Responses Of Mesenteric Arteries Research Articles

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography.

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 μm) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Effect of magnesium supplementation on blood pressure and vascular reactivity in nitric oxide synthase inhibition-induced hypertension model

The aim of this study was to assess the effect of oral magnesium supplementation (Mg-supp) on blood pressure (BP) and possible mechanism in nitric oxide synthase (NOS) inhibition-induced hypertension model. Hypertension and/or Mg-supp were created by N-nitro-l-arginine methyl ester (25 mg/kg/day by drinking water) and magnesium-oxide (0.8% by diet) for 6 weeks. Systolic BP was measured weekly by tail-cuff method. The effects of hypertension and/or Mg-supp in thoracic aorta and third branch of mesenteric artery constriction and relaxation responses were evaluated. NOS-inhibition produced a gradually developing hypertension and the magnitude of the BP was significantly attenuated after five weeks of Mg-supp. The increased phenylephrine-induced contractile and decreased acetylcholine (ACh)-induced dilation responses were found in both artery segments of hypertensive groups. Mg-supp was restored ACh-relaxation response in both arterial segments and also Phe-constriction response in thoracic aorta but not in mesenteric arteries. The contributions of NO, prostaglandins and K+ channels to the dilator response of ACh were similar in the aorta of all the groups. The contribution of the NO to the ACh-mediated relaxation response of mesenteric arteries was suppressed in hypertensive rats, whereas this was corrected by Mg-supp. The flow-mediated dilation response of mesenteric arteries in hypertensive rats failed and could not be corrected by Mg-supp. Whereas, vascular eNOS protein and magnesium levels were not changed and plasma nitrite levels were reduced in hypertensive rats. The results of this study showed that Mg-supp lowered the arterial BP in NOS-inhibition induced hypertension model by restoring the agonist-induced relaxation response of the arteries.

7D.10

Objective: It has been previously demonstrated that inflammation in adipose tissue may be implicated in vascular dysfunction (Circulation 2009; 119(12):1661–1670). A senescence-accelerated prone mouse (SAMP8) is a model of age-related cognitive decline and vascular dysfunction. Several studies demonstrated that SAMP8 suffers from increased oxidative stress and that accelerated senescence was associated with decreased eNOS and nNOS and increased oxygen radicals synthesis. Aim of the study was to investigate functional responses of small mesenteric arteries in a senescence-accelerated prone mouse (SAMP8) before and after chronic treatment with melatonin.Design and method: We investigated 7 SAMP8 and 7 SAMR1 normal controls. Mesenteric small resistance arteries were dissected and mounted on a wire myograph, according to Mulvany-Halpern technique (internal diameter about 200 μm). A concentration-response to norepinephrine (NE, from 10–9 to 10-5 Mol/l) was evaluated in vessels with intact prerivascular fat tissue (WF) and in vessels in which perivascular fat tissue was removed (NoF). Investigations were repeated in 7 SAMP8 and 7 SAMR1 after 54 weeks of chronic treatment with melatonin, an endogenous hormone with antioxidant and vasculoprotective properties. Results: In SAMR1 control mice anticontractile effect of perivascular fat was present (WF vs. NoF: ANOVA p = 0.04), while in aging SAMP8 mice the effect was less pronounced (WF vs. NoF: ANOVA p = NS) (see figure). Long-term treatment with melatonin had no effect in SAMR1 either in WF or NoF vessels, while it decreased the contractile response to norepinephrine in noF vessels of SAMP8 (ANOVA p < 0.001); the effect of melatonin treatment in WF vessels was not statistically significant. Conclusions: The anticontractile effect of perivascular fat is impaired in a senescence-accelerated prone mouse, compared with controls. A long-term treatment with melatonin seems to decrease contractile responses to norepinephrine in NOF mesenteric small arteries of SAMP8, thus restoring an anticontractile effect, probably through antioxidant mechanisms.

