Endothelial Nitric Oxide Synthase Function Research Articles

Passivation protein-adhesion platform promoting stent reendothelialization using two-electron-assisted oxidation of polyphenols

Superhydrophilic surfaces play an important role in nature. Inspired by this, scientists have designed various superhydrophilic materials that are widely used in the field of biomaterials, such as PEG molecular brushes and zwitterionic materials. However, superhydrophilic coatings with only anti-fouling properties do not satisfy the requirements for rapid reendothelialization of cardiovascular stent surfaces. Herein, a novel polyphenol superhydrophilic surface with passivated protein-adsorption properties was developed using two-electron oxidation of dopamine and polyphenols. This coating has a multiscale effects: 1) macroscopically: anti-fouling properties of superhydrophilic; 2) microscopically: protein adhesion properties of active groups (quinone-, amino-, hydroxyphenyl groups and aromatic ring). Polyphenols not only enhance the ability of coating to passivate protein-adsorption, but also make the coating have polyphenol-related biological functions. Therefore, the polyphenol and passivated protein-adsorption platform together maintain the stability of the scaffold microenvironment. This, in turn, provides favorable conditions for the growth of endothelial cells on the scaffold surface. In vivo implantation of the coated stents into the abdominal aorta resulted in uniform and dense endothelial cells covering the surface of the neointima. Moreover, new endothelial cells secreted large amounts of functional endothelial nitric oxide synthase like healthy endothelial cells. These results indicate that the polyphenol superhydrophilic coating potentially resists intra-stent restenosis and promotes surface reendothelialization. Hence, polyphenol superhydrophilic coatings with passivated protein-adsorption properties constructed by two-electron-assisted oxidation are a highly effective and versatile surface-modification strategy for implantable cardiovascular devices.

Effect of Motexafin lutetium-mediated ultrasound-guided red laser photodynamic therapy on endothelial dysfunction using a new automatic ultrasound image processing software

Abstract Background As is well recognized, the majority of atherosclerosis start with an inflammatory process, resulting in endothelial dysfunction. Purpose In this study, we developed an experimental photosensitizer Motexafin lutetium (Lu-Tex)-mediated photodynamic therapy system, and investigated its effectiveness on endothelial dysfunction improvement in an animal model of balloon dilation injury, using a new automatic ultrasound image processing software. Methods Briefly, Golden Syrian hamsters' femoral artery underwent balloon dilatation injury. After four weeks, femoral arteries of the treatment group at the lesion region, treated using Motexafin lutetium - mediated fiber optic catheter-based red low level laser (742 nm, 150 J/cm2) photodynamic therapy. In order to evaluate endothelial-dependent relaxation, acetylcholine-mediated dilation (AMD) was measured during the infusion of acetylcholine at a rate of 0.5 μg/kg/min and endothelial independent relaxation was evaluated by measuring nitroglycerin mediated dilation (NMD) during the infusion of nitroglycerin at a rate of 5μg/kg/min. For off-line analysis, a computerized analysis method for evaluating instantaneous changes in the wall of the hamster femoral artery was used. For automatic measurement of the mean wall thickness and the lumen diameter from longitudinal B-mode ultrasound images, two algorithms, i.e., maximum gradient and dynamic programming were composed and implemented. Reference points and cost function were based on dynamic programming and maximum gradient, respectively. Results Results from computerized B-mode ultrasound image processing software showed significant differences in AMD between the treated and the non-treated hamsters (p<0.05), whereas there were no significant differences in NMD between the treatment and normal groups (p>0.05). Conclusion Cytotoxic and anti-inflammatory effect of Motexafin lutetium, induced by red laser photodynamic therapy accompanied by photobiomodulation effect of red laser, can cause to reduce the immune cells in intimal layer and improve the endothelial dysfunction via increasing the endothelial nitric oxide synthase function. Furthermore, it is concluded that the new automatic method enables accurate and repeated evaluation of the endothelial dysfunction using photodynamic therapy in this model. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Mehrad Research Lab

Abstract 12114: Endothelial Extracellular Signal-Regulated Kinase 2 Contributed to the Development of Pulmonary Arterial Hypertension in Mice Through the Impairment of Endothelial Nitric Oxide Synthase Function

