Endothelial Microparticles From Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity: Role of the Ang II/AT1 Receptor/NADPH Oxidase-Mediated Activation of MAPKs and PI3-Kinase Pathways.

BACKGROUND: Microparticles (MPs) have emerged as a surrogate marker of endothelial dysfunction and cardiovascular risk. This study examined the potential of MPs from senescent endothelial cells (ECs) or from patients with acute coronary syndrome (ACS) to promote premature EC aging and thrombogenicity. METHODS: Primary porcine coronary ECs were isolated from the left circumflex coronary artery. MPs were prepared from ECs and venous blood from patients with ACS (n=30) and from healthy volunteers (n=4) by sequential centrifugation. The level of endothelial senescence was assessed as senescence-associated ß-galactosidase activity using flow cytometry, oxidative stress using the redox-sensitive probe dihydroethidium, tissue factor activity using an enzymatic Tenase assay, the level of target protein expression by Western blot analysis, platelet aggregation using an aggregometer, and shear stress using a cone-and-plate viscometer. RESULTS: Senescence, as assessed by senescence-associated ß-galactosidase activity, was induced by the passaging of porcine coronary artery ECs from passage P1 to P4, and was associated with a progressive shedding of procoagulant MPs. Exposure of P1 ECs to MPs shed from senescent P3 cells or circulating MPs from ACS patients induced increased senescence-associated ß-galactosidase activity, oxidative stress, early phosphorylation of mitogen-activated protein kinases and Akt, and upregulation of p53, p21, and p16. Ex vivo, the prosenescent effect of circulating MPs from ACS patients was evidenced only under conditions of low shear stress. Depletion of endothelial-derived MPs from ACS patients reduced the induction of senescence. Prosenescent MPs promoted EC thrombogenicity through tissue factor upregulation, shedding of procoagulant MPs, endothelial nitric oxide synthase downregulation, and reduced nitric oxide-mediated inhibition of platelet aggregation. These MPs exhibited angiotensin-converting enzyme activity and upregulated AT1 receptors and angiotensin-converting enzyme in P1 ECs. Losartan, an AT1 receptor antagonist, and inhibitors of either mitogen-activated protein kinases or phosphoinositide 3-kinase prevented the MP-induced endothelial senescence. CONCLUSIONS: These findings indicate that endothelial-derived MPs from ACS patients induce premature endothelial senescence under atheroprone low shear stress and thrombogenicity through angiotensin II-induced redox-sensitive activation of mitogen-activated protein kinases and phosphoinositide 3-kinase/Akt. They further suggest that targeting endothelial-derived MP shedding and their bioactivity may be a promising therapeutic strategy to limit the development of an endothelial dysfunction post-ACS.

Antioxidant and vasorelaxant activities induced by northeastern Brazilian fermented grape skins.

BACKGROUND: In northeastern Brazil, grape pomace has become a potential alternative byproduct because of the recover phenolic compounds from the vinification process. Comparative analyses were performed between lyophilized extract of grape skins from pomace, described as fermented (FGS), and fresh, unfermented (UGS) grape skins to show the relevant brand's composition upon the first maceration in winemaking. METHODS: The use of in vitro testing such as Folin-Ciocalteu's, DPPH free radical scavenger and HPLC methods were performed to evidence antioxidant effect and phenolic compounds. Additionally, vascular reactivity studies were performed in third-order branches of rat superior mesenteric arteries, which were obtained and placed in organ baths containing Krebs-Henseleit solution, maintained at 37 °C, gassed with a mixture of 95% O2 and 5% CO2, and maintained at pH 7.4. The in situ formation of reactive oxygen species (ROS) was evaluated in small mesenteric rings using oxidative fluorescent dihydroethidium dye. RESULTS: We found higher phenolic content and antioxidant activity in FGS when compared to UGS. HPLC analyses identified a significant number of phenolic compounds with antioxidant potential in both samples. The vasorelaxant effect induced by FGS was more potent than that induced by UGS, and the activity was attenuated after removal of vascular endothelium or by blockade of endothelium-derived relaxing factors, such as NO and EDHF. CONCLUSIONS: The FGS extract may be a great source of natural polyphenol products with potent antioxidant effects and endothelium-dependent vasodilatory actions involving NO and EDHF pathways.

Vasorelaxation induced by a new naphthoquinone-oxime is mediated by NO-sGC-cGMP pathway.

