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Introduction

Arginase is the enzyme that functions in the last step of the urea cycle to hydrolyzes L-arginine (L-Arg) to L-ornithine and urea. The arginase isoforms, arginase I and II, are encoded by distinct genes, show 60% sequence homology, and exhibit different tissue distribution and subcellular localization patterns. Arginase I is a cytosolic enzyme that is abundantly expressed in the liver, whereas arginase II is a mitochondrial protein that is predominantly expressed in the kidney (1,2). In addition to its primary function in the urea cycle, arginase exhibits various biological activities in vascular smooth muscle cells (VSMCs). In VSMCs of the rat carotid artery, inhibition or knockdown of arginase I led to attenuated medial and neointimal DNA synthesis and neointima formation (3). In mouse VSMCs, arginase II is involved in cell senescence and apoptosis (4). In our previous studies, we demonstrated that the proliferation of rat VSMCs, induced by the inflammatory cytokine, interleukin-1β, was suppressed by inhibition of arginase in a cGMP-dependent manner (5). Inhibition of arginase II also inhibited Ca2+ uptake into the mitochondria of native low-density lipoprotein-stimulated human aortic smooth muscle cells, thus arginase II activity may regulate Ca2+ levels in the cytosol and the mitochondria (6). Furthermore, we recently reported that inhibition of arginase II activated endothelial nitric oxide synthase by increasing the cytosolic Ca2+ level [(Ca2+)] in a p32-dependent manner (7).

The modulation of smooth muscle tone by various factors in the vasculature plays a role in the control of lumen diameter, which associated with blood pressure and organ blood flow (8–10). All smooth muscle cells produce force and contraction through cross-bridge cycling between actin and myosin filaments. The contraction response is the result of myosin-light chain kinase (MLCK) and myosin-light chain phosphatase (MLCP) activation. In VSMCs, this process is dependent on the Ca2+-mediated change in the myosin filaments (9). Increased intracellular Ca2+ is a primary target of the EF-hand Ca2+-binding protein calmodulin, and then the Ca2+/calmodulin complex activates MLCK via Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation. Contraction can also occur independent of Ca2+ through the activation of the RhoA/Rho kinase pathway, which leads to MLCP inactivation and maintenance of the contraction (11).

Because the contractile activity of VSMCs plays an important role in the regulation of vascular tone in the vessels, its dysregulation can result in endothelial dysfunction leading to cardiovascular diseases. Therefore, identifying natural compounds that can effectively inhibit arginase to control this contractile activity would offer a new strategy for the treatment and prevention of vascular conditions. Resveratrol (RSV) is a small polyphenol found in grapes, berries, and peanuts, and is considered to protect against cardiovascular diseases by reducing platelet aggregation, low-density lipoprotein oxidation, prostagladin synthesis, and endothelial cells activation by tumor necrosis factor-α, as well as by activating and inducing endothelial nitric oxide synthase (12–14). RSV has been known to activate to SirT proteins that post-translationally regulate protein activity by deacetylation, and RSV-dependent activation of SirT has showed a marked reduction in the signs of aging. On the other hand, inhibitors of SirT, such as sirtinol and splitomicin, were proposed to treat cancer and human immunodeficiency virus infection (15). Moreover, RSV was found to prevent angiotensin II-stimulated hypertrophy (16) and NADPH oxidase-dependent proliferation (17), as well as to induce differentiation to a contractile phenotype in VSMCs (18). However, the effect of RSV on the regulation of VSMCs contractility remains unknown. We hypothesized that RSV regulates VSMCs contractions by inhibiting arginase. To test this hypothesis, we treated rat VSMCs with RSV and determined the effects on arginase activity and [Ca2+]c, as well as the effecs on contraction by evaluating the change in CaMKII-dependent MLC20 phosphorylation. We further determined the effects of RSV on vascular contractility using an ex vivo vessel tension assay with thoracic aortas of mice. Our results should highlighted the fundamental mechanisms of VSMCs contractility and the potential role of arginase inhibition as a clinical treatment.

Materials and methods

Materials

RSV (trans−1,2-(3,4′, 5-Trihydroxydiphenyl) ethylene; Fig. 1) was purchased from Tokyo Chemical Industry inc. (Tokyo, Japan) and the purity was shown to be ≥99% by gas chromatographic analysis. 2(S)-amino-6-boronohexanoic acid (ABH) was purchased from Calbiochem. Co. (La Jolla, CA, USA). All other reagents were purchased from Sigma unless otherwise stated.

