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Sample records for inhibiting endothelial nitric

  1. Gene Therapy Inhibiting Neointimal Vascular Lesion: In vivo Transfer of Endothelial Cell Nitric Oxide Synthase Gene

    NASA Astrophysics Data System (ADS)

    von der Leyen, Heiko E.; Gibbons, Gary H.; Morishita, Ryuichi; Lewis, Neil P.; Zhang, Lunan; Nakajima, Masatoshi; Kaneda, Yasufumi; Cooke, John P.; Dzau, Victor J.

    1995-02-01

    It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as β-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessel. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor of vascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia.

  2. Rocuronium Bromide Inhibits Inflammation and Pain by Suppressing Nitric Oxide Production and Enhancing Prostaglandin E2 Synthesis in Endothelial Cells

    PubMed Central

    2016-01-01

    Purpose Rocuronium bromide is a nondepolarizing neuromuscular blocking drug and has been used as an adjunct for relaxation or paralysis of the skeletal muscles, facilitation of endotracheal intubation, and improving surgical conditions during general anesthesia. However, intravenous injection of rocuronium bromide induces injection pain or withdrawal movement. The exact mechanism of rocuronium bromide-induced injection pain or withdrawal movement is not yet understood. We investigated whether rocuronium bromide treatment is involved in the induction of inflammation and pain in vascular endothelial cells. Methods For this study, calf pulmonary artery endothelial (CPAE) cells were used, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Western blot, nitric oxide detection, and prostaglandin E2 immunoassay were conducted. Results Rocuronium bromide treatment inhibited endothelial nitric oxide synthase and suppressed nitric oxide production in CPAE cells. Rocuronium bromide activated cyclooxygenase-2, inducible nitric oxide synthase and increased prostaglandin E2 synthesis in CPAE cells. Conclusions Rocuronium bromide induced inflammation and pain in CPAE cells. Suppressing nitric oxide production and enhancing prostaglandin E2 synthesis might be associated with rocuronium bromide-induced injection pain or withdrawal movement. PMID:28043117

  3. Benidipine, a dihydropyridine-calcium channel blocker, inhibits lysophosphatidylcholine-induced endothelial injury via stimulation of nitric oxide release.

    PubMed

    Matsubara, Masahiro; Yao, Kozo; Hasegawa, Kazuhide

    2006-01-01

    Benidipine hydrochloride (benidipine), which is a long-lasting dihydropyridine calcium channel blocker, exerts antihypertensive action via inhibition of Ca(2+) influx through L-type voltage-dependent calcium channels. In addition, benidipine is shown to restore endothelial function. However, the mechanisms whereby benidipine has protective effects on endothelium are poorly defined. Nitric oxide (NO), which is produced by endothelial NO synthase (eNOS), plays important roles in endothelial function. In this study, we examined effects of benidipine on NO production from human umbilical vein endothelial cells. Benidipine (0.3-10 microM) augmented eNOS expression and total eNOS enzymatic activities. Benidipine also promoted the production of NO and the accumulation of cGMP, a second messenger of NO. Lysophosphatidylcholine (lysoPC), a component of oxidized low-density lipoproteins, induced caspase-3 activation followed by apoptosis of endothelial cells. Benidipine (0.3-10 microM) prevented lysoPC-induced caspase-3 activation, which was canceled by Nomega-nitro-L-arginine-methyl ester (L-NAME) (250-2500 microM), an inhibitor of NOS. Moreover, diethylenetetraamine NONOate (30-100 microM), a NO donor, inhibited the caspase-3 activation. These results suggested that the increase in NO production by benidipine might be involved in the inhibition of caspase induction. The direct enhancement of endothelial NO release by benidipine may be in part responsible for amelioration of endothelial dysfunction.

  4. Endothelial nitric oxide synthase activation through obacunone-dependent arginase inhibition restored impaired endothelial function in ApoE-null mice.

    PubMed

    Yoon, Jeongyeon; Park, Minjin; Lee, Jeong hyung; Min, Byung Sun; Ryoo, Sungwoo

    2014-03-01

    Endothelial arginase constrains the activity of endothelial nitric oxide synthase (eNOS) by substrate depletion and reduces nitric oxide bioavailability. During the screening course of arginase inhibitor, we found obacunone as an arginase inhibitor. We tested the hypothesis that obacunone regulates vascular endothelial NO production. Obacunone incubation inhibited arginase I and II activities in liver and kidney lysates, respectively, in dose-dependent manner. Obacunone reciprocally increased nitrite/nitrate (NOx) production in HUVECs. In isolated aortic rings, obacunone increased intracellular l-arginine concentration and enhanced eNOS coupling, leading to increased NO and decreased superoxide production, with no changes in protein expression. Vasoconstriction response to U46619 was attenuated in obacunone-treated aortic vessels compared to that in untreated vessels. Endothelium-dependent vasorelaxant response to acetylcholine was significantly increased in obacunone-treated vessels and was modulated by the NO-dependent signaling cascade. The dose-dependent vasorelaxant response to Ach was reduced in the aortic vessels of ApoE-/- mice fed a high-cholesterol diet. Obacunone incubation increased vasorelaxation to the level of a WT mouse, although the endothelium-independent response to sodium nitroprusside was identical among the groups. Therefore, obacunone may help treat cardiovascular diseases derived from endothelial dysfunction and may be useful for designing pharmaceutical compounds.

  5. Nitric Oxide Inhibits Hetero-adhesion of Cancer Cells to Endothelial Cells: Restraining Circulating Tumor Cells from Initiating Metastatic Cascade

    NASA Astrophysics Data System (ADS)

    Lu, Yusheng; Yu, Ting; Liang, Haiyan; Wang, Jichuang; Xie, Jingjing; Shao, Jingwei; Gao, Yu; Yu, Suhong; Chen, Shuming; Wang, Lie; Jia, Lee

    2014-03-01

    Adhesion of circulating tumor cells (CTCs) to vascular endothelial bed becomes a crucial starting point in metastatic cascade. We hypothesized that nitric oxide (NO) may prevent cancer metastasis from happening by its direct vasodilation and inhibition of cell adhesion molecules (CAMs). Here we show that S-nitrosocaptopril (CAP-NO, a typical NO donor) produced direct vasorelaxation that can be antagonized by typical NO scavenger hemoglobin and guanylate cyclase inhibitor. Cytokines significantly stimulated production of typical CAMs by the highly-purified human umbilical vein endothelial cells (HUVECs). CAP-NO inhibited expression of the stimulated CAMs (particularly VCAM-1) and the resultant hetero-adhesion of human colorectal cancer cells HT-29 to the HUVECs in a concentration-dependent manner. The same concentration of CAP-NO, however, did not significantly affect cell viability, cell cycle and mitochondrial membrane potential of HT-29, thus excluding the possibility that inhibition of the hetero-adhesion was caused by cytotoxicity by CAP-NO on HT-29. Hemoglobin reversed the inhibition of CAP-NO on both the hetero-adhesion between HT-29 and HUVECs and VCAM-1 expression. These data demonstrate that CAP-NO, by directly releasing NO, produces vasorelaxation and interferes with hetero-adhesion of cancer cells to vascular endothelium via down-regulating expression of CAMs. The study highlights the importance of NO in cancer metastatic prevention.

  6. Cyclosporin A inhibits flow-mediated activation of endothelial nitric-oxide synthase by altering cholesterol content in caveolae.

    PubMed

    Lungu, Andreea O; Jin, Zheng-Gen; Yamawaki, Hideyuki; Tanimoto, Tatsuo; Wong, Chelsea; Berk, Bradford C

    2004-11-19

    Fluid shear stress generated by blood flowing over the endothelium is a major determinant of arterial tone, vascular remodeling, and atherogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an essential role in regulation of vascular function and structure by blood flow. Although cyclosporin A (CsA), an inhibitory ligand of cyclophilin A, is a widely used immunosuppressive drug, it causes arterial hypertension in part by impairing eNOS-dependent vasodilation. Here we show that CsA inhibits fluid shear stress-mediated eNOS activation in endothelial cells via decreasing cholesterol content in caveolae. Exposure of cultured bovine aortic endothelial cells to 1 mum CsA for 1 h significantly inhibited NO production and eNOS phosphorylation at Ser-1179 induced by flow (shear stress=dynes/cm2). The effect of CsA was not related to inhibition of two known eNOS kinases, protein kinase B (Akt) and protein kinase A, because CsA did not affect Akt or protein kinase A activation. In rabbit aorta perfused ex vivo, CsA also significantly inhibited flow-induced eNOS phosphorylation at Ser-1179 but had no effect on Akt measured by phosphorylation at Ser-473. However, CsA treatment decreased cholesterol content in caveolae and displaced eNOS from caveolae, which may be caused by CsA disrupting the association of caveolin-1 and cyclophilin A. The magnitude of the cholesterol depleting effect was similar to that of beta-cyclodextrin, a cholesterol-binding molecule, and beta-cyclodextrin had a similar inhibitory effect on flow-mediated eNOS activation. Treating bovine aortic endothelial cells for 24 h with 30 mug/ml cholesterol blocked the CsA effect and restored eNOS phosphorylation in response to flow. These data suggest that decreasing cholesterol content in caveolae by CsA is a potentially important pathogenic mechanism for CsA-induced endothelial dysfunction and hypertension.

  7. Cyclosporin-A inhibits constitutive nitric oxide synthase activity and neuronal and endothelial nitric oxide synthase expressions after spinal cord injury in rats.

    PubMed

    Diaz-Ruiz, Araceli; Vergara, Paula; Perez-Severiano, Francisca; Segovia, Jose; Guizar-Sahagún, Gabriel; Ibarra, Antonio; Ríos, Camilo

    2005-02-01

    Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca2+/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.

  8. Arginase-2 is cooperatively up-regulated by nitric oxide and histone deacetylase inhibition in human umbilical artery endothelial cells.

    PubMed

    Krause, Bernardo J; Hernandez, Cherie; Caniuguir, Andres; Vasquez-Devaud, Paola; Carrasco-Wong, Ivo; Uauy, Ricardo; Casanello, Paola

    2016-01-01

    Arginase-2 counteracts endothelial nitric oxide synthase (eNOS) activity in human endothelium, and its expression is negatively controlled by histone deacetylase (HDAC2). Conversely NO inhibits HDAC and previous studies suggest that arginase-2 is up-regulated by NO. We studied whether NO regulates arginase-2 expression in umbilical artery endothelial cells (HUAEC) increasing ARG2 promoter accessibility. HUAEC exposed to NOC-18 (NO donor, 1-100 μM, 0-24 h) showed an increase in arginase-2 but a decrease in eNOS mRNA levels in a time-dependent manner, with a maximal effect at 100 μM (24 h). Conversely NOS inhibition with L-NAME (100 μM) reduced arginase-2 mRNA and protein levels, an effect reverted by co-incubation with NOC-18. Treatment with TSA paralleled the effects of NO on arginase-2 and eNOS at mRNA and protein levels, with maximal effect at 10 μM. Co-incubation of NOC-18 (100 μM) with a sub-maximal concentration of TSA (1 μM) potentiated the increase in arginase-2 mRNA levels, whilst L-NAME prevented TSA-dependent arginase-2 induction. The effects on arginase-2 mRNA were paralleled by changes in chromatin accessibility, as well as increased levels of H3K9 and H4K12 acetylation, at ARG2 proximal (-579 to -367 and -280 to -73 bp from TSS) and core (-121 to +126 bp from TSS) promoter. Finally NO-dependent arginase-2 induction was prevented by pre-incubation for 10 min with the cysteine blocker MMTS (10 mM). These data showed for the first time that NO up-regulates arginase-2 expression in primary cultured human endothelial cells by an epigenetic-mediated mechanism increasing ARG2 promoter accessibility suggesting a negative regulatory loop for eNOS activity.

  9. Functional inhibition of urea transporter UT-B enhances endothelial-dependent vasodilatation and lowers blood pressure via L-arginine-endothelial nitric oxide synthase-nitric oxide pathway.

    PubMed

    Sun, Yi; Lau, Chi-Wai; Jia, Yingli; Li, Yingjie; Wang, Weiling; Ran, Jianhua; Li, Fei; Huang, Yu; Zhou, Hong; Yang, Baoxue

    2016-01-07

    Mammalian urea transporters (UTs), UT-A and UT-B, are best known for their role in urine concentration. UT-B is especially distributed in multiple extrarenal tissues with abundant expression in vascular endothelium, but little is known about its role in vascular function. The present study investigated the physiological significance of UT-B in regulating vasorelaxations and blood pressure. UT-B deletion in mice or treatment with UT-B inhibitor PU-14 in Wistar-Kyoto rats (WKYs) and spontaneous hypertensive rats (SHRs) reduced blood pressure. Acetylcholine-induced vasorelaxation was significantly augmented in aortas from UT-B null mice. PU-14 concentration-dependently produced endothelium-dependent relaxations in thoracic aortas and mesenteric arteries from both mice and rats and the relaxations were abolished by N(ω)-nitro-L-arginine methyl ester. Both expression and phosphorylation of endothelial nitric oxide synthase (eNOS) were up-regulated and expression of arginase I was down-regulated when UT-B was inhibited both in vivo and in vitro. PU-14 induced endothelium-dependent relaxations to a similar degree in aortas from 12 weeks old SHRs or WKYs. In summary, here we report for the first time that inhibition of UT-B plays an important role in regulating vasorelaxations and blood pressure via up-regulation of L-arginine-eNOS-NO pathway, and it may become another potential therapeutic target for the treatment of hypertension.

  10. Functional inhibition of urea transporter UT-B enhances endothelial-dependent vasodilatation and lowers blood pressure via L-arginine-endothelial nitric oxide synthase-nitric oxide pathway

    PubMed Central

    Sun, Yi; Lau, Chi-Wai; Jia, Yingli; Li, Yingjie; Wang, Weiling; Ran, Jianhua; Li, Fei; Huang, Yu; Zhou, Hong; Yang, Baoxue

    2016-01-01

    Mammalian urea transporters (UTs), UT-A and UT-B, are best known for their role in urine concentration. UT-B is especially distributed in multiple extrarenal tissues with abundant expression in vascular endothelium, but little is known about its role in vascular function. The present study investigated the physiological significance of UT-B in regulating vasorelaxations and blood pressure. UT-B deletion in mice or treatment with UT-B inhibitor PU-14 in Wistar-Kyoto rats (WKYs) and spontaneous hypertensive rats (SHRs) reduced blood pressure. Acetylcholine-induced vasorelaxation was significantly augmented in aortas from UT-B null mice. PU-14 concentration-dependently produced endothelium-dependent relaxations in thoracic aortas and mesenteric arteries from both mice and rats and the relaxations were abolished by Nω-nitro-L-arginine methyl ester. Both expression and phosphorylation of endothelial nitric oxide synthase (eNOS) were up-regulated and expression of arginase I was down-regulated when UT-B was inhibited both in vivo and in vitro. PU-14 induced endothelium-dependent relaxations to a similar degree in aortas from 12 weeks old SHRs or WKYs. In summary, here we report for the first time that inhibition of UT-B plays an important role in regulating vasorelaxations and blood pressure via up-regulation of L-arginine-eNOS-NO pathway, and it may become another potential therapeutic target for the treatment of hypertension. PMID:26739766

  11. Delphinidin, an active compound of red wine, inhibits endothelial cell apoptosis via nitric oxide pathway and regulation of calcium homeostasis.

    PubMed

    Martin, Sophie; Giannone, Grégory; Andriantsitohaina, Ramaroson; Martinez, M Carmen

    2003-07-01

    1. Epidemiological studies have suggested that moderate consumption of natural dietary polyphenolic compounds might reduce the risk of cardiovascular disease and also protect against cancer. The present study investigates the effects of delphinidin, an anthocyanin present in red wine, on bovine aortic endothelial cells apoptosis. 2. Based on flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis and detection of mitochondrial cytochrome c release, we show that delphinidin (10(-2) g l(-1)) alone had no effect either on necrosis or on apoptosis, but it significantly reduced apoptosis elicited by actinomycin D (1 micro g ml(-1), 24 h) and 7beta-hydroxycholesterol (10 micro g ml(-1), 18 h). 3. The protective effect of delphinidin was abolished by inhibitors of nitric oxide-synthase (NOS) (L-NA, 100 micro M and SMT, 100 micro M), guanylyl cyclase (ODQ, 100 micro M) and MAP kinase (PD98059, 30 micro M). 4. Western blot analysis and protein detection by confocal microscopy demonstrate that the antiapoptotic effect of delphinidin was associated with an increased endothelial NOS expression mediated by a MAP kinase pathway. 5. Finally, delphinidin alone had no effect on cytosolic-free calcium ([Ca(2+)](i)), but normalized the changes in [Ca(2+)](i) produced by actinomycin D towards the control values, suggesting that the antiapoptotic effect of delphinidin is associated with the maintenance of [Ca(2+)](i) in the physiological range. 6. All of the observed effects of delphinidin may preserve endothelium integrity, the alteration of which lead to pathologies including cardiovascular diseases, such as atherosclerosis, and is often associated with cancers. In conclusion, the protective effect of delphinidin against endothelial cell apoptosis contributes to understand the potential benefits of a consumption rich in polyphenols.

  12. Activation of mTOR/p70S6 kinase by ANG II inhibits insulin-stimulated endothelial nitric oxide synthase and vasodilation

    PubMed Central

    Jang, Hyun-Ju; Martinez-Lemus, Luis A.; Sowers, James R.

    2012-01-01

    Elevated tissue levels of angiotensin II (ANG II) are associated with impairment of insulin actions in metabolic and cardiovascular tissues. ANG II-stimulated activation of mammalian target of rapamycin (mTOR)/p70 S6 kinase (p70S6K) in cardiovascular tissues is implicated in cardiac hypertrophy and vascular remodeling. However, the role of ANG II-stimulated mTOR/p70S6K in vascular endothelium is poorly understood. In the present study, we observed that ANG II stimulated p70S6K in bovine aortic endothelial cells. ANG II increased phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser636/639 and inhibited the insulin-stimulated phosphorylation of endothelial nitric oxide synthase (eNOS). An inhibitor of mTOR, rapamycin, attenuated the ANG II-stimulated phosphorylation of p70S6K and phosphorylation of IRS-1 (Ser636/639) and blocked the ability of ANG II to impair insulin-stimulated phosphorylation of eNOS, nitric oxide production, and mesenteric-arteriole vasodilation. Moreover, point mutations of IRS-1 at Ser636/639 to Ala prevented the ANG II-mediated inhibition of insulin signaling. From these results, we conclude that activation of mTOR/p70S6K by ANG II in vascular endothelium may contribute to impairment of insulin-stimulated vasodilation through phosphorylation of IRS-1 at Ser636/639. This ANG II-mediated impairment of vascular actions of insulin may help explain the role of ANG II as a link between insulin resistance and hypertension. PMID:22028412

  13. C-reactive protein inhibits insulin activation of endothelial nitric oxide synthase via the immunoreceptor tyrosine-based inhibition motif of FcgammaRIIB and SHIP-1.

    PubMed

    Tanigaki, Keiji; Mineo, Chieko; Yuhanna, Ivan S; Chambliss, Ken L; Quon, Michael J; Bonvini, Ezio; Shaul, Philip W

    2009-06-05

    Insulin promotes the cardiovascular protective functions of the endothelium including NO production by endothelial NO synthase (eNOS), which it stimulates via Akt kinase which phosphorylates eNOS Ser1179. C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk in patients with type 2 diabetes. We previously showed that CRP inhibits eNOS activation by insulin by blunting Ser1179 phosphorylation. We now elucidate the underlying molecular mechanisms. We first show in mice that CRP inhibits insulin-induced eNOS phosphorylation, indicating that these processes are operative in vivo. In endothelial cells we find that CRP attenuates insulin-induced Akt phosphorylation, and CRP antagonism of eNOS is negated by expression of constitutively active Akt; the inhibitory effect of CRP on Akt is also observed in vivo. A requirement for the IgG receptor FcgammaRIIB was demonstrated in vitro using blocking antibody, and reconstitution experiments with wild-type and mutant FcgammaRIIB in NIH3T3IR cells revealed that these processes require the ITIM (immunoreceptor tyrosine-based inhibition motif) of the receptor. Furthermore, we find that endothelium express SHIP-1 (Src homology 2 domain-containing inositol 5'-phosphatase 1), that CRP induces SHIP-1 stimulatory phosphorylation in endothelium in culture and in vivo, and that SHIP-1 knockdown by small interfering RNA prevents CRP antagonism of insulin-induced eNOS activation. Thus, CRP inhibits eNOS stimulation by insulin via FcgammaRIIB and its ITIM, SHIP-1 activation, and resulting blunted activation of Akt. These findings provide mechanistic linkage among CRP, impaired insulin signaling in endothelium, and greater cardiovascular disease risk in type 2 diabetes.

  14. Inhibition of nitric oxide and prostaglandins, but not endothelial-derived hyperpolarizing factors, reduces blood flow and aerobic energy turnover in the exercising human leg.

    PubMed

    Mortensen, Stefan P; González-Alonso, José; Damsgaard, Rasmus; Saltin, Bengt; Hellsten, Ylva

    2007-06-01

    Prostaglandins, nitric oxide (NO) and endothelial-derived hyperpolarizing factors (EDHFs) are substances that have been proposed to be involved in the regulation of skeletal muscle blood flow during physical activity. We measured haemodynamics, plasma ATP at rest and during one-legged knee-extensor exercise (19 +/- 1 W) in nine healthy subjects with and without intra-arterial infusion of indomethacin (Indo; 621 +/- 17 microg min(-1)), Indo + N(G)-monomethyl-L-arginine (L-NMMA; 12.4 +/- 0.3 mg min(-1)) (double blockade) and Indo + L-NMMA + tetraethylammonium chloride (TEA; 12.4 +/- 0.3 mg min(-1)) (triple blockade). Double and triple blockade lowered leg blood flow (LBF) at rest (P<0.05), while it remained unchanged with Indo. During exercise, LBF and vascular conductance were 2.54 +/- 0.10 l min(-1) and 25 +/- 1 mmHg, respectively, in control and they were lower with double (33 +/- 3 and 36 +/- 4%, respectively) and triple (26 +/- 4 and 28 +/- 3%, respectively) blockade (P<0.05), while there was no difference with Indo. The lower LBF and vascular conductance with double and triple blockade occurred in parallel with a lower O(2) delivery, cardiac output, heart rate and plasma [noradrenaline] (P<0.05), while blood pressure remained unchanged and O(2) extraction and femoral venous plasma [ATP] increased. Despite the increased O(2) extraction, leg was 13 and 17% (triple and double blockade, respectively) lower than control in parallel to a lower femoral venous temperature and lactate release (P<0.05). These results suggest that NO and prostaglandins play important roles in skeletal muscle blood flow regulation during moderate intensity exercise and that EDHFs do not compensate for the impaired formation of NO and prostaglandins. Moreover, inhibition of NO and prostaglandin formation is associated with a lower aerobic energy turnover and increased concentration of vasoactive ATP in plasma.

  15. Endothelial nitric oxide synthase in the microcirculation

    PubMed Central

    Shu, Xiaohong; Keller, T.C. Stevenson; Begandt, Daniela; Butcher, Joshua T.; Biwer, Lauren; Keller, Alexander S.; Columbus, Linda; Isakson, Brant E.

    2015-01-01

    Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO) - a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells. PMID:26390975

  16. Endothelial nitric oxide synthase in the microcirculation.

    PubMed

    Shu, Xiaohong; Keller, T C Stevenson; Begandt, Daniela; Butcher, Joshua T; Biwer, Lauren; Keller, Alexander S; Columbus, Linda; Isakson, Brant E

    2015-12-01

    Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.

  17. Inhibition of endothelial nitric oxyde synthase increases capillary formation via Rac1-dependent induction of hypoxia-inducible factor-1α and plasminogen activator inhibitor-1.

    PubMed

    Petry, Andreas; BelAiba, Rachida S; Weitnauer, Michae; Görlach, Agnes

    2012-11-01

    Disruption of endothelial homeostasis results in endothelial dysfunction, characterised by a dysbalance between nitric oxide (NO) and reactive oxygen species (ROS) levels often accompanied by a prothrombotic and proproliferative state. The serine protease thrombin not only is instrumental in formation of the fibrin clot, but also exerts direct effects on the vessel wall by activating proliferative and angiogenic responses. In endothelial cells, thrombin can induce NO as well as ROS levels. However, the relative contribution of these reactive species to the angiogenic response towards thrombin is not completely clear. Since plasminogen activator inhibitor-1 (PAI-1), a direct target of the proangiogenic transcription factors hypoxia-inducible factors (HIFs), exerts prothrombotic and proangiogenic activities we investigated the role of ROS and NO in the regulation of HIF-1α, PAI-1 and capillary formation in response to thrombin. Thrombin enhanced the formation of NO as well as ROS generation involving the GTPase Rac1 in endothelial cells. Rac1-dependent ROS formation promoted induction of HIF-1α, PAI-1 and capillary formation by thrombin, while NO reduced ROS bioavailability and subsequently limited induction of HIF-1α, PAI-1 and the angiogenic response. Importantly, thrombin activation of Rac1 was diminished by NO, but enhanced by ROS. Thus, our findings show that capillary formation induced by thrombin via Rac1-dependent activation of HIF-1 and PAI-1 is limited by the concomitant release of NO which reduced ROS bioavailability. Rac1 activity is sensitive to ROS and NO, thereby playing an essential role in fine tuning the endothelial response to thrombin.

  18. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

    PubMed

    Tillery, Lakeisha C; Epperson, Tenille A; Eguchi, Satoru; Motley, Evangeline D

    2016-03-01

    Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632

  19. Inhibition of mTOR down-regulates scavenger receptor, class B, type I (SR-BI) expression, reduces endothelial cell migration and impairs nitric oxide production.

    PubMed

    Fruhwürth, Stefanie; Krieger, Sigurd; Winter, Katharina; Rosner, Margit; Mikula, Mario; Weichhart, Thomas; Bittman, Robert; Hengstschläger, Markus; Stangl, Herbert

    2014-07-01

    The mammalian target of rapamycin (mTOR) inhibiting drug rapamycin (Sirolimus) has severe side effects in patients including hyperlipidemia, an established risk factor for atherosclerosis. Recently, it was shown that rapamycin decreases hepatic LDL receptor (LDL-R) expression, which likely contributes to hypercholesterolemia. Scavenger receptor, class B, type I (SR-BI) is the major HDL receptor and consequently regulating HDL-cholesterol levels and the athero-protective effects of HDL. By using the mTOR inhibitor rapamycin, we show that SR-BI is down-regulated in human umbilical vein endothelial cells (HUVECs). This reduction of SR-BI protein as well as mRNA levels by about 50% did not alter HDL particle uptake or HDL-derived lipid transfer. However, rapamycin reduced HDL-induced activation of eNOS and stimulation of endothelial cell migration. The effects on cell migration could be counteracted by SR-BI overexpression, indicating that decreased SR-BI expression is in part responsible for the rapamycin-induced effects. We demonstrate that inhibition of mTOR leads to endothelial cell dysfunction and decreased SR-BI expression, which may contribute to atherogenesis during rapamycin treatment.

  20. Nitric Oxide Directly Promotes Vascular Endothelial Insulin Transport

    PubMed Central

    Wang, Hong; Wang, Aileen X.; Aylor, Kevin; Barrett, Eugene J.

    2013-01-01

    Insulin resistance strongly associates with decreased nitric oxide (NO) bioavailability and endothelial dysfunction. In the vasculature, NO mediates multiple processes that affect insulin delivery, including dilating both resistance and terminal arterioles in skeletal muscle in vivo. However, whether NO directly regulates vascular endothelial cell (EC) insulin uptake and its transendothelial transport (TET) is unknown. We report in this article that l-NG-nitro-l-arginine methyl ester (l-NAME) pretreatment blocked, whereas l-arginine and sodium nitroprusside (SNP) each enhanced, EC uptake of fluorescein isothiocyanate (FITC)-labeled insulin. SNP also partly or fully reversed the inhibition of EC insulin uptake caused by l-NAME, wortmannin, the Src inhibitor PP1, and tumor necrosis factor-α. In addition, SNP promoted [125I]TyrA14insulin TET by ∼40%. Treatment with insulin with and without SNP did not affect EC cyclic guanosine monophosphate (cGMP) levels, and the cGMP analog 8-bromo-cGMP did not affect FITC-insulin uptake. In contrast, treatment with insulin and SNP significantly increased EC protein S-nitrosylation, the colocalization of S-nitrosothiol (S-NO) and protein-tyrosine phosphatase 1B (PTP1B), and Akt phosphorylation at Ser473 and inhibited PTP1B activity. Moreover, a high-fat diet significantly inhibited EC insulin-stimulated Akt phosphorylation and FITC-insulin uptake that was partially reversed by SNP in rats. Finally, inhibition of S-nitrosylation by knockdown of thioredoxin-interacting protein completely eliminated SNP-enhanced FITC-insulin uptake. We conclude that NO directly promotes EC insulin transport by enhancing protein S-nitrosylation. NO also inhibits PTP1B activity, thereby enhancing insulin signaling. PMID:23863813

  1. Mechanism of purinergic activation of endothelial nitric oxide synthase in endothelial cells

    PubMed Central

    da Silva, Cleide Gonçalves; Specht, Anke; Wegiel, Barbara; Ferran, Christiane; Kaczmarek, Elzbieta

    2009-01-01

    Background Decreased endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production are critical contributors to endothelial dysfunction and vascular complications observed in many diseases, including diabetes mellitus. Extracellular nucleotides activate eNOS and increase NO generation, however the mechanism of this observation is not fully clarified. Methods and Results To elucidate the signaling pathway(s) leading to nucleotide-mediated eNOS phosphorylation at Ser-1177, human umbilical vein endothelial cells (EC) were treated with several nucleotides including, ATP, UTP, and ADP in the presence or absence of selective inhibitors. These experiments identified P2Y1, P2Y2 and possibly P2Y4 as the purinergic receptors involved in eNOS phosphorylation, and demonstrated that this process was adenosine-independent. Nucleotide-induced eNOS phosphorylation and activity were inhibited by BAPTA-AM (an intracellular free calcium chelator), rottlerin (a protein kinase C (PKC) delta inhibitor) and PKC delta siRNA. In contrast, blockade of AMP-activated protein kinase (AMPK), calcium/calmodulin-dependent kinase (CaMK) II, CaMK kinase (CaMKK), serine/threonine protein kinase B (Akt), protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK) and p38 mitogen-activated protein kinase (MAPK) did not affect nucleotide-mediated eNOS phosphorylation. Conclusions The present study indicates that extracellular nucleotide-mediated eNOS phosphorylation is calcium and PKC delta dependent. This newly identified signaling pathway opens new therapeutic avenues for the treatment of endothelial dysfunction. PMID:19188511

  2. Nitric oxide synthases: structure, function and inhibition.

    PubMed Central

    Alderton, W K; Cooper, C E; Knowles, R G

    2001-01-01

    This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated. PMID:11463332

  3. Jujuboside B Reduces Vascular Tension by Increasing Ca2+ Influx and Activating Endothelial Nitric Oxide Synthase

    PubMed Central

    Zhao, Yixiu; Zhang, Xin; Li, Jiannan; Bian, Yu; Sheng, Miaomiao; Liu, Bin; Fu, Zidong; Zhang, Yan; Yang, Baofeng

    2016-01-01

    Jujuboside B has been reported to have protective effect on many cardiovascular diseases. However, the effects of Jujuboside B on vascular tension and endothelial function are unknown. The present study investigated the effects of Jujuboside B on reducing vascular tension, protecting endothelial function and the potential mechanisms. The tension of isolated rat thoracic aorta ring was measured by Wire myograph system. The concentration of nitric oxide (NO) and the activity of endothelial nitric oxide synthase (eNOS) in human aortic endothelial cells (HAECs) were determined by Griess reagent method and enzyme-linked immune sorbent assay. The protein levels of eNOS and p-eNOS at Serine-1177 were determined by western blot analysis. Intracellular Ca2+ concentration in HAECs was measured by laser confocal imaging microscopy. Results showed that Jujuboside B reduced the tension of rat thoracic aorta rings with intact endothelium in a dose-dependent manner. L-NAME, KN93, EGTA, SKF96365, iberiotoxin and glibenclamide significantly attenuated Jujuboside B-induced vasodilation in endothelium-intact tissues. In contrast, indometacin and 4-DAMP had no such effects. Jujuboside B also promoted NO generation and increased eNOS activity, which were attenuated by L-NAME, EGTA and SKF96365. Moreover, Jujuboside B increased intracellular Ca2+ concentration dose-dependently, which was inhibited by EGTA and SKF96365. Besides, Jujuboside B induced a rapid Ca2+ influx instantaneously after depleting intracellular Ca2+ store, which was significantly inhibited by SKF96365. In conclusion, this study preliminarily confirmed that Jujuboside B reduced vascular tension endothelium-dependently. The underlying mechanisms involved that Jujuboside B increased extracellular Ca2+ influx through endothelial transient receptor potential cation (TRPC) channels, phosphorylated eNOS and promoted NO generation in vascular endothelial cells. In addition, Jujuboside B-induced vasodilation involved

  4. Jujuboside B Reduces Vascular Tension by Increasing Ca2+ Influx and Activating Endothelial Nitric Oxide Synthase.

    PubMed

    Zhao, Yixiu; Zhang, Xin; Li, Jiannan; Bian, Yu; Sheng, Miaomiao; Liu, Bin; Fu, Zidong; Zhang, Yan; Yang, Baofeng

    2016-01-01

    Jujuboside B has been reported to have protective effect on many cardiovascular diseases. However, the effects of Jujuboside B on vascular tension and endothelial function are unknown. The present study investigated the effects of Jujuboside B on reducing vascular tension, protecting endothelial function and the potential mechanisms. The tension of isolated rat thoracic aorta ring was measured by Wire myograph system. The concentration of nitric oxide (NO) and the activity of endothelial nitric oxide synthase (eNOS) in human aortic endothelial cells (HAECs) were determined by Griess reagent method and enzyme-linked immune sorbent assay. The protein levels of eNOS and p-eNOS at Serine-1177 were determined by western blot analysis. Intracellular Ca2+ concentration in HAECs was measured by laser confocal imaging microscopy. Results showed that Jujuboside B reduced the tension of rat thoracic aorta rings with intact endothelium in a dose-dependent manner. L-NAME, KN93, EGTA, SKF96365, iberiotoxin and glibenclamide significantly attenuated Jujuboside B-induced vasodilation in endothelium-intact tissues. In contrast, indometacin and 4-DAMP had no such effects. Jujuboside B also promoted NO generation and increased eNOS activity, which were attenuated by L-NAME, EGTA and SKF96365. Moreover, Jujuboside B increased intracellular Ca2+ concentration dose-dependently, which was inhibited by EGTA and SKF96365. Besides, Jujuboside B induced a rapid Ca2+ influx instantaneously after depleting intracellular Ca2+ store, which was significantly inhibited by SKF96365. In conclusion, this study preliminarily confirmed that Jujuboside B reduced vascular tension endothelium-dependently. The underlying mechanisms involved that Jujuboside B increased extracellular Ca2+ influx through endothelial transient receptor potential cation (TRPC) channels, phosphorylated eNOS and promoted NO generation in vascular endothelial cells. In addition, Jujuboside B-induced vasodilation involved

  5. Oleic acid increases mitochondrial reactive oxygen species production and decreases endothelial nitric oxide synthase activity in cultured endothelial cells.

    PubMed

    Gremmels, Hendrik; Bevers, Lonneke M; Fledderus, Joost O; Braam, Branko; van Zonneveld, Anton Jan; Verhaar, Marianne C; Joles, Jaap A

    2015-03-15

    Elevated plasma levels of free fatty acids (FFA) are associated with increased cardiovascular risk. This may be related to FFA-induced elevation of oxidative stress in endothelial cells. We hypothesized that, in addition to mitochondrial production of reactive oxygen species, endothelial nitric oxide synthase (eNOS)-mediated reactive oxygen species production contributes to oleic acid (OA)-induced oxidative stress in endothelial cells, due to eNOS uncoupling. We measured reactive oxygen species production and eNOS activity in cultured endothelial cells (bEnd.3) in the presence of OA bound to bovine serum albumin, using the CM-H2DCFDA assay and the L-arginine/citrulline conversion assay, respectively. OA induced a concentration-dependent increase in reactive oxygen species production, which was inhibited by the mitochondrial complex II inhibitor thenoyltrifluoroacetone (TTFA). OA had little effect on eNOS activity when stimulated by a calcium-ionophore, but decreased both basal and insulin-induced eNOS activity, which was restored by TTFA. Pretreatment of bEnd.3 cells with tetrahydrobiopterin (BH4) prevented OA-induced reactive oxygen species production and restored inhibition of eNOS activity by OA. Elevation of OA levels leads to both impairment in receptor-mediated stimulation of eNOS and to production of mitochondrial-derived reactive oxygen species and hence endothelial dysfunction.

  6. Methamphetamine-induced nitric oxide promotes vesicular transport in blood-brain barrier endothelial cells.

    PubMed

    Martins, Tânia; Burgoyne, Thomas; Kenny, Bridget-Ann; Hudson, Natalie; Futter, Clare E; Ambrósio, António F; Silva, Ana P; Greenwood, John; Turowski, Patric

    2013-02-01

    Methamphetamine's (METH) neurotoxicity is thought to be in part due to its ability to induce blood-brain barrier (BBB) dysfunction. Here, we investigated the effect of METH on barrier properties of cultured rat primary brain microvascular endothelial cells (BMVECs). Transendothelial flux doubled in response to METH, irrespective of the size of tracer used. At the same time, transendothelial electrical resistance was unchanged as was the ultrastructural appearance of inter-endothelial junctions and the distribution of key junction proteins, suggesting that METH promoted vesicular but not junctional transport. Indeed, METH significantly increased uptake of horseradish peroxidase into vesicular structures. METH also enhanced transendothelial migration of lymphocytes indicating that the endothelial barrier against both molecules and cells was compromised. Barrier breakdown was only observed in response to METH at low micromolar concentrations, with enhanced vesicular uptake peaking at 1 μM METH. The BMVEC response to METH also involved rapid activation of endothelial nitric oxide synthase and its inhibition abrogated METH-induced permeability and lymphocyte migration, indicating that nitric oxide was a key mediator of BBB disruption in response to METH. This study underlines the key role of nitric oxide in BBB function and describes a novel mechanism of drug-induced fluid-phase transcytosis at the BBB.

  7. Tipping off endothelial tubes: nitric oxide drives tip cells.

    PubMed

    Priya, Mani Krishna; Sahu, Giriraj; Soto-Pantoja, David R; Goldy, Naga; Sundaresan, Abaya Meenakshi; Jadhav, Vivek; Barathkumar, T R; Saran, Uttara; Jaffar Ali, B M; Roberts, David D; Bera, Amal Kanti; Chatterjee, Suvro

    2015-04-01

    Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a complex process that warrants cell migration, proliferation, tip cell formation, ring formation, and finally tube formation. Angiogenesis is initiated by a single leader endothelial cell called "tip cell," followed by vessel elongation by "stalk cells." Tip cells are characterized by their long filopodial extensions and expression of vascular endothelial growth factor receptor-2 and endocan. Although nitric oxide (NO) is an important modulator of angiogenesis, its role in angiogenic sprouting and specifically in tip cell formation is poorly understood. The present study tested the role of endothelial nitric oxide synthase (eNOS)/NO/cyclic GMP (cGMP) signaling in tip cell formation. In primary endothelial cell culture, about 40% of the tip cells showed characteristic sub-cellular localization of eNOS toward the anterior progressive end of the tip cells, and eNOS became phosphorylated at serine 1177. Loss of eNOS suppressed tip cell formation. Live cell NO imaging demonstrated approximately 35% more NO in tip cells compared with stalk cells. Tip cells showed increased level of cGMP relative to stalk cells. Further, the dissection of NO downstream signaling using pharmacological inhibitors and inducers indicates that NO uses the sGC/cGMP pathway in tip cells to lead angiogenesis. Taken together, the present study confirms that eNOS/NO/cGMP signaling defines the direction of tip cell migration and thereby initiates new blood vessel formation.

  8. Endothelial nitric oxide synthase interactions with G-protein-coupled receptors.

    PubMed Central

    Marrero, M B; Venema, V J; Ju, H; He, H; Liang, H; Caldwell, R B; Venema, R C

    1999-01-01

    The endothelial nitric oxide synthase (eNOS) is activated in response to stimulation of endothelial cells by a number of vasoactive substances including, bradykinin (BK), angiotensin II (Ang II), endothelin-1 (ET-1) and ATP. In the present study we have used in vitro activity assays of purified eNOS and in vitro binding assays with glutathione S-transferase fusion proteins to show that the capacity to bind and inhibit eNOS is a common feature of membrane-proximal regions of intracellular domain 4 of the BK B2, the Ang II AT1 and the ET-1 ETB receptors, but not of the ATP P2Y2 receptor. Phosphorylation of serine or tyrosine residues in the eNOS-interacting region of the B2 receptor results in a loss of eNOS inhibition due to a decrease in the binding affinity of the receptor domain for the eNOS enzyme. Furthermore, the B2 receptor is transiently phosphorylated on tyrosine residues in cultured endothelial cells in response to BK stimulation. Phosphorylation occurs during the time in which eNOS transiently dissociates from the receptor accompanied by a transient increase in nitric oxide production. Taken together, these data support the hypotheses that eNOS is regulated in endothelial cells by reversible and inhibitory interactions with G-protein-coupled receptors and that these interactions can be modulated by receptor phosphorylation. PMID:10510297

  9. Endothelial nitric oxide synthase-enhancing G-protein coupled receptor antagonist inhibits pulmonary artery hypertension by endothelin-1-dependent and endothelin-1-independent pathways in a monocrotaline model.

    PubMed

    Liu, Chung-Pin; Dai, Zen-Kong; Huang, Chein-Heng; Yeh, Jwu-Lai; Wu, Bin-Nan; Wu, Jiunn-Ren; Chen, Ing-Jun

    2014-06-01

    This study investigates whether endothelin-1 (ET-1) mediates monocrotaline (MCT)-induced pulmonary artery hypertension (PAH) and right ventricular hypertrophy (RVH), and if so, whether the G-protein coupled receptor antagonist KMUP-1 (7-{2-[4-(2-chlorobenzene)piperazinyl]ethyl}-1,3-dimethylxanthine) inhibits ET-1-mediated PA constriction and the aforementioned pathological changes. In a chronic rat model, intraperitoneal MCT (60 mg/kg) induced PAH and increased PA medial wall thickening and RV/left ventricle + septum weight ratio on Day 21 after MCT injection. Treatment with sublingual KMUP-1 (2.5 mg/kg/day) for 21 days prevented these changes and restored vascular endothelial nitric oxide synthase (eNOS) immunohistochemical staining of lung tissues. Western blotting analysis demonstrated that KMUP-1 enhanced eNOS, soluble guanylate cyclase, and protein kinase G levels, and reduced ET-1 expression and inactivated Rho kinase II (ROCKII) in MCT-treated lung tissue over long-term administration. In MCT-treated rats, KMUP-1 decreased plasma ET-1 on Day 21. KMUP-1 (3.6 mg/kg) maximally appeared at 0.25 hours in the plasma and declined to basal levels within 24 hours after sublingual administration. In isolated PA of MCT-treated rats, compared with control and pretreatment with l-NG-nitroarginine methyl ester (100 μM), KMUP-1 (0.1-100 μM) inhibited ET-1 (0.01 μM)-induced vasoconstriction. Endothelium-denuded PA sustained higher contractility in the presence of KMUP-1. In a 24-hour culture of smooth muscle cells (i.e., PA smooth muscle cells or PASMCs), KMUP-1 (0.1-10 μM) inhibited RhoA- and ET-1-induced RhoA activation. KMUP-1 prevented MCT-induced PAH, PA wall thickening, and RVH by enhancing eNOS and suppressing ET-1/ROCKII expression. In vitro, KMUP-1 inhibited ET-1-induced PA constriction and ET-1-dependent/independent RhoA activation of PASMCs. In summary, KMUP-1 attenuates ET-1-induced/ET-1-mediated PA constriction, and could thus aid in the treatment of PAH

  10. Syringaresinol causes vasorelaxation by elevating nitric oxide production through the phosphorylation and dimerization of endothelial nitric oxide synthase

    PubMed Central

    Chung, Byung-Hee; Kim, Sookon; Kim, Jong-Dai; Lee, Jung Joon; Baek, Yi-Yong; Jeoung, Dooil; Lee, Hansoo; Choe, Jongseon; Ha, Kwon-Soo; Won, Moo-Ho; Kwon, Young-Guen

    2012-01-01

    Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in vascular functions, including vasorelaxation. We here investigated the pharmacological effect of the natural product syringaresinol on vascular relaxation and eNOS-mediated NO production as well as its underlying biochemical mechanism in endothelial cells. Treatment of aortic rings from wild type, but not eNOS-/- mice, with syringaresinol induced endothelium-dependent relaxation, which was abolished by addition of the NOS inhibitor NG-monomethyl-L-arginine. Treatment of human endothelial cells and mouse aortic rings with syringaresinol increased NO production, which was correlated with eNOS phosphorylation via the activation of Akt and AMP kinase (AMPK) as well as elevation of intracellular Ca2+ levels. A phospholipase C (PLC) inhibitor blocked the increases in intracellular Ca2+ levels, AMPK-dependent eNOS phosphorylation, and NO production, but not Akt activation, in syringaresinol-treated endothelial cells. Syringaresinol-induced AMPK activation was inhibited by co-treatment with PLC inhibitor, Ca2+ chelator, calmodulin antagonist, and CaMKKβ siRNA. This compound also increased eNOS dimerization, which was inhibited by a PLC inhibitor and a Ca2+-chelator. The chemicals that inhibit eNOS phosphorylation and dimerization attenuated vasorelaxation and cGMP production. These results suggest that syringaresinol induces vasorelaxation by enhancing NO production in endothelial cells via two distinct mechanisms, phosphatidylinositol 3-kinase/Akt- and PLC/Ca2+/CaMKKβ-dependent eNOS phosphorylation and Ca2+-dependent eNOS dimerization. PMID:22170035

  11. Dobesilate enhances endothelial nitric oxide synthase-activity in macro- and microvascular endothelial cells

    PubMed Central

    Suschek, Christoph; Kolb, Hubert; Kolb-Bachofen, Victoria

    1997-01-01

    Dobesilate is used for normalizing vascular dysfunction in a number of diseases. In search for an effect on endothelial NO production, macrovascular endothelial cells from rat aorta, microvascular endothelial cells from rat exocrine pancreatic tissue, and capillary endothelial cells from rat islets, were cultured in the presence or absence of Mg-Dobesilate. The activity of constitutive nitric oxide synthase (ecNOS) in resident cells as well as of inducible nitric oxide synthase (iNOS) in cytokine-activated cells was measured indirectly by recording the citrulline concentrations in culture supernatants.In each of the different endothelial cells Mg-Dobesilate incubation (0.25–1 mM) for 24 h led to a significant and concentration-dependent increase in ecNOS-activities. With cytokine-activated endothelial cell cultures only moderate effects were seen with little or no concentration-dependency. Addition of the NOS-inhibitor NG-monomethyl-L-arginine led to a significant suppression of citrulline formation in all cultures as an evidence for the enzyme specificity of these effects.iNOS- and ecNOS-specific reverse transcription and semi-quantitative polymerase chain reaction (RT–PCR) with RNA from resident or cytokine-activated endothelial cells gave no evidence for an increase in NOS-specific mRNA after Mg-Dobesilate-treatment. Furthermore, Dobesilate-mediated enhancement of NO synthesis in resting endothelial cells was not due to iNOS induction in these cells, as no iNOS-specific signal was found by RT–PCR. PMID:9421302

  12. Endothelial nitric oxide signaling regulates Notch1 in aortic valve disease

    PubMed Central

    Bosse, Authors: Kevin; Hans, Chetan P.; Zhao, Ning; Koenig, Sara N.; Huang, Nianyuan; Guggilam, Anuradha; LaHaye, Stephanie; Tao, Ge; Lucchesi, Pamela A.; Lincoln, Joy; Lilly, Brenda; Garg, Vidu

    2013-01-01

    The mature aortic valve is composed of a structured trilaminar extracellular matrix that is interspersed with aortic valve interstitial cells (AVICs) and covered by endothelium. Dysfunction of the valvular endothelium initiates calcification of neighboring AVICs leading to calcific aortic valve disease (CAVD). The molecular mechanism by which endothelial cells communicate with AVICs and cause disease is not well understood. Using a co-culture assay, we show that endothelial cells secrete a signal to inhibit calcification of AVICs. Gain or loss of nitric oxide (NO) prevents or accelerates calcification of AVICs, respectively, suggesting that the endothelial cell-derived signal is NO. Overexpression of Notch1, which is genetically linked to human CAVD, retards the calcification of AVICs that occurs with NO inhibition. In AVICs, NO regulates the expression of Hey1, a downstream target of Notch1, and alters nuclear localization of Notch1 intracellular domain. Finally, Notch1 and NOS3 (endothelial NO synthase) display an in vivo genetic interaction critical for proper valve morphogenesis and the development of aortic valve disease. Our data suggests that endothelial cell-derived NO is a regulator of Notch1 signaling in AVICs in the development of the aortic valve and adult aortic valve disease. PMID:23583836

  13. L-theanine promotes nitric oxide production in endothelial cells through eNOS phosphorylation.

    PubMed

    Siamwala, Jamila H; Dias, Paul M; Majumder, Syamantak; Joshi, Manoj K; Sinkar, Vilas P; Banerjee, Gautam; Chatterjee, Suvro

    2013-03-01

    Consumption of tea (Camellia sinensis) improves vascular function and is linked to lowering the risk of cardiovascular disease. Endothelial nitric oxide is the key regulator of vascular functions in endothelium. In this study, we establish that l-theanine, a non-protein amino-acid found in tea, promotes nitric oxide (NO) production in endothelial cells. l-theanine potentiated NO production in endothelial cells was evaluated using Griess reaction, NO sensitive electrode and a NO specific fluorescent probe (4-amino-5-methylamino-2',7'-difluororescein diacetate). l-Theanine induced NO production was partially attenuated in presence of l-NAME or l-NIO and completely abolished using eNOS siRNA. eNOS activation was Ca(2+) and Akt independent, as assessed by fluo-4AM and immunoblotting experiments, respectively and was associated with phosphorylation of eNOS Ser 1177. eNOS phosphorylation was inhibited in the presence of ERK1/2 inhibitor, PD-98059 and partially inhibited by PI3K inhibitor, LY-294002 and Wortmanin suggesting PI3K-ERK1/2 dependent pathway. Increased NO production was associated with vasodilation in ex ovo (chorioallantoic membrane) model. These results demonstrated that l-theanine administration in vitro activated ERK/eNOS resulting in enhanced NO production and thereby vasodilation in the artery. The results of our experiments are suggestive of l-theanine mediated vascular health benefits of tea.

  14. Functional interplay between endothelial nitric oxide synthase and membrane type 1–matrix metalloproteinase in migrating endothelial cells

    PubMed Central

    Genís, Laura; Gonzalo, Pilar; Tutor, Antonio S.; Gálvez, Beatriz G.; Martínez-Ruiz, Antonio; Zaragoza, Carlos; Lamas, Santiago; Tryggvason, Karl; Apte, Suneel S.

    2007-01-01

    Nitric oxide (NO) is essential for vascular homeostasis and is also a critical modulator of angiogenesis; however, the molecular mechanisms of NO action during angiogenesis remain elusive. We have investigated the potential relationship between NO and membrane type 1–matrix metalloproteinase (MT1-MMP) during endothelial migration and capillary tube formation. Endothelial NO synthase (eNOS) colocalizes with MT1-MMP at motility-associated structures in migratory human endothelial cells (ECs); moreover, NO is produced at these structures and is released into the medium during EC migration. We have therefore addressed 2 questions: (1) the putative regulation of MT1-MMP by NO in migratory ECs; and (2) the requirement for MT1-MMP in NO-induced EC migration and tube formation. NO upregulates MT1-MMP membrane clustering on migratory human ECs, and this is accompanied by increased degradation of type I collagen substrate. MT1-MMP membrane expression and localization are impaired in lung ECs from eNOS-deficient mice, and these cells also show impaired migration and tube formation in vitro. Inhibition of MT1-MMP with a neutralizing antibody impairs NOinduced tube formation by human ECs, and NO-induced endothelial migration and tube formation are impaired in lung ECs from mice deficient in MT1-MMP. MT1-MMP thus appears to be a key molecular effector of NO during the EC migration and angiogenic processes, and is a potential therapeutic target for NO-associated vascular disorders. PMID:17606763

  15. Functional interplay between endothelial nitric oxide synthase and membrane type 1 matrix metalloproteinase in migrating endothelial cells.

    PubMed

    Genís, Laura; Gonzalo, Pilar; Tutor, Antonio S; Gálvez, Beatriz G; Martínez-Ruiz, Antonio; Zaragoza, Carlos; Lamas, Santiago; Tryggvason, Karl; Apte, Suneel S; Arroyo, Alicia G

    2007-10-15

    Nitric oxide (NO) is essential for vascular homeostasis and is also a critical modulator of angiogenesis; however, the molecular mechanisms of NO action during angiogenesis remain elusive. We have investigated the potential relationship between NO and membrane type 1-matrix metalloproteinase (MT1-MMP) during endothelial migration and capillary tube formation. Endothelial NO synthase (eNOS) colocalizes with MT1-MMP at motility-associated structures in migratory human endothelial cells (ECs); moreover, NO is produced at these structures and is released into the medium during EC migration. We have therefore addressed 2 questions: (1) the putative regulation of MT1-MMP by NO in migratory ECs; and (2) the requirement for MT1-MMP in NO-induced EC migration and tube formation. NO upregulates MT1-MMP membrane clustering on migratory human ECs, and this is accompanied by increased degradation of type I collagen substrate. MT1-MMP membrane expression and localization are impaired in lung ECs from eNOS-deficient mice, and these cells also show impaired migration and tube formation in vitro. Inhibition of MT1-MMP with a neutralizing antibody impairs NOinduced tube formation by human ECs, and NO-induced endothelial migration and tube formation are impaired in lung ECs from mice deficient in MT1-MMP. MT1-MMP thus appears to be a key molecular effector of NO during the EC migration and angiogenic processes, and is a potential therapeutic target for NO-associated vascular disorders.

  16. 2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran promotes endothelial nitric oxide synthase activity in human endothelial cells.

    PubMed

    Ladurner, Angela; Atanasov, Atanas G; Heiss, Elke H; Baumgartner, Lisa; Schwaiger, Stefan; Rollinger, Judith M; Stuppner, Hermann; Dirsch, Verena M

    2012-09-15

    Endothelial nitric oxide synthase (eNOS) mediates important vaso-protective and immunomodulatory effects. Aim of this study was to examine whether lignan derivatives isolated from the roots of the anti-inflammatory medicinal plant Krameria lappacea influence eNOS activity and endothelial nitric oxide (NO) release. The study was performed using cultured human umbilical vein endothelial cells (HUVECs) and HUVEC-derived EA.hy926 cells. Among the eleven isolated compounds only 2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran (DPPB) was able to increase eNOS enzyme activity. DPPB (1-10 μM) treatment for 24 h induced a significant and dose-dependent increase in eNOS activity as determined by the [(14)C]L-arginine/[(14)C]L-citrulline conversion assay. Immunoblotting studies further revealed a time-dependent DPPB-induced increase in eNOS-Ser(1177) and decrease in eNOS-Thr(495) phosphorylation, as well as increased AMPK phosphorylation at Thr(172), whereas Akt phosphorylation at Ser(473) was not affected. Si-RNA-mediated knockdown of AMPK and inhibition of CaMKKβ by STO 609, as well as intracellular Ca(2+) chelation by Bapta AM abolished the stimulating effect of DPPB on eNOS-Ser(1177) and AMPK-Thr(172) phosphorylation. Furthermore, we could show that DPPB increases intracellular Ca(2+) concentrations assessed with the fluorescent dye Fluo-3-AM. DPPB enhances eNOS activity and endothelial NO release by raising intracellular Ca(2+) levels and increases signaling through a CaMKKβ-AMPK dependent pathway.

  17. Activation of Endothelial Nitric Oxide (eNOS) Occurs through Different Membrane Domains in Endothelial Cells

    PubMed Central

    Tran, Jason; Magenau, Astrid; Rodriguez, Macarena; Rentero, Carles; Royo, Teresa; Enrich, Carlos; Thomas, Shane R.; Grewal, Thomas; Gaus, Katharina

    2016-01-01

    Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF)-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells. PMID:26977592

  18. Activation of Endothelial Nitric Oxide (eNOS) Occurs through Different Membrane Domains in Endothelial Cells.

    PubMed

    Tran, Jason; Magenau, Astrid; Rodriguez, Macarena; Rentero, Carles; Royo, Teresa; Enrich, Carlos; Thomas, Shane R; Grewal, Thomas; Gaus, Katharina

    2016-01-01

    Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF)-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

  19. Arginase inhibition restores endothelial function in diet-induced obesity.

    PubMed

    Chung, Ji Hyung; Moon, Jiyoung; Lee, Youn Sue; Chung, Hye-Kyung; Lee, Seung-Min; Shin, Min-Jeong

    2014-08-22

    Arginase may play a major role in the regulation of vascular function in various cardiovascular disorders by impairing nitric oxide (NO) production. In the current study, we investigated whether supplementation of the arginase inhibitor N(ω)-hydroxy-nor-l-arginine (nor-NOHA) could restore endothelial function in an animal model of diet-induced obesity. Arginase 1 expression was significantly lower in the aorta of C57BL/6J mice fed a high-fat diet (HFD) supplemented with nor-NOHA (40mgkg(-1)/day) than in mice fed HFD without nor-NOHA. Arginase inhibition led to considerable increases in eNOS expression and NO levels and significant decreases in the levels of circulating ICAM-1. These findings were further confirmed by the results of siRNA-mediated knockdown of Arg in human umbilical vein endothelial cells. In conclusion, arginase inhibition can help restore dysregulated endothelial function by increasing the eNOS-dependent NO production in the endothelium, indicating that arginase could be a therapeutic target for correcting obesity-induced vascular endothelial dysfunction.

  20. Fo Shou San, an ancient Chinese herbal decoction, protects endothelial function through increasing endothelial nitric oxide synthase activity.

    PubMed

    Bi, Cathy W C; Xu, Li; Tian, Xiao Yu; Liu, Jian; Zheng, Ken Y Z; Lau, Chi Wai; Lau, David T W; Choi, Roy C Y; Dong, Tina T X; Huang, Yu; Tsim, Karl W K

    2012-01-01

    Fo Shou San (FSS) is an ancient herbal decoction comprised of Chuanxiong Rhizoma (CR; Chuanxiong) and Angelicae Sinensis Radix (ASR; Danggui) in a ratio of 2:3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh)-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS) scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs), and it also stimulated the production of nitric oxide (NO) as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS) were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca(2+) levels in HUVECs, and the Ca(2+) chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation. The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca(2+) and a reduction of ROS.

  1. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells.

    PubMed Central

    Papapetropoulos, A; García-Cardeña, G; Madri, J A; Sessa, W C

    1997-01-01

    Vascular endothelial growth factor (VEGF) is a regulator of vasculogenesis and angiogenesis. To investigate the role of nitric oxide (NO) in VEGF-induced proliferation and in vitro angiogenesis, human umbilical vein endothelial cells (HUVEC) were used. VEGF stimulated the growth of HUVEC in an NO-dependent manner. In addition, VEGF promoted the NO-dependent formation of network-like structures in HUVEC cultured in three dimensional (3D) collagen gels. Exposure of cells to VEGF led to a concentration-dependent increase in cGMP levels, an indicator of NO production, that was inhibited by nitro-L-arginine methyl ester. VEGF-stimulated NO production required activation of tyrosine kinases and increases in intracellular calcium, since tyrosine kinase inhibitors and calcium chelators attenuated VEGF-induced NO release. Moreover, two chemically distinct phosphoinositide 3 kinase (PI-3K) inhibitors attenuated NO release after VEGF stimulation. In addition, HUVEC incubated with VEGF for 24 h showed an increase in the amount of endothelial NO synthase (eNOS) protein and the release of NO. In summary, both short- and long-term exposure of human EC to VEGF stimulates the release of biologically active NO. While long-term exposure increases eNOS protein levels, short-term stimulation with VEGF promotes NO release through mechanisms involving tyrosine and PI-3K kinases, suggesting that NO mediates aspects of VEGF signaling required for EC proliferation and organization in vitro. PMID:9399960

  2. Endothelial nitric oxide synthase mediates lymphangiogenesis and lymphatic metastasis

    PubMed Central

    Lahdenranta, Johanna; Hagendoorn, Jeroen; Padera, Timothy P.; Hoshida, Tohru; Nelson, Gregory; Kashiwagi, Satoshi; Jain, Rakesh K.; Fukumura, Dai

    2009-01-01

    Lymphatic metastasis is a critical determinant of cancer prognosis. Recently, several lymphangiogenic molecules such as vafscular endothelial growth factor (VEGF)-C and -D were identified. However, the mechanistic understanding of lymphatic metastasis is still in infancy. Nitric oxide (NO) plays a crucial role in regulating blood vessel growth and function as well as lymphatic vessel function. NOS expression correlates with lymphatic metastasis. However, causal relationship between NOS and lymphatic metastasis has not been documented. To this end, we first show that both VEGF receptor-2 and -3 stimulation activate eNOS in lymphatic endothelial cells and that NO donors induce proliferation and/or survival of cultured lymphatic endothelial cells in a dose dependent manner. We find that an NOS inhibitor L-NMMA blocked regeneration of lymphatic vessels. Using intravital microscopy that allows us to visualize the steps of lymphatic metastasis, we show that genetic deletion of eNOS as well as NOS blockade attenuates peritumor lymphatic hyperplasia of VEGF-C-overexpressing T241 fibrosarcomas and decreases the delivery of metastatic tumor cells to the draining lymph nodes. Genetic deletion of eNOS in the host also leads to a decrease in T241 tumor cell dissemination to the lymph nodes and macroscopic lymph node metastasis of B16F10 melanoma. These findings indicate that eNOS mediates VEGF-C induced lymphangiogenesis and, consequently, plays a critical role in lymphatic metastasis. Our findings explain the correlation between NOS and lymphatic metastasis seen in a number of human tumors and open the door for potential therapies exploiting NO signaling to treat diseases of the lymphatic system. PMID:19318557

  3. Endothelial nitric oxide production stimulated by the bioflavonoid chrysin in rat isolated aorta.

    PubMed

    Villar, Inmaculada Concepción; Vera, Rocío; Galisteo, Milagros; O'Valle, Francisco; Romero, Miguel; Zarzuelo, Antonio; Duarte, Juan

    2005-09-01

    In the present study, the effects of the bioflavonoid chrysin (5,7-dihydroxyflavone) were analysed on nitric oxide (NO) production from vascular endothelium. In aortic rings, incubation with chrysin or acetylcholine (both at 10 microM) increased L-NAME-sensitive endothelial NO release as measured using the fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA). Moreover, chrysin increased cGMP accumulation only in aortic rings with endothelium. However, at this concentration, chrysin had no effect either on basal or on NADPH-stimulated vascular superoxide production. Moreover, at this low concentration, chrysin, similar to acetylcholine, induced aortic relaxation, which was abolished by both endothelial deprivation and NO synthase inhibition. Endothelium-dependent relaxation induced by chrysin was unaltered by removal of extracellular calcium and incubation with the intracellular calcium chelator BAPTA, while the phosphatidylinositol (PI)-3 kinase inhibitor wortmannin suppressed the endothelial dependence. In conclusion, chrysin stimulated NO release from endothelial cells leading to vascular cGMP accumulation and subsequent endothelium dependent aortic relaxation. Chrysin-stimulated NO release is calcium independent and possibly mediated via PI3-kinase.

  4. Monocyte-induced downregulation of nitric oxide synthase in cultured aortic endothelial cells.

    PubMed

    Marczin, N; Antonov, A; Papapetropoulos, A; Munn, D H; Virmani, R; Kolodgie, F D; Gerrity, R; Catravas, J D

    1996-09-01

    Since endothelium-dependent vasodilation is altered in atherosclerosis and enhanced monocyte/endothelial interactions are implicated in early atherosclerosis, we evaluated the effects of monocytes on the endothelial nitric oxide (NO) pathway by estimating release of biologically active NO from cultured endothelial cells and levels of constitutive NO synthase (ecNOS). NO release was estimated in a short-term bioassay using endothelial cell-induced cGMP accumulation in vascular smooth muscle (SM) cells. Exposure of SM cells to porcine aortic endothelial cells (PAECs) and human aortic endothelial cells (HAECs) produced large increases in SM cGMP content; this increase was prevented by NG-nitro-L-arginine methyl ester, the inhibitor of endothelial NOS. Confluent monolayers of PAECs and HAECs cocultured with monocytes also stimulated SM cGMP formation; however, NO release from these cultures was attenuated in a coculture time (2 to 48 hours)- and monocyte concentration (20 to 200 x 10(3) per well)-dependent manner. This effect of monocyte adhesion appeared to be selective for NO release since other biochemical pathways, such as atriopeptin-and isoproterenol-induced cyclic nucleotide accumulation within the endothelial cells, were not altered by monocytes. The effects of adherent monocytes on NO release were mimicked by monocyte-derived cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 alpha. Furthermore, the conditioned medium of monocytes contained significant quantities of these cytokines. Conditioned medium, as well as monocytes physically separated from the endothelial cells, attenuated NO release, suggesting that soluble factors may mediate the effects of monocytes. An IL-1 beta neutralizing antibody fully prevented the NO dysfunction in response to directly adherent monocytes. Superoxide dismutase, catalase, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), and exogenous L-arginine failed to improve NO release, suggesting that oxidant stress

  5. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase

    PubMed Central

    Ruisanchez, Éva; Dancs, Péter; Kerék, Margit; Németh, Tamás; Faragó, Bernadett; Balogh, Andrea; Patil, Renukadevi; Jennings, Brett L.; Liliom, Károly; Malik, Kafait U.; Smrcka, Alan V.; Tigyi, Gabor; Benyó, Zoltán

    2014-01-01

    Lysophosphatidic acid (LPA) has been implicated as a mediator of several cardiovascular functions, but its potential involvement in the control of vascular tone is obscure. Here, we show that both LPA (18:1) and VPC31143 (a synthetic agonist of LPA1–3 receptors) relax intact mouse thoracic aorta with similar Emax values (53.9 and 51.9% of phenylephrine-induced precontraction), although the EC50 of LPA- and VPC31143-induced vasorelaxations were different (400 vs. 15 nM, respectively). Mechanical removal of the endothelium or genetic deletion of endothelial nitric oxide synthase (eNOS) not only diminished vasorelaxation by LPA or VPC31143 but converted it to vasoconstriction. Freshly isolated mouse aortic endothelial cells expressed LPA1, LPA2, LPA4 and LPA5 transcripts. The LPA1,3 antagonist Ki16425, the LPA1 antagonist AM095, and the genetic deletion of LPA1, but not that of LPA2, abolished LPA-induced vasorelaxation. Inhibition of the phosphoinositide 3 kinase–protein kinase B/Akt pathway by wortmannin or MK-2206 failed to influence the effect of LPA. However, pharmacological inhibition of phospholipase C (PLC) by U73122 or edelfosine, but not genetic deletion of PLCε, abolished LPA-induced vasorelaxation and indicated that a PLC enzyme, other than PLCε, mediates the response. In summary, the present study identifies LPA as an endothelium-dependent vasodilator substance acting via LPA1, PLC, and eNOS.—Ruisanchez, É., Dancs, P., Kerék, M., Németh, T., Faragó, B., Balogh, A., Patil, R., Jennings, B. L., Liliom, K., Malik, K. U., Smrcka, A. V., Tigyi, G., Benyó, Z. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase. PMID:24249637

  6. Telmisartan activates endothelial nitric oxide synthase via Ser1177 phosphorylation in vascular endothelial cells.

    PubMed

    Myojo, Masahiro; Nagata, Daisuke; Fujita, Daishi; Kiyosue, Arihiro; Takahashi, Masao; Satonaka, Hiroshi; Morishita, Yoshiyuki; Akimoto, Tetsu; Nagai, Ryozo; Komuro, Issei; Hirata, Yasunobu

    2014-01-01

    Because endothelial nitric oxide synthase (eNOS) has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177) in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172) and eNOS and the concentration of intracellular guanosine 3',5'-cyclic monophosphate (cGMP). Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

  7. Telmisartan Activates Endothelial Nitric Oxide Synthase via Ser1177 Phosphorylation in Vascular Endothelial Cells

    PubMed Central

    Myojo, Masahiro; Nagata, Daisuke; Fujita, Daishi; Kiyosue, Arihiro; Takahashi, Masao; Satonaka, Hiroshi; Morishita, Yoshiyuki; Akimoto, Tetsu; Nagai, Ryozo; Komuro, Issei; Hirata, Yasunobu

    2014-01-01

    Because endothelial nitric oxide synthase (eNOS) has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177) in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172) and eNOS and the concentration of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP). Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling. PMID:24827148

  8. Endothelial nitric oxide synthase in the amphibian, Xenopus tropicalis.

    PubMed

    Trajanovska, Sofie; Donald, John A

    2011-04-01

    Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS (nNOS, nos1), inducible NOS (iNOS, nos2), and endothelial NOS (eNOS, nos3). This study utilised the genome of Xenopus tropicalis to sequence a nos3 cDNA and determine if eNOS protein is expressed in blood vessels. A nos3 cDNA was sequenced that encoded a 1177 amino acid protein called XteNOS, which showed closest sequence identity to mammalian eNOS protein. The X. tropicalis nos3 gene and eNOS protein were determined to be an orthologue of mammalian nos3 and eNOS using gene synteny and phylogenetic analyses, respectively. In X. tropicalis, nos3 mRNA expression was highest in lung and skeletal muscle and lower in the liver, gut, kidney, heart and brain. Western analysis of kidney protein using an affinity-purified anti-XteNOS produced a single band at 140kDa. Immunohistochemistry showed XteNOS immunoreactivity in the proximal tubule of the kidney and endocardium of the heart, but not in the endothelium of blood vessels. Thus, X. tropicalis has a nos3 gene that appears not to be expressed in the vascular endothelium.

  9. Human leucocytes in asthenozoospermic patients: endothelial nitric oxide synthase expression.

    PubMed

    Buldreghini, E; Hamada, A; Macrì, M L; Amoroso, S; Boscaro, M; Lenzi, A; Agarwal, A; Balercia, G

    2014-12-01

    In a basic study at the Andrology Unit, Department of Clinical and Molecular Sciences, Polytechnic University of Marche, Ancona, Italy, we evaluated the pattern of mRNA endothelial nitric oxide synthase (eNOS) expression in human blood leucocytes isolated from normozoospermic fertile and asthenozoospermic infertile men to elucidate any pathogenic involvement in sperm cell motility. Forty infertile men with idiopathic asthenozoospermia and 45 normozoospermic fertile donors, age-matched, were included. Semen parameters were evaluated, and expression analysis of mRNA was performed in human leucocytes using reverse transcription polymerase chain reaction. Sperm volume, count, motility and morphology were determined, and eNOS expression and Western blotting analyses were performed. A positive correlation was observed between the concentrations of NO and the percentage of immotile spermatozoa. The mRNA of eNOS was more expressed in peripheral blood leucocytes isolated from asthenozoospermic infertile men versus those of fertile normozoospermic men (7.46 ± 0.38 versus 7.06 ± 0.56, P = 0.0355). A significant up-regulation of eNOS gene in peripheral blood leucocytes was 1.52-fold higher than that of fertile donors. It is concluded that eNOS expression and activity are enhanced in blood leucocytes in men with idiopathic asthenozoospermia.

  10. Endothelial dysfunction in DOCA-salt-hypertensive mice: role of neuronal nitric oxide synthase-derived hydrogen peroxide.

    PubMed

    Silva, Grazielle C; Silva, Josiane F; Diniz, Thiago F; Lemos, Virginia S; Cortes, Steyner F

    2016-06-01

    Endothelial dysfunction is a common problem associated with hypertension and is considered a precursor to the development of micro- and macro-vascular complications. The present study investigated the involvement of nNOS (neuronal nitric oxide synthase) and H2O2 (hydrogen peroxide) in the impaired endothelium-dependent vasodilation of the mesenteric arteries of DOCA (deoxycorticosterone acetate)-salt-hypertensive mice. Myograph studies were used to investigate the endothelium-dependent vasodilator effect of ACh (acetylcholine). The expression and phosphorylation of nNOS and eNOS (endothelial nitric oxide synthase) were studied by Western blot analysis. Immunofluorescence was used to examine the localization of nNOS and eNOS in the endothelial layer of the mesenteric artery. The vasodilator effect of ACh is strongly impaired in mesenteric arteries of DOCA-salt-hypertensive mice. Non-selective inhibition of NOS sharply reduced the effect of ACh in both DOCA-salt-hypertensive and sham mice. Selective inhibition of nNOS and catalase led to a higher reduction in the effect of ACh in sham than in DOCA-salt-hypertensive mice. Production of H2O2 induced by ACh was significantly reduced in vessels from DOCA-salt-hypertensive mice, and it was blunted after nNOS inhibition. The expression of both eNOS and nNOS was considerably lower in DOCA-salt-hypertensive mice, whereas phosphorylation of their inhibitory sites was increased. The presence of nNOS was confirmed in the endothelial layer of mesenteric arteries from both sham and DOCA-salt-hypertensive mice. These results demonstrate that endothelial dysfunction in the mesenteric arteries of DOCA-salt-hypertensive mice is associated with reduced expression and functioning of nNOS and impaired production of nNOS-derived H2O2 Such findings offer a new perspective for the understanding of endothelial dysfunction in hypertension.

  11. Suppression of endothelial nitric oxide synthase expression and endothelial cell proliferation by an intronic 27-ntmiRNA and it's a novel link to AP-1.

    PubMed

    Li, Yumei; Yan, Limei; Zhang, Wenyu; Hu, Nan; Chen, Wei; Wang, Hui; Kang, Min; Ou, Hesheng

    2015-01-01

    This study aims to investigate the role of activator protein 1 (AP-1) in the effects of 27nt-miRNA on expression of endothelial nitric oxide synthase (eNOS) gene and proliferation of endothelial cells. Cell proliferation was analyzed by cell number counting, colony formation assay and MTT assay. Cell migration and invasion was detected by transwell assay and invasion assay. Expression of eNOS and AP-1 was measured by real-time RT-PCR (mRNA level) and Western blotting (protein level). Luciferase reporter assay was performed to detect the binding of 27nt-miRNA to AP-1. Overexpression of 27nt-miRNA significantly inhibited endothelial cells proliferation, invasion and migration in vitro. And, eNOS and AP-1 expression at mRNA and protein levels were down-regulated by overexpression of 27nt-miRNA. Interestingly, overexpression of AP-1 protein partially restored eNOS expression and endothelial cell proliferation. Furthermore, the luciferase reporter assay demonstrated that AP-1 was a direct target of 27nt-miRNA. These data demonstrate that overexpression of 27nt-miRNA inhibits endothelial cell proliferation, invasion, migration, eNOS expression and AP-1 expression. Moreover, AP-1, a direct target of 27nt-miRNA, reverses the inhibitory effects of 27nt-miRNA. Thus, the effects of 27nt-miRNA might be acted through targeting AP-1.

  12. Endothelial surface glycocalyx can regulate flow-induced nitric oxide production in microvessels in vivo.

    PubMed

    Yen, Wanyi; Cai, Bin; Yang, Jinlin; Zhang, Lin; Zeng, Min; Tarbell, John M; Fu, Bingmei M

    2015-01-01

    Due to its unique location, the endothelial surface glycocalyx (ESG) at the luminal side of the microvessel wall may serve as a mechano-sensor and transducer of blood flow and thus regulate endothelial functions. To examine this role of the ESG, we used fluorescence microscopy to measure nitric oxide (NO) production in post-capillary venules and arterioles of rat mesentery under reduced (low) and normal (high) flow conditions, with and without enzyme pretreatment to remove heparan sulfate (HS) of the ESG and in the presence of an endothelial nitric oxide synthase (eNOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA). Rats (SD, 250-300 g) were anesthetized. The mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. An individual post-capillary venule or arteriole was cannulated and loaded for 45 min with 5 μM 4, 5-Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO production was measured for ~10 min under a low flow (~300 μm/s) and for ~60 min under a high flow (~1000 μm/s). In the 15 min after switching to the high flow, DAF-2-NO fluorescence intensity increased to 1.27-fold of its baseline, DAF-2-NO continuously increased under the high flow, to 1.53-fold of its baseline in 60 min. Inhibition of eNOS by 1 mM L-NMMA attenuated the flow-induced NO production to 1.13-fold in 15 min and 1.30-fold of its baseline in 60 min, respectively. In contrast, no significant increase in NO production was observed after switching to the high flow for 60 min when 1 h pretreatment with 50 mU/mL heparanase III to degrade the ESG was applied. Similar NO production was observed in arterioles under low and high flows and under eNOS inhibition. Our results suggest that ESG participates in endothelial cell mechanosensing and transduction through its heparan sulfate to activate eNOS.

  13. Activation of endothelial nitric oxide synthase is dependent on its interaction with globular actin in human umbilical vein endothelial cells.

    PubMed

    Mi, Qiongyu; Chen, Nan; Shaifta, Yasin; Xie, Liping; Lu, Hui; Liu, Zhen; Chen, Qi; Hamid, Colleen; Becker, Silke; Ji, Yong; Ferro, Albert

    2011-09-01

    Endothelial nitric oxide synthase (eNOS) has been reported to associate with globular actin, and this association increases eNOS activity. Adenosine, histamine, salbutamol and thrombin cause activation of eNOS through widely different mechanisms. Whether these eNOS agonists can regulate eNOS activity through affecting its association with actin is unknown. As previously reported, we confirmed in cultured human umbilical vein endothelial cells (HUVEC) that histamine and thrombin increased intracellular Ca(2+) whereas adenosine and salbutamol did not, and that these four agonists caused different effects on actin filament structure. Nevertheless, despite their divergent effects on intracellular Ca(2+) and on actin filament structure, we found by immunoprecipitation that adenosine, histamine, salbutamol and thrombin all caused an increase in association between eNOS and globular actin. This increase of association was inhibited by pre-treatment with phalloidin, an actin filament stabilizer. All of these agonists also increased phosphorylation of eNOS on serine residue 1177, eNOS activity, and cyclic guanosine-3', 5'-monophosphate, and these increases were all attenuated by phalloidin. Agonist-induced phosphorylation of eNOS on serine 1177 was attenuated by Akt inhibition, whereas association of eNOS with actin was not. We also found, in HEK-293 cells transfected with the eNOS mutants eNOS-S1177A or eNOS-S1177D, that the association between eNOS and globular actin was decreased as compared to cells transfected with wild-type eNOS. We conclude that association of globular actin with eNOS plays an essential and necessary role in agonist-induced eNOS activation, through enabling its phosphorylation by Akt at serine residue 1177.

