ACh-induced Relaxation Research Articles

Egg White Hydrolysate: A new putative agent to prevent vascular dysfunction in rats following long-term exposure to aluminum

Aluminum (Al) is toxic for humans and animals. Here, we have tested the potential for Egg White Hydrolysate (EWH) to protect against cardiovascular changes in rats exposed to both high and low dietary levels of Al. Indeed, EWH has been previously shown to improve cardio metabolic dysfunctions induced by chronic exposure to heavy metals. Male Wistar rats received orally: Group 1) Low aluminum level (AlCl3 at a dose of 8.3 mg/kg b.w. during 60 days) with or without EWH treatment (1 g/kg/day); Group 2) High aluminum level (AlCl3 at a dose of 100 mg/kg b.w. during 42 days) with or without EWH treatment. After Al treatment, rats co-treated with EWH did not show vascular dysfunction or increased blood pressure as was observed in non EWH-cotreated animals. Indeed, co-treatment with EWH prevented the following effects observed in both aorta and mesenteric arteries: the increased vascular responses to phenylephrine (Phe), the decreased ACh-induced relaxation, the reduction on endothelial modulation of vasoconstrictor responses and the nitric oxide bioavailability, as well as the increased reactive oxygen species production from NAD(P)H oxidase. Altogether, our results suggest that EWH could be used as a protective agent against the harmful vascular effects after long term exposure to Al.

Acute activation of endothelial AMPK surprisingly inhibits endothelium-dependent hyperpolarization-like relaxations in rat mesenteric arteries.

Endothelium-dependent hyperpolarizations (EDHs) contribute to the regulation of peripheral resistance. They are initiated through opening of endothelial calcium-activated potassium channels (KCa ); the potassium ions released then diffuse to the underlying smooth muscle cells, causing hyperpolarization and thus relaxation. The present study aimed to examine whether or not AMPK modulates EDH-like relaxations in rat mesenteric arteries. Arterial rings were isolated for isometric tension recording. AMPK activity and protein level were measured by ELISA and western blotting respectively. The AMPK activator, AICAR, reduced ACh-induced EDH-like relaxations and increased AMPK activity in preparations with endothelium; these responses were prevented by compound C, an AMPK inhibitor. AICAR inhibited relaxations induced by SKA-31 (opener of endothelial KCa ) but did not affect potassium-induced, hyperpolarization-attributable relaxations or increase AMPK activity in preparations without endothelium. A769662, another AMPK activator, not only caused a similar inhibition of relaxations to ACh and SKA-31 in preparations with endothelium but also inhibited hyperpolarization-attributable relaxations and augmented AMPK activity in rings without endothelium. Protein levels of total AMPKα, AMPKα1, or AMPKβ1/2 were comparable between preparations with and without endothelium. Activation of endothelial AMPK, by either AICAR or A769662, acutely inhibits EDH-like relaxations of rat mesenteric arteries. Furthermore, A769662 inhibits signalling downstream of smooth muscle hyperpolarization. In view of the major blunting effect of AMPK activation on EDH-like relaxations, caution should be applied when administering therapeutic agents that activate AMPK in patients with endothelial dysfunction characterized by reduced production and/or bioavailability of NO.

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.

Endothelial‐Specific Interference with PPARγ Causes Endothelial Dysfunction with Sex‐ Specific Mechanisms in Offspring Born from AVP‐infused Pregnancies