Figure 1. Structure of reversatrol.

VSMCs isolation and maintenance

Vascular smooth muscle cells (VSMCs) were isolated from the thoracic aortas and the upper parts of the abdominal aorta of 10–12-week old Sprague Dawley male rats (n=4, DBL Co., Chungbuk Korea) as previously described (19) with minor modifications. Briefly, the rat were anesthetized with isoflurane, aortas were stripped and cut into 2 mm pieces that were treated with 1 mg/ml of type II collagenase (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 1 h to remove the endothelial cells, and then the cells were washed with the culture medum. The de-endothelialized pieces of aorta were incubated with culture medium in a gelatin (0.1%)-coated culture dish for approximately 10 d. The VSMCs cultures were confirmed to be >95% pure by immunocytochemical staining for α-smooth muscle actin. VSMCs that were passaged 2–5 times were used in the subsequent experiments. The culture medium consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.5X smooth muscle growth supplement (Cascade Biologics, Portland, OR, USA), 100 U/ml penicillin, 100 µg/ml streptomycin, 8 mM HEPES, and 2 mM glutamine. The VSMCs were cultured at 37°C in a humidified 5% CO2 incubator.

Arginase activity assay and intracellular L-Arg quantification

Arginase activity was determined by quantifying the urea generated from L-Arg substrate as previously described (20). Intracellular concentrations of L-Arg were determined using high-performance liquid chromatography (HPLC) with pre-column derivatization with o-phthalaldehyde (OPA) according to a modification of previously published methods (21). Briefly, L-Arg (100 µmol/l) and polyamine (30 µmol/l/each) were added to the cell lysate (0.1 mmol/l) as internal standard. The samples were extracted on solid-phase extraction cartridges (CBA Bond elute, Varian), with a recovery rate of 87.5±3.9% to L-Arg. The eluates were dried over nitrogen and resuspended in double-distilled water for HPLC analysis. HPLC was performed on a computer-controlled Waters chromatography system (M600E) consisting of an automatic injector (M7725i, Waters Co.) and a fluorescence detector (FP-1520, Jasco Co.). Samples were incubated for 1 min with OPA reagent (5.4 mg/ml OPA in borate buffer, pH 8.4, with 0.4% 2-mercaptoethanol) before automatic injection into the HPLC system. The OPA derivatives of L-Arg and polyamine were separated on a 150×4.6 mm-5 µm Zorbax Eclipse XDB-C18 column with the fluorescence detector set at an excitation of 340 nm and an emission of 450 nm. Samples were eluted from the column with 0.96% citric acid:methanol (70:30), pH 6.8, at a flow rate of 1.5 ml/min.

Cytosolic Ca2+ measurement using confocal microscopy and flow cytometry

Direct assessment of the [Ca2+]c was peformed using an established procedure in which cells were loaded with the fluorescent agent Fluo-4 AM (ThermoFisher Scientific Co., Waltham, MA). Briefly, the cells were incubated with 100 nmol/l Fluo-4 AM at 37°C for 1 h in a starvation medium. The cells were then washed to remove excess Fluo-4 AM and incubated in Tyrode's modified solution (150 mmol/l NaCl, 4 mmol/l KCl, 2 mmol/l CaCl2, 2 mmol/l MgCl2, 10 mmol/l HEPES, and 10 mmol/l glucose). For detection of Fluo-4 AM fluorescence, 494 nm excitation, and 506 nm emission filters were used. Intensity values were normalized according to the initial fluorescence values after subtraction of the background signal using the Metamorph program (Molecular Probe). The [Ca2+]c was also determined using flow cytometry on a FACS Calibur system. The fluorescence intensity for each sample was determined using CellQuest software, and the Ca2+ level was calculated by comparing the fluorescence intensities of treated cells vs. control cells.

MTT assay

The cytotoxic activities of the tested compounds were measured using MTT 3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based colorimetric assay. MTT was purchased from Sigma. In brief, 1×104 cells per well were seeded in 96-well plates and allowed to grow for 24 h. The tested compounds were added to the wells at the indicated concentrations and the plates were incubated for an additional 48 h. Then, MTT solution (5 mg/ml) was added and the plates were incubated for an additional 4 h. The experiment was performed in triplicate and cell viability was determined by measuring the optical density at 570 nm.

