Porcine Coronary Artery Rings Research Articles

Calciprotein Particles Induce Endothelial Dysfunction by Impairing Endothelial Nitric Oxide Metabolism.

Calciprotein particles (CPPs) are associated with the development of vascular calcifications in chronic kidney disease. The role of endothelial cells (ECs) in this process is unknown. Here, we investigated the interaction of CPPs and ECs, thereby focusing on endothelial nitric oxide metabolism and oxidative stress. CPPs were generated in calcium- and phosphate-enriched medium. Human umbilical vein endothelial cells were exposed to different concentrations of CPPs (0-100 µg/mL) for 24 or 72 hours. Ex vivo porcine coronary artery rings were used to measure endothelial cell-dependent vascular smooth muscle cell relaxation after CPP exposure. Serum samples from an early chronic kidney disease cohort (n=245) were analyzed for calcification propensity (measure for CPP formation) and nitrate and nitrite levels (NOx). CPP exposure for 24 hours reduced eNOS (endothelial nitric oxide synthase) mRNA expression and decreased nitrite production, indicating reduced nitric oxide bioavailability. Also, 24-hour CPP exposure caused increased mitochondria-derived superoxide generation, together with nitrotyrosine protein residue formation. Long-term (72 hours) exposure of human umbilical vein endothelial cells to CPPs induced eNOS uncoupling and decreased eNOS protein expression, indicating further impairment of the nitric oxide pathway. The ex vivo porcine coronary artery model showed a significant reduction in endothelial-dependent vascular smooth muscle cell relaxation after CPP exposure. A negative association was observed between NOx levels and calcification propensity (r=-0.136; P=0.049) in sera of (early) chronic kidney disease patients. CPPs cause endothelial cell dysfunction by impairing nitric oxide metabolism and generating oxidative stress. Our findings provide new evidence for direct effects of CPPs on ECs and pathways involved.

Abstract 10442: Protective Effect of Inhibition of Sglt2 on Nanoplastics-Induced Premature Endothelial Senescence and Dysfunction

Background: Nanoplastics (NPs) are now widely acknowledged as a vital part of environmental pollution. However, the effects of NPs on the cardiovascular system are not well understood. We previously reported that NPs induce premature endothelial senescence and dysfunction via a redox-sensitive signaling pathway. Increasing evidence has shown that SGLT2 inhibitors (gliflozins) exhibit protective effects against the development of major cardiovascular diseases. Objective: This study aimed to investigate the role of SGLT2 on NPs-induced premature endothelial senescence and dysfunction, and, if so, to assess the effect of SGLT2 inhibition. Methods: Porcine coronary artery (PCA) rings and cultured endothelial cells (ECs) were exposed to NPs in the presence or absence of the SGLT2 inhibitor, Enavogliflozin (ENA), and the level of SGLTs expression, senescence-associated-beta galactosidase (SA-β-gal) activity, senescence markers, oxidative stress level, and vascular function were evaluated. Results: NPs were internalized in ECs in a time- and concentration-dependent manner. Exposure of PCAECs to NPs significantly up-regulated SGLT2 expression and increased SA-β-gal activity, one of the prominent senescence markers. ENA significantly reduced the NPs-induced increase of SA-β-gal activity in PCA and PCAECs. In addition, ENA prevented the NPs-induced up-regulation of senescence markers, p53, and p21 that promoted the inhibition of ECs proliferation. ENA prevented also the NPs-induced oxidative stress and up-regulation of NOX2 and p22phox in ECs. Exposure of coronary artery rings to NPs blunted endothelium-dependent relaxation and decreased the expression level of endothelial nitric oxide synthase level, and both of these effects were prevented by ENA. Conclusion: The present findings indicate that premature endothelial senescence and dysfunction induced by nanoplastics involve, at least in part, an upregulation of SGLT2, which is associated with increased levels of oxidative stress and the down-regulation of eNOS. They further raise the possibility that SGLT2 may be a potential target for preventing and/or treating environment pollution-associated cardiovascular diseases.

