Organ Chambers Research Articles

Involvement of sodium-glucose cotransporters in the deleterious effects of acute hyperglycemia on vascular function: Implication of oxidative stress

Overconsumption of sugar has recently been implicated in the pathogenesis of cardiovascular pathologies. Previously, we managed to confirm that an acute hyperglycemia may decrease vascular function even in healthy young adults and that oxidative stress seems to be the main factor leading to vascular dysfunction. According to recent literature on other tissues, this oxidative stress would be dependent of sodium-glucose cotransporters (SGLT/SMIT) activation by hyperglycemia. Consequently, this study investigated the potential expression of these cotransporters in vascular tissues and their involvement in oxidative stress impairments of vascular function during acute hyperglycemia. First SGLT1, SGLT2 and SMIT1 expressions were evaluated, in aortas of Wistar rats by Western blot. Then vascular function was evaluated in aortic rings from Wistar rats (n = 20) mounted in an organ chamber to evaluate endothelium dependent vasodilatation (ACh 10−10 to 10−5M). This evaluation was performed in normoglycemic (11 mM) and hyperglycemic (100 mM) conditions in presence or not of an antioxidant (NAC 10 μM) or an NADPH inhibitor (Apocynin 10 μM). Implication of SGLT/SMITs and glucose transporter (GLUT) activities were assessed through their inhibition by respectively phlorizin (1 mM) and phloretin (1 mM). Our first results managed to confirm the deleterious effects of hyperglycemia on vascular function and the role of oxidative stress was pointed out through the protective effects of antioxidants. Secondly, we managed to demonstrate that SGLT1/2 and SMIT1 are expressed in aortic tissues. Moreover, SGLT/SMIT inhibition by phlorizin managed to protect endothelial function in hyperglycemic condition. Such protective effect was not observed when GLUT transporters were inhibited. Those results points out the involvement of sodium glucose cotransporters in the oxidative damaged induced by hyperglycemia while excluding GLUT implication.

The Vasorelaxant Effect of Hibiscus sabdariffa Linn. Polyphenol-Rich Extract (HPE) on Rat’s Isolated Aorta

Hibiscus sabdariffa Linn. or also known as roselle which is rich in polyphenols, has been demonstrated to cause lowering of blood pressure in animal and clinical settings. However its exact mechanism of action particularly from polyphenolic compounds is not clearly understood. Therefore, we aimed to determine the effects of H. sabdariffa polyphenol extract (HPE) towards vascular reactivity and its mechanism of action. The HPE was studied on isolated thoracic aortic rings from normal Sprague-Dawley rats, suspended in a 15-ml organ chambers containing Krebs-Henseleit solution. The changes in tension were recorded by isometric transducer connected to data acquisition. HPE relaxed the contraction induced by phenylephrine (PE, 1 μM) in similar pattern for both endothelium-intact and endothelium denuded aortic rings in dose-dependent manner. The pretreatment with atropine (1 μM), a competitive muscarinic antagonist, and propranolol (1 μM), a non-selective beta- blocker did not alter HPEs vasorelaxation response. In addition, HPE did not inhibit the contraction induced by extracellular Ca2+ precontracted by PE (1 μM) or KCl (60mM), in Ca2+ -free solution, suggesting that the relaxation effect of HPE was not via inhibition of calcium channels. In conclusion, HPE demonstrated vasorelaxation effects on rat thoracic aorta although the underlying mechanism is still unknown. The vasorelaxation effect could be via angiotensin type 1 receptor inhibition in the vascular smooth muscle cells or the activation of hyperpolarizing K+ channel. DOI : http://dx.doi.org./10.17576/JSKM-2018-08

The Vasorelaxant Effect of Hibiscus sabdariffa Linn. Polyphenol-Rich Extract (HPE) on Rat’s Isolated Aorta

