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Sample records for inhibiting endothelial nitric

  1. 25-Hydroxycholesterol impairs endothelial function and vasodilation by uncoupling and inhibiting endothelial nitric oxide synthase.

    PubMed

    Ou, Zhi-Jun; Chen, Jing; Dai, Wei-Ping; Liu, Xiang; Yang, Yin-Ke; Li, Yan; Lin, Ze-Bang; Wang, Tian-Tian; Wu, Ying-Ying; Su, Dan-Hong; Cheng, Tian-Pu; Wang, Zhi-Ping; Tao, Jun; Ou, Jing-Song

    2016-10-01

    Endothelial dysfunction is a key early step in atherosclerosis. 25-Hydroxycholesterol (25-OHC) is found in atherosclerotic lesions. However, whether 25-OHC promotes atherosclerosis is unclear. Here, we hypothesized that 25-OHC, a proinflammatory lipid, can impair endothelial function, which may play an important role in atherosclerosis. Bovine aortic endothelial cells were incubated with 25-OHC. Endothelial cell proliferation, migration, and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation were determined. The expression and phosphorylation of endothelial NO synthase (eNOS) and Akt as well as the association of eNOS and heat shock protein (HSP)90 were detected by immunoblot analysis and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining and caspase-3 activity, and expression of Bcl-2, Bax, cleaved caspase-9, and cleaved caspase-3 were detected by immunoblot analysis. Finally, aortic rings from Sprague-Dawley rats were isolated and treated with 25-OHC, and endothelium-dependent vasodilation was evaluated. 25-OHC significantly inhibited endothelial cell proliferation, migration, and tube formation. 25-OHC markedly decreased NO production and increased superoxide anion generation. 25-OHC reduced the phosphorylation of Akt and eNOS and the association of eNOS and HSP90. 25-OHC also enhanced endothelial cell apoptosis by decreasing Bcl-2 expression and increasing cleaved caspase-9 and cleaved caspase-3 expressions as well as caspase-3 activity. 25-OHC impaired endothelium-dependent vasodilation. These data demonstrated that 25-OHC could impair endothelial function by uncoupling and inhibiting eNOS activity as well as by inducing endothelial cell apoptosis. Our findings indicate that 25-OHC may play an important role in regulating atherosclerosis. Copyright © 2016 the American Physiological Society.

  2. Chlorine Gas Exposure Causes Systemic Endothelial Dysfunction by Inhibiting Endothelial Nitric Oxide Synthase–Dependent Signaling

    PubMed Central

    Honavar, Jaideep; Samal, Andrey A.; Bradley, Kelley M.; Brandon, Angela; Balanay, Joann; Squadrito, Giuseppe L.; MohanKumar, Krishnan; Maheshwari, Akhil; Postlethwait, Edward M.; Matalon, Sadis; Patel, Rakesh P.

    2011-01-01

    Chlorine gas (Cl2) exposure during accidents or in the military setting results primarily in injury to the lungs. However, the potential for Cl2 exposure to promote injury to the systemic vasculature leading to compromised vascular function has not been studied. We hypothesized that Cl2 promotes extrapulmonary endothelial dysfunction characterized by a loss of endothelial nitric oxide synthase (eNOS)-derived signaling. Male Sprague Dawley rats were exposed to Cl2 for 30 minutes, and eNOS-dependent vasodilation of aorta as a function of Cl2 dose (0–400 ppm) and time after exposure (0–48 h) were determined. Exposure to Cl2 (250–400 ppm) significantly inhibited eNOS-dependent vasodilation (stimulated by acetycholine) at 24 to 48 hours after exposure without affecting constriction responses to phenylephrine or vasodilation responses to an NO donor, suggesting decreased NO formation. Consistent with this hypothesis, eNOS protein expression was significantly decreased (∼ 60%) in aorta isolated from Cl2–exposed versus air-exposed rats. Moreover, inducible nitric oxide synthase (iNOS) mRNA was up-regulated in circulating leukocytes and aorta isolated 24 hours after Cl2 exposure, suggesting stimulation of inflammation in the systemic vasculature. Despite decreased eNOS expression and activity, no changes in mean arterial blood pressure were observed. However, injection of 1400W, a selective inhibitor of iNOS, increased mean arterial blood pressure only in Cl2–exposed animals, suggesting that iNOS-derived NO compensates for decreased eNOS-derived NO. These results highlight the potential for Cl2 exposure to promote postexposure systemic endothelial dysfunction via disruption of vascular NO homeostasis mechanisms. PMID:21131444

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities

    PubMed Central

    Martínez-Ruiz, Antonio; Villanueva, Laura; de Orduña, Cecilia González; López-Ferrer, Daniel; Higueras, María Ángeles; Tarín, Carlos; Rodríguez-Crespo, Ignacio; Vázquez, Jesús; Lamas, Santiago

    2005-01-01

    Nitric oxide is implicated in a variety of signaling pathways in different systems, notably in endothelial cells. Some of its effects can be exerted through covalent modifications of proteins and, among these modifications, increasing attention is being paid to S-nitrosylation as a signaling mechanism. In this work, we show by a variety of methods (ozone chemiluminescence, biotin switch, and mass spectrometry) that the molecular chaperone Hsp90 is a target of S-nitrosylation and identify a susceptible cysteine residue in the region of the C-terminal domain that interacts with endothelial nitric oxide synthase (eNOS). We also show that the modification occurs in endothelial cells when they are treated with S-nitroso-l-cysteine and when they are exposed to eNOS activators. Hsp90 ATPase activity and its positive effect on eNOS activity are both inhibited by S-nitrosylation. Together, these data suggest that S-nitrosylation may functionally regulate the general activities of Hsp90 and provide a feedback mechanism for limiting eNOS activation. PMID:15937123

  8. H2S Inhibits Oscillatory Shear Stress-Induced Monocyte Binding to Endothelial Cells Via Nitric Oxide Production

    PubMed Central

    Go, Young-Mi; Lee, Hye-Rim; Park, Heonyong

    2012-01-01

    H2S is a signaling molecule associated with protection against vascular diseases, including atherosclerosis. This protection involves the stimulation of vasorelaxation, but other possible contributing mechanisms have not been extensively explored. In this study, we found that the vascular H2S-producing enzyme, cystathionine-γ-lyase (CSE), was down-regulated by oscillatory shear stress (OSS) among various vaso-regulators. Consistently, NaHS, an H2S donor, appeared to inhibit OSS-induced THP-1 cell adhesion. We also found that NaHS activated the nitric oxide (NO)-producing Akt/endothelial nitric oxide synthase (eNOS) signaling pathway in response to OSS, whereas NaHS had no effect on IκB, a well-known molecule regulating pro-inflammatory signaling pathways. Moreover, NaHS increased OSS-dependent eNOS expression and decreased expression of intercellular adhesion molecule-1 (ICAM-1). NG-nitro-L-arginine methyl ester (L-NAME), an eNOS inhibitor, abrogated the inhibitory effects of NaHS on OSS-induced endothelial ICAM-1 expression and monocyte adhesion to endothelial cells. These data suggest that down-regulation of CSE resulting in decreased levels of H2S is a key factor for OSS-associated atherogenesis and further suggest that regulation of H2S production can be a potential target for preventing cardiovascular diseases. PMID:23124382

  9. 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.

  10. Diabetic HDL-associated myristic acid inhibits acetylcholine-induced nitric oxide generation by preventing the association of endothelial nitric oxide synthase with calmodulin.

    PubMed

    White, James; Guerin, Theresa; Swanson, Hollie; Post, Steven; Zhu, Haining; Gong, Ming; Liu, Jun; Everson, William V; Li, Xiang-An; Graf, Gregory A; Ballard, Hubert O; Ross, Stuart A; Smart, Eric J

    2008-01-01

    In the current study, we examined whether diabetes affected the ability of HDL to stimulate nitric oxide (NO) production. Using HDL isolated from both diabetic humans and diabetic mouse models, we found that female HDL no longer induced NO synthesis, despite containing equivalent amounts of estrogen as nondiabetic controls. Furthermore, HDL isolated from diabetic females and males prevented acetylcholine-induced stimulation of NO generation. Analyses of both the human and mouse diabetic HDL particles showed that the HDLs contained increased levels of myristic acid. To determine whether myristic acid associated with HDL particles was responsible for the decrease in NO generation, myristic acid was added to HDL isolated from nondiabetic humans and mice. Myristic acid-associated HDL inhibited the generation of NO in a dose-dependent manner. Importantly, diabetic HDL did not alter the levels of endothelial NO synthase or acetylcholine receptors associated with the cells. Surprisingly, diabetic HDL inhibited ionomycin-induced stimulation of NO production without affecting ionomycin-induced increases in intracellular calcium. Further analysis indicated that diabetic HDL prevented calmodulin from interacting with endothelial NO synthase (eNOS) but did not affect the activation of calmodulin kinase or calcium-independent mechanisms for stimulating eNOS. These studies are the first to show that a specific fatty acid associated with HDL inhibits the stimulation of NO generation. These findings have important implications regarding cardiovascular disease in diabetic patients.

  11. 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.

  12. Hydrogen sulfide inhibits endothelial nitric oxide formation and receptor ligand-mediated Ca(2+) release in endothelial and smooth muscle cells.

    PubMed

    Kloesch, Burkhard; Steiner, Guenter; Mayer, Bernd; Schmidt, Kurt

    2016-02-01

    In the vascular system, ATP-sensitive K(+)-channels are a target for H2S. Recent evidence suggests that H2S may also modulate Na(+)- and Ca(2+)-permeable channels and intracellular Ca(2+) stores, but the influence of H2S on endothelial Ca(2+) dynamics and Ca(2+)-dependent activation of endothelial nitric oxide synthase (eNOS) is unclear. In this study, we investigated the effects of H2S on Ca(2+) signaling in endothelial and smooth muscle cells with special emphasis given to the role of H2S in modulating endothelial NO formation. Experiments were performed with endothelial cells from porcine aorta, the human endothelial cell line HMEC-1, and smooth muscle cells from rat aorta and trachea. Mobilization of intracellular Ca(2+) and Ca(2+) entry was monitored with Fura-2. Activity of eNOS was determined as conversion of incorporated l-[(3)H]arginine into l-[(3)H]citrulline. Incubation of endothelial cells with the H2S donors sodium hydrogen sulfide (NaHS) and GYY4137 blocked activation of eNOS by the receptor agonist ATP but not by the Ca(2+) ionophore A23187. Data revealed that H2S inhibited ATP-induced release of Ca(2+) from intracellular stores indicating that H2S attenuates eNOS activity by blocking capacitative Ca(2+) entry. A similar inhibitory effect of H2S on ATP-induced Ca(2+) release and Ca(2+) entry was also observed in human microvascular endothelial cells and smooth muscle cells. H2S antagonized Ca(2+) mobilization by receptor agonists and store-operated Ca(2+) entry thereby limiting eNOS activation and NO formation. The effect of H2S on Ca(2+) stores was not restricted to endothelial cells but was also observed in vascular and tracheal smooth muscle cells. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  13. 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.

  14. 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.

  15. Epigallocatechin gallate inhibits endothelial exocytosis.

    PubMed

    Yamakuchi, Munekazu; Bao, Clare; Ferlito, Marcella; Lowenstein, Charles J

    2008-07-01

    Consumption of green tea is associated with a decrease in cardiovascular mortality. The beneficial health effects of green tea are attributed in part to polyphenols, organic compounds found in tea that lower blood pressure, reduce body fat, decrease LDL cholesterol, and inhibit inflammation. We hypothesized that epigallocatechin gallate (EGCG), the most abundant polyphenol in tea, inhibits endothelial exocytosis, the initial step in leukocyte trafficking and vascular inflammation. To test this hypothesis, we treated human umbilical-vein endothelial cells with EGCG and other polyphenols, and then measured endothelial exocytosis. We found that EGCG decreases endothelial exocytosis in a concentration-dependent manner, with the effects most prominent after 4 h of treatment. Other catechin polyphenols had no effect on endothelial cells. By inhibiting endothelial exocytosis, EGCG decreases leukocyte adherence to endothelial cells. In searching for the mechanism by which EGCG affects endothelial cells, we found that EGCG increases Akt phosphorylation, eNOS phosphorylation, and nitric oxide (NO) production. NOS inhibition revealed that NO mediates the anti-inflammatory effects of EGCG. Our data suggest that polyphenols can decrease vascular inflammation by increasing the synthesis of NO, which blocks endothelial exocytosis.

  16. 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.

  17. 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

  18. Cyclooxygenase-2 inhibition improves vascular endothelial dysfunction in a rat model of endotoxic shock: role of inducible nitric-oxide synthase and oxidative stress.

    PubMed

    Virdis, Agostino; Colucci, Rocchina; Fornai, Matteo; Blandizzi, Corrado; Duranti, Emiliano; Pinto, Stefania; Bernardini, Nunzia; Segnani, Cristina; Antonioli, Luca; Taddei, Stefano; Salvetti, Antonio; Del Tacca, Mario

    2005-03-01

    We investigated whether cyclooxygenase (COX) isoforms (COX-1 and COX-2) and decreased NO availability contribute to endothelial dysfunction in endotoxemic rats. The involvement of reactive oxygen species (ROS) was also evaluated. Rats were injected with Salmonella-derived lipopolysaccharide or saline. After 6 h, endothelial function of mesenteric resistance arteries was evaluated. In controls, acetylcholine (ACh)-induced relaxation was inhibited by the nitric-oxide synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA) and unaffected by 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)-phenyl-2(5H)-furanone (DFU) (COX-2 inhibitor). In lipopolysaccharide (LPS)-treated rats, the response to ACh was blunted compared with controls, less sensitive to l-NMMA, and enhanced by DFU. COX-2 blockade also improved the inhibitory effect of l-NMMA on cholinergic relaxation. SC-560 [5-(4-clorophenyl)-1-(4-metoxyphenyl)-3-trifluoromethylpirazole] (COX-1 inhibitor) did not modify the response to ACh in both groups. LPS-induced endothelial dysfunction was unaffected by the thromboxane A(2) (TxA(2)) receptor antagonist SQ-29548 (7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1] hept-2-yl]-[1S(1alpha,2alpha(Z),3alpha,4alpha)]-5-heptenoic acid). In vivo inducible nitric-oxide synthase (iNOS) inhibition by S-methylisothiourea partly attenuated LPS-induced endothelial dysfunction. The antioxidants ascorbic acid and superoxide dismutase normalized endothelium-dependent relaxation and restored the inhibitory action of l-NMMA on ACh. Responses to sodium nitroprusside were similar in both groups. In LPS-treated rats, reverse transcription-polymerase chain reaction showed a marked increase in mesenteric iNOS and COX-2 expressions, whereas endothelial nitric-oxide synthase and COX-1 were unchanged. LPS-induced COX-2 overexpression was reduced but not abrogated by S-methylisothiourea. LPS-induced COX-2 up-regulation was also documented by immunohistochemistry. In

  19. 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.

  20. 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

  1. Peroxynitrite induces destruction of the tetrahydrobiopterin and heme in endothelial nitric oxide synthase: transition from reversible to irreversible enzyme inhibition.

    PubMed

    Chen, Weiguo; Druhan, Lawrence J; Chen, Chun-An; Hemann, Craig; Chen, Yeong-Renn; Berka, Vladimir; Tsai, Ah-Lim; Zweier, Jay L

    2010-04-13

    Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular and cardiac function. Peroxynitrite (ONOO(-)) inactivates eNOS, but questions remain regarding the mechanisms of this process. It has been reported that inactivation is due to oxidation of the eNOS zinc-thiolate cluster, rather than the cofactor tetrahydrobiopterin (BH(4)); however, this remains highly controversial. Therefore, we investigated the mechanisms of ONOO(-)-induced eNOS dysfunction and their dose dependence. Exposure of human eNOS to ONOO(-) resulted in a dose-dependent loss of activity with a marked destabilization of the eNOS dimer. HPLC analysis indicated that both free and eNOS-bound BH(4) were oxidized during exposure to ONOO(-); however, full oxidation of protein-bound biopterin required higher ONOO(-) levels. Additionally, ONOO(-) triggered changes in the UV/visible spectrum and heme content of the enzyme. Preincubation of eNOS with BH(4) decreased dimer destabilization and heme alteration. Addition of BH(4) to the ONOO(-)-destabilized eNOS dimer only partially rescued enzyme function. In contrast to ONOO(-) treatment, incubation with the zinc chelator TPEN with removal of enzyme-bound zinc did not change the eNOS activity or stability of the SDS-resistant eNOS dimer, demonstrating that the dimer stabilization induced by BH(4) does not require zinc occupancy of the zinc-thiolate cluster. While ONOO(-) treatment was observed to induce loss of Zn binding, this cannot account for the loss of enzyme activity. Therefore, ONOO(-)-induced eNOS inactivation is primarily due to oxidation of BH(4) and irreversible destruction of the heme/heme center.

  2. Hyperfiltration and effect of nitric oxide inhibition on renal and endothelial function in humans with uncomplicated type 1 diabetes mellitus

    PubMed Central

    Reich, Heather N.; Jiang, Shan; Har, Ronnie; Nasrallah, Rania; Hébert, Richard L.; Lai, Vesta; Scholey, James W.; Sochett, Etienne B.

    2012-01-01

    Studies of experimental diabetes mellitus (DM) suggest that increased nitric oxide (NO) bioactivity contributes to renal hyperfiltration. However, the role of NO in mediating hyperfiltration has not been fully elucidated in humans. Our aim was to examine the effect of NO synthase inhibition on renal and peripheral vascular function in normotensive subjects with uncomplicated type 1 DM. Renal function and brachial artery flow-mediated vasodilatation (FMD) were measured before and after an intravenous infusion of the NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NMMA) in 21 healthy control and 37 type 1 DM patients. Measurements in DM participants were made under clamped euglycemic conditions. The effect of l-NMMA on circulating and urinary NO metabolites (NOx) and cGMP and on urinary prostanoids was also determined. Baseline characteristics were similar in the two groups. For analysis, the DM patients were divided into those with hyperfiltration (DM-H, n = 18) and normal glomerular filtration rate (GFR) levels (DM-N, n = 19). Baseline urine NOx and cGMP were highest in DM-H. l-NMMA led to a decline in GFR in DM-H (152 ± 16 to 140 ± 11 ml·min−1·1.73 m−2) but not DM-N or healthy control participants. The decline in effective renal plasma flow in response to l-NMMA (806 ± 112 to 539 ± 80 ml·min−1·1.73 m−2) in DM-H was also exaggerated compared with the other groups (repeated measures ANOVA, P < 0.05), along with declines in urinary NOx metabolites and cGMP. Baseline FMD was lowest in DM-H compared with the other groups and did not change in response to l-NMMA. l-NMMA reduced FMD and plasma markers of NO bioactivity in the healthy control and DM-N groups. In patients with uncomplicated type 1 DM, renal hyperfiltration is associated with increased NO bioactivity in the kidney and reduced NO bioactivity in the systemic circulation, suggesting a paradoxical state of high renal and low systemic vascular NO bioactivity. PMID:22855276

  3. 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.

  4. Protein kinase D activity controls endothelial nitric oxide synthesis.

    PubMed

    Aicart-Ramos, Clara; Sánchez-Ruiloba, Lucía; Gómez-Parrizas, Mónica; Zaragoza, Carlos; Iglesias, Teresa; Rodríguez-Crespo, Ignacio

    2014-08-01

    Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone. © 2014. Published by The Company of Biologists Ltd.

  5. Baicalin Protects the Cardiomyocytes from ER Stress-Induced Apoptosis: Inhibition of CHOP through Induction of Endothelial Nitric Oxide Synthase

    PubMed Central

    Wang, Bo; Guo, Xiaowang; Zeng, Chao; Xu, Yong; Shen, Liangliang; Cheng, Ke; Xia, Yuesheng; Li, Xiumin; Wang, Haichang; Fan, Li; Wang, Xiaoming

    2014-01-01

    Baicalin, the main active ingredient of the Scutellaria root, exerts anti-oxidant and anti-apoptotic effects in cardiovascular diseases. However, the therapeutic mechanism of baicalin remains unknown. Cultured neonatal rat cardiomyocytes were pre-treated with baicalin (0–50 µM) for 24 h, and subsequently treated with tunicamycin (100 ng/ml). Cell viability was detected by MTT assay, and cell damage was determined by LDH release and TUNEL assay. The expression of CHOP, JNK, caspase-3, eNOS was analyzed by western blot. NO was measured by DAF-FM staining. As a result, treatment with baicalin significantly reduced apoptosis induced by ER stress inducer tunicamycin in cardiomyocytes. Molecularly, baicalin ameliorated tunicamycin-induced ER stress by downregulation of CHOP. In addition, baicalin inverted tunicamycin-induced decreases of eNOS mRNA and protein levels, phospho eNOS and NO production through CHOP pathway. However, the protective effects of baicalin were significantly decreased in cardiomyocytes treated with L-NAME, which suppressed activation of nitric oxide synthase. In conclusion, our results implicate that baicalin could protect cardiomyocytes from ER stress-induced apoptosis via CHOP/eNOS/NO pathway, and suggest the therapeutic values of baicalin against ER stress-associated cardiomyocyte apoptosis. PMID:24520378

  6. 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

  7. 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.

  8. Endothelial dihydrofolate reductase: critical for nitric oxide bioavailability and role in angiotensin II uncoupling of endothelial nitric oxide synthase.

    PubMed

    Chalupsky, Karel; Cai, Hua

    2005-06-21

    Recent studies demonstrate that oxidative inactivation of tetrahydrobiopterin (H4B) may cause uncoupling of endothelial nitric oxide synthase (eNOS) to produce superoxide (O2*-). H4B was found recyclable from its oxidized form by dihydrofolate reductase (DHFR) in several cell types. Functionality of the endothelial DHFR, however, remains completely unknown. Here we present findings that specific inhibition of endothelial DHFR by RNA interference markedly reduced endothelial H4B and nitric oxide (NO.) bioavailability. Furthermore, angiotensin II (100 nmol/liter for 24 h) caused a H4B deficiency that was mediated by H2O2-dependent down-regulation of DHFR. This response was associated with a significant increase in endothelial O2*- production, which was abolished by eNOS inhibitor N-nitro-L-arginine-methyl ester or H2O2 scavenger polyethylene glycol-conjugated catalase, strongly suggesting H2O2-dependent eNOS uncoupling. Rapid and transient activation of endothelial NAD(P)H oxidases was responsible for the initial burst production of O2* (Rac1 inhibitor NSC 23766 but not an N-nitro-L-arginine-methyl ester-attenuated ESR O2*- signal at 30 min) in response to angiotensin II, preceding a second peak in O2*- production at 24 h that predominantly depended on uncoupled eNOS. Overexpression of DHFR restored NO. production and diminished eNOS production of O2*- in angiotensin II-stimulated cells. In conclusion, these data represent evidence that DHFR is critical for H4B and NO. bioavailability in the endothelium. Endothelial NAD(P)H oxidase-derived H2O2 down-regulates DHFR expression in response to angiotensin II, resulting in H4B deficiency and uncoupling of eNOS. This signaling cascade may represent a universal mechanism underlying eNOS dysfunction under pathophysiological conditions associated with oxidant stress.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. Targeting Pulmonary Endothelial Hemoglobin α Improves Nitric Oxide Signaling and Reverses Pulmonary Artery Endothelial Dysfunction.

    PubMed

    Alvarez, Roger A; Miller, Megan P; Hahn, Scott A; Galley, Joseph C; Bauer, Eileen; Bachman, Timothy; Hu, Jian; Sembrat, John; Goncharov, Dmitry; Mora, Ana L; Rojas, Mauricio; Goncharova, Elena; Straub, Adam C

    2017-08-11

    Pulmonary hypertension is characterized by pulmonary endothelial dysfunction. Previous work showed that systemic artery endothelial cells express hemoglobin α to control nitric oxide diffusion, but the role of this system in the pulmonary circulation has not been evaluated. We hypothesize that up-regulation of hemoglobin α in pulmonary endothelial cells contributes to nitric oxide depletion and pulmonary vascular dysfunction in pulmonary hypertension. Co-cultures of human pulmonary microvascular endothelial cells and distal pulmonary arterial vascular smooth muscle cells, lung tissue from control and pulmonary hypertensive lungs, and a mouse model of chronic hypoxia-induced pulmonary hypertension were used. Immunohistochemical, immunoblot analyses, spectrophotometry, and blood vessel myography experiments were performed in this study. We find increased expression of hemoglobin α in pulmonary endothelium from humans and mice with pulmonary hypertension compared to controls. In addition, we show up-regulation of hemoglobin α in human pulmonary endothelial cells co-cultured with pulmonary artery smooth muscle cells in hypoxia. We treated pulmonary endothelial cells with a hemoglobin α mimetic peptide that disrupts the association of hemoglobin α with endothelial nitric oxide synthase, and found that cells treated with the peptide exhibited increased nitric oxide signaling compared to a scrambled peptide. Myography experiments using pulmonary arteries from hypoxic mice show that the hemoglobin α mimetic peptide enhanced vasodilation in response to acetylcholine. Our findings reveal that endothelial hemoglobin α functions as an endogenous scavenger of nitric oxide in the pulmonary endothelium. Targeting this pathway may offer a novel therapeutic target to increase endogenous levels of nitric oxide in pulmonary hypertension.

  15. Exogenous nitric oxide activates the endothelial glucocorticoid receptor.

    PubMed

    Ji, Julie Y; Diamond, Scott L

    2004-05-21

    This study investigated the effect of exogenous nitric oxide (NO) on endothelial glucocorticoid receptor (GR) function. The NO donor diethylenetriamine NONOate (DETA, 50-500microM) caused concentration dependent nuclear localization of transfected chimeric green fluorescent protein GFP-GR and elevated expression of secreted alkaline phosphatase (SEAP) from a glucocorticoid response element (GRE) promoter construct in bovine aortic endothelial cells. Other weaker NO donors (S-nitroso-N-acetylpenicillamine and spermine NONOate) failed to induce GFP-GR nuclear localization, but all the NO donors activated GRE-SEAP expression, a response unaffected by the antioxidant N-acetyl-L-cysteine. Overall, exogenous NO from high concentration donors can directly activate GR, suggesting a potential feedback mechanism for NO to regulate endothelial inducible nitric oxide synthase (iNOS) expression.

  16. 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

  17. Nitric oxide inhibition of human sperm motility.

    PubMed

    Weinberg, J B; Doty, E; Bonaventura, J; Haney, A F

    1995-08-01

    To determine the effect of nitric oxide (NO) on sperm motility in vitro. Normal human sperm separated by centrifugation through a discontinuous Percoll gradient and subsequent swim-up were incubated for up to 24 hours with NO donors, with and without the known NO quencher hemoglobin, as well as with agents that raise intracellular cyclic 3',5'-guanosine monophosphate (cGMP). Sperm respiration was determined by a tetrazolium-formazan spectrophotometric assay. Andrology laboratory. Absolute sperm motility and respiration. Sperm incubated with the NO donors 1 mM nitroprusside, 100 to 125 microM 3-morpholinosydnonimine, and 25 to 125 microM pure nitric oxide gas dissolved in buffer were inhibited in motility in a dose-dependent fashion. The inhibition could be reversed by the NO quencher hemoglobin. Agents that raise cellular cGMP (dibutyryl cGMP or 8-bromo-cGMP) did not inhibit motility. Nitric oxide inhibited sperm respiration, as measured by the tetrazolium-formazan assay. Nitric oxide reduces sperm motility, possibly by a mechanism involving inhibition of cellular respiration independent of an elevation of intracellular cGMP. Nitric oxide elaborated in the female or male genital tract in vivo could adversely influence sperm function and fertility.

  18. 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

  19. 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

  20. 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.

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

    PubMed Central

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

    2013-01-01

    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. PMID:22960442

  2. 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.

  3. Endothelial nitric oxide synthase uncoupling: a novel pathway in OSA induced vascular endothelial dysfunction.

    PubMed

    Varadharaj, Saradhadevi; Porter, Kyle; Pleister, Adam; Wannemacher, Jacob; Sow, Angela; Jarjoura, David; Zweier, Jay L; Khayat, Rami N

    2015-02-01

    The mechanism of vascular endothelial dysfunction (VED) and cardiovascular disease in obstructive sleep apnea (OSA) is unknown. We performed a comprehensive evaluation of endothelial nitric oxide synthase (eNOS) function directly in the microcirculatory endothelial tissue of OSA patients who have very low cardiovascular risk status. Nineteen OSA patients underwent gluteal biopsies before, and after effective treatment of OSA. We measured superoxide (O2(•-)) and nitric oxide (NO) in the microcirculatory endothelium using confocal microscopy. We evaluated the effect of the NOS inhibitor l-Nitroarginine-Methyl-Ester (l-NAME) and the NOS cofactor tetrahydrobiopterin (BH4) on endothelial O2(•-) and NO in patient endothelial tissue before and after treatment. We found that eNOS is dysfunctional in OSA patients pre-treatment, and is a source of endothelial O2(•-) overproduction. eNOS dysfunction was reversible with the addition of BH4. These findings provide a new mechanism of endothelial dysfunction in OSA patients and a potentially targetable pathway for treatment of cardiovascular risk in OSA. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. Astrocyte-induced cortical vasodilation is mediated by D-serine and endothelial nitric oxide synthase

    PubMed Central

    Stobart, Jillian L. LeMaistre; Lu, Lingling; Mori, Hisashi; Anderson, Christopher M.

    2013-01-01

    Astrocytes play a critical role in neurovascular coupling by providing a physical linkage from synapses to arterioles and releasing vaso-active gliotransmitters. We identified a gliotransmitter pathway by which astrocytes influence arteriole lumen diameter. Astrocytes synthesize and release NMDA receptor coagonist, D-serine, in response to neurotransmitter input. Mouse cortical slice astrocyte activation by metabotropic glutamate receptors or photolysis of caged Ca2+ produced dilation of penetrating arterioles in a manner attenuated by scavenging D-serine with D-amino acid oxidase, deleting the enzyme responsible for D-serine synthesis (serine racemase) or blocking NMDA receptor glycine coagonist sites with 5,7-dichlorokynurenic acid. We also found that dilatory responses were dramatically reduced by inhibition or elimination of endothelial nitric oxide synthase and that the vasodilatory effect of endothelial nitric oxide synthase is likely mediated by suppressing levels of the vasoconstrictor arachidonic acid metabolite, 20-hydroxy arachidonic acid. Our results provide evidence that D-serine coactivation of NMDA receptors and endothelial nitric oxide synthase is involved in astrocyte-mediated neurovascular coupling. PMID:23386721

  9. 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

  10. Characterization of cationic amino acid transporters and expression of endothelial nitric oxide synthase in human placental microvascular endothelial cells.

    PubMed

    Dye, J F; Vause, S; Johnston, T; Clark, P; Firth, J A; D'Souza, S W; Sibley, C P; Glazier, J D

    2004-01-01

    We investigated the expression and activity of arginine transporters and endothelial nitric oxide synthase (eNOS) in human placental microvascular endothelial cells (HPMEC). Using RT-PCR amplification products for eNOS, CAT1, CAT2A, CAT2B, CAT4, 4F2hc (CD98), rBAT and the light chains y+LAT1, y+LAT2, and b0+T1 were detected in HPMEC, but not B0+. Immunohistochemistry and Western blotting confirmed the presence of 4F2hc and CAT1 protein in HPMEC. 4F2hc-light chain dimers were indicated by a shift in molecular mass detected under nonreducing conditions. L-Arginine transport into HPMEC was independent of Na+ or Cl- and was inhibited by the neutral amino acid glutamine, but not by cystine. The Ki for glutamine inhibition was greater in the absence of Na+. Kinetic analysis supported a two-transporter model attributed to system y+L and system y+. Expression of eNOS in HPMEC was detectable by immunohistochemistry and ELISA but not by Western blotting. Activity of eNOS in HPMEC, measured over 48 h, either as the basal production of nitric oxide (NO) or as the accumulation of intracellular cGMP was not detectable. We conclude that HPMEC transport cationic amino acids by systems y+ and y+L and that basal eNOS expression and activity in these cells is low.

