key: cord-0805011-x1qrlcxc authors: Garate-Carrillo, Alejandra; Navarrete-Yañez, Viridiana; Ortiz-Vilchis, Pilar; Guevara, Gustavo; Castillo, Carmen; Mendoza-Lorenzo, Patricia; Ceballos, Guillermo; Ortiz-Flores, Miguel; Najera, Nayelli; Bustamante-Pozo, Moises Muratt; Rubio-Gayosso, Ivan; Villarreal, Francisco; Ramirez-Sanchez, Israel title: Arginase inhibition by (−)-Epicatechin reverses endothelial cell aging date: 2020-08-11 journal: Eur J Pharmacol DOI: 10.1016/j.ejphar.2020.173442 sha: 5d80ef2e868ddabcd7145a09a885a680945bad26 doc_id: 805011 cord_uid: x1qrlcxc Endothelial dysfunction (EnD) occurs with aging and endothelial nitric oxide (NO) production by NO synthase (NOS) can be impaired. Low NO levels have been linked to increased arginase (Ar) activity as Ar competes with NOS for L-arginine. The inhibition of Ar activity can reverse EnD and (−)-epicatechin (Epi) inhibits myocardial Ar activity. In this study, through in silico modeling we demonstrate that Epi interacts with Ar similarly to its inhibitor Norvaline (Norv). Using in vitro and in vivo models of aging, we examined Epi and Norv-inhibition of Ar activity and its endothelium-protective effects. Bovine coronary artery endothelial cells (BCAEC) were treated with Norv (10 μM), Epi (1 μM) or the combination (Epi + Norv) for 48 h. Ar activity increased in aged BCAEC, with decreased NO generation. Treatment decreased Ar activity to levels seen in young cells. Epi and Epi + Norv decreased nitrosylated Ar levels by ∼25% in aged cells with lower oxidative stress (∼25%) (dihydroethidium) levels. In aged cells, Epi and Epi + Norv restored the eNOS monomer/dimer ratio, protein expression levels and NO production to those of young cells. Furthermore, using 18 month old rats 15 days of treatment with either Epi (1 mg/kg), Norv (10 mg/kg) or combo, decreased hypertension and improved aorta vasorelaxation to acetylcholine, blood NO levels and tetra/dihydribiopterin ratios in cultured rat aortic endothelial cells. In conclusion, results provide evidence that inhibiting Ar with Epi reverses aged-related loss of eNOS function and improves vascular function through the modulation of Ar and eNOS protein levels and activity. Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide (Benjamin et al., 2019) . Aging is one the most important predictors of CVD as major age-related changes occur in the cardiovascular system (Benjamin et al., 2019; Zhou et al., 2018) . CVD in aging is associated to endothelial dysfunction (EnD) (O'Rourke and Nichols, 2005; Santhanam et al., 2008; Ungvari et al., 2019) which may be partly reversed through increased bioavailability of nitric oxide (NO) . EnD is also a hallmark of diseases such as atherosclerosis, hypertension and diabetes and may be an important pathophysiological element of COVID-19 disease (Chen and Li, 2020; Guzik et al., 2020; Whyte et al., 2020) . Preclinical and clinical evidence shows that vasodilation is importantly mediated by endothelium-dependent NO (Crecelius et al., 2011; Diehl et al., 2011; Donato et al., 2011; Manicam et al., 2017) . NO is produced by endothelial NO synthase (eNOS), through oxidation of its substrate L-arginine, under the action of receptor agonists or stimulated mechanoreceptors (Iring et al., 2019; Lu and Kassab, 2015) . Studies have demonstrated that aging associated oxidative stress (OS) induces abnormal elevation of arginase (Ar) activity (Berkowitz et al., 2003; Sakai et al., 2004) , contributes to eNOS uncoupling, impairs expression and/or eNOS activity (Höhn et al., 2017; Passos et al., 2010) therefore promoting EnD (Jae et al., 2009; Shin et al., 2012) . Tetrahydrobiopterin (BH4), an essential cofactor for eNOS dimerization, protein stability, and NO synthesis (Alp and Channon, 2004) decreases in the presence of high OS levels, inducing NOS-uncoupling which triggers the switching from NO to superoxide (O 2 *-dimensional structures were downloaded from Chem Spider (www.chemspider.com) and saved in protein data bank (pdb) format using Discovery Studio. Polar hydrogen atoms were added, the number of torsions was set and Gasteiger charges were assigned using Autodock tools (ADT) 1.5.4. Docking analysis was performed in AutoDock Vina. A blind docking