ACh-induced Vasodilation Research Articles

Involvement of Hydrogen Sulfide in Endothelium-Derived Relaxing Factor-Mediated Responses in Rat Cerebral Arteries

Background/Aim: H₂S is a novel vasoactivator. To verify the hypothesis that H₂S may act as an endothelium-derived hyperpolarizing factor (EDHF) in the rat cerebrovasculature, the role of H₂S in endothelium-derived relaxing factor (EDRF)-mediated responses was investigated. Methods: Cystathionine-γ-lyase (CSE) was knocked down with an siRNA technique. Artery diameter, hyperpolarization and Ca²⁺-activated K⁺ (K_Ca) current were measured. Results: CSE knockdown was indicated by a decrease in protein and mRNA expression in the rat middle cerebral artery (MCA) and cerebral basilar artery (CBA). Acetylcholine (ACh) induced significant hyperpolarization and vasodilation in endothelium-intact MCA and CBA. Removal of the endothelium abolished these responses. The nitric oxide (NO) synthase inhibitor <smlcap>L</smlcap>-NAME, but not the PGI₂ production inhibitor indomethacin, significantly inhibited ACh-induced hyperpolarization and vasodilation in the CBA. In the presence of <smlcap>L</smlcap>-NAME and indomethacin, ACh-induced hyperpolarization and vasodilation in the MCA and CBA were attenuated. The non-NO/PGI₂-mediated responses were abolished by the K_Ca channel blockers charybdotoxin and apamin. In the cerebral arteries from the CSE knockdown rat, non-NO/PGI₂-mediated responses were significantly attenuated, and the remaining responses were abolished by charybdotoxin and apamin or the CSE inhibitor propargylglycine. CSE knockdown did not affect <smlcap>L</smlcap>-NAME-sensitive responses in the CBA. Sodium hydrosulfide (NaHS) augmented the K_Ca current in CBA vascular smooth muscle cells. Conclusion: EDHF-mediated responses in rat cerebral arteries were due to H₂S activating the K_Ca channel.

High glucose impairs acetylcholine-mediated vasodilation in isolated arteries from Mourning doves (Z. macroura)

Normal avian plasma glucose levels are 1.5–2 times greater than mammals of similar size. In mammals, hyperglycemia induces oxidative stress and impaired endothelium-dependent vasodilation. Prior work has shown that mourning doves have high levels of antioxidants and isolated vessels have low endogenous oxidative stress. Therefore, the hypothesis was that endothelium-dependent vasodilation of isolated avian arteries would not be impaired following acute exposure to high glucose. Isolated small resistance cranial tibial arteries (c. tibial) were cannulated and pressurized in a vessel chamber then incubated with either normal or high glucose (20mM vs. 30mM) for 1h at 41°C. Vessels were then pre-constricted to 50% of resting inner diameter with phenylephrine (PE) followed by increasing doses of acetylcholine (ACh; 10−9 to 10−5M, 5min per step). Percent vasodilation was measured by tracking the inner diameter with edge-detection software. Contrary to our hypothesis, ACh-induced vasodilation was impaired with acute exposure to high glucose (p=0.013). The impairment was not related to increased osmolarity since vasodilation of arteries exposed to an equimolar combination of 20mM d-glucose and 10mM l-glucose was not different from controls (p=0.273). Rather, the impaired vasodilation was attributed to oxidative stress since superoxide levels were elevated 168±42% (p=0.02) and pre-exposure of arteries to the superoxide dismutase mimetic tiron (10mM) improved vasodilation (p<0.05). Therefore, isolated arteries from doves do not have endogenous mechanisms to prevent impaired vasodilation resulting from high glucose-mediated increases in oxidative stress.

Low-level lead exposure changes endothelial modulation in rat resistance pulmonary arteries

