Relaxation In Mesenteric Resistance Arteries Research Articles

Vasorelaxant Effect of Trachelospermi caulis Extract on Rat Mesenteric Resistance Arteries.

Background: Trachelospermi caulis (T. caulis) has been used as a traditional herbal medicine in Asian countries. Although it is well known that T. caulis has beneficial effects, no sufficient research data are available on the cardiovascular effect of T. caulis. We investigated whether T. caulis extract has vascular effects in rat resistance arteries in this study. Methods: To examine whether T. caulis extract affects vascular reactivity, we measured isometric tension of rat mesenteric resistance arteries using a multi-wire myograph system. T. caulis extract was administered after arteries were pre-contracted with high K+ (70 mM) or phenylephrine (5 µM). Vanillin, a single active component of T. caulis, was used to treat mesenteric arteries. Results: T. caulis extract caused vascular relaxation in a concentration-dependent manner, which was endothelium-independent. To further identify the mechanism, we incubated the arteries in Ca2+-free solution containing high K+, followed by a cumulative administration of CaCl2 (0.01–2.0 mM) with or without T. caulis extract (250 µg/mL). The treatment of T. caulis extract decreased contractile responses induced by the addition of Ca2+, which suggested that the extracellular Ca2+ influx was inhibited by the T. caulis extract. Moreover, an active compound of T. caulis extract, vanillin, also induced vasodilation in mesenteric resistance arteries. Conclusion: T. caulis extract and its active compound, vanillin, concentration-dependently induced vascular relaxation in mesenteric resistance arteries. These results suggest that the administration of T. caulis extract could help decrease blood pressure.

Hydrogen sulfide as a mediator of endothelium-dependent relaxation evoked by Moringa oleifera leaf extract in mesenteric arterial beds isolated from L-NAME hypertensive rats.

Aqueous extract of Moringa oleifera leaves (MOE) is a potent inducer of endothelium-dependent relaxation of mesenteric resistance arteries of rats induced to be hypertensive using Nω-nitro-L-arginine methyl ester (L-NAME). Hydrogen sulfide (H2S) has been shown to participate in endothelium-dependent relaxation of small resistance arteries. Therefore, this study aimed to investigate whether endothelial H2S-dependent signaling plays a role in the vasorelaxation in response to MOE. Mesenteric arterial beds isolated from L-NAME hypertensive rats were set up in an exvivo perfusion system for measurement of vasoreactivity. All experiments were performed in the presence of the nitric oxide synthase inhibitor, L-NAME (100µM) and the cyclooxygenase inhibitor, indomethacin (10µM) to prevent the formation of nitric oxide and prostanoids, respectively. In the presence of the nitric oxide synthase inhibitor, L-NAME and the cyclooxygenase inhibitor, indomethacin, the endothelium-dependent vasorelaxation induced by MOE (0.001-3mg) was completely inhibited by DL-propargylglycine (100µM), which inhibits the H2Sgenerating enzyme, cystathionine γ-lyase. This H2Sdependent response was reduced by the KATP channel blocker; glibenclamide (10µM), the KCa channel blocker; tetraethylammonium (1µM), and the myo-endothelial gap-junctional uncoupler; 18α-glycyrrhetinic acid (10µM). In contrast, the muscarinic receptor antagonist, atropine (100µM), did not affect the response to MOE. The results may suggest that H2S is the likely mediator of endothelium-dependent relaxation in response to MOE in mesenteric arterial beds of L-NAME-induced hypertensive rats. MOE-induced H2S-dependent vasorelaxation involves activation of KATP and KCa channels and requires myo-endothelial gap-junctional communication.

Postpartum Maternal Vascular Function in a Rat Model of Gestational Obstructive Sleep Apnea

