Vasodilation In Mesenteric Arteries Research Articles

Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation.

Nitric oxide regulates BP by binding the reduced heme iron (Fe2+) in soluble guanylyl cyclase (sGC) and relaxing vascular smooth muscle cells (SMCs). We previously showed that sGC heme iron reduction (Fe3+ → Fe2+) is modulated by cytochrome b5 reductase 3 (CYB5R3). However, the in vivo role of SMC CYB5R3 in BP regulation remains elusive. Here, we generated conditional smooth muscle cell-specific Cyb5r3 KO mice (SMC CYB5R3-KO) to test if SMC CYB5R3 loss affects systemic BP in normotension and hypertension via regulation of the sGC redox state. SMC CYB5R3-KO mice exhibited a 5.84-mmHg increase in BP and impaired acetylcholine-induced vasodilation in mesenteric arteries compared with controls. To drive sGC oxidation and elevate BP, we infused mice with angiotensin II. We found that SMC CYB5R3-KO mice exhibited a 14.75-mmHg BP increase, and mesenteric arteries had diminished nitric oxide-dependent vasodilation but increased responsiveness to sGC heme-independent activator BAY 58-2667 over controls. Furthermore, acute injection of BAY 58-2667 in angiotensin II-treated SMC CYB5R3-KO mice showed greater BP reduction compared with controls. Together, these data provide the first in vivo evidence to our knowledge that SMC CYB5R3 is an sGC heme reductase in resistance arteries and provides resilience against systemic hypertension development.

Abstract 069: Local and Circulating Sphingosine-1-Phosphate in Blood Pressure Regulation

Sphingosine-1-phosphate (S1P), a bioactive lipid, regulates different biological processes in health and disease. Once synthetized, endothelial S1P is transported outside the cells via Spinster-2 (Spns-2), and can signal mainly through the sphingosine-1-phosphate receptor-1 (S1P1) in autocrine/paracrine manner. Alternatively, S1P can bind circulating carriers, including ApoM of HDL or albumin. Recently, we reported a key role of endothelial S1P-S1P1 autocrine signaling in vascular function and blood pressure (BP) homeostasis. To dissect the contribution of endothelium-derived S1P versus circulating HDL-bound S1P on BP regulation, we used ApoM knockout mice (ApoM-/-) and developed mice lacking the endothelial Spinster-2 (ECKO-Spns2). At baseline, systolic BP (SBP) was markedly increased in both ECKO-Spns2 (120.8± 2.1 vs. 106.6±1.3 mmHg, n=8) and ApoM-/- (122.6±1.0 vs. 109.0±1.4 mmHg, n=8) vs control mice. Vasodilation of mesenteric arteries (MA) to acetylcholine was preserved in both mouse models, whereas flow-induced vasodilation was significantly reduced in ECKO-Spns2 (Emax 36.0±1.5 vs. 53.6±3.6 % vasodilation, n=5) but not in ApoM-/- MA, suggesting that autocrine S1P signaling requires the S1P transporter Spns-2. However, both ECKO-Spns2 and ApoM-/- showed a reduced NO production compared to control MA. Moreover, chronic infusion of AngII resulted in higher BP in ECKO-Spns2 (156.0± 1.2 vs. 142.0±1.6 mmHg, n=8) and ApoM-/- (158.5±1.1 vs. 144.1±1.3 mmHg, n=8) vs control mice. Interestingly, chronic AngII strongly reduced vasodilation in response to flow in both ECKO-Spns2 (Emax 19.3±2.5 vs. 35.4±7.1 % vasodilation, n=5) and ApoM-/- (Emax 22.9±1.1 vs. 34.8±6.8 % vasodilation, n=5) compared to control MA, suggesting a protective role of local and circulating S1P. Additionally, basal NO production was further impaired by the AngII infusion (ECKO-Spns2 -32.7% and ApoM-/- -42.8%, vs control MA), confirming the pivotal role of S1P-S1P1-NO pathway in vascular and BP homeostasis.

Angiotensin II Type 1 Receptor Antagonist Azilsartan Restores Vascular Reactivity Through a Perivascular Adipose Tissue-Independent Mechanism in Rats with Metabolic Syndrome.

