Mammalian Arginase Inhibitory Activity of Methanolic Extracts and Isolated Compounds from Cyperus Species.

Polyphenolic enriched extracts from two species of Cyperus, Cyperus glomeratus and Cyperus thunbergii, possess mammalian arginase inhibitory capacities, with the percentage inhibition ranging from 80% to 95% at 100 µg/mL and 40% to 64% at 10 µg/mL. Phytochemical investigation of these species led to the isolation and identification of two new natural stilbene oligomers named thunbergin A-B (1-2), together with three other stilbenes, trans-resveratrol (3), trans-scirpusin A (4), trans-cyperusphenol A (6), and two flavonoids, aureusidin (5) and luteolin (7), which were isolated for the first time from C.thunbergii and C. glomeratus. Structures were established on the basis of the spectroscopic data from MS and NMR experiments. The arginase inhibitory activity of compounds 1-7 was evaluated through an in vitro arginase inhibitory assay using purified liver bovine arginase. As a result, five compounds (1, 4-7) showed significant inhibition of arginase, with IC50 values between 17.6 and 60.6 µM, in the range of those of the natural arginase inhibitor piceatannol (12.6 µM). In addition, methanolic extract from Cyperus thunbergii exhibited an endothelium and NO-dependent vasorelaxant effect on thoracic aortic rings from rats and improved endothelial dysfunction in an adjuvant-induced arthritis rat model.

Polyphenolic compounds of Euphorbia umbellata (Pax) Bruyns (Euphorbiaceae) improved endothelial dysfunction through arginase inhibition.

Euphorbia umbellata is used for its anti-inflammatory properties; however, there are limited data available regarding its effects on vascular function. Its bark is rich in polyphenolic compounds, which potentially improve endothelial dysfunction (ED). This study proposes to investigate the effects of E. umbellata bark extracts and its polyphenolic compounds on arginase (ARG) activity and nitric oxide (NO)-related targets. Chromatographic procedures were used for the chemical characterisation of the extracts. Furthermore, in silico (molecular docking), in vitro (ARG inhibition), in vivo (streptozotocin-induced hyperglycemia model), and ex vivo (l-arginine metabolism, vascular reactivity, western blot, and biochemical) techniques were carried out. Quercetin, gallic acid, and ellagic acid were identified in the extracts. In silico screening predicted that gallic acid and quercetin would have the most promising interactions with ARG -identified cavities. This was confirmed in vitro as both compounds had a direct inhibitory effect on ARG, as was the case regarding the extracts. Oral treatment preserved endothelium-dependent vasodilation through ARG inhibition together with an increase in l-arginine bioavailability and endothelial NO synthase expression. Biochemical parameters determined the lack of toxicity for sub-chronic treatment. E. umbellata bark extracts and its compounds can contribute to ED treatment, at least partly, through the inhibition of vascular ARG.

Arginase inhibitory properties of flavonoid compounds from the leaves of Mulberry (Morus alba, Moraceae).

OBJECTIVES: We aimed to isolate and identify bioactive molecules from Morus alba (Moraceae) leaves having arginase inhibitory activity towards the combat of clinical outcomes related to endothelial dysfunction. METHOD: Extraction and isolation were carried out by successive macerations, prepurification by using a Solid Phase Extraction (SPE) and separation using preparative PLC. The structures of the isolated components were established and confirmed by spectroscopic analyses, including the ESI-HRMS and NMR spectroscopic investigations. Biological evaluation was performed by using an in vitro assay with liver bovine purified arginase and by an ex vivo aortic ring study. KEY FINDINGS: We demonstrated that a phenolic extract from the leaves of M. alba possesses mammalian arginase inhibitory capacities. Investigation of the chemical constituents of its leaves results in the isolation and identification of ten compounds investigated in vitro for their arginase inhibitory capacities. Four compounds showed significant inhibition of arginase, with percentage inhibition ranging from 54% to 83% at 100 µm. In isolated rat aortic rings incubated with NO synthase inhibitor, Luteolin-7-diglucoside compound (2) was able to increase acetylcholine-induced relaxation. CONCLUSIONS: These results demonstrated the attractive ability of M. alba to be a potential source for the discovery of new active products on vascular system.