IntroductionDe novo obstructive sleep apnea (OSA) in pregnancy, is associated with adverse gestational outcomes such as preeclampsia, gestational diabetes, and fetoplacental hypoxia. In addition, exposure to chronic intermittent hypoxia (CIH), a model of OSA, induces endothelial dysfunction and enhanced vasoconstriction in pregnant mice. It is currently unknown whether gestational OSA has a long‐term effect on maternal vascular function. We hypothesized that exposure to gestational CIH during late pregnancy will result in postpartum maternal vascular dysfunction.MethodsTimed pregnant female Long‐Evans rats were randomly assigned to two experimental groups: Normoxia (n=6) and CIH (n=6). The CIH group was exposed to five days (gestational days: 15–20) of intermittent hypoxia [6 min cycles of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2)]. Gestational age at delivery was recorded and neonate’s crown‐rump length, abdominal girth, and body weights were measured within 12–16 hours from birth. At weaning (postnatal day 28), dams were euthanized and third‐order mesenteric arteries, and the main uterine artery, and uterine perivascular adipose tissue (PVAT) were excised. To assess endothelium‐dependent relaxation, we performed concentration‐response curves to acetylcholine (ACh: 10−9 – 3×10−5 M) using wire myography. In uterine arteries, we measured responses to ACh in the presence and absence of PVAT (0.1 g) based on our previous findings that uterine PVAT has vasoactive effects on these arteries.ResultsGestational CIH had no effect on the duration of gestation (Normoxia: 22.3 ± 0.21 days vs. CIH: 22.8 ± 1.16 days, p=0.24), litter size (Normoxia: 12.2 ± 1.64 vs. CIH: 10.3 ± 1.40, p=0.34), and neonatal weight at birth (Normoxia: 6.4 ± 0.24 g vs. CIH: 6.7 ± 0.19 g, p=0.16), while it increased neonatal crown‐rump length (Normoxia: 3.98 ± 0.07 cm vs. CIH: 4.19 ± 0.07 cm, p=0.05) and abdominal girth (Normoxia: 4.72 ± 0.10 cm vs. 4.94 ± 0.03 cm, p=0.07). At weaning, dams exposed to gestational CIH were lighter compared to dams exposed to normoxia (Normoxia: 319 ± 6.5 g vs. CIH: 298 ± 8.7 g, p=0.08). Exposure to gestational CIH had no effect on ACh‐induced relaxation in mesenteric resistance arteries (pEC50, Normoxia: 7.80 ± 0.03 vs. CIH: 7.89 ± 0.06, p=0.19). In the absence of PVAT, uterine arteries from dams exposed to gestational CIH had exaggerated responses to ACh compared to dams exposed to normoxia [(−)PVAT/pEC50, Normoxia: 6.77 ± 0.08 vs. CIH: 7.13 ± 0.09, p<0.01], while PVAT normalized this difference [(+)PVAT/pEC50, Normoxia: 6.67 ± 0.08 vs. CIH: 6.70 ± 0.07, p=0.99]. The effects of PVAT were due to its anti‐dilatory influences on uterine arteries from CIH‐treated rats [CIH (−PVAT) vs. CIH (+PVAT), p = 0.003], whereas it had no effect on arteries from control rats [Normoxia (−PVAT) vs. Normoxia (+PVAT), p = 0.77].ConclusionExposure to CIH during gestation resulted in neonatal macrosomia, reduced maternal weight at weaning, and exaggerated postpartum uterine vascular smooth muscle relaxation responses, while it did not affect small resistance artery reactivity. Gestational OSA may impair maternal vascular recovery after birth.Support or Funding InformationUNTHSC Basic Seed Grant

Moringa oleifera leaf extract induces vasorelaxation via endothelium-dependent hyperpolarization and calcium channel blockade in mesenteric arterial beds isolated from L-NAME hypertensive rats

ABSTRACT Background An aqueous extract of Moringa oleifera leaves (MOE) is known to cause relaxation of mesenteric resistance arteries of rats in which hypertension has been induced by the administration of L-NAME, but the mechanism(s) of action of MOE remains unclear. The purpose of this study was to investigate these mechanisms in mesenteric arterial beds isolated from L-NAME induced hypertensive rats. Methods: An investigation of vascular reactivity was conducted on isolated mesenteric arterial beds by measuring the changes in perfusion pressure using an in vitro system. Results MOE (0.001–3 mg in 0.1 ml injection volume) caused a dose-dependent relaxation in methoxamine (5 µM) pre-contracted arterial beds, which was partially abolished by endothelium removal. The endothelium-dependent component of vasorelaxation was insensitive to both L-NAME (100 µM) and indomethacin (10 µM), while completely inhibited in high KCl (45 mM)-induced contraction. MOE (1 and 3 mg/ml) showed a dose-dependent inhibitory effect on CaCl2-induced contractions of denuded preparations in Ca2+-free medium containing a high KCl (60 mM) or methoxamine (10 µM). In Ca2+-free medium, MOE (3 mg/ml) also inhibited phenylephrine-induced contractions of denuded preparations. Conclusion: These findings suggest that MOE relaxes mesenteric arterial beds of L-NAME hypertensive rats via both endothelium-dependent and endothelium-independent mechanisms. The endothelium-dependent action occurred via endothelium-derived hyperpolarizing factor-mediated hyperpolarization. The endothelium-independent action was related to blocking the entry of extracellular Ca2+ via voltage-operated and receptor-operated Ca2+ channels, and inhibiting mobilization of sarcolemmal Ca2+ via inositol trisphosphate receptor Ca2+ channels. MOE may be potentially useful as a natural vasodilator against hypertension.

β‐Hydroxybutyrate (βHOB) Increases Nitric Oxide Synthase Activity in Resistance Arteries from Dahl Salt‐sensitive Rats