Mutations in the ligand‐activated transcription factor PPARγ result in hypertension, and synthetic agonists of PPARγ reduce blood pressure. Previously we found that mice expressing dominant‐negative (DN) PPARγ driven by an endothelium‐specific promoter (E‐DN) exhibit vascular dysfunction. Preeclampsia (PE) is a hypertensive disorder of pregnancy and carries cardiovascular risk to offspring. PE is also associated with vascular dysfunction. Therefore, we hypothesized a role for endothelial PPARγ in the pathogenesis of PE and its sequelae. C57BL/6J dams were bred with E‐DN sires, and symptoms of PE were induced by infusion of vasopressin (AVP, 24 ng/hr sc) throughout gestation. Phenotypes of PE were first assessed in pregnant dams and then in adult offspring. Compared to saline infusion (SAL), AVP elevated maternal blood pressure (SBP: 116±3 vs 107±3, p<0.05) at gestational day (GD) 14–15 and urine protein (70±6 vs 27±4 mg/mL, p<0.05) at GD17. Offspring were phenotyped in adulthood. Data were stratified to sex, genotype, and exposure to AVP or SAL. Adult male and female offspring were exposed to a sub‐pressor dose of Angiotensin II (ANG; 120ng/kg/hr) for 14 days as a cardiovascular stressor. Male and female ANG‐treated E‐DN born to PE pregnancies, but not control pregnancies, exhibited significant impairment in ACh‐induced relaxation in carotid artery (% relaxation: Males − 56±9 vs 81±11, p<0.05; Females − 68±10 vs 84±11, p<0.05). Endothelial dysfunction in male ANG‐treated E‐DN born to PE pregnancies was attenuated by tempol (relaxation improved from 53±9% vs 84±12%, p<0.05), indicative of a role for oxidative stress. Interestingly, tempol did not rescue endothelial dysfunction in female E‐DN exposed to PE (% relaxation: 69±10% vs 81±11%, ns). In keeping with this, Nrf2 protein levels was significantly elevated in aorta from male E‐DN (1.9±0.2 vs 0.2±0.1, p<0.05), but not females (1.2±0.3 vs 0.6±0.2, ns) compared to genotype‐ and sex‐matched controls, suggesting that the mechanism of endothelial dysfunction might differ between sexes. Blockade of Rho‐kinase (ROCK) signaling with Y‐27632 improved endothelium‐dependent relaxation in male and female E‐DN offspring born from PE pregnancies (% relaxation: Males − 60±10 vs 87±12, p<0.05; Females − 69±10 vs 98±13, p<0.05). We observed a significant 4‐fold increase in ROCK2 protein levels in aorta from both male and female E‐DN mice exposed to PE (Males – 2.3±0.5 vs 0.6±0.4, p<0.05; Females − 1.9±0.5 vs 0.5±0.2, p<0.05). No changes were observed in ROCK1 expression. Further, incubation with a ROCK2 specific inhibitor, SLX‐2119, significantly improved relaxation in male and female E‐DN (Males – relaxation improved from 72±11 vs 91±10, p<0.05; Females – relaxation improved from 68±10 vs 94±12, p<0.05). Our data suggests that interference with endothelial PPARγ in pups born from PE pregnancies increases the risk for endothelial dysfunction upon exposure to a cardiovascular stressor in adulthood. Moreover, potential mechanisms accounting for endothelial dysfunction in offspring born from complicated pregnancies might be sex‐dependent.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 EDHF in Potentiation of ACh‐induced Relaxation of Aorta in Male UC Davis Type 2 Diabetes Mellitus (UCD‐T2DM) Rat: Sex Specific Responses

The UC Davis type 2 diabetes mellitus (UCD‐T2DM) Rat is a validated model of T2D that exhibits an etiology more similar to human disease than other rodent models. In spite of being a validated model, there are no published data on the vascular function in this model. We investigated whether vascular reactivity is altered, and if sex differences exist in aortic endothelial function in the UCD‐T2DM rats. We also examined the potential changes in the relative contributions of endothelium‐derived relaxing factors in modulating aortic reactivity of UCD‐T2DM.Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10−8 to 10−5M) was measured in aortic rings pre‐contracted with phenylephrine (PE, 2μM) before and after pretreatment with indomethacin (Indo; 10 μM), a cyclooxygenase (COX) inhibitor, Indo plus ODQ (10 μM), an inhibitor of soluble guanylyl cyclase (sGC) and ultimately following incubation with Indo, ODQ and L‐NNA (100 μM), a nonselective NO synthase (NOS) inhibitor. Endothelium‐independent vasodilation to sodium nitroprusside (SNP, 10−9 to 10−5M) was assessed in endothelium‐denuded rings pre‐contracted with PE (2μM). Furthermore, constrictor response curves to PE (10−8 to 10−5 M) were also generated. The contribution of K+ to ACh‐induced relaxation was assessed in male animals by obtaining the concentration response curves to ACh before and after treatment with TRAM‐34 (1μM), an intermediate‐conductance Ca2+ activated K+ (IKCa) channel inhibitor; apamin (1 μM), a small‐conductance Ca2+ activated K+ (SKCa) channel inhibitor. Moreover, the expression of K+ channels were evaluated in aortic tissues.Diabetes significantly impaired relaxation responses to ACh and SNP in aortic rings from female UCD‐T2DM Rats, however, potentiated the relaxation in males. The responsiveness to PE was significantly enhanced in diabetic groups, regardless of sex. Blocking of COX, sGC and NOS completely abolished the relaxation response to ACh in all of experimental groups except for the diabetic male rats, indicating the presence of endothelium‐dependent hyperpolarizing factor (EDHF)‐type relaxation in this group. To further study the contribution of EDHF in diabetic males, ACh‐induced relaxation was measured before and after TRAM 34 or Apamin in male tissues. Incubation of male aortic rings with TRAM 34, but not Apamin, significantly blunted the relaxation responses to ACh only in diabetic group. Accordingly, the level of expression of IKca channels was significantly higher in diabetic males than that in controls.These data, for the first time, show that the vascular function in aortic rings of UCD‐T2DM is altered in both sexes. It also suggests that an increased contribution of EDHF, specifically IKca channel, plays a role in potentiated relaxation responses to ACh in male UCD‐T2DM rats.Support or Funding InformationSupported by NHLBIThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PP242, mTOR inhibitor, decreases phenylephrine‐induced vascular contractility in hypertensive and normotensive arteries

