Endothelium-dependent Responses Research Articles

Microparticle miRNAs as Biomarkers of Vascular Function and Inflammation Response to Aerobic Exercise in Obesity?

This study aimed to explore the role of nine microRNAs (miRNAs) in microparticles (MPs) on the efficacy of aerobic exercise in the regulation of inflammation and vascular function in obesity. Sedentary women with normal weight (n = 6, BMI < 25 kg/m2 ) and women with obesity (n = 9, BMI > 30 kg/m2 ) were recruited at F. Hached Hospital (Sousse, Tunisia) and enrolled in an 8-week aerobic program. Vascular function was assessed using laser Doppler flowmetry/iontophoresis, circulating MPs by flow cytometry, miRNAs by real-time polymerase chain reaction, and inflammation by ELISA, before and after exercise. Women with obesity presented with high prevalence of cardiovascular risk factors and a higher circulating MP level compared with healthy subjects. The MP miRNA profile was significantly different in the two groups. Exercise reduced BMI and inflammation in both groups and significantly improved endothelial-dependent response (acetylcholine cutaneous vascular conductance) for healthy subjects, with a trend for women with obesity. Circulating MP level was increased after exercise, and miRNA expression was differentially modulated in both populations. Pearson analysis revealed a correlation between MPs miR-124a and miR150 and adiponectin, TNFα, or IL-6 levels. The relation between MPs and miRNA profile, inflammation, vascular function, and exercise is of particular interest for defining "miRNA biomarker signature" in patients with cardiovascular disease who are potentially susceptible to respond to exercise.

45. Effects of syncytiotrophoblast extracellular vesicles (STBEVs) on endothelium-dependent vasodilation in uterine arteries from lectin-like oxidized ldl receptor-1 (LOX-1) overexpressing mice during pregnancy

Introduction Placental factors, such as syncytiotrophoblast extracellular vesicles (STBEVs), have been suggested to contribute to maternal endothelial vascular dysfunction in women with preeclampsia (PE). The lectin-like oxidized LDL receptor-1 (LOX-1) is a multi-ligand scavenger receptor; and in women with PE both STBEV levels and LOX-1 expression are elevated. We used heterozygous LOX-1 overexpressing (LOX-1OE) mice to further investigate the role of LOX-1 and STBEVs in vascular dysfunction during pregnancy. Hypothesis We hypothesized that STBEVs activate LOX-1 and contribute to vascular dysfunction in mouse uterine arteries of LOX-1OE mice, but not in WT controls. Methods Uterine arteries were obtained from late pregnant (gestational day 18; term = day 19) LOX-1OE mice (carrying a bovine LOX-1 transgene) and WT controls (−/−). Isolated vessels were incubated overnight in the absence (n = 8) or presence of STBEVs (200 μg/ml, n = 5–7) from control pregnant women. Using wire myography, endothelium-dependent vasodilation responses to methylcholine (MCh) were assessed with or without L-NAME (pan nitric oxide synthase inhibitor), superoxide dismutase (SOD) or oxidized LDL (oxLDL). Results Endothelium-dependent vasodilation to MCh was unchanged following STBEV-incubation in both mouse strains. In WT mice, nitric oxide did not contribute to vasodilation, while there was a nitric oxide contribution to vasodilation in STBEV-incubated arteries (p Discussion Interestingly, both LOX-1OE and STBEV-exposure increased nitric oxide contribution to relaxation; perhaps as a compensatory mechanism. OxLDL (a potential contributing factor in women with PE and dyslipidemia) impaired endothelial-dependent relaxation in the presence of increased LOX-1 expression during pregnancy. These data suggest that increased LOX-1 expression in women with PE could contribute to impaired vascular function.

Abstract P132: Everolimus, a Targeted Cancer Therapy, Improves Endothelium Dependent Relaxation in Spontaneously Hypertensive Rat Mesenteric Arteries

