Vasorelaxation In Aortic Rings Research Articles

The Protective Effect of Indole Alkaloid Vincanine Against Hypoxia-Induced Vasorelaxation Model of Rat Aorta

Introduction: Using conventional organ bath procedures, the current study sought to determine how vincanine hydrochloride affected vasorelaxation brought on by hypoxia in rat aortic rings. Methods: To induce hypoxia, we used a glucose-free Krebs solution that was infused with 95% N2 and 5% CO2. After 60 minutes of hypoxia, the effect of vincanine was evaluated on aortic rings that were precontracted with either 50 mM KCl or 1 µM phenylephrine (PE). The effect of vincanine was more noticeable in aortic rings that had been precontracted by PE as opposed to KCl. Additionally, when verapamil, a blocker of L-type VDCCs, was preincubated with endothelium-intact aortic rings and KCI was used for precontraction, the effect of vincanine on hypoxia-induced vasorelaxation was significantly reduced. Results: Vincanine inhibited hypoxia-induced vasorelaxation in aortic rings precontracted with PE in a calcium-free buffer. Furthermore, the presence of glibenclamide, a specific inhibitor of ATP-sensitive K+-channels (KATP), and tetraethylammonium chloride (TEA), a nonspecific inhibitor of calcium-activated large conductance K+-channels (BKca), significantly reduced the effect of vincanine on hypoxia-induced vasorelaxation. The removal of the endothelium also had a significant impact on the effect of vincanine on hypoxia-induced vasorelaxation. Conclusion: The present findings showed that alkaloid vincanine isolated from the leaves of Vinca minor H. significantly abolished the hypoxia-induced vasorelaxation in rat aorta. The obtained results suggest that vincanine may protect the rat aorta against hypoxic injuries in the vasculature.

Use of Nitric Oxide Donor-Loaded Microbubble Destruction by Ultrasound in Thrombus Treatment.

In the presence of a vascular thrombus, the recovery of blood flow and vascular recanalization are very important to prevent tissue damage. An alternative procedure to thrombolysis is required for patients who are unable to receive surgery or thrombolytic drugs due to other physical conditions. Recently, the performance of thrombolysis combined with microbubbles has become an attractive and effective therapeutic procedure. Indeed, in a recent study, we demonstrated that, upon exposure to ultrasound, liposomes loaded with nitric oxide release agonists conjugated to microbubbles; therefore, there is potential to release the agonist in a controlled manner into specific tissues. This means that the effect of the agonist is potentiated, decreasing interactions with other tissues, and reducing the dose required to induce nitric-oxide-dependent vasodilation. In the present study, we hypothesized that a liposome microbubble delivery system can be used as a hydrophilic agonist carrier for the nitric oxide donor spermine NONOate, to elicit femoral vasodilation and clot degradation. Therefore, we used spermine-NONOate-loaded microbubbles to evaluate the effect of ultrasound-mediated microbubble disruption (UMMD) on thromboembolic femoral artery recanalization. We prepared spermine NONOate-loaded microbubbles and tested their effect on ex vivo preparations, hypothesizing that ultrasound-induced microbubble disruption is associated with the vasorelaxation of aortic rings. Thrombolysis was demonstrated in aorta blood-flow recovery after disruption by spermine NONOate-loaded microbubbles via ultrasound application in the region where the thrombus is located. Our study provides an option for the clinical translation of NO donors to therapeutic applications.

Aging Additively Influences Insulin- and Insulin-Like Growth Factor-1-Mediated Endothelial Dysfunction and Antioxidant Deficiency in Spontaneously Hypertensive Rats.

This study aimed to investigate the aging-related endothelial dysfunction mediated by insulin and insulin-like growth factor-1 (IGF-1) and antioxidant deficiency in hypertension. Male spontaneously hypertensive rats (SHRs) and age-matched normotensive Wistar–Kyoto rats (WKYs) were randomly divided into 24-week-old (younger) and 48-week-old (older) groups, respectively. The endothelial function was evaluated by the insulin- and IGF-1-mediated vasorelaxation of aortic rings via the organ bath system. Serum levels of nitric oxide (NO), malondialdehyde (MDA), catalase, and total antioxidant capacity (TAC) were examined. The insulin- and IGF-1-mediated vasorelaxation was significantly impaired in both 24- and 48-week-old SHRs compared with age-matched WKYs and was significantly worse in the 48-week-old SHR than the 24-week-old SHR. After pretreatments of phosphoinositide 3-kinase (PI3K) or NO synthase (NOS) inhibitors, the insulin- and IGF-1-mediated vasorelaxation became similar among four groups. The serum level of MDA was significantly increased, while the NO, catalase, and TAC were significantly reduced in the 48-week-old SHR compared with the 24-week-old SHR. This study demonstrated that the process of aging additively affected insulin- and IGF-1-mediated endothelial dysfunction in SHRs, which could be partly attributed to the reduced NO production and antioxidant deficiency.