Cardiovascular disease is the leading cause of death in cancer survivors. The short‐term and long‐term cardiotoxic effects of targeted chemotherapeutics is not well known. PP242, a dual inhibitor to mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and 2 (mTORC2), is a targeted therapy in clinical trials for various malignancies including bladder, ovarian and gastric cancer. mTORC1 inhibition has been associated with heart failure, while mTORC2 inhibition has been considered cardioprotective. We hypothesized that PP242 leads to enhancedcontractile response to phenylephrine (PE), and impaired Acetylcholine (ACh)‐induced relaxation. We studied the effects of PP242 on the contractility and relaxation of aortic rings and mesenteric resistance arteries (MRA) of male Wistar rats (12–15 weeks old, n=4) and spontaneously hypertensive rats (SHR) (15–18 weeks old, n=6) on a wire myograph. Vessel segments were exposed to PP242 (500nM) for one‐hour incubation. Normotensive aortic rings exposed to PP242 showed a decreased contractile response to PE (LogEC50±SEM, Ctrl:−7.617±0.178 vs Drug: −7.292±0.129) and increased relaxation to ACh (LogEC50±SEM, Ctrl:−6.917±0.164 vs Drug: −7.588±0.164). SHR aortic rings exposed to PP242 (LogEC50±SEM, Ctrl:−7.606±0.129 vs Drug: −7.124±0.118) showed decreased contractility but no difference in ACh‐induced relaxation. No differences were observed in normotensive MRA. SHR MRA showed decreased PE‐induced contraction (LogEC50±SEM, Ctrl:−5.402±0.060 vs Drug:‐−5.322±0.065) and increased ACh‐induced relaxation (LogEC50±SEM, Ctrl:−7.992±0.117 vs Drug:−8.139±0.092). Our data suggest that dual inhibition of mTORC1 and mTORC2 with PP242 may provide a cardioprotective effect by limiting vasoconstriction and may be a good therapeutic agent for hypertensive cancer patients. However, long term treatments with this targeted therapy need to be evaluated for vascular dysfunction induction, as the hypocontractile effect could contribute to failure of the cardiovascular system, which could be associated with cardiotoxicity.Support or Funding InformationNIH P01 HL134604 (RCW), k99GM11888 (CFW)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic administration of sildenafil improves endothelial function in spontaneously hypertensive rats by decreasing COX-2 expression and oxidative stress

AimsSpontaneously hypertensive rats (SHR) exhibit impaired endothelial vasodilation and enhanced vasoconstriction. The phosphodiesterase 5 (PDE5) inhibitor sildenafil (Sild) potentiates the nitric oxide (NO)-mediated effects exerting antioxidative and anti-inflammatory actions. In the present study, we hypothesized that Sild could improve endothelial function in SHR. Materials and methodsMale rats were treated daily for 60 days by oral gavage with Sild (45 mg/kg) before the onset of the hypertensive state (pre-hypertensive protocol). The aortic relaxation to acetylcholine (ACh), sodium nitroprusside (SNP) and the phenylephrine (Phe)-induced contraction was evaluated in SHR. Protein expression of eNOS, p-eNOS, caveolin, COX-1, COX-2, ERK and p-ERK was measured by Western blot. Key findingsResting blood pressure was not modified by Sild administration. Treatment with Sild did not alter the relaxation response to SNP but improved the ACh-induced relaxation and reduced Phe-induced contraction in aortic rings from SHR. This protective effect of Sild could be attributed to reduced superoxide anions (O2−) generation, cyclooxygenase type 2 (COX-2) protein downregulation and increased NO bioavailability. SignificanceSild improves endothelial function in SHR aorta without affecting resting blood pressure values. These results indicate that PDE5 inhibition has a potential role in the improvement of vascular function and could be an adjuvant in the treatment of essential hypertension.

Chronic Treatment With Acetylcholinesterase Inhibitors Attenuates Vascular Dysfunction in Spontaneously Hypertensive Rats.

Acetylcholinesterase inhibition prevents autonomic imbalance, reduces inflammation, and attenuates the development of hypertension. Considering that vascular dysfunction is a crucial feature of arterial hypertension, we investigated the effects of chronic administration of acetylcholinesterase inhibitors-pyridostigmine or donepezil-on vascular reactivity of spontaneously hypertensive rats (SHR). Endothelium-dependent relaxant responses to acetylcholine (ACh) and contractile responses induced by electric field stimulation (EFS) and alpha-adrenergic agonist were studied in mesenteric resistance arteries from SHR and Wistar Kyoto rats. SHR were treated for 16 weeks with vehicle, pyridostigmine (1.5 mg/kg/day) or donepezil (1.4 mg/kg/day). Pyridostigmine and donepezil decreased the vasoconstrictor responses to EFS, which were increased in vehicle-treated SHR. Acetylcholinesterase inhibition increased the modulatory effects of nitric oxide (NO) on SHR vascular reactivity, that is, N(ω)-nitro-(L)-arginine methyl ester (L-NAME) increased EFS-induced contractions and reduced ACh-induced relaxation, with more significant effects in pyridostigmine- and donepezil-treated SHR. The acetylcholinesterase inhibitors also decreased vascular reactive oxygen species levels. This study demonstrates for the first time that long-term administration of acetylcholinesterase inhibitors, pyridostigmine or donepezil, attenuates vascular reactivity dysfunction in SHR by decreasing reactive oxygen species generation and increasing NO bioavailability; possibly via increased endothelial NO synthase activity, and inhibition of NADPH oxidase activity.