Beneficial Effects of Caffeic Acid Phenethyl Ester on Wound Healing in a Diabetic Mouse: Role of VEGF and NO

Cutaneous wound healing is delayed in patients with diabetes. Caffeic acid phenethyl ester (CAPE) has been identified as an effective constituent of propolis with improved wound healing abilities via an oxidative stress decrease. However, its impact on wound healing in diabetic models and its underlying mechanisms remain unclear. Determining the vascular endothelial growth factor (VEGF) contents in a human vascular smooth muscle cell (VSMC)-conditioned medium was assessed using human VEGF immunoassay and vascular reactivity using porcine coronary artery rings. Later, C57BL/6 or db/db mice were anesthetized, after which a 6-mm biopsy punch was manipulated for perforation via the back skin. Subsequently, CAPE was applied to the wound and changed daily. Furthermore, the injury in each mouse was digitally photographed, and the wound area was quantified. We observed that CAPE increased VEGF levels in human VSMC-conditioned medium, improved endothelium-dependent nitric oxide (NO)-mediated vasorelaxation, inhibited U46619-induced vasoconstriction porcine coronary artery, and enhanced cutaneous wound healing in the diabetic mouse model. Hence, we propose that CAPE improves wound healing in diabetic mice, which is aided by increased VEGF and NO expression.

Nanoencapsulation of the omega-3 EPA:DHA 6:1 formulation enhances and sustains NO-mediated endothelium-dependent relaxations in coronary artery rings and NO formation in endothelial cells

• Omega-3fatty acids stimulate the endothelial formation of vasoprotective nitric oxide. • EPA:DHA 6:1 nanoparticles caused greater sustained endothelium-dependent relaxation. • EPA:DHA 6:1 nanoparticles caused sustained formation of NO in endothelial cells. • EPA:DHA 6:1 nanoparticles-treated endothelial cells had greater anti-platelet effect. The eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) formulation with a ratio of 6:1 is a potent stimulator of the endothelial formation of nitric oxide (NO). The aim of the study was to investigate whether nanoencapsulation of EPA:DHA 6:1 followed by coating with gum increases its biological activity. Vascular reactivity was assessed using porcine coronary artery rings, the formation of NO in cultured endothelial cells (ECs) using DAF-FM and indirectly by platelet aggregation studies. Coated EPA:DHA 6:1 nanoparticles induced sustained relaxations of coronary artery rings that were greater in rings with than in those without endothelium, and more pronounced than with the native form. Treatment of ECs with coated EPA:DHA 6:1 nanoparticles caused greater and more sustained formation of NO and enhanced their anti-aggregatory effects. Thus, nanoencapsulation of EPA:DHA 6:1 is an attractive strategy to enhance the beneficial effect at the vascular endothelium.

Endothelium-independent vasorelaxant effect of Asphodelus tenuifolius Cav. via inhibition of myosin light chain kinase activity in the porcine coronary artery

Ethnopharmacological relevanceAsphodelus tenuifolius Cav. (Asphodelaceae), a wild, terrestrial, annual stemless herb, is widely used in traditional medicine for the treatment of hypertension, diabetes, atherosclerosis and circulatory problems. A previous research study from our laboratory revealed that A. tenuifolius has beneficial effects in reducing blood pressure and improves aortic endothelial dysfunction in chronically glucose fed rats. Despite the fact that A. tenuifolius reduces blood pressure and improves endothelial function in vivo, there are no detailed studies about its possible mechanism of action. Aim of the studyThis study was designed to provide pharmacological basis and mechanism of action for the traditional use of A. tenuifolius in hypertension and circulatory problems. We explored the vasorelaxant effect of A. tenuifolius and its underlying vasorelaxation mechanism in porcine coronary artery rings. Materials and methodsAqueous methanolic crude extract of A. tenuifolius was prepared by maceration process and then activity guided fractionation was carried out by using different polarity based solvents. Phytochemical studies were carried out using LC-DAD-MS. Segments of porcine distal coronary artery were set up in a wire myograph for isometric force measurements. Extract/fractions of A. tenuifolius seeds were tested for vasodilator activity by measurement of changes in tone after pre-contraction with the thromboxane mimetic U46619 in the presence or absence of inhibitors of intracellular signaling cascades. ResultsCrude extract/fractions of A. tenuifolius produced dose dependent endothelium independent vasorelaxant response in coronary rings, whereas, the butanol fraction of A. tenuifolius (BS-AT) produced the largest relaxation response with 100% relaxation at 1 mg/ml, therefore the mechanism of relaxation of this fraction was determined. The relaxation to BS-AT was unaffected by removal of the endothelium, pre-contraction with KCl, or the presence of the non-selective potassium channel blocker tetraethylammonium, indicating that the relaxation was endothelium-independent, and does not involve activation of potassium channels. BS-AT (1 mg/ml) inhibited the contractile response to calcium,the L-type calcium channel activator BAY K8664,and ionomycin, indicating that it inhibits calcium-induced contractions. The relaxation response to BS-AT was attenuated in the absence of extracellular calcium. However, relaxations to BS-AT were also reduced after deletion of calcium from intracellular stores with cyclopiazonic acid. Incubation with 1 mg/ml BS-AT also inhibited phosphorylation of myosin light chains in homogenates of coronary artery. ConclusionThe butanol extract of Asphodelus tenuifolius produces a large endothelium-independent relaxation of the porcine coronary artery through inhibition of calcium-induced contractions. The effect appears to be downstream of calcium influx, possibly through inhibition of myosin light chain kinase. This study supports previous studies demonstrating that A. tenuifolius reduces blood pressure. Future studies will aim to determine the active compounds underlying this response.