Hibiscus sabdariffa Linn. or also known as roselle which is rich in polyphenols, has been demonstrated to cause lowering of blood pressure in animal and clinical settings. However its exact mechanism of action particularly from polyphenolic compounds is not clearly understood. Therefore, we aimed to determine the effects of H. sabdariffa polyphenol extract (HPE) towards vascular reactivity and its mechanism of action. The HPE was studied on isolated thoracic aortic rings from normal Sprague-Dawley rats, suspended in a 15-ml organ chambers containing Krebs-Henseleit solution. The changes in tension were recorded by isometric transducer connected to data acquisition. HPE relaxed the contraction induced by phenylephrine (PE, 1 μM) in similar pattern for both endothelium-intact and endothelium denuded aortic rings in dose-dependent manner. The pretreatment with atropine (1 μM), a competitive muscarinic antagonist, and propranolol (1 μM), a non-selective beta- blocker did not alter HPEs vasorelaxation response. In addition, HPE did not inhibit the contraction induced by extracellular Ca2+ precontracted by PE (1 μM) or KCl (60mM), in Ca2+ -free solution, suggesting that the relaxation effect of HPE was not via inhibition of calcium channels. In conclusion, HPE demonstrated vasorelaxation effects on rat thoracic aorta although the underlying mechanism is still unknown. The vasorelaxation effect could be via angiotensin type 1 receptor inhibition in the vascular smooth muscle cells or the activation of hyperpolarizing K+ channel. DOI : http://dx.doi.org./10.17576/JSKM-2018-08

Vasorelaxant and Hypotensive Effects of Cheonwangbosimdan in SD and SHR Rats.

Historically, traditional herbal medicines (THMs) have been the conventional treatment strategy in the Korean medical system for treating many diseases. However, THMs have rarely been used to treat hypertension, and moreover few studies have investigated the interaction of blood pressure with the coadministration of synthetic antihypertensives. We aimed to evaluate the vasorelaxant and hypotensive effects of the traditional herbal prescription Cheonwangbosimdan (CWBSD; “Tianwangbuxindan” in Chinese) and the combination of CWBSD with amlodipine. CWBSD was extracted with distilled water at 100°C for 2 h. To investigate vasorelaxant activities, CWBSD with amlodipine (10 μg/ml) was added cumulatively (10–1,000 μg/ml) to isolated rat aortic rings precontracted using phenylephrine or potassium chloride in organ chambers. To investigate hypotensive effects, CWBSD (2,476 mg/kg) was orally administered with or without amlodipine (5 mg/kg) to spontaneously hypertensive rats (SHRs). CWBSD increased the relaxation of rat aortic rings induced by amlodipine (P < 0.01). In vivo, CWBSD coadministration with amlodipine also significantly decreased the blood pressure of SHRs compared to the amlodipine-treated group. These results suggested that CWBSD could be a useful herbal prescription to treat hypertension and we recommend establishing guidelines for the use of herbal medicines in conjunction with antihypertensive drugs, including amlodipine.

VDR Agonist Prevents Diabetic Endothelial Dysfunction through Inhibition of Prolyl Isomerase-1-Mediated Mitochondrial Oxidative Stress and Inflammation.

Background and aim Upregulation of prolyl isomerase-1 (Pin1) protein expression and activity was associated with the pathogenesis of diabetic vasculopathy through induction of endothelial oxidative stress and inflammation. Moreover, VDR agonist protects against high glucose-induced endothelial apoptosis through the inhibition of oxidative stress. We aimed to explore the effects of the VDR agonist on diabetes-associated endothelial dysfunction and the role of Pin1 in this process. Methods Streptozocin-induced diabetic mice were randomly treated with vehicle, VDR agonist (10 μg/kg/d, i.g., twice a week), or Pin1 inhibitor, Juglone (1 mg/kg/d, i.p., every other day), for eight weeks. In parallel, human umbilical vein endothelial cells (HUVECs) exposed to high-glucose condition were treated with 1,25-dihydroxyvitamin D3 and Juglone or vehicle for 72 hours. Organ chamber experiments were performed to assess endothelium-dependent relaxation to acetylcholine. Circulatory levels of Pin1, SOD, MDA, IL-1β, IL-6, and NO in diabetic mice, Pin1 protein expression and activity, subcellular distribution of p66Shc, and NF-κB p65 in high glucose-cultured HUVECs were determined. Results Both VDR agonist and Juglone significantly improved diabetes-associated endothelial dysfunction and reduced high glucose-induced endothelial apoptosis. Mechanistically, the circulatory levels of SOD and NO were increased compared with those of vehicle-treated diabetic mice. Additionally, Pin1 protein expression and activity, p66Shc mitochondrial translocation, and NF-κB p65 in high glucose-cultured HUVECs were also inhibited by VDR agonist and Juglone. Knockdown of VDR abolished the inhibitory effects of VDR agonist on high glucose-induced upregulation of Pin1 protein expression and activity. Conclusions VDR agonist prevents diabetic endothelial dysfunction through inhibition of Pin1-mediated mitochondrial oxidative stress and inflammation.