The new organic nitrate 2-nitrate-1,3-diocthanoxypropan (NDOP) induces nitric oxide production and vasorelaxation via activation of inward-rectifier potassium channels (KIR)

IntroductionCardiovascular diseases are coupled to decreased nitric oxide (NO) bioavailability, and there is a constant search for novel and better NO-donors. Here we synthesized and characterized the cardiovascular effects of the new organic nitrate 2-nitrate-1,3-dioctanoxypropan (NDOP). MethodsA combination of in vitro and in vivo experiments was performed in C57BL/6 mice and Wistar rats. Thus, the ability of NDOP in donating NO in a cell-free system and in vascular smooth muscles cells (VSMC) and its ability to induce vasorelaxation in aortic rings from mice were evaluated. In addition, changes in blood pressure and heart rate to different doses of NDOP were evaluated in conscious rats. Finally, acute pre-clinical toxicity to oral administration of NDOP was assessed in mice. ResultsIn cell-free system, NDOP increased NO levels, which was dependent on xanthine oxidoreductase (XOR). NDOP also increased NO levels in VSMC, which was not influenced by endothelial NO synthase. Furthermore, incubation with the XOR inhibitor febuxostat blunted the vasorelaxation in aortic ring preparations. In conscious rats, NDOP elicited dose-dependent reduction in blood pressure accompanied with increased heart rate. In vessel preparations, NDOP (10−8-10−3 mol/L) induced endothelium-independent vasorelaxation, which was inhibited by the NO scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and hydroxocobalamin or by inhibition of soluble guanylyl cyclase using H- [1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one. To investigate if NDOP acts through potassium channels, selective blockers were used. Inhibition of BKCa, Kv or KATP subtypes of potassium channels had no effect, but inhibition of inward-rectifier potassium channels (KIR) significantly reduced NDOP-mediated vasorelaxation. Lastly, NDOP showed low toxicity (LD50 ~5000 mg/kg). ConclusionBioactivation of NDOP involves functional XOR, and this new organic nitrate elicits vasorelaxation via NO-cGMP-PKG signaling and activation of KIR channels. Future studies should further characterize the underlying mechanism and evaluate the therapeutic benefits of chronic NDOP treatment in relevant cardiovascular disease models.

BJ-PRO-7A and BJ-PRO-10C induce vasodilatation and inotropic effects in normotensive and hypertensive rats: Role of nitric oxide and muscarinic receptors

Bj-PRO-7a and Bj-PRO-10c belong to a family of proline-rich oligopeptides (PROs) identified in Bothrops jararaca (Bj) crude venom. Previous studies have shown an antihypertensive effect evoked by theses peptides. However, the mechanisms underlying the direct effects on vessels and heart remain to be unraveled. Thus, we investigated the effect of the Bj-PRO-7a and Bj-PRO-10c in the aorta and coronary arteries and in cardiac contractility in normotensive (Wistar) and hypertensive (SHR) rats. Pre-constricted aortic rings were exposed to increasing concentrations of Bj-PROs in presence or absence of muscarinic type 1 receptor antagonist (Pirenzepine), nonselective muscarinic receptor antagonist (Atropine), nitric oxide synthase inhibitor (L-NAME), guanylyl cyclase inhibitor (ODQ), adenylyl cyclase inhibitor (MDL), or argininosuccinate synthetase inhibitor (MDLA). The effects of Bj-PROs in the cardiac contractility and coronary vasomotricity were evaluated using Langendorff perfused heart preparation. The rat hearts were perfused with Bj-PRO-7a or Bj-PRO-10c in absence or presence of L-NAME, ODQ or MDL. Both Bj-PROs induced endothelium-dependent vasorelaxation in aortic rings from Wistars and SHRs. These effects were inhibited by L-NAME, ODQ or MDL. Atropine and Pirenzepine blocked the vasorelaxant effect of Bj-PRO-7a in aorta from both strains. MDLA inhibited the Bj-PRO-10c-induced vasorelaxation in aortic rings from SHR, but not Wistar. The Bj-PRO-7a induced coronary vasodilation only in SHR. L-NAME, ODQ and MDL inhibited this effect. Bj-PRO-10c induced coronary vasodilatation in both strains, which was blocked by L-NAME, ODQ and MDL. Bj-PRO-7a decreased the dP/dt max in Wistar hearts and the dP/dt min in Wistar and SHR hearts. These effects were abolished by L-NAME. Bj-PRO-10c decreased dP/dt max and dP/dt min in hearts from normotensive and hypertensive animals, which were abolished in the presence of L-NAME, MDL and ODQ. In summary, the Bj-PROs induced endothelium-dependent vasorelaxation in rat thoracic aorta, coronary vasodilation and negative inotropic effects through mechanisms mediated by activation of nitric oxide pathway.

