Contraction Of Aorta Research Articles

Linolenic Acid Attenuates the Vasodilation Induced by Acetylcholine in Isolated Rat Aortae.

This study aims to examine the effect of linolenic acid on the vasodilation or vasoconstriction induced by acetylcholine and bupivacaine in isolated rat aortae and its underlying mechanism. The effect of linolenic acid on the vasodilation induced by acetylcholine, the calcium ionophore A23187, sodium nitroprusside, and 8-bromoguanosine 3′,5′-cyclic monophosphate sodium salt (bromo-cyclic guanosine monophosphate [bromo-cGMP]) in endothelium-intact and endothelium-denuded aortae was examined. Linolenic acid inhibited vasodilation induced by acetylcholine, calcium ionophore A23187, and sodium nitroprusside. However, this fatty acid increased bromo-cGMP-induced vasodilation in endothelium-denuded aortae. Linolenic acid increased bupivacaine-induced contraction in endothelium-intact aortae, whereas it decreased bupivacaine-induced contraction in endothelium-intact aortae with Nω-nitro-l-arginine methyl ester and endothelium-denuded aortae. Linolenic acid inhibited acetylcholine- and bupivacaine-induced phosphorylation of endothelial nitric oxide synthase. Sodium nitroprusside increased cGMP in endothelium-denuded aortic strips, whereas bupivacaine decreased cGMP in endothelium-intact aortic strips. Linolenic acid decreased cGMP levels produced by bupivacaine and sodium nitroprusside. Together, these results suggest that linolenic acid inhibits acetylcholine-induced relaxation by inhibiting a step just prior to nitric oxide-induced cGMP formation. In addition, linolenic acid-mediated inhibition of vasodilation induced by a toxic concentration (3 × 10−4 M) of bupivacaine seems to be partially associated with inhibition of the nitric oxide–cGMP pathway.

Perivascular adipose tissue contributes to lethal sepsis-induced vasoplegia in rats

It is well established that sepsis induces vascular hyporesponsiveness to vasoconstrictors. Perivascular adipose tissue (PVAT) displays anti-contractile action in various blood vessels. We hypothesized that sepsis would increase the anti-contractile effect of PVAT aggravating sepsis-induced vasoplegia. Male Wistar Hannover rats were subjected to lethal sepsis by cecal ligation and puncture (CLP) method. Aorta or PVAT were collected for functional or biochemical assays 6 h after CLP surgery. Functional experiments showed that sepsis increased the anti-contractile action of PVAT in both endothelium-intact and endothelium-denuded aortas. Carboxy-PTIO, L-NAME and ODQ reversed the hypocontractility mediated by PVAT in aortas from septic rats. Inhibition of nNOS and iNOS with 7-nitroindazole and 1400 W attenuated PVAT-mediated hypocontractility during sepsis. Similar results were found in the presence of indomethacin and Ro1138452, a selective prostacyclin IP receptor antagonist. However, neither tiron nor catalase affected phenylephrine-induced contraction in aortas from septic rats. Increased levels of superoxide anion (O2•-) and 6-keto-prostaglandin F1α (stable product of prostacyclin) were detected in PVAT from septic rats. In situ quantification of reactive oxygen species and nitric oxide (NO) using fluorescent dyes revealed increased levels of both in PVAT from septic rats. The novelty of our study is that PVAT contributes to sepsis-induced vasoplegia by releasing NO and prostacyclin. These findings suggested that signaling pathways in PVAT may be considered as potential novel pharmacological therapeutic targets during sepsis-induced vasoplegia.

The Role of Two-Pore Channels in Norepinephrine-Induced [Ca2+]i Rise in Rat Aortic Smooth Muscle Cells and Aorta Contraction.

Second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) triggers Ca2+ release via two-pore channels (TPCs) localized in endolysosomal vesicles. The aim of the present work is to evaluate the role of TPCs in the action of norepinephrine (NE), angiotensin II (AngII), vasopressin (AVP), and 5-hydroxytriptamine (5-HT) on free cytoplasmic calcium concentration ([Ca2+]i) in smooth muscle cells (SMCs) isolated from rat aorta and on aorta contraction. To address this issue, the NAADP structural analogue and inhibitor of TPCs, NED 19, was applied. We have demonstrated a high degree of colocalization of the fluorescent signals of cis-NED 19 and endolysosmal probe LysoTracker in SMCs. Both cis- or trans-NED 19 inhibited the rise of [Ca2+]i in SMCs induced by 100 μM NE by 50–60%. IC50 for cis- and trans-NED 19 were 2.7 and 8.9 μM, respectively. The inhibition by NED 19 stereoisomers of the effects of AngII, AVP, and 5-HT was much weaker. Both forms of NED 19 caused relaxation of aortic rings preconstricted by NE, with relative potency of cis-NED 19 several times higher than that of trans-NED 19. Inhibition by cis-NED 19 of NE-induced contraction was maintained after intensive washing and slowly reversed within an hour of incubation. Cis- and trans-NED 19 did not cause decrease in the force of aorta contraction in response to Ang II and AVP, and only slightly relaxed aorta preconstricted by 5-HT and by KCl. Suppression of TPC1 in SMCs with siRNA caused a 40% decrease in [Ca2+]i in response to NE, whereas siRNA against TPC2 did not change NE calcium signaling. These data suggest that TPC1 is involved in the NE-stimulated [Ca2+]i rise in SMCs. Inhibition of TPC1 activity by NED 19 could be the reason for partial inhibition of aortic rings contraction in response to NE.