Responsiveness of internal thoracic arteries to nitroglycerin in patients with renal failure

Nitroglycerin is commonly used as an antispasmodic for treating spasm of coronary artery bypass grafts. This study investigated whether the presence of renal failure affects reactivity to nitroglycerin in internal thoracic arteries obtained from patients undergoing coronary bypass surgery. The patients were divided into three groups according to estimated glomerular filtration rate (eGFR, mL/min/1.73m2): without renal failure (60≤eGFR, n=13), with moderate renal failure (30≤eGFR<60, n=10), and with severe renal failure (eGFR<30, n=10). Organ chamber technique was used to evaluate concentration-related responses of isolated internal thoracic arteries to vasodilators. Nitroglycerin induced a concentration-dependent relaxation, which was significantly augmented in patients with severe but not moderate renal failure than in those without renal failure. In addition, there was a negative correlation between eGFR and the relaxant efficacy of nitroglycerin (P=0.016). On the other hand, relaxant responses to BAY 60-2770 (which enhances cGMP generation as with nitroglycerin) were similar among three grades of renal function. An inverse relationship of eGFR to the relaxant efficacy of BAY 60-2770 was not observed, either (P=0.314). These findings suggest that severe renal failure specifically potentiates nitroglycerin-induced relaxation in internal thoracic artery grafts.

Downregulation of circulating MOTS-c levels in patients with coronary endothelial dysfunction

BackgroundMOTS-c is one of the newly identified mitochondrial-derived peptides which play a role in regulating metabolic homeostasis. The current study aimed to investigate whether circulating MOTS-c levels are also associated with endothelial dysfunction(ED) in patients without significant structural coronary lesions. MethodsForty patients undergoing coronary angiography and endothelial function testing for clinical indications of recurrent angina with no structural coronary lesions were included in the study. They were divided into two groups based on coronary blood flow response to intracoronary acetylcholine (ACh) as normal endothelial function (≥ 50% increase from baseline) or ED, (n=20 each). Aortic plasma samples were collected at the time of catheterization for analysis of circulating MOTS-c levels by ELISA. The effect of MOTS-c on vascular reactivity was assessed in organ chambers using aortic rings collected from rats and renal artery stenosis (RAS) mice. ResultsBaseline characteristics were similar between the two groups. MOTS-c plasma levels were lower in patients with ED compared with patients with normal endothelial function (p=0.007). Furthermore, plasma MOTS-c levels were positively correlated with microvascular (p=0.01) and epicardial (p=0.02) coronary endothelial function. Although MOTS-c did not have direct vasoactive effects, pretreating aortic rings from rats or RAS mice with MOTS-c (2μg/ml) improved vessel responsiveness to ACh compared with vessels without MOTS-c treatment. ConclusionLower circulating endogenous MOTS-c levels in human subjects are associated with impaired coronary endothelial function. In rodents, MOTS-c improves endothelial function in vitro. Thus, MOTS-c represents a novel potential therapeutic target in patients with ED.

Effects of aqueous leaf extract of Tridax procumbens on contractile activity of corpus cavernosum in N-nitro-l-arginine methyl ester-induced hypertensive male rats

ObjectiveThis study investigated the effects of aqueous leaf extract of Tridax procumbens (ALETP) on contractile activity of corpus cavernosum in N-nitro-l-arginine methyl ester (l-NAME)-induced hypertensive male rats. MethodsTwenty normal, adult male rats (130–150 g) were divided into four groups of five rats each. Group I (control) was given normal saline (0.6 mL/kg) and group II was given l-NAME (40 mg/kg) for 6 weeks. Groups III and IV also received l-NAME (40 mg/kg) for 6 weeks but were further co-treated with 100 and 200 mg/kg of ALETP, respectively, from week 4 to week 6. All treatments were given orally. Strips of corpus cavernosum from each of the four groups were exposed to increasing concentrations of acetylcholine (ACh) and sodium nitroprusside (SNP) (10−9–10−5mol/L) after contraction with phenylephrine (10−7 mol/L) to test for a dose–response effect. Response to potassium and calcium was also measured after cumulatively adding potassium and calcium (10–50 mmol/L) to potassium- and calcium-free organ chamber. Isometric contractions were recorded through an Ugo Basile data capsule acquisition system. ResultsMean arterial blood pressure was significantly reduced in the ALETP co-treated group compared to the control and l-NAME-only groups (P < 0.05). Cavernosa strips from ALETP co-treated rats exhibited significant inhibition of contraction in response to phenylephrine, potassium chloride, and calcium chloride (P < 0.05). Relaxation in response to Ach and SNP was also significantly impaired in cavernosa strips from the l-NAME-only treated group (P < 0.05), while ALETP co-treated groups showed enhanced percentage relaxation. ConclusionALETP treatment of l-NAME-induced hypertensive rats promotes a relaxant effect on isolated cavernosa strips. ALETP shows potential in correcting erectile dysfunction in hypertension.

