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Sample records for endothelial nos estrogen

  1. Endothelial NOS, estrogen receptor β, and HIFs cooperate in the activation of a prognostic transcriptional pattern in aggressive human prostate cancer

    PubMed Central

    Nanni, Simona; Benvenuti, Valentina; Grasselli, Annalisa; Priolo, Carmen; Aiello, Aurora; Mattiussi, Stefania; Colussi, Claudia; Lirangi, Vittoria; Illi, Barbara; D’Eletto, Manuela; Cianciulli, Anna Maria; Gallucci, Michele; De Carli, Piero; Sentinelli, Steno; Mottolese, Marcella; Carlini, Paolo; Strigari, Lidia; Finn, Stephen; Mueller, Elke; Arcangeli, Giorgio; Gaetano, Carlo; Capogrossi, Maurizio C.; Donnorso, Raffaele Perrone; Bacchetti, Silvia; Sacchi, Ada; Pontecorvi, Alfredo; Loda, Massimo; Farsetti, Antonella

    2009-01-01

    The identification of biomarkers that distinguish between aggressive and indolent forms of prostate cancer (PCa) is crucial for diagnosis and treatment. In this study, we used cultured cells derived from prostate tissue from patients with PCa to define a molecular mechanism underlying the most aggressive form of PCa that involves the functional activation of eNOS and HIFs in association with estrogen receptor β (ERβ). Cells from patients with poor prognosis exhibited a constitutively hypoxic phenotype and increased NO production. Upon estrogen treatment, formation of ERβ/eNOS, ERβ/HIF-1α, or ERβ/HIF-2α combinatorial complexes led to chromatin remodeling and transcriptional induction of prognostic genes. Tissue microarray analysis, using an independent cohort of patients, established a hierarchical predictive power for these proteins, with expression of eNOS plus ERβ and nuclear eNOS plus HIF-2α being the most relevant indicators of adverse clinical outcome. Genetic or pharmacologic modulation of eNOS expression and activity resulted in reciprocal conversion of the transcriptional signature in cells from patients with bad or good outcome, respectively, highlighting the relevance of eNOS in PCa progression. Our work has considerable clinical relevance, since it may enable the earlier diagnosis of aggressive PCa through routine biopsy assessment of eNOS, ERβ, and HIF-2α expression. Furthermore, proposing eNOS as a therapeutic target fosters innovative therapies for PCa with NO inhibitors, which are employed in preclinical trials in non-oncological diseases. PMID:19363294

  2. Estrogen-induced DNA synthesis in vascular endothelial cells is mediated by ROS signaling

    PubMed Central

    Felty, Quentin

    2006-01-01

    Background Since estrogen is known to increase vascular endothelial cell growth, elevated estrogen exposure from hormone replacement therapy or oral contraceptives has the potential to contribute in the development of abnormal proliferative vascular lesions and subsequent thickening of the vasculature. How estrogen may support or promote vascular lesions is not clear. We have examined in this study whether estrogen exposure to vascular endothelial cells increase the formation of reactive oxygen species (ROS), and estrogen-induced ROS is involved in the growth of endothelial cells. Methods The effect of estrogen on the production of intracellular oxidants and the role of estrogen-induced ROS on cell growth was studied in human umbilical vein endothelial cells. ROS were measured by monitoring the oxidation of 2'7'-dichlorofluorescin by spectrofluorometry. Endothelial cell growth was measured by a colorimetric immunoassay based on BrdU incorporation into DNA. Results Physiological concentrations of estrogen (367 fmol and 3.67 pmol) triggered a rapid 2-fold increase in intracellular oxidants in endothelial cells. E2-induced ROS formation was inhibited to basal levels by cotreatment with the mitochondrial inhibitor rotenone (2 μM) and xanthine oxidase inhibitor allopurinol (50 μM). Inhibitors of NAD(P)H oxidase, apocynin and DPI, did not block E2-induced ROS formation. Furthermore, the NOS inhibitor, L-NAME, did not prevent the increase in E2-induced ROS. These findings indicate both mitochondria and xanthine oxidase are the source of ROS in estrogen treated vascular endothelial cells. E2 treated cells showed a 2-fold induction of BrdU incorporation at 18 h which was not observed in cells exposed to vehicle alone. Cotreatment with ebselen (20 μM) and NAC (1 mM) inhibited E2-induced BrdU incorporation without affecting the basal levels of DNA synthesis. The observed inhibitory effect of NAC and ebselen on E2-induced DNA synthesis was also shown to be dose dependent

  3. Estrogen-Responsive nitroso-Proteome in Uterine Artery Endothelial Cells: Role of Endothelial Nitric Oxide Synthase and Estrogen Receptor-β

    PubMed Central

    Zhang, Hong-hai; Feng, Lin; Wang, Wen; Magness, Ronald R.; Chen, Dong-bao

    2011-01-01

    Covalent adduction of a NO moiety to cysteines (S-nitrosylation or SNO) is a major route for NO to directly regulate protein functions. In uterine artery endothelial cells (UAEC), estradiol-17β (E2) rapidly stimulated protein SNO that maximized within 10-30 min post-E2 exposure. E2-bovine serum albumin stimulated protein SNO similarly. Stimulation of SNO by both was blocked by ICI 182, 780, implicating mechanisms linked to specific estrogen receptors (ERs) localized on the plasma membrane. E2-induced protein SNO was attenuated by selective ERβ, but not ERα, antagonists. A specific ERβ but not ERα agonist was able to induce protein SNO. Overexpression of ERβ, but not ERα, significantly enhanced E2-induced SNO. Overexpression of both ERs increased basal SNO, but did not further enhance E2-stimulated SNO. E2-induced SNO was inhibited by N-nitro-L-arginine-methylester and specific endothelial NO synthase (eNOS) siRNA. Thus, estrogen-induced SNO is mediated by endogenous NO via eNOS and mainly ERβ in UAEC. We further analyzed the nitroso-proteomes by CyDye switch technique combined with two dimensional (2D) fluorescence difference gel electrophoresis. Numerous nitrosoprotein (spots) were visible on the 2D gel. Sixty spots were chosen and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Among the 54 identified, 9 were novel SNO-proteins, 32 were increased, 8 were decreased, and the rest were unchanged by E2. Tandom MS identified Cys139 as a specific site for SNO in GAPDH. Pathway analysis of basal and estrogen-responsive nitroso-proteomes suggested that SNO regulates diverse protein functions, directly implicating SNO as a novel mechanism for estrogen to regulate uterine endothelial function and thus uterine vasodilatation. PMID:21374595

  4. Plasma membrane localization and function of the estrogen receptor α variant (ER46) in human endothelial cells

    PubMed Central

    Li, Lei; Haynes, M. Page; Bender, Jeffrey R.

    2003-01-01

    Estrogen receptor (ER) α variants have been identified in an array of nonendothelial cells. We previously demonstrated that estrogen rapidly induces nitric oxide release via a phosphatidylinositol 3-kinase/Akt/endothelial nitric-oxide synthase (eNOS) pathway in EA.hy926 cells (immortalized human endothelial cells), which express a 46-kDa ER. We now confirm that, due to alternative splicing, the 46-kDa endothelial cell protein (ER46) is an amino-terminal truncated product of full-length ERα (ER66). ER46 is expressed in the plasma membrane, cytosol, and nucleus of resting, estrogen-deprived cells. Flow cytometric and immunofluorescence microscopic analyses demonstrated that the ER46 C but not N terminus is Ab-accessible in the plasma membrane. Inhibition of palmitoylation with tunicamycin and [3H]palmitic acid labeling demonstrated an estrogen-induced, palmitoylation-dependent plasma membrane ER46 recruitment, with reorganization into caveolae. In reconstituted, estrogen-stimulated COS-7 (ER-null) cells, membrane ER46 more efficiently triggered membrane eNOS phosphorylation than ER66. Conversely, ER66 more efficiently mediated estrogen response element reporter-gene transactivation than ER46. These results demonstrate that ER46 is localized and further dynamically targeted to the plasma membrane in a palmitoylation-dependent manner. ER46 more efficiently modulates membrane-initiated estrogen actions, including eNOS activation, than full-length ER66. These findings may have important implications in vascular-specific targeting of estrogen receptor agonists. PMID:12682286

  5. Estrogen Stimulates Homing of Endothelial Progenitor Cells to Endometriotic Lesions.

    PubMed

    Rudzitis-Auth, Jeannette; Nenicu, Anca; Nickels, Ruth M; Menger, Michael D; Laschke, Matthias W

    2016-08-01

    The incorporation of endothelial progenitor cells (EPCs) into microvessels contributes to the vascularization of endometriotic lesions. Herein, we analyzed whether this vasculogenic process is regulated by estrogen. Estrogen- and vehicle-treated human EPCs were analyzed for migration and tube formation. Endometriotic lesions were induced in irradiated FVB/N mice, which were reconstituted with bone marrow from FVB/N-TgN (Tie2/green fluorescent protein) 287 Sato mice. The animals were treated with 100 μg/kg β-estradiol 17-valerate or vehicle (control) over 7 and 28 days. Lesion growth, cyst formation, homing of green fluorescent protein(+)/Tie2(+) EPCs, vascularization, cell proliferation, and apoptosis were analyzed by high-resolution ultrasonography, caliper measurements, histology, and immunohistochemistry. Numbers of blood circulating EPCs were assessed by flow cytometry. In vitro, estrogen-treated EPCs exhibited a higher migratory and tube-forming capacity when compared with controls. In vivo, numbers of circulating EPCs were not affected by estrogen. However, estrogen significantly increased the number of EPCs incorporated into the lesions' microvasculature, resulting in an improved early vascularization. Estrogen further stimulated the growth of lesions, which exhibited massively dilated glands with a flattened layer of stroma. This was mainly because of an increased glandular secretory activity, whereas cell proliferation and apoptosis were not markedly affected. These findings indicate that vasculogenesis in endometriotic lesions is dependent on estrogen, which adds a novel hormonally regulated mechanism to the complex pathophysiology of endometriosis. PMID:27315780

  6. 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

  7. Estrogen Enhances Linkage in the Vascular Endothelial Calmodulin Network via a Feedforward Mechanism at the G Protein-coupled Estrogen Receptor 1.

    PubMed

    Tran, Quang-Kim; Firkins, Rachel; Giles, Jennifer; Francis, Sarah; Matnishian, Vahe; Tran, Phuong; VerMeer, Mark; Jasurda, Jake; Burgard, Michelle Ann; Gebert-Oberle, Briana

    2016-05-13

    Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca(2+) signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17β-estradiol (E2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca(2+) and Ca(2+)-CaM indicated that E2 also increases free Ca(2+)-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor α, the plasma membrane Ca(2+)-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor α and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-Gβγ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca(2+) efflux is reversed by E2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca(2+) signals and promote Ca(2+)-dependent CaM interactions with other CaM targets. Consequently, E2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E2 generates a feedforward loop via GPER/GPR30, which enhances Ca(2+)/CaM signals and functional linkage in the endothelial CaM target network. PMID:26987903

  8. 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.

  9. Nox2-dependent glutathionylation of endothelial NOS leads to uncoupled superoxide production and endothelial barrier dysfunction in acute lung injury

    PubMed Central

    Wu, Feng; Szczepaniak, William S.; Shiva, Sruti; Liu, Huanbo; Wang, Yinna; Wang, Ling; Wang, Ying; Kelley, Eric E.; Chen, Alex F.; Gladwin, Mark T.

    2014-01-01

    Microvascular barrier integrity is dependent on bioavailable nitric oxide (NO) produced locally by endothelial NO synthase (eNOS). Under conditions of limited substrate or cofactor availability or by enzymatic modification, eNOS may become uncoupled, producing superoxide in lieu of NO. This study was designed to investigate how eNOS-dependent superoxide production contributes to endothelial barrier dysfunction in inflammatory lung injury and its regulation. C57BL/6J mice were challenged with intratracheal LPS. Bronchoalveolar lavage fluid was analyzed for protein accumulation, and lung tissue homogenate was assayed for endothelial NOS content and function. Human lung microvascular endothelial cell (HLMVEC) monolayers were exposed to LPS in vitro, and barrier integrity and superoxide production were measured. Biopterin species were quantified, and coimmunoprecipitation (Co-IP) assays were performed to identify protein interactions with eNOS that putatively drive uncoupling. Mice exposed to LPS demonstrated eNOS-dependent increased alveolar permeability without evidence for altered canonical NO signaling. LPS-induced superoxide production and permeability in HLMVEC were inhibited by the NOS inhibitor nitro-l-arginine methyl ester, eNOS-targeted siRNA, the eNOS cofactor tetrahydrobiopterin, and superoxide dismutase. Co-IP indicated that LPS stimulated the association of eNOS with NADPH oxidase 2 (Nox2), which correlated with augmented eNOS S-glutathionylation both in vitro and in vivo. In vitro, Nox2-specific inhibition prevented LPS-induced eNOS modification and increases in both superoxide production and permeability. These data indicate that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to LPS. Furthermore, the results implicate Nox2-mediated eNOS-S-glutathionylation as a mechanism underlying LPS-induced eNOS uncoupling in the lung microvasculature. PMID:25326583

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

    SciTech Connect

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

    1994-01-15

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

  11. 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

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

    PubMed

    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; Berkowitz, Dan E

    2009-10-01

    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 N(G)-nitro-l-arginine methyl ester, and arginase, with 2S-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

  13. ER Alpha Rapid Signaling Is Required for Estrogen Induced Proliferation and Migration of Vascular Endothelial Cells

    PubMed Central

    Lu, Qing; Schnitzler, Gavin R.; Ueda, Kazutaka; Iyer, Lakshmanan K.; Diomede, Olga I.; Andrade, Tiffany; Karas, Richard H.

    2016-01-01

    Estrogen promotes the proliferation and migration of vascular endothelial cells (ECs), which likely underlies its ability to accelerate re-endothelialization and reduce adverse remodeling after vascular injury. In previous studies, we have shown that the protective effects of E2 (the active endogenous form of estrogen) in vascular injury require the estrogen receptor alpha (ERα). ERα transduces the effects of estrogen via a classical DNA binding, “genomic” signaling pathway and via a more recently-described “rapid” signaling pathway that is mediated by a subset of ERα localized to the cell membrane. However, which of these pathways mediates the effects of estrogen on endothelial cells is poorly understood. Here we identify a triple point mutant version of ERα (KRR ERα) that is specifically defective in rapid signaling, but is competent to regulate transcription through the “genomic” pathway. We find that in ECs expressing wild type ERα, E2 regulates many genes involved in cell migration and proliferation, promotes EC migration and proliferation, and also blocks the adhesion of monocytes to ECs. ECs expressing KRR mutant ERα, however, lack all of these responses. These observations establish KRR ERα as a novel tool that could greatly facilitate future studies into the vascular and non-vascular functions of ERα rapid signaling. Further, they support that rapid signaling through ERα is essential for many of the transcriptional and physiological responses of ECs to E2, and that ERα rapid signaling in ECs, in vivo, may be critical for the vasculoprotective and anti-inflammatory effects of estrogen. PMID:27035664

  14. Therapeutic effect of enhancing endothelial nitric oxide synthase (eNOS) expression and preventing eNOS uncoupling

    PubMed Central

    Förstermann, Ulrich; Li, Huige

    2011-01-01

    Nitric oxide (NO) produced by the endothelium is an important protective molecule in the vasculature. It is generated by the enzyme endothelial NO synthase (eNOS). Similar to all NOS isoforms, functional eNOS transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH), via the flavins flavin adenine dinucleotide and flavin mononucleotide in the carboxy-terminal reductase domain, to the heme in the amino-terminal oxygenase domain. Here, the substrate L-arginine is oxidized to L-citrulline and NO. Cardiovascular risk factors such as diabetes mellitus, hypertension, hypercholesterolaemia or cigarette smoking reduce bioactive NO. These risk factors lead to an enhanced production of reactive oxygen species (ROS) in the vessel wall. NADPH oxidases represent major sources of this ROS and have been found upregulated in the presence of cardiovascular risk factors. NADPH-oxidase-derived superoxide avidly reacts with eNOS-derived NO to form peroxynitrite (ONOO-). The essential NOS cofactor (6R-)5,6,7,8-tetrahydrobiopterin (BH4) is highly sensitive to oxidation by this ONOO-. In BH4 deficiency, oxygen reduction uncouples from NO synthesis, thereby converting NOS to a superoxide-producing enzyme. Among conventional drugs, compounds interfering with the renin-angiotensin-aldosterone system and statins can reduce vascular oxidative stress and increase bioactive NO. In recent years, we have identified a number of small molecules that have the potential to prevent eNOS uncoupling and, at the same time, enhance eNOS expression. These include the protein kinase C inhibitor midostaurin, the pentacyclic triterpenoids ursolic acid and betulinic acid, the eNOS enhancing compounds AVE9488 and AVE3085, and the polyphenolic phytoalexin trans-resveratrol. Such compounds enhance NO production from eNOS also under pathophysiological conditions and may thus have therapeutic potential. PMID:21198553

  15. Disrupted NOS signaling in lymphatic endothelial cells exposed to chronically increased pulmonary lymph flow.

    PubMed

    Datar, Sanjeev A; Gong, Wenhui; He, Youping; Johengen, Michael; Kameny, Rebecca J; Raff, Gary W; Maltepe, Emin; Oishi, Peter E; Fineman, Jeffrey R

    2016-07-01

    Associated abnormalities of the lymphatic circulation are well described in congenital heart disease. However, their mechanisms remain poorly elucidated. Using a clinically relevant ovine model of a congenital cardiac defect with chronically increased pulmonary blood flow (shunt), we previously demonstrated that exposure to chronically elevated pulmonary lymph flow is associated with: 1) decreased bioavailable nitric oxide (NO) in pulmonary lymph; and 2) attenuated endothelium-dependent relaxation of thoracic duct rings, suggesting disrupted lymphatic endothelial NO signaling in shunt lambs. To further elucidate the mechanisms responsible for this altered NO signaling, primary lymphatic endothelial cells (LECs) were isolated from the efferent lymphatic of the caudal mediastinal node in 4-wk-old control and shunt lambs. We found that shunt LECs (n = 3) had decreased bioavailable NO and decreased endothelial nitric oxide synthase (eNOS) mRNA and protein expression compared with control LECs (n = 3). eNOS activity was also low in shunt LECs, but, interestingly, inducible nitric oxide synthase (iNOS) expression and activity were increased in shunt LECs, as were total cellular nitration, including eNOS-specific nitration, and accumulation of reactive oxygen species (ROS). Pharmacological inhibition of iNOS reduced ROS in shunt LECs to levels measured in control LECs. These data support the conclusion that NOS signaling is disrupted in the lymphatic endothelium of lambs exposed to chronically increased pulmonary blood and lymph flow and may contribute to decreased pulmonary lymphatic bioavailable NO. PMID:27199125

  16. 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. PMID:22819553

  17. Endothelial function in women of the Kronos Early Estrogen Prevention Study

    PubMed Central

    Kling, J. M.; Lahr, B. A.; Bailey, K. R.; Harman, S. M.; Mulvagh, S. L.

    2015-01-01

    Objective Endothelial dysfunction occurs early in the atherosclerotic disease process, often preceding clinical symptoms. Use of menopausal hormone treatment (MHT) to reduce cardiovascular risk is controversial. This study evaluated effects of 4 years of MHT on endothelial function in healthy, recently menopausal women. Methods Endothelial function was determined by pulse volume digital tonometry providing a reactive hyperemia index (RHI) in a subset of women enrolled in the Kronos Early Estrogen Prevention Study. RHI was measured before and annually after randomization to daily oral conjugated equine estrogen (oCEE, 0.45 mg), weekly transdermal 17β-estradiol (tE2, 50 μg) each with intermittent progesterone (200 mg daily 12 days of the month) or placebo pills and patch. Results At baseline, RHI averaged 2.39 ± 0.69 (mean ± standard deviation; n = 83), and over follow-up did not differ significantly among groups: oCEE, 2.26 ± 0.48 (n = 26); tE2, 2.26 ± 0.45 (n = 24); and placebo, 2.37 ± 0.37 (n = 33). Changes in RHI did not correlate with changes in traditional cardiovascular risk factors, but may inversely correlate with carotid intima medial thickness (Spearman correlation coefficient ρ = −0.268, p = 0.012). Conclusion In this 4-year prospective assessment of recently menopausal women, MHT did not significantly alter RHI when compared to placebo. PMID:25417709

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

    PubMed

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

    2005-09-15

    The application of fluid shear stress to endothelial cells elicits the formation of nitric oxide (NO) and phosphorylation of the endothelial NO synthase (eNOS). Shear stress also elicits the enhanced tyrosine phosphorylation of endothelial proteins, especially of those situated in the vicinity of cell-cell contacts. Since a major constituent of these endothelial cell-cell contacts is the platelet endothelial cell adhesion molecule-1 (PECAM-1) we assessed the role of PECAM-1 in the activation of eNOS. In human endothelial cells, shear stress induced the tyrosine phosphorylation of PECAM-1 and enhanced the association of PECAM-1 with eNOS. Endothelial cell stimulation with shear stress elicited the phosphorylation of Akt and eNOS as well as of the AMP-activated protein kinase (AMPK). While the shear-stress-induced tyrosine phosphorylation of PECAM-1 as well as the serine phosphorylation of Akt and eNOS were abolished by the pre-treatment of cells with the tyrosine kinase inhibitor PP1 the phosphorylation of AMPK was unaffected. Down-regulation of PECAM-1 using a siRNA approach attenuated the shear-stress-induced phosphorylation of Akt and eNOS, as well as the shear-stress-induced accumulation of cyclic GMP levels while the shear-stress-induced phosphorylation of AMPK remained intact. A comparable attenuation of Akt and eNOS (but not AMPK) phosphorylation and NO production was also observed in endothelial cells generated from PECAM-1-deficient mice. These data indicate that the shear-stress-induced activation of Akt and eNOS in endothelial cells is modulated by the tyrosine phosphorylation of PECAM-1 whereas the shear-stress-induced phosphorylation of AMPK is controlled by an alternative signaling pathway. PMID:16118242

  19. Endothelial Function and Insulin Resistance in Early Postmenopausal Women with Cardiovascular Risk Factors: Importance of ESR1 and NOS3 Polymorphisms

    PubMed Central

    Clapauch, Ruth; Mourão, André Felipe; Mecenas, Anete S.; Maranhão, Priscila A.; Rossini, Ana; Bouskela, Eliete

    2014-01-01

    Cardiovascular benefits from estradiol activation of nitric oxide endothelial production may depend on vascular wall and on estrogen receptor alpha (ESR1) and nitric oxide synthase (NOS3) polymorphisms. We have evaluated the microcirculation in vivo through nailfold videocapillaroscopy, before and after acute nasal estradiol administration at baseline and after increased sheer stress (postocclusive reactive hyperemia response) in 100 postmenopausal women, being 70 controls (healthy) and 30 simultaneously hypertensive and diabetic (HD), correlating their responses to PvuII and XbaI ESR1 polymorphisms and to VNTR, T-786C and G894T NOS3 variants. In HD women, C variant allele of ESR1 Pvull was associated to higher vasodilatation after estradiol (1.72 vs 1.64 mm/s, p = 0.01 compared to TT homozygotes) while G894T and T-786C NOS3 polymorphisms were connected to lower increment after shear stress (15% among wild type and 10% among variant alleles, p = 0.02 and 0.04). The G variant allele of ESR1 XbaI polymorphism was associated to higher HOMA-IR (3.54 vs. 1.64, p = 0.01) in HD and higher glucose levels in healthy women (91.8 vs. 87.1 mg/dl, p = 0.01), in which increased waist and HOMA-IR were also related to the G allele in NOS3 G894T (waist 93.5 vs 88.2 cm, p = 0.02; HOMA-IR 2.89 vs 1.48, p = 0.05). ESR1 Pvull, NOS3 G894T and T-786C polymorphism analysis may be considered in HD postmenopausal women for endothelial response prediction following estrogen therapy but were not discriminatory for endothelial response in healthy women. ESR1 XbaI and G894T NOS3 polymorphisms may be useful in accessing insulin resistance and type 2 diabetes risks in all women, even before menopause and occurrence of metabolic disease. PMID:25077953

  20. Extensive ethnogenomic diversity of endothelial nitric oxide synthase (eNOS) polymorphisms.

    PubMed

    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

  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. Effect of long-term piceatannol treatment on eNOS levels in cultured endothelial cells.

    PubMed

    Kinoshita, Yosuke; Kawakami, Shinpei; Yanae, Koji; Sano, Shoko; Uchida, Hiroko; Inagaki, Hiroyuki; Ito, Tatsuhiko

    2013-01-18

    Piceatannol (3, 3', 4, 5'-tetrahydroxy-trans-stilbene) is a naturally occurring phytochemical found in passion fruit (Passiflora edulis) seeds. Previously, we demonstrated that piceatannol has acute vasorelaxant effects in rat thoracic aorta. It was suggested that endothelial NO synthase (eNOS) might be involved in piceatannol-induced acute vasorelaxation. Here, we investigated the expression of eNOS in EA.hy926 human umbilical vein cells after long-term treatment with piceatannol, and compared this effect with that of resveratrol, an analog of piceatannol. Long-term treatment with piceatannol up-regulated eNOS mRNA expression and increased eNOS protein expression in a dose-dependent manner. Moreover, piceatannol increased the levels of phosphorylated eNOS. Treatment with resveratrol also increased eNOS expression, but to a lesser degree than piceatannol. These findings indicate that piceatannol may improve vascular function by up-regulating eNOS expression. PMID:23246837

  3. Endothelial nitric oxide synthase (NOS) deficiency affects energy metabolism pattern in murine oxidative skeletal muscle.