Lead exposure induces hypertension and endothelial dysfunction. However, the effects on the pulmonary vasculature have not been explored. In this study, rats exposed to lead acetate for seven days (4μg/100g on the 1st day and 0.05μg/100g/day i.m. subsequently) had lead blood level of 3.9±0.7μg/dL and increased right ventricular pressures. There was an increased Pb deposition and superoxide anions production in the pulmonary arteries, associated with reduced vasoconstriction but unchanged endothelium-dependent vasodilatation to acetylcholine (ACh). In both groups, inhibition of the nitric oxide (NO) synthase with L-NAME blocked the response to ACh, while indomethacin (cycloxygenase inhibitor) had no effect. Incubation with nonspecific potassium channel blocker (tetraethylammonium) reduced the ACh-induced vasodilatation only in the Pb group. Apamin (SKCa channel blocker) and 4-aminopyridine (Kv channel blocker), but not iberiotoxin (BKCa channel blocker), also inhibited this response in the Pb group. The vasodilatation to exogenous NO was reduced by Pb, while relaxation to the cGMP analogue was similar between groups. Concordantly, the protein level of soluble guanylate cyclase (sGC) was reduced. In conclusion, short-term and low-level exposure to Pb changes pulmonary haemodynamic and increases oxidative stress. The pulmonary vasculature exhibited increased hyperpolarization by the Kv and SKCa channels, probably as a compensatory mechanism to the decreased responsiveness to NO.

Improvement of Acetylcholine-Induced Vasodilation by Acute Exercise in Ovariectomized Hypertensive Rats.

Postmenopause is associated with the development of cardiovascular disease, such as hypertension. However, limited information is available regarding effects of exercise on cardiovascular responses and its underlying mechanisms in the simultaneous postmenopausal and hypertensive status. We aimed to investigate whether acute exercise could enhance vasodilation mediated by acetylcholine (ACh) and sodium nitroprusside (SNP) in ovariectomized hypertensive rats. The fifteen-week-old female spontaneously hypertensive rats (SHR) were bilaterally ovariectomized, at the age of twenty-four weeks, and randomly divided into sedentary (SHR-O) and acute exercise (SHR-OE) groups. Age-matched WKY rats were used as the normotensive control group. The SHR-OE group ran on a motor-driven treadmill at a speed of 24 m/min for one hour in a moderate-intensity program. Following a single bout of exercise, rat aortas were isolated for the evaluation of the endothelium-dependent (ACh-induced) and endothelium-independent (SNP-induced) vasodilation by the organ bath system. Also, the serum levels of oxidative stress and antioxidant activities, including malondialdehyde (MDA), superoxide dismutase (SOD), and catalase, were measured after acute exercise among the three groups. We found that acute exercise significantly enhanced the ACh-induced vasodilation, but not the SNP-induced vasodilation, in ovariectomized hypertensive rats. This increased vasodilation was eliminated after the inhibition of nitric oxide synthase (NOS). Also, the activities of SOD and catalase were significantly increased after acute exercise, whereas the level of MDA was comparable among the three groups. These results indicated that acute exercise improved the endothelium-dependent vasodilating response to ACh through the NOS-related pathway in ovariectomized hypertensive rats, which might be associated with increased serum antioxidant activities.

Retrospectively gated MRI for in vivo assessment of endothelium-dependent vasodilatation and endothelial permeability in murine models of endothelial dysfunction.

Endothelial dysfunction is linked to impaired endothelial-dependent vasodilatation and permeability changes. Here, we quantify both of these phenomena associated with endothelial dysfunction by MRI in vivo in mice. Endothelial function was evaluated in the brachiocephalic artery (BCA) and left carotid artery (LCA) in ApoE/LDLR(-/-) and high-fat diet (HFD)-fed mice as compared with control mice (C57BL/6J). The 3D IntraGate® FLASH sequence was used for evaluation of changes in vessels' cross-sectional area (CSA) and volume following acetylcholine (Ach) administration. Evaluation of endothelial permeability after administration of contrast agent (Galbumin, BioPAL) was based on the variable flip angle method for the assessment of parameters based on the relaxation time (T1 ) value. In order to confirm the involvement of nitric oxide (NO) in response to Ach, L-NAME-treated mice were also analyzed. To confirm that endothelial permeability changes accompany the impairment of Ach-dependent vasodilatation, permeability changes were analyzed in isolated, perfused carotid artery. In C57BL/6J mice, Ach-induced vasodilatation led to an approximately 25% increase in CSA in both vessels, which was temporarily dissociated from the effect of Ach on heart rate. In ApoE/LDLR(-/-) or HFD-fed mice Ach induced a paradoxical vasoconstriction that amounted to approximately 30% and 50% decreases in CSA of BCA and LCA respectively. In ApoE/LDLR(-/-) and HFD-fed mice endothelial permeability in BCA was also increased (fall in T1 by about 25%). In L-NAME-treated mice Ach-induced vasodilatation in BCA was lost. In isolated, perfused artery from ApoE/LDLR(-/-) mice endothelial permeability was increased. MRI-based assessment of endothelium-dependent vasodilatation induced by Ach and endothelial permeability using a retrospectively self-gated 3D gradient-echo sequence (IntraGate® FLASH) enables the reliable detection of systemic endothelial dysfunction in mice and provides an important tool for the experimental pharmacology of the endothelium in murine models of diseases in vivo. Copyright © 2016 John Wiley & Sons, Ltd.