Perivascular adipose tissues (PVAT) are involved in the regulation of vascular tone. In mesenteric arteries, the compensatory vasodilatory effects of PVAT appear when vascular relaxation is impaired and disappear at around 23weeks of age in SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats with metabolic syndrome (MetS). The renin-angiotensin system is involved in the development of endothelium and vascular dysfunction. Therefore, we investigated whether azilsartan, a potent angiotensin II type 1 (AT1) receptor antagonist, can protect against the deterioration of the PVAT compensatory vasodilator function that occurs with aging in MetS. Two age groups of SHRSP.ZF rats (13 and 20weeks of age) were administered azilsartan or vehicle through oral gavage once daily for 10weeks. The vasodilation response of the isolated superior-mesenteric arteries upon addition of endothelium-dependent and -independent agonists was determined in the presence or absence of PVAT using organ bath methods. In vivo treatment with azilsartan improved the acetylcholine-induced vasodilation in mesenteric arteries with and without PVAT at both time-points. The mRNA levels of AT1 receptor and AT1 receptor-associated protein were unchanged in PVAT upon azilsartan treatment. Furthermore, in vitro treatment with azilsartan (0.1 and 0.3μM for 30min) did not affect the compensatory effect of PVAT on vasodilation in response to acetylcholine in SHRSP.ZF rat mesenteric arteries. Our results provide evidence supporting the use of azilsartan for the long-term protection against vascular dysfunctions in MetS. Azilsartan did not improve the dysfunction of PVAT-mediated modulation of vascular tone during MetS. The protective effect of azilsartan is mediated by restoring the endothelium- and vascular smooth muscle-mediated mechanisms.

Hypermetabolism and impaired endothelium-dependent vasodilation in mesenteric arteries of type 2 diabetes mellitus db/db mice.

Besides being a metabolic disease, diabetes is considered a vascular disease as many of the complications relate to vascular pathologies. The aim of this study was to investigate how vascular tone and reactivity and vascular cell metabolism were affected in type 2 diabetes mellitus and whether β-hydroxybutyrate could have a positive effect as alternative energy substrate. Isolated mesenteric arteries of db/db and control mice were incubated in media containing [U-13C]glucose or [U-13C]β-hydroxybutyrate, and tissue extracts were analysed by mass spectrometry. Functional characterization was performed by wire myography to assess vasodilation and vasocontraction. Hypermetabolism of glucose and β-hydroxybutyrate was observed for mesenteric arteries of db/db mice; however, hypermetabolism was significant only with β-hydroxybutyrate as energy substrate. The functional characterization showed impaired endothelial-dependent vasodilation in mesenteric arteries of the db/db mice, whereas the contractility was unaffected. This study provides evidence that the endothelial cells are impaired, whereas the vascular smooth muscle cells are more robust and seemed less affected in the db/db mouse. Furthermore, the results indicate that hypermetabolism of energy substrates may be due to adaptive changes in the mesenteric arteries.

Secreted Monocyte miR-27a, via Mesenteric Arterial Mas Receptor-eNOS Pathway, Causes Hypertension.

Essential hypertension is associated with increased plasma concentrations of extracellular vesicles (EVs). We aimed to determine the role of monocyte miR-27a in EVs on arterial Mas receptor expression, and its involvement in the pathogenesis of hypertension. THP-1 cells were transfected with miR-27a mimic and miR-27a inhibitor, and EVs were collected. Mas receptor expression and endothelial nitric oxide synthase (eNOS) phosphorylation were determined by immunoblotting. Sprague-Dawley (SD) rats received EVs via tail-vein injection. Blood pressure (BP) was measured with the tail-cuff method. The vasodilatory response of mesenteric arteries was measured using a small vessel myograph. EVs from THP-1 cells increased rat BP by impairing Ang-(1-7)-mediated vasodilation in mesenteric arteries, which was further exaggerated by EVs from lipopolysaccharides-treated THP-1 cells. As the receptor and key signaling of Ang-(1-7), next experiments found that Mas receptor expression and eNOS phosphorylation were decreased in mesenteric arteries from EVs-treated SD rats. Screening studies found miR-27a in EVs may be involved in this process. Through transfection with miR-27a inhibitor or miR-27a mimic, we found that miR-27a downregulates Mas receptor expression in endothelial cells. Injection of EVs from miR-27a-transfected HEK-293 cells decreased Mas receptor and eNOS phosphorylation in mesenteric arteries, impaired Ang-(1-7)-mediated vasodilation and increased BP. Earlier effects were reversed using cells with downregulation of miR-27 in EVs. Monocyte miR-27a in EVs decreases Mas receptor expression and eNOS phosphorylation in endothelium, impairs Ang-(1-7)-mediated vasodilation, and causes hypertension. Understanding the contributions of EVs in the pathogenesis of hypertension may facilitate their use as a diagnostic biomarker.

Inherited risk plus prenatal insult caused malignant dysfunction in mesenteric arteries in adolescent SHR offspring.