Vasorelaxant effects of Crataegus pentagyna: Links with arginase inhibition and phenolic profile.

ETHNOPHARMACOLOGICAL RELEVANCE: Crataegus leaves, flowers and fruits have been traditionally used to improve blood circulation, numerous preclinical and clinical studies supporting the cardiovascular benefits of Crataegus preparations. In this respect, there is very limited data on Crataegus pentagyna; in addition, the chemical profile of this species is still incompletely elucidated. AIM OF THE STUDY: The objective of this study was to examine the cardiovascular benefits of Crataegus pentagyna Waldst. et Kit. ex Willd. (small-flowered black hawthorn, Rosaceae) extracts (leaf, flower and fruit ethyl acetate extracts) and the underlying mechanisms. We hypothesized that C. pentagyna extracts might exert vasodilatory effects and inhibit arginase activity due, in large part, to their polyphenolic constituents. MATERIALS AND METHODS: C. pentagyna extracts induced-relaxation and the mechanisms involved were studied ex vivo in isolated aortic rings from Sprague-Dawley rats. The inhibitory effects on bovine liver arginase I were assessed by an in vitro assay. Metabolite profiling of C. pentagyna extracts was performed and the most endothelium- and nitric oxide synthase-dependent; flower extract additionally reduced Ca2+ entry and, to a lesser extent, Ca2+ release from the sarcoplasmic reticulum. C. pentagyna proved to be an important source of arginase inhibitors with potential benefits in endothelial dysfunction that remains to be explored.

Vascular Arginase Is a Relevant Target to Improve Cerebrovascular Endothelial Dysfunction in Rheumatoid Arthritis: Evidence from the Model of Adjuvant-Induced Arthritis.

Emerging data revealed that rheumatoid arthritis (RA) is associated with higher risk of cerebrovascular diseases. Whereas cerebral endothelial dysfunction is acknowledged as a critical aspect of cerebrovascular diseases, its presence in RA and the mechanisms involved are currently unknown. By using the model of rat adjuvant-induced arthritis (AIA), the present study investigated cerebrovascular reactivity in pressurized middle cerebral arteries (MCA) on day 33 post-immunization. The results revealed that arthritis induced a dramatic decrease in the vasodilatory response to acetylcholine (ACh), ADP, and bradykinin (n = 7-9 arteries, p < 0.0001). By using nor-NOHA, L-NAME, BH4, and Tempol, the results showed that the reduced response to ACh relied on arginase overactivation (n = 8), low NOS activity (n = 8), BH4 deficiency (n = 9), and excessive superoxide production (n = 9). Immunohistological analysis revealed an endothelial upregulation of arginase 2 (p < 0.05, n = 5-6) and NADPH oxidase (p < 0.05, n = 5-7) while eNOS expression was unchanged in AIA (n = 6). To assess whether arginase inhibition may be a relevant therapeutic, AIA rats were treated with an arginase inhibitor (nor-NOHA, 40 mg/kg/day, i.p., n = 20 rats) daily from day 10 to day 33 post-immunization. The treatment alleviated the impaired response of MCA to endothelium-dependent agonists, through an increase in NOS signaling and a suppression of BH4 deficiency and superoxide overproduction. By contrast, it did not change the course of arthritis. In conclusion, arthritis induced a cerebrovascular endothelial dysfunction involving an imbalance in the arginase/NOS pathway. Arginase inhibition appears as a promising therapy beyond anti-rheumatic drugs for reducing the risk of cerebrovascular diseases in RA.

Metabolite profiling, arginase inhibition and vasorelaxant activity of Cornus mas, Sorbus aucuparia and Viburnum opulus fruit extracts.