Diet supplementation with rice bran enzymatic extract restores endothelial impairment and wall remodelling of ApoE−/− mice microvessels

Background and aimsSmall mesenteric artery resistance and functionality are key factors for the maintenance of blood homeostasis. We attained to evaluate the effects of a rice bran enzymatic extract (RBEE) on structural, mechanic and myogenic alterations and endothelial dysfunction secondary to atherosclerosis disease. MethodsSeven week-old ApoE−/− mice were fed on standard (ST) or high fat (HF) diets supplemented or not with 1 or 5% RBEE (w/w) for 23 weeks. Wild-type C57BL/6J mice fed on ST diet served as controls. Small mesenteric arteries were mounted in a pressure myograph in order to evaluate structural, mechanical and myogenic properties. Vascular reactivity was assessed in the presence of different combinations of inhibitors: l-NAME, indometacin, apamin and charybdotoxin. ResultsApoE−/− mice fed on ST and HF diets showed different structural and mechanical alterations, alleviated by RBEE supplementation of ST and HF diets. C57BL/6J was characterized by increased expression of IKCa (199.3%, p = 0.023) and SKCa (133.2%, p = 0.026), resulting in higher EDHF participation (p = 0.0001). However, NO release was more relevant to ApoE−/− mice vasodilatation. HF diet reduced the amount of NO released due to 2-fold increase of eNOS phosphorylation in the inhibitory residue Thr495 (p = 0.034), which was fully counteracted by RBEE supplementation (p = 0.028), restoring ACh-induced vasodilatation (p = 0.00006). Dihydroethidium fluorescence of superoxide and picrosirius red staining of collagen were reduced by RBEE supplementation of HF diet by 76.91% (p = 0.022) and 65.87% (p = 0.030), respectively. ConclusionRBEE supplemented diet reduced vessel remodeling and oxidative stress. Moreover, RBEE supplemented diet increased NO release by downregulating p-eNOSThr495, thus, protecting the endothelial function.

Context-dependent effects of SOCS3 in angiotensin II-induced vascular dysfunction and hypertension in mice: mechanisms and role of bone marrow-derived cells.

Carotid artery disease is a major contributor to stroke and cognitive deficits. Angiotensin II (Ang II) promotes vascular dysfunction and disease through mechanisms that include the IL-6/STAT3 pathway. Here, we investigated the importance of suppressor of cytokine signaling 3 (SOCS3) in models of Ang II-induced vascular dysfunction. We examined direct effects of Ang II on carotid arteries from SOCS3-deficient (SOCS3(+/-)) mice and wild-type (WT) littermates using organ culture and then tested endothelial function with acetylcholine (ACh). A low concentration of Ang II (1 nmol/l) did not affect ACh-induced vasodilation in WT but reduced that of SOCS3(+/-) mice by ∼50% (P < 0.05). In relation to mechanisms, effects of Ang II in SOCS3(+/-) mice were prevented by inhibitors of STAT3, IL-6, NF-κB, or superoxide. Systemic Ang II (1.4 mg/kg per day for 14 days) also reduced vasodilation to ACh in WT. Surprisingly, SOCS3 deficiency prevented most of the endothelial dysfunction. To examine potential underlying mechanisms, we performed bone marrow transplantation. WT mice reconstituted with SOCS3(+/-) bone marrow were protected from Ang II-induced endothelial dysfunction, whereas reconstitution of SOCS3(+/-) mice with WT bone marrow exacerbated Ang II-induced effects. The SOCS3 genotype of bone marrow-derived cells did not influence direct effects of Ang II on vascular function. These data provide new mechanistic insight into the influence of SOCS3 on the vasculature, including divergent effects depending on the source of Ang II. Bone marrow-derived cells deficient in SOCS3 protect against systemic Ang II-induced vascular dysfunction.

Contribution of K(+) channels to endothelium-derived hypolarization-induced renal vasodilation in rats in vivo and in vitro.

