Mesenteric Resistance Arteries Research Articles

Testosterone modulates vasodilation in mesenteric arteries of hypertensive rats

AimsTo evaluate the effects of testosterone on endothelium-dependent vasodilation and oxidative stress in mesenteric resistance arteries. Main methodsSpontaneously hypertensive rats (SHR), aged 8 to 10 weeks, were divided into four groups: intact (SHAM), intact treated with testosterone (TTO; 3 mg/kg/day) via subcutaneous route (s.c.), intact treated with testosterone and anastrozole [aromatase enzyme inhibitor (TTO + ANA; 0.1 mg/kg/day, s.c.)] and intact treated with testosterone and finasteride [5 α-reductase enzyme inhibitor (TTO + FIN; 5 mg/kg/day, s.c.)] for four weeks. Concentration-response curves to acetylcholine (ACh, 0.1 nmol/L - 10 μmol/L) were obtained in mesenteric resistance arteries previously contracted with phenylephrine (PE, 3 μmol/L), before and after the use of selective inhibitors. Reactive oxygen species (ROS) levels were assessed in the vessels and the endothelium analyzed by scanning electron microscopy. Key findingsTTO group showed a lower participation of nitric oxide (NO), increased oxidative stress, and participation of prostanoids and endothelium-dependent hyperpolarization (EDH), possibly to maintain the vasodilator response. Lower participation of NO and prostanoids, combined to an increased participation of EDH, were observed in the TTO + ANA group, in addition to higher levels of ROS and altered endothelial morphology. The vasodilation to ACh was impaired in TTO + FIN, along increased participation of NO, reduction of prostanoids, and greater EDH-dependent vasodilation. SignificanceTestosterone contributes to endothelial vasodilation by enhancing EDH through an increased participation of epoxyeicosatrienoic acids. While the decrease in NO appears to involve the participation of dihydrotestosterone, 17 β-estradiol seems to stimulate the action of the NO pathway and prostanoids.

Histamine H2-receptor antagonism improves conduit artery endothelial function and reduces plasma aldosterone level without lowering arterial blood pressure in angiotensin II–hypertensive mice

Aldosterone through the mineralocorticoid receptor MR has detrimental effects on cardiovascular disease. It reduces the bioavailability of nitric oxide and impairs endothelium-dependent vasodilatation. In resistance arteries, aldosterone impairs the sensitivity of vascular smooth muscle cells to nitric oxide by promoting the local secretion of histamine which activates H2 receptors. The present experiments tested in vivo and ex vivo the hypothesis that systemic H2-receptor antagonism reduces arterial blood pressure and improves vasodilatation in angiotensin II–induced chronic hypertension. Hypertension was induced by intravenous infusion of angiotensin II (60 ng kg−1 min−1) in conscious, unrestrained mice infused concomitantly with the H2-receptor antagonist ranitidine (27.8 µg kg−1 min−1) or vehicle for 24 days. Heart rate and arterial blood pressure were recorded by indwelling arterial catheter. Resistance (mesenteric) and conductance (aortae) arteries were harvested for perfusion myography and isometric tension recordings by wire myography, respectively. Plasma was analyzed for aldosterone concentration. ANGII infusion resulted in elevated arterial blood pressure and while in vivo treatment with ranitidine reduced plasma aldosterone concentration, it did not reduce blood pressure. Ranitidine improved ex vivo endothelial function (acetylcholine 10−9 to 10−6 mol L−1) in mesenteric resistance arteries. This was abolished by ex vivo treatment with aldosterone (10−9 mol L−1, 1 h). In aortic segments, in vivo ranitidine treatment impaired relaxation. Activation of histamine H2 receptors promotes aldosterone secretion, does not affect arterial blood pressure, and protects endothelial function in conduit arteries but promotes endothelial dysfunction in resistance arteries during angiotensin II–mediated hypertension. Aldosterone contributes little to angiotensin II–induced hypertension in mice.

Nitric oxide, endothelium-derived hyperpolarizing factor, and smooth muscle-dependent mechanisms contribute to magnesium-dependent vascular relaxation in mouse arteries.

Magnesium (Mg2+ ) is a vasorelaxant. The underlying physiological mechanisms driving this vasorelaxation remain unclear. Studies were designed to test the hypothesis that multiple signaling pathways including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in endothelial cells as well as Ca2+ antagonization and TRPM7 channels in vascular smooth muscle cells mediate Mg2+ -dependent vessel relaxation. To uncover these mechanisms, force development was measured exvivo in aorta rings from mice using isometric wire myography. Concentration responses to Mg2+ were studied in intact and endothelium-denuded aortas. Key findings were confirmed in second-order mesenteric resistance arteries perfused exvivo using pressure myography. Effects of Mg2+ on NO formation were measured in Chinese Hamster Ovary (CHO) cells, isolated mesenteric vessels, and mouse urine. Mg2+ caused a significant concentration-dependent relaxation of aorta rings. This relaxation was attenuated significantly in endothelium-denuded aortas. The endothelium-dependent portion was inhibited by NO and cGMP blockade but not by cyclooxygenase inhibition. Mg2+ stimulated local NO formation in CHO cells and isolated mesenteric vessels without changing urinary NOx levels. High extracellular Mg2+ augmented acetylcholine-induced relaxation. SKCa and IKCa channel blockers apamin and TRAM34 inhibited Mg2+ -dependent relaxation. The endothelium-independent relaxation in aorta rings was inhibited by high extracellular Ca2+ . Combined blockade of NO, SKCa , and IKCa channels significantly reduced Mg2+ -dependent dilatation in mesenteric resistance vessels. In mouse conductance and resistance arteries Mg2+ -induced relaxation is contributed by endothelial NO formation, EDHF pathways, antagonism of Ca2+ in smooth muscle cells, and additional unidentified mechanisms.