Introduction: Pulmonary arterial hypertension (PAH) is a progressive disease, characterized by abnormal muscularization and endothelial dysfunction in pulmonary artery (PA). Previous study showed that endothelial extracellular signal-regulated kinase 2 (eERK2) released vasoconstrictive factors and decreased NO bioactivity resulting in obese-induced hypertension and endothelial dysfunction. However, the role of eERK2 in the pathogenesis of PAH remains to be elucidated. Hypothesis: We hypothesized eERK2 impaired eNOS function and progressed the pathogenesis of PAH. Methods and Results: We generated eERK2 knock out mice (EE2KO) crossing Tie2-Cre mice and ERK2 flox mice and induced PAH by exposing mice to chronic hypoxia (10%) for 4 weeks. The littermate ERK2 flox mice without Cre expression were used as Control (Ctr). Immuno-histochemical staining showed the deficiency of eERK2 expression in the PA of EE2KO. We assessed the medial thickness of pulmonary arteries (MTPA) and measured right ventricular systolic pressure (RVSP). Although Ctr exhibited the progression of PA muscularization with an increase in MTPA, EE2KO revealed the significantly attenuated MTPA under hypoxic condition. Moreover, we found the lower RVSPs in EE2KO than those in Ctr under hypoxia in spite of similar RVSPs in normoxia. In RT-PCR, lung endothelin1 (ET1) gene expression was significantly lower in EE2KO than Ctr under hypoxia. Interestingly, hypoxia increased eNOS protein expression both in Ctr and EE2KO with Western blot analysis. The phosphorylation of AMPKα and eNOS were up-regulated in the lungs of EE2KO relative to Ctr under hypoxia. In addition, eNOS dimer/monomer ratios with non redusing SDS-PAGE was significantly increased in EE2KO compared to the Ctr under hypoxia (1.29±0.10 vs. 0.87±0.02, P=0.02, dimer / monomer ratios), suggesting improved eNOS uncoupling. Conclusions: These findings suggest that hypoxia increased eNOS expression and function, and that eERK2 impaired eNOS function by decreasing the phosphorylation of AMPKα and eNOS, and inducing eNOS uncoupling. In addition, ET1 synthesis through eERK2 might contribute to these pathogenesis of PAH with hypoxia.

CD38 Genetic Knockout Confers Marked Protection Against In Vivo Myocardial Ischemia/Reperfusion Injury

Background CD38 is a multifunctional membrane protein with the major NAD(P)ase activity in human tissues. It is highly expressed in endothelial cells and some inflammatory cells. Using ex vivo perfused hearts and cell models, we have shown that ischemia/reperfusion (I/R) in the heart leads to CD38 activation with subsequent NAD(P)H depletion and impaired endothelial nitric oxide synthase function, triggering diminished coronary flow and decreased contractile function. Here we test if genetic knockout (KO) of CD38 protects against in vivo myocardial I/R in mice. Methods Male CD38 KO (n=7) and wild type (WT) mice (n=11) in C57BL/6 background were subjected to I/R surgery at the age of 12–16 weeks. The mice were anesthetized, endotracheal intubated, and ventilated with a rodent ventilator. A left lateral thoracotomy was performed, and the left anterior descending coronary artery was visualized and ligated with a slip knot. Then, mice were subjected to 30 minutes of ischemia followed by 24 h of reperfusion. The left ventricular area at risk (AAR) and infarct areas were visualized by Evans blue and TTC staining, respectively. The hearts were sliced and digitally photographed. Left ventricular AAR and infarct area were contoured using Image J software. (P<0.05) was considered significant in statistical testing. Results In CD38 KO and WT mice the AAR/LV % was similar with values of 56.2±6.8 % and 54.6±4.4 %, respectively. However, CD38 KO mice exhibited marked protection against myocardial I/R injury with decreased infarction. Infarct area/AAR was only 10.3±3.2 % in CD38 KO mice compared to 36.7±6.8 % in WT, p=0.0097. Conclusions Thus, abrogation of CD38 expression was observed to be highly protective against in vivo myocardial I/R injury with major reduction in infarction. As new highly potent CD38 inhibitors have recently been developed, these may hold great promise as therapeutics in the treatment of unstable coronary syndromes and acute myocardial infarction.

Inhibition of CD38 with the Thiazoloquin(az)olin(on)e 78c Protects the Heart against Postischemic Injury.

Inhibition of and genetic deletion of the NAD(P)+ hydrolase [NAD(P)ase] CD38 have been shown to protect against ischemia/reperfusion (I/R) injury in rat and mouse hearts. CD38 has been shown to enhance salvage of NADP(H), which in turn prevents impairment of endothelial nitric oxide synthase function, a hallmark of endothelial dysfunction. Despite growing evidence for a role of CD38 in postischemic injury, until recently there had been a lack of potent CD38 inhibitors. Recently, a new class of thiazoloquin(az)olin(on)e compounds were identified as highly potent and specific CD38 inhibitors. Herein, we investigate the ability of one of these compounds, 78c, to inhibit CD38 and protect the heart in an ex vivo model of myocardial I/R injury. The potency and mechanism of CD38 inhibition by 78c was assessed in vitro using recombinant CD38. The dose-dependent tissue uptake of 78c in isolated mouse hearts was determined, and high tissue permeability of 78c was observed when delivered in perfusate. Treatment of hearts with 78c was protective against both postischemic endothelial and cardiac myocyte injury, with preserved nitric oxide synthase-dependent vasodilatory and contractile function, respectively. Myocardial infarction was also significantly decreased in 78c-treated hearts, with preserved levels of high-energy phosphates. Protective effects peaked at 10 μM treatment, and similar protection without toxicity was seen at 5-fold higher doses. Overall, 78c was shown to be a potent and biologically active CD38 inhibitor with favorable tissue uptake and marked protective effects against I/R injury with enhanced preservation of contractile function, coronary flow, and decreased infarction.