Western blotting analysis

Cells were lysed in SDS sample buffer (62.5 mmol/l Tris, pH 6.8, 2% SDS, and 10% glycerol) and sonicated for 5 sec to reduce sample viscosity. Each sample was resolved by 10% SDS-PAGE, and transferred to PVDF membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were incubated with primary antibodies against phospho-CaMKII, CaMKII, phospho-MLC20, and MLC-20 (BD Biosciences, San Jose, CA) according to the manufacturer's protocol and were visualized with peroxidase and enhanced chemiluminescence (Thermo Fisher Scientific, Inc.). The phosphorylation levels of the proteins were normalized to the total protein levels. Densitometry analysis of bands was performed using ImageJ software (National Institute of Health, Bethesda, MD, USA).

Vascular tension assay

Thoracic aortas were dissected from C57BL/6 mice that had been anesthetized with isoflurane, and the fat and connective tissues were removed from the aortas. The aortas were cut into 1.5-mm rings, and endothelia were gently removed using a wooden stick. The aorta sections were then suspended between two wire stirrups (150 µm) in 10 ml Krebs-ringer solution (95% O2, 5% CO2, pH 7.4, 37°C) in a myograph (Multi myograph system DMT-620). One stirrup was connected to a three-dimensional micromanipulator, and the other was connected to a force transducer. The rings were passively stretched in 100-mg increments at 10-min intervals to reach the optimal tone (600 mg). After the arterial rings had been stretched to their optimal resting tone, the contractile response to 100 mM KCl was determined. Responses to the maximal KCl dose were used to normalize the agonist responses across vessel rings. Dose responses to the vasoconstrictor phenylephrine (PE; 10−10−10−6 M) were also determined.

Statistical analysis

Data are reported as the mean ± SD. Statistical significance was determined using the Bonferroni-corrected unpaired t test for unpaired values. A P-value <0.05 was considered statistically significance. Dose responses were analyzed using two-way or one-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test, using Graphpad prism 5.0 software.

Results

RSV shows arginase inhibitory activity

Arginase I and II solutions were prepared from liver and kidney lysates, respectively, and ability of RSV to inhibit arginase activity was measured. RSV inhibited arginase I and II activities in a dose-dependent manner (Fig. 2A and B). At 50 µmol/l of RSV, the residual activities of arginase I and arginase II were 75.1±1.8 and 82.5±5.8%, respectively. However, ABH, a known arginase inhibitor, strongly inhibited arginase I and II activity at only 10 µmol/l (residual arginase I and II activities were 48.9±1.6 and 43.1±2.6%, respectively). Because effects have preiously been observed with 20% inhibition of arginase activity (20), we used 50 µmol/l RSV for subsequent experiments. In VSMCs culture and in de-endothelialized aortic vessels of mice, 50 µmol/l RSV inhibited arginase activity 38.7±3.1 and 32.0±1.3%, respectively (Fig. 2C and D).

Figure 2. Inhibition of arginase activity by reversatrol. (A) Liver and (B) kidney lysates were prepared and used as arginase I and II enzyme solutions, respectively. Arginase activity was measured in the presence of different concentrations of RSV (P<0.01). Arginase activity was measured in (C) VSMCs and (D) de-endothelialized aortic vessels treated with 50 µmol/l of RSV and 10 µmol/l ABH, a known arginase inhibitor, for 12 h. *P<0.01 vs. untreated control. n=4 independent experiments.

Treatment with RSV increased the intracellular level of L-Arg, thereby raising the [Ca2+]c in VSMCs