  11. 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

  12. Regulation of staphylococcal enterotoxin B-elicited nitric oxide production by endothelial cells.

    PubMed Central

    LeClaire, R D; Kell, W M; Sadik, R A; Downs, M B; Parker, G W

    1995-01-01

    The effect of staphylococcal enterotoxin B (SEB)-elicited inducible nitric oxide synthase (iNOS) in mouse endothelial cells was investigated. Results showed that SEB stimulated the same level of NO production in gamma interferon (IFN-gamma)-primed cells as did trichloroacetic acid-extracted lipopolysaccharide. The kinetics of induced NO production and expression of mRNA for iNOS differed markedly in endothelial and macrophage cells. Induced endothelial nitrite production was transient and was 15 to 20% of that generated by macrophage cells; mRNA levels peaked by 2 h and then steadily declined, whereas macrophage message levels continually increased. The ability of endothelial cells to produce SEB-induced NO depended on priming with IFN-gamma, although detectable mRNA could be elicited by SEB alone. Induction of endothelial iNOS mRNA was inhibited by cycloheximide, which indicated a requirement for de novo protein synthesis. Niacinamide and interleukin-10 significantly reduced SEB-induced endothelial NO production. Both are reported to affect IFN-gamma-induced class II major histocompatibility complex (MHC) expression on antigen-presenting cells. Niacinamide reduced iNOS mRNA levels and markedly reduced IFN-gamma induction of endothelial class II MHC surface antigen. Interleukin-10 did not consistently reduce iNOS mRNA expression and had no effect on IFN-gamma induction of endothelial class II MHC surface antigen. These results suggest that SEB interacts with IFN-gamma-primed endothelial cells to elicit induced NO and that this induction can be effectively modulated at the receptor or transcriptional level. PMID:7529748

  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

    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.

  15. 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

  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. Restoration of autophagy in endothelial cells from patients with diabetes mellitus improves nitric oxide signaling.

    PubMed

    Fetterman, Jessica L; Holbrook, Monica; Flint, Nir; Feng, Bihua; Bretón-Romero, Rosa; Linder, Erika A; Berk, Brittany D; Duess, Mai-Ann; Farb, Melissa G; Gokce, Noyan; Shirihai, Orian S; Hamburg, Naomi M; Vita, Joseph A

    2016-04-01

    Endothelial dysfunction contributes to cardiovascular disease in diabetes mellitus. Autophagy is a multistep mechanism for the removal of damaged proteins and organelles from the cell. Under diabetic conditions, inadequate autophagy promotes cellular dysfunction and insulin resistance in non-vascular tissue. We hypothesized that impaired autophagy contributes to endothelial dysfunction in diabetes mellitus. We measured autophagy markers and endothelial nitric oxide synthase (eNOS) activation in freshly isolated endothelial cells from diabetic subjects (n = 45) and non-diabetic controls (n = 41). p62 levels were higher in cells from diabetics (34.2 ± 3.6 vs. 20.0 ± 1.6, P = 0.001), indicating reduced autophagic flux. Bafilomycin inhibited insulin-induced activation of eNOS (64.7 ± 22% to -47.8 ± 8%, P = 0.04) in cells from controls, confirming that intact autophagy is necessary for eNOS signaling. In endothelial cells from diabetics, activation of autophagy with spermidine restored eNOS activation, suggesting that impaired autophagy contributes to endothelial dysfunction (P = 0.01). Indicators of autophagy initiation including the number of LC3-bound puncta and beclin 1 expression were similar in diabetics and controls, whereas an autophagy terminal phase indicator, the lysosomal protein Lamp2a, was higher in diabetics. In endothelial cells under diabetic conditions, the beneficial effect of spermidine on eNOS activation was blocked by autophagy inhibitors bafilomycin or 3-methyladenine. Blocking the terminal stage of autophagy with bafilomycin increased p62 (P = 0.01) in cells from diabetics to a lesser extent than in cells from controls (P = 0.04), suggesting ongoing, but inadequate autophagic clearance. Inadequate autophagy contributes to endothelial dysfunction in patients with diabetes and may be a target for therapy of diabetic vascular disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. 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

  19. 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.

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

    PubMed

    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

    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. 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. eNOS-/- mice induce NASH development, and these findings provide new insights into the therapeutic approach for fatty liver disease and related disorders.

  1. 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.

  2. Placental endothelial nitric oxide synthase in multiple and single dose betamethasone exposed pregnancies.

    PubMed

    Mertz, Heather L; Mele, Lisa; Spong, Catherine Y; Dudley, Donald J; Wapner, Ronald J; Iams, Jay D; Sorokin, Yoram; Peaceman, Alan; Leveno, Kenneth J; Caritis, Steve N; Miodovnik, Menachem; Mercer, Brian M; Thorp, John M; O'Sullivan, Mary J; Ramin, Susan M; Carpenter, Marshall; Rouse, Dwight J; Sibai, Baha

    2011-06-01

    To compare endothelial nitric oxide synthase expression and capillary density (CDS) in placentas exposed to single or multiple courses of betamethasone. Placental specimens exposed to single vs repeat courses of betamethasone were analyzed through immunohistochemistry and digital image quantification for endothelial nitric oxide synthase and CD34. Quantified endothelial nitric oxide synthase staining, calculated capillary density, ratio of endothelial nitric oxide synthase to capillary density, and clinical characteristics were compared. Linear regression was performed with these as dependent variables. Mean and maximum capillary density were increased (P = .013 and .005) and the ratio of endothelial nitric oxide synthase to capillary density decreased (P = .016) in specimens exposed to 4 courses of betamethasone compared with 1 to 3 courses. Exposure to 4 courses of betamethasone was associated with increased capillary density, but not with endothelial nitric oxide synthase expression. Exposure to 4 courses of betamethasone is associated with increased placental capillary density. The placental effects of multiple courses of betamethasone are unrelated to endothelial nitric oxide synthase expression. Copyright © 2011 Mosby, Inc. All rights reserved.

  3. 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.

  4. 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

  5. Zinc regulates iNOS-derived nitric oxide formation in endothelial cells

    PubMed Central

    Cortese-Krott, Miriam M.; Kulakov, Larissa; Opländer, Christian; Kolb-Bachofen, Victoria; Kröncke, Klaus-D.; Suschek, Christoph V.

    2014-01-01

    Aberrant production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of endothelial dysfunction and vascular disease. Mechanisms responsible for the fine-tuning of iNOS activity in inflammation are still not fully understood. Zinc is an important structural element of NOS enzymes and is known to inhibit its catalytical activity. In this study we aimed to investigate the effects of zinc on iNOS activity and expression in endothelial cells. We found that zinc down-regulated the expression of iNOS (mRNA+protein) and decreased cytokine-mediated activation of the iNOS promoter. Zinc-mediated regulation of iNOS expression was due to inhibition of NF-κB transactivation activity, as determined by a decrease in both NF-κB-driven luciferase reporter activity and expression of NF-κB target genes, including cyclooxygenase 2 and IL-1β. However, zinc did not affect NF-κB translocation into the nucleus, as assessed by Western blot analysis of nuclear and cytoplasmic fractions. Taken together our results demonstrate that zinc limits iNOS-derived high output NO production in endothelial cells by inhibiting NF-κB-dependent iNOS expression, pointing to a role of zinc as a regulator of iNOS activity in inflammation. PMID:25180171

  6. Lipopolysaccharide impairs endothelial nitric oxide synthesis in rat renal arteries.

    PubMed

    Piepot, H A; Boer, C; Groeneveld, A B; Van Lambalgen, A A; Sipkema, P

    2000-06-01

    Impaired endothelium-dependent vasodilation may contribute to hypoperfusion and failure of abdominal organs, including the kidneys during endotoxin or septic shock. In this study, the short-term (2 h) effects of bacterial lipopolysaccharide (LPS) on endothelium-dependent vasodilation in rat renal and superior mesenteric arteries were documented. Rat renal and mesenteric arteries were dissected and exposed in vitro to LPS for two hours. The effects of LPS on vascular reactivity were determined and compared with time-matched controls. Endothelial nitric oxide (NO) release was determined using an NO microsensor in adjacent vessel segments. LPS impaired maximal acetylcholine (ACh)-induced endothelium-dependent vasodilation in renal arteries (62.5 +/- 8.8% vs. 34.4 +/- 7.5% in controls and LPS-exposed arteries), but not in mesenteric arteries. LPS did not alter the sensitivity of renal arteries to exogenous NO. ACh-dependent vasodilation was abolished after blocking NO synthesis with 10-4 mol/L L-NA in control and LPS-incubated renal arteries. When compared with controls, NO release induced by ACh and the receptor-independent calcium ionophore A23187 was significantly decreased (P < 0.05) in LPS-exposed renal segments and was fully abolished in endothelium-denuded segments, indicating that LPS attenuated receptor-dependent as well as receptor-independent endothelial NO release. In contrast, ACh- and A23187-induced NO release was normal in LPS-exposed mesenteric arteries. These results indicate that LPS-induced selective impairment of ACh-induced endothelium-dependent relaxation in rat renal arteries is caused by decreased endothelial NO release. This may contribute to the propensity for acute renal failure during septic shock.

  7. 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

  8. Endothelial Nitric Oxide Synthase Is Present in Dendritic Spines of Neurons in Primary Cultures.

    PubMed

    Caviedes, Ariel; Varas-Godoy, Manuel; Lafourcade, Carlos; Sandoval, Soledad; Bravo-Alegria, Javiera; Kaehne, Thilo; Massmann, Angela; Figueroa, Jorge P; Nualart, Francisco; Wyneken, Ursula

    2017-01-01

    Nitric oxide exerts important regulatory functions in various brain processes. Its synthesis in neurons has been most commonly ascribed to the neuronal nitric oxide synthase (nNOS) isoform. However, the endothelial isoform (eNOS), which is significantly associated with caveolae in different cell types, has been implicated in synaptic plasticity and is enriched in the dendrites of CA1 hippocampal neurons. Using high resolution microscopy and co-distribution analysis of eNOS with synaptic and raft proteins, we now show for the first time in primary cortical and hippocampal neuronal cultures, virtually devoid of endothelial cells, that eNOS is present in neurons and is localized in dendritic spines. Moreover, eNOS is present in a postsynaptic density-enriched biochemical fraction isolated from these neuronal cultures. In addition, qPCR analysis reveals that both the nNOS as well as the eNOS transcripts are present in neuronal cultures. Moreover, eNOS inhibition in cortical cells has a negative impact on cell survival after excitotoxic stimulation with N-methyl-D-aspartate (NMDA). Consistent with previous results that indicated nitric oxide production in response to the neurotrophin BDNF, we could detect eNOS in immunoprecipitates of the BDNF receptor TrkB while nNOS could not be detected. Taken together, our results show that eNOS is located at excitatory synapses where it could represent a source for NO production and thus, the contribution of eNOS-derived nitric oxide to the regulation of neuronal survival and function deserves further investigations.

  9. Endothelial Nitric Oxide Synthase Is Present in Dendritic Spines of Neurons in Primary Cultures

    PubMed Central

    Caviedes, Ariel; Varas-Godoy, Manuel; Lafourcade, Carlos; Sandoval, Soledad; Bravo-Alegria, Javiera; Kaehne, Thilo; Massmann, Angela; Figueroa, Jorge P.; Nualart, Francisco; Wyneken, Ursula

    2017-01-01

    Nitric oxide exerts important regulatory functions in various brain processes. Its synthesis in neurons has been most commonly ascribed to the neuronal nitric oxide synthase (nNOS) isoform. However, the endothelial isoform (eNOS), which is significantly associated with caveolae in different cell types, has been implicated in synaptic plasticity and is enriched in the dendrites of CA1 hippocampal neurons. Using high resolution microscopy and co-distribution analysis of eNOS with synaptic and raft proteins, we now show for the first time in primary cortical and hippocampal neuronal cultures, virtually devoid of endothelial cells, that eNOS is present in neurons and is localized in dendritic spines. Moreover, eNOS is present in a postsynaptic density-enriched biochemical fraction isolated from these neuronal cultures. In addition, qPCR analysis reveals that both the nNOS as well as the eNOS transcripts are present in neuronal cultures. Moreover, eNOS inhibition in cortical cells has a negative impact on cell survival after excitotoxic stimulation with N-methyl-D-aspartate (NMDA). Consistent with previous results that indicated nitric oxide production in response to the neurotrophin BDNF, we could detect eNOS in immunoprecipitates of the BDNF receptor TrkB while nNOS could not be detected. Taken together, our results show that eNOS is located at excitatory synapses where it could represent a source for NO production and thus, the contribution of eNOS-derived nitric oxide to the regulation of neuronal survival and function deserves further investigations. PMID:28725180

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. Genetic association between endothelial nitric oxide synthase and Alzheimer disease.

    PubMed

    Akomolafe, A; Lunetta, K L; Erlich, P M; Cupples, L A; Baldwin, C T; Huyck, M; Green, R C; Farrer, L A

    2006-07-01

    Evidence suggests that vascular and inflammatory factors may be important in the etiology of Alzheimer disease (AD). The Glu/Glu genotype at the Glu298Asp variant of the endothelial nitric oxide synthase (NOS3) gene has been tested for association with AD in several Caucasian and Asian populations, with conflicting results. We tested the Glu298Asp variant for association in African American and Caucasian AD patients, unaffected siblings, and unrelated controls from the MIRAGE Study. To explore whether the inconsistent results in previous studies might be due to linkage disequilibrium with a polymorphism or haplotype not previously tested, we genotyped 10 additional NOS3 single nucleotide polymorphisms (SNPs) spanning 25.3 kb. Finally, we compiled results of previous studies of Glu298Asp using meta-analysis, to determine whether the aggregate studies support an association between Glu298Asp and AD. We found that the Glu298 allele was associated with higher risk of AD in the MIRAGE African American (p = 0.002) but not Caucasian (p = 0.9) groups. None of the additional SNPs were associated with AD in the Caucasians, whereas two showed evidence for association in the African Americans. The meta-analysis showed a small effect of the Glu298Asp GG genotype on AD risk across all studies (summary odds ratio = 1.15, 95% confidence interval: 0.97-1.35) and significant heterogeneity of this association among studies (p = 0.02).

  17. 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.

  18. Aortic valve sclerosis in mice deficient in endothelial nitric oxide synthase

    PubMed Central

    El Accaoui, Ramzi N.; Gould, Sarah T.; Hajj, Georges P.; Chu, Yi; Davis, Melissa K.; Kraft, Diane C.; Lund, Donald D.; Brooks, Robert M.; Doshi, Hardik; Zimmerman, Kathy A.; Kutschke, William; Anseth, Kristi S.; Heistad, Donald D.

    2014-01-01

    Risk factors for fibrocalcific aortic valve disease (FCAVD) are associated with systemic decreases in bioavailability of endothelium-derived nitric oxide (EDNO). In patients with bicuspid aortic valve (BAV), vascular expression of endothelial nitric oxide synthase (eNOS) is decreased, and eNOS−/− mice have increased prevalence of BAV. The goal of this study was to test the hypotheses that EDNO attenuates profibrotic actions of valve interstitial cells (VICs) in vitro and that EDNO deficiency accelerates development of FCAVD in vivo. As a result of the study, coculture of VICs with aortic valve endothelial cells (vlvECs) significantly decreased VIC activation, a critical early phase of FCAVD. Inhibition of VIC activation by vlvECs was attenuated by NG-nitro-l-arginine methyl ester or indomethacin. Coculture with vlvECs attenuated VIC expression of matrix metalloproteinase-9, which depended on stiffness of the culture matrix. Coculture with vlvECs preferentially inhibited collagen-3, compared with collagen-1, gene expression. BAV occurred in 30% of eNOS−/− mice. At age 6 mo, collagen was increased in both bicuspid and trileaflet eNOS−/− aortic valves, compared with wild-type valves. At 18 mo, total collagen was similar in eNOS−/− and wild-type mice, but collagen-3 was preferentially increased in eNOS−/− mice. Calcification and apoptosis were significantly increased in BAV of eNOS−/− mice at ages 6 and 18 mo. Remarkably, these histological changes were not accompanied by physiologically significant valve stenosis or regurgitation. In conclusion, coculture with vlvECs inhibits specific profibrotic VIC processes. In vivo, eNOS deficiency produces fibrosis in both trileaflet and BAVs but produces calcification only in BAVs. PMID:24610917

  19. Increased nitric oxide production in lymphatic endothelial cells causes impairment of lymphatic drainage in cirrhotic rats.

    PubMed

    Ribera, Jordi; Pauta, Montse; Melgar-Lesmes, Pedro; Tugues, Sònia; Fernández-Varo, Guillermo; Held, Kara F; Soria, Guadalupe; Tudela, Raúl; Planas, Anna M; Fernández-Hernando, Carlos; Arroyo, Vicente; Jiménez, Wladimiro; Morales-Ruiz, Manuel

    2013-01-01

    The lymphatic network plays a major role in maintaining tissue fluid homoeostasis. Therefore several pathological conditions associated with oedema formation result in deficient lymphatic function. However, the role of the lymphatic system in the pathogenesis of ascites and oedema formation in cirrhosis has not been fully clarified. The aim of this study was to investigate whether the inability of the lymphatic system to drain tissue exudate contributes to the oedema observed in cirrhosis. Cirrhosis was induced in rats by CCl(4) inhalation. Lymphatic drainage was evaluated using fluorescent lymphangiography. Expression of endothelial nitric oxide synthase (eNOS) was measured in primary lymphatic endothelial cells (LyECs). Inhibition of eNOS activity in cirrhotic rats with ascites (CH) was carried out by L-N(G)-methyl-L-arginine (L-NMMA) treatment (0.5 mg/kg/day). The (CH) rats had impaired lymphatic drainage in the splanchnic and peripheral regions compared with the control (CT) rats. LyECs isolated from the CH rats showed a significant increase in eNOS and nitric oxide (NO) production. In addition, the lymphatic vessels of the CH rats showed a significant reduction in smooth muscle cell (SMC) coverage compared with the CT rats. CH rats treated with L-NMMA for 7 days showed a significant improvement in lymphatic drainage and a significant reduction in ascites volume, which were associated with increased plasma volume. This beneficial effect of L-NMMA inhibition was also associated with a significant increase in lymphatic SMC coverage. The upregulation of eNOS in the LyECs of CH rats causes long-term lymphatic remodelling, which is characterised by a loss of SMC lymphatic coverage. The amelioration of this lymphatic abnormality by chronic eNOS inhibition results in improved lymphatic drainage and reduced ascites.

  20. Intrauterine pulmonary hypertension impairs angiogenesis in vitro: role of vascular endothelial growth factor nitric oxide signaling.

    PubMed

    Gien, Jason; Seedorf, Gregory J; Balasubramaniam, Vivek; Markham, Neil; Abman, Steven H

    2007-12-01

    Mechanisms that impair angiogenesis in neonatal persistent pulmonary hypertension (PPHN) are poorly understood. To determine if PPHN alters fetal pulmonary artery endothelial cell (PAEC) phenotype and impairs growth and angiogenesis in vitro, and if altered vascular endothelial growth factor-nitric oxide (VEGF-NO) signaling contributes to this abnormal phenotype. Proximal PAECs were harvested from fetal sheep that had undergone partial ligation of the ductus arteriosus in utero (PPHN) and age-matched control animals. Growth and tube formation +/- VEGF and NO stimulation and inhibition were studied in normal and PPHN PAECs. Western blot analysis was performed for VEGF, VEGF receptor-2 (VEGF-R2), and endothelial NO synthase (eNOS) protein content. NO production with VEGF administration was measured in normal and PPHN PAECs. PPHN PAECs demonstrate decreased growth and tube formation in vitro. VEGF and eNOS protein expression were decreased in PPHN PAECs, whereas VEGF-R2 protein expression was not different. VEGF and NO increased PPHN PAEC growth and tube formation to values achieved in normal PAECs. VEGF inhibition decreased growth and tube formation in normal and PPHN PAECs. NOS inhibition decreased growth in normal and PPHN PAECs, but tube formation was only reduced in normal PAECs. NO reversed the inhibitory effects of VEGF-R2 inhibition on tube formation in normal and PPHN PAECs. VEGF increased NO production in normal and PPHN PAECs. PPHN in utero causes sustained impairment of PAEC phenotype in vitro, with reduced PAEC growth and tube formation and down-regulation of VEGF and eNOS protein. VEGF and NO enhanced growth and tube formation of PPHN PAECs.

  1. Suppression of endothelin-3-induced nitric oxide synthesis by triglyceride in human endothelial cells.

    PubMed

    Minami, M; Yokokawa, K; Kohno, M; Yasunari, K; Yoshikawa, J

    1998-01-01

    Reduced endothelium-derived nitric oxide (NO) production characterizes several vascular diseases. This study examined the effect of triglyceride on NO production induced by endothelin-3 (ET-3) in cultured human umbilical vein endothelial cells. Triglyceride-rich human plasma obtained after a high-carbohydrate diet with white wine was used in an ex vivo study. The plasma triglyceride fraction was found to consist of large amounts of palmitic and oleic acids detected by gas-liquid chromatography. Therefore, the effect of synthetic tripalmitin and triolein emulsion on NO production was also examined. ET-3 stimulated NO and guanosine 3',5'-cyclic monophosphate production and increased cytosolic Ca2+ levels in the endothelial cells (ECs). After incubation of the ECs with the triglyceride-rich plasma for 2 h, these responses to ET-3 were ameliorated in a triglyceride concentration-dependent manner (50-200 mg/dl). A synthesized emulsion of tripalmitin (100 mg/dl) and triolein (100 mg/dl) also blunted the responses to ET-3. Neither endothelial constitutive NO synthase mRNA expression nor its protein level was affected by treatment with triglycerides. These results suggest that triglyceride suppresses ET-3-induced NO synthesis in human ECs by inhibiting cytosolic Ca2+ elevation.

  2. Pleiotrophin Induces Nitric Oxide Dependent Migration of Endothelial Progenitor Cells

    PubMed Central

    Heiss, Christian; Wong, Maelene L.; Block, Vanessa I.; Lao, David; Real, Wendy May; Yeghiazarians, Yerem; Lee, Randall J.; Springer, Matthew L.

    2009-01-01

    Pleiotrophin (PTN) is produced under ischemic conditions and has been shown to induce angiogenesis in vivo. We studied whether or not PTN exerts chemotaxis of pro-angiogenic early endothelial progenitor cells (EPCs), a population of circulating cells that have been reported to participate in and stimulate angiogenesis. Chemotaxis of EPCs, isolated from blood of healthy humans (n=5), was measured in transwell assays. PTN at 10–500 ng/mL elicited dose-dependent chemotaxis of both EPCs and human umbilical vein endothelial cells (HUVECs), but not of human coronary artery smooth muscle cells (CASMCs) and T98G glioblastoma cells that lack PTN receptors. The degree of chemotaxis was comparable to that induced by the angiogenic factors VEGF and SDF-1α. Chemotaxis to PTN was blocked by the NOS inhibitors L-NNA and L-NMMA, the NO scavenger PTIO, the phosphoinositide-3 kinase inhibitor wortmannin, and the guanylyl cyclase inhibitor ODQ, suggesting dependence of EPC chemotaxis on these pathways. PTN induced NOS-dependent production of NO to a similar degree as did VEGF, as indicated by the NO indicator DAF-2. PTN increased proliferation in EPCs and HUVECs to a similar extent as VEGF, but did not induce proliferation of CASMCs. While L-NNA abolished PTN-induced migration in EPCs and HUVECs, it did not inhibit PTN- and VEGF-enhanced proliferation and also caused proliferation by itself. These data suggest that PTN may mediate its pro-angiogenic effects by increasing the local number of not only endothelial cells but also early EPCs at angiogenic sites. PMID:17960557

  3. 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.

  4. Placental Vesicles Carry Active Endothelial Nitric Oxide Synthase and Their Activity is Reduced in Preeclampsia.

    PubMed

    Motta-Mejia, Carolina; Kandzija, Neva; Zhang, Wei; Mhlomi, Vuyane; Cerdeira, Ana Sofia; Burdujan, Alexandra; Tannetta, Dionne; Dragovic, Rebecca; Sargent, Ian L; Redman, Christopher W; Kishore, Uday; Vatish, Manu

    2017-08-01

    Preeclampsia, a multisystem hypertensive disorder of pregnancy, is associated with increased systemic vascular resistance. Placentae from patients with preeclampsia have reduced levels of endothelial nitric oxide synthase (eNOS) and, thus, less nitric oxide (NO). Syncytiotrophoblast extracellular vesicles (STBEV), comprising microvesicles (STBMV) and exosomes, carry signals from the syncytiotrophoblast to the mother. We hypothesized that STBEV-bound eNOS (STBEV-eNOS), capable of producing NO, are released into the maternal circulation. Dual-lobe ex vivo placental perfusion and differential centrifugation was used to isolate STBEV from preeclampsia (n=8) and normal pregnancies (NP; n=11). Plasma samples of gestational age-matched preeclampsia and NP (n=6) were used to isolate circulating STBMV. STBEV expressed placental alkaline phosphatase, confirming placental origin. STBEV coexpressed eNOS, but not inducible nitric oxide synthase, confirmed using Western blot, flow cytometry, and immunodepletion. STBEV-eNOS produced NO, which was significantly inhibited by N  (G)-nitro-l-arginine methyl ester (eNOS inhibitor; P<0.05) but not by N-(3-(aminomethyl) bezyl) acetamidine) (inducible nitric oxide synthase inhibitor). STBEV-eNOS catalytic activity was confirmed by visualizing eNOS dimerization. STBEV-eNOS was more abundant in uterine vein compared with peripheral blood, indicating placental origin. STBEV isolated from preeclampsia-perfused placentae had lower levels of STBEV-eNOS (STBMV; P<0.05) and overall lower NO activity (STBMV, not significant; syncytiotrophoblast extracellular exosomes, P<0.05) compared with those from NP. Circulating plasma STBMV from preeclampsia women had lower STBEV-eNOS expression compared with that from NP women (P<0.01). This is the first observation of functional eNOS expressed on STBEV from NP and preeclampsia placentae, as well as in plasma. The lower STBEV-eNOS NO production seen in preeclampsia may contribute to the decreased NO

  5. Activation of AP-1 Transcription Factors Differentiates FGF2 and Vascular Endothelial Growth Factor Regulation of Endothelial Nitric-oxide Synthase Expression in Placental Artery Endothelial Cells*

    PubMed Central

    Mata-Greenwood, Eugenia; Liao, Wu-xiang; Wang, Wen; Zheng, Jing; Chen, Dong-bao

    2010-01-01

    FGF2 (fibroblast growth factor 2), but not vascular endothelial growth factor (VEGF), stimulates sustained activation of ERK2/1 for endothelial NOS3 (nitric-oxide synthase 3) protein expression in ovine fetoplacental artery endothelial cells (oFPAEC). We deciphered herein the downstream signaling of ERK2/1 responsible for NOS3 expression by FGF2 in oFPAEC. FGF2, but not VEGF, increased NOS3 mRNA levels without altering its degradation. FGF2, but not VEGF, trans-activated sheep NOS3 promoter, and this was dependent on ERK2/1 activation. FGF2 did not trans-activate NOS3 promoters with deletions upstream of the consensus AP-1 site (TGAGTC A, −678 to −685). Trans-activation of wild-type NOS3 promoter by FGF2 was significantly inhibited when either the AP-1 or the cAMP-response element (CRE)-like sequence (TGCGTCA, −752 to −758) was mutated and was completely blocked when both were mutated. EMSA analyses showed that FGF2, but not VEGF, stimulated AP-1 and CRE DNA-protein complexes primarily composed of JunB and Fra1. Chromatin immunoprecipitation assays confirmed JunB/Fra1 binding to NOS3 promoter AP-1 and CRE elements in intact cells. FGF2, but not VEGF, stimulated JunB and Fra1 expressions; all preceded NOS3 up-regulation and were inhibited by PD98059. Down-regulation of JunB or Fra-1, but not c-Jun, blocked FGF2 stimulation of NOS3 expression and NO production. AP-1 inhibition suppressed FGF2 stimulation of NOS3 expression in human umbilical vein EC and uterine artery endothelial cells. Thus, FGF2 induction of NOS3 expression is mainly mediated by AP-1-dependent transcription involving JunB and Fra1 up-regulation via sustained ERK2/1 activation in endothelial cells. PMID:20371606

  6. 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.

  7. Contribution of endothelial nitric oxide to blood pressure in humans.

    PubMed

    Gamboa, Alfredo; Shibao, Cyndya; Diedrich, André; Choi, Leena; Pohar, Bojan; Jordan, Jens; Paranjape, Sachin; Farley, Ginnie; Biaggioni, Italo

    2007-01-01

    Impaired endothelial-derived NO (eNO) is invoked in the development of many pathological conditions. Systemic inhibition of NO synthesis, used to assess the importance of NO to blood pressure (BP) regulation, increases BP by approximately 15 mm Hg. This approach underestimates the importance of eNO, because BP is restrained by baroreflex mechanisms and does not account for a role of neurally derived NO. To overcome these limitations, we induced complete autonomic blockade with trimethaphan in 17 normotensive healthy control subjects to eliminate baroreflex mechanisms and contribution of neurally derived NO. Under these conditions, the increase in BP reflects mostly blockade of tonic eNO. N(G)-Monomethyl-l-arginine (250 microg/kg per minute IV) increased mean BP by 6+/-3.7 mm Hg (from 77 to 82 mm Hg) in intact subjects and by 21+/-8.4 mm Hg (from 75 to 96 mm Hg) during autonomic blockade. We did not find a significant contribution of neurally derived NO to BP regulation after accounting for baroreflex buffering. To further validate this approach, we compared the effect of NOS inhibition during autonomic blockade in 10 normotensive individuals with that of 6 normotensive smokers known to have endothelial dysfunction but who were otherwise normal. As expected, normotensive smokers showed a significantly lower increase in systolic BP during selective eNO blockade (11+/-4.5 versus 30+/-2.3 mm Hg in normotensive individuals; P<0.005). Thus, we report a novel approach to preferentially evaluate the role of eNO on BP control in normal and disease states. Our results suggest that eNO is one of the most potent metabolic determinants of BP in humans, tonically restraining it by approximately 30 mm Hg.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. Activation of AMP-Activated Protein Kinase Inhibits the Proliferation of Human Endothelial Cells

    PubMed Central

    Peyton, Kelly J.; Liu, Xiao-ming; Yu, Yajie; Yates, Benjamin

    2012-01-01

    AMP-activated protein kinase (AMPK) is an evolutionary conserved energy-sensing enzyme that regulates cell metabolism. Emerging evidence indicates that AMPK also plays an important role in modulating endothelial cell function. In the present study, we investigated whether AMPK modulates endothelial cell growth. Treatment of cultured human umbilical vein endothelial cells with the AMPK activators 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), 6,7-dihydro-4-hydroxy-3-(2′-hydroxy[1,1′-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile (A-769662), or metformin inhibited cell proliferation and DNA synthesis. The antiproliferative action of AICAR was largely prevented by the adenosine kinase inhibitor 5′-iodotubercidin and mimicked by infecting endothelial cells with an adenovirus expressing constitutively active AMPK. In contrast, pharmacological blockade of endothelial nitric oxide synthase or heme oxygenase-1 activity failed to reverse the inhibition of endothelial cell growth by AICAR. Flow cytometry experiments revealed that pharmacological activation of AMPK arrested endothelial cells in the G0/G1 phase of the cell cycle, and this was associated with increases in p53 phosphorylation and p53, p21, and p27 protein expression and decreases in cyclin A protein expression and retinoblastoma protein phosphorylation. In addition, silencing p21 and p27 expression partially restored the mitogenic response of AMPK-activated cells. Finally, activation of AMPK by AICAR blocked the migration of endothelial cells after scrape injury and stimulated tube formation by endothelial cells plated onto Matrigel-coated plates. In conclusion, these studies demonstrate that AMPK activation inhibits endothelial cell proliferation by elevating p21 and p27 expression. In addition, they show that AMPK regulates endothelial cell migration and differentiation and identify AMPK as an attractive therapeutic target in treating diseases associated with aberrant

  14. 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

  15. 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. © 2015 Lu and Kassab.