We investigated the mechanisms behind the endothelial-derived hyperpolarization (EDH)-induced renal vasodilation in vivo and in vitro in rats. We assessed the role of Ca(2+)-activated K(+) channels and whether K(+) released from the endothelial cells activates inward rectifier K(+) (Kir) channels and/or the Na(+)/K(+)-ATPase. Also, involvement of renal myoendothelial gap junctions was evaluated in vitro. Isometric tension in rat renal interlobar arteries was measured using a wire myograph. Renal blood flow was measured in isoflurane anesthetized rats. The EDH response was defined as the ACh-induced vasodilation assessed after inhibition of nitric oxide synthase and cyclooxygenase using L-NAME and indomethacin, respectively. After inhibition of small conductance Ca(2+)-activated K(+) channels (SKCa) and intermediate conductance Ca(2+)-activated K(+) channels (IKCa) (by apamin and TRAM-34, respectively), the EDH response in vitro was strongly attenuated whereas the EDH response in vivo was not significantly reduced. Inhibition of Kir channels and Na(+)/K(+)-ATPases (by ouabain and Ba(2+), respectively) significantly attenuated renal vasorelaxation in vitro but did not affect the response in vivo. Inhibition of gap junctions in vitro using carbenoxolone or 18α-glycyrrhetinic acid significantly reduced the endothelial-derived hyperpolarization-induced vasorelaxation. We conclude that SKCa and IKCa channels are important for EDH-induced renal vasorelaxation in vitro. Activation of Kir channels and Na(+)/K(+)-ATPases plays a significant role in the renal vascular EDH response in vitro but not in vivo. The renal EDH response in vivo is complex and may consist of several overlapping mechanisms some of which remain obscure.

The Gatekeepers in the Mouse Ophthalmic Artery: Endothelium-Dependent Mechanisms of Cholinergic Vasodilation.

Cholinergic regulation of arterial luminal diameter involves intricate network of intercellular communication between the endothelial and smooth muscle cells that is highly dependent on the molecular mediators released by the endothelium. Albeit the well-recognized contribution of nitric oxide (NO) towards vasodilation, the identity of compensatory mechanisms that maintain vasomotor tone when NO synthesis is deranged remain largely unknown in the ophthalmic artery. This is the first study to identify the vasodilatory signalling mechanisms of the ophthalmic artery employing wild type mice. Acetylcholine (ACh)-induced vasodilation was only partially attenuated when NO synthesis was inhibited. Intriguingly, the combined blocking of cytochrome P450 oxygenase (CYP450) and lipoxygenase (LOX), as well as CYP450 and gap junctions, abolished vasodilation; demonstrating that the key compensatory mechanisms comprise arachidonic acid metabolites which, work in concert with gap junctions for downstream signal transmission. Furthermore, the voltage-gated potassium ion channel, Kv1.6, was functionally relevant in mediating vasodilation. Its localization was found exclusively in the smooth muscle. In conclusion, ACh-induced vasodilation of mouse ophthalmic artery is mediated in part by NO and predominantly via arachidonic acid metabolites, with active involvement of gap junctions. Particularly, the Kv1.6 channel represents an attractive therapeutic target in ophthalmopathologies when NO synthesis is compromised.

Skin blood flow responses to locally applied acetylcholine in Caucasian and African descent individuals with and without cyclooxygenase inhibition

Individuals of African descent (AFD) are more susceptible to non-freezing cold injury (NFCI) than Caucasians (CAU) [1]. This may be a consequence of lower skin blood flow during local cold exposure and subsequent rewarming in AFD [2], possibly due to a difference in endothelium function as acetylcholine (ACh)-induced vasodilatation is smaller in AFD than CAU on the non-glabrous finger and toe skin sites [3]. It is known that prostaglandins produced by the enzyme cyclooxygenase (COX) mediate part of the ACh-induced vasodilator response [4] however in hypertensive individuals, COX inhibition results in augmented vasodilatation in response to ACh [5] demonstrating that COX can also promote vasoconstriction. Whether COX products are involved in the attenuated vasodilator response to ACh in healthy AFD [3] is not known. Therefore, the aim of the present study was to investigate the contribution of COX in both CAU and AFD to local application of ACh in foot and finger skin sites which are susceptible to NFCI.