173 Infused silica nanoparticles compromise vascular function in small mesenteric arteries

<h3>Introduction</h3> The emergence of nanomedicine, involving the intravenous injection of tracking agents (such as dye-encapsulated silica nanoparticles [SiNPs]) for imaging diagnostic and therapeutic purposes may hold a biosafety concern specifically to the vascular system. Our previous findings suggest that SiNPs of 100 and 200 nm size have no detrimental effect on conduit arterial function. However, their effect on small size vessels, which play an important role in controlling blood perfusion into tissues, has not been investigated. <h3>Methods and results</h3> Vasoconstrictor and vasodilator responses of mesenteric arteries (MAs) from male Wistar rats were assessed using pressure myography before and 30 min after intravascular infusion of SiNPs. Our data show that SiNPs (100 nm size at 5.32 × 10¹¹ NPs/mL dose) compromised the contractile responses to phenylephrine (Phe) (n = 5; p &lt; 0.01). SiNPs also attenuated the endothelium-dependent (acetylcholine) dilator responses in high potassium (KPSS) and Phe-pre-contracted vessels following their rapid uptake into the cytoplasm of endothelial cells <i>ex vivo</i> as well as <i>in vivo</i> (At 100 nM Ach, the mean percentage dilation was 78.77 ± 8.58% vs. 36.30 ± 5.07% after incubation in PSS and SiNPs respectively, p &lt; 0.001). The acute exposure to SiNPs did not alter the endothelium-independent relaxation to sodium nitroprusside (n = 4). SiNPs were biodistributed in trace amounts into different tissues; including MAs, aortic vessel, liver and spleen <i>in vivo</i>. The physiological changes were accompanied by a decrease in the phosphorylated levels of both extracellular-signal-regulated kinase (ERK) and protein kinase Akt demonstrated by proteomic analysis. <h3>Conclusions</h3> Findings suggest that SiNPs may affect microvessel function <i>via </i>modulation of the ERK/AKT pathway. Our data highlight the need to identify strategies that can help minimise possible detrimental effects of nanoparticles on arterial function.

The GPR55 agonist lysophosphatidylinositol relaxes rat mesenteric resistance artery and induces Ca(2+) release in rat mesenteric artery endothelial cells.

Lysophosphatidylinositol (LPI), a lipid signalling molecule, activates GPR55 and elevates intracellular Ca(2+). Here, we examine the actions of LPI in the rat resistance mesenteric artery and Ca(2+) responses in endothelial cells isolated from the artery. Vascular responses were studied using wire myographs. Single-cell fluorescence imaging was performed using a MetaFluor system. Hypotensive effects of LPI were assessed using a Biopac system. In isolated arteries, LPI-induced vasorelaxation was concentration- and endothelium-dependent and inhibited by CID 16020046, a GPR55 antagonist. The CB1 receptor antagonist AM 251 had no effect, whereas rimonabant and O-1918 significantly potentiated LPI responses. Vasorelaxation was reduced by charybdotoxin and iberiotoxin, alone or combined. LPI decreased systemic arterial pressure. GPR55 is expressed in rat mesenteric artery. LPI caused biphasic elevations of endothelial cell intracellular Ca(2+). Pretreatment with thapsigargin or 2-aminoethoxydiphenyl borate abolished both phases. The PLC inhibitor U73122 attenuated the initial phase and enhanced the second phase, whereas the Rho-associated kinase inhibitor Y-27632 abolished the late phase but not the early phase. LPI is an endothelium-dependent vasodilator in the rat small mesenteric artery and a hypotensive agent. The vascular response involves activation of Ca(2+)-sensitive K(+) channels and is not mediated by CB1 receptors, but unexpectedly enhanced by antagonists of the 'endothelial anandamide' receptor. In endothelial cells, LPI utilizes PLC-IP3 and perhaps ROCK-RhoA pathways to elevate intracellular Ca(2+). Overall, these findings support an endothelial site of action for LPI and suggest a possible role for GPR55 in vasculature.

Soluble epoxide hydrolase modulates adenosine receptor‐induced vascular response in mouse mesenteric arteries

