Omentin, a novel adipokine, induces vasodilation in rat isolated blood vessels

Omentin is a recently identified adipose tissue-derived cytokine and is implicated in obesity-related cardiovascular disorders. In the present study, we tested the hypothesis that omentin could directly affect vascular reactivity of isolated blood vessels. In endothelium-intact rat isolated aorta, pretreatment with omentin (300 ng/ml, 30 min) inhibited noradrenaline (NA; 1 nM-1 μM)-induced concentration-dependent contraction. In NA (100 nM)-pre-contracted aorta, omentin (1-300 ng/ml) directly induced an endothelium-dependent relaxation. While a nitric oxide (NO) synthase (NOS) inhibitor, N^G-nitro-l-arginine methyl ester (100 μM, 30 min) inhibited the relaxation, a PI3 K/Akt inhibitor, LY294002 (10 μM, 30 min) or a tyrosine kinase inhibitor, genistein (30 μM, 30 min) was ineffective. Omentin (300 ng/ml, 5 min) induced a phosphorylation of endothelial NOS at serine 1177 but not a phosphorylation of Akt at serine 473. Omentin (1-300 ng/ml) also relaxed NA pre-contracted mesenteric artery. Present study for the first time demonstrated that omentin has a vasodilating effect on isolated blood vessels, which is mediated through endothelium-derived NO.     

Pomolic acid of Licania pittieri elicits endothelium-dependent relaxation in rat aortic rings

Pomolic acid has recently shown hypotensive effect in rats. The purpose of this investigation was to determine the vascular effects of this triterpenoid and to examine its mode of action. Functional experiments in rat aortic rings precontracted with norepinephrine were performed to evaluate the vasorelaxant effect of pomolic acid. This triterpenoid induced a vasorelaxation (IC₅₀ = 2.45 μM) in a concentration- and endothelium-dependent manner and showed no effect on contractions evoked by KCl (25 mM). Pre-treatment of aortic rings with l-NAME (100 μM), methylene blue (100 μM) or glibenclamide (10 μM), totally prevented the vasorelaxation induced by pomolic acid, while indomethacin (10 μM) had no effect on this response. Additionally, pomolic acid relaxation was unaffected under the muscarinic- and β-adrenergic-receptor blocked ensured for atropine and propanolol respectively (10 μM each). In contrast, the vasorelaxant effect of pomolic acid was abolished under the purinergic-receptor blocked ensured for suramin (10 μM). Finally, apyrase (0.8 U/ml) an enzyme which hydrolyses ATP and ADP did not affect pomolic acid relaxation. In summary, pomolic acid has a potent endothelium-dependent vasorelaxant effect, possibly acting through the direct activation of endothelial purinergic receptors via NO-cGMP signaling pathway, which could be part of the mechanism underlying its hypotensive effect.     

Inhibitory effect of schisandrin on spontaneous contraction of isolated rat colon

This study examined the effect of schisandrin, one of the major lignans isolated from Schisandra chinensis, on spontaneous contraction in rat colon and its possible mechanisms. Schisandrin produced a concentration-dependent inhibition (EC₅₀ = 1.66 μM) on the colonic spontaneous contraction. The relaxant effect of schisandrin could be abolished by the neuronal Na⁺ channel blocker tetrodotoxin (1 μM) but not affected by propranolol (1 μM), phentolamine (1 μM), atropine (1 μM) or nicotine desensitization, suggesting possible involvement of non-adrenergic non-cholinergic (NANC) transmitters released from enteric nerves. N^ω-nitro-l-arginine methyl ester (100-300 μM), a nitric oxide synthase inhibitor, attenuated the schisandrin response. The role of nitric oxide (NO) was confirmed by an increase in colonic NO production after schisandrin incubation, and the inhibition on the schisandrin responses by soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-α]-quinoxalin-1-one (1-30 μM). Non-nitrergic NANC components may also be involved in the action of schisandrin, as suggested by the significant inhibition of apamin on the schisandrin-induced responses. Pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate (100 μM), a selective P2 purinoceptor antagonist, markedly attenuated the responses to schisandrin. In contrast, neither 8-cyclopentyl-1,3-dipropylxanthine, an antagonist for adenosine A₁ receptors, nor chymotrypsin, a serine endopeptidase, affected the responses. All available results have demonstrated that schisandrin produced NANC relaxation on the rat colon, with the involvement of NO and acting via cGMP-dependent pathways. ATP, but not adenosine and VIP, likely plays a role in the non-nitrergic, apamin-sensitive component of the response.     