Cardiovascular disease is the leading cause of death in cancer survivors. The short-term and long-term cardiotoxic effects of targeted chemotherapeutics is not well known. Everolimus, an inhibitor to mammalian target of rapamycin (mTOR) complex 1, is a targeted therapy approved for the treatment of metastatic breast, colon, renal cell and pancreatic cancer. Clinical retrospective reviews suggest heart failure and hypertension as cardiotoxic effects of everolimus. Our lab previously showed that everolimus exposure on normotensive vessels increased sensitivity to adrenergic stimuli in a time and concentration dependent manner. We hypothesized that everolimus leads to enhanced contractility and impaired relaxation in hypertensive vessels. We studied the effects of everolimus on the contractility and relaxation of mesenteric resistance arteries (MRA) of male Wistar rats (12-15 weeks old, n=4) and spontaneously hypertensive rats (SHR) (15-18 weeks old, n=2) on a wire myograph. Two concentrations of everolimus were evaluated, 0.1μM and 0.1nM, during a one-hour incubation. Normotensive MRA exposed to everolimus (0.1μM) showed a decreased contractile response to phenylephrine (LogEC50 + SEM, Ctrl:-5.627 + 0.07 vs Drug: -5.390 + 0.10) but no difference when exposed to 0.1nM. SHR MRA showed no difference in PE-induced contraction at 0.1μM (LogEC50 + SEM, Ctrl:-5.638 + 0.08 vs Drug:-6.286 + 0.36) or 0.1nM (LogEC50 + SEM, Ctrl:-5.638 + 0.08 vs Drug:-5.332 + 0.16). No differences were observed in endothelium dependent relaxation response to acetylcholine (ACh) for either drug concentration in the normotensive vessels; however, SHR MRA exhibited enhanced sensitivity to ACh with everolimus exposure (LogEC50 + SEM, Ctrl: -6.851 + 0.15 vs Drug:-9.233 + 0.37 at 0.1nM; LogEC50 + SEM, Ctrl: -6.851 + 0.15 vs Drug:-8.244 + 0.34 at 0.1μM). Our data show that everolimus affects contractility in normotensive vessels, while affecting the dilatory response in SHR vessels with an improvement in endothelium-dependent relaxation. Everolimus may be a good therapeutic agent for hypertensive cancer patients. However, long term treatments with this targeted therapy need to be evaluated for vascular dysfunction induction, which could be associated with cardiotoxicity.

Apolipoprotein E favours the blunting by high-fat diet of prostacyclin receptor activation in the mouse aorta.

NO-mediated, endothelium-dependent relaxations of isolated arteries are blunted by ageing and high-fat diets, as well as by apolipoprotein E deletion. The present study was designed to test the hypothesis that apolipoprotein E deletion impairs endothelium-dependent responses to prostacyclin (IP) receptor activation. Five-week-old ApoE+/+ and ApoE-/- mice were fed normal chow or high-fat diet for 29weeks. The aortae were isolated for the measurements of isometric tension in Halpern-Mulvany myographs. Levels of proteins were assessed by Western blotting and immunofluorescence, and cyclic nucleotide levels by elisa. The IP receptor agonist, iloprost, induced endothelium-, NO-synthase- and IP-dependent relaxations in aortae of young ApoE+/+ mice. High-fat diet favoured activation of thromboxane receptors by iloprost, causing contraction. Apolipoprotein E was present in aortae of ApoE+/+ mice, especially in endothelium. Its presence was augmented by high-fat diet. Its deletion potentiated iloprost-induced relaxations in aortae of young mice and prevented the blunting of this response by high-fat diet. Levels of cAMP were higher, but those of cGMP were lower in the aorta of ApoE-/- than in ApoE+/+ mice of the same age. The levels of IP receptor protein were not different between ApoE+/+ and ApoE-/- mice. Iloprost induced an endothelium-dependent relaxation in the aorta of young healthy mice which involved both the cGMP and cAMP pathways. This response was blunted by prolonged exposure to a high-fat diet. Apolipoprotein E deletion potentiated relaxations to IP receptor activation, independently of age and diet.

Endothelium-dependent responses in the microcirculation observed invivo.

Endothelium-dependent responses were first demonstrated 40years ago in the aorta. Since then, extensive research has been conducted invitro using conductance vessels and materials derived from them. However, the microcirculation controls blood flow to vital organs and has been the focus of invivo studies of endothelium-dependent dilation beginning immediately after the first invitro report. Initial invivo studies employed a light/dye technique for selectively damaging the endothelium to unequivocally prove, invivo, the existence of endothelium-dependent dilation and in the microvasculature. Endothelium-dependent constriction was similarly proven. Endothelium-dependent agonists include acetylcholine (ACh), bradykinin, arachidonic acid, calcium ionophore A-23187, calcitonin gene-related peptide (CGRP), serotonin, histamine and endothelin-1. Normal and disease states have been studied. Endothelial nitric oxide synthase, cyclooxygenase and cytochrome P450 have been shown to generate the mediators of the responses. Some of the key enzyme systems generate reactive oxygen species (ROS) like superoxide which may prevent EDR. However, one ROS, namely H2 O2 , is one of a number of hyperpolarizing factors that cause dilation initiated by endothelium. Depending upon microvascular bed, a single agonist may use different pathways to elicit an endothelium-dependent response. Interpretation of studies using inhibitors of eNOS is complicated by the fact that these inhibitors may also inhibit ATP-sensitive potassium channels. Other invivo observations of brain arterioles failed to establish nitric oxide as the mediator of responses elicited by CGRP or by ACh and suggest that a nitrosothiol may be a better fit for the latter.