Asymmetric Dimethylarginine Enables Depolarizing Spikes and Vasospasm in Mesenteric and Coronary Resistance Arteries.

Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca2+-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease. We hypothesized ADMA might enable VSM spikes and vasospasm by reducing NO bioavailability, which is opposed by DDAH1 activity and L-arginine. Rat isolated small mesenteric arteries and myogenic rat-isolated intraseptal coronary arteries (RCA) were studied using myography, VSM intracellular recording, Ca2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, NG-(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism. ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α1-adrenoceptor agonist, phenylephrine by enabling T-type voltage-gated calcium channel-dependent depolarizing spikes. However, some endothelium-dependent NO-vasorelaxation remained, which was sensitive to DDAH1-inhibition with NG-(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca2+ spikes and marked vasoconstriction, while NO vasorelaxation was abolished. DDAH1 expression was greater in rat-isolated small mesenteric arteries endothelium compared with RCA, but low in VSM of both arteries. L-arginine prevented depolarizing spikes and protected NO-vasorelaxation in rat-isolated small mesenteric artery and RCA. ADMA increases VSM electrical excitability enhancing vasoreactivity. Endothelial DDAH1 reduces this effect, and low levels of DDAH1 in RCAs may render them susceptible to endothelial dysfunction contributing to vasospasm, changes opposed by L-arginine.

Normotensive placental extracellular vesicles provide long-term protection against hypertension and cardiovascular disease

BackgroundWomen with normotensive pregnancy are at a reduced risk of developing cardiovascular disease postpartum compared to those who experience hypertensive conditions during pregnancy. However, the underlying mechanisms remain poorly understood. During normotensive pregnancy, vast numbers of placental extracellular vesicles are released into the maternal circulation, which protect endothelial cells from activation and alter maternal vascular tone. We hypothesized that placental extracellular vesicles play a mechanistic role in lowering risk of cardiovascular disease following normotensive pregnancy. ObjectiveThis study aimed to investigate the long-term effects of placental extracellular vesicles derived from normotensive term placentae on the cardiovascular system and explore the mechanisms underlying their biological effects. Study DesignSpontaneously hypertensive rats were injected with placental EVs from normotensive term pregnancies (2 mg /kg each time, n=8) or vehicle control (n=9) at 3 months of age. Blood pressure and cardiac function were regularly monitored from 3 months to 15 months of age. The response of mesenteric resistance arteries to vasoactive substances was investigated to evaluate vascular function. Cardiac remodelling, small artery remodelling, and renal function were investigated to comprehensively assess the impact of placental extracellular vesicles on cardiovascular and renal health. ResultsCompared to vehicle-treated control animals, rats treated with normotensive placental extracellular vesicles exhibited a significantly lower increase in blood pressure and improved cardiac function. Furthermore, the vasodilator response to the endothelium-dependent agonist acetylcholine was significantly enhanced in the normotensive placental extracellular vesicle-treated spontaneously hypertensive rats compared to the control. Moreover, treatment with placental extracellular vesicles reduced wall thickening of small renal vessels and attenuated renal fibrosis. ConclusionPlacental extracellular vesicles from normotensive term pregnancies have long-lasting protective effects reducing hypertension and mitigating cardiovascular damage in vivo.

The Melatonin Derivative ITH13001 Prevents Hypertension and Cardiovascular Alterations in Angiotensin II-Infused Mice.

Inflammatory mechanisms and oxidative stress seem to contribute to the pathogenesis of hypertension. ITH13001 is a melatonin-phenyl-acrylate hybrid that moderately induces the antioxidant transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) and has a potent oxidant scavenging effect compared with other derivatives of its family. Here we investigated the effect of ITH13001 on hypertension and the associated cardiovascular alterations. Angiotensin II (AngII)-infused mice were treated with ITH13001 (1 mg/kg per day, i.p.) for 2 weeks. The ITH13001 treatment prevented: 1) the development of hypertension, cardiac hypertrophy, and increased collagen and B-type natriuretic peptide (Bnp) expression in the heart; 2) the reduction of elasticity, incremental distensibility, fenestrae area, intraluminal diameter, and endothelial cell number in mesenteric resistance arteries (MRA); 3) the endothelial dysfunction in aorta and MRA; 4) the plasma and cardiovascular oxidative stress and the reduced aortic nitric oxide (NO) bioavailability; 5) the increased cardiac levels of the cytokines interleukin (IL)-1β, IL-6, and C-C motif chemokine ligand 2 (Ccl2), the T cell marker cluster of differentiation 3 (Cd3), the inflammasome NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), the proinflammatory enzymes inducible nitric oxide synthase (iNOS) and COX-2, the toll-like receptor 4 (TLR4) adapter protein myeloid differentiation primary response 88 (MyD88), and the nuclear factor kappa B (NF-κB) subunit p65; 6) the greater aortic expression of the cytokines tumor necrosis factor alpha (Tnf-α), Ccl2 and IL-6, Cd3, iNOS, MyD88, and NLRP3. Although ITH13001 increased nuclear Nrf2 levels and heme oxygenase 1 (HO-1) expression in vascular smooth muscle cells, both cardiac and vascular Nrf2, Ho-1, and NADPH quinone dehydrogenase 1 (Nqo1) levels remained unmodified irrespective of AngII infusion. Summarizing, ITH13001 improved hypertension-associated cardiovascular alterations independently of Nrf2 pathway activation, likely due to its direct antioxidant and anti-inflammatory properties. Therefore, ITH13001 could be a useful therapeutic strategy in patients with resistant hypertension. SIGNIFICANCE STATEMENT: Despite the existing therapeutic arsenal, only half of the patients treated for hypertension have adequately controlled blood pressure; therefore, the search for new compounds to control this pathology and the associated damage to end-target organs (cerebral, cardiac, vascular, renal) is of particular interest. The present study demonstrates that a new melatonin derivative, ITH13001, prevents hypertension development and the associated cardiovascular alterations due to its antioxidant and anti-inflammatory properties, making this compound a potential candidate for treatment of resistant hypertensive patients.

