INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 31: 803-810, 2013 Arginase inhibition by piceatannol-3'-O-β-D-glucopyranoside improves endothelial dysfunction via activation of endothelial nitric oxide synthase in ApoE-null mice fed a high-cholesterol diet AINIENG WOO1*, WOOSUNG SHIN1*, TO DAO CUONG3, BYUNGSUN MIN3, JEONG HYUNG LEE2, BYUNG HWA JEON4 and SUNGWOO RYOO1 Departments of 1Biology, 2Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701; 3College of Pharmacy, Catholic University, Daegu 712-702; 4Department of Physiology, School of Medicine, Chungnam National University, Daejeon 301-131, Republic of Korea Received October 31, 2012; Accepted December 28, 2012 DOI: 10.3892/ijmm.2013.1261 Abstract. Elevated plasma cholesterol is a hallmark of numerous incubation neither altered the contractile response to a high K+ cardiovascular diseases that are closely linked to endothelial solution nor the relaxation response to SNP. When analyzing dysfunction indicating decreased nitric oxide (NO) production the L-arginine content using high-performance liquid in the endothelium. It has been previously demonstrated that chromatography, PG incubation increased the intracellular piceatannol-3'-O-β-D-glucopyranoside (PG) inhibits arginase L-arginine concentration. PG administration in the drinking activity and reciprocally regulates NO production. Here, we water significantly reduced fatty streak formation in ApoE-/- aimed to ascertain whether PG ameliorates vascular function mice fed an HCD. These data indicate that PG improves the in wild-type (WT) and atherogenic model mice [apolipo- pathophysiology of cholesterol-mediated endothelial dysfunc- protein E-null mice (ApoE-/-)] and to investigate the possible tion. Therefore, we conclude that the development of PG as a underlying mechanism. Preincubation of aortic vessels from novel effective therapy for preventing atherosclerotic diseases WT mice fed a normal diet (ND) with PG attenuated vaso- is warranted. constriction response to U46619 and phenylephrine (PE), while the vasorelaxant response to acetylcholine (Ach) was Introduction markedly enhanced in an endothelium-dependent manner. However, the endothelium-independent NO donor, sodium Elevated plasma cholesterol, such as native low-density nitroprusside (SNP), did not change vessel reactivity. In lipoprotein (LDL) and oxidized LDL (oxLDL), is a hallmark thoracic aorta from ApoE-/- mice, a high-cholesterol diet of numerous cardiovascular diseases including hypercholes- (HCD) induced an increase in arginase activity, a decrease in terolemia, atherosclerosis, hypertension, heart failure, and NO release and an increase in reactive oxygen species genera- diabetes. These diseases are closely linked with endothelial tion that was reversed by treatment with PG. The effect of PG dysfunction indicating decreased nitric oxide (NO) production was associated with enhanced stability of the eNOS dimer and in the endothelium. In the vasculature, NO is a vasoprotective was not dependent on the expression levels of arginase II and molecule and plays a central role in vascular homeostasis by eNOS proteins, although eNOS expression was increased in regulating vasoreactivity, platelet activation, leukocyte adhe- ApoE-/- mice fed an HCD. Furthermore, PG treatment attenu- sion and smooth muscle cell migration and proliferation (1). ated the PE-dependent contractile response, and significantly It is well established that endothelial arginase constrains improved the Ach-dependent vasorelaxation response in aortic the activity of endothelial nitric oxide synthase (eNOS) by rings from ApoE-/- mice fed an HCD. On the other hand, PG substrate depletion, thereby reducing NO bioavailability and contributing to vascular diseases. oxLDL, the primary patho- genic lipid in atherogenesis, activates human endothelial cell arginase II by stimulating the dissociation of arginase II from Correspondence to: Professor Sungwoo Ryoo, Department of microtubules and also by inducing arginase II mRNA tran- Biology, Kangwon National University, Kangwondae-gil 1, Chuncheon, scription (2). Furthermore, atherogenic-prone apolipoprotein Kangwon-do 200-701, Republic of Korea E-null (ApoE-/-) mice treated with an arginase inhibitor exhibit E-mail: [email protected] restored NO bioavailability and endothelial function, reactive *Contributed equally oxygen species (ROS) production, and an arterial compliance similar to that observed in wild-type (WT) mice (3). Therefore, Key words: arginase, piceatannol-3'-O-β-D-glucopyranoside, endo- endothelial arginase may be a novel target for therapeutic drug thelial dysfunction, endothelial nitric oxide synthase, apolipoprotein design for vascular diseases such as atherosclerosis (3). E-null mice Rhubarb is the rhizome of