Endothelial Nitric Oxide Activity Research Articles

Effect of seabuckthorn seed protein and its arginine-enriched peptides on combating memory impairment in mice

The current study characterized the combating memory impairment effect of seabuckthorn seed protein (SSP) and the arginine (Arg)-enriched peptides (SSPP) on d-galactose-induced brain aging in mice. The Arg content in SSP and SSPP were 10.11 and 17.82 g/100 g, respectively. Seven Arg peptides (Ile/Leu-Arg, Arg-Glu, Asp-Arg-Pro, Arg-Try-Ala, Glu-Arg-Ser, Val-Gly-Arg-Pro, and Lys-Thr-Glu-Arg) were identified from SSPP. The animal experiments of the Morris water maze and the step-down test indicated that the oral administration of SSP (0.25, 0.5, 1.0 mg/g·d) and SSPP (0.25, 0.5, 1.0 mg/g·d) significantly (p < 0.05) reversed the learning and memory impairment symptoms. The activation of endothelial nitric oxide (NO) synthase and neuronal NO synthase were increased, and inducible NO synthase decreased after SSP and SSPP in the hippocampus compared to the model group, with the SSPP being quite effective. Moreover, the treatment significantly exhibited the ability to normalize the serum inflammatory cytokine levels (NF-ĸB, TNF-α, IL-6) and suppress the Arg-inducible nitric oxide (Arg-iNO) pathway. Therefore, SSP and SSPP ingestion reversed the behavioral learning and memory impairment symptoms possibly associated with the anti-inflammation and Arg-iNO pathway. Consumption of SSP and SSPP diets can be beneficial to memory impairment.

Aprotinin does not Impair Vascular Function in Patients Undergoing Coronary Artery Bypass Graft Surgery.

Bleeding is a major complication in coronary artery bypass graft surgery. Antifibrinolytic agents like serine protease inhibitor aprotinin can decrease postoperative bleeding and complications of cardiac surgery. However, the effects of aprotinin on vascular function are not completely elucidated. We compared the ex vivo vascular function of left internal mammary arteries from patients undergoing coronary artery bypass graft surgery with and without intraoperative application of aprotinin using a Mulvany Myograph. Human internal mammary arteries were treated with aprotinin ex vivo and tested for changes in vascular function. We analyzed the impact of aprotinin on vascular function in rat aortic rings. Finally, impact of aprotinin on expression and activity of endothelial nitric oxide synthase was tested in human endothelial cells. Intraoperative application of aprotinin did not impair ex vivo vascular function of internal mammary arteries of patients undergoing coronary artery bypass graft surgery. Endothelium-dependent and -independent relaxations were not different in patients with or without aprotinin after nitric oxide synthase blockade. A maximum vasorelaxation of 94.5%±11.4vs. 96.1%±5.5% indicated a similar vascular smooth muscle function in both patient groups (n=13 each). Long-term application of aprotinin under physiological condition preserved vascular function of the rat aorta. In vitro application of increasing concentrations of aprotinin on human endothelial cells resulted in a similar expression and activity of endothelial nitric oxide synthase. In conclusion, intraoperative and ex vivo application of aprotinin does not impair the endothelial function in human internal mammary arteries and experimental models.

HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages.

Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two innate immune cell types are activated by bitter products, including those secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells. NO also increases phagocytosis by macrophages. Using biochemistry and live-cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). Immunofluorescence showed that H441 cells express eNOS and T2Rs and that the bitter agonist denatonium benzoate activates NO production in a Ca2+- and HSP90-dependent manner in cells grown either as submerged cultures or at the air–liquid interface. In primary sinonasal epithelial cells, we determined that HSP90 inhibition reduces T2R-stimulated NO production and ciliary beating, which likely limits pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves as an innate immune modulator by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream Ca2+ signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases such as chronic rhinosinusitis, asthma, or cystic fibrosis.

Loss of Alpha Globin Genes in Human Subjects Is Associated with Improved Nitric Oxide-Mediated Vascular Perfusion