Soluble epoxide hydrolase (sEH) degrades epoxyeicosatrienoic acids (EETs) to less active diols (dihydroxyeicosatrienoic acids; DHETs). EETs are endothelium-derived hyperpolarizing factors (EDHFs) which play an important role in protection from ischaemic injury and have anti-inflammatory actions. Previously, we have reported upregulation of A2A adenosine receptor (AR), CYP2J2, and PPARg, and the downregulation of A1AR and PPARa in mouse aorta from sEH−/− mice with enhanced vascular relaxation. Additionally, A1AR stimulation inhibits BK channel activity involving PKCa in mouse aorta. In present study, we investigated whether over-expression of sEH in endothelium (Tie2-sEH Tr) regulates AR dependent vascular response in mouse mesenteric arteries (MAs). We performed DMT myograph muscle tension measurements and western blot (WB) in WT and Tie2-sEH Tr mice. The data show that endothelial response was comparable between Tie2-sEH Tr vs. WT mice. Phenylephrine induced contraction was significantly higher in Tie2-sEH Tr vs. WT mice. Importantly, NECA (non-selective AR agonist)-induced relaxation was significantly reduced in Tie2-sEH Tr vs. WT mice. WB analysis revealed significantly higher expression of cyp4a and upregulation of A1AR in Tie2-sEH Tr vs. WT mice. Notably, pinacidil (KATP channel opener) induced relaxation was also significantly reduced in Tie2-sEH Tr vs. WT mice. In conclusion our data suggest that over-expression of sEH reduces relaxation possibly due to upregulation of A1AR, that may show a possible interaction between sEH and ARs (HL-114559, Z01-ES025034, HL027339 & HL094447).

Cytochrome P‐450 epoxygenase 2J2 modulates adenosine receptor‐mediated vascular response in mouse mesenteric arteries

Cytochrome P‐450 epoxygenases including CYP2J2 are expressed in vascular endothelial cells that generate epoxyeicosatrienoic acids (EETs) through epoxidation of arachidonic acid. EETs are endothelium‐derived hyperpolarizing factors which are protective in nature and are degraded by soluble epoxide hydrolase (sEH). In aorta from sEH−/− mice, we reported upregulation of A2A adenosine receptor (AR), CYP2J2, and PPARg, and the downregulation of A1AR and PPARa with enhanced vascular relaxation. In this study, we explored whether over‐expression of CYP2J2 in endothelium (Tie2‐CYP2J2 Tr) regulates AR dependent vascular responses in mouse mesenteric arteries (MAs). We performed DMT myograph experiments in WT and Tie2‐CYP2J2 Tr mice. Our findings revealed that acetylcholine‐dependent response was unchanged. Phenylephrine and KCl induced contractions were significantly higher in Tie2‐CYP2J2 Tr vs. WT mice (p&lt;0.05). Importantly, CCPA (selective A1AR agonist)‐induced contractions were significantly reduced in Tie2‐CYP2J2 Tr vs. WT mice (p&lt;0.05). NECA (non‐selective AR agonist)‐induced relaxation was similar Tie2‐sEH Tr vs. WT mice. Especially, pinacidil (KATP channel opener) induced relaxation was also significantly enhanced in Tie2‐CYP2J2 Tr vs. WT mice. Our data suggest that over‐expression of CYP2J2 enhances vascular relaxation possibly due to the combination of reduction in A1AR‐dependent contraction and enhancement of relaxation through KATP channel, indicating a possible interaction among CYP2J2, ARs and KATP channels (HL‐114559, Z01‐ES025034, HL027339 &amp; HL094447).

Beneficial effect of cyclosporine A on traumatic hemorrhagic shock

BackgroundVascular hyporeactivity plays an important role in severe trauma and shock. We investigated the beneficial effect of cyclosporine A (CsA) on traumatic shock and its relationship to vascular reactivity improvement and mitochondrial permeability transition pore (MPTP). Materials and methodsSodium pentobarbital-anesthetized rats were used to induce traumatic hemorrhagic shock by left femur fracture and hemorrhage, the beneficial effects of CsA (1, 5, and 10 mg/kg, intravenously) on animal survival, cardiovascular function, tissue blood perfusion, and mitochondrial function of vital organs were observed. In addition, hypoxia-treated vascular smooth muscle cells from normal rats were used to investigate the relationship of this beneficial effect of CsA to Rho-associated serine/threonine kinase (ROCK) and protein kinase C. ResultsCsA prolonged the survival time and increased the 24-h survival rate of traumatic hemorrhagic shock (31%, 56%, and 56% in 1, 5, and 10 mg/kg CsA group versus 25% in lactated Ringer solution group). Five milligrams per kilogram of CsA had the best effect, which stabilized and improved the hemodynamics, increased the tissue blood flow, and improved the liver and kidney function including its mitochondrial function in shock rats. CsA had no significant influences on the production of inflammatory mediators and cardiac output after traumatic hemorrhagic shock. Further results indicated that CsA significantly improved the vascular constriction and dilation reactivity of superior mesenteric artery to norepinephrine and acetylcholine, which was antagonized by ROCK inhibitor, Y27632, but not by protein kinase C inhibitor, staurosporine. Further studies showed that CsA restored hypoxia-induced decrease of ROCK activity and inhibited the opening of MPTP in hypoxia-treated vascular smooth muscle cells. ConclusionsCsA is beneficial for the treatment of traumatic hemorrhagic shock. The mechanism is mainly through improving the vascular reactivity, stabilizing the hemodynamics, and increasing tissue perfusion. This beneficial effect of CsA is related to the inhibitory effect of CsA on MPTP opening. ROCK is an important regulator molecule in this process.