The deleterious link between overconsumption of dietary salt and hypertension is unequivocal. This dogma has recently expanded with the report that a high salt diet lowers the circulating levels of the ketone metabolite, beta‐hydroxybutyrate (βHOB), in Dahl salt‐sensitive rats (Dahl S‐HS). While stimulators of ketogenesis are well known for improving cardiovascular health (e.g., exercise and caloric restriction), less is known about negative‐regulation of ketone body metabolism and how it may impact hypertension and vascular aging. Therefore, we hypothesized that raising the circulating levels of βHOB would improve endothelium‐dependent relaxation in mesenteric resistance arteries (MRA) of Dahl S‐HS. To test this hypothesis, 1,3‐butanediol (20% v/v), which is a precursor of βHOB, was supplemented in the drinking water of 5 week old, male Dahl S‐HS for 8 weeks. Subsequently MRA function was analyzed using wire myographs. Although relaxation to endothelium‐dependent vasodilator acetylcholine (ACh) was not different [%Relaxation, Dahl S‐low salt (LS): 40±11 vs. Dahl S‐HS: 52±11 vs. Dahl S‐HS+βHOB: 44±11, p>0.05], the mechanism of relaxation was different between the groups. Specifically, while nitric oxide synthase inhibitor (L‐NAME; 100 μM) completely prevented relaxation in Dahl S‐LS and Dahl S‐HS+βHOB groups, MRA from Dahl S‐HS still partially relaxed in the presence of L‐NAME (%Relaxation, Dahl S‐LS: 1±4 vs. Dahl S‐HS: 33±5 vs. Dahl S‐HS+βHOB: 6±4, p<0.05; Figure 1A–C). These data suggest that MRA from Dahl S‐HS relax through nitric oxide‐independent mechanisms and that βHOB treatment restores nitric oxide synthase activity, despite consumption of a high salt diet. To test whether 1,3‐butanediol treatment had direct effects on arterial function, concentrations‐response curves were performed to βHOB and 1,3‐butanediol in MRA from naïve 5 week old, male Dahl S‐LS rats. We observed that βHOB and 1,3‐butanediol caused triphasic‐quadriphasic responses after pre‐contraction to phenylephrine (PE; Figure 2A–B). Specifically, we observed that low concentrations (<3 nM) of βHOB caused pronounced vasodilation and this was inhibited by both L‐NAME and tetraethylammonium (TEA; potassium channel inhibitor; both, p<0.05; Figure 3A–B). Intermediate concentrations of βHOB (3 nM–100 nM) caused contraction and this tended to be inhibited by indomethacin (cyclooxygenase inhibitor; p=0.08; Figure 3C). Finally, high concentrations (100 nM–30 μM) of βHOB caused relaxation again and this was predominately mediated by nitric oxide (p<0.05; Figure 3A). Overall, these data suggest that βHOB stimulates the production of endothelium‐derived factors and low concentrations of βHOB could be a novel therapy against hypertension‐associated vascular aging by increasing nitric oxide synthesis.Support or Funding InformationAmerican Heart Association (18POST34060003) and National Institutes of Health (K99GM118885 and R01HL143082).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P132: Everolimus, a Targeted Cancer Therapy, Improves Endothelium Dependent Relaxation in Spontaneously Hypertensive Rat Mesenteric 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. Everolimus, an inhibitor to mammalian target of rapamycin (mTOR) complex 1, is a targeted therapy approved for the treatment of metastatic breast, colon, renal cell and pancreatic cancer. Clinical retrospective reviews suggest heart failure and hypertension as cardiotoxic effects of everolimus. Our lab previously showed that everolimus exposure on normotensive vessels increased sensitivity to adrenergic stimuli in a time and concentration dependent manner. We hypothesized that everolimus leads to enhanced contractility and impaired relaxation in hypertensive vessels. We studied the effects of everolimus on the contractility and relaxation of mesenteric resistance arteries (MRA) of male Wistar rats (12-15 weeks old, n=4) and spontaneously hypertensive rats (SHR) (15-18 weeks old, n=2) on a wire myograph. Two concentrations of everolimus were evaluated, 0.1μM and 0.1nM, during a one-hour incubation. Normotensive MRA exposed to everolimus (0.1μM) showed a decreased contractile response to phenylephrine (LogEC50 + SEM, Ctrl:-5.627 + 0.07 vs Drug: -5.390 + 0.10) but no difference when exposed to 0.1nM. SHR MRA showed no difference in PE-induced contraction at 0.1μM (LogEC50 + SEM, Ctrl:-5.638 + 0.08 vs Drug:-6.286 + 0.36) or 0.1nM (LogEC50 + SEM, Ctrl:-5.638 + 0.08 vs Drug:-5.332 + 0.16). No differences were observed in endothelium dependent relaxation response to acetylcholine (ACh) for either drug concentration in the normotensive vessels; however, SHR MRA exhibited enhanced sensitivity to ACh with everolimus exposure (LogEC50 + SEM, Ctrl: -6.851 + 0.15 vs Drug:-9.233 + 0.37 at 0.1nM; LogEC50 + SEM, Ctrl: -6.851 + 0.15 vs Drug:-8.244 + 0.34 at 0.1μM). Our data show that everolimus affects contractility in normotensive vessels, while affecting the dilatory response in SHR vessels with an improvement in endothelium-dependent relaxation. Everolimus 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, which could be associated with cardiotoxicity.