  14. Receptor-mediated activation of nitric oxide synthesis by arginine in endothelial cells.

    PubMed

    Joshi, Mahesh S; Ferguson, T Bruce; Johnson, Fruzsina K; Johnson, Robert A; Parthasarathy, Sampath; Lancaster, Jack R

    2007-06-12

    Arginine contains the guanidinium group and thus has structural similarity to ligands of imidazoline and alpha-2 adrenoceptors (alpha-2 AR). Therefore, we investigated the possibility that exogenous arginine may act as a ligand for these receptors in human umbilical vein endothelial cells and activate intracellular nitric oxide (NO) synthesis. Idazoxan, a mixed antagonist of imidazoline and alpha-2 adrenoceptors, partly inhibited L-arginine-initiated NO formation as measured by a Griess reaction. Rauwolscine, a highly specific antagonist of alpha-2 AR, at very low concentrations completely inhibited NO formation. Like L-arginine, agmatine (decarboxylated arginine) also activated NO synthesis, however, at much lower concentrations. We found that dexmedetomidine, a specific agonist of alpha-2 AR was very potent in activating cellular NO, thus indicating a possible role for alpha-2 AR in L-arginine-mediated NO synthesis. D-arginine also activated NO production and could be inhibited by imidazoline and alpha-2 AR antagonists, thus indicating nonsubstrate actions of arginine. Pertussis toxin, an inhibitor of G proteins, attenuated L-arginine-mediated NO synthesis, thus indicating mediation via G proteins. L-type Ca(2+) channel blocker nifedipine and phospholipase C inhibitor U73122 inhibited NO formation and thus implicated participation of a second messenger pathway. Finally, in isolated rat gracilis vessels, rauwolscine completely inhibited the L-arginine-initiated vessel relaxation. Taken together, these data provide evidence for binding of arginine to membrane receptor(s), leading to the activation of endothelial NO synthase (eNOS) NO production through a second messenger pathway. These findings provide a previously unrecognized mechanistic explanation for the beneficial effects of L-arginine in the cardiovascular system and thus provide new potential avenues for therapeutic development.

  15. Receptor-mediated activation of nitric oxide synthesis by arginine in endothelial cells

    PubMed Central

    Joshi, Mahesh S.; Ferguson, T. Bruce; Johnson, Fruzsina K.; Johnson, Robert A.; Parthasarathy, Sampath; Lancaster, Jack R.

    2007-01-01

    Arginine contains the guanidinium group and thus has structural similarity to ligands of imidazoline and α-2 adrenoceptors (α-2 AR). Therefore, we investigated the possibility that exogenous arginine may act as a ligand for these receptors in human umbilical vein endothelial cells and activate intracellular nitric oxide (NO) synthesis. Idazoxan, a mixed antagonist of imidazoline and α-2 adrenoceptors, partly inhibited l-arginine-initiated NO formation as measured by a Griess reaction. Rauwolscine, a highly specific antagonist of α-2 AR, at very low concentrations completely inhibited NO formation. Like l-arginine, agmatine (decarboxylated arginine) also activated NO synthesis, however, at much lower concentrations. We found that dexmedetomidine, a specific agonist of α-2 AR was very potent in activating cellular NO, thus indicating a possible role for α-2 AR in l-arginine-mediated NO synthesis. d-arginine also activated NO production and could be inhibited by imidazoline and α-2 AR antagonists, thus indicating nonsubstrate actions of arginine. Pertussis toxin, an inhibitor of G proteins, attenuated l-arginine-mediated NO synthesis, thus indicating mediation via G proteins. l-type Ca2+ channel blocker nifedipine and phospholipase C inhibitor U73122 inhibited NO formation and thus implicated participation of a second messenger pathway. Finally, in isolated rat gracilis vessels, rauwolscine completely inhibited the l-arginine-initiated vessel relaxation. Taken together, these data provide evidence for binding of arginine to membrane receptor(s), leading to the activation of endothelial NO synthase (eNOS) NO production through a second messenger pathway. These findings provide a previously unrecognized mechanistic explanation for the beneficial effects of l-arginine in the cardiovascular system and thus provide new potential avenues for therapeutic development. PMID:17535904

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

    PubMed Central

    Li, Peng; Yin, Ya-Ling; Guo, Tao; Sun, Xue-Ying; Ma, Hui; Zhu, Mo-Li; Zhao, Fan-Rong; Xu, Ping; Chen, Yuan; Wan, Guang-Rui; Jiang, Fan; Peng, Qi-Sheng; Liu, Chao; Liu, Li-Ying

    2016-01-01

    Background: 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. Methods: 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. Results: 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. Conclusions: 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. PMID:27765794

  17. Endothelial nitric oxide synthase activation and nitric oxide function: new light through old windows.

    PubMed

    Bird, Ian M

    2011-09-01

    The principle mechanisms operating at the level of endothelial nitric oxide synthase (eNOS) itself to control its activity are phosphorylation, the auto-regulatory properties of the protein itself, and Ca(2)(+)/calmodulin binding. It is now clear that activation of eNOS is greatest when phosphorylation of certain serine and threonine residues is accompanied by elevation of cytosolic [Ca2+](i). While eNOS also contains an autoinhibitory loop, Rafikov et al. (2011) present the evidence for a newly identified 'flexible arm' that operates in response to redox state. Boeldt et al. (2011) also review the evidence that changes in the nature of endothelial Ca(2)(+) signaling itself in different physiologic states can extend both the amplitude and duration of NO output, and a failure to change these responses in pregnancy is associated with preeclampsia. The change in Ca(2)(+) signaling is mediated through altering capacitative entry mechanisms inherent in the cell, and so many agonist responses using this mechanism are altered. The term 'adaptive cell signaling' is also introduced for the first time to describe this phenomenon. Finally NO is classically regarded as a regulator of vascular function, but NO has other actions. One proposed role is regulation of steroid biosynthesis but the physiologic relevance was unclear. Ducsay & Myers (2011) now present new evidence that NO may provide the adrenal with a mechanism to regulate cortisol output according to exposure to hypoxia. One thing all three of these reviews show is that even after several decades of study into NO biosynthesis and function, there are clearly still many things left to discover.

  18. Integrins mediate mechanical compression-induced endothelium-dependent vasodilation through endothelial nitric oxide pathway.

    PubMed

    Lu, Xiao; Kassab, Ghassan S

    2015-09-01

    Cardiac and skeletal muscle contraction lead to compression of intramuscular arterioles, which, in turn, leads to their vasodilation (a process that may enhance blood flow during muscle activity). Although endothelium-derived nitric oxide (NO) has been implicated in compression-induced vasodilation, the mechanism whereby arterial compression elicits NO production is unclear. We cannulated isolated swine (n = 39) myocardial (n = 69) and skeletal muscle (n = 60) arteriole segments and exposed them to cyclic transmural pressure generated by either intraluminal or extraluminal pressure pulses to simulate compression in contracting muscle. We found that the vasodilation elicited by internal or external pressure pulses was equivalent; moreover, vasodilation in response to pressure depended on changes in arteriole diameter. Agonist-induced endothelium-dependent and -independent vasodilation was used to verify endothelial and vascular smooth muscle cell viability. Vasodilation in response to cyclic changes in transmural pressure was smaller than that elicited by pharmacological activation of the NO signaling pathway. It was attenuated by inhibition of NO synthase and by mechanical removal of the endothelium. Stemming from previous observations that endothelial integrin is implicated in vasodilation in response to shear stress, we found that function-blocking integrin α5β1 or αvβ3 antibodies attenuated cyclic compression-induced vasodilation and NOx (NO(-)2 and NO(-)3) production, as did an RGD peptide that competitively inhibits ligand binding to some integrins. We therefore conclude that integrin plays a role in cyclic compression-induced endothelial NO production and thereby in the vasodilation of small arteries during cyclic transmural pressure loading.

  19. Integrins mediate mechanical compression–induced endothelium-dependent vasodilation through endothelial nitric oxide pathway

    PubMed Central

    Lu, Xiao

    2015-01-01

    Cardiac and skeletal muscle contraction lead to compression of intramuscular arterioles, which, in turn, leads to their vasodilation (a process that may enhance blood flow during muscle activity). Although endothelium-derived nitric oxide (NO) has been implicated in compression-induced vasodilation, the mechanism whereby arterial compression elicits NO production is unclear. We cannulated isolated swine (n = 39) myocardial (n = 69) and skeletal muscle (n = 60) arteriole segments and exposed them to cyclic transmural pressure generated by either intraluminal or extraluminal pressure pulses to simulate compression in contracting muscle. We found that the vasodilation elicited by internal or external pressure pulses was equivalent; moreover, vasodilation in response to pressure depended on changes in arteriole diameter. Agonist-induced endothelium-dependent and -independent vasodilation was used to verify endothelial and vascular smooth muscle cell viability. Vasodilation in response to cyclic changes in transmural pressure was smaller than that elicited by pharmacological activation of the NO signaling pathway. It was attenuated by inhibition of NO synthase and by mechanical removal of the endothelium. Stemming from previous observations that endothelial integrin is implicated in vasodilation in response to shear stress, we found that function-blocking integrin α5β1 or αvβ3 antibodies attenuated cyclic compression–induced vasodilation and NOx (NO−2 and NO−3) production, as did an RGD peptide that competitively inhibits ligand binding to some integrins. We therefore conclude that integrin plays a role in cyclic compression–induced endothelial NO production and thereby in the vasodilation of small arteries during cyclic transmural pressure loading. PMID:26324675

  20. Endothelial nitric oxide synthase mediates the nitric oxide component of reflex cutaneous vasodilatation during dynamic exercise in humans.

    PubMed

    McNamara, Tanner C; Keen, Jeremy T; Simmons, Grant H; Alexander, Lacy M; Wong, Brett J

    2014-12-01

    Recent data suggests neuronal nitric oxide synthase (nNOS) mediates the NO component of reflex cutaneous vasodilatation with passive heat stress. We tested the hypothesis that nNOS inhibition would attenuate reflex cutaneous vasodilatation during sustained dynamic exercise in young healthy humans. All subjects first performed an incremental V̇O2, peak test to exhaustion on a custom-built supine cycle ergometer. On a separate day, subjects were instrumented with four intradermal microdialysis fibres on the forearm and each randomly assigned as: (1) lactated Ringer's (control); (2) 20 mm Nω-nitro-l-arginine methyl ester hydrochloride (non-selective NOS inhibitor); (3) 5 mm N-propyl-l-arginine (nNOS inhibitor); and (4) 10 mm N(5)-(1-iminoethyl)-l-ornithine dihydrochloride [endothelial NOS (eNOS) inhibitor]. Following microdialysis placement, subjects performed supine cycling with the experimental arm at heart level at 60% V̇O2, peak for a period sufficient to raise core temperature 0.8°C. At the end of cycling, all microdialysis sites were locally heated to 43°C and sodium nitroprusside was perfused to elicit maximal vasodilatation. Mean arterial pressure, skin blood flow via laser-Doppler flowmetry and core temperature via ingestible telemetric pill were measured continuously; cutaneous vascular conductance (CVC) was calculated as laser-Doppler flowmetry/mean arterial pressure and normalized to maximum. There was no significant difference between control (58 ± 2%CVCmax) and nNOS-inhibited (56 ± 3%CVCmax) sites in response to exercise-induced hyperthermia. The increase in CVC at eNOS-inhibited (41 ± 3%CVCmax) and non-selective NOS-inhibited (40 ± 4%CVCmax) sites were significantly attenuated compared to control and nNOS-inhibited (P < 0.001 all conditions) but there was no difference between eNOS-inhibited and non-selective NOS-inhibited sites. These data suggest eNOS, not nNOS, mediate NO synthesis during reflex cutaneous vasodilatation with

  1. Copper enhances EDNO (endothelium-derived nitric oxide) activity by cultured human vascular endothelial cells.

    PubMed

    Kishimoto, T; Oguri, T; Ueda, D; Tada, M

    1996-06-01

    The effect of copper sulfate (Cu) on viable cell number, endothelium-derived nitric oxide (EDNO), and nitric oxide synthase (NOS) in cultured human umbilical vascular endothelial cells (HUVEC) was investigated. The viable cell number was not affected by the addition of Cu (1.0-500.0 microM). To assess the effect of EDNO by HUVEC, platelet aggregation experiments were performed, using cuvettes lined with HUVEC. Thrombin (0.05 units/ml)-induced platelet aggregation was markedly inhibited in the presence of HUVEC compared with aggregation in the absence of HUVEC. The HUVEC-dependent anti-platelet aggregatory effect was slightly reduced when HUVEC were pretreated with indomethacin (IND; 1.0 micro M), an inhibitor of the cyclo-oxygenase pathway. However, the thrombin-induced platelet aggregation in the presence of HUVEC pretreated with IND was smaller than that in the absence of HUVEC, which is dependent on EDNO. The anti-platelet aggregatory effect of HUVEC pretreated with IND was increased dose-dependently by 48-hour pretreatment of HUVEC with Cu (1.0-100.0 microM). To assess the effect of Cu on NOS, HUVEC were stained with NOS/NADPH diaphorase. However, there were no significant differences in the NOS-positive HUVEC cell count between cells without Cu and those with various concentrations of Cu. These findings suggest that Cu stimulates the activity of EDNO, which action may be dependent on Cu decreasing EDNO-oxidative damage.

  2. Angiotensin II activates endothelial constitutive nitric oxide synthase via AT1 receptors.

    PubMed

    Saito, S; Hirata, Y; Emori, T; Imai, T; Marumo, F

    1996-09-01

    To determine whether angiotensin (ANG) II, a vasoconstrictor hormone, activates constitutive nitric oxide synthase (cNOS) in endothelial cells (ECs), we investigated the cellular mechanism by which ANG II induces nitric oxide (NO) formation in cultured bovine ECs. ANG II rapidly (within 1 min) and dose-dependently (10(-9)-10(-6) M) increased nitrate/nitrite (NOx) production. This effect of ANG II was abolished by a NOS inhibitor, NG-monomethyl-L-arginine. An ANG II type 1 (AT1) receptor antagonist (DuP 753), but not an ANG II type 2 (AT2) receptor antagonist (PD 123177), dose-dependently inhibited ANG II-induced NOx production. A Ca(2+)-channel blocker (barnidipine) failed to affect ANG II-induced NOx production, whereas an intracellular Ca2+ chelator (BAPTA) and a calmodulin inhibitor (W-7) abolished NOx production induced by ANG II. A protein kinase C (PKC) inhibitor (H-7) and down-regulation of endogenous PKC after pretreatment with phorbol ester decreased NOx production stimulated by ANG II. ANG II transiently stimulated inositol 1,4,5-trisphosphate (IP3) formation, and increased cytosolic free Ca2+ concentrations; these effects were blocked by DuP 753. Our data demonstrate that ANG II stimulates NO release by activation of Ca2+/calmodulin-dependent cNOS via AT1 receptors in bovine ECs.

  3. Suppressive Role of PPARγ-Regulated Endothelial Nitric Oxide Synthase in Adipocyte Lipolysis

    PubMed Central

    Yamada, Yoko; Eto, Masato; Ito, Yuki; Mochizuki, Satoru; Son, Bo-Kyung; Ogawa, Sumito; Iijima, Katsuya; Kaneki, Masao; Kozaki, Koichi; Toba, Kenji; Akishita, Masahiro; Ouchi, Yasuyoshi

    2015-01-01

    Introduction Metabolic syndrome causes insulin resistance and is associated with risk factor clustering, thereby increasing the risk of atherosclerosis. Recently, endothelial nitric oxide synthase deficient (eNOS-/-) mice have been reported to show metabolic disorders. Interestingly, eNOS has also been reported to be expressed in non-endothelial cells including adipocytes, but the functions of eNOS in adipocytes remain unclear. Methods and Results The eNOS expression was induced with adipocyte differentiation and inhibition of eNOS/NO enhanced lipolysis in vitro and in vivo. Furthermore, the administration of a high fat diet (HFD) was able to induce non-alcoholic steatohepatitis (NASH) in eNOS-/- mice but not in wild type mice. A PPARγ antagonist increased eNOS expression in adipocytes and suppressed HFD-induced fatty liver changes. Conclusions eNOS-/- mice induce NASH development, and these findings provide new insights into the therapeutic approach for fatty liver disease and related disorders. PMID:26317347

  4. Pomegranate juice reduces oxidized low-density lipoprotein downregulation of endothelial nitric oxide synthase in human coronary endothelial cells.

    PubMed

    de Nigris, Filomena; Williams-Ignarro, Sharon; Botti, Chiara; Sica, Vincenzo; Ignarro, Louis J; Napoli, Claudio

    2006-11-01

    We examined the hypothesis that pomegranate juice (PJ) can revert the potent downregulation of the expression of endothelial nitric-oxide synthase (NOSIII) induced by oxidized low-density liporotein (oxLDL) in human coronary endothelial cells. Western blot and Northern blot analyses showed a significant decrease of NOSIII expression after a 24-h treatment with oxLDL. Accordingly, we observed a significant dose-dependent reduction in nitric oxide bioactivity represented by both basal and bradykinin-stimulated cellular cGMP accumulation. These phenomena were corrected significantly by the concomitant treatment with PJ. Our data suggest that PJ can exert beneficial effects on the evolution of clinical vascular complications, coronary heart disease, and atherogenesis in humans by enhancing the NOSIII bioactivity.

  5. Nitric Oxide Inhibits Coxiella burnetii Replication and Parasitophorous Vacuole Maturation

    PubMed Central

    Howe, Dale; Barrows, Lorraine F.; Lindstrom, Nicole M.; Heinzen, Robert A.

    2002-01-01

    Nitric oxide is a recognized cytotoxic effector against facultative and obligate intracellular bacteria. This study examined the effect of nitric oxide produced by inducible nitric oxide synthase (iNOS) up-regulated in response to cytokine stimulation, or by a synthetic nitric oxide donor, on replication of obligately intracellular Coxiella burnetii in murine L-929 cells. Immunoblotting and nitrite assays revealed that C. burnetii infection of L-929 cells augments expression of iNOS up-regulated in response to gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Infection in the absence of cytokine stimulation did not result in demonstrable up-regulation of iNOS expression or in increased nitrite production. Nitrite production by cytokine-treated cells was significantly inhibited by the iNOS inhibitor S-methylisothiourea (SMT). Treatment of infected cells with IFN-γ and TNF-α or the synthetic nitric oxide donor 2,2′-(hydroxynitrosohydrazino)bis-ethanamine (DETA/NONOate) had a bacteriostatic effect on C. burnetii replication. Inhibition of replication was reversed upon addition of SMT to the culture medium of cytokine-treated cells. Microscopic analysis of infected cells revealed that nitric oxide (either cytokine induced or donor derived) inhibited formation of the mature (large) parasitophorous vacuole that is characteristic of C. burnetii infection of host cells. Instead, exposure of infected cells to nitric oxide resulted in the formation of multiple small, acidic vacuoles usually containing one C. burnetii cell. Removal of nitrosative stress resulted in the coalescence of small vacuoles to form a large vacuole harboring multiple C. burnetii cells. These experiments demonstrate that nitric oxide reversibly inhibits replication of C. burnetii and formation of the parasitophorous vacuole. PMID:12183564

  6. Hypoxic relaxation of penile arteries: involvement of endothelial nitric oxide and modulation by reactive oxygen species

    PubMed Central

    Kaminski, Pawel M.; Bagi, Zsolt; Ahmad, Mansoor; Wolin, Michael S.

    2010-01-01

    Although obesity-related cardiovascular disease and hypoxia are associated with erectile dysfunction, little is known about the direct effects of hypoxia on penile arteries. In the present study, the effects of acute hypoxia (Po2 = ∼10 Torr, 20 min) were investigated in isolated penile arteries to determine the influence of endothelium removal, nitric oxide (NO) synthase (NOS), cyclooxygenase (COX), NADPH oxidase, changes in reactive oxygen species (ROS), and a high-fat diet. Hypoxia-relaxed penile arteries contracted with phenylephrine by ∼50%. Relaxation to hypoxia and acetylcholine was reduced by endothelium removal and by inhibition of NOS (Nω-nitro-l-arginine) and COX (indomethacin) but was enhanced by Tempol and by NADPH oxidase inhibition with apocynin and gp91ds-tat. Basal superoxide levels detected by lucigenin chemiluminescence were reduced by Tempol and gp91ds-tat and were enhanced by NOS blockade. Hypoxic relaxant responses were enhanced by catalase and ebselen. Exogenous peroxide evoked relaxations of penile arteries, which were partially inhibited by endothelium removal and by the inhibition of COX and extracellular signal-regulated mitogen-activated protein kinase (MAPK) but enhanced by p38 MAPK blockade. The NO-dependent component of relaxation to hypoxia was impaired in penile arteries from high-fat diet-fed, obese rats associated with increased superoxide production. Thus hypoxic relaxation of penile arteries is partially mediated by endothelial NO in a manner that is normally attenuated by endogenous ROS production. Obesity further increases superoxide production and impairs the influence of NO. Therefore, cardiovascular disease involving decreased NO bioavailability and/or enhanced ROS generation may contribute to erectile dysfunction through impairing the relaxation of penile arteries to hypoxia. PMID:20581086

  7. Delta- and gamma-tocotrienol isomers are potent in inhibiting inflammation and endothelial activation in stimulated human endothelial cells

    PubMed Central

    Muid, Suhaila; Froemming, Gabriele R. Anisah; Rahman, Thuhairah; Ali, A. Manaf; Nawawi, Hapizah M.

    2016-01-01

    Background Tocotrienols (TCTs) are more potent antioxidants than α-tocopherol (TOC). However, the effectiveness and mechanism of the action of TCT isomers as anti-atherosclerotic agents in stimulated human endothelial cells under inflammatory conditions are not well established. Aims 1) To compare the effects of different TCT isomers on inflammation, endothelial activation, and endothelial nitric oxide synthase (eNOS). 2) To identify the two most potent TCT isomers in stimulated human endothelial cells. 3) To investigate the effects of TCT isomers on NFκB activation, and protein and gene expression levels in stimulated human endothelial cells. Methods Human umbilical vein endothelial cells were incubated with various concentrations of TCT isomers or α-TOC (0.3–10 µM), together with lipopolysaccharides for 16 h. Supernatant cells were collected and measured for protein and gene expression of cytokines (interleukin-6, or IL-6; tumor necrosis factor-alpha, or TNF-α), adhesion molecules (intercellular cell adhesion molecule-1, or ICAM-1; vascular cell adhesion molecule-1, or VCAM-1; and e-selectin), eNOS, and NFκB. Results δ-TCT is the most potent TCT isomer in the inhibition of IL-6, ICAM-1, VCAM-1, and NFκB, and it is the second potent in inhibiting e-selectin and eNOS. γ-TCT isomer is the most potent isomer in inhibiting e-selectin and eNOS, and it is the second most potent in inhibiting is IL-6, VCAM-1, and NFκB. For ICAM-1 protein expression, the most potent is δ-TCT followed by α-TCT. α- and β-TCT inhibit IL-6 at the highest concentration (10 µM) but enhance IL-6 at lower concentrations. γ-TCT markedly increases eNOS expression by 8–11-fold at higher concentrations (5–10 µM) but exhibits neutral effects at lower concentrations. Conclusion δ- and γ-TCT are the two most potent TCT isomers in terms of the inhibition of inflammation and endothelial activation whilst enhancing eNOS, possibly mediated via the NFκB pathway. Hence, there is a

  8. Tissue-engineered endothelial cell layers on surface-modified Ti for inhibiting in vitro platelet adhesion

    NASA Astrophysics Data System (ADS)

    Wang, Xiupeng; He, Fupo; Li, Xia; Ito, Atsuo; Sogo, Yu; Maruyama, Osamu; Kosaka, Ryo; Ye, Jiandong

    2013-06-01

    A tissue-engineered endothelial layer was prepared by culturing endothelial cells on a fibroblast growth factor-2 (FGF-2)-l-ascorbic acid phosphate magnesium salt n-hydrate (AsMg)-apatite (Ap) coated titanium plate. The FGF-2-AsMg-Ap coated Ti plate was prepared by immersing a Ti plate in supersaturated calcium phosphate solutions supplemented with FGF-2 and AsMg. The FGF-2-AsMg-Ap layer on the Ti plate accelerated proliferation of human umbilical vein endothelial cells (HUVECs), and showed slightly higher, but not statistically significant, nitric oxide release from HUVECs than on as-prepared Ti. The endothelial layer maintained proper function of the endothelial cells and markedly inhibited in vitro platelet adhesion. The tissue-engineered endothelial layer formed on the FGF-2-AsMg-Ap layer is promising for ameliorating platelet activation and thrombus formation on cardiovascular implants.

  9. Role of Rutin on Nitric Oxide Synthesis in Human Umbilical Vein Endothelial Cells

    PubMed Central

    Zakaria, Zaiton; Chua, Kien Hui; Megat Mohd Nordin, Nor Anita; Abdullah Mahdy, Zaleha

    2014-01-01

    Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is a major antiatherogenic factor in the blood vessel. Oxidative stress plays an important role in the pathogenesis of various cardiovascular diseases, including atherosclerosis. Decreased availability of endothelial NO promotes the progression of endothelial dysfunction and atherosclerosis. Rutin is a flavonoid with multiple cardiovascular protective effects. This study aimed to investigate the effects of rutin on eNOS and NO production in cultured human umbilical vein endothelial cells (HUVEC). HUVEC were divided into four groups: control; oxidative stress induction with 180 μM H2O2; treatment with 300 μM rutin; and concomitant induction with rutin and H2O2 for 24 hours. HUVEC treated with rutin produced higher amount of NO compared to control (P < 0.01). In the oxidative stress-induced HUVEC, rutin successfully induced cells' NO production (P < 0.01). Rutin promoted NO production in HUVEC by inducing eNOS gene expression (P < 0.05), eNOS protein synthesis (P < 0.01), and eNOS activity (P < 0.05). Treatment with rutin also led to increased gene and protein expression of basic fibroblast growth factor (bFGF) in HUVEC. Therefore, upregulation of eNOS expression by rutin may be mediated by bFGF. The results showed that rutin may improve endothelial function by augmenting NO production in human endothelial cells. PMID:25093198

  10. Hypoxia and Reoxygenation Induce Endothelial Nitric Oxide Synthase Uncoupling in Endothelial Cells through Tetrahydrobiopterin Depletion and S-Glutathionylation

    PubMed Central

    2015-01-01

    Ischemia-reperfusion injury is accompanied by endothelial hypoxia and reoxygenation that trigger oxidative stress with enhanced superoxide generation and diminished nitric oxide (NO) production leading to endothelial dysfunction. Oxidative depletion of the endothelial NO synthase (eNOS) cofactor tetrahydrobiopterin can trigger eNOS uncoupling, in which the enzyme generates superoxide rather than NO. Recently, it has also been shown that oxidative stress can induce eNOS S-glutathionylation at critical cysteine residues of the reductase site that serves as a redox switch to control eNOS coupling. While superoxide can deplete tetrahydrobiopterin and induce eNOS S-glutathionylation, the extent of and interaction between these processes in the pathogenesis of eNOS dysfunction in endothelial cells following hypoxia and reoxygenation remain unknown. Therefore, studies were performed on endothelial cells subjected to hypoxia and reoxygenation to determine the severity of eNOS uncoupling and the role of cofactor depletion and S-glutathionylation in this process. Hypoxia and reoxygenation of aortic endothelial cells triggered xanthine oxidase-mediated superoxide generation, causing both tetrahydrobiopterin depletion and S-glutathionylation with resultant eNOS uncoupling. Replenishing cells with tetrahydrobiopterin along with increasing intracellular levels of glutathione greatly preserved eNOS activity after hypoxia and reoxygenation, while targeting either mechanism alone only partially ameliorated the decrease in NO. Endothelial oxidative stress, secondary to hypoxia and reoxygenation, uncoupled eNOS with an altered ratio of oxidized to reduced glutathione inducing eNOS S-glutathionylation. These mechanisms triggered by oxidative stress combine to cause eNOS dysfunction with shift of the enzyme from NO to superoxide production. Thus, in endothelial reoxygenation injury, normalization of both tetrahydrobiopterin levels and the glutathione pool are needed for maximal

  11. Danggui Buxue Tang, Chinese Herbal Decoction Containing Astragali Radix and Angelicae Sinensis Radix, Induces Production of Nitric Oxide in Endothelial Cells: Signaling Mediated by Phosphorylation of Endothelial Nitric Oxide Synthase.

    PubMed

    Gong, Amy G W; Lau, K M; Zhang, Laura M L; Lin, H Q; Dong, Tina T X; Tsim, Karl W K

    2016-03-01

    Danggui Buxue Tang, an ancient Chinese herbal decoction containing Astragali Radix and Angelicae Sinensis Radix at the weight ratio of 5:1, is used to mitigate menopausal syndromes in women. The pharmacological properties of Danggui Buxue Tang have been illustrated in bone development, blood enhancement, and immune stimulation. Here, we extended the possible pharmacological role of Danggui Buxue Tang in cardiovascular function. In cultured human umbilical vein endothelial cells, the application of Danggui Buxue Tang induced the release of nitric oxide and the phosphorylation of endothelial nitric oxide synthase and Akt kinase in time- and dose-dependent manners. The robust activation of nitric oxide signaling, however, required the boiling of Astragali Radix and Angelicae Sinensis Radix together, i.e., as Danggui Buxue Tang instead of other herbal extracts. The Danggui Buxue Tang-induced phosphorylation of endothelial nitric oxide synthase and Akt kinase in human umbilical vein endothelial cells were fully blocked by treatment with an endothelial nitric oxide synthase inhibitor (L-NAME), a PI3K/Akt inhibitor (LY294002), and a Ca(2+) chelator (BAPTA-AM). In parallel, the blockage of endothelial nitric oxide synthase and Akt activation subsequently fully abolished the Danggui Buxue Tang-induced nitric oxide production.

  12. Effects of venous needle turbulence during ex vivo hemodialysis on endothelial morphology and nitric oxide formation.

    PubMed

    Huynh, Thanh N; Chacko, Balu K; Teng, Xinjun; Brott, Brigitta C; Allon, Michael; Kelpke, Stacey S; Thompson, John A; Patel, Rakesh P; Anayiotos, Andreas S

    2007-01-01

    Arteriovenous grafts used for hemodialysis frequently develop intimal hyperplasia (IH), which ultimately leads to graft failure. Although the turbulent jet from the dialysis needle may contribute to vessel wall injury, its role in the pathogenesis of IH is relatively unexplored. In the current study, using bovine aortic endothelial cells (BAEC) cultured on the inner surface of a compliant tube, we evaluated the effects of simulated hemodialysis conditions on morphology and nitric oxide (NO) production. The flows via the graft and needle were 500 ml/min (Reynolds number=819) and 100ml/min (Reynolds number=954), respectively. In the presence of the needle jet for 6h, 19.3% (+/-1.53%) of BAEC were sheared off, whereas no loss of BAEC was observed in the presence of graft flow alone (P<0.05). In the presence of graft flow alone, assessment of cell orientation by the Saltykov method revealed that BAEC were oriented along the flow direction. This alignment, however, was lost in the presence of needle flow. Finally, NO production was also significantly decreased in the presence of the needle flow compared to the presence of graft flow alone (16+/-3.1 vs 34.7+/-1.9 nmol/10(6)cells/h, P<0.05). NO is a key player in vascular homeostasis mechanisms modulating vasomotor tone, inhibiting inflammation and smooth muscle cell proliferation. Thus, the loss of NO signaling and the loss of endothelial integrity caused by needle jet turbulence may contribute to the cascade of events leading to IH formation during hemodialysis.

  13. Pim1 kinase promotes angiogenesis through phosphorylation of endothelial nitric oxide synthase at Ser-633

    PubMed Central

    Chen, Ming; Yi, Bing; Zhu, Ni; Wei, Xin; Zhang, Guan-Xin; Huang, Shengdong; Sun, Jianxin

    2016-01-01

    Aims Posttranslational modification, such as phosphorylation, plays an essential role in regulating activation of endothelial NO synthase (eNOS). In the present study, we aim to determine whether eNOS could be phosphorylated and regulated by a novel serine/threonine–protein kinase Pim1 in vascular endothelial cells (ECs). Methods and results Using immunoprecipitation and protein kinase assays, we demonstrated that Pim1 specifically interacts with eNOS, which leads to a marked phosphorylation of eNOS at Ser-633 and increased production of nitric oxide (NO). Intriguingly, in response to VEGF stimulation, eNOS phosphorylation at Ser-633 exhibits two distinct phases: transient phosphorylation occurring between 0 and 60 min and sustained phosphorylation occurring between 2 and 24 h, which are mediated by the protein kinase A (PKA) and Pim1, respectively. Inhibiting Pim1 by either pharmacological inhibitor SMI-4a or the dominant-negative form of Pim1 markedly attenuates VEGF-induced tube formation, while Pim1 overexpression significantly increases EC tube formation and migration in an NO-dependent manner. Importantly, Pim1 expression and eNOS phosphorylation at Ser-633 were substantially decreased in high glucose-treated ECs and in the aorta of db/db diabetic mice. Increased Pim1 expression ameliorates impaired vascular angiogenesis in diabetic mice, as determined by an ex vivo aortic ring assay. Conclusion Our findings demonstrate Pim1 as a novel kinase that is responsible for the phosphorylation of eNOS at Ser-633 and enhances EC sprouting of aortic rings from diabetic mice, suggesting that Pim1 could potentially serve as a novel therapeutic target for revascularization strategies. PMID:26598507

  14. Immunohistochemical localization of endothelial nitric oxide synthase in endometrial tissue of women with unexplained infertility

    PubMed Central

    Najafi, Tohid; Ghaffari Novin, Marefat; Pakravesh, Jalil; Foghi, Khadijeh; Fadayi, Fatemeh; Rahimi, Gelareh

    2012-01-01

    Background: Nitric oxide (NO) is a molecule that incorporates in many physiological processes of female reproductive system. Recent studies suggested the possible role of endothelial isoform of nitric oxide synthase (eNOS) enzyme in female infertility. Objective: The aim of this study is to evaluate the expression of endothelial nitric oxide synthase in endometrial tissue of women with unexplained infertility. Materials and Methods: In this case-control study a total of 18 endometrial tissues obtained from 10 women with unexplained infertility and 8 normal and fertile women by endometrial biopsy, 6 to 10 days after LH surge. Specimens were fixed in 4% paraformaldhyde fixative and frozen sectioned for semi-quantitative immunohistochemical evaluation using monoclonal anti-human eNOS antibody. Hematoxilin and Eosin was used for Histological dating. Results: Localization of endothelial nitric oxide synthase was seen in glandular and luminal epithelium, vascular endothelium and stroma in both fertile women and women with unexplained infertility. Although there were differences in immunoreactivity of glandular epithelium (p=0.44), vascular endothelium (p=0.60) and stroma (p=0.63) but only over-expression of eNOS in luminal epithelium (p=0.045) of women with unexplained infertility compared to fertile women was statistically significant (p<0.05). Conclusion: This study suggests that changes in luminal expression of eNOS may influence receptivity of endometrium. PMID:25242984

  15. Activation of endothelial nitric oxide synthase in contralateral testis during unilateral testicular torsion in rats.

    PubMed

    Shiraishi, K; Yoshida, K; Naito, K

    2003-01-01

    There are controversies about the injury of the contralateral testis during unilateral testicular torsion (UTT). An autonomic reflex arc between bilateral testes has been proposed. The authors focused on the involvement of nitric oxide (NO) in the contralateral testis during UTT. Eight-week-old male Wistar rats underwent unilateral torsion (1 h)-detorsion (up to 24 h). NO synthase (NOS) activity was detected as NADPH-diaphorase activity after fixation by paraformaldehyde. N-nitro-L-Arginine methyl ester (L-NAME, 20 mg/kg) was injected intravenously to the other group of rats. To evaluate the testicular injury, proteolysis of alpha-fodrin production was detected by Western blotting. Apoptosis of the germ cells was evaluated by TUNEL. Long-term effect on spermatogenesis was evaluated by flow cytometry at 60 days after UTT. Transient activation of NOS was detected following the proteolysis of alpha-fodrin in the contralateral testis. L-NAME inhibited these alterations. NADPH-diaphorase activity and eNOS immunoreactivity were co-localized in the endothelial cells. These reactions were not observed in other organs. There was neither enhanced apoptosis nor deteriorated spermatogenesis in the contralateral testis during and 60 days after UTT. In the contralateral testis, eNOS-derived NO regulates the vasomotor function against unilateral testicular torsion, whereas it acts slightly cytotoxic. These results suggest the possible involvement of a testis-specific neurovasomotor reflex between the bilateral testes.

  16. Organochlorine Pesticide-Mediated Induction of NADPH Oxidase and Nitric-Oxide Synthase in Endothelial Cell

    PubMed Central

    Ghosh, Rishila; Siddharth, Manushi; Singh, Neeru; Kare, Pawan Kumar; Banerjee, Basu Dev; Wadhwa, Neelam

    2017-01-01

    Introduction Organochlorine Pesticides (OCPs) are detected ubiquitously in human and have been shown to be associated with Cardiovascular Disease (CVD) and atherosclerosis. Aim To find out the effect of organochlorine pesticides in endothelial cell with regard to oxidative stress and associated expression of enzymes producing superoxide and Nitric Oxide (NO). Materials and Methods Human Umbilical Vein Endothelial Cells (HUVEC) were cultured and treated with four OCPs which were found in human blood at a concentration of 0.1μM. The cells were tested for Reactive Oxygen Species (ROS) generation, NO production and mRNA expression of NAPDH oxidase (p47phox) and endothelial Nitric Oxide Synthase (eNOS). ROS generation was measured by using 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA) method. NO was analysed by Bioxytech nitric oxide assay kit method and mRNA of NADPH oxidase and eNOS was quantified by real time PCR. Data were expressed as the mean±SEM. Comparison between the groups were made by student’s t-test (2-tailed) or one-way ANOVA with Tukey’s post-hoc analysis depending on number of groups. For all statistical tests, p< 0.05 was considered to be significant. Results All the four pesticides generated ROS accompanied by enhanced expression of NADPH oxidase. Maximum effect was observed with β-endosulfan. Level of NO was found to be decreased significantly in endothelial cells treated with these pesticides accompanied by enhanced expression of eNOS. The antioxidant N-acetylcysteine (NAC) reduced ROS generation and enhanced NO formation. Pesticide-mediated ROS generation possibly reacts with NO forming peroxinitrite and thereby reducing the bioavailability of NO although eNOS expression is increased. Conclusion OCPs induce endothelial dysfunction through increased ROS generation via NADPH oxidase expression and reduced bioavailability of nitric oxide. PMID:28273962

  17. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: Essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase

    SciTech Connect

    Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj; Oh, Seok Jeong; Lee, Moo Yeol; Kang, Keon Wook

    2010-08-01

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10 {mu}g/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  18. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase.

    PubMed

    Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj; Oh, Seok Jeong; Lee, Moo Yeol; Kang, Keon Wook

    2010-08-01

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10μg/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  19. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    SciTech Connect

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K. )

    1990-11-15

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling.

  20. CHOP deficiency inhibits methylglyoxal-induced endothelial dysfunction.

    PubMed

    Choi, Yoon Young; Kim, Suji; Han, Jung-Hwa; Nam, Dae-Hwan; Park, Kwon Moo; Kim, Seong Yong; Woo, Chang-Hoon

    2016-11-18

    Epidemiological studies suggested that diabetic patients are susceptible to develop cardiovascular complications along with having endothelial dysfunction. It has been suggested that methylglyoxal (MGO), a glycolytic metabolite, has more detrimental effects on endothelial dysfunction rather than glucose itself. Here, we investigated the molecular mechanism by which MGO induces endothelial dysfunction via the regulation of ER stress. Biochemical data showed that 4-PBA significantly inhibited MGO-induced protein cleavages of PARP-1 and caspase-3. In addition, it was found that high glucose-induced endothelial apoptosis was enhanced in the presence of GLO1 inhibitor, suggesting the role of endogenous MGO in high glucose-induced endothelial dysfunction. MGO-induced endothelial apoptosis was significantly diminished by the depletion of CHOP with si-RNA against human CHOP, but not by SP600125, a specific inhibitor of JNK. The physiological relevance of this signaling pathway was demonstrated in CHOP deficiency mouse model, in which instillation of osmotic pump containing MGO led to aortic endothelial dysfunction. Notably, the aortic endothelial dysfunction response to MGO infusion was significantly improved in CHOP deficiency mice compared to littermate control. Taken together, these findings indicate that MGO specifically induces endothelial dysfunction in a CHOP-dependent manner, suggesting the therapeutic potential of CHOP inhibition in diabetic cardiovascular complications.

  1. Alpha-1-antitrypsin inhibits nitric oxide production.

    PubMed

    Chan, Edward D; Pott, Gregory B; Silkoff, Philip E; Ralston, Annemarie H; Bryan, Courtney L; Shapiro, Leland

    2012-12-01

    NO is an endogenously produced gas that regulates inflammation, vascular tone, neurotransmission, and immunity. NO production can be increased by exposing cells to several endogenous and exogenous proinflammatory mediators, including IFN-γ, TNF-α, IL-1β, and LPS. As AAT has been shown to inhibit cell activation and suppress cytokine production associated with proinflammatory stimulation, we examined AAT for NO-suppressive function. In RAW 264.7 murine macrophagic cells, physiological AAT concentrations significantly inhibited combined LPS- and IFN-γ-induced NO synthesis, and NO synthesis inhibition was associated with decreased expression of iNOS, suppressed NF-κB activation, and reduced translocation of extracellular AAT into the interior of RAW 264.7 cells. CE-2072, a synthetic inhibitor of serine proteases, also suppressed NO production, iNOS expression, and NF-κB activation. However, AAT did not alter activation of intracellular MAPKs. In subjects with genetic AAT deficiency, exhaled NO was increased significantly compared with exhaled NO in healthy controls. These in vitro and in vivo studies suggest that AAT is an endogenous inhibitor of NO production. Administering AAT or AAT-like molecules may have use as a treatment for diseases associated with excessive NO production.

  2. Estrogen-like effects of wine extracts on nitric oxide synthesis in human endothelial cells.

    PubMed

    Simoncini, Tommaso; Lenzi, Elena; Zöchling, Alfred; Gopal, Santhosh; Goglia, Lorenzo; Russo, Eleonora; Polak, Kinga; Casarosa, Elena; Jungbauer, Alois; Genazzani, Alessandro D; Genazzani, Andrea R

    2011-10-01

    Endothelial dysfunction frequently ensues during the climacteric due to hormonal and metabolic changes. Non-pharmacological interventions such as lifestyle and dietary modifications are emerging as valuable strategies to counteract the cardiovascular consequences of ageing. A number of chemical components of wine, including alcohol and some polyphenols, are known to be active on the vessels. However, the molecular mechanisms through which they modulate endothelial function are largely unclear. The aim of this study was to investigate the effects of non-alcoholic wine fractions from five different wines on the synthesis of nitric oxide (NO) via the expression and enzymatic activation of the endothelial nitric oxide synthase (eNOS) in human endothelial cells. All non-alcoholic fractions studied increased NO synthesis, although with different potencies. All wine extracts maximally enhanced NO production at doses in the range achieved with a moderate wine intake, with decreasing effects with further increases of the dose. Interestingly, a part of these actions was recruited via estrogen receptors (ERs). Within the polyphenols with known binding activity for ERs contained in the tested wines, resveratrol, epicatechin, syringic acid, apigenin, malvidin and ellagic acid were largely responsible for eNOS activation. These findings show that some of the non-alcoholic components of wine enhance the production of NO by the vessels acting on ERs, and suggest that a moderate intake of wine may benefit the cardiovascular system through estrogen-like effects.

  3. Fat-derived factor omentin stimulates endothelial cell function and ischemia-induced revascularization via endothelial nitric oxide synthase-dependent mechanism.

    PubMed

    Maruyama, Sonomi; Shibata, Rei; Kikuchi, Ryosuke; Izumiya, Yasuhiro; Rokutanda, Taku; Araki, Satoshi; Kataoka, Yoshiyuki; Ohashi, Koji; Daida, Hiroyuki; Kihara, Shinji; Ogawa, Hisao; Murohara, Toyoaki; Ouchi, Noriyuki

    2012-01-02

    Obesity-related diseases are associated with vascular dysfunction and impaired revascularization. Omentin is a fat-derived secreted protein, which is down-regulated in association with obese complications. Here, we investigated whether omentin modulates endothelial cell function and revascularization processes in vitro and in vivo. Systemic delivery of an adenoviral vector expressing omentin (Ad-omentin) enhanced blood flow recovery and capillary density in ischemic limbs of wild-type mice in vivo, which were accompanied by increased phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). In cultured human umbilical vein endothelial cells (HUVECs), a physiological concentration of recombinant omentin protein increased differentiation into vascular-like structures and decreased apoptotic activity under conditions of serum starvation. Treatment with omentin protein stimulated the phosphorylation of Akt and eNOS in HUVECs. Inhibition of Akt signaling by treatment with dominant-negative Akt or LY294002 blocked the stimulatory effects of omentin on differentiation and survival of HUVECs and reversed omentin-stimulated eNOS phosphorylation. Pretreatment with the NOS inhibitor also reduced the omentin-induced increase in HUVEC differentiation and survival. Omentin protein also stimulated the phosphorylation of AMP-activated protein kinase in HUVECs. Transduction with dominant-negative AMP-activated protein kinase diminished omentin-induced phosphorylation of Akt and omentin-stimulated increase in HUVEC differentiation and survival. Of importance, in contrast to wild-type mice, systemic administration of Ad-omentin did not affect blood flow in ischemic muscle in eNOS-deficient mice in vivo. These data indicate that omentin promotes endothelial cell function and revascularization in response to ischemia through its ability to stimulate an Akt-eNOS signaling pathway.

  4. Trans Fatty Acids Induce Vascular Inflammation and Reduce Vascular Nitric Oxide Production in Endothelial Cells

    PubMed Central

    Iwata, Naomi G.; Pham, Matilda; Rizzo, Norma O.; Cheng, Andrew M.; Maloney, Ezekiel; Kim, Francis

    2011-01-01

    Intake of trans fatty acids (TFA), which are consumed by eating foods made from partially hydrogenated vegetable oils, is associated with a higher risk of cardiovascular disease. This relation can be explained by many factors including TFA's negative effect on endothelial function and reduced nitric oxide (NO) bioavailability. In this study we investigated the effects of three different TFA (2 common isomers of C18 found in partially hydrogenated vegetable oil and a C18 isomer found from ruminant-derived—dairy products and meat) on endothelial NF-κB activation and nitric oxide (NO) production. Human endothelial cells were treated with increasing concentrations of Elaidic (trans-C18:1 (9 trans)), Linoelaidic (trans-C18:2 (9 trans, 12 trans)), and Transvaccenic (trans-C18:1 (11 trans)) for 3 h. Both Elaidic and Linoelaidic acids were associated with increasing NF-κB activation as measured by IL-6 levels and phosphorylation of IκBα, and impairment of endothelial insulin signaling and NO production, whereas Transvaccenic acid was not associated with these responses. We also measured superoxide production, which has been hypothesized to be necessary in fatty acid-dependent activation of NF-κB. Both Elaidic acid and Linoelaidic acid are associated with increased superoxide production, whereas Transvaccenic acid (which did not induce inflammatory responses) did not increase superoxide production. We observed differential activation of endothelial superoxide production, NF-κB activation, and reduction in NO production by different C18 isomers suggesting that the location and number of trans double bonds effect endothelial NF-κB activation. PMID:22216328

  5. Expression and regulation of endothelial nitric oxide synthase by vascular endothelial growth factor in ECV 304 cells.

    PubMed Central

    Park, Jong Seon; Hong, Gu Ru; Baek, Suk Whan; Shin, Dong Gu; Kim, Young Jo; Shim, Bong Sup

    2002-01-01

    Nitric oxide (NO) seems to play a pivotal role in the vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation. This study was designed to investigate the role and intracellular signal pathway of endothelial nitric oxide synthase (eNOS) activation induced by VEGF. ECV 304 cells were treated with VEGF(165) and then cell proliferation, eNOS protein and mRNA expression levels were analyzed to elucidate the functional role of eNOS in cell proliferation induced by VEGF. After exposure of cells to VEGF(165), eNOS activity and cell growth were increased by approximately two-fold in the VEGF(165) -treated cells compared to the untreated cells. In addition, VEGF stimulated eNOS expression at both the mRNA and protein levels in a dose-dependent manner. Phosphatidylinositol-3 kinase (PI-3K) inhibitors were used to assess PI-3K involvement in eNOS regulation. LY294002 was found to attenuate VEGF-stimulated eNOS expression. Wortmannin was not as effective as LY294002, but the reduction effect was detectable. Cells activated by VEGF showed increased ERK1/2 levels. Moreover, the VEGF-induced eNOS expression was reduced by the PD98059, MAPK pathway inhibitor. This suggests that eNOS expression might be regulated by PI-3K and the ERK1/2 signaling pathway. In conclusion, VEGF(165) induces ECV 304 cell proliferation via the NO produced by eNOS. In addition, eNOS may be regulated by the PI-3K or mitogen-activated protein kinase pathway. PMID:11961297

  6. Globular adiponectin improves high glucose-suppressed endothelial progenitor cell function through endothelial nitric oxide synthase dependent mechanisms.

    PubMed

    Huang, Po-Hsun; Chen, Jia-Shiong; Tsai, Hsiao-Ya; Chen, Yung-Hsiang; Lin, Feng-Yen; Leu, Hsin-Bang; Wu, Tao-Cheng; Lin, Shing-Jong; Chen, Jaw-Wen

    2011-07-01

    Plasma levels of adiponectin, an adipose-specific protein with putative anti-atherogenic properties, could be down-regulated in obese and diabetic subjects. Recent insights suggest that the injured endothelial monolayer is regenerated by circulating endothelial progenitor cells (EPCs), but high glucose reduces number and functions of EPCs. Here, we tested the hypothesis that globular adiponectin can improve high glucose-suppressed EPC functions by restoration of endothelial nitric oxide synthase (eNOS) activity. Late EPCs isolated from healthy subjects appeared with cobblestone shape at 2-4 weeks. EPCs were incubated with high glucose (25 mM) and treatment with globular adiponectin for functional study. Migration and tube formation assays were used to evaluate the vasculogenetic capacity of EPCs. The activities of eNOS, Akt and concentrations of nitric oxide (NO) were also determined. Administration of globular adiponectin at physiological concentrations promoted EPC migration and tube formation, and dose-dependently upregulated phosphorylation of eNOS, Akt and augmented NO production. Chronic incubation of EPCs in high-glucose medium significantly impaired EPC function and induced cellular senescence, but these suppression effects were reversed by treatment with globular adiponectin. Globular adiponectin reversed high glucose-impaired EPC functions through NO- and p38 MAPK-related mechanisms. In addition, nude mice that received EPCs treated with adiponectin in high glucose medium showed a significant improvement in blood flow than those received normal saline and EPCs incubated in high glucose conditions. The administration of globular adiponectin improved high glucose-impaired EPC functions in vasculogenesis by restoration of eNOS activity. These beneficial effects may provide some novel rational to the vascular protective properties of adiponectin.

  7. Regional differences in the arterial response to vasopressin: role of endothelial nitric oxide.

    PubMed Central

    García-Villalón, A. L.; Garcia, J. L.; Fernández, N.; Monge, L.; Gómez, B.; Diéguez, G.

    1996-01-01

    different intensity; (b) the vasoconstriction to this peptide is mediated mainly by stimulation of V1 vasopressin receptors, and (c) endothelial nitric oxide may inhibit the vasoconstriction to this peptide, especially in coronary and renal vasculatures. PMID:8842453

  8. Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells.

    PubMed Central

    Hernández-Perera, O; Pérez-Sala, D; Navarro-Antolín, J; Sánchez-Pascuala, R; Hernández, G; Díaz, C; Lamas, S

    1998-01-01

    Endothelial dysfunction associated with atherosclerosis has been attributed to alterations in the L-arginine-nitric oxide (NO)-cGMP pathway or to an excess of endothelin-1 (ET-1). The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to ameliorate endothelial function. However, the physiological basis of this observation is largely unknown. We investigated the effects of Atorvastatin and Simvastatin on the pre-proET-1 mRNA expression and ET-1 synthesis and on the endothelial NO synthase (eNOS) transcript and protein levels in bovine aortic endothelial cells. These agents inhibited pre-proET-1 mRNA expression in a concentration- and time-dependent fashion (60-70% maximum inhibition) and reduced immunoreactive ET-1 levels (25-50%). This inhibitory effect was maintained in the presence of oxidized LDL (1-50 microg/ml). No significant modification of pre-proET-1 mRNA half-life was observed. In addition, mevalonate, but not cholesterol, reversed the statin-mediated decrease of pre-proET-1 mRNA levels. eNOS mRNA expression was reduced by oxidized LDL in a dose-dependent fashion (up to 57% inhibition), whereas native LDL had no effect. Statins were able to prevent the inhibitory action exerted by oxidized LDL on eNOS mRNA and protein levels. Hence, these drugs might influence vascular tone by modulating the expression of endothelial vasoactive factors. PMID:9637705

  9. Hyperhomocysteinaemia in rats is associated with erectile dysfunction by impairing endothelial nitric oxide synthase activity

    PubMed Central

    Jiang, Weijun; Xiong, Lei; Bin Yang; Li, Weiwei; Zhang, Jing; Zhou, Qing; Wu, Qiuyue; Li, Tianfu; Zhang, Cui; Zhang, Mingchao; Xia, Xinyi

    2016-01-01

    To investigate the effect of hyperhomocysteinaemia (HHCy) on penile erectile function in a rat model, a methionine-rich diet was used in which erectile function, the reproductive system, and nitric oxide synthase were characterized. The intracavernous pressure, apomorphine experiments, measurement of oxidative stress, hematoxylin and eosin staining, immunohistochemistry analysis, reverse transcription-polymerase chain reactions and measurement of endothelial nitric oxide synthase activity were utilized. Our results showed that erections in the middle-dose, high-dose, and interference (INF) groups were significantly lower than the control (P < 0.05). INF group, being fed with vitamins B and folic acid, demonstrated markedly improved penile erections compared with the middle-dose group (P < 0.05). HHCy-induced eNOS and phospho-eNOS protein expression was reduced and the antioxidant effect was markedly impaired. The data of the present data provide evidence that HHCy is a vascular risk factor for erectile dysfunction by impairing cavernosa endothelial nitric oxide synthase activity. Intake of vitamins B can alleviate this abnormality. PMID:27221552

  10. Inhibition of cyclooxygenase (COX)-2 affects endothelial progenitor cell proliferation

    SciTech Connect

    Colleselli, Daniela; Bijuklic, Klaudija; Mosheimer, Birgit A.; Kaehler, Christian M. . E-mail: C.M.Kaehler@uibk.ac.at

    2006-09-10

    Growing evidence indicates that inducible cyclooxygenase-2 (COX-2) is involved in the pathogenesis of inflammatory disorders and various types of cancer. Endothelial progenitor cells recruited from the bone marrow have been shown to be involved in the formation of new vessels in malignancies and discussed for being a key point in tumour progression and metastasis. However, until now, nothing is known about an interaction between COX and endothelial progenitor cells (EPC). Expression of COX-1 and COX-2 was detected by semiquantitative RT-PCR and Western blot. Proliferation kinetics, cell cycle distribution and rate of apoptosis were analysed by MTT test and FACS analysis. Further analyses revealed an implication of Akt phosphorylation and caspase-3 activation. Both COX-1 and COX-2 expression can be found in bone-marrow-derived endothelial progenitor cells in vitro. COX-2 inhibition leads to a significant reduction in proliferation of endothelial progenitor cells by an increase in apoptosis and cell cycle arrest. COX-2 inhibition leads further to an increased cleavage of caspase-3 protein and inversely to inhibition of Akt activation. Highly proliferating endothelial progenitor cells can be targeted by selective COX-2 inhibition in vitro. These results indicate that upcoming therapy strategies in cancer patients targeting COX-2 may be effective in inhibiting tumour vasculogenesis as well as angiogenic processes.

  11. Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

    NASA Technical Reports Server (NTRS)

    Boo, Yong Chool; Jo, Hanjoong

    2003-01-01

    Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.

  12. Hyperglycemic switch from mitochondrial nitric oxide to superoxide production in endothelial cells.

    PubMed

    Brodsky, Sergey V; Gao, Shujuan; Li, Hong; Goligorsky, Michael S

    2002-11-01

    The accumulated ultrastructural and biochemical evidence is highly suggestive of the existence of mitochondrial nitric oxide (NO) synthase (mtNOS), where local production of NO regulates the electron transport along the respiratory chain. Here, the functional competence of mtNOS in situ in a living cell was examined using an intravital fluorescent NO indicator, 4,5-diaminofluorescein, employing a new procedure for loading it into the mitochondria to demonstrate local NO generation in undisrupted endothelial cells and in isolated mitochondria as well as in human embryonic kidney cells stably expressing endothelial NOS. With the use of this approach, we showed that endothelial cells incubated in the presence of high concentration of D-glucose (but not L-glucose) are characterized by the reduced NO synthetic function of mitochondria despite the unaltered abundance of the enzyme. In parallel, mitochondrial generation of superoxide was augmented in endothelial cells incubated in the presence of a high concentration of D-glucose. Both the NO generation and superoxide production in hyperglycemic environment could be restored to control levels by treating cells with a cell-permeable superoxide dismutase mimetic. In addition, enhanced mitochondrial superoxide production could be suppressed with an inhibitor of NOS in stimulated endothelial cells. In conclusion, the data 1) provide direct evidence of mitochondrial NO production in endothelial cells, 2) demonstrate its suppression and enhanced superoxide generation in hyperglycemic environment, and 3) provide evidence that "uncoupled" mtNOS represents an important source of superoxide anions in endothelial cells incubated in high glucose-containing medium.

  13. Post-translational regulation of endothelial nitric oxide synthase in vascular endothelium

    PubMed Central

    Qian, Jin; Fulton, David

    2013-01-01

    Nitric oxide (NO) is a short-lived gaseous signaling molecule. In blood vessels, it is synthesized in a dynamic fashion by endothelial nitric oxide synthase (eNOS) and influences vascular function via two distinct mechanisms, the activation of soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)-dependent signaling and the S-nitrosylation of proteins with reactive thiols (S-nitrosylation). The regulation of eNOS activity and NO bioavailability is critical to maintain blood vessel function. The activity of eNOS and ability to generate NO is regulated at the transcriptional, posttranscriptional, and posttranslational levels. Post-translational modifications acutely impact eNOS activity and dysregulation of these mechanisms compromise eNOS activity and foster the development of cardiovascular diseases (CVDs). This review will intergrate past and current literature on the post-translational modifications of eNOS in both health and disease. PMID:24379783

  14. Spironolactone Prevents Endothelial Nitric Oxide Synthase Uncoupling and Vascular Dysfunction Induced by β-Adrenergic Overstimulation

    PubMed Central

    Victorio, Jamaira A.; Clerici, Stefano P.; Palacios, Roberto; Alonso, María J.; Vassallo, Dalton V.; Jaffe, Iris Z.; Rossoni, Luciana V.

    2016-01-01

    Sustained stimulation of β-adrenoceptors (β-ARs) and activation of renin–angiotensin–aldosterone system are common features of cardiovascular diseases with rising sympathetic activation, including essential hypertension, myocardial infarction, and heart failure. In this study, we investigated the role of AT1 receptor and mineralocorticoid receptor (MR) in the vascular alterations caused by β-AR overstimulation. β-AR overstimulation with associated cardiac hypertrophy and increased vasoconstrictor response to phenylephrine in aorta were modeled in rats by 7-day isoproterenol treatment. The increased vasoconstrictor response to phenylephrine in this model was blunted by the MR antagonist spironolactone, but not by the AT1 receptor antagonist losartan, despite the blunting of cardiac hypertrophy with both drugs. Spironolactone, but not losartan, restored NO bioavailability in association with lower endothelial nitric oxide synthase–derived superoxide production, increased endothelial nitric oxide synthase dimerization, and aortic HSP90 upregulation. MR genomic and nongenomic functions were activated in aortas from isoproterenol-treated rats. Isoproterenol did not modify plasma levels of MR ligands aldosterone and corticosterone but rather increased perivascular adipose tissue–derived corticosterone in association with increased expression of 11β-hydroxysteroid dehydrogenase type 1. The anticontractile effect of aortic perivascular adipose tissue was impaired by β-AR overstimulation and restored by MR blockade. These results suggest that activation of vascular MR signaling contributes to the vascular dysfunction induced by β-AR overstimulation associated with endothelial nitric oxide synthase uncoupling. These findings reveal an additional explanation for the protective effects of MR antagonists in cardiovascular disorders with sympathetic activation. PMID:27432866

  15. Angiotensin II AT1 receptor antagonists inhibit platelet adhesion and aggregation by nitric oxide release.

    PubMed

    Kalinowski, Leszek; Matys, Tomasz; Chabielska, Ewa; Buczko, Włodzimierz; Malinski, Tadeusz

    2002-10-01

    This study investigated the process of nitric oxide (NO) release from platelets after stimulation with different angiotensin II type 1 (AT1)-receptor antagonists and its effect on platelet adhesion and aggregation. Angiotensin II AT1-receptor antagonist-stimulated NO release in platelets was compared with that in human umbilical vein endothelial cells by using a highly sensitive porphyrinic microsensor. In vitro and ex vivo effects of angiotensin II AT1-receptor antagonists on platelet adhesion to collagen and thromboxane A2 analog U46619-induced aggregation were evaluated. Losartan, EXP3174, and valsartan alone caused NO release from platelets and endothelial cells in a dose-dependent manner in the range of 0.01 to 100 micro mol/L, which was attenuated by NO synthase inhibitor N(G)-nitro-L-arginine methyl ester. The angiotensin II AT1-receptor antagonists had more than 70% greater potency in NO release in platelets than in endothelial cells. The degree of inhibition of platelet adhesion (collagen-stimulated) and aggregation (U46619-stimulated) elicited by losartan, EXP3174, and valsartan, either in vitro or ex vivo, closely correlated with the NO levels produced by each of these drugs alone. The inhibiting effects of angiotensin II AT1-receptor antagonists on collagen-stimulated adhesion and U46619-stimulated aggregation of platelets were significantly reduced by pretreatment with N(G)-nitro-L-arginine methyl ester. Neither the AT2 receptor antagonist PD123319, the cyclooxygenase synthase inhibitor indomethacin, nor the selective thromboxane A2/prostaglandin H2 receptor antagonist SQ29,548 had any effect on angiotensin II AT1-receptor antagonist-stimulated NO release in platelets and endothelial cells. The presented studies clearly indicate a crucial role of NO in the arterial antithrombotic effects of angiotensin II AT1-receptor antagonists.

  16. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage.

    PubMed

    Gokay, Nevzat Selim; Yilmaz, Ibrahim; Komur, Baran; Demiroz, Ahu Senem; Gokce, Alper; Dervisoglu, Sergülen; Gokay, Banu Vural

    2016-01-01

    The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg), inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg), or nitric oxide precursor L-arginine (200 mg/kg). After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P = 0.044) positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  17. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    PubMed Central

    Gokay, Nevzat Selim; Yilmaz, Ibrahim; Demiroz, Ahu Senem; Gokce, Alper; Dervisoglu, Sergülen; Gokay, Banu Vural

    2016-01-01

    The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg), inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg), or nitric oxide precursor L-arginine (200 mg/kg). After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P = 0.044) positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders. PMID:27382570

  18. Endothelial-like nitric oxide synthase immunolocalization by using gold nanoparticles and dyes

    PubMed Central

    Gary, Ramla; Amelio, Daniela; Garofalo, Filippo; Petriashvili, Gia; De Santo, Maria Penelope; Ip, Yuen Kwong; Barberi, Riccardo

    2015-01-01

    Immunofluorescence is a biological technique that allows displaying the localization of the target molecule through a fluorescent microscope. We used a combination of gold nanoparticles and the fluorescein isothiocianate, FITC, as optical contrast agents for laser scanning confocal microscopy imaging to localize the endothelial-like nitric oxide synthase in skeletal muscle cells in a three-dimensional tissue phantom at the depth of 4µm. The FITC detected fluorescence intensity from gold-nanoparticles-labelled cells was brighter than the emission intensity from unlabelled cells. PMID:26713190

  19. Effects of a natural extract of Aronia Melanocarpa berry on endothelial cell nitric oxide production.

    PubMed

    Varela, Claudia Elena; Fromentin, Emilie; Roller, Marc; Villarreal, Francisco; Ramirez-Sanchez, Israel

    2016-08-01

    The effects of acute and chronic treatment with Aronia extracts on NO production and endothelial nitric oxide synthase (eNOS) phosphorylation in bovine coronary artery endothelial cells were investigated. Acute time-course and concentration-response experiments were performed to determine the time and concentration at which Aronia induced maximal NO synthesis and eNOS phosphorylation. The findings indicate that relatively low concentrations (0.1 μg/mL) of Aronia extract significantly induced NO synthesis and eNOS phosphorylation after 10 min of treatment. Increased sensitivity of eNOS and a significant increase in NO synthesis resulted from longer-term stimulation with Aronia (48 hr) and an acute re-treatment of the cells (10 min).

  20. Endothelial Nitric Oxide Mediates Caffeine Antagonism of Alcohol-Induced Cerebral Artery Constriction

    PubMed Central

    Chang, Jennifer; Fedinec, Alexander L.; Kuntamallappanavar, Guruprasad; Leffler, Charles W.; Bukiya, Anna N.

    2016-01-01

    Despite preventive education, the combined consumption of alcohol and caffeine (particularly from “energy drinks”) continues to rise. Physiologic perturbations by separate intake of ethanol and caffeine have been widely documented. However, the biologic actions of the alcohol-caffeine combination and their underlying subcellular mechanisms have been scarcely studied. Using intravital microscopy on a closed-cranial window and isolated, pressurized vessels, we investigated the in vivo and in vitro action of ethanol-caffeine mixtures on cerebral arteries from rats and mice, widely recognized models to address cerebrovascular pathophysiology and pharmacology. Caffeine at concentrations found in human circulation after ingestion of one to two cups of coffee (10 µM) antagonized the endothelium-independent constriction of cerebral arteries evoked by ethanol concentrations found in blood during moderate-heavy alcohol intoxication (40–70 mM). Caffeine antagonism against alcohol was similar whether evaluated in vivo or in vitro, suggesting independence of systemic factors and drug metabolism, but required a functional endothelium. Moreover, caffeine protection against alcohol increased nitric oxide (NO•) levels over those found in the presence of ethanol alone, disappeared upon blocking NO• synthase, and could not be detected in pressurized cerebral arteries from endothelial nitric-oxide synthase knockout (eNOS−/−) mice. Finally, incubation of de-endothelialized cerebral arteries with the NO• donor sodium nitroprusside (10 µM) fully restored the protective effect of caffeine. This study demonstrates for the first time that caffeine antagonizes ethanol-induced cerebral artery constriction and identifies endothelial NO• as the critical caffeine effector on smooth muscle targets. Conceivably, situations that perturb endothelial function and/or NO• availability will critically alter caffeine antagonism of alcohol-induced cerebrovascular constriction without

  1. Gomisin J from Schisandra chinensis induces vascular relaxation via activation of endothelial nitric oxide synthase.

    PubMed

    Park, Ji Young; Choi, Young Whan; Yun, Jung Wook; Bae, Jin Ung; Seo, Kyo Won; Lee, Seung Jin; Park, So Youn; Kim, Chi Dae

    2012-01-01

    Gomisin J (GJ) is a lignan contained in Schisandra chinensis (SC) which is a well-known medicinal herb for improvement of cardiovascular symptoms in Korean. Thus, the present study examined the vascular effects of GJ, and also determined the mechanisms involved. Exposure of rat thoracic aorta to GJ (1-30μg/ml) resulted in a concentration-dependent vasorelaxation, which was more prominent in the endothelium (ED)-intact aorta. ED-dependent relaxation induced by GJ was markedly attenuated by pretreatment with L-NAME, a nitric oxide synthase (NOS) inhibitor. In the intact endothelial cells of rat thoracic aorta, GJ also enhanced nitric oxide (NO) production. In studies using human coronary artery endothelial cells, GJ enhanced phosphorylation of endothelial NOS (eNOS) at Ser(1177) with increased cytosolic translocation of eNOS, and subsequently increased NO production. These effects of GJ were attenuated not only by calcium chelators including EGTA and BAPTA-AM, but also by LY294002, a PI3K/Akt inhibitor, indicating calcium- and PI3K/Akt-dependent activation of eNOS by GJ. Moreover, the levels of intracellular calcium were increased immediately after GJ administration, but Akt phosphorylation was started to increase at 20min of GJ treatment. Based on these results with the facts that ED-dependent relaxation occurred rapidly after GJ treatment, it was suggested that the ED-dependent vasorelaxant effects of GJ were mediated mainly by calcium-dependent activation of eNOS with subsequent production of endothelial NO.

  2. Endothelial Nitric Oxide Mediates Caffeine Antagonism of Alcohol-Induced Cerebral Artery Constriction.

    PubMed

    Chang, Jennifer; Fedinec, Alexander L; Kuntamallappanavar, Guruprasad; Leffler, Charles W; Bukiya, Anna N; Dopico, Alex M

    2016-01-01

    Despite preventive education, the combined consumption of alcohol and caffeine (particularly from "energy drinks") continues to rise. Physiologic perturbations by separate intake of ethanol and caffeine have been widely documented. However, the biologic actions of the alcohol-caffeine combination and their underlying subcellular mechanisms have been scarcely studied. Using intravital microscopy on a closed-cranial window and isolated, pressurized vessels, we investigated the in vivo and in vitro action of ethanol-caffeine mixtures on cerebral arteries from rats and mice, widely recognized models to address cerebrovascular pathophysiology and pharmacology. Caffeine at concentrations found in human circulation after ingestion of one to two cups of coffee (10 µM) antagonized the endothelium-independent constriction of cerebral arteries evoked by ethanol concentrations found in blood during moderate-heavy alcohol intoxication (40-70 mM). Caffeine antagonism against alcohol was similar whether evaluated in vivo or in vitro, suggesting independence of systemic factors and drug metabolism, but required a functional endothelium. Moreover, caffeine protection against alcohol increased nitric oxide (NO•) levels over those found in the presence of ethanol alone, disappeared upon blocking NO• synthase, and could not be detected in pressurized cerebral arteries from endothelial nitric-oxide synthase knockout (eNOS(-/-)) mice. Finally, incubation of de-endothelialized cerebral arteries with the NO• donor sodium nitroprusside (10 µM) fully restored the protective effect of caffeine. This study demonstrates for the first time that caffeine antagonizes ethanol-induced cerebral artery constriction and identifies endothelial NO• as the critical caffeine effector on smooth muscle targets. Conceivably, situations that perturb endothelial function and/or NO• availability will critically alter caffeine antagonism of alcohol-induced cerebrovascular constriction without

  3. Houttuynia cordata Extract Improves Physical Endurance Performance by Regulating Endothelial Production of Nitric Oxide.

    PubMed

    Yang, Ui-Jeong; Maeng, Hyojin; Park, Tae-Sik; Shim, Soon-Mi

    2015-09-01

    Vascular function is mediated by various regulatory molecules, including endothelial nitric oxide (NO), which regulates the vasodilation of smooth muscle cells. We investigated whether standardized Houttuynia cordata extract (SHCE) could improve physical endurance performance by regulating the endothelial production of NO. For the standardization of Houttuynia cordata (HC) extract, its bioactive components were identified and quantified using ultraperformance liquid chromatography-mass spectrometry. Bioaccessibility and biological activity were measured by the in vitro digestion model system and free radical scavenging capacity, respectively. The vascular function in the endothelium was assessed by the phosphorylation of endothelial nitric oxide synthase (eNOS). A preliminary clinical trial was carried out to assess the physical endurance performance. HC extract was standardized to bioactive components, including chlorogenic acid, rutin, and quercitrin, with the concentration of 5.53, 6.09, and 16.15 mg from 1 g of dry weight, respectively. Bioaccessibility was 33.17%, 31.67%, and 11.18% for chlorogenic acid, rutin, and quercitrin, respectively. Antioxidant activities of SHCE were expressed as vitamin C equivalent antioxidant capacity in 55.81 and 17.23 mg/g of HC extract using ABTS and DPPH scavenging assay, respectively. In human aortic endothelial cells, insulin-mediated phosphorylation of eNOS was increased by SHCE in the presence of palmitate. However, the expression of blood pressure-regulating genes was not altered. The level of blood lactate concentration and the heart rate of subjects who drank SHCE were lower than those of subjects who drank plain water. Oxygen uptake from subjects drinking SHCE was slightly higher than that from those who drank plain water. This study demonstrated that SHCE decreased heart rate and blood lactate, increased oxygen uptake, and improved physical performance, presumably due to the increased NO production.

  4. Sestrin 2 and AMPK Connect Hyperglycemia to Nox4-Dependent Endothelial Nitric Oxide Synthase Uncoupling and Matrix Protein Expression

    PubMed Central

    Eid, Assaad A.; Lee, Doug-Yoon; Roman, Linda J.; Khazim, Khaled

    2013-01-01

    Mesangial matrix accumulation is an early feature of glomerular pathology in diabetes. Oxidative stress plays a critical role in hyperglycemia-induced glomerular injury. Here, we demonstrate that, in glomerular mesangial cells (MCs), endothelial nitric oxide synthase (eNOS) is uncoupled upon exposure to high glucose (HG), with enhanced generation of reactive oxygen species (ROS) and decreased production of nitric oxide. Peroxynitrite mediates the effects of HG on eNOS dysfunction. HG upregulates Nox4 protein, and inhibition of Nox4 abrogates the increase in ROS and peroxynitrite generation, as well as the eNOS uncoupling triggered by HG, demonstrating that Nox4 functions upstream from eNOS. Importantly, this pathway contributes to HG-induced MC fibronectin accumulation. Nox4-mediated eNOS dysfunction was confirmed in glomeruli of a rat model of type 1 diabetes. Sestrin 2-dependent AMP-activated protein kinase (AMPK) activation attenuates HG-induced MC fibronectin synthesis through blockade of Nox4-dependent ROS and peroxynitrite generation, with subsequent eNOS uncoupling. We also find that HG negatively regulates sestrin 2 and AMPK, thereby promoting Nox4-mediated eNOS dysfunction and increased fibronectin. These data identify a protective function for sestrin 2/AMPK and potential targets for intervention to prevent fibrotic injury in diabetes. PMID:23816887

  5. Role of folic acid in nitric oxide bioavailability and vascular endothelial function.

    PubMed

    Stanhewicz, Anna E; Kenney, W Larry

    2017-01-01

    Folic acid is a member of the B-vitamin family and is essential for amino acid metabolism. Adequate intake of folic acid is vital for metabolism, cellular homeostasis, and DNA synthesis. Since the initial discovery of folic acid in the 1940s, folate deficiency has been implicated in numerous disease states, primarily those associated with neural tube defects in utero and neurological degeneration later in life. However, in the past decade, epidemiological studies have identified an inverse relation between both folic acid intake and blood folate concentration and cardiovascular health. This association inspired a number of clinical studies that suggested that folic acid supplementation could reverse endothelial dysfunction in patients with cardiovascular disease (CVD). Recently, in vitro and in vivo studies have begun to elucidate the mechanism(s) through which folic acid improves vascular endothelial function. These studies, which are the focus of this review, suggest that folic acid and its active metabolite 5-methyl tetrahydrofolate improve nitric oxide (NO) bioavailability by increasing endothelial NO synthase coupling and NO production as well as by directly scavenging superoxide radicals. By improving NO bioavailability, folic acid may protect or improve endothelial function, thereby preventing or reversing the progression of CVD in those with overt disease or elevated CVD risk.

  6. Slow and sustained nitric oxide releasing compounds inhibit multipotent vascular stem cell proliferation and differentiation without causing cell death

    SciTech Connect

    Curtis, Brandon M.; Leix, Kyle Alexander; Ji, Yajing; Glaves, Richard Samuel Elliot; Ash, David E.; Mohanty, Dillip K.

    2014-07-18

    Highlights: • Multipotent vascular stem cells (MVSCs) proliferate and differentiate. • Nitric oxide inhibits proliferation of MVSCs. • Nitric oxide inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs). • Smooth muscle cells (SMCs) neither de-differentiate nor proliferate. - Abstract: Atherosclerosis is the leading cause of cerebral and myocardial infarction. It is believed that neointimal growth common in the later stages of atherosclerosis is a result of vascular smooth muscle cell (SMC) de-differentiation in response to endothelial injury. However, the claims of the SMC de-differentiation theory have not been substantiated by monitoring the fate of mature SMCs in response to such injuries. A recent study suggests that atherosclerosis is a consequence of multipotent vascular stem cell (MVSC) differentiation. Nitric oxide (NO) is a well-known mediator against atherosclerosis, in part because of its inhibitory effect on SMC proliferation. Using three different NO-donors, we have investigated the effects of NO on MVSC proliferation. Results indicate that NO inhibits MVSC proliferation in a concentration dependent manner. A slow and sustained delivery of NO proved to inhibit proliferation without causing cell death. On the other hand, larger, single-burst NO concentrations, inhibits proliferation, with concurrent significant cell death. Furthermore, our results indicate that endogenously produced NO inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs) and subsequently to SMC as well.