Abstract P1123: The Pharmacological Basis for Vasodilator Effect of Sisymbrium Irio Linn. Validates it's Folkloric Use in Hypertension

Traditionally seeds of Sisymbrium irio Linn has been used in different regions of Pakistan for the treatment of gastrointestinal, airways and vascular system ailments. To insight the pharmacological basis in vitro study was conducted in order to validate its folkloric uses in hypertension. 70% aqueous-methanolic extract of seeds from Sisymbrium irio (Si.MEs) was tested on isolated rabbit aorta, jejunum and trachea strip hanged in tissue bath having physiological solutions aerated with carbogen and their responses were measured and recorded via Power Lab. The Si.MEs exhibited the transient spasmogenic effect (0.01-1.0 mg/mL) on spontaneous jejunum contractions, followed by the spasmolytic effect. The addition of atropine resulted in blocking in spasmogenic effect while the spasmolytic effect was originated, suggesting the presence of an antimuscarinic effect. Likewise verapamil, Si.MEs (0.03-5 mg/mL) repressed the high concentration K + (80 mM)-induced contraction and also drifted the Ca 2+ concentration-response curves toward right (0.3-3.0 mg/mL), possibly signifying the Ca 2+ channel blockade. Furthermore, Si.MEs exhibited nonspecific relaxant effect on carbachol (1 μM)- and high concentration K + (80 mM)-induced tracheal contractions in a way comparable to dicyclomine, suggesting the coexistence of Ca 2+ antagonistic and/or antimuscarinic properties. Additionally, Si.MEs also relaxed the phenylephrine(1 μM)- and high concentration K + (80 mM)-induced aortic contraction (0.01-3 mg/mL), suggesting blockade of Ca 2+ channel. Moreover, oral administration of Si.MEs, as high as 6 g per kg, did not produce lethality among the treated groups of mice. Aqueous-methanolic extract of seeds from S. irio (Si.MEs) exhibited the bronchodilator and gut modulator (spasmogenic and spasmolytic) activities, probably through dual blockade of muscarinic receptors and Ca 2+ channels, whereas, vasodilator effect may be due to Ca 2+ channels blockade validates its use in hypertension.

A Review of Endothelium-Dependent and -Independent Vasodilation Induced by Phytochemicals in Isolated Rat Aorta.

This review discusses the contribution of the use of the isolated rat aorta (IRA) as a model for the evaluation of extracts and metabolites produced by plants with a vasodilator effect in animals. This model continues to be a valuable approach for the search and development of new phytochemicals consumed as medicinal plants or foods. In most cases, the sources of phytochemicals have been used in folk medicine to treat ailments that include hypertension. In this model, the endothelium is emphasized as a key component that modulates the vessel contractility, and therefore the basal tone and blood pressure. Based on the functional nature of the model, we focused on studies that determined the endothelium-dependent and -independent vasodilatory activity of phytochemicals. We describe the mechanisms that account for aorta contraction and relaxation, and subsequently show the vasoactive effect of a series of phytochemicals acting as vasodilators and its endothelium dependence. We highlight information regarding the cardiovascular benefits of phytochemicals, especially their potential antihypertensive effect. On this basis, we discuss the advantages of the IRA as a predictive model to support the research and development of new drugs that may be of help in the prevention and treatment of cardiovascular diseases, the number one cause of death worldwide.

Comparative effects of quercetin and hydroalcoholic extract of Otostegia persica boiss with atorvastatin on atherosclerosis complication in male wistar rats.

The use of herbal remedies is significantly considered in the atherosclerosis treatment, reduction of fatty elements, and prevention of activity of oxidative stress factors. The present study was conducted on 48 rats in 6 groups. The experimental and sham groups were fed with 2% cholesterol for 40 days; and experimental groups were separately treated by atorvastatin, quercetin, and hydroalcoholic extract for 4 weeks. After treatment procedure, some serum factors such as low‐density lipoprotein (LDL), total cholesterol (TC), malondialdehyde (MDA), and reactive oxygen species (ROS) were evaluated. Serum levels of LDL, TC, MDA, and ROS were significantly lower in experimental groups than sham group (p < .01). There was a significant decrease in serum MDA levels of these two groups in comparison with the atorvastatin‐treated group (p < .05). Blood pressure parameters were decreased in treated with quercetin and hydroalcoholic extract in comparison with the sham group (p < .05). Quercetin and hydroalcoholic extract similar to atorvastatin could decrease serum lipids [except high‐density lipoprotein (HDL)], oxidative stress factors, aorta contraction, weight gain, and blood pressure. These reagents improved the vascular structure and prevented the plaque formation.