Prenatal hypoxia can induce cardiovascular diseases in the offspring. This study determined whether and how prenatal hypoxia may cause malignant hypertension and impaired vascular functions in spontaneous hypertension rat (SHR) offspring at adolescent stage. Pregnant SHR were placed in a hypoxic chamber (11% O2) or normal environment (21% O2) from gestational day 6 until birth. Body weight and blood pressure (BP) of SHR offspring were measured every week from 5 weeks old. Mesenteric arteries were tested. Gestational hypoxia resulted in growth restriction during 6–12 weeks and a significant elevation in systolic pressure in adolescent offspring at 12 weeks old. Notably, endothelial vasodilatation of mesenteric arteries was impaired in SHR adolescent offspring exposed to prenatal hypoxia, vascular responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were reduced, as well as plasma nitric oxide levels and expression of endothelial nitric oxide synthase (eNOS) in vessels were decreased. Moreover, mesenteric arteries in SHR offspring following prenatal hypoxia showed enhanced constriction responses to phenylephrine (PE), associated with up-regulated activities of L-type calcium channel (Ca2+-dependent), RhoA/Rock pathway signaling (Ca2+-sensitization), and intracellular Ca2+ flow. Pressurized myograph demonstrated altered mechanical properties with aggravated stiffness in vessels, while histological analysis revealed vascular structural disorganization in prenatal hypoxia offspring. The results demonstrated that blood pressure and vascular function in young SHR offspring were affected by prenatal hypoxia, providing new information on development of hypertension in adolescent offspring with inherited hypertensive backgrounds.

Cucurbitacins extracted from Cucumis melo L. (CuEC) exert a hypotensive effect via regulating vascular tone.

As an effective medicine for jaundice in traditional Chinese medicine, Cucumis melo L. has been widely used in China. However, its effect on vascular function is still unclear. In this study, we extracted the compounds of Cucumis melo L., and the major ingredients were identified as cucurbitacins (CuEC, cucurbitacins extracted from Cucumis melo L.), especially cucurbitacin B. We replicated the toxicity in mice by intraperitoneal injection of a high dose of CuEC (2 mg/kg) and demonstrated that the cause of death was CuEC-induced impairment of the endothelial barrier and, thus, increased vascular permeability via decreasing VE-cadherin conjunction. The administration of low doses of CuEC (1 mg/kg) led to a decline in systolic blood pressure (SBP) without causing toxicity in mice. More importantly, CuEC dramatically suppressed angiotensin II (Ang II)-induced SBP increase. Further studies demonstrated that CuEC facilitated acetylcholine-mediated vasodilation in mesenteric arteries of mice. In vitro studies showed that CuEC induced vasodilation in a dose-dependent manner in mesenteric arteries of both mice and rats. Pretreatment with CuEC inhibited phenylephrine-mediated vasoconstriction. In summary, a moderate dose of CuEC reduced SBP by improving blood vessel tension. Therefore, our study provides new experimental evidence for developing new antihypertensive drugs.

Age‐Related Changes in Skeletal Muscle and Small Mesenteric Arterial Function in Spontaneously Hypertensive Rats

Essential hypertension is a chronic idiopathic medical condition that is characterized by elevated arterial blood pressure and is associated with increased risk of cardiovascular disease. Endothelial dysfunction is associated with increased age and a casual factor for the development of hypertension. Therefore, we hypothesized that endothelial dysfunction would be exacerbated by age to a greater extent in spontaneously hypertensive rats (SHRs) compared to Wistar‐Kyoto rats (WKY) control rats. Vascular function was assessed using pressure myography in skeletal muscle and mesenteric resistance arteries isolated from SHR and WKY across three age groups: young (16–17 weeks, n = 4/group), aged (62–64 weeks, n = 4/group) and old (88–100 weeks, n = 5/group). Phenylephrine‐induced vasoconstriction responses in either of the resistance arteries were not significantly different between WKY and SHR at any of the age groups. Endothelial‐dependent vasodilation in mesenteric arteries was not significantly different between WKY and SHR across age groups either. However, endothelium‐dependent vasodilation in the skeletal muscle resistance arteries was significantly enhanced in the young SHR compared to WKY, but not in the aged or old rats. Moreover, endothelial‐independent vasodilation in mesenteric arteries was significantly enhanced in SHR compared to WKY across all age groups, while this difference was only observed in the skeletal muscle resistance arteries of the young rats. Contrary to our hypothesis, these data suggest that endothelial dysfunction is not exacerbated by age in the resistance arteries of SHR.Support or Funding InformationResearch support: National Institute of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Renal vasodilation induced by Glucagon‐like peptide‐1 is mediated only by the known receptor