It has been established that oximes cause endothelium-independent relaxation in blood vessels. In the present study, the cardiovascular effects of the new oxime 3-hydroxy-4-(hydroxyimino)-2-(3-methylbut-2-enylnaphtalen-1(4H)-one (Oxime S1) derived from lapachol were evaluated. In normotensive rats, administration of Oxime S1 (10, 15, 20 and 30 mg/Kg, i.v.) produced dose-dependent reduction in blood pressure. In isolated aorta and superior mesenteric artery rings, Oxime S1 induced endothelium-independent and concentration-dependent relaxations (10(-8) M to 10(-4) M). In addition, Oxime S1-induced vasorelaxations were attenuated by hydroxocobalamin or methylene blue in aorta and by PTIO or ODQ in mesenteric artery rings, suggesting a role for the nitric oxide (NO) pathway. Additionally, Oxime S1 (30 and 100 µM) significantly increased NO concentrations (13.9 ± 1.6 nM and 17.9 ± 4.1 nM, respectively) measured by nitric oxide microsensors. Furthermore, pre-contraction with KCl (80 mM) prevented Oxime S1-derived vasorelaxation in endothelium-denuded aortic rings. Of note, combined treatment with potassium channel inhibitors also reduced Oxime S1-mediated vasorelaxation suggesting a role for potassium channels, more precisely Kir, Kv and KATP channels. We observed the involvement of BKCa channels in Oxime S1-induced relaxation in mesenteric artery rings. In conclusion, these data suggest that the Oxime S1 induces hypotension and vasorelaxation via NO pathway by activating soluble guanylate cyclase (sGC) and K+ channels.

Cardiovascular effects induced by linalool in normotensive and hypertensive rats.

Linalool is a monoterpene alcohol and constituent of several Brazilian aromatic medicinal plants, popularly used against hypertension. Cardiovascular effects induced by linalool were evaluated. In normotensive rats, (+/-)-linalool [1, 5, 10, and 20 mg/kg body weight (BW); intravenous (i.v.)]-induced hypotension was associated with tachycardia, which was attenuated by atropine (2 mg/kg BW) and N(G)-nitro-L-arginine methyl ester (20 mg/kg BW), but was not modified after indomethacin (5 mg/kg BW) administration. In hypertensive rats, linalool [200 mg/kg BW; oral (v.o.)] reduced blood pressure without changing the heart rate. In intact rings of rat mesenteric artery precontracted with 10 microM phenylephrine, linalool (from 6.4 x 10(-6) to 6.4 x 10(-3) M) induced relaxations in a concentration-dependent manner [E(max) = (115 +/- 13)%] that were not changed after atropine administration [E(max) = (105 +/- 2)%], and were not different from those obtained in endothelium-denuded rings precontracted with phenylephrine [E(max) = (108 +/- 7)%] or 80 mM KCl [E(max) = (113 +/- 7)%] or tetraethylammonium incubation [E(max) = (105 +/- 12)%]. Linalool (1.9 x 10(-3) M) antagonized the contractions induced by CaCl2 (3 x 10(-6)-10(-2) M) (maximal inhibition, 81%). Furthermore, linalool inhibited the contractions induced by 10 microM phenylephrine or 20 mM caffeine. In conclusion, these results demonstrate that linalool reduces blood pressure probably due to a direct effect on the vascular smooth muscle leading to vasodilation.

Uncovering the vasorelaxant effect induced by Vale do São Francisco red wine: a role for nitric oxide.

The aim of this study was to investigate the mechanisms underlying the vasorelaxant effect induced by the polyphenolic compounds found in red wine from Vale do São Francisco. In phenylephrine (10 µM) precontracted mesenteric artery rings, the red wine caused a concentration-dependent relaxation (maximum response to phenylephrine 10 µM = 87.5% ± 6.5%, n = 10). After endothelium removal, the vasorelaxant effect elicited by red wine was attenuated (28.4% ± 4.9%, n = 10). In addition, the vasorelaxant effect induced by red wine in rings pretreated with 100 µM of N(w)-nitro-l-arginine methyl ester and 10 µM of 1H-[1,2,4] oxadiazolo-[4,3-a]-quinoxalin-1-one was attenuated (23.4% ± 5.1%, n = 7 and 11.8% ± 2.7%, n = 6, respectively). Pretreatment with atropine did not affect the vasorelaxant effect induced by red wine (81% ± 3.9%, n = 6). Furthermore, in rabbit aortic endothelial cell line, red wine 100 and 300 µg/mL caused concentration-dependent increases in nitric oxide levels (58 ± 1; 82 ± 7.9; Δ% of fluorescence, n = 5, respectively). In conclusion, we suggest that the alcohol free-lyophilized red wine induces an endothelium-dependent vasorelaxant effect due, at least in part, to a secondary increase in the concentration of nitric oxide and that this effect might be associated with phenolic compounds found in the red wine.