Abstract P523: Time and Concentration Dependent Vascular Changes of Cancer Therapy Everolimus in Mesenteric Resistance Arteries

The leading cause of death in cancer survivors is cardiovascular disease, in part due to cancer therapy-induced cardiotoxicity. The short- and long-term cardiovascular side effects of a newer class of targeted chemotherapy, mTOR inhibitors, have not been well established as they have been with anthracyclines, anti-VEGF therapy and tyrosine kinase inhibitors. mTOR inhibitor Everolimus, used as an agent in transplant immunosuppression, is now indicated for the treatment of metastatic breast, colon, renal cell and pancreatic cancer. Retrospective clinical data indicate the possibility of heart failure and hypertension as cardiotoxic effects of Everolimus, although data are inconclusive and no vascular function studies are available. We studied the effects of Everolimus on the contractility and relaxation of mesenteric resistance arteries (MRA) of male Wistar rats (12-15 weeks old) at different drug incubation time frames (1, 12, and 24 hours) on a tension wire myograph. We hypothesized that the longer exposure to Everolimus (i.e. longer incubation time) leads to enhanced contractility to phenylephrine (PE) and impaired relaxation to acetylcholine (ACh). Two concentrations of Everolimus were evaluated, 0.1 μM and 0.1 nM. MRA exposed to Everolimus 0.1 μM showed a decreased contractile response to PE at 1 hour incubation (LogEC 50 , Ctrl: -5.627±0.07 vs. Drug: -5.390±0.10, *p=0.03) and at 12 hours incubation (LogEC 50 , Ctrl: -5.295±0.16 vs. Drug: -5.645±0.08, *p=0.04), while no difference was observed at 24 hours (p>0.05). MRA exposed to Everolimus 0.1 nM for 24 hours showed enhanced contractility to PE (LogEC 50 , Ctrl: -5.295±0.16 vs. Drug=-5.740±0.07, *p=0.005) while no difference at 1 hour or 12 hour incubation times (p>0.05). No differences were observed in the endothelium-dependent relaxation response to ACh for either drug concentration or any of the incubation times. Our data suggest that the effects of Everolimus on resistance arteries from normotensive rats are a time and concentration-dependent: short-term incubation (i.e., < 12hours) increases sensitivity of MRA to adrenergic stimuli. However, long-term treatment with this drug may lead to vascular dysfunction, which could be associated with cardiotoxicity observed in cancer survivals.

GPER agonist dilates mesenteric arteries via PI3K-Akt-eNOS and potassium channels in both sexes

AimThe action of oestrogen has traditionally been attributed to the activation of nuclear receptors (ERα and ERβ). A third receptor, the G protein-coupled oestrogen receptor (GPER), has been described as mediator of the rapid action of oestrogen. Based on the possible protective role of oestrogen in the cardiovascular system, the present study was designed to determine whether selective GPER activation induces relaxation of mesenteric resistance arteries in both sexes and which signalling pathways are involved. Main methodsThird-order mesenteric arteries were isolated, and concentration-response curves were plotted following the cumulative addition of the selective GPER agonist G-1 (1nM−10μM) following induction of contraction with phenylephrine (3μM). The vasodilatory effects of G-1 were assessed before and after removal of the endothelium or incubation for 30min with nitric oxide synthase (Nω-nitro-L-arginine methyl ester - L-NAME, 300μM) and cyclooxygenase (indomethacin – INDO, 10μM) inhibitors alone or combined, PI3K-Akt pathway inhibitor (LY-294,002, 2.5μM) or a potassium channel blocker (tetraethylammonium – TEA, 5mM). GPER immunolocalisation was also performed on the investigated arteries. Key findingsThe tested GPER agonist induced concentration-dependent relaxation of the mesenteric resistance arteries without differences related to sex that were partially endothelium dependent, mainly mediated by the PI3K-Akt-eNOS pathway and attenuated by nonspecific potassium channel blockade. In addition, the endothelial GPER immunolocalisation was stronger among females. SignificanceThis evidence provides a new perspective for understanding the mechanisms involved in the vascular responses triggered by oestrogen via GPER in both sexes.

Chronic fluoxetine treatment increases NO bioavailability and calcium-sensitive potassium channels activation in rat mesenteric resistance arteries

Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), has effects beyond its antidepressant properties, altering, e.g., mechanisms involved in blood pressure and vasomotor tone control. Although many studies have addressed the acute impact of fluoxetine on the cardiovascular system, there is a paucity of information on the chronic vascular effects of this SSRI. We tested the hypothesis that chronic fluoxetine treatment enhances the vascular reactivity to vasodilator stimuli by increasing nitric oxide (NO) signaling and activation of potassium (K+) channels. Wistar rats were divided into two groups: (I) vehicle (water for 21 days) or (II) chronic fluoxetine (10mg/kg/day in the drinking water for 21 days). Fluoxetine treatment increased endothelium-dependent and independent vasorelaxation (analyzed by mesenteric resistance arteries reactivity) as well as constitutive NO synthase (NOS) activity, phosphorylation of eNOS at Serine1177 and NO production, determined by western blot and fluorescence. On the other hand, fluoxetine treatment did not alter vascular expression of neuronal and inducible NOS or guanylyl cyclase (GC). Arteries from fluoxetine-treated rats exhibited increased relaxation to pinacidil. Increased acetylcholine vasorelaxation was abolished by a calcium-activated K+ channel (KCa) blocker, but not by an inhibitor of KATP channels. On the other hand, vascular responses to Bay 41-2272 and 8-bromo-cGMP were similar between the groups. In conclusion, chronic fluoxetine treatment increases endothelium-dependent and independent relaxation of mesenteric resistance arteries by mechanisms that involve increased eNOS activity, NO generation, and KCa channels activation. These effects may contribute to the cardiovascular effects associated with chronic fluoxetine treatment.