Diosgenin regulates adipokine expression in perivascular adipose tissue and ameliorates endothelial dysfunction via regulation of AMPK

Perivascular adipose tissue (PVAT) has been recognized as an active contributor to vascular function due to its paracrine effects on cells contained within vascular wall. The present study was designed to investigate the effect of diosgenin on adipokine expression in PVAT with emphasis on the regulation of endothelial function. Palmitic acid (PA) stimulation induced inflammation and dysregulation of adipokine expression in PVAT. Diosgenin treatment inhibited IKKβ phosphorylation and downregulated mRNA expressions of proinflammatory cytokines/proteins including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein (MCP-1), and inducible nitric oxide synthase (iNOS), while reduced gene expressions for adiponectin, PPARγ, and arginase 1 (Arg-1) were reversed by diosgenin treatment. Diosgenin enhanced AMPK phosphorylation under basal and inflammatory conditions in PVAT, whereas knockdown of AMPK by SiRNA diminished its modulatory effect, indicating that diosgenin inhibited inflammation in an AMPK-dependent manner. We prepared conditioned medium from PA-stimulated PVAT to induce endothelial dysfunction and found that pre-treatment of PVAT with diosgenin effectively restored the loss of ACh-induced vasodilation and increased eNOS phosphorylation in rat aorta. High-fat diet feeding in rats induced inflammation in PVAT and the impairment of endothelium-dependent vasodilation, whereas these alterations were prevented by oral administration of diosgenin at doses of 20 and 40mg/kg. In conclusion, the obtained data showed that diosgenin ameliorated inflammation-associated adipokine dysregulation, and thereby prevented endothelial dysfunction. Our findings would shed a novel insight into the potential mechanism by which diosgenin protected endothelial function against inflammatory insult.

Differential effects of short- and long-term bupivacaine treatment on α1-adrenoceptor-mediated contraction of isolated rat aorta rings and the reversal effect of lipid emulsion.

Arterial function is significantly influenced by bupivacaine at both clinically relevant concentrations and toxic concentrations, but the underlying mechanisms are not fully understood. In the present study we investigated the role of α1-adrenoceptors in bupivacaine effects on isolated rat aortas. Isolated aortic rings were prepared from rats and suspended in an organ bath. Phenylephrine (Phe)-induced vasoconstriction and acetylcholine (ACh)-induced vasodilation were recorded through an isometric force transducer connected to a data acquisition system. Administration of bupivacaine (30-300 μmol/L) produced mild vasoconstriction, and this response declined with repeated administrations. Treatment of the aortic rings with bupivacaine (3-30 μmol/L) for 20 min enhanced Phe-induced vasoconstriction, while treatment for 40 min suppressed Phe-induced vasoconstriction. Both the short- and long-term bupivacaine treatment suppressed ACh-induced vasodilation. Incubation of the aortic rings with 0.2%-0.6% lipid emulsion (LE) for 100 min significantly increased the pD2 and Emax values of Phe-induced vasoconstriction, and incubation with 0.4% LE for 100 min reversed the inhibition of bupivacaine on vasoconstriction induced by Phe (30 μmol/L). In contrast, incubation with LE suppressed ACh-induced vasodilation, even at a lower concentration and with a 5-min incubation. Bupivacaine exerts dual effects on α1-adrenoceptor-mediated vasoconstriction of isolated rat aortic rings: short-term treatment enhances the response, while long-term treatment inhibits it; the inhibition may be reversed via long-term incubation with LE.

PP.41.10

Objective: The primary aim of this study was to evaluate the effectiveness of laser speckle contrast imaging (LSCI), coupled with the transdermal iontophoretic delivery of acetylcholine (ACh), in the assessment of penile microvascular function. Additionally, we tested systemic microvascular function using both LSCI and ACh iontophoresis in the forearm. Design and method: We assessed cutaneous endothelium-dependent microvascular reactivity in the penis and forearm of healthy volunteers (aged 56.6 ± 1.0 years, n = 26), at rest and 60 minutes following the oral administration of the phosphodiesterase type 5 inhibitor, sildenafil (SIL, 100 mg). LSCI was coupled with the iontophoresis of ACh, using increasing anodal currents of 30, 60, 90, 120, 150 and 180 μA, during 10 second intervals spaced 1 minute apart. Results: Basal skin microvascular flow in the penis increased significantly following SIL (P = 0.0014) administration. The endothelium-dependent skin microvascular vasodilator responses induced by ACh were also significantly enhanced following SIL administration for each of the following parameters: the peak values of cutaneous vascular conductance (CVC, P < 0.0001), the increases in CVC (P < 0.0001) and the area under the curve for ACh-induced vasodilation (P = 0.0094). Conclusions: LSCI appears to be a promising noninvasive technique in the evaluation of penile microvascular endothelial function. This methodology may be valuable in the evaluation of penile microvascular reactivity among patients with cardiovascular and metabolic diseases, as well as the effectiveness of drugs used to treat vasculogenic erectile dysfunction.