  7. Expression of endothelial nitric oxide synthase and vascular endothelial growth factor in association with neovascularization in human primary astrocytoma*

    PubMed Central

    Pan, Jian-wei; Zhan, Ren-ya; Tong, Ying; Zhou, Yong-qing; Zhang, Ming

    2005-01-01

    Objective: To investigate the relationship between the expression of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and angiogenesis in primary astrocytoma. Methods: Thirty-seven primary astrocytomas and 4 astrocytic hyperplasia samples were collected and divided into three groups according to histological grade. The expression of eNOS, VEGF and factor VIII related antigen (FVIIIRAg) were assayed by immunohistochemistry. Microvascular density was assessed by FVIIIRAg immunoreactivity. The intensity of immunoreactivity was graded according to the percentage of positive tumor cells. Results: No eNOS and VEGF were expressed in the astrocytes and vascular endothelium in astrocytic hyperplasia. The expression of eNOS or VEGF was light in low-grade astrocytoma and strong in glioblastoma. eNOS expression in astrocytoma was very positively correlated with VEGF. eNOS and VEGF expression in anaplastic astrocytoma was median in contrast to the low grade astrocytoma and glioblastoma. Lower microvascular density was found in low grade astrocytoma than that in higher grade malignant ones. The expressions of eNOS and VEGF were correlated with microvascular density and tumor malignancy. Conclusion: This finding suggests that eNOS and VEGF may have cooperative effect in tumor angiogenesis and play an important role in the pathogenesis of primary astrocytoma. PMID:15973775

  8. H2S regulates endothelial nitric oxide synthase protein stability by promoting microRNA-455-3p expression

    PubMed Central

    Li, Xing-Hui; Xue, Wen-Long; Wang, Ming-Jie; Zhou, Yu; Zhang, Cai-Cai; Sun, Chen; Zhu, Lei; Liang, Kun; Chen, Ying; Tao, Bei-Bei; Tan, Bo; Yu, Bo; Zhu, Yi-Chun

    2017-01-01

    The aims of the present study are to determine whether hydrogen sulfide (H2S) is involved in the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production, and to identify the role of microRNA-455-3p (miR-455-3p) during those processes. In cultured human umbilical vein endothelial cells (HUVECs), the expression of miR-455-3p, eNOS protein and the NO production was detected after administration with 50 μM NaHS. The results indicated that H2S could augment the expression of miR-455-3p and eNOS protein, leading to the increase of NO level. We also found that overexpression of miR-455-3p in HUVECs increased the protein levels of eNOS whereas inhibition of miR-455-3p decreased it. Moreover, H2S and miR-455-3p could no longer increase the protein level of eNOS in the presence of proteasome inhibitor, MG-132. In vivo, miR-455-3p and eNOS expression were considerably increased in C57BL/6 mouse aorta, muscle and heart after administration with 50 μmol/kg/day NaHS for 7 days. We also identified that H2S levels and miR-455-3p expression increased in human atherosclerosis plaque while H2S levels decreased in plasma of atherosclerosis patients. Our data suggest that the stability of eNOS protein and the NO production could be regulated by H2S through miR-455-3p. PMID:28322298

  9. Busulphan-Cyclophosphamide Cause Endothelial Injury, Remodeling of Resistance Arteries and Enhanced Expression of Endothelial Nitric Oxide Synthase

    PubMed Central

    Al-Hashmi, Sulaiman; Boels, Piet J. M.; Zadjali, Fahad; Sadeghi, Behnam; Sällström, Johan; Hultenby, Kjell; Hassan, Zuzana; Arner, Anders; Hassan, Moustapha

    2012-01-01

    Stem cell transplantation (SCT) is a curative treatment for malignant and non malignant diseases. However, transplantation-related complications including cardiovascular disease deteriorate the clinical outcome and quality of life. We have investigated the acute effects of conditioning regimen on the pharmacology, physiology and structure of large elastic arteries and small resistance-sized arteries in a SCT mouse model. Mesenteric resistance arteries and aorta were dissected from Balb/c mice conditioned with busulphan (Bu) and cyclophosphamide (Cy). In vitro isometric force development and pharmacology, in combination with RT-PCR, Western blotting and electron microscopy were used to study vascular properties. Compared with controls, mesenteric resistance arteries from the Bu-Cy group had larger internal circumference, showed enhanced endothelium mediated relaxation and increased expression of endothelial nitric oxide synthase (eNOS). Bu-Cy treated animals had lower mean blood pressure and signs of endothelial injury. Aortas of treated animals had a higher reactivity to noradrenaline. We conclude that short-term consequences of Bu-Cy treatment divergently affect large and small arteries of the cardiovascular system. The increased noradrenaline reactivity of large elastic arteries was not associated with increased blood pressure at rest. Instead, Bu-Cy treatment lowered blood pressure via augmented microvascular endothelial dependent relaxation, increased expression of vascular eNOS and remodeling toward a larger lumen. The changes in the properties of resistance arteries can be associated with direct effects of the compounds on vascular wall or possibly indirectly induced via altered translational activity associated with the reduced hematocrit and shear stress. This study contributes to understanding the mechanisms that underlie the early effects of conditioning regimen on resistance arteries and may help in designing further investigations to understand the late

  10. Inhibitor-κB kinase attenuates Hsp90-dependent endothelial nitric oxide synthase function in vascular endothelial cells

    PubMed Central

    Konopinski, Ryszard; Krishnan, Manickam; Roman, Linda; Bera, Alakesh; Hongying, Zheng; Habib, Samy L.; Mohan, Sumathy

    2015-01-01

    Endothelial nitric oxide (NO) synthase (eNOS) is the predominant isoform that generates NO in the blood vessels. Many different regulators, including heat shock protein 90 (Hsp90), govern eNOS function. Hsp90-dependent phosphorylation of eNOS is a critical event that determines eNOS activity. In our earlier study we demonstrated an inhibitor-κB kinase-β (IKKβ)-Hsp90 interaction in a high-glucose environment. In the present study we further define the putative binding domain of IKKβ on Hsp90. Interestingly, IKKβ binds to the middle domain of Hsp90, which has been shown to interact with eNOS to stimulate its activity. This new finding suggests a tighter regulation of eNOS activity than was previously assumed. Furthermore, addition of purified recombinant IKKβ to the eNOS-Hsp90 complex reduces the eNOS-Hsp90 interaction and eNOS activity, indicating a competition for Hsp90 between eNOS and IKKβ. The pathophysiological relevance of the IKKβ-Hsp90 interaction has also been demonstrated using in vitro vascular endothelial growth factor-mediated signaling and an Ins2Akita in vivo model. Our study further defines the preferential involvement of α- vs. β-isoforms of Hsp90 in the IKKβ-eNOS-Hsp90 interaction, even though both Hsp90α and Hsp90β stimulate NO production. These studies not only reinforce the significance of maintaining a homeostatic balance of eNOS and IKKβ within the cell system that regulates NO production, but they also confirm that the IKKβ-Hsp90 interaction is favored in a high-glucose environment, leading to impairment of the eNOS-Hsp90 interaction, which contributes to endothelial dysfunction and vascular complications in diabetes. PMID:25652452

  11. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    SciTech Connect

    Hwang, Yong Pil; Kim, Hyung Gyun; Hien, Tran Thi; Jeong, Myung Ho; Jeong, Tae Cheon; Jeong, Hye Gwang

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

  12. Activation of nuclear factor erythroid 2-related factor 2 coordinates dimethylarginine dimethylaminohydrolase/PPAR-γ/endothelial nitric oxide synthase pathways that enhance nitric oxide generation in human glomerular endothelial cells.

    PubMed

    Luo, Zaiming; Aslam, Shakil; Welch, William J; Wilcox, Christopher S

    2015-04-01

    Dimethylarginine dimethylaminohydrolase (DDAH) degrades asymmetric dimethylarginine, which inhibits nitric oxide (NO) synthase (NOS). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that binds to antioxidant response elements and transcribes many antioxidant genes. Because the promoters of the human DDAH-1 and DDAH-2, endothelial NOS (eNOS) and PPAR-γ genes contain 2 to 3 putative antioxidant response elements, we hypothesized that they were regulated by Nrf2/antioxidant response element. Incubation of human renal glomerular endothelial cells with the Nrf2 activator tert-butylhydroquinone (20 μmol·L(-1)) significantly (P<0.05) increased NO and activities of NOS and DDAH and decreased asymmetric dimethylarginine. It upregulated genes for hemoxygenase-1, eNOS, DDAH-1, DDAH-2, and PPAR-γ and partitioned Nrf2 into the nucleus. Knockdown of Nrf2 abolished these effects. Nrf2 bound to one antioxidant response element on DDAH-1 and DDAH-2 and PPAR-γ promoters but not to the eNOS promoter. An increased eNOS and phosphorylated eNOS (P-eNOSser-1177) expression with tert-butylhydroquinone was prevented by knockdown of PPAR-γ. Expression of Nrf2 was reduced by knockdown of PPAR-γ, whereas PPAR-γ was reduced by knockdown of Nrf2, thereby demonstrating 2-way positive interactions. Thus, Nrf2 transcribes HO-1 and other genes to reduce reactive oxygen species, and DDAH-1 and DDAH-2 to reduce asymmetric dimethylarginine and PPAR-γ to increase eNOS and its phosphorylation and activity thereby coordinating 3 pathways that enhance endothelial NO generation.

  13. Targeting endothelial connexin40 inhibits tumor growth by reducing angiogenesis and improving vessel perfusion

    PubMed Central

    Alonso, Florian; Domingos-Pereira, Sonia; Le Gal, Loïc; Derré, Laurent; Meda, Paolo; Jichlinski, Patrice; Nardelli-Haefliger, Denise; Haefliger, Jacques-Antoine

    2016-01-01

    Endothelial connexin40 (Cx40) contributes to regulate the structure and function of vessels. We have examined whether the protein also modulates the altered growth of vessels in tumor models established in control mice (WT), mice lacking Cx40 (Cx40−/−), and mice expressing the protein solely in endothelial cells (Tie2-Cx40). Tumoral angiogenesis and growth were reduced, whereas vessel perfusion, smooth muscle cell (SMC) coverage and animal survival were increased in Cx40−/− but not Tie2-Cx40 mice, revealing a critical involvement of endothelial Cx40 in transformed tissues independently of the hypertensive status of Cx40−/− mice. As a result, Cx40−/− mice bearing tumors survived significantly longer than corresponding controls, including after a cytotoxic administration. Comparable observations were made in WT mice injected with a peptide targeting Cx40, supporting the Cx40 involvement. This involvement was further confirmed in the absence of Cx40 or by peptide-inhibition of this connexin in aorta-sprouting, matrigel plug and SMC migration assays, and associated with a decreased expression of the phosphorylated form of endothelial nitric oxide synthase. The data identify Cx40 as a potential novel target in cancer treatment. PMID:26883111

  14. Role of endothelial nitric oxide synthase (eNOS) in chronic stress-promoted tumour growth

    PubMed Central

    Barbieri, Antonio; Palma, Giuseppe; Rosati, Alessandra; Giudice, Aldo; Falco, Antonia; Petrillo, Antonella; Petrillo, Mario; Bimonte, Sabrina; Benedetto, Maria Di; Esposito, Giuseppe; Stiuso, Paola; Abbruzzese, Alberto; Caraglia, Michele; Arra, Claudio

    2012-01-01

    Abstract Accumulating evidence suggests that chronic stress can be a cofactor for the initiation and progression of cancer. Here we evaluated the role of endothelial nitric oxide synthase (eNOS) in stress-promoted tumour growth of murine B16F10 melanoma cell line in C57BL/6 mice. Animals subjected to restraint stress showed increased levels adrenocorticotropic hormone, enlarged adrenal glands, reduced thymus weight and a 3.61-fold increase in tumour growth in respect to no-stressed animals. Tumour growth was significantly reduced in mice treated with the β-antagonist propranolol. Tumour samples obtained from stressed mice displayed high levels of vascular endothelial growth factor (VEGF) protein in immunohistochemistry. Because VEGF can induce eNOS increase, and nitric oxide is a relevant factor in angiogenesis, we assessed the levels of eNOS protein by Western blot analysis. We found a significant increase in eNOS levels in tumour samples from stressed mice, indicating an involvement of this enzyme in stress-induced tumour growth. Accordingly, chronic stress did not promote tumour growth in eNOS−/− mice. These results disclose for the first time a pivotal role for eNOS in chronic stress-induced initiation and promotion of tumour growth. PMID:21722303

  15. Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic grafts.

    PubMed Central

    Akyürek, L. M.; Fellström, B. C.; Yan, Z. Q.; Hansson, G. K.; Funa, K.; Larsson, E.

    1996-01-01

    In the vascular system, distinct isoforms of nitric oxide synthase (NOS) generate nitric oxide (NO), which acts as a biological messenger. Its role in the development of transplant arteriosclerosis (TA) is still unclear. To investigate whether NO is involved in TA, we studied the expression of NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), by immunohistochemistry and in situ hybridization during the first two post-transplantation months and their relation with cold ischemia (1 to 24 hours) and reperfusion injury using an aortic transplantation model in the rat. We found an increased iNOS expression in the intima and adventitia and a decreased expression in the media, whereas eNOS expression was not significantly altered during the development of TA. Co-localization studies suggested that iNOS-positive cells were vascular smooth muscle cells, monocyte-derived macrophages, and endothelial cells. Prolonged ischemic storage time resulted in an increase in eNOS expression in the neointima. In situ hybridization showed iNOS mRNA expression by vascular cells in the neointima and media. NO produced by iNOS and eNOS may be involved, at least in part, in the pathogenesis of TA in aortic grafts. Additional studies are needed to confirm the modulatory mechanism of NO during the development of TA. Images Figure 3 Figure 4 Figure 6 PMID:8952533

  16. Inhaled nitric oxide decreases pulmonary endothelial nitric oxide synthase expression and activity in normal newborn rat lungs.

    PubMed

    Hua-Huy, Thông; Duong-Quy, Sy; Pham, Hoa; Pansiot, Julien; Mercier, Jean-Christophe; Baud, Olivier; Dinh-Xuan, Anh Tuan

    2016-01-01

    Inhaled nitric oxide (iNO) is commonly used in the treatment of very ill pre-term newborns. Previous studies showed that exogenous NO could affect endothelial NO synthase (eNOS) activity and expression in vascular endothelial cell cultures or adult rat models, but this has never been fully described in newborn rat lungs. We therefore aimed to assess the effects of iNO on eNOS expression and activity in newborn rats. Rat pups, post-natal day (P) 0 to P7, and their dams were placed in a chamber containing NO at 5 ppm (iNO-5 ppm group) or 20 ppm (iNO-20 ppm group), or in room air (control group). Rat pups were sacrificed at P7 and P14 for evaluation of lung eNOS expression and activity. At P7, eNOS protein expression in total lung lysates, in bronchial and arterial sections, was significantly decreased in the iNO-20 ppm versus control group. At P14, eNOS expression was comparable among all three groups. The amounts of eNOS mRNA significantly differed at P7 between the iNO-20 ppm and control groups. NOS activity decreased in the iNO-20 ppm group at P7 and returned to normal levels at P14. There was an imbalance between superoxide dismutase and NOS activities in the iNO-20 ppm group at P7. Inhalation of NO at 20 ppm early after birth decreases eNOS gene transcription, protein expression and enzyme activity. This decrease might account for the rebound phenomenon observed in patients treated with iNO.

  17. Correction of hypertension by normalization of endothelial levels of fibroblast growth factor and nitric oxide synthase in spontaneously hypertensive rats.

    PubMed

    Cuevas, P; García-Calvo, M; Carceller, F; Reimers, D; Zazo, M; Cuevas, B; Muñoz-Willery, I; Martínez-Coso, V; Lamas, S; Giménez-Gallego, G

    1996-10-15

    Acidic and basic fibroblast growth factors (FGFs) share a wide range of diverse biological activities. To date, low levels of FGF have not been correlated with a pathophysiologic state. We report that blood vessels of spontaneously hypertensive rats are shown to be associated with a marked decrement in endothelial basic FGF content. This decrement correlates both with hypertension and with a decrease in the endothelial content of nitric oxide synthase. Restoration of FGF to physiological levels in the vascular wall, either by systemic administration or by in vivo gene transfer, significantly augmented the number of endothelial cells with positive immunostaining for nitric oxide synthase, corrected hypertension, and ameliorated endothelial-dependent responses to vasoconstrictors. These results suggest an important role for FGFs in blood pressure homeostasis and open new avenues for the understanding of the etiology and treatment of hypertension.

  18. Correction of Hypertension by Normalization of Endothelial Levels of Fibroblast Growth Factor and Nitric Oxide Synthase in Spontaneously Hypertensive Rats

    NASA Astrophysics Data System (ADS)

    Cuevas, Pedro; Garcia-Calvo, Margarita; Carceller, Fernando; Reimers, Diana; Zazo, Mercedes; Cuevas, Begona; Munoz-Willery, Isabel; Martinez-Coso, Victoria; Lamas, Santiago; Gimenez-Gallego, Guillermo

    1996-10-01

    Acidic and basic fibroblast growth factors (FGFs) share a wide range of diverse biological activities. To date, low levels of FGF have not been correlated with a pathophysiologic state. We report that blood vessels of spontaneously hypertensive rats are shown to be associated with a marked decrement in endothelial basic FGF content. This decrement correlates both with hypertension and with a decrease in the endothelial content of nitric oxide synthase. restoration of FGF to physiological levels in the vascular wall, either by systemic administration or by in vivo gene transfer, significantly augmented the number of endothelial cells with positive immunostaining for nitric oxide synthase, corrected hypertension, and ameliorated endothelial-dependent responses to vasoconstrictors. These results suggest an important role for FGFs in blood pressure homeostasis and open new avenues for the understanding of the etiology and treatment of hypertension.

  19. Endothelial nitric oxide synthase is protective in the initiation of caerulein-induced acute pancreatitis in mice.

    PubMed

    DiMagno, Matthew J; Williams, John A; Hao, Yibai; Ernst, Stephen A; Owyang, Chung

    2004-07-01

    The effect of inhibiting nitric oxide (NO) synthase (NOS) or enhancing NO on the course of acute pancreatitis (AP) is controversial, in part because three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). We investigated whether inhibition or selective gene deletion of NOS isoforms modified the initiation phase of caerulein-induced AP in mice and explored whether this affected pancreatic microvascular blood flow (PMBF). We investigated the effects of nonspecific NOS inhibition with N(omega)-nitro-l-arginine (l-NNA; 10 mg/kg ip) or targeted deletion of eNOS, nNOS, or iNOS genes on the initiation phase of caerulein-induced AP in mice using in vivo and in vitro models. Western blot analysis was performed to assess eNOS phosphorylation status, an indicator of enzyme activity, and microsphere studies were used to measure PMBF. l-NNA and eNOS deletion, but not nNOS or iNOS deletion, increased pancreatic trypsin activity and serum lipase during the initiation phase of in vivo caerulein-induced AP. l-NNA and eNOS did not affect trypsin activity in caerulein-hyperstimulated isolated acini, suggesting that nonacinar events mediate the effect of NOS blockade in vivo. The initiation phase of AP in wild-type mice was associated with eNOS Thr(495) residue dephosphorylation, which accompanies eNOS activation, and a 178% increase in PMBF; these effects were absent in eNOS-deleted mice. Thus eNOS is the main isoform influencing the initiation of caerulein-induced AP. eNOS-derived NO exerts a protective effect through actions on nonacinar cell types, most likely endothelial cells, to produce greater PMBF.

  20. Expression and activity of inducible nitric oxide synthase and endothelial nitric oxide synthase correlate with ethanol-induced liver injury

    PubMed Central

    Yuan, Guang-Jin; Zhou, Xiao-Rong; Gong, Zuo-Jiong; Zhang, Pin; Sun, Xiao-Mei; Zheng, Shi-Hua

    2006-01-01

    AIM: To study the expression and activity of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-κB (NF-κB) and tumor necrosis factor-α (TNF-α) expression in the liver. METHODS: Female Sprague-Dawley rats were given fish oil (0.5 mL) along with ethanol or isocaloric dextrose daily via gastrogavage for 4 or 6 wk. Liver injury was assessed using serum alanine aminotransferase (ALT) activity and pathological analysis. Liver malondialdehyde (MDA), nitric oxide contents, iNOS and eNOS activity were determined. NF-κB p65,iNOS, eNOS and TNF-α protein or mRNA expression in the liver were detected by immunohistochemistry or reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Chronic ethanol gavage for 4 wk caused steatosis, inflammation and necrosis in the liver, and elevated serum ALT activity. Prolonged ethanol administration (6 wk) enhanced the liver damage. These responses were accompanied with increased lipid peroxidation, NO contents, iNOS activity and reduced eNOS activity. NF-κB p65, iNOS and TNF-α protein or mRNA expression were markedly induced after chronic ethanol gavage, whereas eNOS mRNA expression remained unchanged. The enhanced iNOS activity and expression were positively correlated with the liver damage, especially the necro-inflammation, activation of NF-κB, and TNF-α mRNA expression. CONCLUSION: iNOS expression and activity are induced in the liver after chronic ethanol exposure in rats, which are correlated with the liver damage, especially the necro-inflammation, activation of NF-κB and TNF-α expression. eNOS activity is reduced, but its mRNA expression is not affected. PMID:16688828

  1. Interplay of myosin phosphatase and protein phosphatase-2A in the regulation of endothelial nitric-oxide synthase phosphorylation and nitric oxide production

    PubMed Central

    Bátori, Róbert; Bécsi, Bálint; Nagy, Dénes; Kónya, Zoltán; Hegedűs, Csaba; Bordán, Zsuzsanna; Verin, Alexander; Lontay, Beáta; Erdődi, Ferenc

    2017-01-01

    The inhibitory phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) at Thr497 (eNOSpThr497) by protein kinase C or RhoA-activated kinase is a major regulatory determinant of eNOS activity. The signalling mechanisms involved in the dephosphorylation of eNOSpThr497 have not yet been clarified. This study identifies myosin phosphatase (MP) holoenzyme consisting of protein phosphatase-1 catalytic subunit (PP1c) and MP target subunit-1 (MYPT1) as an eNOSpThr497 phosphatase. In support of this finding are: (i) eNOS and MYPT1 interacts in various endothelial cells (ECs) and in in vitro binding assays (ii) MYPT1 targets and stimulates PP1c toward eNOSpThr497 substrate (iii) phosphorylation of MYPT1 at Thr696 (MYPT1pThr696) controls the activity of MP on eNOSpThr497. Phosphatase inhibition suppresses both NO production and transendothelial resistance (TER) of ECs. In contrast, epigallocatechin-3-gallate (EGCG) signals ECs via the 67 kDa laminin-receptor (67LR) resulting in protein kinase A dependent activation of protein phosphatase-2A (PP2A). PP2A dephosphorylates MYPT1pThr696 and thereby stimulates MP activity inducing dephosphorylation of eNOSpThr497 and the 20 kDa myosin II light chains. Thus an interplay of MP and PP2A is involved in the physiological regulation of EC functions implying that an EGCG dependent activation of these phosphatases leads to enhanced NO production and EC barrier improvement. PMID:28300193

  2. Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation.

    PubMed

    Peng, Hu; Zhuang, Yugang; Harbeck, Mark C; He, Donghong; Xie, Lishi; Chen, Weiguo

    2015-01-01

    Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O2-.) in the absence of the cofactor tetrahydrobiopterin (BH4). Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human) phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D) to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca2+, and calmodulin (CAM). S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS). The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca2+ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (P<0.01). VEGF-mediated Serine 1179 phosphorylation further decreased the cellular viability in eNOS-overexpressing 293 cells (P<0.01). Our data demonstrate that eNOS serine 1179 phosphorylation, in addition to enhancing NO production, also profoundly affects superoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity.

  3. Short-term calorie restriction reverses vascular endothelial dysfunction in old mice by increasing nitric oxide and reducing oxidative stress.

    PubMed

    Rippe, Catarina; Lesniewski, Lisa; Connell, Melanie; LaRocca, Thomas; Donato, Anthony; Seals, Douglas

    2010-06-01

    To determine if short-term calorie restriction reverses vascular endothelial dysfunction in old mice, old (O, n = 30) and young (Y, n = 10) male B6D2F1 mice were fed ad libitum (AL) or calorie restricted (CR, approximately 30%) for 8 weeks. Ex vivo carotid artery endothelium-dependent dilation (EDD) was impaired in old ad libitum (OAL) vs. young ad libitum (YAL) (74 +/- 5 vs. 95 +/- 2% of maximum dilation, P < 0.05), whereas old calorie-restricted (OCR) and YCR did not differ (96 +/- 1 vs. 94 +/- 3%). Impaired EDD in OAL was mediated by reduced nitric oxide (NO) bioavailability associated with decreased endothelial NO synthase expression (aorta) (P < 0.05), both of which were restored in OCR. Nitrotyrosine, a cellular marker of oxidant modification, was markedly elevated in OAL (P < 0.05), whereas OCR was similar to Y. Aortic superoxide production was 150% greater in OAL vs. YAL (P < 0.05), but normalized in OCR, and TEMPOL, a superoxide dismutase (SOD) mimetic that restored EDD in OAL (to 97 +/- 2%), had no effect in Y or OCR. OAL had increased expression and activity of the oxidant enzyme, NADPH oxidase, and its inhibition (apocynin) improved EDD, whereas NADPH oxidase in OCR was similar to Y. Manganese SOD activity and sirtuin1 expression were reduced in OAL (P < 0.05), but restored to Y in OCR. Inflammatory cytokines were greater in OAL vs. YAL (P < 0.05), but unaffected by CR. Carotid artery endothelium-independent dilation did not differ among groups. Short-term CR initiated in old age reverses age-associated vascular endothelial dysfunction by restoring NO bioavailability, reducing oxidative stress (via reduced NADPH oxidase-mediated superoxide production and stimulation of anti-oxidant enzyme activity), and upregulation of sirtuin-1.

  4. N-terminal domain of soluble epoxide hydrolase negatively regulates the VEGF-mediated activation of endothelial nitric oxide synthase

    PubMed Central

    Hou, Hsin-Han; Hammock, Bruce D.; Su, Kou-Hui; Morisseau, Christophe; Kou, Yu Ru; Imaoka, Susumu; Oguro, Ami; Shyue, Song-Kun; Zhao, Jin-Feng; Lee, Tzong-Shyuan

    2012-01-01

    Aims The mammalian soluble epoxide hydrolase (sEH) has both an epoxide hydrolase and a phosphatase domain. The role of sEH hydrolase activity in the metabolism of epoxyeicosatrienoic acids (EETs) and the activation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) has been well defined. However, far less is known about the role of sEH phosphatase activity in eNOS activation. In the present study, we investigated whether the phosphatase domain of sEH was involved in the eNOS activation in ECs. Methods and results The level of eNOS phosphorylation in aortas is higher in the sEH knockout (sEH−/−) mice than in wild-type mice. In ECs, pharmacological inhibition of sEH phosphatase or overexpressing sEH with an inactive phosphatase domain enhanced vascular endothelial growth factor (VEGF)-induced NO production and eNOS phosphorylation. In contrast, overexpressing the phosphatase domain of sEH prevented the VEGF-mediated NO production and eNOS phosphorylation at Ser617, Ser635, and Ser1179. Additionally, treatment with VEGF induced a c-Src kinase-dependent increase in transient tyrosine phosphorylation of sEH and the formation of a sEH–eNOS complex, which was abolished by treatment with a c-Src kinase inhibitor, PP1, or the c-Src dominant-negative mutant K298M. We also demonstrated that the phosphatase domain of sEH played a key role in VEGF-induced angiogenesis by detecting the tube formation in ECs and neovascularization in Matrigel plugs in mice. Conclusion In addition to epoxide hydrolase activity, phosphatase activity of sEH plays a pivotal role in the regulation of eNOS activity and NO-mediated EC functions. PMID:22072631

  5. Short-term Calorie Restriction Reverses Vascular Endothelial Dysfunction in Old Mice by Increasing Nitric Oxide and Reducing Oxidative Stress

    PubMed Central

    Rippe, Catarina; Lesniewski, Lisa; Connell, Melanie; LaRocca, Thomas; Donato, Anthony; Seals, Douglas

    2010-01-01

    Summary To determine if short-term calorie restriction reverses vascular endothelial dysfunction in old mice, old (O, n=30) and young (Y, n=10) male B6D2F1 mice were fed ad libitum (AL) or calorie restricted (CR, ∼30%) for 8 weeks. Ex vivo carotid artery endothelium-dependent dilation (EDD) was impaired in OAL vs. YAL (74±5 vs. 95±2% of maximum dilation, P<0.05), whereas OCR and YCR did not differ (96±1 vs. 94±3%). Impaired EDD in OAL was mediated by reduced nitric oxide (NO) bioavailability associated with decreased endothelial NO synthase expression (aorta) (P<0.05), both of which were restored in OCR. Nitrotyrosine, a cellular marker of oxidant modification, was markedly elevated in OAL (P<0.05), whereas OCR was similar to Y. Aortic superoxide production was 150% greater in OAL vs. YAL (P<0.05), but normalized in OCR, and TEMPOL, a superoxide dismutase (SOD) mimetic that restored EDD in OAL (to 97±2%), had no effect in Y or OCR. OAL had increased expression and activity of the oxidant enzyme, NADPH oxidase, and its inhibition (apocynin) improved EDD, whereas NADPH oxidase in OCR was similar to Y. Manganese SOD activity and sirtuin1 expression were reduced in OAL (P<0.05), but restored to Y in OCR. Inflammatory cytokines were greater in OAL vs. YAL (P<0.05), but unaffected by CR. Carotid artery endothelium-independent dilation did not differ among groups. Short-term CR initiated in old age reverses age-associated vascular endothelial dysfunction by restoring NO bioavailability and reducing oxidation stress via reduced NADPH oxidase-mediated superoxide production and stimulation of anti-oxidant enzyme activity, and upregulates sirtuin1. PMID:20121721

  6. Nitric oxide inhibition of coxsackievirus replication in vitro.

    PubMed Central

    Zaragoza, C; Ocampo, C J; Saura, M; McMillan, A; Lowenstein, C J

    1997-01-01

    Nitric oxide is a radical molecule with antibacterial, -parasitic, and -viral properties. We investigated the mechanism of NO inhibition of Coxsackievirus B3 (CVB3) replication in vitro by determining the effect of NO upon a single replicative cycle of CVB3 grown in HeLa cells. Transfection of inducible NO synthase cDNA into HeLa cells reduces the number of viral particles produced during a single cycle of growth. Similarly, a noncytotoxic concentration of the NO donor S-nitroso-amino-penicillamine reduces the number of viral particles in a dose-dependent manner. To explore the mechanisms by which NO exerts its antiviral effect, we assayed the attachment, replication, and translation steps of the CVB3 life cycle. NO does not affect the attachment of CVB3 to HeLa cells. However, NO inhibits CVB3 RNA synthesis, as shown by a [3H]uridine incorporation assay, reverse transcription-PCR, and Northern analysis. In addition, NO inhibits CVB3 protein synthesis, as shown by [35S]methionine protein labeling and Western blot analysis of infected cells. Thus, NO inhibits CVB3 replication in part by inhibiting viral RNA synthesis by an unknown mechanism. PMID:9312175

  7. En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries.

    PubMed

    Yu, Yi; Xiong, Yuyan; Montani, Jean-Pierre; Yang, Zhihong; Ming, Xiu-Fen

    2016-02-25

    Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2(.-)) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2(.-) levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2(.-), it is difficult to measure NO and O2(.-) directly in a blood vessel. Numerous methods have been developed to measure NO and O2(.-) production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2(.-) using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2(.-) levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2(.-) in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

  8. Mechanical perturbations trigger endothelial nitric oxide synthase activity in human red blood cells

    PubMed Central

    Nagarajan, Shunmugan; Raj, Rajendran Kadarkarai; Saravanakumar, Venkatesan; Balaguru, Uma Maheswari; Behera, Jyotirmaya; Rajendran, Vinoth Kumar; Shathya, Yogarajan; Ali, B. Mohammed Jaffar; Sumantran, Venil; Chatterjee, Suvro

    2016-01-01

    Nitric oxide (NO), a vascular signaling molecule, is primarily produced by endothelial NO synthase. Recently, a functional endothelial NO synthase (eNOS) was described in red blood cells (RBC). The RBC-eNOS contributes to the intravascular NO pool and regulates physiological functions. However the regulatory mechanisms and clinical implications of RBC-eNOS are unknown. The present study investigated regulation and functions of RBC-eNOS under mechanical stimulation. This study shows that mechanical stimuli perturb RBC membrane, which triggers a signaling cascade to activate the eNOS. Extracellular NO level, estimated by the 4-Amino-5-Methylamino-2′, 7′-Difluorofluorescein Diacetate probe, was significantly increased under mechanical stimuli. Immunostaining and western blot studies confirmed that the mechanical stimuli phosphorylate the serine 1177 moiety of RBC-eNOS, and activates the enzyme. The NO produced by activation of RBC-eNOS in vortexed RBCs promoted important endothelial functions such as migration and vascular sprouting. We also show that mechanical perturbation facilitates nitrosylation of RBC proteins via eNOS activation. The results of the study confirm that mechanical perturbations sensitize RBC-eNOS to produce NO, which ultimately defines physiological boundaries of RBC structure and functions. Therefore, we propose that mild physical perturbations before, after, or during storage can improve viability of RBCs in blood banks. PMID:27345770

  9. Repeated thermal therapy upregulates arterial endothelial nitric oxide synthase expression in Syrian golden hamsters.

    PubMed

    Ikeda, Y; Biro, S; Kamogawa, Y; Yoshifuku, S; Eto, H; Orihara, K; Kihara, T; Tei, C

    2001-05-01

    It has been previously reported that sauna therapy, a thermal therapy, improves the hemodynamics and clinical symptoms in patients with chronic heart failure and also improves endothelial function, which is impaired in such patients. The present study investigated whether the improvements observed with sauna therapy are through modulation of arterial endothelial nitric oxide synthase (eNOS) expression. Eight male Syrian golden hamsters underwent sauna therapy, using an experimental far infrared-ray dry sauna system, at 39 degrees C for 15 min followed by 30 degrees C for 20 min daily for 4 weeks. Control group hamsters were placed in the sauna system switched off at room temperature of 24 degrees C for 35 min. Immunohistochemistry found greater amounts of the immunoreactive products of eNOS in the endothelial cells of the aorta and carotid, femoral and coronary arteries in the sauna group than in the control group. Western blot analysis also revealed that 4-week sauna therapy significantly increased eNOS expression in aortas by 50% in 4 series of independent experiments with an identical protocol (p<0.01). In reverse transcription polymerase chain reaction assay, the eNOS mRNA in aortas was greater in the sauna group than in controls, with a peak at 1-week of sauna therapy (approximately 40-fold increase). In conclusion, repeated thermal therapy upregulates eNOS expression in arterial endothelium.

  10. Signaling pathway for nitric oxide generation with simulated ischemia in flow-adapted endothelial cells.

    PubMed

    Wei, Z; Al-Mehdi, A B; Fisher, A B

    2001-11-01

    Ischemia in the intact ventilated lung (oxygenated ischemia) leads to endothelial generation of reactive oxygen species (ROS) and nitric oxide (NO). This study investigated the signaling pathway for NO generation with oxygenated ischemia in bovine pulmonary artery endothelial cells (BPAEC) that were flow adapted in vitro. BPAECs were cultured in an artificial capillary system and subjected to abrupt cessation of flow (ischemia) under conditions where cellular oxygenation was maintained. Immunoblotting and dichlorofluorescein/triazolofluorescein fluorescence were used to assess extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation and ROS/NO generation, respectively. ERK1/2 phosphorylation significantly increased during ischemia, whereas total ERK1/2 did not change. ERK1/2 phosphorylation was suppressed by an inhibitor of tyrosine phosphorylation (genestein), cholesterol-binding reagents (filipin or cyclodextrin), or inhibitors of ROS (diphenyleneiodonium, N-acetylcysteine, or catalase), suggesting a role for both membrane cholesterol and ROS in ERK1/2 activation. Ischemia resulted in a 1.8-fold increase in NO generation that was suppressed by inhibitors of ERK1/2 activation (PD-98059 or U-0126). A calmodulin inhibitor (calmidizolium) or removal of Ca2+ from the medium also blocked NO generation, indicating that endothelial NO synthase (eNOS) is the activated isoform. These results indicate ischemia induces NO generation (possibly through a membrane cholesterol-sensitive flow sensor), the ERK1/2 cascade mediates signaling from the sensor to eNOS, and ROS are required for ERK activation.

  11. Role of Heat Shock Protein 90 and Endothelial Nitric Oxide Synthase during Early Anesthetic and Ischemic Preconditioning

    PubMed Central

    Amour, Julien; Brzezinska, Anna K.; Weihrauch, Dorothee; Billstrom, Amie R.; Zielonka, Jacek; Krolikowski, John G.; Bienengraeber, Martin W.; Warltier, David C.; Pratt, Philip F.; Kersten, Judy R.

    2009-01-01

    Background Nitric oxide is known to be essential for early anesthetic (APC) and ischemic (IPC) preconditioning of myocardium. Heat shock protein 90 (Hsp90) regulates endothelial nitric oxide synthase (eNOS) activity. In this study, we tested the hypothesis that Hsp90-eNOS interactions modulate APC and IPC. Methods Myocardial infarct size was measured in rabbits after coronary occlusion and reperfusion in the absence or presence of preconditioning with 30 min of isoflurane (APC) or 5 min of coronary artery occlusion (IPC), and with or without pre-treatment with geldanamycin or radicicol, two chemically distinct Hsp90 inhibitors, or NG-nitro-L-arginine methylester, a non-specific NOS inhibitor. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells or mouse cardiomyocytes, in the absence or presence of Hsp90 inhibitors or NG-nitro-L-arginine methylester. Interactions between Hsp90 and eNOS, and eNOS activation were assessed with immunoprecipitation, immunoblotting, and confocal microscopy. Results APC and IPC decreased infarct size (50% and 59%, respectively) and this action was abolished by Hsp90 inhibitors. NG-nitro-L-arginine methylester blocked APC but not IPC. Isoflurane increased nitric oxide production in human coronary artery endothelial cells, concomitantly with an increase in Hsp90-eNOS interaction (immunoprecipitation, immunoblotting, and immunohistochemistry). Pretreatment with Hsp90 inhibitors abolished isoflurane-dependent nitric oxide production and decreased Hsp90-eNOS interactions. Isoflurane did not increase nitric oxide production in mouse cardiomyocytes and eNOS was below the level of detection. Conclusion The results indicate that Hsp90 plays a critical role in mediating APC and IPC through protein-protein interactions, and suggest that endothelial cells are important contributors to nitric oxide-mediated signalling during APC. PMID:19194158

  12. Nitric Oxide Interacts with Caveolin-1 to Facilitate Autophagy-Lysosome-Mediated Claudin-5 Degradation in Oxygen-Glucose Deprivation-Treated Endothelial Cells

    PubMed Central

    Liu, Jie; Weaver, John; Jin, Xinchun; Zhang, Yuan; Xu, Ji; Liu, Ke J.; Li, Weiping; Liu, Wenlan

    2017-01-01

    Using in vitro oxygen-glucose deprivation (OGD) model, we have previously demonstrated that 2-h OGD induces rapid, caveolin-1-mediated dissociation of claudin-5 from the cellular cytoskeletal framework and quick endothelial barrier disruption. In this study, we further investigated the fate of translocated claudin-5 and the mechanisms by which OGD promotes caveolin-1 translocation. Exposure of bEND3 cells to 4-h OGD, but not 2-h OGD plus 2-h reoxygenation, resulted in claudin-5 degradation. Inhibition of autophagy or the fusion of autophagosome with lysosome, but not proteasome, blocked OGD-induced claudin-5 degradation. Moreover, knockdown of caveolin-1 with siRNA blocked OGD-induced claudin-5 degradation. Western blot analysis showed a transient colocalization of caveolin-1, claudin-5, and LC3B in autolysosome or lipid raft fractions at 2-h OGD. Of note, inhibiting autophagosome and lysosome fusion sustained the colocalization of caveolin-1, claudin-5, and LC3B throughout the 4-h OGD exposure. EPR spin trapping showed increased nitric oxide (NO) generation in 2-h OGD-treated cells, and inhibiting NO with its scavenger C-PTIO or inducible nitric oxide synthase (iNOS) inhibitor 1400W prevented OGD-induced caveolin-1 translocation and claudin-5 degradation. Taken together, our data provide a novel mechanism underlying endothelial barrier disruption under prolonged ischemic conditions, in which NO promotes caveolin-1-mediated delivery of claudin-5 to the autophagosome for autophagy-lysosome-dependent degradation. PMID:26515186

  13. Nitric Oxide Interacts with Caveolin-1 to Facilitate Autophagy-Lysosome-Mediated Claudin-5 Degradation in Oxygen-Glucose Deprivation-Treated Endothelial Cells.

    PubMed

    Liu, Jie; Weaver, John; Jin, Xinchun; Zhang, Yuan; Xu, Ji; Liu, Ke J; Li, Weiping; Liu, Wenlan

    2016-11-01

    Using in vitro oxygen-glucose deprivation (OGD) model, we have previously demonstrated that 2-h OGD induces rapid, caveolin-1-mediated dissociation of claudin-5 from the cellular cytoskeletal framework and quick endothelial barrier disruption. In this study, we further investigated the fate of translocated claudin-5 and the mechanisms by which OGD promotes caveolin-1 translocation. Exposure of bEND3 cells to 4-h OGD, but not 2-h OGD plus 2-h reoxygenation, resulted in claudin-5 degradation. Inhibition of autophagy or the fusion of autophagosome with lysosome, but not proteasome, blocked OGD-induced claudin-5 degradation. Moreover, knockdown of caveolin-1 with siRNA blocked OGD-induced claudin-5 degradation. Western blot analysis showed a transient colocalization of caveolin-1, claudin-5, and LC3B in autolysosome or lipid raft fractions at 2-h OGD. Of note, inhibiting autophagosome and lysosome fusion sustained the colocalization of caveolin-1, claudin-5, and LC3B throughout the 4-h OGD exposure. EPR spin trapping showed increased nitric oxide (NO) generation in 2-h OGD-treated cells, and inhibiting NO with its scavenger C-PTIO or inducible nitric oxide synthase (iNOS) inhibitor 1400W prevented OGD-induced caveolin-1 translocation and claudin-5 degradation. Taken together, our data provide a novel mechanism underlying endothelial barrier disruption under prolonged ischemic conditions, in which NO promotes caveolin-1-mediated delivery of claudin-5 to the autophagosome for autophagy-lysosome-dependent degradation.

  14. Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta.

    PubMed

    Soucy, Kevin G; Lim, Hyun Kyo; Attarzadeh, David O; Santhanam, Lakshmi; Kim, Jae Hyung; Bhunia, Anil K; Sevinc, Baris; Ryoo, Sungwoo; Vazquez, Marcelo E; Nyhan, Daniel; Shoukas, Artin A; Berkowitz, Dan E

    2010-05-01

    Radiation exposure is associated with the development of various cardiovascular diseases. Although irradiation is known to cause elevated oxidant stress and chronic inflammation, both of which are detrimental to vascular function, the molecular mechanisms remain incompletely understood. We previously demonstrated that radiation causes endothelial dysfunction and increased vascular stiffness by xanthine oxidase (XO) activation. In this study, we investigated whether dietary inhibition of XO protects against radiation-induced vascular injury. We exposed 4-mo-old rats to a single dose of 0 or 5 Gy gamma radiation. These rats received normal drinking water or water containing 1 mM oxypurinol, an XO inhibitor. We measured XO activity and superoxide production in rat aorta and demonstrated that both were significantly elevated 2 wk after radiation exposure. However, oxypurinol treatment in irradiated rats prevented aortic XO activation and superoxide elevation. We next investigated endothelial function through fluorescent measurement of nitric oxide (NO) and vascular tension dose responses. Radiation reduced endothelium-dependent NO production in rat aorta. Similarly, endothelium-dependent vasorelaxation in the aorta of irradiated rats was significantly attenuated compared with the control group. Dietary XO inhibition maintained NO production at control levels and prevented the development of endothelial dysfunction. Furthermore, pulse wave velocity, a measure of vascular stiffness, increased by 1 day postirradiation and remained elevated 2 wk after irradiation, despite unchanged blood pressures. In oxypurinol-treated rats, pulse wave velocities remained unchanged from baseline throughout the experiment, signifying preserved vascular health. These findings demonstrate that XO inhibition can offer protection from radiation-induced endothelial dysfunction and cardiovascular complications.

  15. Contribution of central nervous system endothelial nitric oxide synthase to neurohumoral activation in heart failure rats.

    PubMed

    Biancardi, Vinicia C; Son, Sook J; Sonner, Patrick M; Zheng, Hong; Patel, Kaushik P; Stern, Javier E

    2011-09-01

    Neurohumoral activation, a hallmark in heart failure (HF), is linked to the progression and mortality of HF patients. Thus, elucidating its precise underlying mechanisms is of critical importance. Other than its classic peripheral vasodilatory actions, the gas NO is a pivotal neurotransmitter in the central nervous system control of the circulation. While accumulating evidence supports a contribution of blunted NO function to neurohumoral activation in HF, the precise cellular sources, and NO synthase (NOS) isoforms involved, remain unknown. Here, we used a multidisciplinary approach to study the expression, cellular distribution, and functional relevance of the endothelial NOS isoform within the hypothalamic paraventricular nucleus in sham and HF rats. Our results show high expression of endothelial NOS in the paraventricular nucleus (mostly confined to astroglial cells), which contributes to constitutive NO bioavailability, as well as tonic inhibition of presympathetic neuronal activity and sympathoexcitatory outflow from the paraventricular nucleus. A diminished endothelial NOS expression and endothelial NOS-derived NO availability were found in the paraventricular nucleus of HF rats, resulting, in turn, in blunted NO inhibitory actions on neuronal activity and sympathoexcitatory outflow. Taken together, our study supports blunted central nervous system endothelial NOS-derived NO as a pathophysiological mechanism underlying neurohumoral activation in HF.

  16. Endothelial nitric oxide synthase levels and their response to exercise in patients with slow coronary flow

    PubMed Central

    Taşolar, Hakan; Aktürk, Erdal; Eyyüpkoca, Ferhat; Cansel, Mehmet; Yağmur, Jülide; Pekdemir, Hasan; Karakuş, Yasin; Özyalin, Fatma; Altun, Burak

    2013-01-01

    Summary Background Endothelial dysfunction plays a key role in the aetiopathogenesis of slow coronary flow (SCF) even if there is no obstructive epicardial lesion. Reduced plasma levels of endothelial nitric oxide synthase (eNOS) are an important indicator of endothelial dysfunction. We aimed to determine plasma levels of eNOS and their relationship with exercise in patients with SCF. Methods Twenty-two patients with SCF in at least one coronary artery and 17 healthy individuals were included in this study. The TIMI frame count method was used to determine SCF. Plasma levels of eNOS before and after effort were determined in the patient and control groups. Results Basal eNOS levels in the patient group were lower than in the control group (p = 0.040), and plasma eNOS levels after exercise decreased more significantly in the patient group compared to the control group (p = 0.002). Median decreases of eNOS in response to exercise were higher in the SCF group than in the control group (p < 0.001), and the decrease observed in the control group was not statistically significant (p = 0.35). There were significantly negative correlations between TIMI frame count and plasma levels of eNOS at baseline and after exercise (r = –0.51, p = 0.015, r = –0.58, p = 0.005, respectively). Moreover, there was also a positive correlation between the rate–pressure product and plasma levels of eNOS after exercise in patients with SCF (r = 0.494, p = 0.019). Conclusion Our findings indicate an important pathophysiological relationship between the severity of SCF in which endothelial dysfunction plays a role in its pathogenesis and the level of circulating plasma levels of eNOS. PMID:24337211

  17. Erythropoietin-mobilized endothelial progenitors enhance reendothelialization via Akt-endothelial nitric oxide synthase activation and prevent neointimal hyperplasia.

    PubMed

    Urao, Norifumi; Okigaki, Mitsuhiko; Yamada, Hiroyuki; Aadachi, Yasushi; Matsuno, Kuniharu; Matsui, Akihiro; Matsunaga, Shinsaku; Tateishi, Kento; Nomura, Tetsuya; Takahashi, Tomosaburo; Tatsumi, Tetsuya; Matsubara, Hiroaki

    2006-06-09

    We investigated whether the mobilization of endothelial progenitor cells (EPCs) by exogenous erythropoietin (Epo) promotes the repair of injured endothelium. Recombinant human Epo was injected (1000 IU/kg for the initial 3 days) after wire injury of the femoral artery of mice. Neointimal formation was inhibited by Epo to 48% of the control (P<0.05) in an NO-dependent manner. Epo induced a 1.4-fold increase in reendothelialized area of day 14 denuded vessels, 55% of which was derived from bone marrow (BM) cells. Epo increased the circulating Sca-1(+)/Flk-1(+) EPCs (2.0-fold, P<0.05) with endothelial properties NO dependently. BM replacement by GFP- or beta-galactosidase-overexpressing cells showed that Epo stimulated both differentiation of BM-derived EPCs and proliferation of resident ECs. BM-derived ECs increased 2.2- to 2.7-fold (P<0.05) in the Epo-induced neoendothelium, where the expression of Epo receptor was upregulated. Epo induced Akt/eNOS phosphorylation and NO synthesis on EPCs and exerted an antiapoptotic action on wire-injured arteries. In conclusion, Epo treatment inhibits the neointimal hyperplasia after arterial injury in an NO-dependent manner by acting on the injured vessels and mobilizing EPCs to the neo-endothelium.

  18. Protein kinase C activators inhibit capillary endothelial cell growth

    SciTech Connect

    Doctrow, S.R.

    1986-05-01

    Phorbol 12,13-dibutyrate (PDBu) binds specifically to bovine capillary endothelial (BCE) cells (K/sub d/ = 8nM) and inhibits the proliferation (K/sub 50/ = 6 +/- 4 nM). Under similar conditions, PDBu does not inhibit the growth of bovine aortic endothelial or smooth muscle cells. PDBu markedly attenuates the response of BCE cells to purified human hepatoma-derived growth factor which, in the absence of PDBu, stimulates BCE cell growth by about 3-fold. Several observations suggest that the inhibition of BCE cell growth by PDBu is mediated by protein kinase C: (1) different phorbol compounds inhibit BCE cell growth according to the relative potencies as protein kinase C activators (12-tetradecanoylphorbol 13-acetate > PDBu >> phorbol 12,13-diacetate >>>..beta..-phorbol; ..cap alpha..-phorbol 12,13-didecanoate). (2) Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol (diC/sub 8/), a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. (3) A cytosolic extract from BCE cells contains a Ca/sup 2 +//phosphatidylserine-dependent kinase that is activated by diC/sub 8/ and PDBu, but not by ..beta..-phorbol. These results support a role for protein kinase C in suppressing capillary endothelial cell growth and may therefore have implications in the intracellular regulation of angiogenesis.

  19. Nitric oxide inhibits viral replication in murine myocarditis.

    PubMed Central

    Lowenstein, C J; Hill, S L; Lafond-Walker, A; Wu, J; Allen, G; Landavere, M; Rose, N R; Herskowitz, A

    1996-01-01

    Nitric oxide (NO) is a radical molecule that not only serves as a vasodilator and neurotransmitter but also acts as a cytotoxic effector molecule of the immune system. The inducible enzyme making NO, inducible NO synthase (iNOS), is transcriptionally activated by IFN-gamma and TNF-alpha, cytokines which are produced during viral infection. We show that iNOS is induced in mice infected with the Coxsackie B3 virus. Macrophages expressing iNOS are identified in the hearts and spleens of infected animals with an antibody raised against iNOS. Infected mice have increased titers of virus and a higher mortality when fed NOS inhibitors. Thus, viral infection induces iNOS in vivo, and NO inhibits viral replication. NO is a novel, nonspecific immune defense against viruses in vivo. PMID:8621766

  20. Immunohistochemical localization of endothelial and inducible nitric oxide synthase within neurons of cattle with rabies.

    PubMed

    Shin, Taekyun; Weinstock, Daniel; Castro, Marlene D; Hamir, Amir N; Wampler, Thomas; Walter, Mark; Kim, Hyun Young; Acland, Helen

    2004-05-01

    The expression of constitutive endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) in the brains of cattle with natural rabies was studied. Increased expression of eNOS was detected in neurons of the brain stem and Purkinje cells of cerebellum. By contrast, iNOS was diffusely localized in the cytoplasm of affected neurons, and some inflammatory cells were positive. eNOS and rabies antigen were co-localized in inclusion bodies (Negri bodies) in neurons. The specific localization of eNOS, but not iNOS, in the Negri bodies suggests that eNOS is involved in the formation of rabies virus inclusion bodies.

  1. Cloricromene inhibits the induction of nitric oxide synthase.

    PubMed

    Zingarelli, B; Carnuccio, R; Di Rosa, M

    1993-10-19

    The effect of cloricromene, a coumarin derivative, was investigated on the lipopolysaccharide-stimulated nitric oxide (NO) synthase induction in intact aortas from endotoxin shocked rats and in the murine macrophage cell line J774. Rings of thoracic aortas from lipopolysaccharide (4 mg/kg, i.v.)-shocked rats, contracted with phenylephrine, showed a progressive decrease in tone, that was of a greater magnitude than that of aortas from naive rats. Moreover, a decreased response to the constrictor effect of phenylephrine was observed in aortas from shocked rats. In vivo treatment with cloricromene (2 mg/kg, i.v.) 30 min before lipopolysaccharide administration partially prevented the loss in tone of aortic rings and improved their reactivity to phenylephrine. Murine J774 macrophages activated with lipopolysaccharide (100 ng/ml) produced significant amounts of nitrites (NO2-; 28.2 +/- 3.5 nmol/10(6) cells per 24 h). Cloricromene (2, 20 or 200 microM) added to the cells concomitantly with lipopolysaccharide inhibited NO2- production in a concentration-dependent manner. Maximum inhibition (84.0 +/- 8.0%) was observed when cloricromene (200 microM) was added to the cells 6 h before lipopolysaccharide, whereas it was ineffective when given 6 h after endotoxin. These results demonstrate that cloricromene inhibits the expression but not the activity of the inducible NO synthase.

  2. Endothelial nitric oxide synthase gene polymorphism is associated with sickle cell disease patients in India.

    PubMed

    Nishank, Sudhansu Sekhar; Singh, Mendi Prema Shyam Sunder; Yadav, Rajiv; Gupta, Rasik Bihari; Gadge, Vijay Sadashiv; Gwal, Anil

    2013-12-01

    Patients with sickle cell disease (SCD) produce significantly low levels of plasma nitric oxide (NO) during acute vaso-occlusive crisis. In transgenic sickle cell mice, NO synthesized by endothelial nitric oxide synthase (eNOS) enzyme of vascular endothelial cells has been found to protect the mice from vaso-occlusive events. Therefore, the present study aims to explore possible association of eNOS gene polymorphism as a potential genetic modifier in SCD patients. A case control study involving 150 SCD patients and age- and ethnicity-matched 150 healthy controls were genotyped by PCR-restriction fragment length polymorphism techniques for three important eNOS gene polymorphisms-eNOS 4a/b, eNOS 894G>T and eNOS -786T>C. It was observed that SCD patients had significantly higher frequencies of mutant alleles besides heterozygous and homozygous mutant genotypes of these three eNOS gene polymorphisms and low levels of plasma nitrite (NO2) as compared with control groups. The SCD severe group had significantly lower levels of plasma NO2 and higher frequencies of mutant alleles of these three SNPs of eNOS gene in contrast to the SCD mild group of patients. Haplotype analysis revealed that frequencies of one mutant haplotype '4a-T-C' (alleles in order of eNOS 4a/b, eNOS 894G>T and eNOS -786T>C) were significantly high in the severe SCD patients (P<0.0001), whereas the frequency of a wild haplotype '4b-G-T' was found to be significantly high (P<0.0001) in the SCD mild patients, which indicates that eNOS gene polymorphisms are associated with SCD patients in India and may act as a genetic modifier of the phenotypic variation of SCD patients.

  3. Regulation of endothelial nitric oxide synthase by agmatine after transient global cerebral ischemia in rat brain.

    PubMed

    Mun, Chin Hee; Lee, Won Taek; Park, Kyung Ah; Lee, Jong Eun

    2010-09-01

    Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) plays a protective role in cerebral ischemia by maintaining vascular permeability, whereas NO derived from neuronal and inducible NOS is neurotoxic and can participate in neuronal damage occurring in ischemia. Matrix metalloproteinases (MMPs) are up-regulated by ischemic injury and degrade the basement membrane if brain vessels to promote cell death and tissue injury. We previously reported that agmatine, synthesized from L-arginine by arginine decarboxylase (ADC) which is expressed in endothelial cells, has shown a direct increased eNOS expression and decreased MMPs expression in bEnd3 cells. But, there are few reports about the regulation of eNOS by agmatine in ischemic animal model. In the present study, we examined the expression of eNOS and MMPs by agmatine treatment after transient global ischemia in vivo. Global ischemia was induced with four vessel occlusion (4-VO) and agmatine (100 mg/kg) was administered intraperitoneally at the onset of reperfusion. The animals were euthanized at 6 and 24 hours after global ischemia and prepared for other analysis. Global ischemia led severe neuronal damage in the rat hippocampus and cerebral cortex, but agmatine treatment protected neurons from ischemic injury. Moreover, the level and expression of eNOS was increased by agmatine treatment, whereas inducible NOS (iNOS) and MMP-9 protein expressions were decreased in the brain. These results suggest that agmatine protects microvessels in the brain by activation eNOS as well as reduces extracellular matrix degradation during the early phase of ischemic insult.

  4. Arginase inhibition enhances angiogenesis in endothelial cells exposed to hypoxia.

    PubMed

    Wang, Lin; Bhatta, Anil; Toque, Haroldo A; Rojas, Modesto; Yao, Lin; Xu, Zhimin; Patel, Chintan; Caldwell, Ruth B; Caldwell, R William

    2015-03-01

    Hypoxia-induced arginase elevation plays an essential role in several vascular diseases but influence of arginase on hypoxia-mediated angiogenesis is completely unknown. In this study, in vitro network formation in bovine aortic endothelial cells (BAEC) was examined after exposure to hypoxia for 24h with or without arginase inhibition. Arginase activity, protein levels of the two arginase isoforms, eNOS, and VEGF as well as production of NO and ROS were examined to determine the involvement of arginase in hypoxia-mediated angiogenesis. Hypoxia elevated arginase activity and arginase 2 expression but reduced active p-eNOS(Ser1177) and NO levels in BAEC. In addition, both VEGF protein levels and endothelial elongation and network formation were reduced with continued hypoxia, whereas ROS levels increased and NO levels decreased. Arginase inhibition limited ROS, restored NO formation and VEGF expression, and prevented the reduction of angiogenesis. These results suggest a fundamental role of arginase activity in regulating angiogenic function.

  5. Metformin attenuates ventricular hypertrophy by activating the AMP-activated protein kinase-endothelial nitric oxide synthase pathway in rats.

    PubMed

    Zhang, Cheng-Xi; Pan, Si-Nian; Meng, Rong-Sen; Peng, Chao-Quan; Xiong, Zhao-Jun; Chen, Bao-Lin; Chen, Guang-Qin; Yao, Feng-Juan; Chen, Yi-Li; Ma, Yue-Dong; Dong, Yu-Gang

    2011-01-01

    1. Metformin is an activator of AMP-activated protein kinase (AMPK). Recent studies suggest that pharmacological activation of AMPK inhibits cardiac hypertrophy. In the present study, we examined whether long-term treatment with metformin could attenuate ventricular hypertrophy in a rat model. The potential involvement of nitric oxide (NO) in the effects of metformin was also investigated. 2. Ventricular hypertrophy was established in rats by transaortic constriction (TAC). Starting 1 week after the TAC procedure, rats were treated with metformin (300 mg/kg per day, p.o.), N(G)-nitro-L-arginine methyl ester (L-NAME; 50 mg/kg per day, p.o.) or both for 8 weeks prior to the assessment of haemodynamic function and cardiac hypertrophy. 3. Cultured cardiomyocytes were used to examine the effects of metformin on the AMPK-endothelial NO synthase (eNOS) pathway. Cells were exposed to angiotensin (Ang) II (10⁻⁶ mol/L) for 24 h under serum-free conditions in the presence or absence of metformin (10⁻³ mol/L), compound C (10⁻⁶ mol/L), L-NAME (10⁻⁶ mol/L) or their combination. The rate of incorporation of [³H]-leucine was determined, western blotting analyses of AMPK-eNOS, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) were undertaken and the concentration of NO in culture media was determined. 4. Transaortic constriction resulted in significant haemodynamic dysfunction and ventricular hypertrophy. Myocardial fibrosis was also evident. Treatment with metformin improved haemodynamic function and significantly attenuated ventricular hypertrophy. Most of the effects of metformin were abolished by concomitant L-NAME treatment. L-NAME on its own had no effect on haemodynamic function and ventricular hypertrophy in TAC rats. 5. In cardiomyocytes, metformin inhibited AngII-induced protein synthesis, an effect that was suppressed by the AMPK inhibitor compound C or the eNOS inhibitor L-NAME. The improvement in cardiac structure and

  6. Inhibitory effects of NO on carotid body: contribution of neural and endothelial nitric oxide synthase isoforms.

    PubMed

    Valdés, Viviana; Mosqueira, Matías; Rey, Sergio; Del Rio, Rodrigo; Iturriaga, Rodrigo

    2003-01-01

    We tested the hypothesis that nitric oxide (NO) produced within the carotid body is a tonic inhibitor of chemoreception and determined the contribution of neuronal and endothelial nitric oxide synthase (eNOS) isoforms to the inhibitory NO effect. Accordingly, we studied the effect of NO generated from S-nitroso-N-acetylpenicillamide (SNAP) and compared the effects of the nonselective inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) and the selective nNOS inhibitor 1-(2-trifluoromethylphenyl)-imidazole (TRIM) on chemosensory dose-response curves induced by nicotine and NaCN and responses to hypoxia (Po(2) approximately 30 Torr). CBs excised from pentobarbitone-anesthetized cats were perfused in vitro with Tyrode at 38 degrees C and pH 7.40, and chemosensory discharges were recorded from the carotid sinus nerve. SNAP (100 microM) reduced the responses to nicotine and NaCN. l-NAME (1 mM) enhanced the responses to nicotine and NaCN by increasing their duration, but TRIM (100 microM) only enhanced the responses to high doses of NaCN. The amplitude of the response to hypoxia was enhanced by l-NAME but not by TRIM. Our results suggest that both isoforms contribute to the NO action, but eNOS being the main source for NO in the cat CB and exerting a tonic effect upon chemoreceptor activity.

  7. Endothelial nitric oxide synthase: From biochemistry and gene structure to clinical implications of NOS3 polymorphisms.

    PubMed

    Oliveira-Paula, Gustavo H; Lacchini, Riccardo; Tanus-Santos, Jose E

    2016-01-10

    Nitric oxide (NO) is an important vasodilator with a well-established role in cardiovascular homeostasis. While mediator is synthesized from L-arginine by neuronal, endothelial, and inducible nitric oxide synthases (NOS1,NOS3 and NOS2 respectively), NOS3 is the most important isoform for NO formation in the cardiovascular system. NOS3 is a dimeric enzyme whose expression and activity are regulated at transcriptional, posttranscriptional,and posttranslational levels. The NOS3 gene, which encodes NOS3, exhibits a number of polymorphic sites including single nucleotide polymorphisms (SNPs), variable number of tandem repeats (VNTRs), microsatellites, and insertions/deletions. Some NOS3 polymorphisms show functional effects on NOS3 expression or activity, thereby affecting NO formation. Interestingly, many studies have evaluated the effects of functional NOS3 polymorphisms on disease susceptibility and drug responses. Moreover, some studies have investigated how NOS3 haplotypes may impact endogenous NO formation and disease susceptibility. In this article,we carried out a comprehensive review to provide a basic understanding of biochemical mechanisms involved in NOS3 regulation and how genetic variations in NOS3 may translate into relevant clinical and pharmacogenetic implications.

  8. Role of Carnitine Acetyl Transferase in Regulation of Nitric Oxide Signaling in Pulmonary Arterial Endothelial Cells

    PubMed Central

    Sharma, Shruti; Sun, Xutong; Agarwal, Saurabh; Rafikov, Ruslan; Dasarathy, Sridevi; Kumar, Sanjiv; Black, Stephen M.

    2013-01-01

    Congenital heart defects with increased pulmonary blood flow (PBF) result in pulmonary endothelial dysfunction that is dependent, at least in part, on decreases in nitric oxide (NO) signaling. Utilizing a lamb model with left-to-right shunting of blood and increased PBF that mimics the human disease, we have recently shown that a disruption in carnitine homeostasis, due to a decreased carnitine acetyl transferase (CrAT) activity, correlates with decreased bioavailable NO. Thus, we undertook this study to test the hypothesis that the CrAT enzyme plays a major role in regulating NO signaling through its effect on mitochondrial function. We utilized the siRNA gene knockdown approach to mimic the effect of decreased CrAT activity in pulmonary arterial endothelial cells (PAEC). Our data indicate that silencing the CrAT gene disrupted cellular carnitine homeostasis, reduced the expression of mitochondrial superoxide dismutase-and resulted in an increase in oxidative stress within the mitochondrion. CrAT gene silencing also disrupted mitochondrial bioenergetics resulting in reduced ATP generation and decreased NO signaling secondary to a reduction in eNOS/Hsp90 interactions. Thus, this study links the disruption of carnitine homeostasis to the loss of NO signaling observed in children with CHD. Preserving carnitine homeostasis may have important clinical implications that warrant further investigation. PMID:23344032

  9. Extensive Ethnogenomic Diversity of Endothelial Nitric Oxide Synthase (eNOS) Polymorphisms

    PubMed Central

    Thomas, Bolaji N.; Thakur, Tanya J.; Yi, Li; Guindo, Aldiouma; Diallo, Dapa A.; Ott, Jurg

    2013-01-01

    Nitric oxide (NO) is highly reactive, produced in endothelial cells by endothelial NO synthase (eNOS) and has been implicated in sickle cell pathophysiology. We evaluated the distribution of functionally significant eNOS variants (the T786C variant in the promoter region, the Glu298Asp variant in exon 7, and the variable number of tandem repeats (VNTR) in intron 4) in Africans, African Americans and Caucasians. The C-786 variant was more common in Caucasians than in Africans and African Americans. Consistent with other findings, the Asp-298 variant had the highest frequency in Caucasians followed by African Americans, but was completely absent in Africans. The very rare intron 4 allele, eNOS 4c, was found in some Africans and African Americans, but not in Caucasians. eNOS 4d allele was present in 2 Africans. These findings suggest a consistent and widespread genomic diversity in the distribution of eNOS variants in Africans, comparative to African Americans and Caucasians. PMID:23400313

  10. Amphotericin B severely affects expression and activity of the endothelial constitutive nitric oxide synthase involving altered mRNA stability

    PubMed Central

    Suschek, Christoph Viktor; Bonmann, Eckhard; Kleinert, Hartmut; Wenzel, Michael; Mahotka, Csaba; Kolb, Hubert; Förstermann, Ulrich; Gerharz, Claus-Dieter; Kolb-Bachofen, Victoria

    2000-01-01

    The therapeutic use of the antifungal drug amphotericin B (AmB) is limited due to severe side effects like glomerular vasoconstriction and risk of renal failure during AmB administration. As nitric oxide (NO) has substantial functions in renal autoregulation, we have determined the effects of AmB on endothelial constitutive NO synthase (ecNOS) expression and activity in human and rat endothelial cell cultures.AmB used at concentrations of 0.6 to 1.25 μg ml−1 led to increases in ecNOS mRNA and protein expression as well as NO production. This was the result of an increased ecNOS mRNA half-life. In contrast, incubation of cells with higher albeit subtoxic concentrations of AmB (2.5–5.0 μg ml−1) resulted in a decrease or respectively in completely abolished ecNOS mRNA and protein expression with a strongly reduced or inhibited ecNOS activity, due to a decrease of ecNOS mRNA half-life. None of the AmB concentrations affected promoter activity as found with a reporter gene construct stably transfected into ECV304 cells.Thus, our experiments show a concentration-dependent biphasic effect of AmB on expression and activity of ecNOS, an effect best explained by AmB influencing ecNOS mRNA stability. In view of the known renal accumulation of this drug the results reported here could help to elucidate its renal toxicity. PMID:11015297

  11. Role of phosphatase activity of soluble epoxide hydrolase in regulating simvastatin-activated endothelial nitric oxide synthase.

    PubMed

    Hou, Hsin-Han; Liao, Yi-Jen; Hsiao, Sheng-Huang; Shyue, Song-Kun; Lee, Tzong-Shyuan

    2015-08-25

    Soluble epoxide hydrolase (sEH) has C-terminal epoxide hydrolase and N-terminal lipid phosphatase activity. Its hydrolase activity is associated with endothelial nitric oxide synthase (eNOS) dysfunction. However, little is known about the role of sEH phosphatase in regulating eNOS activity. Simvastatin, a clinical lipid-lowering drug, also has a pleiotropic effect on eNOS activation. However, whether sEH phosphatase is involved in simvastatin-activated eNOS activity remains elusive. We investigated the role of sEH phosphatase activity in simvastatin-mediated activation of eNOS in endothelial cells (ECs). Simvastain increased the phosphatase activity of sEH, which was diminished by pharmacological inhibitors of sEH phosphatase. In addition, pharmacological inhibition of sEH phosphatase or overexpressing the inactive phosphatase domain of sEH enhanced simvastatin-induced NO bioavailability, tube formation and phosphorylation of eNOS, Akt, and AMP-activated protein kinase (AMPK). In contrast, overexpressing the phosphatase domain of sEH limited the simvastatin-increased NO biosynthesis and eNOS phosphorylation at Ser1179. Simvastatin evoked epidermal growth factor receptor-c-Src-increased Tyr phosphorylation of sEH and formation of an sEH-Akt-AMPK-eNOS complex, which was abolished by the c-Src kinase inhibitor PP1 or c-Src dominant-negative mutant K298M. These findings suggest that sEH phosphatase activity negatively regulates simvastatin-activated eNOS by impeding the Akt-AMPK-eNOS signaling cascade.

  12. Role of phosphatase activity of soluble epoxide hydrolase in regulating simvastatin-activated endothelial nitric oxide synthase

    PubMed Central

    Hou, Hsin-Han; Liao, Yi-Jen; Hsiao, Sheng-Huang; Shyue, Song-Kun; Lee, Tzong-Shyuan

    2015-01-01

    Soluble epoxide hydrolase (sEH) has C-terminal epoxide hydrolase and N-terminal lipid phosphatase activity. Its hydrolase activity is associated with endothelial nitric oxide synthase (eNOS) dysfunction. However, little is known about the role of sEH phosphatase in regulating eNOS activity. Simvastatin, a clinical lipid-lowering drug, also has a pleiotropic effect on eNOS activation. However, whether sEH phosphatase is involved in simvastatin-activated eNOS activity remains elusive. We investigated the role of sEH phosphatase activity in simvastatin-mediated activation of eNOS in endothelial cells (ECs). Simvastain increased the phosphatase activity of sEH, which was diminished by pharmacological inhibitors of sEH phosphatase. In addition, pharmacological inhibition of sEH phosphatase or overexpressing the inactive phosphatase domain of sEH enhanced simvastatin-induced NO bioavailability, tube formation and phosphorylation of eNOS, Akt, and AMP-activated protein kinase (AMPK). In contrast, overexpressing the phosphatase domain of sEH limited the simvastatin-increased NO biosynthesis and eNOS phosphorylation at Ser1179. Simvastatin evoked epidermal growth factor receptor–c-Src–increased Tyr phosphorylation of sEH and formation of an sEH–Akt–AMPK–eNOS complex, which was abolished by the c-Src kinase inhibitor PP1 or c-Src dominant-negative mutant K298M. These findings suggest that sEH phosphatase activity negatively regulates simvastatin-activated eNOS by impeding the Akt–AMPK–eNOS signaling cascade. PMID:26304753

  13. Tannin 1-alpha-O-galloylpunicalagin induces the calcium-dependent activation of endothelial nitric-oxide synthase via the phosphatidylinositol 3-kinase/Akt pathway in endothelial cells.

    PubMed

    Chen, Lih-Geeng; Liu, Yen-Chin; Hsieh, Chia-Wen; Liao, Being-Chyuan; Wung, Being-Sun

    2008-10-01

    Many polyphenols have been found to increase endothelial nitric oxide (NO) production. In our present study, we investigated the effects of 1-alpha-O-galloylpunicalagin upon endothelial nitric oxide synthase (eNOS) activity in endothelial cells (ECs). Both 1-alpha-O-galloylpunicalagin and punicalagin induced NO production in a dose-dependent manner in ECs. Despite having similar chemical structures, punicalagin induced lower levels of NO production than 1-alpha-O-galloylpunicalagin. After 1-alpha-O-galloylpunicalagin addition, a rise in the intracellular Ca(2+) concentration preceded NO production. The Ca(2+) ionophore A23187 stimulated eNOS phosphorylation and augmented NO production. Pretreatment with Ca(2+) chelators inhibited 1-alpha-O-galloylpunicalagin-induced eNOS phosphorylation and NO production. Treatment with 1-alpha-O-galloylpunicalagin did not alter the eNOS protein levels but, unlike punicalagin, induced a sustained activation of eNOS Ser(1179) phosphorylation. 1-alpha-O-galloylpunicalagin was also found to activate ERK1/2, JNK and Akt in ECs. Moreover, simultaneous treatment of these cells with specific phosphatidylinositol-3-kinase inhibitors significantly inhibited the observed increases in eNOS activity and phosphorylation levels. In contrast, the inhibition of (ERK)1/2, JNK and p38 had no influence on eNOS Ser(1179) phosphorylation. Our present results thus indicate that the 1-alpha-O-galloylpunicalagin-induced calcium-dependent activation of eNOS is primarily mediated via a phosphatidylinositol 3-kinase/Akt-dependent increase in eNOS activity, and occurs independently of the eNOS protein content.

  14. Endothelial Nitric Oxide Synthase G894T Polymorphism Associates with Disease Severity in Puumala Hantavirus Infection

    PubMed Central

    Koskela, Sirpa; Laine, Outi; Mäkelä, Satu; Pessi, Tanja; Tuomisto, Sari; Huhtala, Heini; Karhunen, Pekka J.; Pörsti, Ilkka; Mustonen, Jukka

    2015-01-01

    Introduction Hantavirus infections are characterized by both activation and dysfunction of the endothelial cells. The underlying mechanisms of the disease pathogenesis are not fully understood. Here we tested the hypothesis whether the polymorphisms of endothelial nitric oxide synthase, eNOS G894T, and inducible nitric oxide synthase, iNOS G2087A, are associated with the severity of acute Puumala hantavirus (PUUV) infection. Patients and Methods Hospitalized patients (n = 172) with serologically verified PUUV infection were examined. Clinical and laboratory variables reflecting disease severity were determined. The polymorphisms of eNOS G894T (Glu298Asp, rs1799983) and iNOS G2087A (Ser608Leu, rs2297518) were genotyped. Results The rare eNOS G894T genotype was associated with the severity of acute kidney injury (AKI). The non-carriers of G-allele (TT-homozygotes) had higher maximum level of serum creatinine than the carriers of G-allele (GT-heterozygotes and GG-homozygotes; median 326, range 102–1041 vs. median 175, range 51–1499 μmol/l; p = 0.018, respectively). The length of hospital stay was longer in the non-carriers of G-allele than in G-allele carriers (median 8, range 3–14 vs. median 6, range 2–15 days; p = 0.032). The rare A-allele carriers (i.e. AA-homozygotes and GA-heterozygotes) of iNOS G2087A had lower minimum systolic and diastolic blood pressure than the non-carriers of A-allele (median 110, range 74–170 vs.116, range 86–162 mmHg, p = 0.019, and median 68, range 40–90 vs. 72, range 48–100 mmHg; p = 0.003, respectively). Conclusions Patients with the TT-homozygous genotype of eNOS G894T had more severe PUUV-induced AKI than the other genotypes. The eNOS G894T polymorphism may play role in the endothelial dysfunction observed during acute PUUV infection. PMID:26561052

  15. Nitric oxide scavenging causes remodeling of the endoplasmic reticulum, Golgi apparatus and mitochondria in pulmonary arterial endothelial cells.

    PubMed

    Lee, Jason E; Yuan, Huijuan; Liang, Feng-Xia; Sehgal, Pravin B

    2013-09-01

    The dependence of the structure and function of cytoplasmic organelles in endothelial cells on constitutively produced intracellular nitric oxide (NO) remains largely unexplored. We previously reported fragmentation of the Golgi apparatus in cells exposed to NO scavengers or after siRNA-mediated knockdown of eNOS. Others have reported increased mitochondrial fission in response to an NO donor. Functionally, we previously reported that bovine pulmonary arterial endothelial cells (PAECs) exposed to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) developed a prosecretory phenotype characterized by prolonged secretion of soluble proteins. In the present study, we investigated whether NO scavenging led to remodeling of the endoplasmic reticulum (ER). Live-cell DAF-2DA imaging confirmed the presence of intracellular NO in association with the BODIPY C5-ceramide-labeled Golgi apparatus. Untreated human PAECs displayed a pattern of peripheral tubulo-reticular ER with a juxtanuclear accumulation of ER sheets. Cells exposed to c-PTIO showed a dramatic increase in ER sheets as assayed using immunofluorescence for the ER structural protein reticulon-4b/Nogo-B and the ER-resident GTPase atlastin-3, live-cell fluorescence assays using RTN4-GFP and KDEL-mCherry, and electron microscopy methods. These ER changes were inhibited by the NO donor diethylamine NONOate, and also produced by L-NAME, but not D-NAME or 8-br-cGMP. This ER remodeling was accompanied by Golgi fragmentation and increased fibrillarity and function of mitochondria (uptake of tetramethyl-rhodamine, TMRE). Despite Golgi fragmentation the functional ER/Golgi trafficking unit was preserved as seen by the accumulation of Sec31A ER exit sites adjacent to the dispersed Golgi elements and a 1.8-fold increase in secretion of soluble cargo. Western blotting and immunopanning data showed that RTN4b was increasingly ubiquitinated following c-PTIO exposure, especially in the

  16. Nitric oxide scavenging causes remodeling of the endoplasmic reticulum, Golgi apparatus and mitochondria in pulmonary arterial endothelial cells

    PubMed Central

    Lee, Jason E.; Yuan, Huijuan; Liang, Feng-Xia; Sehgal, Pravin B.

    2013-01-01

    The dependence of the structure and function of cytoplasmic organelles in endothelial cells on constitutively produced intracellular nitric oxide (NO) remains largely unexplored. We previously reported fragmentation of the Golgi apparatus in cells exposed to NO scavengers or after siRNA-mediated knockdown of eNOS. Others have reported increased mitochondrial fission in response to an NO donor. Functionally, we previously reported that bovine pulmonary arterial endothelial cells (PAECs) exposed to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) developed a prosecretory phenotype characterized by prolonged secretion of soluble proteins. In the present study, we investigated whether NO scavenging led to remodeling of the endoplasmic reticulum (ER). Live-cell DAF-2DA imaging confirmed the presence of intracellular NO in association with the BODIPY C5- ceramide-labelled Golgi apparatus. Untreated human PAECs displayed a pattern of peripheral tubulo-reticular ER with a juxtanuclear accumulation of ER sheets. Cells exposed to c-PTIO showed a dramatic increase in ER sheets as assayed using immunofluoresence for the ER structural protein reticulon-4b/Nogo-B and the ER-resident GTPase atlastin-3, live-cell fluorescence assays using RTN4-GFP and KDEL-mCherry, and electron microscopy methods. These ER changes were inhibited by the NO donor diethylamine NONOate, and also produced by L-NAME, but not D-NAME or 8-br-cGMP. This ER remodeling was accompanied by Golgi fragmentation and increased fibrillarity and function of mitochondria (uptake of tetramethyl- rhodamine, TMRE). Despite Golgi fragmentation the functional ER/Golgi trafficking unit was preserved as seen by the accumulation of Sec31A ER exit sites adjacent to the dispersed Golgi elements and a 1.8-fold increase in secretion of soluble cargo. Western blotting and immunopanning data showed that RTN4b was increasingly ubiquitinated following c-PTIO exposure, especially in the

  17. Rapid nontranscriptional activation of endothelial nitric oxide synthase mediates increased cerebral blood flow and stroke protection by corticosteroids

    PubMed Central

    Limbourg, Florian P.; Huang, Zhihong; Plumier, Jean-Christophe; Simoncini, Tommaso; Fujioka, Masayuki; Tuckermann, Jan; Schütz, Günther; Moskowitz, Michael A.; Liao, James K.