Increased mitochondrial NADPH oxidase 4 (NOX4) expression in aging is a causative factor in aortic stiffening

Aging is characterized by increased aortic stiffness, an early, independent predictor and cause of cardiovascular disease. Oxidative stress from excess reactive oxygen species (ROS) production increases with age. Mitochondria and NADPH oxidases (NOXs) are two major sources of ROS in cardiovascular system. We showed previously that increased mitochondrial ROS levels over a lifetime induce aortic stiffening in a mouse oxidative stress model. Also, NADPH oxidase 4 (NOX4) expression and ROS levels increase with age in aortas, aortic vascular smooth muscle cells (VSMCs) and mitochondria, and are correlated with age-associated aortic stiffness in hypercholesterolemic mice. The present study investigated whether young mice (4 months-old) with increased mitochondrial NOX4 levels recapitulate vascular aging and age-associated aortic stiffness. We generated transgenic mice with low (Nox4TG605; 2.1-fold higher) and high (Nox4TG618; 4.9-fold higher) mitochondrial NOX4 expression. Young Nox4TG618 mice showed significant increase in aortic stiffness and decrease in phenylephrine-induced aortic contraction, but not Nox4TG605 mice. Increased mitochondrial oxidative stress increased intrinsic VSMC stiffness, induced aortic extracellular matrix remodeling and fibrosis, a leftward shift in stress-strain curves, decreased volume compliance and focal adhesion turnover in Nox4TG618 mice. Nox4TG618 VSMCs phenocopied other features of vascular aging such as increased DNA damage, increased premature and replicative senescence and apoptosis, increased proinflammatory protein expression and decreased respiration. Aortic stiffening in young Nox4TG618 mice was significantly blunted with mitochondrial-targeted catalase overexpression. This demonstration of the role of mitochondrial oxidative stress in aortic stiffness will galvanize search for new mitochondrial-targeted therapeutics for treatment of age-associated vascular dysfunction.

Betulinic acid attenuates lipopolysaccharide-induced vascular hyporeactivity in the rat aorta by modulating Nrf2 antioxidative function

Betulinic acid (BA), a pentacyclic triterpenoid, has been reported to inhibit cardiovascular dysfunction under sepsis-induced oxidative stress. Nuclear factor erythroid-2 related factor-2 (Nrf2) is regarded as a key transcription factor regulating expression of endogenous antioxidative genes. To explore the preventive effects of BA against vascular hyporeactivity and the related antioxidative mechanism in sepsis, contraction and relaxation in aortas isolated from lipopolysaccharide (LPS)-challenged rats were performed. Male Sprague-Dawley rats were pretreated with brusatol (Bru, 0.4mg/kg/2days, i.p.), an inhibitor of Nrf2, and BA (10, 25, 50mg/kg/day, i.g.) for 3days and injected with LPS (10mg/kg, i.p.) at the 4th day. Rats were anesthetized and killed by cervical dislocation after they were treated with LPS for 4h. Thoracic aortas were immediately dissected out to determine contraction and relaxation using the organ bath system. Pro-inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) and oxidative stress were measured in aortic tissues and plasma. mRNA expression of Nrf2-regulated antioxidative enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), and heme oxygenase-1 (HO-1), in rat aortas was determined. Increases of IL-1β, TNF-α, nitric oxide, and malondialdehyde and the decrease of glutathione induced by LPS were significantly attenuated by pretreatment with different doses of BA in plasma and aortas (p < 0.05 versus LPS), all of which were blocked by Bru (p < 0.01). Inhibition of phenylephrine (PE)- and KCl-induced contractions and acetylcholine (ACh)-induced vasodilatation in aortas from LPS-challenged rats was dose-dependently reduced by BA (p < 0.05; percentage improvements by BA in PE-induced contraction were 55.38%, 96.41%, and 104.33%; those in KCl-induced contraction were 15.11%, 23.96%, and 22.96%; and those in ACh-induced vasodilatation were 16.08%, 42.99%, and 47.97%), all of which were reversed by Bru (p < 0.01). Improvements of SOD, GPx, and HO-1 mRNA expression conferred by BA in LPS-challenged rat aortas were inhibited by Bru (p < 0.01; 145.45% versus 17.42%, 160.69% versus 22.76%, and 166.88% versus 23.57%). These findings suggest that BA attenuates impairments of aortic contraction and relaxation in LPS-challenged rats by activating Nrf2-regulated antioxidative pathways.

Butyrlycholine esterase inhibitory activity and effects of extracts (fruit, bark and leaf) from Zanthoxylum armatum DC in gut, airways and vascular smooth muscles