Aerobic Exercise Training Prevents the Onset of Endothelial Dysfunction via Increased Nitric Oxide Bioavailability and Reduced Reactive Oxygen Species in an Experimental Model of Menopause.

ObjectivePrevious studies have shown that estrogen deficiency, arising in postmenopause, promotes endothelial dysfunction. This study evaluated the effects of aerobic exercise training on endothelial dependent vasodilation of aorta in ovariectomized rats, specifically investigating the role of nitric oxide (NO) and reactive oxygen species (ROS).MethodsFemale Wistar rats ovariectomized (OVX – n=20) or with intact ovary (SHAM – n=20) remained sedentary (OVX and SHAM) or performed aerobic exercise training on a treadmill 5 times a week for a period of 8 weeks (OVX-TRA and SHAM-TRA). In the thoracic aorta the endothelium-dependent and –independent vasodilation was assessed by acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. Certain aortic rings were incubated with L-NAME to assess the NO modulation on the ACh-induced vasodilation. The fluorescence to dihydroethidium in aortic slices and plasma nitrite/nitrate concentrations were measured to evaluate ROS and NO bioavailability, respectively.ResultsACh-induced vasodilation was reduced in OVX rats as compared SHAM (Rmax: SHAM: 86±3.3 vs. OVX: 57±3.0%, p<0.01). Training prevented this response in OVX-TRA (Rmax: OVX-TRA: 88±2.0%, p<0.01), while did not change it in SHAM-TRA (Rmax: SHAM-TRA: 80±2.2%, p<0.01). The L-NAME incubation abolished the differences in ACh-induced relaxation among groups. SNP-induced vasodilation was not different among groups. OVX reduced nitrite/nitrate plasma concentrations and increased ROS in aortic slices, training as effective to restore these parameters to the SHAM levels.ConclusionsExercise training, even in estrogen deficiency conditions, is able to improve endothelial dependent vasodilation in rat aorta via enhanced NO bioavailability and reduced ROS levels.

The evaluation of penile microvascular endothelial function using laser speckle contrast imaging in healthy volunteers

ObjectiveThe primary aim of this study was to evaluate the effectiveness of laser speckle contrast imaging (LSCI) coupled with transdermal iontophoretic delivery of acetylcholine (ACh) for the assessment of penile microvascular function. Additionally, we tested systemic microvascular function using both LSCI and ACh iontophoresis on the forearm. MethodsWe assessed cutaneous, endothelium-dependent microvascular reactivity on the penis and forearm of healthy volunteers (aged 56.6±1.0years, n=26) at rest and 60min following the oral administration of the phosphodiesterase type 5 inhibitor (sildenafil: SIL, 100mg). LSCI was coupled with the iontophoresis of ACh using increasing anodal currents of 30, 60, 90, 120, 150 and 180μA during 10-second intervals spaced 1min apart. ResultsBasal skin microvascular flow in the penis increased significantly following SIL administration. The endothelium-dependent skin microvascular vasodilator responses induced by ACh were also significantly enhanced following SIL administration for each of the following parameters: peak values of cutaneous vascular conductance (CVC); increases in CVC; and the area under the curve for ACh-induced vasodilation. ACh-induced microvascular vasodilation in the forearm was not modified by SIL. Finally, the administration of electric current alone did not affect penile microvascular flow. ConclusionLSCI appears to be a promising non-invasive technique for the evaluation of penile microvascular endothelial function. This methodology may be valuable for the evaluation of penile microvascular reactivity among patients with cardiovascular and metabolic diseases and to test the effectiveness of drugs used to treat vasculogenic erectile dysfunction.