    PubMed Central

    Momken, Iman; Fortin, Dominique; Serrurier, Bernard; Bigard, Xavier; Ventura-Clapier, Renée; Veksler, Vladimir

    2002-01-01

    Oxidative capacity of muscles correlates with capillary density and with microcirculation, which in turn depend on various regulatory factors, including NO generated by endothelial nitric oxide synthase (eNOS). To determine the role of eNOS in patterns of regulation of energy metabolism in various muscles, we studied mitochondrial respiration in situ in saponin-permeabilized fibres as well as the energy metabolism enzyme profile in the cardiac, soleus (oxidative) and gastrocnemius (glycolytic) muscles isolated from mice lacking eNOS (eNOS(-/-)). In soleus muscle, the absence of eNOS induced a marked decrease in both basal mitochondrial respiration without ADP (-32%; P <0.05) and maximal respiration in the presence of ADP (-29%; P <0.05). Furthermore, the eNOS(-/-) soleus muscle showed a decrease in total creatine kinase (-29%; P <0.05), citrate synthase (-31%; P <0.01), adenylate kinase (-27%; P <0.05), glyceraldehyde-3-phosphate dehydrogenase (-43%; P <0.01) and pyruvate kinase (-26%; P <0.05) activities. The percentage of myosin heavy chains I (slow isoform) was significantly increased from 24.3+/-1.5% in control to 30.1+/-1.1% in eNOS(-/-) soleus muscle ( P <0.05) at the expense of a slight non-significant decrease in the three other (fast) isoforms. Besides, eNOS(-/-) soleus showed a 28% loss of weight. Interestingly, we did not find differences in any parameters in cardiac and gastrocnemius muscles compared with respective controls. These results show that eNOS knockout has an important effect on muscle oxidative capacity as well on the activities of energy metabolism enzymes in oxidative (soleus) muscle. The absence of such effects in cardiac and glycolytic (gastrocnemius) muscle suggests a specific role for eNOS-produced NO in oxidative skeletal muscle. PMID:12123418

  4. Constitutive eNOS-derived nitric oxide is a determinant of endothelial junctional integrity.

    PubMed

    Predescu, Dan; Predescu, Sanda; Shimizu, Jun; Miyawaki-Shimizu, Kayo; Malik, Asrar B

    2005-09-01

    Basal vascular endothelial permeability is normally kept low in part by the restrictiveness of interendothelial junctions (IEJs). We investigated the possible role of nitric oxide (NO) in controlling IEJ integrity and thereby regulating basal vascular permeability. We determined the permeability of continuous endothelia in multiple murine vascular beds, including lung vasculature, of wild-type mice, endothelial nitric oxide synthase (eNOS) null mice, and mice treated with NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME). Light and electron microscopic studies revealed that L-NAME treatment resulted in IEJs opening within a few minutes with a widespread response within 30 min. We observed a 35% increase in transendothelial transport of albumin, using as tracer dinitrophenylated albumin in mouse lungs and other organs studied. To rule out the involvement of blood cells in the mechanism of increased endothelial permeability, vascular beds were flushed free of blood, treated with L-NAME, and perfused with the tracer. The open IEJs observed in these studies indicated a direct role for NO in preserving the normal structure of endothelial junctions. We also used the electron-opaque tracer lanthanum chloride to assess vascular permeability. Lanthanum chloride was presented by perfusion to various vascular beds of mice lacking NO. Open IEJs were seen only in capillary and venular endothelial segments of mice lacking NO, and there was a concomitant increase in vascular permeability to the tracer. Together, these data demonstrate that constitutive eNOS-derived NO is a crucial determinant of IEJ integrity and thus serves to maintain the low basal permeability of continuous endothelia. PMID:16093363

  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. Anti-inflammatory effects of a Chinese herbal medicine in atherosclerosis via estrogen receptor β mediating nitric oxide production and NF-κB suppression in endothelial cells

    PubMed Central

    Wang, L; Qiu, X-M; Hao, Q; Li, D-J

    2013-01-01

    Bu-Shen-Ning-Xin Decoction (BSNXD) administration has alleviated the early pathologic damage of atherosclerosis by inhibiting the adhesion molecule expression and upregulating the estrogen receptor (ER) β expression in endothelial cells, and increasing the serum nitric oxide (NO) level without any effect on serum lipid status, endometrium and fat deposition in liver in ovariectomized rabbits. The BSNXD-derived serum increases ER β expression in the human umbilical vein endothelial cells (HUVECs), and decreases malondialdehyde (MDA) production, and upregulates eNOS expression then increases NO synthesis through ERβ-dependent pathway. NO not only suppresses the LPS-induced NF-κB transcription in HUVECs, but also decreases apoptosis of endothelial cells. The BSNXD-derived serum decreases monocyte chemoattractant protein-1 production, and suppresses cell adhesion molecules (ICAM-1, VCAM-1 and E-selectin) expression in HUVECs injured by oxidized low-density lipoproteins (ox-LDL), and these effects can be abolished by ERβ antagonist (R,RTHC) and NO synthase inhibitor (L-NAME). The BSNXD-derived serum-treated HUVECs supernatant reduces CCR2, LFA-1 and VLA-4 expression in monocytes cell line U937 cells, which in turn inhibits adherence of U937 to injured endothelial cells. NO synthesis increases, and MDA production decreases through ERβ-mediated pathway that suppresses apoptosis and NF-κB activity in endothelial cells that downregulates adhesion molecules expression on endothelial cells via ERβ/NO/NF-κB pathway, and in turn leukocyte adhesion, which suggests BSNXD potential value in prophylaxis atherosclerosis. PMID:23519120

  7. Ivabradine Prevents Low Shear Stress Induced Endothelial Inflammation and Oxidative Stress via mTOR/eNOS Pathway

    PubMed Central

    Li, Bing; Zhang, Junxia; Wang, Zhimei; Chen, Shaoliang

    2016-01-01

    Ivabradine not only reduces heart rate but has other cardiac and vascular protective effects including anti-inflammation and anti-oxidation. Since endothelial nitric oxide synthase (eNOS) is a crucial enzyme in maintaining endothelial activity, we aimed to investigate the impact of ivabradine in low shear stress (LSS) induced inflammation and endothelial injury and the role of eNOS played in it. Endothelial cells (ECs) were subjected to LSS at 2dyne/cm2, with 1 hour of ivabradine (0.04μM) or LY294002 (10μM) pre-treatment. The mRNA expression of IL-6, VCAM-1 along with eNOS were measured by QPCR. Reactive oxygen species (ROS) was detected by dihydroethidium (DHE) and DCF, and protein phosphorylation was detected by western blot. It demonstrated that ivabradine decreased LSS induced inflammation and oxidative stress in endothelial cells. Western blot showed reduced rictor and Akt-Ser473 as well as increased eNOS-Thr495 phosphorylation. However, mTORC1 pathway was only increased when LSS applied within 30 minutes. These effects were reversed by ivabradine. It would appear that ivabradine diminish ROS generation by provoking mTORC2/Akt phosphorylation and repressing mTORC1 induced eNOS-Thr495 activation. These results together suggest that LSS induced endothelial inflammation and oxidative stress are suppressed by ivabradine via mTORC2/Akt activation and mTORC1/eNOS reduction. PMID:26890696

  8. Activation of eNOS in endothelial cells exposed to ionizing radiation involves components of the DNA damage response pathway

    SciTech Connect

    Nagane, Masaki; Yasui, Hironobu; Sakai, Yuri; Yamamori, Tohru; Niwa, Koichi; Hattori, Yuichi; Kondo, Takashi; Inanami, Osamu

    2015-01-02

    Highlights: • eNOS activity is increased in BAECs exposed to X-rays. • ATM is involved in this increased eNOS activity. • HSP90 modulates the radiation-induced activation of ATM and eNOS. - Abstract: In this study, the involvement of ataxia telangiectasia mutated (ATM) kinase and heat shock protein 90 (HSP90) in endothelial nitric oxide synthase (eNOS) activation was investigated in X-irradiated bovine aortic endothelial cells. The activity of nitric oxide synthase (NOS) and the phosphorylation of serine 1179 of eNOS (eNOS-Ser1179) were significantly increased in irradiated cells. The radiation-induced increases in NOS activity and eNOS-Ser1179 phosphorylation levels were significantly reduced by treatment with either an ATM inhibitor (Ku-60019) or an HSP90 inhibitor (geldanamycin). Geldanamycin was furthermore found to suppress the radiation-induced phosphorylation of ATM-Ser1181. Our results indicate that the radiation-induced eNOS activation in bovine aortic endothelial cells is regulated by ATM and HSP90.

  9. Adenoviral gene transfer of endothelial nitric-oxide synthase (eNOS) partially restores normal pulmonary arterial pressure in eNOS-deficient mice

    PubMed Central

    Champion, Hunter C.; Bivalacqua, Trinity J.; Greenberg, Stanley S.; Giles, Thomas D.; Hyman, Albert L.; Kadowitz, Philip J.

    2002-01-01

    It has been shown that mice deficient in the gene coding for endothelial nitric-oxide synthase (eNOS) have increased pulmonary arterial pressure and pulmonary vascular resistance. In the present study, the effect of transfer to the lung of an adenoviral vector encoding the eNOS gene (AdCMVeNOS) on pulmonary arterial pressure and pulmonary vascular resistance was investigated in eNOS-deficient mice. One day after intratracheal administration of AdCMVeNOS to eNOS−/− mice, there was an increase in eNOS protein, cGMP levels, and calcium-dependent conversion of l-arginine to l-citrulline in the lung. The increase in eNOS protein and activity in eNOS−/− mice was associated with a reduction in mean pulmonary arterial pressure and pulmonary vascular resistance when compared with values in eNOS-deficient mice treated with vehicle or a control adenoviral vector coding for β-galactosidase, AdCMVβgal. These data suggest that in vivo gene transfer of eNOS to the lung in eNOS−/− mice can increase eNOS staining, eNOS protein, calcium-dependent NOS activity, and cGMP levels and partially restore pulmonary arterial pressure and pulmonary vascular resistance to near levels measured in eNOS+/+ mice. Thus, the major finding in this study is that in vivo gene transfer of eNOS to the lung in large part corrects a genetic deficiency resulting from eNOS deletion and may be a useful therapeutic intervention for the treatment of pulmonary hypertensive disorders in which eNOS activity is reduced. PMID:12237402

  10. Ambient ultrafine particles reduce endothelial nitric oxide production via S-glutathionylation of eNOS

    PubMed Central

    Du, Yunfeng; Navab, Mohamad; Shen, Melody; Hill, James; Pakbin, Payam; Sioutas, Constantinos; Hsiai, Tzung; Li, Rongsong

    2013-01-01

    Exposure to airborne particulate pollutants is intimately linked to vascular oxidative stress and inflammatory responses with clinical relevance to atherosclerosis. Particulate matter (PM) has been reported to induce endothelial dysfunction and atherosclerosis. Here, we tested whether ambient ultrafine particles (UFP, diameter < 200 nm) modulate eNOS activity in terms of nitric oxide (NO) production via protein S-glutathionylation. Treatment of human aortic endothelial cells (HAEC) with UFP significantly reduced NO production. UFP-mediated reduction in NO production was restored in the presence of JNK inhibitor (SP600125), NADPH oxidase inhibitor (Apocynin), anti-oxidant (N-acetyl cysteine), and superoxide dismutase mimetics (Tempol and MnTMPyP). UFP exposure increased the GSSG/GSH ratio and eNOS S-glutathionylation, whereas over-expression of Glutaredoxin-1 (to inhibit S-glutathionylation) restored UFP-mediated reduction in NO production by nearly 80%. Thus, our findings suggest that eNOS S-glutathionylation is a potential mechanism underlying ambient UFP-induced reduction of NO production. PMID:23751346

  11. Estrogen down-regulates nicotine-induced adhesion molecule expression via nongenomic signal pathway in endothelial cells.

    PubMed

    Wang, Yajing; Wang, Zhaoxia; Wang, Lianyun; Zhou, Ying; Zhao, Yangxing; Liu, Liming; Yao, Chenjiang; Qiao, Zhongdong

    2006-06-01

    Although gonadal hormone mostly causes genotropic actions through the members of nuclear receptor family, it also can regulate these actions via membrane receptor. To explore the possibility of plasma membrane estrogen receptors (mER) mediating genotropic events, we have investigated estrogen's effect on nicotine-stimulated adhesion molecule expression and evaluated whether this effect depends on calcium, MAPK signal pathway. Fluorescence Spectroscopy analysis of Ca2+ from human umbilical vein endothelial cells (HUVECs) showed through mER, estrogen induced a rapid rise of intracellular free Ca2+ concentration and this rise could not be inhibited by tamoxifen (classic ER inhibitor). In the context of nicotine stimulating, however, estrogen attenuated phosphorylation of mitogen-activated protein kinase (MAPK) family members, extracellular signal regulated kinase 1/2 (ERK1/2), p38 but not c-Jun-N-terminal kinase (JNK) in HUVECs and this effect could not still be prevented by tamoxifen. In the meantime, estrogen also down-regulated surface/soluble vascular cell adhesion molecule (VCAM-1, sVCAM-1) and endothelial selectin (E-selectin, sE-selectin) levels, which was not abolished by tamoxifen either. Moreover, calcium chelator BAPTA, ERK1/2 inhibitor PD98059, p38 inhibitor SB203580 significantly reduced the production of nicotine-activated surface/soluble VCAM-1 and E-selectin and both of the remained levels were no longer regulated by estrogen. Our study here provides the information of decrease effect of mER-mediated estrogen through Ca2+ and ERK1/2, p38 MAPK signaling pathway on nicotine-stimulated expression of surface/soluble VCAM-1 and E-selectin in HUVECs. PMID:16644474

  12. PGC-1α ameliorates AngiotensinII-induced eNOS dysfunction in human aortic endothelial cells.

    PubMed

    Li, Jie; Geng, Xiao-Yong; Cong, Xiao-Liang

    2016-08-01

    Increasing evidences support that PGC-1α participates in regulating endothelial homeostasis, in part by mediating endothelial nitric oxide (NO) synthase (eNOS) activity and NO production. However, the molecular mechanisms by which PGC-1α regulates eNOS activity are not completely understood. In the present study, we investigated the effects of PGC-1α on eNOS dysfunction and further explore the underlying mechanisms. The results showed that PGC-1α expression was downregulated after AngiotensinII (AngII) treatment and paralleled with the decreased NO generation in human aortic endothelial cells. Overexpression of PGC-1α with adenovirus or pharmacological agonist ameliorated AngII-induced the decrease of NO generation, evidenced by the restoration of cGMP and nitrite concentration. Rather than affecting eNOS expression and uncoupling, PGC-1α inhibited AngII-induced decrease of eNOS serine 1177 phosphorylation through activation of PI3K/Akt signaling. In addition, PGC-1α overexpression suppressed AngII-induced the increase of PP2A-A/eNOS interaction and PP2A phosphatase activity, with a concomitant decrease in PP2A phosphorylation, leading to eNOS serine 1177 phosphorylation. However, pharmacological inhibition of PI3K/Akt signaling blunted the observed effect of PGC-1α on PP2A activity. Taken together, our findings suggest that PGC-1α overexpression improves AngII-induced eNOS dysfunction and that improved eNOS dysfunction is associated with activated PI3K/Akt pathway, impaired PP2A activity and reduced PP2A-A/eNOS association. These date indicate that forced PGC-1α expression may be a novel therapeutic approach for endothelial dysfunction. PMID:27235860

  13. Estrogen

    MedlinePlus

    ... estrogen tablets. If you will be taking Estrace® brand tablets, tell your doctor and pharmacist if you are allergic to aspirin or tartrazine (a food color additive). Ask your pharmacist or check the manufacturer's ...

  14. Estrogen

    MedlinePlus

    Estrogen is used to treat hot flushes ('hot flashes'; sudden strong feelings of heat and sweating) in ... and cause problems with the skin or nervous system), very high or very low levels of calcium ...

  15. Reversal of SIN-1-induced eNOS dysfunction by the spin trap, DMPO, in bovine aortic endothelial cells via eNOS phosphorylation

    PubMed Central

    Das, Amlan; Gopalakrishnan, Bhavani; Druhan, Lawrence J; Wang, Tse-Yao; De Pascali, Francesco; Rockenbauer, Antal; Racoma, Ira; Varadharaj, Saradhadevi; Zweier, Jay L; Cardounel, Arturo J; Villamena, Frederick A

    2014-01-01

    Background and Purpose Nitric oxide (NO) derived from eNOS is mostly responsible for the maintenance of vascular homeostasis and its decreased bioavailability is characteristic of reactive oxygen species (ROS)-induced endothelial dysfunction (ED). Because 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a commonly used spin trap, can control intracellular nitroso-redox balance by scavenging ROS and donating NO, it was employed as a cardioprotective agent against ED but the mechanism of its protection is still not clear. This study elucidated the mechanism of protection by DMPO against SIN-1-induced oxidative injury to bovine aortic endothelial cells (BAEC). Experimental Approach BAEC were treated with SIN-1, as a source of peroxynitrite anion (ONOO−), and then incubated with DMPO. Cytotoxicity following SIN-1 alone and cytoprotection by adding DMPO was assessed by MTT assay. Levels of ROS and NO generation from HEK293 cells transfected with wild-type and mutant eNOS cDNAs, tetrahydrobiopterin bioavailability, eNOS activity, eNOS and Akt kinase phosphorylation were measured. Key Results Post-treatment of cells with DMPO attenuated SIN-1-mediated cytotoxicity and ROS generation, restoration of NO levels via increased in eNOS activity and phospho-eNOS levels. Treatment with DMPO alone significantly increased NO levels and induced phosphorylation of eNOS Ser1179 via Akt kinase. Transfection studies with wild-type and mutant human eNOS confirmed the dual role of eNOS as a producer of superoxide anion (O2−) with SIN-1 treatment, and a producer of NO in the presence of DMPO. Conclusion and Implications Post-treatment with DMPO of oxidatively challenged cells reversed eNOS dysfunction and could have pharmacological implications in the treatment of cardiovascular diseases. PMID:24405159

  16. Resveratrol Prevented Lipopolysaccharide-Induced Endothelial Dysfunction in Rat Thoracic Aorta Through Increased eNOS Expression

    PubMed Central

    Uğurel, Seda Sultan; Kuşçu, Nilay; Özenci, Çiler Çelik; Dalaklıoğlu, Selvinaz; Taşatargil, Arda

    2016-01-01

    Background: The cardiovascular benefits of Resveratrol (RVT) have been well established by previous experimental and clinical studies. Aims: The goal of this study was to test the effectiveness of RVT administration on the impaired endothelial function induced by lipopolysaccharide (LPS), and to elucidate the role of endothelial nitric oxide synthase (eNOS)/Sirtuin 1 (SIRT1) pathway. Study Design: Animal experiment. Methods: Endotoxemia was induced by intraperitoneal injection of 10 mg/kg LPS, and the thoracic aorta was isolated six hours later. RVT was injected intraperitoneally 15 minutes before LPS administration. Six hours after LPS injection, potassium chloride (KCl), phenylephrine (Phe), acetylcholine (ACh), and sodium nitroprusside (SNP) were used to examine to vascular reactivity and endothelial function. eNOS, phospho-eNOS (p-eNOS) (Ser 1177), and SIRT1 expressions in thoracic aorta were evaluated by Western blot. Results: LPS administration significantly inhibited the relaxation response induced by ACh, while the relaxation to SNP was not significantly altered. Phe- and KCl-induced contractile responses in the thoracic aorta significantly decreased in LPS-injected group. eNOS and p-eNOS expression decreased significantly in arteries obtained from LPS group rats. The impaired vasoreactivity as well as decreased expressions of eNOS, p-eNOS, and SIRT1 in vessels from LPS-injected rats were improved by RVT treatment. Conclusion: The endothelium-dependent vasodilatation of the thoracic aorta was significantly inhibited by LPS administration, and RVT treatment may improve vascular endothelial function. The protective effect of RVT might be associated with increased eNOS expression and activity.

  17. Endothelial function does not improve with high-intensity continuous exercise training in SHR: implications of eNOS uncoupling.

    PubMed

    Battault, Sylvain; Singh, François; Gayrard, Sandrine; Zoll, Joffrey; Reboul, Cyril; Meyer, Grégory

    2016-02-01

    Exercise training is a well-recognized way to improve vascular endothelial function by increasing nitric oxide (NO) bioavailability. However, in hypertensive subjects, unlike low- and moderate-intensity exercise training, the beneficial effects of continuous high-intensity exercise on endothelial function are not clear, and the underlying mechanisms remain unknown. The aim of this study was to investigate the impact of high-intensity exercise on vascular function, especially on the NO pathway, in spontaneous hypertensive rats (SHR). These effects were studied on WKY, sedentary SHR and SHR that exercised at moderate (SHR-MOD) and high intensity (SHR-HI) on a treadmill (1 h per day; 5 days per week for 6 weeks at 55% and 80% of their maximal aerobic velocity, respectively). Endothelial function and specific NO contributions to acetylcholine-mediated relaxation were evaluated by measuring the aortic ring isometric forces. Endothelial nitric oxide synthase (eNOS) expression and phosphorylation (ser1177) were evaluated by western blotting. The total aortic and eNOS-dependent reactive oxygen species (ROS) production was assessed using electron paramagnetic resonance in aortic tissue. Although the aortas of SHR-HI had increased eNOS levels without alteration of eNOS phosphorylation, high-intensity exercise had no beneficial effect on endothelium-dependent vasorelaxation, unlike moderate exercise. This result was associated with increased eNOS-dependent ROS production in the aortas of SHR-HI. Notably, the use of the recoupling agent BH4 or a thiol-reducing agent blunted eNOS-dependent ROS production in the aortas of SHR-HI. In conclusion, the lack of a positive effect of high-intensity exercise on endothelial function in SHR was mainly explained by redox-dependent eNOS uncoupling, resulting in a switch from NO to O2(-) generation. PMID:26537830

  18. The effects of (-)-epicatechin on endothelial cells involve the G protein-coupled estrogen receptor (GPER).

    PubMed

    Moreno-Ulloa, Aldo; Mendez-Luna, David; Beltran-Partida, Ernesto; Castillo, Carmen; Guevara, Gustavo; Ramirez-Sanchez, Israel; Correa-Basurto, José; Ceballos, Guillermo; Villarreal, Francisco

    2015-10-01

    We have provided evidence that the stimulatory effects of (-)-epicatechin ((-)-EPI) on endothelial cell nitric oxide (NO) production may involve the participation of a cell-surface receptor. Thus far, such entity(ies) has not been fully elucidated. The G protein-coupled estrogen receptor (GPER) is a cell-surface receptor that has been linked to protective effects on the cardiovascular system and activation of intracellular signaling pathways (including NO production) similar to those reported with (-)-EPI. In bovine coronary artery endothelial cells (BCAEC) by the use of confocal imaging, we evidence the presence of GPER at the cell-surface and on F-actin filaments. Using in silico studies we document the favorable binding mode between (-)-EPI and GPER. Such binding is comparable to that of the GPER agonist, G1. By the use of selective blockers, we demonstrate that the activation of ERK 1/2 and CaMKII by (-)-EPI is dependent on the GPER/c-SRC/EGFR axis mimicking those effects noted with G1. We also evidence by the use of siRNA the role that GPER has on mediating ERK1/2 activation by (-)-EPI. GPER appears to be coupled to a non Gαi/o or Gαs, protein subtype. To extrapolate our findings to an ex vivo model, we employed phenylephrine pre-contracted aortic rings evidencing that (-)-EPI can mediate vasodilation through GPER activation. In conclusion, we provide evidence that suggests the GPER as a potential mediator of (-)-EPI effects and highlights the important role that GPER may have on cardiovascular system protection. PMID:26303816

  19. Osthole relaxes pulmonary arteries through endothelial phosphatidylinositol 3-kinase/Akt-eNOS-NO signaling pathway in rats.

    PubMed

    Yao, Li; Lu, Ping; Li, Yumei; Yang, Lijing; Feng, Hongxuan; Huang, Yong; Zhang, Dandan; Chen, Jianguo; Zhu, Daling

    2013-01-15

    Pulmonary arterial hypertension is a life-threatening disease lacking effective therapies. Osthole is a natural coumarin compound isolated from Angelica pubescens Maxim., which possesses hypotensive effect. Although its effects on isolated thoracic aorta (systemic circulating system) are clarified, it remains unclear whether Osthole relaxes isolated pulmonary arteries (PAs) (pulmonary circulating system). The aim of this study was to investigate the effects of Osthole on isolated PAs and the underlying mechanisms. We examined PA relaxation induced by Osthole in isolated human and rat PA rings with force-electricity transducers, the expression and activity of endothelial nitric oxide synthase (eNOS) and protein kinase B (Akt) with western blot, and nitric oxide (NO) production using DAF-FM DA fluorescent indicator. The results showed that Osthole elicited a dose-dependent vasorelaxation activity with phenylephrine-precontracted human and rat PA rings, which can be diminished by endothelium denudation and inhibition of eNOS, while having no effect on rat mesenteric arteries. Osthole increased NO release as well as activation of Akt and eNOS, indicated with increased phosphorylations of Akt at Ser-473 and eNOS at Ser-1177 in endothelial cells. PI3K inhibitor LY294002 also blocked Osthole induced vasodilation. In summary, dilative effect of Osthole was dependent on endothelial integrity and NO production, and was mediated by endothelial PI3K/Akt-eNOS-NO pathway. These may provide a new pulmonary vasodilator for the therapy of pulmonary arterial hypertension. PMID:23220709

  20. Retinal hypoxia induces vascular endothelial growth factor through induction of estrogen-related receptor γ.

    PubMed

    Do, Ji Yeon; Choi, Young Keun; Kook, Hyun; Suk, Kyoungho; Lee, In-Kyu; Park, Dong Ho

    2015-05-01

    Ischemic retinopathies causing overexpression of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), are the most common cause of blindness. Thus, understanding the pathophysiology of targetable pathways that regulate retinal VEGF is of great interest. A conserved binding site for estrogen-related receptor γ (ERRγ) has been identified in the promoter of the Vegfa gene. ERRγ is a constitutively active orphan nuclear receptor and its expression is increased by hypoxic stimuli in metabolically active tissues. This study evaluated the role of ERRγ in the ischemic retina and the anti-VEGF potential of GSK5182, a selective inverse agonist of ERRγ. In an oxygen-induced retinopathy (OIR) mouse model, immunohistochemistry showed significantly increased ERRγ expression in the ganglion cell layer at postnatal day (P) 17. In a ganglion cell line (RGC-5), mRNA and protein levels of ERRγ were increased by desferrioxamine treatment and hypoxic conditions (1% O2). Transient transfection of RGC-5 cells revealed that ERRγ regulated Vegfa expression and this was inhibited by GSK5182. Intravitreal injection of GSK5182 into the OIR model at P14 inhibited retinal Vegfa mRNA expression at P17. GSK5182 suppresses hypoxia-induced VEGF expression via ERRγ; therefore, ERRγ could be a treatment target for ischemic retinopathies. PMID:25796334

  1. Shear-induced Endothelial NOS Activation and Remodeling via Heparan Sulfate, Glypican-1, and Syndecan-1

    PubMed Central

    Ebong, Eno E; Lopez-Quintero, Sandra V; Rizzo, Victor; Spray, David C; Tarbell, John M

    2014-01-01

    Mammalian epithelial cells are coated with a multifunctional surface glycocalyx (GCX). On vascular endothelial cells (EC), intact GCX is atheroprotective. It is degraded in many vascular diseases. GCX heparan sulfate (HS) is essential for healthy flow-induced EC nitric oxide (NO) release, elongation, and alignment. The HS core protein mechanisms involved in these processes are unknown. We hypothesized that the glypican-1 (GPC1) HS core protein mediates flow-induced EC NO synthase (eNOS) activation because GPC1 is anchored to caveolae where eNOS resides. We also hyphothesized that the HS core protein syndecan-1 (SDC1) mediates flow-induced EC elongation and alignment because SDC1 is linked to the cytoskeleton which impacts cell shape. We tested our hypotheses by exposing EC monolayers treated with HS degrading heparinase III (HepIII), and monolayers with RNA-silenced GPC1, or SDC1, to 3 to 24 hours of physiological shear stress. Shear-conditioned EC with intact GCX exhibited characteristic eNOS activation in short-term flow conditions. After long-term exposure, EC with intact GCX were elongated and aligned in the direction of flow. HS removal and GPC1 inhibition, not SDC1 reduction, blocked shear-induced eNOS activation. EC remodeling in response to flow was attenuated by HS degradation and in the absence of SDC1, but preserved with GPC1 knockdown. These findings clearly demonstrate that HS is involved in both centralized and decentralized GCX-mediated mechanotransduction mechanisms, with GPC1 acting as a centralized mechanotransmission agent and SDC1 functioning in decentralized mechanotransmission. This foundational work demonstrates how EC can transform fluid shear forces into diverse biomolecular and biomechanical responses. PMID:24480876

  2. Development of confocal immunofluorescence FRET microscopy to Investigate eNOS and GSNOR localization and interaction in pulmonary endothelial cells

    NASA Astrophysics Data System (ADS)

    Rehman, Shagufta; Brown-Steinke, Kathleen; Palmer, Lisa; Periasamy, Ammasi

    2015-03-01

    Confocal FRET microscopy is a widely used technique for studying protein-protein interactions in live or fixed cells. Endothelial nitric oxide synthase (eNOS) and S-nitrosoglutathione reductase (GSNOR) are enzymes involved in regulating the bioavailability of S-nitrosothiols (SNOs) in the pulmonary endothelium and have roles in the development of pulmonary arterial hypertension. Labeling of endogenous proteins to better understand a disease process can be challenging. We have used immunofluorescence to detect endogenous eNOS and GSNOR in primary pulmonary endothelial cells to co-localize these proteins as well as to study their interaction by FRET. The challenge has been in selecting the right immunofluorescence labeling condition, right antibody, the right blocking reagent, the right FRET pair and eliminating cross-reactivity of secondary antibodies. We have used Alexa488 and Alexa568 as a FRET pair. After a series of optimizations, the data from Confocal Laser Scanning Microscopy (CLSM) demonstrate co-localization of eNOS and GSNOR in the perinuclear region of the pulmonary endothelial cell primarily within the cis-Golgi with lower levels of co-localization seen within the trans-Golgi. FRET studies demonstrate, for the first time, interaction between eNOS and GSNOR in both murine and bovine pulmonary endothelial cells. Further characterization of eNOSGSNOR interaction and the subcellular location of this interaction will provide mechanistic insight into the importance of S-nitrosothiol signaling in pulmonary biology, physiology and pathology.

  3. Nitrotyrosinylation, Remodeling and Endothelial-Myocyte Uncoupling in iNOS, Cystathionine Beta Synthase (CBS) Knockouts and iNOS/CBS Double Knockout Mice

    PubMed Central

    Kundu, Soumi; Kumar, Munish; Sen, Utpal; Mishra, Paras K.; Tyagi, Neetu; Metreveli, Naira; Lominadze, David; Rodriguez, Walter; Tyagi, Suresh C.

    2009-01-01

    Increased levels of homocysteine (Hcy), recognized as hyperhomocysteinemia (HHcy), were associated with cardiovascular diseases. There was controversy regarding the detrimental versus cardio protective role of inducible nitric oxide synthase (iNOS) in ischemic heart disease. The aim of this study was to test the hypothesis that the Hcy generated nitrotyrosine by inducing the endothelial nitric oxide synthase, causing endothelial-myocyte (E–M) coupling. To differentiate the role of iNOS versus constitutive nitric oxide synthase (eNOS and nNOS) in Hcy-mediated nitrotyrosine generation and matrix remodeling in cardiac dysfunction, left ventricular (LV) tissue was analyzed from cystathionine beta synthase (CBS) heterozygote knockout, iNOS homozygote knockout, CBS−/+/iNOS−/− double knockout, and wild-type (WT) mice. The levels of nitrotyrosine, MMP-2 and -9 (zymographic analysis), and fibrosis (by trichrome stain) were measured. The endothelial-myocyte function was determined in cardiac rings. In CBS−/+ mice, homocysteine was elevated and in iNOS−/− mice, nitric oxide was significantly reduced. The nitrotyrosine and matrix metalloproteinase-9 (MMP-9) levels were elevated in double knockout and CBS−/+ as compared to WT mice. Although MMP-2 levels were similar in CBS−/+, iNOS−/−, and CBS−/+/iNOS−/−, the levels were three- to fourfold higher than WT. The levels of collagen were similar in CBS−/+ and iNOS−/−, but they were threefold higher than WT. Interesting, the levels of collagen increased sixfold in double knockouts, compared to WT, suggesting synergism between high Hcy and lack of iNOS. Left ventricular hypertrophy was exaggerated in the iNOS−/− and double knockout, and mildly increased in the CBS−/+, compared to WT mice. The endothelial-dependent relaxation was attenuated to the same extent in the CBS−/+ and iNOS−/−, compared to WT, but it was robustly blunted in double knockouts. The results concluded that homocysteine

  4. Endothelial NOS-dependent activation of c-Jun NH(2)- terminal kinase by oxidized low-density lipoprotein

    NASA Technical Reports Server (NTRS)

    Go, Y. M.; Levonen, A. L.; Moellering, D.; Ramachandran, A.; Patel, R. P.; Jo, H.; Darley-Usmar, V. M.