method was used with the coordinate of origin set at x = 11.946, y = 20.979 and z = 0.033, at the centre of the protein. The box size was set at x = 70, y = 70 and z = 70. Docking simulations to analyze binding affinities and binding sites were run with the number of modes set to 8. The Discovery Studio was used to produce two-dimensional docking representations of the interactions. To evaluate changes in Norv or Epi's free energy (∆G, kcal/mol), as well as amino acid interactions with Epi, a pdbqt file was created using PyMolwin software. The docking was performed in AutoDock Vina, with the coordinate of origin at x = 2.017, y = − 7.457 and z = − 0.116. The box size was set at x = 70, y = 70 and z = 70 (Ortiz-vilchis et al., 2018) . BCAEC were grown in a complete medium supplemented with 10% FBS, 1% antibiotic/antimycotic solution, and 1% nonessential amino acids. Cells were maintained under a humidified atmosphere at 37 °C with 5% CO 2 and 95% O 2 . As per our previous publication , passages 8-13 were used as a model for young endothelial cells (Y) while passages 31-35 were used as a model for aged endothelial cells (A). Cells were used for experiments at 75% confluence. For all experiments 24 h before treatment, growth medium was replaced with 1% serum medium, phenol red free, 1% antibiotic/antimycotic solution and 1% nonessential amino acids (starving medium). Treatment was provided as follows: vehicle applied to the cells in the control J o u r n a l P r e -p r o o f group (C), Norv (N) 10 µM, Epi (E) 1 µM, or both Epi+Norv (E+N) for 48 h. Fresh starving medium and compounds were reapplied every 24 h. Cells were washed three times with cold buffer (4 ml per plate) and lysed in 80 μl of ice cold lysis buffer (RIPA ThermoFisher Scientific) with protease and phosphatase inhibitor cocktails (Sigma-Aldrich). Homogenates were sonicated for 15 min at 4 °C, and centrifuged at 13 000g for 15 min to remove cell debris. The total protein concentration was measured in the supernatant using the Bradford micro method (Bio-Rad) at 595nm D.O. using a BioQuant 800 spectrophotometer (BioTek Inc.). After treatment, cells were homogenized in 100 µl of solution A (sucrose 2 M, EDTA 0.01 M, HEPES 0.5 M; pH 7.4) and samples centrifuged for 10 min (12000 g) at 4°C. The supernatant was collected and protein concentration on it was determined using the Bradford method. To evaluate Ar activity, 100 μg of protein were added to 25 mM Tris-HCl pH 7.4 and 5 mM MnCl 2 (100 μl final volume) and activated at 55°C for 10 min. The enzymatic reaction was started by the addition of 100 μl of 0.5M L-arginine as substrate (pH 9.7). The mixture reaction was incubated at 37°C for 60 min and stopped by the addition of 200 μl of acidic mixture containing H 2 SO 4 , H 3 PO 4 and H 2 O (1:3:7) followed by the addition of 25 μl of 9% α isonitrosopropiophenone then heated at 100 °C for 45 min. The urea concentration was quantified by measuring O.D. at 540 nm in a BioQuant spectrophotometer (Biotek, Inc). Ar activity levels were calculated by extrapolating the data onto urea standard curve (Ortiz-vilchis et al., 2018) . J o u r n a l P r e -p r o o f After 48 h of treatment with compounds, 100 μl of medium was collected to test NO levels using a fluorescent kit according to the manufacturer's instructions . Briefly, as the final products of NO in vivo are nitrite (NO 2 −) and nitrate (NO 3 −), the best estimation of total NO production is the sum of both NO 2 − and NO 3 −. We used a nitrate/nitrite fluorometric assay as an accurate method for the measurement of the total nitrate/nitrite concentration. In the assay, the conversion of nitrate to nitrite utilizes nitrate reductase followed by the addition of an acidic solution of diaminonaphthalene (DAN) and NaOH, which enhances the detection of the fluorescent product, 1(H)-naphthotriazole. Measurement of the fluorescence generated was quantified using a fluorometer (FLx800, Bio-Tek Instruments). Nitrate/nitrite values were normalized against the total protein after the cells were scrapped from the plate and lysed as described previously. Plasma (50 μl) was collected from treated and control animals and NO levels were measured as above. DHE was evaluated as a surrogate