Vasorelaxant Effect of Boesenbergia rotunda and Its Active Ingredients on an Isolated Coronary Artery.

Cardiovascular diseases are a major cause of death in developed countries. The regulation of vascular tone is a major approach to prevent and ameliorate vascular diseases. As part of our ongoing screening for cardioprotective natural compounds, we investigated the vasorelaxant effect of rhizomes from Boesenbergia rotunda (L.) Mansf. [Boesenbergia pandurata (Roxb.) Schltr.] used as a spice and herbal medicine in Asian countries. The methanol extract of B. rotunda rhizomes (BRE) exhibited significant vasorelaxation effects ex vivo at EC50 values of 13.4 ± 6.1 μg/mL and 40.9 ± 7.9 μg/mL, respectively, with and without endothelium in the porcine coronary artery ring. The intrinsic mechanism was evaluated by treating with specific inhibitors or activators that typically affect vascular reactivity. The results suggested that BRE induced relaxation in the coronary artery rings via an endothelium-dependent pathway involving NO-cGMP, and also via an endothelium-independent pathway involving the blockade of Ca2+ channels. Vasorelaxant principles in BRE were identified by subsequent chromatographic methods, which revealed that flavonoids regulate vasorelaxant activity in BRE. One of the flavonoids was a Diels-Alder type adduct, 4-hydroxypanduratin A, which showed the most potent vasorelaxant effect on porcine coronary artery with an EC50 of 17.8 ± 2.5 μM. Our results suggest that rhizomes of B. rotunda might be of interest as herbal medicine against cardiovascular diseases.

Tomoh Masaki

Tomoh Masaki

Nanoencapsulation of EPA:DHA 6:1 potentiates the endothelium-dependent relaxation of coronary artery rings compared to native form: role of NO, endothelium-dependent hyperpolarization and prostanoids

The omega 3 polyunsaturated fatty acid (PUFAs) formulation containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) with a 6:1 ratio is a potent inducer of endothelium-dependent nitric oxide (NO)-mediated vasorelaxation that also improves ageing-related and angiotensin II-induced endothelial dysfunction in rats. Since PUFAs are unstable, the possibility that nanoencapsulation of EPA:DHA 6:1 followed by coating potentiates the bioactivity was evaluated. EPA:DHA 6:1 was emulsified in water phase and coated with proteins and gum derivatives or used in native form. Changes in isometric tension of porcine coronary artery rings were determined using organ chambers. The role of NO was assessed using Nw-nitro-L-arginine (L-NA, NO synthase inhibitor), prostanoids using indomethacin (Indo, COX inhibitor) and endothelium-derived hyperpolarization (EDH) using TRAM-34 and UCL-1684 (Ca2 + -dependent potassium channel inhibitors). Coated EPA:DHA 6:1 nanoparticles (NP) caused in a concentration-dependent manner endothelium-dependent relaxations that were more sustained than those of the native form. At 0.3%, coated and native forms induced 56.6 ± 2.8% and 51.1 ± 1.5%relaxations at 10 min, and 93.8 ± 2.1% and 35.1 ± 1.7% at 60 min, respectively. The response to the coated EPA:DHA 6:1 NP was inhibited by L-NA and not affected by Indo, TRAM-34 plus UCL-1684, and by the previous incubation of rings with another oil (corn oil 0.3%). In contrast, L-NA, Indo, and TRAM-34 plus UCL-1684 inhibited the relaxation to EPA:DHA 6:1. Nanoencapsulation of EPA:DHA 6:1 followed by coating perpetuated their ability to cause endothelium-dependent relaxation of coronary artery rings that is exclusively mediated by NO. In contrast, the relaxing activity of the native form involved NO, EDH and vasorelaxant prostanoids. Therefore, coated EPA:DHA 6:1 NP appear to be an attractive approach to enhance the vasoprotective effect of omega 3 PUFAs.