Glucagon‐like peptide‐1 (GLP‐1) stimulates insulin release after a meal. The GLP‐1 receptor is found in many tissues including the renal vasculature. GLP‐1 increases renal blood flow (RBF) and glomerular filtration rate by inducing a renal vasodilation. The GLP‐1 receptor knock‐out (KO) mouse has increased glomerular filtration rate (GFR) suggesting a dilation of the afferent arteriole. Moreover, GLP‐1 elicits a vasodilation in mesenteric arteries from the GLP‐1 receptor KO mouse suggesting that GLP‐1 can induce vasodilation through other mechanisms that by the known receptor. Also, the metabolite of GLP‐1 (GLP‐1(9–36)) has been suggested to have vasodilatory effects.Using wire‐myography and isolated segmental arteries from GLP‐1 receptor KO and wild‐type (WT) mice we show that GLP‐1 only elicits renal vasodilation via the known receptor and not by any indirect pathways. In preconstricted segmental arteries GLP‐1 induces a vasodilation that can be inhibited by exendin(9–39) (a GLP‐1 receptor antagonist). Also, GLP‐1(9–36) has no vasodilatory effect on isolated renal vessels from WT and KO showing that in the renal vasculature the metabolite has no effect either directly on the GLP‐1 receptor or through any other pathway.In the isolated juxtamedullary nephron preparation the autoregulatory response in afferent arterioles measured during acute pressure increases in isolated kidneys from WT and GLP‐1 receptor KO mice is normal. This indicates that the pre‐glomerular vasculature in the kidney is not affected functionally by the loss of the GLP‐1 receptor. GLP‐1 only attenuates the autoregulatory response in afferent arterioles from WT mice showing that also in afferent arterioles there is no alternative signaling pathway. GLP‐1 primarily attenuates the myogenic response and not the tubuloglomerular feedback (TGF).Using pressure myography we show that GLP‐1 reduces the myogenic response in WT segmental arteries but has no effect in the GLP‐1 receptor KO vessels. GLP‐1 increases the diameter of WT segmental arteries but a further diameter increase is still found during passive conditions (Ca2+ free media) suggesting that GLP‐1 dose not completely remove all vascular tension.ConclusionThe renal vascular effects of GLP‐1 are elicited only via the known GLP‐1 receptor. The metabolite GLP‐1(9–36) has no effect on the renal GLP‐1 receptor or through alternative pathways. Afferent arterioles and segmental arteries from GLP‐1 receptor KO mice respond normally to acute pressure increases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prenatal hypoxia affected endothelium-dependent vasodilation in mesenteric arteries of aged offspring via increased oxidative stress

Prenatal hypoxia can affect vascular functions in young offspring. However, there is limited knowledge regarding whether and how prenatal hypoxia influences vascular functions in aged offspring. This study compared the effects of prenatal hypoxia on the mesenteric arteries (MA) between a young adult and aged offspring and investigated the underlying mechanisms. Pregnant rats were randomly divided into the control and prenatal hypoxia groups. The vascular functions and molecular levels were assessed in 5-month-old (5 M) or 20-month-old (20 M) offspring. Prenatal hypoxia decreased acetylcholine-mediated vascular relaxations in 20-M but not 5-M offspring. Sodium nitroprusside-mediated relaxation curves were not altered by prenatal hypoxia in 5- and 20-M offspring. Prenatal hypoxia enhanced the contractile responses caused by phenylephrine, phorbol 12,13-dibutyrate, and 5-hydroxytryptamine only in 5-M offspring. The endothelial NO synthase (eNOS) activities were decreased along with downregulated eNOS mRNA expression and phosphorylated eNOS/total eNOS protein expression in 20-M offspring with prenatal hypoxia. The NADPH oxidase (NOX) inhibitor apocynin and superoxide dismutase (SOD) mimetic tempol restored the acetylcholine-mediated weaker relaxations in 20-M offspring with prenatal hypoxia. Enzyme-linked immunosorbent and dihydroethidium assay showed that prenatal hypoxia enhanced oxidative stress in 20-M offspring. Transmission electron microscopy showed that prenatal hypoxia damaged mitochondrial structures in the MA endothelial cells of 20-M offspring. Increased NOX2 protein expression and decreased SOD3 expression were found in 20-M offspring. The results demonstrated that endothelial dysfunction induced by intrauterine hypoxia occurred with aging via enhanced oxidative stress and decreased nitric oxide activities in aged offspring.

Potassium Channel Activation Is Involved in the Cardiovascular Effects Induced by Freeze Dried Syzygium jambolanum (Lam.) DC Fruit Juice.