Visfatin in pregnancy: proposed mechanism of peptide delivery

Visfatin is a newly identified 52 kD adipocytokine that appears to have insulinomimetic properties. We examined visfatin expression in visceral fat from lean and pregnant women. Visfatin gene expression was seven times higher in omental fat of pregnant women than in lean women. Both immunohistochemistry and immunoblot confirmed that visfatin protein was much higher in pregnant women than in nonpregnant women. However, serum visfatin was 20.8 ± 7.7 ng/ml (n = 7) in lean women as compared to only a slight increase to 40.3 ng/ml in pregnant women (n = 4). We measured visfatin mRNA content of human placenta and found that placenta expresses high levels of visfatin mRNA and protein. At a concentration of 2 nM, visfatin and insulin produced nearly identical increase in glucose transport. The discrepancy between the elevated visfatin expression and tissue visfatin compared to only a small increase in serum visfatin is a matter of controversy. The data on serum visfatin concentrations are replete with contradictory data. Taken together, we suggest that visfatin is not a hormone. Instead, we propose that visfatin acts in either a paracrine or autocrine mode. This hypothesis would explain what various laboratories have found widely discrepant values for serum visfatin. Since visfatin potently and efficaciously induced glucose transport in a cell culture model, any hypothetical role for visfatin in pregnancy should include the possibility that it may play a role in maternal/fetal glucose metabolism or distribution and that it may do so by acting locally.     

Pharmacological evidence that Ca²+ channels and, to a lesser extent, K+ channels mediate the relaxation of testosterone in the canine basilar artery

Testosterone induces vasorelaxation through non-genomic mechanisms in several isolated blood vessels, but no study has reported its effects on the canine basilar artery, an important artery implicated in cerebral vasospasm. Hence, this study has investigated the mechanisms involved in testosterone-induced relaxation of the canine basilar artery. For this purpose, the vasorelaxant effects of testosterone were evaluated in KCl- and/or PGF_2α-precontracted arterial rings in vitro in the absence or presence of several antagonists/inhibitors/blockers; the effect of testosterone on the contractile responses to CaCl₂ was also determined. Testosterone (10-180 μM) produced concentration-dependent relaxations of KCl- or PGF_2α-precontracted arterial rings which were: (i) unaffected by flutamide (10 μM), dl-aminoglutethimide (10 μM), actinomycin D (10 μM), cycloheximide (10 μM), SQ 22,536 (100 μM) or ODQ (30 μM); and (ii) significantly attenuated by the blockers 4-aminopyridine (K_V; 1 mM), BaCl₂ (K_IR; 30 μM), iberiotoxin (BKCa2+; 20 nM), but not by glybenclamide (K_ATP; 10 μM). In addition, testosterone (31, 56 and 180 μM) and nifedipine (0.01-1 μM) produced a concentration-dependent blockade of the contraction to CaCl₂ (10 μM to 10 mM) in arterial rings depolarized by 60 mM KCl. These results, taken together, show that testosterone relaxes the canine basilar artery mainly by blockade of voltage-dependent Ca²⁺ channels and, to a lesser extent, by activation of K⁺ channels (K_IR, K_V and BKCa2+). This effect does not involve genomic mechanisms, production of cAMP/cGMP or the conversion of testosterone to 17β-estradiol.     

Measurement of phenol and p-cresol in urine and feces using vacuum microdistillation and high-performance liquid chromatography

In this article, we describe a simple, sensitive, accurate, and repeatable method for the measurement of phenol and p-cresol (4-methylphenol) in human urine and feces. We examined a number of parameters to identify an optimal extraction protocol. Purification of sample extracts was achieved by low-temperature vacuum microdistillation. Separation was achieved in approximately 15 min by high-performance liquid chromatography (HPLC) with quantification by fluorescence at 284/310 nm. Limits of detection for phenol were 2 ng/ml for urine and 20 ng/g for feces, and those for p-cresol were 10 ng/ml for urine and 100 ng/g for feces. For comparison, approximate mean values for urine are 3 μg/ml for phenol and 30 μg/ml for p-cresol, and those for feces are 1 μg/g for phenol and 50 μg/g for p-cresol. An experienced analyst can process 60 samples each day using this method.     

Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit

We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN₂) and then preserved in the LN₂. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN₂. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.     