Small molecule activators of Ca2+-activated K+ channels can enhance endothelium-dependent responses in isolated resistance arteries

Small molecule activators of Ca2+-activated K+ channels can enhance endothelium-dependent responses in isolated resistance arteries

Neuroprotection Mediated by Upregulation of Endothelial Nitric Oxide Synthase in Rho-Associated, Coiled-Coil-Containing Kinase 2 Deficient Mice.

Rho-associated kinases (ROCK1 and ROCK2) are important regulators of the actin cytoskeleton and endothelial nitric oxide synthase (eNOS). Because the phosphorylation of eukaryotic elongation factor-1A1 (eEF1A1) by ROCK2 is critical for eNOS expression, we hypothesized that this molecular pathway may play a critical role in neuroprotection following focal cerebral ischemia.Methods and Results:Adult male wild-type (WT) and mutant ROCK2 and eNOS-/-mice were subjected to middle cerebral artery occlusion (MCAO), and cerebral infarct size, neurological deficit and absolute cerebral blood flow were measured. In addition, aortic endothelium-dependent response to acetylcholine, NG-nitro-L-arginine methyl ester (L-NAME) and sodium nitroprusside were assessed ex vivo. Endothelial cells from mouse brain or heart were used to measure eNOS and eEF1A activity, as well as NO production and eNOS mRNA half-life. In global hemizygous ROCK2+/-and endothelial-specific EC-ROCK2-/-mice, eNOS mRNA stability and eNOS expression were increased, which correlated with enhanced endothelium-dependent relaxation and neuroprotection following focal cerebral ischemia. Indeed, when ROCK2+/-mice were place on an eNOS-/-background, the neuroprotective effects observed in ROCK2+/-mice were abolished. These findings indicate that the phosphorylation of eEF1A1 by ROCK2 is physiologically important for eNOS expression and NO-mediated neuroprotection, and suggest that targeting endothelial ROCK2 and eEF1A may have therapeutic benefits in ischemic stroke and cardiovascular disease.

The Effect on Health of Some Cardiovascular Risk Factors

Arterial endothelium produces a large ramge of active factors which are indispensable for modulation of vasomotor tone and maintenance of vascular wall integrity. From these factors, nitric oxide (NO), wich is released by the endothelial cells as a response to acetylcholine or adenosine action on specific receptors, plays an important role.NO is the result of oxidation process of L-arginine into L-citrulline, under the action of endothelial nitric oxide synthase (NOSe), wich is activated by intracelluar Ca2+ - calmodulin complex . Our study, performed in isolated organ bath, analyzed vascular reactivity of 12 guinea pigs� thoracic aorta rings. After phenylephrine -PHE 10-5 mol/L precontraction, the dose-effect curves for acetylcoline � ACH, adenosine 5� phosphate - 5�ADP and sodium nitroprusside � SNP were determined, before and after incubation of preparation, for 1 hour, with 5% hydrosoluble cigarettes smoke extract (CSE). Statistic analysis, performed with the use of t pair test and ANOVA parametric test, showed that incubation of vascular preparation with 5% CSE has increased the contractile response to PHE 10-5 mol/L (p[0.05), has reduced the endothelium-dependent relaxing response to ATP 10-5 mol/L (p[0.001) and 5�ADP 10-5 molo/L (p[0.001), but has not significantly modified the endothelium-independent relaxing response to SNP 10-5 mol/L (p=0.05). As a conclusion, vascular rings incubation with 5% CSE induced a decrease of endothelium NO synthesis under the action of AXH and 5�ADP, but did not change the smooth muscle fiber respomse in the presence of NO released by SNP.

Abstract P525: Mtor Inhibitor Everolimus Decreases Aortic Sensitivity To Phenylephrine In Normotensive Rats