Acute Hyperbaric Oxygenation, Contrary to Intermittent Hyperbaric Oxygenation, Adversely Affects Vasorelaxation in Healthy Sprague-Dawley Rats due to Increased Oxidative Stress.

The present study was aimed at assessing endothelium-dependent vasorelaxation, at measuring superoxide production in the aorta and femoral artery, and at determining antioxidative enzyme expression and activity in aortas of male Sprague-Dawley rats (N = 135), randomized to an A-HBO2 group exposed to a single hyperbaric oxygenation session (120′ of 100% O2 at 2.0 bars), a 24H-HBO2 group (single session, examined 24 h after exposure), a 4D-HBO2 group (4 consecutive days of single sessions), and a CTRL group (untreated group). Vasorelaxation of aortic rings in response to acetylcholine (AChIR) and to reduced pO2 (HIR) was tested in vitro in the absence/presence of NOS inhibitor L-NAME and superoxide scavenger TEMPOL. eNOS, iNOS, antioxidative enzyme, and NADPH oxidase mRNA expression was assessed by qPCR. Serum oxidative stress markers and enzyme activity were assessed by spectrometry, and superoxide production was determined by DHE fluorescence. Impaired AChIR and HIR in the A-HBO2 group were restored by TEMPOL. L-NAME inhibited AChIR in all groups. Serum oxidative stress and superoxide production were increased in the A-HBO2 group compared to all other groups. The mRNA expression of iNOS was decreased in the A-HBO2 and 24H-HBO2 groups while SOD1 and 3 and NADPH oxidase were increased in the 4D-HBO2 group. The expression and activity of catalase and glutathione peroxidase were increased in the 4D-HBO2 group as well. AChIR was NO dependent. Acute HBO2 transiently impaired vasorelaxation due to increased oxidative stress. Vasorelaxation was restored and oxidative stress was normalized 24 h after the treatment.

Exercise modulates the aortic renin-angiotensin system independently of estrogen therapy in ovariectomized hypertensive rats

The renin-angiotensin-system is an important component of cardiovascular control and is up-regulated under various conditions, including hypertension and menopause. The aim of this study was to evaluate the effects of swimming training and estrogen therapy (ET) on angiotensin-II (ANG II)-induced vasoconstriction and angiotensin-(1-7) [ANG-(1-7)]-induced vasorelaxation in aortic rings from ovariectomized spontaneously hypertensive rats. Animals were divided into Sham (SH), Ovariectomized (OVX), Ovariectomized treated with E2 (OE2), Ovariectomized plus swimming (OSW) and Ovariectomized treated with E2 plus swimming (OE2+SW) groups. ET entailed the administration of 5μg of 17β-Estradiol three times per week. Swimming was undertaken for sixty minutes each day, five times per week. Both, training and ET were initiated seven days following ovariectomy. Forty-eight hours after the last treatment or training session, the animals’ systolic blood pressures were measured, and blood samples were collected to measure plasma ANG II and ANG-(1-7) levels via radioimmunoassay. In aortic rings, the vascular reactivity to ANG II and ANG-(1-7) was assessed. Expression of ANG-(1-7) in aortic wall was analyzed by immunohistochemistry. The results showed that both exercise and ET increased plasma ANG II levels despite attenuating systolic blood pressure. Ovariectomy increased constrictor responses to ANG II and decreased dilatory responses to ANG-(1-7), which were reversed by swimming independently of ET. Moreover, it was observed an apparent increase in ANG-(1-7) content in the aorta of the groups subjected to training and ET. Exercise training may play a cardioprotective role independently of ET and may be an alternative to ET in hypertensive postmenopausal women.