This work aimed to explore the cardiovascular effects induced by freeze-dried juice from Syzygium jambolanum (Lam.) DC fruits (JSJ). JSJ presented high polyphenol content and steroids. HPLC analysis revealed that 2,5-dihydroxybenzoic and caffeic acid were present in higher amounts in the JSJ extract. In rat, JSJ induces hypotension and vasodilatation in mesenteric arteries, with or without vascular endothelium. JSJ-mediated vasodilation response against contractions induced with KCl (60 mM) depolarizing solution was significantly lower than the responses induced by JSJ when evaluated against phenylephrine-induced contractions. To investigate the involvement of potassium channels we used Tyrode's solution with KCl (20 mM) or tetraethylammonium (1.0, 3.0, or 5.0 mM). In these conditions JSJ-induced effects were significantly attenuated. To investigate the potassium channel subtypes involved in the response, we used 4-aminopyridine, glibenclamide, BaCl2, and iberiotoxin. In the presence (simultaneous) of different potassium channel blockers we observed a significant attenuation of JSJ-induced effect. Inhibition was also observed when using BaCl2, glibenclamide, or 4-aminopyridine, separately. However, incubation with iberiotoxin did not promote changes in either maximum effect, or potency. We also evidenced a discrete participation of CaV channels in the JSJ-induced vasorelaxant effect. In addition, patch-clamp studies demonstrated that JSJ could activate potassium channels. In conclusion, JSJ promotes hypotension and vasorelaxation in rats, involving, at least, the activation of potassium channels.

A11957 Irisin Lowers Blood Pressure by Improvement of Endothelial Dysfunction via AMPK-Akt-eNOS-NO Pathway in the Spontaneously Hypertensive Rat

Objectives: Exercise is a major nonpharmacological treatment for hypertension, but its underlying mechanisms are still not completely elucidated. Irisin, a polypeptide containing 112 amino acids, which is secreted mainly by skeletal muscle cells during exercise, exerts a protective role in metabolic diseases, such as diabetes and obesity. Due to the close relationship between irisin and metabolic diseases, we hypothesized that irisin may play a role in the regulation of blood pressure. Methods: Blood pressures of male WKY rats and SHRs were monitored via the carotid artery. The vascular response was checked by tension measurement of the artery rings. Protein expression was tested by immunoblot. Results: Our study found that the acute intravenous injection of irisin reduced blood pressure in SHRs but not WKY rats. Irisin, by itself, had no direct vasorelaxing effect in phenylephrine-pre-constricted mesenteric arteries from SHRs. However, irisin augmented the acetylcholine-induced vasorelaxation in mesenteric arteries from SHRs that could be reversed by Nω-nitro-L-arginine-methyl ester (L-NAME, 100 μM), indicating a role of NO in this action. Indeed, irisin increased NO production and phosphorylation of eNOS in endothelial cells. 5¢-AMP-activated protein kinase (AMPK) was involved in the vasorelaxing effect of irisin because compound C (20 μM), an AMPK inhibitor, blocked the irisin-mediated increase in phosphorylation of eNOS and Akt in endothelial cells and vasodilation in mesenteric arteries. Conclusion: We conclude that acute administration of irisin lowers blood pressure of SHRs by amelioration of endothelial dysfunction of the mesenteric artery via AMPK-Akt-eNOS-NO signaling pathway.

Extract of Aquilaria crassna leaves and mangiferin are vasodilators while showing no cytotoxicity

The leaves of Aquilaria spp. promote “physiological balance”, and are “cardiotonic and provide blood nourishment”. In Asia, these leaves are increasingly consumed as tea and claimed to provide benefits to cardiovascular function, albeit without any scientific proof. Therefore, this study sought to evaluate the action of Aquilaria crassna leaf aqueous extract (AE) on vascular function and vascular smooth muscle cytotoxicity. AE and a main constituent, mangiferin were investigated for their vasorelaxation of rat mesenteric arteries and aortae in vitro. Acute cytotoxicity of AE (0.1–1000 μg/ml) and mangiferin (0.1–100 μM) on rat enzymatically isolated vascular smooth muscle cells was assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. AE dilated rat mesenteric arteries (EC50∼107 μg/ml, Emax∼95%) more than aorta (EC50∼265 μg/ml, Emax∼76%, p < 0.05).AE-induced vasodilation in mesenteric artery was reduced by endothelial removal (EC50∼202 μg/ml, p < 0.05), incubation with endothelial nitric oxide synthase (eNOS) (100 μM, L-NAME) (EC50∼309 μg/ml, p < 0.05), and partly reduced by L-type Ca2+ channel blockade at higher concentrations. Likewise, mangiferin (1–100 μM) dilated the mesenteric artery more potently than the aorta. However, its maximum relaxation was less than with AE (41% in the mesenteric artery and <10% in the aorta). Isolated vascular smooth muscle cells incubated in AE or mangiferin for 1 h showed no cytotoxicity. Thus, AE is a vasorelaxant while being free of acute cytotoxicity towards vascular smooth muscle, thus potentially ameliorating human vascular dysfunction.