Different mechanisms involved in liraglutide and glucagon-like peptide-1 vasodilatation in rat mesenteric small arteries.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates insulin biosynthesis and secretion in a glucose-dependent manner and has been reported to induce vasodilatation. Here, we examined the possible vasorelaxant effect of GLP-1 and its underlying mechanisms. Rat mesenteric arteries (diameter≈200-400μm) and human s.c. arteries were mounted in microvascular myographs for isometric tension recordings. The effect of GLP-1 on vascular responses was examined under normoglycaemic conditions and at high glucose concentrations. In rat mesenteric arteries and human s.c. arteries without branches, physiological concentrations (1-100nM) of GLP-1(7-36) and liraglutide failed to cause relaxation or affect contractions evoked by electrical field stimulation. In contrast to GLP-1(7-36), liraglutide induced relaxations antagonized by the GLP-1 receptor antagonist, exendin-(9-39), in branched mesenteric arteries. In contrast to liraglutide, GLP-1 leftward shifted the concentration relaxation curves for bradykinin in s.c. arteries from patients with peripheral arterial disease, an effect resistant to exendin-(9-39). Under normoglycaemic conditions, neither GLP-1 nor liraglutide affected ACh relaxation in rat mesenteric arteries. In arteries exposed to 40mM glucose, GLP-1, in contrast to liraglutide, potentiated ACh-induced relaxation by a mechanism that was not antagonized by exendin-(9-39). GLP-1 decreased superoxide levels measured with dihydroethidium in rat mesenteric arteries exposed to 40mM glucose. GLP-1 receptors are involved in the liraglutide-induced relaxation of branched arteries, under normoglycaemic conditions, while GLP-1 inhibition of vascular superoxide levels contributes to GLP-1 receptor-independent potentiation of endothelium-dependent vasodilatation in hyperglycaemia.

Abstract 133: Endothelial-Specific Interference With PPARγ Increases the Susceptibility to Angiotensin II-Induced Endothelial Dysfunction in Adult Offspring Born from AVP-Infused Pregnancies

Mutations in the ligand-activated transcription factor PPARγ result in hypertension, and synthetic agonists of PPARγ reduce blood pressure. Previously we found that mice expressing dominant-negative (DN) PPARγ driven by an endothelium-specific promoter (E-DN) exhibit vascular dysfunction. Preeclampsia (PE) is a hypertensive disorder of pregnancy which carries cardiovascular risk to offspring. PE is also associated with vascular dysfunction. We hypothesized a role for endothelial PPARγ in the pathogenesis of PE and its sequelae. C57BL/6J dams were bred with E-DN sires, and symptoms of PE were induced by infusion of vasopressin (AVP, 24 ng/hr sc) throughout gestation. Phenotypes of PE were first assessed in pregnant dams and then in adult offspring. Compared to saline infusion (SAL), AVP elevated maternal blood pressure (SBP: 116±3 vs 107±3, p<0.05) at gestational day (GD) 14-15 and urine protein (70±6 vs 27±4 mg/mL, p<0.05) at GD17. Offspring were phenotyped in adulthood. Data were stratified to sex, genotype, and exposure to AVP or SAL. At 20 weeks of age, SBP and vasorelaxation responses to acetylcholine were similar in offspring exposed to PE compared to mice born from SAL pregnancies. Adult offspring were next exposed to a sub-pressor dose of Angiotensin II (ANG; 120ng/kg/hr) for 14 days. Adult male ANG-treated E-DN born to PE pregnancies, but not NT mice exhibited significant impairment in ACh-induced relaxation in carotid artery (% relaxation: 62±10 vs 86±11, p<0.05). Adult female ANG-treated E-DN exposed to PE also exhibited a trend for impaired endothelial function compared to NT controls (% relaxation: 63±9 vs 72±10). Endothelial dysfunction in male ANG-treated E-DN born to PE pregnancies was attenuated by tempol (relaxation improved from 56±9% vs 81±12%, p<0.05), indicative of oxidative stress, and by a Rho-kinase inhibitor (relaxation improved from 73±11% vs 89±12%, p<0.05). This data suggests that interference with endothelial PPARγ in pups born from PE pregnancies increases the risk for endothelial dysfunction upon exposure to a cardiovascular stressor in adulthood. Thus, conditions which impair PPARγ activity, such as obesity and diabetes, may predispose adult offspring born from PE pregnancy to cardiovascular disease.