Unravelling the cardiovascular effects induced by alpha-terpineol: a role for the nitric oxide-cGMP pathway.

1. Alpha-terpineol is a monoterpene found in the essential oils of several aromatic plant species. In the present study, we investigated the mechanisms underlying the cardiovascular changes induced by alpha-terpineol in rats. 2. In normotensive rats, administration of alpha-terpineol (1, 5, 10, 20 and 30 mg/kg, i.v.) produced a dose-dependent hypotension (-10 +/- 3, -20 +/- 8, -39 +/- 16, -52 +/- 21 and -57 +/- 23 mmHg, respectively; n = 5) followed by tachycardia. The hypotensive responses to 1, 5, 10, 20 and 30 mg/kg, i.v., alpha-terpineol were significantly attenuated following the administration of N(G)-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg, i.v.; -2 +/- 1, -5 +/- 2, -7 +/- 3, -22 +/- 9 and -22 +/- 10 mmHg, respectively; P < 0.05; n = 5). 3. In 10 micromol/L phenylephrine (PE)-precontracted mesenteric artery rings, alpha-terpineol (10(-12) to 10(-5) mol/L) caused a concentration-dependent relaxation (maximum relaxation 61 +/- 6%; n = 7). After removal of the endothelium, the vasorelaxation elicited by alpha-terpineol was attenuated (maximum relaxation 20 +/- 1%; P < 0.05; n = 7). In addition, vasorelaxation induced by alpha-terpineol in rings pretreated with 100 or 300 micromol/L l-NAME, 30 micromol/L hydroxocobalamin or 10 micromol/L 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one was attenuated (maximum relaxation 18 +/- 3, 23 +/- 3, 24 +/- 7 and 21 +/- 1%, respectively; n = 6; P < 0.05). 4. Furthermore, in a rabbit aortic endothelial cell line, 10(-6), 10(-5) and 10(-4) mol/L alpha-terpineol induced concentration-dependent increases in nitric oxide (NO) levels (12 +/- 6, 18 +/- 9 and 34 +/- 12%Delta fluorescence, respectively; n = 3). 5. In conclusion, using combined functional and biochemical approaches in the present study, we were able to demonstrate that alpha-terpineol-induced hypotension and vasorelaxation are mediated, at least in part, by the endothelium, most likely via NO release and activation of the NO-cGMP pathway.

Potassium Channel Activation Is Involved in the Cardiovascular Effects Induced by Freeze Dried Syzygium jambolanum (Lam.) DC Fruit Juice.

This work aimed to explore the cardiovascular effects induced by freeze-dried juice from Syzygium jambolanum (Lam.) DC fruits (JSJ). JSJ presented high polyphenol content and steroids. HPLC analysis revealed that 2,5-dihydroxybenzoic and caffeic acid were present in higher amounts in the JSJ extract. In rat, JSJ induces hypotension and vasodilatation in mesenteric arteries, with or without vascular endothelium. JSJ-mediated vasodilation response against contractions induced with KCl (60 mM) depolarizing solution was significantly lower than the responses induced by JSJ when evaluated against phenylephrine-induced contractions. To investigate the involvement of potassium channels we used Tyrode's solution with KCl (20 mM) or tetraethylammonium (1.0, 3.0, or 5.0 mM). In these conditions JSJ-induced effects were significantly attenuated. To investigate the potassium channel subtypes involved in the response, we used 4-aminopyridine, glibenclamide, BaCl2, and iberiotoxin. In the presence (simultaneous) of different potassium channel blockers we observed a significant attenuation of JSJ-induced effect. Inhibition was also observed when using BaCl2, glibenclamide, or 4-aminopyridine, separately. However, incubation with iberiotoxin did not promote changes in either maximum effect, or potency. We also evidenced a discrete participation of CaV channels in the JSJ-induced vasorelaxant effect. In addition, patch-clamp studies demonstrated that JSJ could activate potassium channels. In conclusion, JSJ promotes hypotension and vasorelaxation in rats, involving, at least, the activation of potassium channels.

The 2-nitrate-1,3-dibuthoxypropan, a new nitric oxide donor, induces vasorelaxation in mesenteric arteries of the rat.