Because inhibition of arginase induced CaMKII phosphorylation (22), we determined whether treatment of RSV would change the [Ca2+]c level. Both RSV and ABH treatments increased the [Ca2+]c in VSMCs as observed by microscopy (Fig. 3A; *vs. untreated, P<0.05; untreated, 60.6±10.6%; RSV 50 µmol/l, 100±2.8%) and real-time measurement (Fig. 3B; *P<0.05). Treatment with RSV also increased the intracellular L-Arg concentration in VSMCs (Fig. 3C; *vs. untreated, P<0.05; untreated, 100.0±2.8%; RSV 50 µmol/l, 128.5±9.6%). To confirm the effect of arginase inhibition on the [Ca2+]c, we examined the change in [Ca2+]c following treatment with L-Arg using flow cytometry. Incubation with L-Arg significantly increased the [Ca2+]c in a dose-dependent manner, and BAPTA-AM significantly attenuated the fluorescent signal of Fluo-4 AM (Fig. 3D; *vs. untreated, P<0.05, untreated vs. L-Arg at 10−6 mol/l, 37.0±5.0 vs. 72.0±3.5%). These results indicated that the increased intracellular L-Arg concentration, which results from arginase inhibition by RSV, plays a crucial role in the augmentation of [Ca2+]c. In addition, RSV, ABH, and L-Arg, did not show cytoxic effects on VSMCs (Fig. 3E, ns, not significant).

Figure 3. RSV-induced inhibition of arginase led to increase in [Ca2+]c in an L-Arg-dependent manner. VSMCs were pre-treated with RSV and ABH for 30 min and then stained with Fluo-4 AM to measure [Ca2+]c. Representative image (A) and real-time measurement scale bar 10 µM (B) to Fluo-4 fluorescence intensity are shown. (C) Intracellular L-Arg concentration was analyzed using HPLC and presented as a bar graph. (D) [Ca2+]c was examined in cells that were pre-treated with L-Arg (30 min) by FACS analysis. BAPTA-AM (5 µmol/l, 30 min) was used as an intracellular Ca2+ chelating agent. (E) Cell viability was measured using the MTT assay after cells were treated with various agents for 24 h. *P<0.05 vs. untreated control. ns, not significant, n=4 independent experiments.

CaMKII activation by RSV associated with MLC20 phosphorylation

Because RSV increased the [Ca2+]c by an L-Arg-dependent mechanism, we examined whether RSV may activate CaMKII. Indeed, 50 µmol/l RSV induced CaMKII phosphorylation at Thr286 (Fig. 4A; *vs. untreated, 1.0±0.1 vs. 3.5±0.2 AU, P<0.05), which associated with phosphorylation of MLC20 (Fig. 4B, *vs. untreated, 1.0±0.2 vs. 3.6±0.3 AU, P<0.05; Fig. 4C, *vs. untreated, 1.0±0.1 vs. 2.2±0.2 AU, P<0.05; #vs. RSV, 1.8±0.1 vs. 2.2±0.2 AU, P<0.01). Consistently, incubation of VSMCs with L-Arg also increased the phosphorylation of CaMKII Thr286 (Fig. 4D, *L-Arg (1 mmol/l) vs. untreated, 1.6±0.2 vs. 1.0±0.1 AU, P<0.05; #vs. L-Arg (1 mmol/l), 0.57±0.1 vs. 1.6±0.2 AU, P<0.01) and MLC20 (Fig. 4E; *L-Arg (1 mmol/l) vs. untreated, 1.6±0.2 vs. 1.0±0.01 AU, P<0.05). We also tested whether the effect of RSV may be derived from activation of Sirt proteins by treating in VSMCs with RSV and sirtinol, an inhibitor of Sirt proteins, and we found that the RSV-induced changes in CaMKII and MLC20 phosphorylation levels were not influenced by sirtinol (Fig. 4F; phospho-CaMKII, *vs. untreated control, 1.7±0.2 vs. 1.0±0.1 AU, P<0.01, #vs. RSV, 1.7±0.2 vs. 1.5±0.15 AU, not significant; phospho-MLC20, *vs. untreated control, 1.8±0.1 vs. 1.0±0.01 AU, P<0.01, #vs. RSV, 1.8±0.1 vs. 1.8±0.1 AU, not significant).

Figure 4. RSV and L-Arg induced MLC-20 phosphorylation. Western blots of CaMKII activation (A) and MLC-20 phosphorylation (B) in lysates of VSMCs that were preincubated with RSV for 30 min. MLC-20 phosphoryaltion was also tested in the presence of KN93 (10 µmol/l, 30 min), an inhibitor of CaMKII. (C) Different concentrations of L-Arg were incubated with VSMCs for 30 min, and CaMKII activation (D) and MLC-20 phosphorylation (E) were analyzed. BAPTA-AM (5 µmol/l, 30 min) was used as a control. The data in the bar graphs represent the results of 4 independent experiments. *vs. untreated control, P<0.05. #vs. RSV, P<0.01. (F) VSMCs were pre-incubated with the Sirt inhibitor sirtinol for 30 min, and the effect of RSV on CaMKII-dependent MLC20 phosphorylation was examined. *P<0.01 vs. untreated control. n=4 independent experiments. AU, arbitrary unit.