The present study investigated the effects of Cornus mas, Sorbus aucuparia and Viburnum opulus fruit extracts on arginase activity and arterial vasodilation. V. opulus fruit extract exerted the highest vasorelaxant activity in phenylephrine precontracted rat aortic rings (EC50 = 6.31 ±â€¯1.61 µg/mL) and a significant inhibition of arginase (IC50 = 71.02 ±â€¯3.06 µg/mL). By contrast, S. aucuparia and C. mas fruit extracts showed no important anti-arginase activity and a significantly weaker activity in the rat aortic rings relaxation assay (EC50 = 100.9 ±â€¯11.63 and 78.52 ±â€¯8.59 µg/mL, respectively). For all extracts, the main mechanism of vasodilation was proven to be endothelium-dependent. HPLC-ESI-Q-TOF-MS/MS studies revealed a very complex metabolite profiling in all three extracts with chlorogenic acid accounting for 30.89, 0.72 and 2.03 mg/g in V. opulus, C. mas and S. aucuparia fruit extracts, respectively. All extracts were declared non-toxic in the brine shrimp acute toxicity test. Our study highlights potential benefits of V. opulus fruit extract in diseases associated with endothelial dysfunction and impaired vasodilation.

Methotrexate did not improve endothelial function in rheumatoid arthritis: a study in rats with adjuvant-induced arthritis.

OBJECTIVES: Rheumatoid arthritis is associated with an increased cardiovascular risk, secondary to endothelial dysfunction. There is accumulating evidence that methotrexate reduces cardiovascular risk in rheumatoid arthritis, but the mechanisms involved are still unknown. In this study, we aimed to determine the effect of methotrexate on endothelial function and traditional cardiovascular risk factors in the adjuvant-induced arthritis (AIA) rat model. METHODS: On the first signs of arthritis, methotrexate (1 mg/kg/week, s.c.) or saline (Vehicle) was administered to AIA for 3 weeks. Endothelial function was studied in aortic rings relaxed with acetylcholine in the presence or not of inhibitors of nitric oxide synthase, cyclooxygenase-2, arginase, EDHF and superoxide anions production. Arthritis and radiological scores, blood pressure and blood levels of cytokines, triglycerides, cholesterol, homocysteine and BMP-4 were measured. RESULTS: Although methotrexate significantly reduced the arthritis score, it had no effect on Ach-induced relaxation. As regards mechanisms, methotrexate increased nitric oxide synthase activity and reduced the superoxide anions production but did not change arginase, cyclooxygenase-2 and EDHF pathways. Methotrexate did not change the radiological score or blood pressure, lipid, glucose and homocysteine levels. By contrast, methotrexate significantly reduced plasma IL-1ß and TNF-α levels and increased serum BMP-4 level. CONCLUSIONS: Despite a reduction of clinical and biological inflammation, methotrexate did not improve endothelial function in AIA rats. Overall data suggest that mechanisms other than the ED reduction are likely involved, and remain to be elucidated to better understand the cardiovascular benefits of methotrexate in rheumatoid arthritis.

Eulophia macrobulbon extract relaxes rat isolated pulmonary artery and protects against monocrotaline-induced pulmonary arterial hypertension.

BACKGROUND: Extract of the wild orchid, Eulophia macrobulbon (EM) inhibits phosphodiesterase5 (PDE5) suggesting it could preferentially dilate the pulmonary vasculature. PURPOSE AND STUDY DESIGN: To pharmacologically characterize the vascular actions of EM ethanolic extract and its active compound, 1-(4'-hydroxybenzyl)-4,8-dimethoxyphenanthrene-2,7-diol using isolated pulmonary arteries (PA) from rats having pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT). PA were fixed and prepared for histology. RESULTS: EM extract relaxed PA (EC50 = 0.17  mg/ml, Emax ∼ 94%) but less so for aorta (EC50 = 0.51 mg/ml, Emax ∼ 62%), suggesting some selectivity towards the pulmonary circulation. PA vasorelaxation was reduced by endothelial removal or NG-nitro-L-arginine methyl ester, but unaffected by indomethacin, apamin +charybdotoxin, 4-aminopyridine, glibenclamide, iberiotoxin, or 1H - [1,2,4]oxadiazolo[4,3-a]quinoxalin -1- one. Sodium nitroprusside-induced relaxation was enhanced by EM extract, probably via PDE5 inhibition. EM extract reduced contractions evoked by extracellular Ca2+application, and inhibited intracellular Ca2+release activated by phenylephrine. The phenanthrene relaxed PA independently of the endothelium. MCT thickened walls and decreased lumens of PA, and hypertrophied right ventricular myocytes, effects ameliorated by 3 weeks of oral sildenafil (20  mg/kg) or EM extract (15, 450 or 1000  mg/kg). CONCLUSION: PAH is improved by EM extract acting through PA relaxation mediated through endothelial NO, reduced Ca2+-mobilization, and reduced PA wall thickness and right ventricular hypertrophy.