The reduced availability of nitric oxide (NO) is associated with cardiovascular diseases. Therefore, NO donors such as organic nitrates are useful for the treatment of these disorders. The 2-nitrate-1,3-dibuthoxypropan (NDBP) is an organic nitrate synthesized from glycerin, which the pharmacological effects have not been investigated. In this study we evaluated the vasorelaxant effect induced by NDBP in superior mesenteric artery from rats. In phenylephrine pre-contracted artery rings, NDBP (10(-8)-10(-4)M) elicited concentration-dependent and endothelium-independent relaxation, which were attenuated by hydroxocobalamin-HDX (30 µM), a NO extracellular scavenger, and 1-H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one-ODQ (10 µM), an inhibitor of soluble guanylyl cyclase (sGC). In addition, the NDBP-induced relaxation was reduced by non-selective K(+) channels blocker KCl (20 mM) or selective K(+) channels blockers such as tetraethylammonium-TEA (B(KCa), 1 mM), charybdotoxin-ChTX (B(KCa), 100 nM), glibenclamide (K(ATP), 1µM) and 4-aminopyridine-4-AP (K(V), 1mM). In preparations with ODQ (10 µM) plus TEA (1 mM), the response was virtually abolished. In rat smooth muscle cells culture, NDBP (10(-6)-10(-4)M) caused concentration-dependent increases in NO levels. These findings suggest that NDBP causes vasorelaxation through NO generation and activation of the sCG/cGMP/PKG pathway.

Cardiovascular effects elicited by milonine, a new 8,14-dihydromorphinandienone alkaloid.

The mechanisms underlying the cardiovascular responses evoked by milonine (i.v.), an alkaloid, were investigated in rats. In normotensive rats, milonine injections produced hypotension and tachycardia, which were attenuated after N(w) -nitro-L-arginine methyl esther (L-NAME; 20 mg/kg, i.v.). In phenylephrine (10 µM), pre-contracted mesenteric artery rings, milonine (10⁻¹° M to 3 × 10⁻4 M) caused a concentration-dependent relaxation (EC50 = 1.1 × 10⁻6 M, E(max) = 100 ± 0.0%) and this effect was rightward shifted after either removal of the vascular endothelium (EC50 = 1.6 × 10⁻5, p < 0.001), or after L-NAME 100 µM (EC50 = 6.2 × 10⁻5, p < 0.001), hydroxocobalamin 30 µM (EC50 = 1.1 × 10⁻4, p < 0.001) or ODQ 10 µM (EC50 = 1.9 × 10⁻4 p < 0.001). In addition, in rabbit aortic endothelial cells, milonine increased NO3⁻ levels. The relaxant effect induced by milonine was attenuated in the presence of KCl (20 mM), a modulator efflux K(+) (EC50 = 1.2 × 10⁻5, p < 0.001), or different potassium channel blockers such as glibenclamide (10 µM) (EC50 = 6.3 × 10⁻5, p < 0.001), TEA (1 mM) (EC50 = 2.3 × 10⁻5 M, n = 6) or Charybdotoxin (0.2 µM) plus apamin (0.2 µM) (EC50 = 3.9 × 10⁻4 M, n = 7). In addition, pre-contraction with high extracellular potassium concentration prevented milonine-induced vasorelaxation (EC50 = 1.0 × 10⁻4, p < 0.001). Milonine also reduced CaCl2 -induced contraction in Ca²(+) -free solution containing KCl (60 mM). In conclusion, using combined functional and biochemical approaches, we demonstrated that the hypotensive and vasorelaxant effects produced by milonine are, at least in part, mediated by the endothelium, likely via nitric oxide release, activation of nitric oxide-cGMP pathway and opening of K(+) channels.

Superoxide scavenging in the rostral ventrolateral medulla blunts the pressor response to peripheral chemoreflex activation.

