ⫽ 3) is compared with IRA segments after 1 hour of thrombosis untreated and treated with arginase inhibitors (thrombosed IRA, clear squares; n ⫽ 3) and treated with nonspecific arginase inhibitor difluoromethylornithine (DFMO IRA, filled squares; n ⫽ 9). Treatment with arginase inhibitor restores the EDR in thrombosed IRA. *P ⬍ .05, **P ⬍ .001 DFMO vs thrombosed IRA; #P ⬍ .01, control vs thrombosed IRA by twoway analysis of variance. Data are presented with the standard error. Fig 3. A, Western blot analysis of arginase I (ARGI) in rat infrarenal arterial segments. Arginase I protein levels were upregulated in endothelium intact thrombosed infrarenal aortic ring segments compared with control (n ⫽ 3). B, Western blot analysis of denuded aortic segments. Arginase I protein is not detected (arrow indicates location of arginase I protein by size). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

from 24% ⫾ 2% (SE) at the acetylcholine 10⫺9M dose to 108% ⫾ 4% of maximal EDR at acetylcholine 10⫺5M dose. However, thrombosed rat aortic segments had significantly lower EDR at all doses between acetylcholine 10⫺8M and acetylcholine 10⫺5M and achieved a maximal EDR of 63% ⫾ 6% (P ⬍ .001, two-way ANOVA). Arterial ring segments responded uniformly to sodium nitroprusside with a 100% EIR at the 10⫺7M doses of sodium nitroprusside. Of note, all arterial ring segments constricted similarly with norepinephrine and high potassium Krebs buffer. Thrombosis increases arginase I protein expression. Arginase protein expression was examined in infrarenal aortic ring segments from nonthrombosed and thrombosed aortic segments (n ⫽ 3). Thrombosed aortic segments had higher (3.8-fold) arginase I protein expression compared with control (Fig 3, A). Arginase II levels were undetectable in thrombosed as well as in control segments. To determine the cellular source of arginase protein, we denuded the endothelium from the aortic segments by gentle rubbing of the inner surface. Arginase I levels were undetectable in these denuded aortic segments (n ⫽ 3), implying that the endothelium was the source of arginase I (Fig 3, B). Endothelial dysfunction is ameliorated by arginase inhibitors. Because arginase protein expression was enhanced by the exposure to thrombus, we explored the

effect of pharmacologic inhibitors of arginase on endothelial function. A single, fixed dose of norepinephrine (10⫺6M) was used to preconstrict infrarenal aortic segments in these experiments. The response of the control and the thrombosed infrarenal aortic segments to norepinephrine 10⫺6M was similar (1.0 ⫾ 0.7 g and 0.96 ⫾ 0.1 g, respectively). However, maximum EDR was significantly diminished (110% ⫾ 8.2% vs 48% ⫾ 7.3%, P ⬍ .001), reconfirming the observation that the acute arterial thrombosis causes endothelial dysfunction. Incubation of thrombosed arterial rings using DFMO (10⫺7M), a nonspecific arginase inhibitor, was performed for 1 hour. DFMOtreated arterial rings (n ⫽ 7) had significantly increased EDR compared with untreated thrombosed infrarenal aortic segments (maximum EDR, 104% ⫾ 5.2% vs 53% ⫾ 7.0%, P ⬍ .001), as displayed in Fig 4. We also tested the effects of a specific arginase blocker, BEC, which has no effects on eNOS. Incubation of thrombosed aortic segments (n ⫽ 6) with BEC (10⫺7M), also significantly increased EDR compared with untreated thrombosed infrarenal aortic segments (108% ⫾ 7.6% vs 53% ⫾ 7.0%, P ⬍ .001, Fig 5). DFMO and BEC treatment of thrombosed infrarenal aortic segments both led to normalization of EDR with curves superimposed on the nonthrombosed control curves. Vasoconstriction with norepinephrine in all groups was similar (P ⬎ .5), indicating uniform vasoconstricting capacity. The EIR, a measure of smooth muscle function, was similar in control infrarenal aortic segments, untreated thrombosed segments, and in those incubated with either DFMO or BEC with increasing doses of sodium nitroprusside, 10⫺9M to 10⫺7M (105% ⫾