Mechanism underlying the vasodilation induced by diosmetin in porcine coronary artery

Diosmetin is a flavonoid present naturally in citrus fruit. Plants containing diosmetin have been reported to have anti-hypertensive and vasorelaxant effects. Therefore, experiments were carried out to study the effects of diosmetin in segments of the porcine coronary artery (PCA).PCA rings were mounted for isometric tension recording in isolated tissue baths and pre-contracted with the thromboxane A2 mimetic U46619 or KCl. Cumulative concentration response curves to diosmetin were then carried out in the presence or absence of inhibitors or activators of different signaling pathways. The effect on calcium channels was determined by investigating the effect of a single concentration of diosmetin (30 μM) on calcium-induced contractions or contractions to BAY K8644.Diosmetin caused a concentration-dependent relaxation after pre-contraction with U46619 or KCl, which was unaffected by removal of the endothelium. Tetraethylammonium (TEA), and 4-aminopyridine (4-AP), but not barium chloride, caused significant inhibition of the diosmetin-mediated vasorelaxation, indicating a role for potassium channels. Diosmetin inhibited calcium-induced contractions and contractions to the L-type calcium channel opener BAY K8644. Furthermore, diosmetin inhibited the contractions in response to caffeine, cyclopiazonic acid and ionomycin, indicating a general effect on calcium-induced contractions. Contractions in response to the protein kinase C (PKC) activator Phorbol 12-myristate 13-acetate (PMA) were also inhibited by diosmetin, suggesting that it may inhibit a calcium-activated PKC isoform.In summary, diosmetin produced significant vasodilatory effects. The data indicate a role for potassium channels as well as an effect on calcium-induced contractile pathways, possible through inhibition of PKC.

English

Anogeissus leiocarpa (DC) Guill. et Perr. belongs to the Combretaceae family and was previously named A. leiocarpa (DC) Guill. Et Perr. It has been widely used in Burkina Faso by traditional medicine for the treatment of hypertension. Previous study showed that the dichloromethanolic fraction from the barks of trunk of A. leiocarpa (ALF) has induced an endothelium-independent and endothelium-dependent vasodilation effect and had the capacity to inhibit in vitro, purified cyclic nucleotide phosphodiesterases (PDEs) activity. The aims of this study were to better underline ALF-induced endothelium-independent vasorelaxation in an organ model. The results showed that ALF significantly reduce the contractile response to U46619 in porcine coronary artery rings without endothelium that were in concentration-dependent manner. In denuded rings, pretreatment by ALF (10, 30 and 100 µg/mL) did not affect relaxation to sodium nitroprusside (SNP), suggesting that relaxation to ALF was not due to its ability to be a nitric oxide donor. Moreover, SNP-induced relaxation had not been affected in the presence of Nimodipine (PDE1 inhibitor), EHNA (PDE2 inhibitor) or DMPPO (PDE5 inhibitor). In addition, the results showed a relaxation effect to isoproterenol in endothelium-denuded artery rings pretreated with ALF (3, 10, and 30 µg/mL) which were significantly affected suggesting a possible membrane hyperpolarization leading to the vasodilation. In the presence of the PDE3 specific inhibitor Cilostamide and ALF (30 and 100 µg/mL), the vasodilation effects of isoproterenol was enhanced and comparable. Moreover, various potassium channels were not involved in ALF-induced relaxation since tetraethylammonium chloride (non-selective K+ channels inhibitor), iberiotoxin (voltage-sensitive potassium channels inhibitor) and Glibenclamide (KATP channels inhibitor) did not notably affect the relaxation effect to ALF in rings without endothelium. Taken together, ALF-induced endothelium-independent relaxation mainly involves a sustained decrease in [Ca2+]i and may be due to PDE1, 3 and 5 inhibitions localized in the vascular smooth muscle cells. While, the involvement of the ions channels have not been clearly revealed in this experiment. Key words: Anogeissus leiocarpa, U46619, phosphodiesterases (PDEs), calcium, porcine coronary artery, sodium nitroprusside (SNP), isoproterenol.