    2002-01-01

    Many cellular responses to corticosteroids involve the transcriptional modulation of target genes by the glucocorticoid receptor (GR). A rapid, non-nuclear effect of GR was found to mediate neuroprotection. High-dose corticosteroids (20 mg/kg intraperitoneally), given within 2 hours of transient cerebral ischemia, acutely increased endothelial nitric oxide synthase (eNOS) activity, augmented regional cerebral blood flow (CBF) by 40% to 50%, and reduced cerebral infarct size by 32%. These neuroprotective effects of corticosteroids were abolished by the GR antagonist RU486 and by inhibition of phosphatidylinositol 3-kinase (PI3K), and were absent in eNOS–/– mice. To determine the mechanism by which GR activated eNOS, we measured the effect of corticosteroids on PI3K and the protein kinase Akt. In a ligand-dependent manner, GR activated PI3K and Akt in vitro and in vivo caused NO-dependent vasodilation, which was blocked by cotreatment with RU486 or the PI3K inhibitor LY294002 but not by transcriptional inhibitors. Indeed, a mutant GR, which cannot dimerize and bind to DNA, still activated PI3K and Akt in response to corticosteroids. These findings indicate that non-nuclear GR rapidly activates eNOS through the PI3K/Akt pathway and suggest that this mechanism mediates the acute neuroprotective effects of corticosteroids through augmentation of CBF. PMID:12464678

  18. Role of PECAM-1 in the shear-stress-induced activation of Akt and the endothelial nitric oxide synthase (eNOS) in endothelial cells.

    PubMed

    Fleming, Ingrid; Fisslthaler, Beate; Dixit, Madhulika; Busse, Rudi

    2005-09-15

    The application of fluid shear stress to endothelial cells elicits the formation of nitric oxide (NO) and phosphorylation of the endothelial NO synthase (eNOS). Shear stress also elicits the enhanced tyrosine phosphorylation of endothelial proteins, especially of those situated in the vicinity of cell-cell contacts. Since a major constituent of these endothelial cell-cell contacts is the platelet endothelial cell adhesion molecule-1 (PECAM-1) we assessed the role of PECAM-1 in the activation of eNOS. In human endothelial cells, shear stress induced the tyrosine phosphorylation of PECAM-1 and enhanced the association of PECAM-1 with eNOS. Endothelial cell stimulation with shear stress elicited the phosphorylation of Akt and eNOS as well as of the AMP-activated protein kinase (AMPK). While the shear-stress-induced tyrosine phosphorylation of PECAM-1 as well as the serine phosphorylation of Akt and eNOS were abolished by the pre-treatment of cells with the tyrosine kinase inhibitor PP1 the phosphorylation of AMPK was unaffected. Down-regulation of PECAM-1 using a siRNA approach attenuated the shear-stress-induced phosphorylation of Akt and eNOS, as well as the shear-stress-induced accumulation of cyclic GMP levels while the shear-stress-induced phosphorylation of AMPK remained intact. A comparable attenuation of Akt and eNOS (but not AMPK) phosphorylation and NO production was also observed in endothelial cells generated from PECAM-1-deficient mice. These data indicate that the shear-stress-induced activation of Akt and eNOS in endothelial cells is modulated by the tyrosine phosphorylation of PECAM-1 whereas the shear-stress-induced phosphorylation of AMPK is controlled by an alternative signaling pathway.

  19. Effects of Vascular-Endothelial Protein Tyrosine Phosphatase Inhibition on Breast Cancer Vasculature and Metastatic Progression

    PubMed Central

    2013-01-01

    Background The solid tumor microvasculature is characterized by structural and functional abnormality and mediates several deleterious aspects of tumor behavior. Here we determine the role of vascular endothelial protein tyrosine phosphatase (VE-PTP), which deactivates endothelial cell (EC) Tie-2 receptor tyrosine kinase, thereby impairing maturation of tumor vessels. Methods AKB-9778 is a first-in-class VE-PTP inhibitor. We examined its effects on ECs in vitro and on embryonic angiogenesis in vivo using zebrafish assays. We studied the impact of AKB-9778 therapy on the tumor vasculature, tumor growth, and metastatic progression using orthotopic models of murine mammary carcinoma as well as spontaneous and experimental metastasis models. Finally, we used endothelial nitric oxide synthase (eNOS)–deficient mice to establish the role of eNOS in mediating the effects of VE-PTP inhibition. All statistical tests were two-sided. Results AKB-9778 induced ligand-independent Tie-2 activation in ECs and impaired embryonic zebrafish angiogenesis. AKB-9778 delayed the early phase of mammary tumor growth by maintaining vascular maturity (P < .01, t test); slowed growth of micrometastases (P < .01, χ2 test) by preventing extravasation of tumor cells (P < 0.01, Fisher exact test), resulting in a trend toward prolonged survival (27.0 vs 36.5 days; hazard ratio of death = 0.33, 95% confidence interval = 0.11 to 1.03; P = .05, Mantel–Cox test); and stabilized established primary tumor blood vessels, enhancing tumor perfusion (P = .03 for 4T1 tumor model and 0.05 for E0771 tumor model, by two-sided t tests) and, hence, radiation response (P < .01, analysis of variance; n = 7 mice per group). The effects of AKB-9778 on tumor vessels were mediated in part by endothelial nitric oxide synthase activation. Conclusions Our results demonstrate that pharmacological VE-PTP inhibition can normalize the structure and function of tumor vessels through Tie-2 activation, which delays tumor

  20. Nitric oxide released from activated platelets inhibits platelet recruitment.

    PubMed Central

    Freedman, J E; Loscalzo, J; Barnard, M R; Alpert, C; Keaney, J F; Michelson, A D

    1997-01-01

    Vessel injury and thrombus formation are the cause of most ischemic coronary syndromes and, in this setting, activated platelets stimulate platelet recruitment to the growing thrombus. Recently, a constitutive nitric oxide synthase (NOS) has been identified in human platelets. To further define the capacity of platelets to produce nitric oxide (NO), as well as to study the role of this NO in platelet recruitment, we adapted a NO-selective microelectrode for use in a standard platelet aggregometer, thereby permitting simultaneous measurement of platelet aggregation and NO production. Treatment of platelets with the NO synthase inhibitor -NG-nitroarginine methyl ester (L-NAME), reduced NO production by 92+/-8% in response to 5 microM ADP compared to control but increased aggregation by only 15+/-2%. In contrast, L-NAME had a more pronounced effect on platelet recruitment as evidenced by a 35+/-5% increase in the extent of aggregation, a 33+/-3% decrease in cyclic GMP content, and a 31+/-5% increase in serotonin release from a second recruitable population of platelets added to stimulated platelets at the peak of NO production. To study platelet recruitment accurately, we developed an assay that monitors two platelet populations simultaneously. Nonbiotinylated platelets were incubated with L-NAME or vehicle and activated with ADP. At peak NO production, biotinylated platelets were added. As measured by three-color flow cytometry, there was a 56+/-11% increase in the number of P selectin- positive platelets in the nonbiotinylated population treated with L-NAME as compared to control. When biotinylated platelets were added to the L-NAME-treated nonbiotinylated population, the number of P selectin positive biotinylated plate-lets increased by 180+/-32% as compared to biotinylated platelets added to the control. In summary, stimulated platelets produce NO that modestly inhibits platelet activation but markedly inhibits additional platelet recruitment. These data suggest

  1. Effects of cocoa extract and dark chocolate on angiotensin-converting enzyme and nitric oxide in human endothelial cells and healthy volunteers--a nutrigenomics perspective.

    PubMed

    Persson, Ingrid A L; Persson, Karin; Hägg, Staffan; Andersson, Rolf G G

    2011-01-01

    Evidence suggests that cocoa from the bean of Theobroma cacao L. has beneficial effects on cardiovascular disease. The aim of this study was to investigate if cocoa extract and dark chocolate influence angiotensin-converting enzyme (ACE) and nitric oxide (NO) in human endothelial cells (in vitro) and in healthy volunteers (in vivo). ACE activity was analyzed with a commercial radioenzymatic assay and measured in human endothelial cells from umbilical veins (HUVEC) after 10 minutes of incubation with cocoa extract. NO was measured after 24 hours of incubation. ACE activity and NO were measured at baseline and after 30, 60, and 180 minutes in 16 healthy volunteers after a single intake of 75 g of dark chocolate containing 72% cocoa. Significant inhibition of ACE activity (P < 0.01) and significant increase of NO (P < 0.001) were seen in HUVEC. In the study subjects, a significant inhibition of ACE activity (mean 18%) 3 hours after intake of dark chocolate was seen, but no significant change in NO was seen. According to ACE genotype, significant inhibition of ACE activity was seen after 3 hours in individuals with genotype insertion/insertion and deletion/deletion (mean 21% and 28%, respectively). Data suggest that intake of dark chocolate containing high amount of cocoa inhibits ACE activity in vitro and in vivo.

  2. ACTIVATION OF VASCULAR ENDOTHELIAL NITRIC OXIDE SYNTHASE AND HEME OXYGENASE-1 EXPRESSION BY ELECTROPHILIC NITRO-FATTY ACIDS

    PubMed Central

    Khoo, Nicholas K.H.; Rudolph, Volker; Cole, Marsha P.; Golin-Bisello, Franca; Schopfer, Francisco J.; Woodcock, Steven R.; Batthyany, Carlos; Freeman, Bruce A.

    2010-01-01

    Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated byproducts of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yield electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO2) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO2 via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO2-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO2-FAs stimulated the phosphorylation of eNOS at Ser1179. These post-translational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO2-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders. PMID:19857569

  3. Endothelial proteoglycans inhibit bFGF binding and mitogenesis.

    PubMed

    Forsten, K E; Courant, N A; Nugent, M A

    1997-08-01

    Basic fibroblast growth factor (bFGF) is a known mitogen for vascular smooth muscle cells and has been implicated as having a role in a number of proliferative vascular disorders. Binding of bFGF to heparin or heparan sulfate has been demonstrated to both stimulate and inhibit growth factor activity. The activity, towards bFGF, of heparan sulfate proteoglycans present within the vascular system is likely related to the chemical characteristics of the glycosaminoglycan as well as the structure and pericellular location of the intact proteoglycans. We have previously shown that endothelial conditioned medium inhibits both bFGF binding to vascular smooth muscle cells and bFGF stimulated cell proliferation in vitro. In the present study, we have isolated proteoglycans from endothelial cell conditioned medium and demonstrated that they are responsible for the bFGF inhibitory activity. We further separated endothelial secreted proteoglycans into two fractions, PG-A and PG-B. The large sized fraction (PG-A) had greater inhibitory activity than did PG-B for both bFGF binding and bFGF stimulation of vascular smooth muscle cell proliferation. The increased relative activity of PG-A was attributed, in part, to larger heparan sulfate chains which were more potent inhibitors of bFGF binding than the smaller heparan sulfate chains on PG-B. Both proteoglycan fractions contained perlecan-like core proteins; however, PG-A contained an additional core protein (approximately 190 kDa) that was not observed in PG-B. Both proteoglycan fractions bound bFGF directly, and PG-A bound a significantly greater relative amount of bFGF than did PG-B. Thus the ability of endothelial heparan sulfate proteoglycans to bind bFGF and prevent its association with vascular smooth muscle cells appears essential for inhibition of bFGF-induced mitogenesis. The production of potent bFGF inhibitory heparan sulfate proteoglycans by endothelial cells might contribute to the maintenance of vascular homeostasis.

  4. Mechanisms of albuminuria in the chronic nitric oxide inhibition model.

    PubMed

    Arcos, M I; Fujihara, C K; Sesso, A; de Almeida Prado, E B; de Almeida Prado, M J; de Nucci, G; Zatz, R

    2000-12-01

    Chronic nitric oxide (NO) inhibition causes hypertension and renal injury. Concomitant salt overload promotes massive albuminuria. We investigated the mechanisms whereby these treatments impair glomerular permselectivity. Adult male Munich-Wistar rats received either a standard-salt (SS; 0.5% Na) or high-salt (HS; 3.1% Na) diet and either no treatment or the NO inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). At 30 days, albuminuria was moderate, the density of fixed anionic sites at the glomerular basement membrane (GBM), estimated by cationic ferritin binding, declined by approximately 35%, and the fractional clearance of 70-kDa neutral dextran (phi) rose moderately in rats receiving L-NAME and SS. Rats given L-NAME and HS exhibited massive albuminuria, whereas phi was nearly tripled. Depletion of GBM anionic sites was also seen in these rats. The GBM was thickened in both L-NAME-treated groups. These abnormalities were largely reversed after cessation of treatments. These results indicate that chronic L-NAME treatment promotes reversible albuminuria by impairing both glomerular size and charge selectivity. These effects likely reflect functional rather than structural disruption of the glomerular wall.

  5. Resveratrol analog piceatannol restores the palmitic acid-induced impairment of insulin signaling and production of endothelial nitric oxide via activation of anti-inflammatory and antioxidative heme oxygenase-1 in human endothelial cells.

    PubMed

    Jeong, Sun-Oh; Son, Yong; Lee, Ju Hwan; Cheong, Yong-Kwan; Park, Seong Hoon; Chung, Hun-Taeg; Pae, Hyun-Ock

    2015-07-01

    Growing evidence suggests that the elevation of free fatty acids, including palmitic acid (PA), are associated with inflammation and oxidative stress, which may be involved in endothelial dysfunction, characterized by the reduced bioavailability of nitric oxide (NO) synthesized from endothelial NO synthase (eNOS). Heme oxygenase-1 (HO-1) is important in the preservation of NO bioavailability. Piceatannol (Pic), with similar chemical structure to resveratrol, is suggested to possess similar protective effects as resveratrol. In the present study, human umbilical vein endothelial cells (HUVECs), stimulated with PA, were used to examine the endothelial protective effects of Pic. Pic increased the expression of HO-1 via nuclear factor erythroid-2-related factor-2 activation in the HUVECs, and decreased the PA-induced secretions of interleukin-6 and tumor necrosis factor-α, and the formation of reactive oxygen species ROS via inhibition of NF-κB activation. Notably, following inhibition of HO-1 activity by tin protoporphryin-IX, Pic did not prevent cytokine secretion, ROS formation, and NF-κB activation in the PA-stimulated HUVECs. PA attenuated insulin-mediated insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, leading to decreased glucose uptake, and phosphorylation of eNOS, leading to a reduction in the production of NO. Pic effectively mitigated the inhibitory effects of PA on the insulin-mediated phosphorylation of IRS-1 and eNOS, which was not observed following inhibition of HO‑1 activity. The results of the present study suggested that Pic may have the potential to prevent PA-induced impairment of insulin signaling and eNOS function, by inducing the expression of the anti-inflammatory and antioxidant, HO-1.

  6. Calcimimetic R-568 and Its Enantiomer S-568 Increase Nitric Oxide Release in Human Endothelial Cells

    PubMed Central

    Morabito, Caterina; Di Silvestre, Sara; Di Cesare, Moreno; Di Pietro, Natalia; Sirolli, Vittorio; Formoso, Gloria; Amoroso, Luigi; Mariggiò, Maria Addolorata; Pandolfi, Assunta

    2012-01-01

    Background Calcimimetics, such as R-568, are thought to activate G protein-linked Ca2+-sensing receptor (CaSR) by allosterically increasing the affinity of the receptor for Ca2+ allowing for efficient control of uremic hyperparathyroidism. Several recent studies suggest they possess additional vascular actions. Although it has been postulated that calcimimetics may have a direct effect on CaSR in the blood vessels, further studies are needed to elucidate their vascular CaSR-dependent versus CaSR-independent effects. Methodology/Principal Findings Focusing on human umbilical vein endothelial cells (HUVECs), we studied the CaSR expression and distribution by Immunofluorescence and Western Blot analysis. CaSR function was evaluated by measuring the potential effect of calcimimetic R-568 and its enantiomer S-568 upon the modulation of intracellular Ca2+ levels (using a single cell approach and FURA-2AM), in the presence or absence of Calhex-231, a negative modulator of CaSR. To address their potential vascular functions, we also evaluated R- and S-568-stimulated enzymatic release of Nitric Oxide (NO) by DAF-2DA, by Nitric Oxide Synthase (NOS) radiometric assay (both in HUVECs and in Human Aortic Endothelial Cells) and by measuring eNOS-ser1177 phosphorylation levels (Immunoblotting). We show that, although the CaSR protein was expressed in HUVECs, it was mainly distributed in cytoplasm while the functional CaSR dimers, usually localized on the plasma membrane, were absent. In addition, regardless of the presence or absence of Calhex-231, both R- and S-568 significantly increased intracellular Ca2+ levels by mobilization of Ca2+ from intracellular stores, which in turn augmented NO release by a time- and Ca2+-dependent increase in eNOS-ser1177 phosphorylation levels. Conclusions/Significance Taken together, these data indicate that in human endothelium there is no stereoselectivity in the responses to calcimimetics and that CaSR is probably not involved in the action of

  7. Arsenic toxicity induced endothelial dysfunction and dementia: Pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors

    SciTech Connect

    Sharma, Bhupesh Sharma, P.M.

    2013-11-15

    Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate and brain GSH levels along with increase in serum and brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. - Highlights: • As has induced endothelial dysfunction (Edf) and vascular dementia (VaD). • As has increased oxidative stress, AChE activity and decreased serum NO. • Inhibitors of HDAC and iNOS have attenuated As induced Edf and VaD. • Both the inhibitors have attenuated As induced biochemical changes. • Inhibitor of HDAC and iNOS has shown good potential in

  8. Homocysteine injures vascular endothelial cells by inhibiting mitochondrial activity

    PubMed Central

    Yang, Fengyong; Qi, Xiujing; Gao, Zheng; Yang, Xingju; Zheng, Xingfeng; Duan, Chonghao; Zheng, Jian

    2016-01-01

    The aim of the present study was to investigate the role of homocysteine (Hcy) in the pathogenesis of pulmonary embolism (PE) and the associated molecular mechanisms in human umbilical vein endothelial cells (HUVECs). Hcy contents were detected with high-performance liquid chromatography. Apoptosis was detected by flow cytometry using Annexin-V staining. Cytochrome c oxidase (COX) activity was assessed with an enzyme activity assay, and the expression levels of COX 17 were determined by western blot analysis. Intracellular reactive oxygen species levels were measured using a microplate reader with a fluorescence probe. The results demonstrated that, compared with the control group, the serum Hcy levels were significantly elevated in the PE group, suggesting that Hcy may be an indicator for PE. Following treatment with Hcy, the apoptosis rate was markedly elevated in HUVECs. Moreover, Hcy decreased COX activity and downregulated the expression of COX 17 in HUVECs. Furthermore, Hcy increased the ROS levels in these endothelial cells. However, all the above-mentioned physiopathological changes induced by Hcy in HUVECs could be restored by folic acid. In conclusion, the results of the present study demonstrated that Hcy inhibited COX activity, downregulated COX 17 expression, increased intracellular ROS levels and enhanced apoptosis in endothelial cells. PMID:27698720

  9. The red-vine-leaf extract AS195 increases nitric oxide synthase-dependent nitric oxide generation and decreases oxidative stress in endothelial and red blood cells.

    PubMed

    Grau, Marijke; Bölck, Birgit; Bizjak, Daniel Alexander; Stabenow, Christina Julia Annika; Bloch, Wilhelm

    2016-02-01

    The red-vine-leaf extract AS195 improves cutaneous oxygen supply and the microcirculation in patients suffering from chronic venous insufficiency. Regulation of blood flow was associated to nitric oxide synthase (NOS)-dependent NO (nitric oxide) production, and endothelial and red blood cells (RBC) have been shown to possess respective NOS isoforms. It was hypothesized that AS195 positively affects NOS activation in human umbilical vein endothelial cells (HUVECs) and RBC. Because patients with microvascular disorders show increased oxidative stress which limits NO bioavailability, it was further hypothesized that AS195 increases NO bioavailability by decreasing the content of reactive oxygen species (ROS) and increasing antioxidant capacity. Cultured HUVECs and RBCs from healthy volunteers were incubated with AS195 (100 μmol/L), tert-butylhydroperoxide (TBHP, 1 mmol/L) to induce oxidative stress and with both AS195 and TBHP. Endothelial and red blood cell-nitric oxide synthase (RBC-NOS) activation significantly increased after AS195 incubation. Nitrite concentration, a marker for NO production, increased in HUVEC but decreased in RBC after AS195 application possibly due to nitrite scavenging potential of flavonoids. S-nitrosylation of RBC cytoskeletal spectrins and RBC deformability were increased after AS195 incubation. TBHP-induced ROS were decreased by AS195, and antioxidative capacity was significantly increased in AS195-treated cells. TBHP also reduced RBC deformability, but reduction was attenuated by parallel incubation with AS195. Adhesion of HUVEC was also reduced after AS195 treatment. Red-vine-leaf extract AS195 increases NOS activation and decreases oxidative stress. Both mechanisms increase NO bioavailability, improve cell function, and may thus account for enhanced microcirculation in both health and disease.

  10. HMG-CoA reductase inhibitor improves endothelial dysfunction in spontaneous hypertensive rats via down-regulation of caveolin-1 and activation of endothelial nitric oxide synthase.

    PubMed

    Suh, Jung-Won; Choi, Dong-Ju; Chang, Hyuk-Jae; Cho, Young-Seok; Youn, Tae-Jin; Chae, In-Ho; Kim, Kwang-Il; Kim, Cheol-Ho; Kim, Hyo-Soo; Oh, Buyng-Hee; Park, Young-Bae

    2010-01-01

    Hypertension is associated with endothelial dysfunction and increased cardiovascular risk. Caveolin-1 regulates nitric oxide (NO) signaling by modulating endothelial nitric oxide synthase (eNOS). The purpose of this study was to examine whether HMG-CoA reductase inhibitor improves impaired endothelial function of the aorta in spontaneous hypertensive rat (SHR) and to determine the underlying mechanisms involved. Eight-week-old male SHR were assigned to either a control group (CON, n=11) or a rosuvastatin group (ROS, n=12), rosuvastatin (10 mg/kg/day) administered for eight weeks. Abdominal aortic rings were prepared and responses to acetylcholine (10(-9)-10(-4) M) were determined in vitro. To evaluate the potential role of NO and caveolin-1, we examined the plasma activity of NOx, eNOS, phosphorylated-eNOS and expression of caveolin-1. The relaxation in response to acetylcholine was significantly enhanced in ROS compared to CON. Expression of eNOS RNA was unchanged, whereas NOx level and phosphorylated-eNOS at serine-1177 was increased accompanied with depressed level of caveolin-1 in ROS. We conclude that 3-Hydroxy-3-methylglutaryl Coenzyme-A (HMG-CoA) reductase inhibitor can improve impaired endothelial dysfunction in SHR, and its underlying mechanisms are associated with increased NO production. Furthermore, HMG-CoA reductase inhibitor can activate the eNOS by phosphorylation related to decreased caveolin-1 abundance. These results imply the therapeutic strategies for the high blood pressure-associated endothelial dysfunction through modifying caveolin status.

  11. Endothelial Nitric Oxide Synthase Gene Single Nucleotide Polymorphism Predicts Cerebral Vasospasm following Aneurysmal Subarachnoid Hemorrhage

    PubMed Central

    Starke, Robert M.; Kim, Grace H.; Komotar, Ricardo J.; Hickman, Zachary L.; Black, Eric M.; Rosales, Maritza B.; Kellner, Christopher P.; Hahn, David K.; Otten, Marc L.; Edwards, John; Wang, Tao; Russo, James J.; Mayer, Stephan A.; Connolly, E. Sander

    2009-01-01

    Summary Vasospasm is a major cause of morbidity and mortality following aneurysmal subarachnoid hemorrhage (aSAH). Studies have demonstrated a link between single nucleotide polymorphisms (SNP) in the endothelial nitric oxide synthase (eNOS) gene and the incidence of coronary spasm and aneurysms. Alterations in the eNOS T-786 SNP may lead to an increased risk of post-aSAH cerebral vasospasm. In this prospective clinical study, 77 aSAH patients provided genetic material and were followed for the occurrence of vasospasm. In multivariate logistic regression analysis, genotype was the only factor predictive of vasospasm. The odds ratio for symptomatic vasospasm in patients with one T allele was 3.3 (95% CI 1.1–10.0, p=0.034) and 10.9 for TT. Patients with angiographic spasm were 3.6 times more likely to have a T allele (95% CI 1.3–9.6, p=0.013, TT OR 12.6). Patients with severe vasospasm requiring endovascular therapy were more likely to have a T allele (OR 3.5, 95% CI 1.3–9.5, p=0.016, TT OR 12.0). Patients with the T allele of the eNOS gene are more likely have severe vasospasm. Presence of this genotype may allow the identification of individuals at high risk for post-aSAH vasospasm and lead to early treatment and improved outcome. PMID:18319732

  12. Endothelial nitric oxide synthase gene polymorphism and elite endurance athlete status: the Genathlete study.

    PubMed

    Wolfarth, B; Rankinen, T; Mühlbauer, S; Ducke, M; Rauramaa, R; Boulay, M R; Pérusse, L; Bouchard, C

    2008-08-01

    In the Genathlete study, we examined the contribution of three polymorphisms in the endothelial nitric oxide synthase (NOS3) gene to discriminate elite endurance athletes (EEA) from sedentary controls (SC). The EEA group included a total of 316 Caucasian males with a VO2max >75 mL/kg. The SC group comprised 299 unrelated sedentary Caucasian males who had VO2max values below 50 mL/kg. The polymerase chain reaction technique was used to amplify a microsatellite (CA)(n) repeat in intron 13, a 27 bp repeat in intron 4 and a third fragment in exon 7 containing the Glu298Asp SNP. No difference was found between the EEA and SC groups for the 27 bp repeat and the Glu298Asp polymorphism. Chi-square analysis of the overall allelic distribution of the (CA)(n) repeat revealed no significant difference between the two groups (P=0.135). However, comparing carriers and non-carriers for the most common (CA)(n) repeat alleles, we found significant differences between SC and EEA, with more EEA subjects carrying the 164 bp allele (P=0.007). In summary, we found suggestive evidence that the 164 bp allele of the (CA)(n) repeat in intron 13 is associated with EEA status and may account for some of the differences between EEA and SC.

  13. Method of measuring nitric oxide release by vascular endothelial cells grown in microfluidic channels

    NASA Astrophysics Data System (ADS)

    Hosseinpour, S.; Liu, A. C.; Barakat, A. I.; Choy, J. C.; Gray, B. L.

    2014-03-01

    In this paper, a simple and versatile method is presented which enables detection of nitric oxide (NO) released from vascular endothelial cells (ECs) cultured in microfluidic structures. The culturing system and NO measurement method allow cell shape to be controlled in a non-invasive manner using microfluidic structures while NO release is monitored for cell shape versus function studies. The culturing system consists of arrays of polydimethylsiloxane (PDMS) fluidic channels 120 micrometers in depth and ranging from 100 micrometers to 3 mm in width. The number of channels in each array is varied to yield a constant cell culture surface area (75 mm2) independent of channel width. The channel surfaces are collagen-coated and ECs are cultured to confluence within the channels. A cell scraper is then used to scrape extraneous cells cultured between channels, and NO measurements are made 18 to 24 hours later. A chemiluminescence-based sensor system (NOA 280i, Sievers NO Analyzer) is utilized to measure sample NO. Initial results indicate that NO concentrations can be measured from different microfluidic channel-containing samples using this method. It is shown that there is no significant difference in NO concentration derived from channels of different widths even though the degree of cell elongation varies due to physical constraint by microfluidic channel walls. However, cells treated with TNFα release more NO than untreated cells in fluidic channels, which is comparable to the function of ECs cultured in conventional culturing systems such as culturing dishes.

  14. Asymmetric dimethylarginine does not inhibit arginase activity and is pro-proliferative in pulmonary endothelial cells.

    PubMed

    Chen, Bernadette; Strauch, Krista; Jin, Yi; Cui, Hongmei; Nelin, Leif D; Chicoine, Louis G

    2014-07-01

    Asymmetric dimethylarginine (ADMA) is an endogenously produced nitric oxide synthase (NOS) inhibitor. L-Arginine can be metabolised by NOS and arginase, and arginase is the first step in polyamine production necessary for cellular proliferation. We tested the hypothesis that ADMA would inhibit NOS but not arginase activity and that this pattern of inhibition would result in greater L-arginine bioavailability to arginase, thereby increasing viable cell number. Bovine arginase was used in in vitro activity assays with various concentrations of substrate (L-arginine, ADMA, N(G) -monomethyl-L-arginine (L-NMMA) and N(G) -nitro-L-arginine methyl ester (L-NAME)). Only L-arginine resulted in measurable urea production (Km = 6.9 ± 0.8 mmol/L; Vmax = 6.6 ± 0.3 μmol/mg protein per min). We then incubated bovine arginase with increasing concentrations of ADMA, L-NMMA and L-NAME in the presence of 1 mmol/L l-arginine and found no effect of any of the tested compounds on arginase activity. Using bovine pulmonary arterial endothelial cells (bPAEC) we determined the effects of ADMA on nitric oxide (NO) and urea production and found significantly lower NO production and greater urea production (P < 0.003) with ADMA, without changes in arginase protein levels. In addition, ADMA treatment resulted in an approximately 30% greater number of viable cells after 48 h than in control bPAEC. These results demonstrate that ADMA is neither a substrate nor an inhibitor of arginase activity and that in bPAEC ADMA inhibits NO production and enhances urea production, leading to more viable cells. These results may have pathophysiological implications in disorders associated with higher ADMA levels, such as pulmonary hypertension.

  15. Asbestos induces nitric oxide synthesis in mesothelioma cells via Rho signaling inhibition.

    PubMed

    Riganti, Chiara; Orecchia, Sara; Silvagno, Francesca; Pescarmona, Gianpiero; Betta, Pier Giacomo; Gazzano, Elena; Aldieri, Elisabetta; Ghigo, Dario; Bosia, Amalia

    2007-06-01

    We have observed that in three human malignant mesothelioma cell lines, crocidolite asbestos induced the activation of the transcription factor NF-kappaB and the synthesis of nitric oxide (NO) by inhibiting the RhoA signaling pathway. The incubation with crocidolite decreased the level of GTP-bound RhoA and the activity of Rho-dependent kinase, and induced the activation of Akt/PKB and IkBalpha kinase, leading to the nuclear translocation of NF-kappaB. The effects of crocidolite fibers on NF-kappaB activation and NO synthesis were mimicked by Y27632 (an inhibitor of the Rho-dependent kinases) and toxin B (an inhibitor of RhoA GTPase activity), while they were reverted by mevalonic acid, the product of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. Furthermore, crocidolite, similarly to mevastatin, inhibited the synthesis of cholesterol and ubiquinone and the prenylation of RhoA: these effects were prevented in the presence of mevalonic acid. This suggests that crocidolite fibers might inhibit the synthesis of isoprenoid molecules at the level of the HMGCoA reductase reaction or of an upstream step, thus impairing the prenylation and subsequent activation of RhoA. Akt can stimulate NO synthesis via a double mechanism: it can activate the inducible NO synthase via the NF-kappaB pathway and the endothelial NO synthase via a direct phosphorylation. Our results suggest that crocidolite increases the NO levels in mesothelioma cells by modulating both NO synthase isoforms.

  16. 15-Deoxy-{delta}{sup 12,14}-prostaglandin J{sub 2}-induced down-regulation of endothelial nitric oxide synthase in association with HSP70 induction

    SciTech Connect

    Hwang, Jinah; Lee, Hyun-Il; Chang, Young-Sun; Lee, Soo Jae; Kim, Kwang Pyo; Park, Sang Ick . E-mail: parksi@nih.go.kr

    2007-05-25

    A natural ligand of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), 15-deoxy-{delta}{sup 12,14}-prostaglandin J{sub 2} (15d-PGJ{sub 2}), decreases endothelial nitric oxide synthase (eNOS) expression by an unknown mechanism. Here we found that 15d-PGJ{sub 2}-induced eNOS reduction is inversely associated with heat shock protein 70 (HSP70) induction in endothelial cells. Treatment of cells with 15d-PGJ{sub 2} decreased eNOS protein expression in a concentration- and time-dependent manner, but independently of PPAR{gamma} with no effect on mRNA levels. Although 15d-PGJ{sub 2} elicited endothelial apoptosis, inhibition of both pan-caspases and cathepsins failed to reverse reduction of eNOS protein. Interestingly, we observed that 15d-PGJ{sub 2} induced HSP70 in a dose-dependent manner. Immunoprecipitation and heat shock treatment demonstrated that eNOS reduction was strongly related to HSP70 induction. Cellular fractionation revealed that treatment with 15d-PGJ{sub 2} increased eNOS distribution 2.5-fold from soluble to insoluble fractions. These findings provide new insights into mechanisms whereby eNOS regulation by 15d-PGJ{sub 2} is related to HSP70 induction.

  17. Determinants of shear stress-stimulated endothelial nitric oxide production assessed in real-time by 4,5-diaminofluorescein fluorescence.

    PubMed

    Qiu, W; Kass, D A; Hu, Q; Ziegelstein, R C

    2001-08-17

    The extremely short biological half-life of endothelial-derived nitric oxide (NO) has impeded real-time measurements of NO synthesis. We used the membrane-permeable fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA) to study determinants of NO synthesis in bovine aortic endothelial cells (BAECs). A step increase in shear stress (SS) from 0.3 to 3.4 dyne/cm(2) triggered an increase in DAF-2 fluorescence starting 3.0 +/- 0.5 min after the flow rise and peaking at 44.7 +/- 7.2 min. This was abolished by intracellular Ca(2+) chelation, but was unaffected by blocking extracellular Ca(2+) influx or by inhibiting SS-related changes in intracellular pH. The increase in DAF-2 fluorescence occurred significantly earlier in BAECs transfected with either superoxide dismutase (SOD) or catalase (CAT), indicating concomitant reactive oxygen species (ROS) generation by SS and "competition" between ROS- and DAF-2-NO interactions. These data provide novel insights into several NO signaling determinants and reveal that DAF-2 can assess real-time SS-stimulated NO synthesis in endothelial cells. This should facilitate the analysis of NO-signaling pathways.

  18. Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells

    NASA Technical Reports Server (NTRS)

    Koh, T. J.; Tidball, J. G.

    2000-01-01

    We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore on C(2)C(12) muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachment, and loss of cellular protein. These results indicate that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore-induced proteolysis was primarily calpain mediated. The activity of m-calpain in a casein zymogram was inhibited by sodium nitroprusside, and this inhibition was reversed by dithiothreitol. Previous incubation with the active site-targeted calpain inhibitor I prevented most of the sodium nitroprusside-induced inhibition of m-calpain activity. These data suggest that nitric oxide inhibited m-calpain activity via S-nitrosylation of the active site cysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.

  19. Selective Irreversible Inhibition of Neuronal and Inducible Nitric-oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide*

    PubMed Central

    Heine, Christian L.; Schmidt, Renate; Geckl, Kerstin; Schrammel, Astrid; Gesslbauer, Bernd; Schmidt, Kurt; Mayer, Bernd; Gorren, Antonius C. F.

    2015-01-01

    Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible NOS was inhibited by the hydrogen sulfide (H2S) donor Na2S with IC50 values of ∼2.4·10−5 and ∼7.9·10−5 m, respectively, whereas human endothelial NOS was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of l-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin, indicating that H2S does not interfere with substrate or cofactor binding. The IC50 decreased to ∼1.5·10−5 m at pH 6.0 and increased to ∼8.3·10−5 m at pH 8.0. Preincubation of concentrated nNOS with H2S under turnover conditions decreased activity after dilution by ∼70%, suggesting irreversible inhibition. However, when calmodulin was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H2S and NO. Likewise, NADPH oxidation was inhibited with an IC50 of ∼1.9·10−5 m in the presence of Arg and BH4 but exhibited much higher IC50 values (∼1.0–6.1·10−4 m) when Arg and/or BH4 was omitted. Moreover, the relatively weak inhibition of nNOS by Na2S in the absence of Arg and/or BH4 was markedly potentiated by the NO donor 1-(hydroxy-NNO-azoxy)-l-proline, disodium salt (IC50 ∼ 1.3–2.0·10−5 m). These results suggest that nNOS and inducible NOS but not endothelial NOS are irreversibly inhibited by H2S/NO at modest concentrations of H2S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H2S formation. PMID:26296888

  20. Widdrol, a sesquiterpene isolated from Juniperus chinensis, inhibits angiogenesis by targeting vascular endothelial growth factor receptor 2 signaling.

    PubMed

    Jin, Soojung; Yun, Hee Jung; Jeong, Hyun Young; Oh, You Na; Park, Hyun-Jin; Yun, Seung-Geun; Kim, Byung Woo; Kwon, Hyun Ju

    2015-09-01

    Widdrol is an odorous compound derived from Juniperus chinensis that is widely used in traditional medicine to treat fever, inflammation and cancer. It was previously reported that widdrol has antitumor activity by apoptosis induction in cancer cells in vitro. However, its anti-angiogenic activity remains elusive. In the present study, we investigated the anti‑angiogenic activity of widdrol and the molecular mechanisms involved. Widdrol inhibited cell proliferation via G1 phase arrest induction in human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Additionally, it was associated with a decreased expression of cyclin-dependent kinase 2 (CDK2) and an increased expression of p21, a CDK inhibitor. Widdrol significantly inhibited the cell migration and tube formation of HUVECs using an in vitro angiogenesis assay. The results showed that widdrol suppressed phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream proteins, such as AKT, focal adhesion kinase (FAK) and endothelial nitric oxide synthase (eNOS). Moreover, widdrol effectively reduced tumor growth and blood vessel formation in colon tumor xenograft mice. Collectively, these results suggested that widdrol may act as a potential anti-angiogenic agent by inhibiting vessel sprouting and growth, which may have implications for angioprevention.

  1. Nitric oxide therapies for local inhibition of platelets' activitation on blood-contacting surfaces

    NASA Astrophysics Data System (ADS)

    Amoako, Kagya Agyeman

    Blood-contacting devices interact with blood during their function much like the endothelium that modulates hemostasis. The surfaces of these devices however, lack endothelial-like properties, and consequently, upon blood contact, activate clotting factors to form clots. Systemic heparinization for inhibiting clot formation can cause bleeding and surface coatings show insignificant benefits. This research investigated nitric oxide (NO) production mimicry of the endothehum on artificial lungs (ALs) and pediatric catheters. Their surfaces were functionalized either by (1) entrapping NO donors inside their bulk, (2) incorporating catalysts to generate NO from NO-donors or (3) supplementing NO into sweep gas of artificial lungs. Pediatric catheters functionalized with NO-donor thin coats using method 1 is limited by short NO release duration. Method 2 has not been applied to large surface-area, low-flow devices like the AL. In this work NO-generating silicone membranes were synthesized and characterized to determine the relationship between surface properties, NO flux, and blood clotting time. These outcomes helped develop and optimize NO-generating gas-exchange silicone fibers that represent the majority of ALs surface area. The first NO-generating AL prototypes, using those fibers, were manufactured, incorporated into NO-generating circuits and tested for their non-thrombogenicity. To test for NO-release duration and non-thrombogenicity, catheters were fabricated to incorporate NO-donors inside their walls, characterized for NO flux and release duration by chemilumincscence, and tested for patency using a thrombogenicity model in rabbits. Methods 1-2 involve material modification using complicated and expensive chemical formulations and/or manufacturing. Method 3 however, functionalizes ALs by only adding NO into sweep gas. Decade-long anti-clotting testing using a wide range of NO concentrations has been conducted without knowledge of what concentration yields

  2. Arginase reciprocally regulates nitric oxide synthase activity and contributes to endothelial dysfunction in aging blood vessels

    NASA Technical Reports Server (NTRS)

    Berkowitz, Dan E.; White, Ron; Li, Dechun; Minhas, Khalid M.; Cernetich, Amy; Kim, Soonyul; Burke, Sean; Shoukas, Artin A.; Nyhan, Daniel; Champion, Hunter C.; Hare, Joshua M.

    2003-01-01

    BACKGROUND: Although abnormal L-arginine NO signaling contributes to endothelial dysfunction in the aging cardiovascular system, the biochemical mechanisms remain controversial. L-arginine, the NO synthase (NOS) precursor, is also a substrate for arginase. We tested the hypotheses that arginase reciprocally regulates NOS by modulating L-arginine bioavailability and that arginase is upregulated in aging vasculature, contributing to depressed endothelial function. METHODS AND RESULTS: Inhibition of arginase with (S)-(2-boronoethyl)-L-cysteine, HCl (BEC) produced vasodilation in aortic rings from young (Y) adult rats (maximum effect, 46.4+/-9.4% at 10(-5) mol/L, P<0.01). Similar vasorelaxation was elicited with the additional arginase inhibitors N-hydroxy-nor-L-arginine (nor-NOHA) and difluoromethylornithine (DFMO). This effect required intact endothelium and was prevented by 1H-oxadiazole quinoxalin-1-one (P<0.05 and P<0.001, respectively), a soluble guanylyl cyclase inhibitor. DFMO-elicited vasodilation was greater in old (O) compared with Y rat aortic rings (60+/-6% versus 39+/-6%, P<0.05). In addition, BEC restored depressed L-arginine (10(-4) mol/L)-dependent vasorelaxant responses in O rings to those of Y. Arginase activity and expression were increased in O rings, whereas NOS activity and cyclic GMP levels were decreased. BEC and DFMO suppressed arginase activity and restored NOS activity and cyclic GMP levels in O vessels to those of Y. CONCLUSIONS: These findings demonstrate that arginase modulates NOS activity, likely by regulating intracellular L-arginine availability. Arginase upregulation contributes to endothelial dysfunction of aging and may therefore be a therapeutic target.

  3. Ethyl-p-methoxycinnamate isolated from kaempferia galanga inhibits inflammation by suppressing interleukin-1, tumor necrosis factor-α, and angiogenesis by blocking endothelial functions

    PubMed Central

    Umar, Muhammad Ihtisham; Asmawi, Mohd Zaini; Sadikun, Amirin; Majid, Amin Malik Shah Abdul; Al-Suede, Fouad Saleih R.; Hassan, Loiy Elsir Ahmed; Altaf, Rabia; Ahamed, Mohamed B. Khadeer

    2014-01-01

    OBJECTIVE: The present study aimed to investigate the mechanisms underlying the anti-inflammatory and anti-angiogenic effects of ethyl-p-methoxycinnamate isolated from Kaempferia galanga. METHODS: The anti-inflammatory effects of ethyl-p-methoxycinnamate were assessed using the cotton pellet granuloma assay in rats, whereby the levels of interleukin-1 and tumor necrosis factor-α were measured in the animals' blood. In addition, the levels of interleukin, tumor necrosis factor, and nitric oxide were measured in vitro using the human macrophage cell line (U937). The analgesic effects of ethyl-p-methoxycinnamate were assessed by the tail flick assay in rats. The anti-angiogenic effects were evaluated first by the rat aortic ring assay and, subsequently, by assessing the inhibitory effects of ethyl-p-methoxycinnamate on vascular endothelial growth factor, proliferation, migration, and tube formation in human umbilical vein endothelial cells. RESULTS: Ethyl-p-methoxycinnamate strongly inhibited granuloma tissue formation in rats. It prolonged the tail flick time in rats by more than two-fold compared with the control animals. The inhibition of interleukin and tumor necrosis factor by ethyl-p-methoxycinnamate was significant in both in vivo and in vitro models; however, only a moderate inhibition of nitric oxide was observed in macrophages. Furthermore, ethyl-p-methoxycinnamate considerably inhibited microvessel sprouting from the rat aorta. These mechanistic studies showed that ethyl-p-methoxycinnamate strongly inhibited the differentiation and migration of endothelial cells, which was further confirmed by the reduced level of vascular endothelial growth factor. CONCLUSION: Ethyl-p-methoxycinnamate exhibits significant anti-inflammatory potential by inhibiting pro-inflammatory cytokines and angiogenesis, thus inhibiting the main functions of endothelial cells. Thus, ethyl-p-methoxycinnamate could be a promising therapeutic agent for the treatment of inflammatory and

  4. Nitric oxide inhibition of adenylyl cyclase type 6 activity is dependent upon lipid rafts and caveolin signaling complexes.

    PubMed

    Ostrom, Rennolds S; Bundey, Richard A; Insel, Paul A

    2004-05-07

    Several cell types, including cardiac myocytes and vascular endothelial cells, produce nitric oxide (NO) via both constitutive and inducible isoforms of NO synthase. NO attenuates cardiac contractility and contributes to contractile dysfunction in heart failure, although the precise molecular mechanisms for these effects are poorly defined. Adenylyl cyclase (AC) isoforms type 5 and 6, which are preferentially expressed in cardiac myocytes, may be inhibited via a direct nitrosylation by NO. Because endothelial NO synthase (eNOS and NOS3), beta-adrenergic (betaAR) receptors, and AC6 all can localize in lipid raft/caveolin-rich microdomains, we sought to understand the role of lipid rafts in organizing components of betaAR-G(s)-AC signal transduction together with eNOS. Using neonatal rat cardiac myocytes, we found that disruption of lipid rafts with beta-cyclodextrin inhibited forskolin-stimulated AC activity and cAMP production, eliminated caveolin-3-eNOS interaction, and increased NO production. betaAR- and G(s)-mediated activation of AC activity were inhibited by beta-cyclodextrin treatment, but prostanoid receptor-stimulated AC activity, which appears to occur outside caveolin-rich microdomains, was unaffected unless eNOS was overexpressed and lipid rafts were disrupted. An NO donor, SNAP, inhibited basal and forskolin-stimulated cAMP production in both native cardiac myocytes and cardiac myocytes and pulmonary artery endothelial cells engineered to overexpress AC6. These effects of SNAP were independent of guanylyl cyclase activity and were mimicked by overexpression of eNOS. The juxtaposition of eNOS with betaAR and AC types 5 and 6 results in selective regulation of betaAR by eNOS activity in lipid raft domains over other G(s)-coupled receptors localized in nonraft domains. Thus co-localization of multiple signaling components in lipid rafts provides key spatial regulation of AC activity.

  5. AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2.

    PubMed

    Cho, Kwang-Jin; Casteel, Darren E; Prakash, Priyanka; Tan, Lingxiao; van der Hoeven, Dharini; Salim, Angela A; Kim, Choel; Capon, Robert J; Lacey, Ernest; Cunha, Shane R; Gorfe, Alemayehu A; Hancock, John F

    2016-12-15

    K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function.

  6. Cyclic ADP ribose-mediated Ca2+ signaling in mediating endothelial nitric oxide production in bovine coronary arteries.

    PubMed

    Zhang, Guo; Teggatz, Eric G; Zhang, Andrew Y; Koeberl, Matthew J; Yi, Fan; Chen, Li; Li, Pin-Lan

    2006-03-01

    The present study tested the hypothesis that cyclic ADP ribose (cADPR) serves as a novel second messenger to mediate intracellular Ca2+ mobilization in coronary arterial endothelial cells (CAECs) and thereby contributes to endothelium-dependent vasodilation. In isolated and perfused small bovine coronary arteries, bradykinin (BK)-induced concentration-dependent vasodilation was significantly attenuated by 8-bromo-cADPR (a cell-permeable cADPR antagonist), ryanodine (an antagonist of ryanodine receptors), or nicotinamide (an ADP-ribosyl cyclase inhibitor). By in situ simultaneously fluorescent monitoring, Ca2+ transient and nitric oxide (NO) levels in the intact coronary arterial endothelium preparation, 8-bromo-cADPR (30 microM), ryanodine (50 microM), and nicotinamide (6 mM) substantially attenuated BK (1 microM)-induced increase in intracellular [Ca2+] by 78%, 80%, and 74%, respectively, whereas these compounds significantly blocked BK-induced NO increase by about 80%, and inositol 1,4,5-trisphosphate receptor blockade with 2-aminethoxydiphenyl borate (50 microM) only blunted BK-induced Ca2+-NO signaling by about 30%. With the use of cADPR-cycling assay, it was found that inhibition of ADP-ribosyl cyclase by nicotinamide substantially blocked BK-induced intracellular cADPR production. Furthermore, HPLC analysis showed that the conversion rate of beta-nicotinamide guanine dinucleotide into cyclic GDP ribose dramatically increased by stimulation with BK, which was blockable by nicotinamide. However, U-73122, a phospholipase C inhibitor, had no effect on this BK-induced increase in ADP-ribosyl cyclase activity for cADPR production. In conclusion, these results suggest that cADPR importantly contributes to BK- and A-23187-induced NO production and vasodilator response in coronary arteries through its Ca2+ signaling mechanism in CAECs.

  7. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    SciTech Connect

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi; Zhang, Qunye; Li, Guorong

    2015-03-13

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.

  8. Citrus Polyphenol Hesperidin Stimulates Production of Nitric Oxide in Endothelial Cells while Improving Endothelial Function and Reducing Inflammatory Markers in Patients with Metabolic Syndrome

    PubMed Central

    Rizza, Stefano; Muniyappa, Ranganath; Iantorno, Micaela; Kim, Jeong-a; Chen, Hui; Pullikotil, Philomena; Senese, Nicoletta; Tesauro, Manfredi; Lauro, Davide; Cardillo, Carmine

    2011-01-01

    Context: Hesperidin, a citrus flavonoid, and its metabolite hesperetin may have vascular actions relevant to their health benefits. Molecular and physiological mechanisms of hesperetin actions are unknown. Objective: We tested whether hesperetin stimulates production of nitric oxide (NO) from vascular endothelium and evaluated endothelial function in subjects with metabolic syndrome on oral hesperidin therapy. Design, Setting, and Interventions: Cellular mechanisms of action of hesperetin were evaluated in bovine aortic endothelial cells (BAEC) in primary culture. A randomized, placebo-controlled, double-blind, crossover trial examined whether oral hesperidin administration (500 mg once daily for 3 wk) improves endothelial function in individuals with metabolic syndrome (n = 24). Main Outcome Measure: We measured the difference in brachial artery flow-mediated dilation between placebo and hesperidin treatment periods. Results: Treatment of BAEC with hesperetin acutely stimulated phosphorylation of Src, Akt, AMP kinase, and endothelial NO synthase to produce NO; this required generation of H2O2. Increased adhesion of monocytes to BAEC and expression of vascular cell adhesion molecule-1 in response to TNF-α treatment was reduced by pretreatment with hesperetin. In the clinical study, when compared with placebo, hesperidin treatment increased flow-mediated dilation (10.26 ± 1.19 vs. 7.78 ± 0.76%; P = 0.02) and reduced concentrations of circulating inflammatory biomarkers (high-sensitivity C-reactive protein, serum amyloid A protein, soluble E-selectin). Conclusions: Novel mechanisms for hesperetin action in endothelial cells inform effects of oral hesperidin treatment to improve endothelial dysfunction and reduce circulating markers of inflammation in our exploratory clinical trial. Hesperetin has vasculoprotective actions that may explain beneficial cardiovascular effects of citrus consumption. PMID:21346065

  9. Dexmedetomidine Inhibits Phenylephrine-induced Contractions via Alpha-1 Adrenoceptor Blockade and Nitric Oxide Release in Isolated Rat Aortae

    PubMed Central

    Byon, Hyo-Jin; Ok, Seong-Ho; Lee, Soo Hee; Kang, Sebin; Cho, Youngil; Han, Jeong Yeol; Sohn, Ju-Tae

    2017-01-01

    The goal of this in vitro study was to examine the effect of the alpha-2 adrenoceptor agonist dexmedetomidine on phenylephrine (alpha-1 adrenoceptor agonist)-induced contraction in isolated rat aortae and to elucidate the associated cellular mechanisms, with a particular focus on alpha-1 adrenoceptor antagonism. Dexmedetomidine dose-response curves were generated in isolated endothelium-intact and endothelium-denuded rat aortae precontracted with phenylephrine or 5-hydroxytryptamine. Endothelium-denuded aortic rings were pretreated with either dexmedetomidine or the reversible alpha-1 adrenoceptor antagonist phentolamine, followed by post-treatment with the irreversible alpha-1 adrenoceptor blocker phenoxybenzamine. Control rings were treated with phenoxybenzamine alone. All rings were repeatedly washed with Krebs solution to remove all pretreatment drugs, including phenoxybenzamine, phentolamine and dexmedetomidine. Phenylephrine dose-response curves were then generated. The effect of rauwolscine on the dexmedetomidine-mediated change in phenylephrine-induced endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein endothelial cells was examined using western blotting. The magnitude of the dexmedetomidine-mediated inhibition of phenylephrine-induced contraction was higher in endothelium-intact aortae than in endothelium-denuded aortae or endothelium-intact aortae treated with Nω-nitro-L-arginine methyl ester. However, dexmedetomidine did not significantly alter 5-hydroxytryptamine-induced contraction. In further experiments, prazosin attenuated dexmedetomidine-induced contraction. Additionally, pretreatment with either dexmedetomidine plus phenoxybenzamine or phentolamine plus phenoxybenzamine produced greater phenylephrine-induced contraction than phenoxybenzamine alone, suggesting that dexmedetomidine protects aortae from the alpha-1 adrenoceptor blockade induced by phenoxybenzamine. Rauwolscine attenuated the dexmedetomidine

  10. Calcium-activated potassium channels in cultured human endothelial cells are not directly modulated by nitric oxide.

    PubMed

    Haburcák, M; Wei, L; Viana, F; Prenen, J; Droogmans, G; Nilius, B

    1997-04-01

    Nitric oxide has been proposed to directly activated large conductance Ca(2+)-dependent K+ channels (BKCa) [Bolotina V.M., Najibi S., Palacino J.J., Pagano P.J., Cohen R.A. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 1994; 368: 850-853]. The nitric oxide (NO) donor S-nitrosocysteine (SNOC) was used to evaluate a possible direct modulation of BKCa by NO in EAhy926 (EA cells), a cultured human umbilical vein derived endothelial cell line, using the whole-cell, cell-attached and inside-out configuration of the patch-clamp technique, together with simultaneous amperometric measurement of NO and the concentration of free intracellular calcium [Ca2+]i. BKCa channels with a large conductance of approximately 190 pS, voltage-dependent activation and a reversal potential close to -80 mV have been identified in EA cells. Exposure of EA cells in the experimental chamber to 1 mM SNOC delivered approximately 5 microM NO, as recorded by an amperometric probe in situ. SNOC produced a modest increases in [Ca2+]i that was insufficient to activate BKCa channels. NO alone neither activated BKCa channels directly nor modulated preactivated BKCa channels in EA cells. These results do not support a direct modulatory effect of NO on large conductance BKCa channels in cultured endothelial cells.

  11. Packed Red Blood Cells Are an Abundant and Proximate Potential Source of Nitric Oxide Synthase Inhibition

    PubMed Central

    Zwemer, Charles F.; Davenport, Robertson D.; Gomez-Espina, Juan; Blanco-Gonzalez, Elisa; Whitesall, Steven E.; D'Alecy, Louis G.

    2015-01-01

    Objective We determined, for packed red blood cells (PRBC) and fresh frozen plasma, the maximum content, and ability to release the endogenous nitric oxide synthase (NOS) inhibitors asymmetric dimethylarginine (ADMA) and monomethylarginine (LNMMA). Background ADMA and LNMMA are near equipotent NOS inhibitors forming blood’s total NOS inhibitory content. The balance between removal from, and addition to plasma determines their free concentrations. Removal from plasma is by well-characterized specific hydrolases while formation is restricted to posttranslational protein methylation. When released into plasma they can readily enter endothelial cells and inhibit NOS. Fresh rat and human whole blood contain substantial protein incorporated ADMA however; the maximum content of ADMA and LNMMA in PRBC and fresh frozen plasma has not been determined. Methods We measured total (free and protein incorporated) ADMA and LNMMA content in PRBCs and fresh frozen plasma, as well as their incubation induced release, using HPLC with fluorescence detection. We tested the hypothesis that PRBC and fresh frozen plasma contain substantial inhibitory methylarginines that can be released chemically by complete in vitro acid hydrolysis or physiologically at 37°C by enzymatic blood proteolysis. Results In vitro strong-acid-hydrolysis revealed a large PRBC reservoir of ADMA (54.5 ± 9.7 µM) and LNMMA (58.9 ± 28.9 μM) that persisted over 42-d at 6° or -80°C. In vitro 5h incubation at 37°C nearly doubled free ADMA and LNMMNA concentration from PRBCs while no change was detected in fresh frozen plasma. Conclusion The compelling physiological ramifications are that regardless of storage age, 1) PRBCs can rapidly release pathologically relevant quantities of ADMA and LNMMA when incubated and 2) PRBCs have a protein-incorporated inhibitory methylarginines reservoir 100 times that of normal free inhibitory methylarginines in blood and thus could represent a clinically relevant and proximate

  12. Endothelial Nitric Oxide Synthase Deficient Mice Are Protected from Lipopolysaccharide Induced Acute Lung Injury

    PubMed Central

    Gross, Christine M.; Rafikov, Ruslan; Kumar, Sanjiv; Aggarwal, Saurabh; Ham III, P. Benson; Meadows, Mary Louise; Cherian-Shaw, Mary; Kangath, Archana; Sridhar, Supriya; Lucas, Rudolf; Black, Stephen M.

    2015-01-01

    Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS-/-) are protected against ALI. In both wild-type and eNOS-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr34. Finally, we found that the reduction in NOS uncoupling in eNOS-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury. PMID:25786132

  13. Endothelial Nitric Oxide Synthase and Angiotensin Converting Enzyme Gene Polymorphisms in Migraine Patients

    PubMed Central

    SİPAHİ, Tammam; GÜLDİKEN, Babürhan; KABAYEL, Levent; PALABIYIK, Orkide; ÖZKAN, Hülya; KILIÇ, Tülay Okman; SÜT, Necdet; TURGUT, Nilda

    2013-01-01

    Introduction In this study, we investigated the association of migraine with the Variable Number of Tandem Repeats (VNTR), repeated as 27 base pair, gene polymorphism in intron 4 of the endothelial nitric oxide synthase (eNOS) and the insertion/deletion of angiotensin converting enzyme (ACE) gene polymorphisms. Methods One hundred and five migraine and ninety seven healthy female control subjects were enrolled in the study. The patients were subdivided as migraine with aura and without aura, and the frequency and severity of migraine headaches were recorded. The eNOS VNTR (eNOS 4 a/b) and ACE insertion/deletion gene polymorphisms (ACE I/D) were assessed by polymerase chain reactions. Result The allele and genotype frequencies of eNOS 4 a/b gene polymorphism showed no difference between the migraine and control groups. The genotypic distribution of the ACE I/D gene polymorphism in the migraine group significantly differed from that in the control group. The DD and ID genotype increased the risk of migraine as much as 2.571 (95% CI-1.138–5.811) and 4.453 (95% CI-2.006–9.883) compared to the II genotype. The same increased risk sustained for both genotypes in the migraine with aura subgroup, but only the ID genotype remained as the risk factor in the migraine without aura subgroup (OR-3.750, 95% CI-1.493–9.420). No association of gene polymorphisms with migraine frequency and severity was observed. Conclusion Our findings support the relationship between migraine and the ACE I/D gene polymorphism. However, no association was found between migraine and the eNOS 4 a/b gene polymorphism.

  14. Vascular endothelial growth factor and nitric oxide synthase expression in human tooth germ development.

    PubMed

    Mastrangelo, F; Sberna, M T; Tettamanti, L; Cantatore, G; Tagliabue, A; Gherlone, E

    2016-01-01

    Vascular Endothelia Growth Factor (VEGF) and Nitric Oxide Synthase (NOS) expression, were evaluated in human tooth germs at two different stages of embryogenesis, to clarify the role of angiogenesis during tooth tissue differentiation and growth. Seventy-two third molar germ specimens were selected during oral surgery. Thirty-six were in the early stage and 36 in the later stage of tooth development. The samples were evaluated with Semi-quantitative Reverse Transcription-Polymerase chain Reaction analyses (RT-PcR), Western blot analysis (WB) and immunohistochemical analysis. Western blot and immunohistochemical analysis showed a VEGF and NOS 1-2-3 positive reaction in all samples analysed. VEGF high positive decrease reaction was observed in stellate reticulum cells, ameloblast and odontoblast clusters in early stage compared to later stage of tooth germ development. Comparable VEGF expression was observed in endothelial cells of early and advanced stage growth. NOS1 and NOS3 expressions showed a high increased value in stellate reticulum cells, and ameloblast and odontoblast clusters in advanced stage compared to early stage of development. The absence or only moderate positive reaction of NOS2 was detected in all the different tissues. Positive NOS2 expression showed in advanced stage of tissue development compared to early stage. The action of VEGF and NOS molecules are important mediators of angiogenesis during dental tissue development. VEGF high positive expression in stellate reticulum cells in the early stage of tooth development compared to the later stage and the other cell types, suggests a critical role of the stellate reticulum during dental embryo-morphogenesis.

  15. Prolactin alters blood pressure by modulating the activity of endothelial nitric oxide synthase

    PubMed Central

    Chang, Albert S.; Grant, Ruriko; Tomita, Hirofumi; Kim, Hyung-Suk; Smithies, Oliver; Kakoki, Masao

    2016-01-01

    Increased levels of a cleaved form of prolactin (molecular weight 16 kDa) have been associated with preeclampsia. To study the effects of prolactin on blood pressure (BP), we generated male mice with a single-copy transgene (Tg; inserted into the hypoxanthine-guanine phosphoribosyltransferase locus) that enables inducible hepatic production of prolactin and its cleavage product. The Tg is driven by the indole-3-carbinol (I3C)-inducible rat cytochrome P450 1A1 promoter. When the Tg mice were fed normal chow (NC), plasma prolactin concentrations were comparable to those in female WT mice in the last third of pregnancy, and BP was lower than in WT mice (∼95 mm Hg vs. ∼105 mm Hg). When the Tg mice were fed chow containing IC3, plasma prolactin concentrations increased threefold, BP increased to ∼130 mm Hg, and cardiac function became markedly impaired. IC3 chow did not affect the WT mice. Urinary excretion of nitrite/nitrate and the amount of Ser1177-phosphorylated endothelial nitric oxide (NO) synthase (eNOS) were significantly greater in the Tg mice fed NC than in WT mice, as they are during pregnancy. However, when I3C was fed, these indicators of NO production became significantly less in the Tg mice than in WT mice. The effects of increased plasma prolactin were abolished by a genetic absence of eNOS. Thus, a threefold increase in plasma prolactin is sufficient to increase BP significantly and to markedly impair cardiac function, with effects mediated by NO produced by eNOS. We suggest that pregnant women with abnormally high prolactin levels may need special attention. PMID:27791173

  16. Endothelial nitric oxide synthase tagging single nucleotide polymorphisms and recovery from aneurysmal subarachnoid hemorrhage.

    PubMed

    Alexander, Sheila; Poloyac, Samuel; Hoffman, Leslie; Gallek, Matthew; Dianxu Ren; Balzer, Jeffrey; Kassam, Amin; Conley, Yvette

    2009-07-01

    Aneurysmal subarachnoid hemorrhage (SAH) is a hemorrhagic stroke subtype with a poor recovery profile. Cerebral vasospasm (CV), a narrowing of the cerebral vasculature, significantly contributes to the poor recovery profile. Variation in the endothelial nitric oxide (NO) synthase (eNOS) gene has been implicated in CV and outcome after SAH. The purpose of this project was to explore the potential association between three eNOS tagging single nucleotide polymorphisms (SNPs) and recovery from SAH. We included 195 participants with a diagnosis of SAH and DNA and 6-month outcome data available but without preexisting neurologic disease/deficit. Genotyping was performed using an ABI Prism 7000 Sequence Detection System and TaqMan assays. CV was verified by cerebral angiogram independently read by a neurosurgeon on 118 participants. Modified Rankin Scores (MRS) and Glasgow Outcome Scale (GOS) scores were collected 6 months posthemorrhage. Data were analyzed using descriptive statistics, analysis of variance (ANOVA) and chi-square analysis as appropriate. The sample was primarily female (n=147; 75.4%) and White (n=178; 91.3%) with a mean age of 54.6 years. Of the participants with CV data, 56 (47.5%) developed CV within 14 days of SAH. None of the SNPs individually were associated with CV presence; however, a combination of the three variant SNPs was significantly associated with CV (p=.017). Only one SNP (rs1799983, variant allele) was associated with worse 6-month GOS scores (p<.001) and MRS (p<.001). These data indicate that the eNOS gene plays a role in the response to SAH, which may be explained by an influence on CV.

  17. Structural Studies of a Complex Between Endothelial Nitric Oxide Synthase and Calmodulin at Physiological Calcium Concentration.

    PubMed

    Piazza, Michael; Dieckmann, Thorsten; Guillemette, Joseph Guy

    2016-10-04

    The small acidic protein Calmodulin (CaM) serves as a Ca(2+) sensor and control element for many enzymes including nitric oxide synthase (NOS) enzymes that play major roles in key physiological and pathological processes. CaM binding causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. In this report, NMR spectroscopy was used to determine the solution structure of the endothelial NOS (eNOS) peptide in complex with CaM at the lowest Ca(2+) concentration (225 nM) required for CaM to bind to eNOS and corresponds to a physiological elevated Ca2+ level found in mammalian cells. Under these conditions, the CaM-eNOS complex has a Ca(2+)-replete C-terminal lobe bound the eNOS peptide and a Ca(2+) free N-terminal lobe loosely associated to the eNOS peptide. With increasing Ca(2+) concentration, the binding of Ca(2+) by the N-lobe of CaM results in a stronger interaction with the C-terminal region of the eNOS peptide and increased α-helical structure of the peptide that may be part of the mechanism resulting in electron transfer from the FMN to the heme in the oxygenase domain of the enzyme. SPR studies performed under the same conditions show Ca(2+) concentration dependent binding kinetics were consistent with the NMR structural results. This investigation shows that structural studies performed under more physiological relevant conditions provide information on subtle changes in structure that may not be apparent when experiments are performed in excess Ca(2+) concentrations.

  18. Modulation of endothelial nitric oxide synthase expression by red blood cell aggregation.

    PubMed

    Baskurt, Oguz K; Yalcin, Ozlem; Ozdem, Sadi; Armstrong, Jonathan K; Meiselman, Herbert J

    2004-01-01

    The effects of enhanced red blood cell (RBC) aggregation on nitric oxide (NO)-dependent vascular control mechanisms have been investigated in a rat exchange transfusion model. RBC aggregation for cells in native plasma was increased via a novel method using RBCs covalently coated with a 13-kDa poloxamer copolymer (Pluronic F-98); control experiments used RBCs coated with a nonaggregating 8.4-kDa poloxamer (Pluronic F-68). Rats exchange transfused with aggregating RBC suspensions demonstrated significantly enhanced RBC aggregation throughout the 5-day follow-up period, with mean arterial blood pressure increasing gradually over this period. Arterial segments ( approximately 300 microm in diameter) were isolated from gracilis muscle on the fifth day and mounted between two glass micropipettes in a special chamber equipped with pressure servo-control system. Dose-dependent dilation by ACh and flow-mediated dilation of arterial segments pressurized to 30 mmHg and preconstricted to 45-55% of the original diameter by phenylephrine were significantly blunted in rats with enhanced RBC aggregation. Both responses were totally abolished by nonspecific NO synthase (NOS) inhibitor (Nomega-nitro-l-arginine methyl ester) treatment of arterial segments, indicating that the responses were NO related. Additionally, expression of endothelial NOS protein was found to be decreased in muscle samples obtained from rats exchanged with aggregating cell suspensions. These results imply that enhanced RBC aggregation results in suppressed expression of NO synthesizing mechanisms, thereby leading to altered vasomotor tonus; the mechanisms involved most likely relate to decreased wall shear stresses due to decreased blood flow and/or increased axial accumulation of RBCs.

  19. Nitric oxide scavengers differentially inhibit ammonia oxidation in ammonia-oxidizing archaea and bacteria.

    PubMed

    Sauder, Laura A; Ross, Ashley A; Neufeld, Josh D

    2016-04-01

    Differential inhibitors are important for measuring the relative contributions of microbial groups, such as ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), to biogeochemical processes in environmental samples. In particular, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) represents a nitric oxide scavenger used for the specific inhibition of AOA, implicating nitric oxide as an intermediate of thaumarchaeotal ammonia oxidation. This study investigated four alternative nitric oxide scavengers for their ability to differentially inhibit AOA and AOB in comparison to PTIO. Caffeic acid, curcumin, methylene blue hydrate and trolox were tested onNitrosopumilus maritimus, two unpublished AOA representatives (AOA-6f and AOA-G6) as well as the AOB representative Nitrosomonas europaea All four scavengers inhibited ammonia oxidation by AOA at lower concentrations than for AOB. In particular, differential inhibition of AOA and AOB by caffeic acid (100 μM) and methylene blue hydrate (3 μM) was comparable to carboxy-PTIO (100 μM) in pure and enrichment culture incubations. However, when added to aquarium sponge biofilm microcosms, both scavengers were unable to inhibit ammonia oxidation consistently, likely due to degradation of the inhibitors themselves. This study provides evidence that a variety of nitric oxide scavengers result in differential inhibition of ammonia oxidation in AOA and AOB, and provides support to the proposed role of nitric oxide as a key intermediate in the thaumarchaeotal ammonia oxidation pathway.

  20. NG-methyl-L-arginine inhibits tumor necrosis factor-induced hypotension: implications for the involvement of nitric oxide.

    PubMed Central

    Kilbourn, R G; Gross, S S; Jubran, A; Adams, J; Griffith, O W; Levi, R; Lodato, R F

    1990-01-01

    Clinical assessment of the activity of tumor necrosis factor (TNF) against human cancer has been limited by a dose-dependent cardiovascular toxicity, most frequently hypotension. TNF is also thought to mediate the vascular collapse resulting from bacterial endotoxin. The present studies address the mechanism by which TNF causes hypotension and provide evidence for elevated production of nitric oxide, a potent vasodilator initially characterized as endothelium-derived relaxing factor. Nitric oxide is synthesized by several cell types, including endothelial cells and macrophages, from the guanidino nitrogen of L-arginine; the enzymatic pathway is competitively inhibited by NG-methyl-L-arginine. We found that hypotension induced in pentobarbital-anesthetized dogs by TNF (10 micrograms/kg, i.v., resulting in a fall in mean systemic arterial pressure from 124.7 +/- 7 to 62.0 +/- 22.9 mmHg; 1 mmHg = 133 Pa) was completely reversed within 2 min following administration of NG-methyl-L-arginine (4.4 mg/kg, i.v.). In contrast, NG-methyl-L-arginine failed to reverse the hypotensive response to an equivalent depressor dose of nitroglycerin, a compound that acts by forming nitric oxide by a nonenzymatic, arginine-independent mechanism. The effect of NG-methyl-L-arginine on TNF-induced hypotension was antagonized, and the hypotension restored, by administration of excess L-arginine (100 mg/kg, i.v.). Our findings suggest that excessive nitric oxide production mediates the hypotensive effect of TNF. PMID:2333306

  1. Nitric oxide inhibition sustains vasopressin-induced vasoconstriction.

    PubMed Central

    Dworkin, M. J.; Carnochan, P.; Allen-Mersh, T. G.

    1995-01-01

    Hepatic parenchymal vasoconstriction increases cytotoxic drug uptake into hepatic metastases by increasing the tumour to liver blood flow ratio. Prolonged infusion of the vasoconstrictor vasopressin does not result in sustained vasoconstriction, and this may limit the benefit of vasopressin in infusional chemotherapy. We have assessed whether loss of vasopressin-induced vasoconstriction is mediated by nitric oxide. Hepatic and tumour blood flow were continuously monitored, in an animal hepatic tumour model, by laser Doppler flowmetry. The response to regionally infused vasopressin and the nitric oxide inhibitor N-nitro-L-arginine methyl ester (L-NAME) were assessed over a 30 min infusion period. The vasopressin-induced vasoconstrictor effect diminished after 15 min despite continued infusion. Vasoconstriction was significantly prolonged when L-NAME was infused in addition to vasopressin. The increase in tumour to normal blood flow ratio was greater over the infusion period when L-NAME was co-administered with vasopressin. Our results suggest that the loss of vasopressin-induced vasoconstriction seen in liver parenchyma after regional infusion is prevented by the nitric oxide synthase inhibitor L-name and may be mediated by nitric oxide. PMID:7734317

  2. Androgens inhibit tumor necrosis factor-α-induced cell adhesion and promote tube formation of human coronary artery endothelial cells.

    PubMed

    Liao, Chun-Hou; Lin, Feng-Yen; Wu, Yi-No; Chiang, Han-Sun

    2012-06-01

    Endothelial cells contribute to the function and integrity of the vascular wall, and a functional aberration may lead to atherogenesis. There is increasing evidence on the atheroprotective role of androgens. Therefore, we studied the effect of the androgens-testosterone and dihydrotestosterone-and estradiol on human coronary artery endothelial cell (HCAEC) function. We found by MTT assay that testosterone is not cytotoxic and enhances HCAEC proliferation. The effect of testosterone (10-50 nM), dihydrotestosterone (5-50 nM), and estradiol (0.1-0.4 nM) on the adhesion of tumor necrosis factor-α (TNF-α)-stimulated HCAECs was determined at different time points (12-96 h) by assessing their binding with human monocytic THP-1 cells. In addition, the expression of adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), was determined by ELISA and Western blot analysis. Both testosterone and dihydrotestosterone attenuated cell adhesion and the expression of VCAM-1 and ICAM-1 in a dose- and time-dependent manner. Furthermore, androgen treatment for a longer duration inhibited cell migration, as demonstrated by wound-healing assay, and promoted tube formation on a Matrigel. Western blot analysis demonstrated that the expression of phosphorylated endothelial nitric oxide synthase (eNOS) increased, whereas that of inducible nitric oxide synthase (iNOS) decreased following the 96-h steroid treatment of TNF-α-stimulated HCAECs. Our findings suggest that androgens modulate endothelial cell functions by suppressing the inflammatory process and enhancing wound-healing and regenerative angiogenesis, possibly through an androgen receptor (AR)-dependent mechanism.

  3. Estradiol augments while progesterone inhibits arginine transport in human endothelial cells through modulation of cationic amino acid transporter-1.

    PubMed

    Bentur, Ohad S; Schwartz, Doron; Chernichovski, Tamara; Ingbir, Merav; Weinstein, Talia; Chernin, Gil; Schwartz, Idit F

    2015-08-15

    Decreased generation of nitric oxide (NO) by endothelial NO synthase (eNOS) characterizes endothelial dysfunction (ECD). Delivery of arginine to eNOS by cationic amino acid transporter-1 (CAT-1) was shown to modulate eNOS activity. We found in female rats, but not in males, that CAT-1 activity is preserved with age and in chronic renal failure, two experimental models of ECD. In contrast, during pregnancy CAT-1 is inhibited. We hypothesize that female sex hormones regulate arginine transport. Arginine uptake in human umbilical vein endothelial cells (HUVEC) was determined following incubation with either 17β-estradiol (E2) or progesterone. Exposure to E2 (50 and 100 nM) for 30 min resulted in a significant increase in arginine transport and reduction in phosphorylated CAT-1 (the inactive form) protein content. This was coupled with a decrease in phosphorylated MAPK/extracellular signal-regulated kinase (ERK) 1/2. Progesterone (1 and 100 pM for 30 min) attenuated arginine uptake and increased phosphorylated CAT-1, phosphorylated protein kinase Cα (PKCα), and phosphorylated ERK1/2 protein content. GO-6976 (PKCα inhibitor) prevented the progesterone-induced decrease in arginine transport. Coincubation with both progesterone and estrogen for 30 min resulted in attenuated arginine transport. While estradiol increases arginine transport and CAT-1 activity through modulation of constitutive signaling transduction pathways involving ERK, progesterone inhibits arginine transport and CAT-1 via both PKCα and ERK1/2 phosphorylation, an effect that predominates over estradiol.

  4. Contribution of insulin and Akt1 signaling to endothelial nitric oxide synthase in the regulation of endothelial function and blood pressure.

    PubMed

    Symons, J David; McMillin, Shawna L; Riehle, Christian; Tanner, Jason; Palionyte, Milda; Hillas, Elaine; Jones, Deborah; Cooksey, Robert C; Birnbaum, Morris J; McClain, Donald A; Zhang, Quan-Jiang; Gale, Derrick; Wilson, Lloyd J; Abel, E Dale

    2009-05-08

    Impaired insulin signaling via phosphatidylinositol 3-kinase/Akt to endothelial nitric oxide synthase (eNOS) in the vasculature has been postulated to lead to arterial dysfunction and hypertension in obesity and other insulin resistant states. To investigate this, we compared insulin signaling in the vasculature, endothelial function, and systemic blood pressure in mice fed a high-fat (HF) diet to mice with genetic ablation of insulin receptors in all vascular tissues (TTr-IR(-/-)) or mice with genetic ablation of Akt1 (Akt1-/-). HF mice developed obesity, impaired glucose tolerance, and elevated free fatty acids that was associated with endothelial dysfunction and hypertension. Basal and insulin-mediated phosphorylation of extracellular signal-regulated kinase 1/2 and Akt in the vasculature was preserved, but basal and insulin-stimulated eNOS phosphorylation was abolished in vessels from HF versus lean mice. In contrast, basal vascular eNOS phosphorylation, endothelial function, and blood pressure were normal despite absent insulin-mediated eNOS phosphorylation in TTr-IR(-/-) mice and absent insulin-mediated eNOS phosphorylation via Akt1 in Akt1-/- mice. In cultured endothelial cells, 6 hours of incubation with palmitate attenuated basal and insulin-stimulated eNOS phosphorylation and NO production despite normal activation of extracellular signal-regulated kinase 1/2 and Akt. Moreover, incubation of isolated arteries with palmitate impaired endothelium-dependent but not vascular smooth muscle function. Collectively, these results indicate that lower arterial eNOS phosphorylation, hypertension, and vascular dysfunction following HF feeding do not result from defective upstream signaling via Akt, but from free fatty acid-mediated impairment of eNOS phosphorylation.

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

    PubMed Central

    Polhemus, David J.; Gao, Juan; Scarborough, Amy L.; Trivedi, Rishi; McDonough, Kathleen H.; Goodchild, Traci T.; Smart, Frank

    2016-01-01

    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

  6. The loss of sustained Ca(2+) signaling underlies suppressed endothelial nitric oxide production in preeclamptic pregnancies: implications for new therapy.