Abstract P277: Ceramide Modulates GPCR And TK Receptor Signaling To Impact Endothelial Functions And Blood Pressure

Ceramides are sphingolipids that modulate a variety of cellular processes, via two major modes of actions: by functioning as second messengers and by regulating the formation of lipid raft, important signaling platform. Receptor-mediated signaling orchestrates multiple pathophysiological processes, hence more than 50% of drugs clinically prescribed target mainly GPCRs. Alterations of ceramide levels are implicated in endothelial dysfunction, a key event in many cardiovascular diseases, including hypertension and atherosclerosis. However, specific molecular mechanisms of how ceramides modulate the structure and the function of GPCRs and TK receptors to impact endothelial functions remains unknown and unexplored. Thus, we generated mice lacking the endothelial sphingolipid de novo biosynthesis, by deleting the serine palmitoyltransferase long subunit 2, of the first enzyme of the pathway, named ECKO-Sptlc2. Systolic blood pressure was markedly increased in ECKO-Sptlc2 vs. control mice (122.1± 1.9 vs. 103.6±0.7 mmHg, n=9). Vasodilation of mesenteric arteries to acetylcholine and histamine (Gq-coupled receptors) and the NO-signaling downstream was preserved suggesting that altered membrane sphingolipid levels did not affect Gq-coupled receptor activation. On the contrary, vasodilation induced by the activation of tyrosine kinase receptors, i.e. VEGFR2 (Emax 32.8±3.5 vs. 55.7±3.6 % vasodilation, n=5), Gi-coupled GPCRs, i.e. S1PR1 (Emax 9.0±11.0 vs. 58.7 ±3.5 % vasodilation, n=5) and flow were markedly reduced. C16-, C24- and C24:1-ceramide are the most abundant ceramides in the endothelium and were markedly reduced by the loss of Sptlc2. Interestingly, the treatment of the mice and endothelial cells in culture with C16-, C24- and C24:1-ceramides, showed that C16-ceramide play a specific role in VEGFR2, IR, S1PR1 and flow mediated vasodilation. Mechanistically, C16-ceramide modulates VEGFR2 phosphorylation and downstream signaling in a concentration-dependent manner. The finding that C16-ceramide affects specific the function of Gi-coupled, TK but not Gq-coupled receptor is of great significance considering that alterations in C16-ceramide strongly correlate with CV conditions, such as CAD and heart failure.

Microvascular vasodilator properties of the angiotensin II type 2 receptor in a mouse model of type 1 diabetes

Diabetes Mellitus is associated with severe cardiovascular disorders involving the renin-angiotensin system, mainly through activation of the angiotensin II type 1 receptor (AT1R). Although the type 2 receptor (AT2R) opposes the effects of AT1R, with vasodilator and anti-trophic properties, its role in diabetes is debatable. Thus we investigated AT2R-mediated dilatation in a model of type 1 diabetes induced by streptozotocin in 5-month-old male mice lacking AT2R (AT2R−/y). Glucose tolerance was reduced and markers of inflammation and oxidative stress (cyclooxygenase-2, gp91phox p22phox and p67phox) were increased in AT2R−/y mice compared to wild-type (WT) animals. Streptozotocin-induced hyperglycaemia was higher in AT2R−/y than in WT mice. Arterial gp91phox and MnSOD expression levels in addition to blood 8-isoprostane and creatinine were further increased in diabetic AT2R−/y mice compared to diabetic WT mice. AT2R-dependent dilatation in both isolated mesenteric resistance arteries and perfused kidneys was greater in diabetic mice than in non-diabetic animals. Thus, in type 1 diabetes, AT2R may reduce glycaemia and display anti-oxidant and/or anti-inflammatory properties in association with greater vasodilatation in mesenteric arteries and in the renal vasculature, a major target of diabetes. Therefore AT2R might represent a new therapeutic target in diabetes.

Irisin Lowers Blood Pressure by Improvement of Endothelial Dysfunction via AMPK-Akt-eNOS-NO Pathway in the Spontaneously Hypertensive Rat.