Abstract T P225: cGMP-Dependent Protein Kinase I in Smooth Muscle Cells Protects Against Stroke Injury in Mice

The signaling pathways by which NO protects against stroke injury are mediated by cyclic guanosine monophosphate (cGMP) formation. However, the mechanisms of downstream signaling from cGMP to smooth muscle cells (SMC) remain incompletely understood. The cGMP-dependent protein kinase I (cGKI) is a key mediator of cGMP signaling. The goal of this study is to test the hypothesis that cGKI in SMC protects against stroke injury. Animals: We used the novel mouse line for highly efficient tamoxifen-inducible SMC-specific gene knockout, SMA-CreERT2, which expresses the CreERT2 recombinase under the control of the smooth muscle alpha-actin promoter (cGKI KO mice). The litter mate wild-type (WT) mice (no Cre gene) receiving the same tamoxifen treatment were used as control (cGKI WT) mice. Methods and Results: Mean blood pressure measured acutely from the common carotid artery under anesthesia (30% oxygen, 70% nitrous oxide and 1.5% isoflurane) was higher in cGKI KO mice (107±8 mmHg, Mean±SD) than in cGKI WT mice (95±1 mmHg, n=4/group, P<0.05). One hour of middle cerebral artery (MCA) occlusion by filament with 23 hours of reperfusion produced increased cerebral infarct volume (TTC staining, indirect calculation) in cGKI KO mice (123±39 mm3) as compared with cGKI WT (85±17 mm3, n=5/group, p< 0.05). Neurological deficit by 5 point scale was worse in cGKI KO mice (4.0 points) as compared with cGKI WT (2.4 points). Cerebral blood flow monitored by laser Doppler flowmetry above the core of the MCA territory indicated that reperfusion was less in cGKI KO mice than in cGKI WT mice, suggesting that cGKI mediates cerebrovascular SMC relaxation and protects against ischemic stroke. Vascular relaxation of mesenteric resistance arteries to acetylcholine, and the NO donor, NONOate after preconstriction with phenylephrine was dramatically impaired in cGKI KO mice. It suggests the importance of cGKI in the vascular SMC for NO-induced relaxation. In conclusion, stroke injury was more pronounced in cGKI SMC specific inducible conditional knockout mice than in cGKI WT mice, suggesting a protective role for cGKI in SMC against cerebral reperfusion injury. These studies identify the cGKI in cerebral vasculature as a potential target for therapies aimed at reducing stroke injury.

The new NO donor Terpy induces similar relaxation in mesenteric resistance arteries of renal hypertensive and normotensive rats

The present work aimed to investigate the cellular mechanisms involved on the vasorelaxation induced by the new nitric oxide donor [Ru(terpy)(bdq)NO]3+ (Terpy) in isolated mesenteric resistance artery and to compare the vascular responses in isolated vessels from 2K and 2K-1C hypertensive rats. We have used this artery because it is important to the control of vascular resistance and consequently to the blood pressure control. The NO donor Terpy induced relaxation in a concentration-dependent way in mesenteric resistance arteries. There were no differences between renal hypertensive (2K-1C) and normotensive (2K) in Terpy-induced relaxation neither in NO released. The relaxation induced by Terpy was inhibited by the soluble guanylyl-cyclase (sGC) inhibitor ODQ both in 2K and in 2K-1C with similar amplitude. In agreement with these data, the protein expression of the subunits α1 and β1 of the enzyme sGC was not different between 2K-1C and 2K mesenteric bed. The relaxation induced by Terpy was inhibited by the cGMP-dependent protein kinase (G kinase) inhibitor or by the non-selective K+ channel blocker tetraethylamonium (TEA), but with no difference between 2K-1C and 2K arteries. The relaxation induced by Terpy was also inhibited by the SERCA inhibitor thapsigargin in both groups. Taken together, these results show that the vascular relaxation induced by the NO donor [Ru(terpy)(bdq)NO]3+ involves the activation of NO/sGC/cGMP/GK pathway, activation of K+ channels sensitive to TEA and SERCA in normotensive and renal hypertensive rat mesenteric resistance arteries. Surprisingly, Terpy-induced vasorelaxation is similar in mesenteric resistance arteries of renal hypertensive and normotensive rats.

Abstract 642: GPR30-Mediated Relaxation of Mesenteric Resistance Arteries is Diminished in Males and Aging Females