Introduction Absence of alpha globin genes has long been known to influence the physiology of sickle cell disease (SCD). Individuals with SCD who are missing one or two alpha globin genes have decreased rates of cerebral vasculopathy, stroke, acute chest syndrome, and leg ulcers (Bernaudin, Blood 2008; Flanagan, Blood 2011; Nolan, Br J Haematol 2006). Although there is laboratory evidence of decreased hemolytic rate in these patients (Higgs, N Engl J Med 1982), the mechanism for their improved clinical outcomes has not been identified. Recently, the alpha globin protein has been shown to be present in the endothelial wall of human arterioles, where it modulates nitric oxide (NO) scavenging during vasoconstriction (Straub, Nature 2012). In mice, pharmacological inhibition of alpha globin leads to increased endothelial NO activity, independently of NO production, and results in increased blood perfusion, reduced systemic hypertension, and increased pulmonary artery vasodilation (Keller, Hypertension 2016; Alvarez, Am J Respir Cell Mol Biol 2017). The relationship between absence of alpha globin and arterial vasodilation, and the role of alpha globin in NO-mediated vascular signaling are potential mechanisms that could explain the beneficial effect of missing alpha globin genes in SCD. Using alpha thalassemia as a naturally occurring human model of alpha globin gene knockout, we hypothesized that loss of alpha globin genes leads to improvement in microvascular blood flow in thalassemia trait subjects without hemolysis. Methods Alpha thalassemia trait subjects missing one or two alpha globin genes, and healthy controls were recruited to the study, which was approved by the Children's Hospital Los Angeles Institutional Review Board. Blood samples were obtained from all subjects to test for hemoglobin, mean corpuscular volume (MCV), reticulocyte count, plasma hemoglobin, lactate dehydrogenase, and alpha globin genotype. We assessed flow-mediated dilation (FMD) of the brachial artery following distal forearm occlusion (Detterich, Blood 2015) simultaneously with laser Doppler flowmetry (LDF) and photoplethysmography (PPG) in the fingertip. We also measured the increase in microvascular perfusion with a thermal stimulus. The maximal change in vascular perfusion after provocation indicates vasodilatory capacity. Statistical analysis was performed in JMP® version 14 (SAS Institute Inc., USA). Results Twenty-seven subjects were enrolled, including 12 controls (4 alpha globin genes), 10 patients with 3 alpha globin genes and 5 with 2. The mean MCV was lower in subjects missing alpha globin genes than in controls (p=0.0099). Importantly, hemoglobin levels and markers of hemolysis were normal in both groups. There was no detectable difference in FMD between individuals missing one and two alpha globin genes; thus, these groups were combined and labeled as alpha trait for further analyses. FMD was significantly higher in alpha trait subjects after adjusting for age (Figure 1, p=0.0357). Missing alpha globin genes had no effect on microvascular flow by LDF or PPG (data not shown). Discussion FMD is an established and specific predictor of NO bioavailability (Thijssen, Am J Physiol Heart Circ Physiol 2011), and, in addition to shear-mediated NO circulation in conduit vessels, it reflects the sum of flow in multiple arteriolar networks downstream of the conduit artery. Using this method, a difference in endothelial function between control and alpha thalassemia trait was easily detected (Figure 1). Because endothelial alpha globin is present in arterioles rather than conduit vessels (Butcher, Free Radic Biol Med 2014), we measured microvascular flow in a 1-mm3 volume in the skin using a laser Doppler sensor, and in the fingertip by PPG, but were unable to detect an effect of alpha trait. As none of the subjects had anemia or evidence of hemolysis, the significantly increased FMD associated with loss of alpha globin genes is most likely due to increased NO as a result of decreased scavenging by alpha globin. The finding reported here that lower alpha globin gene number is associated with increased NO-related perfusion in humans may explain the beneficial effect of alpha thalassemia trait in SCD and suggests that the presence of alpha thalassemia trait may also play a role in other types of vascular disease. Disclosures Wood: BiomedInformatics: Consultancy; Imago Biosciences: Consultancy; BluebirdBio: Consultancy; Celgene: Consultancy; WorldcareClinical: Consultancy; Philips Medical Systems: Research Funding. Coates:apo pharma (Chiesi Pharma): Consultancy, Honoraria; Sangamo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios pharma: Consultancy, Honoraria; Vifor Pharma: Consultancy, Honoraria; Celgene, BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Pleiotropic Activation of Endothelial Function by Angiotensin II Receptor Blockers is Crucial to Their Protective Anti‐vascular Remodeling Effects