Abstract P281: Alamandine-induced Vasorelaxation is Selectively Increased in Sp-shr

The renin angiotensin system is implicated in hypertension and cardiovascular diseases. It’s actions are dependent of counter-regulatory modulation of its vasopressor and vasodepressor axes. Recently, our laboratory described and characterized a new component the RAS, alamandine and its receptor, the MrgD. Alamandine can be formed from angiotensin A through the action ACE2 or from angiotensin-(1-7), by a still unknown decarboxylase. Among the actions of alamandine, a endothelium-dependent vasorelaxation in aortic rings of mice and rats has been described. The aim of this study was to investigate the vasorelaxing effect of alamandine in aortic rings of hypertensive rats. The vasorelaxing effect of alamandine was tested in aortic rings taken from SP-SHR, Wistar and SHR, pre-contracted with phenylephrine (0.1 μM) with and without enthotelium or in the presence of L-Name (100 μM ) or Indomethacin ( 10 μM). In aortic rings from SP-SHR alamandine produced a pronounced dose-dependent relaxation when compared with Wistar (Emax= 80± 6,0 vs 45 ± 4). This response was diminished in the presence of L-Name (Emax= 39 ± 7 in SP-SHR and 1,0 ± 3 in Wistar). Indomethacin also attenuated the vasorelaxation produced by alamandine in aortic rings from SP-SHR (Emax = 40 ± 4,0), while a smaller effect was observed in Wistar rats (Emax= 31 ± 4). The vasorelaxing effect of alamandine in SP-SHR was abolished in endothelium desnude rings (Emax= -9 ± 4). When we compare two models of animals with hypertension, SHR-SP and SHR the vasorelaxing effect of alamandine was also more pronounced in SP-SHR ( Emax = 80± 6,0 and 38 ± 4 respectively).Taken together these results suggest that the vasorelaxing effect of alamandine is selectively increased in SP-SHR. The mechanism of it effect in SP-SHR appears to involve NO and prostaglandins release.

Endothelial NO Production Is Mandatory for Epigallocatechin-3-Gallate–induced Vasodilation

:The underlying mechanisms for the vasodilating effects of the tea catechin epigallocatechin-3-gallate (EGCG) are still not fully understood. Besides nitric oxide (NO)-dependent effects, other modes of action are discussed. To elucidate whether the NO pathway is a prerequisite in mediating vasodilating effects, we investigated EGCG-induced vasorelaxation in isolated aortic rings of endothelial nitric oxide knockout (eNOS−/−) mice. Vasodilation to acetylcholine was fully prevented in aortic rings of eNOS−/− mice, confirming lack of vascular NO production. Vasodilation to the exogenous NO donor sodium nitroprusside was preserved in eNOS−/− mice aortic rings. Low concentrations of EGCG (5–15 µM) resulted in strong vasorelaxation in aortic rings of wild type mice, whereas it was completely absent in eNOS−/− mice. In corroboration, relaxation in response to green tea was significantly inhibited in aortic rings of eNOS−/− mice. These results demonstrate that EGCG-induced vasodilation strongly relies on functional NO synthase in endothelial cells and subsequent stimulation of NO production in vessels.

Icariin regulates PRMT/ADMA/DDAH pathway to improve endothelial function

Oxidative stress may affect PRMT/ADMA/DDAH (protein arginine methyltransferases/asymmetric dimethylarginine/dimethylarginine dimethylaminohydrolase) pathway to impair endothelial dysfunction. The present study was carried out to test the effect of icariin on endothelial function and the mechanisms responsible for this. Eighty mice at 12 weeks of age were separated randomly into four groups (n = 20): C57BL/6J control, untreated apolipoprotein E-deficient (ApoE(-/-)), two groups of icariin-treated (10 or 30 mg/kg body wt/day, intragastrically) ApoE(-/-). Primary human umbilical vein endothelial cells (HUVECs) were randomly divided into 7 groups: control group, vehicle of icariin (10 μmol/L) group, icariin (10 μmol/L) group, lysophosphatidylcholine (LPC) (10 μg/mL) group, LPC plus icariin (1 μmol/L) group, LPC plus icariin (3 μmol/L) group, and LPC plus icariin (10 μmol/L) group. In ApoE(-/-) mice and primary HUVECs, icariin treatment decreased reactive oxygen species production, PRMT I expression, ADMA level, half-maximum effective concentration of ApoE(-/-) mice aortic rings. Icariin increased DDAH II expression, DDAH activity, maximal relaxation value and endothelium-dependent vasorelaxation in aortic rings from ApoE(-/-) mice (p < 0.05 or p < 0.01). The present results suggest that icariin regulates PRMT/ADMA/DDAH pathway to improve endothelial function.