ACUTE VASODILATOR TESTING: AN OPPORTUNITY TO REFINE STUDY DESIGN AND PROVIDE PRECISION CARE IN PULMONARY HYPERTENSION

SESSION TITLE: Pulmonary Vascular Disease SESSION TYPE: Fellow Case Reports PRESENTED ON: 10/07/2018 10:45 AM - 11:45 AM INTRODUCTION: Inhaled nitric oxide is currently used to acutely evaluate pulmonary vascular reactivity and identify patients with pulmonary arterial hypertension who may be appropriate candidates for long-term treatment with a calcium channel blocker (CCB). However, some patients with an acute decrease in mean pulmonary arterial pressure (MPAP) with nitric oxide fail to improve, or progressively worsen over time, with a CCB alone. Here we present a patient with severe pulmonary arterial hypertension who develops a normal MPAP while breathing nitric oxide. We describe the acute and long-term effects of other agents on her MPAP. CASE PRESENTATION: The patient presented at 8 years of age in 2003 with decreased exercise tolerance and syncope. Non-invasive studies were consistent with a diagnosis of idiopathic pulmonary arterial hypertension. Her hemodynamic measurements during heart catheterization and acute vasodilator testing are shown along with interim therapy in the table. See tableThe patient's MPAP has consistently decreased to normal during acute vasodilator testing with nitric oxide. There was little or no acute improvement in MPAP with nitroglycerin and sildenafil, and a corresponding failure with therapy long-term. There was moderate acute improvement with diltiazem and riociguat, and a corresponding moderate improvement with amlodipine long-term. The dose of amlodipine and the dose of riociguat have been limited by adverse side effects (gingival hyperplasia and gastroesophageal reflux). Nighttime nitric oxide therapy was safely used in her home for 4 months when an increase in MPAP was temporally associated with an adverse reaction to minocycline. Unfortunately, a reliable delivery system was not available to continue long-term therapy at that time.She has a polymorphism in endothelial nitric oxide synthase (Glu298Asp), a normal cytogenomic microarray and elevated levels of asymmetric dimethylarginine. Genetic testing for specific variants associated with pulmonary arterial hypertension has not been performed. DISCUSSION: Our patient's response to nitric oxide is consistent with a phenotype of decreased endothelial nitric oxide synthase function. Her clinical course illustrates how acute vasodilator testing may be used to guide therapy, even if a corresponding genotype is unknown. In this case, long-term inhaled nitric oxide may be more effective and less likely to cause adverse systemic effects than other agents. Any agent may fail to demonstrate a beneficial effect in a heterogeneous population of patients, but may have a meaningful effect in a subset of responsive patients. For example, a combination of inhaled nitric oxide and a CCB might prevent long-term treatment failures that occur with a CCB alone. CONCLUSIONS: Acute vasodilator testing identified appropriate medications for the long-term therapy of our patient. There is a need to design trials with a focus on precision care. Reference #1: Day, RW. Differences in the acute pulmonary vascular effects of oxygen with nitric oxide and diltiazem: Implications for the long-term treatment of pulmonary arterial hypertension. Congenit Heart Dis 2013;8:71-77. DISCLOSURES: No relevant relationships by Meghan Cirulis, source=Web Response No relevant relationships by Nicole Day, source=Web Response No relevant relationships by Ronald Day, source=Web Response No relevant relationships by Lynda Jorgensen, source=Web Response

Identification of eNOS‐based Megakaryocyte Subpopulations and Their Pharmacological Characterization by IFNγ and IL‐10