BackgroundExercise is a major nonpharmacological treatment for hypertension, but its underlying mechanisms are still not completely elucidated. Irisin, a polypeptide containing 112 amino acids, which is secreted mainly by skeletal muscle cells during exercise, exerts a protective role in metabolic diseases, such as diabetes mellitus and obesity. Because of the close relationship between irisin and metabolic diseases, we hypothesized that irisin may play a role in the regulation of blood pressure.Methods and ResultsBlood pressures of male Wistar‐Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were monitored through the carotid artery. Our study found that acute intravenous injection of irisin reduced blood pressure in SHRs, but not WKY rats. Irisin, by itself, had no direct vasorelaxing effect in phenylephrine‐preconstricted mesenteric arteries from SHRs. However, irisin augmented the acetylcholine‐induced vasorelaxation in mesenteric arteries from SHRs that could be reversed by Nω‐nitro‐l‐arginine‐methyl ester (L‐NAME; 100 μmol/L), indicating a role of nitric oxide (NO) in this action. Indeed, irisin increased NO production and phosphorylation of endothelial nirtic oxide synthase (eNOS) in endothelial cells. 5′‐AMP‐activated protein kinase (AMPK) was involved in the vasorelaxing effect of irisin because compound C (20 μmol/L), an AMPK inhibitor, blocked the irisin‐mediated increase in phosphorylation of eNOS and protein kinase B (Akt) in endothelial cells and vasodilation in mesenteric arteries.ConclusionsWe conclude that acute administration of irisin lowers blood pressure of SHRs by amelioration of endothelial dysfunction of the mesenteric artery through the AMPK‐Akt‐eNOS‐NO signaling pathway.

Inhibition of epoxyeicosatrienoic acid production in rats with cirrhosis has beneficial effects on portal hypertension by reducing splanchnic vasodilation.

In cirrhosis, 11,12-epoxyeicosatrienoic acid (EET) induces mesenteric arterial vasodilation, which contributes to the onset of portal hypertension. We evaluated the hemodynamic effects of in vivo inhibition of EET production in experimental cirrhosis. Sixteen control rats and 16 rats with carbon tetrachloride-induced cirrhosis were studied. Eight controls and eight rats with cirrhosis were treated with the specific epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH; 20 mg/kg/day) for 3 consecutive days. Portal blood flow and renal and splenic resistive indexes were calculated through echographic measurements, while portal and systemic pressures were measured through polyethylene-50 catheters. Small resistance mesenteric arteries were connected to a pressure servo controller in a video-monitored perfusion system, and concentration-response curves to phenylephrine and acetylcholine were evaluated. EET levels were measured in tissue homogenates of rat liver, kidney, and aorta, using an enzyme-linked immunosorbent assay. Urinary Na(+) excretion function was also evaluated. In rats with cirrhosis, treatment with MS-PPOH significantly reduced portal blood flow and portal pressure compared to vehicle (13.6 ± 5.7 versus 25.3 ± 7.1 mL/min/100 g body weight, P < 0.05; 9.6 ± 1.1 versus 12.2 ± 2.3 mm Hg, P < 0.05; respectively) without effects on systemic pressure. An increased response to acetylcholine of mesenteric arteries from rats with cirrhosis (50% effect concentration -7.083 ± 0.197 versus -6.517 ± 0.73 in control rats, P < 0.05) was reversed after inhibition of EET production (-6.388 ± 0.263, P < 0.05). In liver, kidney, and aorta from animals with cirrhosis, treatment with MS-PPOH reversed the increase in EET levels. In both controls and rats with cirrhosis, MS-PPOH increased urinary Na(+) excretion. In rats with cirrhosis, in vivo inhibition of EET production normalizes the response of mesenteric arteries to vasodilators, with beneficial effects on portal hypertension. (Hepatology 2016;64:923-930).

Age Impaired endothelium-dependent vasodilation is improved by resveratrol in rat mesenteric arteries.

[Purpose]To determine whether resveratrol improves the adverse effects age on vascular function in mesenteric arteries (MAs), and diminishes the hyperactivity in adrenal gland with age.[Methods]Male F344 x Brown Norway rats were assigned to 6-month control (YC), 6-month resveratrol (YR), 24-month control (OC) and 24-month resveratrol (OR). Resveratrol (15 mg/kg) was provided to resveratrol groups in drinking water for 14 days.[Results]Concentration response curves to phenylephrine (PE, 10-9-10-5M), acetylcholine (Ach, 10-9-10-5M) and resveratrol (10-8-10-4M) were evaluated in pressurized isolated MAs. The Ach concentration-response curve was right shifted with maximal response diminished in OC compared with YC rats. These effects were reversed by resveratrol treatment. The resveratrol-mediated relaxant responses were unchanged with age or resveratrol suggesting an endothelium-independent mechanism. Resveratrol tended to increase endothelial nitric oxide synthase; caused no effect on copper-zinc superoxide dismutase; and normalized the age-related elevatation in DβH and NPY levels in adrenal medulla, two indicators of sympathetic activity[Conclusion]These data indicate that resveratrol reverses age-related dysfunction in endothelium-dependent vasodilation in MAs and partially reverses hyperactivity of adrenomedullary function with age. This treatment may have a therapeuticpotential in the treatment of cardiovascular diseases or hypertension in the elderly.