Tambulin is a major active compound of a methanolic extract of fruits of Zanthoxylum armatum DC causing endothelium-independent relaxations in porcine coronary artery rings via the cyclic AMP and cyclic GMP relaxing pathways

BackgroundZanthoxylum armatum DC (Z. armatum), belonging to Rutaceae family, has been traditionally used for the treatment of various diseases such as hypertension, abdominal pain, headache, fever, high altitude sickness, diarrhea, dysentery, and as a tonic, condiment, and an anthelmintic treatment. HypothesisThe present study aims to evaluate the vasorelaxant effect of a methanolic extract of the fruits of Z. armatum, isolate the active components and characterize the underlying mechanism. Study designA methanolic extract of fruits of Z. armatum was prepared and its vasorelaxant effect was studied using porcine coronary artery rings. Thereafter, the methanolic extract was analyzed, and a major compound was isolated and its structure elucidated (tambulin). Different pharmacological tools were used to characterize the vasorelaxant effect of tambulin. ResultsThe methanolic extract and the isolated tambulin caused similar endothelium-independent relaxations of porcine coronary artery rings with and without endothelium indicating a direct relaxing effect at the vascular smooth muscle. Tambulin did not affect the relaxation curves to the endothelium-dependent vasodilators, bradykinin and the calcium ionophore A23187 in rings with endothelium. Tambulin (1 µM) slightly but significantly shifted leftwards the concentration-relaxation curve to the endothelium-independent vasodilators, sodium nitroprusside (SNP), forskolin (FC) and isoproterenol but not those to soluble guanylyl cyclase activators (YC-1 and BAY 41-2272) and K+ channel openers (levcromakalim and 1-EBIO). Pretreatment with tambulin inhibited, in a concentration-dependent manner, contractions to KCl, serotonin (5-HT), CaCl2 and U46619 in coronary artery rings without endothelium. Both the protein kinase A (H-89, 10 µM) and the protein kinase G (Rp-8-br-cyclic GMPS, 30 µM) inhibitors significantly reduced relaxations to tambulin in coronary artery rings without endothelium. ConclusionThe present findings indicate that tambulin isolated from Z. armatum (fruits) is a major active principle inducing vasorelaxation through a direct effect at the vascular smooth muscle and involving both the cyclic AMP and/or cyclic GMP relaxing pathways.

Mechanisms underlying vasorelaxation induced in the porcine coronary arteries by Thymus linearis, Benth

Ethnopharmacological relevanceThymus linearis, Benth indigenous to Pakistan has been traditionally used for the treatment of various diseases including hypertension. Aim of the studyPresent study aims to investigate vasorelaxant effect of Thymus linearis and its underlying vasorelaxation mechanisms in porcine coronary artery rings. Materials and methodsAqueous-methanolic extract of aerial parts of Thymus linearis was prepared by maceration process and then bio-guided fractionation was carried out using different solvents. The effects of extract and subsequent fractions were assessed on coronary artery rings with intact and denuded endothelium. The mechanisms of vasorelaxant effect were investigated using different pharmacological tools. The in-vitro inhibitory effects of the test fractions were also assessed on purified phophodiestrases using radioenzymatic assay. Phytochemical studies were carried out using GCMS. ResultsThe aqueous-methanolic extract elicited similar relaxations in coronary artery rings with and without endothelium in dose dependent fashion and removal of endothelium did not alter this response. Further, n-butanolic fraction of Thymus liniaris (TLB) was found to be the most potent among other derived fractions. TLB did not alter the relaxation produced by endothelium dependent vasodilators in rings with intact endothelium. However, TLB significantly potentiated the relaxation elicited by cyclic AMP and cyclic GMP elevating drugs but not those to soluble guanylyl cyclase activators (YC-1 and BAY 41–2272) and K+ channel openers (levcromakalim and 1-EBIO). Pretreatment with TLB inhibited in a concentration-dependent manner contractions to KCl, CaCl2 and U46619 in coronary artery rings without endothelium. Further, TLB was found to non-selectively inhibit the PDE activity in concentration manner. Conclusionn-Butanolic fraction of Thymus linearis possesses endothelium independent vasorelaxant effects in coronary artery by direct acting on the smooth muscles. These effects involve the elevation of the cyclic AMP and cyclic GMP possibly through the inhibition of various PDEs. GCMS analysis revel presence of thymole and carvacrol as major constituents. Furthermore, these investigations also support the folklore use of Thymus linearis in hypertension.

An aqueous extract of the Anogeissus leiocarpus bark (AEAL) induces the endothelium-dependent relaxation of porcine coronary artery rings involving predominantly nitric oxide.