PPAR-α agonists acutely inhibit Ca2+-independent PLA2 to reduce H2O2-induced contractions in aortae of spontaneously hypertensive rats.

Hypertension is associated with endothelial dysfunction, which favors the release of endothelium-derived contracting factors, including vasoconstrictor prostanoids and reactive oxygen species. Peroxisome proliferator-activated receptor-α (PPAR-α) agonists, clinically used as lipid-lowering drugs, possess antioxidant properties and exert beneficial effects in the vascular system. The present study aimed to identify the mechanism(s) underlying the acute effects of the PPAR-α agonists Wy14643 and fenofibate on endothelium-dependent contractions, in particular those related to oxidative stress, in the aorta of the spontaneously hypertensive rat (SHR). Aortic rings with and without endothelium of male SHRs and normotensive Wistar-Kyoto rats were suspended in organ chambers for isometric tension measurements and homogenized for enzyme activity assays. Contractions to acetylcholine in quiescent SHR aortae with endothelium were reduced by tiron (superoxide anion scavenger), diethyldithiocarbamic acid (superoxide dismutase inhibitor), and acute treatment with either Wy14643 or fenofibrate. Similarly to contractions evoked by acetylcholine, H2O2-induced increases in tension in SHR aortae involved, in succession, phospholipase A2 (PLA2), cyclooxygenase, and thromboxane-prostanoid receptors. Wy14643 or fenofibrate, by decreasing the activity of endothelial Ca2+-independent PLA2, attenuated the contractions to H2O2. In conclusion, the increased oxidative stress in the SHR aorta (mainly increased production of H2O2 and its partially reduced product, hydroxyl radical) contributed to acetylcholine-induced, endothelium-dependent contractions; PPAR-α agonists likely inhibit the H2O2-mediated contractions by inhibiting endothelial Ca2+-independent PLA2. The present study highlights the prospective therapeutic effects of PPAR-α agonists in improving endothelial function in hypertension and other vascular implications due to oxidative stress. NEW & NOTEWORTHY Peroxisome proliferator-activated receptor-α agonists, which are used clinically as lipid-lowering drugs, acutely reduce H2O2-induced contractions in aortae of hypertensive rats by inhibiting the activity of endothelial Ca2+-independent phospholipase A2. These vascular effects of peroxisome proliferator-activated receptor-α agonists suggest that they may help to prevent vascular complications under pathological conditions associated with oxidative stress.

Effects of erythropoietin on spontaneous and oxytocin induced myometrial contractions in the nonpregnant rat.

Erythropoietin (EPO) is a glycoprotein hormone that regulates erythropoiesis. EPO activity has also been detected in a variety of tissue including the nervous system, and female and male reproductive organs. It has been shown that EPO causes relaxation in vascular smooth muscle. In the present study, we have investigated effects of EPO on spontaneous and oxytocin-induced contractions of non-pregnant rat myometrium. Myometrial stripes were obtained from adult Wistar rats at the oestrous stage. The samples were placed in an isolated organ chamber under physiological conditions and 1 g passive tension. Epoetin beta (rEPO) was added cumulatively at 0.1, 1 and 10 IU/ml concentrations to the myometrial samples showing regular spontaneous contractions for periods of 30 min. Frequency and amplitude of contractions were electrophysiologically recorded and analyzed by using a BIOPAC data acquisition system. rEPO inhibited both area under curve and frequency of spontaneous contractions (ANOVA, n1, 2 = 9, f1 = 20.938, f2 = 20.492, p1,2 = 0.000). The inhibitory effect was insignificant at 0.1 mIU/ml rEPO level (Tukey HSD, p1 = 0.051, p2 = 0.581). In the oxytocin treated myometrial samples, a single dose of 1 IU/ml rEPO was studied. The area under curve and frequency values of these samples were inhibited by rEPO (Student's t-test, n = 9, t1 = 4.776, p1 = 0.000; t2 = 2.835, p2 = 0.012, respectively). rEPO inhibited spontaneous and oxytocin-induced rat myometrial contractions at 1 and 10 IU/ml concentrations. It appeared that the effect was dose-dependent.