PurposeRecently, our laboratory identified in human blood two platelet subpopulations based on the presence or absence of a functional endothelial nitric oxide synthase (eNOS)‐signalling pathway and the ability to produce nitric oxide (NO). We have found that eNOS‐negative (eNOSneg) platelets, although less abundant are more reactive than eNOS‐positive (eNOSpos) platelets and initiate aggregate/thrombus formation, while eNOS‐positive platelets through their ability to generate NO limit aggregate size. As platelets derive from bone marrow megakaryocytes, we hypothesized that eNOSneg and eNOSpos subpopulations of megakaryocytes exist and give rise to their respective eNOS‐based platelet subpopulations. Additionally, we hypothesized that interferon‐γ (IFNγ) and interleukin‐10 (IL‐10), which are known to counter‐regulate eNOS expression and megakaryocyte differentiation, would determine in a concentration manner the ratio of eNOSneg to eNOSpos megakaryocytes.MethodsThe human megakaryoblastic cell line (Meg‐01) and megakaryocytes isolated from eNOS‐GFP transgenic mice were studied. RT‐PCR and DNA sequencing were performed to validate eNOS presence within Meg‐01. Flow cytometry was used to identify eNOSneg and eNOSpos megakaryocytes, and NO production was measured using DAF‐FM, a cell‐permeable fluorescent probe. Further, Meg‐01 cells were treated for 48 hours with IFNγ (0 – 100 ng/ml) and IL‐10 (0 – 100 ng/ml), and eNOS as well as inducible NOS (iNOS) were measured by flow cytometry and immunoblot, respectively.ResultsThe presence of eNOS was confirmed in Meg‐01 and eNOS‐GFP transgenic mice. Similar to human platelets, the majority of Meg‐01 cells were eNOSpos (91.5%±1.2 vs. 8.5%±1.2 eNOSneg, P&lt;0.05) and NO‐producing (92.0%±3.34 vs. 8.0%±6.68 non‐producing, P &lt; 0.05). Conversely, similar to mouse platelets, the majority of mouse megakaryocytes were eNOSneg (95.8%±0.7 vs. 4.2%±0.7 eNOSpos, P &lt; 0.05). The proinflammatory cytokine IFNγ decreased the percent eNOSpos Meg‐01 in a concentration‐dependent manner and also increased the level of iNOS within Meg‐01. IL‐10 in a concentration‐dependent manner (10–100ng/ml) reversed IFNγ‐induced iNOS expression, but not the decrease in percent eNOSpos Meg‐01. The changes in eNOS expression within Meg‐01 were further confirmed by qPCR within the total Meg‐01 population and demonstrated that IFNγ (10 ng/ml) caused a 6.4 fold decrease of eNOS mRNA but only 2.2 fold decrease in the presence IL‐10 (100ng/ml).ConclusionseNOS‐based megakaryocyte subpopulations exist within the Meg‐01 cell line and in eNOS‐GFP transgenic mice. Similar to previously shown for platelets, major species differences exist in eNOSpos to eNOSneg ratios between human and mouse megakaryocytes. IFNγ decreases eNOS expression and percent eNOSpos Meg‐01; while increasing iNOS expression. However, high concentrations of IL‐10 attenuated this effect. Our data show that pro‐inflammatory IFN‐γ may promote growth/differentiation of megakaryocytes with decreased levels/absence of eNOS, which may lead to formation of higher numbers of more reactive eNOSneg platelets potentially leading to higher risk of a pro‐thrombotic state. Further experiments are required to confirm whether eNOSpos and eNOSneg megakaryocytes give rise to their respective platelet subtypes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hydrogen Sulfide Preserves Endothelial Nitric Oxide Synthase Function by Inhibiting Proline-Rich Kinase 2: Implications for Cardiomyocyte Survival and Cardioprotection.

Hydrogen sulfide (H2S) exhibits beneficial effects in the cardiovascular system, many of which depend on nitric oxide (NO). Proline-rich tyrosine kinase 2 (PYK2), a redox-sensitive tyrosine kinase, directly phosphorylates and inhibits endothelial NO synthase (eNOS). We investigated the ability of H2S to relieve PYK2-mediated eNOS inhibition and evaluated the importance of the H2S/PYK2/eNOS axis on cardiomyocyte injury in vitro and in vivo. Exposure of H9c2 cardiomyocytes to H2O2 or pharmacologic inhibition of H2S production increased PYK2 (Y402) and eNOS (Y656) phosphorylation. These effects were blocked by treatment with Na2S or by overexpression of cystathionine γ-lyase (CSE). In addition, PYK2 overexpression reduced eNOS activity in a H2S-reversible manner. The viability of cardiomyocytes exposed to Η2Ο2 was reduced and declined further after the inhibition of H2S production. PYK2 downregulation, l-cysteine supplementation, or CSE overexpression alleviated the effects of H2O2 on H9c2 cardiomyocyte survival. Moreover, H2S promoted PYK2 sulfhydration and inhibited its activity. In vivo, H2S administration reduced reactive oxygen species levels, as well as PYK2 (Y402) and eNOS (Y656) phosphorylation. Pharmacologic blockade of PYK2 or inhibition of PYK2 activation by Na2S reduced myocardial infarct size in mice. Coadministration of a PYK2 inhibitor and Na2S did not result in additive effects on infarct size. We conclude that H2S relieves the inhibitory effect of PYK2 on eNOS, allowing the latter to produce greater amounts of NO, thereby affording cardioprotection. Our results unravel the existence of a novel H2S-NO interaction and identify PYK2 as a crucial target for the protective effects of H2S under conditions of oxidative stress.