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Fig 5. Effect of specific arginase inhibition on endothelialdependent relaxation (EDR) of thrombosed infrarenal aorta (IRA) is shown. EDR plotted as percentage relaxation of baseline, with log increases from 10⫺9M to 10⫺4M in the dose of acetylcholine (Ach). EDR of nonthrombosed (control IRA, triangle; n ⫽ 3) is compared with IRA after 1 hour of thrombosis untreated with arginase inhibitors (Thrombosed IRA, clear square; n ⫽ 3) and treated with the specific arginase inhibitor [S]-[2-boronoethyl]-LCysteine-HCl (BEC IRA, black square; n ⫽ 6). Treatment with arginase inhibitor restores the EDR in thrombosed IRA. **P ⬍ .001 BEC vs thrombosed IRA analysis of variance. #P ⬍ .01, ##P ⬍ .001 control vs thrombosed IRA by two-way analysis of variance. Data are presented with the standard error.

4.8%, 104% ⫾ 1.3%, 105% ⫾ 2.3%, and 102% ⫾ 0.4%, respectively, sodium nitroprusside 10⫺7M, P ⫽ NS). DISCUSSION The importance of an intact endothelium to balance hemostasis, but prevent thrombosis, is well known. Although most studies focus on the effect of endothelium on thrombus generation, the effect of acute thrombosis on the vessel wall remains under-appreciated. Endothelial dysfunction after acute thrombosis has been observed in multiple studies.6,10-12 Enhanced arginase activity has been implicated as a cause of endothelial dysfunction in various disease models. In agerelated vascular dysfunction, Berkowitz et al2 showed that older animals have increased arginase activity and expression compared with younger animals. Inhibition of arginase pharmacologically by using arginase inhibitors or by antisense oligonucleotide restored the endothelial NO production. Arginase activity is increased in spontaneous as well as saltsensitive hypertensive rats.1,13 Arginase activity was associated with increase in Rho A activity in atherosclerotic aortas in apolipoprotein E⫺/⫺ mice.3 For this study, we hypothesized that thrombin can lead to increased arginase activity and that this may contribute to the endothelial dysfunction in arteries exposed to acute thrombus. We used cell culture of endothelial cells derived from rat aortas to elucidate the mechanism of possible impaired NO generation in endothelial cells. Based on our work,12,14 and results from others,15 we evaluated the response of RAECs to thrombin, a key enzyme of the coagulation cascade and a potent component of arterial thrombus. We found that thrombin increases the expression of arginase mRNA determined by RT-PCR by nearly