Spinal cord injury increases the reactivity of rat tail artery to angiotensin II.

Studies in individuals with spinal cord injury (SCI) suggest the vasculature is hyperreactive to angiotensin II (Ang II). In the present study, the effects of SCI on the reactivity of the rat tail and mesenteric arteries to Ang II have been investigated. In addition, the effects of SCI on the facilitatory action of Ang II on nerve-evoked contractions of these vessels were determined. Isometric contractions of artery segments from T11 (tail artery) or T4 (mesenteric arteries) spinal cord-transected rats and sham-operated rats were compared 6–7 weeks postoperatively. In both tail and mesenteric arteries, SCI increased nerve-evoked contractions. In tail arteries, SCI also greatly increased Ang II-evoked contractions and the facilitatory effect of Ang II on nerve-evoked contractions. By contrast, SCI did not detectably change the responses of mesenteric arteries to Ang II. These findings provide the first direct evidence that SCI increases the reactivity of arterial vessels to Ang II. In addition, in tail artery, the findings indicate that Ang II may contribute to modifying their responses following SCI.

116-POS

Objectives We evaluated role of sFlt-1 and VEGF in a preeclampsia model driven by RAS activation. We hypothesized as transgenic crossbreeds develop preeclampsia, features of hypertension, proteinuria, kidney and placenta end-organ damage will occur concurrently with (1) sFlt1 rising higher in preeclamptic mice, (2) VEGF remaining high in preeclamptic mice but equivalent to controls; and, (3) preeclamptic kidneys demonstrating increased VEGF binding. Methods Transgenic hAGT and hREN, or wild type (WT) mice were used. Non-gravid females underwent telemetric transmitter implantation for blood pressure (BP) measurements. Mice were cross-bred: hAGT × hREN for RAS preeclamptic model and pregnant controls (WT × WT). Blood and urine collected on days (GD) 12, 15, and 18. Vascular reactivity in mesenteric arteries investigated by wire myography using KCl, acetylcholine (ACH), phenylephrine (PE). ELISA performed on urine for albumin and on plasma for VEGF and sFlt-1. Necropsies were performed GD 18-19. Kidneys and placenta were sectioned for HE and immunostain. Results 11 RAS pregnancies and 9 WT pregnancies (WTP) compared. 12,414 h of BP data analyzed. RAS mice demonstrated higher BP and proteinuria. RAS mice had glomerular endotheliosis (80% vs. 11%; p = 0.02), and placental necrosis (60% vs. 0%; p Conclusions While RAS model of preeclampsia recapitulates human preeclamptic state with high fidelity, renal injury may actually be mediated by increased VEGF binding. Disclosures J.M. Denney: None. C.E. Shaw: None. A. Gendron-Fitzpatrick: None. I. Bird: None. D. Shah: None.

TRPM4 inhibitor 9-phenanthrol activates endothelial cell intermediate conductance calcium-activated potassium channels in rat isolated mesenteric artery.