Anogeissus leiocarpus is a Sahel tree traditionally used by the residents of Burkina Faso for its antihypertensive properties. In this study, experiments were conducted to evaluate whether an aqueous extract of the Anogeissus leiocarpus (AEAL) trunk bark induces a vasorelaxant effect on porcine coronary artery rings and to investigate the underlying mechanism. AEAL-induced relaxations were assessed using porcine coronary artery rings suspended in organ chambers. The phosphorylation levels of Src, Akt and endothelial nitric oxide synthase (eNOS) were assessed in a primary endothelial cell culture by Western blot. The reactive oxygen species (ROS) formation was assessed using dihydroethidine. In porcine coronary artery rings, AEAL at 0.1-300 μg/mL induced endothelium-dependent relaxations, which were inhibited in the presence of inhibitors of nitric oxide (NO) and the endothelium-derived hyperpolarization pathways. Moreover, the AEAL-induced NO-mediated relaxations were significantly reduced by the inhibitors of Src and PI3-kinase as well as by the membrane-permeant ROS scavengers. In cultured porcine coronary artery endothelial cells, treatment with AEAL is associated with an intracellular generation of ROS. Moreover, the AEAL induced the phosphorylations of Akt (Ser473), eNOS (Ser1177) and a transient phosphorylation of Src (Ser17) in a time-dependent manner. These findings indicate that AEAL is a potent inducer of endothelium-dependent NO-mediated relaxations in porcine coronary arteries through the redox-sensitive Src/PI3-kinase/Akt pathway-dependent activation of eNOS.

P-119 - Ex-vivo vasorelaxation induced by polyphenol-rich extracts from plant, fruits and berries on porcine coronary artery rings

The aim of the present study was to investigate if the polyphenol content of various commercial plant, fruits and berries extracts was linked to vasorelaxation properties. A total of 24 commercial extracts (including black currant fruit, green tea, grape seed, leucocyanidins of grape seed, pine bark, apple, pomegranate, bilberry, red vine, citrus pericarp, bitter orange, cocoa, aronia, raspberry leaf, hibiscus, olive leaf, olive fruit and a mix of olive fruit and leaf) was assessed on porcine coronary artery rings. Among all extracts, the most important endothelium-dependent vasorelaxation were obtained with grape seed (EC50=0.87 µg/ml and 2.0 µg/ml), leucocyanidins of grape seed (EC50=1.6 µg/ml), pine bark (EC50=2.2 µg/ml and 3.1 µg/ml), apple (EC50=2.2 µg/ml), cocoa (EC50=3.6 µg/ml), black currant (EC50=6.6 µg/ ml), pomegranate (EC50=6.6 µg/ml) and aronia EC50=12.7 µg/ml). The efficiency of all extracts (EC50) were significantly correlated with their content in total polyphenols (r=0.70; p=0.0002; n=23), in flavanols (r=0.65; p=0.031; n=11) and in flavonols (r=0.57; p=0.016; n=17). Our results suggest that some polyphenol-rich extracts could be potentially interesting to maintain a good cardiovascular health.

The potency of commercial blackcurrant juices to induce relaxation in porcine coronary artery rings is not correlated to their antioxidant capacity but to their anthocyanin content.

Polyphenol-rich products such as fruit juices have been found to have strong antioxidant capacities and to induce potent endothelium-dependent relaxation. We evaluated whether the commercial blackcurrant juices induced endothelium-dependent relaxation of isolated coronary arteries can be related to their antioxidant capacity and/or phenolic content. Six different commercial blackcurrant juices were selected. Their main phenolic compounds were measured by ultra-performance liquid chromatography and antioxidant capacity was evaluated by spectrometric methods. Vascular reactivity studies with these juices were done using isolated porcine coronary arteries. The six different commercial blackcurrant juices induced relaxation ranging from 21% to 100% at the concentration of 0.5% volume per volume (v/v). The relaxation induced at 0.5% v/v was not correlated to their antioxidant capacity measured by either oxygen radical antioxidant capacity or DPPH (2,2-diphenyl-1-picrylhydrazyl) assays and also not to the ascorbic acid, total polyphenols, total flavanols, and total phenolic acid contents. In contrast, the amplitude of the relaxation was correlated to the total anthocyanins content and the individual anthocyanin concentration. Correlations between relaxation amplitude and total anthocyanin or individual anthocyanin contents are of interest for the development of functional blackcurrant beverages with the potential to promote vascular protection.