    PubMed

    Krupp, Jennifer; Boeldt, Derek S; Yi, Fu-Xian; Grummer, Mary A; Bankowski Anaya, Heather A; Shah, Dinesh M; Bird, Ian M

    2013-10-01

    Approximately 8% of pregnancies are complicated by preeclampsia (PE), a hypertensive condition characterized by widespread endothelial dysfunction. Reduced nitric oxide (NO) output in PE subjects has been inferred but not directly measured, and there is little understanding of why this occurs. To address this we have used direct imaging of changes in intracellular Ca(2+) concentration ([Ca(2+)]i) and NO in umbilical vein endothelium of normal and PE subjects that is still intact and on the vessel luminal surface. This was achieved by dissection and preloading with fura 2 and DAF-2 imaging dyes, respectively, before subsequent challenge with ATP (100 μM, 30 min). As a control to reveal the content of active endothelial nitric oxide synthase (eNOS) per vessel segment, results were compared with a maximal stimulus with ionomycin (5 μM, 30 min). We show for the first time that normal umbilical vein endothelial cells respond to ATP with sustained bursting that parallels sustained NO output. Furthermore, in subjects with PE, a failure of sustained [Ca(2+)]i bursting occurs in response to ATP and is associated with blunted NO output. In contrast, NO responses to maximal [Ca(2+)]i elevation using ionomycin and the levels of eNOS protein are more similar between groups than the responses to ATP. When the endothelial cells from PE subjects are isolated and allowed to recover in culture, they regain the ability under fura 2 imaging to show multiple [Ca(2+)]i bursts otherwise seen in the cells from normal subjects. Thus novel clinical therapy aimed at restoring function in vivo may be possible.

  7. Nitric oxide inhibits isoproterenol-stimulated adipocyte lipolysis through oxidative inactivation of the beta-agonist.

    PubMed Central

    Klatt, P; Cacho, J; Crespo, M D; Herrera, E; Ramos, P

    2000-01-01

    Nitric oxide has been implicated in the inhibition of catecholamine-stimulated lipolysis in adipose tissue by as yet unknown mechanisms. In the present study, it is shown that the nitric oxide donor, 2,2-diethyl-1-nitroso-oxyhydrazine, antagonized isoproterenol (isoprenaline)-induced lipolysis in rat adipocytes, freshly isolated from white adipose tissue, by decreasing the potency of the beta-agonist without affecting its efficacy. These data suggest that nitric oxide did not act downstream of the beta-adrenoceptor but reduced the effective concentration of isoproterenol. In support of the latter hypothesis, we found that pre-treatment of isoproterenol with nitric oxide abolished the lipolytic activity of the catecholamine. Spectroscopic data and HPLC analysis confirmed that the nitric oxide-mediated inactivation of isoproterenol was in fact because of the modification of the catecholamine through a sequence of oxidation reactions, which apparently involved the generation of an aminochrome. Similarly, aminochrome was found to be the primary product of isoproterenol oxidation by 3-morpholinosydnonimine and peroxynitrite. Finally, it was shown that nitric oxide released from cytokine-stimulated adipocytes attenuated the lipolytic effect of isoproterenol by inactivating the catecholamine. In contrast with very recent findings, which suggest that nitric oxide impairs the beta-adrenergic action of isoproterenol through intracellular mechanisms and not through a chemical reaction between NO and the catecholamine, we showed that nitric oxide was able to attenuate the pharmacological activity of isoproterenol in vitro as well as in a nitric oxide-generating cellular system through oxidation of the beta-agonist. These findings should be taken into account in both the design and interpretation of studies used to investigate the role of nitric oxide as a modulator of isoproterenol-stimulated signal transduction pathways. PMID:11023835

  8. Nitric oxide inhibits isoproterenol-stimulated adipocyte lipolysis through oxidative inactivation of the beta-agonist.

    PubMed

    Klatt, P; Cacho, J; Crespo, M D; Herrera, E; Ramos, P

    2000-10-15

    Nitric oxide has been implicated in the inhibition of catecholamine-stimulated lipolysis in adipose tissue by as yet unknown mechanisms. In the present study, it is shown that the nitric oxide donor, 2,2-diethyl-1-nitroso-oxyhydrazine, antagonized isoproterenol (isoprenaline)-induced lipolysis in rat adipocytes, freshly isolated from white adipose tissue, by decreasing the potency of the beta-agonist without affecting its efficacy. These data suggest that nitric oxide did not act downstream of the beta-adrenoceptor but reduced the effective concentration of isoproterenol. In support of the latter hypothesis, we found that pre-treatment of isoproterenol with nitric oxide abolished the lipolytic activity of the catecholamine. Spectroscopic data and HPLC analysis confirmed that the nitric oxide-mediated inactivation of isoproterenol was in fact because of the modification of the catecholamine through a sequence of oxidation reactions, which apparently involved the generation of an aminochrome. Similarly, aminochrome was found to be the primary product of isoproterenol oxidation by 3-morpholinosydnonimine and peroxynitrite. Finally, it was shown that nitric oxide released from cytokine-stimulated adipocytes attenuated the lipolytic effect of isoproterenol by inactivating the catecholamine. In contrast with very recent findings, which suggest that nitric oxide impairs the beta-adrenergic action of isoproterenol through intracellular mechanisms and not through a chemical reaction between NO and the catecholamine, we showed that nitric oxide was able to attenuate the pharmacological activity of isoproterenol in vitro as well as in a nitric oxide-generating cellular system through oxidation of the beta-agonist. These findings should be taken into account in both the design and interpretation of studies used to investigate the role of nitric oxide as a modulator of isoproterenol-stimulated signal transduction pathways.

  9. Impaired Endothelial Nitric Oxide Synthase Homodimer Formation Triggers Development of Transplant Vasculopathy - Insights from a Murine Aortic Transplantation Model

    PubMed Central

    Oberhuber, Rupert; Riede, Gregor; Cardini, Benno; Bernhard, David; Messner, Barbara; Watschinger, Katrin; Steger, Christina; Brandacher, Gerald; Pratschke, Johann; Golderer, Georg; Werner, Ernst R.; Maglione, Manuel

    2016-01-01

    Transplant vasculopathy (TV) represents a major obstacle to long-term graft survival and correlates with severity of ischemia reperfusion injury (IRI). Donor administration of the nitric oxide synthases (NOS) co-factor tetrahydrobiopterin has been shown to prevent IRI. Herein, we analysed whether tetrahydrobiopterin is also involved in TV development. Using a fully allogeneic mismatched (BALB/c to C57BL/6) murine aortic transplantation model grafts subjected to long cold ischemia time developed severe TV with intimal hyperplasia (α-smooth muscle actin positive cells in the neointima) and endothelial activation (increased P-selectin expression). Donor pretreatment with tetrahydrobiopterin significantly minimised these changes resulting in only marginal TV development. Severe TV observed in the non-treated group was associated with increased protein oxidation and increased occurrence of endothelial NOS monomers in the aortic grafts already during graft procurement. Tetrahydrobiopterin supplementation of the donor prevented all these early oxidative changes in the graft. Non-treated allogeneic grafts without cold ischemia time and syngeneic grafts did not develop any TV. We identified early protein oxidation and impaired endothelial NOS homodimer formation as plausible mechanistic explanation for the crucial role of IRI in triggering TV in transplanted aortic grafts. Therefore, targeting endothelial NOS in the donor represents a promising strategy to minimise TV. PMID:27883078

  10. eNOS-derived nitric oxide regulates endothelial barrier function through VE-cadherin and Rho GTPases

    PubMed Central

    Di Lorenzo, Annarita; Lin, Michelle I.; Murata, Takahisa; Landskroner-Eiger, Shira; Schleicher, Michael; Kothiya, Milankumar; Iwakiri, Yasuko; Yu, Jun; Huang, Paul L.; Sessa, William C.

    2013-01-01

    Summary Transient disruption of endothelial adherens junctions and cytoskeletal remodeling are responsible for increases in vascular permeability induced by inflammatory stimuli and vascular endothelial growth factor (VEGF). Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is crucial for VEGF-induced changes in permeability in vivo; however, the molecular mechanism by which endogenous NO modulates endothelial permeability is not clear. Here, we show that the lack of eNOS reduces VEGF-induced permeability, an effect mediated by enhanced activation of the Rac GTPase and stabilization of cortical actin. The loss of NO increased the recruitment of the Rac guanine-nucleotide-exchange factor (GEF) TIAM1 to adherens junctions and VE-cadherin (also known as cadherin 5), and reduced Rho activation and stress fiber formation. In addition, NO deficiency reduced VEGF-induced VE-cadherin phosphorylation and impaired the localization, but not the activation, of c-Src to cell junctions. The physiological role of eNOS activation is clear given that VEGF-, histamine- and inflammation-induced vascular permeability is reduced in mice bearing a non-phosphorylatable knock-in mutation of the key eNOS phosphorylation site S1176. Thus, NO is crucial for Rho GTPase-dependent regulation of cytoskeletal architecture leading to reversible changes in vascular permeability. PMID:24046447

  11. Association of endothelial nitric oxide synthase gene T-786C promoter polymorphism with gastric cancer

    PubMed Central

    Krishnaveni, Devulapalli; Amar Chand, Bhayal; Shravan Kumar, Porika; Uma Devi, Malladi; Ramanna, Macherla; Jyothy, Akka; Pratibha, Nallari; Balakrishna, N; Venkateshwari, Ananthapur

    2015-01-01

    AIM: To investigate the role of endothelial nitric oxide synthase -786T > C promoter polymorphism in the etiology of gastric cancer (GC). METHODS: A total of 150 GC patients and 150 control subjects were included in the study. The information on demographic features was elicited with an informed consent from all the patients and control subjects using a structured questionnaire. Helicobacter pylori (H. pylori) infectivity status was tested in antral biopsies from all the subjects by rapid urease test following the method of Vaira et al. Genomic DNA was isolated from whole blood samples following the salting out method of Lahiri et al. Genotype analysis of the rs2070744 polymorphism was carried out by allele-specific polymerase chain reaction method. The genotypes were determined based on the appearance of bands on an agarose gel stained with ethidium bromide under ultraviolet gel documentation with the help of 100 bp ladder. Odds ratios and corresponding 95%CIs were determined using java stat online software. RESULTS: There was a significant difference in the distribution of C allele (C vs T; P = 0.000, OR = 5.038) in patient group compared to the control subjects exhibiting a fivefold increased risk for GC. When the T/T and C/C genotypes were compared, there was an enhanced GC risk for individuals with C/C genotype (T/T vs C/C; P = 0.000). Among the demographic factors, smoking and alcoholism were the common risk factors in patients compared to the control subjects (P < 0.05). Patients with smoking and alcoholism developed cancer even in heterozygous T/C condition (smoking: P = 0.020 and alcoholism: P = 0.005). Individuals with H. pylori infection showed seven fold increased risk for cancer. All the patients with C/C genotype revealed a significant association between H. pylori infection and GC. Among the patients 2.4% of them revealed familial incidence of GC. No significant difference was noticed between cases and controls with regard to consanguinity (P = 0

  12. Vascular endothelial-derived semaphorin 3 inhibits sympathetic axon growth.

    PubMed

    Damon, Deborah H

    2006-03-01

    Vascular sympathetic innervation is an important determinant of blood pressure and blood flow. The mechanisms that determine vascular sympathetic innervation are not well understood. Recent studies indicate that vascular endothelial cells (EC) express semaphorin 3A, a repulsive axon guidance cue. This suggests that EC would inhibit the growth of axons to blood vessels. The present study tests this hypothesis. RT-PCR and Western analyses confirmed that rat aortic vascular ECs expressed semaphorin 3A as well as other class 3 semaphorins (sema 3s). To determine the effects of EC-derived sema 3 on sympathetic axons, axon outgrowth was assessed in cultures of neonatal sympathetic ganglia grown for 72 h in the absence and presence of vascular EC. Nerve growth factor-induced axon growth in the presence of ECs was 50 +/- 4% (P < 0.05) of growth in the absence of ECs. ECs did not inhibit axon growth in the presence of an antibody that neutralized the activity of sema 3 (P > 0.05). RT-PCR and Western analyses also indicated that sema 3s were expressed in ECs of intact arteries. To assess the function of sema 3s in arteries, sympathetic ganglia were grown in the presence of arteries for 72 h, and the percentage of axons that grew toward the artery was determined: 44 +/- 4% of axons grew toward neonatal carotid arteries. Neutralization of sema 3s or removal of EC increased the percentage of axons that grew toward the artery (71 +/- 8% and 72 +/- 8%, respectively). These data indicate that vascular EC-derived sema 3s inhibit sympathetic axon growth and may thus be a determinant of vascular sympathetic innervation.

  13. Association of Nitric Oxide Levels and Endothelial Nitric Oxide Synthase G894T Polymorphism with Coronary Artery Disease in the Iranian Population

    PubMed Central

    Mahmoodi, Khalil; Nasehi, Leila; Karami, Elham; Soltanpour, Mohammad Soleiman

    2016-01-01

    Purpose: The endothelial nitric oxide synthase (eNOS) G894T polymorphism has been reported to cause endothelial dysfunction and may have a role in the development of coronary artery disease (CAD). The aim of the present study was to investigate the association of eNOS G894T genetic polymorphism and plasma levels of nitric oxide (NO) with CAD risk in an Iranian population. Materials and Methods: We studied 200 patients with angiographically documented CAD and 100 matched controls. Analysis of G894T genetic polymorphism of eNOS was performed by polymerase chain reaction-restriction fragment length polymorphism method. Plasma levels of NO were determined using Griess method. Biochemical analysis was conducted by routine colorimetric methods. Results: Plasma levels of NO were significantly lower in CAD patients than control subjects (41.60±12.70 vs. 55.48±16.57, P=0.001). Also, the mean plasma levels of NO were significantly lower in T allele carriers of eNOS G894T polymorphism than G allele carriers (P<0.001). The genotype distribution and minor T allele frequency of eNOS G894T polymorphism significantly differed between CAD patients and control subjects (P<0.05). However, no significant association was found between the eNOS G894T polymorphism and the severity of CAD (number of diseased vessel) or the lipid profile of CAD patients (P>0.05). Conclusion: Reduced plasma level of NO is associated with increased risk of CAD in our population. Moreover, eNOS G894T polymorphism is a significant risk factor for CAD development via reducing the plasma levels of NO. However, eNOS G894T polymorphism is not a contributing factor for the severity of CAD. PMID:27699157

  14. Far-infrared radiation acutely increases nitric oxide production by increasing Ca(2+) mobilization and Ca(2+)/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179.

    PubMed

    Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

    2013-07-12

    Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser(1179)) in a time-dependent manner (up to 40min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca(2+) levels. Treatment with KN-93, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. This study suggests that FIR radiation increases NO production via increasing CaMKII-mediated eNOS-Ser(1179) phosphorylation but TRPV channels may not be involved in this pathway. Our results may provide the molecular mechanism by which FIR radiation improves endothelial function.

  15. Nitric oxide synthases activation and inhibition by metallacarborane-cluster-based isoform-specific affectors.

    PubMed

    Kaplánek, Robert; Martásek, Pavel; Grüner, Bohumír; Panda, Satya; Rak, Jakub; Masters, Bettie Sue Siler; Král, Vladimír; Roman, Linda J

    2012-11-26

    A small library of boron-cluster- and metallacarborane-cluster-based ligands was designed, prepared, and tested for isoform-selective activation or inhibition of the three nitric oxide synthase isoforms. On the basis of the concept of creating a hydrophobic analogue of a natural substrate, a stable and nontoxic basic boron cluster system, previously used for boron neutron capture therapy, was modified by the addition of positively charged moieties to its periphery, providing hydrophobic and nonclassical hydrogen bonding interactions with the protein. Several of these compounds show efficacy for inhibition of NO synthesis with differential effects on the various nitric oxide synthase isoforms.

  16. P2Y12 receptor blockade synergizes strongly with nitric oxide and prostacyclin to inhibit platelet activation

    PubMed Central

    Chan, Melissa V.; Knowles, Rebecca B. M.; Lundberg, Martina H.; Tucker, Arthur T.; Mohamed, Nura A.; Kirkby, Nicholas S.; Armstrong, Paul C. J.; Mitchell, Jane A.

    2016-01-01

    Aims In vivo platelet function is a product of intrinsic platelet reactivity, modifiable by dual antiplatelet therapy (DAPT), and the extrinsic inhibitory endothelial mediators, nitric oxide (NO) and prostacyclin (PGI2), that are powerfully potentiated by P2Y12 receptor blockade. This implies that for individual patients endothelial mediator production is an important determinant of DAPT effectiveness. Here, we have investigated this idea using platelets taken from healthy volunteers treated with anti‐platelet drugs. Methods Three groups of male volunteers (n = 8) received either prasugrel (10 mg), aspirin (75 mg) or DAPT (prasugrel + aspirin) once daily for 7 days. Platelet reactivity in the presence of diethylammonium (Z)‐1‐(N,N‐diethylamino)diazen‐1‐ium‐1,2‐diolate (DEA/NONOate) and PGI2 was studied before and following treatment. Results Ex vivo, PGI2 and/or DEA/NONOate had little inhibitory effect on TRAP‐6‐induced platelet reactivity in control conditions. However, in the presence of DAPT, combination of DEA/NONOate + PGI2 reduced platelet aggregation (74 ± 3% to 19 ± 6%, P < 0.05). In vitro studies showed even partial (25%) P2Y12 receptor blockade produced a significant (67 ± 2% to 39 ± 10%, P < 0.05) inhibition when DEA/NONOate + PGI2 was present. Conclusions We have demonstrated that PGI2 and NO synergize with P2Y12 receptor antagonists to produce powerful platelet inhibition. Furthermore, even with submaximal P2Y12 blockade the presence of PGI2 and NO greatly enhances platelet inhibition. Our findings highlight the importance of endothelial mediator in vivo modulation of P2Y12 inhibition and introduces the concept of refining ex vivo platelet function testing by incorporating an assessment of endothelial function to predict thrombotic outcomes better and adjust therapy to prevent adverse outcomes in individual patients. PMID:26561399

  17. Vildagliptin stimulates endothelial cell network formation and ischemia-induced revascularization via an endothelial nitric-oxide synthase-dependent mechanism.

    PubMed

    Ishii, Masakazu; Shibata, Rei; Kondo, Kazuhisa; Kambara, Takahiro; Shimizu, Yuuki; Tanigawa, Tohru; Bando, Yasuko K; Nishimura, Masahiro; Ouchi, Noriyuki; Murohara, Toyoaki

    2014-09-26

    Dipeptidyl peptidase-4 inhibitors are known to lower glucose levels and are also beneficial in the management of cardiovascular disease. Here, we investigated whether a dipeptidyl peptidase-4 inhibitor, vildagliptin, modulates endothelial cell network formation and revascularization processes in vitro and in vivo. Treatment with vildagliptin enhanced blood flow recovery and capillary density in the ischemic limbs of wild-type mice, with accompanying increases in phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS). In contrast to wild-type mice, treatment with vildagliptin did not improve blood flow in ischemic muscles of eNOS-deficient mice. Treatment with vildagliptin increased the levels of glucagon-like peptide-1 (GLP-1) and adiponectin, which have protective effects on the vasculature. Both vildagliptin and GLP-1 increased the differentiation of cultured human umbilical vein endothelial cells (HUVECs) into vascular-like structures, although vildagliptin was less effective than GLP-1. GLP-1 and vildagliptin also stimulated the phosphorylation of Akt and eNOS in HUVECs. Pretreatment with a PI3 kinase or NOS inhibitor blocked the stimulatory effects of both vildagliptin and GLP-1 on HUVEC differentiation. Furthermore, treatment with vildagliptin only partially increased the limb flow of ischemic muscle in adiponectin-deficient mice in vivo. GLP-1, but not vildagliptin, significantly increased adiponectin expression in differentiated 3T3-L1 adipocytes in vitro. These data indicate that vildagliptin promotes endothelial cell function via eNOS signaling, an effect that may be mediated by both GLP-1-dependent and GLP-1-independent mechanisms. The beneficial activity of GLP-1 for revascularization may also be partially mediated by its ability to increase adiponectin production.

  18. Gene therapy techniques for the delivery of endothelial nitric oxide synthase to the lung for pulmonary hypertension.

    PubMed

    Deng, W; Bivalacqua, T J; Champion, H C; Hellstrom, W J; Murthy, Subramanyam N; Kadowitz, Philip J

    2010-01-01

    Pulmonary hypertension (PH) is a serious, often fatal disease characterized by remodeling of the pulmonary vascular bed, increased pulmonary arterial pressure, and right heart failure. The increased vascular resistance in the pulmonary circulation is due to structural changes and increased vasoconstrictor tone. Although current therapies have prolonged survival, the long-term outcome is not favorable. Nitric oxide (NO) is synthesized by endothelial nitric oxide synthase (eNOS) and is important in regulating vascular resistance and in vascular remodeling in the lung. NO deficiency due to endothelial dysfunction plays an important role in the pathogenesis of PH. Therefore, local eNOS gene delivery to the lung is a promising approach for the treatment of PH. Adenoviral-mediated in vivo gene therapy and adult stem cell-based ex vivo gene therapy are two attractive current gene therapies for the treatment of cardiovascular and pulmonary diseases. In this chapter we describe the use of two gene transfer techniques, i.e., adenoviral gene transfer of eNOS and eNOS gene-modified rat marrow stromal cells, for eNOS gene delivery to the lung of laboratory animals for the treatment of PH.

  19. Cuminum cyminum, a dietary spice, attenuates hypertension via endothelial nitric oxide synthase and NO pathway in renovascular hypertensive rats.

    PubMed

    Kalaivani, Periyathambi; Saranya, Ramesh Babu; Ramakrishnan, Ganapathy; Ranju, Vijayan; Sathiya, Sekar; Gayathri, Veeraraghavan; Thiyagarajan, Lakshmi Kantham; Venkhatesh, Jayakothanda Ramaswamy; Babu, Chidambaram Saravana; Thanikachalam, Sadagopan

    2013-01-01

    Cuminum cyminum (CC) is a commonly used spice in South Indian foods. It has been traditionally used for the treatment and management of sleep disorders, indigestion, and hypertension. The present study was carried out to scientifically evaluate the anti-hypertensive potential of standardized aqueous extract of CC seeds and its role in arterial endothelial nitric oxide synthase expression, inflammation, and oxidative stress in renal hypertensive rats. Renal hypertension was induced by the two-kidney one-clip (2K/1C) method in rats. Systolic blood pressure (SBP), plasma nitrate/nitrite, carotid-eNOS, renal-TNF-α, IL-6, Bax, Bcl-2, thioredoxin 1 (TRX1), and thioredoxin reductase 1 (TRXR1) mRNA expressions were studied to demonstrate the anti-hypertensive action of CC. Cuminum cyminum was administered orally (200 mg/kg b.wt) for a period of 9 weeks; it improved plasma nitric oxide and decreased the systolic blood pressure in hypertensive rats. It also up-regulated the gene expression of eNOS, Bcl-2, TRX1, and TRXR1; and down-regulated Bax, TNF-α, and IL-6. These data reveal that CC seeds augment endothelial functions and ameliorate inflammatory and oxidative stress in hypertensive rats. The present report is the first of its kind to demonstrate the mechanism of anti-hypertensive action of CC seeds in an animal model of renovascular hypertension.

  20. The Traditional Japanese Formula Keishibukuryogan Inhibits the Production of Inflammatory Cytokines by Dermal Endothelial Cells

    PubMed Central

    Yoshihisa, Yoko; Furuichi, Megumi; Ur Rehman, Mati; Ueda, Chieko; Makino, Teruhiko; Shimizu, Tadamichi

    2010-01-01

    Keishibukuryogan (KBG) is one of the traditional herbal formulations widely administered to patients with blood stagnation for improving blood circulation; currently, it is the most frequently prescribed medicine in Japan. KBG has been reported to improve conjunctional microcirculation. The aim of this study was to evaluate the role of KBG and paeoniflorin, a bioactive compound of KBG, in inhibiting the production of inflammatory cytokines using human dermal microvessel endothelial cells (HDMECs). The authors observed that lipopolysaccharide (LPS; 1 μg/mL) stimulated the secretion of proinflammatory cytokines in HDMECs. KBG treatment (10 mg/mL) significantly suppressed the mRNA levels of migration inhibitory factor (MIF), interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in LPS-stimulated cultured HDMECs. Similarly, paeoniflorin significantly suppressed the mRNA levels of these cytokines in LPS-stimulated cultured HDMECs. ELISA showed that KBG and paeoniflorin suppressed the production of MIF, IL-6, IL-8, and TNF-α in LPS-stimulated HDMECs. Moreover, KBG and paeoniflorin decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase (iNOS) in these cells. These results suggest that KBG may be useful for improving microvascular inflammation in patients with skin diseases. PMID:21253500

  1. Reduced nitric oxide-mediated relaxation and endothelial nitric oxide synthase expression in the tail arteries of streptozotocin-induced diabetic rats.

    PubMed

    Mokhtar, Siti Safiah; Vanhoutte, Paul M; Leung, Susan Wai Sum; Suppian, Rapeah; Yusof, Mohd Imran; Rasool, Aida Hanum Ghulam

    2016-02-15

    Diabetes is associated with endothelial dysfunction, which is characterized by impaired endothelium-dependent relaxations. The present study aimed to examine the role of nitric oxide (NO), prostacyclin and endothelium-dependent hyperpolarization (EDH), in the relaxation of ventral tail arteries of rats under diabetic conditions. Relaxations of tail arteries of control and diabetic rats were studied in wire myograph. Western blotting and immunostaining were used to determine the presence of proteins. Acetylcholine-induced relaxations were significantly smaller in arteries of diabetic compared to control rats (Rmax; 70.81 ± 2.48% versus 85.05 ± 3.15%). Incubation with the combination of non-selective cyclooxygenase (COX) inhibitor, indomethacin and potassium channel blockers, TRAM 34 and UCL 1684, demonstrated that NO-mediated relaxation was attenuated significantly in diabetic compared to control rats (Rmax; 48.47 ± 5.84% versus 68.39 ± 6.34%). EDH-type (in the presence of indomethacin and NO synthase inhibitor, LNAME) and prostacyclin-mediated (in the presence of LNAME plus TRAM 34 and UCL 1684) relaxations were not significantly reduced in arteries of diabetic compared to control rats [Rmax: (EDH; 17.81 ± 6.74% versus 34.16 ± 4.59%) (prostacyclin; 15.85 ± 3.27% versus 17.23 ± 3.75%)]. Endothelium-independent relaxations to sodium nitroprusside, salbutamol and prostacyclin were comparable in the two types of preparations. Western blotting and immunostaining indicated that diabetes diminished the expression of endothelial NO synthase (eNOS), while increasing those of COX-1 and COX-2. Thus, since acetylcholine-induced NO-mediated relaxation was impaired in diabetes because of reduced eNOS protein expression, pharmacological intervention improving NO bioavailability could be useful in the management of diabetic endothelial dysfunction.

  2. Intact mitochondrial Ca2+ uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS)

    PubMed Central

    Charoensin, Suphachai; Eroglu, Emrah; Opelt, Marissa; Bischof, Helmut; Madreiter-Sokolowski, Corina T.; Kirsch, Andrijana; Depaoli, Maria R.; Frank, Saša; Schrammel, Astrid; Mayer, Bernd; Waldeck-Weiermair, Markus; Graier, Wolfgang F.; Malli, Roland

    2017-01-01

    Mitochondrial Ca2+ uptake regulates diverse endothelial cell functions and has also been related to nitric oxide (NO•) production. However, it is not entirely clear if the organelles support or counteract NO• biosynthesis by taking up Ca2+. The objective of this study was to verify whether or not mitochondrial Ca2+ uptake influences Ca2+-triggered NO• generation by endothelial NO• synthase (eNOS) in an immortalized endothelial cell line (EA.hy926), respective primary human umbilical vein endothelial cells (HUVECs) and eNOS-RFP (red fluorescent protein) expressing human embryonic kidney (HEK293) cells. We used novel genetically encoded fluorescent NO• probes, the geNOps, and Ca2+ sensors to monitor single cell NO• and Ca2+ dynamics upon cell treatment with ATP, an inositol 1,4,5-trisphosphate (IP3)-generating agonist. Mitochondrial Ca2+ uptake was specifically manipulated by siRNA-mediated knock-down of recently identified key components of the mitochondrial Ca2+ uniporter machinery. In endothelial cells and the eNOS-RFP expressing HEK293 cells we show that reduced mitochondrial Ca2+ uptake upon the knock-down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca2+-triggered NO• increase independently of global cytosolic Ca2+ signals. The knock-down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca2+ sequestration and Ca2+-induced NO• signals. The positive correlation between mitochondrial Ca2+ elevation and NO• production was independent of eNOS phosphorylation at serine1177. Our findings emphasize that manipulating mitochondrial Ca2+ uptake may represent a novel strategy to control eNOS-mediated NO• production. PMID:27923677

  3. Salidroside inhibits endogenous hydrogen peroxide induced cytotoxicity of endothelial cells.

    PubMed

    Zhao, Xingyu; Jin, Lianhai; Shen, Nan; Xu, Bin; Zhang, Wei; Zhu, Hongli; Luo, Zhengli

    2013-01-01

    Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L., shows potent antioxidant property. Herein, we investigated the protective effects of salidroside against hydrogen peroxide (H2O2)-induced oxidative damage in human endothelial cells (EVC-304). EVC-304 cells were incubated in the presence or absence of low steady states of H2O2 (3-4 µM) generated by glucose oxidase (GOX) with or without salidroside. 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) assays were performed, together with Hoechst 33258 staining and flow cytometric analysis using Annexin-V and propidium iodide (PI) label. The results indicated that salidroside pretreatment attenuated endogenous H2O2 induced apoptotic cell death in EVC-304 cells in a dose-dependent pattern. Furthermore, Western blot data revealed that salidroside inhibited activation of caspase-3, 9 and cleavage of poly(ADP-ribose) polymerase (PARP) induced by endogenous H2O2. It also decreased the expression of Bax and rescued the balance of pro- and anti-apoptotic proteins. All these results demonstrated that salidroside may present a potential therapy for oxidative stress in cardiovascular and cerebrovascular diseases.

  4. Antioxidant and nitric oxide inhibition activities of Thai medicinal plants.

    PubMed

    Makchuchit, Sunita; Itharat, Arunporn; Tewtrakul, Supinya

    2010-12-01

    Nineteen Thai medicinal plants used in Thai traditional medicine preparation to treat colds, asthma and fever were studied for their antioxidant and NO inhibitory activities. Three extracts were obtained from each plant. First extract obtained by macerating the plant part in 95% ethanol (Et) residue was boiled in water, where water extract (EW) was obtained. The third extract (HW) was obtained by boiling each plant in water similar to that of Thai traditional medicine practice. These extracts were tested for their antioxidant activity using DPPH assay, and anti-inflammatory activity by determination of inhibitory activity on lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW 264.7 cell lines using Griess reagent. Results indicated that Et, EW and HW of Syzygium aromaticum showed the highest antioxidant activity (EC50 = 6.56, 4.73 and 5.30 microg/ml, respectively). Et of Atractylodes lancea exhibited the most potent inhibitory activity on lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW 264.7 cells, with IC50 value of 9.70 microg/ml, followed by Et of Angelica sinensis and Cuminum cyminum (IC50 = 12.52 and 13.56 microg/ml, respectively) but water extract (EW, HW) of all plants were apparently inactive. These results of anti-inflammatory activity of these plants correspond with the traditional use for fever; cold, allergic-related diseases and inflammatory-related diseases.

  5. Asiaticoside Inhibits TNF-α-Induced Endothelial Hyperpermeability of Human Aortic Endothelial Cells.

    PubMed

    Fong, Lai Yen; Ng, Chin Theng; Zakaria, Zainul Amiruddin; Baharuldin, Mohamad Taufik Hidayat; Arifah, Abdul Kadir; Hakim, Muhammad Nazrul; Zuraini, Ahmad

    2015-10-01

    The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor-alpha (TNF-α), a pro-atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF-α in human aortic endothelial cells (HAEC). TNF-α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)-dextran. Asiaticoside pretreatment significantly suppressed TNF-α-induced increased permeability. Asiaticoside also prevented TNF-α-induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF-α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti-hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D-induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF-α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F-actin organization.

  6. Genistein, a soy phytoestrogen, upregulates the expression of human endothelial nitric oxide synthase and lowers blood pressure in spontaneously hypertensive rats.

    PubMed

    Si, Hongwei; Liu, Dongmin

    2008-02-01

    Genistein, a soy phytoestrogen, may improve vascular function, but the mechanism of this effect is unclear. Endothelial-derived nitric oxide (NO) is a key regulator of vascular tone and atherogenesis. Previous studies have established that estrogen can act directly on vascular endothelial cells (EC) to enhance NO synthesis through genomic stimulation of endothelial NO synthase (eNOS) expression. However, it is unknown whether genistein has a similar effect. We therefore investigated whether genistein directly regulates NO synthesis in primary human aortic EC (HAEC) and human umbilical vein EC (HUVEC). Genistein, at physiologically achievable concentrations in individuals consuming soy products, enhanced the expression of eNOS and subsequently elevated NO synthesis in both HAEC and HUVEC, with 1-10 micromol/L genistein inducing the maximal effects. However, the effects of genistein on eNOS and NO were not mediated by activation of estrogen signaling or inhibition of tyrosine kinases, 2 known biological actions of genistein. Genistein (1-10 micromol/L) increased eNOS gene expression (1.8- to 2.6-fold of control) and significantly increased eNOS promoter activity of the human eNOS gene in HAEC and HUVEC, suggesting that genistein activates eNOS transcription. Dietary supplementation of genistein to spontaneously hypertensive rats restored aortic eNOS levels, improved aortic wall thickness, and alleviated hypertension, confirming the biological relevance of the in vitro findings. Our data suggest that genistein has direct genomic effects on the vascular wall that are unrelated to its known actions, leading to increased eNOS expression and NO synthesis, thereby improving hypertension.

  7. Progesterone increases nitric oxide synthesis in human vascular endothelial cells through activation of membrane progesterone receptor-α.

    PubMed

    Pang, Yefei; Dong, Jing; Thomas, Peter

    2015-05-15

    Progesterone exerts beneficial effects on the human cardiovascular system by inducing rapid increases in nitric oxide (NO) production in vascular endothelial cells, but the receptors mediating these nongenomic progesterone actions remain unclear. Using human umbilical vein endothelial cells (HUVECs) as a model, we show that progesterone binds to plasma membranes of HUVECs with the characteristics of membrane progesterone receptors (mPRs). The selective mPR agonist Org OD 02-0 had high binding affinity for the progesterone receptor on HUVEC membranes, whereas nuclear PR (nPR) agonists R5020 and medroxyprogesterone acetate displayed low binding affinities. Immunocytochemical and Western blot analyses confirmed that mPRs are expressed in HUVECs and are localized on their plasma membranes. NO levels increased rapidly after treatment with 20 nM progesterone, Org OD 02-0, and a progesterone-BSA conjugate but not with R5020, suggesting that this progesterone action is at the cell surface and initiated through mPRs. Progesterone and Org OD 02-0 (20 nM) also significantly increased endothelial nitric oxide synthase (eNOS) activity and eNOS phosphorylation. Knockdown of mPRα expression by treatment with small-interfering RNA (siRNA) blocked the stimulatory effects of 20 nM progesterone on NO production and eNOS phosphorylation, whereas knockdown of nPR was ineffective. Treatment with PI3K/Akt and MAP kinase inhibitors blocked the stimulatory effects of progesterone, Org OD 02-0, and progesterone-BSA on NO production and eNOS phosphorylation and also prevented progesterone- and Org OD 02-0-induced increases in Akt and ERK phosphorylation. The results suggest that progesterone stimulation of NO production in HUVECs is mediated by mPRα and involves signaling through PI3K/Akt and MAP kinase pathways.

  8. An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

    1998-04-01

    Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

  9. Flavonoids from artichoke (Cynara scolymus L.) up-regulate endothelial-type nitric-oxide synthase gene expression in human endothelial cells.

    PubMed

    Li, Huige; Xia, Ning; Brausch, Isolde; Yao, Ying; Förstermann, Ulrich

    2004-09-01

    Nitric oxide (NO) produced by endothelial nitric-oxide synthase (eNOS) represents an antithrombotic and anti-atherosclerotic principle in the vasculature. Hence, an enhanced expression of eNOS in response to pharmacological interventions could provide protection against cardiovascular diseases. In EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVECs), an artichoke leaf extract (ALE) increased the activity of the human eNOS promoter (determined by luciferase reporter gene assay). An organic subfraction from ALE was more potent in this respect than the crude extract, whereas an aqueous subfraction of ALE was without effect. ALE and the organic subfraction thereof also increased eNOS mRNA expression (measured by an RNase protection assay) and eNOS protein expression (determined by Western blot) both in EA.hy 926 cells and in native HUVECs. NO production (measured by NO-ozone chemiluminescence) was increased by both extracts. In organ chamber experiments, ex vivo incubation (18 h) of rat aortic rings with the organic subfraction of ALE enhanced the NO-mediated vasodilator response to acetylcholine, indicating that the up-regulated eNOS remained functional. Caffeoylquinic acids and flavonoids are two major groups of constituents of ALE. Interestingly, the flavonoids luteolin and cynaroside increased eNOS promoter activity and eNOS mRNA expression, whereas the caffeoylquinic acids cynarin and chlorogenic acid were without effect. Thus, in addition to the lipid-lowering and antioxidant properties of artichoke, an increase in eNOS gene transcription may also contribute to its beneficial cardiovascular profile. Artichoke flavonoids are likely to represent the active ingredients mediating eNOS up-regulation.

  10. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A

    NASA Technical Reports Server (NTRS)

    Boo, Yong Chool; Sorescu, George; Boyd, Nolan; Shiojima, Ichiro; Walsh, Kenneth; Du, Jie; Jo, Hanjoong

    2002-01-01

    Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction

  11. Genistein ameliorated endothelial nitric oxidase synthase uncoupling by stimulating sirtuin-1 pathway in ox-LDL-injured HUVECs.

    PubMed

    Zhang, Hua-ping; Zhao, Jia-hui; Yu, Hai-xia; Guo, Dong-xing

    2016-03-01

    Endothelial nitric oxidase synthase (eNOS) uncoupling plays a causal role in endothelial dysfunction in atherosclerosis. Genistein consumption has been associated with the prevention of atherosclerosis. However, the effect of genistein on eNOS uncoupling has not been reported. A model of oxidized low-density lipoprotein (ox-LDL)-induced injury on human umbilical vein endothelial cells (HUVECs) was established to evaluate the effect of genistein on eNOS uncoupling. We investigated the effect of genistein on NADPH oxidase-dependent superoxide production, NOX4 expression, BH4 synthesis and oxidation, the expression of GTP cyclohydrolase 1 (GCH1) and dihydrofolate reductase (DHFR). The results showed that genistein decreased superoxide production and NOX4 expression, enhanced the ratio of BH4/BH2, augmented the expressions of GCH1 and DHFR. Accompanied with genistein ameliorating eNOS uncoupling, genistein elevated the expression of sirtuin-1; furthermore, the effects of genistein on eNOS uncoupling were blunted with sirtuin-1 siRNA. The present study indicated that genistein ameliorated eNOS uncoupling was concerned with sirtuin-1 pathway in ox-LDL-injured HUVECs.

  12. Hydrostatic pressure and shear stress affect endothelin-1 and nitric oxide release by endothelial cells in bioreactors.

    PubMed

    Vozzi, Federico; Bianchi, Francesca; Ahluwalia, Arti; Domenici, Claudio

    2014-01-01

    Abundant experimental evidence demonstrates that endothelial cells are sensitive to flow; however, the effect of fluid pressure or pressure gradients that are used to drive viscous flow is not well understood. There are two principal physical forces exerted on the blood vessel wall by the passage of intra-luminal blood: pressure and shear. To analyze the effects of pressure and shear independently, these two stresses were applied to cultured cells in two different types of bioreactors: a pressure-controlled bioreactor and a laminar flow bioreactor, in which controlled levels of pressure or shear stress, respectively, can be generated. Using these bioreactor systems, endothelin-1 (ET-1) and nitric oxide (NO) release from human umbilical vein endothelial cells were measured under various shear stress and pressure conditions. Compared to the controls, a decrease of ET-1 production by the cells cultured in both bioreactors was observed, whereas NO synthesis was up-regulated in cells under shear stress, but was not modulated by hydrostatic pressure. These results show that the two hemodynamic forces acting on blood vessels affect endothelial cell function in different ways, and that both should be considered when planning in vitro experiments in the presence of flow. Understanding the individual and synergic effects of the two forces could provide important insights into physiological and pathological processes involved in vascular remodeling and adaptation.

  13. Traditional Chinese medicine xin-mai-jia recouples endothelial nitric oxide synthase to prevent atherosclerosis in vivo.

    PubMed

    Yin, Ya-Ling; Zhu, Mo-Li; Wan, Jia; Zhang, Chong; Pan, Guo-Pin; Lu, Jun-Xiu; Ping, Song; Chen, Yuan; Zhao, Fan-Rong; Yu, Hai-Ya; Guo, Tao; Jian, Xu; Liu, Li-Ying; Zhang, Jia-Ning; Wan, Guang-Rui; Wang, Shuang-Xi; Li, Peng

    2017-03-02

    Endothelial dysfunction, which is caused by endothelial nitric oxide synthase (eNOS) uncoupling, is an initial step in atherosclerosis. This study was designed to explore whether Chinese medicine xin-mai-jia (XMJ) recouples eNOS to exert anti-atherosclerotic effects. Pretreatment of XMJ (25, 50, 100 μg/ml) for 30 minutes concentration-dependently activated eNOS, improved cell viabilities, increased NO generations, and reduced ROS productions in human umbilical vein endothelial cells incubated with H2O2 for 2 hours, accompanied with restoration of BH4. Importantly, these protective effects produced by XMJ were abolished by eNOS inhibitor L-NAME or specific eNOS siRNA in H2O2-treated cells. In ex vivo experiments, exposure of isolated aortic rings from rats to H2O2 for 6 hours dramatically impaired acetylcholine-induced vasorelaxation, reduced NO levels and increased ROS productions, which were ablated by XMJ in concentration-dependent manner. In vivo analysis indicated that administration of XMJ (0.6, 2.0, 6.0 g/kg/d) for 12 weeks remarkably recoupled eNOS and reduced the size of carotid atherosclerotic plaque in rats feeding with high fat diet plus balloon injury. In conclusion, XMJ recouples eNOS to prevent the growth of atherosclerosis in rats. Clinically, XMJ is potentially considered as a medicine to treat patients with atherosclerosis.

  14. Traditional Chinese medicine xin-mai-jia recouples endothelial nitric oxide synthase to prevent atherosclerosis in vivo

    PubMed Central

    Yin, Ya-Ling; Zhu, Mo-Li; Wan, Jia; Zhang, Chong; Pan, Guo-Pin; Lu, Jun-Xiu; Ping, Song; Chen, Yuan; Zhao, Fan-Rong; Yu, Hai-Ya; Guo, Tao; Jian, Xu; Liu, Li-Ying; Zhang, Jia-Ning; Wan, Guang-Rui; Wang, Shuang-Xi; Li, Peng

    2017-01-01

    Endothelial dysfunction, which is caused by endothelial nitric oxide synthase (eNOS) uncoupling, is an initial step in atherosclerosis. This study was designed to explore whether Chinese medicine xin-mai-jia (XMJ) recouples eNOS to exert anti-atherosclerotic effects. Pretreatment of XMJ (25, 50, 100 μg/ml) for 30 minutes concentration-dependently activated eNOS, improved cell viabilities, increased NO generations, and reduced ROS productions in human umbilical vein endothelial cells incubated with H2O2 for 2 hours, accompanied with restoration of BH4. Importantly, these protective effects produced by XMJ were abolished by eNOS inhibitor L-NAME or specific eNOS siRNA in H2O2-treated cells. In ex vivo experiments, exposure of isolated aortic rings from rats to H2O2 for 6 hours dramatically impaired acetylcholine-induced vasorelaxation, reduced NO levels and increased ROS productions, which were ablated by XMJ in concentration-dependent manner. In vivo analysis indicated that administration of XMJ (0.6, 2.0, 6.0 g/kg/d) for 12 weeks remarkably recoupled eNOS and reduced the size of carotid atherosclerotic plaque in rats feeding with high fat diet plus balloon injury. In conclusion, XMJ recouples eNOS to prevent the growth of atherosclerosis in rats. Clinically, XMJ is potentially considered as a medicine to treat patients with atherosclerosis. PMID:28252100

  15. CCK receptors-related signaling involved in nitric oxide production caused by gastrin 17 in porcine coronary endothelial cells.

    PubMed

    Grossini, Elena; Caimmi, Philippe; Molinari, Claudio; Uberti, Francesca; Mary, David; Vacca, Giovanni

    2012-03-05

    In anesthetized pigs gastrin-17 increased coronary blood flow through CCK1/CCK2 receptors and β(2)-adrenoceptors-related nitric oxide (NO) release. Since the intracellular pathway has not been investigated the purpose of this study was to examine in coronary endothelial cells the CCK1/CCK2 receptors-related signaling involved in the effects of gastrin-17 on NO release. Gastrin-17 caused a concentration-dependent increase of NO production (17.3-62.6%; p<0.05), which was augmented by CCK1/CCK2 receptors agonists (p<0.05). The effect of gastrin-17 was amplified by the adenylyl-cyclase activator and β(2)-adrenoceptors agonist (p<0.05), abolished by cAMP/PKA and β(2)-adrenoceptors and CCK1/CCK2 receptors blockers, and reduced by PLC/PKC inhibitor. Finally, Western-blot revealed the preferential involvement of PKA vs. PKC as downstream effectors of CCK1/CCK2 receptors activation leading to Akt, ERK, p38 and endothelial NOS (eNOS) phosphorylation. In conclusion, in coronary endothelial cells, gastrin-17 induced eNOS-dependent NO production through CCK1/CCK2 receptors- and β(2)-adrenoceptors-related pathway. The intracellular signaling involved a preferential PKA pathway over PKC.

  16. Influence of radiographic contrast media on the nitric oxide release from human arterial and venous endothelial cells on extracellular matrix.

    PubMed

    Franke, R P; Fuhrmann, R; Jung, F

    2013-01-01

    Radiographic contrast media (RCM) can vary widely in their physicochemical properties, e.g. the iodine concentration, osmolality, molecule structure, chemotoxicity, hydrophilicity, electric charge and viscosity. Besides the necessary effect of Roentgen ray absorption, which provides contrast-rich images of vessels, RCMs can have varying adverse effects. As one possible cause of microcirculatory disorders, changes in morphology and function of endothelial cells are discussed. Therefore, RCM media-induced release of nitric oxide from arterial as well as from venous endothelial cells in contact with two commercially available RCMs (Iodixanol and Iomeprol) was investigated. NO concentrations started to increase slightly in the HUVEC control cultures after 3 min incubation time, however, NO concentrations in the cultures incubated with Iomeprol 350 and Iodixanol 320 did not change over time (Iomeprol 350: p = 0.4905; Iodixanol 320: p = 0.784). On the whole, the time-dependent NO release differed for the three groups (RCM × time: p = 0.00224). This difference was due to the fact that, after incubation with the two contrast agents (Iodixanol 320: p = 0.0003; Iomeprol 350: p = 0.0168), less NO was released by the exposed HUVEC at 3 minutes and after 12 hours than by the control cells. In the control cultures of arterial endothelial cells as well as in cultures incubated with 30% v/v Iodixanol supplemented culture medium the NO release did not change. In those cultures of arterial endothelial cells supplemented with 30% v/v Iomeprol the NO release was significantly less than in control cultures and in cultures supplemented with Iodixanol (p = 0.021; p = 0.043). Inspite of a missing shear stress in our static plane vessel wall model there was a RCM-dependent difference in NO release from endothelial cells in vitro. The NO release from venous endothelial cells differed significantly from the NO release from arterial endothelial cells. While the administration of Iomeprol

  17. Pharmacological inhibition of S-nitrosoglutathione reductase improves endothelial vasodilatory function in rats in vivo

    PubMed Central

    Chen, Qiumei; Sievers, Richard E.; Varga, Monika; Kharait, Sourabh; Haddad, Daniel J.; Patton, Aaron K.; Delany, Christopher S.; Mutka, Sarah C.; Blonder, Joan P.; Dubé, Gregory P.; Rosenthal, Gary J.

    2013-01-01

    Nitric oxide (NO) exerts a wide range of cellular effects in the cardiovascular system. NO is short lived, but S-nitrosoglutathione (GSNO) functions as a stable intracellular bioavailable NO pool. Accordingly, increased levels can facilitate NO-mediated processes, and conversely, catabolism of GSNO by the regulatory enzyme GSNO reductase (GSNOR) can impair these processes. Because dysregulated GSNOR can interfere with processes relevant to cardiovascular health, it follows that inhibition of GSNOR may be beneficial. However, the effect of GSNOR inhibition on vascular activity is unknown. To study the effects of GSNOR inhibition on endothelial function, we treated rats with a small-molecule inhibitor of GSNOR (N6338) that has vasodilatory effects on isolated aortic rings and assessed effects on arterial flow-mediated dilation (FMD), an NO-dependent process. GSNOR inhibition with a single intravenous dose of N6338 preserved FMD (15.3 ± 5.4 vs. 14.2 ± 6.3%, P = nonsignificant) under partial NO synthase inhibition that normally reduces FMD by roughly 50% (14.1 ± 2.9 vs. 7.6 ± 4.4%, P < 0.05). In hypertensive rats, daily oral administration of N6338 for 14 days reduced blood pressure (170.0 ± 5.3/122.7 ± 6.4 vs. 203.8 ± 1.9/143.7 ± 7.5 mmHg for vehicle, P < 0.001) and vascular resistance index (1.5 ± 0.4 vs. 3.2 ± 1.0 mmHg·min·l−1 for vehicle, P < 0.001), and restored FMD from an initially impaired state (7.4 ± 1.7%, day 0) to a level (13.0 ± 3.1%, day 14, P < 0.001) similar to that observed in normotensive rats. N6338 also reversed the pathological kidney changes exhibited by the hypertensive rats. GSNOR inhibition preserves FMD under conditions of impaired NO production and protects against both microvascular and conduit artery dysfunction in a model of hypertension. PMID:23349456

  18. eNOS-dependent S-nitrosylation of β-catenin prevents its association with TCF4 and inhibits proliferation of endothelial cells by Wnt3a.

    PubMed

    Zhang, Ying; Chidiac, Rony; Delisle, Chantal; Gratton, Jean-Philippe

    2017-03-20

    Nitric oxide (NO) produced by endothelial NO synthase (eNOS) modulates many functions in endothelial cells. S-nitrosylation (SNO) of cysteine residues on β-catenin by eNOS-derived NO has been shown to influence intercellular contacts between endothelial cells. However, the implication of SNO in the regulation of β-catenin transcriptional activity is ill-defined. Here we report that NO inhibits the transcriptional activity of β-catenin and endothelial cell proliferation induced by activation of Wnt/β-catenin signaling. Interestingly, induction by Wnt3a of β-catenin target genes, such as Axin2, is repressed in an eNOS-dependent manner by VEGF. We identify Cys466 of β-catenin as a target for SNO by eNOS-derived NO and as the critical residue for the repressive effects of NO on β-catenin transcriptional activity. Furthermore, we observed that Cys466 of β-catenin, located at the binding interface of the β-catenin/TCF4 transcriptional complex, is essential for disruption of this complex by NO. Importantly, Cys466 of β-catenin is necessary for the inhibitory effects of NO on Wnt3a-stimulated proliferation of endothelial cells. Thus our data define the mechanism responsible for the repressive effects of NO on the transcriptional activity of β-catenin and link eNOS-derived NO to the modulation by VEGF of Wnt/β-catenin-induced endothelial cell proliferation.

  19. Serine 1179 phosphorylation of endothelial nitric oxide synthase caused by 2,4,6-trinitrotoluene through PI3K/Akt signaling in endothelial cells

    SciTech Connect

    Sun Yang; Sumi, Daigo; Kumagai, Yoshito . E-mail: yk-em-tu@md.tsukuba.ac.jp

    2006-07-01

    Although 2,4,6-trinitrotoluene (TNT) has been found to uncouple nitric oxide synthase (NOS), thereby leading to reactive oxygen species (ROS), cellular response against TNT still remains unclear. Exposure of bovine aortic endothelial cells (BAECs) to TNT (100 {mu}M) resulted in serine 1179 phosphorylation of endothelial NOS (eNOS). With specific inhibitors (wortmannin and LY294002), we found that PI3K/Akt signaling participated in the eNOS phosphorylation caused by TNT, whereas the ERK pathway did not. ROS were generated following exposure of BAECs to TNT. However, TNT-mediated phosphorylation of either eNOS or Akt was drastically blocked by NAC and PEG-CAT. Interestingly, pretreatment with apocynin, a specific inhibitor for NADPH oxidase, diminished the phosphorylation of eNOS and Akt. These results suggest that TNT affects NADPH oxidase, thereby generating hydrogen peroxide, which is capable of activating PI3K/Akt signaling associated with eNOS Ser 1179 phosphorylation.

  20. Human recombinant erythropoietin alters the flow-dependent vasodilatation of in vitro perfused rat mesenteric arteries with unbalanced endothelial endothelin-1 / nitric oxide ratio.

    PubMed

    Barhoumi, Tlili; Jallat, Isabelle; Berthelot, Alain; Laurant, Pascal

    2011-06-01

    Chronic use of human recombinant erythropoietin (r-HuEPO) is accompanied by serious vascular side effects related to the rise in blood viscosity and shear stress. We investigated the direct effects of r-HuEPO on endothelium and nitric oxide (NO)-dependent vasodilatation induced by shear stress of cannulated and pressurized rat mesenteric resistance arteries. Intravascular flow was increased in the presence or absence of the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME; 10(-4) mol/L). In the presence of r-HuEPO, the flow-dependent vasodilatation was attenuated, while L-NAME completely inhibited it. The association of r-HuEPO and L-NAME caused a vasoconstriction in response to the rise in intravascular flow. Bosentan (10(-5) mol/L), an inhibitor of endothelin-1 (ET-1) receptors, corrected the attenuated vasodilatation observed with r-HuEPO and inhibited the vasoconstriction induced by flow in the presence of r-HuEPO and L-NAME. r-HuEPO and L-NAME exacerbated ET-1 vasoconstriction. At shear stress values of 2 and 14 dyn/cm(2) (1 dyn = 10(-5) N), cultured EA.hy926 endothelial cells incubated with r-HuEPO, L-NAME, or both released greater ET-1 than untreated cells. In conclusion, r-HuEPO diminishes flow-induced vasodilatation. This inhibitory effect seems to implicate ET-1 release. NO withdrawal exacerbates the vascular effects of ET-1 in the presence of r-HuEPO. These findings support the importance of a balanced endothelial ET-1:NO ratio to avoid the vasopressor effects of r-HuEPO.

  1. Role of microRNAs 221/222 on Statin Induced Nitric Oxide Release in Human Endothelial Cells

    PubMed Central

    Cerda, Alvaro; Fajardo, Cristina Moreno; Basso, Rodrigo Gouveia; Hirata, Mario Hiroyuki; Hirata, Rosario Dominguez Crespo

    2015-01-01

    Background Nitric oxide (NO) has been largely associated with cardiovascular protection through improvement of endothelial function. Recently, new evidence about modulation of NO release by microRNAs (miRs) has been reported, which could be involved with statin-dependent pleiotropic effects, including anti-inflammatory properties related to vascular endothelium function. Objective To evaluate the effects of cholesterol-lowering drugs including the inhibitors of cholesterol synthesis, atorvastatin and simvastatin, and the inhibitor of cholesterol absorption ezetimibe on NO release, NOS3 mRNA expression and miRs potentially involved in NO bioavailability. Methods Human umbilical vein endothelial cells (HUVEC) were exposed to atorvastatin, simvastatin or ezetimibe (0 to 5.0 μM). Cells were submitted to total RNA extraction and relative quantification of NOS3 mRNA and miRs -221, -222 and -1303 by qPCR. NO release was measured in supernatants by ozone-chemiluminescence. Results Both statins increased NO levels and NOS3 mRNA expression but no influence was observed for ezetimibe treatment. Atorvastatin, simvastatin and ezetimibe down-regulated the expression of miR-221, whereas miR-222 was reduced only after the atorvastatin treatment. The magnitude of the reduction of miR-221 and miR-222 after treatment with statins correlated with the increment in NOS3 mRNA levels. No influence was observed on the miR-1303 expression after treatments. Conclusion NO release in endothelial cells is increased by statins but not by the inhibitor of cholesterol absorption, ezetimibe. Our results provide new evidence about the participation of regulatory miRs 221/222 on NO release induction mediated by statins. Although ezetimibe did not modulate NO levels, the down-regulation of miR-221 could involve potential effects on endothelial function. PMID:25517390

  2. Effects of Acute Nitric Oxide Synthase Inhibition on Lower Leg Vascular Function in Chronic Tetraplegia

    PubMed Central

    La Fountaine, Michael F; Radulovic, Miroslav; Cardozo, Christopher P; Spungen, Ann M; DeMeersman, Ronald E; Bauman, William A

    2009-01-01

    Background/Objective: To improve our understanding of the lower-leg vascular responses of nitric oxide synthase inhibition in persons with tetraplegia. Participants: Six people with chronic tetraplegia and 6 age-matched controls. Methods: Lower-leg relative vascular resistance and venous volume variation were obtained by venous occlusion plethysmography and blood pressure by auscultation at baseline. Postintravenous infusion of the nitric oxide synthase inhibitor NG-nitro-l-arginine-methyl-ester (1 mg·kg−1) or placebo on separate days. Results: At baseline in the group with tetraplegia compared with controls, mean arterial pressure and relative vascular resistance of the leg were significantly lower. After nitric oxide synthase inhibition, mean arterial pressure and lower leg vascular resistance were significantly elevated in both groups. There were no group or intervention differences in venous volume variation. Conclusion: These preliminary results suggest that nitric oxide synthase inhibition with 1 mg·kg−1 NG-nitro-l-arginine-methyl-ester normalizes seated blood pressure and lower leg vascular resistance to control group baseline levels. PMID:20025149

  3. Nitric oxide inhibits c-Jun DNA binding by specifically targeted S-glutathionylation.

    PubMed

    Klatt, P; Molina, E P; Lamas, S

    1999-05-28

    This study addresses potential molecular mechanisms underlying the inhibition of the transcription factor c-Jun by nitric oxide. We show that in the presence of the physiological sulfhydryl glutathione nitric oxide modifies the two cysteine residues contained in the DNA binding module of c-Jun in a selective and distinct way. Although nitric oxide induced the formation of an intermolecular disulfide bridge between cysteine residues in the leucine zipper site of c-Jun monomers, this same radical directed the covalent incorporation of stoichiometric amounts of glutathione to a single conserved cysteine residue in the DNA-binding site of the protein. We found that covalent dimerization of c-Jun apparently did not affect its DNA binding activity, whereas the formation of a mixed disulfide with glutathione correlated well with the inhibition of transcription factor binding to DNA. Furthermore, we provide experimental evidence that nitric oxide-induced S-glutathionylation and inhibition of c-Jun involves the formation of S-nitrosoglutathione. In conclusion, our results support the reversible formation of a mixed disulfide between glutathione and c-Jun as a potential mechanism by which nitrosative stress may be transduced into a functional response at the level of transcription.

  4. Ozone inhibits endothelial cell cyclooxygenase activity through formation of hydrogen peroxide

    SciTech Connect

    Madden, M.C.; Eling, T.E.; Friedman, M.

    1987-09-01

    We have previously demonstrated that a 2H exposure of cultured pulmonary endothelial cells to ozone (0.0-1.0 ppm) in-vitro resulted in a concentration-dependent reduction of endothelial prostacyclin production (90% decrease at the 1.0 ppm level). Ozone-exposed endothelial cells, incubated with 20 uM arachidonate, also demonstrated a significant inhibition of prostacyclin synthesis. To further examine the mechanisms of the inhibition of prostacyclin synthesis, bovine pulmonary endothelial cells were exposed to 1.0 ppm ozone for 2H. A significant decrease in prostacyclin synthesis was found within 5 min of exposure (77 +/- 36% of air-exposed control values, p less than 0.05). Endothelial prostacyclin synthesis returned to baseline levels by 12H after ozone exposure, a time point which was similar to the recovery time of unexposed endothelium treated with 0.5 uM acetylsalicylic acid. Incubation of endothelial cells, previously exposed to 1.0 ppm ozone for 2 hours, with 4 uM PGH2 resulted in restoration of essentially normal prostacyclin synthesis. When endothelial cells were co-incubated with catalase (5 U/ml) during ozone exposure, no inhibition of prostacyclin synthesis was observed. Co-incubation with either heat-inactivated catalase or superoxide dismutase (10 U/ml) did not affect the ozone-induced inhibition of prostacyclin synthesis. These data suggest that H/sub 2/O/sub 2/ is a major toxic species produced in endothelial cells during ozone exposure and responsible for the inhibition of endothelial cyclooxygenase activity.

  5. Ozone inhibits endothelial cell cyclooxygenase activity through formation of hydrogen peroxide.

    PubMed

    Madden, M C; Eling, T E; Friedman, M

    1987-09-01

    We have previously demonstrated that a 2H exposure of cultured pulmonary endothelial cells to ozone (0.0-1.0 ppm) in-vitro resulted in a concentration-dependent reduction of endothelial prostacyclin production (90% decrease at the 1.0 ppm level). Ozone-exposed endothelial cells, incubated with 20 uM arachidonate, also demonstrated a significant inhibition of prostacyclin synthesis. To further examine the mechanisms of the inhibition of prostacyclin synthesis, bovine pulmonary endothelial cells were exposed to 1.0 ppm ozone for 2H. A significant decrease in prostacyclin synthesis was found within 5 min of exposure (77 +/- 36% of air-exposed control values, p less than 0.05). Endothelial prostacyclin synthesis returned to baseline levels by 12H after ozone exposure, a time point which was similar to the recovery time of unexposed endothelium treated with 0.5 uM acetylsalicylic acid. Incubation of endothelial cells, previously exposed to 1.0 ppm ozone for 2 hours, with 4 uM PGH2 resulted in restoration of essentially normal prostacyclin synthesis. When endothelial cells were co-incubated with catalase (5 U/ml) during ozone exposure, no inhibition of prostacyclin synthesis was observed. Co-incubation with either heat-inactivated catalase or superoxide dismutase (10 U/ml) did not affect the ozone-induced inhibition of prostacyclin synthesis. These data suggest that H2O2 is a major toxic species produced in endothelial cells during ozone exposure and responsible for the inhibition of endothelial cyclooxygenase activity.

  6. Effect of chronic renal medullary nitric oxide inhibition on blood pressure.

    PubMed

    Mattson, D L; Lu, S; Nakanishi, K; Papanek, P E; Cowley, A W

    1994-05-01

    The effects of chronic nitric oxide inhibition in the renal medulla on renal cortical and medullary blood flow, sodium balance, and blood pressure were evaluated in conscious uninephrectomized Sprague-Dawley rats. During a 5-day renal medullary interstitial infusion of the nitric oxide inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 120 micrograms/h) in saline (0.5 ml/min), renal medullary blood flow was selectively decreased by 30% after 2 h and was maintained at that level for the entire infusion. The decrease in medullary blood flow was associated with sodium retention and increased blood pressure. After the cessation of L-NAME infusion, medullary blood flow returned to control, and the sodium balance became negative as blood pressure returned to baseline. These data indicate that renal medullary nitric oxide plays an important role in the regulation of renal blood flow, sodium excretion, and blood pressure.

  7. CCN1 acutely increases nitric oxide production via integrin αvβ3-Akt-S6K-phosphorylation of endothelial nitric oxide synthase at the serine 1177 signaling axis.

    PubMed

    Hwang, Soojin; Lee, Hyeon-Ju; Kim, Gyungah; Won, Kyung-Jong; Park, Yoon Shin; Jo, Inho

    2015-12-01

    Although CCN1 (also known as cysteine-rich, angiogenic inducer 61, CYR61) has been reported to promote angiogenesis and neovascularization in endothelial cells (ECs), its effects on endothelial nitric oxide (NO) production have never been studied. Using human umbilical vein ECs, we investigated whether and how CCN1 regulates NO production. CCN1 acutely increased NO production in a time- and dose-dependent manner, which was accompanied by increased phosphorylation of endothelial NO synthase (eNOS) at serine 1177 (eNOS-Ser(1177)), but not that of eNOS-Thr(495) or eNOS-Ser(114). The level of total eNOS expression was unaltered. Treatment with either LY294002, a selective inhibitor of phosphoinositide 3-kinase known as an upstream kinase of Akt, or H-89, an inhibitor of protein kinase A, mitogen- and stress-activated protein kinase 1, Rho-associated protein kinase 2, and ribosomal protein S6 kinase (S6K), inhibited CCN1-stimulated eNOS-Ser(1177) phosphorylation and subsequent NO production. Ectopic expression of small interfering RNA against Akt and S6K significantly inhibited the effects of CCN1. Consistently, CCN1 increased the phosphorylation of Akt-Ser(473) and S6K-Thr(389). However, CCN1 did not alter the expression or secretion of VEGF, a known downstream factor of CCN1 and a potential upstream factor of Akt-mediated eNOS-Ser(1177) phosphorylation. Furthermore, neutralization of integrin αvβ3 with corresponding antibody completely reversed all of the observed effects of CCN1. Moreover, CCN1 increased acetylcholine-induced relaxation in the rat aortas. Finally, we also found that CCN1-stimulated eNOS-Ser(1177) phosphorylation and NO production are true for other types of EC tested. In conclusion, CCN1 acutely increases NO production via activation of a signaling axis in integrin αvβ3-Akt-S6K-eNOS-Ser(1177) phosphorylation, suggesting an important role for CCN1 in vasodilation.

  8. Synthesis, nitric oxide release, and α-glucosidase inhibition of nitric oxide donating apigenin and chrysin derivatives.

    PubMed

    Wang, Qi-Qin; Cheng, Ning; Yi, Wen-Bing; Peng, Sheng-Ming; Zou, Xiao-Qing

    2014-03-01

    α-Glucosidase (AG) play crucial roles in the digestion of carbohydrates. Inhibitors of α-glucosidase (AGIs) are promising candidates for the development of anti-diabetic drugs. Here, five series of apigenin and chrysin nitric oxide (NO)-donating derivatives were synthesised and evaluated for their AG inhibitory activity and NO releasing capacity in vitro. Except for 9a-c, twelve compounds showed remarkable inhibitory activity against α-glucosidase, with potency being better than that of acarbose and 1-deoxynojirimycin. All organic nitrate derivatives released low concentrations of NO in the presence of l-cysteine. Structure activity relationship studies indicated that 5-OH, hydrophobic coupling chain, and carbonyl groups of the coupling chain could enhance the inhibitory activity. Apigenin and chrysin derivatives therefore represents a new class of promising compounds that can inhibit α-glucosidase activity and supply moderate NO for preventing the development of diabetic complications.

  9. Sulfhydryl angiotensin-converting enzyme inhibitor promotes endothelial cell survival through nitric-oxide synthase, fibroblast growth factor-2, and telomerase cross-talk.

    PubMed

    Donnini, Sandra; Terzuoli, Erika; Ziche, Marina; Morbidelli, Lucia

    2010-03-01

    The protective effect exerted by angiotensin-converting enzyme inhibitors (ACEI) in cardiovascular diseases caused by endothelial injury and aging has been attributed to the restoration of endothelial cell functions. Recently, we demonstrated a central role of the fibroblast growth factor-2 (FGF-2)/FGF receptor-1 system in mediating the acquisition of an angiogenic phenotype in coronary microvascular endothelium exposed to ACEI. Here, we report on the rescuing effect of ACEI on impaired endothelium and the intracellular signaling mechanisms that lead endothelial cells to enter apoptosis and to senesce. Conditions mimicking pathological cell damage (serum deprivation) lead to endothelial apoptosis as evidenced by increased caspase-3 activity. ACEI enhanced cell survival through activation of prosurvival and antiaging signals involving Akt phosphorylation, endothelial nitric-oxide synthase (eNOS) expression and activation, FGF-2 and telomerase catalytic subunit (TERT) up-regulation, and delayed senescence. In microvascular endothelial cells exposed to ACEI, Akt/eNOS pathway-dependent FGF-2 was necessary for gene transcription of TERT. These protective effects were particularly evident for sulfhydryl-containing ACEI (zofenoprilat), which were reported to exhibit potent antioxidant effects. In conclusion, ACEI with antioxidant properties up-regulate eNOS, FGF-2, and TERT mRNA, which favor endothelial cell survival and prolong their lifespan, thus restoring endothelial cell functions after vascular damage. These effects could explain the beneficial effects of these drugs in various cardiovascular diseases associated with endothelial injury and aging.

  10. β-amyloid decreases detectable endothelial nitric oxide synthase in human erythrocytes: a role for membrane acetylcholinesterase.

    PubMed

    Misiti, Francesco; Carelli-Alinovi, Cristiana; Sampaolese, Beatrice; Giardina, Bruno

    2012-08-01

    Until few years ago, many studies of Alzheimer's disease investigated the effects of this syndrome in the central nervous system. Only recently, the detection of amyloid beta peptide (Aβ) in the blood has evidenced the necessity to extend studies on extraneuronal cells, particularly on erythrocytes. Aβ is also present in brain capillaries, where it interacts with the erythrocytes, inducing several metabolic and functional alterations. Recently, functionally active endothelial type nitric oxide synthase (eNOS) was discovered in human erythrocytes. The goal of the present study was to evidence the effect of Aβ on erythrocyte eNOS. We found that Aβ following to 24-h exposure causes a decrease in the immune staining of erythrocyte eNOS. Concurrently, Aβ alters erythrocyte cell morphology, decreases nitrites and nitrates levels, and affects membrane acetylcholinesterase activity. Propidium, an acetylcholinesterase inhibitor, was able to reverse the effects elicited by Aβ. These events could contribute to the vascular alterations associated with Alzheimer's disease disease.

  11. Could thiamine pyrophosphate be a regulator of the nitric oxide synthesis in the endothelial cell of diabetic patients?

    PubMed

    Alcázar-Leyva, Susana; Alvarado-Vásquez, Noé

    2011-05-01

    Thiamine (Vitamin B1) is considered an essential micronutrient for humans; its deficient intake brings about the Wernicke-Korsakoff syndrome (encephalopathy and psychosis) or beriberi (a neurological and cardiovascular disease). Once thiamine enters the cells it is phosphorylated by thiamine pyrophosphokinase (TPPK), and converted into the coenzyme thiamine pyrophosphate (TPP), the active form of thiamine. TPP is a relevant cofactor for transketolase (TK), α-ketoglutarate dehydrogenase (αKDH), and pyruvate dehydrogenase (PDH), all these enzymes are fundamental for glucose metabolism. Diabetes mellitus (DM), however, is considered both a deficient thiamine and deficient energy state, as a consequence of the limited TPP synthesis. Recent evidences have shown that the administration of thiamine or lipid-soluble derivatives, such as benfotiamine (developed to improve the bioavailability of thiamine), has positive effects in the diabetic patient (after thiamine is transformed into TPP). For this reason, administration of supplements with TPP in the diabetic patients is recommended to avoid complications, like neuropathy and nephropathy. It has been suggested that these beneficial effects are a consequence of the activation of TK (pentose pathway) or the PDH complex in mitochondria. Nitric oxide (NO) is synthesized by the endothelial cell and is also an important element for the viability and functionality of this cell type. However, in the DM patient, a deficient synthesis of NO has been reported. It is relevant to mention that recent evidences have led to propose mitochondrial activity as an important regulator of nitric oxide synthesis (ON). We consider that the exogenous administration of TPP facilitates the utilization of this molecule, regulating some metabolic processes such as phosphorylation of thiamine by TPPK, energy consumption (ATP), as well as mitochondrial activity, inducing eventually NO synthesis. If this is confirmed, the administration of TPP to the

  12. Folic Acid Promotes Recycling of Tetrahydrobiopterin and Protects Against Hypoxia-Induced Pulmonary Hypertension by Recoupling Endothelial Nitric Oxide Synthase

    PubMed Central

    Chalupsky, Karel; Kračun, Damir; Kanchev, Ivan; Bertram, Katharina

    2015-01-01

    Abstract Aims: Nitric oxide (NO) derived from endothelial NO synthase (eNOS) has been implicated in the adaptive response to hypoxia. An imbalance between 5,6,7,8-tetrahydrobiopterin (BH4) and 7,8-dihydrobiopterin (BH2) can result in eNOS uncoupling and the generation of superoxide instead of NO. Dihydrofolate reductase (DHFR) can recycle BH2 to BH4, leading to eNOS recoupling. However, the role of DHFR and eNOS recoupling in the response to hypoxia is not well understood. We hypothesized that increasing the capacity to recycle BH4 from BH2 would improve NO bioavailability as well as pulmonary vascular remodeling (PVR) and right ventricular hypertrophy (RVH) as indicators of pulmonary hypertension (PH) under hypoxic conditions. Results: In human pulmonary artery endothelial cells and murine pulmonary arteries exposed to hypoxia, eNOS was uncoupled as indicated by reduced superoxide production in the presence of the nitric oxide synthase inhibitor, L-(G)-nitro-L-arginine methyl ester (L-NAME). Concomitantly, NO levels, BH4 availability, and expression of DHFR were diminished under hypoxia. Application of folic acid (FA) restored DHFR levels, NO bioavailability, and BH4 levels under hypoxia. Importantly, FA prevented the development of hypoxia-induced PVR, right ventricular pressure increase, and RVH. Innovation: FA-induced upregulation of DHFR recouples eNOS under hypoxia by improving BH4 recycling, thus preventing hypoxia-induced PH. Conclusion: FA might serve as a novel therapeutic option combating PH. Antioxid. Redox Signal. 23, 1076–1091. PMID:26414244

  13. Endothelial Cell-Derived Nitric Oxide Mobilization is Attenuated in Copper-Deficient Rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The attenuation of endothelium-dependent nitric oxide (NO)-mediated vasodilation is a consistent finding in both conduit and resistance vessels during dietary Cu deficiency. While the effect is well established, evidence for the mechanism is still circumstantial. This study was designed to determine...

  14. Nitric Oxide Donor Molsidomine Positively Modulates Myogenic Differentiation of Embryonic Endothelial Progenitors

    PubMed Central

    Tirone, Mario; Conti, Valentina; Manenti, Fabio; Nicolosi, Pier Andrea; D’Orlando, Cristina; Azzoni, Emanuele

    2016-01-01

    Embryonic VE-Cadherin-expressing progenitors (eVE-Cad+), including hemogenic endothelium, have been shown to generate hematopoietic stem cells and a variety of other progenitors, including mesoangioblasts, or MABs. MABs are vessel-associated progenitors with multilineage mesodermal differentiation potential that can physiologically contribute to skeletal muscle development and regeneration, and have been used in an ex vivo cell therapy setting for the treatment of muscular dystrophy. There is currently a therapeutic need for molecules that could improve the efficacy of cell therapy protocols; one such good candidate is nitric oxide. Several studies in animal models of muscle dystrophy have demonstrated that nitric oxide donors provide several beneficial effects, including modulation of the activity of endogenous cell populations involved in muscle repair and the delay of muscle degeneration. Here we used a genetic lineage tracing approach to investigate whether the therapeutic effect of nitric oxide in muscle repair could derive from an improvement in the myogenic differentiation of eVE-Cad+ progenitors during embryogenesis. We show that early in vivo treatment with the nitric oxide donor molsidomine enhances eVE-Cad+ contribution to embryonic and fetal myogenesis, and that this effect could originate from a modulation of the properties of yolk sac hemogenic endothelium. PMID:27760216

  15. Endothelial repair in stented arteries is accelerated by inhibition of Rho-associated protein kinase

    PubMed Central

    Hsiao, Sarah T.; Spencer, Tim; Boldock, Luke; Prosseda, Svenja Dannewitz; Xanthis, Ioannis; Tovar-Lopez, Francesco J.; Van Beusekom, Heleen M. M.; Khamis, Ramzi Y; Foin, Nicolas; Bowden, Neil; Hussain, Adil; Rothman, Alex; Ridger, Victoria; Halliday, Ian; Perrault, Cecile; Gunn, Julian; Evans, Paul C.

    2016-01-01

    Aims Stent deployment causes endothelial cells (EC) denudation, which promotes in-stent restenosis and thrombosis. Thus endothelial regrowth in stented arteries is an important therapeutic goal. Stent struts modify local hemodynamics, however the effects of flow perturbation on EC injury and repair are incompletely understood. By studying the effects of stent struts on flow and EC migration, we identified an intervention that promotes endothelial repair in stented arteries. Methods and Results In vitro and in vivo models were developed to monitor endothelialization under flow and the influence of stent struts. A 2D parallel-plate flow chamber with 100 μm ridges arranged perpendicular to the flow was used. Live cell imaging coupled to computational fluid dynamic simulations revealed that EC migrate in the direction of flow upstream from the ridges but subsequently accumulate downstream from ridges at sites of bidirectional flow. The mechanism of EC trapping by bidirectional flow involved reduced migratory polarity associated with altered actin dynamics. Inhibition of Rho-associated protein kinase (ROCK) enhanced endothelialization of ridged surfaces by promoting migratory polarity under bidirectional flow (P < 0.01). To more closely mimic the in vivo situation, we cultured EC on the inner surface of polydimethylsiloxane tubing containing Coroflex Blue stents (65 μm struts) and monitored migration. ROCK inhibition significantly enhanced EC accumulation downstream from struts under flow (P < 0.05). We investigated the effects of ROCK inhibition on re-endothelialization in vivo using a porcine model of EC denudation and stent placement. En face staining and confocal microscopy revealed that inhibition of ROCK using fasudil (30 mg/day via osmotic minipump) significantly increased re-endothelialization of stented carotid arteries (P < 0.05). Conclusions Stent struts delay endothelial repair by generating localized bidirectional flow which traps migrating EC. ROCK

  16. Matrix metalloproteinase 13 mediates nitric oxide activation of endothelial cell migration

    PubMed Central

    López-Rivera, Esther; Lizarbe, Tania R.; Martínez-Moreno, Mónica; López-Novoa, José Miguel; Rodríguez-Barbero, Alicia; Rodrigo, José; Fernández, Ana Patricia; Álvarez-Barrientos, Alberto; Lamas, Santiago; Zaragoza, Carlos

    2005-01-01

    To explore the mechanisms by which NO elicits endothelial cell (EC) migration we used murine and bovine aortic ECs in an in vitro wound-healing model. We found that exogenous or endogenous NO stimulated EC migration. Moreover, migration was significantly delayed in ECs derived from endothelial NO synthase-deficient mice compared with WT murine aortic EC. To assess the contribution of matrix metalloproteinase (MMP)-13 to NO-mediated EC migration, we used RNA interference to silence MMP-13 expression in ECs. Migration was delayed in cells in which MMP-13 was silenced. In untreated cells MMP-13 was localized to caveolae, forming a complex with caveolin-1. Stimulation with NO disrupted this complex and significantly increased extracellular MMP-13 abundance, leading to collagen breakdown. Our findings show that MMP-13 is an important effector of NO-activated endothelial migration. PMID:15728377

  17. Resveratrol increases nitric oxide synthase, induces accumulation of p53 and p21(WAF1/CIP1), and suppresses cultured bovine pulmonary artery endothelial cell proliferation by perturbing progression through S and G2.