BackgroundFruit, bark and leaves of Zanthoxylum armatum DC are popular remedies for gastrointestinal, cardiovascular and respiratory disorders in the subcontinent traditional practices. The aim of the study was to individually probe the profile of methanol extracts from three different parts of Zanthoxylum armatum.MethodsThe ex-vivo muscle relaxant effects of extracts were assessed in the isolated intestine, trachea and thoracic aortic rings and were compared with the positive controls and CRC were constructed. The anti-diarrheal effect of extracts was evaluated in mice by inducing diarrhea with castor oil. The extracts were also studied for acute toxicity and butyrylcholine esterase inhibition.ResultsThe extracts from fruit, bark and leaves of Z. armatum showed inhibitory effect against the butyrylcholine esterase enzyme with percent inhibition of 50.75 ± 1.23, 82.57 ± 1.33, and 37.52 ± 1.11respectively, compared to standard serine (IC50: 0.04 ± 0.001 μmol/L). The fruit and bark extracts provided 75, and 52% diarrheal protection, compared to verapamil (96%). In isolated rabbit jejunum strips, increasing addition of the extracts inhibited the spontaneous and high K+ precontractions with EC50 values of 0.71 and 3 mg/mL for fruit, EC50 values of 0.61 and 0.5 mg/mL for bark, EC50 0.81 and 3.1 mg/mL for leaves, like verapamil. The extracts induced a concentration-dependent relaxation of the carbachol (1 μM) and high K+ (80 mM) precontractions with EC50 values of 2.4 and 0.9 mg/mL for fruit, EC50 values of 1.2 and 3 for leaves. The bark extract was equipotent against both contractions with EC50 3.1 and 0.7 mg/mL, respectively. In the aortic rings, the fruit extract completely relaxed the phenylephrine (1 μM)-induced contractions with (EC50 value = 0.8 mg/ml) and a partial inhibition of high K+ induced contractions. The leaves extract completely relaxed the aortic contractions with (EC50 values = 1.0 and 8.5 mg/ml). The extracts caused no acute toxicity up to 3 g/kg dose.ConclusionsThe experiments revealed that the extracts of aerial parts of Z. armatum have antidiarrheal properties in vivo and showed spasmolytic effect in intestinal and tracheal preparations with possible mechanism involving the blockage of Ca++ channels. These experiments provide enough justification for use of this plant in ethnomedicine in diarrhea, gut and bronchial spasms.

Urotensin-#receptor antagonist SB-706375 protected isolated rat heart from ischaemia-reperfusion injury by attenuating myocardial necrosis via RhoA/ROCK/RIP3 signalling pathway.

SB-706375 is a selective receptor antagonist of human urotensin-II (hU-II), which can block the aorta contraction induced by hU-II in rats. The effect of SB-706375 on myocardial ischaemia-reperfusion (I/R) injury is unclear. The major objective of this study was to investigate whether SB-706375 has a protective effect on myocardial I/R injury in rats and explore its possible mechanisms. Isolated hearts of Adult Sprague-Dawley were perfused in a Langendorff apparatus, and haemodynamic parameters, lactate dehydrogenase (LDH), creatine phosphokinase-MB (CK-MB), cardiac troponin I (cTnI), RhoA, and the protein expressions of U-II receptor (UTR), receptor-interacting protein 3 (RIP3), Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) and Rho-associated coiled-coil-containing protein kinase 2 (ROCK2) were assessed. We found that SB-706375 (1 × 10-6 and 1 × 10-5mol/L) significantly inhibited the changes of haemodynamic parameters and reduced LDH and CK-MB activities and also cTnI level in the coronary effluents in the heart subjected to myocardial I/R injury. Further experiments studies showed that SB-706375 obviously prevented myocardial I/R increased RhoA activity and UTR, RIP3, ROCK1, and ROCK2 protein expressions. ROCK inhibition abolished the improving effect of SB-706375 on myocardial I/R-induced haemodynamic change in the isolated perfused rat heart. These findings suggested that SB-706375 provides cardio-protection against I/R injury in isolated rats by blocking UTR-RhoA/ROCK-RIP3 pathway.

Protein Tyrosine Phosphatase Inhibitor, Orthovanadate, Induces Contraction &lt;i&gt;via&lt;/i&gt; Rho Kinase Activation in Mouse Thoracic Aortas

Orthovanadate (OVA), a protein tyrosine phosphatase inhibitor, induces contraction in endothelium-denuded mouse thoracic aortas. OVA-induced contraction was significantly (vs. control rings) suppressed by Rho kinase (Y-27632, 10 µM), extracellular signal-regulated kinase 1 and 2 (Erk1/2, FR180204, 10 µM), Erk1/2 kinase (MEK, PD98059, 10 µM), epidermal growth factor receptor (EGFR, AG1478, 10 µM), and Src inhibitors, and was partially suppressed by c-Jun N-terminal kinase (JNK, AS601245, 10 µM) and p38 (SB203580, 10 µM) inhibitors. However, a myosin light chain kinase inhibitor (ML-7, 10 µM) and a metalloproteinase inhibitor (TAPI-0, 10 µM) had no effect on OVA-induced contraction in mouse thoracic aortas. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1) was abolished by inhibitors of Src, EGFR, MEK, Erk1/2, and Rho kinase, but not by inhibitors of JNK and p38. Erk1/2 phosphorylation by OVA was blocked by inhibitors of EGFR, Src, MEK, and Erk1/2, but not by Rho kinase inhibition. Src phosphorylation at Tyr-416 was abrogated by only Src inhibitor. EGFR phosphorylation at Tyr-1173 was suppressed by a Src inhibitor. These findings suggest that OVA induces contraction via activation of Src, EGFR, MEK, Erk1/2, and Rho kinase, leading to inactivation of myosin light chain phosphatase via MYPT1 phosphorylation.

Ethyl Acetate Fraction of Lannea microcarpa Engl. and K. Krause (Anacardiaceae) Trunk Barks Corrects Angiotensin II-Induced Hypertension and Endothelial Dysfunction in Mice.