While targeted chemotherapeutics have improved cancer clinical outcomes, many of these agents have the drawback of cardiovascular complications. It has been established that the mTOR complexes have a pivotal role in many types of cancer, promoting cell growth, proliferation, and survival. Inhibitors of this pathway such as first generation Everolimus, and second generation PP242, have inhibited cancer cell proliferation and cancer progression. When compared with other target chemotherapeutics such as VEGF and tyrosine kinase inhibitors, first generation mTOR inhibitors have demonstrated decreased incidence of cardiovascular complications, however the specific vascular effects of mTOR inhibitors have not been investigated. Therefore, Everolimus, an inhibitor of mTORC1, and PP242, an inhibitor of mTORC1 and mTORC2, were evaluated for their effects on vascular reactivity of rat aorta. Previous studies have demonstrated that mTORC2 displayed cardioprotective effects. Therefore, we hypothesize that Everolimus, which does not inhibit mTORC2, will have a decreased contractile response to Phenylephrine (PE) and increased Acetylcholine (Ach)-induced relaxation. On the contrary, since PP242 inhibits mTORC2, we hypothesize that PP242 will increase contractile response to PE, and will decrease ACh-induced relaxation. Concentration response curves to PE (1nM - 30μM), and Ach (1nM - 30μM) were conducted in isolated aortic rings from male normotensive Wistar rats (12-15 weeks old) on a pin myograph ex vivo after 1 hour of incubation with each drug. Aortic rings treated with 31 μM of Everolimus showed decreased sensitivity to PE (LogEC 50 = -7.322 ±0.078 vs. treated: -6.578 ± 0.1116 p&lt;0.05). No differences were observed in the contractile responses to PE for Everolimus (0.1 nM) or PP242 (245 nM) (p&gt;0.05). Similarly, no differences were observed in the endothelium-dependent relaxation response to Ach for all three treatments. These results suggest that Everolimus may be a good therapeutic choice for cancer patients also suffering from cardiovascular dysfunction.

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

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

Long-term endothelial dysfunction after trans-radial catheterization: A meta-analytic approach.

Following cardiac catheterization using radial artery (RA) access, persistent endothelial dysfunction may limit the use of RA as a conduit during coronary artery bypass graft (CABG) surgery. We reviewed published literature to investigate the effects of transradial coronary catheterization on RA endothelial function. We searched PubMed from inception to April 2017 for published studies assessing RA endothelial function late (≥1 month) after coronary catheterization. A total of 12 eligible published studies (n = 490 patients) were included in the final quantitative synthesis. Statistical heterogeneity among studies was assessed by the I2 . A random effects model was used to calculate the pooled estimate for standardized mean difference (SMD). Meta-regression analysis was used to explore predictors of change in RA endothelial function following catheterization. In all studies, a significant reduction in endothelium dependent response was observed post-catheterization (SMD = -0.53, 95% confidence interval [CI]: -0.93 to -0.13, P = 0.01) and a marginal, non-significant, reduction in endothelium independent response (SMD = -0.38, 95%CI: -0.77, 0.01, P < 0.059). In controlled studies, using the contralateral RA as a control, a significant impairment in endothelial function was confirmed (SMD = -6.26, 95%CI: -9.71 to -2.81, P < 0.0001), while the change in endothelium-independent response was not significant (SMD = -4.46, 95%CI: -13.3 to 4.37, P = 0.32). In meta-regression analysis male gender (z = 2.36, P = 0.018) and increasing time following catheterization (z = 2.62, P = 0.009) were associated with less RA endothelial dysfunction. Transradial catheterization impairs endothelium dependent vasodilatory properties of the cannulated RA, which do not recover even several months post-catheterization. Non-recovery of vasomotor function of cannulated RAs may limit their use as arterial grafts during CABG surgery.

O38 The omega-3 EPA:DHA 6:1 formulation improves ageing-related blunted endothelium-dependent relaxations and increased contractile responses in the mesenteric artery: Role of oxidative stress and cyclooxygenases

Introduction Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to cause endothelium-dependent nitric oxide (NO)-mediated relaxations of isolated blood vessels, with EPA:DHA 6:1 being a superior formulation. The aim of the present study was to determine whether EPA:DHA 6:1 improves ageing-related endothelial dysfunction, and, if so, to determine the underlying mechanism. Methods Male Wistar rats (20 months-old) received daily 500 mg/kg of either EPA:DHA 6:1 (omega 3), corn oil (control), or water for 2 weeks. Young male Wistar rats (12 weeks-old) were used as a control. Results In the main mesenteric artery, ageing was associated with an endothelial dysfunction characterized by a blunted NO-mediated component, an abolished endothelium-dependent hyperpolarization (EDH)-mediated component, and also by the induction of endothelium-dependent contractile responses (EDCF, in the presence of NO and EDH inhibitors) sensitive to indomethacin (a cyclooxygenase inhibitor) in response to acetylcholine. Endothelial dysfunction was associated with an increased level of vascular oxidative stress and expression of COX-2, but not of COX-1, in the mesenteric artery. The EPA:DHA 6:1 treatment improved both the NO- and EDH-mediated relaxations, and reduced EDCF, vascular oxidative stress and expression of COX-2 in the old mesenteric artery. Conclusion The present findings indicate that intake of EPA:DHA 6:1 prevented the development of the ageing-related endothelial dysfunction in rats. The beneficial effect involves an improvement of both the NO- and the EDH-mediated relaxations as well as a reduction of endothelium-dependent contractile response most likely by preventing vascular oxidative stress.

Genetic Deletion of NADPH Oxidase 1 Rescues Microvascular Function in Mice With Metabolic Disease.