    PubMed

    Hsieh, T C; Juan, G; Darzynkiewicz, Z; Wu, J M

    1999-06-01

    Epidemiological studies have shown that the regular consumption of red wine may in part account for the apparent compatibility of a high fat diet with a low incidence of coronary atherosclerosis. This phenomenon, commonly referred to as the French paradox, may be associated with red wine constituents that exhibit tumor-preventive properties as well as inhibit reactions that increase the risk of coronary heart disease. Here we show that resveratrol, a polyphenol in red wine, induces nitric oxide synthase, the enzyme responsible for the biosynthesis of NO, in cultured pulmonary artery endothelial cells, suggesting that resveratrol could afford cardioprotection by affecting the expression of nitric oxide synthase. We also show that resveratrol inhibits the proliferation of pulmonary artery endothelial cells, which, based on flow cytometric analysis, correlates with the suppression of cell progression through S and G2 phases of the cell cycle. Western blot analysis and immunocytochemical protein detection combined with multiparameter flow cytometry further demonstrate that the perturbed progression through S and G2 phases is accompanied by an increase in the expression of tumor suppressor gene protein p53 and elevation of the level of cyclin-dependent kinase inhibitor p21(WAF1/CIP1). All of the observed effects of resveratrol, including induction of apoptosis at its higher concentration, are also compatible with its putative chemopreventive and/or antitumor activity.

  18. Alteration of syncytiotrophoblast mitochondria function and endothelial nitric oxide synthase expression in the placenta of rural residents.

    PubMed

    Rivero Osimani, Valeria L; Valdez, Susana R; Guiñazú, Natalia; Magnarelli, Gladis

    2016-06-01

    The impact of environmental organophosphate (OP) pesticide exposure on respiratory complexes, enzymatic antioxidant defense activities, and oxidative damage markers in the syncytiotrophoblast and cytotrophoblast mitochondria was evaluated. Placental progesterone (PG) levels and endothelial nitric oxide synthase (eNOS) expression were studied. Samples from women non-exposed (control group-CG) and women living in a rural area (rural group-RG) were collected during pesticide spraying season (RG-SS) and non-spraying season (RG-NSS). In RG-SS, the exposure biomarker placental carboxylesterase decreased and syncytiotrophoblast cytochrome c oxidase activity increased, while 4-hydroxynonenal levels decreased. PG levels decreased in RG-SS and in the RG. Nitric oxide synthase expression decreased in RG, RG-SS and RG-NSS. No significant changes in mitochondrial antioxidant enzyme activities were found. These results suggest that the alteration of syncytiotrophoblast mitochondrial complex IV activity and steroidogenic function may be associated to pesticide exposure. Reduction in placental PG and eNOS expression may account for low newborn weight in RG.

  19. Effect of high soy diet on the cerebrovasculature and endothelial nitric oxide synthase in the ovariectomized rat

    PubMed Central

    Schreihofer, Derek A.; Deutsch, Christian; Lovekamp-Swan, Tara; Sullivan, Jennifer C.; Dorrance, Anne M.

    2010-01-01

    High soy (HS) diets are neuroprotective and promote vascular dilatation in the periphery. We hypothesized that a HS diet would promote vascular dilatation in the cerebrovasculature by mimicking estradiol's actions on the endothelial nitric oxide synthase (eNOS) system including increasing eNOS expression and decreasing caveolin-1 expression to increase nitric oxide (NO) production. Ovariectomized rats were fed HS or a soy-free diet (SF) ± low physiological estradiol (E2) for 4 weeks. Neither E2 nor HS altered middle cerebral artery (MCA) structure or vascular responses to acetylcholine, serotonin, or phenylephrine. Estradiol enhanced bradykinin-induced relaxation in an eNOS-dependent manner. Although E2 and HS increased eNOS mRNA expression in the brain and cerebrovasculature, they had no effect on eNOS protein expression or phosphorylation in the MCA. However, E2 decreased caveolin-1 protein in the MCA. In MCAs neither E2 nor HS altered estrogen receptor (ER) alpha expression, but E2 did reduce ER beta levels. These data suggest that HS diets have no effect on vascular NO production, and that E2 may modulate basal NO production by reducing the expression of caveolin-1, an allosteric inhibitor of NOS activity. However, the effects of E2 and HS on the cerebrovasculature are small and may not underlie their protective actions in pathological states. PMID:20197113

  20. The volatile oil of Nardostachyos Radix et Rhizoma induces endothelial nitric oxide synthase activity in HUVEC cells.

    PubMed

    Maiwulanjiang, Maitinuer; Bi, Cathy W C; Lee, Pinky S C; Xin, Guizhong; Miernisha, Abudureyimu; Lau, Kei M; Xiong, Aizhen; Li, Ning; Dong, Tina T X; Aisa, Haji A; Tsim, Karl W K

    2015-01-01

    Nardostahyos Radix et Rhizoma (NRR; the root and rhizome of Nardostachys jatamansi DC.) is a widely used medicinal herb. Historically, NRR is being used for the treatment of cardiovascular and neurological diseases. To search for active ingredients of NRR, we investigated the vascular benefit of NRR volatile oil in (i) the vasodilation in rat aorta ring, and (ii) the release of nitric oxide (NO) and the phosphorylation of endothelial NO synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVECs). By measuring the fluorescence signal in cultures, application of NRR volatile oil resulted in a rapid activation of NO release as well as the phosphorylation of eNOS: both inductions were markedly reduced by L-NAME. In parallel, the phosphorylation level of Akt kinase was markedly increased by the oil treatment, which was partially attenuated by PI3K/Akt inhibitor LY294002. This inhibitor also blocked the NRR-induced NO production and eNOS phosphorylation. In HUVECs, application of NRR volatile oil elevated the intracellular Ca(2+) level, and BAPTA-AM, a Ca(2+) chelator, reduced the Ca(2+) surge: the blockage were also applied to NRR-induced eNOS phosphorylation and NO production. These findings suggested the volatile oil of NRR was the major ingredient in triggering the vascular dilatation, and which was mediated via the NO production.

  1. The Volatile Oil of Nardostachyos Radix et Rhizoma Induces Endothelial Nitric Oxide Synthase Activity in HUVEC Cells

    PubMed Central

    Maiwulanjiang, Maitinuer; Bi, Cathy W. C.; Lee, Pinky S. C.; Xin, Guizhong; Miernisha, Abudureyimu; Lau, Kei M.; Xiong, Aizhen; Li, Ning; Dong, Tina T. X.; Aisa, Haji A.; Tsim, Karl W. K.

    2015-01-01

    Nardostahyos Radix et Rhizoma (NRR; the root and rhizome of Nardostachys jatamansi DC.) is a widely used medicinal herb. Historically, NRR is being used for the treatment of cardiovascular and neurological diseases. To search for active ingredients of NRR, we investigated the vascular benefit of NRR volatile oil in (i) the vasodilation in rat aorta ring, and (ii) the release of nitric oxide (NO) and the phosphorylation of endothelial NO synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVECs). By measuring the fluorescence signal in cultures, application of NRR volatile oil resulted in a rapid activation of NO release as well as the phosphorylation of eNOS: both inductions were markedly reduced by L-NAME. In parallel, the phosphorylation level of Akt kinase was markedly increased by the oil treatment, which was partially attenuated by PI3K/Akt inhibitor LY294002. This inhibitor also blocked the NRR-induced NO production and eNOS phosphorylation. In HUVECs, application of NRR volatile oil elevated the intracellular Ca2+ level, and BAPTA-AM, a Ca2+ chelator, reduced the Ca2+ surge: the blockage were also applied to NRR-induced eNOS phosphorylation and NO production. These findings suggested the volatile oil of NRR was the major ingredient in triggering the vascular dilatation, and which was mediated via the NO production. PMID:25643147

  2. Nelfinavir Suppresses Insulin Signaling and Nitric Oxide Production by Human Aortic Endothelial Cells: Protective Effects of Thiazolidinediones

    PubMed Central

    Mondal, Debasis; Liu, Kai; Hamblin, Milton; Lasky, Joseph A.; Agrawal, Krishna C.

    2013-01-01

    ABSTRACT Background In human immunodeficiency virus 1 (HIV-1)–infected individuals, exposure to a protease inhibitor (PI)-based highly active antiretroviral therapy (HAART) regimen increases cardiovascular disease and endothelial dysfunction. However, the mechanisms of PI-induced effects on endothelial cells (ECs) are not known. Furthermore, strategies to suppress these deleterious outcomes of PIs need to be developed. Insulin-induced PI3K/Akt signaling and endothelial nitric oxide (NO)-synthase (eNOS) phosphorylation regulates NO production by ECs that maintain vascular homeostasis. We evaluated whether nelfinavir (NEL), a potent HIV-1 PI that suppresses Akt phosphorylation, can alter insulin-induced NO production in human aortic endothelial cells (HAECs) and whether insulin sensitization of HAECs via the peroxisome proliferator-activated receptor-gamma agonists, thiazolidinediones, can ameliorate these side effects. Methods Real-time NO production in HAECs was monitored by fluorimetric dyes DAF-FM DA and DAF-2 DA. Immunodetection studies were used to determine the phosphorylation of Akt, eNOS, insulin receptor-β (IR-β), insulin receptor substrate-1 (IRS-1), and PI3K/p85α. Expression of eNOS messenger RNA was measured by reverse transcription polymerase chain reaction. Results In vitro exposure (72 hours) of HAECs to NEL (0.25-2 μg/mL) decreased both basal (2.5-fold) and insulin-induced NO production (4- to 5-fold). NEL suppressed insulin-induced phosphorylation of both Akt and eNOS at serine residues 473 and 1177, respectively. NEL decreased tyrosine phosphorylation of IR-β, IRS-1, and PI3K. Coexposure to troglitazone (TRO; 250 nM) ameliorated the suppressive effects of NEL on insulin signaling and NO production. Coexposure to TRO also increased eNOS expression in NEL-treated HAECs. Conclusion Our findings indicate that treatment with potent insulin sensitizers may protect against PI-mediated endothelial dysfunction during long-term HAART. PMID:23533049

  3. Imeglimin prevents human endothelial cell death by inhibiting mitochondrial permeability transition without inhibiting mitochondrial respiration

    PubMed Central

    Detaille, D; Vial, G; Borel, A-L; Cottet-Rouselle, C; Hallakou-Bozec, S; Bolze, S; Fouqueray, P; Fontaine, E

    2016-01-01

    Imeglimin is the first in a new class of oral glucose-lowering agents, having recently completed its phase 2b trial. As Imeglimin did show a full prevention of β-cell apoptosis, and since angiopathy represents a major complication of diabetes, we studied Imeglimin protective effects on hyperglycemia-induced death of human endothelial cells (HMEC-1). These cells were incubated in several oxidative stress environments (exposure to high glucose and oxidizing agent tert-butylhydroperoxide) which led to mitochondrial permeability transition pore (PTP) opening, cytochrome c release and cell death. These events were fully prevented by Imeglimin treatment. This protective effect on cell death occurred without any effect on oxygen consumption rate, on lactate production and on cytosolic redox or phosphate potentials. Imeglimin also dramatically decreased reactive oxygen species production, inhibiting specifically reverse electron transfer through complex I. We conclude that Imeglimin prevents hyperglycemia-induced cell death in HMEC-1 through inhibition of PTP opening without inhibiting mitochondrial respiration nor affecting cellular energy status. Considering the high prevalence of macrovascular and microvascular complications in type 2 diabetic subjects, these results together suggest a potential benefit of Imeglimin in diabetic angiopathy. PMID:27551496

  4. Structural Basis for Isoform-Selective Inhibition in Nitric Oxide Synthase

    PubMed Central

    LI, HUIYING

    2013-01-01

    CONSPECTUS Nitric oxide synthase (NOS) converts L-arginine into L-citrulline and releases the important signaling molecule nitric oxide (NO). In the cardiovascular system NO produced by endothelial NOS (eNOS) relaxes smooth muscle which controls vascular tone and blood pressure.Neuronal NOS (nNOS) produces NO in the brain, where it influences a variety of neural functions such as neural transmitter release. NO can also support immune system, serving as a cytotoxic agent during infections. Even with all of these important functions, NO is a free radical, and, when overproduced, it can cause tissue damage. This mechanism can operate in many neurodegenerative diseases, and as a result, the development of drugs targeting nNOS is a desirable therapeutic goal. However, the active sites of all 3 human isoforms are very similar, and designing inhibitors specific for nNOS is a challenging problem. It is critically important, for example, not to inhibit eNOS owing to its central role in controlling blood pressure. In this Account we summarize our efforts in collaboration with Rick Silverman at Northwestern University to develop drug candidates that specifically target NOS using crystallography, computational chemistry, and organic synthesis. As a result we have developed aminopyridine compounds that are 3,800 fold more selective for nNOS than eNOS, some of which show excellent neuro-protective effects in animal models. Our group has solved approximately 130 NOS-inhibitor crystal structures which have provided the structural basis for our design efforts. Initial crystal structures of nNOS and eNOS bound to selective dipeptide inhibitors showed that a single amino acid difference (Asp in nNOS and Asn in eNOS) results in much tighter binding to nNOS. The NOS active site is open and rigid, which produces few large structural changes when inhibitors bind. However, we have found that relatively small changes in the active site and inhibitor chirality can account for large

  5. Increased activity of guanylate cyclase in the atherosclerotic rabbit aorta: role of non-endothelial nitric oxide synthases.

    PubMed Central

    Rupin, A.; Behr, D.; Verbeuren, T. J.

    1996-01-01

    1. Experiments were performed to examine the effects of putative non-endothelial nitric oxide on the soluble guanylate cyclase activity of severe atherosclerotic aortae from hypercholesterolaemic rabbits fed a cholesterol rich diet for 45 weeks. 2. The guanosine 3':5'-cyclic monophosphate (cyclic GMP) content of aortae from rabbits fed either a control diet or a diet containing 0.3% cholesterol for 45 weeks was quantified in saline extracts or in trichloracetic acid/either extracts by use of a competitive immunoenzymatic assay. Rabbit anti-cyclic GMP immunoglobulin G was covalently linked to the solid phase, in order to avoid false positive results due to high rabbit immunoglobulin G concentrations in the atherosclerotic saline extracts. 3. Saline extracts of atherosclerotic aortae which were harvested immediately after death (intact aortae) contained about 6 fold more cyclic GMP than control aortae when expressed in pmol cyclic GMP mg-1 protein. The cyclic GMP concentrations in trichloracetic acid/ether extracts of atherosclerotic and control aortae expressed in pmol mg-1 fresh tissue were not significantly different. 4. Neointimal-medial explants from atherosclerotic and control aortae were placed in a physiological saline solution and incubated at 37 degrees C for six hours in an incubator gassed with 5% CO2. Before the incubation, the cyclic GMP concentrations in saline extracts of atherosclerotic explants (0.74 +/- 0.27 pmol mg-1) were found to be 17 fold higher than those of control explants (0.043 +/- 0.008 pmol mg-1). The cyclic GMP content of control explants decreased significantly after 6 h of incubation, while that of atherosclerotic explants remained elevated. 5. Chronic administration of NG-nitro-L-arginine methyl ester, a non selective inhibitor of nitric oxide synthases, at 12 mg kg-1 day-1 subcutaneously for one month did not reduce the cyclic GMP concentration of intact atherosclerotic aortae, while that of intact aortae from control rabbits

  6. Overexpression of endothelial nitric oxide synthase improves endothelium-dependent vasodilation in arteries infused with helper-dependent adenovirus.

    PubMed

    Jiang, Bo; Du, Liang; Flynn, Rowan; Dronadula, Nagadhara; Zhang, Jingwan; Kim, Francis; Dichek, David

    2012-11-01

    Adenoviral vectors (Ad) are useful tools for in vivo gene transfer into endothelial cells. However, endothelium-dependent vasodilation is impaired after Ad infusion, and this impairment is not prevented by use of advanced-generation "helper-dependent" (HD) Ad that lack all viral genes. We hypothesized that endothelium-dependent vasodilation could be improved in Ad-infused arteries by overexpression of endothelial nitric oxide synthase (eNOS). We tested this hypothesis in hyperlipidemic, atherosclerosis-prone rabbits because HDAd will likely be used for treating and preventing atherosclerosis. Moreover, the consequences of eNOS overexpression might differ in normal and atherosclerosis-prone arteries and could include atherogenic effects, as reported in transgenic mice. We cloned rabbit eNOS and constructed an HDAd that expresses it. HDAdeNOS increased NO production by cultured endothelial cells and increased arterial eNOS mRNA in vivo by ∼10-fold. Compared to arteries infused with a control HDAd, HDAdeNOS-infused arteries of hyperlipidemic rabbits had significantly improved endothelium-dependent vasodilation, and similar responses to phenylephrine and nitroprusside. Moreover, infusion of HDAdeNOS had local atheroprotective effects including large, significant decreases in intimal lipid accumulation and arterial tumor necrosis factor (TNF)-α expression (p≤0.04 for both). HDAdeNOS infusion yields a durable (≥2 weeks) increase in arterial eNOS expression, improves vasomotor function, and reduces artery wall inflammation and lipid accumulation. Addition of an eNOS expression cassette improves the performance of HDAd, has no harmful effects, and may reduce atherosclerotic lesion growth.

  7. Differential regulation of inducible and endothelial nitric oxide synthase by kinin B1 and B2 receptors

    PubMed Central

    Kuhr, F.; Lowry, J.; Zhang, Y.; Brovkovych, V.; Skidgel, R.A.

    2010-01-01

    Kinins are vasoactive peptides that play important roles in cardiovascular homeostasis, pain and inflammation. After release from their precursor kininogens, kinins or their C-terminal des-Arg metabolites activate two distinct G protein-coupled receptors (GPCR), called B2 (B2R) or B1 (B1R). The B2R is expressed constitutively with a wide tissue distribution. In contrast, the B1R is not expressed under normal conditions but is upregulated by tissue insult or inflammatory mediators. The B2R is considered to mediate many of the acute effects of kinins while the B1R is more responsible for chronic responses in inflammation. Both receptors can couple to Gαi and Gαq families of G proteins to release mediators such as nitric oxide (NO), arachidonic acid, prostaglandins, leukotrienes and endothelium derived hyperpolarizing factor and can induce the release of other inflammatory agents. The focus of this review is on the different transduction events that take place upon B2R and B1R activation in human endothelial cells that leads to generation of NO via activation of different NOS isoforms. Importantly, B2R-mediated eNOS activation leads to a transient (~ 5 min) output of NO in control endothelial cells whereas in cytokine-treated endothelial cells, B1R activation leads to very high and prolonged (~90 min) NO production that is mediated by a novel signal transduction pathway leading to post-translational activation of iNOS. PMID:20045558

  8. Evidence against a role of inducible nitric oxide synthase in the endothelial protective effects of delayed preconditioning

    PubMed Central

    Laude, Karine; Richard, Vincent; Henry, Jean-Paul; Lallemand, Françoise; Thuillez, Christian

    2000-01-01

    Preconditioning the heart with brief periods of ischaemia induces delayed endothelial protection against reperfusion injury, but the precise mechanisms involved in this endogenous protein are still unclear. Induction of the type II-nitric oxide synthase (iNOS) acts as a mediator of the preconditioning against myocardial infarction and stunning. The present study was designed to assess whether iNOS also contributes to the delayed endothelial protective effects of preconditioning. Rats were subjected to 20 min ischaemia followed by 60 min reperfusion 24 h after sham surgery or preconditioning (one cycle or 2 min ischaemia/5 min reperfusion and two cycles of 5 min ischaemia/5 min reperfusion). At the end of the reperfusion, coronary segments were removed distal to the site of occlusion and mounted in wire myographs. Ischaemia-reperfusion (I/R) decreased the endothelium-dependent relaxations to acetylcholine (maximal relaxations: sham, 66±5%; I/R, 40±1%; P<0.05) and this impairment was prevented by preconditioning (maximal relaxation: 61±6%). Administration of N-(3-aminomethyl)benzyl)acetaminide (1400W), a highly selective inhibitor for iNOS, 10 min before prolonged ischaemia did not modify the beneficial effect of preconditioning (maximal relaxation: 66±5%). These data show that preconditioning induces delayed protection against reperfusion-injury. However, in contrast to the myocytes, these endothelial protective effects of delayed preconditioning do not involve iNOS. PMID:10928956

  9. Direct, real-time measurement of shear stress-induced nitric oxide produced from endothelial cells in vitro.

    PubMed

    Andrews, Allison M; Jaron, Dov; Buerk, Donald G; Kirby, Patrick L; Barbee, Kenneth A

    2010-12-15

    Nitric oxide (NO) produced by the endothelium is involved in the regulation of vascular tone. Decreased NO production or availability has been linked to endothelial dysfunction in hypercholesterolemia and hypertension. Shear stress-induced NO release is a well-established phenomenon, yet the cellular mechanisms of this response are not completely understood. Experimental limitations have hindered direct, real-time measurements of NO under flow conditions. We have overcome these challenges with a new design for a parallel-plate flow chamber. The chamber consists of two compartments, separated by a Transwell® membrane, which isolates a NO recording electrode located in the upper compartment from flow effects. Endothelial cells are grown on the bottom of the membrane, which is inserted into the chamber flush with the upper plate. We demonstrate for the first time direct real-time NO measurements from endothelial cells with controlled variations in shear stress. Step changes in shear stress from 0.1 dyn/cm(2) to 6, 10, or 20 dyn/cm(2) elicited a transient decrease in NO followed by an increase to a new steady state. An analysis of NO transport suggests that the initial decrease is due to the increased removal rate by convection as flow increases. Furthermore, the rate at which the NO concentration approaches the new steady state is related to the time-dependent cellular response rather than transport limitations of the measurement configuration. Our design offers a method for studying the kinetics of the signaling mechanisms linking NO production with shear stress as well as pathological conditions involving changes in NO production or availability.

  10. Prostaglandin E2 reduces swine myocardial ischemia reperfusion injury via increased endothelial nitric oxide synthase and vascular endothelial growth factor expression levels

    PubMed Central

    Zhou, Ying; Yang, Peng; Li, Aili; Ye, Xiaojun; Ren, Shiyan; Li, Xianlun

    2017-01-01

    Prostaglandin E2 (PGE2) has been demonstrated to attenuate cardiac ischemia-reperfusion (I/R) injury. However, the underlying mechanism of PGE2 in cardiac I/R injury remains unknown. Upregulated expression levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) were reported in acute myocardial infarction (AMI), and were demonstrated to diminish I/R injury. In the current study the involvement of VEGF and eNOS in the myocardial protective effect of PGE2 were investigated in a catheter-based porcine model of AMI. Twenty-two Chinese miniature pigs were randomized into sham-surgery (n=6), control (n=8) and PGE2 (n=8) groups. PGE2 (1 µg/kg) was injected from 10 min prior to left anterior descending occlusion up to 1 h after reperfusion in the PGE2 group. Subsequently, the hemodynamic parameters were evaluated. Thioflavin-S and Evans Blue double staining were performed to evaluate the extent of the myocardial reperfusion area (RA) and no-reflow area (NRA). Immunohistochemical and western blot analysis were used to evaluate protein expression levels of VEGF and eNOS. Left ventricular (LV) systolic pressure significantly improved and LV end-diastolic pressure significantly decreased in the PGE2 group when compared with the control group 2 h after occlusion and 3 h after reperfusion (P<0.05, respectively). The RA and NRA were smaller in the PGE2 group than in the control group (P<0.05, respectively). Furthermore, PGE2 treatment increased the myocardial content of VEGF and eNOS when compared with the control group (P<0.05, respectively). Thus, the results of the present study demonstrate the cardio-protective mechanisms of PGE2, which may protect the heart from I/R injury via enhancement of VEGF and eNOS expression levels. PMID:28357071

  11. Hypoxia-inducible factor-1 modulates the expression of vascular endothelial growth factor and endothelial nitric oxide synthase induced by eccentric exercise.

    PubMed

    Rodriguez-Miguelez, Paula; Lima-Cabello, Elena; Martínez-Flórez, Susana; Almar, Mar; Cuevas, María J; González-Gallego, Javier

    2015-04-15

    The present study investigated the effects of acute and chronic eccentric exercise on the hypoxia-inducible factor (HIF)-1α activation response and the concomitant modulation of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression in rat skeletal muscle. Twenty-four male Wistar rats were randomly assigned to three experimental groups: rested control group, acutely exercised group after an intermittent downhill protocol for 90 min, and acutely exercise group with a previous eccentric training of 8 wk. HIF-1α activation, VEGF and eNOS gene expression, protein content, and promoter activation were assessed in vastus lateralis muscle biopsies. Acute eccentric exercise induced a marked activation of HIF-1α and resulted in increased VEGF and eNOS mRNA level and protein concentration. The binding of HIF-1α to the VEGF and eNOS promoters, measured by a chromatin immunoprecipitation assay, was undetectable in rested rats, whereas it was evident in acutely exercised animals. Acute exercise also increased myeloperoxidase, toll-like receptor-4, tumor necrosis factor-α, and interleukin-1β protein content, suggesting a contribution of proinflammatory stimuli to HIF-1α activation and VEGF overexpression. All of these effects were partially abolished by training. Moreover, training resulted in an increased capillary density. In summary, our findings indicate that eccentric exercise prompts an HIF-1α response in untrained skeletal muscle that contributes to the upregulation of VEGF and eNOS gene expression and is attenuated after an eccentric training program.

  12. Isolation and chromosomal localization of the human endothelial nitric oxide synthase (NOS3) gene

    SciTech Connect

    Robinson, L.J.; Michel, T.; Weremowicz, S.; Morton, C.C. )

    1994-01-15

    Endothelial NOS activity is a major determinant of vascular tone and blood pressure, and in several important (and sometimes hereditary) disease states, such as hypertension, diabetes, and atherosclerosis, the endothelial NO signaling system appears to be abnormal. To explore the relationship of the endothelial NOS activity, the authors isolated the human gene encoding the endothelial NOS. Genomic clones containing the 5[prime] end of this gene were identified in a human genomic library by applying a polymerase chain reaction (PCR)-based approach. Identification of the human gene for endothelial NOS (NOS3) was confirmed by nucleotide sequence analysis of the first coding exon, which was found to be identical to its cognate cDNA. The NOS3 gene spans at least 20 kb and appears to contain multiple introns. The transcription start site and promoter region of the NOS3 gene were identified by primer extension and ribonuclease protection assays. Sequencing of the putative promoter revealed consensus sequences for the shear stress-response element, as well as cytokine-responsive cis regulatory sequences, both possible important to the roles played by NOS3 in the normal and the diseased cardiovascular system. The authors also mapped the chromosomal location of the NOS3 gene. First, a chromosomal panel of human-rodent somatic cell hybrids was screened using PCR with oligonucleotide primers derived from the NOS3 genomic clone. The specificity of the amplified PCR product was confirmed by human and hamster genomic DNA controls, as well as by Southern blot analysis, using the NOS3 cDNA as probe. Definitive chromosomal assignment of the NOS3 gene to human chromosome 7 was based upon 0% discordancy; fluorescence in situ hybridization sublocalized the NOS3 gene to 7q36. The identification and characterization of the NOS3 gene may lead to further insights into heritable disease states associated with this gene product. 41 refs., 3 figs., 1 tab.

  13. Inhibition of astroglia-induced endothelial differentiation by inorganic lead: a role for protein kinase C.

    PubMed Central

    Laterra, J; Bressler, J P; Indurti, R R; Belloni-Olivi, L; Goldstein, G W

    1992-01-01

    Microvascular endothelial function in developing brain is particularly sensitive to lead toxicity, and it has been hypothesized that this results from the modulation of protein kinase C (PKC) by lead. We examined the effects of inorganic lead on an in vitro model of central nervous system endothelial differentiation in which astroglial cells induce central nervous system endothelial cells to form capillary-like structures. Capillary-like structure formation within C6 astroglial-endothelial cocultures was inhibited by lead acetate with 50% maximal inhibition at 0.5 microM total lead. Inhibition was independent of effects on cell viability or growth. Under conditions that inhibited capillary-like structure formation, we found that lead increased membrane-associated PKC in both C6 astroglial and endothelial cells. Prolonged exposure of C6 cells to 5 microM lead for up to 16 h resulted in a time-dependent increase in membranous PKC as determined by immunoblot analysis. Membranous PKC increased after 5-h exposures to as little as 50 nM lead and was maximal at approximately 1 microM. Phorbol esters were used to determine whether PKC modulation was causally related to the inhibition of endothelial differentiation by lead. Phorbol 12-myristate 13-acetate (10 nM) inhibited capillary-like structure formation by 65 +/- 5%, whereas 4 alpha-phorbol 12,13-didecanoate was without effect. These findings suggest that inorganic lead induces cerebral microvessel dysfunction by interfering with PKC modulation in microvascular endothelial or perivascular astroglial cells. Images PMID:1438272

  14. Nitric oxide regulates nitric oxide synthase-2 gene expression by inhibiting NF-kappaB binding to DNA.

    PubMed Central

    Park, S K; Lin, H L; Murphy, S

    1997-01-01

    Treatment of astroglial cells with interleukin 1beta and interferon gamma transcriptionally activates the nitric oxide synthase (NOS)-2 gene. The duration of mRNA expression is brief because of transcript instability. In addition, NO donors reduce the expression of NOS-2 mRNA dramatically by reducing the rate of transcription. In this study we observed that the NO donor, spermine NONOate did not inhibit the activation and translocation of NF-kappaB, a key transcription factor in the induction of NOS-2, but inhibited formation of the NF-kappaB-DNA complex. This effect was reversed by methaemoglobin (acting as an NO trap) and by the reducing agent dithiothreitol. Formation of the interferon-regulatory factor-DNA complex was unaffected by NO. These results suggest that NO can modulate its own production by interfering with NF-kappaB interaction with the promoter region of the NOS gene, a negative feedback effect that may be important for limiting NO production in vivo. PMID:9065784

  15. Ascorbate inhibits NADPH oxidase subunit p47phox expression in microvascular endothelial cells.

    PubMed

    Wu, Feng; Schuster, David P; Tyml, Karel; Wilson, John X

    2007-01-01

    The production of reactive oxygen species (ROS) is central to the etiology of endothelial dysfunction in sepsis. Endothelial cells respond to infection by activating NADPH oxidases that are sources of intracellular ROS and potential targets for therapeutic administration of antioxidants. Ascorbate is an antioxidant that accumulates in these cells and improves capillary blood flow, vascular reactivity, arterial blood pressure, and survival in experimental sepsis. Therefore, the present study tested the hypothesis that ascorbate regulates NADPH oxidases in microvascular endothelial cells exposed to septic insult. We observed that incubation with Escherichia coli lipopolysaccharide (LPS) and interferon-gamma (IFNgamma) increased NADPH oxidase activity and expression of the enzyme subunit p47phox in mouse microvascular endothelial cells of skeletal muscle origin. Pretreatment of the cells with ascorbate prevented these increases. Polyethylene glycol-conjugated catalase and selective inhibitors of Jak2 also abrogated induction of p47phox. Exogenous hydrogen peroxide induced p47phox expression that was prevented by pretreatment of the cells with ascorbate. LPS+IFNgamma or hydrogen peroxide activated the Jak2/Stat1/IRF1 pathway and this effect was also inhibited by ascorbate. In conclusion, ascorbate blocks the stimulation by septic insult of redox-sensitive Jak2/Stat1/IRF1 signaling, p47phox expression, and NADPH oxidase activity in microvascular endothelial cells. Because endothelial NADPH oxidases produce ROS that can cause endothelial dysfunction, their inhibition by ascorbate may represent a new strategy for sepsis therapy.

  16. Interferon-alpha and dexamethasone inhibit adhesion of T cells to endothelial cells and synovial cells

    PubMed Central

    Eguchi, K.; Kawakami, A.; Nakashima, M.; Ida, H.; Sakito, S.; Matsuoka, N.; Terada, K.; Sakai, M.; Kawabe, Y.; Fukuda, T.; Ishimaru, T.; Kurouji, K.; Fujita, N.; Aoyagi, T.; Maeda, K.; Nagataki, S.

    1992-01-01

    We investigated whether interferon-gamma (IFN-γ), interferon-alpha (IFN-α) and glucocorticoids affected the adhesion of T cells to human umbilical endothelial cells or human synovial cells. About 30% of peripheral blood T cells could bind to unstimulated endothelial cells, but only a few T cells could bind to unstimulated synovial cells. When both endothelial cells and synovial cells were cultured with recombinant IFN-γ (rIFN-γ), the percentage of T cell binding to both types of cells increased in a dose-dependent manner. rIFN-α and dexamethasone blocked the T cell binding to unstimulated endothelial cells. Furthermore, rIFN-α and dexamethasone suppressed T cell binding to both endothelial cells and synovial cells stimulated by IFN-γ, and also inhibited intercellular adhesion molecule-1 (ICAM-1) expression on both endothelial cells and synovial cells stimulated by IFN-γ. These results suggest that IFN-α and glucocorticoids may inhibit T cell binding to endothelial cells or synovial cells by modulating adhesion molecule expression on these cells. PMID:1606729

  17. Conditioned Media from Microvascular Endothelial Cells Cultured in Simulated Microgravity Inhibit Osteoblast Activity

    PubMed Central

    Cazzaniga, Alessandra; Castiglioni, Sara; Maier, Jeanette A. M.

    2014-01-01

    Background and Aims. Gravity contributes to the maintenance of bone integrity. Accordingly, weightlessness conditions during space flight accelerate bone loss and experimental models in real and simulated microgravity show decreased osteoblastic and increased osteoclastic activities. It is well known that the endothelium and bone cells cross-talk and this intercellular communication is vital to regulate bone homeostasis. Because microgravity promotes microvascular endothelial dysfunction, we anticipated that the molecular cross-talk between endothelial cells exposed to simulated microgravity and osteoblasts might be altered. Results. We cultured human microvascular endothelial cells in simulated microgravity using the rotating wall vessel device developed by NASA. Endothelial cells in microgravity show growth inhibition and release higher amounts of matrix metalloproteases type 2 and interleukin-6 than controls. Conditioned media collected from microvascular endothelial cells in simulated microgravity were used to culture human osteoblasts and were shown to retard osteoblast proliferation and inhibit their activity. Discussion. Microvascular endothelial cells in microgravity are growth retarded and release high amounts of matrix metalloproteases type 2 and interleukin-6, which might play a role in retarding the growth of osteoblasts and impairing their osteogenic activity. Conclusions. We demonstrate that since simulated microgravity modulates microvascular endothelial cell function, it indirectly impairs osteoblastic function. PMID:25210716

  18. Efficacy of statins on sirtuin 1 and endothelial nitric oxide synthase expression: the role of sirtuin 1 gene variants in human coronary atherosclerosis.

    PubMed

    Kilic, Ulkan; Gok, Ozlem; Elibol-Can, Birsen; Uysal, Omer; Bacaksiz, Ahmet

    2015-04-01

    Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and are used to reduce the risk of coronary artery disease (CAD) due to their pleiotropic effects. Recently, greater focus has been placed on the role of sirtuin 1 (SIRT1) in cardiovascular disease research. However, insufficient data exist on the relationships between statins, SIRT1 protein levels, and SIRT1 gene variants. In the present study, we investigated the effects of statins, atorvastatin and rosuvastatin, in CAD patients by analysing the associations between SIRT1 gene variants, rs7069102C>G and rs2273773C>T, and SIRT1/endothelial nitric oxide (eNOS) expression, as well as total antioxidant and oxidant status, and the oxidative stress index. SIRT1 expression was significantly higher, and eNOS expression was significantly lower in CAD patients when compared with controls. Statin treatment reduced SIRT1 expression and increased eNOS expression, similar to the levels found in the control population, independent from the studied SIRT1 gene variants. Oxidative stress parameters were significantly increased in CAD patients, and were decreased by statin treatment, demonstrating the antioxidative effects of statins on atherosclerosis. These results indicate that statin treatment could produce its protective effect on cardiovascular disease through the inhibition of SIRT1 expression. This is the first study reporting on the effect of statins, specifically atorvastatin and rosuvastatin, on SIRT1 expression in CAD patients.

  19. Pretreatment with light-emitting diode therapy reduces ischemic brain injury in mice through endothelial nitric oxide synthase-dependent mechanisms.

    PubMed

    Lee, Hae In; Lee, Sae-Won; Kim, So Young; Kim, Nam Gyun; Park, Kyoung-Jun; Choi, Byung Tae; Shin, Yong-Il; Shin, Hwa Kyoung

    2017-03-24

    Photostimulation with low-level light emitting diode therapy (LED-T) modulates neurological and psychological functions. The purpose of this study was to evaluate the effects of LED-T pretreatment on the mouse brain after ischemia/reperfusion and to investigate the underlying mechanisms. Ischemia/reperfusion brain injury was induced by middle cerebral artery occlusion. The mice received LED-T twice a day for 2 days prior to cerebral ischemia. After reperfusion, the LED-T group showed significantly smaller infarct and edema volumes, fewer behavioral deficits compared to injured mice that did not receive LED-T and significantly higher cerebral blood flow compared to the vehicle group. We observed lower levels of endothelial nitric oxide synthase (eNOS) phosphorylation in the injured mouse brains, but significantly higher eNOS phosphorylation in LED-T-pretreated mice. The enhanced phospho-eNOS was inhibited by LY294002, indicating that the effects of LED-T on the ischemic brain could be attributed to the upregulation of eNOS phosphorylation through the phosphoinositide 3-kinase (PI3K)/Akt pathway. Moreover, no reductions in infarct or edema volume were observed in LED-T-pretreated eNOS-deficient (eNOS(-/-)) mice. Collectively, we found that pretreatment with LED-T reduced the amount of ischemia-induced brain damage. Importantly, we revealed that these effects were mediated by the stimulation of eNOS phosphorylation via the PI3K/Akt pathway.

  20. Soy Isoflavone Protects Myocardial Ischemia/Reperfusion Injury through Increasing Endothelial Nitric Oxide Synthase and Decreasing Oxidative Stress in Ovariectomized Rats

    PubMed Central

    Tang, Yan; Li, Shuangyue; Zhang, Ping; Zhu, Jinbiao; Meng, Guoliang; Xie, Liping; Yu, Ying; Ji, Yong; Han, Yi

    2016-01-01

    There is a special role for estrogens in preventing and curing cardiovascular disease in women. Soy isoflavone (SI), a soy-derived phytoestrogen, has similar chemical structure to endogenous estrogen-estradiol. We investigate to elucidate the protective mechanism of SI on myocardial ischemia/reperfusion (MI/R) injury. Female SD rats underwent bilateral ovariectomy. One week later, rats were randomly divided into several groups, sham ovariectomy (control group), ovariectomy with MI/R, or ovariectomy with sham MI/R. Other ovariectomy rats were given different doses of SI or 17β-estradiol (E2). Four weeks later, they were exposed to 30 minutes of left coronary artery occlusion followed by 6 or 24 hours of reperfusion. SI administration significantly reduced myocardial infarct size and improved left ventricle function and restored endothelium-dependent relaxation function of thoracic aortas after MI/R in ovariectomized rats. SI also decreased serum creatine kinase and lactate dehydrogenase activity, reduced plasma malonaldehyde, and attenuated oxidative stress in the myocardium. Meanwhile, SI increased phosphatidylinositol 3 kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) signal pathway. SI failed to decrease infarct size of hearts with I/R in ovariectomized rats if PI3K was inhibited. Overall, these results indicated that SI protects myocardial ischemia/reperfusion injury in ovariectomized rats through increasing PI3K/Akt/eNOS signal pathway and decreasing oxidative stress. PMID:27057277

  1. Nitric oxide modulates chromatin folding in human endothelial cells via protein phosphatase 2A activation and class II histone deacetylases nuclear shuttling.

    PubMed

    Illi, Barbara; Dello Russo, Claudio; Colussi, Claudia; Rosati, Jessica; Pallaoro, Michele; Spallotta, Francesco; Rotili, Dante; Valente, Sergio; Ragone, Gianluca; Martelli, Fabio; Biglioli, Paolo; Steinkuhler, Christian; Gallinari, Paola; Mai, Antonello; Capogrossi, Maurizio C; Gaetano, Carlo

    2008-01-04

    Nitric oxide (NO) modulates important endothelial cell (EC) functions and gene expression by a molecular mechanism which is still poorly characterized. Here we show that in human umbilical vein ECs (HUVECs) NO inhibited serum-induced histone acetylation and enhanced histone deacetylase (HDAC) activity. By immunofluorescence and Western blot analyses it was found that NO induced class II HDAC4 and 5 nuclear shuttling and that class II HDACs selective inhibitor MC1568 rescued serum-dependent histone acetylation above control level in NO-treated HUVECs. In contrast, class I HDACs inhibitor MS27-275 had no effect, indicating a specific role for class II HDACs in NO-dependent histone deacetylation. In addition, it was found that NO ability to induce HDAC4 and HDAC5 nuclear shuttling involved the activation of the protein phosphatase 2A (PP2A). In fact, HDAC4 nuclear translocation was impaired in ECs expressing small-t antigen and exposed to NO. Finally, in cells engineered to express a HDAC4-Flag fusion protein, NO induced the formation of a macromolecular complex including HDAC4, HDAC3, HDAC5, and an active PP2A. The present results show that NO-dependent PP2A activation plays a key role in class II HDACs nuclear translocation.

  2. Far-infrared radiation acutely increases nitric oxide production by increasing Ca{sup 2+} mobilization and Ca{sup 2+}/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

    SciTech Connect

    Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

    2013-07-12

    Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser{sup 1179} phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser{sup 1179} phosphorylation. •FIR increases intracellular Ca{sup 2+} levels. •Thermo-sensitive TRPV Ca{sup 2+} channels are unlikely to be involved in the FIR-mediated eNOS-Ser{sup 1179} phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser{sup 1179}) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca{sup 2+} levels. Treatment with KN-93, a selective inhibitor of Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. This

  3. Syntaxin-binding protein STXBP5 inhibits endothelial exocytosis and promotes platelet secretion

    PubMed Central

    Zhu, Qiuyu; Yamakuchi, Munekazu; Ture, Sara; de la Luz Garcia-Hernandez, Maria; Ko, Kyung Ae; Modjeski, Kristina L.; LoMonaco, Michael B.; Johnson, Andrew D.; O’Donnell, Christopher J.; Takai, Yoshimi; Morrell, Craig N.; Lowenstein, Charles J.

    2014-01-01

    In humans, vWF levels predict the risk of myocardial infarction and thrombosis; however, the factors that influence vWF levels are not completely understood. Recent genome-wide association studies (GWAS) have identified syntaxin-binding protein 5 (STXBP5) as a candidate gene linked to changes in vWF plasma levels, though the functional relationship between STXBP5 and vWF is unknown. We hypothesized that STXBP5 inhibits endothelial cell exocytosis. We found that STXBP5 is expressed in human endothelial cells and colocalizes with and interacts with syntaxin 4. In human endothelial cells reduction of STXBP5 increased exocytosis of vWF and P-selectin. Mice lacking Stxbp5 had higher levels of vWF in the plasma, increased P-selectin translocation, and more platelet-endothelial interactions, which suggests that STXBP5 inhibits endothelial exocytosis. However, Stxbp5 KO mice also displayed hemostasis defects, including prolonged tail bleeding times and impaired mesenteric arteriole and carotid artery thrombosis. Furthermore, platelets from Stxbp5 KO mice had defects in platelet secretion and activation; thus, STXBP5 inhibits endothelial exocytosis but promotes platelet secretion. Our study reveals a vascular function for STXBP5, validates the functional relevance of a candidate gene identified by GWAS, and suggests that variation within STXBP5 is a genetic risk for venous thromboembolic disease. PMID:25244095

  4. Nafamostat Mesilate Inhibits TNF-α-Induced Vascular Endothelial Cell Dysfunction by Inhibiting Reactive Oxygen Species Production.

    PubMed

    Kang, Min-Woong; Song, Hee-Jung; Kang, Shin Kwang; Kim, Yonghwan; Jung, Saet-Byel; Jee, Sungju; Moon, Jae Young; Suh, Kwang-Sun; Lee, Sang Do; Jeon, Byeong Hwa; Kim, Cuk-Seong

    2015-05-01

    Nafamostat mesilate (NM) is a serine protease inhibitor with anticoagulant and anti-inflammatory effects. NM has been used in Asia for anticoagulation during extracorporeal circulation in patients undergoing continuous renal replacement therapy and extra corporeal membrane oxygenation. Oxidative stress is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial function. We investigated whether NM could inhibit endothelial dysfunction induced by tumor necrosis factor-α (TNF-α). Human umbilical vein endothelial cells (HUVECs) were treated with TNF-α for 24 h. The effects of NM on monocyte adhesion, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) protein expression, p38 mitogen-activated protein kinase (MAPK) activation, and intracellular superoxide production were then examined. NM (0.01~100 µg/mL) did not affect HUVEC viability; however, it inhibited the increases in reactive oxygen species (ROS) production and p66shc expression elicited by TNF-α (3 ng/mL), and it dose dependently prevented the TNF-α-induced upregulation of endothelial VCAM-1 and ICAM-1. In addition, it mitigated TNF-α-induced p38 MAPK phosphorylation and the adhesion of U937 monocytes. These data suggest that NM mitigates TNF-α-induced monocyte adhesion and the expression of endothelial cell adhesion molecules, and that the anti-adhesive effect of NM is mediated through the inhibition of p66shc, ROS production, and p38 MAPK activation.

  5. Cell-permeable iron inhibits vascular endothelial growth factor receptor-2 signaling and tumor angiogenesis

    PubMed Central

    Kir, Devika; Saluja, Manju; Modi, Shrey; Venkatachalam, Annapoorna; Schnettler, Erica; Roy, Sabita; Ramakrishnan, Sundaram

    2016-01-01

    Angiogenesis is important for tumor growth and metastasis. Hypoxia in tumors drives this angiogenic response by stabilizing Hypoxia Inducible Factors (HIF) and target genes like Vascular Endothelial Growth Factor (VEGF). HIF stability is regulated by Prolylhydroxylases (PHD)-mediated modification. Iron is an important cofactor in regulating the enzymatic activity of PHDs. Reducing intracellular iron, for instance, mimics hypoxia and induces a pro-angiogenic response. It is hypothesized that increasing the intracellular iron levels will have an opposite, anti-angiogenic effect. We tested this hypothesis by perturbing iron homeostasis in endothelial cells using a unique form of iron, Ferric Ammonium Citrate (FAC). FAC is a cell-permeable form of iron, which can passively enter into cells bypassing the transferrin receptor mediated uptake of transferrin-bound iron. Our studies show that FAC does not decrease the levels of HIF-1α and HIF-2α in endothelial cells but inhibits the autocrine stimulation of VEGF-Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) system by blocking receptor tyrosine kinase phosphorylation. FAC inhibits VEGF-induced endothelial cell proliferation, migration, tube formation and sprouting. Finally, systemic administration of FAC inhibits VEGF and tumor cell-induced angiogenesis in vivo. In conclusion, our studies show that cell-permeable iron attenuates VEGFR-2 mediated signaling and inhibits tumor angiogenesis. PMID:27589831

  6. Inhibition of inducible nitric oxide synthase and osteoclastic differentiation by Atractylodis Rhizoma Alba extract

    PubMed Central

    Choi, Sung-Ho; Kim, Sung-Jin

    2014-01-01

    Background: Atractylodis Rhizoma Alba (ARA) has been used in Korean folk medicine for constipation, dizziness, and anticancer agent. In the present study, we performed to test whether the methanolic extract of ARA has antioxidant and antiosteoclastogenesis activity in RAW 264.7 macrophage cells. Materials and Methods: Antioxidant capacities were tested by measuring free radical scavenging activity, nitric oxide (NO) levels, reducing power, and inducible nitric oxide synthase (iNOS) expression in response to lipopolysaccharides (LPS). Antiosteoclastogenesis activity was evaluated by performing tartrate-resistant acid phosphatase assay in RAW 264.7 macrophage cells. Results: The extract exerted significant 1,1-diphenyl-2-picrylhydrazyl and NO radical scavenging activity, and it exerted dramatic reducing power. Induction of iNOS and NO by LPS in RAW 264.7 cells was significantly inhibited by the extract, suggesting that the ARA extract inhibits NO production by suppressing iNOS expression. Strikingly, the ARA extracts substantially inhibited the receptor activator of NF-κB ligand-induced osteclastic differentiation of LPS-activated RAW 264.7 cells. The ARA extract contains a significant amount of antioxidant components, including phenolics, flavonoids and anthocyanins. Conclusion: These results suggest that the methanolic extract of ARA exerts significant antioxidant activities potentially via inhibiting free radicals and iNOS induction, thereby leading to the inhibition of osteoclastogenesis. PMID:25298665

  7. Chemical Structures of 4-Oxo-Flavonoids in Relation to Inhibition of Oxidized Low-Density Lipoprotein (LDL)-Induced Vascular Endothelial Dysfunction

    PubMed Central

    Yi, Long; Jin, Xin; Chen, Chun-Ye; Fu, Yu-Jie; Zhang, Ting; Chang, Hui; Zhou, Yong; Zhu, Jun-Dong; Zhang, Qian-Yong; Mi, Man-Tian

    2011-01-01

    Vascular endothelial dysfunction induced by oxidative stress has been demonstrated to be the initiation step of atherosclerosis (AS), and flavonoids may play an important role in AS prevention and therapy. Twenty-three flavonoids categorized into flavones, flavonols, isoflavones, and flavanones, all with 4-oxo-pyronenucleus, were examined for what structural characteristics are required for the inhibitory effects on endothelial dysfunction induced by oxidized low-density lipoprotein (oxLDL). Human vascular endothelial cells EA.hy926 were pretreated with different 4-oxo-flavonoids for 2 hs, and then exposed to oxLDL for another 24 hs. Cell viability and the level of malondialdehyde (MDA), nitric oxide (NO) and soluble intercellular adhesion molecule-1 (sICAM-1) were measured, respectively. Then, correlation analysis and paired comparison were used to analyze the structure–activity relationships. Significant correlations were observed between the number of −OH moieties in total or in B-ring and the inhibitory effectson endothelial dysfunction. Furthermore, 3′,4′-ortho-dihydroxyl on B-ring, 3-hydroxyl on C-ring and 2,3-double bondwere correlated closely to the inhibitory effects of flavonolson cell viability decrease and lipid peroxidation. 5,7-meta-dihydroxyl group on A-ring was crucial for the anti-inflammatory effects of flavones and isoflavones in endothelial cells. Moreover, the substituted position of B-ring on C3 rather than C2 was important for NO release. Additionally, hydroxylation at C6 position significantly attenuated the inhibitory effects of 4-oxo-flavonoids on endothelial dysfunction. Our findings indicated that the effective agents in inhibiting endothelial dysfunction include myricetin, quercetin, luteolin, apigenin, genistein and daidzein. Our work might provide some evidence for AS prevention and a strategy for the design of novel AS preventive agents. PMID:22016603

  8. Chemical structures of 4-oxo-flavonoids in relation to inhibition of oxidized low-density lipoprotein (LDL)-induced vascular endothelial dysfunction.

    PubMed

    Yi, Long; Jin, Xin; Chen, Chun-Ye; Fu, Yu-Jie; Zhang, Ting; Chang, Hui; Zhou, Yong; Zhu, Jun-Dong; Zhang, Qian-Yong; Mi, Man-Tian

    2011-01-01

    Vascular endothelial dysfunction induced by oxidative stress has been demonstrated to be the initiation step of atherosclerosis (AS), and flavonoids may play an important role in AS prevention and therapy. Twenty-three flavonoids categorized into flavones, flavonols, isoflavones, and flavanones, all with 4-oxo-pyronenucleus, were examined for what structural characteristics are required for the inhibitory effects on endothelial dysfunction induced by oxidized low-density lipoprotein (oxLDL). Human vascular endothelial cells EA.hy926 were pretreated with different 4-oxo-flavonoids for 2 hs, and then exposed to oxLDL for another 24 hs. Cell viability and the level of malondialdehyde (MDA), nitric oxide (NO) and soluble intercellular adhesion molecule-1 (sICAM-1) were measured, respectively. Then, correlation analysis and paired comparison were used to analyze the structure-activity relationships. Significant correlations were observed between the number of -OH moieties in total or in B-ring and the inhibitory effectson endothelial dysfunction. Furthermore, 3',4'-ortho-dihydroxyl on B-ring, 3-hydroxyl on C-ring and 2,3-double bondwere correlated closely to the inhibitory effects of flavonolson cell viability decrease and lipid peroxidation. 5,7-meta-dihydroxyl group on A-ring was crucial for the anti-inflammatory effects of flavones and isoflavones in endothelial cells. Moreover, the substituted position of B-ring on C3 rather than C2 was important for NO release. Additionally, hydroxylation at C6 position significantly attenuated the inhibitory effects of 4-oxo-flavonoids on endothelial dysfunction. Our findings indicated that the effective agents in inhibiting endothelial dysfunction include myricetin, quercetin, luteolin, apigenin, genistein and daidzein. Our work might provide some evidence for AS prevention and a strategy for the design of novel AS preventive agents.

  9. Inhibition of UVA-mediated melanogenesis by ascorbic acid through modulation of antioxidant defense and nitric oxide system.

    PubMed

    Panich, Uraiwan; Tangsupa-a-nan, Vanida; Onkoksoong, Tasanee; Kongtaphan, Kamolratana; Kasetsinsombat, Kanda; Akarasereenont, Pravit; Wongkajornsilp, Adisak

    2011-05-01

    Ascorbic acid (AA) has been well known as a skin whitening agent, although attempts have been made to evaluate its protective role against ultraviolet (UV)-induced skin hyperpigmentation or increased melanin production. While melanogenesis is a defense mechanism of the skin against UV irradiation, melanin overproduction may also contribute to melanoma initiation. UVA might play a role in melanogenesis through promoting oxidative stress, which occurs as the result of increased formation of oxidants and/or reactive nitrogen species (RNS) including nitric oxide (NO). Therefore, we investigated the antimelanogenic effect of AA (7.5-120 μM) in association with its inhibitory effect on UVA-induced oxidant formation, NO production through endothelial and inducible NO synthases (eNOS and iNOS) activation and impairment of antioxidant defense using G361 human melanoma cells. Our study demonstrated a comparable ability of AA with that of kojic acid, a well-known tyrosinase inhibitor in inhibiting mushroom tyrosinase. Melanin content was reduced by AA, but neither tyrosinase activity nor mRNA levels were reduced by AA at non-cytotoxic concentrations in UVA-irradiated G361 cells. AA was shown to inhibit UVA-mediated catalase (CAT) inactivation, glutathione (GSH) depletion, oxidant formation and NO production through suppression of eNOS and iNOS mRNA. We report herein that AA can protect against UVA-dependent melanogenesis possibly through the improvement of antioxidant defense capacity and inhibition of NO production through down-regulation of eNOS and iNOS mRNA.

  10. Nitric oxide inhibits irreversibly P815 cell proliferation: involvement of potassium channels.

    PubMed

    Costa, R S A; Assreuy, J

    2002-12-01

    Nitric oxide (NO) has been shown to inhibit both normal and cancer cell proliferation. Potassium channels are involved in cell proliferation and, as NO activates these channels, we investigated the effect of NO on the proliferation of murine mastocytoma cell lines and the putative involvement of potassium channels. NO (in the form of NO donors) caused dose-dependent inhibition of cell proliferation in the P815 cell line inducing growth arrest in the mitosis phase. Incubation with NO donor for 4 or 24 h had a similar inhibitory effect on cell proliferation, indicating that this effect is irreversible. The inhibitory effect of NO was completely prevented by the blockade of voltage- and calcium-dependent potassium channels, but not by blockade of ATP-dependent channels. NO inhibition of cell proliferation was unaffected by guanylate cyclase and by cytoskeleton disruptors. Therefore, NO inhibits cell proliferation irreversibly via a potassium channel-dependent but guanylate cyclase-independent pathway in murine mastocytoma cells.

  11. Nitric oxide mediates low magnesium inhibition of osteoblast-like cell proliferation.

    PubMed

    Leidi, Marzia; Dellera, Federica; Mariotti, Massimo; Banfi, Giuseppe; Crapanzano, Calogero; Albisetti, Walter; Maier, Jeanette A M

    2012-10-01

    An adequate intake of magnesium (Mg) is important for bone cell activity and contributes to the prevention of osteoporosis. Because (a) Mg is mitogenic for osteoblasts and (b) reduction of osteoblast proliferation is detected in osteoporosis, we investigated the influence of different concentrations of extracellular Mg on osteoblast-like SaOS-2 cell behavior. We found that low Mg inhibited SaOS-2 cell proliferation by increasing the release of nitric oxide through the up-regulation of inducible nitric oxide synthase (iNOS). Indeed, both pharmacological inhibition with the iNOS inhibitor l-N(6)-(iminoethyl)-lysine-HCl and genetic silencing of iNOS by small interfering RNA restored the normal proliferation rate of the cells. Because a moderate induction of nitric oxide is sufficient to potentiate bone resorption and a relative deficiency in osteoblast proliferation can result in their inadequate activity, we conclude that maintaining Mg homeostasis is relevant to ensure osteoblast function and, therefore, to prevent osteoporosis.

  12. Influence of nitric oxide synthase inhibition on vasopressin and corticosterone secretion during water deprivation in rats.

    PubMed

    Mornagui, Bessem; Rezg, Raja; Grissa, Abir; Duvareille, Monique; Gharib, Claude; Kamoun, Abdelaziz; El-Fazaa, Saloua; Gharbi, Najoua

    2010-12-01

    Nitric oxide (NO) is a short-lived radical that functions as a neurotransmitter in the central nervous system and plays a physiological role in the regulation of hypothalamic-pituitary-adrenal axis and vasopressinergic axis. In the present study, we aimed to investigate the interaction between the generation of NO and vasopressin (AVP) and corticosterone release after 3 days of water deprivation in rats. Animals were previously treated with intraperitoneal (i.p.) saline or L-nitro-arginine methyl ester (L-NAME) injection. L-NAME is a nonspecific inhibitor of nitric oxide synthases. In control rats given i.p. saline or L-NAME, hypothalamic, pituitary, and plasma AVP levels and plasma corticosterone did not change from baseline levels (p>0.05). Three days of water deprivation increased significantly the corticosterone levels in plasma (p<0.01) and AVP levels in hypothalamus and plasma (p<0.01), but not in pituitary, which showed a significant decrease. These variations were concomitant with the elevation of nitrates/nitrates in plasma. L-NAME injection abolished significantly (p<0.01) the elevation of plasma corticosterone and hypothalamic AVP levels induced by water deprivation. These findings showed that in water-deprived rats, nitric oxide synthase inhibition by L-NAME inhibits corticosterone and vasopressin release, suggesting a potent stimulatory role of NO.

  13. Evidence for antiviral effect of nitric oxide. Inhibition of herpes simplex virus type 1 replication.

    PubMed Central

    Croen, K D

    1993-01-01

    Nitric oxide (NO) has antimicrobial activity against a wide spectrum of infectious pathogens, but an antiviral effect has not been reported. The impact of NO, from endogenous and exogenous sources, on herpes simplex virus type 1 (HSV 1) replication was studied in vitro. HSV 1 replication in RAW 264.7 macrophages was reduced 1,806-fold in monolayers induced to make NO by activation with gamma IFN and LPS. A competitive and a noncompetitive inhibitor of nitric oxide synthetase substantially reduced the antiviral effect of activated RAW macrophages. S-nitroso-L-acetyl penicillamine (SNAP) is a donor of NO and was added to the media of infected monolayers to assess the antiviral properties of NO in the absence of gamma IFN and LPS. A single dose of S-nitroso-L-acetyl penicillamine 3 h after infection inhibited HSV 1 replication in Vero, HEp2, and RAW 264.7 cells in a dose-dependent manner. Neither virucidal nor cytocidal effects of NO were observed under conditions that inhibited HSV 1 replication. Nitric oxide had inhibitory effects, comparable to that of gamma IFN/LPS, on protein and DNA synthesis as well as on cell replication. This report demonstrates that, among its diverse properties, NO has an antiviral effect. PMID:8390481

  14. Neuronal and Endothelial Nitric Oxide Synthases in the Paraventricular Nucleus Modulate Sympathetic Overdrive in Insulin-Resistant Rats

    PubMed Central

    Lu, Qing-Bo; Feng, Xue-Mei; Tong, Ning; Sun, Hai-Jian; Ding, Lei; Wang, Yu-Jiao; Wang, Xuan; Zhou, Ye-Bo

    2015-01-01

    A central mechanism participates in sympathetic overdrive during insulin resistance (IR). Nitric oxide synthase (NOS) and nitric oxide (NO) modulate sympathetic nerve activity (SNA) in the paraventricular nucleus (PVN), which influences the autonomic regulation of cardiovascular responses. The aim of this study was to explore whether the NO system in the PVN is involved in the modulation of SNA in fructose-induced IR rats. Control rats received ordinary drinking water, whereas IR rats received 12.5% fructose-containing drinking water for 12 wks to induce IR. Basal SNA was assessed based on the changes in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in response to chemicals administered to the PVN. We found an increased plasma norepinephrine level but significantly reduced NO content and neuronal NOS (nNOS) and endothelial NOS (eNOS) protein expression levels in the PVN of IR rats compared to Control rats. No difference in inducible NOS (iNOS) protein expression was observed between the two groups. In anesthetized rats, the microinjection of sodium nitroprusside (SNP), an NO donor, or Nω-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of NOS, into the PVN significantly decreased and increased basal SNA, respectively, in both normal and IR rats, but these responses to SNP and L-NAME in IR rats were smaller than those in normal rats. The administration of selective inhibitors of nNOS or eNOS, but not iNOS, to the PVN significantly increased basal SNA in both groups, but these responses were also smaller in IR rats. Moreover, IR rats exhibited reduced nNOS and eNOS activity in the PVN. In conclusion, these data indicate that the decreased protein expression and activity levels of nNOS and eNOS in the PVN lead to a reduction in the NO content in the PVN, thereby contributing to a subsequent enhancement in sympathoexcitation during IR. PMID:26485682

  15. Association between increased expression of endothelial isoform of nitric oxide synthase in the human fallopian tube and tubal ectopic pregnancy

    PubMed Central

    Fath Bayati, Leyla; Ghaffari Novin, Marefat; Fadaei Fathabadi, Fatemeh; Piryaei, Abbas; Heidari, Mohammad Hasan; Bandehpour, Mozhgan; Norouzian, Mohsen; Alizadeh Parhizgar, Mahdi; Shakooriyan Fard, Mahmood

    2014-01-01

    Background: Tubal ectopic pregnancy (tEP) is the most common type of extra-uterine pregnancy and the most common cause of maternal mortality. Nitric oxide (NO) is a molecule that incorporates in many physiological processes of female reproductive system. Recent studies have demonstrated the possible role of endothelial isoform of nitric oxide synthase (eNOS) enzyme in the regulation of many reproductive events that occur in the fallopian tube (FT). Objective: The aim of this study was to evaluate the expression of eNOS in the FTs of women with tEP. Materials and Methods: In this case-control study, a total number of 30FTs samples were obtained from three groups including: 10 FTs of women that bearing an EP, 10 FTs from the non-pregnant women at luteal phase of the menstrual cycle, and 10 FTs of healthy pregnant women (n=10). Samples were fixed in 10% buffered formalin and then were evaluated by immunohistochemistry. Results: Localization of eNOS was seen in secretory and ciliated luminal epithelium and vascular endothelium of all groups. However, we did not observed the expression of eNOS in smooth muscle cells of all groups. Expression of eNOS in luminal epithelium of women with EP compared to non-pregnant women at luteal phase of menstrual cycle and healthy pregnant group showed statistically significant increase (p=0.00). Significant difference in expression of eNOS was not observed in luminal epithelium of FTs of women at luteal phase compared to healthy pregnant groups (p=0.78). Conclusion: This study indicates that changes in expression of eNOS in luminal epithelium of FT may lead to development of EP. This article extracted from M.Sc. thesis. (Leyla Fath Bayati) PMID:24799858

  16. The activation of nitric oxide synthase by copper ion is mediated by intracellular Ca2+ mobilization in human pulmonary arterial endothelial cells

    PubMed Central

    Demura, Yoshiki; Ishizaki, Takeshi; Ameshima, Shingo; Okamura, Seitarou; Hayashi, Takio; Matsukawa, Shigeru; Miyamori, Isamu

    1998-01-01

    The aim of the study was to elucidate the vasodilatory mechanism due to Cu2+ by assessing nitric oxide (NO) production as determined by NOx (NO, NO2−, and NO3−) that is released from human pulmonary arterial endothelial cell (HPAEC) monolayers using a NO chemiluminescence analyzer, and also to assess Ca2+ movement using 45Ca and fura 2 in HPAEC.Cu2+ (10−6–10−4 M) significantly increased NO production in a dose-dependent manner when extracellular Ca2+ was present.45Ca influx into the adherent cells was dose-dependently enhanced by Cu2+ (10−6–10−4 M), but not by Mn2+, Zn2+ or Fe2+.[Ca2+]i, measured by monitoring the fluorescence changes of fura 2, was significantly elevated in the presence of Cu2+.The increase in [Ca2+]i induced by Cu2+ was inhibited by either diethyldithiocarbamate (DDC) or the depletion of extracellular Ca2+.The dihydropyridine receptor agonist, BayK8644, significantly attenuated the Cu2+-induced increase in [Ca2+]i in a dose dependent manner and nitrendipine or nifedipine, the dihydropyridine receptor antagonists, dose-dependently inhibited a Cu2+-induced increase in [Ca2+]i.These results suggest that Cu2+ activates eNOS through the mechanism of [Ca2+]i elevation due to Ca2+ influx into HPAEC and that the Cu2+-induced [Ca2+]i elevation in HPAEC is likely due to activation of the dihydropyridine-like receptors. PMID:9863645

  17. Myocardial and coronary endothelial protective effects of acetylcholine after myocardial ischaemia and reperfusion in rats: role of nitric oxide.

    PubMed Central

    Richard, V.; Blanc, T.; Kaeffer, N.; Tron, C.; Thuillez, C.

    1995-01-01

    1. Recent experiments suggest that acetylcholine (ACh) may exert myocardial protective effects during ischaemia (I) and reperfusion (R). The present study was designed (i) to assess whether ACh limits infarct size and protects coronary endothelial cells in a rat model of I and R, (ii) to evaluate the role of ATP-sensitive potassium (KATP) channels and nitric oxide (NO) in the beneficial effect of ACh (iii) to evaluate whether the protective effect of ACh also extends to coronary endothelial cells and (iv) to assess whether ACh contributes to the beneficial effect of preconditioning. 2. Anaesthetized rats were subjected to 20 min I (left coronary artery occlusion) and 2 h of R. Infarct size was assessed by triphenyltetrazolium (TTC) staining and expressed as a % of the area at risk (India ink injection). Vascular studies were performed on 1.5-2 mm coronary segments (internal diameter 250-300 micros) removed distal to the site of occlusion and mounted in wire myographs. 3. ACh limited infarct size (from 59 +/- 3 to 26 +/- 5%, P < 0.01), and this was prevented by atropine (46 +/- 7%; P < 0.05 vs ACh), but not by the inhibitor of KATP channels, glibenclamide (29 +/- 8%). The inhibitor of NO synthesis NG-nitro L-arginine did not affect infarct size (54 +/- 5%) but abolished the beneficial effect of ACh (59 +/- 8%; P < 0.05 vs ACh), whereas the NO donor 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1 limited infarct size to the same extent as ACh (28 +/- 6%).(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8564215

  18. Therapeutic concentrations of tacrolimus do not interfere with endothelial nitric oxide synthesis in rat thoracic aortas and coronary arteries.

    PubMed

    Can, Cenk; Erol, Ayşe; Cinar, Mehtap; Olukman, Murat; Ulker, Sibel; Evinç, Akgün

    2007-10-01

    This study aimed to investigate the potential effect of in vivo administration of immunosuppressive agent FK-506 (tacrolimus) on the endothelial function of rat thoracic aortas with respect to nitric oxide (NO) synthesis. In vitro effect of the drug on NO synthesis in cultured rat coronary microvascular endothelial cells (CMEC) was also studied.In vivo administration of tacrolimus (1 mg/kg/d, intramuscular) to rats for 14 days resulted in decreased relaxant responses to the higher concentrations (1 to 30 muM) of acetylcholine in the aortas; however, responses to calcium ionophore A23187, sodium nitroprusside, L-arginine, and L-NAME did not change significantly. No changes were observed in phenylephrine-induced contractions in endothelium-denuded or -intact preparations. Administration of the vehicle for 14 days did not affect these parameters. In order to evaluate the in vitro effect of tacrolimus on NO release, CMEC isolated from rat hearts were incubated with either tacrolimus (0.01, 0.1 microM) or the vehicle. Basal, calcium ionophore-stimulated, or interleukin-1 beta-induced NO synthesis was determined by measuring total nitrite in the media. Neither tacrolimus nor the vehicle changed nitrite accumulation. It has been concluded that therapeutic concentrations of tacrolimus do not alter NO production in rat thoracic aorta or cultured CMEC; however, it impairs relaxant responses of rat aorta induced by higher concentrations of acetylcholine, possibly through changes in the downstream of receptor activation or through an imbalance between endothelium-dependent relaxant and contracting factors within the endothelium in favor of the contracting factor(s).

  19. Endothelial nitric oxide synthase in red blood cells: Key to a new erythrocrine function?☆

    PubMed Central

    Cortese-Krott, Miriam M.; Kelm, Malte

    2014-01-01

    Red blood cells (RBC) have been considered almost exclusively as a transporter of metabolic gases and nutrients for the tissues. It is an accepted dogma that RBCs take up and inactivate endothelium-derived NO via rapid reaction with oxyhemoglobin to form methemoglobin and nitrate, thereby limiting NO available for vasodilatation. Yet it has also been shown that RBCs not only act as “NO sinks”, but exert an erythrocrine function – i.e an endocrine function of RBC – by synthesizing, transporting and releasing NO metabolic products and ATP, thereby potentially controlling systemic NO bioavailability and vascular tone. Recent work from our and others laboratory demonstrated that human RBCs carry an active type 3, endothelial NO synthase (eNOS), constitutively producing NO under normoxic conditions, the activity of which is compromised in patients with coronary artery disease. In this review we aim to discuss the potential role of red cell eNOS in RBC signaling and function, and to critically revise evidence to this date showing a role of non-endothelial circulating eNOS in cardiovascular pathophysiology. PMID:24494200

  20. Idesolide inhibits the adipogenic differentiation of mesenchymal cells through the suppression of nitric oxide production.

    PubMed

    Hwang, Jun-Ha; Moon, Sung Ah; Lee, Cham Han; Byun, Mi Ran; Kim, A Rum; Sung, Mi Kyung; Park, Hyun-Jin; Hwang, Eun Sook; Sung, Sang Hyun; Hong, Jeong-Ho

    2012-06-15

    Obesity is a major health problem worldwide and can increase the risk for several chronic diseases, including diabetes and cardiovascular disease. In this study, we screened small compounds isolated from natural products for the development of an anti-obesity drug. Among them, idesolide, a spiro compound isolated from the fruits of Idesia polycarpa Maxim, showed a significant suppression of the adipogenic differentiation in mesenchymal cells, as indicated by the decrease in fat droplets and expression of adipogenic marker genes such as aP2 and adiponectin. Idesolide inhibits the PPARγ-mediated gene transcription in a dose-dependent manner, revealed by luciferase reporter gene assay. During adipogenic differentiation, idesolide inhibits nitric oxide production through the suppression of iNOS expression, and the increased adipogenic differentiation by arginine, the substrate for NOS, is significantly inhibited by idesolide, suggesting that the inhibition of nitric oxide production plays a major role in idesolide-induced adipogenic suppression. Taken together, the results reveal that idesolide has anti-adipogenic activity and highlight its potential in the prevention and treatment of obesity.

  1. Factors Associated with Nitric Oxide-mediated β2 Integrin Inhibition of Neutrophils*

    PubMed Central

    Bhopale, Veena M.; Yang, Ming; Yu, Kevin; Thom, Stephen R.

    2015-01-01

    This investigation explored the mechanism for inhibition of β2 integrin adhesion molecules when neutrophils are exposed to nitric oxide (•NO). Roles for specific proteins were elucidated using chemical inhibitors, depletion with small inhibitory RNA, and cells from knock-out mice. Optimal inhibition occurs with exposures to a •NO flux of ∼28 nmol/min for 2 min or more, which sets up an autocatalytic cascade triggered by activating type 2 nitric-oxide synthase (NOS-2) and NADPH oxidase (NOX). Integrin inhibition does not occur with neutrophils exposed to a NOX inhibitor (Nox2ds), a NOS-2 inhibitor (1400W), or with cells from mice lacking NOS-2 or the gp91phox component of NOX. Reactive species cause S-nitrosylation of cytosolic actin that enhances actin polymerization. Protein cross-linking and actin filament formation assays indicate that increased polymerization occurs because of associations involving vasodilator-stimulated phosphoprotein, focal adhesion kinase, and protein-disulfide isomerase in proximity to actin filaments. These effects were inhibited in cells exposed to ultraviolet light which photo-reverses S-nitrosylated cysteine residues and by co-incubations with cytochalasin D. The autocatalytic cycle can be arrested by protein kinase G activated with 8-bromo-cyclic GMP and by a high •NO flux (∼112 nmol/min) that inactivates NOX. PMID:26032418

  2. RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border

    PubMed Central

    Marcos-Ramiro, Beatriz; García-Weber, Diego; Barroso, Susana; Feito, Jorge; Ortega, María C.; Cernuda-Morollón, Eva; Reglero-Real, Natalia; Fernández-Martín, Laura; Durán, Maria C.; Alonso, Miguel A.; Correas, Isabel; Cox, Susan; Ridley, Anne J.

    2016-01-01

    Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation. PMID:27138256

  3. Inhibition of matrix metalloproteinase-2 improves endothelial function and prevents hypertension in insulin-resistant rats

    PubMed Central

    Nagareddy, PR; Rajput, PS; Vasudevan, H; McClure, B; Kumar, U; MacLeod, KM; McNeill, JH

    2012-01-01

    BACKGROUND AND PURPOSE Insulin resistance is often found to be associated with high blood pressure. We propose that in insulin-resistant hypertension, endothelial dysfunction is the consequence of increased activity of vascular MMP-2. As MMP-2 proteolytically cleaves a number of extracellular matrix proteins, we hypothesized that MMP-2 impairs endothelial function by proteolytic degradation of endothelial NOS (eNOS) or its cofactor, heat shock protein 90 (HSP90). EXPERIMENTAL APPROACH We tested our hypothesis in bovine coronary artery endothelial cells and fructose-fed hypertensive rats (FHR), a model of acquired systolic hypertension and insulin resistance. KEY RESULTS Treatment of FHRs with the MMP inhibitor doxycycline, preserved endothelial function as well as prevented the development of hypertension, suggesting that MMPs impair endothelial function. Furthermore, incubating endothelial cells in vitro with a recombinant MMP-2 decreased NO production in a dose-dependent manner. Using substrate cleavage assays and immunofluorescence microscopy studies, we found that MMP-2 not only cleaves and degrades HSP90, an eNOS cofactor but also co-localizes with both eNOS and HSP90 in endothelial cells, suggesting that MMPs functionally interact with the eNOS system. Treatment of FHRs with doxycycline attenuated the decrease in eNOS and HSP90 expression but did not improve insulin sensitivity. CONCLUSIONS AND IMPLICATIONS Our data suggest that increased activity of MMP-2 in FHRs impairs endothelial function and promotes hypertension. Inhibition of MMP-2 could be a potential therapeutic strategy for the management of hypertension. PMID:21740410

  4. Insulin reverses D-glucose-increased nitric oxide and reactive oxygen species generation in human umbilical vein endothelial cells.

    PubMed

    González, Marcelo; Rojas, Susana; Avila, Pía; Cabrera, Lissette; Villalobos, Roberto; Palma, Carlos; Aguayo, Claudio; Peña, Eduardo; Gallardo, Victoria; Guzmán-Gutiérrez, Enrique; Sáez, Tamara; Salsoso, Rocío; Sanhueza, Carlos; Pardo, Fabián; Leiva, Andrea; Sobrevia, Luis

    2015-01-01

    Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose-alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A2 mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH4) level was determined by HPLC, and L-arginine transport (0-1000 μmol/L) was measured in response to 5-25 mmol/L D-glucose (0-36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose-increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose-increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O2•-) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O2•- generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose-increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose-reduced BH4 level. Insulin and

  5. Endothelial nitric oxide synthase reduces crescentic and necrotic glomerular lesions, reactive oxygen production, and MCP1 production in murine lupus nephritis.