Ferrochelatase is a therapeutic target for ocular neovascularization.

Ocular neovascularization underlies major blinding eye diseases such as “wet” age‐related macular degeneration (AMD). Despite the successes of treatments targeting the vascular endothelial growth factor (VEGF) pathway, resistant and refractory patient populations necessitate discovery of new therapeutic targets. Using a forward chemical genetic approach, we identified the heme synthesis enzyme ferrochelatase (FECH) as necessary for angiogenesis in vitro and in vivo. FECH is overexpressed in wet AMD eyes and murine choroidal neovascularization; siRNA knockdown of Fech or partial loss of enzymatic function in the Fech m1Pas mouse model reduces choroidal neovascularization. FECH depletion modulates endothelial nitric oxide synthase function and VEGF receptor 2 levels. FECH is inhibited by the oral antifungal drug griseofulvin, and this compound ameliorates choroidal neovascularization in mice when delivered intravitreally or orally. Thus, FECH inhibition could be used therapeutically to block ocular neovascularization.

Contribution of maternal hypertension to autism etiology in a murine model; cerebellar gene expression

Aim: To study the contribution of maternal hypertension to autism spectrum disorders’ (ASD) phenotype, and gene expression, in a murine model. Materials &amp; methods: To examine the effects of maternal hypertension, we used a well-described transgenic mouse model lacking functional endothelial nitric oxide synthase (eNOS or NOS3). Behavioral testing was performed on male offspring between 8 and 10 weeks of age. Cerebella underwent shotgun transcriptome RNA sequencing. Differentially expressed genes were examined for Gene Ontology enrichment. 2-way-RM-ANOVA, 1-way-ANOVA and Student's t-test were used for statistical analysis. Results &amp; conclusion: Our findings revealed that a deficit in social behavior, the hallmark of ASD, is differentially present in offspring born to hypertensive mothers. Novel ASD-related genes were differentially expressed in the cerebellum, implicating its possible role in ASD etiology. Condensation: Altered uterine environment resulting from maternal hypertension contributes to ASD phenotype, and modifies expression of novel ASD-related genes in cerebella of eNOS heterozygous offspring.

Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo.

GTP cyclohydrolase 1 (GCH1) deficiency is critical for endothelial nitric oxide synthase uncoupling in endothelial dysfunction. MicroRNAs (miRs) are a class of regulatory RNAs that negatively regulate gene expression. We investigated whether statins prevent endothelial dysfunction via miR-dependent GCH1 upregulation. Endothelial function was assessed by measuring acetylcholine-induced vasorelaxation in the organ chamber. MiR-133a expression was assessed by quantitative reverse transcription polymerase chain reaction and fluorescence in situ hybridization. We first demonstrated that GCH1 mRNA is a target of miR-133a. In endothelial cells, miR-133a was robustly induced by cytokines/oxidants and inhibited by lovastatin. Furthermore, lovastatin upregulated GCH1 and tetrahydrobiopterin, and recoupled endothelial nitric oxide synthase in stressed endothelial cells. These actions of lovastatin were abolished by enforced miR-133a expression and were mirrored by a miR-133a antagomir. In mice, hyperlipidemia- or hyperglycemia-induced ectopic miR-133a expression in the vascular endothelium, reduced GCH1 protein and tetrahydrobiopterin levels, and impaired endothelial function, which were reversed by lovastatin or miR-133a antagomir. These beneficial effects of lovastatin in mice were abrogated by in vivo miR-133a overexpression or GCH1 knockdown. In rats, multiple cardiovascular risk factors including hyperglycemia, dyslipidemia, and hyperhomocysteinemia resulted in increased miR-133a vascular expression, reduced GCH1 expression, uncoupled endothelial nitric oxide synthase function, and induced endothelial dysfunction, which were prevented by lovastatin. Statin inhibits aberrant miR-133a expression in the vascular endothelium to prevent endothelial dysfunction by targeting GCH1. Therefore, miR-133a represents an important therapeutic target for preventing cardiovascular diseases.

Fetal programming of blood pressure in a transgenic mouse model of altered intrauterine environment.