Early vascular changes in metabolic disease that precipitate the development of cardiovascular complications are largely driven by reactive oxygen species accumulation, yet the extent to which excess reactive oxygen species derive from specific NADPH oxidase isoforms remains ill defined. Identify the role of Nox1 in the development of microvascular dysfunction in metabolic disease. Four genotypes were generated by breeding Nox1 knockout mice with db/db mice: lean (HdbWnox1), lean Nox1 knockout (HdbKnox1), obese (KdbWnox1), and obese KK (KdbKnox1). The degree of adiposity, insulin resistance, and dyslipidemia in KW mice was not influenced by Nox1 deletion as determined by nuclear magnetic resonance spectroscopy, glucose tolerance tests, and plasma analyses. Endothelium-dependent responses to acetylcholine in pressurized mesenteric arteries were reduced in KW versus HW (P<0.01), whereas deletion of Nox1 in KW mice normalized dilation. Vasodilator responses after inhibition of NO synthase blunted acetylcholine responses in KK and lean controls, but had no impact in KW, attributing recovered dilatory capacity in KK to normalization of NO. Acetylcholine responses were improved (P<0.05) with Tempol, and histochemistry revealed oxidative stress in KW animals, whereas Tempol had no impact and reactive oxygen species staining was negligible in KK. Blunted dilatory responses to an NO donor and loss of myogenic tone in KW animals were also rescued with Nox1 deletion. Nox1 deletion reduces oxidant load and restores microvascular health in db/db mice without influencing the degree of metabolic dysfunction. Therefore, targeted Nox1 inhibition may be effective in the prevention of vascular complications.

2K1C-activated Angiotensin II (Ang II) exacerbates vascular damage in a rat model of arthritis through the ATR/ERK1/2 signaling pathway.

To explore the role and mechanism of the two-kidney one-clip (2K1C)-activated Angiotensin II (Ang II) in the development of vascular damage in adjuvant-induced arthritis (AA) rats. 2K1C rats were established in normal and AA rats for 35days. Hypertension, endothelial dysfunction, and vascular hypertrophy induced by 2K1C-activated Ang II in systemic inflammation rats were evaluated. The levels of Ang II and TNF-α in serum were observed by ELISA kits. Expressions of Ang II/ATR/ERK1/2 signaling pathway molecules in the aorta were tested by immunohistochemistry or western blot. The migration and capillary tube formation abilities of human umbilical vein endothelial cells (HUVECs) were tested by migration chamber and capillary tube formation assays. The level of Ang II in serum was significantly increased in 2K1C rats. Compared with AA rats, the high level of Ang II activated by 2K1C reduced the endothelium-dependent vasodilator responses to acetylcholine (ACh) in the thoracic aorta and exacerbated endothelial dysfunction and vascular hypertrophy. Expressions of ATR, GRK2, p-ERK1/2, and p-NF-κB were significantly increased in the aorta of AA combined with 2K1C rats. The migration and capillary tube formation abilities of HUVECs were significantly enhanced by Ang II and TNF-α co-stimulations in vitro through the ATR/ERK1/2 signaling pathway compared to those stimulated with TNF-α. 2K1C-activated Ang II is involved in aggravated vascular injury and endothelial dysfunction through the ATR/ERK1/2 signaling pathway in AA rats.

CYP epoxygenase-derived H2O2 is involved in the endothelium-derived hyperpolarization (EDH) and relaxation of intrarenal arteries

Reactive oxygen species (ROS) like hydrogen peroxide (H2O2) are involved in the in endothelium-derived hyperpolarization (EDH)-type relaxant responses of coronary and mesenteric arterioles. The role of ROS in kidney vascular function has mainly been investigated in the context of harmful ROS generation associated to kidney disease. The present study was sought to investigate whether H2O2 is involved in the endothelium–dependent relaxations of intrarenal arteries as well the possible endothelial sources of ROS generation involved in these responses. Under conditions of cyclooxygenase (COX) and nitric oxide (NO) synthase inhibition, acetylcholine (ACh) induced relaxations and stimulated H2O2 release that were reduced by catalase and by the glutathione peroxidase (GPx) mimetic ebselen in rat renal interlobar arteries, suggesting the involvement of H2O2 in the endothelium-dependent responses. ACh relaxations were also blunted by the CYP2C inhibitor sulfaphenazole and by the NADPH oxidase inhibitor apocynin. Acetylcholine stimulated both superoxide (O2•−) and H2O2 production that were reduced by sulfaphenazole and apocynin. Expression of the antioxidant enzyme CuZnSOD and of the H2O2 reducing enzymes catalase and GPx-1 was found in both intrarenal arteries and renal cortex. On the other hand, exogenous H2O2 relaxed renal arteries by decreasing vascular smooth muscle (VSM) intracellular calcium concentration [Ca2+]i and markedly enhanced endothelial KCa currents in freshly isolated renal endothelial cells. CYP2C11 and CYP2C23 epoxygenases were highly expressed in interlobar renal arteries and renal cortex, respectively, and were co-localized with eNOS in renal endothelial cells. These results demonstrate that H2O2 is involved in the EDH-type relaxant responses of renal arteries and that CYP 2C epoxygenases are physiologically relevant endothelial sources of vasodilator H2O2 in the kidney.