Vasorelaxant and Hypotensive Effects of an Ethanolic Extract of Eulophia macrobulbon and Its Main Compound 1-(4'-Hydroxybenzyl)-4,8-Dimethoxyphenanthrene-2,7-Diol.

Background: Ethnopharmacological studies demonstrated the potential for Eulophia species to treat inflammation, cancer, and cardio-metabolic diseases. The aim of the study was to investigate the vasorelaxant effect of ethanolic Eulophia macrobulbon (EM) extract and its main phenanthrene on rat isolated mesenteric artery and to investigate the hypotensive effect of EM. Methods: The vasorelaxant effects of EM extract or phenanthrene and the underlying mechanisms were evaluated on second-order mesenteric arteries from Sprague Dawley rats. In addition, the acute hypotensive effect was evaluated in anesthetized rats infused with cumulative concentrations of the EM extract. Results: Both EM extract (10-4-1 mg/ml) and phenanthrene (10-7-10-4 M) relaxed endothelium-intact arteries, an effect that was partly reduced by endothelium removal (p < 0.001). A significant decrease in the relaxant effect of the extract and the phenanthrene was observed with L-NAME and apamin/charybdotoxin in endothelium-intact vessels, and with iberiotoxin in denuded vessels. SNP (sodium nitroprusside)-induced relaxation was significantly enhanced by EM extract and phenanthrene. By contrast, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one), 4-aminopyridine and glibenclamide (endothelium-denuded vessels) and indomethacin (endothelium-intact vessels) had no effect. In calcium-free solution, both the EM extract and phenanthrene inhibited extracellular Ca2+-induced contraction in high KCl and phenylephrine (PE) pre-contracted rings. They also inhibited the intracellular Ca2+ release sensitive to PE. The acute infusion of EM extract (20 and 70 mg/kg) induced an immediate and transient dose-dependent hypotensive effect. Conclusion: The ethanolic extract of EM tubers and its main active compound, 1-(4'-hydroxybenzyl)-4,8-dimethoxyphenanthrene-2,7-diol (phenanthrene) induced vasorelaxant effects on rat resistance vessels, through pleiotropic effects including endothelium-dependent effects (NOS activation, enhanced EDH production) and endothelium-independent effects (opening of KCa channels, inhibition of Ca2+ channels, inhibition of intracellular Ca2+ release and PDE inhibition).

Brain-derived neurotrophic factor in adjuvant-induced arthritis in rats. Relationship with inflammation and endothelial dysfunction.

OBJECTIVES: Both peripheral and central brain-derived neurotrophic factor (BDNF) levels are decreased in depression and normalized by efficient anti-depressive therapies. While depression symptoms are frequent in rheumatoid arthritis, BDNF has been poorly investigated in this pathology. Therefore, the present study explored cerebral and peripheral BDNF in arthritis rats as well as the link between brain BDNF and the two factors recently involved in the pathogenesis of depression and present in rheumatoid arthritis namely inflammation and endothelial dysfunction. METHODS: The brain (hippocampus and frontal cortex) and blood (serum) were collected in rats subjected to adjuvant-induced arthritis (AIA) when inflammatory symptoms and endothelial dysfunction are fully developed. Anhedonia as a core symptom of depression symptom was assessed from preference for a saccharin drinking solution. Inflammation was assessed from the arthritis score and serum levels of TNFα and IL-1ß. Treatment with the arginase inhibitor N(w)-hydroxy-nor-l-arginine (nor-NOHA) was used as a strategy to prevent endothelial dysfunction without improving inflammatory symptoms. RESULTS: As compared to controls, AIA rats displayed decreased brain BDNF levels that coexisted with anhedonia but contrasted with increased BDNF levels in serum. Brain BDNF deficiency correlated neither with arthritis score nor with pro-inflammatory cytokines levels, while it was mitigated by nor-NOHA treatment. A positive correlation was observed between serum BDNF and TNFα levels. CONCLUSIONS: Our study reveals that arthritis decreases BDNF levels in the brain and that endothelial dysfunction rather than inflammation contributes to the decrease. It also identifies a disconnection between serum and brain BDNF levels in arthritis.