Effect of nitric oxide synthase inhibitor Nω-Nitro-L-arginine on the penile erectile function of healthy male rats

Ojective To investigate the direct effect of exogenous NOS inhibitor Nω-Nitro-L-arginine (L-NNA) on the erectile function of healthy male rats in order to clarify the mechanism of the erectile dysfunction of type 2 diabetic rats. Methods L-NNA (50 mg/kg) was administered by oral gavage to Sprague Dawley (SD) rats for 16 weeks to induce rat model of NOS inhibition. By the end of the experiment, corpora cavernosa was isolated from the rats under anesthetization and fixed in an organ chamber for the examination of relaxation function response to acetylcholine (ACh) to reflect erectile function. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the content of cyclic guanosine monophosphate (cGMP) in cavernosal tissue. NOS activity and nitric oxide (NO) content were measured by colorimetric method. Western blotting was employed to detect the protein expression of NOS and phosphodiesterase 5 (PDE5). The activity of the antioxidative enzyme superoxide dismutase (SOD) and content of the lipid peroxidative product malondialdehyde (MDA) were analyzed to reflect oxidative stress. Results In comparison with control group, the relaxation response to acetylcholine of corpus cavernosum was significan-tly impaired in L-NNA model rats, indicating penile erectile dysfunction. Either decreased contents of NO and cGMP or suppressed activity of NOS were observed in the corpus cavernosum of L-NNA model rats and in accompany with the down-regulation of endothelial NOS (eNOS) and neuronal NOS (nNOS) expression, up-regulation of inducible NOS (iNOS) and PDE5 expression as well as an increase of oxidative stress. Incubation of corpus cavernosum from control rats with L-NNA (1-10 μmol/L) ex vivo for 30 min could also inhibit the relaxation function responses to acetylcholine in a dose-dependent manner. Treatment with L-arginine ex vivo for 45 min could improve the impairments of relaxation function responses to acetylcholine of corpus cavernosum induced by L-NNA in vivo and ex vivo. Conclusions Exogenous NOS inhibitor L-NNA could induce penile erectile dysfunction in healthy male rats and the mechanism may be related to reducing NO content and increasing oxidative stress. Key words: N′-Nitro-L-arginine; Corpus cavernosum; Erectile function; Nitric oxide; Oxidative stress

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.

O10 Beneficial effect of Phyllanthus amarus (Euphorbiaceae) on DOCA-salt-induced left ventricle cardiac hypertrophy and endothelial dysfunction in rats

Introduction Phyllanthus amarus is used in African traditional medicine to treat cardiovascular disorders such as hypertension. This study has evaluated the effect of an aqueous extract of Phyllanthus amarus (AEPA) on blood pressure, cardiac and endothelial function in the deoxycorticosterone acetate and high salt diet (DOCA-salt)-induced hypertensive rats. Methods Male Wistar rats were assigned into five groups including control, AEPA (100 mg/kg), DOCA-salt (50 mg/kg), and DOCA-salt + AEPA(100 or 300 mg/kg). Systolic blood pressure (SBP) was determined by tail-cuff plethysmography, left ventricle cardiac structure and function by echocardiography, vascular reactivity of main mesenteric artery rings using organ chambers, the vascular level of oxidative stress using dihydroethidine staining, and the level of proteins by immunofluorescence methods. Results After 5 weeks, the AEPA treatments (100 or 300 mg/kg/day) significantly lowered SBP in DOCA-salt hypertensive rats by about 24 and 21 mmHg, respectively. Echocardiography indicated that AEPA affected slightly cardiac function and reduced significantly the increased posterior and septum diastolic wall thickness and the left ventricle mass in hypertensive rats. AEPA improved the DOCA-salt-induced endothelial dysfunction, and the blunted nitric oxide- and endothelium-dependent hyperpolarization-mediated relaxations in mesenteric artery rings. AEPA treatments reduced the level of vascular oxidative stress and the expression level of target proteins (eNOS, COX-2, p22 phox ) in DOCA-salt rats. Conclusion The Phyllanthus amarus aqueous extract induced an antihypertensive effect associated with an improved cardiac structure and endothelial dysfunction in DOCA-salt hypertensive rats. Such a beneficial effect might involve the normalization of the level of vascular oxidative stress.