Traditional remedies prepared from Lannea microcarpa leaves, barks, roots, and fruits are used to treat many diseases including hypertension. This study investigated whether oral administration of the ethyl acetate fraction of Lannea microcarpa trunk barks (LMAE) corrects angiotensin (Ang) II-induced hypertension in mice. Its effects on vascular function were specifically investigated. Experiments explored hemodynamic and echocardiographic parameters in vivo and vascular reactivity to acetylcholine (ACh) and CaCl2 ex vivo on isolated aortas. Mice received LMAE for 3 weeks (50 mg/kg/day) by oral gavage. In the last two weeks of treatment, mice were implanted with osmotic minipumps delivering NaCl (0.9%) or Ang II (0.5 mg/kg/day). LMAE completely prevented the increase in systolic and diastolic blood pressure induced by Ang II. Echocardiographic and kidney parameters were not affected by the different conditions. LMAE abrogated Ang II-induced impairment of ACh-induced relaxation without affecting that of sodium nitroprusside. LMAE also completely prevented CaCl2-induced contraction in KCl-exposed aorta ex vivo. The extract alone did not modify superoxide (O2 −) and nitric oxide (NO·) production in femoral arteries from control mice but significantly limited Ang II-induced O2 − production. These effects were associated with reduced expression of inducible isoform of cyclooxygenase- (COX-) 2 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoform NOX-2 in aortas. Finally, phytochemical analysis showed that LMAE contains sterols, triterpenes, coumarins, and anthraquinone. These results showed that LMAE prevents Ang II-induced hypertension and vascular dysfunction through a reduction of oxidative stress linked to COX-2 and NOX-2 pathway and inhibition of calcium entry. This study provides pharmacological basis of the empirical use of Lannea microcarpa trunk bark extract against hypertension.

Endothelium-independent vasodilator effects of nobiletin in rat aorta

Nobiletin is a one of the polymethoxyflavones contained in the peel of citrus fruits, such as Citrus depressa. In this study, the effect of nobiletin-induced relaxation on phenylephrine (PE)-induced contraction of endothelium-denuded rat aorta was investigated. Nobiletin inhibited PE- or KCl-induced contractions in a concentration-dependent manner in endothelium-intact and -denuded aortas. However, this relaxation was stronger in PE-induced contractions than in KCl-induced contractions; moreover, the nobiletin-induced relaxation was significantly increased on PE-induced contraction in endothelium-intact aorta. ODQ significantly inhibited the nobiletin-induced relaxation in endothelium-denuded aorta; however, SQ22536 did not affect the relaxation. In addition, IBMX synergistically enhanced the nobiletin-induced relaxation. Nobiletin increased cGMP levels in aorta. Also, IBMX significantly increased cGMP content in aorta, and ODQ significantly reduced cGMP levels. Nobiletin-induced relaxation was significantly inhibited by the Ca2+-activated K+ (BK) channel inhibitor iberiotoxin (IbTX) and the ATP-sensitive K+ (KATP) channel inhibitor glybenclamide. Sodium nitroprusside-induced relaxation was suppressed by IbTX, but not by glybenclamide. These results suggest that nobiletin inhibits PE-induced contractions of endothelium-denuded rat aorta by increasing cGMP levels via GC activation. Moreover, the present findings indicate the possibility that nobiletin opened BK channels by a cGMP-related signal, but KATP channels were opened by a cGMP-nonrelated signal in rat aorta.

Endothelial-Dependent and Independent Vascular Relaxation Effect of Tetrahydropalmatine on Rat Aorta.

Tetrahydropalmatine (THP) is an active natural alkaloid isolated from Corydalis yanhusuo W.T. Wang which has been widely used for treating pain and cardiovascular disease in traditional Chinese medicine. Previous studies suggested THP have various pharmacological effects in neural and cardio tissue while the vascular reactivity of THP was not fully established. The present study found that THP relaxed rat aorta which contracted by phenylephrine (Phe), KCl, and U46619. The vascular relaxation effect of THP was partially attenuated by PI3K inhibitor wortmannin, Akt inhibitor IV, endothelial nitric oxide synthetase (eNOS) inhibitor L-NAME, guanylate cyclase inhibitors and the mechanical removal of endothelium. Also, the eNOS substrate L-arginine reversed the inhibition effect of L-NAME on THP-induced vascular relaxation. THP also induced intracellular NO production in human umbilical vein endothelial cells. However, Pre-incubation with β-adrenergic receptor blocker propranolol, angiotensin II receptor 1 (AT1) inhibitor losartan, angiotensin II receptor 2 (AT1) inhibitor PD123319 or angiotensin converting enzyme inhibitor enalapril enhanced the vascular relaxation effect of THP. THP did not affect the angiotensin II induced vascular contraction. Cyclooxygenase-2 (COX2) inhibitor indomethacin did not affect the vascular relaxation effect of THP. Furthermore, pre-treatment THP attenuated KCl and Phe induced rat aorta contraction in standard Krebs solution. In Ca2+ free Krebs solution, THP inhibited the Ca2+ induced vascular contraction under KCl or Phe stress and reduced KCl stressed Ca2+ influx in rat vascular smooth muscle cells. THP also inhibited intracellular Ca2+ release induced vascular contraction by blocking Ryr or IP3 receptors. In addition, the voltage-dependent K+ channel (Kv) blocker 4-aminopyridine, ATP-sensitive K+ channel (KATP) blocker glibenclamide and inward rectifying K+ channel blocker BaCl2 attenuated THP induced vascular relaxation regardless of the Ca2+-activated K+ channel (KCa) blocker tetraethylammonium. Thus, we could conclude that THP relaxed rat aorta in an endothelium-dependent and independent manner. The underlying mechanism of THP relaxing rat aorta involved PI3K/Akt/eNOS/NO/cGMP signaling path-way, Ca2+ channels and K+ channels rather than COX2, β-adrenergic receptor and renin-angiotensin system (RAS). These findings indicated that THP might be a potent treatment of diseases with vascular dysfunction like hypertension.