    PubMed

    Gilkeson, Gary S; Mashmoushi, Ahmad K; Ruiz, Phillip; Caza, Tiffany N; Perl, Andras; Oates, Jim C

    2013-01-01

    Systemic lupus erythematosus, in both animal models and in humans, is characterized by autoantibody production followed by immune complex deposition in target tissues. Ensuing target organ damage is modulated by reactive intermediates, including reactive nitrogen and oxygen species, through as of now incompletely understood mechanisms. Endothelial nitric oxide synthase is known to impact vascular reactivity; however its impact on reactive intermediate production and inflammatory renal disease is less well defined. In this study, we assessed the impact of endothelial nitric oxide synthase (eNOS) on disease in lupus prone MRL/lpr mice. Mice lacking eNOS developed earlier more severe disease with decreased survival. eNOS deficient mice died sooner and developed significantly more glomerular crescents, necrosis, inflammatory infiltrates and vasculitis, indicating a role for eNOS in modulating these renal lesions. Immune complex deposition was similar between groups, indicating the impact of eNOS is distal to antibody/complement glomerular deposition. Urinary nitric oxide production was decreased in the eNOS deficient mice, while proteinuria was increased. Urinary monocyte chemotactic protein-1 was also increased in the knockout mice. CD4+ T cells from MRL/lpr mice demonstrated mitochondrial hyperpolarization, increased nitric oxide and superoxide production and increased calcium flux compared to B6 control mice. Deficiency of eNOS resulted in decreased nitric oxide and mitochondrial calcium levels but had no effect on mitochondrial hyperpolarization. Renal cortices from MRL/lpr mice that are eNOS deficient demonstrated increased superoxide production, which was blocked by both nitric oxide synthase and NADPH oxidase inhibitors. These studies thus demonstrate a key role for eNOS in modulating renal disease in lupus prone MRL/lpr mice. The impact appears to be mediated by effects on superoxide production in the kidney, impacting downstream mediators such as monocyte

  6. Sinapic Acid Prevents Hypertension and Cardiovascular Remodeling in Pharmacological Model of Nitric Oxide Inhibited Rats

    PubMed Central

    Silambarasan, Thangarasu; Manivannan, Jeganathan; Krishna Priya, Mani; Suganya, Natarajan; Chatterjee, Suvro; Raja, Boobalan

    2014-01-01

    Objectives Hypertensive heart disease is a constellation of abnormalities that includes cardiac fibrosis in response to elevated blood pressure, systolic and diastolic dysfunction. The present study was undertaken to examine the effect of sinapic acid on high blood pressure and cardiovascular remodeling. Methods An experimental hypertensive animal model was induced by L-NAME intake on rats. Sinapic acid (SA) was orally administered at a dose of 10, 20 and 40 mg/kg body weight (b.w.). Blood pressure was measured by tail cuff plethysmography system. Cardiac and vascular function was evaluated by Langendorff isolated heart system and organ bath studies, respectively. Fibrotic remodeling of heart and aorta was assessed by histopathologic analyses. Oxidative stress was measured by biochemical assays. mRNA and protein expressions were assessed by RT-qPCR and western blot, respectively. In order to confirm the protective role of SA on endothelial cells through its antioxidant property, we have utilized the in vitro model of H2O2-induced oxidative stress in EA.hy926 endothelial cells. Results Rats with hypertension showed elevated blood pressure, declined myocardial performance associated with myocardial hypertrophy and fibrosis, diminished vascular response, nitric oxide (NO) metabolites level, elevated markers of oxidative stress (TBARS, LOOH), ACE activity, depleted antioxidant system (SOD, CAT, GPx, reduced GSH), aberrant expression of TGF-β, β-MHC, eNOS mRNAs and eNOS protein. Remarkably, SA attenuated high blood pressure, myocardial, vascular dysfunction, cardiac fibrosis, oxidative stress and ACE activity. Level of NO metabolites, antioxidant system, and altered gene expression were also repaired by SA treatment. Results of in vitro study showed that, SA protects endothelial cells from oxidative stress and enhance the production of NO in a concentration dependent manner. Conclusions Taken together, these results suggest that SA may have beneficial role in the

  7. Ultrafast dynamics of ligand and substrate interaction in endothelial nitric oxide synthase under Soret excitation.

    PubMed

    Hung, Chih-Chang; Yabushita, Atsushi; Kobayashi, Takayoshi; Chen, Pei-Feng; Liang, Keng S

    2016-01-01

    Ultrafast transient absorption spectroscopy of endothelial NOS oxygenase domain (eNOS-oxy) was performed to study dynamics of ligand or substrate interaction under Soret band excitation. Photo-excitation dissociates imidazole ligand in <300fs, then followed by vibrational cooling and recombination within 2ps. Such impulsive bond breaking and late rebinding generate proteinquakes, which relaxes in several tens of picoseconds. The photo excited dynamics of eNOS-oxy with L-arginine substrate mainly occurs at the local site of heme, including ultrafast internal conversion within 400fs, vibrational cooling, charge transfer, and complete ground-state recovery within 1.4ps. The eNOS-oxy without additive is partially bound with water molecule, thus its photoexcited dynamics also shows ligand dissociation in <800fs. Then it followed by vibrational cooling coupled with charge transfer in 4.8ps, and recombination of ligand to distal side of heme in 12ps.

  8. Simvastatin Attenuation of Cerebral Vasospasm After Subarachnoid Hemorrhage in Rats Via Increased Phosphorylation of Akt and Endothelial Nitric Oxide Synthase

    PubMed Central

    Sugawara, Takashi; Ayer, Robert; Jadhav, Vikram; Chen, Wanqiu; Tsubokawa, Tamiji; Zhang, John H.

    2009-01-01

    The mechanisms involved in simvastatin-mediated attenuation of cerebral vasospasm after subarachnoid hemorrhage (SAH) are unclear. We investigated the role of the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway and endothelial nitric oxide synthase (eNOS) in the cerebral vasculature in statin-mediated attenuation of cerebral vasospasm using wortmannin, an irreversible pharmacological PI3K inhibitor, and a rat SAH endovascular perforation model. Simvastatin was administered intraperitoneally in two dosages (1 mg/kg and 20 mg/kg) at 0.5, 24, and 48 hr after SAH and histological parameters of ipsilateral intracranial carotid artery (ICA) were assessed at 24 and 72 hr. SAH significantly decreased ICA diameter and perimeter while increasing wall thickness at both 24 and 72 hr. High-dosage simvastatin prevented the reduction of ICA diameter and perimeter following SAH, whereas both high and low dosages reduced wall thickness significantly at 24 and 72 hr. The effects of simvastatin were significantly reversed by wortmannin. High-dosage simvastatin increased pAkt and peNOS (phosphorylated forms) levels without increasing Akt and eNOS expression compared with the SAH group and also improved neurological deficits at 24 and 72 hr. Simvastatin did not affect protein levels by itself compared with untreated sham group. The present study elucidates the critical role of the PI3K activation leading to phosphorylation of Akt and eNOS in simvastatin-mediated attenuation of cerebral vasospasm after SAH. PMID:18683242

  9. Investigation of the effect of tanshinone IIA on nitric oxide production in human vascular endothelial cells by fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Huang, Ke-Jing; Wang, Hong; Xie, Wan-Zhen; Zhang, Hua-Shan

    2007-12-01

    Nitric oxide (NO) has been proved to be a potent vasodilator that played an important role in regulating vascular tones. Tanshinone, one of the active components of Radix Salvia miltiorrhiza, was used widely in clinics in China for treating cardiovascular diseases. The objective of this study was to sensitively and specifically investigate the effects of tanshinone IIA, one important pharmacological constituent of tanshinone, on the release of NO from human vascular endothelial cells (HVECs) by fluorescence imaging with an excellent fluorescent probe 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3',4'-diaminophenyl)-difluoroboradiaza- s-indacence (TMDCDABODIPY). After cells were incubated with tanshinone IIA, TMDCDABODIPY was employed to label NO. Following the tagging, real-time imaging of NO release from the cells was performed with inverted fluorescence microscope. The results of the experiments showed that tanshinone IIA could induce NO production significantly enhanced in HVECs. The activation of NO by tanshinone IIA may be employed therapeutically in modulating NO production in HVECs.

  10. Activation of endothelial nitric oxide synthase by dietary isoflavones: role of NO in Nrf2-mediated antioxidant gene expression.

    PubMed

    Mann, Giovanni E; Rowlands, David J; Li, Francois Y L; de Winter, Patricia; Siow, Richard C M

    2007-07-15

    The endothelium plays a key role in the maintenance of vascular homeostasis, and increased oxidative stress in vascular disease leads to reduced nitric oxide bioavailability and impaired endothelium-dependent relaxation of resistance vessels. Although epidemiological evidence suggests that diets containing high amounts of natural antioxidants afford protection against coronary heart disease (CHD), antioxidant supplementation trials have largely reported only marginal health benefits. There is controversy concerning the cardiovascular benefits of prolonged estrogen/progestin or soy isoflavone therapy for postmenopausal women and patients with an increased risk of CHD. Research on the potential health benefits of soy isoflavones and other polyphenols contained in red wine, green and black tea and dark chocolate developed rapidly during the 1990's, and recent clinical trials and studies in animal models and cultured endothelial cells provide important and novel insights into the mechanisms by which dietary polyphenols afford protection against oxidative stress. In this review, we highlight that NO and reactive oxygen radicals may mediate dietary polyphenol induced activation of Nrf2, which in turn triggers antioxidant response element (ARE) driven transcription of phase II detoxifying and antioxidant defense enzymes in vascular cells.

  11. Distinctive expression patterns of hypoxia-inducible factor-1α and endothelial nitric oxide synthase following hypergravity exposure

    PubMed Central

    Yoon, Gun; Oh, Choong Sik; Kim, Hyun-Soo

    2016-01-01

    This study was designed to examine the expression of hypoxia-inducible factor-1α (HIF-1α) and the level and activity of endothelial nitric oxide synthase (eNOS) in the hearts and livers of mice exposed to hypergravity. Hypergravity-induced hypoxia and the subsequent post-exposure reoxygenation significantly increased cardiac HIF-1α levels. Furthermore, the levels and activity of cardiac eNOS also showed significant increase immediately following hypergravity exposure and during the reoxygenation period. In contrast, the expression of phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase (p-ERK) showed significant elevation only during the reoxygenation period. These data raise the possibility that the increase in cardiac HIF-1α expression induced by reoxygenation involves a cascade of signaling events, including activation of the Akt and ERK pathways. In the liver, HIF-1α expression was significantly increased immediately after hypergravity exposure, indicating that hypergravity exposure to causes hepatocellular hypoxia. The hypergravity-exposed livers showed significantly higher eNOS immunoreactivity than did those of control mice. Consistent with these results, significant increases in eNOS activity and nitrate/nitrite levels were also observed. These findings suggest that hypergravity-induced hypoxia plays a significant role in the upregulation of hepatic eNOS. PMID:27191892

  12. Nitric oxide inhibits the formation of zinc protoporphyrin IX and protoporphyrin IX.

    PubMed

    Wakamatsu, Jun-ichi; Hayashi, Nobutaka; Nishimura, Takanori; Hattori, Akihito

    2010-01-01

    The aim of this study was to elucidate the mechanism by which curing agents, especially nitrite, inhibit the formation of zinc protoporphyrin IX (ZPP) in dry-cured hams such as Parma ham. The oxidation-reduction potential of model solutions was increased by the addition of nitrite, but it was not clear whether the formation of ZPP is inhibited by the oxidizing property of nitrite. The effect of nitric oxide (NO) produced from nitrite on the formation of ZPP was examined. The amount of ZPP formed was decreased by the addition of NO donors. The amount of protoporphyrin IX (PPIX), which is the precursor of ZPP, was also decreased by the addition of NO donors. It is concluded that NO produced from nitrite inhibited the formation of PPIX and ZPP was therefore not formed in cured meat products with the addition of nitrite or nitrate.

  13. Inhibition of dipeptidyl peptidase 4 regulates microvascular endothelial growth induced by inflammatory cytokines

    SciTech Connect

    Takasawa, Wataru; Ohnuma, Kei; Hatano, Ryo; Endo, Yuko; Dang, Nam H.

    2010-10-08

    Research highlights: {yields} TNF-{alpha} or IL-1{beta} induces EC proliferation with reduction of CD26 expression. {yields} CD26 siRNA or DPP-4 inhibition enhances TNF-{alpha} or IL-1{beta}-induced EC proliferation. {yields} Loss of CD26/DPP-4 enhances aortic sprouting induced by TNF-{alpha} or IL-1{beta}. {yields} Capillary formation induced by TNF-{alpha} or IL-1{beta} is enahced in the CD26{sup -/-} mice. -- Abstract: CD26/DPP-4 is abundantly expressed on capillary of inflamed lesion as well as effector T cells. Recently, CD26/dipeptidyl peptidase 4 (DPP-4) inhibition has been used as a novel oral therapeutic approach for patients with type 2 diabetes. While accumulating data indicate that vascular inflammation is a key feature of both micro- and macro-vascular complications in diabetes, the direct role of CD26/DPP-4 in endothelial biology is to be elucidated. We herein showed that proinflammatory cytokines such as tumor necrosis factor or interleukin-1 reduce expression of CD26 on microvascular endothelial cells, and that genetical or pharmacological inhibition of CD26/DPP-4 enhances endothelial growth both in vitro and in vivo. With DPP-4 inhibitors being used widely in the treatment of type 2 diabetes, our data strongly suggest that DPP-4 inhibition plays a pivotal role in endothelial growth and may have a potential role in the recovery of local circulation following diabetic vascular complications.

  14. Nitric oxide synthase inhibition reduces muscle inflammation and necrosis in modified muscle use

    NASA Technical Reports Server (NTRS)

    Pizza, F. X.; Hernandez, I. J.; Tidball, J. G.

    1998-01-01

    The objective of this study was to determine the role of nitric oxide in muscle inflammation, fiber necrosis, and apoptosis of inflammatory cells in vivo. The effects of nitric oxide synthase (NOS) inhibition on the concentrations of neutrophils, ED1+ and ED2+ macrophages, apoptotic inflammatory cells, and necrotic muscle fibers in rats subjected to 10 days of hindlimb unloading and 2 days of reloading were determined. Administration of NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) significantly reduced the concentrations of neutrophils, ED1+ and ED2+ macrophages, and necrotic fibers in soleus muscle relative to water-treated controls. The concentration of apoptotic inflammatory cells was also significantly lower for L-NAME-treated animals compared with water-treated controls. However, the proportion of the inflammatory cell population that was apoptotic did not differ between L-NAME-treated and control animals, suggesting that L-NAME treatment did not decrease inflammatory cell populations by increasing the frequency of apoptosis. Thus, nitric oxide or one of its intermediates promotes muscle inflammation and fiber necrosis during modified muscle use and plays no more than a minor role in the resolution of muscle inflammation by inducing apoptosis of inflammatory cells.

  15. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide.

    PubMed Central

    Beckman, J S; Beckman, T W; Chen, J; Marshall, P A; Freeman, B A

    1990-01-01

    Superoxide dismutase reduces injury in many disease processes, implicating superoxide anion radical (O2-.) as a toxic species in vivo. A critical target of superoxide may be nitric oxide (NO.) produced by endothelium, macrophages, neutrophils, and brain synaptosomes. Superoxide and NO. are known to rapidly react to form the stable peroxynitrite anion (ONOO-). We have shown that peroxynitrite has a pKa of 7.49 +/- 0.06 at 37 degrees C and rapidly decomposes once protonated with a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition generates a strong oxidant with reactivity similar to hydroxyl radical, as assessed by the oxidation of deoxyribose or dimethyl sulfoxide. Product yields indicative of hydroxyl radical were 5.1 +/- 0.1% and 24.3 +/- 1.0%, respectively, of added peroxynitrite. Product formation was not affected by the metal chelator diethyltriaminepentaacetic acid, suggesting that iron was not required to catalyze oxidation. In contrast, desferrioxamine was a potent, competitive inhibitor of peroxynitrite-initiated oxidation because of a direct reaction between desferrioxamine and peroxynitrite rather than by iron chelation. We propose that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO. under pathological conditions by preventing the formation of peroxynitrite. PMID:2154753

  16. Nitric oxide/cyclic guanosine monophosphate signaling in the central complex of the grasshopper brain inhibits singing behavior.

    PubMed

    Wenzel, Beate; Kunst, Michael; Günther, Cornelia; Ganter, Geoffrey K; Lakes-Harlan, Reinhard; Elsner, Norbert; Heinrich, Ralf

    2005-07-25

    Grasshopper sound production, in the context of mate finding, courtship, and rivalry, is controlled by the central body complex in the protocerebrum. Stimulation of muscarinic acetylcholine receptors in the central complex has been demonstrated to stimulate specific singing in various grasshoppers including the species Chorthippus biguttulus. Sound production elicited by stimulation of muscarinic acetylcholine receptors in the central complex is inhibited by co-applications of various drugs activating the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway. The nitric oxide-donor sodium nitroprusside caused a reversible suppression of muscarine-stimulated sound production that could be blocked by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxaline-1-one (ODQ), which prevents the formation of cGMP by specifically inhibiting soluble guanylyl cyclase. Furthermore, injections of both the membrane-permeable cGMP analog 8-Br-cGMP and the specific inhibitor of the cGMP-degrading phosphodiesterase Zaprinast reversibly inhibited singing. To identify putative sources of nitric oxide, brains of Ch. biguttulus were subjected to both nitric oxide synthase immunocytochemistry and NADPH-diaphorase staining. Among other areas known to express nitric oxide synthase, both procedures consistently labeled peripheral layers in the upper division of the central body complex, suggesting that neurons supplying this neuropil contain nitric oxide synthase and may generate nitric oxide upon activation. Exposure of dissected brains to nitric oxide and 3-(5'hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) induced cGMP-associated immunoreactivity in both the upper and lower division. Therefore, both the morphological and pharmacological data presented in this study strongly suggest a contribution of the nitric oxide/cGMP signaling pathway to the central control of grasshopper sound production.

  17. Adiporedoxin suppresses endothelial activation via inhibiting MAPK and NF-κB signaling

    PubMed Central

    He, Hui; Guo, Fang; Li, Yong; Saaoud, Fatma; Kimmis, Brooks D.; Sandhu, Jeena; Fan, Michelle; Maulik, Dev; Lessner, Susan; Papasian, Christopher J.; Fan, Daping; Jiang, Zhisheng; Fu, Mingui

    2016-01-01

    Adiporedoxin (Adrx) is a recently discovered redox regulatory protein that is preferentially expressed in adipose tissue and plays a critical role in the regulation of metabolism via its modulation of adipocyte protein secretion. We here report that Adrx suppresses endothelial cell activation via inhibiting MAPK and NF-kB signaling pathways. Adrx is constitutively expressed in human vascular endothelial cells, and significantly induced by a variety of stimuli such as TNFα, IL-1β, H2O2 and OxLDL. Overexpression of Adrx significantly attenuated TNFα-induced expression of VCAM-1 and ICAM-1, and thus reduced monocyte adherence to human umbilical vein endothelial cells (HUVECs). Conversely, siRNA-mediated knockdown of Adrx increased TNFα-induced expression of adhesion molecules and monocyte adherence to HUVECs. Furthermore, forced expression of Adrx decreased TNFα-induced activation of ERK1/2, JNK, p38 and IKKs in HUVECs. Adrx mutant in the CXXC motif that lost its anti-redox activity is less efficient than the wild-type Adrx, suggesting that Adrx-mediated inhibition of endothelial activation is partially dependent on its antioxidant activity. Finally, Adrx expression was markedly increased in human atheroma compared with normal tissue from the same carotid arteries. These results suggest that Adrx is an endogenous inhibitor of endothelial activation, and might be a therapeutic target for vascular inflammatory diseases. PMID:27941911

  18. The monocyte locomotion inhibitory factor produced by Entamoeba histolytica inhibits induced nitric oxide production in human leukocytes.

    PubMed

    Rico, G; Leandro, E; Rojas, S; Giménez, J A; Kretschmer, R R

    2003-07-01

    The monocyte locomotion inhibitory factor, an anti-inflammatory pentapeptide produced by Entamoeba histolytica, inhibits the in vitro production of nitric oxide induced by cytokines (INF-gamma, TNF-alpha) or PMA in human leukocytes. This can be added to the other previously reported functional effects of this factor, such as the inhibition of monocyte locomotion and the synthesis of reactive oxygen intermediates in both monocytes and neutrophils. The decreased nitric oxide production may interfere with the killing of amebas by neutrophils in the early invasive stages of amebiasis, when oxidative mechanisms are used [reactive oxygen and nitrogen intermediates either individually or synergistically via peroxynitrite (ONOO(-))], and in the advanced stages, when both non-oxidative and oxidative (including nitric oxide) mechanisms are employed by macrophages. Diminished nitric oxide production by leukocytes may also contribute to the paucity of late inflammatory components in amebic abscess of the liver and other amebic lesions.

  19. Synergistic Inhibition of Endothelial Cell Proliferation, Tube Formation, and Sprouting by Cyclosporin A and Itraconazole

    PubMed Central

    Nacev, Benjamin A.; Liu, Jun O.

    2011-01-01

    Pathological angiogenesis contributes to a number of diseases including cancer and macular degeneration. Although angiogenesis inhibitors are available in the clinic, their efficacy against most cancers is modest due in part to the existence of alternative and compensatory signaling pathways. Given that angiogenesis is dependent on multiple growth factors and a broad signaling network in vivo, we sought to explore the potential of multidrug cocktails for angiogenesis inhibition. We have screened 741 clinical drug combinations for the synergistic inhibition of endothelial cell proliferation. We focused specifically on existing clinical drugs since the re-purposing of clinical drugs allows for a more rapid and cost effective transition to clinical studies when compared to new drug entities. Our screen identified cyclosporin A (CsA), an immunosuppressant, and itraconazole, an antifungal drug, as a synergistic pair of inhibitors of endothelial cell proliferation. In combination, the IC50 dose of each drug is reduced by 3 to 9 fold. We also tested the ability of the combination to inhibit endothelial cell tube formation and sprouting, which are dependent on two essential processes in angiogenesis, endothelial cell migration and differentiation. We found that CsA and itraconazole synergistically inhibit tube network size and sprout formation. Lastly, we tested the combination on human foreskin fibroblast viability as well as Jurkat T cell and HeLa cell proliferation, and found that endothelial cells are selectively targeted. Thus, it is possible to combine existing clinical drugs to synergistically inhibit in vitro models of angiogenesis. This strategy may be useful in pursuing the next generation of antiangiogenesis therapy. PMID:21969860

  20. Chronic Inhibition of PPAR-γ Signaling Induces Endothelial Dysfunction In The Juvenile Lamb

    PubMed Central

    Sharma, Shruti; Barton, Jubilee; Rafikov, Ruslan; Aggarwal, Saurabh; Kuo, Hsuan-Chang; Oishi, Peter E.; Datar, Sanjeev A.; Fineman, Jeffrey R; Black, Stephen M.

    2013-01-01

    We have recently shown that the development of endothelial dysfunction in lambs with increased pulmonary blood flow (PBF) correlates with a decrease in peroxisome proliferator activated receptor-γ (PPAR-γ) signaling. Thus, in this study we determined if the loss of PPAR-γ signaling is necessary and sufficient to induce endothelial dysfunction by exposing lambs with normal PBF to the PPAR-γ antagonist, GW9662. Two-weeks of exposure to GW9662 significantly decreased both PPAR-γ protein and activity. In addition, although eNOS protein and nitric oxide metabolites (NOx) were significantly increased, endothelial dependent pulmonary vasodilation in response to acetylcholine was attenuated, indicative of endothelial dysfunction. To elucidate whether downstream mediators of vasodilation were impaired we examined soluble guanylate cyclase (sGC)- α and β subunit protein, cGMP levels, and phosphodiesterase 5 (PDE5) protein and activity, but we found no significant changes. However, we found that peroxynitrite levels were significantly increased in GW9662-treated lambs and this correlated with a significant increase in protein kinase G-1α (PKG-1α) nitration and a reduction in PKG activity. Peroxynitrite is formed by the interaction of NO with superoxide and we found that there was a significant increase in superoxide generation in GW9662-treated lambs. Further, we identified dysfunctional mitochondria as the primary source of the increased superoxide. Finally, we found that the mitochondrial dysfunction was due to a disruption in carnitine metabolism. We conclude that loss of PPAR-γ signaling is sufficient to induce endothelial dysfunction confirming its important role in maintaining a healthy vasculature. PMID:23257346

  1. Curcumin protects endothelial cells against homocysteine induced injury through inhibiting inflammation

    PubMed Central

    Li, Jian; Luo, Ming; Xie, Nanzi; Wang, Jianxin; Chen, Li

    2016-01-01

    Objective: This study aimed to investigate the protective effects of curcumin on the homocysteine (HCY) induced injury to the endothelial cells. Methods: Endothelial cells were treated with HCY at different concentrations, and MTT assay was employed to determine an optimal concentration of HCY. Cells were divided into 3 groups: normal control group, HCY group and HCY + curcumin group. In curcumin group, cells were pretreated with 2.5 mmol/L HCY for 2 h and then incubated with curcumin at different concentrations. MTT assay was employed to detect the cell viability. ELISA was performed to detect the content of IL-8 in the supernatant. Western blotting was used to detect NF-κB expression in cells. Results: (1) Endothelial cells were polygonal or stone-like, or aggregated to form masses, and then gradually became long spindle shaped, cell body enlarged, cells were rich in cytoplasm, and immunohistochemistry for factor VIII showed positive. (2) MTT assay showed HCY at ≥2.5 mmol/L caused significant damage to endothelial cells as compared to control group. Thus, 2.5 mmol/L HCY was used in following experiments. (3) ELISA showed IL-8 in the supernatant increased significantly in a time dependent manner after HCY treatment (P<0.01), but curcumin could significantly inhibit the IL-8 secretion in endothelial cells after HCY treatment. (4) Western blotting showed HCY was able to markedly increase NF-κB expression, which, however, was significantly inhibited by curcumin. Conclusion: Curcumin is able to protect the endothelial cells against HCY induced injury through inhibiting NF-κB activation and down-regulating IL-8 expression. PMID:27904665

  2. Autocrine inhibition of Na+/K(+)-ATPase by nitric oxide in mouse proximal tubule epithelial cells.

    PubMed Central

    Guzman, N J; Fang, M Z; Tang, S S; Ingelfinger, J R; Garg, L C

    1995-01-01

    An inducible nitric oxide synthase has recently been described in proximal tubule epithelium. To investigate the effects of proximal tubule NO on Na+/K(+)-ATPase, we induced NO production in mouse proximal tubule epithelial cells by treatment with lipopolysaccharide (LPS) and interferon-gamma (IFN gamma) followed by determinations of ouabain-sensitive ATPase activity. Na+/K(+)-ATPase activity decreased after 4 h of LPS/IFN gamma treatment, reaching maximal inhibition after 24 h (34% reduction in activity). The inhibition of Na+/K(+)-ATPase activity by LPS/IFN gamma was prevented by simultaneous incubation with N omega-nitro L-arginine and markedly blunted by removal of L-arginine from the medium. The NO donors sodium nitroprusside and SIN-1 also inhibited Na+/K(+)-ATPase activity to a similar extent than LPS/IFN gamma. However, treatment with 8-pCPT-cGMP only modestly reduced Na+/K(+)-ATPase activity. Interestingly, superoxide dismutase prevented the inhibitory effects of NO on Na+/K(+)-ATPase activity, suggesting a role for peroxynitrite in this inhibition. We conclude that NO generated by mouse proximal tubule epithelial cell iNOS inhibits Na/K ATPase activity in an autocrine fashion and that this inhibition is accompanied by a reduction in Na-dependent solute transport. PMID:7537754

  3. Direct vasorelaxation by a novel phytoestrogen tanshinone IIA is mediated by nongenomic action of estrogen receptor through endothelial nitric oxide synthase activation and calcium mobilization.

    PubMed

    Fan, Guanwei; Zhu, Yan; Guo, Hao; Wang, Xiaoying; Wang, Hong; Gao, Xiumei

    2011-03-01

    Salvia miltiorrhiza (Danshen) has been widely used in China and other Asian countries for treating various cardiovascular diseases resulting from its ability to improve coronary microcirculation and increase coronary blood flow. Tanshinone IIA (Tan IIA), the major active lipophilic ingredient responsible for the beneficial actions of Salvia miltiorrhiza, has been shown to induce vasodilation in coronary arteries. Because our recent study identified Tan IIA as a new member of the phytoestrogens, we hypothesized that its action might be mediated by estrogen receptor (ER) in vascular endothelial cells. The aim of the present study was to assess whether cardiovascular protection exerted by Tan IIA is mediated by the ER signal pathway and whether the genomic or nongenomic action of ER is involved within arteries and vascular endothelial cells. The effect of Tan IIA on blood vessels was investigated by vascular ring assay using endothelium-intact and endothelium-denuded rat aortas. Similar to estrogen, Tan IIA caused an nitric oxide- and endothelium-dependent relaxation, which was blocked by ER antagonist ICI 182,780. Primary cardiac microvascular endothelial cells were used as a model to study the cellular and molecular mechanisms of Tan IIA-induced vasorelaxation. We demonstrate that Tan IIA is capable of activating the estrogen receptor signal pathway, leading to increased endothelial nitric oxide synthase gene expression, nitric oxide production, ERK1/2 phosphorylation, and Ca mobilization. Collectively, these effects contribute to Tan IIA's vasodilative activity effects of y ER antagonist Cnt of cardiovascular diseases. Our findings support a continued effort in discovering and developing novel phytoestrogens as an alternative hormone replacement therapy for safer and more effective treatment of cardiovascular diseases.

  4. Upregulation of microRNA‑335 and microRNA‑584 contributes to the pathogenesis of severe preeclampsia through downregulation of endothelial nitric oxide synthase.

    PubMed

    Jiang, Feng; Li, Jipeng; Wu, Guojun; Miao, Zhuo; Lu, Linshan; Ren, Guoping; Wang, Xiaohong

    2015-10-01

    The aim of the present study was to identify the differentially expressed microRNAs (miRNAs) in placenta from patients with preeclampsia, and examine their roles in the pathogenesis of preeclampsia in vivo and ex vivo. The placental expression levels of miRNAs were examined in tissue samples harvested from 20 patients with preeclampsia and 20 healthy control individuals. A total of 18 miRNAs were differentially expressed (12 upregulated and six downregulated) among the preeclampsia cases, compared with the controls. By further functional/pathway analysis, two significantly upregulated miRNAs, miR‑335 and miR‑584, were identified. These target endothelial nitric oxide synthase (eNOS), which has been repeatedly reported to be involved in the development of preeclampsia. The present study then verified eNOS as a target gene of miR‑335 and miR‑584 using a luceriferase assay, and confirmed the expression patterns of the two miRNAs and eNOS in preeclampsic and normal placentas. Additionally, to examine the function of miR‑584 and miR‑335 in human placenta, the present study transiently transfected the HTR8/Svneo cell line with miR‑584 and miR‑335 mimics or their inhibitors, and the results of a subsequent Transwell insert invasion assay revealed that miR‑584 and miR‑335 inhibited the migratory ability of the trophoblast cells, and that the effect was 'rescued' by overexpressed eNOS. These data revealed a negative regulatory role of miR‑584 and miR‑335 in the migration of HTR‑8/SVneo cells by targeting eNOS, and identified miR‑584 and miR‑335 as potential novel therapeutic targets in preeclampsia.

  5. Nox4 NADPH Oxidase Mediates Peroxynitrite-dependent Uncoupling of Endothelial Nitric-oxide Synthase and Fibronectin Expression in Response to Angiotensin II

    PubMed Central

    Lee, Doug-Yoon; Wauquier, Fabien; Eid, Assaad A.; Roman, Linda J.; Ghosh-Choudhury, Goutam; Khazim, Khaled; Block, Karen; Gorin, Yves

    2013-01-01

    Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces endothelial nitric-oxide synthase (eNOS) uncoupling with enhanced generation of reactive oxygen species (ROS) and decreased production of NO. Ang II promotes a rapid increase in 3-nitrotyrosine formation, and uric acid attenuates Ang II-induced decrease in NO bioavailability, demonstrating that peroxynitrite mediates the effects of Ang II on eNOS dysfunction. Ang II rapidly up-regulates Nox4 protein. Inhibition of Nox4 abolishes the increase in ROS and peroxynitrite generation as well as eNOS uncoupling triggered by Ang II, indicating that Nox4 is upstream of eNOS. This pathway contributes to Ang II-mediated fibronectin accumulation in MCs. Ang II also elicits an increase in mitochondrial abundance of Nox4 protein, and the oxidase contributes to ROS production in mitochondria. Overexpression of mitochondrial manganese superoxide dismutase prevents the stimulatory effects of Ang II on mitochondrial ROS production, loss of NO availability, and MC fibronectin accumulation, whereas manganese superoxide dismutase depletion increases mitochondrial ROS, NO deficiency, and fibronectin synthesis basally and in cells exposed to Ang II. This work provides the first evidence that uncoupled eNOS is responsible for Ang II-induced MC fibronectin accumulation and identifies Nox4 and mitochondrial ROS as mediators of eNOS dysfunction. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal fibrosis. PMID:23940049

  6. The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells*

    PubMed Central

    Nacev, Benjamin A.; Grassi, Paola; Dell, Anne; Haslam, Stuart M.; Liu, Jun O.

    2011-01-01

    Itraconazole is a safe and widely used antifungal drug that was recently found to possess potent antiangiogenic activity. Currently, there are four active clinical trials evaluating itraconazole as a cancer therapeutic. Tumor growth is dependent on angiogenesis, which is driven by the secretion of growth factors from the tumor itself. We report here that itraconazole significantly inhibited the binding of vascular endothelial growth factor (VEGF) to VEGF receptor 2 (VEGFR2) and that both VEGFR2 and an immediate downstream substrate, phospholipase C γ1, failed to become activated after VEGF stimulation. These effects were due to a defect in VEGFR2 trafficking, leading to a decrease in cell surface expression, and were associated with the accumulation of immature N-glycans on VEGFR2. Small molecule inducers of lysosomal cholesterol accumulation and mammalian target of rapamycin (mTOR) inhibition, two previously reported itraconazole activities, failed to recapitulate itraconazole's effects on VEGFR2 glycosylation and signaling. Likewise, glycosylation inhibitors did not alter cholesterol trafficking or inhibit mTOR. Repletion of cellular cholesterol levels, which was known to rescue the effects of itraconazole on mTOR and cholesterol trafficking, was also able to restore VEGFR2 glycosylation and signaling. This suggests that the new effects of itraconazole occur in parallel to those previously reported but are downstream of a common target. We also demonstrated that itraconazole globally reduced poly-N-acetyllactosamine and tetra-antennary complex N-glycans in endothelial cells and induced hypoglycosylation of the epidermal growth factor receptor in a renal cell carcinoma line, suggesting that itraconazole's effects extend beyond VEGFR2. PMID:22025615

  7. Phloroglucinol Inhibits the in vitro Differentiation Potential of CD34 Positive Cells into Endothelial Progenitor Cells

    PubMed Central

    Kwon, Yi-Hong; Lee, Jun-Hee; Jung, Seok-Yun; Kim, Jae-Won; Lee, Sang-Hun; Lee, Dong-Hyung; Lee, Kyu-Sup; Lee, Boo-Yong; Kwon, Sang-Mo

    2012-01-01

    Inhibiting the bioactivities of circulating endothelial progenitor cells (EPCs) results in significant inhibition of neovessel formation during tumor angiogenesis. To investigate the potential effect of phloroglucinol as an EPC inhibitor, we performed several in vitro functional assays using CD34+ cells isolated from human umbilical cord blood (HUCB). Although a high treatment dose of phloroglucinol did not show any cell toxicity, it specifically induced the cell death of EPCs under serum free conditions through apoptosis. In the EPC colony-forming assay (EPC-CFA), we observed a significant decreased in the small EPC-CFUs for the phloroglucinol group, implying that phloroglucinol inhibited the early stage of EPC commitment. In addition, in the in vitro expansion assay using CD34+ cells, treatment with phloroglucinol was shown to inhibit endothelial lineage commitment, as demonstrated by the decrease in endothelial surface markers of EPCs including CD34+, CD34+/CD133+, CD34+/CD31+ and CD34+/CXCR4+. This is the first report to demonstrate that phloroglucinol can inhibit the functional bioactivities of EPCs, indicating that phloroglucinol may be used as an EPC inhibitor in the development of biosafe anti-tumor drugs that target tumor angiogenesis. PMID:24116289

  8. TGF-{beta}2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

    SciTech Connect

    Lu Jiawei; Lu Zhenyu; Reinach, Peter

    2006-11-01

    The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-{beta}2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-{beta}2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-{beta}2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-{beta}2 and FGF-2 oppositely affect BCE cell proliferation and TGF-{beta}2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-{beta}2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-{beta}2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-{beta}2-induced suppression of the PI3-kinase/AKT signaling pathway.

  9. Hop derived flavonoid xanthohumol inhibits endothelial cell functions via AMPK activation.

    PubMed

    Gallo, Cristina; Dallaglio, Katiuscia; Bassani, Barbara; Rossi, Teresa; Rossello, Armando; Noonan, Douglas M; D'Uva, Gabriele; Bruno, Antonino; Albini, Adriana

    2016-09-13

    Angiogenesis, a process characterized by the formation of new blood vessels from pre-existing ones, is a crucial step in tumor growth and dissemination. Recently, increased attention has been addressed to the ability of flavonoids to prevent cancer by suppressing angiogenesis, strategy that we named "angioprevention". Several natural compounds exert their anti-tumor properties by activating 5' adenosine monophosphate-activated protein kinase (AMPK), a key regulator of metabolism in cancer cells. Drugs with angiopreventive activities, in particular metformin, regulate AMPK in endothelial cells. Here we investigated the involvement of AMPK in the anti-angiogenic effects of xanthohumol (XN), the major prenylated flavonoid of the hop plant, and mechanisms of action. The anti-angiogenic activity of XN was more potent than epigallocatechin-3-gallate (EGCG). Treatment of endothelial cells with XN led to increased AMPK phosphorylation and activity. Functional studies using biochemical approaches confirmed that AMPK mediates XN anti-angiogenic activity. AMPK activation by XN was mediated by CAMMKβ, but not LKB1. Analysis of the downstream mechanisms showed that XN-induced AMPK activation reduced nitric oxide (NO) levels in endothelial cells by decreasing eNOS phosphorylation. Finally, AKT pathway was inactivated by XN as part of its anti-angiogenic activity, but independently from AMPK, suggesting that these two signaling pathways proceed autonomously. Our study dissects the molecular mechanism by which XN exerts its potent anti-angiogenic activity, pointing out AMPK as a crucial signal transducer.

  10. Hop derived flavonoid xanthohumol inhibits endothelial cell functions via AMPK activation

    PubMed Central

    Gallo, Cristina; Dallaglio, Katiuscia; Bassani, Barbara; Rossi, Teresa; Rossello, Armando; Noonan, Douglas M.; D'Uva, Gabriele; Bruno, Antonino; Albini, Adriana

    2016-01-01

    Angiogenesis, a process characterized by the formation of new blood vessels from pre-existing ones, is a crucial step in tumor growth and dissemination. Recently, increased attention has been addressed to the ability of flavonoids to prevent cancer by suppressing angiogenesis, strategy that we named “angioprevention”. Several natural compounds exert their anti-tumor properties by activating 5′ adenosine monophosphate-activated protein kinase (AMPK), a key regulator of metabolism in cancer cells. Drugs with angiopreventive activities, in particular metformin, regulate AMPK in endothelial cells. Here we investigated the involvement of AMPK in the anti-angiogenic effects of xanthohumol (XN), the major prenylated flavonoid of the hop plant, and mechanisms of action. The anti-angiogenic activity of XN was more potent than epigallocatechin-3-gallate (EGCG). Treatment of endothelial cells with XN led to increased AMPK phosphorylation and activity. Functional studies using biochemical approaches confirmed that AMPK mediates XN anti-angiogenic activity. AMPK activation by XN was mediated by CAMMKβ, but not LKB1. Analysis of the downstream mechanisms showed that XN-induced AMPK activation reduced nitric oxide (NO) levels in endothelial cells by decreasing eNOS phosphorylation. Finally, AKT pathway was inactivated by XN as part of its anti-angiogenic activity, but independently from AMPK, suggesting that these two signaling pathways proceed autonomously. Our study dissects the molecular mechanism by which XN exerts its potent anti-angiogenic activity, pointing out AMPK as a crucial signal transducer. PMID:27494895

  11. Lycopene inhibits LPS-induced proinflammatory mediator inducible nitric oxide synthase in mouse macrophage cells.

    PubMed

    Rafi, Mohamed M; Yadav, Prem Narayan; Reyes, Marynell

    2007-01-01

    Lycopene is a fat-soluble red-orange carotenoid found primarily in tomatoes and tomato-derived products, including tomato sauce, tomato paste, and ketchup, and other dietary sources, including dried apricots, guava, watermelon, papaya, and pink grapefruit. In this study, we have demonstrated the molecular mechanism underlying the anti-inflammatory properties of lycopene using a mouse macrophage cell line (RAW 264.7). Treatment with lycopene (10 microM) inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (40% compared with the control). Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that lycopene treatment decreased LPS-induced inducible nitric oxide synthase (iNOS) protein and mRNA expression in RAW 264.7 cells, respectively. These results suggest that lycopene has anti-inflammatory activity by inhibiting iNOS proteins and mRNA expressions in mouse macrophage cell lines. Furthermore, cyclooxygenase-2 (COX-2) protein and mRNA expression were not affected by treatment with lycopene.

  12. Potentiation of osteoclast bone-resorption activity by inhibition of nitric oxide synthase.

    PubMed Central

    Kasten, T P; Collin-Osdoby, P; Patel, N; Osdoby, P; Krukowski, M; Misko, T P; Settle, S L; Currie, M G; Nickols, G A

    1994-01-01

    We have examined the effects of modulating nitric oxide (NO) levels on osteoclast-mediated bone resorption in vitro and the effects of nitric oxide synthase (NOS) inhibitors on bone mineral density in vivo. Diaphorase-based histochemical staining for NOS activity of bone sections or highly enriched osteoclast cultures suggested that osteoclasts exhibit substantial NOS activity that may account for basal NO production. Chicken osteoclasts were cultured for 36 hr on bovine bone slices in the presence or absence of the NO-generating agent sodium nitroprusside or the NOS inhibitors N-nitro-L-arginine methyl ester and aminoguanidine. Nitroprusside markedly decreased the number of bone pits and the average pit area in comparison with control cultures. On the other hand, NOS inhibition by N-nitro-L-arginine methyl ester or aminoguanidine dramatically increased the number of bone pits and the average resorption area per pit. In a model of osteoporosis, aminoguanidine potentiated the loss of bone mineral density in ovariectomized rats. Aminoguanidine also caused a loss of bone mineral density in the sham-operated rats. Inhibition of NOS activity in vitro and in vivo resulted in an apparent potentiation of osteoclast activity. These findings suggest that endogenous NO production in osteoclast cultures may regulate resorption activity. The modulation of NOS and NO levels by cells within the bone microenvironment may be a sensitive mechanism for local control of osteoclast bone resorption. Images PMID:7513424

  13. Human astrocytes inhibit Cryptococcus neoformans growth by a nitric oxide-mediated mechanism

    PubMed Central

    1994-01-01

    Cryptococcus neoformans is an opportunistic fungus that causes life- threatening meningoencephalitis in 5-10% of patients with acquired immune deficiency syndrome. Cryptococcal meningoencephalitis is characterized by a lymphohistiocytic infiltrate, accumulation of encapsulated forms of C. neoformans, and varying degrees of glial reaction. Little is known about the contribution of endogenous central nervous system cells to the pathogenesis of cryptococcal infections. In this study, we investigated the role of astrocytes as potential effector cells against C. neoformans. Primary cultures of human fetal astrocytes, activated with interleukin 1 beta plus interferon gamma inhibited the growth of C. neoformans. The inhibition of C. neoformans growth was paralleled by production of nitrite, and reversed by the inhibitors of nitric oxide (NO.) synthase, NG-methyl-mono-arginine and NG-nitro-arginine methyl ester. The results suggest a novel function for human astrocytes in host defence and provide a precedent for the use of NO. as an antimicrobial effector molecule by human cells. PMID:8006595

  14. Chitinous materials inhibit nitric oxide production by activated RAW 264.7 macrophages.

    PubMed

    Hwang, S M; Chen, C Y; Chen, S S; Chen, J C

    2000-04-29

    Chitinous materials have been studied in wound healing and artificial skin substitutes for many years. Nitric oxide (NO) has been shown to contribute to cytotoxicity in cell proliferation during inflammation of wound healing. In this study, we examined the effect of chitin and its derivatives on NO production by activated RAW 264.7 macrophages. Chitin and chitosan showed a significantly inhibitory effect on NO production by the activated macrophages. Hexa-N-acetylchitohexaose and penta-N-acetylchitopentaose also inhibited NO production but with less potency. However, N-acetylchitotetraose, -triose, -biose, and monomer of chitin, N-acetylglucosamine and glucosamine had little effect on NO production by the activated cells. These results suggest that the promotive effect of chitinous material on wound healing be related, at least partly, to inhibit NO production by the activated macrophages.

  15. [Knockdown of RUNX3 inhibits hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells].

    PubMed

    Liu, Yanhua; Li, Bingong; Wang, Yuqin; Wang, Delong; Zou, Jin; Ke, Xuan; Hao, Yanqin

    2016-12-01

    Objective To investigate the effects of Runt-related transcription factor 3 (RUNX3) knockdown on hypoxia-induced endothelial-to-mesenchymal transition (EndoMT) of human cardiac microvascular endothelial cells (HCMECs), and elucidate the underlying molecular mechanism. Methods HCMECs were cultured in hypoxic conditions and infected with RUNX3-RNAi lentivirus to knock-down the expression of RUNX3. Reverse transcription PCR was performed to detect the mRNA expressions of RUNX3 and EndoMT related genes such as CD31, vascular endothelial cadherin (VE-cadherin), α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1); Western blotting was used to determine the protein expressions of RUNX3, CD31, α-SMA and another molecules involved in EndoMT; and immunofluorescence cytochemistry was applied to observe the colocalization of CD31 and α-SMA. Results Hypoxia induced the transition of HCMECs to mesenchymal cells. Hypoxia up-regulated the expression of TGF-β2, Smad2/3, phosphorylation of Smad2/3 (p-Smad2/3), Notch-1, Hes1, and Hey1; knockdown of RUNX3 down-regulated the levels of Smad2/3, p-Smad2/3, Hes1, and Hey1 to different extents, and raised the levels of TGF-β2 and Notch-1. Conclusion Knockdown of RUNX3 in HCMECs attenuates hypoxia-induced EndoMT via partially inhibiting TGF-β and Notch signaling pathway.

  16. TRAF6 inhibits proangiogenic signals in endothelial cells and regulates the expression of vascular endothelial growth factor

    SciTech Connect

    Bruneau, Sarah; Datta, Dipak; Flaxenburg, Jesse A.; Pal, Soumitro; Briscoe, David M.

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer TNF-receptor associated factors (TRAFs) function in the angiogenesis response. Black-Right-Pointing-Pointer TRAF6 regulates basal and inducible expression of VEGF in endothelial cells (EC). Black-Right-Pointing-Pointer TRAF6 is an endogenous inhibitor of EC proliferation and migration in EC. Black-Right-Pointing-Pointer TRAF6 inhibits VEGF expression in part via its ability to regulate Src signaling. -- Abstract: TNF-family molecules induce the expression Vascular Endothelial Growth Factor (VEGF) in endothelial cells (EC) and elicit signaling responses that result in angiogenesis. However, the role of TNF-receptor associated factors (TRAFs) as upstream regulators of VEGF expression or as mediators of angiogenesis is not known. In this study, HUVEC were cotransfected with a full-length VEGF promoter-luciferase construct and siRNAs to TRAF 1, -2, -3, -5, -6, and promoter activity was measured. Paradoxically, rather than inhibiting VEGF expression, we found that knockdown of TRAF6 resulted in a 4-6-fold increase in basal VEGF promoter activity compared to control siRNA-transfected EC (P < 0.0001). In addition, knockdown of TRAF 1, -2, -3 or -5 resulted in a slight increase or no change in VEGF promoter activation. Using [{sup 3}H]thymidine incorporation assays as well as the in vitro wound healing assay, we also found that basal rates of EC proliferation and migration were increased following TRAF6 knockdown; and this response was inhibited by the addition of a blocking anti-VEGF antibody into cell cultures. Using a limited protein array to gain insight into TRAF6-dependent intermediary signaling responses, we observed that TRAF6 knockdown resulted in an increase in the activity of Src family kinases. In addition, we found that treatment with AZD-0530, a pharmacological Src inhibitor, reduced the regulatory effect of TRAF6 knockdown on VEGF promoter activity. Collectively, these findings define a novel pro-angiogenic signaling

  17. Inhibition of NF-kappaB DNA binding by nitric oxide.

    PubMed Central

    Matthews, J R; Botting, C H; Panico, M; Morris, H R; Hay, R T

    1996-01-01

    It has been suggested that the NF-kappaB transcription factor family may mediate expression of the gene encoding the cytokine-inducible form of nitric oxide synthase (iNOS). To establish if nitric oxide (NO) could in turn affect activity of NF-kappaB, the ability of NO-donor compounds to influence NF-kappaB DNA binding activity in vitro was investigated. NO-donor compounds sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) both inhibited the DNA binding activity of recombinant NF-kappaB p50 and p65 homodimers and of p50-p65 heterodimers. Inhibition of NF-kappaB p50 DNA binding by NO-donor compounds involved modification of the conserved redox-sensitive C62 residue, as a C62S p50 mutant was significantly more resistant to SNP-mediated inactivation. Non-reducing SDS-polyacrylamide gel electrophoresis demonstrated that SNP could inhibit p50 DNA binding by mechanisms other than the formation of intersubunit disulphide bonds involving p50 residue C62. Electrospray ionization mass spectrometry of a synthetic NF-kappaB p5O peptide containing the C62 residue suggested that NO gas can modify C62 by S-nitrosylation. This study indicates that NO-donors can directly inhibit the DNA binding activity of NF-kappaB family proteins, suggesting that cellular NO provides another control mechanism for modulating the expression of NF-kappaB-responsive genes. PMID:8710491

  18. Oxidants downstream from superoxide inhibit nitric oxide production by vascular endothelium--a key role for selenium-dependent enzymes in vascular health.

    PubMed

    McCarty, M F

    1999-10-01

    Although superoxide can directly quench endothelium-generated nitric oxide (NO), there is considerable evidence that oxidants derived from superoxide--notably peroxides and their further derivatives--can also impair NO bioactivity. In part, this reflects inhibition of NO synthase activity, perhaps mediated by the oxidation of labile sulfhydryl groups, as well as the activation of protein kinase C. Selenium deficiency exacerbates these effects, presumably owing to the crucial role of selenium-dependent thioredoxin reductase and glutathione peroxidases in preventing and reversing oxidant damage to proteins. High-normal homocyst(e)ine levels may induce an 'effective selenium deficiency' by suppressing glutathione peroxidase transcription in endothelial cells. Considerable epidemiology, primarily of European origin, points to mediocre selenium nutrition as a significant vascular risk factor; the risk associated with elevated plasma homocyst(e)ine levels is now well established. In addition to preventing LDL oxidation, vitamin E can be expected to minimize the contribution of lipid peroxides to endothelial dysfunction. Lipoic acid, which can function in vivo as a versatile antioxidant and sulfhydryl reductant, may have particular value for protecting endothelium from oxidants; its clinical utility in diabetic neuropathy may reflect this benefit. Good selenium status, as well as supra-nutritional intakes of lipoic acid, may down-regulate cytokine-mediated endothelial activation by helping to maintain the proper structure of oxidant-labile proteins--such as tyrosine phosphatases--that modulate this signaling. It can be concluded that a number of supplemental nutrients--including selenium, vitamin E, lipoic acid, and the vitamins that promote catabolism of homocysteine--have the potential to promote vascular health by mitigating the adverse impact of superoxide-derived oxidants on endothelial function.

  19. Linagliptin but not Sitagliptin inhibited transforming growth factor-β2-induced endothelial DPP-4 activity and the endothelial-mesenchymal transition.

    PubMed

    Shi, Sen; Kanasaki, Keizo; Koya, Daisuke

    2016-02-26

    Dipeptidyl peptidase (DPP)-4 plays an important role in endothelial cell biology. We have shown that the DPP-4 inhibitor Linagliptin can inhibit the endothelial-mesenchymal transition (EndMT) and ameliorate diabetic kidney fibrosis associated with the suppression of DPP-4 protein levels via the induction of miR-29. The current study demonstrated that such effects of Linagliptin on endothelial cell profibrotic programs were drug-specific but not class effects. In the cell-free system, both Linagliptin and Sitagliptin inhibited recombinant DPP-4 activity in a concentration-dependent manner. Linagliptin can inhibit all of the following: DPP-4 activity and protein level, integrin β1 protein levels, EndMT, and DPP-4 3'UTR activity; Sitagliptin, however, inhibited none of these in the current study. Additionally, TGF-β2 induced both the induction of VEGF-R1 and the suppression of VEGF-R2 levels in endothelial cells, and both were inhibited by Linagliptin but not by Sitagliptin. miR-29, the miR that negatively regulates the 3'UTR of DPP-4 mRNA, was suppressed by TGF-β2 and restored by Linagliptin but not by Sitagliptin. Following the overexpression of pCMV-DPP-4-GFP and pCMV6-Myc-DPP-4 in endothelial cells, the proximity of Myc-DPP-4 and DPP-4-GFP was suppressed by Linagliptin but not by Sitagliptin, suggesting that only Linagliptin inhibited the homo-dimer formation of DPP-4 in endothelial cells; this difference in activity between the two gliptins could explain their diverse effects on endothelial cell biology. In conclusion, each of the DPP-4 inhibitors may have unique drug-specific effects.

  20. Ascorbate inhibits platelet-endothelial adhesion in an in-vitro model of sepsis via reduced endothelial surface P-selectin expression.

    PubMed

    Secor, Dan; Swarbreck, Scott; Ellis, Christopher G; Sharpe, Michael D; Feng, Qingping; Tyml, Karel

    2017-01-01

    Plugging of the capillary bed can lead to organ failure and mortality in sepsis. We have reported that intravenous ascorbate injection reduces platelet adhesion to the capillary wall and capillary plugging in septic mice. Both platelet adhesion and capillary plugging require P-selectin, a key adhesion molecule. To elucidate the beneficial effect of ascorbate, we hypothesized that ascorbate reduces platelet-endothelial adhesion by reducing P-selectin surface expression in endothelial cells. We used mouse platelets, and monolayers of cultured microvascular endothelial cells (mouse skeletal muscle origin) stimulated with lipopolysaccharide, to examine platelet-endothelial adhesion. P-selectin mRNA expression in endothelial cells was determined by real-time PCR and P-selectin protein expression at the surface of these cells by immunofluorescence. Secretion of von Willebrand factor from cells into the supernatant (a measure of P-selectin-containing granule exocytosis) was determined by ELISA. Lipopolysaccharide (10 μg/ml, 1 h) increased platelet-endothelial adhesion. P-selectin-blocking antibody inhibited this adhesion. Lipopolysaccharide also increased P-selectin mRNA in endothelial cells, P-selectin expression at the endothelial surface, and von Willebrand factor secretion. Ascorbate pretreatment (100 μmol/l, 4 h) inhibited the increased platelet adhesion, surface expression of P-selectin, and von Willebrand factor secretion, but not the increase in P-selectin mRNA. The lipopolysaccharide-induced increase in platelet-endothelial adhesion requires P-selectin presence at the endothelial surface. Ascorbate's ability to reduce this presence could be important in reducing both platelet adhesion to the capillary wall and capillary plugging in sepsis.

  1. Curcumin supplementation improves vascular endothelial function in healthy middle-aged and older adults by increasing nitric oxide bioavailability and reducing oxidative stress

    PubMed Central

    Santos-Parker, Jessica R.; Strahler, Talia R.; Bassett, Candace J.; Bispham, Nina Z.; Chonchol, Michel B.; Seals, Douglas R.

    2017-01-01

    We hypothesized that curcumin would improve resistance and conduit artery endothelial function and large elastic artery stiffness in healthy middle-aged and older adults. Thirty-nine healthy men and postmenopausal women (45-74 yrs) were randomized to 12 weeks of curcumin (2000 mg/day Longvida®; n=20) or placebo (n=19) supplementation. Forearm blood flow response to acetylcholine infusions (FBFACh; resistance artery endothelial function) increased 37% following curcumin supplementation (107±13 vs. 84±11 AUC at baseline, P=0.03), but not placebo (P=0.2). Curcumin treatment augmented the acute reduction in FBFACh induced by the nitric oxide synthase inhibitor NG monomethyl-L-arginine (L-NMMA; P=0.03), and reduced the acute increase in FBFACh to the antioxidant vitamin C (P=0.02), whereas placebo had no effect (both P>0.6). Similarly, brachial artery flow-mediated dilation (conduit artery endothelial function) increased 36% in the curcumin group (5.7±0.4 vs. 4.4±0.4% at baseline, P=0.001), with no change in placebo (P=0.1). Neither curcumin nor placebo influenced large elastic artery stiffness (aortic pulse wave velocity or carotid artery compliance) or circulating biomarkers of oxidative stress and inflammation (all P>0.1). In healthy middle-aged and older adults, 12 weeks of curcumin supplementation improves resistance artery endothelial function by increasing vascular nitric oxide bioavailability and reducing oxidative stress, while also improving conduit artery endothelial function. PMID:28070018

  2. Nitric oxide generation by endothelial cells exposed to shear stress in glass tubes perfused with red blood cell suspensions: role of aggregation.

    PubMed

    Yalcin, Ozlem; Ulker, Pinar; Yavuzer, Ugur; Meiselman, Herbert J; Baskurt, Oguz K

    2008-05-01

    Endothelial function is modulated by wall shear stress acting on the vessel wall, which is determined by fluid velocity and the local viscosity near the vessel wall. Red blood cell (RBC) aggregation may affect the local viscosity by favoring axial migration. The aim of this study was to investigate the role of RBC aggregation, with or without altered plasma viscosity, in the mechanically induced nitric oxide (NO)-related mechanisms of endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured on the inner surface of cylindrical glass capillaries that were perfused with RBC suspensions having normal and increased aggregation at a nominal shear stress of 15 dyn/cm(2). RBC aggregation was enhanced by two different approaches: 1) poloxamer-coated RBC suspended in normal, autologous plasma, resulting in enhanced aggregation but unchanged plasma viscosity and 2) normal RBC suspended in autologous plasma containing 0.5% dextran (mol mass 500 kDa), with a similar level of RBC aggregation but higher plasma viscosity. Compared with normal cells in unmodified plasma, perfusion with suspensions of poloxamer-coated RBC in normal plasma resulted in decreased levels of NO metabolites and serine 1177 phosphorylation of endothelial nitric oxide synthase (eNOS). Perfusion with normal RBC in plasma containing dextran resulted in a NO level that remained elevated, whereas only a modest decrease of phosphorylated eNOS level was observed. The results of this study suggest that increases of RBC aggregation tendency affect endothelial cell functions by altering local blood composition, especially if the alterations of RBC aggregation are due to modified cellular properties and not to plasma composition changes.

  3. Aldosterone Inactivates the Endothelin-B Receptor via a Cysteinyl Thiol Redox Switch to Decrease Pulmonary Endothelial Nitric Oxide Levels and Modulate Pulmonary Arterial Hypertension

    PubMed Central

    Maron, Bradley A.; Zhang, Ying-Yi; White, Kevin; Chan, Stephen Y.; Handy, Diane E.; Mahoney, Christopher E.; Loscalzo, Joseph; Leopold, Jane A.

    2012-01-01

    Background Pulmonary arterial hypertension (PAH) is characterized, in part, by decreased endothelial nitric oxide (NO•) production and elevated levels of endothelin-1. Endothelin-1 is known to stimulate endothelial nitric oxide synthase (eNOS) via the endothelin-B receptor (ETB), suggesting that this signaling pathway is perturbed in PAH. Endothelin-1 also stimulates adrenal aldosterone synthesis; in systemic blood vessels, hyperaldosteronism induces vascular dysfunction by increasing endothelial reactive oxygen species (ROS) generation and decreasing NO• levels. We hypothesized that aldosterone modulates PAH by disrupting ETB-eNOS signaling through a mechanism involving increased pulmonary endothelial oxidant stress. Methods and Results In rats with PAH, elevated endothelin-1 levels were associated with elevated aldosterone levels in plasma and lung tissue and decreased lung NO• metabolites in the absence of left heart failure. In human pulmonary artery endothelial cells (HPAECs), endothelin-1 increased aldosterone levels via PGC-1α/steroidogenesis factor-1-dependent upregulation of aldosterone synthase. Aldosterone also increased ROS production, which oxidatively modified cysteinyl thiols in the eNOS-activating region of ETB to decrease endothelin-1-stimulated eNOS activity. Substitution of ETB-Cys405 with alanine improved ETB-dependent NO• synthesis under conditions of oxidant stress, confirming that Cys405 is a redox sensitive thiol that is necessary for ETB-eNOS signaling. In HPAECs, mineralocorticoid receptor antagonism with spironolactone decreased aldosterone-mediated ROS generation and restored ETB-dependent NO• production. Spironolactone or eplerenone prevented or reversed pulmonary vascular remodeling and improved cardiopulmonary hemodynamics in two animal models of PAH in vivo. Conclusions Our findings demonstrate that aldosterone modulates an ETB cysteinyl thiol redox switch to decrease pulmonary endothelium-derived NO• and promote PAH

  4. Endothelial nitric oxide synthase regulates white matter changes via the BDNF/TrkB pathway after stroke in mice.

    PubMed

    Cui, Xu; Chopp, Michael; Zacharek, Alex; Ning, Ruizhuo; Ding, Xiaoshuang; Roberts, Cynthia; Chen, Jieli

    2013-01-01

    Stroke induced white matter (WM) damage is associated with neurological functional deficits, but the underlying mechanisms are not well understood. In this study, we investigate whether endothelial nitric oxide synthase (eNOS) affects WM-damage post-stroke. Adult male wild-type (WT) and eNOS knockout (eNOS(-/-)) mice were subjected to middle cerebral artery occlusion. Functional evaluation, infarct volume measurement, immunostaining and primary cortical cell culture were performed. To obtain insight into the mechanisms underlying the effects of eNOS(-/-) on WM-damage, measurement of eNOS, brain-derived neurotrophic factor (BDNF) and its receptor TrkB in vivo and in vitro were also performed. No significant differences were detected in the infarction volume, myelin density in the ipsilateral striatal WM-bundles and myelin-based protein expression in the cerebral ischemic border between WT and eNOS(-/-) mice. However, eNOS(-/-) mice showed significantly: 1) decreased functional outcome, concurrent with decreases of total axon density and phosphorylated high-molecular weight neurofilament density in the ipsilateral striatal WM-bundles. Correlation analysis showed that axon density is significantly positive correlated with neurological functional outcome; 2) decreased numbers of oligodendrocytes / oligodendrocyte progenitor cells in the ipsilateral striatum; 3) decreased synaptophysin, BDNF and TrkB expression in the ischemic border compared with WT mice after stroke (n = 12/group, p<0.05). Primary cortical cell culture confirmed that the decrease of neuronal neurite outgrowth in the neurons derived from eNOS(-/-) mice is mediated by the reduction of BDNF/TrkB (n = 6/group, p<0.05). Our data show that eNOS plays a critical role in WM-damage after stroke, and eNOS(-/-)-induced decreases in the BDNF/TrkB pathway may contribute to increased WM-damage, and thereby decrease functional outcome.

  5. Endothelial nitric oxide synthase gene variants and haplotypes associated with an increased risk of idiopathic recurrent miscarriage.

    PubMed

    Almawi, W Y; Guarino, B D; Al-Sulaiti, M A; Al-Busaidi, A S; Racoubian, E; Finan, R R

    2013-09-01

    We investigated the association of endothelial nitric oxide synthase (NOS3) polymorphisms rs2070744 (-786T> C), 27-bp repeat 4b/4a, rs1799983 (Glu298Asp), rs3918188 (-734C> A), and rs743507 (113G> A) with idiopathic recurrent miscarriage (IRM). This was a case-control study involving women with confirmed IRM (n = 296), and 305 age- and ethnically matched control women. NOS3 rs2070744, rs1799983, rs3918188, and rs743507 genotyping was done by TaqMan assays; NOS3 4b/4a genotyping was done by PCR-ASA. A higher frequency of -786C and 298Asp alleles was seen in IRM cases, which remained associated independently with IRM on multivariate analysis. Allele and genotype distribution of 4b/4a, rs3918188 (-734C> A) and rs743507 (113A> G) were comparable between IRM cases and control women. Taking homozygous wild-type genotype as a reference, regression analysis confirmed the association of Glu298Asp and -786T/C, and rs743507 homozygous carriers with IRM risk. Marked linkage disequilibrium was seen between tested NOS3 variants, thus allowing the construction of 5-locus [-786T> C/4b4a/Glu298Asp/-734C> A/113G> A] haplotypes. Taking the common T4bGCA haplotype as a reference, multivariate analysis confirmed the positive association of C4bTCG haplotype with IRM, after controlling for traditional covariates. Genetic variation at the NOS3 locus represents a genetic risk factor for increased susceptibility to IRM.

  6. MicroRNA-335 and -543 suppress bone metastasis in prostate cancer via targeting endothelial nitric oxide synthase.

    PubMed

    Fu, Qizhong; Liu, Xianfeng; Liu, Ying; Yang, Jianxun; Lv, Guangyao; Dong, Shengfang

    2015-11-01

    Skeletal metastasis is the major problem in the management of prostate cancer (PCa). Even though the regulatory role of microRNAs (miRNAs) in the control of tumor metastases has been well described in numerous types of cancer, the importance in bone metastasis of PCa remains largely unknown. In the present study, the differentially expressed miRNAs were identified between the primary PCa and bone metastatic PCa samples by comparing their expression profiling using miRNA microarray, and 4 miRNAs (miR‑335, ‑543, ‑196 and ‑19a) were noted to be significantly downregulated in bone metastasis compared with primary PCa. Among those, the downregulation of 2 miRNAs (miR‑335 and ‑543) was confirmed in a total of 20 paired primary PCa and bone metastasis samples using reverse transcription‑quantitative polymerase chain reaction. Using the online target prediction tool, endothelial nitric oxide synthase (eNOS) was found to be a shared target of miR‑335 and ‑543, which was further verified using the luciferase assay. By examining the expression pattern of eNOS in primary PCa and skeletal metastatic samples, the mRNA and protein expression levels of eNOS were markedly upregulated in the metastatic samples. Furthermore, exogenous overexpression of miR‑335 and ‑543 significantly downregulated the expression level of eNOS, and substantially compromised the ability of migration and invasion in vitro. These findings suggested that miR‑335 and ‑543 are associated with bone metastasis of PCa and indicated that they may have important roles in the bone metastasis, which may also be clinically used as novel biomarkers in discriminating the different stages of human PCa and predicting bone metastasis.

  7. Endothelin-1 but not Endothelial Nitric Oxide Synthase Gene Polymorphism is Associated with Sickle Cell Disease in Africa

    PubMed Central

    Thakur, Tanya J; Guindo, Aldiouma; Cullifer, Londyn R; Li, Yi; Imumorin, Ikhide G; Diallo, Dapa A; Thomas, Bolaji N

    2014-01-01

    Sickle cell disease shows marked variability in severity and pathophysiology among individuals, probably linked to differential expression of various adhesion molecules. In this study, we investigated the differential distribution, genomic diversity and haplotype frequency of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) polymorphisms, recently implicated as important in modification of disease severity. One hundred and forty five sickle cell disease patients (HbSS) and 244 adult and pediatric controls, without sickle cell disease (HbAA), were recruited from Mali. Genotypic analysis of the functionally significant eNOS variants (T786C, G894T and intron 4) and endothelin-1 (G5665T) was carried out with a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Our results show that the wild type alleles are the most frequent for all eNOS variants between cases and controls. Allelic and genotypic frequencies of eNOS polymorphic groups are not significantly different between cases and controls (P > 0.05). In addition, there is no association between eNOS variants and sickle cell disease, contrary to published reports. On the other hand, we report that endothelin-1 (G5665T) mutant variant had the lowest allelic frequency, and is significantly associated with sickle cell disease in Africa (P < 0.05). Similarly, haplotype frequencies were the same between cases and controls, except for the haplotype combining all mutant variants (T, C, 4a; P = 0.01). eNOS polymorphic variants are less frequent, with no significance with sickle cell disease in Africa. On the other hand, endothelin-1 is associated with sickle cell disease, and has the capacity to redefine pathophysiology and possibly serve as modulator of disease phenotype. PMID:24932102

  8. Intrauterine growth retardation in endothelial nitric oxide synthase-deficient mice is established from early stages of pregnancy.

    PubMed

    Pallares, Pilar; Gonzalez-Bulnes, Antonio

    2008-06-01

    The current study aimed to determine effects of deficiencies in nitric oxide synthase (NOS) 3 on embryo and fetal development by in vivo, noninvasive, real-time ultrasonographic assessment of phenotypic changes in Nos3-knockout pregnant mice and their wild-type counterparts. From Day 4.5 of pregnancy onwards, embryonic vesicle diameters, crown-rump lengths, and trunk diameters were obtained by serial scanning of seven adult pregnant female mice, strain B6.129P2-Nos3(tm1Unc)/J, N9 generation backcrossing with C57BL/6J mice, homozygous for the disruption of the endothelial NOS gene (group Nos3(-/-)), and 12 pregnant, wild-type C57BL/6J mice (group Nos3(+/+)). All the measurements increased in both genotypes throughout gestation. However, embryo length and width were significantly larger in Nos3(+/+) than in Nos3(-/-) mice from Day 8.5, and both longitudinal and transverse diameters of the entire gestational sacs were larger in Nos3(+/+) mice from Day 10.5. Assessment of the relative growth of embryos/fetuses and gestational annexes showed different patterns among Nos3(-/-) and Nos3(+/+) mice. Throughout pregnancy, the distance between the external limit of the gestational sac and the embryo in Nos3(+/+) mice diminished in longitudinal sections, or remained unaffected in transverse sections. In Nos3(-/-) mice, there were significant increases (P < 0.005) in the differences between embryo and gestational vesicle measurements in both longitudinal and transversal curves from Days 5.5 to 14.5, but from Day 14.5 of pregnancy onward, the changes were not significant. The results demonstrate that the processes of fetal growth retardation in the Nos3(-/-) mice are established from early pregnancy stages.

  9. The Endothelial Nitric Oxide Synthase Gene T-786C Polymorphism Increases Myocardial Infarction Risk: A Meta-Analysis.

    PubMed

    Kong, Xiang-Zhen; Zhang, Zheng-Yi; Wei, Lian-Hua; Li, Rui; Yu, Jing

    2017-02-11

    BACKGROUND Polymorphisms of the endothelial nitric oxide synthase (eNOS) gene are reportedly associated with myocardial infarction (MI) risk. However, definitive evidence of this association is lacking. In this study, we investigated the potential association of eNOS gene polymorphisms with MI risk by conducting a meta-analysis of studies evaluating this association. MATERIAL AND METHODS PubMed, Web of Knowledge, ScienceDirect, China National Knowledge Infrastructure (CNKI), WanFang, and Database of Chinese Scientific and Technical Periodicals (VIP) were searched for relevant studies. Pooled odds ratios (OR) with 95% confidence interval (CI) were calculated to evaluate the association of eNOS gene T-786C and 4b4a polymorphisms with MI risk. RESULTS Fifteen studies with 8,067 controls and 4,923 MI cases were included in the final meta-analysis. In the overall analysis, T-786C (rs2070744) polymorphism was associated with MI risk (p<0.05, OR=1.69, 95% CI: 1.53-1.86 for T vs. C; p<0.05, OR=2.76, 95% CI: 2.03-3.75 for TT vs. CC; p<0.05, OR=1.74, 95% CI 1.56-1.95 for TT vs. (CT + CC); p<0.05, OR=2.43, 95% CI: 1.79-3.30 for (CT + TT) vs. CC). In addition, a significant association between 4b4a VNTR polymorphism and MI risk was observed. On sub-group analyses by ethnicity, a significant increase in MI risk was observed separately for Asian and Caucasian populations for T-786C polymorphism, but not for the 4b4a polymorphism. CONCLUSIONS In this meta-analysis, T-786C polymorphism of the eNOS gene was associated with the risk of MI, especially in the Asian populations.

  10. The Endothelial Nitric Oxide Synthase Gene T-786C Polymorphism Increases Myocardial Infarction Risk: A Meta-Analysis

    PubMed Central

    Kong, Xiang-Zhen; Zhang, Zheng-Yi; Wei, Lian-Hua; Li, Rui; Yu, Jing

    2017-01-01

    Background Polymorphisms of the endothelial nitric oxide synthase (eNOS) gene are reportedly associated with myocardial infarction (MI) risk. However, definitive evidence of this association is lacking. In this study, we investigated the potential association of eNOS gene polymorphisms with MI risk by conducting a meta-analysis of studies evaluating this association. Material/Methods PubMed, Web of Knowledge, ScienceDirect, China National Knowledge Infrastructure (CNKI), WanFang, and Database of Chinese Scientific and Technical Periodicals (VIP) were searched for relevant studies. Pooled odds ratios (OR) with 95% confidence interval (CI) were calculated to evaluate the association of eNOS gene T-786C and 4b4a polymorphisms with MI risk. Results Fifteen studies with 8,067 controls and 4,923 MI cases were included in the final meta-analysis. In the overall analysis, T-786C (rs2070744) polymorphism was associated with MI risk (p<0.05, OR=1.69, 95% CI: 1.53–1.86 for T vs. C; p<0.05, OR=2.76, 95% CI: 2.03–3.75 for TT vs. CC; p<0.05, OR=1.74, 95% CI 1.56–1.95 for TT vs. (CT + CC); p<0.05, OR=2.43, 95% CI: 1.79–3.30 for (CT + TT) vs. CC). In addition, a significant association between 4b4a VNTR polymorphism and MI risk was observed. On sub-group analyses by ethnicity, a significant increase in MI risk was observed separately for Asian and Caucasian populations for T-786C polymorphism, but not for the 4b4a polymorphism. Conclusions In this meta-analysis, T-786C polymorphism of the eNOS gene was associated with the risk of MI, especially in the Asian populations. PMID:28188309

  11. Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells

    PubMed Central

    Ibiza, Sales; Pérez-Rodríguez, Andrea; Ortega, Ángel; Martínez-Ruiz, Antonio; Barreiro, Olga; García-Domínguez, Carlota A.; Víctor, Víctor M.; Esplugues, Juan V.; Rojas, José M.; Sánchez-Madrid, Francisco; Serrador, Juan M.

    2008-01-01

    Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys118, suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys118 contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments. PMID:18641128

  12. Low intensity exercise prevents disturbances in rat cardiac insulin signaling and endothelial nitric oxide synthase induced by high fructose diet.

    PubMed

    Stanišić, Jelena; Korićanac, Goran; Ćulafić, Tijana; Romić, Snježana; Stojiljković, Mojca; Kostić, Milan; Pantelić, Marija; Tepavčević, Snežana

    2016-01-15

    Increase in fructose consumption together with decrease in physical activity contributes to the development of metabolic syndrome and consequently cardiovascular diseases. The current study examined the preventive role of exercise on defects in cardiac insulin signaling and function of endothelial nitric oxide synthase (eNOS) in fructose fed rats. Male Wistar rats were divided into control, sedentary fructose (received 10% fructose for 9 weeks) and exercise fructose (additionally exposed to low intensity exercise) groups. Concentration of triglycerides, glucose, insulin and visceral adipose tissue weight were determined to estimate metabolic syndrome development. Expression and/or phosphorylation of cardiac insulin receptor (IR), insulin receptor substrate 1 (IRS1), tyrosine-specific protein phosphatase 1B (PTP1B), Akt, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and eNOS were evaluated. Fructose overload increased visceral adipose tissue, insulin concentration and homeostasis model assessment index. Exercise managed to decrease visceral adiposity and insulin level and to increase insulin sensitivity. Fructose diet increased level of cardiac PTP1B and pIRS1 (Ser307), while levels of IR and ERK1/2, as well as pIRS1 (Tyr 632), pAkt (Ser473, Thr308) and pERK1/2 were decreased. These disturbances were accompanied by reduced phosphorylation of eNOS at Ser1177. Exercise managed to prevent most of the disturbances in insulin signaling caused by fructose diet (except phosphorylation of IRS1 at Tyr 632 and phosphorylation and protein expression of ERK1/2) and consequently restored function of eNOS. Low intensity exercise could be considered as efficient treatment of cardiac insulin resistance induced by fructose diet.

  13. Endothelin-1 but not Endothelial Nitric Oxide Synthase Gene Polymorphism is Associated with Sickle Cell Disease in Africa.

    PubMed

    Thakur, Tanya J; Guindo, Aldiouma; Cullifer, Londyn R; Li, Yi; Imumorin, Ikhide G; Diallo, Dapa A; Thomas, Bolaji N

    2014-01-01

    Sickle cell disease shows marked variability in severity and pathophysiology among individuals, probably linked to differential expression of various adhesion molecules. In this study, we investigated the differential distribution, genomic diversity and haplotype frequency of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) polymorphisms, recently implicated as important in modification of disease severity. One hundred and forty five sickle cell disease patients (HbSS) and 244 adult and pediatric controls, without sickle cell disease (HbAA), were recruited from Mali. Genotypic analysis of the functionally significant eNOS variants (T786C, G894T and intron 4) and endothelin-1 (G5665T) was carried out with a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Our results show that the wild type alleles are the most frequent for all eNOS variants between cases and controls. Allelic and genotypic frequencies of eNOS polymorphic groups are not significantly different between cases and controls (P > 0.05). In addition, there is no association between eNOS variants and sickle cell disease, contrary to published reports. On the other hand, we report that endothelin-1 (G5665T) mutant variant had the lowest allelic frequency, and is significantly associated with sickle cell disease in Africa (P < 0.05). Similarly, haplotype frequencies were the same between cases and controls, except for the haplotype combining all mutant variants (T, C, 4a; P = 0.01). eNOS polymorphic variants are less frequent, with no significance with sickle cell disease in Africa. On the other hand, endothelin-1 is associated with sickle cell disease, and has the capacity to redefine pathophysiology and possibly serve as modulator of disease phenotype.

  14. Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

    SciTech Connect

    Vallon, Mario; Rohde, Franziska; Janssen, Klaus-Peter; Essler, Markus

    2010-02-01

    Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

  15. Nitric oxide synthase inhibition attenuates cutaneous vasodilation during the post-menopausal hot flash

    PubMed Central

    Hubing, Kimberly A.; Wingo, Jonathan E.; Brothers, R. Matthew; Coso, Juan Del; Low, David A.; Crandall, Craig G.

    2010-01-01

    Objective The purpose of this study was to test the hypothesis that local inhibition of nitric oxide and prostaglandin synthesis attenuates cutaneous vasodilator responses during post-menopausal hot flashes. Methods Four microdialysis membranes were inserted into forearm skin (dorsal surface) of 8 post-menopausal women (mean ± SD, 51±7 y). Ringers solution (control), 10mM Ketorolac (Keto) to inhibit prostaglandin synthesis, 10mM NG-L-arginine methyl ester (L-NAME) to inhibit nitric oxide synthase, and a combination of 10mM Keto + 10mM L-NAME were each infused at the separate sites. Skin blood flow at each site was indexed using laser-Doppler flowmetry. Cutaneous vascular conductance (CVC) was calculated as laser-Doppler flux/mean arterial blood pressure and was expressed as a percentage of the maximal calculated CVC (CVCmax) obtained following infusion of 50mM sodium nitropruside at all sites at the end of the study. Data from 13 hot flashes were analyzed. Results At the control site, the mean ± SD peak increase in CVC was 15.5±6% CVCmax units. This value was not different relative to the peak increase in CVC at the Keto site (13.0±5 % CVCmax units, P = 0.09). However, the peak increase in CVC during the flash was attenuated at the L-NAME and L-NAME + Keto sites (7.4±4 % CVCmax units and 8.7±7 % CVCmax units, respectively) relative to both the control and the Keto sites (P<0.05 for both comparisons). There were no significant differences in the peak increases in sweat rate between any of the sites (P = 0.24). Conclusions These data demonstrate that cutaneous vasodilation during a hot flash has a nitric oxide component. Increases in CVC despite the inhibition of prostaglandin synthesis suggest prostaglandins do not contribute to cutaneous vasodilation during a hot flash. PMID:20505548

  16. Chlorogenic acid improves ex vivo vessel function and protects endothelial cells against HOCl-induced oxidative damage, via increased production of nitric oxide and induction of Hmox-1.