RSV augmented vessel constriction in de-endothelialized aortas

Because RSV treatment increased MLC20 phosphorylation in VSMCs, we measured the effect of RSV on vascular tension using de-endothelialized aortas of wild-type (WT) mice. The de-endothelialized aortic vessels did not respond to the endothelium-dependent vasorelaxant acetylcholine (Ach, Fig. 5A). However, dose responses to PE (Fig. 5B), an endothelium-independent vasoconstrictor, were enhanced in RSV-treated vessels (Emax, WT + de-endo vs. WT + RSV + de-endo, Emax, 179.2±6.6 vs. 253.4±9.8%, logEC50, −7.3±0.1 vs. −7.2±0.1 mol/l, *P<0.05), and preincubation with 10 µmol/l of sirtinol did not change the RSV-induced effects (Emax, WT + RSV vs. WT + RSV + sitinol, Emax, 253.4±9.8 vs. 275.4±9.7%, logEC50,-7.2±0.1 vs. −7.2±0.1 mol/l, not significant). Interestingly, treatment of de-endothelialized aortic vessels of WT mice with L-Arg induced vasoconstriction [Fig. 5C; L-Arg (10−5 mol/l) vs. PE-preconstriction, 86.6±12.0 vs. 100±0.1%; L-Arg (10−4 mol/l) vs. PE-preconstriction, 79.4±13.2 vs. 100±0.1%, *P<0.05]. However, there was no significant difference in the dose responses to the nitric oxide (NO) donor sodium nitroprusside (SNP) among the treatment groups (Fig. 5D). Additionally, the vasoconstrictive effect of RSV did not differ upon short-term incubation (4 h) and long-term incubation (16 h).

Figure 5. RSV increased vasoconstrictive activity in de-endothelialized vessels. De-endothelialized aortic vessels from WT mice (A) were incubated with RSV (50 µmol/l, 16 h), and contractile responses to phenylephrine (PE) were analyzed (B). *P<0.05. n=8 vessels from 3 mice. (C) De-endothelialized aortic rings were pre-constrited with PE (10−6 mol/l) for 10 min and then treated with different concentration of L-Arg for 1 h. The final responses were showed. *P<0.05 vs. untreated control, n=16 rings from 8 mice. (D) Endothelium-independent relaxation responses were measured with SNP treatment. n=9 rings from 4 mice.

Discussion

In this study, we demonstrated that RSV at relatively higher concentrations inhibited arginase I and II activities and consequently increased the concentration of the substrate, L-Arg. Interestingly, RSV also increased [Ca2+]c in VSMCs in a manner dependent on the L-Arg concentration. The increased [Ca2+]c led to CaMKII-dependent MLC20 phosphorylation, which was mimicked with L-Arg stimulation in VSMCs. These RSV-induced effects were maintained when VSMCs were treated with sirtinol, an inhibitor of Sirt proteins. Moreover, in a vascular tension assay with de-endothelialized aortic vessels, vasoconstrictor responses to PE were significantly enhanced in both RSV- and L-Arg-treated vessels.

An increase in the level of L-Arg, which results from arginase inhibition, is beneficial because L-Arg augments endothelial NO production, which regulates vasoreactivity, platelet activation, adhesion molecules expression, monocytes infiltration into the intima, apoptosis of endothelial cells, and proliferation and migration of VSMCs. We previously reported that inhibition of arginase II suppresses proliferation of VSMCs, which were stimulated with native low-density lipoprotein, through NADPH oxidase inactivation (23) and interleukin-8 production through p38 MAPK inactivation (6). However, L-Arg supplementation exhibited harmful effects for phathophysiological conditions, such as diabetic nephropathy (24), hypertension (25), and peripheral artery disease (26). Consistent with these findings, in this study, we found that arginase inhibition with RSV led to vessels constriction, which may cause aggravation of a pathological condition in vasculature with endothelial dysfunction.