  16. 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

  17. 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.

  18. 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.

  19. Phosphorylated endothelial nitric oxide synthase mediates vascular endothelial growth factor-induced penile erection.

    PubMed

    Musicki, Biljana; Palese, Michael A; Crone, Julie K; Burnett, Arthur L

    2004-02-01

    The objective of the present study was to evaluate whether vascular endothelial growth factor (VEGF)-induced penile erection is mediated by activation of endothelial nitric oxide synthase (eNOS) through its phosphorylation. We assessed the role of constitutively activated eNOS in VEGF-induced penile erection using wild-type (WT) and eNOS-knockout (eNOS(-/-)) mice with and without vasculogenic erectile dysfunction. Adult WT and eNOS(-/-) mice were subjected to sham operation or bilateral castration to induce vasculogenic erectile dysfunction. At the time of surgery, animals were injected intracavernosally with a replication-deficient adenovirus expressing human VEGF145 (10(9) particle units) or with empty virus (Ad.Null). After 7 days, erectile function was assessed in response to cavernous nerve electrical stimulation. Total and phosphorylated protein kinase B (Akt) as well as total and phosphorylated eNOS were quantitatively assessed in mice penes using Western immunoblot and immunohistochemistry. In intact WT mice, VEGF145 significantly increased erectile responses, and in WT mice after castration, it completely recovered penile erection. However, VEGF145 failed to increase erectile responses in intact eNOS(-/-) mice and only partially recovered erectile function in castrated eNOS(-/-) mice. In addition, VEGF145 significantly increased phosphorylation of eNOS at Serine 1177 by approximately 2-fold in penes of both intact and castrated WT mice. The data provide a molecular explanation for VEGF stimulatory effect on penile erection, which involves phosphorylated eNOS (Serine 1177) mediation.

  20. 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

  1. Cavin-2 regulates the activity and stability of endothelial nitric oxide synthase (eNOS) in angiogenesis.

    PubMed

    Boopathy, Gandhi T K; Kulkarni, Madhura; Ho, Sze Yuan; Boey, Adrian; Chua, Edmond Wei Min; Barathi, Veluchamy A; Carney, Tom J; Wang, Xiaomeng; Hong, Wanjin

    2017-09-14

    Angiogenesis is a highly regulated process for formation of new blood vessels from pre-existing ones. Angiogenesis is dysregulated in various pathologies, including age-related macular degeneration, arthritis, and cancer. Inhibiting pathological angiogenesis therefore represents a promising therapeutic strategy for treating these disorders, highlighting the need to study angiogenesis in more detail. To this end, identifying the genes essential for blood vessel formation and elucidating their function are crucial for a complete understanding of angiogenesis. Here, focusing on potential candidate genes for angiogenesis, we performed a morpholino-based genetic screen in zebrafish and identified Cavin-2, a membrane-bound phosphatidylserine-binding protein and critical organizer of caveolae (small microdomains in the plasma membrane), as a regulator of angiogenesis. Using endothelial cells, we show that Cavin-2 is required for in vitro angiogenesis and also for endothelial cell proliferation, migration, and invasion. We noted a high level of Cavin-2 expression in the neovascular tufts in the mouse model of oxygen-induced retinopathy, suggesting a role for Cavin-2 in pathogenic angiogenesis. Interestingly, we also found that Cavin-2 regulates the production of nitric oxide (NO) in endothelial cells by controlling the stability and activity of the endothelial nitric oxide synthase (eNOS) and that Cavin-2 knockdown cells produce much less NO than WT cells. Also, mass spectrometry, flow cytometry, and electron microscopy analyses indicated that Cavin-2 is secreted in endothelial microparticles (EMPs) and is required for EMP biogenesis. Taken together, our results indicate that in addition to its function in caveolae biogenesis, Cavin-2 plays a critical role in endothelial cell maintenance and function by regulating eNOS activity. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  2. 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

  3. 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.

  4. Platelet aggregation responses are critically regulated in vivo by endogenous nitric oxide but not by endothelial nitric oxide synthase.

    PubMed

    Tymvios, C; Moore, C; Jones, S; Solomon, A; Sanz-Rosa, D; Emerson, M

    2009-12-01

    Although exogenous nitric oxide (NO) clearly modifies platelet function, the role and the source of endogenous NO in vivo remain undefined. In addition, endothelial NO synthase (NOS-3) critically regulates vessel tone but its role in modulating platelet function is unclear. In this paper we have investigated the roles of endogenous NO and NOS-3 in regulating platelet function in vivo and determined the functional contribution made by platelet-derived NO. We used a mouse model for directly assessing platelet functional responses in situ in the presence of an intact vascular endothelium with supporting in vitro and molecular studies. Acute NOS inhibition by N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME) enhanced platelet aggregatory responses to thrombin and platelets were shown to be regulated primarily by NO sources external to the platelet. Elevation of endogenous NOS inhibitors to mimic effects reported in patients with cardiovascular diseases did not enhance platelet responses. Platelet responsiveness following agonist stimulation was not modified in male or female NOS-3(-/-) mice but responses in NOS-3(-/-) mice were enhanced by L-NAME. Platelets are regulated by endogenous NO in vivo, primarily by NO originating from the environment external to the platelet with a negligible or undetectable role of platelet-derived NO. Raised levels of endogenous NOS inhibitors, as reported in a range of diseases were not, in isolation, sufficient to enhance platelet activity and NOS-3 is not essential for normal platelet function in vivo due to the presence of bioactive NO following deletion of NOS-3.

  5. Traumatic Brain Injury Disrupts Cerebrovascular Tone Through Endothelial Inducible Nitric Oxide Synthase Expression and Nitric Oxide Gain of Function

    PubMed Central

    Villalba, Nuria; Sonkusare, Swapnil K.; Longden, Thomas A.; Tran, Tram L.; Sackheim, Adrian M.; Nelson, Mark T.; Wellman, George C.; Freeman, Kalev

    2014-01-01

    Background Traumatic brain injury (TBI) has been reported to increase the concentration of nitric oxide (NO) in the brain and can lead to loss of cerebrovascular tone; however, the sources, amounts, and consequences of excess NO on the cerebral vasculature are unknown. Our objective was to elucidate the mechanism of decreased cerebral artery tone after TBI. Methods and Results Cerebral arteries were isolated from rats 24 hours after moderate fluid‐percussion TBI. Pressure‐induced increases in vasoconstriction (myogenic tone) and smooth muscle Ca2+ were severely blunted in cerebral arteries after TBI. However, myogenic tone and smooth muscle Ca2+ were restored by inhibition of NO synthesis or endothelium removal, suggesting that TBI increased endothelial NO levels. Live native cell NO, indexed by 4,5‐diaminofluorescein (DAF‐2 DA) fluorescence, was increased in endothelium and smooth muscle of cerebral arteries after TBI. Clamped concentrations of 20 to 30 nmol/L NO were required to simulate the loss of myogenic tone and increased (DAF‐2T) fluorescence observed following TBI. In comparison, basal NO in control arteries was estimated as 0.4 nmol/L. Consistent with TBI causing enhanced NO‐mediated vasodilation, inhibitors of guanylyl cyclase, protein kinase G, and large‐conductance Ca2+‐activated potassium (BK) channel restored function of arteries from animals with TBI. Expression of the inducible isoform of NO synthase was upregulated in cerebral arteries isolated from animals with TBI, and the inducible isoform of NO synthase inhibitor 1400W restored myogenic responses following TBI. Conclusions The mechanism of profound cerebral artery vasodilation after TBI is a gain of function in vascular NO production by 60‐fold over controls, resulting from upregulation of the inducible isoform of NO synthase in the endothelium. PMID:25527626

  6. Critical Role of the Nitric Oxide/Reactive Oxygen Species Balance in Endothelial Progenitor Dysfunction

    PubMed Central

    Fleissner, Felix

    2011-01-01

    Abstract Endothelial injury and dysfunction are critical events in the pathogenesis of cardiovascular disease. During these processes, an impaired balance of nitric oxide bioavailability and oxidative stress is mechanistically involved. Circulating angiogenic cells (including early and late outgrowth endothelial progenitor cells (EPC)) contribute to formation of new blood vessels, neovascularization, and homeostasis of the vasculature, and are highly sensitive for misbalance between NO and oxidative stress. We here review the role of the endothelial nitric oxide synthase and oxidative stress producing enzyme systems in EPC during cardiovascular disease. We also focus on the underlying molecular mechanisms and potential emerging drug- and gene-based therapeutic strategies to improve EPC function in cardiovascular diseased patients. Antioxid. Redox Signal. 15, 933–948. PMID:20712407

  7. 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

  8. Traditional Chinese medicine's intervention in endothelial nitric oxide synthase activation and nitric oxide synthesis in cardiovascular system.

    PubMed

    Zhu, Jin-Qiang; Song, Wan-Shan; Hu, Zhen; Ye, Qiao-Feng; Liang, Yu-Bin; Kang, Li-Yuan

    2015-02-10

    Cardiovascular disease (CVD) is one of the most dangerous diseases which has become a major cause of human death. Many researches evidenced that nitric oxide (NO)/endothelial nitric oxide synthase (eNOS) system plays a significant role in the occurrence and development of CVD. NO, an important signaling molecule, closely associated with the regulation of vasodilatation, blood rheology, blood clotting and other physiological and pathological processes. The synthesis of NO in the endothelial cells primarily depends on the eNOS activity, thus the exploration of the mechanisms and effects of the eNOS activation on NO production is of great significance. Recently, studies on the effects of traditional Chinese medicine (TCM) and its extracts on eNOS activation and NO synthesis have gradually attracted more and more attentions. In this paper, we reviewed the mechanisms of NO synthesis and eNOS activation in the vascular endothelial cells (VECs) and intervention of TCM, so as to provide reference and train of thought to the intensive study of NO/eNOS system and the research and development of new drug for the treatment of CVD.

  9. Endothelial Nitric Oxide Synthase–Related Mechanotransduction Changes in Aged Porcine Angular Aqueous Plexus Cells

    PubMed Central

    Lei, Yuan; Stamer, William Daniel; Wu, Jihong; Sun, Xinghuai

    2014-01-01

    Purpose. To investigate effects of aging on endothelial nitric oxide synthase (eNOS) expression and signaling in angular aqueous plexus (AAP) (functional equivalent to human Schlemm's canal) cells subjected to shear stress. Methods. The AAP cells were isolated differentially from porcine outflow tissues using puromycin selection. Cell aging was induced by culturing cells in hyperoxia condition (40% oxygen and 5% carbon dioxide) for 14 days. The AAP cells grown in chamber slides were exposed to a shear stress of 8 dynes/cm2 for 24 hours. Expression of eNOS, eNOS-phospho Thr495, eNOS-phospho Ser1177, and Akt-phospho was tested by Western blot analysis and immunofluorescence staining. Nitric oxide (NO) levels were measured using the Griess assay. Results. Compared with control, eNOS levels in aged cells were significantly reduced by 60% (P < 0.05; n = 6). Phosphorylation of eNOS at Ser1177 and Akt at Ser473 was 63% and 80% lower in aged cells, respectively, whereas phosphorylation of the eNOS inhibition site (Thr495) increased by 6.1-fold (P < 0.05; n = 6). Shear stress (8 dynes/cm2 for 24 hours) increased eNOS abundance (total protein and at cell borders) and phosphorylation at Ser1177 by 1.7-fold and 1.8-fold, respectively (P < 0.05; n = 6), whereas aged cells were unresponsive. In control cells exposed to shear stress, the NO concentration was 1.8-fold higher than in the static group (P < 0.05; n = 4); however, aged cells were unresponsive to shear stress (mean ± SD, 4.3 ± 1.3 vs. 4.1 ± 1.4 μM). Conclusions. Aged AAP cells appear compromised in their mechanotransduction machinery involving eNOS, the protein product of the gene, NOS3, polymorphisms of which impart a risk for the development of glaucoma. PMID:25377220

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. Modulation of Local and Systemic Heterocellular Communication by Mechanical Forces: A Role of Endothelial Nitric Oxide Synthase

    PubMed Central

    Erkens, Ralf; Suvorava, Tatsiana; Kramer, Christian M.; Diederich, Lukas D.; Kelm, Malte

    2017-01-01

    Abstract Significance: In this review, we discuss the role of nitric oxide (NO) as a key physiological mechanotransducer modulating both local and systemic heterocellular communication and contributing to the integrated (patho)physiology of the cardiovascular system. A deeper understanding of mechanotransduction-mediated local and systemic nodes controlling heterocellular communication between the endothelium, blood cells, and other cell types (e.g., cardiomyocytes) may suggest novel therapeutic strategies for endothelial dysfunction and cardiovascular disease. Recent Advances: Mechanical forces acting on mechanoreceptors on endothelial cells activate the endothelial NO synthase (eNOS) to produce NO. NO participates in (i) abluminal heterocellular communication, inducing vasorelaxation, and thereby regulating vascular tone and blood pressure; (ii) luminal heterocellular communication, inhibiting platelet aggregation, and controlling hemostasis; and (iii) systemic heterocellular communication, contributing to adaptive physiological processes in response to exercise and remote ischemic preconditioning. Interestingly, shear-induced eNOS-dependent activation of vascular heterocellular communication constitutes the molecular basis of all methods applied in the clinical routine for evaluation of endothelial function. Critical Issues and Future Directions: The integrated physiology of heterocellular communication is still not fully understood. Dedicated experimental models are needed to analyze messengers and mechanisms underpinning heterocellular communication in response to physical forces in the cardiovascular system (and elsewhere). Antioxid. Redox Signal. 26, 917–935. PMID:27927026

  19. 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.

  20. Dietary inorganic nitrate reverses features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice.

    PubMed

    Carlström, Mattias; Larsen, Filip J; Nyström, Thomas; Hezel, Michael; Borniquel, Sara; Weitzberg, Eddie; Lundberg, Jon O

    2010-10-12

    The metabolic syndrome is a clustering of risk factors of metabolic origin that increase the risk for cardiovascular disease and type 2 diabetes. A proposed central event in metabolic syndrome is a decrease in the amount of bioavailable nitric oxide (NO) from endothelial NO synthase (eNOS). Recently, an alternative pathway for NO formation in mammals was described where inorganic nitrate, a supposedly inert NO oxidation product and unwanted dietary constituent, is serially reduced to nitrite and then NO and other bioactive nitrogen oxides. Here we show that several features of metabolic syndrome that develop in eNOS-deficient mice can be reversed by dietary supplementation with sodium nitrate, in amounts similar to those derived from eNOS under normal conditions. In humans, this dose corresponds to a rich intake of vegetables, the dominant dietary nitrate source. Nitrate administration increased tissue and plasma levels of bioactive nitrogen oxides. Moreover, chronic nitrate treatment reduced visceral fat accumulation and circulating levels of triglycerides and reversed the prediabetic phenotype in these animals. In rats, chronic nitrate treatment reduced blood pressure and this effect was also present during NOS inhibition. Our results show that dietary nitrate fuels a nitrate-nitrite-NO pathway that can partly compensate for disturbances in endogenous NO generation from eNOS. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against cardiovascular disease and type 2 diabetes.

  1. Dietary inorganic nitrate reverses features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice

    PubMed Central

    Carlström, Mattias; Larsen, Filip J.; Nyström, Thomas; Hezel, Michael; Borniquel, Sara; Weitzberg, Eddie; Lundberg, Jon O.

    2010-01-01

    The metabolic syndrome is a clustering of risk factors of metabolic origin that increase the risk for cardiovascular disease and type 2 diabetes. A proposed central event in metabolic syndrome is a decrease in the amount of bioavailable nitric oxide (NO) from endothelial NO synthase (eNOS). Recently, an alternative pathway for NO formation in mammals was described where inorganic nitrate, a supposedly inert NO oxidation product and unwanted dietary constituent, is serially reduced to nitrite and then NO and other bioactive nitrogen oxides. Here we show that several features of metabolic syndrome that develop in eNOS-deficient mice can be reversed by dietary supplementation with sodium nitrate, in amounts similar to those derived from eNOS under normal conditions. In humans, this dose corresponds to a rich intake of vegetables, the dominant dietary nitrate source. Nitrate administration increased tissue and plasma levels of bioactive nitrogen oxides. Moreover, chronic nitrate treatment reduced visceral fat accumulation and circulating levels of triglycerides and reversed the prediabetic phenotype in these animals. In rats, chronic nitrate treatment reduced blood pressure and this effect was also present during NOS inhibition. Our results show that dietary nitrate fuels a nitrate–nitrite–NO pathway that can partly compensate for disturbances in endogenous NO generation from eNOS. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against cardiovascular disease and type 2 diabetes. PMID:20876122

  2. 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

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

    PubMed

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

    2017-01-01

    Organochlorine Pesticides (OCPs) are detected ubiquitously in human and have been shown to be associated with Cardiovascular Disease (CVD) and atherosclerosis. 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). 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. 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. OCPs induce endothelial dysfunction through increased ROS generation via NADPH oxidase expression and reduced bioavailability of nitric oxide.

  4. 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.

  5. 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.

  6. Activation of NAD(P)H oxidases by thromboxane A2 receptor uncouples endothelial nitric oxide synthase.

    PubMed

    Zhang, Miao; Song, Ping; Xu, Jian; Zou, Ming-Hui

    2011-01-01

    The thromboxane receptor (TPr) and multiple TPr ligands, including thromboxane A(2) (TxA(2)) and prostaglandin H(2), are elevated during vascular and atherothrombotic diseases. How TPr stimulation causes vascular injury remains poorly defined. This study was conducted to investigate the mechanism by which TPr stimulation leads to vascular injury. Exposure of bovine aortic endothelial cells to either [1S-(1α,2β(5Z),3α(1E,3R),4α]-7-[3-(3-hydroxy-4-(d'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1] heptan-2-yl]-5'-heptenoic acid (IBOP) or U46619, 2 structurally related TxA(2) mimetics, for 24 hours markedly increased the release of superoxide anions (O(2)(·-)) and peroxynitrite (ONOO(-)) but reduced cyclic GMP, an index of nitric oxide bioactivity. IBOP also significantly suppressed activity of endothelial nitric oxide synthase (eNOS), increased enzyme-inactive eNOS monomers, and reduced levels of tetrahydrobiopterin, an essential eNOS cofactor. IBOP- and U46619-induced increases in O(2)(·-) were accompanied by the membrane translocation of the p67(phox) subunit of NAD(P)H oxidase. Pharmacological or genetic inhibition of either NAD(P)H oxidase or TPr abolished IBOP-induced O(2)(·-) formation. Furthermore, TPr activation significantly increased protein kinase C-ζ (PKC-ζ) in membrane fractions and PKC-ζ phosphorylation at Thr410. Consistently, PKC-ζ inhibition abolished TPr activation-induced membrane translocation of p67(phox) and O(2)(·-) production. Finally, exposure of isolated mouse aortae to IBOP markedly increased O(2)(·-) in wild-type but not in those from gp91(phox) knockout mice. We conclude that TPr activation via PKC-ζ-mediated NAD(P)H oxidase activation increases both O(2)(·-) and ONOO(-), resulting in eNOS uncoupling in endothelial cells.

  7. 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.

  8. Arginase inhibition restores NOS coupling and reverses endothelial dysfunction and vascular stiffness in old rats

    PubMed Central

    Kim, Jae Hyung; Bugaj, Lukasz J.; Oh, Young Jun; Bivalacqua, Trinity J.; Ryoo, Sungwoo; Soucy, Kevin G.; Santhanam, Lakshmi; Webb, Alanah; Camara, Andre; Sikka, Gautam; Nyhan, Daniel; Shoukas, Artin A.; Ilies, Monica; Christianson, David W.; Champion, Hunter C.

    2009-01-01

    There is increasing evidence that upregulation of arginase contributes to impaired endothelial function in aging. In this study, we demonstrate that arginase upregulation leads to endothelial nitric oxide synthase (eNOS) uncoupling and that in vivo chronic inhibition of arginase restores nitroso-redox balance, improves endothelial function, and increases vascular compliance in old rats. Arginase activity in old rats was significantly increased compared with that shown in young rats. Old rats had significantly lower nitric oxide (NO) and higher superoxide (O2−) production than young. Acute inhibition of both NOS, with NG-nitro-l-arginine methyl ester, and arginase, with 2(S)-amino- 6-boronohexanoic acid (ABH), significantly reduced O2− production in old rats but not in young. In addition, the ratio of eNOS dimer to monomer in old rats was significantly decreased compared with that shown in young rats. These results suggest that eNOS was uncoupled in old rats. Although the expression of arginase 1 and eNOS was similar in young and old rats, inducible NOS (iNOS) was significantly upregulated. Furthermore, S-nitrosylation of arginase 1 was significantly elevated in old rats. These findings support our previously published finding that iNOS nitrosylates and activates arginase 1 (Santhanam et al., Circ Res 101: 692–702, 2007). Chronic arginase inhibition in old rats preserved eNOS dimer-to-monomer ratio and significantly reduced O2− production and enhanced endothelial-dependent vasorelaxation to ACh. In addition, ABH significantly reduced vascular stiffness in old rats. These data indicate that iNOS-dependent S-nitrosylation of arginase 1 and the increase in arginase activity lead to eNOS uncoupling, contributing to the nitroso-redox imbalance, endothelial dysfunction, and vascular stiffness observed in vascular aging. We suggest that arginase is a viable target for therapy in age-dependent vascular stiffness. PMID:19661445

  9. Nitric oxide deficiency and endothelial dysfunction in pulmonary arterial hypertension.

    PubMed

    Klinger, James R; Abman, Steven H; Gladwin, Mark T

    2013-09-15

    Nitric oxide (NO) signaling plays a major role in modulating vascular tone and remodeling in the pulmonary circulation, but its role in the pathogenesis of pulmonary vascular diseases is still not completely understood. Numerous abnormalities of NO synthesis and signaling have been identified in animal models of pulmonary vascular disease and in humans with pulmonary hypertension. Many of these abnormalities have become targets of new therapies for the treatment of pulmonary hypertension. However, it is unclear to what extent alterations in NO signaling contribute to pulmonary hypertensive responses or merely reflect abnormalities induced by the underlying disease. This perspective examines the current understanding of altered NO signaling in pulmonary hypertensive diseases and discusses how these alterations may contribute to the pathogenesis of pulmonary hypertension. The efficacy and limitations of presently available therapies for pulmonary hypertension that target NO signaling are reviewed along with an update on investigational therapies that use this pathway to reverse pulmonary hypertensive changes.

  10. Glycated human serum albumin induces NF-κB activation and endothelial nitric oxide synthase uncoupling in human umbilical vein endothelial cells.

    PubMed

    Rodiño-Janeiro, Bruno K; Paradela-Dobarro, Beatriz; Raposeiras-Roubín, Sergio; González-Peteiro, Mercedes; González-Juanatey, José R; Álvarez, Ezequiel

    2015-01-01

    Non-enzymatic glycated proteins could mediate diabetes vascular complications, but the molecular mechanisms are unknown. Our objective was to find new targets involved in the glycated human serum albumin (gHSA)-enhanced extracellular reactive oxygen species (ROS) production in human endothelial cells. Some nuclear factors and phosphorylation cascades were analysed. gHSA activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which up-regulated NOX4 and P22PHOX and enhanced ROS production. Pharmacological inhibition of NF-κB reversed gHSA-enhanced NOX4 expression and decreased gHSA-induced ROS production in extra- and intracellular spaces. The inhibition of activator protein-1 (AP-1) induced a rise in NOX4 and P22PHOX subunit expression and a down-regulation of endothelial nitric oxide synthase (eNOS). AP-1 inhibition also enhanced extracellular ROS production in the presence of serum albumin, but not with gHSA. These results were explained by the eNOS uncoupling induced by gHSA, also demonstrated in this study. Phosphatidylinositol 3-kinase or mitogen-activated protein kinase kinase 1/2 did not show to be involved in gHSA-induced ROS production. All together, the results suggested that gHSA-enhanced ROS production in endothelium is mediated by: 1) NF-κB activation and subsequence up-regulation of NADPH oxidase, 2) eNOS uncoupling. AP-1, although is not directly affected by gHSA, is another target for regulating NADPH oxidase and eNOS expression in endothelial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. 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.

  12. 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.

  13. Inhibition of soluble epoxide hydrolase attenuates endothelial dysfunction in animal models of diabetes, obesity and hypertension.

    PubMed

    Zhang, Le-Ning; Vincelette, Jon; Chen, Dawn; Gless, Richard D; Anandan, Sampath-Kumar; Rubanyi, Gabor M; Webb, Heather K; MacIntyre, D Euan; Wang, Yi-Xin Jim

    2011-03-01

    Endothelial dysfunction is a hallmark of, and plays a pivotal role in the pathogenesis of cardiometabolic diseases, including type II diabetes, obesity, and hypertension. It has been well established that epoxyeicosatrienoic acids (EETs) act as an endothelial derived hyperpolarization factor (EDHF). Soluble epoxide hydrolase (s-EH) rapidly hydrolyses certain epoxylipids (e.g. EETs) to less bioactive diols (DHETs), thereby attenuating the evoked vasodilator effects. The aim of the present study was to examine if inhibition of s-EH can restore impaired endothelial function in three animal models of cardiometabolic diseases. Isolated vessel rings of the aorta and/or mesenteric artery from mice or rats were pre-contracted using phenylephrine or U46619. Endothelium-dependent and independent vasorelaxation to acetylcholine and sodium nitroprusside (SNP) were measured using wire myography in vessels isolated from db/db or diet-induced obesity (DIO) mice, and angiotensin II-induced hypertensive rats treated chronically with s-EH inhibitors AR9281 or AR9276 or with vehicle. Vasorelaxation to acetylcholine, but not to SNP was severely impaired in all three animal models. Oral administration of AR9281 or AR9276 abolished whole blood s-EH activity, elevated epoxy/diol lipid ratio, and abrogated endothelial dysfunction in all three models. Incubating the mesenteric artery of db/db mice with L-NAME and indomethacin to block nitric oxide (NO) and prostacyclin formation did not affect AR9821-induced improvement of endothelial function. These data indicate that inhibition of s-EH ameliorates endothelial dysfunction and that effects in the db/db model are independent of the presence of NO and cyclooxygenase derived prostanoids. Thus, preserving vasodilator EETs by inhibition of s-EH may be of therapeutic benefit by improving endothelial function in cardiometabolic diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

  14. Effect of Interleukin-10 and Laminar Shear Stress on Endothelial Nitric Oxide Synthase and Nitric Oxide in African American Human Umbilical Vein Endothelial Cells.

    PubMed

    Babbitt, Dianne M; Kim, Ji-Seok; Forrester, Steven J; Brown, Michael D; Park, Joon-Young

    2015-11-05

    African Americans have a predisposition to heightened systemic inflammation and a high prevalence of hypertension. The purpose of this study was to evaluate the influence of interleukin-10 (IL-10) and laminar shear stress (LSS) on African American endothelial cells by measuring total endothelial nitric oxide synthase (eNOS) protein expression and its phosphorylated form (p-eNOS) at Serine 1177, and nitric oxide (NO) levels, in response to IL-10 incubation and high physiological levels of LSS, used as an in vitro mimetic for aerobic exercise training (AEXT). Human umbilical vein endothelial cells (HUVEC) from an African American donor were cultured. The experimental conditions included Static, Static with IL-10 Incubation, LSS at 20 dynes/cm², and LSS at 20 dynes/cm² with IL-10 Incubation. Western blotting was used to measure eNOS and p-eNOS protein expression in the cells. A modified Griess assay was used to measure NO metabolites in the cell culture media. There were significant increases in p-eNOS, eNOS, and NO in the LSS at 20 dynes/cm² and LSS at 20 dynes/cm² with IL-10 Incubation experimental conditions when compared to the Static experimental condition. There were no other statistically significant differences demonstrating that IL-10 did not have an additive effect on eNOS activity in our study. The significant increases in p-eNOS, eNOS, and NO as a result of LSS in African American HUVECs suggest that AEXT may be a viable, nonpharmacologic method to improve vascular inflammation status and vasodilation, and thereby contribute to hypertension reduction in the African American population.

  15. 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.

  16. Aged garlic extract restores nitric oxide bioavailability in cultured human endothelial cells even under conditions of homocysteine elevation.

    PubMed

    Weiss, Norbert; Papatheodorou, Louisa; Morihara, Naoaki; Hilge, Robert; Ide, Nagatoshi

    2013-01-09

    Supplementation with aged garlic extract (AGE) has been shown to restore impaired endothelium-dependent vasodilator response in subjects with acutely elevated plasma homocysteine (Hcy) levels after an oral methionine load and in patients with chronic coronary artery disease. Moreover, AGE has been shown to inhibit the progression of coronary calcifications in patients with coronary artery disease. The molecular mechanisms, by which AGE preserves endothelial function is unknown. Our objective was to explore whether AGE preserves endothelial nitric oxide (NO) output even under conditions of elevated Hcy levels by preventing oxidative inactivation of the NO synthase cofactor tetrahydrobiopterin. Endothelial (EA.hy 926) cells were incubated with hypoxanthine, aminopterin, thymidine and methionine (HAT/MET) to increase cellular Hcy levels, and with and without AGE. Agonist stimulated NO output was measured using the fluorescent probe DAF-2, and cellular thiol levels (Hcy, cysteine, reduced and oxidized glutathione) and cellular tetrahydrobiopterin levels were measured by high performance liquid chromatography. HAT/MET incubation resulted in significantly increased cellular Hcy levels, unaffected by coincubation with AGE. Elevated Hcy went along with significantly decreased NO output (to 34.4 ± 4.4% of control) and levels of tetrahydrobiopterin (from 4.67 ± 2.17 to 2.17 ± 0.97 pmol/mg). Incubation with AGE (5mg/mL) in HAT/MET-treated cells prevented the declines in NO output and tetrahydrobiopterin levels. AGE increased cellular levels of cysteine and total glutathione, and prevented glutathione and tetrahydrobiopterin oxidation induced by elevated Hcy. Incubation with AGE preserved normal NO output from endothelial cells even under conditions of elevated Hcy levels by increasing cellular thiol antioxidant and prevention of tetrahydrobiopterin oxidation. This suggests that AGE might be useful in the prevention of endothelial dysfunction. Copyright © 2012 Elsevier

  17. Geranylgeranylacetone, heat shock protein 90/AMP-activated protein kinase/endothelial nitric oxide synthase/nitric oxide pathway, and endothelial function in humans.