Nitric oxide is essential in the vascular adaptation to pregnancy, as knockout mice lacking nitric oxide synthase (NOS3) have abnormal utero-placental perfusion, hypertension and growth restriction. We previously showed with ex vivo studies on transgenic animals lacking NOS3 that adverse intrauterine environment alters fetal programming of vascular reactivity in adult offspring. The current research shows that altered vascular reactivity correlates with higher blood pressure in vivo. Our data suggest that higher blood pressure depends on both genetic background (NOS3 deficiency) and uterine environment, becomes more evident with age (> 7 postnatal weeks), activity and stress, is gender specific (preponderant among males), and can be affected by the sleep-awake cycle. In utero or early postnatal life (< 7weeks), before onset of hypertension, may represent a potential window for intervention to prevent future cardiovascular disorders. Nitric oxide is involved in the vascular adaptation to pregnancy. Using transgenic animals, we previously showed that adverse intrauterine environment alters vascular reactivity in adult offspring. The aim of our study was to determine if altered vascular programming is associated with abnormal blood pressure (BP) profiles in vivo. Mice lacking a functional endothelial nitric oxide synthase (KO, NOS3-/- ) and wild-type mice (WT, NOS3+/+ ) were crossbred to generate homozygous NOS3-/- (KO), maternally derived heterozygous NOS3+/- (KOM: mother with adverse intrauterine environment from NOS3 deficiency), paternally derived heterozygous NOS3+/- (KOP: mother with normal in utero milieu) and NOS3+/+ (WT) litters. BP was measured in vivo at 7, 14 and 21weeks of age. After univariate analysis, multivariate population-averaged linear regression models were used to identify factors affecting BP. When compared to WT offspring, systolic (SBP), diastolic (DBP) and mean (MAP) BP progressively increased from KOP, to KOM, and peaked among KO (P <0.001), although significance was not reached for KOP. Higher BP was also associated with male gender, older age (> 7 postnatal weeks), higher locomotor activity, daytime recordings, and recent blood pressure transducer insertion (P <0.001). Post hoc analysis showed that KOM had higher SBP than KOP (P <0.05). Our study indicates that adverse intrauterine environment contributes, along with multiple other factors, to account for hypertension; moreover, in utero or early postnatal life may represent a possible therapeutic window for prevention of cardiovascular disease later in life.

Radiofrequency Renal Denervation Protects the Ischemic Heart via Inhibition of GRK2 and Increased Nitric Oxide Signaling.

Rationale:Catheter-based renal denervation (RDN) is currently under development for the treatment of resistant hypertension and is thought to reduce blood pressure via interruption of sympathetic pathways that modulate cardiovascular function. The sympathetic nervous system also plays a critical role in the pathogenesis of acute myocardial infarction and heart failure.Objective:We examined whether treatment with radiofrequency (RF)-RDN would protect the heart against subsequent myocardial ischemia/reperfusion injury via direct effects on the myocardium.Methods and Results:Spontaneously hypertensive rats received either bilateral RF-RDN or sham-RDN. At 4 weeks after RF-RDN (n=14) or sham-RDN (n=14) treatment, spontaneously hypertensive rats were subjected to 30 minutes of transient coronary artery occlusion and 24 hours –7 days reperfusion. Four weeks after RF-RDN, myocardial oxidative stress was markedly attenuated, and transcription and translation of antioxidants, superoxide dismutase 1 and glutathione peroxidase-1, were significantly upregulated compared with sham-RDN spontaneously hypertensive rats. RF-RDN also inhibited myocardial G protein–coupled receptor kinase 2 pathological signaling and enhanced myocardial endothelial nitric oxide synthase function and nitric oxide signaling. RF-RDN therapy resulted in a significant reduction in myocardial infarct size per area at risk compared with sham-RDN (26.8 versus 43.9%; P<0.01) at 24 hours postreperfusion and significantly improved left ventricular function at 7 days after myocardial ischemia/reperfusion.Conclusions:RF-RDN reduced oxidative stress, inhibited G protein–coupled receptor kinase 2 signaling, increased nitric oxide bioavailability, and ameliorated myocardial reperfusion injury in the setting of severe hypertension. These findings provide new insights into the remote cardioprotective effects of RF-RDN acting directly on cardiac myocytes to attenuate cell death and protect against ischemic injury.

(-)-Epicatechin-induced recovery of mitochondria from simulated diabetes: Potential role of endothelial nitric oxide synthase.

(-)-Epicatechin increases indicators associated with mitochondrial biogenesis in endothelial cells and myocardium. We investigated endothelial nitric oxide synthase involvement on (-)-epicatechin-induced increases in indicators associated with mitochondrial biogenesis in human coronary artery endothelial cells cultured in normal-glucose and high-glucose media, as well as to restore indicators of cardiac mitochondria from the effects of simulated diabetes. Here, we demonstrate the role of endothelial nitric oxide synthase on (-)-epicatechin-induced increases in mitochondrial proteins, transcription factors and sirtuin 1 under normal-glucose conditions. In simulated diabetes endothelial nitric oxide synthase function, mitochondrial function-associated and biogenesis-associated indicators were adversely impacted by high glucose, effects that were reverted by (-)-epicatechin. As an animal model of type 2 diabetes, 2-month old C57BL/6 mice were fed a high-fat diet for 16 weeks. Fasting and fed blood glucose levels were increased and NO plasma levels decreased. High-fat-diet-fed mice myocardium revealed endothelial nitric oxide synthase dysfunction, reduced mitochondrial activity and markers of mitochondrial biogenesis. The administration of 1 mg/kg (-)-epicatechin for 15 days by oral gavage shifted these endpoints towards control mice values. Results suggest that endothelial nitric oxide synthase mediates (-)-epicatechin-induced increases of indicators associated with mitochondrial biogenesis in endothelial cells. (-)-Epicatechin also counteracts the negative effects that high glucose or simulated type 2 diabetes has on endothelial nitric oxide synthase function.