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sevenfold. This results in more than a twofold increase in arginase I protein concentration by Western blot analysis. This is consistent with other reports in aging rats4 and hypertensive rats,1,13 where enhanced arginase activity was found along with increased arginase protein concentration, which was responsible for the endothelial dysfunction. Thrombin regulation of arginase protein could be occurring at multiple levels, including translational as well as post-translational levels. At the translational level, increased message and its translation can lead to increased protein concentrations in endothelial cells exposed to thrombus. Ryoo et al16 found oxidized low-density lipoprotein causes an increase in the arginase message as well as protein levels in human aortic endothelial cells. Post-translational modification leading to release of arginase from cellular storage could be another possibility if microtubules served as storage for arginase. Recently, other investigators found that human umbilical vein endothelial cells exposed to thrombin (25nM) for 10 minutes caused destabilization of 44% of tubulins.17 Destabilization of the microtubule architecture by depolymerization can lead to decreases in NO generation.18 Arginase II, the predominant isoform in human endothelial cells, colocalizes with tubulins in human aortic cells experiments. This intriguing mechanism could partially explain the increase in arginase protein levels observed after 1 hour of acute arterial thrombosis. The results of the endothelial function studies using arterial ring samples are compelling. Endothelial function (EDR) diminished with thrombosis. All the vessels retained the capacity to dilate to the NO donor sodium nitroprusside, indicating normal smooth muscle cell responsiveness to NO. Furthermore, arginase I protein levels were increased in the thrombosed specimens. Denudation of the endothelial layer in the thrombosed as well as in control rat infrarenal arteries made arginase undetectable in these vessels, confirming the tissue source as endothelial cell layer. This is consistent with the White et al4 finding of arginase I to be present predominately in the endothelial layer in rat artery. In contrast, Zhang et al19 reported the presence of arginase in endothelial as well as smooth muscle layers of the porcine coronary artery. Buga et al20 has showed arginase I and II both to be present in rat aorta and found arginase I to be constitutively expressed, whereas arginase II was induced by lipo polysaccharide. Distribution of isomers of arginase enzyme in vascular tissue appears to be species specific. In porcine artery and rat aorta, arginase I is predominant and constitutively active,19,20 whereas arginase II is found in mouse aorta.3 Both isomers are found in human umbilical vein endothelial cells.14,21 Of note, previous experiments on arginase isoforms may have been complicated by smooth muscle contamination of endothelial specimens. The use of the arginase inhibitors DFMO and BEC restored endothelial function in arteries exposed to thrombosis. DFMO is a nonspecific but efficient arginase inhibitor as well as an inhibitor of ornithine decarboxylase, the rate-limiting enzyme for polyamine synthesis from ornithine. Because DFMO also is an inhibitor of ornithine

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decarboxylase, which by increasing L-arginine utilization in urea cycle can interfere in NO production, we repeated these experiments with BEC, a boric acid analogue of L-arginine and a specific arginase inhibitor. BEC is a potent arginase inhibitor and has no effect on eNOS function. These data indicate that DFMO and BEC through inhibition of arginase can restore endothelial function in thrombosed aortic rings, perhaps by increasing NO bioavailability. This work has several limitations. Other factors in thrombus may be playing a role in endothelial dysfunction. We chose to study thrombin and its relationship to arginase, but thrombosis involves multiple mechanisms. Our model of acute thrombosis is not ideally designed to study the inflammatory component of the thrombosis cascade. Lipo polysaccharide–activated endothelial cells induce a different arginase isoform, inducible NOS upregulation, and relatively large increases in NO production with native arginase suppression, which maybe more relevant to inflammation.20 The question remains of whether thrombin’s effects are receptor-dependent or through a global, nonspecific effect. We chose to use a single dose of thrombin based on previous studies.14,22 Additional mechanistic studies are needed and are currently underway, including the effects of thrombin on the arginase promoter complex and examining L-arginine metabolites. The relevance of these findings to the clinical scenario of thrombosis in diseased arteries and endothelium is unknown. Presumably, the younger patient with an embolus causing acute ischemia in otherwise normal arteries would be the closest clinical correlate. Human clinical studies in this arena would be compelling. Finally, the role of arginase inhibitors as a clinical adjunct remains unknown.

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CONCLUSIONS This study shows that thrombin increases arginase protein and message levels in endothelial cells. Similarly, thrombosed rat aortas develop endothelial dysfunction, increase arginase protein levels, and restore endothelial function with arginase inhibitors. Further research in this area is warranted.

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AUTHOR CONTRIBUTIONS

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Conception and design: CL, PD, VK Analysis and interpretation: CL, WZ, PD, VK Data collection: CL, WZ, MP, CK Writing the article: CL, VK Critical revision of the article: WZ, MP, CK, PD, VK Final approval of the article: CL, WZ, MP, CK, PD, VK Statistical analysis: CL, VK Obtained funding: VK Overall responsibility: VK REFERENCES 1. Demougeot C, Prigent-Tessier A, Marie C, Berthelot A. Arginase inhibition reduces endothelial dysfunction and blood pressure rising in spontaneously hypertensive rats. J Hypertens 2005;23:971-8. 2. Berkowitz DE, White R, Li D, Minhas KM, Cernetich A, Kim S, et al. Arginase reciprocally regulates nitric oxide synthase activity and contrib-

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Submitted Nov 30, 2007; accepted Feb 16, 2008.