Endothelin Receptor Blockade Improves Cerebral Blood Flow-Mediated Dilation in a Mouse Model of Alzheimer’s Disease

Introduction: Cerebral blood flow (CBF) is reduced in patients with Alzheimer’s disease (AD). Flow-mediated dilation (FMD), which plays a key role in the regulation of blood flow, is attenuated by endothelin-1. We hypothesized that endothelin receptor blockade may improve CBF in AD. Methods: We investigated cerebrovascular reactivity in a mouse model of AD (APP-PS1; 5-6-month-old male subjects). We assessed the in vivo response to normoxic hypercapnia and in vitro FMD in isolated cerebral and mesenteric resistance arteries before and after endothelin receptor blockade (bosentan). Results: Normoxic hypercapnia increased basilar trunk blood flow velocity (+12.3 ± 2.4%; p = 0.006, n = 6) in wild-type (WT) mice but reduced blood flow in APP-PS1 mice (−11.4 ± 1.2%; p < 0.0001, n = 8). Bosentan (50 mg/kg, acute intraperitoneal injection) restored cerebrovascular reactivity in APP-PS1 mice (+10.2 ± 2.2%; p < 0.0001, n = 8) but had no effect in WT. FMD was reduced in the posterior cerebral artery of APP-PS1 compared to WT and was normalized by bosentan (1 μmol/L, 30 min, or 50 mg/kg/day for 28 days). FMD was similar in the mesenteric artery of APPS-PS1 and WT. Conclusion: APP-PS1 mice exhibited cerebrovascular endothelial dysfunction. Acute and chronic blockade of endothelin receptors restored endothelial vasomotor function, suggesting a promising therapeutic approach to restoring cerebral vasoreactivity in AD.

Abstract 12477: ER Stress Chop Depletion in Endothelial Cells Protects From Renovascular Hypertension Pathogenesis

Introduction: Renovascular hypertension poses a significant risk for cardiovascular diseases, with endoplasmic reticulum (ER) stress playing a critical role in their development. However, the specific impact of ER stress-induced C/EBP homologous protein (CHOP) expression in endothelial cells on renovascular hypertension-induced cardiovascular complications remains unexplored. This study investigates the effects of disrupting ER stress CHOP in endothelial cells on microvascular dysfunction associated with renovascular hypertension. Hypothesis: We hypothesize that deleting ER stress CHOP in endothelial cells (EC) can mitigate renovascular hypertension-induced vascular endothelial dysfunction. Methods: Eight-week-old male and female mice (CHOP flx/flx and EC CHOP-/- ) were divided into eight groups: control groups undergoing a sham operation for 4 weeks and renovascular hypertension groups subjected to 2-kidney-1-clip (2K1C) surgery for 4 weeks. Body weight, blood pressure, running performance, cardiac hypertrophy and fibrosis, lung edema, inflammation, vascular endothelial function, and signaling were assessed. Results: Male and female CHOP flx/flx mice subjected to 2K1C for four weeks exhibited hypertension, cardiac hypertrophy and fibrosis, lung edema, impaired running performance, and endothelium-dependent vascular relaxation dysfunction. In contrast, male and female EC CHOP-/- mice subjected to 2K1C for four weeks were protected against the pathogenesis of renovascular hypertension. Furthermore, mesenteric resistance arteries from CHOP flx/flx mice displayed reduced endothelial nitric oxide synthase (eNOS) phosphorylation, while EC HOP-/- mice showed no such effect. Conclusions: These findings emphasize the significance of targeting ER stress CHOP in endothelial cells of male and female mice to protect against the development and pathogenesis of renovascular hypertension.

Myoendothelial feedback in mouse mesenteric resistance arteries is similar between the sexes, dependent on nitric oxide synthase, and independent of TPRV4.