    PubMed

    Fujimura, Noritaka; Jitsuiki, Daisuke; Maruhashi, Tatsuya; Mikami, Shinsuke; Iwamoto, Yumiko; Kajikawa, Masato; Chayama, Kazuaki; Kihara, Yasuki; Noma, Kensuke; Goto, Chikara; Higashi, Yukihito

    2012-01-01

    Geranylgeranylacetone (GGA) induces expression of heat shock protein 90 (Hsp90), an adaptor molecule for assembly of endothelial nitric oxide synthase (eNOS) phosphorylation complex. The purpose of this study was to determine whether GGA enhances Hsp90 expression and augments endothelium-dependent vasodilation via upregulation of eNOS in humans. We evaluated the effects of GGA on human umbilical vein endothelial cells (HUVECs) and on forearm blood flow (FBF) responses to acetylcholine and sodium nitroprusside in 40 healthy young men. Hsp90, eNOS, AMP-activated protein kinase (AMPK), and Akt expression in HUVECs and peripheral blood mononuclear cells was detected by Western blot analysis. GGA increased Hsp90 expression and phosphorylation of eNOS and AMPK but not Akt in HUVECs and increased Hsp90 expression in peripheral blood mononuclear cells. Oral administration of GGA (600 mg) augmented the FBF response to acetylcholine. Infusion of N(G)-monomethyl-l-arginine, an NO synthase inhibitor, completely abolished GGA-induced augmentation of the FBF response to acetylcholine. GGA also augmented the acetylcholine-stimulated NO release in smokers. These findings suggest that GGA-induced activation of Hsp90/AMPK significantly increased NO-mediated vasodilation in healthy subjects, as well as in smokers. The use of GGA may be a new therapeutic approach for improving endothelial dysfunction.

  18. Gene variations of nitric oxide synthase regulate the effects of a saturated fat rich meal on endothelial function

    USDA-ARS?s Scientific Manuscript database

    Objective: Endothelial nitric oxide synthase gene variations have been linked to a higher risk for cardiovascular diseases by unknown mechanisms. Our aim was to determine if two SNPs located in NOS3 (E298D and i19342) interfere with microvascular endothelial function (MEF) and/or oxidative stress du...

  19. Pore-forming bacterial toxins potently induce release of nitric oxide in porcine endothelial cells

    PubMed Central

    1993-01-01

    Nitric oxide (NO) is believed to play an important role in sepsis- related hypotension. We examined the effects of two pore-forming bacterial exotoxins, Escherichia coli hemolysin and Staphylococcus aureus alpha-toxin, on NO formation in cultured porcine pulmonary artery endothelial cells. NO was quantified using a difference- spectrophotometric method based on the rapid and stoichiometric reaction of NO with oxyhemoglobin. Endothelial cyclic guanosine monophosphate levels were also monitored. Both exotoxins increased NO synthesis in endothelial cells in a time- and dose-dependent manner to an extent exceeding that observed with the ionophore A23187 or thrombin. The capacity of exotoxins to induce NO formation may be relevant in patients with severe local or systemic bacterial infections. PMID:8391061

  20. 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.

  1. 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

  2. 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

  3. Lack of Endothelial Nitric Oxide Synthase Aggravates Murine Accelerated Anti-Glomerular Basement Membrane Glomerulonephritis

    PubMed Central

    Heeringa, Peter; van Goor, Harry; Itoh-Lindstrom, Yoshie; Maeda, Nobuyo; Falk, Ronald J.; Assmann, Karel J. M.; Kallenberg, Cees G. M.; Jennette, J. Charles

    2000-01-01

    Nitric oxide (NO) radicals generated by endothelial nitric oxide synthase (eNOS) are involved in the regulation of vascular tone. In addition, NO radicals derived from eNOS inhibit platelet aggregation and leukocyte adhesion to the endothelium and, thus, may have anti-inflammatory effects. To study the role of eNOS in renal inflammation, the development of accelerated anti-glomerular basement membrane (GBM) glomerulonephritis was examined in mice lacking a functional gene for eNOS and compared with wild-type (WT) C57BL/B6j mice. WT C57BL/6j mice (n = 12) and eNOS knockout (−/−) mice (n = 12) were immunized intraperitoneally with sheep IgG (0.2 mg in complete Freund’s adjuvant). At day 6.5 after immunization, mice received a single i.v. injection of sheep anti-mouse GBM (1 mg in 200 μl PBS). Mice were sacrificed at day 1 and 10 after induction of the disease. All WT mice survived until day 10, whereas 1 eNOS−/− mouse died and 2 more became moribund, requiring sacrifice. At day 10, eNOS−/− mice had higher levels of blood urea nitrogen than WT mice (P < 0.02), although proteinuria was comparable. Immunofluorescence microscopy documented similar IgG deposition in both WT and eNOS−/− mice, but eNOS−/− mice had more extensive glomerular staining for fibrin at day 10 (P < 0.007). At day 10, light microscopy demonstrated that eNOS−/− mice had more severe glomerular thrombosis (P < 0.003) and influx of neutrophils (P < 0.006), but similar degrees of overall glomerular endocapillary hypercellularity and crescent formation. In conclusion, accelerated anti-GBM glomerulonephritis is severely aggravated in eNOS−/− mice, especially with respect to glomerular capillary thrombosis and neutrophil infiltration. These results indicate that NO radicals generated by eNOS play a protective role during renal inflammation. PMID:10702405

  4. 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.

  5. Resistin decreases expression of endothelial nitric oxide synthase through oxidative stress in human coronary artery endothelial cells

    PubMed Central

    Jiang, Jun; Lü, Jian-Ming; Chai, Hong; Wang, Xinwen; Lin, Peter H.; Yao, Qizhi

    2010-01-01

    Resistin is a newly discovered adipocyte-derived cytokine that may play an important role in insulin resistance, diabetes, adipogenesis, inflammation, and cardiovascular disease. However, it is largely unknown whether resistin impairs endothelial functions by affecting the endothelial nitric oxide synthase (eNOS) system. In this study, we determined the effect of human recombinant resistin protein on eNOS expression and regulation in human coronary artery endothelial cells (HCAECs). When cells were treated with clinically relevant concentrations of resistin (40 or 80 ng/ml) for 24 h, the levels of eNOS mRNA, protein, and activity and eNOS mRNA stability were significantly reduced. Cellular nitric oxide levels were also decreased. In addition, the cellular levels of reactive oxygen species (ROS), including superoxide anion, were significantly increased in resistin-treated HCAECs. Mitochondrial membrane potential and the activities of catalase and superoxide dismutase were reduced. Three antioxidants, seleno-l-methionine, ginsenoside Rb1, and MnTBAP (superoxide dismutase mimetic), effectively blocked resistin-induced eNOS downregulation. Meanwhile, resistin activated the mitogen-activated protein kinases p38 and c-Jun NH2-terminal kinase (JNK), and the specific p38 inhibitor SB-239063 effectively blocked resistin-induced ROS production and eNOS downregulation. Furthermore, immunoreactivity of resistin was increased in atherosclerotic regions of human aorta and carotid arteries. Thus resistin directly induces eNOS downregulation through overproduction of ROS and activation of p38 and JNK in HCAECs. Resistin-induced mitochondrial dysfunction and imbalance in cellular redox enzymes may be the underlying mechanisms of oxidative stress. PMID:20435848

  6. 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

  7. Alveolar macrophage inducible nitric oxide synthase-dependent pulmonary microvascular endothelial cell septic barrier dysfunction.

    PubMed

    Farley, K S; Wang, L F; Law, C; Mehta, S

    2008-11-01

    Inducible nitric oxide (NO) synthase (iNOS) from neutrophils and alveolar macrophages (AM) contributes to the pathophysiology of murine septic acute lung injury (ALI). It is not known if AM iNOS has a direct effect on septic pulmonary microvascular endothelial cell (PMVEC) permeability. We hypothesized that AM iNOS mediates PMVEC permeability in vitro under septic conditions through NO and peroxynitrite. 100,000 confluent PMVEC on cell-culture inserts were co-incubated with iNOS+/+ vs. iNOS-/- AM, in various ratios of AM to PMVEC. PMVEC injury was assessed by trans-PMVEC Evans Blue-labelled albumin flux in the presence or absence of cytomix (equimolar TNF-alpha, IL-1beta and IFN-gamma). Cytomix stimulation dose-dependently increased trans-PMVEC EB-albumin flux, which was exaggerated (1.4+/-0.1% vs. 0.4+/-0.1% in unstimulated PMVEC, p<0.05) in the presence of iNOS+/+, but not iNOS-/-, AM in the upper compartment. Similarly, iNOS+/+, but not iNOS-/-, AM in the lower compartment also enhanced septic trans-PMVEC albumin leak. The mechanism of iNOS-dependent septic PMVEC permeability was pursued through pharmacologic studies with inhibitors of NOS, and scavengers of NO, superoxide, and peroxynitrite, and treatment of PMVEC with the NO donor, DETA-NONOate. Septic iNOS+/+ AM-dependent trans-PMVEC albumin leak was significantly attenuated by pharmacologic iNOS inhibition (L-NAME and 1400W), and scavenging of either NO (oxyhemoglobin), superoxide (PEG-SOD), or peroxynitrite (FeTPPS). Exogenous NO (DETA-NONOate) had no effect on PMVEC permeability. These data are consistent with a direct role of AM iNOS in septic PMVEC barrier dysfunction, which is likely mediated, in part, through peroxynitrite.

  8. Glucocorticoid response elements and 11β-hydroxysteroid dehydrogenases in the regulation of endothelial nitric oxide synthase expression

    PubMed Central

    Liu, Yong; Mladinov, Domagoj; Pietrusz, Jennifer L.; Usa, Kristie; Liang, Mingyu

    2009-01-01

    Aims Hypertensive and other effects of excess glucocorticoids might be in part mediated by the suppression of endothelial nitric oxide synthase (eNOS) expression. We studied the transcriptional and biochemical mechanisms that mediate or modulate the suppression of eNOS expression by glucocorticoids. Methods and results We found that a mere three-fold increase in the concentration of the natural glucocorticoid cortisol (from 30 to 100 nmol/L) significantly decreased the expression level of eNOS in human endothelial cells. Deletion analysis of the eNOS promoter indicated that the segment within −119 bp upstream from the transcription start site was significantly involved in the effect of cortisol. Site-directed mutagenesis and chromatin immunoprecipitation analyses demonstrated the presence of a suppressive glucocorticoid response element (GRE) at −111 to −105 bp. 11β-hydroxysteroid dehydrogenases (11β-HSD) catalyse the interconversion of active and inactive glucocorticoids. The suppression of 11β-HSD2 using small interfering RNA markedly exacerbated the inhibition of eNOS by cortisol. The suppression of 11β-HSD1 abolished the inhibition of eNOS expression by cortisol. Conclusion We identified the first GRE in the eNOS promoter region and demonstrated that endogenous 11β-HSD1 and 11β-HSD2 play significant and distinct roles in modulating the effect of glucocorticoids on eNOS expression. PMID:18716005

  9. Reversible inhibition of cellular respiration by nitric oxide in vascular inflammation.

    PubMed

    Borutaite, V; Matthias, A; Harris, H; Moncada, S; Brown, G C

    2001-12-01

    Incubation of rat aortas with endotoxin and interferon-gamma for 24 h resulted in an aortic oxygen consumption that was substantially inhibited and strongly oxygen dependent (37% inhibition at 160 microM O(2) and 62% inhibition at 80 microM O(2) relative to untreated aortas). This respiratory inhibition was reversed by a nitric oxide (NO) scavenger (oxyhemoglobin) or by an inhibitor of inducible NO synthase [N-(3-(aminomethyl)benzyl)acetamide x 2HCl, 1400W], but not by an inhibitor of soluble guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one). Addition of 1 microM NO to untreated aortas caused rapid and reversible inhibition of oxygen consumption that was greater at lower oxygen concentrations. Incubation of endothelial cells isolated from rat aortas with endotoxin and interferon-gamma for 24 h resulted in a steady-state NO concentration of approximately 0.5 microM and 90% inhibition of cellular oxygen consumption that was immediately reversed by an NO scavenger (oxyhemoglobin). These results suggest that during inflammation and sepsis, tissue respiration may be substantially reduced due to inhibition by NO of cytochrome oxidase.

  10. 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

  11. Impaired Endothelial Repair Capacity of Early Endothelial Progenitor Cells in Hypertensive Patients With Primary Hyperaldosteronemia: Role of 5,6,7,8-Tetrahydrobiopterin Oxidation and Endothelial Nitric Oxide Synthase Uncoupling.

    PubMed

    Chen, Long; Ding, Mei-Lin; Wu, Fang; He, Wen; Li, Jin; Zhang, Xiao-Yu; Xie, Wen-Li; Duan, Sheng-Zhong; Xia, Wen-Hao; Tao, Jun

    2016-02-01

    Although hyperaldosteronemia exerts detrimental impacts on vascular endothelium in addition to elevating blood pressure, the effects and molecular mechanisms of hyperaldosteronemia on early endothelial progenitor cell (EPC)-mediated endothelial repair after arterial damage are yet to be determined. The aim of this study was to investigate the endothelial repair capacity of early EPCs from hypertensive patients with primary hyperaldosteronemia (PHA). In vivo endothelial repair capacity of early EPCs from PHAs (n=20), age- and blood pressure-matched essential hypertension patients (n=20), and age-matched healthy subjects (n=20) was evaluated by transplantation into a nude mouse carotid endothelial denudation model. Endothelial function was evaluated by flow-mediated dilation of brachial artery in human subjects. In vivo endothelial repair capacity of early EPCs and flow-mediated dilation were impaired both in PHAs and in essential hypertension patients when compared with age-matched healthy subjects; however, the early EPC in vivo endothelial repair capacity and flow-mediated dilation of PHAs were impaired more severely than essential hypertension patients. Oral spironolactone improved early EPC in vivo endothelial repair capacity and flow-mediated dilation of PHAs. Increased oxidative stress, oxidative 5,6,7,8-tetrahydrobiopterin degradation, endothelial nitric oxide synthase uncoupling and decreased nitric oxide production were found in early EPCs from PHAs. Nicotinamide adenine dinucleotide phosphate oxidase subunit p47(phox) knockdown or 5,6,7,8-tetrahydrobiopterin supplementation attenuated endothelial nitric oxide synthase uncoupling and enhanced in vivo endothelial repair capacity of early EPCs from PHAs. In conclusion, PHAs exhibited more impaired endothelial repair capacity of early EPCs than did essential hypertension patients independent of blood pressure, which was associated with mineralocorticoid receptor-dependent oxidative stress and subsequently 5

  12. 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

  13. Metabolites of flavonoid compounds preserve indices of endothelial cell nitric oxide bioavailability under glucotoxic conditions.

    PubMed

    Qian, Y; Babu, P V A; Symons, J D; Jalili, T

    2017-09-11

    We hypothesized that metabolites of dietary flavonoids attenuate impairments in nitric oxide (NO) bioavailability evoked by glucotoxic conditions mimicking Type 1 or 2 diabetes. To test this, human aortic endothelial cells were treated with either vehicle control, quercetin-3-O-glucoronide, piceatannol or 3-(3-hydroxyphenyl)propionoic acid for 24 h. These are metabolites of quercetin, resveratrol and proanthocyanidin, respectively. Next, cells were exposed to control (5 mM) or high (25 mM) glucose conditions for 48 h, followed by insulin treatment (100 nM, 10 min) to stimulate NO production. In control glucose conditions NO production, phosphorylated to total endothelial nitric oxide synthase (p-eNOS(ser1177): eNOS), and phosphorylated to total Akt (p-Akt(Ser473): Akt) were all increased by insulin stimulation. This response was abolished during high glucose conditions. Pretreatment of cells with flavonoid metabolites prior to high glucose challenge preserved insulin stimulated increases in NO production, p-Akt(Ser473): Akt and p-eNOS(Ser1177): eNOS. These effects may be secondary to oxidative stress as pretreatment with all flavonoid metabolites prevented elevations in reactive oxygen and nitrogen species in response to high glucose. These data support the hypothesis that beneficial effects of flavonoids on endothelial cell function in the context of glucotoxicity, at least in part, are secondary to their metabolites.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Piper sarmentosum increases nitric oxide production in oxidative stress: a study on human umbilical vein endothelial cells.

    PubMed

    Ugusman, Azizah; Zakaria, Zaiton; Hui, Chua Kien; Nordin, Nor Anita Megat Mohd

    2010-07-01

    Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs). HUVECS WERE DIVIDED INTO FOUR GROUPS: control, treatment with 180 microM hydrogen peroxide (H(2)O(2)), treatment with 150 microg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H(2)O(2) for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction. Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs. Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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.

  4. Endothelial thrombomodulin induces Ca2+ signals and nitric oxide synthesis through epidermal growth factor receptor kinase and calmodulin kinase II.

    PubMed

    David-Dufilho, Monique; Millanvoye-Van Brussel, Elisabeth; Topal, Gokce; Walch, Laurence; Brunet, Annie; Rendu, Francine

    2005-10-28

    Endothelial membrane-bound thrombomodulin is a high affinity receptor for thrombin to inhibit coagulation. We previously demonstrated that the thrombin-thrombomodulin complex restrains cell proliferation mediated through protease-activated receptor (PAR)-1. We have now tested the hypothesis that thrombomodulin transduces a signal to activate the endothelial nitric-oxide synthase (NOS3) and to modulate G protein-coupled receptor signaling. Cultured human umbilical vein endothelial cells were stimulated with thrombin or a mutant of thrombin that binds to thrombomodulin and has no catalytic activity on PAR-1. Thrombin and its mutant dose dependently activated NO release at cell surface. Pretreatment with anti-thrombomodulin antibody suppressed NO response to the mutant and to low thrombin concentration and reduced by half response to high concentration. Thrombin receptor-activating peptide that only activates PAR-1 and high thrombin concentration induced marked biphasic Ca2+ signals with rapid phosphorylation of PLC(beta3) and NOS3 at both serine 1177 and threonine 495. The mutant thrombin evoked a Ca2+ spark and progressive phosphorylation of Src family kinases at tyrosine 416 and NOS3 only at threonine 495. It activated rapid phosphatidylinositol-3 kinase-dependent NO synthesis and phosphorylation of epidermal growth factor receptor and calmodulin kinase II. Complete epidermal growth factor receptor inhibition only partly reduced the activation of phospholipase Cgamma1 and NOS3. Prestimulation of thrombomodulin did not affect NO release but reduced Ca2+ responses to thrombin and histamine, suggesting cross-talks between thrombomodulin and G protein-coupled receptors. This is the first demonstration of an outside-in signal mediated by the cell surface thrombomodulin receptor to activate NOS3 through tyrosine kinase-dependent pathway. This signaling may contribute to thrombomodulin function in thrombosis, inflammation, and atherosclerosis.

  5. 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

  6. Endothelial Nitric Oxide Synthase Prevents Heparanase Induction and the Development of Proteinuria.

    PubMed

    Garsen, Marjolein; Rops, Angelique L; Li, Jinhua; van Beneden, Katrien; van den Branden, Christiane; Berden, Jo Hm; Rabelink, Ton J; van der Vlag, Johan

    2016-01-01

    Endothelial nitric oxide synthase (eNOS) deficiency exacerbates proteinuria and renal injury in several glomerular diseases, but the underlying mechanism is not fully understood. We recently showed that heparanase is essential for the development of experimental diabetic nephropathy and glomerulonephritis, and hypothesize that heparanase expression is regulated by eNOS. Here, we demonstrate that induction of adriamycin nephropathy (AN) in C57BL/6 eNOS-deficient mice leads to an increased glomerular heparanase expression accompanied with overt proteinuria, which was not observed in the AN-resistant wild type counterpart. In vitro, the eNOS inhibitor asymmetric dimethylarginine (ADMA) induced heparanase expression in cultured mouse glomerular endothelial cells. Moreover, ADMA enhanced transendothelial albumin passage in a heparanase-dependent manner. We conclude that eNOS prevents heparanase induction and the development of proteinuria.

  7. Endothelial Nitric Oxide Synthase Prevents Heparanase Induction and the Development of Proteinuria

    PubMed Central

    Garsen, Marjolein; Rops, Angelique L.; Li, Jinhua; van Beneden, Katrien; van den Branden, Christiane; Berden, Jo HM; Rabelink, Ton J.

    2016-01-01

    Endothelial nitric oxide synthase (eNOS) deficiency exacerbates proteinuria and renal injury in several glomerular diseases, but the underlying mechanism is not fully understood. We recently showed that heparanase is essential for the development of experimental diabetic nephropathy and glomerulonephritis, and hypothesize that heparanase expression is regulated by eNOS. Here, we demonstrate that induction of adriamycin nephropathy (AN) in C57BL/6 eNOS-deficient mice leads to an increased glomerular heparanase expression accompanied with overt proteinuria, which was not observed in the AN-resistant wild type counterpart. In vitro, the eNOS inhibitor asymmetric dimethylarginine (ADMA) induced heparanase expression in cultured mouse glomerular endothelial cells. Moreover, ADMA enhanced transendothelial albumin passage in a heparanase-dependent manner. We conclude that eNOS prevents heparanase induction and the development of proteinuria. PMID:27505185

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Histone Deacetylase-3 antagonizes Aspirin-stimulated Endothelial Nitric Oxide production by reversing Aspirin-induced lysine acetylation of Endothelial Nitric Oxide Synthase

    PubMed Central

    Jung, Saet-Byel; Kim, Cuk-Seong; Naqvi, Asma; Yamamori, Tohru; Mattagajasingh, Ilwola; Hoffman, Timothy A; Cole, Marsha P; Kumar, Ajay; DeRicco, Jeremy S.; Jeon, Byeong Hwa; Irani, Kaikobad

    2010-01-01

    Rationale Low-dose acetylsalicylic acid (aspirin) is widely used in the treatment and prevention of vascular atherothrombosis. Cardiovascular doses of aspirin also reduce systemic blood pressure and improve endothelium-dependent vasorelaxation in patients with atherosclerosis or risk factors for atherosclerosis. Aspirin can acetylate proteins, other than its pharmacological target cyclooxygenase (COX), at lysine residues. The role of lysine acetylation in mediating the effects of low-dose aspirin on the endothelium is not known. Objective To determine the role of lysine acetylation of eNOS in the regulation of endothelial NO production by low-dose aspirin, and to examine whether the lysine deacetylase Histone Deacetylase-3 (HDAC3) antagonizes the effect of low-dose aspirin on endothelial NO production by reversing acetylation of functionally critical eNOS lysine residues. Methods and results Low concentrations of aspirin induce lysine acetylation of eNOS, stimulating eNOS enzymatic activity and endothelial NO production in a cyclooxygenase-1 (COX-1)-independent fashion. Low-dose aspirin in vivo also increases bioavailable vascular NO in an eNOS-dependent and COX-1-independent manner. Low-dose aspirin promotes the binding of eNOS to calmodulin. Lysine 609 in the calmodulin autoinhibitory domain of bovine eNOS mediates aspirin-stimulated binding of eNOS to calmodulin and eNOS-derived NO production. Overexpression of HDAC3 inhibits aspirin-stimulated lysine acetylation of eNOS, increase in eNOS enzymatic activity, eNOS-derived NO, and binding of eNOS to calmodulin. Similarly, downregulation of HDAC3 promotes lysine acetylation of eNOS, and endothelial NO generation. Conclusions Lysine acetylation of eNOS is a post-translational protein modification supporting low-dose aspirin-induced vasoprotection. HDAC3, by deacetylating aspirin-acetylated eNOS, antagonizes aspirin-stimulated endothelial production of NO. PMID:20705923

  15. Decursin inhibits vasculogenesis in early tumor progression by suppression of endothelial progenitor cell differentiation and function.

    PubMed

    Jung, Seok Yun; Choi, Jin Hwa; Kwon, Sang-Mo; Masuda, Haruchika; Asahara, Takayuki; Lee, You-Mie

    2012-05-01

    Endothelial progenitor cells (EPCs) contribute to the tumor vasculature during tumor progression. Decursin isolated from the herb Angelica gigas is known to possess potent anti-inflammatory activities. Recently, we reported that decursin is a novel candidate for an angiogenesis inhibitor [Jung et al., 2009]. In this study, we investigated whether decursin regulates EPC differentiation and function to inhibit tumor vasculogenesis. We isolated AC133+ cells from human cord blood and decursin significantly decreased the number of EPC colony forming units of human cord blood-derived AC133+ cells that produce functional EPC progenies. Decursin dose-dependently decreased the cell number of EPC committing cells as demonstrated by EPC expansion studies. Decursin inhibited EPC differentiation from progenitor cells into spindle-shaped EPC colonies. Additionally, decursin inhibited proliferation and migration of early EPCs isolated from mouse bone marrow. Furthermore, decursin suppressed expression of angiopoietin-2, angiopoietin receptor Tie-2, Flk-1 (vascular endothelial growth factor receptor-2), and endothelial nitric oxide synthase in mouse BM derived EPCs in a dose-dependent manner. Decursin suppressed tube formation ability of EPCs in collaboration with HUVEC. Decursin (4 mg/kg) inhibited tumor-induced mobilization of circulating EPCs (CD34 + /VEGFR-2+ cells) from bone marrow and early incorporation of Dil-Ac-LDL-labeled or green fluorescent protein (GFP)+ EPCs into neovessels of xenograft Lewis lung carcinoma tumors in wild-type- or bone-marrow-transplanted mice. Accordingly, decursin attenuated EPC-derived endothelial cells in neovessels of Lewis lung carcinoma tumor masses grown in mice. Together, decursin likely affects EPC differentiation and function, thereby inhibiting tumor vasculogenesis in early tumorigenesis. Copyright © 2012 Wiley Periodicals, Inc.

  16. 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. © The Author(s) 2016. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. 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

  18. 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

  19. MicroRNA-221 regulates endothelial nitric oxide production and inflammatory response by targeting adiponectin receptor 1.

    PubMed

    Chen, Chao-Feng; Huang, Jinyu; Li, Hong; Zhang, Chu; Huang, Xurui; Tong, Guoxin; Xu, Yi-Zhou

    2015-07-10

    Adiponectin exerts anti-atherosclerosis property through its 2 receptors (AdipoR1 and AdipoR2). The mechanism regulating the expression of adiponectin receptors is unclear. Bioinformatics analysis showed that miR-221 targeted the 3'-untranslated region (3'UTR) of the AdipoR1 mRNA. The protein level and the mRNA level of AdipoR1 were reduced when miR-221 was expressed in human umbilical vein endothelial cells (HUVECs). Meanwhile, miR-221 repressed the activity of luciferase reporter containing the 3'UTR of AdipoR1. The inhibitory effect of miR-221 was abolished when the miR-221 binding site within the AdipoR1 3'UTR was deleted. Overexpression of miR-221 inhibited adiponectin-stimulated nitric oxide (NO) production in HUVECs. Furthermore, miR-221 abolished the inhibitory effect of adiponectin on NF-kB activation and the expression of adhesion molecules. Altogether, these results indicated that miR-221 targets AdipoR1 to regulate endothelial inflammatory response. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. 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

  1. 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

  2. Simvastatin combined with nifedipine enhances endothelial cell protection by inhibiting ROS generation and activating Akt phosphorylation

    PubMed Central

    Chen, Xiao-niao; Xu, Jun; Feng, Zhe; Fan, Ming; Han, Jing-yao; Yang, Zhuo

    2010-01-01

    Aim: To investigate the protective effects of simvastatin (Sim) combined with nifedipine (Nif) on endothelial cells and elucidate the action mechanism. Methods: Human umbilical vein endothelial cells (HUVEC) were used. mRNA and protein levels were measured by using reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. Intracellular calcium and reactive oxygen species (ROS) were detected using confocal microscopy. The Griess assay was used to evaluate nitric oxide (NO) release. Results: Treatment of HUVEC with H2O2 100 μmol/L for 30 min inhibited the mRNA and protein expression of endothelial nitric oxide synthase (eNOS). With increased concentrations of Nif, eNOS mRNA and protein levels increased (P<0.05). Combined treatment with Sim 1.0 μmol/L and Nif 1.0 μmol/L significantly increased the mRNA and protein expression of eNOS and NO release compared with Sim or Nif alone (P<0.05). The combination significantly lowered the intracellular ROS level (P<0.05), which was correlated with the increase in eNOS and NO, but there was no visible change in intracellular calcium (P>0.05). Compared with individual drug treatment, Akt phosphorylation and the ratio of p-eNOS/eNOS were up-regulated in the combination group, and this effect was inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Conclusion: The Sim-Nif combination effectively protects HUVEC against H2O2 injury by inhibiting intracellular ROS generation, increasing the ratio of p-eNOS/eNOS and up-regulating Akt phosphorylation. PMID:20562903

  3. 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.

  4. Estrogen increases the severity of anaphylaxis in female mice through enhanced endothelial nitric oxide synthase expression and nitric oxide production.

    PubMed

    Hox, Valerie; Desai, Avanti; Bandara, Geethani; Gilfillan, Alasdair M; Metcalfe, Dean D; Olivera, Ana

    2015-03-01

    Clinical observations suggest that anaphylaxis is more common in adult women compared with adult men, although the mechanistic basis for this sex bias is not well understood. We sought to document sex-dependent differences in a mouse model of anaphylaxis and explore the role of female sex hormones and the mechanisms responsible. Passive systemic anaphylaxis was induced in female and male mice by using histamine, as well as IgE or IgG receptor aggregation. Anaphylaxis was assessed by monitoring body temperature, release of mast cell mediators and/or hematocrit, and lung weight as a measure of vascular permeability. A combination of ovariectomy, estrogen receptor antagonism, and estrogen administration techniques were used to establish estrogen involvement. Anaphylactic responses were more pronounced in female than male mice. The enhanced severity of anaphylaxis in female mice was eliminated after pretreatment with an estrogen receptor antagonist or ovariectomy but restored after administration of estradiol in ovariectomized mice, demonstrating that the sex-specific differences are due to the female steroid estradiol. Estrogen did not affect mast cell responsiveness or anaphylaxis onset. Instead, it increased tissue expression of endothelial nitric oxide synthase (eNOS). Blockage of NOS activity with the inhibitor L-NG-nitroarginine methyl ester or genetic eNOS deficiency abolished the sex-related differences. Our study defines a contribution of estrogen through its regulation of eNOS expression and nitric oxide production to vascular hyperpermeability and intensified anaphylactic responses in female mice, providing additional mechanistic insights into risk factors and possible implications for clinical management in the further exploration of human anaphylaxis. Published by Elsevier Inc.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

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

    PubMed Central

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

    1996-01-01

    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. Images Fig. 2 Fig. 4 PMID:8876251

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. Osteopontin protects against hyperoxia-induced lung injury by inhibiting nitric oxide synthases.