RationaleEndothelial function is strongly associated with vascular health and homeostasis. However, therapeutically relevant approaches capable of stimulating the protective properties of the endothelium remain elusive.ObjectiveTo determine whether modulation of the Angiotensin II (AngII) system with Angiotensin receptor blockers (ARBs) can improve endothelial function and promote downstream vascular homeostasis.Methods and ResultsWe show that all 4 ARBs tested (losartan, telmisartan, olmesartan, and valsartan) can promote robust chronic activation of endothelial nitric oxide (NO) in mice. With telmisartan, a well‐known pleiotropic ARB, in vivo treatment lead to a 58% inhibition of vascular contractility after two weeks of treatment and up to 77% at 6 months, in a fully L‐NAME sensitive fashion. Endothelial NO synthase (eNOS) knockout (KO) mice showed complete resistance against the endothelial function effects of telmisartan, whereas the Angiotensin converting enzyme inhibitor (ACEi) captopril showed little to no endothelial function activity at a similar blood pressure (BP) lowering dose. In a myograph chamber, direct stimulation of aortic vessels with telmisartan in the absence of AngII stimulated endothelial function, confirming pleiotropism. Moreover, in vitro stimulation of cultured endothelial cells caused rapid intracellular Calcium ([Ca2+]i) mobilization, which is a known upstream event of eNOS activation. In vivo, telmisartan completely abolished aortic wall remodeling in aging animals as well as a well‐established mouse model of Marfan syndrome (MFS), which causes accelerated and pre‐mature vascular aging and remodeling of the aortic root. In addition, in vivo inhibition of NOS activity with L‐NAME rendered telmisartan inactive in a BP‐independent manner.ConclusionARBs have unique and robust endothelial function activation properties independent of their inhibitory effects on the AngII system. Direct stimulation of the endothelial NO system with ARBs results in vascular protection and homeostasis independent of BP lowering effects, which supports a broader prophylactic use of this class of medication.Support or Funding InformationSupported by CIHR, HSFC, BCKDF, Marfan Foundation, SPH Foundation and Rare Disease Foundation.

Nitric oxide maintains endothelial redox homeostasis through PKM2 inhibition.

Decreased nitric oxide (NO) bioavailability and oxidative stress are hallmarks of endothelial dysfunction and cardiovascular diseases. Although numerous proteins are S-nitrosated, whether and how changes in protein S-nitrosation influence endothelial function under pathophysiological conditions remains unknown. We report that active endothelial NO synthase (eNOS) interacts with and S-nitrosates pyruvate kinase M2 (PKM2), which reduces PKM2 activity. PKM2 inhibition increases substrate flux through the pentose phosphate pathway to generate reducing equivalents (NADPH and GSH) and protect against oxidative stress. In mice, the Tyr656 to Phe mutation renders eNOS insensitive to inactivation by oxidative stress and prevents the decrease in PKM2 S-nitrosation and reducing equivalents, thereby delaying cardiovascular disease development. These findings highlight a novel mechanism linking NO bioavailability to antioxidant responses in endothelial cells through S-nitrosation and inhibition of PKM2.

Caveolin-1 Regulates Atherogenesis by Attenuating Low-Density Lipoprotein Transcytosis and Vascular Inflammation Independently of Endothelial Nitric Oxide Synthase Activation.

Atherosclerosis is driven by synergistic interactions between pathological, biomechanical, inflammatory, and lipid metabolic factors. Our previous studies demonstrated that absence of caveolin-1 (Cav1)/caveolae in hyperlipidemic mice strongly inhibits atherosclerosis, which was attributed to activation of endothelial nitric oxide (NO) synthase (eNOS) and increased production of NO and reduced inflammation and low-density lipoprotein trafficking. However, the contribution of eNOS activation and NO production in the athero-protection of Cav1 and the exact mechanisms by which Cav1/caveolae control the pathogenesis of diet-induced atherosclerosis are still not clear. Triple-knockout mouse lacking expression of eNOS, Cav1, and Ldlr were generated to explore the role of NO production in Cav1-dependent athero-protective function. The effects of Cav1 on lipid trafficking, extracellular matrix remodeling, and vascular inflammation were studied both in vitro and in vivo with a mouse model of diet-induced atherosclerosis. The expression of Cav1 and distribution of caveolae regulated by flow were analyzed by immunofluorescence staining and transmission electron microscopy. We found that absence of Cav1 significantly suppressed atherogenesis in Ldlr-/-eNOS-/- mice, demonstrating that athero-suppression is independent of increased NO production. Instead, we find that the absence of Cav1/caveolae inhibited low-density lipoprotein transport across the endothelium and proatherogenic fibronectin deposition and disturbed flow-mediated endothelial cell inflammation. Consistent with the idea that Cav1/caveolae may play a role in early flow-dependent inflammatory priming, distinct patterns of Cav1 expression and caveolae distribution were observed in athero-prone and athero-resistant areas of the aortic arch even in wild-type mice. These findings support a role for Cav1/caveolae as a central regulator of atherosclerosis that links biomechanical, metabolic, and inflammatory pathways independently of endothelial eNOS activation and NO production.