    PubMed

    Jiang, Rujia; Hodgson, Jonathan M; Mas, Emilie; Croft, Kevin D; Ward, Natalie C

    2016-01-01

    Dietary polyphenols are potential contributors toward improved cardiovascular health. Coffee is one of the richest sources of dietary polyphenols in a coffee-drinking population, the most abundant form being chlorogenic acid (CGA). Endothelial dysfunction is an early and major risk factor for cardiovascular disease. Nitric oxide (NO) is a key factor in regulation of endothelial function. Heme oxygenase-1 (Hmox-1), an inducible isoform of heme oxygenase that is produced in response to stressors such as oxidative stress, may also play a role in vascular protection. The aim of this study was to investigate the effect of CGA on endothelial function with oxidant-induced damage in isolated aortic rings from C57BL mice. We further examine the mechanism by investigating cell viability, activation of eNOS and induction of Hmox-1 in human aortic endothelial cells (HAECs). We found that pretreatment of isolated aortic rings with 10-μM CGA-protected vessels against HOCl-induced endothelial dysfunction (P<0.05). Pretreatment of cultured HAECs with 10-μM CGA increased endothelial cell viability following exposure to HOCl (P<0.05). Moreover, CGA increased NO production in HAECs in a dose-dependent manner, peaking at 6 h (P<0.05). CGA at 5 μM and 10 μM increased eNOS dimerization at 6 h and induced Hmox-1 protein expression at 6 h and 24 h in HAECs. These results are consistent with the cardiovascular protective effects of coffee polyphenols and demonstrate that CGA can protect vessels and cultured endothelial cells against oxidant-induced damage. The mechanism behind the beneficial effect of CGA appears to be in part via increased production of NO and induction of Hmox-1.

  17. Rhus coriaria suppresses angiogenesis, metastasis and tumor growth of breast cancer through inhibition of STAT3, NFκB and nitric oxide pathways

    PubMed Central

    El Hasasna, Hussain; Saleh, Alaaeldin; Samri, Halima Al; Athamneh, Khawlah; Attoub, Samir; Arafat, Kholoud; Benhalilou, Nehla; Alyan, Sofyan; Viallet, Jean; Dhaheri, Yusra Al; Eid, Ali; Iratni, Rabah

    2016-01-01

    Recently, we reported that Rhus coriaria exhibits anticancer activities by promoting cell cycle arrest and autophagic cell death of the metastatic triple negative MDA-MB-231 breast cancer cells. Here, we investigated the effect of Rhus coriaria on the migration, invasion, metastasis and tumor growth of TNBC cells. Our current study revealed that non-cytotoxic concentrations of Rhus coriaria significantly inhibited migration and invasion, blocked adhesion to fibronectin and downregulated MMP-9 and prostaglandin E2 (PgE2). Not only did Rhus coriaria decrease their adhesion to HUVECs and to lung microvascular endothelial (HMVEC-L) cells, but it also inhibited the transendothelial migration of MDA-MB-231 cells through TNF-α-activated HUVECs. Furthermore, we found that Rhus coriaria inhibited angiogenesis, reduced VEGF production in both MDA-MB-231 and HUVECs and downregulated the inflammatory cytokines TNF-α, IL-6 and IL-8. The underlying mechanism for Rhus coriaria effects appears to be through inhibiting NFκB, STAT3 and nitric oxide (NO) pathways. Most importantly, by using chick embryo tumor growth assay, we showed that Rhus coriaria suppressed tumor growth and metastasis in vivo. The results described in the present study identify Rhus coriaria as a promising chemopreventive and therapeutic candidate that modulate triple negative breast cancer growth and metastasis. PMID:26888313

  18. TSG attenuates LPC-induced endothelial cells inflammatory damage through notch signaling inhibition.

    PubMed

    Zhao, Jing; Liang, Yuan; Song, Fan; Xu, Shouzhu; Nian, Lun; Zhou, Xuanxuan; Wang, Siwang

    2016-01-01

    Lysophosphatidylcholine (LPC) induces inflammation in endothelial cells (ECs) but the mechanism is not fully understood. The Notch signaling pathway is involved in chronic EC inflammation, but its functions in LPC-induced endothelial inflammatory damage and 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside's (TSG) protective effect during LPC-induced inflammatory damage in human umbilical vein endothelial cells (HUVECs) is largely unknown. We report that Notch signaling activation contributed to LPC-induced injury in HUVECs, and that TSG protected HUVECs from LPC-induced injury by antagonizing Notch signaling activation by LPC. γ-secretase inhibitor (DAPT), a specific inhibitor of the Notch signaling pathway, and Notch1 siRNA were used to inhibit Notch activity. HUVECs were exposed to LPC in the presence or absence of TSG, DAPT, and Notch1 siRNA. LPC treatment of HUVECs resulted in reduced cell viability, and Notch1 and Hes1 upregulation. Either silencing of Notch1 by siRNA or pharmacological inhibition of Notch signaling by DAPT prevented the loss of cell viability, and induction of apoptosis, and enhanced expression Notch1, Hes1 and MCP-1 by LPC in HUVECs. Similarly, TSG reduced LPC stimulation of Notch1, Hes1, and MCP-1 expression, prevented the release of IL-6 and CRP and rescued HUVECs from LPC-induced cell damage. Our data indicate that the Notch signaling pathway is a crucial mediator of endothelial inflammatory damage and that TSG protects against endothelial inflammatory damage by inhibiting the Notch signaling pathway. Our findings suggest that targeting Notch signaling by natural products such as TSG is a promising strategy for the prevention and treatment of chronic inflammation associated diseases, including atherosclerosis. © 2015 IUBMB Life, 68(1):37-50, 2016.

  19. Copper induces--and copper chelation by tetrathiomolybdate inhibits--endothelial activation in vitro.

    PubMed

    Wei, Hao; Zhang, Wei-Jian; Leboeuf, Renee; Frei, Balz

    2014-01-01

    Endothelial activation with increased expression of cellular adhesion molecules and chemokines critically contributes to vascular inflammation and atherogenesis. Redox-active transition metal ions play an important role in vascular oxidative stress and inflammation. Therefore, the goal of the present study was to investigate the role of copper in endothelial activation and the potential anti-inflammatory effects of copper chelation by tetrathiomolybdate (TTM) in human aortic endothelial cells (HAECs). Incubating HAECs with cupric sulfate dose- and time-dependently increased mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and monocyte chemotactic protein-1 (MCP-1). Copper also activated the redox-sensitive transcription factors, nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1), which was inhibited by pretreatment of the cells with TTM. Furthermore, TTM dose-dependently inhibited tumor necrosis factor α (TNFα)-induced activation of NF-κB and AP-1, as well as mRNA and protein expression of VCAM-1, ICAM-1, and MCP-1, which was abolished by preincubating the cells with 5 µM TTM and 15 µM cupric sulfate. The inhibitory effect of TTM on TNFα-induced NF-κB activation was associated with decreased phosphorylation and degradation of IκBα. These data suggest that intracellular copper causes activation of redox-sensitive transcription factors and upregulation of inflammatory mediators in endothelial cells. Copper chelation by TTM may attenuate TNFα-induced endothelial activation and, hence, inhibit vascular inflammation and atherosclerosis.

  20. Andrographolide, a Novel NF-κB Inhibitor, Inhibits Vascular Smooth Muscle Cell Proliferation and Cerebral Endothelial Cell Inflammation

    PubMed Central

    Chang, Chao-Chien; Duann, Yeh-Fang; Yen, Ting-Lin; Chen, Yu-Ying; Jayakumar, Thanasekaran; Ong, Eng-Thiam; Sheu, Joen-Rong

    2014-01-01

    Background Aberrant vascular smooth muscle cell (VSMC) proliferation and cerebral endothelial cell (CEC) dysfunction contribute significantly in the pathogenesis of cardiovascular diseases. Therefore, inhibition of these cellular events would be by candidate agents for treating these diseases. In the present study, the mechanism of anti-proliferative and anti-inflammatory effects of andrographolides, a novel nuclear factor-κB inhibitor, was investigated in VSMC and CEC cells. Methods VSMCs and CECs were isolated from rat artery and mouse brain, respectively, and cultured before experimentation. The effect of andro on platelet-derived growth factor-BB (PDGF-BB) induced VSMC cell proliferation was evaluated by cell number, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of extracellular signal regulated kinase 1/2 (ERK1/2), proliferating cell nuclear antigen (PCNA), and the effects on lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and, cyclooxygenase-2 (COX2) were detected by Western blotting. Results Andro significantly inhibited PDGF-BB (10 ng/ml) induced cell proliferation in a concentration (20-100 μM) dependent manner, which may be due to reducing the expression of ERK1/2, and by inhibiting the expression of PCNA. Andro also remarkably diminished LPS-induced iNOS and COX2 expression. Conclusions The results of this study suggested that the effects of andro against VSMCs proliferation and CECs dysfunction may represent a promising approach for treatment of vascular diseases. PMID:27122804

  1. 5-HT1D receptor inhibits renal sympathetic neurotransmission by nitric oxide pathway in anesthetized rats.

    PubMed

    García-Pedraza, José-Ángel; García, Mónica; Martín, María-Luisa; Morán, Asunción

    2015-09-01

    Although serotonin has been shown to inhibit peripheral sympathetic outflow, serotonin regulation on renal sympathetic outflow has not yet been elucidated. This study investigated which 5-HT receptor subtypes are involved. Wistar rats were anesthetized (sodium pentobarbital; 60mg/kg, i.p.), and prepared for in situ autoperfused rat kidney, which allows continuous measurement of systemic blood pressure (SBP), heart rate (HR) and renal perfusion pressure (PP). Electrical stimulation of renal sympathetic nerves resulted in frequency-dependent increases in PP (18.3±1.0, 43.7±2.7 and 66.7±4.0 for 2, 4 and 6Hz, respectively), without altering SBP or HR. 5-HT, 5-carboxamidotryptamine (5-HT1/7 agonist) (0.00000125-0.1μg/kg each) or l-694,247 (5-HT1D agonist; 0.0125μg/kg) i.a. bolus inhibited vasopressor responses by renal nerve electrical stimulation, unlike i.a. bolus of agonists α-methyl-5-HT (5-HT2), 1-PBG (5-HT3), cisapride (5-HT4), AS-19 (5-HT7), CGS-12066B (5-HT1B) or 8-OH-DPAT (5-HT1A) (0.0125μg/kg each). The effect of l-694,247 did not affect the exogenous norepinephrine-induced vasoconstrictions, whereas was abolished by antagonist LY310762 (5-HT1D; 1mg/kg) or l-NAME (nitric oxide; 10mg/kg), but not by indomethacin (COX1/2; 2mg/kg) or glibenclamide (ATP-dependent K(+) channel; 20mg/kg). These results suggest that 5-HT mechanism-induced inhibition of rat vasopressor renal sympathetic outflow is mainly mediated by prejunctional 5-HT1D receptors via nitric oxide release.

  2. Interleukin-13 inhibits inducible nitric oxide synthase expression in human mesangial cells.

    PubMed Central

    Saura, M; Martínez-Dalmau, R; Minty, A; Pérez-Sala, D; Lamas, S

    1996-01-01

    The synthesis of nitric oxide in inflammatory situations requires the expression of an inducible isoform of nitric oxide synthase (iNOS). Human mesangial cells (HMC) express an iNOS enzyme after exposure to multiple co-stimuli. In this study we have observed that while tumour necrosis factor-alpha, interleukin (IL)-1 beta, interferon-gamma and bacterial lipopolysaccharide (LPS) were unable to significantly induce NO synthesis when used alone, they induced an evident stimulation of NO synthesis when used in various combinations. A mixture of the three cytokines (CM) and LPS resulted in a 10-15-fold stimulation of NO synthesis over control values which started to be significant after 16 h. The addition of IL-13, a cytokine with anti-inflammatory properties, inhibited CM/LPS-induced NO synthesis in a concentration-dependent manner. A marked inhibitory effect (60-65%) could be observed when HMC were treated with IL-13 (10 ng/ml) 24 h before, at the same time as, or even 4 h after the addition of CM/LPS. This inhibitory effect was still significant (25%) when IL-13 was added 16 h after CM/LPS. Northern analysis showed that IL-13-mediated iNOS inhibition was closely correlated with the suppression of iNOS mRNA expression. These results identify IL-13 as a powerful regulatory tool for the inhibition of NO synthesis in human cells, a property which may be pathophysiologically relevant in NO-related inflammatory processes. PMID:8573104

  3. Inhibition of NADPH oxidase activation in endothelial cells by ortho-methoxy-substituted catechols.

    PubMed

    Johnson, David K; Schillinger, Kurt J; Kwait, David M; Hughes, Chambers V; McNamara, Erin J; Ishmael, Fauod; O'Donnell, Robert W; Chang, Ming-Mei; Hogg, Michael G; Dordick, Jonathan S; Santhanam, Lakshmi; Ziegler, Linda M; Holland, James A

    2002-01-01

    NADPH oxidase is a major enzymatic source of oxygen free radicals in stimulated endothelial cells (ECs). The ortho-methoxy-substituted catechol, apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa, inhibits the release of superoxide anion (O2*-) by this enzyme. The compound acts by blocking the assembly of a functional NADPH oxidase complex. The underlying chemistry of this inhibitory activity, and its physiological significance to EC proliferation, have been investigated. A critical event is the reaction of ortho-methoxy-substituted catechols with reactive oxygen species (ROS) and peroxidase. Analysis of this reaction reveals that apocynin is converted to a symmetrical dimer through the formation of a 5,5' carbon-carbon bond. Both reduced glutathione and L-cysteine inhibit this dimerization process. Catechols without the ortho-methoxy-substituted group do not undergo this chemical reaction. Superoxide production by an endothelial cell-free system incubated with apocynin was nearly completely inhibited after a lagtime for inhibition of ca. 2 min. Conversely, O2*- production was nearly completely inhibited, without a lagtime, by incubation with the dimeric form of apocynin. The apocynin dimer undergoes a two-electron transfer reaction with standard redox potentials of -0.75 and -1.34 V as determined by cyclic voltammetry. Inhibition of endothelial NADPH oxidase by apocynin caused a dose-dependent inhibition of cell proliferation. These findings identify a metabolite of an ortho-methoxy-substituted catechol, which may be the active compound formed within stimulated ECs that prevents NADPH oxidase complex assembly and activation.

  4. Anticoagulation and inhibition of nitric oxide synthase influence hepatic hypoxia after monocrotaline exposure.

    PubMed

    Copple, Bryan L; Roth, Robert A; Ganey, Patricia E

    2006-08-15

    Monocrotaline (MCT) is a pyrrolizidine alkaloid plant toxin that produces hepatotoxicity in humans and animals. Administration of MCT to rats causes rapid sinusoidal endothelial cell (SEC) injury, hemorrhage, pooling of blood and fibrin deposition in centrilobular regions of liver. These events precede hepatic parenchymal cell (HPC) injury and produce marked changes in the microvasculature of the liver, which could interrupt blood flow and produce hypoxia in affected regions. To test the hypothesis that hypoxia occurs in liver after MCT exposure, rats were treated with 300mgMCT/kg, and hypoxia was detected immunohistochemically. MCT produced significant hypoxia in centrilobular regions of livers by 8h after treatment. Inasmuch as fibrin deposition can impair oxygen delivery by reducing blood flow, the effect of anticoagulant treatment on MCT-induced hypoxia was determined. Administration of warfarin to MCT-treated rats reduced hypoxia in the liver by approximately 70%, suggesting that fibrin deposition plays a causal role in the development of hypoxia in the liver. Conversely, administration of l-NAME, a nonspecific inhibitor of nitric oxide synthases (NOSs), enhanced MCT-induced hypoxia and HPC injury. l-NAME did not, however, affect SEC injury or coagulation system activation. Results from these studies show that hypoxia occurs in the liver after MCT exposure. Furthermore, hypoxia precedes HPC injury, and manipulations that modify hypoxia also modulate HPC injury.

  5. Depolymerization of macrophage microfilaments prevents induction and inhibits activity of nitric oxide synthase.

    PubMed

    Fernandes, P D; Araujo, H M; Riveros-Moreno, V; Assreuy, J

    1996-12-01

    We have investigated the relationship between peritoneal murine macrophage cytoskeleton and nitric oxide (NO) synthase (NOS). Activation of the cells with lipopolysaccharide plus interferon-gamma (LI) induced iNOS, detected by nitrite or by labeled L-citrulline production and by a specific antibody against macrophage iNOS. Addition of cytochalasin B (a microfilament-depolymerizing agent) caused a dose-dependent inhibition in NO production by macrophages, whereas colchicine (a microtubule depolymerizing agent) inhibited it only by 20% and not dose-dependently. Addition of cytochalasin B together with LI abolished nitrite and L-citrulline accumulation as well as the amount of iNOS antigen in activated macrophage. Moreover, addition of cytochalasin B 6 or 12 h after stimulus, also decreased the nitrite and L-citrulline production by macrophages although iNOS antigen content by Western blot was the same in the presence or in the absence of cytochalasin B added 12 h after activation. Since cytochalasin B failed to inhibit iNOS activity directly, its inhibitory effects on NO production by macrophages is likely to be indirect, through microfilament network in central regions of cells, but not in filaments seen at pseudopodia or edging processes. Our findings demonstrate that disruption of microfilaments but not of microtubules prevents the iNOS induction process and inhibits its enzymatic activity in activated macrophages.

  6. Arginase inhibition in airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin

    SciTech Connect

    Bratt, Jennifer M.; Franzi, Lisa M.; Linderholm, Angela L.; O'Roark, Erin M.; Kenyon, Nicholas J.; Last, Jerold A.

    2010-01-01

    Arginase1 and nitric oxide synthase2 (NOS2) utilize L-arginine as a substrate, with both enzymes expressed at high levels in the asthmatic lung. Inhibition of arginase in ovalbumin-exposed C57BL/6 mice with the transition state inhibitor N{sup o}mega-hydroxy-nor-L-arginine (nor-NOHA) significantly increased total L-arginine content in the airway compartment. We hypothesized that such an increase in L-arginine content would increase the amount of nitric oxide (NO) being produced in the airways and thereby decrease airway hyperreactivity and eosinophilic influx. We further hypothesized that despite arginase inhibition, NOS2 knockout (NOS2-/-) mice would be unable to up-regulate NO production in response to allergen exposure and would demonstrate higher amounts of airway hyperreactivity and eosinophilia under conditions of arginase inhibition than C57BL/6 animals. We found that administration of nor-NOHA significantly decreased airway hyperreactivity and eosinophilic airway inflammation in ovalbumin-exposed C57BL/6 mice, but these parameters were unchanged in ovalbumin-exposed NOS2-/- mice. Arginase1 protein content was increased in mice exposed to ovalbumin, an effect that was reversed upon nor-NOHA treatment in C57BL/6 mice. Arginase1 protein content in the airway compartment directly correlated with the degree of airway hyperreactivity in all treatment groups. NOS2-/- mice had significantly greater arginase1 and arginase2 concentrations compared to their respective C57BL/6 groups, indicating that inhibition of arginase may be dependent upon NOS2 expression. Arginase1 and 2 content were not affected by nor-NOHA administration in the NOS2-/- mice. We conclude that L-arginine metabolism plays an important role in the development of airway hyperreactivity and eosinophilic airway inflammation. Inhibition of arginase early in the allergic inflammatory response decreases the severity of the chronic inflammatory phenotype. These effects appear to be attributable to NOS2

  7. Inhibition of inducible Nitric Oxide Synthase by a mustard gas analog in murine macrophages

    PubMed Central

    Qui, Min; Paromov, Victor M; Yang, Hongsong; Smith, Milton; Stone, William L

    2006-01-01

    Background 2-Chloroethyl ethyl sulphide (CEES) is a sulphur vesicating agent and an analogue of the chemical warfare agent 2,2'-dichlorodiethyl sulphide, or sulphur mustard gas (HD). Both CEES and HD are alkylating agents that influence cellular thiols and are highly toxic. In a previous publication, we reported that lipopolysaccharide (LPS) enhances the cytotoxicity of CEES in murine RAW264.7 macrophages. In the present investigation, we studied the influence of CEES on nitric oxide (NO) production in LPS stimulated RAW264.7 cells since NO signalling affects inflammation, cell death, and wound healing. Murine macrophages stimulated with LPS produce NO almost exclusively via inducible nitric oxide synthase (iNOS) activity. We suggest that the influence of CEES or HD on the cellular production of NO could play an important role in the pathophysiological responses of tissues to these toxicants. In particular, it is known that macrophage generated NO synthesised by iNOS plays a critical role in wound healing. Results We initially confirmed that in LPS stimulated RAW264.7 macrophages NO is exclusively generated by the iNOS form of nitric oxide synthase. CEES treatment inhibited the synthesis of NO (after 24 hours) in viable LPS-stimulated RAW264.7 macrophages as measured by either nitrite secretion into the culture medium or the intracellular conversion of 4,5-diaminofluorescein diacetate (DAF-2DA) or dichlorofluorescin diacetate (DCFH-DA). Western blots showed that CEES transiently decreased the expression of iNOS protein; however, treatment of active iNOS with CEES in vitro did not inhibit its enzymatic activity Conclusion CEES inhibits NO production in LPS stimulated macrophages by decreasing iNOS protein expression. Decreased iNOS expression is likely the result of CEES induced alteration in the nuclear factor kappa B (NF-κB) signalling pathway. Since NO can act as an antioxidant, the CEES induced down-regulation of iNOS in LPS-stimulated macrophages could elevate

  8. α-1 Antitrypsin Inhibits Caspase-3 Activity, Preventing Lung Endothelial Cell Apoptosis

    PubMed Central

    Petrache, Irina; Fijalkowska, Iwona; Medler, Terry R.; Skirball, Jarrett; Cruz, Pedro; Zhen, Lijie; Petrache, Horia I.; Flotte, Terence R.; Tuder, Rubin M.

    2006-01-01

    α-1 Antitrypsin (A1AT) is an abundant circulating serpin with a postulated function in the lung of potently inhibiting neutrophil-derived proteases. Emphysema attributable to A1AT deficiency led to the concept that a protease/anti-protease imbalance mediates cigarette smoke-induced emphysema. We hypothesized that A1AT has other pathobiological relevant functions in addition to elastase inhibition. We demonstrate a direct prosurvival effect of A1AT through inhibition of lung alveolar endothelial cell apoptosis. Primary pulmonary endothelial cells internalized human A1AT, which co-localized with and inhibited staurosporine-induced caspase-3 activation. In cell-free studies, native A1AT, but not conformers lacking an intact reactive center loop, inhibited the interaction of recombinant active caspase-3 with its specific substrate. Furthermore, overexpression of human A1AT via replication-deficient adeno-associated virus markedly attenuated alveolar wall destruction and oxidative stress caused by caspase-3 instillation in a mouse model of apoptosis-dependent emphysema. Our findings suggest that direct inhibition of active caspase-3 by A1AT may represent a novel anti-apoptotic mechanism relevant to disease processes characterized by excessive structural cell apoptosis, oxidative stress, and inflammation, such as pulmonary emphysema. PMID:17003475

  9. Tyrosol attenuates ischemia-reperfusion-induced kidney injury via inhibition of inducible nitric oxide synthase.

    PubMed

    Wang, Pengqi; Zhu, Qingjun; Wu, Nan; Siow, Yaw L; Aukema, Harold; O, Karmin

    2013-04-17

    Tyrosol is a natural phenolic antioxidant compound. Oxidative stress represents one of the important mechanisms underlying ischemia-reperfusion-induced kidney injury. The aim of this study was to investigate the effect of tyrosol against ischemia-reperfusion-induced acute kidney injury. The left kidney of Sprague-Dawley rats was subjected to 45 min of ischemia followed by reperfusion for 6 h. Ischemia-reperfusion caused an increase in peroxynitrite formation and lipid peroxidation. The level of nitric oxide (NO) metabolites and the mRNA of inducible nitric oxide synthase (iNOS) were elevated in ischemia-reperfused kidneys. Administration of tyrosol (100 mg/kg body weight) to rats prior to the induction of ischemia significantly reduced peroxynitrite formation, lipid peroxidation, and the level of NO metabolites. Tyrosol administration also attenuated ischemia-reperfusion-induced NF-κB activation and iNOS expression. Such a treatment improved kidney function. Results suggest that tyrosol may have a protective effect against acute kidney injury through inhibition of iNOS-mediated oxidative stress.

  10. Experimental inhibition of nitric oxide increases Plasmodium relictum (lineage SGS1) parasitaemia.

    PubMed

    Bichet, Coraline; Cornet, Stéphane; Larcombe, Stephen; Sorci, Gabriele

    2012-12-01

    Malaria is a widespread vector-borne disease infecting a wide range of terrestrial vertebrates including reptiles, birds and mammals. In addition to being one of the most deadly infectious diseases for humans, malaria is a threat to wildlife. The host immune system represents the main defence against malaria parasites. Identifying the immune effectors involved in malaria resistance has therefore become a major focus of research. However, this has mostly involved humans and animal models (rodents) and how the immune system regulates malaria progression in non-model organisms has been largely ignored. The aim of the present study was to investigate the role of nitric oxide (NO) as an immune effector contributing to the control of the acute phase of infection with the avian malaria agent Plasmodium relictum. We used experimental infections of domestic canaries in conjunction with the inhibition of the enzyme inducible nitric oxide synthase (iNOS) to assess the protective function of NO during the infection, and the physiological costs paid by the host in the absence of an effective NO response. Our results show that birds treated with the iNOS inhibitor suffered from a higher parasitaemia, but did not pay a higher cost of infection (anaemia). While these findings confirm that NO contributes to the resistance to avian malaria during the acute phase of the infection, they also suggest that parasitaemia and costs of infection can be decoupled.

  11. Dlitiazem inhibits the oxidative stress induced by angiotensin II through growth hormone secretagogue receptor type 1a in human umbilicus vein endothelial cells.

    PubMed

    Zhou, Lingyun; Yang, Meng; Zuo, Shanru; Guan, Xiaofeng; Wang, Jianglin; Chen, Qingjie; Zuo, Xiaocong; Jia, Sujie; Guo, Ren

    2017-02-16

    Diltiazem has been used for post-transplant hypertension, but the mechanism underlying its protective effect of endothelial cells against angiotensin II (Ang II) - induced impairment remains unclear. Human umbilicus vein endothelial cells (HUVECs) were cultured and divided into seven groups: control, Ang II (10(-6)M), diltiazem (10(-6)M), [D-Lys3]-GHRP-6(25μM), diltiazem (10(-6)M)+Ang II (10(-6)M), losartan (10(-6)M)+Ang II (10(-6)M), [D-Lys3]-GHRP-6 (25μM) + Dil(10(-6)M)+Ang II (10(-6)M) groups. Nitric oxide (NO) production, intracellular reactive oxygen species (ROS) levels, protein and mRNA expressions of endothelial nitric oxide synthase (eNOS) and p47 phox subunit of NADPH were evaluated. Results indicated that pre- treatment with diltiazem significantly decreased the intracellular ROS levels and increased NO production. Treatment with 10(-6)M Ang II for 24h induced a significant decrease in the mRNA and protein levels of eNOS, which was significantly increased by the pre-incubated with diltiazem (10(-6)M). Treatment with 10(-6)M Ang II for 24h induced a significant increase in the mRNA and protein levels of p47 phox subunit of NADHP oxidase, which was significantly decreased by the pre-incubated with diltiazem. However, all of these protective roles of diltiazem were attenuated by pre-incubation of [D-Lys3]-GHRP-6. The results reveal that diltiazem inhibits the Ang II - induced oxidative stress in HUVECs, which may be partly mediated by GHSR1a.

  12. Flavonoids from Citrus unshiu Marc. inhibit cancer cell adhesion to endothelial cells by selective inhibition of VCAM-1.

    PubMed

    Jin, Hana; Lee, Won Sup; Yun, Jeong Won; Jung, Ji Hyun; Yi, Sang Mi; Kim, Hye Jung; Choi, Yung Hyun; Kim, Gonsup; Jung, Jin-Myung; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan

    2013-11-01

    Citrus fruits have been used as edible fruit and a component of traditional medicine for various diseases including cancer since ancient times. Herein, we investigated the anticancer activity of flavonoids of Citrus unshiu Marc. (FCM) focusing on anti-metastatic effects. We prepared FCM and performed experiments using MDA-MB-231 human breast cancer cells. FCM inhibited TNF-induced cancer cell adhesion to human umbilical vein endothelial cells (HUVECs) without showing any toxicity. FCM inhibited the expression of VCAM-1, but not of ICAM-1, on MDA-MB-231 cells as well as HUVECs. FCM inhibited protein kinase C (PKC) phosphorylation, but not Akt phosphorylation. FCM also inhibited cancer cell invasion in a dose-dependent manner, but not MMP-9 expression. In conclusion, this study suggested that FCM inhibits TNF-induced cancer cell adhesion to HUVECs by inhibiting VCAM-1 through inhibition of PKC, providing evidence that FCM have anti-metastatic activity by inhibiting adhesion molecules and invasion on human breast cancer cells.

  13. Mechanism study of endothelial protection and inhibits platelet activation of low molecular weight fucoidan from Laminaria japonica

    NASA Astrophysics Data System (ADS)

    Chen, Anjin; Zhang, Fang; Shi, Jie; Zhao, Xue; Yan, Meixing

    2016-10-01

    Several studies have indicated that fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica could inhibit the activation of platelets directly by reducing the platelet aggregation. To explore the direct effect of LMW fucoidan on the platelet system furthermore and examine the possible mechanism, the endothelial protection and inhibits platelet activation effects of two LMW fucoidan were investigated. In the present study, Endothelial injury model of rats was made by injection of adrenaline (0.4 mg kg-1) and human umbilical vein endothelial cells were cultured. vWF level was be investigated in vivo and in vitro as an important index of endothelial injury. LMW fucoidan could significantly reduce vWF level in vascular endothelial injury rats and also significantly reduce vWF level in vitro. The number of EMPs was be detected as another important index of endothelial injury. The results showed that LMW fucoidan reduced EMPs stimulated by tumor necrosis factor. In this study, it was found that by inhibiting platelet adhesion, LMW fucoidan played a role in anti-thrombosis and the specific mechanism of action is to inhibit the flow of extracellular Ca2+. All in a word, LMW fucoidan could inhibit the activation of platelets indirectly by reducing the concentration of EMPs and vWF, at the same time; LMW fucoidan inhibited the activation of platelets directly by inhibiting the flow of extracellular Ca2+.

  14. The chemokine CXCL13 (BCA-1) inhibits FGF-2 effects on endothelial cells.

    PubMed

    Spinetti, G; Camarda, G; Bernardini, G; Romano Di Peppe, S; Capogrossi, M C; Napolitano, M

    2001-11-23

    Several chemokines, belonging to both the CXC and CC classes, act as positive or negative regulators of angiogenesis. We sought to investigate the role of CXCL13, B cell-attracting chemokine 1 (BCA-1), also known as B-lymphocyte chemoattractant (BLC), on endothelial cell functions. We tested the effect of CXCL13 on HUVEC chemotaxis and proliferation in the presence of fibroblast growth factor (FGF)-2 and found that such chemokine inhibits FGF-2-induced functions, while is not active by itself. To test whether other FGF-2-mediated biological activities may be affected, we evaluated the ability of CXCL13 to rescue HUVEC from starvation-induced apoptosis, as FGF-2 is a survival factor for endothelial cells, and found that CXCL13 partially inhibits such rescue. Multiple mechanisms may be responsible for these biological activities as CXCL13 displaces FGF-2 binding to endothelial cells, inhibits FGF-2 homodimerization, and induces the formation of CXCL13-FGF-2 heterodimers. Our data suggest that CXCL13 may modulate angiogenesis by interfering with FGF-2 activity.

  15. Inhibition of nitric oxide-induced apoptosis by nicotine in oral epithelial cells.

    PubMed

    Banerjee, Abhijit G; Gopalakrishnan, Velliyur K; Vishwanatha, Jamboor K

    2007-11-01

    Development of oral cancer is clearly linked to the usage of smokeless tobacco. The molecular mechanisms involved in this process are however not well understood. Toward this goal, we investigated the effect of smokeless tobacco exposure on apoptosis of oral epithelial cells. Exposure of oral epithelial cells to smokeless tobacco extract (STE) induces apoptosis in a dose-dependent manner, until a threshold level of nicotine is achieved upon which apoptosis is inhibited. 1 mM of nicotine is able to inhibit apoptosis significantly induced by STE in these oral cells. Exposure of cells to nicotine alone has no effect on apoptosis, but nicotine inhibits apoptosis induced by other agents present in STE. In this study we show that, the anti-apoptotic action of nicotine is specifically associated with down-regulation of nitric oxide (NO) production. Using specific inducers of NO, we have demonstrated that inhibition of apoptosis by nicotine is through down-regulation of NO production. Further, we observed that nicotine clearly acts as a sink of NO radicals, shown using peroxynitrite generator (SIN-1) in conjunction or absence of radical scavengers. Nicotine thus causes most damage in transformed epithelial cells as depicted by accumulation of nitrotyrosine in a 3-NT ELISA assay. Inhibition of apoptosis is a hallmark in tumor progression and propels development of cancer. It may further result in functional loss of apoptotic effector mechanisms in the transformed cells. Thus, our data clearly indicates that inhibition of NO-induced apoptosis by nicotine may lead to tobacco-induced oral carcinogenesis, and implies careful development of modalities in tobacco cessation programs.

  16. CONTRARY EFFECTS OF THE RTK INHIBITOR VANDETANIB ON CONSTITUTIVE AND FLOW-STIMULATED NITRIC OXIDE ELABORATION IN HUMANS

    PubMed Central

    Mayer, Erica L.; Dallabrida, Susan M.; Rupnick, Maria A.; Redline, Whitney M.; Hannagan, Keri; Ismail, Nesreen S.; Burstein, Harold J.; Beckman, Joshua A.

    2015-01-01

    Vascular endothelial growth factor regulates neoplastic angiogenesis through production of endothelium-derived nitric oxide. We performed a prospective evaluation of vascular function during treatment with vandetanib, a vascular endothelial growth receptor 2 and 3 receptor tyrosine kinase inhibitor, to determine the effects of vascular endothelial growth receptor signal interruption on endothelial function in humans. Seventeen patients with stage IV breast cancer received dose escalated vandetanib in combination with low-dose oral chemotherapy. We measured blood pressure, systemic nitrate/nitrite levels, and brachial artery vascular function. In vitro analyses of cultured endothelial cells were performed to determine the effect of vandetanib on nitric oxide production, akt473 phosphorylation, and endothelial nitric oxide synthase protein content and membrane localization. Vandetanib treatment for 6 weeks significantly increased blood pressure, decreased resting brachial artery diameter, and decreased plasma systemic nitrate/nitrite levels compared to baseline. Flow-mediated vasodilation was preserved, and no change was noted in nitroglycerin-mediated vasodilation. In vitro, endothelial cell nitrite levels and akt473 phosphorylation were reduced, vascular endothelial growth receptor 2 levels did not change, but endothelial nitric oxide synthase membrane concentration doubled. Vandetanib reduces constitutive nitric oxide production and increases blood pressure, yet flow-stimulated nitric oxide bioavailability was preserved. Changes in vascular function with tyrosine kinase inhibition are complex and require further study in humans. PMID:21482957

  17. Rapamycin antagonizes TNF induction of VCAM-1 on endothelial cells by inhibiting mTORC2

    PubMed Central

    Wang, Chen; Qin, Lingfeng; Manes, Thomas D.; Kirkiles-Smith, Nancy C.; Tellides, George

    2014-01-01

    Recruitment of circulating leukocytes into inflamed tissues depends on adhesion molecules expressed by endothelial cells (ECs). Here we report that rapamycin pretreatment reduced the ability of TNF-treated ECs to capture T cells under conditions of venular flow. This functional change was caused by inhibition of TNF-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and could be mimicked by knockdown of mammalian target of rapamycin (mTOR) or rictor, but not raptor, implicating mTORC2 as the target of rapamycin for this effect. Mechanistically, mTORC2 acts through Akt to repress Raf1-MEK1/2-ERK1/2 signaling, and inhibition of mTORC2 consequently results in hyperactivation of ERK1/2. Increased ERK1/2 activity antagonizes VCAM-1 expression by repressing TNF induction of the transcription factor IRF-1. Preventing activation of ERK1/2 reduced the ability of rapamycin to inhibit TNF-induced VCAM-1 expression. In vivo, rapamycin inhibited mTORC2 activity and potentiated activation of ERK1/2. These changes correlated with reduced endothelial expression of TNF-induced VCAM-1, which was restored via pharmacological inhibition of ERK1/2. Functionally, rapamycin reduced infiltration of leukocytes into renal glomeruli, an effect which was partially reversed by inhibition of ERK1/2. These data demonstrate a novel mechanism by which rapamycin modulates the ability of vascular endothelium to mediate inflammation and identifies endothelial mTORC2 as a potential therapeutic target. PMID:24516119

  18. Resveratrol Ameliorates Clonidine-Induced Endothelium-Dependent Relaxation Involving Akt and Endothelial Nitric Oxide Synthase Regulation in Type 2 Diabetic Mice.

    PubMed

    Taguchi, Kumiko; Hida, Mari; Matsumoto, Takayuki; Kobayashi, Tsuneo

    2015-01-01

    Diabetic vascular complication is one of the manifestations of endothelial dysfunction. Resveratrol (RV) is considered to be beneficial in protecting endothelial function. However, the exact protective effect and mechanisms involved have not been fully clarified. In this study, we investigated the relationship between Akt/endothelial nitric oxide synthase (eNOS) activation and RV in diabetes-induced endothelial dysfunction. Aortas were dissected and placed in organ chambers, and nitric oxide (NO) production in response to acetylcholine (ACh) and RV was measured. ACh-induced endothelium-dependent relaxation was markedly increased in controls by RV pretreatment. Furthermore, RV caused NO-dependent relaxation via the Akt signaling pathway, which was weaker in the aortas of diabetic mice than age-matched controls. To further examine the underlying mechanisms, we measured the phosphorylation of Akt and eNOS by Western blotting. RV caused the phosphorylation of Akt and eNOS in aortas, which was decreased in diabetic mice. However, RV augmented the impaired clonidine-induced relaxation in diabetic mice. Interestingly, the phosphorylation of Akt and eNOS was increased under stimulation with RV and clonidine only in diabetic mice. Thus, either RV or clonidine causes Akt-dependent NO-mediated relaxation, which is weaker in diabetic mice than controls. However, additional exposure to RV and clonidine has an augmenting effect on the Akt/eNOS signaling pathway under diabetic conditions. RV-induced Akt/eNOS activity may be a common link involved in the clonidine-induced Akt/eNOS activity, so RV and clonidine may have a synergistic effect.

  19. Plasma from pre-pubertal obese children impairs insulin stimulated Nitric Oxide (NO) bioavailability in endothelial cells: Role of ER stress.

    PubMed

    Di Pietro, Natalia; Marcovecchio, M Loredana; Di Silvestre, Sara; de Giorgis, Tommaso; Cordone, Vincenzo Giuseppe Pio; Lanuti, Paola; Chiarelli, Francesco; Bologna, Giuseppina; Mohn, Angelika; Pandolfi, Assunta

    2017-03-05

    Childhood obesity is commonly associated with early signs of endothelial dysfunction, characterized by impairment of insulin signaling and vascular Nitric Oxide (NO) availability. However, the underlying mechanisms remain to be established. Hence, we tested the hypothesis that endothelial insulin-stimulated NO production and availability was impaired and related to Endoplasmic Reticulum (ER) in human umbilical vein endothelial cells (HUVECs) cultured with plasma obtained from pre-pubertal obese (OB) children. OB children (N = 28, age: 8.8 ± 2.2; BMI z-score: 2.15 ± 0.39) showed impaired fasting glucose, insulin and HOMA-IR than normal weight children (CTRL; N = 28, age: 8.8 ± 1.7; BMI z-score: 0.17 ± 0.96). The in vitro experiments showed that OB-plasma significantly impaired endothelial insulin-stimulated NO production and bioavailability compared to CTRL-plasma. In parallel, in HUVECs OB-plasma increased GRP78 and activated PERK, eIF2α, IkBα and ATF6 (all ER stress markers). Moreover, OB-plasma increased NF-κB activation and its nuclear translocation. Notably, all these effects proved to be significantly restored by using PBA and TUDCA, known ER stress inhibitors. Our study demonstrate for the first time that plasma from obese children is able to induce in vitro endothelial insulin resistance, which is characterized by reduced insulin-stimulated NO production and bioavailability, endothelial ER stress and increased NF-κB activation.

  20. Nitric oxide signaling pathway activation inhibits the immune escape of pancreatic carcinoma cells

    PubMed Central

    LU, YEBIN; HU, JUANJUAN; SUN, WEIJIA; DUAN, XIAOHUI; CHEN, XIONG

    2014-01-01

    The aim of the present study was to investigate the effect of the nitric oxide signaling pathway on immune escape; thus, a tumorigenesis model was established using nude mice. The mice were inoculated with pancreatic carcinoma cells and divided into two groups, a glyceryl trinitrate (GTN) and a placebo group. When tumor volumes reached 150 mm3, the mice in the GTN group were treated with GTN transdermal patches (dose, 7.3 μg/h) while the mice in the placebo group were administered untreated patches. Following treatment, the tumor volume was recorded every 3–4 days and after 28 days, the tumors were analyzed. The results indicated that GTN treatment may reduce the levels of soluble major histocompatibility complex class I chain-related molecules, and natural killer group 2 member D, as well as inhibiting tumor growth. PMID:25364398

  1. Lignans from Arctium lappa and their inhibition of LPS-induced nitric oxide production.

    PubMed

    Park, So Young; Hong, Seong Su; Han, Xiang Hua; Hwang, Ji Sang; Lee, Dongho; Ro, Jai Seup; Hwang, Bang Yeon

    2007-01-01

    A new butyrolactone sesquilignan, isolappaol C (1), together with four known lignans, lappaol C (2), lappaol D (3), lappaol F (4), and diarctigenin (5), were isolated from the methanolic extract of the seeds from the Arctium lappa plant. The structure of isolappaol C (1) was determined by spectral analysis including 1D- and 2D-NMR. All the isolates were evaluated for their inhibitory effects on the LPS-induced nitric oxide production using murine macrophage RAW264.7 cells. Lappaol F (4) and diarctigenin (5) strongly inhibited NO production in the LPS-stimulated RAW264.7 cells with IC(50) values of 9.5 and 9.6 microM, respectively.

  2. Nitric oxide donors preferentially inhibit neuronally mediated rat gastric acid secretion.

    PubMed

    Barrachina, D; Calatayud, S; Esplugues, J; Whittle, B J; Moncada, S; Esplugues, J V

    1994-09-01

    Continuous i.v. infusion of the nitric oxide (NO) donors, S-nitroso-glutathione (10-50 micrograms kg-1 min-1) and S-nitroso-N-acetyl-penicillamine (10 micrograms kg-1 min-1) inhibited neuronally mediated gastric acid secretion, as induced by gastric distension (20 cm water) or i.v. bolus administration of 2-deoxy-D-glucose (150 mg kg-1) in the anaesthetized rat. By contrast, gastric acid responses to i.v. infusion of submaximal doses of pentagastrin (8 micrograms kg-1 h-1) or histamine (1 mg kg-1 h-1) were not influenced by these NO donors. These findings suggest that NO does not directly influence acid secretion in vivo but could play an inhibitory modulator role in neuronally mediated acid responses.

  3. Inhibition of proliferation of retinal vascular endothelial cells more effectively than choroidal vascular endothelial cell proliferation by bevacizumab

    PubMed Central

    Mynampati, Bharani Krishna; Sambhav, Kumar; Grover, Sandeep; Chalam, Kakarla V.

    2017-01-01

    AIM To evaluate the differential inhibitory effects of bevacizumab on cell proliferation of vascular endothelial growth factor (VEGF)-stimulated choroidal vascular endothelial cells (CVECs) and retinal vascular endothelial cells (RVECs) in vitro. METHODS VEGF (400 ng/mL) enriched CVECs and RVECs were treated with escalating doses of bevacizumab (0.1, 0.5, 1, 1.5 and 2 mg/mL). Cell proliferation changes were analyzed with WST-1 assay and trypan blue exclusion assay at 48, 72h and 1wk. Morphological changes were recorded with bright field microscopy. RESULTS VEGF enriched RVECs showed significantly more decline of cell viability than CVECs after bevacizumab treatment. One week after treatment, RVEC cell proliferation decreased by 29.7%, 37.5%, 52.8%, 35.9% and 45.6% at 0.1, 0.5, 1.0, 1.5 and 2 mg/mL bevacizumab respectively compared to CVEC proliferation decrease of 4.1%, 7.7%, 2.4%, 4.1% and 17.7% (P<0.05) by WST-1 assay. Trypan blue exclusion assay also revealed similar decrease in RVEC proliferation of 20%, 60%, 73.3%, 80% and 93.3% compared to CVEC proliferation decrease of 4%, 12%, 22.9%, 16.7% and 22.2% respectively (P<0.05). The maximum differential effect between the two cell types was observed at bevacizumab doses of 1.0 and 1.5 mg/mL at all time points. RVECs were 22 fold more sensitive (P<0.01) compared to CVECs (52.8% vs 2.4%) at concentration of 1.0 mg/mL, and 8.7 fold more at 1.5 mg/mL (35.9% vs 4.1%) 1wk after treatment (P<0.05 respectively). CONCLUSION VEGF-enriched RVECs are more susceptible to bevacizumab inhibition than CVECs at clinically used dosage of 1.25 mg and this differential sensitivity between two cell types should be taken into consideration in dosage selection. PMID:28149771

  4. Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress.

    PubMed Central

    Topper, J N; Cai, J; Falb, D; Gimbrone, M A

    1996-01-01

    Early atherosclerotic lesions develop in a topographical pattern that strongly suggests involvement of hemodynamic forces in their pathogenesis. We hypothesized that certain endothelial genes, which exhibit differential responsiveness to distinct fluid mechanical stimuli, may participate in the atherogenic process by modulating, on a local level within the arterial wall, the effects of systemic risk factors. A differential display strategy using cultured human endothelial cells has identified two genes, manganese superoxide dismutase and cyclooxygenase-2, that exhibit selective and sustained up-regulation by steady laminar shear stress (LSS). Turbulent shear stress, a nonlaminar fluid mechanical stimulus, does not induce these genes. The endothelial form of nitric oxide synthase also demonstrates a similar LSS-selective pattern of induction. Thus, three genes with potential atheroprotective (antioxidant, antithrombotic, and antiadhesive) activities manifest a differential response to distinct fluid mechanical stimuli, providing a possible mechanistic link between endothelial gene expression and early events in atherogenesis. The activities of these and other LSS-responsive genes may have important implications for the pathogenesis and prevention of atherosclerosis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8816815

  5. ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor

    SciTech Connect

    Inagaki, Junko; Takahashi, Katsuyuki; Ogawa, Hiroko; Asano, Keiichi; Faruk Hatipoglu, Omer; Zeynel Cilek, Mehmet; Obika, Masanari; Ohtsuki, Takashi; Hofmann, Matthias; Kusachi, Shozo; Ninomiya, Yoshifumi; Hirohata, Satoshi

    2014-05-01

    Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angiogenesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy. - Highlights: • ADAMTS1 significantly inhibited tube formation and cell proliferation in HMVEC-dLy. • Reduced lymph endothelial cell migration in ADAMTS1 transduced co-culture systems. • VEGFC-stimulated phosphorylation of VEGFR3 is attenuated by ADAMTS1. • Reduced phosphorylation of Akt and ERK1/2 in ADAMTS1 treated HMVEC-dLy. • ADAMTS1 binds directly to VEGFC.

  6. Wnt5a inhibits K(+) currents in hippocampal synapses through nitric oxide production.

    PubMed

    Parodi, Jorge; Montecinos-Oliva, Carla; Varas, Rodrigo; Alfaro, Iván E; Serrano, Felipe G; Varas-Godoy, Manuel; Muñoz, Francisco J; Cerpa, Waldo; Godoy, Juan A; Inestrosa, Nibaldo C

    2015-09-01

    Hippocampal synapses play a key role in memory and learning processes by inducing long-term potentiation and depression. Wnt signaling is essential in the development and maintenance of synapses via several mechanisms. We have previously found that Wnt5a induces the production of nitric oxide (NO), which modulates NMDA receptor expression in the postsynaptic regions of hippocampal neurons. Here, we report that Wnt5a selectively inhibits a voltage-gated K(+) current (Kv current) and increases synaptic activity in hippocampal slices. Further supporting a specific role for Wnt5a, the soluble Frizzled receptor protein (sFRP-2; a functional Wnt antagonist) fully inhibits the effects of Wnt5a. We additionally show that these responses to Wnt5a are mediated by activation of a ROR2 receptor and increased NO production because they are suppressed by the shRNA-mediated knockdown of ROR2 and by 7-nitroindazole, a specific inhibitor of neuronal NOS. Together, our results show that Wnt5a increases NO production by acting on ROR2 receptors, which in turn inhibit Kv currents. These results reveal a novel mechanism by which Wnt5a may regulate the excitability of hippocampal neurons.

  7. Endothelial nitric oxide synthase mediates cutaneous vasodilation during local heating and is attenuated in middle-aged human skin.

    PubMed

    Bruning, Rebecca S; Santhanam, Lakshmi; Stanhewicz, Anna E; Smith, Caroline J; Berkowitz, Dan E; Kenney, W Larry; Holowatz, Lacy A

    2012-06-01

    Local skin heating is used to assess microvascular function in clinical populations because NO is required for full expression of the response; however, controversy exists as to the precise NO synthase (NOS) isoform producing NO. Human aging is associated with attenuated cutaneous vasodilation but little is known about the middle aged, an age cohort used for comparison with clinical populations. We hypothesized that endothelial NOS (eNOS) is the primary isoform mediating NO production during local heating, and eNOS-dependent vasodilation would be reduced in middle-aged skin. Vasodilation was induced by local heating (42°C) and during acetylcholine dose-response (ACh-DR: 0.01, 0.1, 1.0, 5.0, 10.0, 50.0, 100.0 mmol/l) protocols. Four microdialysis fibers were placed in the skin of 24 men and women; age cohorts were 12 middle-aged (53 ± 1 yr) and 12 young (23 ± 1 yr). Sites served as control, nonselective NOS inhibited [N(G)-nitro-l-arginine methyl ester (l-NAME)], inducible NOS (iNOS) inhibited (1400W), and neuronal NOS (nNOS) inhibited (N(ω)-propyl-l-arginine). After full expression of the local heating response, l-NAME was perfused at all sites. Cutaneous vascular conductance was measured and normalized to maximum (%CVC(max): Nitropress). l-NAME reduced %CVCmax at baseline, all phases of the local heating response, and at all ACh concentrations compared with all other sites. iNOS inhibition reduced the initial peak (53 ± 2 vs. 60 ± 2%CVC(max); P < 0.001); however, there were no other differences between control, nNOS-, and iNOS-inhibited sites during the phases of local heating or ACh-DR. When age cohorts were compared, NO-dependent vasodilation during local heating (52 ± 6 vs. 68 ± 4%CVC(max); P = 0.013) and ACh perfusion (50 mmol/l: 83 ± 3 vs. 93 ± 2%CVC(max); 100 mmol/l: 83 ± 4 vs. 92 ± 3%CVC(max); both P = 0.03) were reduced in middle-aged skin. There were no differences in NOS isoform expression obtained from skin biopsy samples between groups (all

  8. Association of Endothelial Nitric Oxide Synthase Gene Polymorphisms with Coronary Artery Disease: An Updated Meta-Analysis and Systematic Review

    PubMed Central

    Parveen, Farah; Kapoor, Aditya; Sinha, Nakul

    2014-01-01

    Several association studies of endothelial nitric oxide synthase (NOS3) gene polymorphisms with respect to coronary artery disease (CAD) have been published in the past two decades. However, their association with the disease, especially among different ethnic subgroups, still remains controversial. This prompted us to conduct a systematic review and an updated structured meta-analysis, which is the largest so far (89 articles, 132 separate studies, and a sample size of 69,235), examining association of three polymorphic forms of the NOS3 gene (i.e. Glu298Asp, T786-C and 27bp VNTR b/a) with CAD. In a subgroup analysis, we tested their association separately among published studies originating predominantly from European, Middle Eastern, Asian, Asian-Indian and African ancestries. The pooled analysis confirmed the association of all the three selected SNP with CAD in three different genetic models transcending all ancestries worldwide. The Glu298Asp polymorphism showed strongest association (OR range = 1.28–1.52, and P<0.00001 for all comparisons), followed by T786-C (OR range = 1.34–1.42, and P<0.00001 for all comparisons) and 4b/a, (OR range = 1.19–1.41, and P≤0.002 for all comparisons) in our pooled analysis. Subgroup analysis revealed that Glu298Asp (OR range = 1.54–1.87, and P<0.004 for all comparisons) and 4b/a (OR range = 1.71–3.02, and P<0.00001 for all comparisons) have highest degree of association amongst the Middle Easterners. On the other hand, T786-C and its minor allele seem to carry a highest risk for CAD among subjects of Asian ancestry (OR range = 1.61–1.90, and P≤0.01 for all comparisons). PMID:25409023

  9. Redox function of tetrahydrobiopterin and effect of L-arginine on oxygen binding in endothelial nitric oxide synthase.

    PubMed

    Berka, Vladimir; Yeh, Hui-Chun; Gao, De; Kiran, Farheen; Tsai, Ah-Lim

    2004-10-19

    Tetrahydrobiopterin (BH(4)), not dihydrobiopterin or biopterin, is a critical element required for NO formation by nitric oxide synthase (NOS). To elucidate how BH(4) affects eNOS activity, we have investigated BH(4) redox functions in the endothelial NOS (eNOS). Redox-state changes of BH(4) in eNOS were examined by chemical quench/HPLC analysis during the autoinactivation of eNOS using oxyhemoglobin oxidation assay for NO formation at room temperature. Loss of NO formation activity linearly correlated with BH(4) oxidation, and was recovered by overnight incubation with fresh BH(4). Thus, thiol reagents commonly added to NOS enzyme preparations, such as dithiothreitol and beta-mercaptoethanol, probably preserve enzyme activity by preventing BH(4) oxidation. It has been shown that conversion of L-arginine to N-hydroxy-L-arginine in the first step of NOS catalysis requires two reducing equivalents. The first electron that reduces ferric to the ferrous heme is derived from flavin oxidation. The issue of whether BH(4) supplies the second reducing equivalent in the monooxygenation of eNOS was investigated by rapid-scan stopped-flow and rapid-freeze-quench EPR kinetic measurements. In the presence of L-arginine, oxygen binding kinetics to ferrous eNOS or to the ferrous eNOS oxygenase domain (eNOS(ox)) followed a sequential mechanism: Fe(II) <--> Fe(II)O(2) --> Fe(III) + O(2)(-). Without L-arginine, little accumulation of the Fe(II)O(2) intermediate occurred and essentially a direct optical transition from the Fe(II) form to the Fe(III) form was observed. Stabilization of the Fe(II)O(2) intermediate by L-arginine has been established convincingly. On the other hand, BH(4) did not have significant effects on the oxygen binding and decay of the oxyferrous intermediate of the eNOS or eNOS oxygenase domain. Rapid-freeze-quench EPR kinetic measurements in the presence of L-arginine showed a direct correlation between BH(4) radical formation and decay of the Fe(II)O(2

  10. Inhibition of bacterial adhesion and biofilm formation by dual functional textured and nitric oxide releasing surfaces.

    PubMed

    Xu, Li-Chong; Wo, Yaqi; Meyerhoff, Mark E; Siedlecki, Christopher A

    2017-03-15

    In separate prior studies, physical topographic surface modification or nitric oxide (NO) release has been demonstrated to each be an effective approach to inhibit and control bacterial adhesion and biofilm formation on polymeric surfaces. Such approaches can prevent biomaterial-associated infection without causing the antibiotic resistance of the strain. In this work, both techniques were successfully integrated and applied to a polyurethane (PU) biomaterial surface that bears ordered pillar topographies (400/400nm and 500/500nm patterns) at the top surface and a S-nitroso-N-acetylpenicillamine (SNAP, NO donor) doped sub-layer in the middle, via a soft lithography two-stage replication process. Upon placing the SNAP textured PU films into PBS at 37°C, the decomposition of SNAP within polymer film initiates NO release with a lifetime of up to 10days at flux levels >0.5×10(-10)molmin(-1)cm(-2) for a textured polyurethane layer containing 15wt% SNAP. The textured surface reduces the accessible surface area and the opportunity of bacteria-surface interaction, while the NO release from the same surface further inhibits bacterial growth and biofilm formation. Such dual functionality surfaces are shown to provide a synergistic effect on inhibition of Staphylococcus epidermidis bacterial adhesion that is significantly greater than the inhibition of bacterial adhesion achieved by either single treatment approach alone. Longer term experiments to observe biofilm formation demonstrate that the SNAP doped-textured PU surface can inhibit the biofilm formation for >28d and provide a practical approach to improve the biocompatibility of current biomimetic biomaterials and thereby reduce the risk of pathogenic infection.

  11. You're Only as Old as Your Arteries: Translational Strategies for Preserving Vascular Endothelial Function with Aging

    PubMed Central

    Kaplon, Rachelle E.; Gioscia-Ryan, Rachel A.; LaRocca, Thomas J.

    2014-01-01

    Endothelial dysfunction develops with age and increases the risk of age-associated vascular disorders. Nitric oxide insufficiency, oxidative stress, and chronic low-grade inflammation, induced by upregulation of adverse cellular signaling processes and imbalances in stress resistance pathways, mediate endothelial dysfunction with aging. Healthy lifestyle behaviors preserve endothelial function with aging by inhibiting these mechanisms, and novel nutraceutical compounds that favorably modulate these pathways hold promise as a complementary approach for preserving endothelial health. PMID:24985329

  12. Coniferyl Aldehyde Attenuates Radiation Enteropathy by Inhibiting Cell Death and Promoting Endothelial Cell Function

    PubMed Central

    Son, Yeonghoon; Jang, Jun-Ho; Lee, Yoon-Jin; Kim, Sung-Ho; Ko, Young-Gyo; Lee, Yun-Sil; Lee, Hae-June

    2015-01-01

    Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA), an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR) to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function. PMID:26029925

  13. Coniferyl aldehyde attenuates radiation enteropathy by inhibiting cell death and promoting endothelial cell function.

    PubMed

    Jeong, Ye-Ji; Jung, Myung Gu; Son, Yeonghoon; Jang, Jun-Ho; Lee, Yoon-Jin; Kim, Sung-Ho; Ko, Young-Gyo; Lee, Yun-Sil; Lee, Hae-June

    2015-01-01

    Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA), an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR) to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function.

  14. Inhibition of constitutive endothelial NO-synthase activity by tannin and quercetin.

    PubMed

    Chiesi, M; Schwaller, R

    1995-02-14

    The effect of natural polyphenols on three isoforms of NO-synthase was investigated. Among the compounds tested, tannin was the most potent, inhibiting endothelial constitutive NO synthase (eNOS) with an IC50 of 2.2 microM. Other NOS isoforms (i.e. neuronal constitutive NOS and smooth muscle inducible NOS) were also inhibited but at much higher concentrations (selectivity ratio of approx. 20-30). Quercetin was also an effective but less potent inhibitor of eNOS (IC50 = 220 microM). The kinetics of tannin inhibition were investigated to gather information on the mechanism of action. Tannin did not interfere with the interaction of the enzyme with the co-substrates L-arginine and NADPH nor with the cofactor tetrahydrobiopterin. The inhibition level was also independent of free Ca2+ concentration as well as of the presence of high exogenous calmodulin concentrations.

  15. Role of the transient receptor potential vanilloid type 1 receptor and stretch-activated ion channels in nitric oxide release from endothelial cells of the aorta and heart in rats

    PubMed Central

    Torres-Narváez, Juan Carlos; Mondragón, Leonardo del Valle; Varela López, Elvira; Pérez-Torres, Israel; Díaz Juárez, Julieta Anabell; Suárez, Jorge; Hernández, Gustavo Pastelín

    2012-01-01

    Shear stress stimulates nitric oxide (NO) release in endothelial cells. Stretch-activated ion channels (SACs) and the transient receptor potential vanilloid type 1 (TRPV1) receptor respond to mechanical stimulus and are permeable to Na+, Ca2+ and K+. The influence of SACs and the TRPV1 receptor on NO release on the heart and on the vascular reactivity of the thoracic aorta (TA) was studied. Experiments were performed in isolated perfused heart, cultured endothelial cells and TA rings from Wistar rats. Capsaicin (10 μM, 30 μM) was used as a NO release stimulator, capsazepine (6 μM, 10 μM) was used as a capsaicin antagonist and gadolinium (3 μM, 5 μM) was used as an inhibitor of SACs. NO was measured by the Kelm and Tenorio methods. Left ventricular pressure was recorded and coronary vascular resistance was calculated. Capsaicin increased NO release in the heart by 58% (395±8 pmol/mL to 627±23 pmol/mL). Capsazepine and gadolinium inhibited NO release by 74% and 82%, respectively. This tendency was similar in all experimental models. Capsaicin attenuated the effects of norepinephrine (10 M to 7 M) on TA and had no effect in the presence of Nω-nitro-L-arginine methyl ester. Therefore, the authors conclude that SACs and the TRPV1 receptor are both present in the coronary endothelium and that both participate in Ca2+-dependent NO release. PMID:23620694

  16. Endostatin lowers blood pressure via nitric oxide and prevents hypertension associated with VEGF inhibition

    PubMed Central

    Sunshine, Sarah B.; Dallabrida, Susan M.; Durand, Ellen; Ismail, Nesreen S.; Bazinet, Lauren; Birsner, Amy E.; Sohn, Regina; Ikeda, Sadakatsu; Pu, William T.; Kulke, Matthew H.; Javaherian, Kashi; Zurakowski, David; Rupnick, Maria

    2012-01-01

    Antiangiogenesis therapy has become a vital part of the armamentarium against cancer. Hypertension is a dose-limiting toxicity for VEGF inhibitors. Thus, there is a pressing need to address the associated adverse events so these agents can be better used. The hypertension may be mediated by reduced NO bioavailability resulting from VEGF inhibition. We proposed that the hypertension may be prevented by coadministration with endostatin (ES), an endogenous angiogenesis inhibitor with antitumor effects shown to increase endothelial NO production in vitro. We determined that Fc-conjugated ES promoted NO production in endothelial and smooth muscle cells. ES also lowered blood pressure in normotensive mice and prevented hypertension induced by anti-VEGF antibodies. This effect was associated with higher circulating nitrate levels and was absent in eNOS-knockout mice, implicating a NO-mediated mechanism. Retrospective study of patients treated with ES in a clinical trial revealed a small but significant reduction in blood pressure, suggesting that the findings may translate to the clinic. Coadministration of ES with VEGF inhibitors may offer a unique strategy to prevent drug-related hypertension and enhance antiangiogenic tumor suppression. PMID:22733742

  17. Lysophosphatidylcholine and 7-oxocholesterol modulate Ca2+ signals and inhibit the phosphorylation of endothelial NO synthase and cytosolic phospholipase A2.

    PubMed Central

    Millanvoye-Van Brussel, Elisabeth; Topal, Gökce; Brunet, Annie; Do Pham, Thuc; Deckert, Valérie; Rendu, Francine; David-Dufilho, Monique

    2004-01-01

    The oxidation of plasma LDLs (low-density lipoproteins) is a key event in the pathogenesis of atherosclerosis. LPC (lysophosphatidylcholine) and oxysterols are major lipid constitutents of oxidized LDLs. In particular, 7-oxocholesterol has been found in plasma from cardiac patients and atherosclerotic plaque. In the present study, we investigated the ability of 7-oxocholesterol and LPC to regulate the activation of eNOS (endothelial nitric oxide synthase) and cPLA2 (cytosolic phospholipase A2) that synthesize two essential factors for vascular wall integrity, NO (nitric oxide) and arachidonic acid. In endothelial cells from human umbilical vein cords, both 7-oxocholesterol (150 microM) and LPC (20 microM) decreased histamine-induced NO release, but not the release activated by thapsigargin. The two lipids decreased NO release through a PI3K (phosphoinositide 3-kinase)-dependent pathway, and decreased eNOS phosphorylation. Their mechanisms of action were, however, different. The NO release reduction was dependent on superoxide anions in LPC-treated cells and not in 7-oxocholesterol-treated ones. The Ca2+ signals induced by histamine were abolished by LPC, but not by 7-oxocholesterol. The oxysterol also inhibited (i) the histamine- and thapsigargin-induced arachidonic acid release, and (ii) the phosphorylation of both cPLA2 and ERK1/2 (extracellular-signal-regulated kinases 1/2). The results show that 7-oxocholesterol inhibits eNOS and cPLA2 activation by altering a Ca2+-independent upstream step of PI3K and ERK1/2 cascades, whereas LPC desensitizes eNOS by interfering with receptor-activated signalling pathways. This suggests that 7-oxocholesterol and LPC generate signals which cross-talk with heterologous receptors, effects which could appear at early stage of atherosclerosis. PMID:14992685

  18. Dual NEP/ECE inhibition improves endothelial function in mesenteric resistance arteries of 32-week-old SHR.

    PubMed

    Lemkens, Pieter; Spijkers, Leon Ja; Meens, Merlijn J; Nelissen, Jelly; Janssen, Ben; Peters, Stephan Lm; Schiffers, Paul Mh; De Mey, Jo Gr

    2017-03-16

    Endothelin 1 (ET-1), a potent vasoconstrictor, pro-mitogenic and pro-inflammatory peptide, may promote development of endothelial dysfunction and arterial remodeling. ET-1 can be formed through cleavage of big-ET-1 by endothelin-converting enzyme (ECE) or neutral endopeptidase (NEP). We investigated whether chronic treatment with the novel dual NEP/ECE inhibitor SOL1 improves functional and structural properties of resistance-sized arteries of 32-week-old male spontaneously hypertensive rats (SHR). SHR received a chronic 4-week treatment with SOL1, losartan or hydralazine. We then compared effects of inhibition of NO synthase (NOS) (100 μM l-NAME), blockade of ETA- and ETB-receptors (10 μM bosentan) and stimulation of the endothelium with 0.001-10 μM acetylcholine (ACh) in isolated third-order mesenteric resistance arteries. Losartan and hydralazine significantly lowered blood pressure. Losartan decreased the media-to-lumen ratio of resistance arteries. l-NAME (1) increased arterial contractile responses to K(+) (5.9-40 mM) in the losartan, SOL1 and vehicle group and (2) increased the sensitivity to phenylephrine (PHE; 0.16-20 μM) in the SOL1 group but not in the losartan, hydralazine and vehicle group. Relaxing responses to ACh in the absence or presence of l-NAME during contractions induced by either 10 μM PHE or 40 mM K(+) were not altered by any in vivo treatment. Acute treatment with bosentan did, however, significantly improve maximal relaxing responses involving endothelium-derived nitric oxide and -hyperpolarizing factors in the SOL1 group but not in the losartan, hydralazine or vehicle group. Thus, chronic inhibition of NEP/ECE improved basal endothelial function but did not alter blood pressure, resistance artery structure and stimulated endothelium-dependent relaxing responses in 32-week-old SHR.Hypertension Research advance online publication, 16 March 2017; doi:10.1038/hr.2017.38.

  19. Jin Fu Kang Oral Liquid Inhibits Lymphatic Endothelial Cells Formation and Migration

    PubMed Central

    Wang, Dan; Tang, Jie

    2016-01-01

    Lung cancer is the leading cause of cancer-related deaths worldwide. Jin Fu Kang (JFK), an oral liquid prescription of Chinese herbal drugs, has been clinically available for the treatment of non-small cell lung cancer (NSCLC). Lymphangiogenesis is a primary event in the process of cancer development and metastasis, and the formation and migration of lymphatic endothelial cells (LECs) play a key role in the lymphangiogenesis. To assess the activity of stromal cell-derived factor-1 (SDF-1) and the coeffect of SDF-1 and vascular endothelial growth factor-C (VEGF-C) on the formation and migration of LECs and clarify the inhibitory effects of JFK on the LECs, the LECs were differentiated from CD34+/VEGFR-3+ endothelial progenitor cells (EPCs), and JFK-containing serums were prepared from rats. SDF-1 and VEGF-C both induced the differentiation of CD34+/VEGFR-3+ EPCs towards LECs and enhanced the LECs migration. Couse of SDF-1 and VEGF-C displayed an additive effect on the LECs formation but not on their migration. JFK inhibited the formation and migration of LECs, and the inhibitory effects were most probably via regulation of the SDF-1/CXCR4 and VEGF-C/VEGFR-3 axes. The current finding suggested that JFK might inhibit NSCLC through antilymphangiogenesis and also provided a potential to discover antilymphangiogenesis agents from natural resources. PMID:27698675

  20. Nitric oxide inhibits neointimal hyperplasia following vascular injury via differential, cell-specific modulation of SOD-1 in the arterial wall.

    PubMed

    Bahnson, Edward S M; Koo, Nathaniel; Cantu-Medellin, Nadiezhda; Tsui, Aaron Y; Havelka, George E; Vercammen, Janet M; Jiang, Qun; Kelley, Eric E; Kibbe, Melina R

    2015-01-30

    Superoxide (O2(•-)) promotes neointimal hyperplasia following arterial injury. Conversely, nitric oxide ((•)NO) inhibits neointimal hyperplasia through various cell-specific mechanisms, including redox regulation. What remains unclear is whether (•)NO exerts cell-specific regulation of the vascular redox environment following arterial injury to inhibit neointimal hyperplasia. Therefore, the aim of the present study was to assess whether (•)NO exerts cell-specific, differential modulation of O2(•-) levels throughout the arterial wall, establish the mechanism of such modulation, and determine if it regulates (•)NO-dependent inhibition of neointimal hyperplasia. In vivo, (•)NO increased superoxide dismutase-1 (SOD-1) levels following carotid artery balloon injury in a rat model. In vitro, (•)NO increased SOD-1 levels in vascular smooth muscle cells (VSMC), but had no effect on SOD-1 in endothelial cells or adventitial fibroblasts. This SOD-1 increase was associated with an increase in sod1 gene expression, increase in SOD-1 activity, and decrease in O2(•-) levels. Lastly, to determine the role of SOD-1 in (•)NO-mediated inhibition of neointimal hyperplasia, we performed the femoral artery wire injury model in wild type and SOD-1 knockout (KO) mice, with and without (•)NO. Interestingly, (•)NO inhibited neointimal hyperplasia only in wild type mice, with no effect in SOD-1 KO mice. In conclusion, these data show the cell-specific modulation of O2(•-) by (•)NO through regulation of SOD-1 in the vasculature, highlighting its importance on the inhibition of neointimal hyperplasia. These results also shed light into the mechanism of (•)NO-dependent redox balance, and suggest a novel VSMC redox target to prevent neointimal hyperplasia.

  1. Proliferation of macrophages due to the inhibition of inducible nitric oxide synthesis by oxidized low-density lipoproteins

    PubMed Central

    Brunner, Monika; Gruber, Miriam; Schmid, Diethart; Baran, Halina; Moeslinger, Thomas

    2015-01-01

    Oxidized low-density lipoprotein (ox-LDL) is assumed to be a major causal agent in hypercholesteraemia-induced atherosclerosis. Because the proliferation of lipid-loaden macrophages within atherosclerotic lesions has been described, we investigated the dependence of macrophage proliferation on the inhibition of inducible nitric oxide synthase (iNOS) by hypochlorite oxidized LDL. Ox-LDL induces a dose dependent inhibition of inducible nitric oxide synthesis in lipopolysaccharide-interferon stimulated mouse macrophages (J774.A1) with concomitant macrophage proliferation as assayed by cell counting, tritiated-thymidine incorporation and measurement of cell protein. Native LDL did not influence macrophage proliferation and inducible nitric oxide synthesis. iNOS protein and mRNA was reduced by HOCl-oxidized LDL (0-40 µg/ml) as revealed by immunoblotting and competitive semiquantitative PCR. Macrophage proliferation was increased by the addition of the iNOS inhibitor L-NAME. The addition of ox-LDL to L-NAME containing incubations induced no further statistically significant increase in cell number. Nitric oxide donors decreased ox-LDL induced macrophage proliferation and nitric oxide scavengers restored macrophage proliferation to the initial values achieved by ox-LDL. The decrease of cytosolic DNA fragments in stimulated macrophages incubated with ox-LDL demonstrates that the proliferative actions of ox-LDL are associated with a decrease of NO-induced apoptosis. Our data show that inhibition of iNOS dependent nitric oxide production caused by hypochlorite oxidized LDL enhances macrophage proliferation. This might be a key event in the pathogenesis of atherosclerotic lesions. PMID:26600745

  2. Improvement of blood flow, expression of nitric oxide, and vascular endothelial growth factor by low-energy shockwave therapy in random-pattern skin flap model.

    PubMed

    Yan, Xiaoyu; Zeng, Bingfang; Chai, Yimin; Luo, Congfeng; Li, Xiaolin

    2008-12-01

    Extracorporeal shock wave therapy (ESWT) can improve flap survival, but its mechanism remains unclear. In this study, we aim to investigate whether ESWT can improve blood flow in ischemic skin flaps and the possible mechanism. Cranially based random-pattern flap (3 x 10 cm) model was established, and its ischemic portion was treated with or without ESWT at 0.09 mJ/mm2 with 750 impulses (1.5 Hz), immediately after operation. Survival area, blood flow, vessel distribution, microvessel density, and expression of nitric oxide and vascular endothelial growth factor were evaluated at 1, 3, and 10 days postoperatively. The results showed that blood perfusion, expression of nitric oxide and vascular endothelial growth factor, vasodilatation of pre-existing vessels at early postoperative stage, neovascularization at late stage, and flap survival were all significantly promoted in treatment group. In conclusion, ESWT can improve skin flap surviving rate through enhanced vasodilatation at early postoperative stage and neovascularization at late stage via modulation of angio-active factors expression.

  3. Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide.

    PubMed

    Kwon, N S; Stuehr, D J; Nathan, C F

    1991-10-01

    Macrophage-derived nitric oxide (NO) is cytostatic to tumor cells and microbial pathogens. We tested whether one molecular target for the cytostatic action of NO may be ribonucleotide reductase (RR), a rate-limiting enzyme in DNA synthesis. In a concentration-dependent manner, NO gas and lysates of activated macrophages that generated comparable amounts of NO led to the same degree of inhibition of partially purified RR from L1210 mouse lymphoma cells. Lysates from nonactivated macrophages, which do not produce NO, were noninhibitory. With lysates from activated macrophages, RR was protected by omitting L-arginine or by adding the NO synthase inhibitors diphenyleneiodonium, N omega-methyl-L-arginine, or N omega-amino-L-arginine. L-Arginine, but not D-arginine, abolished the protective effect of N omega-amino-L-arginine. The prototypic pharmacologic inhibitor of RR is hydroxyurea. Its structural resemblance to N omega-hydroxy-L-arginine, a reaction intermediate of NO synthase, prompted us to test if hydroxyurea can generate NO. In the presence of H2O2 and CuSO4, hydroxyurea produced NO2-/NO3-, aerobic reaction products of NO. Addition of morpholine blocked NO2-/NO3- generation from hydroxyurea and led to formation of nitrosomorpholine, as detected by gas chromatography/mass spectrometry. Thus, hydroxyurea can produce an NO-like, nitrosating rectant. L1210 cell DNA synthesis was inhibited completely by activated macrophages or by hydroxyurea, and was partially restored to the same degree in both settings by providing deoxyribonucleosides to bypass the block in RR. Thus, both NO gas and NO generated by activated macrophage lysates inhibit tumor cell RR. The RR inhibitor hydroxyurea can also generate an NO-like species. Similar, partial restoration of tumor cell DNA synthesis by deoxyribonucleosides in the presence of activated macrophages or hydroxyurea suggests that cytostasis by activated macrophages and by hydroxyurea has comparable mechanisms, including, but

  4. G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition.

    PubMed

    Zhang, Panpan; He, Qiuping; Chen, Dongbo; Liu, Weixiao; Wang, Lu; Zhang, Chunxia; Ma, Dongyuan; Li, Wei; Liu, Bing; Liu, Feng

    2015-10-01

    In vertebrates, embryonic hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of endothelial cells, the hemogenic endothelium (HE), through the endothelial-to-hematopoietic transition (EHT). Notch signaling is essential for HSPC development during embryogenesis across vertebrates. However, whether and how it regulates EHT remains unclear. Here, we show that G protein-coupled receptor 183 (Gpr183) signaling serves as an indispensable switch for HSPC emergence by repressing Notch signaling before the onset of EHT. Inhibition of Gpr183 significantly upregulates Notch signaling and abolishes HSPC emergence. Upon activation by its ligand 7α-25-OHC, Gpr183 recruits β-arrestin1 and the E3 ligase Nedd4 to degrade Notch1 in specified HE cells and then facilitates the subsequent EHT. Importantly, 7α-25-OHC stimulation promotes HSPC emergence in vivo and in vitro, providing an attractive strategy for enhancing the in vitro generation of functional HSPCs.

  5. Olive oil compounds inhibit vascular endothelial growth factor receptor-2 phosphorylation

    SciTech Connect

    Lamy, Sylvie Ouanouki, Amira; Béliveau, Richard; Desrosiers, Richard R.

    2014-03-10

    Vascular endothelial growth factor (VEGF) triggers crucial signaling processes that regulate tumor angiogenesis and, therefore, represents an attractive target for the development of novel anticancer therapeutics. Several epidemiological studies have confirmed that abundant consumption of foods from plant origin is associated with reduced risk of developing cancers. In the Mediterranean basin, the consumption of extra virgin olive oil is an important constituent of the diet. Compared to other vegetable oils, the presence of several phenolic antioxidants in olive oil is believed to prevent the occurrence of a variety of pathological processes, such as cancer. While the strong antioxidant potential of these molecules is well characterized, their antiangiogenic activities remain unknown. The aim of this study is to investigate whether tyrosol (Tyr), hydroxytyrosol (HT), taxifolin (Tax), oleuropein (OL) and oleic acid (OA), five compounds contained in extra virgin olive oil, can affect in vitro angiogenesis. We found that HT, Tax and OA were the most potent angiogenesis inhibitors through their inhibitory effect on specific autophosphorylation sites of VEGFR-2 (Tyr951, Tyr1059, Tyr1175 and Tyr1214) leading to the inhibition of endothelial cell (EC) signaling. Inhibition of VEGFR-2 by these olive oil compounds significantly reduced VEGF-induced EC proliferation and migration as well as their morphogenic differentiation into capillary-like tubular structures in Matrigel. Our study demonstrates that HT, Tax and OA are novel and potent inhibitors of the VEGFR-2 signaling pathway. These findings emphasize the chemopreventive properties of olive oil and highlight the importance of nutrition in cancer prevention. - Highlights: • We investigated five compounds contained in extra virgin olive oil on angiogenesis. • Hydroxytyrosol, taxifolin and oleic acid are the best angiogenesis inhibitors. • Olive oil compounds affect endothelial cell functions essential for