    PubMed

    Zhang, Xiang-Feng; Liu, Shuang; Zhou, Yu-Jie; Zhu, Guang-Fa; Foda, Hussein D

    2010-04-05

    Exposure of adult mice to more than 95% O(2) produces a lethal injury by 72 hours. Nitric oxide synthase (NOS) is thought to contribute to the pathophysiology of murine hyperoxia-induced acute lung injury (ALI). Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase (iNOS), which generates large amounts of nitric oxide production. However, the relationship between nitric oxide and endogenous OPN in lung tissue during hyperoxia-induced ALI has not yet been elucidated, thus we examined the role that OPN plays in the hyperoxia-induced lung injury and its relationships with NOS. One hundred and forty-four osteopontin knock-out (KO) mice and their matched wild type background control (WT) were exposed in sealed cages > 95% oxygen or room air for 24- 72 hours, and the severity of lung injury was assessed; expression of OPN, endothelial nitric oxide synthase (eNOS) and iNOS mRNA in lung tissues at 24, 48 and 72 hours of hyperoxia were studied by reverse transcription-polymerase chain reaction (RT-PCR); immunohistochemistry (IHC) was performed for the detection of iNOS, eNOS, and OPN protein in lung tissues. OPN KO mice developed more severe acute lung injury at 72 hours of hyperoxia. The wet/dry weight ratio increased to 6.85 +/- 0.66 in the KO mice at 72 hours of hyperoxia as compared to 5.31 +/- 0.92 in the WT group (P < 0.05). iNOS mRNA (48 hours: 1.04 +/- 0.08 vs. 0.63 +/- 0.09, P < 0.01; 72 hours: 0.89 +/- 0.08 vs. 0.72 +/- 0.09, P < 0.05) and eNOS mRNA (48 hours: 0.62 +/- 0.08 vs. 0.43 +/- 0.09, P < 0.05; 72 hours: 0.67 +/- 0.08 vs. 0.45 +/- 0.09, P < 0.05) expression was more significantly increased in OPN KO mice than their matched WT mice when exposed to hyperoxia. IHC study showed higher expression of iNOS (20.54 +/- 3.18 vs. 12.52 +/- 2.46, P < 0.05) and eNOS (19.83 +/- 5.64 vs. 9.45 +/- 3.82, P < 0.05) in lung tissues of OPN KO mice at 72 hours of hyperoxia. OPN can protect against

  2. Voltage-Dependent Anion Channel-2 Interaction with Nitric Oxide Synthase Enhances Pulmonary Artery Endothelial Cell Nitric Oxide Production

    PubMed Central

    Alvira, Cristina M.; Umesh, Anita; Husted, Cristiana; Ying, Lihua; Hou, Yanli; Lyu, Shu-Chen; Nowak, Jeffrey

    2012-01-01

    Increased pulmonary artery endothelial cell (PAEC) endothelium-dependent nitric oxide synthase (eNOS) activity mediates perinatal pulmonary vasodilation. Compromised eNOS activity is central to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Voltage-derived anion channel (VDAC)-1 was recently demonstrated to bind eNOS in the systemic circulation. We hypothesized that VDAC isoforms modulate eNOS activity in the pulmonary circulation, and that decreased VDAC expression contributes to PPHN. In PAECs derived from an ovine model of PPHN: (1) there is eNOS activity, but not expression; and (2) VDAC1 and -2 proteins are decreased. Immunocytochemistry, coimmunoprecipitation, and in situ proximity ligation assays in human PAECs (hPAECs) demonstrate binding between eNOS and both VDAC1 and -2, which increased upon stimulation with NO agonists. The ability of agonists to increase the eNOS/VDAC interaction was significantly blunted in hypertensive, compared with normotensive, ovine PAECs. Depletion of VDAC2, but not VDAC1, blocked the agonist-induced increase in eNOS activity in hPAECs. Overexpression of VDAC2 in hypertensive PAECs increased eNOS activity. Binding of VDAC2 enhances eNOS activity in the pulmonary circulation, and diminished VDAC2 constrains eNOS in PAECs derived from fetal lambs with chronic intrauterine pulmonary hypertension. We speculate that decreases in VDAC2 may contribute to the limited eNOS activity that characterizes pulmonary hypertension. PMID:22842492

  3. Voltage-dependent anion channel-2 interaction with nitric oxide synthase enhances pulmonary artery endothelial cell nitric oxide production.

    PubMed

    Alvira, Cristina M; Umesh, Anita; Husted, Cristiana; Ying, Lihua; Hou, Yanli; Lyu, Shu-Chen; Nowak, Jeffrey; Cornfield, David N

    2012-11-01

    Increased pulmonary artery endothelial cell (PAEC) endothelium-dependent nitric oxide synthase (eNOS) activity mediates perinatal pulmonary vasodilation. Compromised eNOS activity is central to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Voltage-derived anion channel (VDAC)-1 was recently demonstrated to bind eNOS in the systemic circulation. We hypothesized that VDAC isoforms modulate eNOS activity in the pulmonary circulation, and that decreased VDAC expression contributes to PPHN. In PAECs derived from an ovine model of PPHN: (1) there is eNOS activity, but not expression; and (2) VDAC1 and -2 proteins are decreased. Immunocytochemistry, coimmunoprecipitation, and in situ proximity ligation assays in human PAECs (hPAECs) demonstrate binding between eNOS and both VDAC1 and -2, which increased upon stimulation with NO agonists. The ability of agonists to increase the eNOS/VDAC interaction was significantly blunted in hypertensive, compared with normotensive, ovine PAECs. Depletion of VDAC2, but not VDAC1, blocked the agonist-induced increase in eNOS activity in hPAECs. Overexpression of VDAC2 in hypertensive PAECs increased eNOS activity. Binding of VDAC2 enhances eNOS activity in the pulmonary circulation, and diminished VDAC2 constrains eNOS in PAECs derived from fetal lambs with chronic intrauterine pulmonary hypertension. We speculate that decreases in VDAC2 may contribute to the limited eNOS activity that characterizes pulmonary hypertension.

  4. 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.

  5. 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.

  6. 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.

  7. 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). These in vitro results may be translated into potential future clinical applications where Aronia extracts may be used for prevention and coadjuvant treatment of cardiovascular diseases via increases in endothelial NO synthesis and related improvements in vascular functions. Given the dose-response effect of Aronia extract in vitro and metabolism of polyphenols that occurs in humans, dose-response studies would be necessary to define the optimal daily amount to be consumed.

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

    PubMed Central

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

    2015-01-01

    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). PRACTICAL APPLICATIONS These in vitro results may be translated into potential future clinical applications where Aronia extracts may be used for prevention and coadjuvant treatment of cardiovascular diseases via increases in endothelial NO synthesis and related improvements in vascular functions. Given the dose-response effect of Aronia extract in vitro and metabolism of polyphenols that occurs in humans, dose-response studies would be necessary to define the optimal daily amount to be consumed. PMID:27616799

  9. Cytosolic calcium microdomains by arachidonic acid and nitric oxide in endothelial cells.

    PubMed

    Tomatis, Cristiana; Fiorio Pla, Alessandra; Munaron, Luca

    2007-03-01

    Intracellular calcium signals activated by growth factors in endothelial cells during angiogenesis regulate cytosolic and nuclear events involved in survival, proliferation and motility. Among the intracellular messengers released after proangiogenic stimulation (bFGF, VEGF), arachidonic acid (AA), nitric oxide (NO) and their metabolites play a key role and their effects are strictly related to calcium homeostasis. Recently, we showed that AA and NO are able to stimulate the opening of store-independent calcium-permeable channels in the plasmamembrane of bovine aortic endothelial cells (BAECs). Here, we studied the intracellular spatiotemporal dynamics of AA- and NO-induced calcium increases following store-independent calcium entry from extracellular medium. Using confocal calcium imaging, we show that calcium entry is preferentially restricted to peripheral cytosolic microdomains and does not necessarily invade the nuclear region. These results support the existence of local mitogen-activated calcium signals. Several factors could account for this spatial restriction, including the geometry of the cells and the clusterization of calcium channels and other signalling molecules. Intracellular calcium fingerprints could contribute to the specificity of endothelial cell responses to angiogenic factors.

  10. 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

  11. Effects of Chinese yellow wine on nitric oxide synthase and intercellular adhesion molecule-1 expressions in rat vascular endothelial cells.

    PubMed

    Zhao, Fei; Ji, Zheng; Chi, Jufang; Tang, Weiliang; Zhai, Xiaoya; Meng, Liping; Guo, Hangyuan

    2016-02-01

    The objective of this study was to determine similarities in the effect of yellow wine as compared to statin and the possibility that yellow wine inhibits tumour necrosis factor-α (TNF-α)-induced nitric oxide (NO) synthesis, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1) in cultured rat vascular endothelial cells (VECs). We isolated VECs, and cultivated and purified Sprague Dawley (SD) rat thoracic aortas in vitro. We selected the optimal wine concentration using clonogenic and MTT assays to measure cell survival. Next, we divided the cells into 9 groups: (1) control, (2) TNF-α, (3) TNF-α + rosuvastatin (10 μmol/L), (4) TNF-α + ethanol 0.5%, (5) TNF-α + yellow wine 0.5%, (6) TNF-α + ethanol 1.0%, (7) TNF-α + yellow wine 1.0%, (8) TNF-α + ethanol 1.5%, and (9) TNF-α + yellow wine 1.5% and they were given the corresponding treatment for 24 h. We determined NO production with nitrate reductase. We then measured eNOS activity, and detected eNOS, iNOS, and ICAM-1 protein levels by Western blotting. Compared with the TNF-α group, NO production, eNOS activity, and eNOS protein expression in the rosuvastatin, and yellow wine 1.0%, and 1.5% groups were significantly increased. Protein expression of iNOS and ICAM-1 in the rosuvastatin, yellow wine 1.0%, and 1.5% groups were significantly decreased. Compared with the rosuvastatin group, eNOS, iNOS, and ICAM-1 protein expression in the yellow wine (0.5% -1.5%) groups were significantly different. Treatment with yellow wine increased NO production, eNOS activity, and eNOS protein expression, which decreases iNOS and ICAM-1 protein expression. We conclude that yellow wine may have similar beneficial effects as rosuvastatin on the cardiovascular system. These effects may be attributed to their anti-atherosclerotic actions.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. Sepiapterin improves angiogenesis of pulmonary artery endothelial cells with in utero pulmonary hypertension by recoupling endothelial nitric oxide synthase

    PubMed Central

    Du, Jianhai; Xu, Hao; Bakhutashvili, Ivane; Eis, Annie; Shi, Yang; Pritchard, Kirkwood A.; Konduri, Girija G.

    2011-01-01

    Persistent pulmonary hypertension of the newborn (PPHN) is associated with decreased blood vessel density that contributes to increased pulmonary vascular resistance. Previous studies showed that uncoupled endothelial nitric oxide (NO) synthase (eNOS) activity and increased NADPH oxidase activity resulted in marked decreases in NO bioavailability and impaired angiogenesis in PPHN. In the present study, we hypothesize that loss of tetrahydrobiopterin (BH4), a critical cofactor for eNOS, induces uncoupled eNOS activity and impairs angiogenesis in PPHN. Pulmonary artery endothelial cells (PAEC) isolated from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were used to investigate the cellular mechanisms impairing angiogenesis in PPHN. Cellular mechanisms were examined with respect to BH4 levels, GTP-cyclohydrolase-1 (GCH-1) expression, eNOS dimer formation, and eNOS-heat shock protein 90 (hsp90) interactions under basal conditions and after sepiapterin (Sep) supplementation. Cellular levels of BH4, GCH-1 expression, and eNOS dimer formation were decreased in HTFL-PAEC compared with NFL-PAEC. Sep supplementation decreased apoptosis and increased in vitro angiogenesis in HTFL-PAEC and ex vivo pulmonary artery sprouting angiogenesis. Sep also increased cellular BH4 content, NO production, eNOS dimer formation, and eNOS-hsp90 association and decreased the superoxide formation in HTFL-PAEC. These data demonstrate that Sep improves NO production and angiogenic potential of HTFL-PAEC by recoupling eNOS activity. Increasing BH4 levels via Sep supplementation may be an important therapy for improving eNOS function and restoring angiogenesis in PPHN. PMID:21622842

  17. 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.

  18. Overproduction of nitric oxide by endothelial cells and macrophages contributes to mitochondrial oxidative stress in adrenocortical cells and adrenal insufficiency during endotoxemia.

    PubMed

    Wang, Chang-Nan; Duan, Guo-Li; Liu, Yu-Jian; Yu, Qing; Tang, Xiao-Lu; Zhao, Wei; Li, Xiao-Han; Zhu, Xiao-Yan; Ni, Xin

    2015-06-01

    We have recently demonstrated that lipopolysaccharide (LPS) causes mitochondrial oxidative stress and dysfunction in adrenal glands, thereby leading to adrenocortical insufficiency. Since nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) leads to mitochondrial damage in various tissues, the present study aims to investigate whether NO contributes to mitochondrial oxidative stress in adrenal cortex and adrenocortical insufficiency during endotoxemia. Systemic administration of LPS increased iNOS expression and NO production in adrenal glands of mice. The specific iNOS inhibitor 1400 W significantly attenuated the LPS-induced mitochondrial superoxide production and dysfunction in adrenal glands, and reversed the LPS-induced adrenocortical hyporesponsiveness to adrenocorticotropic hormone (ACTH). In contrast, administration of the NO donor sodium nitroprusside (SNP) led to mitochondrial oxidative stress and dysfunction in adrenal glands, which resulted in a blunted corticosterone response to ACTH. Using double immunofluorescence staining for iNOS with the vascular endothelial cell marker CD31 or the macrophage marker CD68, we found that increased iNOS expression was found in vascular endothelial cells and macrophages, but not adrenocortical cells in the adrenal gland during endotoxemia. Administration of the hydrogen sulfide (H2S) donor GYY4137 inhibited NO production and reversed LPS-induced adrenocortical hyporesponsiveness. Our data suggest that overproduction of NO, which is mainly generated by endothelial cells and macrophages during endotoxemia, contributes to mitochondrial oxidative stress in adrenocortical cells and subsequently leads to adrenal insufficiency. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Overexpression of steroidogenic acute regulatory protein in rat aortic endothelial cells attenuates palmitic acid-induced inflammation and reduction in nitric oxide bioavailability

    PubMed Central

    2012-01-01

    Background Endothelial dysfunction is a well documented evidence for the onset of atherosclerosis and other cardiovascular diseases. Lipids disorder is among the main risk factors for endothelial dysfunction in these diseases. Steroidogenic acute regulatory protein (StAR), one of the cholesterol transporters, plays an important role in the maintenance of intracellular lipid homeostasis. However, the effect of StAR on endothelial dysfunction is not well understood. Palmitic acid (PA) has been shown to decrease eNOS activity and induce inflammation, both are the causes of endothelial dysfunction, in an endothelial cell culture model. Methods StAR gene was introduced into primary rat aortic endothelial cells by adenovirus infection. Real-time PCR and Western blotting were performed to determine the relative genes and proteins expression level to elucidate the underlying mechanism. The free fatty acid and cholesterol quantification kits were used to detect total cellular free fatty acid and cholesterol. The levels of inflammatory factors and nitric oxide were determined by ELISA and classic Griess reagent methods respectively. Results We successfully overexpressed StAR in primary rat aortic endothelial cells. Following StAR overexpression, mRNA levels of IL-1β, TNFα, IL6 and VCAM-1 and protein levels of IL-1β, , TNFα and IL-6 in culture supernatant were significantly decreased, which duing to blocke NFκB nuclear translocation and activation. Moreover, StAR overexpression attenuated the PA-induced reduction of nitric oxide bioavailability by protecting the bioactivity of pAkt/peNOS/NO pathway. Furthermore, the key genes involved in lipid metabolism were greatly reduced following StAR overexpression. In order to investigate the underlying mechanism, cerulenin and lovastatin, the inhibitor of fatty acid and cholesterol synthase, were added prior to PA treatment. The results showed that both cerulenin and lovastatin had a similar effect as StAR overexpression. On the

  20. 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

  1. 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

  2. Inhibition of calcifying nodule formation in cultured porcine aortic valve cells by nitric oxide donors.

    PubMed

    Kennedy, Jennifer A; Hua, Xiang; Mishra, Kumaril; Murphy, Geraldine A; Rosenkranz, Anke C; Horowitz, John D

    2009-01-05

    Calcific aortic stenosis displays some similarities to atherosclerosis including evidence of endothelial dysfunction. Whether nitric oxide (NO), which is produced by valvular endothelium, has direct protective effects extending to calcification processes in aortic valve cells has not previously been examined. In vitro calcifying nodules in porcine aortic valve interstitial cell cultures, formed in response to transforming growth factor-beta1 (TGF-beta1) 5 ng/ml, were inhibited by NO donors DETA-NONOate 5-100 microM, and sodium nitroprusside (SNP) 3 microM. Raising intracellular cGMP concentrations, via 8-bromo cGMP 1 mM or via brain natiuretic peptide and C-type natiuretic peptide 0.1 microM, inhibited TGF-beta1-induced nodule formation, potentially implicating the cGMP pathway in the NO effect. Stimulation of interstitial cells with substance P or calcium ionophone (A23187) caused NO release and increased intracellular cGMP respectively. However in the presence of TGF-beta1 basal levels of NO production via nitric oxide synthase (NOS) were insufficient to affect nodule formation. Increased dihydroethidium (DHE) fluorescence in response to TGF-beta1, which was inhibited by DETA-NONOate and TEMPOL, suggested a role for intracellular superoxide in TGF-beta1 signalling. Moreover, nodule formation was suppressed by superoxide scavengers TEMPOL, hydralazine and polyethylene glycol-superoxide dismutase (PEG-SOD), but not SOD. In conclusion, NO donors, or agents raising intracellular cGMP levels, may protect aortic valve interstitial cells from early events leading to calcification.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Association between 894G>T endothelial nitric oxide synthase gene polymorphisms and metabolic syndrome.

    PubMed

    Piccoli, Jacqueline C Escobar; Gottlieb, Maria Gabriela Valle; Castro, Luciano; Bodanese, Luiz Carlos; Manenti, Euler Roberto Fernandes; Bogo, Mauricio Reis; Peres, Alessandra; Rocha, Maria Izabel U M da; Cruz, Ivana Beatrice Mânica da

    2008-11-01

    Metabolic syndrome (MS) is a cluster of cardiovascular risk factors such as hypertension, dyslipidemia, obesity and type II diabetes. Here, we performed a case-control study analyzing the association between 894G>T endothelial nitric oxide synthase gene polymorphism (NOS3) and MS in 616 subjects. Genotype frequencies were TT= 9.3%, GG= 37.2 and TG= 53.6% and the allelic frequencies were T=0.36 and G= 0.64. We observed a higher TT genotype frequency in the male MS group than control subjects (p=0.02), independent of other variables. We found an association between hypertension and TT genotype in females. Our data suggests that 894G>T plays a significant role in the mechanistic interaction between metabolic risk such as hypertension and MS, although sex-related differences may exist.

  8. Lack of association between endothelial nitric oxide synthase (NOS3) gene polymorphisms and suicide attempts

    PubMed Central

    Sáiz, Pilar A; García-Portilla, Maria P; Paredes, Begoña; Arango, Celso; Morales, Blanca; Alvarez, Victoria; Coto E, Eliecer; Bascarán, Teresa; Bousoño, Manuel; Bobes, Julio

    2007-01-01

    Objective The aim of this study is to investigate the association between two polymorphisms of endothelial nitric oxide synthase (NOS3) and suicide attempts. Methods We genotyped 186 suicide attempters and 420 unrelated healthy controls. The following polymorphisms were analysed: T-786C and 27-bp repeat in intron 4. Results No significant differences were found in genotype or in allelic distribution of the aforesaid polymorphisms. There were also no differences in the genotype distribution or allelic frequencies when separately assessing males and females or impulsive and non-impulsive attempters and normal controls. Estimated haplotype frequencies were similar in both groups. Conclusion Our data do not support the hypothesis that genetically determined changes in the NOS3 gene confer increased susceptibility for suicidal behavior. PMID:17605790

  9. Overexpression of Endothelial Nitric Oxide Synthase Prevents Diet-Induced Obesity and Regulates Adipocyte Phenotype

    PubMed Central

    Sansbury, Brian E.; Cummins, Timothy D.; Tang, Yunan; Hellmann, Jason; Holden, Candice R.; Harbeson, Matthew A.; Chen, Yang; Patel, Rakesh P.; Spite, Matthew; Bhatnagar, Aruni; Hill, Bradford G.

    2013-01-01

    Rationale Endothelial dysfunction is a characteristic feature of diabetes and obesity in animal models and humans. Deficits in nitric oxide production by endothelial nitric oxide synthase (eNOS) are associated with insulin resistance, which is exacerbated by high fat diet. Nevertheless, the metabolic effects of increasing eNOS levels have not been studied. Objective The current study was designed to test whether overexpression of eNOS would prevent diet-induced obesity and insulin resistance. Methods and Results In db/db mice and in high fat-fed wild-type (WT) C57BL/6J mice, the abundance of eNOS protein in adipose tissue was decreased without significant changes in eNOS levels in skeletal muscle or aorta. Mice overexpressing eNOS (eNOS-TG mice) were resistant to diet-induced obesity and hyperinsulinemia, although systemic glucose intolerance remained largely unaffected. In comparison with WT mice, high fat-fed eNOS-TG mice displayed a higher metabolic rate and attenuated hypertrophy of white adipocytes. Overexpression of eNOS did not affect food consumption or diet-induced changes in plasma cholesterol or leptin levels, yet plasma triglycerides and fatty acids were decreased. Metabolomic analysis of adipose tissue indicated that eNOS overexpression primarily affected amino acid and lipid metabolism; subpathway analysis suggested changes in fatty acid oxidation. In agreement with these findings, adipose tissue from eNOS-TG mice showed higher levels of PPAR-α and PPAR–γ gene expression, elevated abundance of mitochondrial proteins, and a higher rate of oxygen consumption. Conclusions These findings demonstrate that increased eNOS activity prevents the obesogenic effects of high fat diet without affecting systemic insulin resistance, in part, by stimulating metabolic activity in adipose tissue. PMID:22896587

  10. Endothelial Nitric Oxide Synthase Polymorphism Is Associated with Delayed Cerebral Ischemia Following Aneurysmal Subarachnoid Hemorrhage.

    PubMed

    Hendrix, Philipp; Foreman, Paul M; Harrigan, Mark R; Fisher, Winfield S; Vyas, Nilesh A; Lipsky, Robert H; Lin, Minkuan; Walters, Beverly C; Tubbs, R Shane; Shoja, Mohammadali M; Pittet, Jean-Francois; Mathru, Mali; Griessenauer, Christoph J

    2017-05-01

    Nitric oxide is critical in the regulation of cerebral blood flow and smooth muscle proliferation. It is synthesized by 3 nitric oxide synthase (NOS) isoforms: neuronal, inducible, and endothelial NOS (eNOS). Aneurysmal subarachnoid hemorrhage (aSAH) causes endothelial dysfunction that, in turn, contributes to pathophysiologic processes surrounding aSAH. Previous studies reported an association of an eNOS single nucleotide polymorphism (SNP) with the clinical sequelae of aSAH. Here, we further elucidate the impact of this eNOS SNP on the clinical course after aSAH. The Cerebral Aneurysm Renin Angiotensin System study prospectively enrolled aSAH patients at 2 academic institutions in the United States from 2012-2015. Blood samples from all patients enrolled in the study were used for genetic evaluation using 5'exonuclease (Taqman) genotyping assays. Associations between the eNOS SNP rs2070744 (786 T->C) and clinical course after aSAH were analyzed. Samples from 149 aSAH patients were available for analysis. The C allele of the eNOS SNP independently predicted an increased risk for delayed cerebral ischemia (OR = 2.936, 95% CI 1.048-8.226, P = 0.040). The eNOS SNP rs2070744 was not associated with functional outcome or size of aneurysm at the time of rupture. The present study is the first to demonstrate that the C allele of the eNOS SNP 786 T->C rs2070744 is independently associated with an increased risk for delayed cerebral ischemia following aSAH. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. Gamma-Glutamylcysteine Inhibits Oxidative Stress in Human Endothelial Cells

    DTIC Science & Technology

    2012-01-01

    γ-Glutamylcysteine inhibits oxidative stress in human endothelial cells Yukiko K. Nakamura a, Michael A. Dubick b, Stanley T. Omaye a,⁎ a Department...n f o Article history: Received 12 July 2011 Accepted 16 October 2011 Keywords: γ-Glutamylcysteine Glutathione Glutathione synthetase Oxidative stress...include reducing risks of oxidative stress-related injuries and diseases. The ob- jective of this studywas to investigate the efficacy of GGC on GSH

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

    PubMed

    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 & brain GSH levels along with increase in serum & 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. © 2013.

  19. 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

  20. Effects of rotational culture on morphology, nitric oxide production and cell cycle of endothelial cells.

    PubMed

    Tang, Chaojun; Wu, Xue; Ye, Linqi; Xie, Xiang; Wang, Guixue

    2012-12-01

    Devices for the rotational culture of cells and the study of biological reactions have been widely applied in tissue engineering. However, there are few reports exploring the effects of rotational culture on cell morphology, nitric oxide (NO) production, and cell cycle of the endothelial cells from human umbilical vein on the stent surface. This study focuses on these parameters after the cells are seeded on the stents. Results showed that covering of stents by endothelial cells was improved by rotational culture. NO production decreased within 24 h in both rotational and static culture groups. In addition, rotational culture significantly increased NO production by 37.9% at 36 h and 28.9% at 48 h compared with static culture. Flow cytometry showed that the cell cycle was not obviously influenced by rotational culture. Results indicate that rotational culture may be helpful for preparation of cell-seeded vascular grafts and intravascular stents, which are expected to be the most frequently implanted materials in the future.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. Nitric oxide dynamics and endothelial dysfunction in type II model of genetic diabetes.

    PubMed

    Bitar, Milad S; Wahid, Sabah; Mustafa, Seham; Al-Saleh, Eyad; Dhaunsi, Gursev S; Al-Mulla, Fahd

    2005-03-21

    Although diabetes is a major risk factor for vascular diseases, e.g., hypertension and atherosclerosis, mechanisms that underlie the "risky" aspects of diabetes remain obscure. The current study is intended to examine the notion that diabetic endothelial dysfunction stems from a heightened state of oxidative stress induced by an imbalance between vascular production and scavenging of reactive oxygen/nitrogen species. Goto-Kakizaki (GK) rats were used as a genetic animal model for non-obese type II diabetes. Nitric oxide (NO) bioavailability and O2- generation in aortic tissues of GK rats were assessed using the Griess reaction and a lucigenin-chemiluminescence-based technique, respectively. Organ chamber-based isometric tension studies revealed that aortas from GK rats had impaired relaxation responses to acetylcholine whereas a rightward shift in the dose-response curve was noticed in the endothelium-independent vasorelaxation exerted by the NO donor sodium nitroprusside. An enhancement in superoxide (O2-) production and a diminuation in NO bioavailability were evident in aortic tissues of GK diabetic rats. Immunoblotting and high-performance liquid chromatography (HPLC)-based techniques revealed, respectively, that the above inverse relationship between O2- and NO was associated with a marked increase in the protein expression of nitric oxide synthase (eNOS) and a decrease in the level of its cofactor tetrahydrobiopterin (BH4) in diabetic aortas. Endothelial denudation by rubbing or the addition of pharmacological inhibitors of eNOS (e.g. N(omega)-nitro-L-arginine methyl ester (L-NAME)), and NAD(P)H oxidase (e.g. diphenyleneiodonium, apocynin) strikingly reduced the diabetes-induced enhancement in vascular O2- production. Aortic contents of key markers of oxidative stress (isoprostane F2alpha III, protein-bound carbonyls, nitrosylated protein) in connection with the protein expression of superoxide generating enzyme NAD(P)H oxidase (e.g. p47phox, pg91phox), a

  6. 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

  7. Inhibition of inflammation may enhance nitric oxide availability in patients undergoing bariatric surgery for weight loss.

    PubMed

    Blum, A; Ginat-Maimon, L; Yehuda, H; Geron, N; Ben Ami, M; Tamir, S

    2015-10-01

    Weight loss surgery is the most effective treatment for morbid obesity. The mechanisms underlying the beneficial cardiovascular effects are poorly understood, although inhibition of inflammatory markers has been demonstrated. We hypothesized that anti-inflammatory and antioxidative stress reactions are responsible for the beneficial effects of bariatric surgery that have been shown in clinical trials. The inflammatory system was studied by measuring mRNA levels of E-selectin, tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and in a cell line (HUVEC-CS) of human umbilical vein endothelial cells that were incubated for 4 h with pools of serum, collected before and 3 months after surgery, from 20 women who underwent bariatric surgery for weight loss. The oxidative stress pathway was examined by mRNA expression of NADPH oxidase (P22(phox) ), paraoxonase (PON2), superoxide dismutase 2 (SOD2), glutathione peroxidase (GPx) and catalase following incubation of the cells for 4 h with serum pools. The nitric oxide (NO) pathway was studied by measuring mRNA levels of inducible NOS and endothelial NOS and by determining nitrite and nitrate levels. To study the functional behaviour of endothelial cells under stress, primary human umbilical vein endothelial cells (PECs) were incubated with the serum pools for 48 h, with lipopolysaccharide (LPS) for the last 4 h. The inflammatory system: incubation of HUVEC-CS cells with serum from women who underwent bariatric surgery led to a significant decrease in mRNA expression of E-selectin and IL-6 postsurgery. Stimulation of PECs with LPS in the presence of serum from women who underwent bariatric surgery caused a more significant difference in E-selectin and TNF-α mRNA expression before and after surgery. The antioxidant system: incubation of HUVEC-CS cells with serum from women who underwent bariatric surgery did not lead to any difference in mRNA expression of P22(phox) , PON2, SOD2, GPx or catalase. Stimulation of

  8. 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.

  9. 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

  10. 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

  11. Endothelial dysfunction in rats with ligature-induced periodontitis: Participation of nitric oxide and cycloxygenase-2-derived products.

    PubMed

    Campi, Paula; Herrera, Bruno Schneider; de Jesus, Flavia Neto; Napolitano, Mauro; Teixeira, Simone Aparecida; Maia-Dantas, Aline; Spolidorio, Luis Carlos; Akamine, Eliana Hiromi; Mayer, Marcia Pinto Alves; de Carvalho, Maria Helena Catelli; Costa, Soraia Katia Pereira; Muscara, Marcelo Nicolas

    2016-03-01

    Considering the evident relationship between periodontitis and cardiovascular diseases in humans, we aimed to study the in vitro vascular reactivity of aorta rings prepared from rats with ligature-induced periodontitis. Seven days after the induction of unilateral periodontitis, the animals were euthanised; rings were prepared from the descending abdominal aortas and mounted in tissue baths for the in vitro measurement of the isometric force responses to norepinephrine (NE) and acetylcholine (ACh), as well as in the presence of inhibitors of nitric oxide synthase (NOS) and cycloxygenase (COX) isoenzymes. Aortic COX and NOS gene expressions were analysed by RT-PCR, as well as protein COX-2 expression by Western blot. Periodontitis resulted in significant alveolar bone loss and did not affect arterial pressure. However, both NE-induced contraction and ACh-induced relaxation were significantly decreased and related to the presence of endothelium. Diminished eNOS and augmented COX-2 and iNOS expressions were found in the aortas from rats with periodontitis, and the pharmacological inhibition of COX-2 or iNOS improved the observed vasomotor deficiencies. We can thus conclude that periodontitis induces significant endothelial dysfunction in rat aorta which is characterized by decreased eNOS expression and mediated by upregulated iNOS and COX-2 products. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. 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

  13. High concentration of glucose inhibits glomerular endothelial eNOS through a PKC mechanism.

    PubMed

    Chu, Shaoyou; Bohlen, H Glenn

    2004-09-01

    Kidney glomeruli are important targets of diabetic nephropathy. We hypothesized a high concentration of glucose could suppress glomerular endothelial nitric oxide synthase (eNOS) by a protein kinase C (PKC) mechanism, as has been found in other tissues. Mouse kidney slices (150-200 microm) were bathed in Hanks' solution with 100 microM L-arginine and exposed to either 5 or 20-30 mM D-glucose. Immunofluorescence identified only eNOS in normal mouse glomeruli. Measurements of glomerular NO concentration with NO-sensitive fluorescent dye (4,5-diaminofluorescein diacetate) using confocal microscopy and NO-sensitive microelectrodes verified that resting glomeruli had active production of NO that was inhibited by N(G)-nitro-L-arginine methyl ester. High-concentration (20-30 mM) D-glucose inhibited 60-70% of the NO production within 15-30 min; L-glucose at the same concentration did not have any effect. Inhibition of PKC-beta with 100 nM ruboxistaurin prevented eNOS suppression in high-glucose media. Activation of PKC with 100 nM phorbol ester also suppressed the glomerular NO concentration. We concluded that eNOS in the renal glomerular capillary endothelial cells is suppressed by activity of PKC at high-glucose concentrations comparable to those in diabetic animals and humans. The consequence is a rapid decline in the generation of NO in the glomerular endothelial cells in the presence of a high concentration of glucose.