Myoendothelial feedback (MEF), the endothelium-dependent vasodilation following sympathetic vasoconstriction (mediated by smooth muscle to endothelium gap junction communication), has been well studied in resistance arteries of males, but not females. We hypothesized that MEF responses would be similar between the sexes, but different in the relative contribution of the underlying nitric oxide and hyperpolarization mechanisms, given that these mechanisms differ between the sexes in agonist-induced endothelium-dependent dilation. We measured MEF responses (diameter changes) of male and female first- to second-order mouse mesenteric arteries to phenylephrine (10 µM) over 30 min using isolated pressure myography ± blinded inhibition of nitric oxide synthase (NOS) using Nω-nitro-l-arginine methyl ester (l-NAME; 0.1-1.0 mM), hyperpolarization using 35 mM KCl, or transient receptor potential vanilloid 4 (TRPV4) channels using GSK219 (0.1-1.0 µM) or RN-1734 (30 µM). MEF was similar [%dilation (means ± SE): males = 26.7 ± 2.0 and females = 26.1 ± 1.9 at 15 min] and significantly inhibited by l-NAME (1.0 mM) at 15 min [%dilation (means ± SE): males = 8.2 ± 3.3, P < 0.01; females = 6.8 ± 1.9, P < 0.001] and over time (P < 0.01) in both sexes. l-NAME (0.1 mM) + 35 mM KCl nearly eliminated MEF in both sexes (P < 0.001-0.0001). Activation of TRPV4 with GSK101 (0.1-10 µM) induced similar dilation between the sexes. Inhibition of TRPV4, which is reportedly involved in the hyperpolarization mechanism, did not inhibit MEF in either sex. Similar expression of eNOS was found between the sexes with Western blot. Thus, MEF is prominent and similar in murine first- and second-order mesenteric resistance arteries of both sexes, and reliant primarily on NOS and secondarily on hyperpolarization, but not TRPV4.NEW & NOTEWORTHY We found that female mesenteric resistance arteries have similar postconstriction dilatory responses (i.e., myoendothelial feedback) to a sympathetic neurotransmitter analog as male arteries. Both sexes use nitric oxide synthase (NOS) and hyperpolarization, but not TRPV4, in this response. Moreover, the key protein involved in this pathway (eNOS) is similarly expressed in these arteries between the sexes. These similarities are surprising given that agonist-induced endothelium-dependent dilatory mechanisms differ in these arteries between the sexes.

NTPDase1/CD39 Ectonucleotidase Is Necessary for Normal Arterial Diameter Adaptation to Flow.

NTPDase1/CD39, the major vascular ectonucleotidase, exerts thrombo-immunoregulatory function by controlling endothelial P2 receptor activation. Despite the well-described release of ATP from endothelial cells, few data are available regarding the potential role of CD39 as a regulator of arterial diameter. We thus investigated the contribution of CD39 in short-term diameter adaptation and long-term arterial remodeling in response to flow using Entpd1-/- male mice. Compared to wild-type littermates, endothelial-dependent relaxation was modified in Entpd1-/- mice. Specifically, the vasorelaxation in response to ATP was potentiated in both conductance (aorta) and small resistance (mesenteric and coronary) arteries. By contrast, the relaxing responses to acetylcholine were supra-normalized in thoracic aortas while decreased in resistance arteries from Entpd1-/- mice. Acute flow-mediated dilation, measured via pressure myography, was dramatically diminished and outward remodeling induced by in vivo chronic increased shear stress was altered in the mesenteric resistance arteries isolated from Entpd1-/- mice compared to wild-types. Finally, changes in vascular reactivity in Entpd1-/- mice were also evidenced by a decrease in the coronary output measured in isolated perfused hearts compared to the wild-type mice. Our results highlight a key regulatory role for purinergic signaling and CD39 in endothelium-dependent short- and long-term arterial diameter adaptation to increased flow.

Impaired vascular β-adrenergic relaxation in spontaneously hypertensive rats: The differences between conduit and resistance arteries

It was suggested that impaired β-adrenergic relaxation in spontaneously hypertensive rats (SHR) might contribute to their high blood pressure (BP). Our study was focused on isoprenaline-induced dilatation of conduit femoral or resistance mesenteric arteries and on isoprenaline-induced BP reduction in SHR and Wistar-Kyoto rats (WKY). We confirmed decreased β-adrenergic relaxation of SHR femoral arteries due to the absence of its endothelium-independent component, whereas endothelium-dependent component of β-adrenergic smooth muscle relaxation was similar in both strains. Conversely, isoprenaline-induced relaxation of resistance mesenteric arteries was similar in both strains and this was true for endothelium-dependent and endothelium-independent components. We observed moderately reduced sensitivity of SHR mesenteric arteries to salmeterol (β2-adrenergic agonist) and this strain difference disappeared after endothelium removal. However, there was no difference in mesenteric arteries relaxation by dobutamine (β1-adrenergic agonist) which was independent of endothelium. The increasing isoprenaline doses elicited similar BP decrease in both rat strains, although BP sensitivity to isoprenaline was slightly decreased in SHR. The blockade of cyclooxygenase (indomethacin) and NO synthase (L-NAME) further reduced BP sensitivity to isoprenaline in SHR. On the other hand, salmeterol elicited similar BP decrease in both strains and the blockade of cyclooxygenase and NO synthase increased BP sensitivity to salmeterol in SHR as compared to WKY. In conclusion, attenuated β-adrenergic vasodilatation of conduit arteries of SHR but similar β-adrenergic relaxation of resistance mesenteric arteries from WKY and SHR and their similar BP response to β-adrenergic agonists do not support major role of altered β-adrenergic vasodilatation for high BP in genetic hypertension.