Quaternary ammonium salt of U50,488H elicits protective effects against hypoxic pulmonary hypertension

The present study aimed to investigate the role of quaternary ammonium salt of U50,488H (Q-U50,488H) in hypoxic pulmonary hypertension (HPH) and underlying mechanisms involved. A HPH animal model was established in rats under hypoxia and the mean pulmonary arterial pressure (mPAP) and right ventricular pressure (RVP) were measured. Relaxation of the pulmonary artery in response to Q-U50,488H was determined. In addition, expression and activity of endothelial nitric oxide (NO) synthase (eNOS) and inducible NO synthase (iNOS) with NO content, Akt expression, total antioxidant capacity (T-AOC), and gp91phox were evaluated. Cell viability was determined by the cell counting kit-8 (CCK-8) assay. We demonstrated that both the molecular weight and solubility of Q-U50,488H were higher than that of U50,488H. Q-U50,488H reduced mPAP and RVP and prevented the development of HPH. Moreover, Q-U50,488H relaxed the pulmonary arteries from both normal and HPH rats in a time-dependent manner. Under hypoxic conditions, Q-U50,488H significantly increased Akt phosphorylation, eNOS phosphorylation, NO content in serum, and T-AOC in pulmonary arteries of HPH rats. In addition, the activity of eNOS was elevated, but the activity of iNOS was reduced when Q-U50,488H was given under hypoxia. Q-U50,488H significantly counteracted the increase of gp91phox expression in pulmonary arteries under hypoxia. In addition, in vitro studies suggested that Q-U50,488H inhibited pulmonary artery smooth muscle cells (PASMCs) proliferation under hypoxic conditions and that the effects of Q-U50,488H were blocked by nor-binaltorphimine (nor-BNI). Thus, our results provided evidence that Q-U50,488H plays a protective role against HPH via κ-opioid receptor stimulation.

The Significance of Nitric Oxide as A Signaling Molecule In Regulation of Subclinical Inflammation and Apoptosis in Hypertensive Elderly Patients

Nitric oxide (NO) is a universal regulator of different processes, including vascular tone, hemostasis ?nd apoptosis. One of the important functions of NO as a signaling molecule is determined by a high capacity for diffusion through the cell membrane to synthesize it and penetrate into target cells by controlling their metabolism ?nd realizing intercellular interactions. For the purpose of estimation in elderly persons due to for determine the effect of nitric oxide and its products on the process of subclinical inflammation and apoptosis of endothelial cells in the development of endothelial dysfunction as a target organ in arterial hypertension 66 patients of elderly age with arterial hypertension (AH) of II stage and 24 persons of similar age without cardiovascular diseases were examined. The degree of endothelial dysfunction was examined by dopplerography of brachial artery, the endotheliocytemia level was estimated by the Hladovec and Rossmann method, the concentration of nitric oxide - in the Griess reaction, the levels of C-reactive protein, primary inflammatory mediator of TNF-a, apoptosis markers and nitrotyrosine - were determined by the immune enzyme method. It has been established that the synthesis of basal nitric oxide depends on the activity of endothelial constitutive NO - synthase and maintains the tone of the vessel in a state of light relaxation. In the progression of endothelial dysfunction (ED) a number of phases were identified: a compensation phase with increased secretory activity of endothelial cells, an intermediate phase when the balance is disrupted due to changes in the secretion process of production and inactivation of endothelial factors and a decompensation phase as a result of structural and metabolic disorders of endothelium resulting in its functional failure, death and desquamation. Regulatory effect of NO on subclinical inflammation and apoptosis intensity was confirmed by its strong inverse correlation with the level of TNF-a and caspase-3. It proved the change in the NO concentration, its auto- and paracrine effect on the formation of involutive changes in the vascular wall, its regulatory effect on the processes of subclinical inflammation and apoptosis of endothelial cells in the formation of hypertension, as well as its progression during aging.

Current Literature Review.

Current Literature Review.