PP.41.01

Objective: The endothelial surface layer (ESL) consists of negatively charged glycosaminoglycans that can osmotically inactivate sodium. The ESL may therefore affect sodium homeostasis and blood pressure (BP). In mice, we investigated the consequences of combined heterozygous loss of EXT-1 and EXT-2 on BP after acute and chronic sodium loading. Both EXT genes are involved in polymerization of heparan sulfate, the predominant ESL glycosaminoglycan. Design and method: After a two-week normal sodium (0.3%) diet, 11-week old EXT1+/-EXT2+/- and wildtype mice were fed a normal or high sodium (8.0%) diet and water ad libitum for one week while monitoring BP with tail cuff measurements. We trained all mice one week before performing tail cuff measurements. At the end of the experiment, mice on normal sodium diet were subjected to intravenous saline loading (1.8%, 8 μl/g) while measuring BP invasively in the carotid artery. After exsanguination, we prepared mesenteric arteries for wire myography to study methacholine-induced (10 nmol/l-10 μmol/l) endothelium-dependent vasorelaxation after initial vasoconstriction using U46116. All statistical analyses were performed using non-parametric tests. Results: Baseline mean arterial pressure (MAP) was comparable between wildtype (89.2 (8.0) mmHg, n = 11) and EXT1+/-EXT2+/- mice (88.3 (8.4) mmHg, n = 16; p = 0.79). After a 7-day high sodium diet, MAP change in wildtype (+0.6 (8.4) mmHg, n = 6; p = 0.88) and EXT1+/-EXT2+/- mice (-5.2 (10.5) mmHg, n = 10, p = 0.15) was not significantly different (p = 0.18). Infusion of 220 (16) μl saline in 100 (20) seconds resulted in a significant 15.4 (10.8)% (p = 0.018) MAP decrease in wildtype mice while EXT1+/-EXT2+/- mice showed a non-significant 2.9 (13.4)% increase (p = 0.65).This significant variation in BP response (p = 0.030) was not accompanied by any change in heart rate in wildtype (-4.0 (8.0)%, p = 0.28) or EXT1+/-EXT2+/- mice (-0.7 (1.1)%, p = 0.26). Methacholine dose-response curves showed impaired vasodilation of mesenteric arteries of EXT1+/EXT2+/- mice (41 (10)%) when compared to wildtype mice (65 (22)%; p = 0.030). Conclusions: A defect in heparan sulfate polymerization in mice alters BP response to acute sodium loading, possibly due to impaired vasodilatory capacity. This indicates that heparan sulfates may be pivotal in vascular responses to acute sodium loading.

Impaired NO‐mediated vasodilatation in rat coronary arteries after asphyxial cardiac arrest

Cardiovascular dysfunction after cardiac arrest is a common finding. It is unknown whether altered endothelium-mediated vasoreactivity contributes to this dysfunction. We hypothesised that cardiac arrest and resuscitation results in impaired endothelial function. This was addressed by measurements of inflammatory and endothelial plasma markers and of endothelium-dependent vasodilatation in coronary and mesenteric arteries in rats after cardiac arrest and resuscitation. Male Sprague Dawley rats underwent either asphyxia-induced cardiac arrest for 5 min and subsequent resuscitation (n = 30) or a sham procedure (control animals, n = 39). Animals were euthanised after 30 min or 2 h. Blood was analysed for TNF-α, IL-1β, IL-6, IL-10, sE-selectin, sP-selectin, sVCAM-1, ICAM-1, VEGF-α and vWF. Arterial rings of the left anterior descending coronary artery and mesenteric resistance arteries were mounted in microvascular myographs, and concentration-response curves were constructed. The plasma levels of the endothelial markers, sP-selectin and vWF increased following cardiac arrest at both 30 min and 2 h. Acetylcholine-induced endothelium-dependent and mainly nitric oxide (NO)-mediated vasodilatation was impaired in the coronary arteries at 30 min, but not 2 h after resuscitation. Endothelium-derived hyperpolarisation (EDH)-type vasodilatation induced by NS309 and vasodilatation induced by the NO donor sodium nitroprusside was unaltered. In parallel with systemic hypotension, mesenteric arteries exhibited a larger response to NS309 2 h after resuscitation. The present results show marked endothelial alterations after cardiac arrest and resuscitation reflected by increased endothelial plasma markers, impaired NO-mediated coronary vasodilatation in the early post-resuscitation phase and enhanced EDH-type vasodilatation in mesenteric arteries later in the post-resuscitation phase.