Abstract 14650: A Monoclonal Antibody to a Sodium Pump Inhibitor Marinobufagenin Reversed Aortic Remodeling and Stiffness in Normotensive Rats on a High Salt Intake

Background: Marinobufagenin (MBG), an endogenous cardiotonic steroid, is a Na/K-ATPase inhibitor and a vasoconstrictor. Previously it was demonstrated, that administration of 3E9 anti-MBG-antibody (mAb) reduced blood pressure (BP) and reversed left ventricular fibrosis in animal models of salt-sensitive hypertension and nephropathy. In the present study we investigated whether mAb alleviates BP and vascular remodeling in normotensive rats on a high salt intake. Methods: Wistar rats (5 months old) received normal salt diet (CTRL; n=8) or high salt intake (2% NaCl in drinking water) for 4 weeks. Rats on a high salt were administered vehicle (SALT; n=8) or mAb (50 ug/kg) (SALT-AB; n=8) 3 times during the last week of a high salt diet. BP was measured at baseline, after 3 and 4 weeks of experiment. Na/K-ATPase activity was measured in erythrocytes. Aortas were weighed, and were used to study sensitivity to the vasorelaxant effect of sodium nitroprusside (SNP), and for the histochemistry analysis of collagen deposition. Renal 24-hr MBG excretion was measured at week 4. Results: In SALT vs. CTRL, in the absence of BP changes, elevated levels of MBG (14.1±1.1 vs. 9.0±1.6 pmol/24hr, p&lt;0.05) were associated with inhibition of erythrocyte Na/K-ATPase (12.6±0.3 vs. 14.2±0.35 μmol Pi/ml/hr, p&lt;0.05), increased aortic weights (217±15 vs. 158±9 mg/kg BW, p&lt;0.01), increased levels of collagen in aorta (2.5-fold; p&lt;0.05), and compromised SNP vasorelaxant effect in aortic explants (EC50=167±19.3 nM vs. 99±2.0 nM; P&lt;0.01). Antibody treatment in SALT-AB vs. SALT increased Na/K-ATPase activity (13.93±0.54 μmol Pi/ml/hr, p&lt;0.05), reduced the aortic weight (180±12 mg/kg; P&lt;0.05) and collagen deposition 3-fold (P&lt;0.05), and restored the vasorelaxation of aortic rings by SNP to the levels in CTRL (70±1.5 nM, p&lt;0.01). Conclusion: These findings for the first time demonstrated that in normotensive rats on a high salt intake heightened MBG levels induced vascular fibrosis and impairment of vasorelaxation in the absence of blood pressure changes. Immunoneutralization of MBG reversed these changes. Thus, high dietary NaCl intake in normotensive animals can stimulate vascular fibrosis via pressure-independent/ MBG-dependent mechanisms, and this remodeling is reversible.

Effect of combination treatment of S–amlodipine with peroxisome proliferator-activated receptor agonists on metabolic and cardiovascular parameters in Zucker fa/fa rats