RSV, a polyphenol molecule containing two phenyl rings connected by a methylene bridge (Fig. 1), was the first compound discovered that could mimic calorie restriction that occurs when sirtuins are stimulated (27,28). Treatment of RSV was shown to improves the general health of mice that were fed high-calorie diet (29); these mice showed a marked reduction in the signs of aging, including reduced albuminuria and cataracts formation, decreased inflammation and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, and preserved bone mineral density (30). Furthermore, RSV has been shown to have several effects on VSMCs (16–18), however, these studies used various RSV concentrations and found different concentration-dependent effects. In one study, at a low dose of RSV, VSMCs differentiation was found to be induced by SirT1-dependent Akt2 activation, whereas a high dose of RSV stimulated AMPK, which inhibited the mTORC1 pathway, thereby relieving the S6K1 inhibition and allowing Akt activation and the induction of differentiation (18). Although RSV can induce a multiple signaling cascades, the molecular target of RSV at a high dose has not been clearly demonstrated to date. Here, we demonstrate that arginase II, which regulates the mTORC1/S6K1 cascade and AMPK pathway, may be a target of RSV at a high concentration (31).

In VSMCs, [Ca2+]c is required to maintain the basal vascular tone, which is modulated by Ca2+ release from the intracellular stores in the sarcoplasmic reticulum and mitochondria and by Ca2+ entry from the extracellular space through Ca2+ channels in the plasma membrane. We found that RSV increased the [Ca2+]c and the L-Arg level by inhibiting arginase (Fig. 3). Previously, RSV was associated with intracellular Ca2+ signaling because of the direct rise in [Ca2+]c (32–34), which was proposed to originate from the intracellular Ca2+ store (33). Interestingly, the endoplasmic reticulum (ER) Ca2+ content in prostate cancer cell lines PC3 and DU145 decreased upon treatment with RSV (35). Collectively, these findings suggest that RSV increases in [Ca2+]c through activation of the IP3 receptor in the ER.

Polyphenols, such as RSV (3,4′,5-trihydroxystilbene) and piceatannol (3,3′,4′,5-tetrahydroxystilbene, a hydroxylated analogue of RSV), are secondary metabolites present in many fruits, vegetables, and medicinal plants. Numerous epidemiological and experimental studies strongly indicate their potential in the treatment of chronic diseases, including vascular and cardiac diseases, obesity, diabetes, and cancer. The positive effects of polyphenols are partly due to increase in NO production (36). In our previous studies, piceatannol inhibited the activity of arginase isoforms and reciprocally regulated the NO production by endothelial NO synthase (37). Because piceatannol showed stronger arginase inhibitory activity than RSV, it may be useful to compare two polyphenols for structure-based drug design. RSV is generally accepted as a beneficial natural product to treat cardiovascular disorders because increases production of NO, which is a vasoprotective molecule. The maximal concentration of RSV in commercial red wine was reported to be 11.2 mg/l (37), which is equivalent to 50 µmol/l. However, patients with vascular disorders, such as hypertension, should be careful when taking RSV because RSV may exacerbate blood pressure as shown in this study. However, in healthy obese man, 150 mg/day of a nutraceutical formulation RSV has been shown to have beneficial effects, which mimic the effects of calorie restriction (38).

In conclusion, RSV at a high concentration inhibited arginase activity and increased the L-Arg level leading to enhanced Ca2+ signaling by the activation of CaMKII in VSMCs. In turn, RSV-dependent CaMKII activation elicited MLC20 phosphorylation that augmented vasoconstriction in de-endothelialized vessels; however, Sirt protein activation by RSV was not involved in the Ca2+-dependent constriction. Therefore, although arginase inhibition has shown beneficial effects for various diseases, care is required when considering the administration of an arginase inhibitor to patients with vessels with endothelial dysfunction.

Acknowledgements

The authors would like to thank the Central Laboratory of Kangwon National University (Chuncheon, Korea) for technical assistance with the instruments.

Funding

This study was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (grant nos. 2015M3A9B6066968, 2016M3A9B6903185 and 2018R1D1A1B07047959), and by a 2017 Research Grant from Kangwon National University (grant no. 2017-S.R).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

CIC, BHK, and DH performed the experiments. HJK, KLH, MHW, and YMK analyzed the data and wrote the manuscript. HKL and SR designed the study and wrote the manuscript.

Ethics approval and consent to participate

All experiments were performed with approval of the Ethics Committee of Kangwon National University (Chuncheon, Korea).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References