Effect of changes in pH on wall tension in isolated rat pulmonary artery: role of the RhoA/Rho-kinase pathway

Pulmonary arteries (PA) are resistant to the vasodilator effects of extracellular acidosis in systemic vessels; the mechanism underlying this difference between systemic and pulmonary circulations has not been elucidated. We hypothesized that RhoA/Rho-kinase-mediated Ca2+ sensitization pathway played a greater role in tension development in pulmonary than in systemic vascular smooth muscle and that this pathway was insensitive to acidosis. In arterial rings contracted with the alpha1-agonist phenylephrine (PE), the Rho-kinase inhibitor Y-27632 (< or =3 microM) induced greater relaxation in precontracted PA rings than in aortic rings. In PA rings stimulated by PE, the activation of RhoA was greater than in aorta. Normocapnic acidosis (NA) induced a smaller relaxation in precontracted PA than in aorta. However, in the presence of nifedipine and thapsigargin, when PE-induced contraction was predominantly mediated by Rho-kinase, the relaxant effect of NA was reduced and similar in both vessel types. Furthermore, in the presence of Y-27632, NA induced a greater relaxation in both PA and aorta, which was similar in both vessels. Finally, in alpha-toxin-permeabilized smooth muscle, PE-induced contraction at constant Ca2+ activity was inhibited by Y-27632 and unaffected by acidosis. These results indicate that Ca2+ sensitization induced by the RhoA/Rho-kinase pathway played a greater role in agonist-induced vascular smooth muscle contraction in PA than in aorta and that tension mediated by this pathway was insensitive to acidosis. The predominant role of the RhoA/Rho-kinase pathway in the pulmonary vasculature may account for the resistance of this circulation to the vasodilator effect of acidosis observed in the systemic circulation.     

Pharmacological characterisation of 5-hydroxytryptamine-induced contractile effects in the isolated gut of the lepidopteran caterpillar Spodoptera frugiperda

The indolealkylamine 5-hydroxytryptamine (5-HT, 0.1 nM-1 μM) caused dose-dependent increases in the number of contractions observed in guts isolated from the caterpillar Spodoptera frugiperda. Of the 5-HT analogues tested for agonist action, 2-methyl-5-HT (0.1-10 μM) was a full agonist with reduced potency while α-methyl-5-HT (0.1-100 μM), 5-carboxamidotryptamine (0.1-100 μM), 5-methoxytryptamine (5-MeOT) (10 nM-10 μM), and tryptamine (1-100 μM) were partial agonists. Incubation of isolated guts with proven mammalian 5-HT receptor antagonists showed that cyproheptadine (10 nM-1 μM), MDL 72222 (1-10 μM), tropisetron (1-10 μM) and 5-benzoyloxygramine (1-10 μM) were potent non-competitive antagonists of 5-HT-induced tissue contraction. In comparison, ketanserin (0.1-1 μM) was a competitive antagonist. The mammalian selective serotonin reuptake inhibitors, clomipramine (10 nM-10 μM) and fluoxetine (10 nM-10 μM) also caused non-competitive inhibition of 5-HT-induced contraction while fluvoxamine (10 nM-10 μM) was a weak competitive antagonist. Low doses of clomipramine (0.1 μM) caused potentiation of 5-HT-induced gut contraction thereby suggesting the presence of 5-HT reuptake systems in this tissue. The contractile effects of 5-HT were inhibited by verapamil, Li⁺ and H7 and potentiated by theophylline thereby indicating that L-type Ca²⁺ channels, phosphatidylinositol second messengers and cAMP, respectively, are involved in 5-HT-induced tissue contraction. The 5-HT receptors mediating contractility in the gut of S. frugiperda have properties in common with mammalian 5-HT₂ and Drosophila 5-HT_dro2A/2B receptors. In addition, these data suggest that the tissue also contains receptors that are similar to mammalian 5-ht₆ and 5-HT₇ as well as Drosophila_dro1 receptors. However, the primary amino acid sequence of these lepidopteran 5-HT receptors will have to be elucidated before full comparisons can be made.     

Endothelium-independent vasorelaxation effects of 16-O-acetyldihydroisosteviol on isolated rat thoracic aorta

Aims

The aim of this study is to investigate the vasorelaxant effect of 16-O-acetyldihydroisosteviol (ADIS) and its underlying mechanisms in isolated rat aorta.