    2001-01-01

    Oxidized low-density lipoprotein (oxLDL) is known to activate a number of signal transduction pathways in endothelial cells. Among these are the c-Jun NH(2)-terminal kinase (JNK), also known as stress-activated protein kinase, and extracellular signal-regulated kinase (ERK). These mitogen-activated protein kinases (MAP kinase) determine cell survival in response to environmental stress. Interestingly, JNK signaling involves redox-sensitive mechanisms and is activated by reactive oxygen and nitrogen species derived from both NADPH oxidases, nitric oxide synthases (NOS), peroxides, and oxidized low-density lipoprotein (oxLDL). The role of endothelial NOS (eNOS) in the activation of JNK in response to oxLDL has not been examined. Herein, we show that on exposure of endothelial cells to oxLDL, both ERK and JNK are activated through independent signal transduction pathways. A key role of eNOS activation through a phosphatidylinositol-3-kinase-dependent mechanism leading to phosphorylation of eNOS is demonstrated for oxLDL-dependent activation of JNK. Moreover, we show that activation of ERK by oxLDL is critical in protection against the cytotoxicity of oxLDL.

  5. Retinal hypoxia induces vascular endothelial growth factor through induction of estrogen-related receptor γ

    SciTech Connect

    Do, Ji Yeon; Choi, Young Keun; Kook, Hyun; Suk, Kyoungho; Lee, In-Kyu; Park, Dong Ho

    2015-05-01

    Ischemic retinopathies causing overexpression of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), are the most common cause of blindness. Thus, understanding the pathophysiology of targetable pathways that regulate retinal VEGF is of great interest. A conserved binding site for estrogen-related receptor γ (ERRγ) has been identified in the promoter of the Vegfa gene. ERRγ is a constitutively active orphan nuclear receptor and its expression is increased by hypoxic stimuli in metabolically active tissues. This study evaluated the role of ERRγ in the ischemic retina and the anti-VEGF potential of GSK5182, a selective inverse agonist of ERRγ. In an oxygen-induced retinopathy (OIR) mouse model, immunohistochemistry showed significantly increased ERRγ expression in the ganglion cell layer at postnatal day (P) 17. In a ganglion cell line (RGC-5), mRNA and protein levels of ERRγ were increased by desferrioxamine treatment and hypoxic conditions (1% O{sub 2}). Transient transfection of RGC-5 cells revealed that ERRγ regulated Vegfa expression and this was inhibited by GSK5182. Intravitreal injection of GSK5182 into the OIR model at P14 inhibited retinal Vegfa mRNA expression at P17. GSK5182 suppresses hypoxia-induced VEGF expression via ERRγ; therefore, ERRγ could be a treatment target for ischemic retinopathies. - Highlights: • OIR mice exhibited increased ERRγ expression in the ganglion cell layer. • Hypoxia-induced ERRγ expression was observed in retinal ganglion cells. • ERRγ overexpression increased VEGFA expression in retinal ganglion cells. • An ERRγ inverse agonist suppressed VEGFA expression in retinal ganglion cells. • Intravitreal injection of an ERRγ inverse agonist suppressed VEGFA in OIR mice.

  6. Endothelial AMPK activation induces mitochondrial biogenesis and stress adaptation via eNOS-dependent mTORC1 signaling.

    PubMed

    Li, Chunying; Reif, Michaella M; Craige, Siobhan M; Kant, Shashi; Keaney, John F

    2016-05-01

    Metabolic stress sensors like AMP-activated protein kinase (AMPK) are known to confer stress adaptation and promote longevity in lower organisms. This study demonstrates that activating the metabolic stress sensor AMP-activated protein kinase (AMPK) in endothelial cells helps maintain normal cellular function by promoting mitochondrial biogenesis and stress adaptation. To better define the mechanisms whereby AMPK promotes endothelial stress resistance, we used 5-aminoimidazole-4-carboxamide riboside (AICAR) to chronically activate AMPK and observed stimulation of mitochondrial biogenesis in wild type mouse endothelium, but not in endothelium from endothelial nitric oxide synthase knockout (eNOS-null) mice. Interestingly, AICAR-enhanced mitochondrial biogenesis was blocked by pretreatment with the mammalian target of rapamycin complex 1 (mTORC1) inhibitor, rapamycin. Further, AICAR stimulated mTORC1 as determined by phosphorylation of its known downstream effectors in wild type, but not eNOS-null, endothelial cells. Together these data indicate that eNOS is needed to couple AMPK activation to mTORC1 and thus promote mitochondrial biogenesis and stress adaptation in the endothelium. These data suggest a novel mechanism for mTORC1 activation that is significant for investigations in vascular dysfunction. PMID:26989010

  7. Pretreatment with β-Boswellic Acid Improves Blood Stasis Induced Endothelial Dysfunction: Role of eNOS Activation.

    PubMed

    Wang, Mingming; Chen, Minchun; Ding, Yi; Zhu, Zhihui; Zhang, Yikai; Wei, Peifeng; Wang, Jingwen; Qiao, Yi; Li, Liang; Li, Yuwen; Wen, Aidong

    2015-01-01

    Vascular endothelial cells play an important role in modulating anti-thrombus and maintaining the natural function of vascular by secreting many active substances. β-boswellic acid (β-BA) is an active triterpenoid compound from the extract of boswellia serrate. In this study, it is demonstrated that β-BA ameliorates plasma coagulation parameters, protects endothelium from blood stasis induced injury and prevents blood stasis induced impairment of endothelium-dependent vasodilatation. Moreover, it is found that β-BA significantly increases nitric oxide (NO) and cyclic guanosine 3', 5'-monophosphate (cGMP) levels in carotid aortas of blood stasis rats. To stimulate blood stasis-like conditions in vitro, human umbilical vein endothelial cells (HUVECs) were exposed to transient oxygen and glucose deprivation (OGD). Treatment of β-BA significantly increased intracellular NO level. Western blot and immunofluorescence as well as immunohistochemistry reveal that β-BA increases phosphorylation of enzyme nitric oxide synthase (eNOS) at Ser1177. In addition, β-BA mediated endothelium-dependent vasodilatation can be markedly blocked by eNOS inhibitor L-NAME in blood stasis rats. In OGD treated HUEVCs, the protective effect of β-BA is attenuated by knockdown of eNOS. In conclusion, the above findings provide convincing evidence for the protective effects of β-BA on blood stasis induced endothelial dysfunction by eNOS signaling pathway. PMID:26482008

  8. Pretreatment with β-Boswellic Acid Improves Blood Stasis Induced Endothelial Dysfunction: Role of eNOS Activation

    PubMed Central

    Wang, Mingming; Chen, Minchun; Ding, Yi; Zhu, Zhihui; Zhang, Yikai; Wei, Peifeng; Wang, Jingwen; Qiao, Yi; Li, Liang; Li, Yuwen; Wen, Aidong

    2015-01-01

    Vascular endothelial cells play an important role in modulating anti-thrombus and maintaining the natural function of vascular by secreting many active substances. β-boswellic acid (β-BA) is an active triterpenoid compound from the extract of boswellia serrate. In this study, it is demonstrated that β-BA ameliorates plasma coagulation parameters, protects endothelium from blood stasis induced injury and prevents blood stasis induced impairment of endothelium-dependent vasodilatation. Moreover, it is found that β-BA significantly increases nitric oxide (NO) and cyclic guanosine 3’, 5’-monophosphate (cGMP) levels in carotid aortas of blood stasis rats. To stimulate blood stasis-like conditions in vitro, human umbilical vein endothelial cells (HUVECs) were exposed to transient oxygen and glucose deprivation (OGD). Treatment of β-BA significantly increased intracellular NO level. Western blot and immunofluorescence as well as immunohistochemistry reveal that β-BA increases phosphorylation of enzyme nitric oxide synthase (eNOS) at Ser1177. In addition, β-BA mediated endothelium-dependent vasodilatation can be markedly blocked by eNOS inhibitor L-NAME in blood stasis rats. In OGD treated HUEVCs, the protective effect of β-BA is attenuated by knockdown of eNOS. In conclusion, the above findings provide convincing evidence for the protective effects of β-BA on blood stasis induced endothelial dysfunction by eNOS signaling pathway. PMID:26482008

  9. Regulation of Endothelial Glutathione by ICAM-1 governs VEGF-A mediated eNOS Activity and Angiogenesis

    PubMed Central

    Langston, Will; Chidlow, John H.; Booth, Blake A.; Barlow, Shayne C.; Lefer, David J.; Patel, Rakesh P.; Kevil, Christopher G.

    2007-01-01

    Previous studies suggest that inflammatory cell adhesion molecules may modulate endothelial cell migration and angiogenesis through unknown mechanisms. Using a combination of in vitro and in vivo approaches, herein we reveal a novel redox sensitive mechanism by which ICAM-1 modulates endothelial GSH that controls VEGF-A induced eNOS activity, endothelial chemotaxis, and angiogenesis. In vivo disk angiogenesis assays showed attenuated VEGF-A mediated angiogenesis in ICAM-1−/− mice. Moreover, VEGF-A dependent chemotaxis, eNOS phosphorylation, and nitric oxide (NO) production were impaired in ICAM-1−/− MAEC compared to WT MAEC. Decreasing intracellular GSH in ICAM-1−/− MAEC to levels observed in WT MAEC with 150 μM buthionine sulfoximine (BSO) restored VEGF-A responses. Conversely, GSH supplementation of WT MAEC with 5 mM glutathione ethyl ester (GEE) mimicked defects observed in ICAM-1−/− cells. Deficient angiogenic responses in ICAM-1−/− cells were associated with increased expression of the lipid phosphatase, PTEN, consistent with antagonism of signaling pathways leading to eNOS activation. PTEN expression was also sensitive to GSH status, decreasing or increasing in proportion to intracellular GSH concentrations. These data suggest a novel role for ICAM-1 in modulating VEGF-A induced angiogenesis and eNOS activity through regulation of PTEN expression via modulation of intracellular GSH status. PMID:17291995

  10. ET-1 Stimulates Superoxide Production by eNOS Following Exposure of Vascular Endothelial Cells to Endotoxin.

    PubMed

    Gopalakrishna, Deepak; Pennington, Samantha; Karaa, Amel; Clemens, Mark G

    2016-07-01

    It has been shown that microcirculation is hypersensitized to endothelin1 (ET-1) following endotoxin (lipopolysaccharide [LPS]) treatment leading to an increased vasopressor response. This may be related in part to decreased activation of endothelial nitric oxide synthase (eNOS) by ET-1. eNOS can also be uncoupled to produce superoxide (O2). This aberrant eNOS activity could further contribute to the hyperconstriction and injury caused by ET-1 following LPS. We therefore tested whether LPS affects ROS production by vascular endothelial cells and whether and how this effect is altered by ET-1. Human umbilical vein endothelial cells (HUVEC) or human microvascular endothelial cells (HMEC) were subjected to a 6-h treatment with LPS (250 ng/mL) or LPS and sepiapterin (100 μM) followed by a 30-min treatment with 100 μM L-Iminoethyl Ornithine (L-NIO) an irreversible eNOS inhibitor and 30-min treatment with ET-1 (10 nM). Conversion of [H]L-arginine to [H]L-citrulline was used to measure eNOS activity. Superoxide dismutase (SOD) inhibitable reduction of Cytochrome-C, dihydro carboxy fluorescein (DCF), and Mitosox was used to estimate ROS. LT-SDS PAGE was used to assess the degree of monomerization of the eNOS homodimer. Stimulation of HUVECs with ET-1 significantly increased NO synthesis by 1.4-fold (P < 0.05). ET-1 stimulation of LPS-treated HUVECs failed to increase NO production. Western blot for eNOS protein showed no change in eNOS protein levels. LPS alone resulted in an insignificant increase in ROS production as measured by cytochrome C that was increased 4.6-fold by ET-1 stimulation (P < 0.05). L-NIO significantly decreased ET-1-induced ROS production (P < 0.05). Sepiapterin significantly decreased ROS production in both; unstimulated and ET-1-stimulated LPS-treated groups, but did not restore NO production. DCF experiments confirmed intracellular ROS while Mitosox suggested a non-mitochondrial source. ET-1 treatment following a chronic LPS stress

  11. Endothelial Estrogen Receptor-α Does Not Protect Against Vascular Stiffness Induced by Western Diet in Female Mice.

    PubMed

    Manrique, Camila; Lastra, Guido; Ramirez-Perez, Francisco I; Haertling, Dominic; DeMarco, Vincent G; Aroor, Annayya R; Jia, Guanghong; Chen, Dongqing; Barron, Brady J; Garro, Mona; Padilla, Jaume; Martinez-Lemus, Luis A; Sowers, James R

    2016-04-01

    Consumption of a diet high in fat and refined carbohydrates (Western diet [WD]) is associated with obesity and insulin resistance, both major risk factors for cardiovascular disease (CVD). In women, obesity and insulin resistance abrogate the protection against CVD likely afforded by estrogen signaling through estrogen receptor (ER)α. Indeed, WD in females results in increased vascular stiffness, which is independently associated with CVD. We tested the hypothesis that loss of ERα signaling in the endothelium exacerbates WD-induced vascular stiffening in female mice. We used a novel model of endothelial cell (EC)-specific ERα knockout (EC-ERαKO), obtained after sequential crossing of the ERα double floxed mice and VE-Cadherin Cre-recombinase mice. Ten-week-old females, EC-ERαKO and aged-matched genopairs were fed either a regular chow diet (control diet) or WD for 8 weeks. Vascular stiffness was measured in vivo by pulse wave velocity and ex vivo in aortic explants by atomic force microscopy. In addition, vascular reactivity was assessed in isolated aortic rings. Initial characterization of the model fed a control diet did not reveal changes in whole-body insulin sensitivity, aortic vasoreactivity, or vascular stiffness in the EC-ERαKO mice. Interestingly, ablation of ERα in ECs reduced WD-induced vascular stiffness and improved endothelial-dependent dilation. In the setting of a WD, endothelial ERα signaling contributes to vascular stiffening in females. The precise mechanisms underlying the detrimental effects of endothelial ERα in the setting of a WD remain to be elucidated. PMID:26872089

  12. ICAM-1-activated Src and eNOS signaling increase endothelial cell surface PECAM-1 adhesivity and neutrophil transmigration.

    PubMed

    Liu, Guoquan; Place, Aaron T; Chen, Zhenlong; Brovkovych, Viktor M; Vogel, Stephen M; Muller, William A; Skidgel, Randal A; Malik, Asrar B; Minshall, Richard D

    2012-08-30

    Polymorphonuclear neutrophil (PMN) extravasation requires selectin-mediated tethering, intercellular adhesion molecule-1 (ICAM-1)-dependent firm adhesion, and platelet/endothelial cell adhesion molecule 1 (PECAM-1)-mediated transendothelial migration. An important unanswered question is whether ICAM-1-activated signaling contributes to PMN transmigration mediated by PECAM-1. We tested this concept and the roles of endothelial nitric oxide synthase (eNOS) and Src activated by PMN ligation of ICAM-1 in mediating PECAM-1-dependent PMN transmigration. We observed that lung PMN infiltration in vivo induced in carrageenan-injected WT mice was significantly reduced in ICAM-1(-/-) and eNOS(-/-) mice. Crosslinking WT mouse ICAM-1 expressed in human endothelial cells (ECs), but not the phospho-defective Tyr(518)Phe ICAM-1 mutant, induced SHP-2-dependent Src Tyr530 dephosphorylation that resulted in Src activation. ICAM-1 activation also stimulated phosphorylation of Akt (p-Ser473) and eNOS (p-Ser1177), thereby increasing NO production. PMN migration across EC monolayers was abolished in cells expressing the Tyr(518)Phe ICAM-1 mutant or by pretreatment with either the Src inhibitor PP2 or eNOS inhibitor L-NAME. Importantly, phospho-ICAM-1 induction of Src signaling induced PECAM-1 Tyr686 phosphorylation and increased EC surface anti-PECAM-1 mAb-binding activity. These results collectively show that ICAM-1-activated Src and eNOS signaling sequentially induce PECAM-1-mediated PMN transendothelial migration. Both Src and eNOS inhibition may be important therapeutic targets to prevent or limit vascular inflammation. PMID:22806890

  13. Obligatory Role for Endothelial Heparan Sulphate Proteoglycans and Caveolae Internalization in Catestatin-Dependent eNOS Activation

    PubMed Central

    Fornero, Sara; Bassino, Eleonora; Ramella, Roberta; Mahata, Sushil K.; Tota, Bruno; Alloatti, Giuseppe

    2014-01-01

    The chromogranin-A peptide catestatin modulates a wide range of processes, such as cardiovascular functions, innate immunity, inflammation, and metabolism. We recently found that the cardiac antiadrenergic action of catestatin requires a PI3K-dependent NO release from endothelial cells, although the receptor involved is yet to be identified. In the present work, based on the cationic properties of catestatin, we tested the hypothesis of its interaction with membrane heparan sulphate proteoglycans, resulting in the activation of a caveolae-dependent endocytosis. Experiments were performed on bovine aortic endothelial cells. Endocytotic vesicles trafficking was quantified by confocal microscopy using a water-soluble membrane dye; catestatin colocalization with heparan sulphate proteoglycans and caveolin 1 internalization were studied by fluorimetric measurements in live cells. Modulation of the catestatin-dependent eNOS activation was assessed by immunofluorescence and immunoblot analysis. Our results demonstrate that catestatin (5 nM) colocalizes with heparan sulphate proteoglycans and induces a remarkable increase in the caveolae-dependent endocytosis and caveolin 1 internalization, which were significantly reduced by both heparinase and wortmannin. Moreover, catestatin was unable to induce Ser1179 eNOS phosphorylation after pretreatments with heparinase and methyl-β-cyclodextrin. Taken together, these results highlight the obligatory role for proteoglycans and caveolae internalization in the catestatin-dependent eNOS activation in endothelial cells. PMID:25136621

  14. Uric acid enhances PKC-dependent eNOS phosphorylation and mediates cellular ER stress: A mechanism for uric acid-induced endothelial dysfunction

    PubMed Central

    LI, PENG; ZHANG, LINA; ZHANG, MEI; ZHOU, CHANGYONG; LIN, NAN

    2016-01-01

    The mechanism by which hyperuricemia induced-endothelial dysfunction contributes to cardiovascular diseases (CVDs) is not yet fully understood. In the present study, we used uric acid (UA) to trigger endothelial dysfunction in cultured endothelial cells, and investigated the effects of induced reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress induction, and the protein kinase C (PKC)-dependent endothelial nitric oxide synthase (eNOS) signaling pathway. Human umbilical vein endothelial cells (HUVECs) were incubated with 6, 9 or 12 mg/dl UA, ROS scavenger polyethylene glycol-superoxide dismutase (PEG-SOD), ER stress inhibitor 4-phenylbutyric acid (4-PBA), and PKC inhibitor polymyxin B for 6–48 h. Nitric oxide (NO) production, eNOS activity, intracellular ROS, ER stress levels, and the interaction between eNOS and calmodulin (CaM) and cytosolic calcium levels were assessed using fluorescence microscopy and western blot analysis. Apoptosis was assessed by annexin V staining. UA increased HUVEC apoptosis and reduced eNOS activity and NO production in a dose- and time-dependent manner. Intracellular ROS was elevated after 3 h, while ER stress level increased after 6 h. UA did not alter intracellular Ca2+, CaM, or eNOS concentration, or eNOS Ser1177 phosphorylation. However, PKC-dependent eNOS phosphorylation at Thr495 was greatly enhanced, and consequently interaction between eNOS and CaM was reduced. Cellular ROS depletion, ER stress inhibition and PKC activity reduction inhibited the effect of UA on eNOS activity, NO release and apoptosis in HUVECs. Thus, we concluded that UA induced HUVEC apoptosis and endothelial dysfunction by triggering oxidative and ER stress through PKC/eNOS-mediated eNOS activity and NO production. PMID:26935704

  15. Platelet activating factor-induced ceramide micro-domains drive endothelial NOS activation and contribute to barrier dysfunction.

    PubMed

    Predescu, Sanda; Knezevic, Ivana; Bardita, Cristina; Neamu, Radu Florin; Brovcovych, Viktor; Predescu, Dan

    2013-01-01

    The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles. PMID:24086643

  16. Platelet Activating Factor-Induced Ceramide Micro-Domains Drive Endothelial NOS Activation and Contribute to Barrier Dysfunction

    PubMed Central

    Predescu, Sanda; Knezevic, Ivana; Bardita, Cristina; Neamu, Radu Florin; Brovcovych, Viktor; Predescu, Dan

    2013-01-01

    The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles. PMID:24086643

  17. Endothelial and neuronal nitric oxide synthases variably modulate the estrogen-mediated control of blood pressure and cardiovascular autonomic control

    PubMed Central

    El-Mas, Mahmoud M.; Abdel-Rahman, Abdel A.

    2014-01-01

    Summary We have previously shown that long-term estrogen (E2) replacement lowers blood pressure (BP) and improves the cardiovascular autonomic control in ovariectomized (OVX) rats. In this study, we investigated whether constitutive and/or inducible nitric oxide synthase (NOS) modulate these E2 effects.We evaluated changes in BP, myocardial contractility index (dP/dtmax), and power spectral indices of hemodynamic variability following selective inhibition of eNOS [N5-(1-iminoethyl)-L-ornithine; L-NIO], nNOS (Nω-propyl-L-arginine; NPLA), or iNOS (1400W) in telemetered OVX rats treated for 16 weeks with (OVXE2) or without (control, OVXC) E2.OVXE2 rats exhibited: (i) reduced BP, and increased dP/dtmax, (ii) cardiac parasympathetic dominance as reflected by the reduced low-frequency (LF, 0.25–0.75 Hz)/high-frequency (HF, 0.75–3 Hz) ratio of interbeat intervals (IBILF/HF), and (iii) reduced LF oscillations of systolic BP, suggesting a reduced vasomotor sympathetic tone.eNOS inhibition (L-NIO, 20 mg/kg i.p.) elicited a shorter-lived pressor response in OVXE2, than in OVXC, rats along with reductions in dP/dtmax and increases in the spectral index of spontaneous baroreflex sensitivity (index α). NPLA (1 mg/kg i.p.) reduced BP and increased IBILF/HF ratio in OVXE2, but not OVXC rats. The iNOS inhibitor 1400W (5 mg/kg i.p.) caused no hemodynamic changes in OVXC or OVXE2 rats.Overall, constitutive NOS isoforms exert restraining tonic modulatory BP effects, which encompass eNOS-mediated reduction and nNOS-mediated elevation in BP in OVXE2 rats. Baroreflex facilitation, and dP/dtmax reductions might account for the shorter pressor action of L-NIO in E2-treated, compared with untreated, OVX rats. PMID:24471817

  18. Purinergic glio-endothelial coupling during neuronal activity: role of P2Y1 receptors and eNOS in functional hyperemia in the mouse somatosensory cortex.

    PubMed

    Toth, Peter; Tarantini, Stefano; Davila, Antonio; Valcarcel-Ares, M Noa; Tucsek, Zsuzsanna; Varamini, Behzad; Ballabh, Praveen; Sonntag, William E; Baur, Joseph A; Csiszar, Anna; Ungvari, Zoltan

    2015-12-01

    Impairment of moment-to-moment adjustment of cerebral blood flow (CBF) via neurovascular coupling is thought to play a critical role in the genesis of cognitive impairment associated with aging and pathological conditions associated with accelerated cerebromicrovascular aging (e.g., hypertension, obesity). Although previous studies demonstrate that endothelial dysfunction plays a critical role in neurovascular uncoupling in these conditions, the role of endothelial NO mediation in neurovascular coupling responses is not well understood. To establish the link between endothelial function and functional hyperemia, neurovascular coupling responses were studied in mutant mice overexpressing or deficient in endothelial NO synthase (eNOS), and the role of P2Y1 receptors in purinergic glioendothelial coupling was assessed. We found that genetic depletion of eNOS (eNOS(-/-)) and pharmacological inhibition of NO synthesis significantly decreased the CBF responses in the somatosensory cortex evoked by whisker stimulation and by administration of ATP. Overexpression of eNOS enhanced NO mediation of functional hyperemia. In control mice, the selective and potent P2Y1 receptor antagonist MRS2179 attenuated both whisker stimulation-induced and ATP-mediated CBF responses, whereas, in eNOS(-/-) mice, the inhibitory effects of MRS2179 were blunted. Collectively, our findings provide additional evidence for purinergic glio-endothelial coupling during neuronal activity, highlighting the role of ATP-mediated activation of eNOS via P2Y1 receptors in functional hyperemia. PMID:26453330

  19. Triterpenoic Acids from Apple Pomace Enhance the Activity of the Endothelial Nitric Oxide Synthase (eNOS).

    PubMed

    Waldbauer, Katharina; Seiringer, Günter; Nguyen, Dieu Linh; Winkler, Johannes; Blaschke, Michael; McKinnon, Ruxandra; Urban, Ernst; Ladurner, Angela; Dirsch, Verena M; Zehl, Martin; Kopp, Brigitte

    2016-01-13

    Pomace is an easy-accessible raw material for the isolation of fruit-derived compounds. Fruit consumption is associated with health-promoting effects, such as the prevention of cardiovascular disease. Increased vascular nitric oxide (NO) bioavailability, for example, due to an enhanced endothelial nitric oxide synthase (eNOS) activity, could be one molecular mechanism mediating this effect. To identify compounds from apple (Malus domestica Borkh.) pomace that have the potential to amplify NO bioavailability via eNOS activation, a bioassay-guided fractionation of the methanol/water (70:30) extract has been performed using the (14)C-L-arginine to (14)C-L-citrulline conversion assay (ACCA) in the human endothelium-derived cell line EA.hy926. Phytochemical characterization of the active fractions was performed using the spectrophotometric assessment of the total phenolic content, as well as TLC, HPLC-DAD-ELSD, and HPLC-MS analyses. Eleven triterpenoic acids, of which one is a newly discovered compound, were identified as the main constituents in the most active fraction, accompanied by only minor contents of phenolic compounds. When tested individually, none of the tested compounds exhibited significant eNOS activation. Nevertheless, cell stimulation with the reconstituted compound mixture restored eNOS activation, validating the potential of apple pomace as a source of bioactive components. PMID:26682617

  20. TNFα reduces eNOS activity in endothelial cells through serine 116 phosphorylation and Pin1 binding: Confirmation of a direct, inhibitory interaction of Pin1 with eNOS.

    PubMed

    Kennard, Simone; Ruan, Ling; Buffett, Ryan J; Fulton, David; Venema, Richard C

    2016-06-01

    Production of NO by the endothelial nitric oxide synthase (eNOS) has a major role in blood pressure control and suppression of atherosclerosis. In a previous study, we presented evidence implicating the Pin1 prolyl isomerase in negative modulation of eNOS activity in bovine aortic endothelial cells (BAECs). Pin1 recognizes phosphoserine/phosphothreonine-proline motifs in target proteins and catalyzes prolyl isomerization at the peptide bond. In the present study, we show, first, with purified proteins, that Pin1 binds to eNOS directly via the Pin1 WW domain. Binding is enhanced by mimicking phosphorylation of eNOS at S116. Interaction of Pin1 with eNOS markedly reduces eNOS enzymatic activity. Second, in BAECs, we show that TNFα induces ERK 1/2-mediated S116 phosphorylation of eNOS, accompanied by Pin1 binding. TNFα treatment of BAECs results in a reduction in NO release from the cells in a manner that depends on the activities of both Pin1 and ERK 1/2. Evidence is also presented that this mechanism of eNOS regulation cannot occur in rat and mouse cells because there is no proline residue in the mouse and rat amino acid sequences adjacent to the putative phosphorylation site. Moreover, we find that phosphorylation of this site is not detectable in mouse eNOS. PMID:27073025

  1. Endothelial Nitric Oxide Synthase (eNOS) 4a/b and G894T Polymorphisms and Susceptibility to Preeclampsia

    PubMed Central

    Rahimi, Zohreh; Aghaei, Amir; Rahimi, Ziba; Vaisi-Raygani, Asad

    2013-01-01

    Background Preeclampsia is a pregnancy complication with unknown etiology and its incidence is associated with genetic and environmental factors. There are inconsistent reports related to the role of endothelial nitric oxide synthase (eNOS) 4a/b polymorphism on the risk of preeclampsia development. The aim of the present study was to investigate the possible influence of eNOS 4a/b and its synergism with eNOS G894T polymorphism on the risk of preeclampsia. Methods The present case-control study consisted of 179 unrelated women with preeclampsia including 118 with mild and 61 with severe preeclampsia and 96 unrelated women with normal pregnancy as controls. All studied women were from Kermanshah Province of Iran. eNOS 4a/b and G894T genotypes were detected using polymerase chain reaction (PCR), and PCR-restriction fragment length polymorphism (RFLP) methods, respectively. The categorical variables between groups were compared using χ2 test and the Odds ratios (OR) were obtained by SPSS logistic regression. Statistical significance was assumed at p<0.05 level. Results The frequency of eNOS a allele was slightly higher in both mild (16.5%) and severe (17.2%) preeclamptic women than controls (15.1%). Also, no significant difference was found between early- and late-onset preeclamptic women regarding the distribution of eNOS 4a/b genotypes. The presence of each allele of eNOS a or T was not associated with the risk of preeclampsia. However, the concomitant presence of both eNOS a and T alleles was associated with a non significant increased risk of severe preeclampsia by 1.77-fold (p=0.35). Conclusion The present study indicates the lack of association between eNOS a and T alleles with the risk of mild preeclampsia and a non significant increased risk of severe preeclampsia in the presence of both alleles which needs to be investigated in a study with larger samples. PMID:24551572

  2. Cicletanine stimulates eNOS phosphorylation and NO production via Akt and MAP kinase/Erk signaling in sinusoidal endothelial cells.