Smooth muscle transient receptor potential melastatin 4 (TRPM4) channels play a fundamental role in the development of the myogenic arterial constriction that is necessary for blood flow autoregulation. As TRPM4 channels are present throughout the vasculature, we investigated their potential role in non-myogenic resistance arteries using the TRPM4 inhibitor 9-phenanthrol. Pressure and wire myography were used to assess the reactivity of rat arteries, the latter in combination with measurements of smooth muscle membrane potential. Immunohistochemistry (IHC) and endothelial cell (EC) calcium changes were assessed in pressurized vessels and patch clamp measurements made in isolated ECs. The TRPM4 inhibitor 9-phenanthrol reversibly hyperpolarized mesenteric arteries to circa EK and blocked α1 -adrenoceptor-mediated vasoconstriction. Hyperpolarization was abolished and vasoconstriction re-established by damaging the endothelium. In mesenteric and cerebral artery smooth muscle, 9-phenanthrol hyperpolarization was effectively blocked by the KCa 3.1 inhibitor TRAM-34. 9-Phenanthrol did not increase mesenteric EC [Ca(2+)]i , and Na(+) substitution with N-methyl-D-glucamine only increased the muscle resting potential by 10 mV. Immunolabelling for TRPM4 was restricted to the endothelium and perivascular tissue. These data reveal a previously unrecognized action of the TRPM4 inhibitor 9-phenanthrol - the ability to act as an activator of EC KCa 3.1 channels. They do not indicate a functionally important role for TRPM4 channels in the reactivity of non-myogenic mesenteric arteries.

High-cholesterol diet enriched with onion affects endothelium-dependent relaxation and NADPH oxidase activity in mesenteric microvessels from Wistar rats.

BackgroundThe aim of the present study was to examine the effects of onion as functional ingredient on the oxidative status, lipoprotein levels (total cholesterol-TC, HDL-C, LDL-C), triacylglycerides (TAG) and vascular reactivity of mesenteric arteries in hypercholesterolemic Wistar rats.MethodsTwenty-four animals were fed with three different diets [control, high-cholesterol diet (HC) and high-cholesterol enriched with onion diet (HCO)]. After seven weeks of experimental feeding the rats were euthanized for blood and tissues collection. TC, HDL-C, LDL-C and TAG were measured, and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•+) scavenging capacity and ferric reducing antioxidant power (FRAP) were determined in plasma. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme activities were assayed in erythrocyte lysates. Endothelium-dependent vasodilation to acetylcholine was evaluated in mesenteric arterial segments. NADPH oxidase (NOX) was also measured by lucigenin-derived chemiluminiscence.ResultsThe dietary cholesterol content significantly affected plasma lipoprotein levels, increased superoxide generation from NOX, and caused impaired endothelium-dependent vasodilation in the rat mesenteric arteries. Onion ingredient improved antioxidant status in HCO group, as it was evidenced by ABTS•+ and FRAP values and SOD and GPx enzyme activities compared to the HC-fed group, reduced the increment in NOX activity and reversed endothelial dysfunction promoted by the HC diet. Scavenging of superoxide with TEMPOL or inhibition of NOX with apocynin improved endothelium-dependent vasodilation only in HC-fed rats.ConclusionsEnrichment of diet with onion as functional ingredient could be proposed as a complementary approach to prevent or partially modulate vascular dysfunction, reducing some of the risk indexes linked to initial development of atherosclerosis.

ASSA14-02-03 Maternal exposure to lipopolysaccharide results in augmenting response of mesenteric artery to AT1receptor in adult offspring rats

Objective To explore the mechanism of vascular function by maternal exposure to lipopolysaccharide in adult offspring rats. Methods Nulliparous pregnant and time-matched SD rats were randomly divided into two groups control group and LPS group (n = 6 for each group). Saline or LPS (0.79 mg/kg) were administered intraperitoneally on the pregnant day 8, 10 and 12. All offspring rats were weaned at 4 weeks of age. Bloodpressure was measured noninvasively by the tail - cuff method at 9, 11, 13 and 15 weeks of age. Mesenteric vascular reactivity in response to angiotensin II (Ang II, 10 –10 –10 –5 mol/L) and protein level of angiotensin II1 receptor (AT1R) were determined by Westen blot, respectively. Results The systolic blood pressure in the postnatal offsprings treated with LPS rats was higher than control group at 9, 11, 13 and 15 weeks of age (109.6 ± 2.6 vs 102.9 ± 2.5,117.7 ± 1.5 vs 108.1 ± 3.3,128.1 ± 3.6 vs 117.2 ± 2.1,135.1 ± 3.2 vs 123.0 ± 2.3)mm Hg, respectively, all p 1 receptor expression of mesenteric arteries (1.21 ± 0.08 vs 0.75 ± 0.08) were increased at the age of 15 weeks in rat offsprings after maternal exposure to LPS. Conclusion The increased AT1 receptor expression and augmented response to Ang II are involved in the pathogenesis of hypertension in the adult rat offsprings following maternal exposure to LPS.