  14. Up-regulation of the RhoA/Rho-kinase signaling pathway in corpus cavernosum from endothelial nitric-oxide synthase (NOS), but not neuronal NOS, null mice.

    PubMed

    Priviero, Fernanda B M; Jin, Li-Ming; Ying, Zhekang; Teixeira, Cleber E; Webb, R Clinton

    2010-04-01

    We tested the hypothesis that the basal release of nitric oxide (NO) from endothelial cells modulates contractile activity in the corpus cavernosum (CC) via inhibition of the RhoA/Rho-kinase signaling pathway. Cavernosal strips from wild-type (WT), endothelial nitric-oxide synthase knockout [eNOS(-/-)], and neuronal nitric-oxide synthase knockout [nNOS(-/-)] mice were mounted in myographs, and isometric force was recorded. mRNA and protein expression of key molecules in the RhoA/Rho-kinase pathway were analyzed by real-time polymerase chain reaction and Western blot, respectively. The cGMP levels were determined. The Rho-kinase inhibitors (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl] homopiperazine (H-1152) reduced cavernosal contractions evoked by phenylephrine or electrical field stimulation (EFS) in a concentration-dependent manner, although this inhibition was less effective in tissues from eNOS(-/-) mice. Y-27632 enhanced relaxations induced by sodium nitroprusside, EFS, and NO (administered as acidified NaNO2) without affecting the cGMP content of the cavernosal strips. This enhancement was less prominent in CC from eNOS(-/-). The protein expression of RhoA, Rho-guanine dissociation inhibitor, and Rho-kinase beta did not differ among the strains. However, in eNOS(-/-) CC, the protein expression of Rho-kinase alpha and both mRNA and protein expression of p115-Rho-associated guanine exchange factor (RhoGEF), PDZ-RhoGEF, and leukemia-associated RhoGEF were up-regulated. Phosphorylation of MYPT1 at Thr696 was higher in tissues from eNOS(-/-) mice. A high concentration of Y-27632 significantly enhanced NO release in CC stimulated by EFS. These results suggest a basal release of NO from endothelial cells, which inhibits contractions mediated by the RhoA/Rho-kinase pathway and modulates the expression of proteins related to this pathway in mouse CC. It indicates that

  15. Up-Regulation of the RhoA/Rho-Kinase Signaling Pathway in Corpus Cavernosum from Endothelial Nitric-Oxide Synthase (NOS), but Not Neuronal NOS, Null Mice

    PubMed Central

    Jin, Li-Ming; Ying, Zhekang; Teixeira, Cleber E.; Webb, R. Clinton

    2010-01-01

    We tested the hypothesis that the basal release of nitric oxide (NO) from endothelial cells modulates contractile activity in the corpus cavernosum (CC) via inhibition of the RhoA/Rho-kinase signaling pathway. Cavernosal strips from wild-type (WT), endothelial nitric-oxide synthase knockout [eNOS(−/−)], and neuronal nitric-oxide synthase knockout [nNOS(−/−)] mice were mounted in myographs, and isometric force was recorded. mRNA and protein expression of key molecules in the RhoA/Rho-kinase pathway were analyzed by real-time polymerase chain reaction and Western blot, respectively. The cGMP levels were determined. The Rho-kinase inhibitors (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl] homopiperazine (H-1152) reduced cavernosal contractions evoked by phenylephrine or electrical field stimulation (EFS) in a concentration-dependent manner, although this inhibition was less effective in tissues from eNOS(−/−) mice. Y-27632 enhanced relaxations induced by sodium nitroprusside, EFS, and NO (administered as acidified NaNO2) without affecting the cGMP content of the cavernosal strips. This enhancement was less prominent in CC from eNOS(−/−). The protein expression of RhoA, Rho-guanine dissociation inhibitor, and Rho-kinase β did not differ among the strains. However, in eNOS(−/−) CC, the protein expression of Rho-kinase α and both mRNA and protein expression of p115-Rho-associated guanine exchange factor (RhoGEF), PDZ-RhoGEF, and leukemia-associated RhoGEF were up-regulated. Phosphorylation of MYPT1 at Thr696 was higher in tissues from eNOS(−/−) mice. A high concentration of Y-27632 significantly enhanced NO release in CC stimulated by EFS. These results suggest a basal release of NO from endothelial cells, which inhibits contractions mediated by the RhoA/Rho-kinase pathway and modulates the expression of proteins related to this pathway in mouse CC. It

  16. 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

  17. 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.

  18. 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

  19. Endothelial beta3-adrenoreceptors mediate nitric oxide-dependent vasorelaxation of coronary microvessels in response to the third-generation beta-blocker nebivolol.

    PubMed

    Dessy, Chantal; Saliez, Julie; Ghisdal, Philippe; Daneau, Géraldine; Lobysheva, Irina I; Frérart, Françoise; Belge, Catharina; Jnaoui, Karima; Noirhomme, Philippe; Feron, Olivier; Balligand, Jean-Luc

    2005-08-23

    The therapeutic effects of nonspecific beta-blockers are limited by vasoconstriction, thus justifying the interest in molecules with ancillary vasodilating properties. Nebivolol is a selective beta1-adrenoreceptor antagonist that releases nitric oxide (NO) through incompletely characterized mechanisms. We identified endothelial beta3-adrenoreceptors in human coronary microarteries that mediate endothelium- and NO-dependent relaxation and hypothesized that nebivolol activates these beta3-adrenoreceptors. Nebivolol dose-dependently relaxed rodent coronary resistance microarteries studied by videomicroscopy (10 micromol/L, -86+/-6% of prostaglandin F2alpha contraction); this was sensitive to NO synthase (NOS) inhibition, unaffected by the beta(1-2)-blocker nadolol, and prevented by the beta(1-2-3)-blocker bupranolol (P<0.05; n=3 to 8). Importantly, nebivolol failed to relax microarteries from beta3-adrenoreceptor-deficient mice. Nebivolol (10 micromol/L) also relaxed human coronary microvessels (-71+/-5% of KCl contraction); this was dependent on a functional endothelium and NO synthase but insensitive to beta(1-2)-blockade (all P<0.05). In a mouse aortic ring assay of neoangiogenesis, nebivolol induced neocapillary tube formation in rings from wild-type but not beta3-adrenoreceptor- or endothelial NOS-deficient mice. In cultured endothelial cells, 10 micromol/L nebivolol increased NO release by 200% as measured by electron paramagnetic spin trapping, which was also reversed by NOS inhibition. In parallel, endothelial NOS was dephosphorylated on threonine(495), and fura-2 calcium fluorescence increased by 91.8+/-23.7%; this effect was unaffected by beta(1-2)-blockade but abrogated by beta(1-2-3)-blockade (all P<0.05). Nebivolol dilates human and rodent coronary resistance microarteries through an agonist effect on endothelial beta3-adrenoreceptors to release NO and promote neoangiogenesis. These properties may prove particularly beneficial for the treatment of

  20. 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.

  1. 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

  2. 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.

  3. TRPV1 agonism inhibits endothelial cell inflammation via activation of eNOS/NO pathway.

    PubMed

    Wang, Youping; Cui, Lin; Xu, Hui; Liu, Suxiao; Zhu, Feiyun; Yan, Fengna; Shen, Si; Zhu, Mingjun

    2017-05-01

    Transient receptor potential vanilloid type 1 channel (TRPV1) is found to be expressed in endothelial cells (ECs) and activate endothelial nitric oxide synthase (eNOS). Recent studies implicate TRPV1 in attenuating inflammatory responses. However, the mechanisms underlying the beneficial effects remain unclear. In this study, we investigated whether TRPV1 suppresses inflammatory responses of ECs via eNOS/NO pathway. Human umbilical vein endothelial cells (HUVECs) and renal microvascular endothelial cells (MVECs) isolated from deoxycorticosterone (DOCA)-salt hypertensive mice were cultured in the presence of capsaicin (CAP, a specific TRPV1 agonist) with or without the specific inhibitor of TRPV1, NOS, or Ca(2+)-dependent phosphatidylinositol 3-kinase (PI3K)/Akt pathway, before lipopolysaccharide (LPS) stimulation. NO metabolites, protein expression, and inflammatory molecules were evaluated by Griess assay and immune assay-based multiplex analysis, respectively. Monocyte adhesion was determined by measuring the fluorescently labeled human monocytes attached to LPS-stimulated ECs. In HUVECs, treatment with CAP increased NO production, and CAP-induced NO production was accompanied by increased eNOS(ser1177) phosphorylation. Additionally, CAP attenuated LPS-induced cytokine and chemokine production, adhesion molecule expression, activation of NF-κB, and monocyte adhesion in HUVECs, and these effects were abrogated by the inhibition of TRPV1, NOS, or Ca(2+)-dependent PI3K/Akt pathway. Moreover, these protective actions of TRPV1 were also observed in renal MVECs isolated from DOCA-salt hypertensive mice. Our results indicate that TRPV1 activation suppresses the inflammatory response of ECs via the activation of Ca(2+)/PI3K/Akt/eNOS/NO pathway, the protective effects are also documented in ECs derived from salt-sensitive hypertensive mice. Copyright © 2017. Published by Elsevier B.V.

  4. 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.

  5. The role of endothelial nitric oxide synthase (eNOS) in the pathogenesis of sinonasal polyps.

    PubMed

    Muluk, N Bayar; Arikan, O K; Atasoy, P; Kiliç, R; Yalçinozan, E Tuna

    2014-01-01

    The pathogenesis of sinonasal polyps has not been known completely. We investigated the role of endothelial Nitric Oxide Synthase (eNOS) in the pathogenesis of sinonasal polyps. Study group (Groups 1-3) consisted of nasal polyp samples of patients with sinonasal polyps; and control group consisted of inferior turbinate samples of patients without nasal polyp. In Group 1: 14 specimens from ethmoid sinus; in Group 2: 10 specimens from nasal cavity; in Group 3: 10 specimens from maxillary sinus; and in Group 4 (Control): 9 specimens from inferior turbinate were included. By immunohistochemical staining technique, eNOS Positivity Index in mucosal layers; and in the inflammatory cells were assessed. eNOS Positivity Index was higher at apical layer of epithelium; and perivascular and glandular parts of subepithelial layer. As a rate of mononuclear cells increased, eNOS positivity increased at basal part of epithelium. In eNOS Positivity Index of mononuclear cells increased ones, eNOS values also increased at glands of subepithelial layer. In nasal cavity, eNOS positivity index of all cells was significantly higher than that of the control group. Increased eNOS all cells positivity index values were seen with decreased glandular and endothelial eNOS values. In all cells group, fibroblasts were seen beside the mononuclear cells. It was observed that eNOS was not expressed in PMNC (mainly neutrophils), growing more in acute inflammatory process; and was expressed in MNCs and all cells group with fibroblasts which were the cells of chronic inflammatory process. Especially MNCs and fibroblasts may play a role in the polyp formation process. In males and in patients with longer polyp duration, eNOS values decreased. We concluded that eNOS Positivity Index was higher at apical layer of epithelium; and perivascular and glandular parts of subepithelial layer. eNOS plays role in vascular dilatation, increases in vascular permeability; increases in nasal secretion due to glandular

  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. 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 Central

    JEONG, SUN-OH; SON, YONG; LEE, JU HWAN; CHEONG, YONG-KWAN; PARK, SEONG HOON; CHUNG, HUN-TAEG; PAE, HYUN-OCK

    2015-01-01

    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. PMID:25815690

  8. 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.

  9. Nitric Oxide Plasma Level as a Barometer of Endothelial Dysfunction in Factory Workers.

    PubMed

    Miyata, Seiko; Noda, Akiko; Hara, Yuki; Ueyama, Jun; Kitaichi, Kiyoyuki; Kondo, Takaaki; Koike, Yasuo

    2017-07-27

    Objective Nitric oxide (NO) plays a key role in the regulation of vascular tone and is known as one of the key markers of endothelial dysfunction. We investigated the relationship between NO and risk factors of lifestyle-related disease in factory workers. Methods Our study included 877 factory workers presenting hypertension, dyslipidemia and type 2 diabetes. oxidated forms of NO, NO2-/NO3- (NOx) plasma concentrations were measured using a colorimetric method. Results NOx plasma levels in patients with lifestyle-related disease were significantly lower than those in the controls. The brachial-ankle pulse wave velocity (baPWV) measured in those patients was significantly greater than that of the controls. Multiple regression analysis revealed that LDL cholesterol was an independent risk factor for reducing NOx plasma concentrations. Interestingly, individuals with low NOx plasma concentrations were more likely to present type 2 diabetes compared to those with the highest plasma levels of NOx (odds ratio [OR] [95% confidence interval; CI]=3.65 [1.61-8.28], P=0.002, 2.67 [1.15-6.20], P=0.022, and 3.27 [1.43-7.48], P=0.005). Subjects with the lowest levels of plasma NOx were more likely to present dyslipidemia (OR [95% CI]=1.69 [1.13-2.53], P=0.01). Conclusion Endothelial function evaluated with plasma NOx may be indicative of lifestyle-related diseases independently from the vascular function assessed using baPWV. © Georg Thieme Verlag KG Stuttgart · New York.

  10. Cerebrovascular inflammation after brief episodic hypoxia: modulation by neuronal and endothelial nitric oxide synthase.

    PubMed

    Altay, Tamer; Gonzales, Ernesto R; Park, T S; Gidday, Jeffrey M

    2004-03-01

    Obstructive sleep apnea, apnea of prematurity, and sudden infant death syndrome are associated with a high risk of morbidity and mortality secondary to the neuronal and cerebrovascular consequences of the associated intermittent hypoxia. We hypothesized that episodic hypoxia (EH) promotes inflammation in the cerebral microcirculation and that nitric oxide (NO) produced by the endothelial and neuronal isoforms of NO synthase (eNOS and nNOS, respectively) modulates this response. Anesthetized and ventilated Swiss-Webster ND4 mice, wild-type mice, and NO synthase knockout mice were subjected to a 1-h period of EH (twelve 30-s periods of hypoxia every 5 min). Four, 24, or 48 h later, mice were reanesthetized for imaging of leukocyte dynamics in the cortical venular microcirculation by epifluorescence videomicroscopy through closed cranial windows. In Swiss-Webster ND4 mice, leukocyte adherence increased 2.1-fold at 4 h, 3.4-fold at 24 h, and 1.8-fold at 48 h relative to time-matched, normoxic controls; there was no evidence of delayed hippocampal CA1 pyramidal cell death. A similar response was noted in wild-type mice. However, in eNOS knockouts, leukocyte-endothelial cell adherence was elevated to 4.4-fold over baseline 24 h after EH, and a significant fraction of these animals showed evidence of delayed CA1 cell death. Conversely, in nNOS knockouts, no increase in adherence was noted at 24 h and CA1 viability remained unaffected. We conclude that NO derived from nNOS promotes an inflammatory response in the cerebrovascular microcirculation after short-term EH and that NO produced by eNOS blunts the extent of this response and exerts neuroprotective effects.

  11. 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

  12. 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.

  13. 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

  14. The hyperaemic response to passive leg movement is dependent on nitric oxide: a new tool to evaluate endothelial nitric oxide function

    PubMed Central

    Mortensen, Stefan P; Askew, Christopher D; Walker, Meegan; Nyberg, Michael; Hellsten, Ylva

    2012-01-01

    Passive leg movement is associated with a ∼3-fold increase in blood flow to the leg but the underlying mechanisms remain unknown. The objective of the present study was to examine the role of nitric oxide (NO) for the hyperaemia observed during passive leg movement. Leg haemodynamics and metabolites of NO production (nitrite and nitrate; NOx) were measured in plasma and muscle interstitial fluid at rest and during passive leg movement with and without inhibition of NO formation in healthy young males. The hyperaemic response to passive leg movement and to ACh was also assessed in elderly subjects and patients with peripheral artery disease. Passive leg movement (60 r.p.m.) increased leg blood flow from 0.3 ± 0.1 to 0.9 ± 0.1 litre min−1 at 20 s and 0.5 ± 0.1 litre min−1 at 3 min (P < 0.05). Mean arterial pressure remained unchanged during the trial. When passive leg movement was performed during inhibition of NO formation (NG-mono-methyl-l-arginine; 29–52 mg min−1), leg blood flow and vascular conductance were increased after 20 s (P < 0.05) and then returned to baseline levels, despite an increase in arterial pressure (P < 0.05). Passive leg movement increased the femoral venous NOx levels from 35 ± 5 at baseline to 62 ± 11 μmol l−1 during passive leg movement (P < 0.05), whereas muscle interstitial NOx levels remained unchanged. The hyperaemic response to passive leg movement were correlated with the vasodilatation induced by ACh (r2 = 0.704, P < 0.001) and with age (r2 = 0.612, P < 0.001). Leg blood flow did not increase during passive leg movement in individuals with peripheral arterial disease. These results suggest that the hypaeremia induced by passive leg movement is NO dependent and that the source of NO is likely to be the endothelium. Passive leg movement could therefore be used as a non-invasive tool to evaluate NO dependent endothelial function of the lower limb. PMID:22733658

  15. Clonidine-induced nitric oxide-dependent vasorelaxation mediated by endothelial α2-adrenoceptor activation

    PubMed Central

    Figueroa, Xavier F; Poblete, M Inés; Boric, Mauricio P; Mendizábal, Victoria E; Adler-Graschinsky, Edda; Huidobro-Toro, J Pablo

    2001-01-01

    To assess the involvement of endothelial α2-adrenoceptors in the clonidine-induced vasodilatation, the mesenteric artery of Sprague Dawley rats was cannulated and perfused with Tyrode solution (2 ml min−1). We measured perfusion pressure, nitric oxide (NO) in the perfusate using chemiluminescence, and tissue cyclic GMP by RIA.In phenylephrine-precontracted mesenteries, clonidine elicited concentration-dependent vasodilatations associated to a rise in luminal NO. One hundred nM rauwolscine or 100 μM Lω-nitro-L-arginine antagonized the clonidine-induced vasodilatation. Guanabenz, guanfacine, and oxymetazoline mimicked the clonidine-induced vasorelaxation.In non-contracted mesenteries, 100 nM clonidine elicited a maximal rise of NO (123±13 pmol); associated to a peak in tissue cyclic GMP. Endothelium removal, Lω-nitro-L-arginine, or rauwolscine ablated the rise in NO. One hundred nM aminoclonidine, guanfacine, guanabenz, UK14,304 and oxymetazoline mimicked the clonidine-induced surge of NO. Ten μM ODQ obliterated the clonidine-induced vasorelaxation and the associated tissue cyclic GMP accumulation; 10 – 100 nM sildenafil increased tissue cyclic GMP accumulation without altering the clonidine-induced NO release.α2-Adrenergic blockers antagonized the clonidine-induced rise in NO. Consistent with a preferential α2D-adrenoceptor activation, the KBs for yohimbine, rauwolscine, phentolamine, WB-4101, and prazosin were: 6.8, 24, 19, 165, and 1489 nM, respectively.Rat pretreatment with 100 mg kg−1 6-hydroxydopamine reduced 95% tissue noradrenaline and 60% neuropeptide Y. In these preparations, 100 nM clonidine elicited a rise of 91.9±15.5 pmol NO. Perfusion with 1 μM guanethidine or 1 μM guanethidine plus 1 μM atropine did not modify the NO surge evoked by 100 nM clonidine.Clonidine and congeners activate endothelial α2D-adrenoceptors coupled to the L-arginine pathway, suggesting that the antihypertensive action of

  16. 786T/c endothelial nitric oxide synthase gene polymorphism and coronary collateral circulation.

    PubMed

    Seckin, Satilmis; Emrah, Bozbeyoglu; Biyik, Ismail; Emre, Arugaslan; Burak, Tangurek; Azmi, Sungur; Omer, Celik; Sinan, Dagdelen

    2016-02-11

    In this study, we investigated the association between -786T/C polymorphism of the endothelial nitric oxide (NOS3) gene in which thymidine is replaced by a cytosine at nucleotide -786 (rs 2070744) and coronary collateral circulation (CCC) in patients with stable coronary artery disease. 286 patients having a critical stenosis (> 95%) in at least one major epicardial coronary vessel were included in the study. CCC was defined according to the Rentrop classification (R). Patients with R0-1 CCC were included in the poor CCC group and subjects with R2-3 CCC were assigned to the good CCC group. The polymerase chain reaction method was used for genotyping. 152 patients with poor CCC and 134 patients with good CCC were examined. The frequency of cytosine-cytosine (CC) and thymidine-cytosine (TC) genotypes and allele C were higher in the poor CCC group, but the difference did not reach statistical significance. In the dominant model, the frequency of CC+TC vs. thymidine-thymidine (TT) genotypes was significantly higher in the poor CCC group (67.1% vs. 54.5%, respectively; χ²=4.78; p=0.02). In multivariate regression analysis, the dominant model for -786T/C polymorphism of the NOS3 gene remained as an independent correlate of poor CCC. -786T/C polymorphism of the NOS3 gene (rs 2070744) may be associated with poor angiogenesis and the development of CCC in stable coronary artery disease.

  17. Genetic Variants within Endothelial Nitric Oxide Synthase Gene and Prostate Cancer: A Meta‐Analysis

    PubMed Central

    Nikolić, Zorana Z.; Pavićević, Dušanka Lj. Savić; Romac, Stanka P.

    2014-01-01

    Abstract Several variants within gene‐encoding endothelial isoform of nitric oxide synthase have been reported to confer prostate cancer (PCa) susceptibility and/or progression. Nevertheless, studies referring to this issue have yielded inconsistent results. In order to elucidate the involvement of these variants in prostate carcinogenesis, we have conducted a meta‐analysis of previously published case‐control and relevant case‐only studies. Eleven studies comprising in total 3,806 cases and 4,466 controls were included in the meta‐analysis which yielded evidence of association of rs2070744 (ORCC = 1.43, 95% CI 1.04–1.97; p = 0.03) and intron 4a/b variant (ORab+aa = 1.47, 95% CI 1.00–2.14; p = 0.05) with PCa risk under recessive and dominant model, respectively. Furthermore, PCa patients carrying 4a/b a allele were found to have an increased risk of cancer progression to a less differentiated form, characterized by a high Gleason score (OR = 2.29, 95% CI 1.51–3.49; p < 0.01) and to higher TNM stage (OR = 2.55, 95% CI 1.71–3.81; p < 0.01). These results support the involvement of NOS3 variants in molecular pathogenesis of PCa. PMID:25164276

  18. 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

  19. 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.

  20. Placental Endothelial Nitric Oxide Synthase in Multiple and Single Dose Betamethasone Exposed Pregnancies

    PubMed Central

    MERTZ, Heather L.; MELE, Lisa; SPONG, Catherine Y.; DUDLEY, Donald J.; WAPNER, Ronald J.; IAMS, Jay D.; SOROKIN, Yoram; PEACEMAN, Alan; LEVENO, Kenneth J.; CARITIS, Steve N.; MIODOVNIK, Menachem; MERCER, Brian M.; THORP, John M.; O'SULLIVAN, Mary J.; RAMIN, Susan M.; CARPENTER, Marshall; ROUSE, Dwight J.; SIBAI, Baha

    2011-01-01

    Objective To compare endothelial nitric oxide synthase (eNOS ) expression and capillary density (CDS) in placentas exposed to single or multiple courses of betamethasone. Study design Placental specimens exposed to single versus repeat courses of betamethasone were analyzed through immunohistochemistry and digital image quantification for eNOS and CD34. Quantified eNOS staining, calculated CDS, ratio of eNOS to CDS, and clinical characteristics were compared. Linear regression performed with these as dependent variables. Results Mean and maximum CDS were increased (p=0.013 and 0.005) and the ratio of eNOS to CDS decreased (p=0.016) in specimens exposed to 4 courses of betamethasone compared with 1 to 3 courses. Exposure to 4 courses of betamethasone was associated with increased CDS, but not with eNOS expression. Conclusion Exposure to 4 courses of betamethasone is associated with increased placental CDS. The placental effects of multiple courses of betamethasone are unrelated to eNOS expression. PMID:21529755

  1. Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection

    PubMed Central

    Hurt, K. Joseph; Musicki, Biljana; Palese, Michael A.; Crone, Julie K.; Becker, Robyn E.; Moriarity, John L.; Snyder, Solomon H.; Burnett, Arthur L.

    2002-01-01

    In the penis, nitric oxide (NO) can be formed by both neuronal NO synthase and endothelial NOS (eNOS). eNOS is activated by viscous drag/shear stress in blood vessels to produce NO continuously, a process mediated by the phosphatidylinositol 3-kinase (PI3kinase)/Akt pathway. Here we show that PI3-kinase/Akt physiologically mediates erection. Both electrical stimulation of the cavernous nerve and direct intracavernosal injection of the vasorelaxant drug papaverine cause rapid increases in phosphorylated (activated) Akt and eNOS. Phosphorylation is diminished by wortmannin and LY294002, inhibitors of PI3-kinase, the upstream activator of Akt. The two drugs also reduce erection. Penile erection elicited by papaverine is reduced profoundly in mice with targeted deletion of eNOS. Our findings support a model in which rapid, brief activation of neuronal NOS initiates the erectile process, whereas PI3-kinase/Akt-dependent phosphorylation and activation of eNOS leads to sustained NO production and maximal erection. PMID:11904450

  2. 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.

  3. Nitric oxide inhibits cutaneous vasoconstriction to exogenous norepinephrine

    PubMed Central

    Shibasaki, Manabu; Low, David A.; Davis, Scott L.; Crandall, Craig G.

    2008-01-01

    Previously, we found that nitric oxide (NO) inhibits cutaneous vasoconstrictor responsiveness evoked by whole body cooling, as well as an orthostatic stress in the heat-stressed human (Shibasaki M, Durand S, Davis SL, Cui J, Low DA, Keller DM, Crandall CG. J Physiol 585: 627–634, 2007). However, it remains unknown whether this response occurs via NO acting through presynaptic or postsynaptic mechanisms. The aim of this study was to test the hypothesis that NO is capable of impairing cutaneous vasoconstriction via postsynaptic mechanisms. Skin blood flow was monitored over two forearm sites where intradermal microdialysis membranes were previously placed. Skin blood flow was elevated four- to fivefold through perfusion of the NO donor sodium nitroprusside at one site and through perfusion of adenosine (primarily non-NO mechanisms) at a second site. Once a plateau in vasodilation was evident, increasing concentrations of norepinephrine (1 × 10−8 to 1 × 10−2 M) were administrated through both microdialysis probes, while the aforementioned vasodilator agents continued to be perfused. Cutaneous vascular conductance was calculated by dividing skin blood flow by mean arterial blood pressure. The administration of norepinephrine decreased cutaneous vascular conductance at both sites. However, the dose of norepinephrine at the onset of vasoconstriction (−5.9 ± 1.3 vs. −7.2 ± 0.7 log M norepinephrine, P = 0.021) and the concentration of norepinephrine resulting in 50% of the maximal vasoconstrictor response (−4.9 ± 1.2 vs. −6.1 ± 0.2 log M norepinephrine dose; P = 0.012) occurred at significantly higher norepinephrine concentrations for the sodium nitroprusside site relative to the adenosine site, respectively. These results suggested that NO is capable of attenuating cutaneous vasoconstrictor responsiveness to norepinephrine via postsynaptic mechanisms. PMID:18801956

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Impact of Trans-Resveratrol-Sulfates and -Glucuronides on Endothelial Nitric Oxide Synthase Activity, Nitric Oxide Release and Intracellular Reactive Oxygen Species

    PubMed Central

    Ladurner, Angela; Schachner, Daniel; Schueller, Katharina; Pignitter, Marc; Heiss, Elke H.; Somoza, Veronika; Dirsch, Verena M.

    2015-01-01

    Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenolic natural product mainly present in grape skin, berries and peanuts. In the vasculature resveratrol is thought to boost endothelial function by increasing endothelial nitric oxide synthase (eNOS) expression, by enhancing eNOS activity, and by reduction of reactive oxygen species (ROS) levels. Recent studies show that dietary resveratrol is metabolized in the liver and intestine into resveratrol-sulfate and -glucuronide derivatives questioning the relevance of multiple reported mechanistic in vitro data on resveratrol. In this study, we compare side by side different physiologically relevant resveratrol metabolites (resveratrol sulfates- and -glucuronides) and their parent compound in their influence on eNOS enzyme activity, endothelial NO release, and intracellular ROS levels. In contrast to resveratrol, none of the tested resveratrol metabolites elevated eNOS enzyme activity and endothelial NO release or affected intracellular ROS levels, leaving the possibility that not tested metabolites are active and able to explain in vivo findings. PMID:25329867

  9. The EGCg-induced redox-sensitive activation of endothelial nitric oxide synthase and relaxation are critically dependent on hydroxyl moieties.

    PubMed

    Auger, Cyril; Kim, Jong-Hun; Chabert, Philippe; Chaabi, Mehdi; Anselm, Eric; Lanciaux, Xavier; Lobstein, Annelise; Schini-Kerth, Valérie B

    2010-02-26

    Several rich sources of polyphenols stimulate the endothelial formation of nitric oxide (NO), a potent vasoprotecting factor, via the redox-sensitive activation of the PI3-kinase/Akt pathway leading to the phosphorylation of endothelial NO synthase (eNOS). The present study examined the molecular mechanism underlying the stimulatory effect of epicatechins on eNOS. NO-mediated relaxation was assessed using porcine coronary artery rings in the presence of indomethacin, and charybdotoxin plus apamin, inhibitors of cyclooxygenases and EDHF-mediated responses, respectively. The phosphorylation level of Akt and eNOS was assessed in cultured coronary artery endothelial cells by Western blot, and ROS formation using dihydroethidine. (-)-Epigallocatechin-3-O-gallate (EGCg) caused endothelium-dependent relaxations in coronary artery rings and the phosphorylation of Akt and eNOS in endothelial cells. These responses were inhibited by membrane-permeant analogues of superoxide dismutase and catalase, whereas native superoxide dismutase, catalase and inhibitors of major enzymatic sources of reactive oxygen species including NADPH oxidase, xanthine oxidase, cytochrome P450 and the mitochondrial respiration chain were without effect. The EGCg derivative with all hydroxyl functions methylated induced neither relaxations nor the intracellular formation of ROS, whereas both responses were observed when the hydroxyl functions on the gallate moiety were present. In conclusion, EGCg causes endothelium-dependent NO-mediated relaxations of coronary artery rings through the Akt-dependent activation of eNOS in endothelial cells. This response is initiated by the intracellular formation of superoxide anions and hydrogen peroxide, and is critically dependent on the gallate moiety and on the presence of hydroxyl functions possibly through intracellular auto-oxidation. Copyright 2010 Elsevier Inc. All rights reserved.

  10. 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.