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.

In vivo imaging of murine vasodynamics analyzing different mouse strains by optical coherence tomography

Background and aimsWe tried to circumvent the limitations of standard organ chamber experiments using in vivo optical coherence tomography (OCT) to analyze the vascular function of small arteries in different mouse strains. MethodsOCT images were acquired with a two-axis galvanometer scanner head. Time series (3 frames per second, 300 × 512 pixel per frame) of cross-sectional images were analyzed with image processing software measuring the time course of vessel lumen dynamics. Vascular function of murine saphenous artery of male C57BL/6 (wild-type) and hypercholesterolemic LDLR knockout (LDLR−/−) mice was analyzed at 6 weeks and after 14 weeks feeding a control or high-fat diet containing 21.2% butter fat and 2.1 mg/kg cholesterol. Vasoconstriction and vasodilation was analyzed by OCT in response to 80 mM K+ and 1 mM SNP. ResultsThe OCT technique allowed determination of inner diameter, flow resistance, maximal velocity of diameter change and time to half-maximal diameter change in murine saphenous arteries of wild-type and LDLR−/− mice. LDLR−/− had impaired vasodilation and changes in vasodynamics after 14 weeks on control or high-fat diet, compared to wild-type mice. The diameter of the saphenous artery of LDLR−/− mice was reduced after vasoconstriction (38 ± 7 μm vs 12 ± 6 μm) and vasodilation (245 ± 8 μm vs 220 ± 10 μm) (P < 0.05 vs C57BL/6). ConclusionOCT was used as an innovative method to image vascular function of small arteries of wild-type and hypercholesterolemic LDLR−/− mice after high-fat diet. The method offers the ability to display differences in the vasodynamics at early stages of endothelial dysfunction in vivo.

Abstract 634: Partial Genetic Disruption of mTOR Signaling Does Not Improve Vascular Endothelial Function in Hypercholesterolemic Mice

Endothelial cell senescence promotes inflammation and impairs endothelium-dependent vasodilation. While aging, hypercholesterolemia, and caloric excess are known to drive cellular senescence in part through activation of mTOR, it is unknown whether reducing mTOR signaling can improve vasomotor function in aging mice. Here, we used hypercholesterolemic mice (Ldlr -/- /apoB 100/100 , or LA) carrying intact copies of mTOR (LA-mTOR +/+ ) or were deficient in one copy of mTOR (LA- mTOR +/- ). Mice were fed Western Diet for 6 or 12 months, and vasomotor function in aorta was evaluated using isolated organ chamber baths. Maximum relaxation to acetylcholine (MR ACH ) was progressively impaired from 6 months to 12 months in LA-mTOR +/+ mice (55±4% and 30±4%, respectively). In contrast to our hypothesis, however, MR ACH was not significantly improved in LA-mTOR +/- mice at either time point (62±3% and 35±5%, respectively). Interestingly, a subgroup analysis showed significant improvement in MR ACH in male mice at the 6-month point (male LA-mTOR +/+ mice = 30 ± 9% vs male LA-mTOR +/- mice = 55±4%; p &lt; 0.01), whereas vasomotor function in female mice was nearly identical across groups. There was no significant difference at the 12-month point (male LA-mTOR +/+ mice = 19 ± 3% vs male LA-mTOR +/- mice = 22 ± 5%). Relaxation to acetylcholine was attenuated by L-NAME at 6 months and 12 months (but identical between genotypes) and relaxation to nitroprusside was identical between strains at each time point, suggesting that changes in endothelial function were not masked by compensatory mechanisms (e.g., endothelium-derived hyperpolarizing factors). Furthermore, reduction of mTOR did not elicit changes in maximal tension generated in response to Prostaglandin F 2 α at either time point. Collectively, despite compelling evidence from in vitro model systems, genetic reduction of mTOR signaling in vivo does not appear to be sufficient to improve vasomotor function in our model of aging hypercholesterolemic mice. Our data do, however, shed light on a potential role of sex in dictating phenotypic changes in early to moderate stages of disease, which warrants further investigation.