BackgroundType 2 diabetes is a complex metabolic disorder characterized by hyperglycemia, impaired glucose tolerance and insulin resistance associated with dyslipidemia and hypertension. The available drugs are not sufficiently efficacious in reducing cardiovascular risk and restoring normal glucose metabolism associated with type 2 diabetes as a mono- or a combination therapy. The present study examined the combined effects of an antihypertensive (S-Amlodipine) and an insulin-sensitizing agent, peroxisome proliferator-activated receptor (PPAR) agonists (Pioglitazone and Ragaglitazar), on cardiovascular risk factors in aged diabetic and insulin-resistant Zucker fa/fa rats.MethodsFollowing combination treatment for 14 days, blood pressure (BP), serum glucose, total cholesterol and triglycerides were measured. Aortic ring study was conducted to determine the effect of combination treatments on phenylephrine-induced vasoconstriction and acetylcholine (Ach)-induced vasorelaxation.ResultsIn combination, S-Amlodipine and Pioglitazone significantly reduced blood glucose (115.1 ± 6.6 vs. 81.7 ± 4.2), BP (184.4 ± 5.0 vs. 155.1 ± 5.0), serum triglycerides (362.5 ± 47.5 vs. 211.1 ± 23.7) and glucose intolerance when compared with vehicle treated Zucker fa/fa rats. Similar results were observed with the combination of S-Amlodipine and Ragaglitazar (Triglycerides, 362.5 ± 47.5 vs. 252.34 ± 27.86; BP, 184.4 ± 5.0 vs. 159.0 ± 8.0) except for serum glucose. ACh-induced vasorelaxation in aortic rings was also superior with both of the combinations compared to individual treatment. Furthermore, there was less body weight gain and food intake with S-Amlodipine and Pioglitazone combination in Zucker fa/fa rats. S-Amlodipine itself caused significant reduction in glucose (115.1 ± 6.6 vs. 89.7 ± 2.7) and BP (184.4 ± 5.0 vs. 156.1 ± 4.0) with improvement in insulin sensitivity observed through oral glucose tolerance test.ConclusionsThe results suggest that a combination of PPAR agonists and S-Amlodipine has partial benefits in improving the cardiovascular risk factors such as reduction in triglyceride levels, associated with chronic type 2 diabetes, and therefore may be utilized as an approach for addressing some of these devastating metabolic syndrome complications.

Cannabinoid receptor type 1 activation by arachidonylcyclopropylamide in rat aortic rings causes vasorelaxation involving calcium-activated potassium channel subunit alpha-1 and calcium channel, voltage-dependent, L type, alpha 1C subunit

Cannabinoids are key regulators of vascular tone, some of the mechanisms involved include the activation of cannabinoid receptor types 1 and 2 (CB); the transient receptor potential cation channel, subfamily V, member 1 (TRPV1); and non-(CB1)/non-CB2 receptors. Here, we used the potent, selective CB1 agonist arachidonylcyclopropylamide (ACPA) to elucidate the mechanism underlying vascular tone regulation. Immunohistochemistry and confocal microscopy revealed that CB1 was expressed in smooth muscle and endothelial cells in rat aorta. We performed isometric tension recordings in aortic rings that had been pre-contracted with phenylephrine. In these conditions, ACPA caused vasorelaxation in an endothelium-independent manner. To confirm that the effect of ACPA was mediated by CB1 receptor, we repeated the experiment after blocking these receptors with a selective antagonist, AM281. In these conditions, ACPA did not cause vasorelaxation. We explored the role of K+ channels in the effect of ACPA by applying high-K+ solution to induce contraction in aortic rings. In these conditions, the ACPA-induced vasorelaxation was about half that observed with phenylephrine-induced contraction. Thus, K+ channels were involved in the ACPA effect. Furthermore, the vasorelaxation effect was similarly reduced when we specifically blocked calcium-activated potassium channel subunit alpha-1 (KCa1.1) (MaxiK; BKCa) prior to adding ACPA. Finally, ACPA-induced vasorelaxation was also diminished when we specifically blocked the calcium channel, voltage-dependent, L type, alpha 1C subunit (Cav1.2). These results showed that ACPA activation of CB1 in smooth muscle caused vasorelaxation of aortic rings through a mechanism involving the activation of KCa1.1 and the inhibition of Cav1.2.

Angiotensin-Converting Enzyme 2 Activation Improves Endothelial Function

Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II-induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

105 NOX2 NADPH—oxidase a novel target to prevent insulin resistance related endothelial cell dysfunction

IntroductionInsulin resistance, a central pathophysiological feature of type 2 diabetes is characterised by a deleterious change in endothelial cell phenotype, a hallmark of which is increased generation of reactive oxygen...