PECAM1 regulates flow-mediated Gab1 tyrosine phosphorylation and signaling

Endothelial dysfunction, characterized by impaired activation of endothelial nitric oxide (NO) synthase (eNOS) and ensued decrease of NO production, is a common mechanism of various cardiovascular pathologies, including hypertension and atherosclerosis. Laminar blood flow-mediated specific signaling cascades modulate vascular endothelial cells (ECs) structure and functions. We have previously shown that flow-stimulated Gab1 (Grb2-associated binder-1) tyrosine phosphorylation mediates eNOS activation in ECs, which in part confers laminar flow atheroprotective action. However, the molecular mechanisms whereby flow regulates Gab1 tyrosine phosphorylation and its downstream signaling events remain unclear. Here we show that platelet endothelial cell adhesion molecule-1 (PECAM1), a key molecule in an endothelial mechanosensing complex, specifically mediates Gab1 tyrosine phosphorylation and its downstream Akt and eNOS activation in ECs upon flow rather than hepatocyte growth factor (HGF) stimulation. Small interfering RNA (siRNA) targeting PECAM1 abolished flow- but not HGF-induced Gab1 tyrosine phosphorylation and Akt, eNOS activation as well as Gab1 membrane translocation. Protein-tyrosine phosphatase SHP2, which has been shown to interact with Gab1, was involved in flow signaling and HGF signaling, as SHP2 siRNA diminished the flow- and HGF-induced Gab1 tyrosine phosphorylation, membrane localization and downstream signaling. Pharmacological inhibition of PI3K decreased flow-, but not HGF-mediated Gab1 phosphorylation and membrane localization as well as eNOS activation. Finally, we observed that flow-mediated Gab1 and eNOS phosphorylation in vivo induced by voluntary wheel running was reduced in PECAM1 knockout mice. These results demonstrate a specific role of PECAM1 in flow-mediated Gab1 tyrosine phosphorylation and eNOS signaling in ECs.

Excess maternal salt or fructose intake programmes sex-specific, stress- and fructose-sensitive hypertension in the offspring

The Western diet is typically high in salt and fructose, which have pressor activity. Maternal diet can affect offspring blood pressure, but the extent to which maternal intake of excess salt and fructose may influence cardiovascular function of the offspring is unknown. We sought to determine the effect of moderate maternal dietary intake of salt and/or fructose on resting and stimulated cardiovascular function of the adult male and female offspring. Pregnant rats were fed purified diets (± 4% salt) and water (± 10% fructose) before and during gestation and through lactation. Male and female offspring were weaned onto standard laboratory chow. From 9 to 14 weeks of age, cardiovascular parameters (basal, circadian and stimulated) were assessed continuously by radiotelemetry. Maternal salt intake rendered opposite-sex siblings with a 25-mmHg difference in blood pressure as adults; male offspring were hypertensive (15 mmHg mean arterial pressure (MAP)) and female offspring were hypotensive (10 mmHg MAP) above and below controls, respectively. Sex differences were unrelated to endothelial nitric oxide activity in vivo, but isolation-induced anxiety revealed a significantly steeper coupling between blood pressure and heart rate in salt-exposed male offspring but not in female offspring. MAP of all offspring was refractory to salt loading but sensitive to subsequent dietary fructose, an effect exacerbated in female offspring from fructose-fed dams. Circadian analyses of pressure in all offspring revealed higher mean set-point for heart rate and relative non-dipping of nocturnal pressure. In conclusion, increased salt and fructose in the maternal diet has lasting effects on offspring cardiovascular function that is sex-dependent and related to the offspring's stress-response axis.

Abstract 12231: PECAM1 is Essential for Flow-mediated Gab1 Tyrosine Phosphorylation and Signaling in vitro and in vivo

Introduction: Impaired activation of endothelial nitric oxide (NO) synthase (eNOS) and ensued NO production is a common mechanism of various cardiovascular pathologies, including hypertension and atherosclerosis. Specific signaling cascades, generated by vascular endothelial cells (ECs) in response to laminar flow, modulate EC structure and functions, NO production in particular. We have previously shown that flow-stimulated Gab1 (Grb2-associated binder-1) tyrosine phosphorylation mediates eNOS activation. However, the upstream mechanism that regulates Gab1 tyrosine phosphorylation remains unclear. Hypothesis: We hypothesized that platelet endothelial cell adhesion molecule-1 (PECAM1), a key molecule in an endothelial mechanosensing complex, specifically mediates Gab1 tyrosine phosphorylation and its downstream Akt and eNOS activation in ECs upon flow rather than hepatocyte growth factor (HGF) stimulation. Methods: Western blot, en face staining and voluntary wheel running. Results: Small interfering RNA (siRNA) targeting PECAM1 abolished flow- but not HGF-induced Gab1 tyrosine phosphorylation and Akt, eNOS activation as well as Gab1 membrane translocation. Protein-tyrosine phosphatase SHP2, which has been shown to interact with Gab1, was involved in a flow signaling pathway as well as HGF-induced signaling, as SHP2 siRNA diminished the flow- and HGF-induced Gab1 tyrosine phosphorylation, membrane localization and downstream signaling. Pharmacological inhibition of PI3K by LY294002 decreased flow, but not HGF-mediated Gab1 phosphorylation and membrane localization as well as eNOS activation. Finally, we observed that flow-mediated Gab1 and eNOS phosphorylation in vivo induced by voluntary wheel running was reduced in PECAM1 knockout mice. Conclusions: These results demonstrate a specific role of PECAM1 in flow-mediated Gab1 tyrosine phosphorylation and eNOS signaling in ECs

Uncoupling of endothelial nitric oxide synthase in cerebral vasculature of Tg2576 mice.