We previously reported that chronic activation of estrogen receptor GPR30 by the selective agonist G-1 decreases blood pressure in ovariectomized hypertensive mRen2.Lewis rats but not male littermates. Furthermore, acute G-1 relaxes female mesenteric resistance arteries via both endothelium-dependent and -independent mechanisms. Because of the lack of a blood pressure-lowering effect by G-1 in males and because aging may influence estrogen receptor expression, we hypothesized that GPR30-dependent vasodilation was reduced in males and older females. Mesenteric vessels (∼200 μm I.D.) from 15 wk Lewis and mRen2.Lewis males and 52 wk Lewis females were mounted on a wire myograph and preconstricted with 10 μM phenylephrine before assessing the response to increasing concentrations of 17β-estradiol or G-1 (0.001-3 μM). At the highest concentration, 17β-estradiol vasodilation was greater in 15 wk Lewis females (62±8%, n=5) and mRen2.Lewis females (57±9%, n=6) than age-matched Lewis males (33±7%, n=5, P<0.001) and mRen2.Lewis males (30±7%, n=4, P<0.001). The G-1 response was also greater in Lewis females (60±12%, n=7) and mRen2.Lewis females (57±8%, n=5) than in Lewis males (33±8%, n=7, P<0.01) and mRen2.Lewis males (28±7%, n=4, P<0.01). Pretreatment of male vessels with the nitric oxide (NO) synthase inhibitor L-NAME had no significant effect on the estradiol or G-1 response (n=3-6, P>0.05). In aged females, vasorelaxation was also significantly blunted in response to 17β-estradiol (34±6%, n=5, P<0.01) and G-1 (32±6%, n=8, P<0.01). In contrast to males, L-NAME inhibition or endothelial denuding in older females further reduced vasodilation to both 17β-estradiol (L-NAME: 15±6%, n=4, P<0.05; denuded: 13±2%, n=8, P<0.001) and G-1 (L-NAME: 10±6%, n=3, P<0.01; denuded 8±2%, n=11, P<0.0001). In summary, GPR30-mediated vasodilation was attenuated in Lewis and mRen2.Lewis males, and the ineffectiveness of L-NAME inhibition suggests a loss in NO signaling. G-1 vasorelaxation was also blunted in aging Lewis females, but the intact NO response indicates a smooth muscle cell deficit. We conclude that alterations in GPR30 expression or signaling may contribute to the loss of cardiovascular protection in aging postmenopausal females and in adult males.

The AGE-Breaker ALT-711 Restores High Blood Flow–Dependent Remodeling in Mesenteric Resistance Arteries in a Rat Model of Type 2 Diabetes

Flow-mediated remodeling of resistance arteries is essential for revascularization in ischemic diseases, but this is impaired in diabetes. We hypothesized that breaking advanced glycation end product (AGE) cross-links could improve remodeling in mesenteric resistance arteries in Zucker diabetic fatty (ZDF) rats compared with lean Zucker (LZ) rats. Arteries, exposed to high (HF) or normal (NF) blood flow after alternate arterial ligation in vivo, were collected after 2 weeks. In LZ rats, HF artery diameter was larger than for NF vessels, but this was not the case in ZDF rats. Endothelium-mediated dilation in ZDF rats, which was lower than in LZ rats, was further decreased in HF arteries. Treatment of rats with the AGE-breaker 4,5-dimethyl-3-phenacylthiazolium chloride (ALT-711) (3 mg/kg/day; 3 weeks) reversed diabetes-induced impairment of HF-dependent remodeling. ALT-711 also improved endothelium nitric oxide–dependent relaxation in mesenteric resistance arteries. Reactive oxygen species reduction restored relaxation in ZDF rats but not in LZ or ALT-711–treated rats. AGEs were reduced in ALT-711–treated ZDF rats compared with ZDF rats. Metalloproteinase activity, necessary for HF-dependent remodeling, was reduced in ZDF rats compared with LZ rats and restored by ALT-711. Thus, targeting AGE cross-links may provide a therapeutic potential for overcoming microvascular complications in ischemic disorders occurring in diabetes.

Regulator of G Protein Signaling 2 Deficiency Causes Endothelial Dysfunction and Impaired Endothelium-derived Hyperpolarizing Factor-mediated Relaxation by Dysregulating Gi/o Signaling

Regulator of G protein signaling 2 (RGS2) is a GTPase-activating protein for G(q/11)α and G(i/o)α subunits. RGS2 deficiency is linked to hypertension in mice and humans, although causative mechanisms are not understood. Because endothelial dysfunction and increased peripheral resistance are hallmarks of hypertension, determining whether RGS2 regulates microvascular reactivity may reveal mechanisms relevant to cardiovascular disease. Here we have determined the effects of systemic versus endothelium- or vascular smooth muscle-specific deletion of RGS2 on microvascular contraction and relaxation. Contraction and relaxation of mesenteric resistance arteries were analyzed in response to phenylephrine, sodium nitroprusside, or acetylcholine with or without inhibitors of nitric oxide (NO) synthase or K(+) channels that mediate endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation. The results showed that deleting RGS2 in vascular smooth muscle had minor effects. Systemic or endothelium-specific deletion of RGS2 strikingly inhibited acetylcholine-evoked relaxation. Endothelium-specific deletion of RGS2 had little effect on NO-dependent relaxation but markedly impaired EDHF-dependent relaxation. Acute, inducible deletion of RGS2 in endothelium did not affect blood pressure significantly. Impaired EDHF-mediated vasodilatation was rescued by blocking G(i/o)α activation with pertussis toxin. These findings indicated that systemic or endothelium-specific RGS2 deficiency causes endothelial dysfunction resulting in impaired EDHF-dependent vasodilatation. RGS2 deficiency enables endothelial G(i/o) activity to inhibit EDHF-dependent relaxation, whereas RGS2 sufficiency facilitates EDHF-evoked relaxation by squelching endothelial G(i/o) activity. Mutation or down-regulation of RGS2 in hypertension patients therefore may contribute to endothelial dysfunction and defective EDHF-dependent relaxation. Blunting G(i/o) signaling might improve endothelial function in such patients.