Peripheral chemoreflex activation has been considered the key drive for the overactivity of the sympathetic nervous system observed in some pathological conditions such as sleep obstructive apnea. In addition, increases in angiotensin-II-derived reactive oxygen species found in some autonomic regulatory brain areas have been implicated in hypertension. However, a link between oxidative stress and peripheral chemoreflex integration within the RVLM has never been investigated. Here, we tested the hypothesis that the pressor response induced by peripheral chemoreflex activation involves the angiotensin-II/AT(1)R/superoxide pathway within the rostral ventrolateral medulla (RVLM). Seventeen male Wistar rats (260-300 g) were implanted with bilateral guide cannulae towards the RVLM and were fitted with catheters for blood pressure recordings and drug administration. Peripheral chemoreflex activation with potassium cyanide (80 microg/kg, i.v.) produced a transient increase in blood pressure, which was attenuated 2 minutes after bilateral microinjection of losartan (1 nmol), an AT(1) receptor antagonist, in the RVLM (+54+/-4 vs +19+/-3 Delta mmHg, P<0.05, n=6). Moreover, superoxide scavenging in the RVLM using a superoxide dismutase (SOD) mimetic, Tempol (5 nmol), significantly blunted the pressor response to peripheral chemoreflex activation (+50+/-3 vs +18+/-3 Delta mmHg, P<0.05, n=7). On the other hand, bilateral microinjection of saline (n=4) in the RVLM produced no change in the pressor response to chemoreflex activation. Taken together, these data suggest that the neurotransmission of the peripheral chemoreflex within the RVLM involves, at least in part, the activation of AT(1) receptors and downstream superoxide formation.

Hypotensive and vasorelaxant effects of citronellol, a monoterpene alcohol, in rats.

Citronellol is an essential oil constituent from the medicinal plants Cymbopogon citratus, Cymbopogon winterianus and Lippia alba which are thought to possess antihypertensive properties. Citronellol-induced cardiovascular effects were evaluated in this study. In rats, citronellol (1-20 mg/kg, i.v.) induced hypotension, which was not affected by pre-treatment with atropine, hexamethonium, N(omega)-nitro-L-arginine methyl ester hydrochloride or indomethacin, and tachycardia, which was only attenuated by pre-treatment with atropine and hexamethonium. These responses were less than those obtained for nifedipine, a reference drug. In intact rings of rat mesenteric artery pre-contracted with 10 microM phenylephrine, citronellol induced relaxations (pD(2) = 0.71 +/- 0.11; E(max) = 102 +/- 5%; n = 6) that were not affected by endothelium removal, after tetraethylamonium in rings without endothelium pre-contracted with KCl 80 mM. Citronellol strongly antagonized (maximal inhibition = 97 +/- 4%; n = 6) the contractions induced by CaCl(2) (10(-6) to 3 x 10(-3 )M) and did not induce additional effects on the maximal response of nifedipine (10 microM). Finally, citronellol inhibited the contractions induced by 10 microM phenylephrine or 20 mM caffeine. The present results suggest that citronellol lowers blood pressure by a direct effect on the vascular smooth muscle leading to vasodilation.

Erythroxylum pungens elicits vasorelaxation by reducing intracellular calcium concentration in vascular smooth muscle cells of rats

The cardiovascular effects elicited by the ethanolic extract obtained from the roots of Erythroxylum pungens O.E. Schulz, Erythroxylaceae (EEEP) and the vasorelaxant effect induced by its main tropane alkaloid (pungencine) were investigated. In normotensive rats, administration of EEEP (1, 10, 30 and 60 mg/kg i.v., randomly) produced dose-dependent hypotension (-2±1, -7±0.5 -17.6±1, -24±1 Δ mmHg, n=5) followed by tachycardia (3±0.5, 7±2, 7.1±1, 10±5 Δ bpm, n=5). In intact phenylephrine (Phe, 10 µM)-pre-contracted rings, EEEP (0.01-500 µg/mL) induced concentration-dependent vasorelaxation (EC50 13.7±5.5 µg/mL, Maximal Response= 92±2.6%), and this effect was unchanged after the removal of the vascular endothelium (EC50 27.2±4.7 µg/ml, Maximal Response= 88.3±3.3 %). In KCl (80 mM)-pre-contracted-endothelium-denuded rings, EEEP elicited concentration-dependent relaxation (EC50= 128.2±11.2 µg/mL, Maximal Response 76.8±3.4%). Vasorelaxation has also been achieved with tonic contractions evoked by the L-type Ca2+ channel agonist Bay K 8644 (EC50 80.2±9.1 µg/mL, Maximal Response 86.3±8.3%). In addition, in a depolarizing medium, EEEP inhibited CaCl2 (30-500 µg/mL) induced contractions and caused a concentration-dependent rightward shift of the relaxation curves. Lastly, the tropane alkaloid pungencine caused vasorelaxation in mesenteric arteries resembling to the EEEP responses. These results suggests that EEEP induces hypotension and vasorelaxation, at least in part, due to the reduction in [Ca2+]i in vascular smooth muscle cells.