In this study, we tested the hypothesis that reduced bioavailability of tetrahydrobiopterin (BH4) is a major mechanism responsible for pathogenesis of endothelial dysfunction in cerebral microvessels of transgenic mice expressing the Swedish double mutation of human amyloid precursor protein (APP) (Tg2576 mice). Endothelial nitric oxide synthase (eNOS) protein expression was significantly increased in cerebral vasculature of Tg2576 mice. In contrast, bioavailability of BH4 was significantly reduced (p < 0.05). Moreover, superoxide anion production was increased in cerebral microvessels of Tg2576 mice (p < 0.05). Incubation with NOS inhibitor, Nω-nitro-L-arginine methyl ester, decreased superoxide anion indicating that uncoupled eNOS is most likely the source of superoxide anion. Increasing BH4 bioavailability either exogenously by BH4 supplementation or endogenously by treatment with the selective peroxisome proliferator-activated receptor--delta activator GW501516 (2 mg/kg/day, 14 days) attenuated eNOS uncoupling and decreased superoxide anion production in cerebral microvessels of Tg2576 mice (p < 0.05). Treatment with GW501516 restored the biological activity of endothelial nitric oxide in cerebral microvessels of Tg2576 mice, as indicated by the increased nitrite/nitrate content and 3,5-cyclic guanosine monophosphate levels (p < 0.05). Our studies indicate that sub-optimal BH4 bioavailability in cerebral vasculature is an important contributor to oxidant stress and endothelial dysfunction in Tg2576 mouse model of Alzheimer's disease. Existing evidence suggests that Aβ peptides-induced up-regulation of expression and activity of NADPH oxidase causes increased production of superoxide anion (.O2(-)). .O2(-) can also be converted to hydrogen peroxide (H2O2) by enzymatic activity of superoxide dismutase (SOD) or spontaneous dismutation. Elevation of .O2(-) and H2O2 might cause oxidation of tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2) and subsequent uncoupling of endothelial nitric oxide synthase (eNOS) (a) thus reducing levels of nitric oxide (NO) and 3',5'-cyclic guanosine monophosphate (cGMP). Supplementation of BH4 or activation of PPARδ prevents detrimental effects of eNOS uncoupling by restoring bioavailability of BH4 and scavenging of .O2(-), respectively (b). Activation of PPARδ also increases expression of catalase thereby inactivating H2O2. Generation of H2O2 by uncoupled eNOS in cerebral microvessels of Tg2576 mice is hypothetical.

PP.13.06

Objective: Increased arterial stiffness (AS) is an independent risk factor for cardiovascular disease. Endothelial nitric oxide (NO) is vasoprotective by decreasing not only vascular tone in resistance arteries, but also stiffness in conductance arteries. Asymmetric (ADMA) and symmetric (SDMA) dimethylarginines, endogenous inhibitors of NO synthesis from the precursor L-arginine (ARN), increase in conditions of oxidative stress and contribute to progression of endothelial dysfunction and arteriosclerosis. In essential hypertensives (EH) we investigated the relationship between systemic AS, as assessed by the ratio of pulse pressure (PP) to stroke volume (SV), with circulating levels of ADMA, SDMA, ARN and the activity of the renin-aldosterone system in resting conditions. Design and method: In 25 untreated grade I-II EH (age 50 ± 12 yrs, range 19–71, M/F = 15/10) blood pressure (BP, sphygmomanometer), heart rate (HR, EKG), stroke volume (SV, impedance cardiography) were measured after supine rest; venous blood was simultaneously sampled for plasma ADMA, SDMA, ARN (by HPLC), plasma renin activity (PRA) and aldosterone (Aldo)(by RIA). Results: Table shows means ± sd:Supine BP was 142/96 ± 15/8.4 mmHg, HR = 70.0 ± 9.5 bt/min; PRA was 0.3 ± 0.05 ng/ml/h (range 0.1–0.9) and Aldo 9.8 ± 0.7 ng/dl (range 3.7–17). AS (range 0.89–2.21 mmHg/ml) was directly related to age (r = 0.61, p < 0.05), to SDMA (r = 0.83, p < 0.01) and inversely to ARN/ADMA ratio, an indicator of NO production (r = -0.64, p < 0.05); AS was not related to PRA or Aldo. In a multiple regression analysis, SDMA was the best predictor of AS (p < 0.05). Conclusions: Systemic AS in EH was directly related to circulating levels of endogenous inhibitors of NO production. These data are compatible with an impaired activity of endothelial NO associated with increased stiffness of conductance arteries, a condition at higher cardiovascular risk in uncomplicated hypertensives.