Diclofenac but not celecoxib improves endothelial function in rheumatoid arthritis: A study in adjuvant-induced arthritis.

BACKGROUND AND AIMS: We aimed at investigating the effect of celecoxib (COX-2 selective inhibitor) and diclofenac (non-selective COX inhibitor) on endothelial function, and at identifying the underlying mechanisms in adjuvant-induced arthritis (AIA). METHODS: At the first signs of AIA, diclofenac (5 mg/kg twice a day, i.p), celecoxib (3 mg/kg/day, i.p) or saline (Vehicle) was administered for 3 weeks. Endothelial function was studied in aortic rings relaxed with acetylcholine (Ach) with or without inhibitors of NOS, arginase, EDHF and superoxide anions (O2-°) production. Aortic expression of eNOS, Ser1177-phospho-eNOS, COX-2, arginase-2, p22phox and p47phox was evaluated by Western blotting analysis. Arthritis scores, blood pressure, glycaemia and serum ADMA levels were measured. RESULTS: Diclofenac and celecoxib significantly reduced arthritis score to the same extent (p<0.05). As compared to vehicle-treated AIA, celecoxib did not change whereas diclofenac improved endothelial function (p<0.05) through increased EDHF production, decreased arginase activity and expression, decreased superoxide anions production and expression of p22phox and p47phox. Diclofenac but not celecoxib significantly enhanced blood pressure and serum ADMA levels. Glycaemia was unchanged by both treatments. CONCLUSIONS: Our study reveals that the effect of NSAIDs on endothelial function cannot be extrapolated from their impact on arthritis severity and suggest that changes in blood pressure and plasma ADMA levels may not be useful to predict CV risk of NSAIDs in RA.

Bacopa monnieri extract increases rat coronary flow and protects against myocardial ischemia/reperfusion injury.

BACKGROUND: This study explored Bacopa monnieri, a medicinal Ayurvedic herb, as a cardioprotectant against ischemia/reperfusion injury using cardiac function and coronary flow as end-points. METHODS: In normal isolated rat hearts, coronary flow, left ventricular developed pressure, heart rate, and functional recovery were measured using the Langendorff preparation. Hearts were perfused with either (i) Krebs-Henseleit (normal) solution, (control), or with 30, 100 µg/ml B. monnieri ethanolic extract (30 min), or (ii) with normal solution or extract for 10 min preceding no-perfusion ischemia (30 min) followed by reperfusion (30 min) with normal solution. Infarct volumes were measured by triphenyltetrazolium staining. L-type Ca2+-currents (ICa, L) were measured by whole-cell patching in HL-1 cells, a mouse atrial cardiomyocyte cell line. Cytotoxicity of B. monnieri was assessed in rat isolated ventricular myocytes by trypan blue exclusion. RESULTS: In normally perfused hearts, B. monnieri increased coronary flow by 63 ± 13% (30 µg/ml) and 216 ± 21% (100 µg/ml), compared to control (5 ± 3%) (n = 8-10, p < 0.001). B. monnieri treatment preceding ischemia/reperfusion improved left ventricular developed pressure by 84 ± 10% (30 µg/ml), 82 ± 10% (100 µg/ml) and 52 ± 6% (control) compared to pre- ischemia/reperfusion. Similarly, functional recovery showed a sustained increase. Moreover, B. monnieri (100 µg/ml) reduced the percentage of infarct size from 51 ± 2% (control) to 25 ± 2% (n = 6-8, p < 0.0001). B. monnieri (100 µg/ml) reduced ICa, L by 63 ± 4% in HL-1 cells. Ventricular myocyte survival decreased at higher concentrations (50-1000 µg/ml) B. monnieri. CONCLUSIONS: B. monnieri improves myocardial function following ischemia/reperfusion injury through recovery of coronary blood flow, contractile force and decrease in infarct size. Thus this may lead to a novel cardioprotectant strategy.