of reactive oxygen species (ROS) production in live BCAEC. DHE was used as a fluorescent probe for the detection of superoxide and hydrogen peroxide. Antimycin A, an inhibitor of complex III of the mitochondrial electron transport chain, was included as a positive control for ROS generation. N-acetyl Cysteine was included as an antioxidant control. Breifly, cells were seeded in a 96 well plate at a density of 50 cells/well. After 48 h treatment with compounds cells were washed with PBS and added 150 μl of Cell-Based Assay Buffer and incubated for 10 min at room temp. Cell-Based Assay Buffer, was removed and 20 μl of fresh assay buffer and 130 μl of ROS Staining Buffer per well were added. Then 10 μl of N-acetyl cysteine assay reagent were added to designated negative control wells and incubated for 30 min at 37°C protected from light. Following, we added 10 μl of the Antimycin A reagent to positive control wells and incubated for 1 h at 37°C protected from light. Carefully we J o u r n a l P r e -p r o o f aspirated ROS Staining Buffer and added 100 μl of Cell-Based Assay Buffer. Plate was placed on fluorometer (FLx800, Bio-Tek Instruments) plate reader and fluorescence was measured at an excitation wavelength between 480-520 nm and an emission wavelength between 570-600 nm. ROS generation data was represented as total DHE fluorescence units. BH4 and BH2 were measured separately using competitive ELISA kits from Novus Biologicals. The micro ELISA plate provided in the kit is pre-coated with either BH4 or BH2. Breifly, cells were washed with cool PBS and dissociated with trypsin. Cell suspension was collected and centrifuged for 5 min at 1000 g. Cell pellet was washed 3 times with cool PBS and finally resuspended with 250 μl of cool PBS. Cell suspention was sonicate 15 min at 4°C and centrifuged for 10min at 1500 g at 4°C. The supernatant was collected to carry out the assay according to manufacturer instructions. During the reaction, BH4 or BH2 in the sample or In order to examine protein levels of Ar I and II, and nitrosylated form, eNOS, phospho (p) eNOS, and related proteins (i.e. GTP cyclohydrolase 1 [GCH1]), immunoblotting was J o u r n a l P r e -p r o o f performed. Cells were treated as described above. After treatment, cells were lysed and total protein was extracted. A total of 30 μg of protein was loaded onto a 4-15% gel, electrotransferred, and incubated for 1 h followed by either 1-3 h incubation at room temperature or overnight incubation at 4 °C with primary antibodies. Primary antibodies were typically diluted 1:1000 or 1:2000 in buffer plus 5% bovine serum albumin or 2% non-fat milk. Membranes were washed (3X for 5 min) in buffer and incubated 1 h at room temperature in the presence of conjugated secondary antibodies diluted 1:5000 in a blocking solution. Membranes were again washed three times in buffer and immunoblots were developed. Band intensities were digitally quantified and normalized against GAPDH. After treatment, endothelial cells were lysed with 200 µL of nondenaturing extraction buffer (0.5%, Triton X-100, 50 mmol/l Tris-HCl, pH: 7.4, 0.15 mol/l NaCl, 0.5 mmol/l EDTA) and supplemented with protease and phosphatase inhibitor cocktails plus 2 mmol/l Na 3 VO 4 , and 1 mmol/l NaF. Homogenates were passed through an insulin syringe 3X and incubated on ice with shaking for 25 min and centrifuged at 12,000 g for 15 min at 4°C. A total of 0.5 mg protein was precleared by adding 1 mg of normal rabbit IgG control and 20 µl protein A/G-agarose and mixed for 30 min (4° C) with subsequent centrifugation at 12,000 g for 10 min at 4° C. The supernatant was recovered and incubated at 4° C under mild agitation for 3 h with 10 µl of IP antibody (rabbit nitrotyrosine). Twenty microliters of protein A/G-agarose was added, and the mixture was incubated overnight at 4° C with shaking. The IP mixture was centrifuged at 12,000 g for 15 min at 4° C, and the supernatant was recovered and stored at 4 °C for later analysis. The pellet was washed 3X with extraction buffer under shaking 15 min and centrifuged at 12,000 g for 15 min at 4° C. The IP proteins in the pellet and those remaining in the supernatant J o u r n a l P r e -p r o o f were applied to a precast 