Distinct Role of Endothelial Caveolae in Contraction and Relaxation Responses of Aortas from Pregnant Normotensive and Hypertensive Rats

We evaluated the role of endothelial caveolae in contraction and relaxation of aortas of Wistar and spontaneously hypertensive rats (SHR) at the end of pregnancy. The quantification of the endothelial caveolae number in the absence or presence of methyl‐β‐ciclodextrin (DEX,10 mM/L) in aortas from non‐pregnant (NP) and pregnant (P) Wistar and SHR with 12 weeks old (n= 5) was assessed by electronic microscopy. Aorta rings from different groups (n= 5) were stimulated with cumulative concentrations (1 nM/L to 10 μM/L) of phenylephrine (PE) or acetylcholine (Ach) in the absence or presence of DEX (10 mM/L). We performed the analysis of caveolin‐1 (cav‐1) and p‐cav‐1 expression by Western Blot in aortic homogenates (n= 5). Ethics committee FOA‐UNESP proc. 00730/2015. The results were compared among groups (ANOVA, p &lt;0.05). Hypertensive NP rats present a reduced number of caveolae in aortas when compared to aortas from Wistar NP. Wistar P has a lower number of caveolae in aortas when compared to aortas from Wistar NP, but the number of caveolae was not altered between aortas of SHR P and SHR NP. DEX was able to reduce the number of caveolae in aortas only in the NP groups of normotensive or hypertensive rats. Vascular reactivity assays showed that in the presence of DEX the aortas of the Wistar NP and SHR NP groups had an increase in the contractile response to PE as well as a reduction in the potency of ACh. However, in aortas of the Wistar P and SHR P groups had no change in the contractile response for PE and ACh. p‐cav‐1/cav‐1 expression is decreased in aortas of SHR NP when compared to Wistar NP. Pregnancy does not alter p‐cav‐1/cav‐1 expression in aortas of Wistar rats, but it increases cav‐1 phosphorylation in SHR aortas. These results showed that pregnancy does not alter the reduced number of caveolae in aortas of hypertensive rats. Due to the reduced number of caveolae in aortas of pregnant rats, DEX would have no effect on aortic reactivity to PE or ACh. Although the reduced number of caveolae, the increased p‐cav‐1 expression in aortas of SHR P could be associated with increased eNOS activity and consequently lower NO‐dependent reactivity, from aortas to PE.Support or Funding InformationFAPESP 2015/09373‐0; 2016/22180‐9; 2018/10635‐7 and CAPES.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Calcium Sensitization in 5‐Hudroxytryptamine‐Induced Contraction of Rat Aorta with or without Perivascular Adipose Tissue

We have previously shown that the perivascular adipose tissue (PVAT) exerted an anti‐contractile effect against 5‐hydroxytryptamine (5‐HT)‐induced contraction of aorta segments from pregnant and non‐pregnant rats. This anti‐contractile effect was significantly reduced in aorta segments from pregnant rats. In the present investigation, we have examined the possible role of calcium sensitization involving ROCK and PKC in this loss of anti‐contractile effect of PVAT. Both Y‐27632 (10−6M) and GF 109203X (10−6M), inhibitors of calcium sensitization, significantly reduced 5‐HT‐induced contractions of PVAT‐free aorta segments from non‐pregnant and pregnant rats. However, they were less effective in aorta segments from pregnant rats. PVAT reduced the effectiveness of Y‐27632 and GF 109203X in aorta segments from pregnant and non‐pregnant rats. ROCK‐I and ROCK‐II protein were expressed in aorta segments and PVAT from pregnant and non‐pregnant rats. There was a reduction in the expression of both isoforms in aorta segments but not PVAT from pregnant rats. In addition, there was no significant difference in the expression of ROCK‐I and ROCK‐II in PVAT from pregnant and non‐pregnant rats. We concluded that the loss of anti‐contractile effect of PVAT in aorta segments from pregnant rats could not be explained by altered calcium‐sensitization mechanisms.Support or Funding InformationResearch Sector, Kuwait University, KuwaitThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Testosterone Replacement Enhances Internal Pudendal Artery Relaxation to Reverse Erectile Dysfunction in a Rat Model of Androgen Deprivation Therapy