Abstract 110: Vascular Smooth Muscle RhoBTB1 Protects From Hypertension and Arterial Stiffness by Cullin-3 Dependent Ubiquitination of Phosphodiesterase 5

We previously reported that vascular smooth muscle cell (VSMC) selective expression of hypertension (HT)-causing mutations in either PPARγ or the E3 Ring Ubiquitin Ligase Cullin-3 causes nitric oxide resistance and HT. Here we sought to assess the physiological role of RhoBTB1, a VSMC PPARγ target gene and Cullin-3 substrate adaptor. S-P467L mice which selectively express dominant negative PPARγ-P467L in VSMC exhibit RhoBTB1-deficiency. We bred S-P467L mice with mice inducibly expressing RhoBTB1 in response to Cre-recombinase. Inducible VSMC-specific restoration of RhoBTB1 in S-P467L mice fully corrected the HT (SBP, 141±6 vs 124±3 mmHg, p<0.01, n=8-10), arterial stiffness (Aortic Pulse Wave Velocity, 3.8±0.2 vs 2.5±0.1 mm/ms, p<0.01, n=11-13), and vasodilator function (Aorta, 46±5 vs 80±2% ACh-induced relaxation, p<0.01, n=6-9). Notably, the cardiovascular protection occurred despite preservation of increased agonist-mediated contraction and RhoA/Rho kinase activity, suggesting RhoBTB1 selectively controls vasodilation. Sodium nitroprusside-induced production of cGMP in aorta was severely impaired in S-P467L but restored by RhoBTB1. Consistent with this, phosphodiesterase 5 (PDE5) activity in aorta was augmented 2.5±0.3 fold in S-P467L but was returned to normal by RhoBTB1. PDE5 and RhoBTB1 reciprocally co-immunoprecipitated in HEK293 cells. Ubiquitination of PDE5 by Cullin-3 in HEK293 cells was RhoBTB1-dependent. Consistent with a role for Cullin-3 in mediating turnover of PDE5, PDE5 activity was augmented in MLN4924-treated aorta, a Cullin inhibitor, and abrogated by PDE5 inhibitor. The beneficial cardiovascular effect of RhoBTB1 in S-P467L mice was phenocopied by PDE5 inhibition. Angiotensin-II infusion also causes RhoBTB1-deficiency and HT which was reversed by smooth muscle specific RhoBTB1 restoration. We conclude that RhoBTB1 augments the cGMP response to nitric oxide by restraining the activity of PDE5 by acting as a substrate adaptor delivering PDE5 to the Cullin-3 E3 Ring ubiquitin ligase complex for ubiquitination and proteasomal degradation. RhoBTB1 provides protection from HT, vascular smooth muscle dysfunction, and arterial stiffness in at least two models of HT.

Activation of TP receptors induces high release of PGI2 in coronary arteries of renal hypertensive rats

AimTo investigate the molecular mechanisms and cellular signaling pathways involved in the activation of TP receptors and the consequent induction of contractile responses in coronary arteries of renal hypertensive (2K-1C) rats. Methods and resultsThe coronary perfusion pressure (CPP) was lower in 2K-1C rats during increased coronary flow as measured by the Langendorff technique. The coronary contraction and relaxation were evaluated by vascular reactivity studies, and the molecular mechanisms were investigated on the basis of the protein expression of TP receptors, Cav-1, eNOS, COX-1, and COX-2, as measured by Western blot. The levels of eicosanoids were determined by ELISA immunoassay and analyzed by reverse-phase HPLC coupled to electrospray ionization mass spectrometry (HPLC-MS/MS). The metabolites from NO production were evaluated by the Griess reaction. The coronary arteries of 2K-1C rats expressed COX-2 to a larger extent and TP receptors to a lesser extent than the coronary arteries of normotensive (2K) rats. Selective COX-1 and non-selective COX inhibitors reversed the reduction in the contraction induced by TP receptors in the coronary arteries of 2K-1C rats. U46619, an agonist of TP receptors, induced a contractile response that was relaxed by acetylcholine (ACh). In the coronary arteries of 2K-1C rats, this ACh-induced relaxation depended on COX. The activation of TP receptors increased the production of PGI2 in the coronary arteries of 2K-1C rats. The results demonstrated that increased COX signaling in the coronary arteries of 2K-1C rats mediated the low levels of CPP, the contraction induced by the activation of TP receptors, and the endothelium-dependent relaxation. The vasodilator PGI2 seemed to be the major product. ConclusionActivation of TP receptors increases production of PGI2 in coronary arteries of 2K-1C rats.

Inhibition of ferrochelatase impairs vascular eNOS/NO and sGC/cGMP signaling.