    PubMed

    Liu, Songling; Rockey, Don C

    2013-07-15

    The function of the endothelial isoform of nitric oxide synthase (eNOS) and production of nitric oxide (NO) is altered in a number of disease states. Pharmacological approaches to enhancing NO synthesis and thus perhaps endothelial function could have substantial benefits in patients. We analyzed the effect of cicletanine, a synthetic pyridine with potent vasodilatory characteristics, on eNOS function and NO production in normal (liver) and injured rat sinusoidal endothelial cells, and we studied the effect of cicletanine-induced NO on stellate cell contraction and portal pressure in an in vivo model of liver injury. Sinusoidal endothelial cells were isolated from normal and injured rat livers. After exposure to cicletanine, eNOS phosphorylation, NO synthesis, and the signaling pathway regulating eNOS activation were measured. Cicletanine led to an increase in eNOS (Ser¹¹⁷⁷) phosphorylation, cytochrome c reductase activity, L-arginine conversion to L-citrulline, as well as NO production. The mechanism of the effect of cicletanine appeared to be via the protein kinase B (Akt) and MAP kinase/Erk signaling pathways. Additionally, cicletanine improved NO synthesis in injured sinusoidal endothelial cells. NO production induced by cicletanine in sinusoidal endothelial cells increased protein kinase G (PKG) activity as well as relaxation of stellate cells. Finally, administration of cicletanine to mice with portal hypertension induced by bile duct ligation led to reduction of portal pressure. The data indicate that cicletanine might improve eNOS activity in injured sinusoidal endothelial cells and likely activates hepatic stellate cell NO/PKG signaling. It raises the possibility that cicletanine could improve intrahepatic vascular function in portal hypertensive patients. PMID:23639812

  3. Ursolic acid from the Chinese herb danshen (Salvia miltiorrhiza L.) upregulates eNOS and downregulates Nox4 expression in human endothelial cells.

    PubMed

    Steinkamp-Fenske, Katja; Bollinger, Larissa; Völler, Natalie; Xu, Hui; Yao, Ying; Bauer, Rudolf; Förstermann, Ulrich; Li, Huige

    2007-11-01

    Danshen, the dried root of Salvia miltiorrhiza Bunge (Lamiaceae), is one of the most commonly used traditional Chinese medicines for cardiovascular indications. In EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVEC), an aqueous extract of danshen, and also a methanol extract of the plant, increased eNOS promoter activity, eNOS mRNA and protein expression, as well as endothelial NO production. A dichloromethane extract, in contrast, did not change eNOS gene expression. Thus, the active danshen constituent(s) responsible for eNOS upregulation is (are) hydrophilic and/or alcohol-soluble. One such compound is ursolic acid that significantly increased eNOS expression in EA.hy 926 cells and native HUVEC, and enhanced bioactive NO production measured in terms of its cGMP increasing activity. Other tested hydrophilic and alcohol-soluble compounds isolated from danshen had no effect on eNOS expression. Interestingly, ursolic acid also reduced the expression of the NADPH oxidase subunit Nox4 and suppressed the production of reactive oxygen species in human endothelial cells. Upregulation of eNOS and a parallel downregulation of Nox4 lead to an increase in bioactive NO. This in turn could mediate some of the beneficial effects of danshen. Ursolic acid is a prototypical compound responsible for this effect of the plant. PMID:17481637

  4. Adenosine Prevents TNFα-Induced Decrease in Endothelial Mitochondrial Mass via Activation of eNOS-PGC-1α Regulatory Axis

    PubMed Central

    Kalogeris, Theodore J.; Baines, Christopher; Korthuis, Ronald J.

    2014-01-01

    We tested whether adenosine, a cytoprotective mediator and trigger of preconditioning, could protect endothelial cells from inflammation-induced deficits in mitochondrial biogenesis and function. We examined this question using human microvascular endothelial cells exposed to TNFα. TNFα produced time and dose-dependent decreases in mitochondrial membrane potential, cellular ATP levels, and mitochondrial mass, preceding an increase in apoptosis. These effects were prevented by co-incubation with adenosine, a nitric oxide (NO) donor, a guanylate cyclase (GC) activator, or a cell-permeant cyclic GMP (cGMP) analog. The effects of adenosine were blocked by a nitric oxide synthase inhibitor, a soluble guanylate cyclase inhibitor, a morpholino antisense oligonucleotide to endothelial nitric oxide synthase (eNOS), or siRNA knockdown of the transcriptional coactivator, PGC-1α. Incubation with exogenous NO, a GC activator, or a cGMP analog reversed the effect of eNOS knockdown, while the effect of NO was blocked by inhibition of GC. The protective effects of NO and cGMP analog were prevented by siRNA to PGC-1α. TNFα also decreased expression of eNOS, cellular NO levels, and PGC-1α expression, which were reversed by adenosine. Exogenous NO, but not adenosine, rescued expression of PGC-1α in cells in which eNOS expression was knocked down by eNOS antisense treatment. Thus, TNFα elicits decreases in endothelial mitochondrial function and mass, and an increase in apoptosis. These effects were reversed by adenosine, an effect mediated by eNOS-synthesized NO, acting via soluble guanylate cyclase/cGMP to activate a mitochondrial biogenesis regulatory program under the control of PGC-1α. These results support the existence of an adenosine-triggered, mito-and cytoprotective mechanism dependent upon an eNOS-PGC-1α regulatory pathway, which acts to preserve endothelial mitochondrial function and mass during inflammatory challenge. PMID:24914683

  5. Roles of ROS and PKC-βII in ionizing radiation-induced eNOS activation in human vascular endothelial cells.

    PubMed

    Sakata, Kimimasa; Kondo, Takashi; Mizuno, Natsumi; Shoji, Miki; Yasui, Hironobu; Yamamori, Tohru; Inanami, Osamu; Yokoo, Hiroki; Yoshimura, Naoki; Hattori, Yuichi

    2015-07-01

    Vascular endothelial cells can absorb higher radiation doses than any other tissue in the body, and post-radiation impaired endothelial nitric oxide synthase (eNOS) function may be developed as a potential contributor to the pathogenesis of vascular injury. In this study, we investigated early alterations of eNOS signaling in human umbilical venous endothelial cells (HUVECs) exposed to X-ray radiation. We found that ionizing radiation increased eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in HUVECs in a dose-dependent (≤ 20 Gy) and time-dependent (6-72 h) manner. The total expression levels of eNOS were unchanged by radiation. Although a transient but significant increase in extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation and a biphasic decline in Akt phosphorylation were observed after irradiation, these inhibitors were without effect on the radiation-induced changes in eNOS phosphorylation. There was an increase in protein kinase C-βII (PKC-βII) expression and the ablation of PKC-βII by small interfering RNA (siRNA) negated the radiation effect on the two eNOS phosphorylation events. Furthermore, when the radiation-induced increase in reactive oxygen species (ROS) generation was prevented by the anti-oxidant N-acetyl-L-cysteine, eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation in irradiated HUVECs were significantly reduced. However, transfection of PKC-β siRNA did not alter ROS production after irradiation, and NAC failed to block the radiation-induced increase in PKC-βII expression. Taken together, our results suggest that ionizing radiation-induced eNOS activation in human vascular endothelial cells is attributed to both the up-regulation of PKC-βII and the increase in ROS generation which were independent of each other. PMID:25869503

  6. Antiphospholipid antibodies promote leukocyte-endothelial cell adhesion and thrombosis in mice by antagonizing eNOS via β2GPI and apoER2.

    PubMed

    Ramesh, Sangeetha; Morrell, Craig N; Tarango, Cristina; Thomas, Gail D; Yuhanna, Ivan S; Girardi, Guillermina; Herz, Joachim; Urbanus, Rolf T; de Groot, Philip G; Thorpe, Philip E; Salmon, Jane E; Shaul, Philip W; Mineo, Chieko

    2011-01-01

    In antiphospholipid syndrome (APS), antiphospholipid antibodies (aPL) binding to β2 glycoprotein I (β2GPI) induce endothelial cell-leukocyte adhesion and thrombus formation via unknown mechanisms. Here we show that in mice both of these processes are caused by the inhibition of eNOS. In studies of cultured human, bovine, and mouse endothelial cells, the promotion of monocyte adhesion by aPL entailed decreased bioavailable NO, and aPL fully antagonized eNOS activation by diverse agonists. Similarly, NO-dependent, acetylcholine-induced increases in carotid vascular conductance were impaired in aPL-treated mice. The inhibition of eNOS was caused by antibody recognition of domain I of β2GPI and β2GPI dimerization, and it was due to attenuated eNOS S1179 phosphorylation mediated by protein phosphatase 2A (PP2A). Furthermore, LDL receptor family member antagonism with receptor-associated protein (RAP) prevented aPL inhibition of eNOS in cell culture, and ApoER2-/- mice were protected from aPL inhibition of eNOS in vivo. Moreover, both aPL-induced increases in leukocyte-endothelial cell adhesion and thrombus formation were absent in eNOS-/- and in ApoER2-/- mice. Thus, aPL-induced leukocyte-endothelial cell adhesion and thrombosis are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by β2GPI, apoER2, and PP2A. Our results suggest that novel therapies for APS can now be developed targeting these mechanisms. PMID:21123944

  7. A protective role of ciglitazone in ox-LDL-induced rat microvascular endothelial cells via modulating PPARγ-dependent AMPK/eNOS pathway.

    PubMed

    Xu, Lei; Wang, Shijun; Li, Bingyu; Sun, Aijun; Zou, Yunzeng; Ge, Junbo

    2015-01-01

    Thiazolidinediones, the antidiabetic agents such as ciglitazone, has been proved to be effective in limiting atherosclerotic events. However, the underlying mechanism remains elucidative. Ox-LDL receptor-1 (LOX-1) plays a central role in ox-LDL-mediated atherosclerosis via endothelial nitric oxide synthase (eNOS) uncoupling and nitric oxide reduction. Therefore, we tested the hypothesis that ciglitazone, the PPARγ agonist, protected endothelial cells against ox-LDL through regulating eNOS activity and LOX-1 signalling. In the present study, rat microvascular endothelial cells (RMVECs) were stimulated by ox-LDL. The impact of ciglitazone on cell apoptosis and angiogenesis, eNOS expression and phosphorylation, nitric oxide synthesis and related AMPK, Akt and VEGF signalling pathway were observed. Our data showed that both eNOS and Akt phosphorylation, VEGF expression and nitric oxide production were significantly decreased, RMVECs ageing and apoptosis increased after ox-LDL induction for 24 hrs, all of which were effectively reversed by ciglitazone pre-treatment. Meanwhile, phosphorylation of AMP-activated protein kinase (AMPK) was suppressed by ox-LDL, which was also prevented by ciglitazone. Of interest, AMPK inhibition abolished ciglitazone-mediated eNOS function, nitric oxide synthesis and angiogenesis, and increased RMVECs ageing and apoptosis. Further experiments showed that inhibition of PPARγ significantly suppressed AMPK phosphorylation, eNOS expression and nitric oxide production. Ciglitazone-mediated angiogenesis and reduced cell ageing and apoptosis were reversed. Furthermore, LOX-1 protein expression in RMVECs was suppressed by ciglitazone, but re-enhanced by blocking PPARγ or AMPK. Ox-LDL-induced suppression of eNOS and nitric oxide synthesis were largely prevented by silencing LOX-1. Collectively, these data demonstrate that ciglitazone-mediated PPARγ activation suppresses LOX-1 and moderates AMPK/eNOS pathway, which contributes to endothelial cell

  8. Chronic aerobic exercise associated to dietary modification improve endothelial function and eNOS expression in high fat fed hamsters.

    PubMed

    Boa, Beatriz C S; Souza, Maria das Graças C; Leite, Richard D; da Silva, Simone V; Barja-Fidalgo, Thereza Christina; Kraemer-Aguiar, Luiz Guilherme; Bouskela, Eliete

    2014-01-01

    Obesity is epidemic in the western world and central adipose tissue deposition points to increased cardiovascular morbidity and mortality, independently of any association between obesity and other cardiovascular risk factors. Physical exercise has been used as non-pharmacological treatment to significantly reverse/attenuate obesity comorbidities. In this study we have investigated effects of exercise and/or dietary modification on microcirculatory function, body composition, serum glucose, iNOS and eNOS expression on 120 male hamsters treated for 12 weeks with high fat chow (HF, n = 30) starting on the 21st day of birth. From week 12 to 20, animals were randomly separated in HF (no treatment change), return to standard chow (HFSC, n = 30), high fat chow associated to an aerobic exercise training program (AET) (HFEX, n = 30) and return to standard chow+AET (HFSCEX, n = 30). Microvascular reactivity in response to acetylcholine and sodium nitroprusside and macromolecular permeability increase induced by 30 minutes ischemia followed by reperfusion were assessed on the cheek pouch preparation. Total body fat and aorta eNOS and iNOS expression by immunoblotting assay were evaluated on the experimental day. Compared to HFSC and HFSCEX groups, HF and HFEX ones presented increased visceral fat [(mean±SEM) (HF)4.9±1.5 g and (HFEX)4.7±0.9 g vs. (HFSC)*3.0±0.7 g and (HFSCEX)*1.9±0.4 g/100 g BW]; impaired endothelial-dependent vasodilatation [Ach 10(-8) M (HF)87.9±2.7%; (HFSC)*116.7±5.9%; (HFEX)*109.1±4.6%; (HFSCEX)*105±2.8%; Ach10(-6) M (HF)95.3±3.1%; (HFSC)*126±6.2%; (HFEX)*122.5±2.8%; (HFSCEX)*118.1±4.3% and Ach10(-4) M (HF)109.5±4.8%; (HFSC)*149.6±6.6%; (HFEX)*143.5±5.4% and (HFSCEX)*139.4±5.2%], macromolecular permeability increase after ischemia/reperfusion [(HF)40.5±4.2; (HFSC)*19.0±1.6; (HFEX)*18.6±2.1 and (HFSCEX)* 21.5±3.7 leaks/cm2), decreased eNOS expression, increased leptin and glycaemic levels. Endothelial

  9. Fenofibrate Improves Vascular Endothelial Function by Reducing Oxidative Stress While Increasing eNOS in Healthy Normolipidemic Older Adults

    PubMed Central

    Walker, Ashley E; Kaplon, Rachelle E; Lucking, Sara Marian S; Russell-Nowlan, Molly J; Eckel, Robert H; Seals, Douglas R

    2013-01-01

    Vascular endothelial dysfunction develops with aging, as indicated by impaired endothelium-dependent dilation(EDD), and is related to increased cardiovascular disease risk. We hypothesized that short-term treatment with fenofibrate, a lipid-lowering agent with potential pleiotropic effects, would improve EDD in middle-aged and older normolipidemic adults by reducing oxidative stress. Brachial artery flow-mediated dilation (FMD), a measure of EDD, was assessed in 22healthy adults aged 50-77 years before and after 7days of fenofibrate (145 mg/d; n=12/7M) or placebo (n=10/5M). Brachial FMD was unchanged with placebo, but improved after 2 and 7 days of fenofibrate (5.1±0.7 vs. 2d: 6.0±0.7 and 7d: 6.4±0.6 %Δ; both P<0.005). The improvements in FMD after 7 days remained significant (P<0.05) after accounting for modest changes in plasma total and LDL-cholesterol. Endothelium-independent dilation was not affected by fenofibrate or placebo (P>0.05). Infusion (i.v.) of the antioxidant vitamin C improved brachial FMD at baseline in both groups and during placebo treatment (P<0.05), but not after 2 and 7 days of fenofibrate (P>0.05). Fenofibrate treatment also reduced plasma oxidized LDL, a systemic marker of oxidative stress, compared with placebo (P<0.05). In vascular endothelial cells sampled from peripheral veins of the subjects, endothelial nitric oxide synthase (eNOS) protein expression was unchanged with placebo and after 2 days of fenofibrate, but was increased after 7 days of fenofibrate (0.54±0.03 vs. 2d: 0.52±0.04 and 7d: 0.76±0.11 intensity/HUVEC control; P<0.05 7d). Short-term treatment with fenofibrate improves vascular endothelial function in healthy normolipidemic middle-aged/older adults by reducing oxidative stress and induces increases in eNOS. PMID:23108655

  10. Sodium nitrite exerts an antihypertensive effect and improves endothelial function through activation of eNOS in the SHR.

    PubMed

    Ling, Wei Chih; Murugan, Dharmani Devi; Lau, Yeh Siang; Vanhoutte, Paul M; Mustafa, Mohd Rais

    2016-01-01

    Sodium nitrite (NaNO2) induces relaxation in isolated arteries partly through an endothelium-dependent mechanism involving NO-eNOS-sGC-cGMP pathway. The present study was designed to investigate the effect of chronic NaNO2 administration on arterial systolic blood pressure (SBP) and vascular function in hypertensive rats. NaNO2 (150 mg L-1) was given in drinking water for four weeks to spontaneously (SHR) and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) treated hypertensive SD rats. Arterial SBP and vascular function in isolated aortae were studied. Total plasma nitrate/nitrite and vascular cyclic guanosine monophosphate (cGMP) levels were measured using commercially available assay kits. Vascular nitric oxide (NO) levels were evaluated by DAF-FM fluorescence while the proteins involved in endothelial nitric oxide synthase (eNOS) activation was determined by Western blotting. NaNO2 treatment reduced SBP, improved the impaired endothelium-dependent relaxation, increased plasma total nitrate/nitrite level and vascular tissue NO and cGMP levels in SHR. Furthermore, increased presence of phosphorylated eNOS and Hsp-90 was observed in NaNO2-treated SHR. The beneficial effect of nitrite treatment was not observed in L-NAME treated hypertensive SD rats. The present study provides evidence that chronic treatment of genetically hypertensive rats with NaNO2 improves endothelium-dependent relaxation in addition to its antihypertensive effect, partly through mechanisms involving activation of eNOS. PMID:27616322

  11. eNOS Glu298Asp Polymorphism and Endothelial Dysfunction in Patients with and without End-stage Renal Disease

    PubMed Central

    İlhan, Nevin; Ateş, Kadir; İlhan, Necip; Kaman, Dilara; Çeliker, Hüseyin

    2016-01-01

    Background: Chronic kidney diseases are known to influence nitric oxide metabolites (NOx) and asymmetric dimethylarginine (ADMA), though the exact mechanism is still poorly understood. Aims: The purpose of the present study was to examine eNOS Glu298Asp gene polymorphism, plasma NOx and ADMA concentration in subjects with and without End-stage Renal Disease. Study Design: Case-control study. Methods: In this study, genotype distributions of Glu-298Asp in exon 7 of the eNOS gene polymorphisms in 130 hemodialysis and 64 peritoneal dialysis patients were compared with 92 controls. NOx was measured by using the Griess reaction while arginine, ADMA and SDMA measurements were performed by HPLC. Genotyping for eNOS Glu298Asp polymorphism was detected with the polymerase chain reaction and/or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Results: When the genotype frequencies of TT and GT genes were compared between both groups, there was no detected statistically important difference, even-though a TT genotype frequency was 27 (20.8%) versus 17 (26.6%), GT heterozygote genotype frequency was 52 (40%) versus 22 (34.4%), and GG homozygote genotype frequency was 51 (39.2%) versus 25 (39.1%), respectively (p>0.05). NOx, SDMA and ADMA concentrations were significantly elevated in subjects with hemodialysis patients as compared to their corresponding controls. Whereas nitrite was found to be significantly decreased in the patient with peritoneal dialysis. Conclusion: Not observed any connection between the Glu298Asp polymorphism in the eNOS gene and end-stage Renal Diseases in our study population under different dialysis treatments. However, higher ADMA and SDMA concentrations in subjects with ESRD support the existing hypothesis that NOx overproduction affects endothelial dysfunction. Thus, the reduction of ADMA and SDMA concentrations might play a protective role in ESRD patients. PMID:27403380

  12. Estrogen Defines the Dorsal-Ventral Limit of VEGF Regulation to Specify the Location of the Hemogenic Endothelial Niche

    PubMed Central

    Carroll, Kelli J; Esain, Virginie; Garnaas, Maija K; Cortes, Mauricio; Dovey, Michael C; Nissim, Sahar; Frechette, Gregory; Liu, Sarah; Kwan, Wanda; Cutting, Claire C; Harris, James M; Gorelick, Daniel; Halpern, Marnie; Lawson, Nathan; Goessling, Wolfram; North, Trista E

    2015-01-01

    Summary The genetic control of hematopoietic stem and progenitor cell (HSPC) function is increasingly understood, however less is known about the interactions specifying the embryonic hematopoietic niche. Here, we report 17β-estradiol (E2) influences the production of runx1+ HSPCs in the AGM region by antagonizing VEGF-signaling and subsequent assignment of hemogenic endothelial (HE) identity. Exposure to exogenous E2 during vascular niche development (12–24hpf) significantly disrupted flk1+ vessel maturation, ephrinB2+ arterial identity and specification of scl+ HE by decreasing the expression of VEGFAa and downstream arterial Notch-pathway components; heat-shock induction of VEGFAa/Notch rescued E2-mediated hemato-vascular defects. Conversely, repression of endogenous E2-activity increased somitic VEGF expression and vascular-target regulation, shifting the assignment of arterial/venous fate and HE localization; blocking E2-signaling allowed venous production of scl+/runx1+ cells, independent of arterial identity acquisition. Together, these data suggest yolk-derived estrogen sets the ventral boundary of hemogenic vascular niche specification by antagonizing the dorsal-ventral limits of VEGF regulation. PMID:24871948

  13. Placental villous vascular endothelial growth factor expression and vascularization after estrogen suppression during the last two-thirds of baboon pregnancy

    PubMed Central

    Robb, Victoria A.; Pepe, Gerald J.; Albrecht, Eugene D.

    2009-01-01

    We have recently shown that placental cytotrophoblast vascular endothelial growth factor (VEGF) expression and vessel density were increased by elevating estrogen and decreased by suppressing estrogen in early baboon pregnancy. The present study determined whether the elevation in estrogen which occurs in the last two-thirds of baboon pregnancy also has a role in the regulation of placental villous VEGF expression and angiogenesis. Placentas were obtained on day 170 of gestation (term, 184 days) from baboons untreated or treated with the aromatase inhibitor CGS 20267 or CGS 20267 plus estradiol daily on days 30–169. Serum estradiol levels in CGS 20267-treated baboons were decreased (P < 0.001) by 95%, however, placental cytotrophoblast VEGF mRNA levels (means ± SE, attomoles/μg RNA) were similar in untreated (25,807 ± 5,873), CGS 20267-treated (23,900 ± 1,940) and CGS 20267 plus estradiol-treated (26,885 ± 2,569) baboons. VEGF mRNA levels in the syncytiotrophoblast (2,008 ± 405) and inner villous stromal cell (1,724 ± 287) fractions of untreated baboons also were not altered by CGS 20267. However, whole villous VEGF mRNA levels in CGS 20267-treated baboons (18,590 ± 2,315) were 4-fold greater (P < 0.001) than in untreated animals and restored to normal by estradiol. Percent vascularized area (15.88 ± 0.88%) and vessel density (1,375 ± 71/mm2) of the villous placenta in untreated animals were not altered by estrogen deprivation. We propose that villous cytotrophoblasts lose their responsivity to estrogen and that placental villous cytotrophoblast VEGF expression and angiogenesis are regulated by estrogen in a cell- and gestational age-specific manner, and that factors other than estrogen maintain VEGF expression in the last two-thirds of pregnancy. PMID:17906373

  14. AB037. Icariside II improves human cavernous endothelial cells function by regulating miR-155/eNOS signal pathway

    PubMed Central

    Guan, Ruili; Lei, Hongen; Yang, Bicheng; Li, Huixi; Wang, Lin; Guo, Yinglu; Xin, Zhongcheng

    2016-01-01

    Background To investigate the changes of miR-155/endothelial nitric oxide synthase (eNOS) signal pathway under the stimulation of age-BSA and glucose with or without icariside II (ICAII) intervention inhuman cavernous endothelial cells (HCECs). Methods Purified HCECs were first divided into three groups randomly: normal group + BSA (NC group), age-BSA + glucose group (DM group), ICAII treatment group (DM + ICAII group with different concentrations at 0.1, 1, 10 µM). Western Blot to detect the protein expression of eNOS and RAGE; real time PCR to detect the expression of miR-155 and eNOS; DAF-FM DA fluorescent probes assay and NaNO3/NaNO2 assay to detect the NO concentration. Lentivirus mediated miR-155 over-expression was constructed to observe the changes of eNOS and NO. Results The eNOS and RAGE expression in DM group is significantly reduced and increased respectively compared with that of NC group (P<0.05), while ICAII intervention could reverse this change effectively. The 10 µM of ICAII has the most powerful effect. MiR-155 has the highest fold changes among candidate miRNAs in diabetic like HCECs (P<0.05). MiR-155 increased and eNOS decreased remarkably in DM group, while ICAII intervention could inhibit the miR-155 expression, which led to the significantly higher eNOS expression and NO concentration (P<0.05). In lentivirus mediated miR-155 overexpression with or without ICAII intervention model, we found the similar trend with the above diabetic model. Conclusions MiR-155/eNOS signal pathway may be involved in the process of diabetic HCECs dysfunction. ICAII could promote the recovery of the endothelial dysfunction by regulating the miR-155/eNOS signal pathway.

  15. A selective estrogen receptor modulator inhibits TNF-alpha-induced apoptosis by activating ERK1/2 signaling pathway in vascular endothelial cells.

    PubMed

    Yu, Jing; Eto, Masato; Akishita, Masahiro; Okabe, Tetsuro; Ouchi, Yasuyoshi

    2009-07-01

    Tumor necrosis factor (TNF-alpha) is a pleiotropic cytokine exerting both inflammatory and cell death activity and is thought to play a role in the pathogenesis of atherosclerosis. The present study was designed to examine whether the raloxifene analogue, LY117018 could inhibit TNF-alpha-induced apoptosis in vascular endothelial cells and to clarify the involved mechanisms. Apoptosis of endothelial cells was determined by DNA fragmentation assay and the activation of caspase-3. LY117018 significantly inhibited TNF-alpha-induced caspase-3 activation and cell DNA fragmentation levels in bovine carotid artery endothelial cells. The inhibitory effect of LY117018 was abolished by an estrogen receptor antagonist ICI 182,780. p38 MAPK, JNK, ERK1/2 and Akt have been shown to act as apoptotic or anti-apoptotic signals. TNF-alpha stimulated the phosphorylation levels of p38 MAPK, JNK, ERK1/2 and Akt in vascular endothelial cells. TNF-alpha-induced apoptosis was significantly decreased by SB203580, a p38 MAPK inhibitor or SP600125, a JNK inhibitor, but was enhanced by an ERK1/2 pathway inhibitor, PD98059 or a PI3-kinase/Akt pathway inhibitor, wortmannin. The anti-apoptotic effect of LY117018 was abrogated only by PD98059 but was not affected by the inhibitors for p38 MAPK, JNK, or Akt. LY117018 stimulated the further increase in phosphorylation of ERK1/2 in TNF-alpha treated endothelial cells but it did not affect phosphorylation levels of p38 MAPK, JNK or Akt. These results suggest that LY 110718 prevents caspase-3 dependent apoptosis induced by TNF-alpha in vascular endothelial cells through activation of the estrogen receptors and the ERK1/2 signaling pathway. PMID:19275968

  16. Insulin resistance reduces arterial prostacyclin synthase and eNOS activities by increasing endothelial fatty acid oxidation

    PubMed Central

    Du, Xueliang; Edelstein, Diane; Obici, Silvana; Higham, Ninon; Zou, Ming-Hui; Brownlee, Michael

    2006-01-01

    Insulin resistance markedly increases cardiovascular disease risk in people with normal glucose tolerance, even after adjustment for known risk factors such as LDL, triglycerides, HDL, and systolic blood pressure. In this report, we show that increased oxidation of FFAs in aortic endothelial cells without added insulin causes increased production of superoxide by the mitochondrial electron transport chain. FFA-induced overproduction of superoxide activated a variety of proinflammatory signals previously implicated in hyperglycemia-induced vascular damage and inactivated 2 important antiatherogenic enzymes, prostacyclin synthase and eNOS. In 2 nondiabetic rodent models — insulin-resistant, obese Zucker (fa/fa) rats and high-fat diet–induced insulin-resistant mice — inactivation of prostacyclin synthase and eNOS was prevented by inhibition of FFA release from adipose tissue; by inhibition of the rate-limiting enzyme for fatty acid oxidation in mitochondria, carnitine palmitoyltransferase I; and by reduction of superoxide levels. These studies identify what we believe to be a novel mechanism contributing to the accelerated atherogenesis and increased cardiovascular disease risk occurring in people with insulin resistance. PMID:16528409

  17. Ellagic acid protects endothelial cells from oxidized low-density lipoprotein-induced apoptosis by modulating the PI3K/Akt/eNOS pathway

    SciTech Connect

    Ou, Hsiu-Chung; Lee, Wen-Jane; Lee, Shin-Da; Huang, Chih-Yang; Chiu, Tsan-Hung; Tsai, Kun-Ling; Hsu, Wen-Cheng; Sheu, Wayne Huey-Herng

    2010-10-15

    Endothelial apoptosis is a driving force in atherosclerosis development. Oxidized low-density lipoprotein (oxLDL) promotes inflammatory and thrombotic processes and is highly atherogenic, as it stimulates macrophage cholesterol accumulation and foam cell formation. Previous studies have shown that the phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase/nitric oxide (PI3K/Akt/eNOS/NO) pathway is involved in oxLDL-induced endothelial apoptosis. Ellagic acid, a natural polyphenol found in berries and nuts, has in recent years been the subject of intense research within the fields of cancer and inflammation. However, its protective effects against oxLDL-induced injury in vascular endothelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effect of ellagic acid in human umbilical vein endothelial cells (HUVECs) exposed to oxLDL and explored the possible mechanisms. Our results showed that pretreatment with ellagic acid (5-20 {mu}M) significantly attenuated oxLDL-induced cytotoxicity, apoptotic features, and generation of reactive oxygen species (ROS). In addition, the anti-apoptotic effect of ellagic acid was partially inhibited by a PI3K inhibitor (wortmannin) and a specific eNOS inhibitor (cavtratin) but not by an ERK inhibitor (PD98059). In exploring the underlying mechanisms of ellagic acid action, we found that oxLDL decreased Akt and eNOS phosphorylation, which in turn activated NF-{kappa}B and downstream pro-apoptotic signaling events including calcium accumulation, destabilization of mitochondrial permeability, and disruption of the balance between pro- and anti-apoptotic Bcl-2 proteins. Those alterations induced by oxLDL, however, were attenuated by pretreatment with ellagic acid. The inhibition of oxLDL-induced endothelial apoptosis by ellagic acid is due at least in part to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway.