Main methods

Rat aortic rings were isolated, suspended in organ baths containing Kreb's solution, maintained at 37 °C, and mounted on tungsten wire and continuously bubbled with a mixture of 95% O₂ and 5% CO₂ under a resting tension of 1 g. The vasorelaxant effects of ADIS were investigated by means of isometric tension recording experiment.

Key findings

ADIS (0.1 μM–3 mM) induced relaxation of aortic rings pre-contracted by phenylephrine (PE, 10 μM) and KCl (80 mM) with intact-endothelium (E_max = 79.26 ± 3.74 and 79.88 ± 3.79, respectively) or denuded-endothelium (E_max = 88.05 ± 3.69 and 78.22 ± 6.86, respectively). In depolarization Ca²⁺-free solution, ADIS inhibits calcium chloride (CaCl₂)-induced contraction in endothelium-denuded rings in a concentration-dependent manner. In addition, ADIS attenuates transient contractions in Ca²⁺-free medium containing EGTA (1 mM) induced by PE (10 μM) and caffeine (20 mM). By contrast, relaxation was not affected by tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 1 mM), glibenclamide (10 μM), barium chloride (BaCl₂, 1 mM), and 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 1 μM).

Significance

These findings reveal the vasorelaxant effect of ADIS, through endothelium-independent pathway. It acts as a Ca^2 + channel blocker through both intracellular and extracellular Ca^2 + release.     

CCN2 promotes keratinocyte adhesion and migration via integrin α5β1

Background

CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration.Materials and methods: Adhesion assays were performed by coating wells with 10 μg/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 μl arginine–glycine–aspartic acid (RGD), 500 μM arginine–glycine–glutamate–serine (RGES), and 10 μg/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 μM PD98059 (MEK-1 inhibitor) or 20 μM PF573228 (FAK inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2.Results: CCN2 enhanced keratinocyte adhesion to fibronectin via integrin α5β1. The addition of anti-integrin α5β1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits α5 and β1. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration.Conclusions: Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin α5β1 and activation of the FAK-MAPK signaling cascade.     

Endothelium-dependent vasorelaxant effects of the essential oil from aerial parts of Alpinia zerumbet and its main constituent 1,8-cineole in rats

Vasorelaxant effects of essential oil of Alpinia zerumbet (EOAZ) and its main constituent, 1,8-cineole (CIN) were studied. In rat isolated aorta preparations with intact endothelium, EOAZ (0.01–3000 μg/ml) induced significant but incomplete relaxation of the phenylephrine-induced contraction, an effect that was abolished by removal of vascular endothelium. However, at the same concentrations (0.01–3000 μg/ml corresponding to 0.0000647–19.5 mM), CIN induced a complete vasorelaxant effects (IC₅₀=663.2±63.8 μg/ml) that were significantly reduced in endothelium-denuded rings (IC₅₀=1620.6±35.7 μg/ml). Neither EOAZ nor CIN affected the basal tonus of isolated aorta. Vasorelaxant effects of both EOAZ and CIN remained unaffected by the addition of tetraethylamonium chloride (500 μM) or indomethacin (10 μM) into the bath, but were significantly reduced by N^G-nitro-L-arginine methyl ester (100 μM). It is concluded that EOAZ induces a potent vasorelaxant effect that could not be fully attributed to the actions of the main constituent CIN, and appears totally dependent on the integrity of a functional vascular endothelium. The data is novel and corroborate the popular use of A. zerumbet for the treatment of hypertension.     

Endothelium-dependent vasorelaxant effects of the essential oil from aerial parts of Alpinia zerumbet and its main constituent 1,8-cineole in rats

Vasorelaxant effects of essential oil of Alpinia zerumbet (EOAZ) and its main constituent, 1,8-cineole (CIN) were studied. In rat isolated aorta preparations with intact endothelium, EOAZ (0.01–3000 μg/ml) induced significant but incomplete relaxation of the phenylephrine-induced contraction, an effect that was abolished by removal of vascular endothelium. However, at the same concentrations (0.01–3000 μg/ml corresponding to 0.0000647–19.5 mM), CIN induced a complete vasorelaxant effects (IC₅₀=663.2±63.8 μg/ml) that were significantly reduced in endothelium-denuded rings (IC₅₀=1620.6±35.7 μg/ml). Neither EOAZ nor CIN affected the basal tonus of isolated aorta. Vasorelaxant effects of both EOAZ and CIN remained unaffected by the addition of tetraethylamonium chloride (500 μM) or indomethacin (10 μM) into the bath, but were significantly reduced by N^G-nitro-L-arginine methyl ester (100 μM). It is concluded that EOAZ induces a potent vasorelaxant effect that could not be fully attributed to the actions of the main constituent CIN, and appears totally dependent on the integrity of a functional vascular endothelium. The data is novel and corroborate the popular use of A. zerumbet for the treatment of hypertension.     