Fluoxetine Effect on Aortic Nitric Oxide-Dependent Vasorelaxation in the Unpredictable Chronic Mild Stress Model of Depression in Mice

Major depression is an independent risk factor for the development of cardiovascular diseases. However, the exact mechanism by which depression may induce cardiovascular events is unclear. Endothelial dysfunction has been reported as a possible link between depression and subsequent cardiovascular events as described in depressed subjects. The purpose of this study was to investigate endothelial dysfunction and atherosclerosis formation in the aorta of mice exposed to the unpredictable chronic mild stress (UCMS) procedure. BALB/c mice were exposed to two 7-week UCMS procedures separated by 6 weeks. Treatments (fluoxetine 10 mg/kg; NaCl 0.9%) started at the third week until the end of the seventh week of each procedure. Endothelial function was evaluated by in vitro assessment of acetylcholine-induced vasorelaxation in aortic rings. By using specific inhibitors for nitric oxide (NO)- and prostacyclin-dependent relaxation, we assessed the part played by NO, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF)-like mediators in endothelium-dependent relaxation. Atherosclerosis was evaluated by histological examination. Depression-like behavior was increased in the UCMS versus unstressed group and was reversed by chronic fluoxetine treatment. Vascular reactivity study indicated that UCMS induced a decrease in the NO-dependent relaxation that was partially compensated by an EDHF-like dependent relaxation. Because fluoxetine per se increased the NO-dependent relaxation, fluoxetine was able to reverse UCMS effect on the NO component and abolished the EDHF-like component. Atherosclerotic lesion was found in aorta of UCMS and nonstressed animals. As an independent risk factor, UCMS reproduced the endothelial alterations observed in depression but was not sufficient to provoke morphological alterations.

Tamoxifen and Its Metabolites Cause Acute Vasorelaxation of Aortic Rings by Inducing Vasodilator Prostanoid Synthesis

The vascular effects of tamoxifen (Tam) and its metabolites are poorly known. We compared the vasorelaxation induced by Tam and its metabolites (N-desmethyl-Tam, 4-hydroxy-Tam, and endoxifen) in aortic rings from rats using standardized organ bath procedures, and we investigated the mechanisms involved in this effect. Tam and its metabolite-induced vasorelaxation in a concentration-dependent manner. Although 4-hydroxy-Tam and Tam had similar potency (pD2 = 8.5 ± 0.1 vs. 8.8 ± 0.1, respectively) and maximum effect (Emax = 88.5% ± 1.3% vs. 92.6% ± 1.3%, respectively), N-desmethyl-Tam and endoxifen were more potent and showed higher Emax than Tam did (pD2 = 9.0 ± 0.1 and 8.9 ± 0.1; Emax = 101.1% ± 1.8% and 101.0% ± 1.8% for N-desmethyl-Tam and endoxifen, respectively). Although preincubation of aortic rings with the estrogen receptor antagonist ICI 182780 or with the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride induced no changes in the vasorelaxation induced by Tam or 4-hydroxy-Tam, both drugs significantly reduced Emax in response to N-desmethyl-Tam or to endoxifen. Inhibition of cyclooxygenase with indomethacin or the incubation with the prostaglandin D2 and E2 receptor antagonist AH6809 reduced the vasorelaxation-induced Tam and its metabolites by approximately 50%. Preincubation with Nω-nitro-L-arginine methyl ester hydrochloride combined with indomethacin abolished the vasorelaxation-induced Tam and its metabolites. These results show that Tam and its metabolites cause acute vasorelaxation by inducing vasodilator prostanoids synthesis.

A Selective Akt Inhibitor Produces Hypotension and Bradycardia in Conscious Rats Due to Inhibition of the Autonomic Nervous System

Akt is a serine-threonine kinase that is amplified in a variety of human cancers, and as with other anticancer agents, some Akt inhibitors have produced functional cardiovascular effects such as marked hypotension that may limit their clinical benefit. Although identified in preclinical studies, the mechanism(s) responsible for these effects are often not fully characterized; potential targets include Akt signaling disruption in cardiac tissue, vascular smooth muscle, and/or autonomic system signaling. A selective Akt inhibitor was found to produce a rapid and marked hypotension and bradycardia in conscious rats. Isolated right atrial tissue and isolated thoracic aortic rings were used to examine direct effects of Akt inhibition on cardiac and vascular tissues, respectively. In addition, rats surgically prepared with telemetry units for monitoring blood pressure and heart rate were used to investigate potential effects on the autonomic nervous system (ANS). Whereas this Akt inhibitor did not produce any significant effect on atrial tissue, it did cause vasorelaxation of aortic rings. More significantly, in conscious rats, the Akt inhibitor inhibited the neural pressor response to the known nicotinic acetylcholine receptor (nAchR) agonist dimethylphenylpiperazinium (DMPP). In fact, the response observed was comparable to the response observed with the known ganglionic blocker hexamethonium. Thus, the hypotension and bradycardia produced by the Akt inhibitor is primarily due to blockade of nAchRs in autonomic ganglia. This finding highlights the importance of evaluating the ANS for cardiovascular effects associated with new chemical entities as well as suggesting a novel direct effect of an Akt inhibitor on nAchRs.