  18. Exenatide exerts direct protective effects on endothelial cells through the AMPK/Akt/eNOS pathway in a GLP-1 receptor-dependent manner.

    PubMed

    Wei, Rui; Ma, Shifeng; Wang, Chen; Ke, Jing; Yang, Jin; Li, Weihong; Liu, Ye; Hou, Wenfang; Feng, Xinheng; Wang, Guang; Hong, Tianpei

    2016-06-01

    Glucagon-like peptide-1 (GLP-1) may have direct favorable effects on cardiovascular system. The aim of this study was to investigate the effects of the GLP-1 analog exenatide on improving coronary endothelial function in patients with type 2 diabetes and to investigate the underlying mechanisms. The newly diagnosed type 2 diabetic subjects were enrolled and given either lifestyle intervention or lifestyle intervention plus exenatide treatment. After 12-wk treatment, coronary flow velocity reserve (CFVR), an important indicator of coronary endothelial function, was improved significantly, and serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were remarkably decreased in the exenatide treatment group compared with the baseline and the control group. Notably, CFVR was correlated inversely with hemoglobin A1c (Hb A1c) and positively with high-density lipoprotein cholesterol (HDL-C). In human umbilical vein endothelial cells, exendin-4 (a form of exenatide) significantly increased NO production, endothelial NO synthase (eNOS) phosphorylation, and GTP cyclohydrolase 1 (GTPCH1) level in a dose-dependent manner. The GLP-1 receptor (GLP-1R) antagonist exendin (9-39) or GLP-1R siRNA, adenylyl cyclase inhibitor SQ-22536, AMPK inhibitor compound C, and PI3K inhibitor LY-294002 abolished the effects of exendin-4. Furthermore, exendin-4 reversed homocysteine-induced endothelial dysfunction by decreasing sICAM-1 and reactive oxygen species (ROS) levels and upregulating NO production and eNOS phosphorylation. Likewise, exendin (9-39) diminished the protective effects of exendin-4 on the homocysteine-induced endothelial dysfunction. In conclusion, exenatide significantly improves coronary endothelial function in patients with newly diagnosed type 2 diabetes. The effect may be mediated through activation of AMPK/PI3K-Akt/eNOS pathway via a GLP-1R/cAMP-dependent mechanism. PMID:27072494

  19. G Protein-Coupled Estrogen Receptor-1 Is Involved in the Protective Effect of Protocatechuic Aldehyde against Endothelial Dysfunction

    PubMed Central

    Kong, Byung Soo; Cho, Yoon Hee; Lee, Eun Jig

    2014-01-01

    Protocatechuic aldehyde (PCA), a phenolic aldehyde, has therapeutic potency against atherosclerosis. Although PCA is known to inhibit the migration and proliferation of vascular smooth muscle cells and intravascular thrombosis, the underlying mechanism remains unclear. In this study, we investigated the protective effect of PCA on endothelial cells and injured vessels in vivo in association with G protein-coupled estrogen receptor-1 (GPER-1). With PCA treatment, cAMP production was increased in HUVECs, while GPER-1 expression was increased in both HUVECs and a rat aortic explant. PCA and G1, a GPER-1 agonist, reduced H2O2 stimulated ROS production in HUVECs, whereas, G15, a GPER-1 antagonist, increased ROS production further. These elevations were inhibited by co-treatment with PCA or G1. TNFα stimulated the expression of inflammatory markers (VCAM-1, ICAM-1 and CD40), phospho-NF-κB, phospho-p38 and HIF-1α; however, co-treatment with PCA or G1 down-regulated this expression significantly. Likewise, increased expression of inflammatory markers by treatment with G15 was inhibited by co-treatment with PCA. In re-endothelization, aortic ring sprouting and neointima formation assay, rat aortas treated with PCA or G1 showed accelerated re-endothelization of the endothelium and reduced sprouting and neointima formation. However, aortas from G15-treated rats showed decelerated re-endothelization and increased sprouting and neointima formation. The effects of G15 were restored by co-treatment with PCA or G1. Also, in the endothelia of these aortas, PCA and G1 increased CD31 and GPER-1 and decreased VCAM-1 and CD40 expression. In contrast, the opposite effect was observed in G15-treated endothelium. These results suggest that GPER-1 might mediate the protective effect of PCA on the endothelium. PMID:25411835

  20. Omentin protects against LPS-induced ARDS through suppressing pulmonary inflammation and promoting endothelial barrier via an Akt/eNOS-dependent mechanism.

    PubMed

    Qi, Di; Tang, Xumao; He, Jing; Wang, Daoxin; Zhao, Yan; Deng, Wang; Deng, Xinyu; Zhou, Guoqi; Xia, Jing; Zhong, Xi; Pu, Shenglan

    2016-01-01

    Acute respiratory distress syndrome (ARDS) is characterized by increased pulmonary inflammation and endothelial barrier permeability. Omentin has been shown to benefit obesity-related systemic vascular diseases; however, its effects on ARDS are unknown. In the present study, the level of circulating omentin in patients with ARDS was assessed to appraise its clinical significance in ARDS. Mice were subjected to systemic administration of adenoviral vector expressing omentin (Ad-omentin) and one-shot treatment of recombinant human omentin (rh-omentin) to examine omentin's effects on lipopolysaccharide (LPS)-induced ARDS. Pulmonary endothelial cells (ECs) were treated with rh-omentin to further investigate its underlying mechanism. We found that a decreased level of circulating omentin negatively correlated with white blood cells and procalcitonin in patients with ARDS. Ad-omentin protected against LPS-induced ARDS by alleviating the pulmonary inflammatory response and endothelial barrier injury in mice, accompanied by Akt/eNOS pathway activation. Treatment of pulmonary ECs with rh-omentin attenuated inflammatory response and restored adherens junctions (AJs), and cytoskeleton organization promoted endothelial barrier after LPS insult. Moreover, the omentin-mediated enhancement of EC survival and differentiation was blocked by the Akt/eNOS pathway inactivation. Therapeutic rh-omentin treatment also effectively protected against LPS-induced ARDS via the Akt/eNOS pathway. Collectively, these data indicated that omentin protects against LPS-induced ARDS by suppressing inflammation and promoting the pulmonary endothelial barrier, at least partially, through an Akt/eNOS-dependent mechanism. Therapeutic strategies aiming to restore omentin levels may be valuable for the prevention or treatment of ARDS. PMID:27607575

  1. Salidroside improves endothelial function and alleviates atherosclerosis by activating a mitochondria-related AMPK/PI3K/Akt/eNOS pathway.

    PubMed

    Xing, Sha-Sha; Yang, Xiao-Yan; Zheng, Tao; Li, Wen-Jing; Wu, Dan; Chi, Jiang-Yang; Bian, Fang; Bai, Xiang-Li; Wu, Guang-Jie; Zhang, You-Zhi; Zhang, Cun-Tai; Zhang, Yong-Hui; Li, Yong-Sheng; Jin, Si

    2015-09-01

    Salidroside (SAL) is a phenylpropanoid glycoside isolated from the medicinal plant Rhodiola rosea. A recent study has reported that SAL can efficiently decrease atherosclerotic plaque formation in low-density lipoprotein receptor-deficient mice. This study was to investigate the molecular mechanism of antiatherogenic effects of SAL. Given the importance of endothelial nitric oxide synthase (eNOS) in atherosclerosis, we sought to elucidate whether SAL could stimulate eNOS activation and also to explore its upstream signaling pathway. Six-week old apoE(-/-) male mice were fed a high-fat diet for 8weeks and then were administered with SAL for another 8weeks. SAL significantly improved endothelial function associated with increasing eNOS activation, thus reduced the atherosclerotic lesion area. SAL increased eNOS-Ser1177 phosphorylation and decreased eNOS-Thr495 phosphorylation, indicative of eNOS activation in endothelium. The aortic sinus lesions in SAL treated mice displayed reduced inflammation. SAL significantly activated AMP-activated protein kinase (AMPK). Both AMPK inhibitor and AMPK small interfering RNA (siRNA) abolished SAL-induced Akt-Ser473 and eNOS-Ser1177 phosphorylation. In contrast, LY294002, the PI3k/Akt pathway inhibitor, abolished SAL-induced phosphorylation and expression of eNOS. High performance liquid chromatography (HPLC) analysis revealed that SAL decreased cellular ATP content and increased the cellular AMP/ATP ratio, which was associated with the activation of AMPK. SAL was found to decrease the mitochondrial membrane potential (ΔΨm), which is a likely consequence of reduced ATP production. The action of SAL to reduce atherosclerotic lesion formation may at least be attributed to its effect on improving endothelial function by promoting nitric oxide (NO) production, which was associated with mitochondrial depolarization and subsequent activation of the AMPK/PI3K/Akt/eNOS pathway. Taken together, our data described the effects of SAL on

  2. MiR-21 is induced in endothelial cells by shear stress and modulates apoptosis and eNOS activity

    SciTech Connect

    Weber, Martina; Baker, Meredith B.; Moore, Jeffrey P.; Searles, Charles D.

    2010-03-19

    Mechanical forces associated with blood flow play an important role in regulating vascular signaling and gene expression in endothelial cells (ECs). MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in diverse cell functions, including differentiation, growth, proliferation, and apoptosis. miRNAs are known to have an important role in modulating EC biology, but their expression and functions in cells subjected to shear stress conditions are unknown. We sought to determine the miRNA expression profile in human ECs subjected to unidirectional shear stress and define the role of miR-21 in shear stress-induced changes in EC function. TLDA array and qRT-PCR analysis performed on HUVECs exposed to prolonged unidirectional shear stress (USS, 24 h, 15 dynes/cm{sup 2}) identified 13 miRNAs whose expression was significantly upregulated (p < 0.05). The miRNA with the greatest change was miR-21; it was increased 5.2-fold (p = 0.002) in USS-treated versus control cells. Western analysis demonstrated that PTEN, a known target of miR-21, was downregulated in HUVECs exposed to USS or transfected with pre-miR-21. Importantly, HUVECs overexpressing miR-21 had decreased apoptosis and increased eNOS phosphorylation and nitric oxide (NO{sup {center_dot}}) production. These data demonstrate that shear stress forces regulate the expression of miRNAs in ECs, and that miR-21 influences endothelial biology by decreasing apoptosis and activating the NO{sup {center_dot}} pathway. These studies advance our understanding of the mechanisms by which shear stress forces modulate vascular homeostasis.

  3. Modulation of cardiac contraction, relaxation and rate by the endothelial nitric oxide synthase (eNOS): lessons from genetically modified mice

    PubMed Central

    Massion, P B; Balligand, J-L

    2003-01-01

    The modulatory role of endothelial nitric oxide synthase (eNOS) on heart contraction, relaxation and rate is examined in light of recent studies using genetic deletion or overexpression in mice under specific conditions. Unstressed eNOS-/- hearts in basal conditions exhibit a normal inotropic and lusitropic function, with either decreased or unchanged heart rate. Under stimulation with catecholamines, eNOS-/- mice predominantly show a potentiation in their β-adrenergic inotropic and lusitropic responsiveness. A similar phenotype is observed in β3-adrenoceptor deficient mice, pointing to a key role of this receptor subtype for eNOS coupling. The effect of eNOS on the muscarinic cholinergic modulation of cardiac function probably operates in conjunction with other NO-independent mechanisms, the persistence of which may explain the apparent dispensability of this isoform for the effect of acetylcholine in some eNOS-/- mouse strains. eNOS-/- hearts submitted to short term ischaemia-reperfusion exhibit variable alterations in systolic and diastolic function and infarct size, while those submitted to myocardial infarction present a worsened ventricular remodelling, increased 1 month mortality and loss of benefit from ACE inhibitor or angiotensin II type I receptor antagonist therapy. Although non-conditional eNOS gene deletion may engender phenotypic adaptations (e.g. ventricular hypertrophy resulting from chronic hypertension, or upregulation of the other NOS isoforms) potentially confounding the interpretation of comparative studies, the use of eNOS-/- mice has undoubtedly advanced (and will probably continue to improve) our understanding of the complex role of eNOS (in conjunction with the other NOSs) in the regulation of cardiac function. The challenge is now to confirm the emerging paradigms in human cardiac physiology and hopefully translate them into therapy. PMID:12509479

  4. Effects of Estrogen on Platelet Reactivity After Transient Forebrain Ischemia in Rats

    PubMed Central

    Littleton-Kearney, Marguerite T.; Gaines, Jessica M.; Callahan, Kevin P.; Murphy, Stephanie J.; Hurn, Patricia D.

    2009-01-01

    Estrogen’s prothrombotic effects are of increasing concern, particularly in stroke risk and recovery. Using an ischemic rodent model, the authors sought to determine (a) if estrogen replacement increases post-ischemic platelet reactivity, (b) if changes in estrogen status alter intraplatelet endothelial nitric oxide synthase (eNOS) synthesis, and (c) if estrogen-mediated effects on platelets alter cerebral blood flow during reperfusion. Intact (I), ovariectomized (OVX), and OVX + 17β-estradiol (E50) rats were subjected to 30 min of forebrain ischemia and 60 min of reperfusion. Using the platelet activation marker P-selectin, postischemic platelet reactivity was quantified by flow cytometry. In a separate cohort (I, OVX, E50), the authors quantified platelet eNOS by Western blot. Another cohort (OVX, E50) was subjected to ischemia/reperfusion, and cerebral blood flow was determined using the iodoantipyrine technique. Collagen-stimulated platelet P-selectin expression was increased in the OVX rats at 60 min of reperfusion, and this effect was reversed by estrogen treatment. No differences in platelet eNOS expression were detected among groups. Cerebral blood flow at 60 min reperfusion was comparable between the OVX and the E50 rats. The authors conclude that during reperfusion, estrogen deficiency increases postischemic platelet sensitivity to stimuli in estrogen-deficient rats. Estrogen treatment mitigates effects of estrogen loss on platelets, but this early effect is apparently not caused by intraplatelet eNOS depression. Neither estrogen deficiency nor estrogen treatment changes early postischemic regional brain blood flow. In this rodent global cerebral ischemic model, physiologic doses of estrogen are not deleterious to platelet reactivity and may initially reduce postischemic platelet reactivity. PMID:16267375

  5. Possible involvement of PPARγ-associated eNOS signaling activation in rosuvastatin-mediated prevention of nicotine-induced experimental vascular endothelial abnormalities.

    PubMed

    Kathuria, Sonam; Mahadevan, Nanjaian; Balakumar, Pitchai

    2013-02-01

    Nicotine exposure via cigarette smoking and tobacco chewing is associated with vascular complications. The present study investigated the effect of rosuvastatin in nicotine (2 mg/kg/day, i.p., 4 weeks)-induced vascular endothelial dysfunction (VED) in rats. The development of VED was assessed by employing isolated aortic ring preparation and estimating aortic and serum nitrite/nitrate concentration. Further, scanning electron microscopy and hematoxylin-eosin staining of thoracic aorta were performed to assess the vascular endothelial integrity. Moreover, oxidative stress was assessed by estimating aortic superoxide anion generation and serum thiobarbituric acid-reactive substances. The nicotine administration produced VED by markedly reducing acetylcholine-induced endothelium-dependent relaxation, impairing the integrity of vascular endothelium, decreasing aortic and serum nitrite/nitrate concentration, increasing oxidative stress, and inducing lipid alteration. However, treatment with rosuvastatin (10 mg/kg/day, i.p., 4 weeks) markedly attenuated nicotine-induced vascular endothelial abnormalities, oxidative stress, and lipid alteration. Interestingly, the co-administration of peroxisome proliferator-activated receptor γ (PPARγ) antagonist, GW9662 (1 mg/kg/day, i.p., 2 weeks) submaximally, significantly prevented rosuvastatin-induced improvement in vascular endothelial integrity, endothelium-dependent relaxation, and nitrite/nitrate concentration in rats administered nicotine. However, GW9662 co-administration did not affect rosuvastatin-associated vascular anti-oxidant and lipid-lowering effects. The incubation of aortic ring, isolated from rosuvastatin-treated nicotine-administered rats, with L-NAME (100 μM), an inhibitor of nitric oxide synthase (NOS), significantly attenuated rosuvastatin-induced improvement in acetylcholine-induced endothelium-dependent relaxation. Rosuvastatin prevents nicotine-induced vascular endothelial abnormalities by activating

  6. Icariin stimulates angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways in human endothelial cells

    SciTech Connect

    Chung, Byung-Hee; Kim, Jong-Dai; Kim, Chun-Ki; Kim, Jung Huan; Won, Moo-Ho; Lee, Han-Soo; Dong, Mi-Sook; Ha, Kwon-Soo; Kwon, Young-Geun; Kim, Young-Myeong

    2008-11-14

    We investigated the molecular effect and signal pathway of icariin, a major flavonoid of Epimedium koreanum Nakai, on angiogenesis. Icariin stimulated in vitro endothelial cell proliferation, migration, and tubulogenesis, which are typical phenomena of angiogenesis, as well as increased in vivo angiogenesis. Icariin activated the angiogenic signal modulators, ERK, phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric oxide synthase (eNOS), and increased NO production, without affecting VEGF expression, indicating that icariin may directly stimulate angiogenesis. Icariin-induced ERK activation and angiogenic events were significantly inhibited by the MEK inhibitor PD98059, without affecting Akt and eNOS phosphorylation. The PI3K inhibitor Wortmannin suppressed icariin-mediated angiogenesis and Akt and eNOS activation without affecting ERK phosphorylation. Moreover, the NOS inhibitor NMA partially reduced the angiogenic activity of icariin. These results suggest that icariin stimulated angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways and may be a useful drug for angiogenic therapy.

  7. Impact of Rosuvastatin Treatment on HDL-Induced PKC-βII and eNOS Phosphorylation in Endothelial Cells and Its Relation to Flow-Mediated Dilatation in Patients with Chronic Heart Failure.

    PubMed

    Winzer, Ephraim B; Gaida, Pauline; Höllriegel, Robert; Fischer, Tina; Linke, Axel; Schuler, Gerhard; Adams, Volker; Erbs, Sandra

    2016-01-01

    Background. Endothelial function is impaired in chronic heart failure (CHF). Statins upregulate endothelial NO synthase (eNOS) and improve endothelial function. Recent studies demonstrated that HDL stimulates NO production due to eNOS phosphorylation at Ser(1177), dephosphorylation at Thr(495), and diminished phosphorylation of PKC-βII at Ser(660). The aim of this study was to elucidate the impact of rosuvastatin on HDL mediated eNOS and PKC-βII phosphorylation and its relation to endothelial function. Methods. 18 CHF patients were randomized to 12 weeks of rosuvastatin or placebo. At baseline, 12 weeks, and 4 weeks after treatment cessation we determined lipid levels and isolated HDL. Human aortic endothelial cells (HAEC) were incubated with isolated HDL and phosphorylation of eNOS and PKC-βII was evaluated. Flow-mediated dilatation (FMD) was measured at the radial artery. Results. Rosuvastatin improved FMD significantly. This effect was blunted after treatment cessation. LDL plasma levels were reduced after rosuvastatin treatment whereas drug withdrawal resulted in significant increase. HDL levels remained unaffected. Incubation of HAEC with HDL had no impact on phosphorylation of eNOS or PKC-βII. Conclusion. HDL mediated eNOS and PKC-βII phosphorylation levels in endothelial cells do not change with rosuvastatin in CHF patients and do not mediate the marked improvement in endothelial function. PMID:27563480

  8. Impact of Rosuvastatin Treatment on HDL-Induced PKC-βII and eNOS Phosphorylation in Endothelial Cells and Its Relation to Flow-Mediated Dilatation in Patients with Chronic Heart Failure

    PubMed Central

    Gaida, Pauline; Höllriegel, Robert; Fischer, Tina; Linke, Axel; Schuler, Gerhard; Adams, Volker; Erbs, Sandra

    2016-01-01

    Background. Endothelial function is impaired in chronic heart failure (CHF). Statins upregulate endothelial NO synthase (eNOS) and improve endothelial function. Recent studies demonstrated that HDL stimulates NO production due to eNOS phosphorylation at Ser1177, dephosphorylation at Thr495, and diminished phosphorylation of PKC-βII at Ser660. The aim of this study was to elucidate the impact of rosuvastatin on HDL mediated eNOS and PKC-βII phosphorylation and its relation to endothelial function. Methods. 18 CHF patients were randomized to 12 weeks of rosuvastatin or placebo. At baseline, 12 weeks, and 4 weeks after treatment cessation we determined lipid levels and isolated HDL. Human aortic endothelial cells (HAEC) were incubated with isolated HDL and phosphorylation of eNOS and PKC-βII was evaluated. Flow-mediated dilatation (FMD) was measured at the radial artery. Results. Rosuvastatin improved FMD significantly. This effect was blunted after treatment cessation. LDL plasma levels were reduced after rosuvastatin treatment whereas drug withdrawal resulted in significant increase. HDL levels remained unaffected. Incubation of HAEC with HDL had no impact on phosphorylation of eNOS or PKC-βII. Conclusion. HDL mediated eNOS and PKC-βII phosphorylation levels in endothelial cells do not change with rosuvastatin in CHF patients and do not mediate the marked improvement in endothelial function. PMID:27563480

  9. NFKB1 promoter variation implicates shear-induced NOS3 gene expression and endothelial function in prehypertensives and stage I hypertensives.

    PubMed

    Park, Joon-Young; Farrance, Iain K G; Fenty, Nicola M; Hagberg, James M; Roth, Stephen M; Mosser, David M; Wang, Min Qi; Jo, Hanjoong; Okazaki, Toshihiko; Brant, Steven R; Brown, Michael D

    2007-10-01

    In endothelial cells, NF-kappaB is an important intracellular signaling molecule by which changes in wall shear stress are transduced into the nucleus to initiate downstream endothelial nitric oxide synthase (NOS3) gene expression. We investigated whether NF-kappa light-chain gene enhancer in B cells 1 (NFKB1) promoter polymorphism ((-94)NFKB1 I/D, where I is the insertion allele and D is the deletion allele) was associated with 1) NOS3 gene expression in endothelial cells under physiological levels of unidirectional laminar shear stress (LSS) and 2) endothelial function in prehypertensive and stage I hypertensive individuals before and after a 6-mo supervised endurance exercise intervention. Competitive EMSAs revealed that proteins present in the nuclei of endothelial cells preferentially bound to the I allele NFKB1 promoter compared with the D allele. Reporter gene assays showed that the I allele promoter had significantly higher activity than the D allele. In agreement with these observations, homozygous II genotype cells had higher p50 expression levels than homozygous DD genotype cells. Cells with the homozygous II genotype showed a greater increase in NOS3 protein expression than did homozygous DD genotype cells under LSS. Functional experiments on volunteers confirmed higher baseline reactive hyperemic forearm blood flow, and, furthermore, the subgroup analysis revealed that DD homozygotes were significantly less prevalent in the exercise responder group compared with II and ID genotypes. We conclude that the (-94)NFKB1 I/D promoter variation contributes to the modulation of vascular function and adaptability to exercise-induced flow shear stress, most likely due to differences in NFKB1 gene transactivity. PMID:17644577

  10. The Involvement of Angiotensin Type 1 and Type 2 Receptors in Estrogen-Induced Cell Proliferation and Vascular Endothelial Growth Factor Expression in the Rat Anterior Pituitary

    PubMed Central

    Lawnicka, Hanna; Ptasinska-Wnuk, Dorota; Mucha, Slawomir; Kunert-Radek, Jolanta; Pawlikowski, Marek; Stepien, Henryk

    2012-01-01

    The aim of our study was to examine the involvement of renin-angiotensin system (RAS) in estrogen-induced lactotropes proliferation and vascular endothelial growth factor (VEGF) expression in rat pituitary. The study was performed on Fisher 344 rats underwent 8-day treatment with diethylstilboestrol (DES). The proliferation index (PCNA) and VEGF expression in pituitary sections were estimated using immunohistochemical methods. Treatment with DES increased the number of PCNA-positive cells, VEGF-positive cells, and VEGF-positive blood vessels in pituitary. Stimulatory effect of estrogen on cell proliferation and VEGF expression in blood vessels was attenuated by losartan, PD123319, and captopril. VEGF immunoreactivity in pituitary cells of DES-treated rats was decreased by AT1 antagonist and not changed by AT2 blocker and ACE inhibitor. Our findings suggest the involvement of RAS in DES-induced cell proliferation and VEGF expression in pituitary. Both the AT1 and AT2 receptors appear to mediate the estrogen-dependent mitogenic and proangiogenic effects in rat pituitary. PMID:22645419

  11. Extract from Ribes nigrum leaves in vitro activates nitric oxide synthase (eNOS) and increases CD39 expression in human endothelial cells.

    PubMed

    Luzak, Boguslawa; Boncler, Magdalena; Rywaniak, Joanna; Dudzinska, Dominika; Rozalski, Marek; Krajewska, Urszula; Balcerczak, Ewa; Podsedek, Anna; Redzynia, Malgorzata; Watala, Cezary

    2014-12-01

    The aim of the present study was to evaluate whether blackcurrant leaf extract (BLE) modulates endothelium antithrombotic function, namely increases the expression/activity of ADPase (CD39) and augments the production of nitric oxide in human umbilical vein endothelial cells (HUVEC). It was found that BLE with proanthocyanidins (60 % of the total polyphenol content) increased the CD39-positive endothelial cell fraction (up to 10 % for 2.5 μg/ml, and up to 33 % for 15 μg/ml, p < 0.05 or less) in a concentration-dependent manner, and enhanced endothelial nitric oxide synthase (eNOS) activation (T495 phosphorylation decreased by 31 ± 6 % for 2.5 μg/ml and 48 ± 6 % for 15 μg/ml; S1177 phosphorylation increased by 13 ± 3 % for 2.5 μg/ml and 18 ± 7 % for 15 μg/ml, compared to untreated cells, p < 0.05 or less). Additionally, incubation for 24 or 48 h with BLE at a lower range of polyphenol concentrations, significantly increased cell viability with a maximal effect at 2.5 μg/ml (viability increased by 24.8 ± 1.0 % for 24 h and by 32.5 ± 2.7 % for 48-h time incubation, p < 0.0001). The increased CD39 expression and the increased eNOS activation in HUVEC can be regarded as the beneficial markers of the improvement of antiplatelet action of endothelial cells. Unexpectedly, these assumptions were not confirmed in the experimental model of platelet-endothelial cell interactions. These observations lead to the conclusion that BLE may improve endothelial cell viability at low physiological concentrations without affecting the antiplatelet action of endothelium. PMID:25407137

  12. Apolipoprotein A-I mimetic peptide D-4F promotes human endothelial progenitor cell proliferation, migration, adhesion though eNOS/NO pathway.