TRPV4 Channels Regulate Endothelial Ca Signaling in Rat Maternal Uteroplacental Vasculature

An increase in cytosolic Ca2+ ([Ca2+]i) is a key mechanism translating the effects of agonist stimulation of endothelial cells (ECs) into cellular responses. In this study we explored the role of TRPV4 channels in regulating EC Ca2+ signaling in uteroplacental arteries of pregnant rats. The diameters, SMC membrane potential (MP) and [Ca2+]i responses to activation of TRPV4 channels with GSK 1016790A (GSK) were studied using pressurized arteries. ACh- and GSK-induced EC [Ca2+]i responses were characterized in sheets of ECs. The effects of GSK on MP of ECs were tested using perforated patch clamp. RT PCR was performed to quantify mRNA levels of TRPV4 in uterine vasculature. GSK induced an endothelium-mediated vasodilation (78 ± 7 % at 10 nM, n = 8) that was associated with SMC hyperpolarization (by 22 ± 3 mV) or SMC [Ca2+]i reduction. GSK induced EC [Ca2+]i rise of 418 ± 45 nM (n = 4). HC067047 (HC) and TRAM-34, inhibitors of TRPV4 and IK channels, abolished GSK responses. EC hyperpolarization (by 25 ± 6 mV, n = 5) to GSK was reversed by charybdotoxin. ACh-induced vasodilation or elevation of EC [Ca2+]i was reduced by HC by 76 % (n = 4). TRPV4 expression in radial arteries was two-fold higher than in main uterine artery. TRPV4 channels are essential Ca2+ entry pathways in ECs of uteroplacental arteries. They induce IK-dependent hyperpolarization of ECs resulting in EC and SMC hyperpolarization, SMC [Ca2+]i reduction and vasodilation. Downregulation of these channels may contribute to an impaired EC Ca2+ signaling and uterine endothelial dysfunction in complicated pregnancies. Supported by NIH HL088245 (NIG), P01 HL095488 (ADB)

Lisinopril Improves Cutaneous Microvascular Function in Hypertensive Men and Women

Hypertension is a chronic disease that induces microvascular dysfunction through upregulation of arginase and inducible nitric oxide synthase (iNOS) resulting in a loss of endothelial (e)NOS‐dependent vasodilation. Angiotensin converting enzyme inhibitors may have off‐target beneficial vascular effects on endothelial signaling and microvessel structure. We hypothesized that treatment with Lisinopril (&gt;3 month) would improve cutaneous microvascular function in hypertensive adults. Four intradermal microdialysis fibers were placed in the forearm skin of 6 controlled hypertensive subjects (Lisinopril (LIS): MAP 94 mmHg), 9 hypertensive (HTN: MAP 111 mmHg) and 10 age matched normotensive (NTN: MAP 86 mmHg) subjects. Ringer's solution (control), 20mM L‐NAME (NOS inhibited), 0.1mM 1400w (iNOS inhibited), and 5mM BEC (arginase inhibited) were perfused through the fibers. Red cell flux was measured by laser‐Doppler flowmetry (LDF) during an acetylcholine (ACh) dose‐response (0.01mM‐100mM ACh). Cutaneous vascular conductance (CVC=LDF/MAP) was calculated and normalized to a percent of maximum (%CVCmax: 28mM SNP). SNP‐induced maximum vasodilation (CVCmax) was also assessed. There were no differences in ACh‐induced vasodilation between the LIS and NTN groups. LIS treatment improved %CVCmax responses to ACh compared to unmedicated HTN (all ACh doses 蠅 1mM p&lt;0.05). Neither iNOS nor arginase inhibition altered the %CVCmax response to ACh in the LIS group (vs. control site). CVCmax was attenuated in HTN (1.57 ± 0.6 vs NTN 1.75 ± 0.6, p&lt;0.05) and not improved in LIS (1.64 ± 0.5, p=0.33). These data suggest that pharmacotherapy for essential hypertension with Lisinopril improves eNOS‐dependent but not SNP‐induced maximal vasodilation in hypertensive adults.NIH R01 HL089302

Exercise‐Like Mechanical Stimulation of Soleus Feed Arteries (SFA) Attenuates Age‐Induced Endothelial Dysfunction

ObjectiveTo determine the mechanism of exercise‐induced attenuation of the detrimental effects of aging on endothelial function. We tested the hypothesis that exercise‐like mechanical stimulation of aged SFA with an acute increase in intraluminal pressure in combination with an increase in intraluminal shear stress would induce greater improvements in endothelium‐dependent dilation than an acute increase in intraluminal pressure alone. Methods: SFA from young and old, Fischer 344 rats were pressurized at 90 cmH20 or 130 cmH20 (P130). P130 arteries were exposed to no shear stress (0 dyn/cm2) or high shear stress (~65 dyn/cm2) for 1 h. Following the 1 h treatment period, pressure in the P130 arteries was reduced to 90 cmH2O for examination of endothelium‐dependent [acetylcholine (ACh)] and endothelium‐independent [sodium nitroprusside (SNP)] dilation.ResultsACh‐induced vasodilation was impaired in old SFA compared to young SFA. Mechanical stimulation with increased pressure for 1 h improved ACh‐induced dilation in old SFA. Stimulation of old SFA with an increase in pressure plus shear stress also improved ACh‐induced. There were no between‐group differences in SNP‐induced dilation.ConclusionThese results indicate that stimulation with an acute increase in intraluminal pressure alone or in combination with an acute increase in shear stress improves endothelium‐dependent dilation. Research support: AHA grant 0765043Y, AHA grant 4150031, The National Space Biomedical Research Institute through NCC 9‐58 and a Sydney and JL Huffines Institute of Sports Medicine Graduate Student Research Grant.