Kenneth D. Bloch, MD (1956–2014)

On a sunny, beautiful day in September 2014, we gathered in a funeral home in the town of Brookline, Massachusetts, to say a sad good-bye to our beloved friend and colleague, Dr. Kenneth D. Bloch. Ken was born in New York City on May 17, 1956, and raised in Brookline. After receiving his undergraduate and medical education at Brown University, he began his career as a medical resident at Massachusetts General Hospital (MGH), followed by a cardiology fellowship and then a position on the division staff in 1990. Two years later, he became an investigator in the Cardiovascular Research Center of MGH. Ken was a brilliant researcher who made a number of seminal contributions. He characterized the mechanisms of atrial natriuretic peptide and endothelial nitric oxide synthase function as well as the downstream mechanisms of nitric oxide and cyclic guanosine monophosphate (cGMP) signaling. More recently, Ken and colleagues in his laboratory discovered novel functions and mechanisms of action of the bone morphogenetic protein family in relation to cardiovascular disease and anemia of chronic inflammation, identifying this pathway as an important target in human disease. Ken was appointed the William T. G. Morton Professor of Anesthesia at Harvard Medical School in 2007 and Professor of Medicine in 2008. Ken was much more than a gifted molecular biologist and cardiologist. He had many personal gifts that were truly exceptional and that made him a rare mentor, colleague, and friend. Here are just a few stories. In our lab meetings, he would listen as we analyzed a problem of applied physiology or biochemistry or molecular biology and struggled with the tougher ones. Almost always, as the discussion ensued Ken would come up with a key insight unlocking the path to the next step in the research. If we got off track, Ken was always the one to gently point the way back to the core problem. Ken never failed to remember a past study by us or others. Even in these last years, as he struggled courageously with cancer, never once complaining, his memory was extraordinary and unfailing. Ken was a Pied Piper of scientists. He loved to work with young scientists, always had a flock of young students and physicians/scientists, and spent many hours each day guiding them and counseling them. He did this with the patience of a saint. He also spent untold hours correcting the drafts of papers, and it was not just his fellows’ work. We would send Ken what we considered to be a just-about-perfect draft of a new paper, and in about a week we would get it back with 100 or 200 red editorial balloon comments. Ken was unflappable. He possessed equanimity in the face of any stress. He could logically and sequentially dissect and deal with any issue (and not only the scientific ones) thoughtfully and without losing his calm demeanor. We cannot remember Ken raising his voice, even with the most exasperating people. He dealt sensitively with the toughest problems arising in our lab administration, and we knew he would find a just and proper solution with the fewest ruffled feathers. Ken managed every challenge. Ken spent his happiest hours in the lab. Each day, including Saturday and Sunday, he would arrive early, before anyone else. He loved to get his hands wet with an experiment or clean out the ice from a freezer. When he was happy all of us knew it—because Ken whistled. Not a tune we recognized, but his happiness was evident to all around him. He glowed when an experiment went well or a fellow or student gave a great talk. We sought his compliments, and he often gave them to us after a great presentation or scientific advance. An “attaboy” from Ken was a proud gold star to any recipient. Ken leaves behind him a hundred intellectual children from nations around the globe. He brightened our lives with his wisdom, his amazing grasp of science, his carefully and gently articulated insights and opinions, and his remarkable way of helping us express ourselves. Ken was a bright, steady beacon who lit the way for many of us. So strong and true, his beacon will continue to guide us and give us strength as we carry on our lives, scientific and personal. We can hear him admonishing us to do that and asking us to stop talking about his very private life. Let us go forward, do our science, live our lives and make him proud. To quote Dr. Seuss, that famous American philosopher we often quoted to our children, “Don't cry because it's over, laugh because it happened!”

Endothelial dysfunction abrogates the efficacy of normobaric hyperoxia in stroke.