    PubMed

    Zhang, Zhengang; Qun, Jianhua; Cao, Chunmei; Wang, Jun; Li, Wei; Wu, Yong; Du, Lin; Zhao, Pei; Gong, Kaizheng

    2012-04-01

    Circulating endothelial progenitor cells (EPCs) have a critical role in endothelial maintenance and repair. Apolipoprotein A-I mimetic peptide D-4F has been shown to posses anti-atherogenic properties via sequestration of oxidized phospholipids, induction of remodeling of high density lipoprotein and promotion of cholesterol efflux from macrophage-derived foam cells. In this study, we test the effects of D-4F on EPC biology. EPCs were isolated from the peripheral venous blood of healthy male volunteers and characterized by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated LDL uptake and ulex europaeus agglutinin binding and flow cytometry. Cell proliferation, migration, adhesion, nitric oxide production and endothelial nitric oxide synthase (eNOS) expression in the absence and presence of D-4F or simvastatin (as a positive control), were assayed. We demonstrated that D-4F significantly enhanced EPC proliferation, migration and adhesion in a dose-dependent manner compared with vehicle. However, all of the favorable effects of D-4F on EPCs were dramatically attenuated by preincubation with NOS inhibitor L-NAME. Further, D-4F also increased nitric oxide production in culture supernatant and the levels of eNOS expression and phosphorylation. The stimulatory effects of D-4F (10 μg/ml) on EPC biology were comparable to 0.5 μM simvastatin. These results suggest that eNOS/NO pathway mediates the functional modulation of EPC biology in response to D-4F treatment and support the notion that the beneficial role of D-4F on EPCs may be one of the important components of its anti-atherogenic potential. PMID:21947883

  13. Rumex acetosa L. induces vasorelaxation in rat aorta via activation of PI3-kinase/Akt- AND Ca(2+)-eNOS-NO signaling in endothelial cells.

    PubMed

    Sun, Y Y; Su, X H; Jin, J Y; Zhou, Z Q; Sun, S S; Wen, J F; Kang, D G; Lee, H S; Cho, K W; Jin, S N

    2015-12-01

    Rumex acetosa L. (RA) (Polygonaceae) is an important traditional Chinese medicine (TCM) commonly used in clinic for a long history in China and the aerial parts of RA has a wide variety of pharmacological actions such as diuretic, anti-hypertensive, anti-oxidative, and anti-cancer effects. However, the mechanisms involved are to be defined. The purpose of the present study was to evaluate the vasorelaxant effect and define the mechanism of action of the ethanol extract of Rumex acetosa L. (ERA) in rat aorta. ERA was examined for its vascular relaxant effect in isolated phenylephrine-precontracted rat thoracic aorta and its acute effects on arterial blood pressure. In addition, the roles of the nitric oxide synthase (NOS)-nitric oxide (NO) signaling in the ERA-induced effects were tested in human umbilical vein endothelial cells (HUVECs). The phosphorylation levels of Akt and eNOS were assessed by Western blot analysis in the cultured HUVECs. ERA induced endothelium-dependent vasorelaxation. The ERA-induced vasorelaxation was abolished by L-NAME (an NOS inhibitor) or ODQ (a sGC inhibitor), but not by indomethacin. Inhibition of PI3-kinase/Akt signaling pathway markedly reduced the ERA-induced vasorelaxation. In HUVECs, ERA increased NO formation in a dose-dependent manner, which was inhibited by L-NAME and by removing extracellular Ca(2+). In addition, ERA promoted phosphorylation of Akt and eNOS, which was prevented by wortmannin and LY294002, indicating that ERA induces eNOS phosphorylation through the PI3-kinase/Akt pathway. Further, in anesthetized rats, intravenously administered ERA decreased arterial blood pressure in a dose-dependent manner through an activation of the NOS-NO system. In summary, the ERA- induced vasorelaxation was dependent on endothelial integrity and NO production, and was mediated by activation of both the endothelial PI3-kinase/Akt- and Ca(2+)-eNOS-NO signaling and muscular NO-sGC-cGMP signaling. PMID:26769840

  14. Endothelial glutathione-S-transferase A4-4 protects against oxidative stress and modulates iNOS expression through NF-{kappa}B translocation

    SciTech Connect

    Yang Yongzhen; Yang Yusong; Xu Ya; Lick, Scott D.; Awasthi, Yogesh C.; Boor, Paul J.

    2008-07-15

    Our recent work in endothelial cells and human atherosclerotic plaque showed that overexpression of glutathione-S-tranferases (GSTs) in endothelium protects against oxidative damage from aldehydes such as 4-HNE. Nuclear factor (NF)-{kappa}B plays a crucial role during inflammation and immune responses by regulating the expression of inducible genes such as inducible nitric oxide synthase (iNOS). 4-HNE induces apoptosis and affects NF-{kappa}B mediated gene expression, but conflicting results on 4-HNE's effect on NF-{kappa}B have been reported. We compared the effect of 4-HNE on iNOS and the NF-{kappa}B pathway in control mouse pancreatic islet endothelial (MS1) cells and those transfected with mGSTA4, a {alpha}-class GST with highest activity toward 4-HNE. When treated with 4-HNE, mGSTA4-transfected cells showed significant upregulation of iNOS and nitric oxide (NO) through (NF)-{kappa}B (p65) translocation in comparison with wild-type or vector-transfected cells. Immunohistochemical studies of early human plaques showed lower 4-HNE content and upregulation of iNOS, which we take to indicate that GSTA4-4 induction acts as an enzymatic defense against high levels of 4-HNE, since 4-HNE accumulated in more advanced plaques, when detoxification and exocytotic mechanisms are likely to be overwhelmed. These studies suggest that GSTA4-4 may play an important defensive role against atherogenesis through detoxification of 4-HNE and upregulation of iNOS.

  15. Splice isoform estrogen receptors as integral transmembrane proteins

    PubMed Central

    Kim, Kyung Hee; Toomre, Derek; Bender, Jeffrey R.

    2011-01-01

    In addition to enhancing or repressing transcription, steroid hormone receptors rapidly transduce kinase activation signals. On ligand engagement, an N-terminus–truncated splice isoform of estrogen receptor (ER) α, ER46, triggers membrane-initiated signals, resulting in endothelial nitric oxide synthase (eNOS) activation and endothelial NO production. The orientation of ER46 at the plasma membrane is incompletely defined. With the use of ecliptic pHluorin-fused ER46, total internal reflection fluorescence microscopy in live human endothelial cells illustrates that ER46 can topologically conform to a type I transmembrane protein structure. Mutation of isoleucine-386 at the center of ER46's transmembrane hydrophobic core prevents membrane spanning, obscures the N-terminal ectodomain, and effects a marked reduction in membrane-impermeant estrogen binding with diminished rapid eNOS activation and NO production, despite maintained genomic induction of an estrogen response element–luciferase reporter. Thus there exist pools of transmembrane steroid hormone receptors that are efficient signaling molecules and potential novel therapeutic targets. PMID:21937726

  16. Chronic Cigarette Smoking Impairs Erectile Function through Increased Oxidative Stress and Apoptosis, Decreased nNOS, Endothelial and Smooth Muscle Contents in a Rat Model.

    PubMed

    Huang, Yun-Ching; Chin, Chih-Chien; Chen, Chih-Shou; Shindel, Alan W; Ho, Dong-Ru; Lin, Ching-Shwun; Shi, Chung-Sheng

    2015-01-01

    Cigarette use is an independent risk factor for the development of erectile dysfunction (ED). While the association between chronic smoking and ED is well established, the fundamental mechanism(s) of cigarette-related ED are incompletely understood, partly due to no reliable animal model of smoking-induced ED. The present study was designed to validate an in vivo rat model of chronic cigarette-induced ED. Forty 12-week old male Sprague-Dawley rats were divided into 4 groups. Ten rats served as control group and were exposed only to room air. The remaining 30 rats were passively exposed to cigarette smoke (CS) for 4 weeks (n = 10), 12 weeks (n = 10), and 24 weeks (n = 10). At the 24-week time point all rats were assessed with intracavernous pressure (ICP) during cavernous nerve electrostimulation. Blood and urine were collected to measure serum testosterone and oxidative stress, respectively. Corporal tissue was assessed by Western blot for neuronal nitric oxide synthase (nNOS). Penile tissues were subjected to immunohistochemistry for endothelial, smooth muscle, and apoptotic content. Mean arterial pressure (MAP) was significantly higher in 24-week cigarette exposed animals compared to the control animals. Mean ICP/MAP ratio and cavernosal smooth muscle/endothelial contents were significantly lower in the 12- and 24-week rats compared to control animals. Oxidative stress was significantly higher in the 24-week cigarette exposed group compared to control animals. Mean nNOS expression was significantly lower, and apoptotic index significantly higher, in CS-exposed animals compared to control animals. These findings indicate that the rat model exposure to CS increases apoptosis and oxidative stress and decreases nNOS, endothelial and smooth muscle contents, and ICP in a dose dependent fashion. The rat model is a useful tool for further study of the molecular and cellular mechanisms of CS-related ED. PMID:26491965

  17. Chronic Cigarette Smoking Impairs Erectile Function through Increased Oxidative Stress and Apoptosis, Decreased nNOS, Endothelial and Smooth Muscle Contents in a Rat Model

    PubMed Central

    Huang, Yun-Ching; Chin, Chih-Chien; Chen, Chih-Shou; Shindel, Alan. W.; Ho, Dong-Ru; Lin, Ching-Shwun; Shi, Chung-Sheng

    2015-01-01

    Cigarette use is an independent risk factor for the development of erectile dysfunction (ED). While the association between chronic smoking and ED is well established, the fundamental mechanism(s) of cigarette-related ED are incompletely understood, partly due to no reliable animal model of smoking-induced ED. The present study was designed to validate an in vivo rat model of chronic cigarette-induced ED. Forty 12-week old male Sprague-Dawley rats were divided into 4 groups. Ten rats served as control group and were exposed only to room air. The remaining 30 rats were passively exposed to cigarette smoke (CS) for 4 weeks (n = 10), 12 weeks (n = 10), and 24 weeks (n = 10). At the 24-week time point all rats were assessed with intracavernous pressure (ICP) during cavernous nerve electrostimulation. Blood and urine were collected to measure serum testosterone and oxidative stress, respectively. Corporal tissue was assessed by Western blot for neuronal nitric oxide synthase (nNOS). Penile tissues were subjected to immunohistochemistry for endothelial, smooth muscle, and apoptotic content. Mean arterial pressure (MAP) was significantly higher in 24-week cigarette exposed animals compared to the control animals. Mean ICP/MAP ratio and cavernosal smooth muscle/endothelial contents were significantly lower in the 12- and 24-week rats compared to control animals. Oxidative stress was significantly higher in the 24-week cigarette exposed group compared to control animals. Mean nNOS expression was significantly lower, and apoptotic index significantly higher, in CS-exposed animals compared to control animals. These findings indicate that the rat model exposure to CS increases apoptosis and oxidative stress and decreases nNOS, endothelial and smooth muscle contents, and ICP in a dose dependent fashion. The rat model is a useful tool for further study of the molecular and cellular mechanisms of CS-related ED. PMID:26491965

  18. Pterostilbene, an Active Constituent of Blueberries, Stimulates Nitric Oxide Production via Activation of Endothelial Nitric Oxide Synthase in Human Umbilical Vein Endothelial Cells.

    PubMed

    Park, Seong Hoon; Jeong, Sun-Oh; Chung, Hun-Teag; Pae, Hyun-Ock

    2015-09-01

    Endothelial dysfunction, a key process in development of cardiovascular diseases, is largely due to reduced nitric oxide (NO) derived from endothelial NO synthase (eNOS). Resveratrol has been reported to stimulate NO production via estrogen receptor α (ERα) activation in endothelial cells. Here, we investigated whether two natural methylated analogs of resveratrol, pterostilbene (Pts) and trans-3,5,4'-trimethoxystilbene (TMS), similarly to resveratrol, could influence endothelial NO release in human umbilical vein endothelial cells (HUVECs). In HUVECs exposed to Pts or TMS, NO production and phosphorylation of eNOS, protein kinase B (Akt), and ERα were measured by using a fluorimetric NO assay kit and Western blot analysis, respectively. Dimethylated Pts, but not trimethylated TMS, stimulated dose-dependent NO production via eNOS phosphorylation. Pts also stimulated dose-dependent phosphorylation of Akt, but not of ERα. NO production and eNOS phosphorylation in response to Pts were significantly abolished by the phosphoinositide 3-kinase (PI3K)/Akt inhibitor LY294002, but not by the ERα antagonist ICI182780. Our results suggest that Pts, but not TMS, is capable of inducing eNOS phosphorylation and the subsequent NO release, presumably, by activating PI3K/Akt pathway. The potential efficacy of Pts, an active constituent of blueberries, may aid in the prevention of cardiovascular diseases characterized by endothelial dysfunction. PMID:26008990

  19. Oxidized LDL at low concentration promotes in-vitro angiogenesis and activates nitric oxide synthase through PI3K/Akt/eNOS pathway in human coronary artery endothelial cells

    SciTech Connect

    Yu, Shan; Wong, Siu Ling; Lau, Chi Wai; Huang, Yu; Yu, Cheuk-Man

    2011-04-01

    Research highlights: {yields} Low-concentration oxidized LDL enhances angiogenesis through nitric oxide (NO). {yields} Oxidized LDL increases intracellular NO levels via eNOS phosphorylation. {yields} Akt/PI3K signaling mediates oxidized LDL-induced eNOS phosphorylation. -- Abstract: It has long been considered that oxidized low-density lipoprotein (oxLDL) causes endothelial dysfunction and is remarkably related to the development of atherosclerosis. However, the effect of oxLDL at very low concentration (<10 {mu}g/ml) on the endothelial cells remains speculative. Nitric oxide (NO) has a crucial role in the endothelial cell function. In this study, we investigated the effect of oxLDL at low concentration on NO production and proliferation, migration, tube formation of the human coronary artery endothelial cells (HCAEC). Results showed that oxLDL at 5 {mu}g/ml enhanced HCAEC proliferation, migration and tube formation. These phenomena were accompanied by an increased intracellular NO production. L-NAME (a NOS inhibitor), LY294002 and wortmannin (PI3K inhibitors) could abolish oxLDL-induced angiogenic effects and prevent NO production in the HCAEC. The phosphorylation of Akt, PI3K and eNOS were up-regulated by oxLDL, which was attenuated by LY294002. Our results suggested that oxLDL at low concentration could promote in-vitro angiogenesis and activate nitric oxide synthesis through PI3K/Akt/eNOS pathway in HCAEC.

  20. Genistein activates endothelial nitric oxide synthase in broiler pulmonary arterial endothelial cells by an Akt-dependent mechanism

    PubMed Central

    Yang, Ying; Nie, Wei; Yuan, Jianmin; Zhang, Bingkun; Wang, Zhong

    2010-01-01

    Deregulation of endothelial nitric oxide synthase (eNOS) plays an important role in the development of multiple cardiovascular diseases. Our recent study demonstrated that genistein supplementation attenuates pulmonary arterial hypertension in broilers by restoration of endothelial function. In this study, we investigated the molecular mechanism by using broiler pulmonary arterial endothelial cells (PAECs). Our results showed that genistein stimulated a rapid phosphorylation of eNOS at Ser1179 which was associated with activation of eNOS/NO axis. Further study indicated that the activation of eNOS was not mediated through estrogen receptors or tyrosine kinase inhibition, but via a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent signaling pathway, as the eNOS activity and related NO release were largely abolished by pharmacological inhibitors of PI3K or Akt. Thus, our findings revealed a critical function of Akt in mediating genistein-stimulated eNOS activity in PAECs, partially accounting for the beneficial effects of genistein on the development of cardiovascular diseases observed in animal models. PMID:20926919

  1. Estrogen-related receptor alpha induces the expression of vascular endothelial growth factor in breast cancer cells

    PubMed Central

    Stein, Rebecca A.; Gaillard, Stéphanie; McDonnell, Donald P.

    2009-01-01

    Estrogen-related receptor alpha (ERRα) is an orphan member of the nuclear receptor family of transcription factors. In addition to its function as a metabolic regulator, ERRα has been implicated in the growth and progression of several malignancies. In the setting of breast cancer, not only is ERRα a putative negative prognostic factor, but we have recently found that knockdown of its expression retards tumor growth in a xenograft model of this disease. The specific aspects of ERRα function that are responsible for its actions in breast cancer, however, remain unclear. Using the coactivator PGC-1α as a protein ligand to regulate ERRα activity, we analyzed the effects of this receptor on gene expression in the ERα-positive MCF-7 cell line. This analysis led to the identification of a large number of potential ERRα target genes, many of which were subsequently validated in other breast cancer cell lines. Importantly, we demonstrate in this study that activation of ERRα in several different breast cancer cell lines leads to a significant increase in VEGF mRNA expression, an activity that translates into an increase in VEGF protein secretion. The induction of VEGF results from the interaction of ERRα with specific ERR-responsive elements within the VEGF promoter. These findings suggest that ERRα-dependent induction of VEGF may contribute to the overall negative phenotype observed in tumors in which ERRα is expressed and provide validation for its use as a therapeutic target in cancer. PMID:19429439

  2. Redox-sensitive up-regulation of eNOS by purple grape juice in endothelial cells: role of PI3-kinase/Akt, p38 MAPK, JNK, FoxO1 and FoxO3a.

    PubMed

    Alhosin, Mahmoud; Anselm, Eric; Rashid, Sherzad; Kim, Jong Hun; Madeira, Socorro Vanesca Frota; Bronner, Christian; Schini-Kerth, Valérie B

    2013-01-01

    The vascular protective effect of grape-derived polyphenols has been attributable, in part, to their direct action on blood vessels by stimulating the endothelial formation of nitric oxide (NO). The aim of the present study was to determine whether Concord grape juice (CGJ), which contains high levels of polyphenols, stimulates the expression of endothelial NO synthase (eNOS) in porcine coronary artery endothelial cells and, if so, to determine the signaling pathway involved. CGJ dose- and time-dependently increased eNOS mRNA and protein levels and this effect is associated with an increased formation of NO in endothelial cells. The stimulatory effect of CGJ on eNOS mRNA is not associated with an increased eNOS mRNA stability and inhibited by antioxidants such as MnTMPyP, PEG-catalase, and catalase, and by wortmannin (an inhibitor of PI3-kinase), SB 203580 (an inhibitor of p38 MAPK), and SP 600125 (an inhibitor of JNK). Moreover, CGJ induced the formation of reactive oxygen species (ROS) in endothelial cells and this effect is inhibited by MnTMPyP, PEG-catalase, and catalase. The CGJ-induced the phosphorylation of p38 MAPK and JNK kinases is abolished by MnTMPyP. CGJ induced phosphorylation of transcription factors FoxO1 and FoxO3a, which regulate negatively eNOS expression, and this effect is prevented by MnTMPyP, PEG-catalase, wortmannin, SB203580 and SP600125. Moreover, chromatin immunoprecipitation assay indicated that the FoxO3a protein is associated with the eNOS promoter in control cells and that CGJ induced its dissociation. Thus, the present study indicates that CGJ up-regulates the expression of eNOS mRNA and protein leading to an increased formation of NO in endothelial cells. The stimulatory effect of CGJ is a redox-sensitive event involving PI3-kinase/Akt, p38 MAPK and JNK pathways, and the inactivation of the FoxO transcription factors, FoxO1 and FoxO3a, thereby preventing their repression of the eNOS gene. PMID:23533577

  3. Redox-Sensitive Up-Regulation of eNOS by Purple Grape Juice in Endothelial Cells: Role of PI3-Kinase/Akt, p38 MAPK, JNK, FoxO1 and FoxO3a

    PubMed Central

    Rashid, Sherzad; Kim, Jong Hun; Frota Madeira, Socorro Vanesca; Bronner, Christian; Schini-Kerth, Valérie B.

    2013-01-01

    The vascular protective effect of grape-derived polyphenols has been attributable, in part, to their direct action on blood vessels by stimulating the endothelial formation of nitric oxide (NO). The aim of the present study was to determine whether Concord grape juice (CGJ), which contains high levels of polyphenols, stimulates the expression of endothelial NO synthase (eNOS) in porcine coronary artery endothelial cells and, if so, to determine the signaling pathway involved. CGJ dose- and time-dependently increased eNOS mRNA and protein levels and this effect is associated with an increased formation of NO in endothelial cells. The stimulatory effect of CGJ on eNOS mRNA is not associated with an increased eNOS mRNA stability and inhibited by antioxidants such as MnTMPyP, PEG-catalase, and catalase, and by wortmannin (an inhibitor of PI3-kinase), SB 203580 (an inhibitor of p38 MAPK), and SP 600125 (an inhibitor of JNK). Moreover, CGJ induced the formation of reactive oxygen species (ROS) in endothelial cells and this effect is inhibited by MnTMPyP, PEG-catalase, and catalase. The CGJ-induced the phosphorylation of p38 MAPK and JNK kinases is abolished by MnTMPyP. CGJ induced phosphorylation of transcription factors FoxO1 and FoxO3a, which regulate negatively eNOS expression, and this effect is prevented by MnTMPyP, PEG-catalase, wortmannin, SB203580 and SP600125. Moreover, chromatin immunoprecipitation assay indicated that the FoxO3a protein is associated with the eNOS promoter in control cells and that CGJ induced its dissociation. Thus, the present study indicates that CGJ up-regulates the expression of eNOS mRNA and protein leading to an increased formation of NO in endothelial cells. The stimulatory effect of CGJ is a redox-sensitive event involving PI3-kinase/Akt, p38 MAPK and JNK pathways, and the inactivation of the FoxO transcription factors, FoxO1 and FoxO3a, thereby preventing their repression of the eNOS gene. PMID:23533577

  4. The estrogen-dependent baroreflex dysfunction caused by nicotine in female rats is mediated via NOS/HO inhibition: Role of sGC/PI3K/MAPKERK.

    PubMed

    Fouda, Mohamed A; El-Gowelli, Hanan M; El-Gowilly, Sahar M; El-Mas, Mahmoud M

    2015-12-15

    We have previously reported that estrogen (E2) exacerbates the depressant effect of chronic nicotine on arterial baroreceptor activity in female rats. Here, we tested the hypothesis that this nicotine effect is modulated by nitric oxide synthase (NOS) and/or heme oxygenase (HO) and their downstream soluble guanylate cyclase (sGC)/phosphatidylinositol 3-kinase (PI3K)/mitogen-activated protein kinases (MAPKs) signaling. We investigated the effects of (i) inhibition or facilitation of NOS or HO on the interaction of nicotine (2mg/kg/day i.p., 2 weeks) with reflex bradycardic responses to phenylephrine in ovariectomized (OVX) rats treated with E2 or vehicle, and (ii) central pharmacologic inhibition of sGC, PI3K, or MAPKs on the interaction. The data showed that the attenuation by nicotine of reflex bradycardia in OVXE2 rats was abolished after treatment with hemin (HO inducer) or l-arginine (NOS substrate). The hemin or l-arginine effect disappeared after inhibition of NOS (Nω-Nitro-l-arginine methyl ester hydrochloride, L-NAME) and HO (zinc protoporphyrin IX, ZnPP), respectively, denoting the interaction between the two enzymatic pathways. E2-receptor blockade (ICI 182,780) reduced baroreflexes in OVXE2 rats but had no effect on baroreflex improvement induced by hemin or l-arginine. Moreover, baroreflex enhancement by hemin was eliminated following intracisternal (i.c.) administration of wortmannin, ODQ, or PD98059 (inhibitors of PI3K, sGC, and extracellular signal-regulated kinases, MAPKERK, respectively). In contrast, the hemin effect was preserved after inhibition of MAPKp38 (SB203580) or MAPKJNK (SP600125). Overall, NOS/HO interruption underlies baroreflex dysfunction caused by nicotine in female rats and the facilitation of NOS/HO-coupled sGC/PI3K/MAPKERK signaling might rectify the nicotine effect. PMID:26597895

  5. Estrogen receptors and endothelium.

    PubMed

    Arnal, Jean-François; Fontaine, Coralie; Billon-Galés, Audrey; Favre, Julie; Laurell, Henrik; Lenfant, Françoise; Gourdy, Pierre

    2010-08-01

    Estrogens, and in particular 17beta-estradiol (E2), play a pivotal role in sexual development and reproduction and are also implicated in a large number of physiological processes, including the cardiovascular system. Both acetylcholine-induced and flow-dependent vasodilation are preserved or potentiated by estrogen treatment in both animal models and humans. Indeed, E2 increases the endothelial production of nitric oxide and prostacyclin and prevents early atheroma through endothelial-mediated mechanisms. Furthermore, whereas it prevents endothelial activation, E2 potentiates the ability of several subpopulations of the circulating or resident immune cells to produce proinflammatory cytokines. The balance between these 2 actions could determine the final effect in a given pathophysiological process. E2 also promotes endothelial healing, as well as angiogenesis. Estrogen actions are essentially mediated by 2 molecular targets: estrogen receptor-alpha (ERalpha) and ERbeta. The analysis of mouse models targeted for ERalpha or ERbeta demonstrated a prominent role of ERalpha in vascular biology. ERalpha directly modulates transcription of target genes through 2 activation functions (AFs), AF-1 and AF-2. Interestingly, an AF-1-deficient ERalpha isoform can be physiologically expressed in the endothelium and appears sufficient to mediate most of the vasculoprotective actions of E2. In contrast, AF-1 is necessary for the E2 actions in reproductive targets. Thus, it appears conceivable to uncouple the vasculoprotective and sexual actions with appropriate selective ER modulators. PMID:20631350

  6. Roles of endothelial nitric oxide synthase (eNOS) and mitochondrial permeability transition pore (MPTP) in epoxyeicosatrienoic acid (EET)-induced cardioprotection against infarction in intact rat hearts.

    PubMed

    Gross, Garrett J; Hsu, Anna; Pfeiffer, Adam W; Nithipatikom, Kasem

    2013-06-01

    We previously demonstrated that 11,12 and 14,15-epoxeicosatrienoic acids (EETs) produce cardioprotection against ischemia-reperfusion injury in dogs and rats. Several signaling mechanisms have been implicated in the cardioprotective actions of the EETs; however, their mechanisms remain largely elusive. Since nitric oxide (NO) plays a significant role in cardioprotection and EETs have been demonstrated to induce NO production in various tissues, we hypothesized that NO is involved in mediating the EET actions in cardioprotection. To test this hypothesis, we used an in vivo rat model of infarction in which intact rat hearts were subjected to 30-min occlusion of the left coronary artery and 2-hr reperfusion. 11,12-EET or 14,15-EET (2.5mg/kg) administered 10min prior to the occlusion reduced infarct size, expressed as a percentage of the AAR (IS/AAR), from 63.9±0.8% (control) to 45.3±1.2% and 45.5±1.7%, respectively. A nonselective nitric oxide synthase (NOS) inhibitor, L-NAME (1.0mg/kg) or a selective endothelial NOS inhibitor, L-NIO (0.30mg/kg) alone did not affect IS/AAR but they completely abolished the cardioprotective effects of the EETs. On the other hand, a selective neuronal NOS inhibitor, nNOS I (0.03mg/kg) and a selective inducible NOS inhibitor, 1400W (0.10mg/kg) did not affect IS/AAR or block the cardioprotective effects of the EETs. Administration of 11,12-EET (2.5mg/kg) to the rats also transiently increased the plasma NO concentration. 14,15-EET (10μM) induced the phosphorylation of eNOS (Ser(1177)) as well as a transient increase of NO production in rat cardiomyoblast cell line (H9c2 cells). When 11,12-EET or 14,15-EET was administered at 5min prior to reperfusion, infarct size was also reduced to 42.8±2.2% and 42.6±1.9%, respectively. Interestingly, L-NAME (1.0mg/kg) and a mitochondrial KATP channel blocker, 5-HD (10mg/kg) did not abolish while a sarcolemmal KATP channel blocker, HMR 1098 (6.0mg/kg) and a mitochondrial permeability transition

  7. Endothelial nitric-oxide synthase antisense (NOS3AS) gene encodes an autophagy-related protein (APG9-like2) highly expressed in trophoblast.

    PubMed

    Yamada, Takahiro; Carson, Andrew R; Caniggia, Isabella; Umebayashi, Kyohei; Yoshimori, Tamotsu; Nakabayashi, Kazuhiko; Scherer, Stephen W

    2005-05-01

    Macroautophagy is an intracellular degradation system for the majority of proteins and some organelles that is conserved in all eukaryotic species. The precise role of autophagy in mammalian development and potential involvement in disease remain to be discerned. Yeast Atg9p is the first integral membrane protein shown to be essential for the cytoplasm to vacuole targeting (Cvt) pathway and autophagy, whereas its mammalian functional orthologue has yet to be identified. We have identified two human genes homologous to yeast Atg9p and designated these as APG9L1 and APG9L2. We have previously identified APG9L2 as NOS3AS, which participates in the post-transcriptional regulation of the endothelial nitric-oxide synthase (NOS3) gene on chromosome 7 through its antisense overlap. In human adult tissues, APG9L1 was ubiquitously expressed, whereas APG9L2 was highly expressed in placenta (trophoblast cells) and pituitary gland. In transient transfection assays we found that both proteins were primarily localized to the perinuclear region and also scattered throughout the cytosol as dots, a subset of which colocalized with an autophagosome-specific marker LC3 under starvation conditions. Finally, by the small interfering RNA-mediated knockdown of APG9L1 in HeLa cells, we demonstrated that APG9L1 is essential for starvation-induced autophagosome formation. In addition, APG9L2 can functionally complement APG9L1 in this process. These results, taken together with those of phylogenetic and sequence analyses, suggest that both APG9L1 and APG9L2 are functionally orthologous to the yATG9 in autophagosome formation. Moreover, APG9L2 is a vertebrate-specific gene that may have gained critical roles in mammalian-specific developmental events, such as placentation, through rapid evolution. PMID:15755735

  8. The relationship between inflammation and the anticoagulant pathway: the emerging role of endothelial nitric oxide synthase (eNOS).