Effects of a chronic l-arginine supplementation on the arginase pathway in aged rats.

While ageing is frequently associated with l-arginine deficiency, clinical and experimental studies provided controversial data on the interest of a chronic l-arginine supplementation with beneficial, no or even deleterious effects. It was hypothesized that these discrepancies might relate to a deviation of l-arginine metabolism towards production of l-ornithine rather than nitric oxide as a result of age-induced increase in arginase activity. This study investigated the effect of ageing on arginase activity/expression in target tissues and determined whether l-arginine supplementation modulated the effect of ageing on arginase activity. Arginase activity and expression were measured in the heart, vessel, brain, lung, kidney and liver in young rats (3-months old) and aged Wistar rats (22-24-months-old) with or without l-arginine supplementation (2.25% in drinking water for 6weeks). Plasma levels of l-arginine and l-ornithine were quantified in order to calculate the plasma l-arginine/l-ornithine ratio, considered as a reflection of arginase activity. Cardiovascular parameters (blood pressure, heart rate) and aortic vascular reactivity were also studied. Ageing dramatically reduced plasma l-arginine and l-arginine/l-ornithine ratio, decreased liver and kidney arginase activities but did not change activities in other tissues. l-Arginine supplementation normalized plasma l-arginine and l-arginine/l-ornithine ratio, improved endothelial function and decreased systolic blood pressure. These effects were associated with decreased arginase activity in aorta along with no change in the other tissues except in the lung in which activity was increased. A strong mismatch was therefore observed between arginase activity and expression in analyzed tissues. The present study reveals that ageing selectively changes arginase activity in clearance tissues, but does not support a role of the arginase pathway in the potential deleterious effect of the l-arginine supplementation in aged patients. Moreover, our data argue against the use of the measurement of plasma l-arginine/l-ornithine ratio to estimate arginase activity in aged patients.

The Cerebral Brain-Derived Neurotrophic Factor Pathway, Either Neuronal or Endothelial, Is Impaired in Rats with Adjuvant-Induced Arthritis. Connection with Endothelial Dysfunction.

Cognitive abilities are largely dependent on activation of cerebral tropomyosin-related kinase B receptors (TrkB) by brain-derived neurotrophic factor (BDNF) that is secreted under a bioactive form by both neurons and endothelial cells. In addition, there is mounting evidence for a link between endothelial function and cognition even though the underlying mechanisms are not well known. Therefore, we investigated the cerebral BDNF pathway, either neuronal or endothelial, in rheumatoid arthritis (RA) that combines both endothelial dysfunction (ED) and impaired cognition. Adjuvant-induced arthritis (AIA) in rats was used as a model of RA. Clinical inflammatory symptoms were evaluated from an arthritis score and brains were collected at day 31 ± 2 post-immunization. Neuronal expression of BDNF and TrkB phosphorylated at tyrosine 816 (p-TrkB) was examined in brain slices. Endothelial BDNF and p-TrkB expression was examined on both brain slices (hippocampal arterioles) and isolated cerebral microvessels-enriched fractions (vessels downstream to arterioles). The connection between endothelial nitric oxide (NO) and BDNF production was explored on the cerebrovascular fractions using endothelial NO synthase (eNOS) levels as a marker of NO production, Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) as a NOS inhibitor and glyceryl-trinitrate as a slow releasing NO donor. Brain slices displayed lower BDNF and p-TrkB staining in both neurons and arteriolar endothelial cells in AIA than in control rats. For endothelial cells but not neurons, a strong correlation was observed between BDNF and p-TrkB staining. Of note, a strong correlation was also observed between neuronal p-TrkB and endothelial BDNF staining. In cerebral microvessels-enriched fractions, AIA led to decreased BDNF and eNOS levels with a positive association between the 2 parameters. These effects coincided with decreased BDNF and p-TrkB staining in endothelial cells. The exposure of AIA cerebrovascular fractions to GTN increased BDNF levels while the exposure of control fractions to L-NAME decreased BDNF levels. Changes in the cerebral BDNF pathway were not associated with arthritis score. The present study reveals that AIA impairs the endothelial and neuronal BDNF/TrkB pathway, irrespective of the severity of inflammatory symptoms but dependent on endothelial NO production. These results open new perspectives for the understanding of the link between ED and impaired cognition.