Heterozygous eNOS Deficient Mice as a Model to Examine the Effects of eNOS Haploinsufficiency on the Cerebral Circulation

Nitric oxide derived from endothelial nitric oxide synthase (eNOS) has been shown to be a major mediator of endothelium-dependent responses in cerebral blood vessels. Loss of a single eNOS gene is not associated with any apparent negative consequences on endothelial function in most blood vessels. In contrast, we have recently demonstrated that heterozygous eNOS gene deficiency in combination with a high fat diet is associated with marked impairment of endothelial function. These findings provide an important example of eNOS haploinsufficiency and one that directly impacts the cerebral vasculature. A major mechanism associated with the impairment of endothelial function with eNOS deficiency and a high fat diet appears to be related to increases in plasma IL-6 that serves to further reduce the bioavailability of NO either directly or indirectly via reductions in eNOS expression or activity and via increases in vascular superoxide. Taken together, these findings provide important insights into genetic and molecular mechanisms that promote endothelial dysfunction in response to a high fat diet in cerebral blood vessels with inherent reductions in eNOS gene expression, such as those due to eNOS gene polymorphisms. These findings also highlight the importance of eNOS+/- mice to study the effects of eNOS haploinsufficiency on cerebral blood vessels.

GPR55 agonist lysophosphatidylinositol and lysophosphatidylcholine inhibit endothelial cell hyperpolarization via GPR-independent suppression of Na+-Ca2 + exchanger and endoplasmic reticulum Ca2 + refilling

Lysophosphatidylinositol (LPI) and lysophosphatidylcholine (LPC) are lipid signaling molecules that induce endothelium-dependent vasodilation. In addition, LPC suppresses acetylcholine (Ach)-induced responses. We aimed to determine the influence of LPC and LPI on hyperpolarizing responses in vitro and in situ endothelial cells (EC) and identify the underlying mechanisms. Using patch-clamp method, we show that LPI and LPC inhibit EC hyperpolarization to histamine and suppress Na+/Ca2+ exchanged (NCX) currents in a concentration-dependent manner. The inhibition is non-mode-specific and unaffected by intracellular GDPβS infusion and tempol, a superoxide dismutase mimetic. In excised mouse aorta, LPI strongly inhibits the sustained and the peak endothelial hyperpolarization induced by Ach, but not by SKA-31, an opener of Ca2+-dependent K+ channels of intermediate and small conductance. The hyperpolarizing responses to consecutive histamine applications are strongly reduced by NCX inhibition. In a Ca2+-re-addition protocol, bepridil, a NCX inhibitor, and KB-R7943, a blocker of reversed NCX, inhibit the hyperpolarizing responses to Ca2+-re-addition following Ca2+ stores depletion. These finding indicate that LPC and LPI inhibit endothelial hyperpolarization to Ach and histamine independently of G-protein coupled receptors and superoxide anions. Reversed NCX is critical for ER Ca2+ refilling in EC. The inhibition of NCX by LPI and LPC underlies diminished endothelium-dependent responses and endothelial dysfunction accompanied by increased levels of these lipids in the blood.

Treatment with a New Nitric Oxide Donor, a Hybrid Derived from Thalidomide and Hydroxycarbamide 3-(1,3-dioxoisoindolin-2-yl)Benzyl Nitrate, Reverses Priapism in the Sickle Cell Mouse and the Nitric Oxide-Deficient Mouse