Poly(ADP-Ribose) Polymerase 1 Inhibition Improves Coronary Arteriole Function in Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) is associated with microvascular dysfunction. We hypothesized that increased poly(ADP-ribose) polymerase 1 (PARP-1) activity contributes to microvascular dysfunction in T2DM. T2DM (db(-)/db(-)) and nondiabetic control (db(-)/db(+)) mice were treated with 2 different PARP-1 inhibitors (INO-1001, 5 mg/kg per day and ABT-888, 15 mg/kg per day) for 2 weeks. Isolated coronary arterioles were mounted in an arteriograph. Pressure-induced myogenic tone was significantly potentiated, whereas endothelium-dependent relaxation was significantly attenuated in diabetic mice compared with control mice. These results were associated with decreased endothelial NO synthase phosphorylation and cGMP level and increased PARP-1 activity in coronary arterioles from diabetic mice compared with control mice. Interestingly, PARP-1 inhibitors significantly reduced the potentiation of myogenic tone, improved endothelium-dependent relaxation, restored endothelial NO synthase phosphorylation and cGMP, and attenuated cleaved PARP-1. These results were supported by in vitro studies indicating that downregulation of PARP-1 in mesenteric resistance arteries using PARP-1 short hairpin RNA lentiviral particles significantly improved endothelium-dependent relaxation in mesenteric resistance arteries from diabetic mice compared with control mice. The inhibition of NO synthesis by N(G)-nitro-L-arginine methyl ester (L-NAME) significantly reduced the endothelium-dependent relaxation in coronary arterioles and mesenteric resistance arteries from control and diabetic mice treated with PARP-1 inhibitors and PARP-1 short hairpin RNA lentiviral particles. In addition, we demonstrated that enhanced cleaved PARP-1, its binding to DNA, and DNA damage were reduced after PARP-1 inhibition in cultured endothelial cells stimulated with high glucose. We provide evidence that T2DM impairs microvascular function by an enhanced PARP-1 activity-dependent mechanism. Therefore, PARP-1 could be a potential target for overcoming diabetic microvascular complications.

TRPV1 activation prevents high-salt diet-induced nocturnal hypertension in mice

High dietary salt-caused hypertension is associated with increasing reactive oxygen species generation and reduced nitric oxide (NO) bioavailability. Transient receptor potential vanilloid type 1 (TRPV1), a specific receptor for capsaicin, is proposed to be involved in Dahl salt-sensitive hypertension, as determined in acute or short-term experiments. However, it remains unknown whether activation of TRPV1 by dietary capsaicin could prevent the vascular oxidative stress and hypertension induced by a high-salt diet. Here, we report that consumption of a high-salt diet blunted endothelium-dependent relaxation in mesenteric resistance arteries and elevated nocturnal blood pressure in mice. These effects were associated with increased superoxide anion generation and reduced NO levels in mesenteric vessels in mice on a high-salt diet. However, chronic administration of capsaicin reduced the high-salt diet-induced endothelial dysfunction and nocturnal hypertension in part by preventing the generation of superoxide anions and NO reduction of mesenteric arteries through vascular TRPV1 activation. Our findings provide new insights into the role of TRPV1 channels in the long-term regulation of blood pressure in response to high-salt intake. TRPV1 activation through chronic dietary capsaicin may represent a promising lifestyle intervention in populations with salt-sensitive hypertension.

Aldosterone induces endothelial dysfunction in resistance arteries from normotensive and hypertensive rats by increasing thromboxane A2 and prostacyclin

The present study was designed to assess whether cyclooxygenase-2 (COX-2) activation is involved in the effects of chronic aldosterone treatment on endothelial function of mesenteric resistance arteries (MRA) from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Relaxation to acetylcholine was measured in MRA from both untreated and aldosterone-treated strains. Vasomotor responses to prostacyclin and U46619 were also analysed. Release of 6-oxo-prostaglandin (PG)F1alpha and thromboxane B2 (TxB2) was determined by enzyme immunoassay. COX-2 protein expression was measured by western blot. Aldosterone reduced acetylcholine relaxation in MRA from both strains. In MRA from both aldosterone-treated strains the COX-1/2 or COX-2 inhibitor (indomethacin and NS-398, respectively), TxA2 synthesis inhibitor (furegrelate), prostacyclin synthesis inhibitor (tranylcypromine) or TxA2/ PGH2 receptor antagonist (SQ 29 548), but not COX-1 inhibitor SC-560, increased acetylcholine relaxation. In untreated rats this response was increased only in SHR. Prostacyclin elicited a biphasic vasomotor response: lower concentrations elicited relaxation, whereas higher concentrations elicited contraction that was reduced by SQ 29 548. Aldosterone increased the acetylcholine-stimulated production of 6-oxo-PGF(1alpha) and TxB2 in MRA from both strains. COX-2 expression was higher in both strains of rats treated with aldosterone. Chronic treatment with aldosterone impaired endothelial function in MRA under normotensive and hypertensive conditions by increasing COX-2-derived prostacyclin and thromboxane A2. As endothelial dysfunction participates in the pathogenesis of many cardiovascular disorders we hypothesize that anti-inflammatory drugs, specifically COX-2 inhibitors, could ameliorate vascular damage in patients with elevated aldosterone production.