Impact of type 2 diabetes on vascular reactivity to cGMP generators in human internal thoracic arteries

ObjectiveThe balance between nitric oxide (NO)-sensitive and -insensitive forms of soluble guanylate cyclase (sGC) has been demonstrated to be disrupted in certain lifestyle-related diseases. However, it remains unclear whether type 2 diabetes results in a shift of sGC to the NO-insensitive form. This study addressed this issue in the human blood vessel. MethodsInternal thoracic arteries were obtained from patients undergoing coronary artery bypass grafting. Helically cut strips of the arteries were suspended in organ chambers, and relaxant responses to nitroglycerin (NO-sensitive sGC stimulant) and BAY 60-2770 (NO-insensitive sGC stimulant) were assessed. ResultsThe patients were divided into two groups according to the presence of type 2 diabetes (HbA1c: 7.0±0.3%) or its absence (HbA1c: 5.6±0.1%). Nitroglycerin-induced relaxation was not different in the arteries obtained from type 2 diabetic and non-diabetic patients. In addition, the relaxant response to BAY 60-2770 in type 2 diabetics was comparable to that observed in non-diabetics. Although the patients enrolled often had vascular risk factors other than type 2 diabetes, the relaxant responses were still in the same range in a comparison based on the number of risk factors. However, in separate experiments, the relaxant response to nitroglycerin was attenuated by pre-incubation of the arteries with ODQ (sGC imbalance inducer), whereas the relaxant response to BAY-60-2770 was augmented. ConclusionsThese findings suggest that type 2 diabetes does not affect the balance between NO-sensitive and -insensitive sGC in human internal thoracic artery grafts.

Homocysteine ameliorates the endothelium-independent hypoxic vasoconstriction via the suppression of phosphatidylinositol 3-kinase/Akt pathway in porcine coronary arteries

ObjectiveEndothelium-independent coronary vasoconstriction induced by continuous hypoxia contributes to the development of ischemic heart diseases. Acute elevation of homocysteine (Hcy) has a potent of vasodilation. The present study aims to investigate the role of Hcy in endothelium-independent hypoxic coronary vasoconstriction and its underlying mechanisms. Methods and resultsVessel tension of isolated porcine coronary arteries was measured by organ chamber study and the protein expression were detected by western blot. A sustained contraction of porcine coronary artery was induced when exposed to prolonged hypoxia for more than 15 min, which was significantly reduced by Hcy in a dose-dependent manner but not affected by cysteine or N-acetyl-l-cysteine. Phosphorylated myosin light chain (MLC-p) at Ser19 was decreased when exposure to hypoxia for 15 min, and could be reversed by prolonged hypoxia for 30 and 60 min. The recovery of MLC-p at Ser19 by hypoxia for more than 30 min could be abolished by Hcy. The protein levels of phosphorylated Akt at Ser473 and phosphorylated P85 at Tyr508 were decreased by Hcy in normoxia, and were also reduced exposure to hypoxia for 15 min and then augmented by prolonged hypoxia for more than 30 min, which could be prevented by Hcy. The protein level of P110α was not affected by Hcy or prolonged hypoxia. ConclusionsThis study demonstrates that Hcy can ameliorate the endothelium-independent hypoxic coronary vasoconstriction, in which the inhibition of PI3K/Akt signaling pathway may be involved.

Responsiveness of Coronary Arteries to Nitroglycerin under Hypoxia: The Importance of the Endothelium

Background/Aims: Nitroglycerin is widely used as a coronary vasodilator in the treatment of ischemic heart diseases. This study investigated the influence of hypoxia on nitroglycerin-induced relaxation in endothelium-intact and -denuded rabbit, monkey, and porcine coronary arteries. Methods: Helically cut strips of coronary arteries were suspended in organ chambers, and isometric tension was recorded. Results: Nitroglycerin concentration dependently relaxed endothelium-intact rabbit coronary arteries, which were not different under normoxic and hypoxic conditions. On the other hand, nitroglycerin-induced relaxation of endothelium-denuded arteries was significantly attenuated by hypoxia. Similarly, the relaxant response of endothelium-intact monkey coronary arteries to nitroglycerin was not affected by hypoxia, whereas that of endothelium-denuded arteries was impaired. As is the case with rabbit and monkey coronary arteries, exposure to hypoxia resulted in impaired relaxation by nitroglycerin in endothelium-denuded but not endothelium-intact porcine coronary arteries. Conclusion: These findings suggest that coronary endothelium plays a pivotal role in preventing the hypoxia-induced impairment of nitroglycerin responsiveness, regardless of the animal species.

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.