▪Introduction: Sickle cell disease (SCD) men display priapism. Previous studies showed that endothelial nitric oxide synthase (eNOS) gene-deficient mice and combined eNOS and neuronal nitric oxide synthase double gene-deficient mice (dNOS-/-) display a priapism phenotype associated with phosphodiesterase-5 (PDE5) downregulation. The Berkeley SCD transgenic mice display features of priapism, and, as in SCD human, this appears to be due to decreased nitric oxide (NO) signaling, which leads to lower PDE5 in the penis. Thus, when an erectile stimulus occurs in vivo, cGMP accumulates into the cavernosal smooth muscle cells, rendering the penile vasculature uncontrollably dilated, penile erection persists (i.e. priapism) as cGMP is not degraded as a consequence of PDE5 downregulation. Moreover, oxidative/nitrosative stress have been associated with priapism in SCD mice. In this study, since decreased NO bioavailability leads to lower PDE5 in penis, we aimed to evaluate the effects of new NO donor, a hybrid derived from thalidomide and hydroxycarbamide, 3-(1,3-dioxoisoindolin-2-yl)benzyl nitrate (4C), on functional and molecular alterations of erectile function in SCD and dNOS-/- mice. We have focused on the dysregulated NO-cGMP-PDE5 pathway and oxidative/nitrosative stress in erectile tissue of SCD and dNOS-/-mice.Methods: Wild type (WT, C57BL/6), Berkeley SCD transgenic and dNOS-/-male mice (3-5 mo old) were treated with compound 4C (100 µmol/kg/day) or its vehicle (20% Cremophor) daily for 3 weeks via intraperitoneal injection. The intracavernous pressure (ICP) was assessed following electrical stimulation of cavernous nerve (CN) in anaesthetized mice. ICP were normalized per mean arterial pressure (MAP). In separate protocols, corpus cavernosum (CC) strips were mounted in isolated organ baths, and the relaxing responses to acetylcholine (ACh; endothelium-dependent response) and sodium nitroprusside (SNP; endothelium-independent response), as well as electrical-field stimulation (EFS; nitrergic relaxation) were obtained in CC strips precontracted with the α1-adrenergic receptor agonist phenylephrine (10 µM).Results: The CN stimulation (4 V) caused increases of ICP/MAP ratio in WT, SCD and dNOS-/- mice. However, half detumescence time (sec) and poststimulated ICP/MAP ratio values after 9 min were (P<0.05) higher in SCD compared to the WT group, indicating that SCD mice display priapism. In dNOS-/- group, maximal ICP/MAP ratio values were higher (P<0.05) compared to WT mice, indicating that dNOS-/- mice also display priapism. Compound 4C treatment reversed the priapism in SCD and dNOS-/- mice. The cumulative addition of ACh (0.001-10 µM) produced concentration-dependent CC relaxations in WT and SCD groups, but the ACh potency (pEC50) value was higher (P<0.05) in CC of SCD compared to WT mice, which was reversed by 4C. Likewise, the nitrergic relaxation induced by EFS was also higher (P<0.05) in SCD mice compared to control mice (2 Hz: 12 ± 3 and 37 ± 9 %, respectively; n=5), which was reversed by 4C treatment. Similarly, SNP (0.01- 10 µM) produced concentration-dependent CC relaxations in WT, SCD and dNOS-/- groups, but, again, the maximal relaxations elicited by this agent were higher (P<0.05) in SCD (93 ± 7%) and dNOS-/- (105 ± 1%) groups compared to WT mice (72 ± 4%), which were fully restored to WT values with compound 4C treatment (n=5-9). PDE5 protein expressions were reduced (P<0.05) by approximately 48% and 35% in penile tissues from SCD and dNOS-/- compared to the WT group, respectively. Compound 4C treatment restored (P<0.05) the protein levels of PDE5 in the penises from SCD and dNOS-/- group. The protein expressions for gp91phox and 3-NT were significantly higher (P<0.05) in erectile tissues from SCD and dNOS-/- compared to the WT group, which were reduced (P<0.05) by 4C treatment (n=5-9). In WT mice, PDE5, gp91phox and 3-NT protein expressions were not affected by 4C, as well as CC relaxations induced by ACh, SNP and EFS.Conclusion: Compound 4C treatment reversed the priapism, as well as enhanced NO-mediated CC relaxations and normalized PDE5 expressions in the penises from SCD and dNOS-/- mice. Moreover, increased gp91phox and 3-nitrotyrosine protein expressions were also decreased by 4C in SCD and dNOS-/-mice. Compound 4C may constitute an additional strategy to prevent priapism in SCD, since SCD-related priapism therapies are few.Financial Support: FAPESP DisclosuresNo relevant conflicts of interest to declare.

Abstract 068: Endothelin-1 Exaggerates Type-1 Diabetes-accelerated Atherosclerosis Through NADPH Oxidases 1 and 4