    PubMed

    Hooper, W Craig

    2004-01-01

    Inflammation represents the interaction of the immune and coagulation systems in an attempt to restore normal hemostasis following injury. The underlying basis of the interrelationship between these two physiological systems revolves around the following: a) the activation of coagulation by inflammation, b) the augmentation of the inflammatory response by coagulation, c) the significant attenuation of inflammation by the anticoagulant response and d) the separate influence of the vascular endothelium on coagulation and inflammation as well as its mediation or control of the cross-talk between these two physiological systems. In hemostasis, the protein C anticoagulant pathway is a major mechanism that functions to prevent the development of a pathological thrombus through the regulation of the procoagulant pathway. The endothelium is essential in maintaining a physiological balance between the anticoagulant and procoagulant pathways with proinflammatory cytokines functioning, in part, to regulate endothelial-cell- surface associated coagulation and anticoagulation proteins. In addition to its anticoagulant properties, activated protein C can also function as a regulator of proinflammatory cytokine production. Current evidence suggests that activated protein C may act to control inflammation through NF-kappaB and/or nitric oxide synthase. A better understanding of the relationship between APC and inflammation may provide new targets for drug design. PMID:15032695

  9. Estrogen overdose

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/002584.htm Estrogen overdose To use the sharing features on this page, please enable JavaScript. Estrogen is a female hormone. Estrogen overdose occurs when ...

  10. Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17β-Estradiol

    PubMed Central

    Sud, Neetu; Wiseman, Dean A.; Black, Stephen M.

    2010-01-01

    Evidence suggests that estrogen mediates rapid endothelial nitric oxide synthase (eNOS) activation via estrogen receptor-a (ERα) within the plasma membrane of endothelial cells (EC). ERα is known to colocalize with caveolin 1, the major structural protein of caveolae, and caveolin 1 stimulates the translocation of ERα to the plasma membrane. However, the role played by caveolin 1 in regulating 17β-estradiol-mediated NO signaling in EC has not been adequately resolved. Thus, the purpose of this study was to explore how 17β-estradiol stimulates eNOS activity and the role of caveolin 1 in this process. Our data demonstrate that modulation of caveolin 1 expression using small interfering RNA or adenoviral gene delivery alters ERα localization to the plasma membrane in EC. Further, before estrogen stimulation ERα associates with caveolin 1, whereas stimulation promotes a pp60Src-mediated phosphorylation of caveolin 1 at tyrosine 14, increasing ERα-PI3 kinase interactions and disrupting caveolin 1-ERα interactions. Adenoviral mediated overexpression of a phosphorylation-deficient mutant of caveolin (Y14FCav) attenuated the ERα/PI3 kinase interaction and prevented Akt-mediated eNOS activation. Furthermore, Y14FCav overexpression reduced eNOS phosphorylation at serine1177 and decreased NO generation after estrogen exposure. Using a library of overlapping peptides we identified residues 62–73 of caveolin 1 as the ERα-binding site. Delivery of a synthetic peptide based on this sequence decreased ERα plasma membrane translocation and reduced estrogen-mediated activation of eNOS. In conclusion, caveolin 1 stimulates 17β-estradiol-induced NO production by promoting ERα to the plasma membrane, which facilitates the activation of the PI3 kinase pathway, leading to eNOS activation and NO generation. PMID:20610538

  11. XJP-1 protects endothelial cells from oxidized low-density lipoprotein-induced apoptosis by inhibiting NADPH oxidase subunit expression and modulating the PI3K/Akt/eNOS pathway.

    PubMed

    Fu, Rong; Wang, Qiujuan; Guo, Qinglong; Xu, Jinyi; Wu, Xiaoming

    2013-01-01

    Endothelial apoptosis triggered by oxidized low-density lipoprotein (ox-LDL) can accelerate the progression of endothelial dysfunction in atherosclerosis. (±)7,8-Dihydroxy-3-methyl-isochromanone-4 (XJP-1) is a natural phenolic compound derived from banana peel. In the present study, we investigated the anti-apoptotic effect of XJP-1 in human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL and explored underlying mechanisms. Our results showed that in the presence of ox-LDL, XJP-1 significantly attenuated ox-LDL-mediated cytotoxicity, apoptosis, caspase-3 activation, reactive oxygen species (ROS) generation, and NADPH oxidase subunit (p22phox and p47phox) expression in HUVECs. In addition, the anticytotoxic and anti-apoptotic effect of XJP-1 was partially inhibited by a PI3K inhibitor (LY294002), an Akt inhibitor (SH-6), a specific eNOS inhibitor (l-NAME) and a NADPH oxidase inhibitor (DPI). In exploring the underlying mechanisms of XJP-1 action, we found that XJP-1 eliminated ox-LDL-induced dephosphorylation of Akt and eNOS in a dose-dependent manner. However, XJP-1 alone upregulation of Akt and eNOS phosphorylation were blocked by LY294002 and SH-6. Moreover, XJP-1 increased NO production, but this effect was abolished by LY294002, SH-6 and l-NAME. The inhibition of ox-LDL-induced endothelial dysfunction by XJP-1 is due at least in part to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway. PMID:22980246

  12. Ratio of 5,6,7,8-tetrahydrobiopterin to 7,8-dihydrobiopterin in endothelial cells determines glucose-elicited changes in NO vs. superoxide production by eNOS

    PubMed Central

    Crabtree, Mark J.; Smith, Caroline L.; Lam, George; Goligorsky, Michael S.; Gross, Steven S.

    2009-01-01

    5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH4, in the setting of diabetes and other chronic vasoinflammatory conditions, can cause cofactor insufficiency and uncoupling of endothelial NOS (eNOS), manifest by a switch from nitric oxide (NO) to superoxide production. Here we tested the hypothesis that eNOS uncoupling is not simply a consequence of BH4 insufficiency, but rather results from a diminished ratio of BH4 vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH2). In support of this hypothesis, [3H]BH4 binding studies revealed that BH4 and BH2 bind eNOS with equal affinity (Kd ≈ 80 nM) and BH2 can rapidly and efficiently replace BH4 in preformed eNOS-BH4 complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH2 levels increased from undetectable to 40% of total biopterin. This BH2 accumulation was associated with diminished calcium ionophore-evoked NO activity and accelerated superoxide production. Since superoxide production was suppressed by NOS inhibitor treatment, eNOS was implicated as a principal superoxide source. Importantly, BH4 supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH4: BH2 and not absolute intracellular levels of BH4. Reciprocally, superoxide production was found to negatively correlate with intracellular BH4:BH2. Hyperglycemia-associated BH4 oxidation and NO insufficiency was recapitulated in vivo, in the Zucker diabetic fatty rat model of type 2 diabetes. Together, these findings implicate diminished intracellular BH4:BH2, rather than BH4 depletion per se, as the molecular trigger for NO insufficiency in diabetes. PMID:18192221

  13. An NO Donor Approach to Neuroprotective and Procognitive Estrogen Therapy Overcomes Loss of NO Synthase Function and Potentially Thrombotic Risk

    PubMed Central

    VandeVrede, Lawren; Abdelhamid, Ramy; Qin, Zhihui; Choi, Jaewoo; Piyankarage, Sujeewa; Luo, Jia; Larson, John; Bennett, Brian M.; Thatcher, Gregory R. J.

    2013-01-01

    Selective estrogen receptor modulators (SERMs) are effective therapeutics that preserve favorable actions of estrogens on bone and act as antiestrogens in breast tissue, decreasing the risk of vertebral fractures and breast cancer, but their potential in neuroprotective and procognitive therapy is limited by: 1) an increased lifetime risk of thrombotic events; and 2) an attenuated response to estrogens with age, sometimes linked to endothelial nitric oxide synthase (eNOS) dysfunction. Herein, three 3rd generation SERMs with similar high affinity for estrogen receptors (ERα, ERβ) were studied: desmethylarzoxifene (DMA), FDMA, and a novel NO-donating SERM (NO-DMA). Neuroprotection was studied in primary rat neurons exposed to oxygen glucose deprivation; reversal of cholinergic cognitive deficit was studied in mice in a behavioral model of memory; long term potentiation (LTP), underlying cognition, was measured in hippocampal slices from older 3×Tg Alzheimer's transgenic mice; vasodilation was measured in rat aortic strips; and anticoagulant activity was compared. Pharmacologic blockade of GPR30 and NOS; denudation of endothelium; measurement of NO; and genetic knockout of eNOS were used to probe mechanism. Comparison of the three chemical probes indicates key roles for GPR30 and eNOS in mediating therapeutic activity. Procognitive, vasodilator and anticoagulant activities of DMA were found to be eNOS dependent, while neuroprotection and restoration of LTP were both shown to be dependent upon GPR30, a G-protein coupled receptor mediating estrogenic function. Finally, the observation that an NO-SERM shows enhanced vasodilation and anticoagulant activity, while retaining the positive attributes of SERMs even in the presence of NOS dysfunction, indicates a potential therapeutic approach without the increased risk of thrombotic events. PMID:23976955

  14. Tetramethylpyrazine attenuates TNF-α-induced iNOS expression in human endothelial cells: Involvement of Syk-mediated activation of PI3K-IKK-IκB signaling pathways

    SciTech Connect

    Zheng, Zhen; Li, Zhiliang; Chen, Song; Pan, Jieyi; Ma, Xiaochun

    2013-08-15

    Endothelial cells produce nitric oxide (NO) by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NO synthase (iNOS). We explored the effect of tetramethylpyrazine (TMP), a compound derived from chuanxiong, on tumor necrosis factor (TNF)-α-induced iNOS in human umbilical vein endothelial cells (HUVECs) and explored the signal pathways involved by using RT-PCR and Western blot. TMP suppressed TNF-α-induced expression of iNOS by inhibiting IκB kinase (IKK) phosphorylation, IκB degradation and nuclear factor κB (NF-κB) nuclear translocation, which were required for NO gene transcription. Exposure to wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression, suggesting activation of such a signal pathway might be phosphoinositide-3-kinase (PI3K) dependent. Spleen tyrosine kinase (Syk) inhibitor piceatannol significantly inhibited NO production. Furthermore, piceatannol obviously suppressed TNF-α-induced IκB phosphorylation and the downstream NF-κB activation, suggesting that Syk is an upstream key regulator in the activation of PI3K/IKK/IκB-mediated signaling. TMP significantly inhibited TNF-α-induced phosphorylation of Syk and PI3K. Our data indicate that TMP might repress iNOS expression, at least in part, through its inhibitory effect of Syk-mediated PI3K phosphorylation in TNF-α-stimulated HUVECs. -- Highlights: •TMP suppressed TNF-α-induced expression of iNOS by inhibiting IKK/IκB/NF-κB pathway. •PI3K inhibitor wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression. •Syk inhibitor piceatannol repressed PI3K/IKK/IκB mediated NO production. •Syk is an upstream regulator in the activation of PI3K/IKK/IκB-mediated signaling. •TMP might repress iNOS expression through Syk-mediated PI3K pathway.

  15. VASCULAR ACTIONS OF ESTROGENS: FUNCTIONAL IMPLICATIONS

    PubMed Central

    Miller, Virginia M.; Duckles, Sue P.

    2009-01-01

    The impact of estrogen exposure in preventing or treating cardiovascular disease is controversial. But it is clear that estrogen has important effects on vascular physiology and pathophysiology, with potential therapeutic implications. Therefore, it is the goal of this review to summarize, using an integrated approach, current knowledge of the vascular effects of estrogen, both in humans and in experimental animals. Aspects of estrogen synthesis and receptors, as well as general mechanisms of estrogenic action are reviewed with an emphasis on issues particularly relevant to the vascular system. Recent understanding of the impact of estrogen on mitochondrial function suggests that the longer lifespan of women compared to men may depend in part on the ability of estrogen to decrease production of reactive oxygen species in mitochondria. Mechanisms by which estrogen increases endothelial vasodilator function, promotes angiogenesis and modulates autonomic function are summarized. Key aspects of the relevant pathophysiology of inflammation, atherosclerosis, stroke, migraine and thrombosis are reviewed concerning current knowledge of estrogenic effects. A number of emerging concepts are addressed throughout. These include the importance of estrogenic formulation and route of administration and the impact of genetic polymorphisms, either in estrogen receptors or in enzymes responsible for estrogen metabolism, on responsiveness to hormone treatment. The importance of local metabolism of estrogenic precursors and the impact of timing for initiation of treatment and its duration are also considered. While consensus opinions are emphasized, controversial views are presented in order to stimulate future research. PMID:18579753

  16. Estrogen Injection

    MedlinePlus

    ... forms of estrogen injection are used to treat hot flushes (hot flashes; sudden strong feelings of heat and sweating) ... If you are using estrogen injection to treat hot flushes, your symptoms should improve within 1 to ...

  17. G Protein-coupled Estrogen Receptor Protects from Atherosclerosis

    PubMed Central

    Meyer, Matthias R.; Fredette, Natalie C.; Howard, Tamara A.; Hu, Chelin; Ramesh, Chinnasamy; Daniel, Christoph; Amann, Kerstin; Arterburn, Jeffrey B.; Barton, Matthias; Prossnitz, Eric R.

    2014-01-01

    Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity. PMID:25532911

  18. Mechanisms underlying protective effects of trimetazidine on endothelial progenitor cells biological functions against H2O2-induced injury: involvement of antioxidation and Akt/eNOS signaling pathways.

    PubMed

    Wu, Qinqin; Qi, Benling; Liu, Yun; Cheng, Bei; Liu, Lihua; Li, Yuanyuan; Wang, Qian

    2013-05-01

    Trimetazidine (TMZ) is a widely used drug exerting cardioprotective effects against ischemic heart disease through a number of mechanisms in conditions of oxidative stress. However, there are few data regarding the effects of TMZ on endothelial lineage, especially endothelial progenitor cells (EPCs). Thus, we sought to investigate whether TMZ could protect EPCs against oxidative stress injury induced by H2O2 (100 µM) and the preliminary mechanisms involved in vitro. The results showed that pretreatment of EPCs with TMZ (10 µM) protected the proliferation, adhesion, migration, and apoptosis of EPCs against H2O2, accompanied by an increase in superoxide dismutase (SOD) activity, a decrease in malonaldehyde (MDA) content, and increases in eNOS, Akt phosphorylation, and NO production. These TMZ-mediated beneficial effects on EPCs could be attenuated by pre-incubation with the Akt inhibitor triciribine. In conclusion, the present study demonstrates that TMZ ameliorated H2O2-induced impairment of biological functions in EPCs with the involvement of antioxidation and Akt/eNOS signaling pathway. These findings suggest that TMZ mediating preservation of EPCs may contribute to its cardioprotective effects on ischemic heart disease. PMID:23528356

  19. The phytoestrogen alpha-zearalenol reverses endothelial dysfunction induced by oophorectomy in rats.

    PubMed

    Altavilla, D; Saitta, A; Galeano, M; Squadrito, G; Marino, D; Minutoli, L; Calapai, G; Deodato, B; D'Anna, R; Corrado, F; Caputi, A P; Squadrito, F

    2001-02-01

    It has been shown recently that alpha-zearalenol, a resorcyclic acid lactone, prevents bone loss in a rat model of postmenopausal bone loss. We have therefore investigated the effects of this phytoestrogen on endothelial dysfunction induced by estrogen deficiency in rats. Female mature Sprague-Dawley rats underwent a bilateral oophorectomy (OVX rats). Sham-operated animals (sham OVX rats) were used as controls. Three weeks after surgery, animals were randomized to the following treatments: alpha-zearalenol (1 mg/kg/day, i.m., for 4 weeks), 17beta-estradiol (20 microg/kg/day, i.m., for 4 weeks), or their vehicle (100 microl, i.m., of cottonseed oil). Two other groups of rats were treated with alpha-zearalenol or 17beta-estradiol plus the pure estrogen receptor antagonist ICI 182780 (2.5 mg/kg/day, i.m., for 4 weeks). Mean arterial blood pressure (MAP), heart rate (HR), total plasma cholesterol, plasma estradiol, and plasma alpha-zearalenol were studied. We also investigated endothelial-dependent (acetylcholine, 10 nM to 10 microM) and endothelial-independent (sodium nitroprusside, 15 nM to 30 nM) relaxation of aortic rings, as well as N(G)-methyl-L-arginine (L-NMA: 10 to 100 microM)-induced vasoconstriction and calcium-dependent nitric oxide synthase (cNOS) activity in homogenates of lungs taken from both sham OVX rats and OVX rats. Untreated OVX rats had, compared with sham OVX animals, unchanged body weight, MAP, HR, and plasma cholesterol. In contrast oophorectomy reduced plasma estradiol levels (OVX, 2 +/- 0.5 pg/ml; sham OVX, 35 +/- 6 pg/ml), impaired endothelial-dependent relaxation and blunted L-NMA-induced contraction (L-NMA 100 microM: sham OVX, 2.7 +/- 0.3 g/mg tissue; OVX, 1.3 +/- 0.1 g/mg tissue). Moreover OVX rats showed a reduced calcium-dependent NO synthase (cNOS) activity. Treatment with alpha-zearalenol or with 17beta-estradiol reverted the endothelial dysfunction and increased cNOS activity in lung homogenates. These effects were abolished by the

  20. GPER inhibits diabetes-mediated RhoA activation to prevent vascular endothelial dysfunction.

    PubMed

    Li, Zilin; Cheng, Liang; Liang, Hongliang; Duan, Weixun; Hu, Jing; Zhi, Weiwei; Yang, Jinbao; Liu, Zhenhua; Zhao, Minggao; Liu, Jincheng

    2016-02-01

    The effect of estrogen receptors on diabetes-induced vascular dysfunction is critical, but ambiguous. Individuals with diabetic vascular disease may require estrogen receptor-specific targeted therapy in the future. The G protein-coupled estrogen receptor (GPER) has beneficial effects on vascular function. However, its fundamental mechanisms are unclear. The RhoA/Rho-kinase pathway contributes to diabetic vascular complications, whereas estrogen can suppress Rho-kinase function. Thus, we assumed that GPER inhibits diabetes-mediated RhoA activation to prevent vascular dysfunction. We further investigated the underlying mechanisms involved in this process. Vascular endothelial cells and ex vivo cultured ovariectomized (OVX) C57BL/6 mouse aortae were treated with high glucose (HG) alone or in combination with GPER agonist (G1). G1 treatment was also administered to OVX db/db mice for 8 weeks. An ex-vivo isovolumic myograph was used to analyze the endothelium-dependent vasodilation and endothelium-independent contraction of mouse aortae. Apoptosis, oxidative stress, and inflammation were attenuated in G1-pretreated vascular endothelial cells. G1 significantly decreased the phosphorylation of inhibitory endothelial nitric oxide (NO) synthase residue threonine 495 (eNOS Thr495), inhibited RhoA expression, and increased NO production. Additionally, G1 rescued the impaired endothelium-dependent relaxation and inhibited RhoA activation in the thoracic aorta of OVX db/db mice and ex-vivo cultured OVX C57BL/6 mouse aortae treated with HG. Estrogens acting via GPER could protect vascular endothelium, and GPER activation might elicit ERα-independent effect to inhibit RhoA/Rho-kinase pathway. Additionally, GPER activation might reduce vascular smooth muscle contraction by inhibiting RhoA activation. Thus, the results of the present study suggest a new therapeutic paradigm for end-stage vascular dysfunction by inhibiting RhoA/Rho-kinase pathway via GPER activation. PMID:26785611

  1. The role of NOS2A −954G/C and vascular endothelial growth factor +936C/T polymorphisms in type 2 diabetes mellitus and diabetic nonproliferative retinopathy risk management

    PubMed Central

    Porojan, Mihai Dumitru; Cătană, Andreea; Popp, Radu A; Dumitrascu, Dan L; Bala, Cornelia

    2015-01-01

    Type 2 diabetes mellitus (T2DM) remains one of the major health problems in Europe. Retinopathy is one of the major causes of morbidity in T2DM, strongly influencing the evolution and prognosis of these patients. In the last 2 decades, several studies have been conducted to identify the possible genetic susceptibility factors involved in the pathogenesis of the disease. However, there is little data related to the involvement of vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS) gene polymorphisms in the T2DM Caucasian population. The objective of this study was to identify a possible connection between NOS2A −954G/C (rs2297518) and VEGF +936C/T (rs3025039) polymorphisms and the risk of developing T2DM and nonproliferative diabetic retinopathy in a Caucasian population group. We investigated 200 patients diagnosed with T2DM and 208 controls. Genotypes were determined by multiplex polymerase chain reaction-restriction fragment length polymorphism. Statistical and comparative analyses (Fisher’s exact test) for dominant and recessive models of NOS2A −954G/C and VEGF +936C/T polymorphisms revealed an increased risk of T2DM (χ2=8.14, phi =0.141, P=0.004, odds ratio [OR] =2.795, 95% confidence interval [CI] =1.347–5.801; χ2=18.814, phi =0.215, P<0.001, OR =2.59, 95% CI =1.675–4.006, respectively). Also, comparative analysis for the recessive model (using Pearson’s chi-square test [χ2] and the phi coefficient [phi]) reveals that the variant CC genotype of NOS2A gene is more frequently associated with T2DM without retinopathy (χ2=3.835, phi =−0.138, P=0.05, OR =0.447, 95% CI =0.197–1.015). In conclusion, the results of the study place VEGF +936C/T polymorphisms among the genetic risk factor for T2DM, whereas NOS2A −954G/C polymorphisms act like a protective individual factor for nonproliferative retinopathy. PMID:26664124

  2. Role of estrogen in diastolic dysfunction

    PubMed Central

    Zhao, Zhuo; Wang, Hao; Jessup, Jewell A.; Lindsey, Sarah H.; Chappell, Mark C.

    2014-01-01

    The prevalence of left ventricular diastolic dysfunction (LVDD) sharply increases in women after menopause and may lead to heart failure. While evidence suggests that estrogens protect the premenopausal heart from hypertension and ventricular remodeling, the specific mechanisms involved remain elusive. Moreover, whether there is a protective role of estrogens against cardiovascular disease, and specifically LVDD, continues to be controversial. Clinical and basic science have implicated activation of the renin-angiotensin-aldosterone system (RAAS), linked to the loss of ovarian estrogens, in the pathogenesis of postmenopausal diastolic dysfunction. As a consequence of increased tissue ANG II and low estrogen, a maladaptive nitric oxide synthase (NOS) system produces ROS that contribute to female sex-specific hypertensive heart disease. Recent insights from rodent models that mimic the cardiac phenotype of an estrogen-insufficient or -deficient woman (e.g., premature ovarian failure or postmenopausal), including the ovariectomized congenic mRen2.Lewis female rat, provide evidence showing that estrogen modulates the tissue RAAS and NOS system and related intracellular signaling pathways, in part via the membrane G protein-coupled receptor 30 (GPR30; also called G protein-coupled estrogen receptor 1). Complementing the cardiovascular research in this field, the echocardiographic correlates of LVDD as well as inherent limitations to its use in preclinical rodent studies will be briefly presented. Understanding the roles of estrogen and GPR30, their interactions with the local RAAS and NOS system, and the relationship of each of these to LVDD is necessary to identify new therapeutic targets and alternative treatments for diastolic heart failure that achieve the cardiovascular benefits of estrogen replacement without its side effects and contraindications. PMID:24414072

  3. Role of estrogen in diastolic dysfunction.

    PubMed

    Zhao, Zhuo; Wang, Hao; Jessup, Jewell A; Lindsey, Sarah H; Chappell, Mark C; Groban, Leanne

    2014-03-01

    The prevalence of left ventricular diastolic dysfunction (LVDD) sharply increases in women after menopause and may lead to heart failure. While evidence suggests that estrogens protect the premenopausal heart from hypertension and ventricular remodeling, the specific mechanisms involved remain elusive. Moreover, whether there is a protective role of estrogens against cardiovascular disease, and specifically LVDD, continues to be controversial. Clinical and basic science have implicated activation of the renin-angiotensin-aldosterone system (RAAS), linked to the loss of ovarian estrogens, in the pathogenesis of postmenopausal diastolic dysfunction. As a consequence of increased tissue ANG II and low estrogen, a maladaptive nitric oxide synthase (NOS) system produces ROS that contribute to female sex-specific hypertensive heart disease. Recent insights from rodent models that mimic the cardiac phenotype of an estrogen-insufficient or -deficient woman (e.g., premature ovarian failure or postmenopausal), including the ovariectomized congenic mRen2.Lewis female rat, provide evidence showing that estrogen modulates the tissue RAAS and NOS system and related intracellular signaling pathways, in part via the membrane G protein-coupled receptor 30 (GPR30; also called G protein-coupled estrogen receptor 1). Complementing the cardiovascular research in this field, the echocardiographic correlates of LVDD as well as inherent limitations to its use in preclinical rodent studies will be briefly presented. Understanding the roles of estrogen and GPR30, their interactions with the local RAAS and NOS system, and the relationship of each of these to LVDD is necessary to identify new therapeutic targets and alternative treatments for diastolic heart failure that achieve the cardiovascular benefits of estrogen replacement without its side effects and contraindications. PMID:24414072

  4. A multilevel prediction of physiological response to challenge: Interactions among child maltreatment, neighborhood crime, endothelial nitric oxide synthase gene (eNOS), and GABA(A) receptor subunit alpha-6 gene (GABRA6).

    PubMed

    Lynch, Michael; Manly, Jody Todd; Cicchetti, Dante

    2015-11-01

    Physiological response to stress has been linked to a variety of healthy and pathological conditions. The current study conducted a multilevel examination of interactions among environmental toxins (i.e., neighborhood crime and child maltreatment) and specific genetic polymorphisms of the endothelial nitric oxide synthase gene (eNOS) and GABA(A) receptor subunit alpha-6 gene (GABRA6). One hundred eighty-six children were recruited at age 4. The presence or absence of child maltreatment as well as the amount of crime that occurred in their neighborhood during the previous year were determined at that time. At age 9, the children were brought to the lab, where their physiological response to a cognitive challenge (i.e., change in the amplitude of the respiratory sinus arrhythmia) was assessed and DNA samples were collected for subsequent genotyping. The results confirmed that complex Gene × Gene, Environment × Environment, and Gene × Environment interactions were associated with different patterns of respiratory sinus arrhythmia reactivity. The implications for future research and evidence-based intervention are discussed. PMID:26535938

  5. Bee Venom Accelerates Wound Healing in Diabetic Mice by Suppressing Activating Transcription Factor-3 (ATF-3) and Inducible Nitric Oxide Synthase (iNOS)-Mediated Oxidative Stress and Recruiting Bone Marrow-Derived Endothelial Progenitor Cells.

    PubMed

    Badr, Gamal; Hozzein, Wael N; Badr, Badr M; Al Ghamdi, Ahmad; Saad Eldien, Heba M; Garraud, Olivier

    2016-10-01

    Multiple mechanisms contribute to impaired diabetic wound healing including impaired neovascularization and deficient endothelial progenitor cell (EPC) recruitment. Bee venom (BV) has been used as an anti-inflammatory agent for the treatment of several diseases. Nevertheless, the effect of BV on the healing of diabetic wounds has not been studied. Therefore, in this study, we investigated the impact of BV on diabetic wound closure in a type I diabetic mouse model. Three experimental groups were used: group 1, non-diabetic control mice; group 2, diabetic mice; and group 3, diabetic mice treated with BV. We found that the diabetic mice exhibited delayed wound closure characterized by a significant decrease in collagen production and prolonged elevation of inflammatory cytokines levels in wounded tissue compared to control non-diabetic mice. Additionally, wounded tissue in diabetic mice revealed aberrantly up-regulated expression of ATF-3 and iNOS followed by a marked elevation in free radical levels. Impaired diabetic wound healing was also characterized by a significant elevation in caspase-3, -8, and -9 activity and a marked reduction in the expression of TGF-β and VEGF, which led to decreased neovascularization and angiogenesis of the injured tissue by impairing EPC mobilization. Interestingly, BV treatment significantly enhanced wound closure in diabetic mice by increasing collagen production and restoring the levels of inflammatory cytokines, free radical, TGF-β, and VEGF. Most importantly, BV-treated diabetic mice exhibited mobilized long-lived EPCs by inhibiting caspase activity in the wounded tissue. Our findings reveal the molecular mechanisms underlying improved diabetic wound healing and closure following BV treatment. J. Cell. Physiol. 231: 2159-2171, 2016. © 2016 Wiley Periodicals, Inc. PMID:26825453

  6. Estrogen Receptor Alpha as a Key Target of Red Wine Polyphenols Action on the Endothelium

    PubMed Central

    Chalopin, Matthieu; Tesse, Angela; Martínez, Maria Carmen; Rognan, Didier; Arnal, Jean-François; Andriantsitohaina, Ramaroson

    2010-01-01

    Background A greater reduction in cardiovascular risk and vascular protection associated with diet rich in polyphenols are generally accepted; however, the molecular targets for polyphenols effects remain unknown. Meanwhile evidences in the literature have enlightened, not only structural similarities between estrogens and polyphenols known as phytoestrogens, but also in their vascular effects. We hypothesized that alpha isoform of estrogen receptor (ERα) could be involved in the transduction of the vascular benefits of polyphenols. Methodology/Principal Findings Here, we used ERα deficient mice to show that endothelium-dependent vasorelaxation induced either by red wine polyphenol extract, Provinols™, or delphinidin, an anthocyanin that possesses similar pharmacological profile, is mediated by ERα. Indeed, Provinols™, delphinidin and ERα agonists, 17-beta-estradiol and PPT, are able to induce endothelial vasodilatation in aorta from ERα Wild-Type but not from Knock-Out mice, by activation of nitric oxide (NO) pathway in endothelial cells. Besides, silencing the effects of ERα completely prevented the effects of Provinols™ and delphinidin to activate NO pathway (Src, ERK 1/2, eNOS, caveolin-1) leading to NO production. Furthermore, direct interaction between delphinidin and ERα activator site is demonstrated using both binding assay and docking. Most interestingly, the ability of short term oral administration of Provinols™ to decrease response to serotonin and to enhance sensitivity of the endothelium-dependent relaxation to acetylcholine, associated with concomitant increased NO production and decreased superoxide anions, was completely blunted in ERα deficient mice. Conclusions/Significance This study provides evidence that red wine polyphenols, especially delphinidin, exert their endothelial benefits via ERα activation. It is a major breakthrough bringing new insights of the potential therapeutic of polyphenols against cardiovascular

  7. Estrogen and Osteoporosis.

    ERIC Educational Resources Information Center

    Lindsay, Robert

    1987-01-01

    This article reviews the use of estrogen in the prevention and treatment of osteoporosis. Dosage levels, interactions with other factors, side effects, and the mechanism of estrogen action are discussed. (Author/MT)

  8. Estrogen and Bazedoxifene

    MedlinePlus

    Estrogen and bazedoxifene tablets are used to treat hot flashes (sudden feelings of warmth, especially in the ... women may experience other symptoms and body changes). Estrogen and bazedoxifene tablets are also used to prevent ...