Hyperoxia has been uniformly efficacious in experimental focal cerebral ischemia. However, pilot clinical trials have showed mixed results slowing its translation in patient care. To explain the discordance between experimental and clinical outcomes, we tested the impact of endothelial dysfunction, exceedingly common in stroke patients but under-represented in experimental studies, on the neuroprotective efficacy of normobaric hyperoxia. We used hyperlipidemic apolipoprotein E knock-out and endothelial nitric oxide synthase knock-out mice as models of endothelial dysfunction, and examined the effects of normobaric hyperoxia on tissue perfusion and oxygenation using high-resolution combined laser speckle and multispectral reflectance imaging during distal middle cerebral artery occlusion. In normal wild-type mice, normobaric hyperoxia rapidly and significantly improved tissue perfusion and oxygenation, suppressed peri-infarct depolarizations, reduced infarct volumes, and improved neurological function. In contrast, normobaric hyperoxia worsened perfusion in ischemic brain and failed to reduce infarct volumes or improve neurological function in mice with endothelial dysfunction. These data suggest that the beneficial effects of hyperoxia on ischemic tissue oxygenation, perfusion, and outcome are critically dependent on endothelial nitric oxide synthase function. Therefore, vascular risk factors associated with endothelial dysfunction may predict normobaric hyperoxia nonresponders in ischemic stroke. These data may have implications for myocardial and systemic circulation as well.

209 Beta-catenin Functions as an Endogenous Negative Regulator of Endothelial Nitric Oxide Synthase Function

IntroductionNitric oxide (NO) derived from endothelial NO synthase (eNOS) has multiple anti-atherogenic actions in the vasculature. eNOS activity is controlled by association with a number of regulatory proteins. We here...

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Interactions Between Vascular Wall and Perivascular Adipose Tissue Reveal Novel Roles for Adiponectin in the Regulation of Endothelial Nitric Oxide Synthase Function in Human Vessels

Adiponectin is an adipokine with potentially important roles in human cardiovascular disease states. We studied the role of adiponectin in the cross-talk between adipose tissue and vascular redox state in patients with atherosclerosis. The study included 677 patients undergoing coronary artery bypass graft surgery. Endothelial function was evaluated by flow-mediated dilation of the brachial artery in vivo and by vasomotor studies in saphenous vein segments ex vivo. Vascular superoxide (O2(-)) and endothelial nitric oxide synthase (eNOS) uncoupling were quantified in saphenous vein and internal mammary artery segments. Local adiponectin gene expression and ex vivo release were quantified in perivascular (saphenous vein and internal mammary artery) subcutaneous and mesothoracic adipose tissue from 248 patients. Circulating adiponectin was independently associated with nitric oxide bioavailability and O2(-) production/eNOS uncoupling in both arteries and veins. These findings were supported by a similar association between functional polymorphisms in the adiponectin gene and vascular redox state. In contrast, local adiponectin gene expression/release in perivascular adipose tissue was positively correlated with O2(-) and eNOS uncoupling in the underlying vessels. In ex vivo experiments with human saphenous veins and internal mammary arteries, adiponectin induced Akt-mediated eNOS phosphorylation and increased tetrahydrobiopterin bioavailability, improving eNOS coupling. In ex vivo experiments with human saphenous veins/internal mammary arteries and adipose tissue, we demonstrated that peroxidation products produced in the vascular wall (ie, 4-hydroxynonenal) upregulate adiponectin gene expression in perivascular adipose tissue via a peroxisome proliferator-activated receptor-γ-dependent mechanism. We demonstrate for the first time that adiponectin improves the redox state in human vessels by restoring eNOS coupling, and we identify a novel role of vascular oxidative stress in the regulation of adiponectin expression in human perivascular adipose tissue.

Circulating Microparticles Carry a Functional Endothelial Nitric Oxide Synthase That Is Decreased in Patients With Endothelial Dysfunction

BackgroundMicroparticles (MPs) are circulating membrane particles of less than a micrometer in diameter shed from endothelial and blood cells. Recent literature suggests that MPs are not just functionally inert cell debris but may possess biological functions and mediate the communication between vascular cells. As a significant proportion of MPs originate from platelets and endothelial cells, we hypothesized that MPs may harbor functional enzymes including an endothelial NO synthase (eNOS).Methods and ResultsUsing immunoprecipitation and Western blot analysis, we found that human circulating MPs carry an eNOS. Ca2+ and l‐arginine‐dependent NOS activity of crude enzyme extract from MPs was determined by measuring the conversion of [3H]‐L‐arginine to [3H]‐citrulline and NOS‐dependent nitrite production. NOS‐dependent NO production in intact MPs was assessed by the NO‐specific fluorescent probe MNIP‐Cu. In patients with cardiovascular disease, endothelial dysfunction was associated with an increase in the total number of circulating MPs as well as a significant decrease in the expression and activity of eNOS in MPs. No difference in reactive oxygen species was noted in MPs isolated from either group.ConclusionsOur data further support the concept that circulating MPs may not only retain phenotypic markers but also preserve the functionality of enzymes of the cells they originate from, including eNOS.