Objective: NADPH oxidase (NOX) 1 but not NOX4-dependent oxidative stress plays a role in diabetic vascular disease, including atherosclerosis. Endothelin (ET)-1 has been implicated in diabetes-induced vascular complications. We showed that crossing mice overexpressing ET-1 selectively in endothelium (eET-1) with apolipoprotein E knockout ( Apoe -/- ) mice exaggerated high-fat diet-induced atherosclerosis in part by increasing oxidative stress. We hypothesized that ET-1 overexpression in the endothelium would exaggerate diabetes-accelerated atherosclerosis through a mechanism involving NOX1 but not NOX4. Methods: Six-week-old male Apoe -/- mice, eET-1/ Apoe -/- and eET-1/ Apoe -/- mice deficient in Nox1 (eET-1/ Apoe -/- / Nox1 y/- ) or Nox4 (eET-1/ Apoe -/- / Nox4 -/- ) were rendered diabetic with 55 mg/kg/day streptozotocin (STZ) IP injections for 5 days and studied 14 weeks later. Endothelial function and vascular remodeling were assessed in mesenteric arteries (MA) using pressurized myography. Aortic atherosclerotic lesions were quantified using Oil Red O staining. Plasma cholesterol, HDL and triglycerides were measured. Results: Diabetic Apoe -/- mice presented an impaired endothelium-dependent vasodilatory response to acetylcholine, which was not observed in diabetic eET-1/ Apoe -/- , eET-1/ Apoe -/- / Nox1 y/- or eET-1/ Apoe -/- / Nox4 -/- mice (E max : 20±6 vs 99±1, 98±1 and 100±0%). ET-1 overexpression caused a 1.8-fold increase in MA media/lumen of diabetic Apoe -/- mice (5.3±0.3 vs 2.9±0.2%), which was further increased 1.2-fold by Nox4 (6.4±0.3%) but not Nox1 knockout (5.5±0.3%). ET-1 overexpression exaggerated &gt;2-fold the atherosclerotic lesion area in the aortic sinus in diabetic Apoe -/- mice (plaque area [x10 5 μm 2 ]: 5.3±0.5 vs 2.9±0.6), which was reduced ~40% by Nox1 and Nox4 knockout (plaque area [x10 5 μm 2 ]: 3.3±0.6 and 3.6±0.6). Plasma triglycerides were unaffected by ET-1 overexpression but reduced by Nox1 (2.2±0.4 vs 3.4±0.3 mmol/L) and Nox4 knockout (1.8±0.4 mmol/L). Plasma HDL and cholesterol were similar between groups. Conclusions: Increased levels of ET-1 exaggerate diabetes-accelerated atherosclerosis through NOX1 and NOX4, despite paradoxically improving endothelium-dependent relaxation in small arteries.

Abstract P225: Nox1 or Nox4 Deletion Prevents Type-1 Diabetes-induced Endothelial Dysfunction

Objective: The prognosis of type-1 diabetes is in part related to the increased risk of vascular complications such as atherosclerosis. Overproduction of reactive oxygen species by NADPH oxidase (NOX) is believed to play an important role in diabetes-related vascular injury. NOX1 may play a role in the macrovascular disease, whereas NOX4 may have protective actions. Nevertheless, their role in diabetic vascular injury is less well understood. We hypothesized that deletion of Nox1 would prevent diabetes-induced endothelial dysfunction and vascular remodeling of small arteries whereas Nox4 would exaggerate vascular injury in atherosclerosis-prone apolipoprotein knockout ( Apoe -/- ) mice. Methods: Diabetes was induced by streptozotocin IP injections (STZ, 55 mg/kg/day) for 5 days in 6-week-old male Apoe -/- mice, Apoe -/- mice deficient in Nox1 ( Apoe -/- / Nox1 y/- ) and Nox4 ( Apoe -/- / Nox4 -/- ). Mice were studied 14 weeks later. Endothelial function and vascular remodeling were assessed in mesenteric arteries (MA) using pressurized myography. Results: Apoe -/- mice presented a maximal endothelium-dependent vasodilatory response (E max ) to acetylcholine of 48±8%, which was further decreased by diabetes to 20±6%. In contrast, endothelium-dependent relaxations to acetylcholine were 1.5-fold higher in diabetic Apoe -/- / Nox1 y/- and Apoe -/- / Nox4 -/- mice compared to non-diabetic Apoe -/- mice (E max : 72±9 and 70±7 vs 48±8%). Diabetes decreased MA stiffness in Apoe -/- mice, as indicated by a rightward displacement of the stress-strain curves (strain at 140 mm Hg: 1.02±0.04 vs 0.75±0.04), which was blunted by Nox1 or Nox4 knockout (strain at 140 mm Hg: 0.77±0.02 and 0.81±0.02). MA media/lumen was unaltered by diabetes. Knockout of Nox4 but not Nox1 increased MA media/lumen 1.4-fold in diabetic Apoe -/- mice (4.1±0.4 and 3.5±0.2 vs 2.9±0.2%). Conclusions: These results suggest that NOX1 and NOX4 play a pathophysiological role in diabetes-induced endothelial dysfunction and contribute to potentially maladaptive changes in vascular stiffness. NOX4 seems to have dual actions on the vasculature, as it is also protective against vascular remodeling of small arteries in type 1 diabetes.