Phenolic content of extra virgin olive oil is essential to restore endothelial dysfunction but not to prevent vascular inflammation in atherosclerotic lesions of Apo E deficient mice

Extra Virgin Olive Oil (EVOO) contains monounsaturated fatty acids and multiple minor components with biological properties and health benefits. We evaluate whether a diet supplemented on Oleaster®(OT), an EVOO obtained by a new process aimed to increase phenolic composition, provided additional benefits to those observed in the animals fed a diet rich in traditional EVOO on atherosclerosis development and vasomotor function in aortic vessel of ApoE−/− mice. Results were compared with those obtained in mice fed an EVOO, a standard diet (SD) or a high fat diet (HFD) rich in saturated fatty acids. Both extra-virgin olive oils reduced macrophage accumulation and protein expression of iNOS, ICAM-1 and VCAM-1, TNF-α and NF-κB, independently of its phenolic content toward values found in SD. Superoxide anion production was also reduced to the same extent in Apo E−/− mice fed EVOO or OT. Endothelial function assessed by ACh-induced vasodilatation, and lipid accumulation within the atherosclerotic plaque were restored in OT- but not in EVOO-fed mice. We conclude that EVOO contribute to prevent the inflammatory markers of atherosclerosis plaque progression but the polyphenolic content is not responsible for this effect. However, those compounds are able to restore endothelial function and to reduce lipid accumulation within the atherosclerotic lesion.

Abstract 610: Context-Dependent Effects of Suppressor of Cytokine Signaling 3 (SOCS3) in Angiotensin II-Induced Vascular Dysfunction: Mechanisms and Role of Bone Marrow-Derived Cells

Angiotensin II (Ang II) promotes vascular disease and hypertension, in part, by activating the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway. Although SOCS3 regulates this pathway in the immune system, its role in vascular disease and hypertension is unknown. In this study, we investigated the role of SOCS3 in a model of Ang II-induced vascular disease. To exam direct effects, carotid arteries from wild-type (WT) and SOCS3 haplodeficient (SOCS3 +/- ) mice were incubated with Ang II for 22 hrs, followed by examination of endothelial function using acetylcholine (Ach). Relaxation to Ach was similar in all arteries incubated with vehicle. A low concentration of Ang II (1 nmol/L) did not affect Ach-induced vasodilation in WT mice, but reduced that of SOCS3 +/- mice by ~50% (P&lt;0.05). Ang II-induced impairment was prevented by inhibitors of STAT3, IL-6, NF-κB, or a scavenger of superoxide. Responses to nitroprusside were similar in all groups. We also tested the impact of SOCS3 in vivo by systemically infusing Ang II (1.4 mg/kg per day) for 14 days via osmotic mini-pumps. Ach-induced vasodilation in carotid and resistance arteries in brain from WT mice was reduced by ~60% (P&lt;0.05). Surprisingly, deficiency in SOCS3 prevented the majority of Ang II-induced endothelial dysfunction without affecting the pressor response to Ang II. Lethally irradiated WT mice reconstituted with SOCS3 +/- bone marrow were protected from Ang II-induced endothelial dysfunction (P&lt;0.05), while reconstitution of irradiated SOCS3 +/- mice with WT bone marrow exacerbated Ang II-induced vascular dysfunction (P&lt;0.05). WT into WT and SOCS3 +/- into SOCS3 +/- bone marrow chimeras exhibited vascular function consistent with non-irradiated controls. The pressor response to Ang II was reduced by ~50% in WT mice reconstituted with bone marrow from SOCS3 +/- mice (P&lt;0.05). These data suggest SOCS3 exerts divergent local versus systemic effects on Ang II-induced vascular dysfunction. In the face of SOCS3 deficiency, bone marrow-derived cells protect against Ang II-induced vascular dysfunction and hypertension.