Introduction/ObjectivesAndrogen deprivation therapy (ADT) is frequently used to manage prostate cancer; however, erectile dysfunction (ED) and cardiovascular disease are common side effects. After cessation of ADT, testosterone (T) levels may take years to recover leaving men prone to cardiovascular disease and long‐term ED. The internal pudendal arteries (IPA) supply blood to the penis and vascular injury to these vessels can lead to ED. This study will determine if castration impairs vascular function in systemic (aorta and mesenteric arteries) and penile vasculature (IPA), and if T can restore erectile and vascular physiology. We hypothesize the IPA will develop impaired relaxation prior to systemic arteries following castration, and T therapy will recover both vascular and erectile function.MethodsMale Sprague Dawley rats (12 wks) were divided into 3 groups (n=8/grp): control (CON), CAST (6 wks castration), and CAST+T (6 wks castration+T (3mg/kg) in last 2 wks). Erections were assessed via cavernous nerve stimulation and measurement of intracavernosal to mean arterial pressures (ICP/MAP). Aortas, mesenteric arteries, and IPA were excised, cut (2 mm) and mounted into tissue baths. Contractility to high concentration potassium solution (KCl), electrical field stimulation (EFS), and phenylephrine (PE) were measured. Endothelial dependent acetylcholine (ACh) relaxation and endothelial independent relaxation to DEA NONOate were assessed. Non‐adrenergic non‐cholinergic (NANC) relaxation was evaluated by EFS in the presence of guanethidine and atropine. Serum testosterone levels were measured via ELISA. Androgen receptor protein expression was measured in IPA.ResultsCAST markedly impaired erectile function (ICP/MAP; CON: 0.83±0.03, CAST: 0.31±0.02; p&lt;0.05) and decreased IPA ACh relaxation (CON: 50.4±3.5%, CAST: 31.2±2.3%; p&lt;0.05). DEA NONOate relaxation was unchanged with CAST. CAST diminished IPA NANC relaxation (CON: 40.8±5.1%, CAST: 22.1±3.9%; p&lt;0.05). However, CAST did not impact IPA contraction. Interestingly, CAST did not change vascular contraction or relaxation in aortas or mesenteric arteries. IPA androgen receptor expression decreased after CAST (CON: 1±0.17, CAST: 0.39±0.03; p&lt;0.05). T supplementation restored serum T (T (ng/ml); CON: 4.2±0.8, CAST: 0.1±0.02, CAST+T: 32.9±3.2: p&lt;0.05), erectile function (ICP/MAP; CAST+T 0.74±0.01 p&lt;0.05) and improved ACh relaxation in IPA to values greater than CON (CAST+T: 63.9±2.9%; p&lt;0.05). Similarly, CAST+T enhanced IPA NANC relaxation to levels greater than CON (CAST+T: 69.2±1.4%; p&lt;0.05). T supplementation did not impact IPA contractions and had no effect on aortic or mesenteric vasoreactivity.ConclusionsCastration induces severe IPA vascular damage, yet systemic vasculature is unaffected. T therapy dramatically enhances IPA endothelial dependent and NANC‐mediated relaxation. Though T therapy safety in cancer survivors remains controversial, a growing body of literature is finding no correlation between T therapy and cancer progression. In certain populations of prostate cancer survivors, T replacement is warranted.Support or Funding InformationBSOM start‐up grant, SMSNA, AUAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiotensin ll Type 1 Receptor (AT1) Induces Altered Vascular Responses in Allergic Mice via NOX4 and HIF‐a

Airway diseases like asthma leads to cardiovascular disease (CVD). We have shown that in a mouse model of asthma, airway hypersensitivity is exacerbated by Angiotensin II (Ang II) via AT1 receptor activation (Am J Respir Crit Care Med. 2015;191:A5663). In this study (see graphic for hypothesis), we investigated the contribution of NOX4 to Ang II-mediated aortic responses in a mouse model of allergic asthma developed in this laboratory. The effects of NOX4 inhibitor GKT137831, HIF-1α inhibitor FM19 and thioredoxin (TRX) activator auranofin were studied on Ang-ll induced-contraction in isolated aortic rings from asthmatic mice using ex-vivo organ bath. Mice were divided into control (CON) and allergen sensitized-challenged (SEN) groups. Mice were sensitized (i.p.) on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. AT1 antagonist losartan was given i.p. as a single bolus dose (20mg/kg) on day 14 to SEN mice. Organ bath studies were done on day 14 and Western Blot analyses were done to determine aortic expression of AT1, NOX4 as well as p22phox, the subunits of NOX4. In the SEN group, Ang II (10μM) produced higher aortic contraction (33.48±3.26% vs vs. 18.06±3.07% in CON; p<0.05) that was lowered significantly with the use of inhibitors for HIF-1α (10μM; 13.70%±7.43%) and NOX4 (3.54%±6.61%) respectively. Studies have shown that TRX can inhibit Ang ll, HIF-1α sand NOX 4 in the vascular system. Our data showed that TRX activator (auranofin) inhibited Ang ll-mediated contraction to a greater extent in CON mice than SEN. Aortic AT1 receptor expression was significantly higher in SEN mice (1.74±0.08) than CON (1.00±0.13). The expression of NOX4 (1.27±0.06 vs. 0.99±0.09) and p22phox (3.45±0.35 vs. 1.35±0.62) were significantly higher in SEN mice compared to CON. Treatment with losartan significantly attenuated the increases in NOX4 (from1.27±0.06 to 0.67±0.11) and p22phox (from 3.45±0.35 to 0.26±0.11) in SEN mice. These data suggest that most Ang ll through HIF-1α and upregulation by Nox 4, may contribute to altered vascular responses in allergic mice, while TRX antioxidant effect is lower in SEN mice. AT1 antagonist losartan reverses all of these effects, and thus may prevent altered vascular responses to Ang ll in asthma. Support or Funding Information Institutional Research Grant Long Island University SCHEMATIC REPRESENTATION OF HYPOTHESIS TESTED AND PROPOSED PATHWAY FOR ANG ll-MEDIATED VASCULAR CONTRACTION IN ASTHMA This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fibrocytes are Increased in the Medial Layer of the Aorta from Spontaneously Hypertensive Rats