Ferrochelatase (FECH) is an enzyme necessary for heme synthesis, which is essential for maintaining normal functions of endothelial nitric oxide synthase (eNOS) and soluble guanylyl cyclase (sGC). We tested the hypothesis that inhibition of vascular FECH to attenuate heme synthesis downregulates eNOS and sGC expression, resulting in impaired NO/cGMP-dependent relaxation. To this end, isolated bovine coronary arteries (BCAs) were in vitro incubated without (as controls) or with N-methyl protoporphyrin (NMPP; 10−5–10-7M; a selective FECH antagonist) for 24 and 72 hours respectively. Tissue FECH activity, heme, nitrite/NO and superoxide levels were sequentially measured. Protein expression of FECH, eNOS and sGC was detected by western blot analysis. Vascular responses to various vasoactive agents were evaluated via isometric tension studies. Treatment of BCAs with NMPP initiated a time- and dose-dependent attenuation of FECH activity without changes in its protein expression, followed by significant reduction in the heme level. Moreover, ACh-induced relaxation and ACh-stimulated release of NO were significant reduced, associated with suppression of eNOS protein expression in NMPP-treated groups. Decreased relaxation to NO donor spermine-NONOate reached the statistical significance in BCAs incubated with NMPP for 72 hours, concomitantly with downregulation of sGCβ1 expression that was independent of heat shock protein 90 (HSP90), nor did it significantly affect BCA relaxation caused by BAY 58–2667 that activates sGC in the heme-deficiency. Neither vascular responses to non-NO/sGC-mediators nor production of superoxide was affected by NMPP-treatment. In conclusion, deletion of vascular heme production via inhibiting FECH elicits downregulation of eNOS and sGC expression, leading to an impaired NO-mediated relaxation in an oxidative stress-independent manner.

Impairment of Protease-Activated Receptor 2-Induced Relaxation of Aortas of Aged Spontaneously Hypertensive Rat.

Hypertension is one of the most prevalent diseases worldwide and can cause harmful complications within the vascular system. Further research on vascular responsiveness to different ligands and diverse receptors in various arteries is required to understand the mechanisms underlying the development of these vascular complications. Here, we investigated the vasorelaxant effect of the protease-activated receptor 2 (PAR2) agonist 2-furoyl-LIGRLO-amide (2-Fly) and two commonest agents, namely endothelium-dependent dilator acetylcholine (ACh) and endothelium-independent dilator sodium nitroprusside (SNP), on the thoracic aorta isolated from aged spontaneously hypertensive rats (SHR) (age, 52±1 weeks). The effects of these agents were compared between aortas isolated from SHR and age-matched normotensive Wistar Kyoto (WKY) rats. Compared with the WKY group, in the SHR group, 2-Fly-induced relaxation was impaired, ACh-induced relaxation was slightly decreased at low concentrations, and SNP-induced relaxation was similar. In addition, 2-Fly-induced aortic relaxation was largely decreased by a PAR2 antagonist (FSLLRY), endothelial denudation, and a nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NNA) but not by an Akt inhibitor. These results suggested that PAR2-induced relaxations of aortas of aged SHR was impaired, and this impaired aortic relaxation may be attributed to decreased NO bioavailability rather than altered NO sensitivity unrelated to the Akt activity.

Ergothioneine prevents endothelial dysfunction induced by mercury chloride.

Exposure to mercury has detrimental effects on the cardiovascular system, particularly the vascular endothelium. The present study aimed to investigate the effects of ergothioneine (EGT) on endothelial dysfunction induced by low-dose mercury chloride (HgCl2). Agonist-induced contractions and relaxations were evaluated in isolated aortic rings from 3-month-old male Wistar rats treated by intra-muscular injection to caudal hind leg muscle with HgCl2 (first dose, 4.6 µg/kg; subsequent doses, 0.07 µg/kg/day for 15 days) and optionally with EGT (2 µg/kg for 30 days). Reactive oxygen species (ROS) in aortic rings were measured by means of lucigenin- and luminol-enhanced chemiluminescence. The protein level of endothelial nitric oxide synthase was evaluated by ELISA. Blood glutathione (GSH) and catalase levels, lipid peroxidation and total nitrite were measured spectrophotometrically. The results indicated that low-dose HgCl2 administration impaired acetylcholine (ACh)-induced relaxation and potentiated phenylephrine- and serotonin-induced contractions in rat aortas. In addition, HgCl2 significantly increased the levels of ROS in the aortic tissue. EGT prevented the loss of ACh-induced relaxations and the increase in contractile responses. These effects were accompanied by a significant decrease in ROS levels. EGT also improved the ratio of reduced GSH to oxidized GSH and catalase levels with a concomitant decrease in lipid peroxidation. In conclusion, to the best of our knowledge, the present study was the first to report that EGT prevents endothelial dysfunction induced by low-dose HgCl2 administration. EGT may serve as a therapeutic tool to reduce mercury-associated cardiovascular complications via improving the antioxidant status.