Ezetimibe Induces Vasodilation in Rat Mesenteric Resistance Arteries through Inhibition of Extracellular Ca2+ Influx.

Ezetimibe is a lipid-lowering agent that selectively inhibits cholesterol absorption by binding to the Niemann-Pick C1-like 1 (NPC1L1) protein. Although it is well known that administration of ezetimibe in hypercholesterolemia patients reduces the risk of cardiovascular events through attenuation of atherosclerosis, studies on the direct effect of ezetimibe on vascular function are not sufficient. The aim of the present study was to investigate the vascular effects of ezetimibe in rat mesenteric arteries. In the present study, 12-week-old male Sprague Dawley rats were used. After the rats were sacrificed, the second branches of the mesenteric arteries were isolated and cut into 2-3 mm segments and mounted in a multi-wire myography system to measure isometric tension. Ezetimibe reduced vasoconstriction induced by U46619 (500 nM) in endothelium-intact and endothelium-denuded arteries. Ezetimibe-induced vasodilation was not affected by the endothelial nitric oxide synthase (eNOS) inhibitor Nω-Nitro-L-arginine (L-NNA, 300 μM) or the non-selective potassium channel blocker, tetraethylammonium (TEA, 10 mM). Moreover, ezetimibe also completely blocked the contraction induced by an increase in external calcium concentration. Ezetimibe significantly reduced vascular contraction induced by L-type Ca2+ channel activator (Bay K 8644, 30 nM). Treatment with ezetimibe decreased the phosphorylation level of 20 kDa myosin light chain (MLC20) in vascular smooth muscle cells. In the present study, we found that ezetimibe has a significant vasodilatory effect in rat mesenteric resistance arteries. These results suggest that ezetimibe may have beneficial cardiovascular effects beyond its cholesterol-lowering properties.

Abstract 027: Chronic Unpredictable Stress Affects Mitochondrial Function In The Hypothalamic-Pituitary-Adrenal Axis And Impairs Vascular Reactivity In Male And Female Mice

Chronic stress is associated with hypertension but the underlying mechanisms linking the interplay between the changes in the hypothalamic-pituitary-adrenal axis and vascular dysfunction remain unclear. We hypothesize that chronic unpredictable stress (CUS) induces mitochondrial dysfunction in the HPA axis and in the vasculature, associated with GasderminD pore formation in the mitochondrial membrane. Adult male and female C57Bl6/J mice were exposed to 28 consecutive days of CUS or handling (CTL). Anxiety-like behavior, coping behavior, and systolic blood pressure (SBP) were assessed. Mitochondrial function was assessed using high-resolution respirometry in the hypothalamus, amygdala, and adrenal tissues. The vascular reactivity of mesenteric resistance arteries (MRA), pudendal arteries (PA), and aorta were assessed in response to phenylephrine or acetylcholine. GasderminD protein expression was measured in the MRA. Data are presented as mean ± S.E.M and significance was set at p&lt;0.05. In both sexes, CUS increased SBP by 12.1±4.9%. CUS decreased body weight gain by 53% (p=0.04) and increased anxiety-like behavior (p=0.01) in males (vs CTL) but did not affect females. CUS decreased latency to immobility in the forced swim test by 46% (p=0.0001) in both sexes. Sex-dependent decreases in mitochondrial respiration were observed as follows: in the amygdala of males (20%, p=0.05) and the hypothalamus (20%, p=0.004) and adrenal glands (17%, p=0.06) of females. In the vasculature, CUS led to endothelial dysfunction observed by reduced potency of ACh in the MRA (pEC 50 CUS 6.17±0.09 vs CTL 6.81±0.11; p=0.003) and PA (pEC 50 CUS 6.31 ±0.07 vs CTL 6.65±0.09; p=0.008) of male mice, as well as in the MRA of females (pEC 50 CUS 5.94±0.30 vs CTL 6.98±0.15; p=0.041). Contractile responses to PE were significantly reduced in the aorta of CUS males only (E MAX CTL: 4.70±0.48 mN vs CUS 3.01± 0.23 mN; p=0.007). The expression of gasderminD was increased in the MRA of female mice only. CUS increased SBP in males and females, induced sex-specific changes in the mitochondrial function in key areas of the brain, and caused vascular dysfunction, suggesting that behavioral, brain, and vascular adaptations to CUS are sex-dependent.

Abstract P231: Lipoprotein Lipase Activation Is A Novel Anti-contractile Mechanism That Is Both Endothelium-dependent And Independent.