4-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for Westerns using Ar I and II antibodies. Low-temperature SDS-PAGE was performed for detection of the eNOS monomer and dimer (Benson et al., 2013) . Briefly, cells lysates were prepared as described above. Protein lysates were resolved using a 7.5% Tris-glycine gel (Bio-Rad) under reducing conditions. All gels and buffers were at 4 °C before electrophoresis, and the buffer tank was placed in an ice bath during electrophoresis to maintain the gel temperature below 15 °C. Western blots were developed as described above with rabbit anti-eNOS polyclonal antibody. J o u r n a l P r e -p r o o f 2.14 Blood pressure A non-invasive method to measure systolic blood pressure was employed by using a rat tail cuff pressure transducer with values digitally recorded. Under anesthesia (pentobarbital 60 mg/kg) animals were decapitated and the thoracic aorta from the diaphragm to the aortic arch isolated. Aortas were immediately submerged in cold Krebs solution to remove all adjacent connective tissue, then cut into ring segments (4-5 mm long), which were then mounted on two stainless steel hooks within an isolated organ chamber. One of the hooks was fixed to the bottom of the chamber and the other to a transducer linked to a Biopac System apparatus for registering changes in tension (force). The isolated organ chamber contained 10 ml of Krebs bicarbonate solution. The chamber was maintained at a constant temperature of 37°C, pH of 7.4 and a continuous bubbling with a mixture of 95% O 2 and 5% CO 2 . Aorta rings were pre-contracted using phenylephrine [1 * 10 −6 ], and standard concentration-response curves to acetylcholine [1 * 10 −7 -1 * 10 −4 M] were constructed to analyze the effect of 15 days of Norv, Epi or both E+N treatment on the vascular reactivity of the pre-contracted aortas. Aged rat aorta endothelial cells were isolated and cultured in order to evaluate arginase activity, BH2 and BH4 assays. Briefly, a large section of thoracic aorta was collected from 6 aged rats/group. Lumen of the vessels was exposed, gently washed with Hank's solution and incubated with 0.05% trypsin for 10 min at 37ºC in a petri dish; the total volume of the trypsin solution was recovered, and a 0.1 volumes FBS was added. Cells were harvested and centrifuged at 1200 x g /10 min and the pellet was resuspended in DMEM supplemented with 10% FBS, 2mM L-glutamine, 1% antibiotic-antimycotic mixture, 1% non-esential aminoacids J o u r n a l P r e -p r o o f and 5 mg/ml brain bovine endothelial mitogen (Ramirez-Sanchez et al., 2010) . Endothelial cells were incubated at 37ºC and 5% CO 2 , and four days latter used for Ar activity, BH2 and BH4 assays. The docking analysis of Norv and Epi interaction with Arg 1 documents interactions which occur at different sites. L-Norv interacts in its active pocket with an affinity of − 4.9 kcal/mol and its binding is provided by conventional hydrogen bonds principally (complex 1). Epi was able to interact with the allosteric site with an affinity of − 8.1 kcal/mol by van der Waals interactions predominantly (complex 2). (Fig.1 and Table 1 ). The analysis of Epi binding possibilities when Norv is present (complex 1) shows changes in binding affinity (ΔG = −7.4) while Norv binding analysis when Epi is present (complex 2) shows no changes in binding affinity (ΔG = − 4.4). Additionally, these interactions lead to changes in binding amino acids suggesting conformational changes in Ar. Ar I and Ar II expression and activity were measured in young (Y) and aged (A) BCAEC treated with vehicle (C), Norv (N), Epi (E) or a combination of Norv plus Epi (E+N) (Fig 2) . Ar I ( Fig. 2A) and Ar II (Fig. 2B ) protein levels did not change in cells treated with Norv, Epi or both vs YC or AC. Ar activity, as urea content, was decreased in YN, YE and YE+N (P<0.05). Urea content also was increased (~110%) in AC vs. YC (P<0.05), while AN, AE and AE+N were able to decrease Ar activity almost to YC levels (P<0.05) (Fig. 2C) . Ar activity/Ar protein levels (ratio) was also decreased in Y or A cells treated with Norv, Epi or both compounds (P<0.05) (Fig. 2D ). Nitrosylation of Ar I and Ar II as an indicator of enzyme oxidation/activation was measured