Arginase Inhibitors: A Rational Approach Over One Century.

Arginase (EC 3.5.3.1) is the bimanganese enzyme that converts L-arginine into ornithine and urea. This enzyme was discovered more than a century ago and early α-amino acids were identified as weak inhibitors. It was only during the 90s, after nitric oxide (NO) was reported as one of the most important biological mediators and when tight interrelation of arginase and NO synthase was found, that the development of arginase inhibitors was accelerated. The regulation of arginase activity by the N-hydroxy-L-arginine (3, NOHA) intermediate of the NO synthesis was the starting point of the N-hydroxy-nor-arginine (21, nor-NOHA) that proved to be the first micromolar inhibitor. The previously known manganese and arginase binding by borate inspired the 2(S)-amino-6-boronohexanoic acid (39, ABH) and S-(2-boronoethyl)-L-cysteine (40, BEC) now both considered as reference compounds in arginase inhibition. The high-resolution crystal structure of arginase and molecular modeling has rendered possible the recent design of (53) the strongest α,α-disubstituted derivatives of ABH. Simultaneously, traditional medicinal plants have contributed as a source of molecular diversity to the discovery of arginase inhibitors. This rational, step-by-step approach serves as guide in the present review where emphasis is placed on structure activity relationships. Highlights exhaustive review on arginase inhibitors highlight is made on rational approach to conception and structure activity relationships evaluation model is systematically mentioned with results.

Investigation of Mammal Arginase Inhibitory Properties of Natural Ubiquitous Polyphenols by Using an Optimized Colorimetric Microplate Assay.

Polyphenols are plant secondary metabolites which possess many positive effects on human health. Although these beneficial effects could be mediated through an increase in nitric oxide synthase activity, little is known regarding the inhibitory effect of polyphenols on mammal arginase, an enzyme which competes with nitric oxide synthase for their common substrate, L-arginine. The aim of the present study was to determine the potential of a series of polyphenols as mammalian arginase inhibitors and to identify some structure-activity relationships. For this purpose, we first developed a simple and cost-effective in vitro colorimetric microplate method using commercially-available mammal bovine liver arginase (b-ARG 1). Among the ten tested polyphenolic compounds [chlorogenic acid, piceatannol, resveratrol, (-)-epicatechin, taxifolin, quercetin, fisetin, caffeic acid, quinic acid, and kaempferol], cholorogenic acid and piceatannol exhibited the highest inhibitory activities (IC50 = 10.6 and 12.1 µM, respectively) but were however less active as (S)-(2-Boronoethyl)-L-cysteine (IC50 = 3.3 µM), used as reference compound. Enzyme kinetic studies showed that both chlorogenic acid and piceatannol are competitive arginase inhibitors. Structural data identified the importance of the caffeoyl (3,4-dihydroxycinnamoyl)-part and of the catechol function in the inhibitory activity of the tested compounds. These results identified chlorogenic acid and piceatannol as two potential core structures for the design of new arginase inhibitors.

The Janus face of chlorogenic acid on vascular reactivity: A study on rat isolated vessels.

BACKGROUND: Chlorogenic acid (CGA), the main polyphenol contained in coffee, is a major contributor to dietary polyphenol intake. Few studies reported its anti-hypertensive properties but the mechanisms are still indefinite. PURPOSE: The present study assessed the direct effect of CGA in endothelium denuded or intact aortic rings from male Wistar rats and the mechanisms involved. METHODS/RESULTS: CGA induced a direct endothelium-dependent relaxation that was significantly reduced by L-NAME (10(-4)M), indomethacin (10(-5)M) and combination of apamin (10(-7)M) and charybdotoxin (10(-7)M). Incubation of rings with CGA induced a dual effect on agonist-induced vasorelaxation. At 10(-6)M, it enhanced the relaxant effects of acetylcholine and reduced the contracting effects of phenylephrine due to increased basal and stimulated NOS activity, respectively. At 10(-4)M, CGA blunted acetylcholine and bradykinin-induced vasorelaxation, reduced phenylephrine-induced vasoconstriction but did not change the response to sodium nitroprusside, a NO-donor. CONCLUSION: In summary, CGA induces a direct endothelium-dependent vasodilation by increasing NOS, COX and EDHF signalling pathways. However, this new pharmacological action that can explain some positive effects of CGA in case of hypertension has to be modulated at the light of its deleterious impact on vascular relaxation at high concentrations and incite to be cautious when using high doses of CGA in clinical studies.