The efficacy and safety of acupuncture for cerebral vasospasm after subarachnoid hemorrhage: study protocol for a randomized controlled trial.

BackgroundSubarachnoid hemorrhage (SAH) is a neurological disease with a high mortality rate. Several serious complications frequently arise after successful surgery for this condition. Cerebral vasospasm, one such complication, occurs in 50 to 70% of SAH patients. These patients suffer neurological symptoms known as delayed ischemic neurological deficit (DIND); however, the effect of treatment of vasospasm is limited. The major pathogenesis of cerebral vasospasm is the reduction of nitric oxide (NO) and activation of vasoconstrictors. Acupuncture is known to increase the production and activity of vascular endothelial cell-derived NO and improve endothelium-dependent vasodilatation. A preliminary retrospective case study to investigate the ability of acupuncture to prevent the occurrence of cerebral vasospasm has been conducted. However, no randomized, controlled clinical trials have been carried out to evaluate the efficacy of acupuncture for cerebral vasospasm.Methods/DesignThis trial will be a single-center, randomized, placebo-controlled, parallel group, patient-assessor-blinded clinical trial. A total of 80 patients with SAH will be randomized into two groups: a study group given acupuncture, electroacupuncture, and intradermal acupuncture, and a control group given mock transcutaneous electrical nerve stimulation and sham intradermal acupuncture. Intervention will start within 96 h after SAH, and a total of 12 sessions will be performed during a 2-week period. The primary outcome measure will be the occurrence of DIND, and the secondary outcomes will be vasospasm as measured by cerebral angiography, transcranial Doppler, clinical symptoms, vasospasm-related infarcts, NO and endothelin-1 plasma levels, mortality, and modified Rankin Scale scores.DiscussionThis trial will examine the efficacy and safety of acupuncture for cerebral vasospasm after SAH. The placebo effect will be excluded and the mechanism of action of the treatments will be evaluated through blood testing.Trial registrationClinicalTrials.gov NCT02275949, Registration date: 26 October 2014.

Symmetric dimethylarginine alters endothelial nitric oxide activity in glomerular endothelial cells

Circulating symmetric dimethylarginine (SDMA) is increased in patients with chronic kidney disease. SDMA is considered an inert metabolite, but because it can transported into cells, we studied the effect of SDMA on glomerular endothelial cells. SDMA suppressed VEGF-induced endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide production, but not VEGFR2 activation and signaling leading to eNOS activation. SDMA caused eNOS uncoupling and increased superoxide anion production in response to VEGF. All these effects were blocked by preventing cellular uptake of SDMA with a molar excess of arginine. These data show that SDMA interferes with nitric oxide production by uncoupling eNOS and leads to oxidative stress in glomerular endothelial cells.In conclusion, our data show that SDMA is not an inert metabolite and that it could contribute to oxidative stress in the renal endothelium.

Ergocalciferol and microcirculatory function in chronic kidney disease and concomitant vitamin d deficiency: an exploratory, double blind, randomised controlled trial.

Background and ObjectivesVitamin D deficiency and endothelial dysfunction are non-traditional risk factors for cardiovascular events in chronic kidney disease. Previous studies in chronic kidney disease have failed to demonstrate a beneficial effect of vitamin D on arterial stiffness, left ventricular mass and inflammation but none have assessed the effect of vitamin D on microcirculatory endothelial function.Study DesignWe conducted a randomised controlled trial of 38 patients with non diabetic chronic kidney disease stage 3–4 and concomitant vitamin D deficiency (<16 ng/dl) who received oral ergocalciferol (50,000 IU weekly for one month followed by 50,000 IU monthly) or placebo over 6 months. The primary outcome was change in microcirculatory function measured by laser Doppler flowmetry after iontophoresis of acetylcholine. Secondary endpoints were tissue advanced glycation end products, sublingual functional capillary density and flow index as well as macrovascular parameters. Parallel in vitro experiments were conducted to determine the effect of ergocalciferol on cultured human endothelial cells.ResultsTwenty patients received ergocalciferol and 18 patients received placebo. After 6 months, there was a significant improvement in the ergocalciferol group in both endothelium dependent microcirculatory vasodilatation after iontophoresis of acetylcholine (p = 0.03) and a reduction in tissue advanced glycation end products (p = 0.03). There were no changes in sublingual microcirculatory parameters. Pulse pressure (p = 0.01) but not aortic pulse wave velocity was reduced. There were no significant changes in bone mineral parameters, blood pressure or left ventricular mass index suggesting that ergocalciferol improved endothelial function independently of these parameters. In parallel experiments, expression of endothelial nitric oxide synthase and activity were increased in human endothelial cells in a dose dependent manner.ConclusionsErgocalciferol improved microcirculatory endothelial function in patients with chronic kidney disease and concomitant vitamin D deficiency. This process may be mediated through enhanced expression and activity of endothelial nitric oxide synthase.Trial RegistrationClinical trials.gov NCT00882401