in young and aged BCAEC treated with Norv, Epi and E+N. In both enzyme isoforms, nitrosylation J o u r n a l P r e -p r o o f was increased by ~100% in AC vs YC (P<0.05) (Fig. 3A-C) . Interestingly, AE and A E+N decreased nitrosylated Ar I and Ar II levels more than 50% vs AC (P<0.05). Similarly, DHE as a surrogate of ROS production, was significantly increased in AC (~40%) vs YC (P<0.05) (Fig. 3D ). Treatment with Epi and E+N significantly decreased DHE levels in ~25% vs AC (P<0.05) almost as a YC levels. To address eNOS proper coupling, dimerization levels were measured (Fig. 4A-B) . Of note is that YE and YE+N groups increased dimerization (~80% and ~120% respectively) vs YC (P<0.05). AC decreased dimerization of eNOS, while AE and AE+N demonstrated increased dimerization by ~40 and ~50% respectively (P<0.05). Results demonstrated that Epi and Epi+Norv treatments of aged cells can restore eNOS dimerization levels similar to YC. Additionally, the BH4/BH2 ratio does not shown differences between Y groups (Fig. 4C ). However, in AC BH4:BH2 ratio significantly decreased (~40%) vs YC (P<0.05) (Fig. 4C) , and were restored in AE and AE+N almost as YC levels. Norv, per se, was unable to produce any effects on cells. GCH1, the protein related to BH4 biosynthesis, levels did not show any changes between either Y or A groups (Fig. 4D) . NO production and eNOS activation/phosphorylation was measured in Y and A cells treated with vehicle, Norv, Epi and E+N (Fig. 5) . NO levels were increased in YN, YE and Y E+N treatments vs YC (P<0.05), while AC decreased NO production by 50% vs YC (P<0.05). NO levels in AN, AE and AE+N were elevated as compared vs. AC (P<0.05) (Fig. 5A) . eNOS protein levels increased in YE and Y E+N (~20% and ~25% respectively) vs. YC (P<0.05), while AC decreased protein levels by ~25% vs YC (P<0.05). AE and AE+N groups were able to recover eNOS protein levels as YC (Fig 5B) . eNOS activation (phosphorylation at Serine 1179), J o u r n a l P r e -p r o o f was significantly increased in YN (~40%), YE (~120%), YE+N (~155%) as compared vs YC, (P<0.05) while AC eNOS phosphorylation was decreased by ~55% and increased in AN (~100%), AE (~175%) and AE+N (~185%) (P<0.05) (Fig. 5B, C) . Fig. 5D reports on the phospho eNOS/total eNOS ratio supporting the results in Fig. 5B and C. AC rats demonstrated high blood pressure. However, in AN, AE and AE+N groups blood pressure decreased by ~20%, 30% and 40% respectively (P<0.05) (Fig. 6A) . NO (Nitrite/Nitrates) production was significally decreased in AC rats and increased in AN, AE and AE+N groups between 25 to 70% (P<0.05) (Fig. 6B ). BH4, BH2 and Ar activity levels were evaluated in cultured aortic endothelium from aged rats. BH4:BH2 ratio was significantly decreased in AC, while increased in AE and AE+N ~75% to 100%) (P<0.05) (Fig. 6C ). Endothelial cells from AC animals demonstrated increased Ar activity and a significant decrease was observed in AN, AE (~45%) and E+N (~75%) treatments (P<0.05) (Fig. 6D ). In aortic rings isolated from AC animals, a vasodilatory effect of acetylcholine was documented at concentrations ranging from 10 −7 to 10 −4 mol L −1 . The calculated ED 50 in AC animals was (6.5*10 −7 mol L −1 ). An enhanced vasodilatory response was triggered in the AN (ED 50 1.9*10 −7 mol L −1 ), AE (ED 50 2.1*10 −8 mol L −1 ) and AE+N (ED 50 5.1*10 −9 mol L−1) (P<0.05) treated groups; suggesting an increased vasodilatory effect in the aortic rings, reaching a maximal level of relaxation of ~ 0.4 g in AC vs. ~ 0.75 g in AN group, ~1.0 g in AE group and ~ 1.45 g in the aortas from AE+N rats (P<0.05). Results of vascular reactivity measurements analyzed by area J o u r n a l P r e -p r o o f under the curve (AUC) calculations demonstrated that AN, AE and AE+N treated groups induced significantly more vasodilation of aortas (~55%, 100% and 220% respectively) vs. AC aortas (P<0.05) (Fig. 6E-F ). Unique results from this study demonstrate that Epi has the capacity to bind Ar and inhibit its activity. In aged endothelium, Ar activity is increased, yielding a reduction in NO bioavailability which, is associated with decreased eNOS phosphorylation at activation sites (Ser1179) and higher levels of protein uncoupling (