BackgroundChronic hypertension causes a state of inflammation and leads to vascular remodeling. Vascular remodeling involves alterations in vascular smooth muscle cells, endothelium, and extracellular matrix. Collagen deposition is increased in conductance and resistance vessels of hypertensive animals.Fibrocytes are bone marrow‐derived cells found in the circulation that migrate toward injured tissue. At the injured site, fibrocytes contribute to healing by serving as antigen presenting cells and secrete cytokines such as IL‐6 and IL‐8 to recruit T cells. Fibrocytes also lay down extracellular matrix such as collagen, and its activation is implicated in many fibrotic diseases including cardiac fibrosis. Studies have shown that patients with hypertensive heart disease have increased number of circulating fibrocytes and that hearts of hypertensive patients have increased collagen levels. The role of fibrocytes in vascular remodeling in hypertension have not been investigated.HypothesisThis study tests the hypothesis that fibrocytes are increased in hypertension and play a role in vascular remodeling.MethodsMale spontaneously hypertensive rats (SHR) and Wistar rats 13–16 weeks old were used in this study. Flow cytometry was used to determine the percentage of fibrocytes (CD34+/CD45+/CollagenI+) in the peripheral blood, aorta, mesenteric resistance arteries (MRA), and pudendal arteries. Wire myograph was used to evaluate the differences in vascular function in response to phenylephrine (PE), norepinephrine (NE), acetylcholine (ACh), and sodium nitroprusside (SNP) between SHR and Wistar. Immunohistochemistry was used to detect fibrocytes (CD34+/CD45+/Collagen I+) within the vascular walls of aorta, mesenteric, and pudendal arteries. Statistical analysis performed using nonlinear regression, two‐tailed unpaired t‐test, and two‐way ANOVA.ResultsFlow cytometry did not show a difference between the percentage of fibrocytes in SHR compared to Wistar. Wire myography demonstrated differences between the two groups in aortic contraction to PE (p&lt;0.0001) and NE (p&lt;0.0001), aortic relaxation to ACh (p&lt;0.0001) and SNP (p&lt;0.0001), MRA relaxation to ACh (p&lt;0.0001), and pudendal artery relaxation to SNP (p&lt;0.0001). Immunohistochemistry showed increased number of fibrocytes in the media layer of the SHR aorta (Figure 1) compared to Wistar (Figure 2).ConclusionsThe number of fibrocytes are increased in the media layer in the aorta in SHR compared to Wistar. The increased number of fibrocytes may contribute to increased collagen deposition leading to decreased vascular compliance in hypertensive animals.Support or Funding InformationNIH P01 HL‐134604This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The citrus flavanone hesperetin preferentially inhibits slow-inactivating currents of a long QT syndrome type 3 syndrome Na+ channel mutation.

The citrus flavanone hesperetin has been proposed for the treatment of several human pathologies, but its cardiovascular actions remain largely unexplored. Here, we evaluated the effect of hesperetin on cardiac electrical and contractile activities, on aortic contraction, on the wild-type voltage-gated NaV 1.5 channel, and on a channel mutant (R1623Q) associated with lethal ventricular arrhythmias in the long QT syndrome type 3 (LQT3). We used cardiac surface ECG and contraction force recordings to evaluate the effects of hesperetin in rat isolated hearts and aortic rings. Whole-cell patch clamp was used to record NaV 1.5 currents (INa ) in rat ventricular cardiomyocytes and in HEK293T cells expressing hNaV 1.5 wild-type or mutant channels. Hesperetin increased the QRS interval and heart rate and decreased the corrected QT interval and the cardiac and aortic contraction forces at concentrations equal or higher than 30μmol·L-1 . Hesperetin blocked rat and human NaV 1.5 channels with an effective inhibitory concentration of ≈100μmol·L-1 . This inhibition was enhanced at depolarized holding potentials and higher stimulation frequency and was reduced by the disruption of the binding site for local anaesthetics. Hesperetin increased the rate of inactivation and preferentially inhibited INa during the slow inactivation phase, these effects being more pronounced in the R1623Q mutant. Hesperetin preferentially inhibits the slow inactivation phase of INa , more markedly in the mutant R1623Q. Hesperetin could be used as a template to develop drugs against lethal cardiac arrhythmias in LQT3.