A comparison of responses to raised extracellular potassium and endothelium-derived hyperpolarizing factor (EDHF) in rat pressurised mesenteric arteries.

The present study examined the hypothesis that potassium ions act as an endothelium-derived hyperpolarizing factor (EDHF) released in response to ACh in small mesenteric arteries displaying myogenic tone. Small mesenteric arteries isolated from rats were set up in a pressure myograph at either 60 or 90 mmHg. After developing myogenic tone, responses to raising extracellular potassium were compared to those obtained with ACh (in the presence of nitric oxide synthase and cyclo-oxygenase inhibitors). The effects of barium and oubain, or capsaicin, on responses to raised extracellular potassium or ACh were also determined. The effects of raised extracellular potassium levels and ACh on membrane potential, were measured using sharp microelectrodes in pressurised arteries. Rat small mesenteric arteries developed myogenic tone when pressurised. On the background of vascular tone set by a physiological stimulus (i.e pressure), ACh fully dilated the small arteries in a concentration-dependent manner. This response was relatively insensitive to the combination of barium and ouabain, and insensitive to capsaicin. Raising extracellular potassium produced a more inconsistent and modest vasodilator response in pressurised small mesenteric arteries. Responses to raising extracellular potassium were sensitive to capsaicin, and the combination of barium and ouabain. ACh caused a substantial hyperpolarisation in pressurized arteries, while raising extracellular potassium did not. These data indicate that K+ is not the EDHF released in response to ACh in myogenically active rat mesenteric small arteries. Since the hyperpolarization produced by ACh was sensitive to carbenoxolone, gap junctions are the likely mediator of EDH responses under physiological conditions.

A vasoactive role for endogenous relaxin in mesenteric arteries of male mice.

The peptide hormone relaxin has striking effects on the vascular system. Specifically, endogenous relaxin treatment reduces myogenic reactivity through nitric oxide (NO)-mediated vasorelaxation and increases arterial compliance in small resistance arteries. However, less is known about the vascular roles of endogenous relaxin, particularly in males. Therefore, we used male wild-type (Rln +/+) and relaxin knockout (Rln −/−) mice to test the hypothesis that passive wall properties and vascular reactivity in mesenteric arteries would be compromised in Rln −/− mice. Passive compliance was determined in arteries (n = 8–9) mounted on a pressure myograph and in Ca2+-free Krebs containing 2 mM EGTA. Passive volume compliance was significantly (P = 0.01) decreased in the mesenteric arteries of Rln −/− mice. Vascular reactivity was assessed using wire myography. In mesenteric arteries (n = 5) of Rln −/− mice, there was a significant (P<0.03) increase in sensitivity to the vasoconstrictors phenylephrine and thromboxane-mimetic U41669. This enhanced responsiveness to vasoconstrictors was abolished by endothelial denudation, and attributed to impaired NO and prostanoid pathways in Rln −/− mice. Sensitivity to the endothelial agonist acetylcholine was significantly (n = 7–9, P≤0.03) decreased, and this was abolished in the presence of the cyclooxygenase inhibitor, indomethacin (2 µM). This indicates that prostanoid vasoconstrictor pathways were upregulated in the mesenteric arteries of Rln −/− mice. In summary, we demonstrate endothelial dysfunction and impaired arterial wall remodeling in male mice deficient in relaxin. Thus, our results highlight a role for endogenous relaxin in the maintenance of normal mesenteric artery structure and function in males.

Abstract 344: Endothelin-1 Overexpression Exaggerates Diabetes-induced Endothelial Dysfunction