Risk factors such as hypertriglyceridemia and hypercholesterolemia are commonly attributed to the pathogenesis of atherosclerosis, however, lipoprotein metabolism is also associated with end organ damage and cardiovascular events in hypertensive patients. Lipoprotein lipase (LPL) is an important hydrolase for lipoprotein catabolism. Previous work from our lab has shown that LPL activator, NO-1886 (Ibrolipim), significantly decreased vascular contraction. Physiologically, endothelial function limits contractility and endothelial dysfunction is a hallmark of hypertension. Therefore, we hypothesized that LPL activation would cause endothelial-dependent hypocontractility. To test this hypothesis, mesenteric resistance arteries (MRA) were isolated from male Fischer 344 rats at 3 months of age (n=4-12) and mounted onto a wire myograph. Phenylephrine (PE) concentration-response curves were performed with and without LPL activator, NO-1886 (30 min). Some MRA were denuded of their endothelium or incubated with inhibitors of endothelium-dependent vasodilation. As expected, activating LPL exerted a significant anti-contractile effect in a concentration-dependent manner [KCl (%), Veh: 125±3 vs. NO-1886 (1 uM): 116.0±17 vs. NO-1886 (10 uM): 108±9* vs. NO-1886 (50 uM): 55±4* vs. NO-1886 (100 uM): 29±5*, *p&lt;0.05 vs. Veh]. However, contrary to our hypothesis, denudation only partially restored contraction [KCl (%),Veh: 125±3 vs. NO-1886 (50 uM): 55±4* vs. NO-1886+denuded: 76±4* # , *p&lt;0.05 vs. Veh, # p&lt;0.05 vs. NO-1886]. This suggests that there is both an endothelium-dependent and -independent mechanism by which LPL limits contractility. Endothelial-dependent hypocontractility was found to be mediated by nitric oxide synthase [KCl (%),Veh: 125±3 vs. NO-1886 (50 uM): 55±4 vs. NO-1886+L-NAME (100 uM): 90±5* # , *p&lt;0.05 vs. Veh, # p&lt;0.05 vs. NO-1886]. Endothelium-independent mechanisms by which NO-1886 causes hypocontractility have yet been clarified (data not shown). Overall, this suggests that LPL has an important physiological role in controlling vascular contraction through both endothelial-dependent and -independent means and loss of LPL activity could be a novel mechanism of endothelial dysfunction in hypertension.

Abstract 106: Vascular Dysfunction In The Mesenteric Resistance Arteries Occurs Prior To The Occurrence Of Early-onset Alzheimer’S Disease In A Sex-dependent Manner

Alzheimer’s Disease (AD) and cardiovascular diseases (CVDs) are a major cause ofdisability and death among the older population. Familial Alzheimer’s Disease (FAD) islinked with an early onset AD (EOAD) pathology (&lt;65years) that manifests throughpathogenic mutations in the presenilin (PSEN)1 and PSEN2 genes, leading to theincreased production of central amyloid β (Aβ) peptides. There is increasing evidencethat elevated blood pressure and peripheral arterial stiffness contribute to thedevelopment and increase the risk of β-amyloid deposition in the brain leading to cognitivedecline later in life. We hypothesized that peripheral vascular remodeling and vasculardysfunction in the aorta and mesenteric resistance arteries (MRA) would be observedprior to the development of EOAD. The MRA of male and female C57Bl/6J (control) andAD mice (B6. CApptm1DboTg(APPswe,PSEN1dE9)85Dbo) at 9-weeks-old were isolatedprior to the onset of AD. Acetylcholine-induced relaxation (1pM-30μM) andphenylephrine-induced contraction (0.1nM-30μM) were evaluated using wire myography.Pulse wave velocity (PWV) and H&amp;E staining were used to measure vascular stiffnessand morphology, respectively. Data were analyzed as non-linear curve regression andmaximum response (Rmax) using the Student t test ( p &lt;0.05*). MRA from AD miceshowed impaired relaxation in both males (Rmax: control 90±0.9 vs. AD 59±6.4* %, n=3-4) and females (Rmax: control 90±2.8 vs. AD 70±8.3*%, n=4-6). Interestingly,phenylephrine-induced contraction was reduced in arteries from males (Rmax: control11.7±0.9 vs . AD 7.7±0.8* mN, n=3-4), but not in arteries from females (Rmax: control9.3±1.3 vs. AD 8.1±0.6 mN, n=6-7). However, no differences were observed in the aortawall thickness in the male (control 51.8±2.9 vs. AD 72±9.2 μm, n=3-4), or female (control63.7±9.8 vs. AD 54±4.8μm, n=2-3) AD mice, as well as the PWV analysis from male ADmice (control 2.41±0.44 vs. AD 4.05±1.4 mm/ms, n=6). These data suggest that thechanges observed in the peripheral microvasculature prior to the onset of AD could laterhave direct effects on cerebral blood flow leading to areas of altered blood perfusion andmicrovascular damage, thus contributing to the genesis and maintenance of AD.

Abstract 123: Partial Endothelial Cell In Vivo Reprogramming Reverses The Hypercontractility To Phenylephrine In Resistance Arteries Isolated From Males But Not From Females BPH/2J Hypertensive Mice