Cytochrome P4501A1 Is Required for Vascular Dysfunction and Hypertension Induced by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

National Health and Nutrition Examination Survey data show an association between hypertension and exposure to dioxin-like halogenated aromatic hydrocarbons (HAHs). Furthermore, chronic exposure of mice to the prototypical HAH, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces reactive oxygen species (ROS), endothelial dysfunction, and hypertension. Because TCDD induces cytochrome P4501A1 (CYP1A1) and CYP1A1 can increase ROS, we tested the hypothesis that TCDD-induced endothelial dysfunction and hypertension are mediated by CYP1A1. CYP1A1 wild-type (WT) and knockout (KO) mice were fed one control or TCDD-containing pill (180 ng TCDD/kg, 5 days/week) for 35 days (n = 10-14/genotype/treatment). Blood pressure was monitored by radiotelemetry, and liver TCDD concentration, CYP1A1 induction, ROS, and aortic reactivity were measured at 35 days. TCDD accumulated to similar levels in livers of both genotypes. TCDD induced CYP1A1 in endothelium of aorta and mesentery without detectable expression in the vessel wall. TCDD also induced superoxide anion production, measured by NADPH-dependent lucigenin luminescence, in aorta, heart, and kidney of CYP1A1 WT mice but not KO mice. In contrast, TCDD induced hydrogen peroxide, measured by amplex red assay, to similar levels in aorta of CYP1A1 WT and KO mice but not in heart or kidney. TCDD reduced acetylcholine-dependent vasorelaxation in aortic rings of CYP1A1 WT mice but not in KO mice. Finally, TCDD steadily increased blood pressure after 15 days, which plateaued after 25 days (+20 mmHg) in CYP1A1 WT mice but failed to alter blood pressure in KO mice. These results demonstrate that CYP1A1 is required for TCDD-induced cardiovascular superoxide anion production, endothelial dysfunction, and hypertension.

XNT, A SYNTHETIC ACE2 ACTIVATOR, IMPROVES ENDOTHELIAL FUNCTION: 1D.03

Endothelial cells play an important role in maintaining vascular homeostasis and their dysfunction is associated with most forms of cardiovascular diseases, such as atherosclerosis, diabetes and hypertension. Recent evidences indicate that the protective axis of the renin-angiotensin-system composed by angiotensin-converting enzyme 2 (ACE2), angiotensin-(1-7) [Ang-(1-7)] and receptor Mas, exerts many beneficial vascular effects. Here, we aimed to evaluate the effect of XNT, a synthetic ACE2 activator (Ferreira et al AJRCCM, 2009: 179: 1048–54), on endothelial function. Isolated aortic rings system was used to evaluate the acute and chronic vascular effect of XNT. The effect of XNT on reactive oxigen species (ROS) production was analyzed using dihydroethidium in human aortic endothelial cells (HAEC). Acute XNT treatment caused endothelial-dependent vasorelaxation in aortic rings from Sprague Dawley rats (55% relaxation at maximum dose of 100microM) pre-constricted with phenylephrine. This vasorelaxant effect was attenuated by D-pro7-Ang-(1-7), a Mas receptor antagonist. Chronic treatment with XNT (1 mg/Kg/day during 4 weeks) improved the endothelial function of SHR when evaluated by the vasorelaxant response for acetylcholine/nitroprusside. Ang II induced ROS production in HAEC was attenuated by XNT treatment. These results show that XNT produced an endothelial-dependent vasorelaxant response which was attenuated by D-pro7-Ang-(1-7), a Mas antagonist. Furthermore, chronic ACE2 activation improved the endothelial function of SHR vessels and ROS production stimulated by Ang II was reduced in HAEC by XNT treatment. This study indicates that ACE2 activation by XNT promotes beneficial effects on endothelial function and is a potential target for treating cardiovascular disease.