Etanercept improves endothelial function via pleiotropic effects in rat adjuvant-induced arthritis.

OBJECTIVES: To determine the effect of etanercept on endothelial dysfunction and on traditional cardiovascular (CV) risk factors in the adjuvant-induced arthritis (AIA) rat model. METHODS: At the first signs of arthritis, etanercept (10 mg/kg/3 days, s.c.) or saline was administered for 3 weeks in AIA rats. Body weights and arthritis scores were monitored daily. Endothelial function was studied in aortic rings relaxed with acetylcholine (Ach) with or without inhibitors of nitric oxide synthase (NOS), cyclo-oxygenase (COX-2), arginase, endothelium-derived hyperpolarizing factor and superoxide anions (O2 (-)°) production. Aortic expression of endothelial nitic oxide synthase (eNOS), Ser1177-phospho-eNOS, COX-2, arginase-2, p22(phox) and p47(phox) was evaluated by western blotting analysis. Blood pressure, heart rate and blood levels of triglycerides, cholesterol and glucose were measured. RESULTS: Etanercept significantly reduced arthritis score (P < 0.001). It improved Ach-induced relaxation (P < 0.05) as a result of increased NOS activity, decreased COX-2/arginase activities and decreased O2 (-)° production. These functional effects relied on increased eNOS expression and phosphorylation, and decreased COX-2, arginase-2 and p22(phox) expressions. No correlation was found between arthritis score and Ach-induced relaxation. The treatment did not change triglycerides, cholesterol and glucose levels, but significantly increased systolic blood pressure and heart rate (P < 0.05). CONCLUSION: Our data demonstrated that efficient dosage of etanercept on inflammatory symptoms improved endothelial function in AIA. This beneficial effect on endothelial function is disconnected from its impact on CV risk factors and relates to pleiotropic effects of etanercept on endothelial pathways. These results suggest that etanercept could be a good choice for patients with rheumatoid arthritis at high risk of CV events.

Endothelial Dysfunction in Rheumatoid Arthritis: Mechanistic Insights and Correlation with Circulating Markers of Systemic Inflammation.

OBJECTIVES: To determine mechanisms involved in endothelial dysfunction (ED) during the course of arthritis and to investigate the link between cytokines, chemokines and osteoprotegerin. APPROACH AND RESULTS: Experiments were conducted on aortic rings at day 4 (preclinical), day 11 (onset of disease), day 33 (acute disease) and day 90 (chronic disease) after adjuvant-induced arthritis (AIA) in Lewis rats. At day 4, the unique vascular abnormality was a reduced norepinephrine-induced constriction. At day 11, endothelial function assessed by the relaxation to acetylcholine was normal despite increased cyclo-oxygenase-2 activity (COX-2) and overproduction of superoxide anions that was compensated by increased nitric oxide synthase (NOS) activity. At day 33, ED apparition coincides with the normalization of NOS activity. At day 90, ED was only observed in rats with a persisting imbalance between endothelial NOS and COX-2 pathways and higher plasma levels of IL-1ß and TNFα. Plasma levels of IL-1ß, TNFα and MIP-1α negatively correlated with Ach-induced relaxation throughout the course of AIA. CONCLUSIONS: Our data identified increased endothelial NOS activity as an important compensatory response that opposes the ED in the early arthritis. Thereafter, a cross-talk between endothelial COX-2/NOS pathways appears as an important element for the occurrence of ED. Our results encourage determining the clinical value of IL-1ß, TNFα and MIP-1α as biomarkers of ED in RA.