Sonic hedgehog mediates BDNF-induced neuroprotection against mitochondrial inhibitor 3-nitropropionic acid

Sonic hedgehog (SHH), a morphogen critical for embryogenesis, has also been shown to be neuroprotective. We have recently reported that pretreatment of rat cortical neurons for 8 h with brain-derived neurotrophic factor (BDNF; 100 ng/ml) affords protection against neurotoxicity of 3-nitropropionic acid (3-NP; 2.5 mM for 24 h), a mitochondrial complex II inhibitor. However, whether SHH is involved in BDNF-mediated neuroprotection remains unknown. Herein we tested whether BDNF induces SHH expression and if so, whether BDNF induction of SHH contributes to the observed neuroprotective effects. We found BDNF (100 ng/ml) increased SHH expression at both mRNA and protein levels. BDNF protection against 3-NP was abolished by cyclopamine (CPM; 5 μM), the SHH pathway inhibitor. Preconditioning of cortical neurons with N-terminal fragment of SHH (SHH-N; 0.1-1 ng/ml) was sufficient to confer resistance. These results indicate that BDNF induces SHH expression, which contributes to neuroprotection against 3-NP toxicity in rat cortical neurons.     

FERM domain promotes resveratrol-induced apoptosis in endothelial cells via inhibition of NO production

Focal adhesion kinase (FAK) consists of an N-terminal band 4.1; ezrin, radixin, moesin (FERM) domain; tyrosine kinase domain; and C-terminal FA targeting domain. Here we show that ectopically expressed FERM is largely located in the cytosolic fraction under quiescent conditions. We further found that this ectopically expressed FERM domain aggravates endothelial cell apoptosis triggered by 100 μM resveratrol, whereas FERM had no effect on apoptosis induced by TNF-α. We determined that resveratrol at low doses (<20 μM) promotes phosphorylation (S1177) of eNOS via an AMPK-dependent pathway. The presence of the FERM domain blocked this resveratrol-stimulated eNOS phosphorylation and NO production. Thus, the pro-apoptotic activity of cytosolic FERM domain is at least partially mediated by down-regulation of NO, a critical cell survival factor. Consistently, we found that the apoptosis induced by cytosolic FERM in the presence of resveratrol was reversed by an NO donor, SNAP. In conclusion, FERM located in the cytosolic fraction plays a pivotal role in aggravating cell apoptosis through diminishing NO production.     

Visfatin impairs endothelium-dependent relaxation in rat and human mesenteric microvessels through nicotinamide phosphoribosyltransferase activity

Visfatin, also known as extracellular pre-B-cell colony-enhancing factor (PBEF) and nicotinamide phosphoribosyltransferase (Nampt), is an adipocytokine whose circulating levels are enhanced in metabolic disorders, such as type 2 diabetes mellitus and obesity. Circulating visfatin levels have been positively associated with vascular damage and endothelial dysfunction. Here, we investigated the ability of visfatin to directly impair vascular reactivity in mesenteric microvessels from both male Sprague-Dawley rats and patients undergoing non-urgent, non-septic abdominal surgery. The pre-incubation of rat microvessels with visfatin (50 and 100 ng/mL) did not modify the contractile response to noradrenaline (1 pmol/L to 30 μmol/L), as determined using a small vessel myograph. However, visfatin (10 to 100 ng/mL) concentration-dependently impaired the relaxation to acetylcholine (ACh; 100 pmol/L to 3 μmol/L), without interfering with the endothelium-independent relaxation to sodium nitroprusside (1 nmol/L to 3 μmol/L). In both cultured human umbilical vein endothelial cells and rat microvascular preparations, visfatin (50 ng/mL) stimulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, as determined by lucigenin-derived chemiluminiscence. The relaxation to ACh impaired by visfatin was restored by the NADPH oxidase inhibitor apocynin (10 μmol/L). Additionally, the Nampt inhibitor APO866 (10 mmol/L to 10 μmol/L), but not an insulin receptor-blocking antibody, also prevented the stimulation of NADPH oxidase and the relaxation impairment elicited by visfatin. Accordingly, the product of Nampt activity nicotinamide mononucleotide (100 nmol/L to 1 mmol/L) stimulated endothelial NADPH oxidase activity and concentration-dependently impaired ACh-induced vasorelaxation. In human mesenteric microvessels pre-contracted with 35 mmol/L potassium chloride, the endothelium-dependent vasodilation to bradykinin (1 nmol/L to 3 μmol/L) was equally impaired by visfatin and restored upon co-incubation with APO866. In conclusion, visfatin impairs endothelium-dependent relaxation through a mechanism involving NADPH oxidase stimulation and relying on Nampt enzymatic activity, and therefore arises as a potential new player in the development of endothelial dysfunction.     