The Schlager hypertensive (BPH/2J) mouse strain is a genetic model of spontaneoushypertension. Endothelial dysfunction is a hallmark characteristic of hypertension,including endothelial phenotypic transition. We hypothesized that treating endothelialcells with the transcription factors Oct4, Sox2 and Klf4, will lead to partial reprogramming,reduce non-functional phenotype changes, and prevent vascular dysfunction. Mouseendothelial cells were transduced with a lentiviral vector containing VE-cadherin-Oct4-Sox2-Klf4-eGFP (LVTF), control lentivirus (LVCO), or phosphate buffer saline (PBS).After 72h, cell migration and proliferation, Ki67 marker, and the endothelial progenitormarkers (CD31, CD133 and CD34) were evaluated. Male or female BPH/2J mice (33-42-week-old) and their age-matched controls BPN/3J (normotensive) received a singleinjection of LVCO or LVTF (100μL) via the tail vein and were evaluated after 10 days.Systolic blood pressure (SBP) was measured directly by catheterization of the left carotidartery. Mesenteric resistance arteries were isolated and mounted on wire myographs toevaluate phenylephrine-induced contraction. Data were analyzed using One-way orTwo-way ANOVA, with Bonferroni correction ( p &lt;0.05). Lentivirus-transduced endothelialcells had eGFP fluorescence (LVCO: 45±2%, LVTF:19±1%, n=5), and LVTF increasedOct4 (LVTF: 5.6±0.7% vs. LVCO: 1.8±0.2% vs. PBS: 1.5±0.4%, n=5) and CD133 (LVTF:11.7±0.8% vs. LVCO: 7.6±0.3% vs. PBS: 6.5±0.5%, n=5), but not CD31, CD34 or Ki67.Cell migration was lower in LVTF (71±2%) than LVCO cells (90±2%, n=5). Male andfemale BPH/2J treated with LVCO had higher SBP (male: 103±3mmHg, female:95±4mmHg, n=2-3) compared to BPN/3J mice (male: 88±1mmHg, female: 80±4mmHg,n=2-6), and LVTF treatment tended to decrease SBP in male BPH/2J (86±2mmHg, n=2, p =0.0548) but not in female mice (90±4mmHg, n=3). Phenylephrine-induced contractionwas reduced in arteries from male BPH/2J treated with LVTF (6.77±0.90mN, n=3), butnot in female mice (11.01±1.45mN, n=3) compared to LVCO (male: 12.23±0.15mN;female: 10.77±0.92mN; n=2-3). These data reveal that partial endothelial cellreprogramming in vivo is a new strategy to prevent endothelial dysfunction in malehypertensive mice.1

Abstract 018: Overexpression Of ERα In Adipose Tissue Contributes To The Anti-contractile Effect Of Mesenteric PVAT By NOX4-induced H 2 O 2

Perivascular adipose tissue (PVAT) induces a significant anti-contractile effect on vasculature, secreting relaxing and constricting factors. It is well established that the PVAT anti-contractile function is lost in cardiometabolic diseases, including obesity. Estrogen receptor alpha (ERα) is known to play a crucial role in the function of adipose tissues and its activation has been associated with adipogenesis by redox balance. In addition, NADPH oxidase 4 (NOX4) is an enzyme that contribute to adipose tissue differentiation and intracellular H 2 O 2 generation. While ERα are expressed in adipose tissue, but their potential role in PVAT function is unknown. To test the hypothesis, adipose tissue specific ERα overexpressing female mice at 30 weeks old were used. We aimed to determine whether the overexpression of ERα would enhance the anti-contractile effect of PVAT by NOX4-induced H 2 O 2 . The high-fat diet (40% fat) was administered for 20 weeks and mesenteric resistance arteries, with and without PVAT, were isolated and used for measurements of vascular function, protein levels (Western blot) and redox signaling (Amplex Red). The higher-fat diet increases the weigh in ~ 80% in Wild Type (WT) and ERα (p&gt;0.05). The anti-contractile effect of PVAT was exacerbated in arteries of ERα mice than WT [Area under the curve (AUC): WT 369±2 vs. ERα 230±1 p&lt;0.05). NOX1/4 inhibitor (GSK136901) increased the vasoconstriction only in arteries from ERα mice (WT 348 ±4 vs. ERα 580±5 p&lt;0.05). NOX4 (0.19±0.01 vs 2.3±0.6) and H 2 O 2 (&gt; 70%) levels were higher in PVAT from ERα mice than WT. In addition, 6 uM of H 2 O 2 , concentration corresponding to the observed in PVAT from ERα mice, induced vasodilatation in arteries from WT. On the other hand, a high-fat diet abrogates the anti-contractile effect of PVAT (WT with PVAT 233±1 vs. without PVAT 241±4) and has partially restored in the ERα, analyzing LogEC 50 (with PVAT 5±0.7 and without PVAT 6±0.8 M). In addition, H 2 O 2 levels (&gt;50%) were higher in PVAT from the high fat diet fed ERα mice, but NOX4 levels did not change. In summary, overexpression of ERα increases H 2 O 2 levels derived from NOX4, contributing to the anti-contractile effect of PVAT in healthy animals. This effect is partially restored in mice on a high-fat diet with overexpressed ERα.