  11. Exposure to High or Low Glucose Levels Accelerates the Appearance of Markers of Endothelial Cell Senescence and Induces Dysregulation of Nitric Oxide Synthase

    PubMed Central

    2013-01-01

    To test the hypothesis that aging impairs endothelial cell response to glucose stress, we utilized a human umbilical vein endothelial cell in vitro model in which clinically relevant concentrations of normal (5.5mM), high (25mM), and low (1.5mM) glucose were tested. With advancing population doubling, exposure to normal glucose gradually decreased endothelial nitric oxide synthase expression and activity, resulting in slow, progressive development of markers of cell senescence (by population doubling level [PDL] 44). High or low glucose treatment accelerated the appearance of markers of senescence (by ~PDL 35) along with declines in endothelial nitric oxide synthase expression and activity. Human umbilical vein endothelial cells exposed to alternating low and high glucose gave even more rapid acceleration in the appearance of markers of senescence (by ~PDL 18) and reduction in endothelial nitric oxide synthase levels. Thus, exposure to low and high glucose induces earlier appearance of markers of endothelial cell senescence and dysregulation of the nitric oxide synthase gene and protein expression and function. These findings will help to elucidate endothelial dysfunction associated with glucose intolerance and improve future therapy for diabetic seniors. PMID:23585419

  12. Exogenous nitric oxide inhibits sympathetically mediated vasoconstriction in human skin

    PubMed Central

    Durand, S; Davis, SL; Cui, J; Crandall, CG

    2005-01-01

    Two experiments were performed to identify whether nitric oxide (NO) inhibits sympathetically mediated vasoconstriction in human skin. In eight subjects increasing doses of sodium nitroprusside (SNP; 8.4 × 10−6–8.4 × 10−3 m) were administered via intradermal microdialysis. At each dose of SNP, cutaneous vasoconstrictor responsiveness was assessed during a 3 min whole-body cold stress. The relative reduction in forearm cutaneous vascular conductance (CVC) during the cold stress was significantly attenuated for SNP doses greater than 8.4 × 10−4 m (control: 63.0 ± 4.1%, SNP 8.4 × 10−6 m: 57.1 ± 4.7%, SNP 8.4 × 10−5 m: 57.0 ± 3.6%, SNP 8.4 × 10−4m: 44.5 ± 5.4% and SNP 8.4 × 10−3m: 28.8 ± 7.9%). The second experiment was performed to identify whether this response was due to NO attenuating sympathetically mediated vasoconstriction or due to a non-specific effect of an elevated CVC secondary to SNP administration. In seven subjects forearm CVC during a whole-body cold stress was assessed at two sites: at a site dilated via microdialysis administration of SNP and at a site dilated with isoproterenol (ISO). CVC was not different between sites prior to (SNP: 0.42 ± 0.11; ISO: 0.46 ± 0.11 AU mmHg−1 (AU, arbitrary units), P > 0.05) or following drug infusion (SNP: 1.36 ± 0.21; ISO: 1.27 ± 0.23 AU mmHg−1, P > 0.05). The reduction in CVC during the subsequent cold stress was significantly less at the SNP site (38.1 ± 6.2%) relative to the ISO site (65.0 ± 5.5%; P = 0.007). These data suggest NO is capable of inhibiting sympathetically mediated vasoconstriction in the cutaneous vasculature. PMID:15539401

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.; hide

    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.

  20. 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.

  1. 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.

  2. Ceramide mediates inhibition of the Akt/eNOS pathway by high levels of glucose in human vascular endothelial cells.

    PubMed

    Wang, Aimin; Li, Chun; Liao, Jie; Dong, Min; Xiao, Zhiming; Lei, Minxiang

    2013-01-01

    To investigate how ceramide mediates the effects of high-glucose-induced inhibition of the Akt/endothelial nitric oxide synthase (eNOS) signalling pathway in human vascular endothelial cells (HUVECs). NO levels were determined by ELISA. Endogenous ceramide levels were determined using a liquid chromatography-mass spectrometry assay. Akt and eNOS protein expressions were determined by Western blotting. High-glucose levels induce ceramide accumulation in a dose- and time-dependent manner (p<0.05). We also show that exposure of HUVECs to high-glucose conditions inhibits the insulin-mediated activation of Akt/eNOS signalling and the subsequent NO generation in a dose-dependent manner (p<0.05). Preventing de novo ceramide synthesis attenuated the antagonistic effects of high-glucose levels on the Akt/eNOS signalling pathway (p<0.05); conversely, inducing ceramide build-up augmented the inhibitory effects of high-glucose levels on the Akt/eNOS signalling pathway (p<0.05). Ceramide is both necessary and sufficient for mediating the inhibition of the Akt/eNOS signalling pathway by high-glucose levels in endothelial cells.

  3. 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.

  4. 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.

  5. Mechanism of endothelial nitric oxide synthase phosphorylation and activation by tentacle extract from the jellyfish Cyanea capillata

    PubMed Central

    Wang, Qianqian; Zhang, Hui; Liu, Guoyan

    2017-01-01

    Our previous study demonstrated that tentacle extract (TE) from the jellyfish Cyanea capillata (C. capillata) could cause a weak relaxation response mediated by nitric oxide (NO) using isolated aorta rings. However, the intracellular mechanisms of TE-induced vasodilation remain unclear. Thus, this study was conducted to examine the role of TE on Akt/eNOS/NO and Ca2+ signaling pathways in human umbilical vein endothelial cells (HUVECs). Our results showed that TE induced dose- and time-dependent increases of eNOS activity and NO production. And TE also induced Akt and eNOS phosphorylation in HUVECs. However, treatment with specific PI3-kinase inhibitor (Wortmannin) significantly inhibited the increases in NO production and Akt/eNOS phosphorylation. In addition, TE also stimulated an increase in the intracellular Ca2+ concentration ([Ca2+]i), which was significantly attenuated by either IP3 receptor blocker (Heparin) or PKC inhibitor (PKC 412). In contrast, extracellular Ca2+-free, L-type calcium channel blocker (Nifedipine), or PKA inhibitor (H89) had no influence on the [Ca2+]i elevation. Since calcium ions also play a critical role in stimulating eNOS activity, we next explored the role of Ca2+ in TE-induced Akt/eNOS activation. In consistent with the attenuation of [Ca2+]i elevation, we found that Akt/eNOS phosphorylation was also dramatically decreased by Heparin or PKC 412, but not affected by Nifedipine or H89. However, the phosphorylation level could also be decreased by the removal of extracellular calcium. Taken together, our findings indicated that TE-induced eNOS phosphorylation and activation were mainly through PI3K/Akt-dependent, PKC/IP3R-sensitive and Ca2+-dependent pathways. PMID:28413728

  6. Mechanism of endothelial nitric oxide synthase phosphorylation and activation by tentacle extract from the jellyfish Cyanea capillata.

    PubMed

    Wang, Beilei; Liu, Dan; Wang, Chao; Wang, Qianqian; Zhang, Hui; Liu, Guoyan; Tao, Xia; Zhang, Liming

    2017-01-01

    Our previous study demonstrated that tentacle extract (TE) from the jellyfish Cyanea capillata (C. capillata) could cause a weak relaxation response mediated by nitric oxide (NO) using isolated aorta rings. However, the intracellular mechanisms of TE-induced vasodilation remain unclear. Thus, this study was conducted to examine the role of TE on Akt/eNOS/NO and Ca(2+) signaling pathways in human umbilical vein endothelial cells (HUVECs). Our results showed that TE induced dose- and time-dependent increases of eNOS activity and NO production. And TE also induced Akt and eNOS phosphorylation in HUVECs. However, treatment with specific PI3-kinase inhibitor (Wortmannin) significantly inhibited the increases in NO production and Akt/eNOS phosphorylation. In addition, TE also stimulated an increase in the intracellular Ca(2+) concentration ([Ca(2+)]i), which was significantly attenuated by either IP3 receptor blocker (Heparin) or PKC inhibitor (PKC 412). In contrast, extracellular Ca(2+)-free, L-type calcium channel blocker (Nifedipine), or PKA inhibitor (H89) had no influence on the [Ca(2+)]i elevation. Since calcium ions also play a critical role in stimulating eNOS activity, we next explored the role of Ca(2+) in TE-induced Akt/eNOS activation. In consistent with the attenuation of [Ca(2+)]i elevation, we found that Akt/eNOS phosphorylation was also dramatically decreased by Heparin or PKC 412, but not affected by Nifedipine or H89. However, the phosphorylation level could also be decreased by the removal of extracellular calcium. Taken together, our findings indicated that TE-induced eNOS phosphorylation and activation were mainly through PI3K/Akt-dependent, PKC/IP3R-sensitive and Ca(2+)-dependent pathways.

  7. 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

  8. Mice with gene disruption of both endothelial and neuronal nitric oxide synthase exhibit insulin resistance.

    PubMed

    Shankar, R R; Wu, Y; Shen, H Q; Zhu, J S; Baron, A D

    2000-05-01

    Studies from our laboratory using acute pharmacologic blockade of nitric oxide synthase (NOS) activity have suggested that nitric oxide (NO) has an important role in regulating carbohydrate metabolism. We now report on insulin sensitivity in mice with targeted disruptions in endothelial NOS (eNOS) and neuronal NOS (nNOS) genes compared with their wild-type (WT) counterparts. Mice underwent hyperinsulinemic-euglycemic clamp studies after a 24-h fast, during an insulin infusion of 20 mU x kg(-1) x min(-1). Glucose levels were measured at baseline and every 10 min during the clamp. Insulin levels were measured at baseline and at the end of the clamp study. Glucose infusion rates (GIRs) during the last 30 min of the clamp study were in a steady state. Tritiated glucose infusion was used to measure rates of endogenous glucose output (EGO) both at baseline and during steady-state euglycemia. Glucose disposal rates (GDRs) were computed from the GIR and EGO. Fasting and steady-state glucose and insulin levels were comparable in the 3 groups of mice. No differences in fasting EGO were noted between the groups. GIR was significantly reduced (37%, P = 0.001) in the eNOS knockout (KO) mice compared with the WT mice, with values for the nNOS mice being intermediate. EGO was completely suppressed in the nNOS and WT mice during insulin infusion, but not in the eNOS mice. Even so, the eNOS mice displayed significantly reduced whole-body GDRs compared with those of the WT mice (82.67+/-10.77 vs. 103.67+/-3.47 mg x kg(-1) x min(-1), P = 0.03). eNOS KO mice are insulin resistant at the level of the liver and peripheral tissues, whereas the nNOS KO mice are insulin resistant only in the latter. These data indicate that NO plays a role in modulating insulin sensitivity and carbohydrate metabolism and that the eNOS isoform may play a dominant role relative to nNOS.

  9. The Relationship Between Endothelial Nitric Oxide Synthase Gene (NOS3) Polymorphisms, NOS3 Expression, and Varicocele.

    PubMed

    Kahraman, Cigdem Yuce; Tasdemir, Sener; Sahin, Ibrahim; Marzioglu Ozdemir, Ebru; Yaralı, Oguzhan; Ziypak, Tevfik; Adanur, Senol; Kahraman, Mustafa; Tatar, Abdulgani

    2016-04-01

    Varicocele is an abnormal enlargement of the pampiniform venous plexus in the scrotum. Varicocele is the most common cause of secondary male infertility. Nitric oxide (NO), which has a role on varicocele pathophysiology, is synthesized by endothelial nitric oxide synthase gene (NOS3). In our study, we aimed to explain the relationship between varicocele, three common NOS3 polymorphisms (T-786C, G894T, 4b/a), and NOS3 mRNA expression levels. We investigated NOS3 T-786C, G894T, and 4b/a polymorphisms in 102 patients with varicocele and 100 healthy controls. Twenty-four patients and 17 controls were chosen for expression studies based on polymorphism subgroupings. Subgroup 1 includes patients who have no minor allele polymorphisms, and subgroups 2, 3, and 4 have T-786C, G894T, and 4b/a polymorphisms, respectively. The 4b/a polymorphism demonstrated significantly elevated levels in both allele and genotype analysis in the control group compared to the patient group. The G894T polymorphism was statistically elevated for genotypic frequencies in the patient group compared to the control group, but this finding did not extend to allelic frequencies. There were no statistically significant differences in either the allelic or genotypic frequencies between patients and control groups for the T-786C polymorphism. When patient and control expression levels were compared without considering the subgroups, the NOS3 expression level was found to be statistically higher in the patient group. There were no statistically significant differences in the patient and control group expression levels when stratified by subgroup, nor was there any effect of the polymorphisms under study on expression levels. The 4b/a polymorphism may have a protective effect for varicocelem and G894T polymorphism may contribute to varicocele occurrence by lowering the level of NO. The higher NOS3 expression levels in the patient group may be a kind of dilator compensatory mechanism to protect vascular

  10. Vascular endothelial growth factor inhibitor-induced hypertension: from pathophysiology to prevention and treatment based on long-acting nitric oxide donors.

    PubMed

    Kruzliak, Peter; Novák, Jan; Novák, Miroslav

    2014-01-01

    Hypertension is the most common adverse effect of the inhibitors of vascular endothelial growth factor (VEGF) pathway-based therapy (VEGF pathway inhibitors therapy, VPI therapy) in cancer patients. VPI includes monoclonal antibodies against VEGF, tyrosine kinase inhibitors, VEGF Traps, and so-called aptamers that may become clinically relevant in the future. All of these substances inhibit the VEGF pathway, which in turn causes a decrease in nitric oxide (NO) and an increase in blood pressure, with the consequent development of hypertension and its final events (e.g., myocardial infarction or stroke). To our knowledge, there is no current study on how to provide an optimal therapy for patients on VPI therapy with hypertension. This review summarizes the roles of VEGF and NO in vessel biology, provides an overview of VPI agents, and suggests a potential treatment procedure for patients with VPI-induced hypertension.

  11. 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

  12. Nitric oxide synthesis inhibition induces leukocyte adhesion via superoxide and mast cells.

    PubMed

    Kubes, P; Kanwar, S; Niu, X F; Gaboury, J P

    1993-10-01

    Recent work has demonstrated that inhibition of nitric oxide production with various nitric oxide synthesis inhibitors (L-NAME, L-NMMA) initiate leukocyte adhesion to postcapillary venules. The objective of this study was to elucidate the mechanism (or mechanisms) that promote the L-NAME-induced leukocyte response. Intravital microscopy was used to examine 25-40 microns venules in the rat mesentery. Nitric oxide synthesis was inhibited with L-NAME and leukocyte adhesion was observed over the first 60 min. The fourfold increase in leukocyte adhesion was independent of alterations in venular red blood cell velocity. The adhesion was superoxide-mediated inasmuch as superoxide dismutase (SOD) abolished the rise in leukocyte adhesion associated with nitric oxide synthesis inhibition. Ketotifen, a mast cell stabilizer, also abolished the rise in leukocyte adhesion induced by L-NAME. Histology revealed that mast cell degranulation occurred only in animals treated with L-NAME but not in animals pretreated with SOD or ketotifen. This observation suggests that mast cells become activated in the absence of nitric oxide production and superoxide contributes to the mast cell activation. The L-NAME-induced leukocyte adhesion could be reproduced by infusing hypoxanthine/xanthine oxidase (a superoxide generating system) or compound 48/80 (an activator of mast cells) and both responses were attenuated by ketotifen. These data suggest that inhibition of nitric oxide synthesis results in a superoxide and mast cell-dependent leukocyte adhesion.

  13. Cigarette Smoke Extract Changes Expression of Endothelial Nitric Oxide Synthase (eNOS) and p16(INK4a) and is Related to Endothelial Progenitor Cell Dysfunction.

    PubMed

    He, Zhihui; Chen, Yan; Hou, Can; He, Wenfang; Chen, Ping

    2017-07-02

    BACKGROUND Endothelial dysfunction is an important pathophysiologic feature in many smoke-related diseases. Endothelial progenitor cells (EPCs) are the precursors of endothelial cells and play a fundamental role in the maintenance of endothelial integrity and function. Endothelial nitric oxide synthase (eNOS) is the dominant NOS isoform in the vasculature and plays a central role in the maintenance of endothelial homeostasis. p16(INK4a) is a cyclin-dependent kinase inhibitor and could be regarded as a major dominant senescence gene. The present study aimed to determine whether the expression of eNOS and p16(INK4a) in EPCs is related to EPCs function and the possible epigenetic mechanism, if any. MATERIAL AND METHODS We investigated EPCs capacity for proliferation, adhesion, and secretion, and the expression of eNOS and p16(INK4a) in EPCs which were altered by cigarette smoke extract (CSE) in vitro. Furthermore, Decitabine (Dec), an agent of demethylation, was used to examine whether it could alter the changes induced by CSE. RESULTS The present study demonstrated that EPCs altered by CSE in vitro displayed decreased capacities of proliferation, adhesion, and secretion, which was accompanied by decreased eNOS expression and increased p16(INK4a) expression in EPCs. Furthermore, Dec could alleviate the changes in the expression of eNOS and p16(INK4a), and protect against the EPCs dysfunction caused by CSE. CONCLUSIONS The decreased eNOS expression and increased p16(INK4a) expression was associated with dysfunction of EPCs caused by CSE. The mechanism of methylation, one of the most common epigenetic mechanism, may be involved in the EPCs dysfunction caused by CSE.

  14. 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

  15. 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.

  16. 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

  17. Nitric oxide synthase inhibition and oxidative stress in cardiovascular diseases: possible therapeutic targets?

    PubMed

    Rochette, Luc; Lorin, Julie; Zeller, Marianne; Guilland, Jean-Claude; Lorgis, Luc; Cottin, Yves; Vergely, Catherine

    2013-12-01

    Nitric oxide (NO) is synthetized enzymatically from l-arginine (l-Arg) by three NO synthase isoforms, iNOS, eNOS and nNOS. The synthesis of NO is selectively inhibited by guanidino-substituted analogs of l-Arg or methylarginines such as asymmetric dimethylarginine (ADMA), which results from protein degradation in cells. Many disease states, including cardiovascular diseases and diabetes, are associated with increased plasma levels of ADMA. The N-terminal catalytic domain of these NOS isoforms binds the heme prosthetic group as well as the redox cofactor, tetrahydrobiopterin (BH(4)) associated with a regulatory protein, calmodulin (CaM). The enzymatic activity of NOS depends on substrate and cofactor availability. The importance of BH(4) as a critical regulator of eNOS function suggests that BH(4) may be a rational therapeutic target in vascular disease states. BH(4) oxidation appears to be a major contributor to vascular dysfunction associated with hypertension, ischemia/reperfusion injury, diabetes and other cardiovascular diseases as it leads to the increased formation of oxygen-derived radicals due to NOS uncoupling rather than NO. Accordingly, abnormalities in vascular NO production and transport result in endothelial dysfunction leading to various cardiovascular disorders. However, some disorders including a wide range of functions in the neuronal, immune and cardiovascular system were associated with the over-production of NO. Inhibition of the enzyme should be a useful approach to treat these pathologies. Therefore, it appears that both a lack and excess of NO production in diseases can have various important pathological implications. In this context, NOS modulators (exogenous and endogenous) and their therapeutic effects are discussed. © 2013.

  18. 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.

  19. 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.

  20. Endothelial nitric oxide synthase polymorphisms and erectile dysfunction: a meta-analysis.

    PubMed

    Liu, Chunhui; Lu, Kai; Tao, Tao; Zhang, Lei; Zhang, Xiaowen; Jiang, Liang; Huang, Yeqing; Guan, Han; Chen, Ming; Xu, Bin

    2015-06-01

    Erectile dysfunction (ED) is a frequent disorder in men and has a serious impact on the quality of the patient's life. Recent studies have examined the relationship between endothelial nitric oxide synthase (eNOS) polymorphisms and ED. However, the results remain inconclusive. The present study aimed to offer an actual view of estimating the correlation between eNOS polymorphisms and ED. We performed a meta-analysis to estimate the association between eNOS polymorphisms and ED risk. Databases employed for data mining until December 1, 2014 included PubMed, Web of Science, and the Chinese National Knowledge Infrastructure. Two study investigators independently conducted a literature search and data extraction. Odds ratios (ORs) with 95% confidence intervals for the risk were calculated by using a random effects model or fixed effects model. A total of 20 studies in 13 publications increased ED risk in allele contrast, dominant, heterozygote, and homozygote models (allele contrast: OR = 1.514, 95% confidence interval were included in the meta-analysis. In the overall comparison, the eNOS G984T polymorphism was associated with an [CI]: 1.019-2.248). For 4 VNTR polymorphisms, the overall analysis showed a significant association between homozygote comparison and recessive genetic model (homozygote comparison: OR = 1.917, CI: 1.073-3.424). The eNOS T786C polymorphism increased ED risk in allele contrast, homozygote, and recessive models (allele contrast: OR = 1.588, CI: 1.316-1.915). Significant heterogeneity was mainly observed in studies on the G894T polymorphism. No publication bias was detected in all of the variants. The eNOS polymorphisms G894T, 4 VNTR, and T786C were associated with an increased risk for ED. However, these results are still preliminary. Further studies based on different confounders and using a large population size should be conducted to generate more accurate and reliable conclusions. © 2015 International Society for Sexual Medicine.

  1. Endothelial Nitric Oxide Synthase Gene Variation Associated With Chronic Kidney Disease After Liver Transplant

    PubMed Central

    Bambha, Kiran; Kim, W. Ray; Rosen, Charles B.; Pedersen, Rachel A.; Rys, Cynthia; Kolbert, Christopher P.; Cunningham, Julie M.; Therneau, Terry M.

    2010-01-01

    OBJECTIVE: To identify single nucleotide polymorphisms (SNPs) associated with risk of developing chronic kidney disease (CKD), a prevalent comorbidity, after liver transplant (LT). PATIENTS AND METHODS: This study consists of a cohort of adult (≥18 years) primary-LT recipients who had normal renal function before LT and who survived 1 year or more after LT at a high-volume US LT program between January 1, 1990, and December 31, 2000. Patients with adequate renal function (estimated glomerular filtration rate, ≥40 mL/min per 1.73 m2 during follow-up; n=308) and patients with incident CKD (estimated glomerular filtration rate, <40 mL/min per 1.73 m2 after LT; n=92) were identified. To investigate the association of 6 candidate genes with post-LT CKD, we selected SNPs that have been associated with renal function in the literature. Hazard ratios were estimated using Cox regression, adjusted for potential confounding variables. RESULTS: The variant allele (298Asp) of the Glu298Asp SNP in the endothelial nitric oxide synthase gene (NOS3) was significantly associated with CKD after LT (P=.05; adjusted for multiple comparisons). The 5-year incidence of CKD was 70% among patients homozygous for the NOS3 variant allele (298Asp) compared with 42% among those not homozygous for the NOS3 variant allele. Specifically, homozygosity for the NOS3 variant allele conferred a 2.5-fold increased risk of developing CKD after LT (P=.005, adjusted for confounding variables). CONCLUSION: Homozygosity for the variant allele of NOS3 (298Asp) is associated with CKD after LT and may be useful for identifying recipients at higher risk of post-LT CKD. PMID:20810793

  2. 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.

  3. 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.

  4. 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

  5. Mepivacaine-induced contraction is attenuated by endothelial nitric oxide release in isolated rat aorta.

    PubMed

    Sung, Hui-Jin; Choi, Mun-Jeoung; Ok, Seong-Ho; Lee, Soo Hee; Hwang, Il Jeong; Kim, Hee Sook; Chang, Ki Churl; Shin, Il-Woo; Lee, Heon-Keun; Park, Kyeong-Eon; Chung, Young-Kyun; Sohn, Ju-Tae

    2012-07-01

    Mepivacaine is an aminoamide-linked local anesthetic with an intermediate duration that intrinsically produces vasoconstriction both in vivo and in vitro. The aims of this in-vitro study were to examine the direct effect of mepivacaine in isolated rat aortic rings and to determine the associated cellular mechanism with a particular focus on endothelium-derived vasodilators, which modulate vascular tone. In the aortic rings with or without endothelium, cumulative mepivacaine concentration-response curves were generated in the presence or absence of the following antagonists: N(ω)-nitro-L-arginine methyl ester [L-NAME], indomethacin, fluconazole, methylene blue, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one [ODQ], verapamil, and calcium-free Krebs solution. Mepivacaine produced vasoconstriction at low concentrations (1 × 10(-3) and 3 × 10(-3) mol/L) followed by vasodilation at a high concentration (1 × 10(-2) mol/L). The mepivacaine-induced contraction was higher in endothelium-denuded aortae than in endothelium-intact aortae. Pretreatment with L-NAME, ODQ, and methylene blue enhanced mepivacaine-induced contraction in the endothelium-intact rings, whereas fluconazole had no effect. Indomethacin slightly attenuated mepivacaine-induced contraction, whereas verapamil and calcium-free Krebs solution more strongly attenuated this contraction. The vasoconstriction induced by mepivacaine is attenuated mainly by the endothelial nitric oxide - cyclic guanosine monophosphate pathway. In addition, mepivacaine-induced contraction involves cyclooxygenase pathway activation and extracellular calcium influx via voltage-operated calcium channels.

  6. 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

  7. 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.

  8. Endothelial nitric oxide synthase Glu298Asp gene polymorphism in periodontal diseases.

    PubMed

    Berdeli, Afig; Gürkan, Ali; Emingil, Gülnur; Atilla, Gül; Köse, Timur

    2006-08-01

    Endothelial nitric oxide synthase (eNOS) is involved in key steps of immune response. The aim of the present study was to evaluate genotype distribution and genotype-phenotype association in periodontal disease regarding Glu298Asp polymorphism of the eNOS gene. A total of 272 subjects were included into the study. Genomic DNA was obtained from the peripheral blood of 51 chronic periodontitis (CP) patients, 48 generalized aggressive periodontitis (GAgP), and 173 reference controls. Polymerase chain reaction (PCR) amplification and subsequent BanII restriction fragment length polymorphism (RFLP) analysis were used to detect eNOS Glu298Asp polymorphism. Probing depth, clinical attachment loss, plaque accumulation, and bleeding on probing (BOP) were recorded. The data were analyzed by the chi2 test, logistic regression, and Mann-Whitney U test. The distributions of eNOS Glu298Asp genotypes and alleles were similar among study groups. Subjects with the Asp allele (Asp+) were statistically higher in the CP group compared to the control group (odds ratio [OR] = 1.957; 95% confidence interval [95% CI] = 1.038 to 3.689). In the GAgP group, BOP (%) was significantly higher in patients with the 298Asp allele (Asp+) compared to patients without the Asp allele (Asp-) (P = 0.015). The present study showed that eNOS Glu298Asp polymorphism is associated with BOP in GAgP patients. Moreover, the 298Asp allele of the eNOS gene might be related to CP in the Turkish population.

  9. Impact of nutrient excess and endothelial nitric oxide synthase on the plasma metabolite profile in mice

    PubMed Central

    Sansbury, Brian E.; Bhatnagar, Aruni; Hill, Bradford G.

    2014-01-01

    An increase in calorie consumption is associated with the recent rise in obesity prevalence. However, our current understanding of the effects of nutrient excess on major metabolic pathways appears insufficient to develop safe and effective metabolic interventions to prevent obesity. Hence, we sought to identify systemic metabolic changes caused by nutrient excess and to determine how endothelial nitric oxide synthase (eNOS)—which has anti-obesogenic properties—affects systemic metabolism by measuring plasma metabolites. Wild-type (WT) and eNOS transgenic (eNOS-TG) mice were placed on low fat or high fat diets for 6 weeks, and plasma metabolites were measured using an unbiased metabolomic approach. High fat feeding in WT mice led to significant increases in fat mass, which was associated with significantly lower plasma levels of 1,5-anhydroglucitol, lysophospholipids, 3-dehydrocarnitine, and bile acids, as well as branched chain amino acids (BCAAs) and their metabolites. Plasma levels of several lipids including sphingomyelins, stearoylcarnitine, dihomo-linoleate and metabolites associated with oxidative stress were increased by high fat diet. In comparison with low fat-fed WT mice, eNOS-TG mice showed lower levels of several free fatty acids, but in contrast, the levels of bile acids, amino acids, and BCAA catabolites were increased. When placed on a high fat diet, eNOS overexpressing mice showed remarkably higher levels of plasma bile acids and elevated levels of plasma BCAAs and their catabolites compared with WT mice. Treatment with GW4064, an inhibitor of bile acid synthesis, decreased plasma bile acid levels but was not sufficient to reverse the anti-obesogenic effects of eNOS overexpression. These findings reveal unique metabolic changes in response to high fat diet and eNOS overexpression and suggest that the anti-obesity effects of eNOS are likely independent of changes in the bile acid pool. PMID:25505420

  10. Dietary nitrate ameliorates pulmonary hypertension: cytoprotective role for endothelial nitric oxide synthase and xanthine oxidoreductase

    PubMed Central

    Baliga, Reshma S; Milsom, Alexandra B; Ghosh, Suborno M; Trinder, Sarah L; MacAllister, Raymond J; Ahluwalia, Amrita; Hobbs, Adrian J

    2012-01-01

    Background Pulmonary hypertension (PH) is a multi-factorial disease characterized by increased pulmonary vascular resistance and right ventricular failure; morbidity and mortality remain unacceptably high. Loss of nitric oxide (NO) bioactivity is thought to contribute to the pathogenesis of PH and agents that augment pulmonary NO signaling are clinically effective in the disease. Inorganic nitrate (NO3−) and nitrite (NO2−) elicit a reduction in systemic blood pressure in healthy individuals; this effect is underpinned by endogenous and sequential reduction to NO. Herein, we determined whether dietary nitrate and nitrite might be preferentially reduced to NO by the hypoxia associated with PH, and thereby offer a convenient, inexpensive method of supplementing NO functionality to reduce disease severity. Methods & Results Dietary nitrate reduced the right ventricular pressure and hypertrophy, and pulmonary vascular re-modeling, in wild-type mice exposed to 3 weeks hypoxia; this beneficial activity was mirrored largely by dietary nitrite. The cytoprotective effects of dietary nitrate were associated with increased plasma & lung concentrations of nitrite and cGMP. The beneficial effects of dietary nitrate and nitrite were reduced in mice lacking endothelial NO synthase (eNOS) or treated with the xanthine oxidoreductase (XOR) inhibitor allopurinol. Conclusions These data demonstrate that dietary nitrate, and to a lesser extent dietary nitrite, elicit pulmonary dilatation, prevent pulmonary vascular remodeling, and reduce the RVH characteristic of PH. This favorable pharmacodynamic profile is dependent on eNOS and XOR -catalyzed reduction of nitrite to NO. Exploitation of this mechanism (i.e. dietary nitrate/nitrite supplementation) represents a viable, orally-active therapy for PH. PMID:22572914

  11. Lobe-Specific Calcium Binding in Calmodulin Regulates Endothelial Nitric Oxide Synthase Activation

    PubMed Central

    Wu, Pei-Rung; Kuo, Cheng-Chin; Yet, Shaw-Fang; Liou, Jun-Yang; Wu, Kenneth K.; Chen, Pei-Feng

    2012-01-01

    Background Human endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear. Methodology/Principal Findings Using a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q) resulted in ∼2–3 fold increase in the CaM concentration necessary for half-maximal activation (EC50) of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q) or at C-terminal (B34Q) lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS. Conclusions Our results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding. PMID:22768143

  12. 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

  13. 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

  14. Inhaled nitric oxide reduces endothelial activation and parasite accumulation in the brain, and enhances survival in experimental cerebral malaria.