O34 Endothelium-independent vasorelaxant effect of Thymus linearis via inhibition of phosphodiesterases in porcine coronary artery

Thymus linearis Benth. indigenous to Pakistan has been traditionally used for the treatment of various diseases including hypertension. The aim of the study was to investigate the vasorelaxant effect of Thymus linearis in porcine coronary artery rings. Aqueous-methanolic extract and various fractions (butanol, ethyacetate and aqueous) of Thymus linearis were prepared. The effects of extract and fractions were assessed using coronary artery rings and various pharmacological tools were applied to characterize the underlying mechanism of active fraction (butanol). The inhibitory effect of butanolic fraction was also studied on phosphodiesterases using radio enzymatic assay. Phytochemical studies were also carried out using GCMS. The aqueous-methanolic extract of Thymus linearis elicited similar relaxations in coronary artery rings with and without endothelium in a dose-dependent manner. Among the fractions butanolic fraction of Thymus linearis (TLB) was found to be the most potent. TLB significantly potentiated the relaxation induced by forskolin, isoproterenol, sodium nitroprusside and atrial natriuretic peptide whereas the relaxation induced by YC-1, BAY 41-2272, levcromakalim and 1-EBIO was unaffected by TLB. Pretreatment with TLB inhibited the contraction induced by KCl, CaCl2 and U46619 in endothelium denuded coronary artery rings. Furthermore, TLB significantly inhibited the PDE activity (PDE1CaM activated PDE2, PDE3, PDE4 AND PDE5) in a concentration-dependent manner. GCMS analysis revealed the presence of thymol and Carvacrolas as major constituents. In conclusion, TLB induced endothelium-independent relaxations in porcine coronary artery rings by acting directly at the vascular smooth muscle to increase concentrations of both cyclic AMP and cyclic GMP via inhibition of various PDEs.

O36 Endothelium-dependent contractions depending on biased sGC activation: The Janus face of thymoquinone

Thymoquinone, a major component of black seed oil, has beneficial (vasodilator, antioxidative and anti-inflammatory) effects in the cardiovascular system. Previous experiments in isolated arteries indicate that thymoquinone causes augmentations of contraction. The present study aims to identify the mechanisms underlying the counterintuitive augmentations caused by thymoquinone. Isometric tension was measured in rings of porcine coronary arteries, and rat aortae and mesenteric arteries. Precontracted preparations were exposed to increasing concentrations of thymoquinone, in the absence/presence of endothelium and of pharmacological inhibitors/activators. Cyclic nucleotide levels in rings were measured with ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC–MS/MS). Thymoquinone-induced augmentations in the three arteries were not observed in rings without endothelium, and were abolished by inhibitors of nitric oxide (NO) synthase and soluble guanylyl cyclase (sGC), L-NAME and ODQ respectively. Rho-kinase inhibitor and L-type voltage-dependent calcium channel (VDCC) blockers inhibited the augmentations in porcine, but not in rat arteries, while T-type VDCC blockers did so only in the latter. In L-NAME-treated rings, the NO donor detaNONOate and sGC activator YC-1 restored the augmentations; by contrast, in ODQ-treated rings, cell-permeable cyclic GMP did not. UPLC–MS/MS measurements showed that thymoquinone increased intracellular levels of cyclic IMP. Thymoquinone causes endothelium-dependent augmentations that depend on a biased sGC activation by NO to produce cyclic IMP instead of cyclic GMP. Cyclic IMP causes contraction of the vascular smooth muscle through alteration of calcium handling. Hence, thymoquinone is the first pharmacological agent reported to cause endothelium-dependent contractions with similar characteristics as hypoxic vasoconstrictions.

O11 Endothelium-dependent relaxation by a hydroethanolic extract of Adansonia digitata leaves in porcine coronary artery rings and rat thoracic aorta, mesenteric, carotid artery rings: Role of NO and EDH

Introduction Adansonia digitata leaves are used for the traditional treatment of hypertension in Senegal. This study evaluated the relaxant effect of a hydroethanolic extract of leaves of Adansonia digitata (ADF) in artery rings, and investigated the underlying mechanism. Methods Vascular reactivity was assessed in organ chambers. Rings were either untreated or incubated with a pharmacological tool for 30 min before contraction of coronary artery rings with either U46619 or phenylephrine, and the subsequent addition of ADF. In some experiments, the endothelium was removed mechanically. The phosphorylation level of target proteins was assessed by Western blot analysis. Results ADF induced concentration-dependent relaxations in coronary artery rings with endothelium, which reached 100% at a concentration as low as 10 μg/ml. The relaxation to ADF was significantly reduced by NLA (NO synthase inhibitor) and by the combination of Tram-34 and apamin (inhibitors of intermediate and small conductance calcium-dependent K+ channels, respectively), not affected by indomethacin (cyclooxygenase inhibitor), and abolished by wortmannin (PI3-kinase inhibitor) and MnTMPyP (membrane permeant superoxide dismutase mimetic). ADF caused relaxations in the rat superior mesenteric artery, carotid artery and aortic rings but not in the secondary mesenteric artery and femoral artery rings. ADF induced a sustained phosphorylation of Akt and eNOS at Ser1177 in cultured porcine coronary artery endothelial cells. Conclusion The Adansonia digitata leave extract induced potent endothelium-dependent relaxations, involving predominantly NO and endothelium-dependent hyperpolarization. Such vasorelaxant effects may explain the antihypertensive use of this plant in traditional African medicine.