Echinocystic acid,isolated from Gleditsia sinensis fruit,protects endothelial progenitor cells from damage caused by oxLDL via the Akt/eNOS pathway

Aims

Our previous studies revealed that echinocystic acid (EA) showed obvious attenuation of atherosclerosis in rabbits fed a high-fat diet. However, the underlying mechanisms remain to be elucidated. Considering the importance of endothelial progenitor cells (EPCs) in atherosclerosis, we hypothesise that EPCs may be one of the targets for the anti-atherosclerotic potential of EA.

Main methods

After in vitro cultivation, EPCs were exposed to 100 μg/mL of oxidised low-density lipoprotein (oxLDL) and incubated with or without EA (5 and 20 μM) for 48 h. An additional two groups of EPCs (oxLDL + 20 μM EA) were pre-treated with either wortmannin, an inhibitor of the phosphoinositide 3-kinase (PI3K) pathway, or nitro-l-arginine methyl ester (l-NAME), an endothelial nitric oxide synthase (eNOS)-specific inhibitor. Assessment of EPC apoptosis, adhesion, migration, and nitric oxide (NO) release was performed using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining, cell counting, caspase-3 activity assay, transwell chamber assay, and Griess reagent, respectively. The protein expression of protein kinase B (Akt) and eNOS was detected using Western blot.

Key findings

Treatment of EPCs with oxLDL induced significant apoptosis and impaired adhesion, migration, and NO production. The deleterious effects of oxLDL on EPCs were attenuated by EA. However, when EPCs were pre-treated with wortmannin or l-NAME, the effects of EA were abrogated. Additionally, oxLDL significantly down-regulated eNOS protein expression as well as repression of eNOS and Akt phosphorylation.

Significance

The inhibitory effect of oxLDL on Akt/eNOS phosphorylation was attenuated by EA. Taken together, the results indicate that EA protects EPCs from damage caused by oxLDL via the Akt/eNOS pathway.     

Determination of lipoic acid in human plasma by HPLC-ECD using liquid–liquid and solid-phase extraction: Method development,validation and optimization of experimental parameters

A rapid, inexpensive, sensitive and specific HPLC-ECD method for the determination of lipoic acid in human plasma was developed and validated over the linearity range of 0.001–10 μg/ml using naproxen sodium as an internal standard (IS). Extraction of lipoic acid and IS from plasma (250 μl) was carried out with a simple one step liquid–liquid extraction using dichloromethane. Similarly solid-phase extraction was carried out using dichloromethane as extraction solvent. The separated organic layer was dried under the stream of nitrogen at 40 °C and the residue was reconstituted with the mobile phase. Complete separation of both lipoic acid and IS at 30 °C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved in 6 min using 0.05 M phosphate buffer (pH 2.5 adjusted with phosphoric acid):acetonitrile (50:50, v/v) as a mobile phase pumped at the rate of 1.5 ml/min using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification of lipoic acid were 200 pg/ml and 1 ng/ml, respectively. While on column limit of detection and limit of quantification of lipoic acid were 10 and 50 pg/ml, respectively. The absolute recoveries of lipoic acid with liquid–liquid and solid-phase extraction were 98.43, 95.65, 101.45, and 97.36, 102.73, 100.17% at 0.5, 1 and 5 μg/ml levels, respectively. Coefficient of variations for both intra-day and inter-day were between 0.28 and 4.97%. The method is validated and will be quite suitable for the analysis of lipoic acid in the plasma of human volunteers as well as patients with diabetes and cardiovascular diseases.