Fasudil improves endothelial dysfunction in rats exposed to chronic intermittent hypoxia through RhoA/ROCK/NFATc3 pathway.

Endothelial dysfunction is one of the main pathological changes in Obstructive sleep apnoea (OSA). The Rho kinase (ROCK) pathway is associated with endothelial dysfunction. However, the interaction between ROCK and nuclear factor of activated T cells isoform c3 (NFATc3) in the development of this pathological response under chronic intermittent hypoxia (CIH) is unclear. To simulate the OSA model, we established a moderate CIH rat model by administering the fraction of inspired O2 (FiO2) from 21% to 9%, 20 times/h, 8 h/day for 3 weeks. Fasudil (ROCK inhibitor, 8 mg/kg/d, i.p.) was administrated in the rats exposed to CIH for 3 weeks. Our results demonstrated that CIH caused significantly endothelial dysfunction, accompanying with increased ET-1 level, decreased eNOS expression and NO production, which reduced ACh-induced vascular relaxation responses. Moreover, RhoA/ROCK-2/NFATc3 expressions were up-regulated. Fasudil significantly improved CIH induced endothelial dysfunction. Data suggested that the ROCK activation is necessary for endothelial dysfunction during CIH.

Knocking Out Endothelial TRPV4 Channels Enhances Vasoconstriction and Exacerbates Endothelial Dysfunction in Low‐Flow Vascular Injury

Atherosclerosis remains a leading killer of men and women. It is associated with reduced blood flow, progressive endothelial dysfunction and vascular remodeling. Preventing pathologic endothelial changes is crucial for stopping the disease. Transient receptor potential cation channels (TRPV4) play a key role in endothelial responses to shear stress and local mediators by controlling dynamic endothelial Ca2+ signals. In the current study, we assessed the specific impact of endothelial TRPV4 channels on carotid artery function in both wild type (WT) control and endothelial‐specific TRPV4 knockout transgenic mice (KO). Following tissue harvest, carotid arteries were cut into rings for Isometric force myography. Depolarization‐induced contractions (60 mM KCl) were larger in KO than WT arteries (1.14 ± 0.14 mN vs. 0.52 ± 0.08 mN, P<0.0005, N=6) and a similar trend was noted for contractions induced by the α1 adrenergic receptor agonist phenylephrine (0.68 ± 0.12 mN vs. 0.43 ± 0.08 mN, P<0.1, N=6). The calculated EC50 indicated no significant difference (1.7×10−7 vs 1.5×10−7 M, n=6) in phenylephrine sensitivity. Net relaxation to acetylcholine was increased in KO vs. WT but EC50 values were not significantly different (5.5×10−7 vs 5.9×10−7, n=6). Using confocal imaging, endothelial Ca2+ was evaluated in carotid artery segments that were cut open longitudinally. The dynamic Ca2+ events that drive endothelium dependent vasorelaxation were altered in KO vs. WT arteries, exhibiting a lower frequency of these events but also events with larger mean amplitude and duration. A carotid artery partial ligation model was used to mimic the low flow predicted with atherosclerosis. Following 2 weeks of reduced flow, KO arteries demonstrated enhanced contractility and impaired ACh‐induced relaxation compared to similarly ligated WT arteries. Overall, our findings suggest that loss of endothelial TRPV4 channels inherently augments vasoconstriction and exacerbates endothelial dysfunction under conditions of chronic low flow.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impaired Mesenteric Arterial Function of Male UC Davis Type 2 Diabetes Mellitus (UCD‐T2DM) Rats: Possible Involvement of Small Conductance Calcium‐activated Potassium Channels (SKca)