Abstract P318: INDOLE-3 ACETIC ACID: A NOVEL DUAL-ACTION INHIBITOR OF THE ENDOTHELIN SYSTEM FOR TREATING HYPERTENSION

Elevated levels of endothelin-1 (ET-1) stimulate potent vasoconstriction, free radical production, oxidative stress, and vascular inflammation, which are often present in salt-sensitive hypertension, pulmonary hypertension, drug-resistant hypertension, and hypertension among African Americans. There is currently no known drug that simultaneously targets both ET-1 receptors (ETRs) and the ET-1 synthesizing enzyme ECE-1, even though dual targeting of the ET system could lead to a new generation of drug molecules with improved therapeutic outcome. Using an integrated in vitro, in vivo and in silico approaches, here we report the discovery of indole-3 acetic acid (IAA) as an inhibitor of both ET-1 receptors and ECE-1. IAA is produced in the human body via tryptophan metabolism by gut microbiota. Our data demonstrates that IAA concentration-dependently inhibits ET-1-evoked vasoconstriction in rat resistance mesenteric arteries and ET-1-stimulated reactive oxygen species (ROS) production in smooth muscle cells (SMCs). Furthermore, IAA suppresses ET-1-induced endothelial cell (EC) activation, adhesion molecule overexpression, and leukocyte adhesion. IAA inhibits ET-1-stimulated, ET A R- and ET B R-mediated increases in [Ca 2+ ] i . Intriguingly, IAA produces a concentration-dependent inhibition of purified human ECE-1. Treatment of Dahl salt-sensitive (DSS) hypertensive rats, a model of augmented ET-1 signaling, with an oral extended-release formulation of IAA significantly inhibits blood pressure elevation in high-salt diet-fed (HSD) DSS rats. IAA-treated DSS arteries show intact acetylcholine- and sodium nitroprusside-induced vasodilation, suggesting that IAA preserves endothelial and smooth muscle function, respectively. IAA treatment also prevents arterial remodeling, oxidative stress, and kidney dysfunction in HSD DSS rats. Overall, our study identifies IAA as the first-in-class dual-action inhibitor of ETRs and ECE-1, which produces profound antihypertensive effect in vivo. IAA could also be therapeutically useful for other cardiovascular diseases in which ET-1 signaling is implicated.

Abstract P104: EXAMINING THE EFFECTS OF FOLLISTATIN ON CONTRACTION OF VESSELS FROM NORMOTENSIVE AND HYPERTENSIVE RATS

The lab has previously shown that follistatin (FST), a glycoprotein inhibitor of activins, reduced blood pressure in the Spontaneously Hypertensive Rat (SHR) model of Essential Hypertension. Short-term exposure (30 minutes) to FST also reduced vessel contraction induced by potassium (KCl). This study aims to illustrate the mechanisms through which FST inhibits potassium-induced contraction in SHR and normotensive (WKY) rats. Function of SHR or WKY resistance mesenteric arteries was tested in response to KCl and ion channel modulators using wire myography. Cultures of primary smooth muscle cells (SMC) were also established from these vessels using cell explant technique. The calcium sensor dye Fluo-4 AM was used to examine calcium influx on a cellular level. To test whether FST effects were due to activin neutralization or activin-receptor inhibition, neutralizing antibodies against activin A and B or an activin receptor inhibitor (SB 431542) were used, respectively. Only activin A neutralization in the SHR reduced KCl-induced contraction similarly to FST. Activin receptor inhibition also reduced KCl constriction similarly to FST. When directly stimulated with Activin A, both WKY and SHR vessels displayed augmented KCl contraction compared to controls but the effect was more pronounced in the SHR. Preliminary Fluo-4 results showed that FST decreased KCl-induced calcium influx in SMC. Since activin A was shown to increase L-type voltage-gated Ca2+channel activity, an agonist (FPL 64176) of this channel was used to induce contraction. Although FST reduced KCl-induced contraction, it did not affect FPL-induced contraction in either SHR or WKY. Thus, Activin A neutralization reproduces FST reduction of KCl-induced vessel contraction. FST did not reduce FPL-induced contraction, suggesting that its inhibition of KCl-induced contraction is not through effects on this channel but rather an alternate Ca2+ or K-ATP channel.

The vasoconstrictor activities of prostaglandin D2 via the thromboxane prostanoid receptor and E prostanoid receptor-3 outweigh its concurrent vasodepressor effect mainly through D prostanoid receptor-1 ex vivo and in vivo

Prostaglandin (PG) D2, a commonly considered vasodilator through D prostanoid receptor-1 (DP1), might also evoke vasoconstriction via acting on the thromboxane (Tx)-prostanoid receptor (the original receptor of TxA2; TP) and/or E prostanoid receptor-3 (one of the vasoconstrictor receptors of PGE2; EP3). This study aimed to test the above hypothesis in the mouse renal vascular bed (main renal arteries and perfused kidneys) and/or mesenteric resistance arteries and determine how the vasoconstrictor mechanism influences the overall PGD2 effect on systemic blood pressure under in vivo conditions. Experiments were performed on control wild-type (WT) mice and mice with deficiencies in TP (TP−/−) and/or EP3 (EP3−/−). Here we show that PGD2 indeed evoked vasoconstrictor responses in the above-mentioned tissues of WT mice, which were however not only reduced by TP−/− or EP3−/−, but also reversed by TP−/−/EP3−/− in some of the above tissues (mesenteric resistance arteries or perfused kidneys) to dilator reactions that were reduced by non-selective DP antagonism. A slight or mild pressor response was also observed with PGD2 under in vivo conditions, and this was again reversed to a depressor response in TP−/− or TP−/−/EP3−/− mice. Non-selective DP antagonism reduced the PGD2-evoked depressor response in TP−/−/EP3−/− mice as well. These results thus demonstrate that like other PGs, PGD2 activates TP and/or EP3 to evoke vasoconstrictor activities, which can outweigh its concurrent vasodepressor activity mediated mainly through DP1, and hence result in a pressor response, although the response might only be of a slight or mild extent.