Variable reactive hyperemia in normotensive strains of rat.

Previous studies from our laboratory report variation in nitric oxide (NO)‐dependent arterial pressure within the same strain of normotensive Sprague–Dawley rat dependent upon the commercial vendor supplying the rats. Clinical assessment of endothelial NO activity and endothelial function in general has used postocclusion, flow‐mediated dilation (FMD). Therefore, this study was conducted to determine whether the reactive hyperemic response was different between two normotensive strains from two different suppliers, Sprague–Dawley (SD) and Wistar–Kyoto (WKY) rats from Charles River (CR) and Harlan Laboratories (H), respectively. Rats were anesthetized and the femoral artery was occluded for 5 min, with femoral blood flow measured continuously by use of an ultrasonic perivascular flow probe. The average area under the reactive hyperemic response curve (3‐min duration) was not different between SD rats from CR (80 ± 23 mL/min∙s; n = 6) and H (94 ± 16 mL/min∙s; n = 6). As previously reported, blood pressures were higher in the SD rats from H versus CR. WKY rats from both suppliers had significantly larger hyperemia; 371 ± 67 versus 281 ± 71 mL/min∙s (n = 5) for the CR and H WKY rats, respectively, but again, were not different between vendors. Blood pressures in WKY rats were similar between vendors. These results suggest that differences in NO bioactivity are not discernable with an adapted FMD protocol in the rat and that normotensive strains of rat can have large differences in reactive hyperemia despite having similar blood pressures.

Abstract 18776: Endothelial Function is Not Affected by Fresh or Storage-Aged Autologous Blood Transfusions in Healthy Individuals

Background: Red cells play a modulating role in nitric oxide (NO)-mediated signaling and hypoxic vasodilation that may be influenced by blood transfusions, particularly with storage-aged blood (pRBCs). We have shown that brachial artery endothelial function, measured as flow-mediated vasodilation (FMD) is adversely affected in patients receiving aged compared to fresh pRBCs. Here we investigated endothelial NO activity in healthy subjects receiving autologous pRBCs with the hypothesis that aged pRBCs will impair endothelial conductance vessel and microvascular function. Methods: Sixteen healthy participants (aged 31±10 years, 38% female) were transfused 1 unit of fresh (aged 3-7 days) and subsequently with storage-aged (35-42 days) autologous pRBCs. Endothelial function was measured using FMD and reactive hyperemia index (RHI), a measure of microvascular function, was measured using pulsatile arterial tonometry (Itamar Inc., n=7), before, 1 hour, and 24 hours after transfusion. Data were analyzed by repeated measures ANOVA. Results: Hemoglobin was similar before both transfusions (p=0.066) and increased by 1.02±1.0 g/dL (±SD) and 1.2±1.2 g/dL with fresh and storage-aged transfusions, respectively. The FMD before, 1 hr and 24 hr after transfusion of fresh pRBCs (7.8±6.9%, 8.2±4.8% and 6.9±4.3%) or stored pRBCs (7.1±5.5%, 7.4±4.7% and 8.5±4.9%) was similar,( p=0.45). The RHI before, 1 hr and 24 hr after transfusion of fresh pRBCs ( 2.45±0.23, 2.26±0.08 and 2.26±0.21) showed no significant changes, p=0.87 or stored pRBCs ( 2.25±0.67, 2.27±0.53 and 2.22±0.26, p=0.42). Conclusions: Vascular NO activity and microvascular dilatory capacity are not impaired by transfusion of fresh or storage-aged autologous pRBC in healthy individuals, unlike findings with allogeneic transfusions in patients. These differences suggest that the risks of aged blood transfusions are likely to be greater in patients with vascular disease who have underlying endothelial dysfunction.