Effect of High-Salt Diet on Vascular Relaxation and Oxidative Stress in Mesenteric Resistance Arteries

This study tested the hypothesis that superoxide levels are elevated in isolated mesenteric resistance arteries (100–300 µm) from rats fed a short-term high-salt (HS) diet (4% NaCl for 3 days) compared to controls fed a low-salt (LS) diet (0.4% NaCl). Vascular relaxation induced by the superoxide dismutase mimetic tempol (4-hydroxytetramethylpiperidine-1-oxyl), the NADPH oxidase inhibitor apocynin and the xanthine/xanthine oxidase inhibitor oxypurinol was significantly larger in mesenteric arteries from animals fed HS diet compared to arteries from animals fed LS diet. Basal superoxide levels assessed via dihydroethidine (DHE) fluorescence were significantly elevated in arteries from rats fed HS diet, and were reduced by tempol, apocynin and oxypurinol, but not by L-NAME. Basal and methacholine-induced NO production (assessed by DAF-2T fluorescence) was significantly reduced in arteries from rats fed HS diet versus arteries from rats on LS diet. Impaired methacholine-induced NO release and vascular relaxation were restored by tempol and apocynin, but not by oxypurinol. These data suggest that the elevated production of superoxide by NADPH oxidase and xanthine/xanthine oxidase contribute to elevated basal superoxide levels, reduced NO release and impaired vascular relaxation in mesenteric resistance arteries of rats fed HS diet.

Nitric oxide‐dependent relaxation is modulated by intermediate conductance Ca2+‐activated potassium channels in rat mesenteric resistance arteries

Endothelium-dependent relaxation of isolated rat mesenteric resistance arteries to acetylcholine (ACh) is mediated by the release of nitric oxide (NO) and by endothelium-dependent hyperpolarization. We have investigated the role of Ca2+-activated potassium channels (KCa) in NO-mediated, endothelium-dependent relaxation of mesenteric resistance arteries. Pre-incubation with either 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (Tram-34; 5 μM), an inhibitor of intermediate conductance KCa (IKCa), or L-NG-nitroarginine methylester (L-NAME; 100 μM), an inhibitor of NO synthase, caused significant inhibition of relaxation to ACh in arterial segments pre-contracted with phenylephrine (1–3 μM). The magnitude of inhibition caused by Tram-34 was not significantly different from that caused by L-NAME and the effects of the two inhibitors were not additive. Pre-incubation with apamin (50 nM), an inhibitor of small conductance KCa, did not significantly alter ACh-evoked relaxation. However, in the presence of L-NAME, apamin significantly inhibited relaxations to ACh and the combination of apamin with Tram-34 reduced the maximum relaxation to ACh by 80%. Addition of L-NAME with apamin and Tram-34 did not cause further significant inhibition. Tram-34 and apamin were without effect on relaxations evoked by the NO donor diethylamine NONOate. These data indicate that endothelial IKCa play a key role modulating NO-dependent relaxation to ACh in rat mesenteric resistance arteries. Supported by Heart & Stroke Foundation of Canada, URGC & USRP.

Increased Superoxide Anion Production by Interleukin-1β Impairs Nitric Oxide-Mediated Relaxation in Resistance Arteries

The present study was designed to analyze the effect of long-term incubation with interleukin-1beta (IL-1beta) on endothelium-dependent relaxation in rat mesenteric resistance arteries. Vessels were incubated in culture medium with or without IL-1beta (10 ng/ml, 14 h). Changes in lumen diameter were recorded in a pressure myograph. Protein expression, nitrite, and superoxide anion (O(2)(.)) production were evaluated by either Western blot or immunofluorescence, Griess reaction, and ethidium fluorescence, respectively. IL-1beta impaired acetylcholine (ACh) and sodium nitroprusside (SNP) vasodilation and increased nitrite and O(2)(.) production and inducible nitric-oxide synthase (iNOS), xanthine oxidase, and p22(phox) expression. However, neither endothelial nitric-oxide synthase (NOS) nor soluble guanylate cyclase protein expression were affected by IL-1beta treatment. Polyethylene glycol superoxide dismutase partially reversed the impairment of ACh relaxation and abolished the O(2)(.) production observed in IL-1beta-treated arteries. The impairment of ACh relaxation induced by IL-1beta was also partially reversed by the xanthine oxidase inhibitor allopurinol (1 mM) but not by either the NADPH oxidase inhibitor apocynin (0.3 mM) or the inducible NOS inhibitor N-3-aminomethylbenzylacetamidine (1 microM). However, all these inhibitors improved the impaired SNP response. The results of the present study demonstrate that long-term incubation with IL-1beta induces an impairment of the nitric oxide-mediated relaxation in mesenteric resistance arteries through the production of O(2)(.), mainly from xanthine oxidase.