Objective: Vascular disease associated with endothelial dysfunction is a major cause of morbidity in patients with type-1 diabetes. Endothelin (ET)-1 plays a role in diabetes-induced vascular complications, since ET-1 type A receptor blockade reduces diabetes-induced vascular injury. However, whether ET-1 contributes to diabetes-induced endothelial dysfunction remains unproven. We hypothesized that vascular ET-1 overexpression will exaggerate diabetes-induced endothelial dysfunction. Methods: Diabetes was induced by streptozotocin treatment (STZ, 55 mg/kg/day, ip) for 5 days in 6-week-old male wild-type (WT) mice and in mice overexpressing ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Blood was collected to determine glucose. Mesenteric artery reactivity and remodeling were evaluated using pressurized myography and aortic fibronectin expression by immnunofluorescence. Results: STZ-induced diabetes was confirmed by a 3-fold increase in glycemia in WT and eET-1 ( P &lt;0.001). Diabetes impaired endothelium-dependent relaxation (EDR) reponses to acetylcholine in WT (60.9±6.4% vs 83.9±3.4%, P &lt;0.05) and eET-1 (48.6±5.1% vs 81.5±5.2%, P &lt;0.001). EDR impairment was exaggerated in eET-1 compared to WT ( P &lt;0.05). Meclofenamic acid, an inhibitor of cyclooxygenase, increased EDR in eET-1 compared to WT (78.4±9.4% vs 66.7±3.2%, P &lt;0.01), which was not observed in diabetic mice. L-NAME, an inhibitor of nitric oxide (NO) synthase, completely blocked EDR in WT, eET-1 and diabetic WT, but not in diabetic eET-1 (4.1±1.6%, 6.4±5.7%, 2.2±4.6% and 26.6±4.6%, P &lt;0.05). Apamin plus Tram34, inhibitors of endothelium-dependent hyperpolarization inhibited EDR in the four groups. Endothelium-independent relaxation to sodium nitroprusside, a NO donor, was similar in the four groups. Diabetes reduced media/lumen in WT (2.7±0.3 vs 3.6±0.3, P &lt;0.05) and eET-1 (2.9±0.2 vs 3.8±0.3, P &lt;0.05). Diabetes decreased aortic fibronectin expression in WT (94.0±11.0 vs. 151.9±21.8 RFU/μm 2 , P &lt;0.05) and eET-1 (66.3±8.7 vs. 146.6±20.7 RFU/μm 2 , P &lt;0.05). Conclusion: ET-1 contributes to alterations in several pathways mediating endothelium-dependent relaxation in type-1 diabetes, leading to exaggerated endothelial dysfunction.

Abstract 339: Chronic Treatment With Fluoxetine Increases Relaxation Of Rat Resistance Mesenteric Arteries Via Atp-sensitive Potassium Channels Activation

Fluoxetine, a selective serotonin reuptake inhibitor (SSRI) has properties that go beyond its antidepressant effects and alters mechanisms involved in the regulation of vasomotor tone. While there are many studies demonstrating the acute effects of fluoxetine in the vasculature, studies on the chronic effects of this SSRI are still limited. Here we postulated that chronic treatment with fluoxetine enhances vascular reactivity to vasodilator stimuli by increasing nitric oxide (NO) signaling. The effects of chronic treatment with fluoxetine on vascular reactivity were determined in resistance mesenteric arteries from Wistar rats, which were treated with (I) vehicle (water for 21 days) or (II) fluoxetine (10 mg/kg/day for 21 days in the drinking water). Fluoxetine treatment increased endothelium-dependent (pEC50, Veh = -7.08±0.07; Fluox = -7.4±0.11, p&lt;0.05) and -independent relaxant response (pEC50, Veh = -7.75±0.08; Fluox = -8.5 ± 0.11, p&lt;0.05). Fluoxetine also increased vascular NOx (NO metabolites) levels (Veh = 1.2±0.13 μM/μg; Fluox = 2.0 ± 0.13 μM/μg, p &lt;0.05), nitric oxide sintase (NOS) activity (Fluox = 76%) and phosphorylation of endothelial NOS (eNOS) at serine1177 [arbitrary units (a.u.), Veh = 0.36±0.10; Fluox = 1.2 ± 0.08, p &lt;0.05]. Fluoxetine treatment did not change neuronal NOS (nNOS) or soluble guanylate cyclase (sGC) expression neither vascular responses to cyclic guanosine monophosphate (cGMP) or sGC activators. However, pinacidil- (KATP channels activator)-induced relaxation was increased by fluoxetine treatment (pEC50, Veh = -5.9±0.12; Fluox = -6.5±0.17, p&gt;0.05). In conclusion, chronic treatment with fluoxetine increases endothelium-dependent and -independent relaxation response in resistance mesenteric arteries by mechanisms that involve increased NOS activity, NO generation and KATP channels activation. These effects may contribute to the cardiovascular side effects associated with fluoxetine treatment.