Rheum undulatum and is commonly distributed in Asia. Many components of the 804 WOO et al: PICEATANNOL-3'-O-β-D-GLUCOPYRANOSIDE IMPROVES ENDOTHELIAL DYSFUNCTION rhizome possess diverse biological activities and have been to agonist across vessel rings. The dose response to the vaso- reported as being able to counter allergic (4) and diabetic states constrictors, PE (10-9-10 -4 M) and U46619 (10-9-10 -5 M), was (5), as having anti-oxidant properties (4), and as functioning as performed first. This was followed by the dose response to a vasorelaxant (6). Piceatannol, one of the active components the vasodilators, acetylcholine (Ach, 10-9-10 -5 M) and SNP of rhubarb, was recently found to inhibit lipooxygenase activity (10 -9-10 -5 M) after pre-constriction with PE (10-6 M). At the (7) and VSMC proliferation and migration (8). Recently, we end of the experiments, the NO-dependency of vasorelaxation reported that piceatannol-3'-O-β-D-glucopyranoside (PG) is a was confirmed by adding the inhibitor of guanylate cyclase potent inhibitor of arginase isoforms. PG inhibited the arginase [1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one (ODQ), 10-6 M)]. isoforms in a dose-dependent manner, resulting in augmented NO production by enhancing eNOS dimer stabilization (9). Arginase activity. Tissue lysates were prepared using lysis Based on these data, we hypothesized that PG regulates buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA and protease vascular function. Therefore, we examined whether PG inhibitors) by homogenization at 4˚C followed by centrifuga- improves NO/ROS production and endothelial dysfunction in tion for 20 min at 14,000 x g at 4˚C. The supernatants were ApoE-/- mice fed a high-cholesterol diet (HCD). We also inves- used to assay for arginase activity as previously described (10). tigated whether arginase inhibition by PG restores L-arginine bioavailability and attenuates fatty streak formation in this Estimation of NO or ROS generation using DAF-FM or DHE atherogenic mouse model. in isolated mice aorta. Mice aortic rings were isolated and incubated overnight at 37˚C in 5% CO2 in Dulbecco's modified Materials and methods Eagle's medium containing 2% FBS and antibiotics (1X) in the presence of PG (50 µmol/l) (10). The fluorescence from the Animals. Twenty 10-week-old male wild-type (WT) aortic endothelium was measured at different time intervals (C57BL/6J) and ApoE-/- mice (Dae Han Biolink Co.) were under microscopy (9). studied. The study was approved in accordance with the Guide for the Care and Use of Laboratory Animals (Institutional Determination of intracellular L-arginine concentrations. Review Board, Kangwon National University). The intracellular concentration of L-arginine was determined by high-performance liquid chromatography (HPLC) using Protocol. To determine the effect of PG on vascular reactivity, pre-column derivatization with o-phthalaldehyde (OPA) by we studied aortic rings isolated from 20 male C57BL/6J WT modification of a previously published method (11). L-arginine mice fed a normal diet (ND) and 20 male ApoE-/- mice fed (100 µmol/l) was added to the cell lysate (0.5 ml) as an internal an HCD (D12108C; Research Diet Inc., USA) for 6 weeks. standard. The samples were extracted on solid-phase extraction Aortic rings were incubated with or without PG (50 µmol/l) cartridges (CBA Bond Elute; Varian, Inc.). Recovery rates were for 18 h as previously described (9). For the pathological assay, 87.5±3.9%. Eluates were dried over nitrogen and resuspended PG was administered in the drinking water to ApoE-/- mice in double-distilled water for HPLC analysis. HPLC was for 6 weeks when the mice were started on the HCD. Given performed on a computer-controlled Waters chromatography that each mouse consumed ~10 ml water/day this represented system (M600E) consisting of an automatic injector (M7725i; a daily dose of ~500 µg/mouse/day of PG. Waters Co.) and a fluorescence detector (FP-1520; Jasco Co.) located in the Central Laboratory of Kangwon National Western blot analysis. Aortic vessel lysates were subjected University. Samples were incubated for exactly 1 min with OPA to SDS-PAGE, and densitometry of the bands was conducted reagent (5.4 mg/ml OPA in borate buffer, pH 8.4, containing using NIH ImageJ (9). To analyze the ratio of eNOS dimer 0.4% 2-mercaptoethanol) before automatic injection for the to monomer, proteins were separated using low-temperature HPLC. The OPA derivative of L-arginine was separated on a SDS-PAGE followed by western blot analysis (9). 150x4.6 mm, 3.5-µm Zorbax Eclipse XDB-C18 column with the fluorescence detector set at Ex 340 nm and Em 450 nm. Aortic vascular tension assay. Male C57BL/6J mice fed an Samples were eluted from the column with 0.96% citric acid/