O35 An anthocyanin-rich blackcurrant extract induced NO-mediated relaxation in coronary artery rings and eNOS phosphorylation in cultured endothelial cells: Role of sodium-glucose cotransporters 1 and 2

Introduction The beneficial cardiovascular effect of polyphenol-rich food and beverages has been attributed, at least in part, to an improved vascular function through an increased endothelial formation of nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH), two major vasoprotective mechanisms. Black currant is a rich source of anthocyanins, and, in particular, of glucoside- and rutinoside-conjugated delphinidin and cyanidin. This study evaluated the role of sodium-glucose cotransporters (SGLT) 1 and 2 in the blackcurrant extract (BCE)-induced NO-mediated endothelium-dependent relaxation and activation of endothelial NO synthase (eNOS). Methods The reactivity of porcine coronary artery rings was assessed in organ chambers, and the expression and phosphorylation level of proteins in cultured porcine coronary artery endothelial cells by Western blot analysis. Results BCE (310.0 ± 0.1 mg gallic acid equivalent/g) was prepared from a blackcurrant juice containing 2.7 g gallic acid equivalent/L of polyphenols using a Sephadex LH-20 column. BCE caused potent concentration-dependent relaxations of coronary artery rings with endothelium, which were significantly reduced by the dual SGLT1 and SGLT2 inhibitor LX4211, and also to some extent by the SGLT2 inhibitor canagliflozin but not dapagliflozin. In contrast, LX4211 did not affect relaxations to bradykinin, sodium nitroprusside and epigallocatechin gallate. BCE induced the phosphorylation of eNOS at the activator site Ser1177 in cultured endothelial cells, which was significantly prevented by LX4211, dapagliflozin and canagliflozin. Conclusion These observations indicate that BCE is a potent activator of eNOS and inducer of NO-mediated vasorelaxation possibly subsequent to the entry of conjugated anthocyanins via SGLT1/2 into endothelial cells.

Lipid Emulsions Containing Medium Chain Triacylglycerols Blunt Bradykinin-Induced Endothelium-Dependent Relaxation in Porcine Coronary Artery Rings.

Lipid emulsions for parenteral nutrition are used to provide calories and essential fatty acids for patients. They have been associated with hypertriglyceridemia, hypercholesterolemia, and metabolic stress, which may promote the development of endothelial dysfunction in patients. The aim of the present study was to determine whether five different industrial lipid emulsions may affect the endothelial function of coronary arteries. Porcine coronary artery rings were incubated with lipid emulsions 0.5, 1, or 2% (v/v) for 30min before the determination of vascular reactivity in organ chambers and the level of oxidative stress using electron paramagnetic resonance. Incubation of coronary artery rings with either Lipidem®, Medialipid® containing long- and medium-chain triacylglycerols (LCT/MCT), or SMOFlipid® containing LCT, MCT, omega-9, and -3, significantly reduced the bradykinin-induced endothelium-dependent relaxation, affecting both the nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) components, whereas, Intralipid® containing LCT (soybean oil) and ClinOleic® containing LCT (soybean and olive oil) did not have such an effect. The endothelial dysfunction induced by Lipidem® was significantly improved by indomethacin, a cyclooxygenase (COX) inhibitor, inhibitors of oxidative stress (N-acetylcysteine, superoxide dismutase, catalase) and transition metal chelating agents (neocuproine, tetrathiomolybdate, deferoxamine and L-histidine). Lipidem® significantly increased the arterial level of oxidative stress. The present findings indicate that lipid emulsions containing LCT/MCT induce endothelial dysfunction in coronary artery rings by blunting both NO- and EDH-mediated relaxations. The Lipidem®-induced endothelial dysfunction is associated with increased vascular oxidative stress and the formation of COX-derived vasoconstrictor prostanoids.