UC Davis type‐2 diabetes mellitus (UCD‐T2DM) model is a novel and validated model of type‐2 diabetes exhibiting etiology very similar to clinical T2DM seen in humans. This model is characterized by polygenic adult‐onset obesity and diabetes observed in both sexes along with development of insulin resistance, impaired glucose tolerance, and subsequent β‐cell decompensation. We have previously shown that vascular reactivity in mesenteric arteries (MA) of male UCD‐T2DM is altered. Endothelium‐dependent hyperpolarizing factors (EDHF) is an important regulator of vascular tone, especially in small vessels such as MA. The objective of this study was to investigate the contribution of small or intermediate conductance calcium‐activated potassium channels (SKca & IKca) in modulating vascular function of MA in this model. Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10−8 to 10−5 M) in pre‐contracted MA with phenylephrine (PE, 2μM) was measured before and after pretreatment with indomethacin (Indo, 10μm), a cyclooxygenase inhibitor, followed by addition of L‐NAME (200uM), a nitric oxide synthase inhibitor, and then Apamin (1μm), a selective inhibitor of SKca; and TRAM‐34 (1μm), a selective inhibitor of IKca. We demonstrated that the sensitivity to ACh‐induced relaxation was significantly reduced in MA from male UCD‐T2DM rats compared to controls. Addition of L‐NAME reduced Indo‐insensitive vasorelaxation in both control and diabetic animals. However, when compared with that of control, the effect of L‐NAME was much greater in MA of UCD‐T2DM, indicating a reduced contribution of EDHFtype relaxation or an enhanced role of NO in this disease model. The remaining EDV to ACh which is referred to as the L‐NAME/Indo‐insensitive component, or EDHF‐type relaxation was subsequently blocked by inhibiting intermediate (but not small) conductance calcium‐activated potassium channels in MA of diabetic model. In control animals, however, incubation of MA with apamin (a SKca inhibitor) led to a further reduction of EDV. The subsequent addition of TRAM‐34 eventually blunted the EDHF‐type relaxation in this group. These data, for the first time, show that the contribution of EDHF in EDV is reduced in the MA of UCD‐T2DM group, and the down regulation of SKca may be involved. Clearly, further studies will be needed to document the underlying mechanism(s) of altered vascular function in male UCD‐T2DM rats.Support or Funding InformationSupported by NIH/NHLBIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulation of endothelial function by pro-inflammatory cytokines involved in chronic inflammatory diseases. Focus on IL-17A, IL-20, IL-23 and IL-9

Chronic inflammatory diseases (CID) such as rheumatoid arthritis, systemic lupus erythematosus, spondyloarthritis are associated with increased cardiovascular risk secondary to the presence of endothelial dysfunction. In addition to the well-known cytokines (TNF-α, IL-1β and IL-6), emerging data identified new cytokines such as IL-17A, IL-20, IL-23 and IL-9 key-players of the pathogenesis of CID. To date, whether these cytokines might contribute to CID-associated endothelial dysfunction is not known. This study investigated the effect of IL-17A, IL-20, IL-23 and IL-9 on endothelium-dependent relaxation in response to acetylcholine (Ach) in aortic rings from male Lewis rats. Thoracic aortic rings are incubated for 1 or 24 h at 37 °C with 2 concentrations of each cytokine (IL-17A: 250 pg/mL and 10 ng/mL; IL-20: 500 pg/mL and 5 ng/mL; IL-23: 80 pg/mL and 10 ng/mL; and IL-9: 300 pg/mL and 10 ng/mL). Incubation with 10 ng/ml TNF-α was used as a positive control and with vehicle as negative control. At the end of the incubation period, concentration-response curves to Ach (10 −11 –10 −4 mol/L) were assessed. As expected, a 24 h- but not 1 h-incubation with TNF-α reduced Ach-induced relaxation. The same result was obtained with IL-17A (10 ng/mL). By contrast, IL-20 did not change Ach-induced relaxation whatever the concentration and the incubation time. Impairment in vascular relaxation was observed after exposure to IL-9 (10 ng/mL), both after 1 h- and more severely after 24 h-incubation. As regards IL-23, an effect was observed only after 1 h incubation and with high concentration. Our data demonstrated that IL-17A, IL-23 and IL-9 but not IL-20 induced endothelial dysfunction, with different kinetics profiles. IL-9 exhibited the most important effect thus revealing a new putative role of this pleiotropic cytokine in CID-associated cardiovascular risk. Further studies are needed to confirm these data on animal models of diseases.

Differential participation of calcium-activated potassium channel in endothelium-dependent hyperpolarization-type relaxation in superior mesenteric arteries of spontaneously hypertensive rats.

The purpose of this study was to determine the relationship of KCa channels to endothelium-dependent hyperpolarizing factor (EDHF)-mediated relaxation induced by acetylcholine (ACh) in the superior mesenteric arteries of 7-month-old spontaneously hypertensive rats (SHR). Upon inhibition of nitric oxide synthase and cyclooxygenase, ACh-induced EDHF-mediated relaxation was found to be weaker in SHR than in age-matched Wistar Kyoto rats (WKY). These relaxations in both group were attenuated by combined treatment with small-conductance and intermediate-conductance Ca2+-activated K+ channels (SKCa and IKCa) inhibitors, with the exception of relaxation resistant to inhibition of these channels in SHR (vs. WKY). Treatment with large-conductance Ca2+-activated K+ channels (BKCa) inhibitor specifically attenuated relaxation in SHR, but not in WKY. Protein expression of IKCa and SKCa in the arteries did not differ between the 2 groups, whereas ratio of sloβ1 subunit to α subunit of BKCa was increased in SHR (vs. WKY). These results suggest that EDHF-mediated relaxations in superior mesenteric arteries are impaired in SHR, and utilize components of BKCa in addition to SKCa/IKCa channel activities, that the increased participation of BKCa may be attributable to alterations in α and sloβ1 subunit ratio, and that components unrelated to KCa activity may also contribute to the difference between SHR and WKY arteries.