    PubMed

    Serghides, Lena; Kim, Hani; Lu, Ziyue; Kain, Dylan C; Miller, Chris; Francis, Roland C; Liles, W Conrad; Zapol, Warren M; Kain, Kevin C

    2011-01-01

    The host immune response contributes to the onset and progression of severe malaria syndromes, such as cerebral malaria. Adjunctive immunomodulatory strategies for severe malaria may improve clinical outcome beyond that achievable with artemisinin-based therapy alone. Here, we report that prophylaxis with inhaled nitric oxide significantly reduced systemic inflammation (lower TNF, IFNγ and MCP-1 in peripheral blood) and endothelial activation (decreased sICAM-1 and vWF, and increased angiopoeitin-1 levels in peripheral blood) in an experimental cerebral malaria model. Mice that received inhaled nitric oxide starting prior to infection had reduced parasitized erythrocyte accumulation in the brain, decreased brain expression of ICAM-1, and preserved vascular integrity compared to control mice.Inhaled nitric oxide administered in combination with artesunate, starting as late as 5.5 days post-infection, improved survival over treatment with artesunate alone (70% survival in the artesunate only vs. 100% survival in the artesunate plus iNO group, p = 0.03). These data support the clinical investigation of inhaled nitric oxide as a novel adjunctive therapy in patients with severe malaria.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. Isometric contraction induces the Ca2+-independent activation of the endothelial nitric oxide synthase

    PubMed Central

    Fleming, Ingrid; Bauersachs, Johann; Schäfer, Andreas; Scholz, Dimitri; Aldershvile, Jan; Busse, Rudi

    1999-01-01

    Shear stress and tyrosine phosphatase inhibitors have been shown to activate the endothelial NO synthase (eNOS) in a Ca2+/calmodulin-independent manner. We report here that isometric contraction of rabbit aorta activates eNOS by a pharmacologically identical pathway. Endothelium-intact aortic rings were precontracted under isometric conditions up to 60% of the maximal phenylephrine-induced tone. The NO synthase inhibitor NGnitro-l-arginine (l-NA) and the soluble guanylyl cyclase inhibitor NS 2028 induced an additional contraction, the amplitude of which depended on the level of precontraction. The maximal production of NO by isometrically contracted aortic rings (as estimated by the increase in cGMP in detector smooth muscle cells in a superfusion bioassay) was observed during the initial phase of isometric contraction and was greater than that detected following the application of acetylcholine. The supplementary l-NA-induced increase in vascular tone was inhibited by the nonselective kinase inhibitor staurosporine and the tyrosine kinase inhibitors erbstatin A and herbimycin A. Another tyrosine kinase inhibitor, genistein, the calmodulin antagonist calmidazolium, and the selective protein kinase C inhibitor, Ro 31–8220, had no effect. Coincident with the enhanced NO formation during isometric contraction was an increase in the tyrosine phosphorylation of endothelial proteins, which also correlated with the level of precontraction. Thus, isometric contraction activates eNOS via a Ca2+-independent, tyrosine kinase inhibitor-sensitive pathway and, like shear stress, seems to be an independent determinant of mechanically induced NO formation. PMID:9927704

  2. 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.

  3. Relationship of Cell-Free Hemoglobin to Impaired Endothelial Nitric Oxide Bioavailability and Perfusion in Severe Falciparum Malaria

    PubMed Central

    Yeo, Tsin W.; Lampah, Daniel A.; Tjitra, Emiliana; Gitawati, Retno; Kenangalem, Enny; Piera, Kim; Granger, Donald L.; Lopansri, Bert K.; Weinberg, J. Brice; Price, Ric N.; Duffull, Stephen B.; Celermajer, David S.; Anstey, Nicholas M.

    2013-01-01

    Background Hemolysis causes anemia in falciparum malaria, but its contribution to microvascular pathology in severe malaria (SM) is not well characterized. In other hemolytic diseases, release of cell-free hemoglobin causes nitric oxide (NO) quenching, endothelial activation, and vascular complications. We examined the relationship of plasma hemoglobin and myoglobin to endothelial dysfunction and disease severity in malaria. Methods Cell-free hemoglobin (a potent NO quencher), reactive hyperemia peripheral arterial tonometry (RH-PAT) (a measure of endothelial NO bioavailability), and measures of perfusion and endothelial activation were quantified in adults with moderately severe (n = 78) or severe (n = 49) malaria and control subjects (n = 16) from Papua, Indonesia. Results Cell-free hemoglobin concentrations in patients with SM (median, 5.4 μmol/L; interquartile range [IQR], 3.2–7.4 μmol/L) were significantly higher than in those with moderately severe malaria (2.6 μmol/L; IQR, 1.3–4.5 μmol/L) or controls (1.2 μmol/L; IQR, 0.9–2.4 μmol/L; P < .001). Multivariable regression analysis revealed that cell-free hemoglobin remained inversely associated with RH-PAT, and in patients with SM, there was a significant longitudinal association between improvement in RH-PAT index and decreasing levels of cell-free hemoglobin (P = .047). Cell-free hemoglobin levels were also independently associated with lactate, endothelial activation, and proinflammatory cytokinemia. Conclusions Hemolysis in falciparum malaria results in NO quenching by cell-free hemoglobin, and may exacerbate endothelial dysfunction, adhesion receptor expression and impaired tissue perfusion. Treatments that increase NO bioavailability may have potential as adjunctive therapies in SM. PMID:19803726

  4. 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

  5. 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

  6. In Silico Modeling of Shear-Stress-Induced Nitric Oxide Production in Endothelial Cells through Systems Biology

    PubMed Central

    Koo, Andrew; Nordsletten, David; Umeton, Renato; Yankama, Beracah; Ayyadurai, Shiva; García-Cardeña, Guillermo; Dewey, C. Forbes

    2013-01-01

    Nitric oxide (NO) produced by vascular endothelial cells is a potent vasodilator and an antiinflammatory mediator. Regulating production of endothelial-derived NO is a complex undertaking, involving multiple signaling and genetic pathways that are activated by diverse humoral and biomechanical stimuli. To gain a thorough understanding of the rich diversity of responses observed experimentally, it is necessary to account for an ensemble of these pathways acting simultaneously. In this article, we have assembled four quantitative molecular pathways previously proposed for shear-stress-induced NO production. In these pathways, endothelial NO synthase is activated 1), via calcium release, 2), via phosphorylation reactions, and 3), via enhanced protein expression. To these activation pathways, we have added a fourth, a pathway describing actual NO production from endothelial NO synthase and its various protein partners. These pathways were combined and simulated using CytoSolve, a computational environment for combining independent pathway calculations. The integrated model is able to describe the experimentally observed change in NO production with time after the application of fluid shear stress. This model can also be used to predict the specific effects on the system after interventional pharmacological or genetic changes. Importantly, this model reflects the up-to-date understanding of the NO system, providing a platform upon which information can be aggregated in an additive way. PMID:23708369

  7. Relationships between endothelial nitric oxide synthase gene polymorphisms and osteoporosis in postmenopausal women*

    PubMed Central

    Liu, Shun-zhi; Yan, Hong; Hou, Wei-kun; Xu, Peng; Tian, Juan; Tian, Li-fang; Zhu, Bo-feng; Ma, Jie; Lu, She-min

    2009-01-01

    Objective: To investigate the relationships between endothelial nitric oxide synthases (eNOS) G894T and 27 bp-variable number tandem repeat (VNTR) gene polymorphisms and osteoporosis in the postmenopausal women of Chinese Han nationality. Methods: In the present study, 281 postmenopausal women from Xi’an urban area in West China were recruited, and divided into osteoporosis, osteopenia, and normal groups according to the diagnostic criteria of osteoporosis proposed by World Health Organization (WHO). The bone mineral density (BMD) values of lumbar vertebrae and left hips were determined by QDR-2000 dual energy X-ray absorptiometry. Blood samples were tested for plasma biochemical indicators including testosterone, estradiol, calcitonin, osteocalcin, and procollagen type I amino-terminal propeptide by enzyme-linked immunosorbent assay (ELISA), tartrate-resistant acid phosphatase by spectrophotometric method, and the content of nitric oxide by Griess method. Genome DNA was extracted from whole blood, and G894T polymorphism of eNOS gene was analyzed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and 27 bp-VNTR polymorphism of eNOS gene was genotyped by PCR method. Then the relationships between genotypes and biochemical indicators, genotypes and osteoporosis, and haplotypes and osteoporosis were analyzed. Results: The average BMD values of the femoral neck, ward’s triangle and lumbar vertebrae 1~4 (L1~L4) in the subjects with T/T genotype in eNOS G894T locus were significantly higher than those in the subjects with G/T and G/G genotypes (P<0.05). The average BMD of the femoral neck in the subjects with a/a genotype of eNOS 27 bp-VNTR locus was evidently higher than that in the subjects with b/b genotype (P<0.05). The plasma testosterone and osteocalcin concentrations in the subjects of eNOS G894T G/T genotype were evidently higher than those in the subjects of other genotypes (P<0.05); the plasma estradiol

  8. 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.

  9. Arginase inhibition augments nitric oxide production and facilitates left ventricular systolic function in doxorubicin‐induced cardiomyopathy in mice

    PubMed Central

    Toya, Takumi; Hakuno, Daihiko; Shiraishi, Yasunaga; Kujiraoka, Takehiko; Adachi, Takeshi

    2014-01-01

    Abstract A metabolizing enzyme arginase can decrease nitric oxide (NO) production by competing with NO synthase for arginine as a substrate, but its pathophysiological role in heart failure remains unknown. We aimed to investigate the effect of pharmacological inhibition of arginase on left ventricular function in doxorubicin‐induced cardiomyopathy in mice. Doxorubicin administration for 5 weeks significantly increased protein expression levels or activity of arginase in the lungs and liver, and caused moderate increase in arginase 2 expression in the aorta. In the lungs, accumulated interstitial cells strongly expressed both arginase 1 and arginase 2 by doxorubicin administration. Echocardiography revealed that administration of a potent, reversible arginase inhibitor N‐omega‐hydroxy‐nor‐l‐arginine completely reversed doxorubicin‐induced decrease in the ejection fraction, in parallel with expression levels of BNP mRNA, without affecting apoptosis, hypertrophy, fibrosis, or macrophage infiltration in the left ventricle. Arginase inhibition reversibly lowered systolic blood pressure, and importantly, it recovered doxorubicin‐induced decline in NO concentration in the serum, lungs, and aorta. Furthermore, arginase inhibition stimulated NO secretion from aortic endothelial cells and peritoneal macrophages in vitro. In conclusion, pharmacological inhibition of arginase augmented NO concentration in the serum, lungs, and aorta, promoted NO‐mediated decrease in afterload for left ventricle, and facilitated left ventricular systolic function in doxorubicin‐induced cardiomyopathy in mice. PMID:25263201

  10. 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

  11. Posttranslational inactivation of endothelial nitric oxide synthase in the transgenic sickle cell mouse penis

    PubMed Central

    Musicki, Biljana; Champion, Hunter C.; Hsu, Lewis L.; Bivalacqua, Trinity J.; Burnett, Arthur L.

    2017-01-01

    INTRODUCTION Sickle cell disease (SCD)-associated priapism is characterized by endothelial nitric oxide synthase (eNOS) dysfunction in the penis. However, the mechanism of decreased eNOS function/activation in the penis in association with SCD is not known. AIMS Our hypothesis in the present study was that eNOS is functionally inactivated in the SCD penis in association with impairments in eNOS posttranslational phosphorylation and the enzyme’s interactions with its regulatory proteins. METHODS Sickle cell transgenic (sickle) mice were used as an animal model of SCD. Wild type (WT) mice served as controls. Penes were excised at baseline for molecular studies. eNOS phosphorylation on Ser-1177 (positive regulatory site) and Thr-495 (negative regulatory site), total eNOS, and phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177) expressions, and eNOS interactions with heat shock protein 90 (HSP90) and caveolin-1 were measured by Western blot. Constitutive NOS catalytic activity was measured by conversion of L-[14C]arginine-to-L-[14C]citrulline in the presence of calcium. MAIN OUTCOME MEASURES Molecular mechanisms of eNOS dysfunction in the sickle mouse penis. RESULTS eNOS phosphorylated on Ser-1177, an active portion of eNOS, was decreased in the sickle mouse penis compared to WT penis. eNOS interaction with its positive protein regulator HSP90, but not with its negative protein regulator caveolin-1, and phosphorylated AKT expression, as well as constitutive NOS activity, were also decreased in the sickle mouse penis compared to WT penis. eNOS phosphorylated on Thr-495, total eNOS, HSP90, and caveolin-1 protein expressions in the penis were not affected by SCD. CONCLUSION These findings provide a molecular basis for chronically reduced eNOS function in the penis by SCD, which involves decreased eNOS phosphorylation on Ser-1177 and decreased eNOS-HSP90 interaction. PMID:21143412

  12. Endothelial S100A1 modulates vascular function via nitric oxide.

    PubMed

    Pleger, Sven T; Harris, David M; Shan, Changguang; Vinge, Leif E; Chuprun, J Kurt; Berzins, Brett; Pleger, Wiebke; Druckman, Charles; Völkers, Mirko; Heierhorst, Jörg; Øie, Erik; Remppis, Andrew; Katus, Hugo A; Scalia, Rosario; Eckhart, Andrea D; Koch, Walter J; Most, Patrick

    2008-04-11

    S100A1, a Ca(2+)-binding protein of the EF-hand type, is known to modulate sarcoplasmic reticulum Ca(2+) handling in skeletal muscle and cardiomyocytes. Recently, S100A1 has been shown to be expressed in endothelial cells (ECs). Because intracellular Ca(2+) ([Ca(2+)](i)) transients can be involved in important EC functions and endothelial NO synthase activity, we sought to investigate the impact of endothelial S100A1 on the regulation of endothelial and vascular function. Thoracic aortas from S100A1 knockout mice (SKO) showed significantly reduced relaxation in response to acetylcholine compared with wild-type vessels, whereas direct vessel relaxation using sodium nitroprusside was unaltered. Endothelial dysfunction attributable to the lack of S100A1 expression could also be demonstrated in vivo and translated into hypertension of SKO. Mechanistically, both basal and acetylcholine-induced endothelial NO release of SKO aortas was significantly reduced compared with wild type. Impaired endothelial NO production in SKO could be attributed, at least in part, to diminished agonist-induced [Ca(2+)](i) transients in ECs. Consistently, silencing endothelial S100A1 expression in wild type also reduced [Ca(2+)](i) and NO generation. Moreover, S100A1 overexpression in ECs further increased NO generation that was blocked by the inositol-1,4,5-triphosphate receptor blocker 2-aminoethoxydiphenylborate. Finally, cardiac endothelial S100A1 expression was shown to be downregulated in heart failure in vivo. Collectively, endothelial S100A1 critically modulates vascular function because lack of S100A1 expression leads to decreased [Ca(2+)](i) and endothelial NO release, which contributes, at least partially, to impaired endothelium-dependent vascular relaxation and hypertension in SKO mice. Targeting endothelial S100A1 expression may, therefore, be a novel therapeutic means to improve endothelial function in vascular disease or heart failure.

  13. 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.

  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. 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

  16. 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.

  17. Erection capability is potentiated by long-term sildenafil treatment: role of blood flow-induced endothelial nitric-oxide synthase phosphorylation.

    PubMed

    Musicki, Biljana; Champion, Hunter C; Becker, Robyn E; Liu, Tongyun; Kramer, Melissa F; Burnett, Arthur L

    2005-07-01

    Despite demonstrated clinical efficacy of sildenafil for the temporary treatment of erectile dysfunction, the possibility that sildenafil used long-term durably augments erectile ability remains unclear. We investigated whether continuous long-term administration of sildenafil at clinically relevant levels to aged rats "primes" the penis for improved erectile ability and involves nitric oxide (NO) or RhoA/Rho-kinase signaling pathways. In aged, but not young rats, sildenafil prolonged erection and increased the protein expressions of phosphorylated endothelial NO synthase (eNOS) at serine-1177 and phosphorylated Akt at serine-473 in penes. Only in the young rat penis, protein expressions of phosphodiesterase-5 and phosphomyosin phosphatase target subunit 1, a marker of Rho-kinase activity, were increased by sildenafil. Sildenafil inhibited phosphodiesterase-5 activity in penes of young and aged rats coincident with assayed free plasma levels of the drug equivalent to clinically therapeutic measurements. We conclude that erectile ability can be enhanced under preconditions of erectile impairment by long-term inhibition of phosphodiesterase-5 and that the effect is mediated by Akt-dependent eNOS phosphorylation. The lack of erectile ability enhancement in young rats by long-term phosphodiesterase-5 inhibition may relate to restrained NO signaling by phosphodiesterase-5 up-regulation, lack of incremental Akt and eNOS phosphorylation, and heightened Rho-kinase signaling in the penis.

  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. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. Inhibition of endothelial lipase activity by sphingomyelin in the lipoproteins

    PubMed Central

    Yang, Peng; Belikova, Natalia A.; Billheimer, Jeff; Rader, Daniel J.; Hill, John S.; Subbaiah, Papasani V.

    2014-01-01

    Endothelial lipase (EL) is a major determinant of plasma HDL concentration, its activity being inversely proportional to HDL levels. Although it is known that it preferentially acts on HDL, compared to LDL and VLDL, the basis for this specificity is not known. Here we tested the hypothesis that sphingomyelin, a major phospholipid in lipoproteins is a physiological inhibitor of EL, and that the preference of the enzyme for HDL may be due to low sphingomyelin/ phosphatidylcholine (PtdCho) ratio in HDL, compared to other lipoproteins. Using recombinant human EL, we showed that sphingomyelin inhibits the hydrolysis of PtdCho in the liposomes in a concentration-dependent manner. While the enzyme showed lower hydrolysis of LDL PtdCho, compared to HDL PtdCho, this difference disappeared after the degradation of lipoprotein sphingomyelin by bacterial sphingomyelinase. Analysis of molecular species of PtdCho hydrolyzed by EL in the lipoproteins showed that the enzyme preferentially hydrolyzed PtdCho containing polyunsaturated fatty acids (PUFA) such as 22:6, 20:5, 20:4 at sn-2 position, generating the corresponding PUFA-lyso PtdCho. This specificity for PUFA-PtdCho species was not observed after depletion of sphingomyelin by sphingomyelinase. These results show that sphingomyelin not only plays a role in regulating EL activity, but also influences its specificity towards PtdCho species. PMID:25167836

  6. Inhibition of endothelial lipase activity by sphingomyelin in the lipoproteins.

    PubMed

    Yang, Peng; Belikova, Natalia A; Billheimer, Jeff; Rader, Daniel J; Hill, John S; Subbaiah, Papasani V

    2014-10-01

    Endothelial lipase (EL) is a major determinant of plasma HDL concentration, its activity being inversely proportional to HDL levels. Although it is known that it preferentially acts on HDL compared to LDL and VLDL, the basis for this specificity is not known. Here we tested the hypothesis that sphingomyelin, a major phospholipid in lipoproteins is a physiological inhibitor of EL, and that the preference of the enzyme for HDL may be due to low sphingomyelin/phosphatidylcholine (PtdCho) ratio in HDL, compared to other lipoproteins. Using recombinant human EL, we showed that sphingomyelin inhibits the hydrolysis of PtdCho in the liposomes in a concentration-dependent manner. While the enzyme showed lower hydrolysis of LDL PtdCho, compared to HDL PtdCho, this difference disappeared after the degradation of lipoprotein sphingomyelin by bacterial sphingomyelinase. Analysis of molecular species of PtdCho hydrolyzed by EL in the lipoproteins showed that the enzyme preferentially hydrolyzed PtdCho containing polyunsaturated fatty acids (PUFA) such as 22:6, 20:5, 20:4 at the sn-2 position, generating the corresponding PUFA-lyso PtdCho. This specificity for PUFA-PtdCho species was not observed after depletion of sphingomyelin by sphingomyelinase. These results show that sphingomyelin not only plays a role in regulating EL activity, but also influences its specificity towards PtdCho species.

  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. Methylene blue inhibits angiogenesis in chick chorioallontic membrane through a nitric oxide-independent mechanism

    PubMed Central

    Zacharakis, N; Tone, P; Flordellis, CS; Maragoudakis, ME; Tsopanoglou, NE

    2006-01-01

    Angiogenesis is the process of generating new blood vessels from preexisting vessels and is considered essential in many pathological conditions. The purpose of the present study was to evaluate the effect of methylene blue in chick chorioallantoic membrane angiogenesis model in vivo. In this well characterized model, methylene blue inhibited angiogenesis in a concentration-dependent manner. In addition, when methylene blue was combined with sodium nitroprusside, a spontaneous generator of nitric oxide, an inhibition of angiogenesis was evident which was comparable with that observed by the application of methylene blue alone. Sodium nitroprusside, alone, caused a significant inhibition in basal angiogenesis. These results provide evidence that methylene blue inhibits angiogenesis independently of nitric oxide pathway and suggest that methylene blue may be useful for treating angiogenesis-dependent human diseases. PMID:16796814

  9. 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.

  10. Humic acid induces the endothelial nitric oxide synthase phosphorylation at Ser1177 and Thr495 Via Hsp90α and Hsp90β upregulation in human umbilical vein endothelial cells.

    PubMed

    Tanaka, Masato; Miyajima, Miki; Hishioka, Naoko; Nishimura, Ryo; Kihara, Yusuke; Hosokawa, Toshiyuki; Kurasaki, Masaaki; Tanaka, Shunitz; Saito, Takeshi

    2015-02-01

    Humic acid (HA) has been implicated as a contributory factor for blackfoot disease, which is an endemic peripheral vascular disease. We investigated the effect of HA on the regulation of endothelial nitric oxide (NO) synthase (eNOS) in human umbilical vein endothelial cells (HUVECs) to evaluate the involvement of eNOS and related factors in peripheral vascular impairment with HA exposure. Treatment of HUVECs with HA induced upregulation of eNOS. This result coincides with those of previous studies. Furthermore this is the first study to report that HA induces upregulation of heat shock protein (Hsp)90α, Hsp90β, eNOS phosphorylation at Ser1177, and eNOS phosphorylation at Thr495, as compared to that in the control. In contrast, treatment with BAPTA, an intracellular Ca(2+) chelator, inhibited upregulation of these proteins induced by HA. This study demonstrates that HA treatment leads to increases in both Hsp90α and Hsp90β proteins and indicates that Hsp90α leads to eNOS phosphorylation at Ser1177 and that Hsp90β leads to eNOS phosphorylation at Thr495, respectively. Upregulation of eNOS, Hsp90α, and Hsp90β in HUVECs is regulated by intracellular Ca(2+) accumulation induced by HA. These results suggest that upregulation of eNOS phosphorylation at Ser1177 and eNOS phosphorylation at Thr495 produce NO and superoxide anions, respectively, resulting in generation of peroxynitrite, which causes impairment of vascular endothelial cells. © 2013 Wiley Periodicals, Inc.

  11. 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.

  12. Modulation of cerebral RAGE expression following nitric oxide synthase inhibition in rats subjected to focal cerebral ischemia.

    PubMed

    Greco, Rosaria; Demartini, Chiara; Zanaboni, Anna Maria; Blandini, Fabio; Amantea, Diana; Tassorelli, Cristina

    2017-04-05

    The receptor for advanced glycation endproducts (RAGE) is a key mediator of neuroinflammation following cerebral ischemia. Nitric oxide (NO) plays a dualistic role in cerebral ischemia, depending on whether it originates from neuronal, inducible or endothelial synthase. Although a dynamic interplay between RAGE and NO pathways exists, its relevance in ischemic stroke has not been investigated. The aim of this study is to evaluate the effect of the NO synthase (NOS) inhibition on RAGE expression in rats subjected to transient middle cerebral artery occlusion (tMCAo). Full-length (fl-RAGE) gene expression was elevated in the striatum and, to a lesser extent, in the cortex of rats undergone tMCAo. The exacerbation of cortical damage caused by systemic administration of L-N-(1-iminoethyl)ornithine (L-NIO), a relatively selective inhibitor of endothelial NOS (eNOS), was associated with elevated mRNA levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α and fl-RAGE in both the cortex and the striatum. Conversely, NG-nitro-l-arginine methyl ester (L-NAME), a non-selective NOS inhibitor, decreased cortical damage, did not affect cerebral cytokine mRNA levels, while it increased fl-RAGE mRNA expression only in the striatum. Fl-RAGE striatal protein levels varied accordingly with observed mRNA changes in the striatum, while in the cortex, RAGE protein levels were reduced by tMCAo and further decreased following L-NIO treatment. Modulation of RAGE expression by different inhibitors of NOS may have opposite effects on transient cortical ischemia: the non selective inhibition of NOS activity is protective, while the selective inhibition of eNOS is harmful, probably via the activation of inflammatory pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. The mechanism of cytochrome C oxidase inhibition by nitric oxide.

    PubMed

    Antunes, Fernando; Cadenas, Enrique

    2007-01-01

    The basic biochemistry of the inhibition of cytochrome oxidase by NO is reviewed. Three possible mechanisms that include the binding of NO to the fully reduced Fe(a3)-Cu(B) site, to the semi-reduced Fe(a3)-Cu(B) site, and to the fully oxidized Fe(a3)-Cu(B) site are confronted with the experimental data. Mathematical models are used to facilitate the analysis and to solve puzzling observations concerning the NO inhibition of cytochrome oxidase. It is concluded that the inhibition of cytochrome oxidase by NO is mixed, having both competitive and uncompetitive components, but under physiological electron flows the competitive component is largely predominant. The physiological and pathological relevance of this inhibition is briefly discussed.

  3. Hsp90β inhibition modulates nitric oxide production and nitric oxide-induced apoptosis in human chondrocytes

    PubMed Central

    2011-01-01

    Background Hsp90β is a member of the Hsp90 family of protein chaperones. This family plays essential roles in the folding, maturation and activity of many proteins that are involved in signal transduction and transcriptional regulation. The role of this protein in chondrocytes is not well understood, although its increase in osteoarthritic cells has been reported. The present study aimed to explore the role of Hsp90β in key aspects of OA pathogenesis. Methods Human OA chondrocytes were isolated from cartilage obtained from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with proinflammatory cytokines (IL-1β or TNF-α) and nitric oxide donors (NOC-12 or SNP). For Hsp90β inhibition, two different chemical inhibitors (Geldanamycin and Novobiocin) were employed, or siRNA transfection procedures were carried out. Gene expression was determined by real-time PCR, apoptosis was quantified by flow cytometry and ELISA, and nitric oxide (NO) production was evaluated by the Griess method. Indirect immunofluorescence assays were performed to evaluate the presence of Hsp90β in stimulated cells. Results Hsp90β was found to be increased by proinflammatory cytokines. Inhibition of Hsp90β by the chemicals Geldanamycin (GA) and Novobiocin (NB) caused a dose-dependent decrease of the NO production induced by IL-1β in chondrocytes, up to basal levels. Immunofluorescence analyses demonstrate that the NO donors NOC-12 and SNP also increased Hsp90β. Chemical inhibition or specific gene silencing of this chaperone reduced the DNA condensation and fragmentation, typical of death by apoptosis, that is induced by NO donors in chondrocytes. Conclusions The present results show how Hsp90β modulates NO production and NO-mediated cellular death in human OA chondrocytes. PMID:22004293

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. Role of microRNAs 221/222 on statin induced nitric oxide release in human endothelial cells.

    PubMed

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

    2015-03-01

    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. 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. 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. 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. 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.

  10. Endothelin-2 signaling in the neural retina promotes the endothelial tip cell state and inhibits angiogenesis

    PubMed Central

    Rattner, Amir; Yu, Huimin; Williams, John; Smallwood, Philip M.; Nathans, Jeremy

    2013-01-01

    Endothelin signaling is required for neural crest migration and homeostatic regulation of blood pressure. Here, we report that constitutive overexpression of Endothelin-2 (Edn2) in the mouse retina perturbs vascular development by inhibiting endothelial cell migration across the retinal surface and subsequent endothelial cell invasion into the retina. Developing endothelial cells exist in one of two states: tip cells at the growing front and stalk cells in the vascular plexus behind the front. This division of endothelial cell states is one of the central organizing principles of angiogenesis. In the developing retina, Edn2 overexpression leads to overproduction of endothelial tip cells by both morphologic and molecular criteria. Spatially localized overexpression of Edn2 produces a correspondingly localized endothelial response. Edn2 overexpression in the early embryo inhibits vascular development at midgestation, but Edn2 overexpression in developing skin and brain has no discernible effect on vascular structure. Inhibition of retinal angiogenesis by Edn2 requires expression of Endothelin receptor A but not Endothelin receptor B in the neural retina. Taken together, these observations imply that the neural retina responds to Edn2 by synthesizing one or more factors that promote the endothelial tip cell state and inhibit angiogenesis. The response to Edn2 is sufficiently potent that it overrides the activities of other homeostatic regulators of angiogenesis, such as Vegf. PMID:24043815

  11. 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.

  12. 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.

  13. Iron ions derived from the nitric oxide donor sodium nitroprusside inhibit mineralization.

    PubMed

    Huitema, Leonie F A; van Weeren, P René; Barneveld, Ab; van de Lest, Chris H A; Helms, J Bernd; Vaandrager, Arie B

    2006-08-07

    Sodium nitroprusside (SNP) is a nitric oxide (NO) donor drug, which is therapeutically used as a vasodilating drug in heart transplantations. In our previous study it was found that SNP at a concentration of 100 microM inhibited mineralization in a cell culture system, indicating that the beneficial effects of this drug may also include inhibition of vascular calcification. The aim of this study was to investigate which bioactive compounds generated from SNP inhibit mineralization. ATDC5 cells were grown for 14 days and mineralization was induced by addition of 5 mM phosphate for 24 h. Mineralization was determined by staining precipitated calcium with an alizarin red stain. It was found that the NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine were not able to inhibit mineralization and NO scavengers could not antagonize the inhibiting effect of SNP on mineralization. The iron chelator deferoxamine (200 microM) antagonized the inhibiting effect on mineralization mediated by SNP and ammonium iron sulfate inhibited mineralization in a dose-dependent manner (10-100 microM). Furthermore, iron ions (30 microM) were detected to be released from SNP in the cell culture. These data show that the iron moiety of sodium nitroprusside, rather than nitric oxide inhibits mineralization.

  14. 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.

  15. 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.

  16. Inhibition of Insulin Signaling in Endothelial Cells by Protein Kinase C-induced Phosphorylation of p85 Subunit of Phosphatidylinositol 3-Kinase (PI3K)*

    PubMed Central

    Maeno, Yasuhiro; Li, Qian; Park, Kyoungmin; Rask-Madsen, Christian; Gao, Benbo; Matsumoto, Motonobu; Liu, Yingjie; Wu, I-Hsien; White, Morris F.; Feener, Edward P.; King, George L.

    2012-01-01

    The regulation of endothelial function by insulin is consistently abnormal in insulin-resistant states and diabetes. Protein kinase C (PKC) activation has been reported to inhibit insulin signaling selectively in endothelial cells via the insulin receptor substrate/PI3K/Akt pathway to reduce the activation of endothelial nitric-oxide synthase (eNOS). In this study, it was observed that PKC activation differentially inhibited insulin receptor substrate 1/2 (IRS1/2) signaling of insulin's activation of PI3K/eNOS by decreasing only tyrosine phosphorylation of IRS2. In addition, PKC activation, by general activator and specifically by angiotensin II, increased the phosphorylation of p85/PI3K, which decreases its association with IRS1 and activation. Thr-86 of p85/PI3K was identified to be phosphorylated by PKC activation and confirmed to affect IRS1-mediated activation of Akt/eNOS by insulin and VEGF using a deletion mutant of the Thr-86 region of p85/PI3K. Thus, PKC and angiotensin-induced phosphorylation of Thr-86 of p85/PI3K may partially inhibit the activation of PI3K/eNOS by multiple cytokines and contribute to endothelial dysfunction in metabolic disorders. PMID:22158866

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. Endothelial nitric oxide synthase uncoupling and microvascular dysfunction in the mesentery of mice deficient in α-galactosidase A

    PubMed Central

    Kang, Justin J.; Shu, Liming; Park, James L.; Bodary, Peter F.

    2013-01-01

    A defect in the gene for the lysosomal enzyme α-galactosidase A (Gla) results in globotriaosylceramide (Gb3) accumulation in Fabry disease and leads to premature death from cardiac and cerebrovascular events. However, gastrointestinal symptoms are often first observed during childhood in these patients and are not well understood. In this study, we demonstrate an age-dependent microvasculopathy of the mesenteric artery (MA) in a murine model of Fabry disease (Gla-knockout mice) resulting from dysregulation of the vascular homeostatic enzyme endothelial nitric oxide synthase (eNOS). The progressive accumulation of Gb3 in the MA was confirmed by thin-layer chromatographic analysis. A total absence of endothelium-dependent dilation was observed in MAs from mice at 8 mo of age, while suppression of ACh-mediated vasodilation was evident from 2 mo of age. Endothelium-independent dilation with sodi