Abstract 343: Endothelin-1 Overexpression Preserves Endothelial Function In Mice With Vascular Smooth Muscle Cell-specific Deletion Of Ppar-gamma

Objective: Peroxisome proliferator activated receptor γ (PPARγ) agonists reduce blood pressure (BP) and vascular injury in hypertensive rodents and humans. Pparγ inactivation in vascular smooth muscle cells (VSMC) using a tamoxifen inducible Cre-Lox system enhanced angiotensin II-induced vascular remodeling and endothelial dysfunction. Transgenic mice overexpressing endothelin (ET)-1 selectively in the endothelium (eET-1) exhibit endothelial dysfunction, increased oxidative stress and inflammation. We hypothesized that inactivation of the Ppar gene in VSMC (sm Pparγ -/- ) will exaggerate ET-1-induced vascular damage. Methods: Eleven week-old male control, eET-1, sm Pparγ -/- and eET-1/sm Pparγ -/- mice were used. BP by telemetry, mesenteric artery (MA) reactivity and structure by pressurized myography, reactive oxygen species (ROS) by dihydroethidium (DHE) staining and fibronectin expression by immunofluorescence were determined. Results: Systolic BP was higher in eET-1 and eET-1/sm Pparγ -/- compared to control and sm Pparγ -/- (127±3 and 124±7 vs 109±2 and 114 ± 4 mmHg, respectively, P &lt;0.05). Endothelium-dependent relaxation (EDR) responses to acetylcholine were impaired in sm Pparγ -/- ( P &lt;0.05) but not in eET-1 and sm Pparγ -/- /eET-1 compared with control (E max : 52.0±6.7%, 66.2±7.6 and 87.1±4.4% vs 82.2±4.9%). Endothelium-independent relaxation responses to the nitric oxide donor, sodium nitroprusside, were similar in the four groups. Media/lumen at 45 mmHg was higher only in eET-1/sm Pparγ -/- compared with control (5.5±0.21 vs 4.5±0.21, P &lt;0.05). Increased stiffness was observed in eET-1, sm Pparγ -/- and eET-1/sm Pparγ -/- compared to control, as indicated by a leftward displacement of the stress-strain curves (strain at 140 mmHg: 0.82±0.03, 0.81±0.04, 0.80±0.03 vs 0.97±0.02, P &lt;0.05). ROS levels were increased 1.7±0.2 fold in eET-1, 2.2±0.2 fold in sm Pparγ -/- and 2.8±0.4 fold in eET-1/sm Pparγ -/- compared with control ( P &lt;0.05). Fibronectin staining in MA was similar in the four groups. Conclusion: These results suggest that increased ET-1 paradoxically preserves endothelial function in mice with inactivated Pparγ in vascular smooth muscle, despite presence of enhanced oxidative stress.

Anticontractile activity of perivascular fat in obese mice and the effect of long-term treatment with melatonin.

It has been demonstrated previously that inflammation in perivascular adipose tissue (PVAT) may be implicated in vascular dysfunction. The aim of this study was to investigate the functional responses of small mesenteric arteries in a hyperphagic animal model of obesity after chronic treatment with melatonin, an endogenous hormone with antioxidant and vasculoprotective properties. Ten obese mice (ob/ob) and 10 control lean mice (CLM) were treated with melatonin 100 mg/kg per day in the drinking water for 8 weeks. Mesenteric small resistance arteries were dissected and mounted on a wire myograph and a concentration-response to norepinephrine was evaluated in vessels with intact PVAT and after PVAT was removed and in the presence of iberiotoxin, a selective blocker of BKCA channels as well as under conditions of induced hypoxia in vitro. The presence of PVAT reduced the contractile response to norepinephrine in both ob/ob and CLM; however, the effect was significantly reduced in ob/ob. The anticontractile effect of PVAT completely disappeared with iberiotoxin preincubation. After melatonin treatment, inflammation was significantly ameliorated, and the contractile response in ob/ob and CLM was significantly reduced when PVAT was removed. Anticontractile effect of PVAT that is lost in obesity can be rescued using melatonin. A reduced expression of adiponectin and adiponectin receptor was observed in perivascular fat of ob/ob, whereas significant increase was observed in ob/ob treated with melatonin. Melatonin seems to exert a protective effect on arteries from both ob/ob and CLM, counteracting the adverse effect of hypoxia and iberiotoxin.