  9. Estrogen and cancer.

    PubMed

    Liang, Jing; Shang, Yongfeng

    2013-01-01

    Estrogen exhibits a broad spectrum of physiological functions ranging from regulation of the menstrual cycle and reproduction to modulation of bone density, brain function, and cholesterol mobilization. Despite the beneficial actions of endogenous estrogen, sustained exposure to exogenous estrogen is a well-established risk factor for various cancers. We summarize our current understanding of the molecular mechanisms of estrogen signaling in normal and cancer cells and discuss the major challenges to existing antiestrogen therapies. PMID:23043248

  10. [Estrogens and pharmacological modulation of estrogen receptors].

    PubMed

    Sanidize, T V; Ratiani, L R; Gabuniia, L Iu; Tortladze, M L; Kuridze, N N

    2009-02-01

    Estrogens belong to more or less frequently prescribed preparations. Main fields of application of these preparations (as in monotherapy as well as in combination) are contraception and hormone replacement therapy during menopause. More uncommon indications of estrogens are growth inhibition and hypogonadism (in this case they are prescribed along with gonadotropic hormones). Synthesis and metabolism of estrogens, as well as their intracellular receptors are well studied these days, which allow us to understand physiology and pharmacology of these hormones. In pharmacology the main stage is detection of estrogen receptors inside of cells of targets. There are two types of estrogen receptors alpha- and beta- coded by different genes. A number of steroid and non-steroid compounds have characteristics of estrogens. Likely in the future their popularity will increase, as by the aging of population number of those women, who receive replacement therapy, will increase. Investigations to find an ideal elective modulator of estrogen receptors, that will possess anti-estrogenic activity in connection with mammal gland and develop indifference in connection with endometrium and at the same time will display ability to reduce hot flushes, bone resorption, atrophy of mucous membranes of vagina and urinary bladder, as well as it will favorably effect on metabolism of lipoproteins are carried out. PMID:19276483

  11. Dexamethasone, tetrahydrobiopterin and uncoupling of endothelial nitric oxide synthase

    PubMed Central

    Tobias, Silke; Habermeier, Alice; Siuda, Daniel; Reifenberg, Gisela; Xia, Ning; Closs, Ellen I; Förstermann, Ulrich; Li, Huige

    2015-01-01

    Objective To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase (eNOS). Methods & Results A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin (BH4). Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH4-synthesizing enzymes: GTP cyclohydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH4, dihydrobiopterin (BH2) as well as BH4: BH2 ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH4: eNOS molar ratio in dexamethasone-treated cells. Because the BH4-eNOS stoichiometry rather than the absolute BH4 amount is the key determinant of eNOS functionality (i.e., coupled or uncoupled), the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable. Conclusions Although dexamethasone reduces BH4 levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177. PMID:26512245

  12. Estrogens, inflammation and cognition.

    PubMed

    Au, April; Feher, Anita; McPhee, Lucy; Jessa, Ailya; Oh, Soojin; Einstein, Gillian

    2016-01-01

    The effects of estrogens are pleiotropic, affecting multiple bodily systems. Changes from the body's natural fluctuating levels of estrogens, through surgical removal of the ovaries, natural menopause, or the administration of exogenous estrogens to menopausal women have been independently linked to an altered immune profile, and changes to cognitive processes. Here, we propose that inflammation may mediate the relationship between low levels of estrogens and cognitive decline. In order to determine what is known about this connection, we review the literature on the cognitive effects of decreased estrogens due to oophorectomy or natural menopause, decreased estrogens' role on inflammation - both peripherally and in the brain - and the relationship between inflammation and cognition. While this review demonstrates that much is unknown about the intersection between estrogens, cognition, inflammation, we propose that there is an important interaction between these literatures. PMID:26774208

  13. Regulation of retinal angiogenesis by endothelial nitric oxide synthase signaling pathway

    PubMed Central

    Ha, Jung Min; Jin, Seo Yeon; Lee, Hye Sun; Shin, Hwa Kyoung; Lee, Dong Hyung; Song, Sang Heon; Kim, Chi Dae

    2016-01-01

    Angiogenesis plays an essential role in embryo development, tissue repair, inflammatory diseases, and tumor growth. In the present study, we showed that endothelial nitric oxide synthase (eNOS) regulates retinal angiogenesis. Mice that lack eNOS showed growth retardation, and retinal vessel development was significantly delayed. In addition, the number of tip cells and filopodia length were significantly reduced in mice lacking eNOS. Retinal endothelial cell proliferation was significantly blocked in mice lacking eNOS, and EMG-2-induced endothelial cell sprouting was significantly reduced in aortic vessels isolated from eNOS-deficient mice. Finally, pericyte recruitment to endothelial cells and vascular smooth muscle cell coverage to blood vessels were attenuated in mice lacking eNOS. Taken together, we suggest that the endothelial cell function and blood vessel maturation are regulated by eNOS during retinal angiogenesis. PMID:27610040

  14. Regulation of retinal angiogenesis by endothelial nitric oxide synthase signaling pathway.

    PubMed

    Ha, Jung Min; Jin, Seo Yeon; Lee, Hye Sun; Shin, Hwa Kyoung; Lee, Dong Hyung; Song, Sang Heon; Kim, Chi Dae; Bae, Sun Sik

    2016-09-01

    Angiogenesis plays an essential role in embryo development, tissue repair, inflammatory diseases, and tumor growth. In the present study, we showed that endothelial nitric oxide synthase (eNOS) regulates retinal angiogenesis. Mice that lack eNOS showed growth retardation, and retinal vessel development was significantly delayed. In addition, the number of tip cells and filopodia length were significantly reduced in mice lacking eNOS. Retinal endothelial cell proliferation was significantly blocked in mice lacking eNOS, and EMG-2-induced endothelial cell sprouting was significantly reduced in aortic vessels isolated from eNOS-deficient mice. Finally, pericyte recruitment to endothelial cells and vascular smooth muscle cell coverage to blood vessels were attenuated in mice lacking eNOS. Taken together, we suggest that the endothelial cell function and blood vessel maturation are regulated by eNOS during retinal angiogenesis. PMID:27610040

  15. NOS3 polymorphisms, cigarette smoking, and cardiovascular disease risk: The Atherosclerosis Risk in Communities study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Endothelial nitric oxide synthase (NOS3) activity and cigarette smoking significantly influence endothelial function. We sought to determine whether cigarette smoking modified the association between NOS3 polymorphisms and risk of coronary heart disease or stroke. All 1085 incident coronary heart di...

  16. Endothelial nitric oxide synthase (eNOS) T-786C, 4a4b, and G894T polymorphisms and male infertility: study for idiopathic asthenozoospermia and meta-analysis.

    PubMed

    Song, Pingping; Zou, Shasha; Chen, Tingting; Chen, Jianhua; Wang, Yanan; Yang, Juanjuan; Song, Zhijian; Jiang, Huayu; Shi, Huijuan; Huang, Yiran; Li, Zheng; Shi, Yongyong; Hu, Hongliang

    2015-02-01

    Recent studies on the eNOS gene and male infertility show that expression of eNOS regulates normal spermatogenesis in the testis, and the eNOS gene variants (T-786C, 4a4b, and G894T) are potentially involved in impairment of spermatogenesis and sperm function. Thus, we conducted this association and meta-analysis study to further validate whether variants of those three loci affected the risk of idiopathic asthenozoospermia (AZS) and male infertility. Approximately 340 Chinese idiopathic AZS patients and 342 healthy men were included for this case-control study, genotyped by gel electrophoresis analysis or direct sequencing of PCR products. The eNOS mRNA isolated from the semen of patients was further examined by quantitative real-time PCR. Also, a meta-analysis of association between eNOS gene polymorphisms and male infertility was performed. A significant association was identified on allelic level between 4a4b variant and AZS in our study (chi-squared = 7.53, corrected P = 0.018, odds ratio (OR) = 1.808), while there were no significant difference of T-786C and G894T for asthenozoospermia in both genotype and allele distributions. In addition, expression of eNOS was up-regulated in patients compared with controls (about 2.4-fold, P < 0.001). Furthermore, the results of the meta-analysis support the conclusion that the T-786C and 4a4b loci were associated with male infertility in both Asian and Caucasian populations. Our study provides genetic evidence for the eNOS gene being a risk factor for idiopathic AZS and male infertility. Considering genetic differences among populations and complex pathogenesis of male infertility, more validating studies using independent samples are suggested in the future. PMID:25505202

  17. Is Estrogen a Therapeutic Target for Glaucoma?

    PubMed

    Dewundara, Samantha S; Wiggs, Janey L; Sullivan, David A; Pasquale, Louis R

    2016-01-01

    endothelial nitric oxide synthase, a gene receptive to estrogen regulation, are associated with glaucoma. The study concluded that increasing evidence suggests that lifetime exposure to estrogen may alter the pathogenesis of glaucoma. Estrogen exposure may have a neuroprotective effect on the progression of POAG but further studies need to confirm this finding. The role of sex-specific preventive and therapeutic treatment may be on the horizon. PMID:26959139

  18. Estrogens, cartilage, and osteoarthritis.

    PubMed

    Richette, Pascal; Corvol, Maïté; Bardin, Thomas

    2003-08-01

    A role for estrogens in osteoarthritis is consistent with the larger increases in women than in men in the incidence and prevalence of hip, knee, and finger osteoarthritis after 50 years of age. Furthermore, hormone replacement therapy for the menopause seems to be associated with a decrease in the prevalence of symptoms and radiological alterations related to hip and knee osteoarthritis. The two estrogen receptors alpha and beta (ERalpha and Erbeta) have been identified in normal and osteoarthritic cartilage, indicating that cartilage can respond to estrogens. Finally, in vivo experiments in animals and in vitro studies have shed light on the mechanisms by which estrogens may influence chondrocyte metabolism. PMID:12951307

  19. On the selectivity of neuronal NOS inhibitors

    PubMed Central

    Pigott, B; Bartus, K; Garthwaite, J

    2013-01-01

    Background and Purpose Isoform-selective inhibitors of NOS enzymes are desirable as research tools and for potential therapeutic purposes. Vinyl-l-N-5-(1-imino-3-butenyl)-l-ornithine (l-VNIO) and Nω-propyl-l-arginine (NPA) purportedly have good selectivity for neuronal over endothelial NOS under cell-free conditions, as does N-[(3-aminomethyl)benzyl]acetamidine (1400W), which is primarily an inducible NOS inhibitor. Although used in numerous investigations in vitro and in vivo, there have been surprisingly few tests of the potency and selectivity of these compounds in cells. This study addresses this deficiency and evaluates the activity of new and potentially better pyrrolidine-based compounds. Experimental Approach The inhibitors were evaluated by measuring their effect on NMDA-evoked cGMP accumulation in rodent hippocampal slices, a response dependent on neuronal NOS, and ACh-evoked cGMP synthesis in aortic rings of the same animals, an endothelial NOS-dependent phenomenon. Key Results l-VNIO, NPA and 1400W inhibited responses in both tissues but all showed less than fivefold higher potency in the hippocampus than in the aorta, implying useless selectivity for neuronal over endothelial NOS at the tissue level. In addition, the inhibitors had a 25-fold lower potency in the hippocampus than reported previously, the IC50 values being approximately 1 μM for l-VNIO and NPA, and 150 μM for 1400W. Pyrrolidine-based inhibitors were similarly weak and nonselective. Conclusion and Implications The results suggest that l-VNIO, NPA and 1400W, as well as the newer pyrrolidine-type inhibitors, cannot be used as neuronal NOS inhibitors in cells without stringent verification. The identification of inhibitors with useable selectivity in cells and tissues remains an important goal. PMID:23072468

  20. Allele, Genotype and Haplotype Structures of Functional Polymorphic Variants in Endothelial Nitric Oxide Synthase (eNOS), Angiotensinogen (ACE) and Aldosterone Synthase (CYP11B2) Genes in Healthy Pregnant Women of Indian Ethnicity

    PubMed Central

    Devendran, Anichavezhi; Nampoothiri, Sreekala; Shewade, Deepak Gopal; Chatterjee, Suvro; Jayaraman, Balachandar; Chandrasekharan, Adithan

    2015-01-01

    Background: Variants in the candidate genes eNOS, CYP11B2 and ACE have been implicated as liable biomarkers that can predict complications like hypertension and preeclampsia. Studies on the impact and distribution of these variants on healthy pregnancy have not been done so far in south Indian or in any of the native Indian population. Examining these variants could lay a strong basis in understanding the genetic aspects of preeclampsia and further offer effective means in early risk assessment in a preeclampsia. Methods: Genotyping for 303 unrelated healthy women of Tamilian origin who underwent uncomplicated term pregnancies was executed by PCR-RFLP for eNOS, CYP11B2 and ACE variants. Haplotype assessment and pairwise linkage disequilibrium (LD) investigation were performed by Haploview software. Results: The prevalence of eNOS variants (−786T>C, Glu298Asp and intron 4 VNTR) was 12%, 21.6% and 21.1%, respectively. The incidence of CYP11B2 (−344 C>T) and ACE (287 bp Alu I/D) variants was found to be 43.8% and 42.7%. The observed frequencies of the studied polymorphisms did not diverge from the HWE (p>0.05). Significant LD was observed between 3 eNOS gene polymorphisms. Six different haplotype structures with a frequency of >1% were generated from three eNOS variants. Among the haplotypes generated, the haplotype T-4b-G was the most common with the frequency of 64.4%. There was a statistically significant inconsistency in the study population in comparison to other global races. Conclusion: The outcome of this study could be used for investigating future therapeutic value of the variants in a preeclamptic set-up which could pose a credible diagnostic potential for primary risk assessment of women susceptible to preeclampsia/other pregnancy related complications. PMID:27110515

  1. How mental stress affects endothelial function.

    PubMed

    Toda, Noboru; Nakanishi-Toda, Megumi

    2011-12-01

    Mental stress is an important factor contributing to recognized mechanisms underlying cardiovascular events. Among these, stress-related endothelial dysfunction is an early risk factor that predicts future development of severe cardiovascular disorders. Acute mental stress by a variety of tests impairs endothelial function in humans, although the opposite results have been reported by some investigators. Chronic stress always deteriorates endothelial function in humans and experimental animals. Stress hormones, such as glucocorticoids and pro-inflammatory cytokines, and endothelin-1 liberated in response to mental stress participate in endothelial dysfunction possibly via downregulation of endothelial nitric oxide synthase (eNOS) expression, eNOS inactivation, decreased nitric oxide (NO) actions, and increased NO degradation, together with vasoconstriction counteracting against NO-induced vasodilatation. Catecholamines do not directly affect endothelial function but impair its function when blood pressure elevation by the amines is sustained. Endogenous opioids favorably affect endothelial function, which counteract deteriorating effects of other stress hormones and mediators. Inhibition of cortisol and endothelin-1 production, prevention of pro-inflammatory mediator accumulation, hypnotics, mirthful laughter, humor orientation, and lifestyle modification would contribute to the prevention and treatment for stress-related endothelial dysfunction and future serious cardiovascular disease. PMID:21947555

  2. Growth Hormone Effects in Immune Stress: AKT/eNOS Signaling Module in the Cellular Response

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The activation of the constitutive endothelial nitric-oxide synthase (eNOS) and expression of inducible NOS (iNOS) with subsequent nitric oxide production are among the early cellular responses that follow in a systemic exposure of animals to lipopolysaccharide (LPS). Growth hormone (GH) has been sh...

  3. Endothelial Lessons.

    PubMed

    Vanhoutte, Paul M

    2016-01-01

    This essay focuses on nine important lessons learned during more than thirty years of endothelial research. They include: the danger of hiding behind a word, the confusion generated by abbreviations, the need to define the physiological role of the response studied, the local role of endothelium- dependent responses, the strength of pharmacological analyses, endothelial dysfunction as consequence and cause of disease, the importance of rigorous protocols, the primacy of in vivo studies and the importance of serendipity. PMID:26638800

  4. Arsenite induces endothelial cytotoxicity by down-regulation of vascular endothelial nitric oxide synthase

    SciTech Connect

    Tsou, T.-C. . E-mail: tctsou@nhri.org.tw; Tsai, F.-Y.; Hsieh, Y.-W.; Li, L.-A.; Yeh, S.C; Chang, L.W.

    2005-11-01

    Epidemiological studies have demonstrated a high association of inorganic arsenic exposure with vascular diseases. Recent research has also linked this vascular damage to impairment of endothelial nitric oxide synthase (eNOS) function by arsenic exposure. However, the role of eNOS in regulating the arsenite-induced vascular dysfunction still remains to be clarified. In our present study, we investigated the effect of arsenite on Akt1 and eNOS and its involvement in cytotoxicity of vascular endothelial cells. Our study demonstrated that arsenite decreased the protein levels of both Akt1 and eNOS accompanied with increased levels of ubiquitination of total cell lysates. We found that inhibition of the ubiquitin-proteasome pathway by MG-132 could partially protect Akt1 and eNOS from degradation by arsenite together with a proportional protection from the arsenite-induced cytoxicity. Moreover, up-regulation of eNOS protein expression significantly attenuated the arsenite-induced cytotoxicity and eNOS activity could be significantly inhibited after incubation with arsenite for 24 h in a cell-free system. Our study indicated that endothelial eNOS activity could be attenuated by arsenite via the ubiquitin-proteasome-mediated degradation of Akt1/eNOS as well as via direct inhibition of eNOS activity. Our study also demonstrated that eNOS actually played a protective role in arsenite-induced cytoxicity. These observations supported the hypothesis that the impairment of eNOS function by arsenite is one of the mechanisms leading to vascular changes and diseases.

  5. Ablation of the ATP-binding cassette transporter, Abca2 modifies response to estrogen-based therapies.

    PubMed

    Mack, Jody T; Brown, Carol B; Garrett, Tracy E; Uys, Joachim D; Townsend, Danyelle M; Tew, Kenneth D

    2012-09-01

    The ATP-binding cassette transporter 2 (ABCA2) is an endolysosomal protein expressed in oligodendrocytes and Schwann cells, prostate, ovary and macrophages. In cell cultures, ABCA2 over-expression has been linked with resistance to the anticancer agent, estramustine phosphate (EMP; a nor-nitrogen mustard conjugate of estradiol). The present study shows that Abca2 knockout (KO) mice have greater sensitivity to a variety of side effects induced by EMP treatment. Chronic EMP (12×100 mg/kg body weight) produced mortality in 36% of KO mice, but only 7% of age-matched wild type (WT). Side effects of the drug were also more prevalent in the KO mouse. For example, during the first week of EMP treatments, 67% of KO males (compared to 6% of WT males) responded with episodic erectile events. In WT mice, ABCA2 protein localized within pene corpuscles, (which rely on modified Schwann cells for amplification of tactile signals) suggesting that the transporter may function in the erectile process. Endothelial nitric oxide synthase (eNOS; a source of nitric oxide during erectile response) levels were similar in WT and KO male penile tissue. Treatment with 100 mg/kg EMP (once daily for four days) elevated serum estradiol and estrone in both WT and KO. However, the circulating levels of these estrogens were higher in KO mice implying a reduced plasma clearance of estrogens as a consequence of ABCA2 ablation. Consistent with the pro-convulsant effects of estrogens, KO mice also displayed an increased incidence of seizures following EMP (14% vs. 0%). Taken together, these data indicate that ABCA2 deficiency renders mice more sensitive to EMP treatment-induced effects implying that the transporter has a role in regulating EMP transport and/or metabolism. PMID:22898081

  6. Ablation of the ATP-binding cassette transporter, Abca2 modifies response to estrogen-based therapies

    PubMed Central

    Mack, Jody T.; Brown, Carol B.; Garrett, Tracy E.; Uys, Joachim D.; Townsend, Danyelle M.; Tew, Kenneth D.

    2013-01-01

    The ATP-binding cassette transporter 2 (ABCA2) is an endolysosomal protein expressed in oligodendrocytes and Schwann cells, prostate, ovary and macrophages. In cell cultures, ABCA2 over-expression has been linked with resistance to the anticancer agent, estramustine phosphate (EMP; a nor-nitrogen mustard conjugate of estradiol). The present study shows that Abca2 knockout (KO) mice have greater sensitivity to a variety of side effects induced by EMP treatment. Chronic EMP (12 × 100 mg/kg body weight) produced mortality in 36% of KO mice, but only 7% of age-matched wild type (WT). Side effects of the drug were also more prevalent in the KO mouse. For example, during the first week of EMP treatments, 67% of KO males (compared to 6% of WT males) responded with episodic erectile events. In WT mice, ABCA2 protein localized within pene corpuscles, (which rely on modified Schwann cells for amplification of tactile signals) suggesting that the transporter may function in the erectile process. Endothelial nitric oxide synthase (eNOS; a source of nitric oxide during erectile response) levels were similar in WT and KO male penile tissue. Treatment with 100 mg/kg EMP (once daily for four days) elevated serum estradiol and estrone in both WT and KO. However, the circulating levels of these estrogens were higher in KO mice implying a reduced plasma clearance of estrogens as a consequence of ABCA2 ablation. Consistent with the pro-convulsant effects of estrogens, KO mice also displayed an increased incidence of seizures following EMP (14% vs. 0%). Taken together, these data indicate that ABCA2 deficiency renders mice more sensitive to EMP treatment-induced effects implying that the transporter has a role in regulating EMP transport and/or metabolism. PMID:22898081

  7. The use of a whole animal biophotonic model as a screen for the angiogenic potential of estrogenic compounds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vascular endothelial growth factor (VEGF) is essential for normal vascular growth and development during wound repair. VEGF is estrogen responsive and capable of regulating its own receptor, vascular endothelial growth factor receptor-2 (VEGFR-2). Several agricultural pesticides (e.g., methoxychlor)...

  8. Soy Isoflavone Protects Myocardial Ischemia/Reperfusion Injury through Increasing Endothelial Nitric Oxide Synthase and Decreasing Oxidative Stress in Ovariectomized Rats

    PubMed Central

    Tang, Yan; Li, Shuangyue; Zhang, Ping; Zhu, Jinbiao; Meng, Guoliang; Xie, Liping; Yu, Ying; Ji, Yong; Han, Yi

    2016-01-01

    There is a special role for estrogens in preventing and curing cardiovascular disease in women. Soy isoflavone (SI), a soy-derived phytoestrogen, has similar chemical structure to endogenous estrogen-estradiol. We investigate to elucidate the protective mechanism of SI on myocardial ischemia/reperfusion (MI/R) injury. Female SD rats underwent bilateral ovariectomy. One week later, rats were randomly divided into several groups, sham ovariectomy (control group), ovariectomy with MI/R, or ovariectomy with sham MI/R. Other ovariectomy rats were given different doses of SI or 17β-estradiol (E2). Four weeks later, they were exposed to 30 minutes of left coronary artery occlusion followed by 6 or 24 hours of reperfusion. SI administration significantly reduced myocardial infarct size and improved left ventricle function and restored endothelium-dependent relaxation function of thoracic aortas after MI/R in ovariectomized rats. SI also decreased serum creatine kinase and lactate dehydrogenase activity, reduced plasma malonaldehyde, and attenuated oxidative stress in the myocardium. Meanwhile, SI increased phosphatidylinositol 3 kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) signal pathway. SI failed to decrease infarct size of hearts with I/R in ovariectomized rats if PI3K was inhibited. Overall, these results indicated that SI protects myocardial ischemia/reperfusion injury in ovariectomized rats through increasing PI3K/Akt/eNOS signal pathway and decreasing oxidative stress. PMID:27057277

  9. Stromal cell–derived factor 2 is critical for Hsp90-dependent eNOS activation

    PubMed Central

    Siragusa, Mauro; Fröhlich, Florian; Park, Eon Joo; Schleicher, Michael; Walther, Tobias C.; Sessa, William C.

    2016-01-01

    Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of l-arginine and molecular oxygen into l-citrulline and nitric oxide (NO), a gaseous second messenger that influences cardiovascular physiology and disease. Several mechanisms regulate eNOS activity and function, including phosphorylation at Ser and Thr residues and protein-protein interactions. Combining a tandem affinity purification approach and mass spectrometry, we identified stromal cell–derived factor 2 (SDF2) as a component of the eNOS macromolecular complex in endothelial cells. SDF2 knockdown impaired agonist-stimulated NO synthesis and decreased the phosphorylation of eNOS at Ser1177, a key event required for maximal activation of eNOS. Conversely, SDF2 overexpression dose-dependently increased NO synthesis through a mechanism involving Akt and calcium (induced with ionomycin), which increased the phosphorylation of Ser1177 in eNOS. NO synthesis by iNOS (inducible NOS) and nNOS (neuronal NOS) was also enhanced upon SDF2 overexpression. We found that SDF2 was a client protein of the chaperone protein Hsp90, interacting preferentially with the M domain of Hsp90, which is the same domain that binds to eNOS. In endothelial cells exposed to vascular endothelial growth factor (VEGF), SDF2 was required for the binding of Hsp90 and calmodulin to eNOS, resulting in eNOS phosphorylation and activation. Thus, our data describe a function for SDF2 as a component of the Hsp90-eNOS complex that is critical for signal transduction in endothelial cells. PMID:26286023

  10. The Measurement of Estrogens

    NASA Astrophysics Data System (ADS)

    Holder, Geoff; Makin, Hugh L. J.; Bradlow, H. Leon

    Biologists use the word ‘estrogen' when referring to molecules which have the ability to induce uterine growth or vaginal cornification in the immature or ovariectomized rodent. The word estrogen was derived from two Greek words - oistros meaning frenzy and gennein - to beget. Chemists and biochemists, however, often restrict their use of this term to molecules that contain a characteristic 18-carbon steroid nucleus with an aromatic (phenolic) A-ring, both those that are biologically active estrogens and those without biologic activity but which are of intrinsic interest, such as the estrogen conjugates. This chapter is concerned only with these steroid compounds. The structure and inter-relationship of some common estrogens are given in Fig. 8.1. In addition to the biological estrogens, there are a wide variety of both natural and synthetic compounds which have estrogenic activity when measured by one or another parameter. While many of the assay procedures described in this review are applicable to these compounds, their application to non C18-steroids will not be discussed here. Methodology for these non-steroidal compounds can be found in reviews by Wang et al. (2002), Wu et al. (2004), Muir (2006), and Delmonte and Rader (2006). While not wishing to downgrade the importance of previous work in the estrogen field, the authors have taken a deliberate decision to exclude most publications prior to 1975, not because these do not have value but simply because space is not unlimited and readers of the present chapter might be expected to be seeking information about methodology which is less than 30 years old. Readers seeking pre-1975 information in this area can find it in Oakey and Holder (1995).

  11. Ovine caveolin-1: cDNA cloning, E. coli expression, and association with endothelial nitric oxide synthase.

    PubMed

    Chen, D; Zangl, A L; Zhao, Q; Markley, J L; Zheng, J; Bird, I M; Magness, R R

    2001-04-25

    Caveolin-1 (Cav-1), the principal coat protein of caveolae, plays an obligatory role in regulating the activity of endothelial nitric oxide (NO) synthase (eNOS). We propose that Cav-1 may be critical to eNOS-NO mediated uterine vasodilatation during pregnancy and estrogen replacement therapy. To test this hypothesis in the sheep model, we isolated the full-length cDNA of ovine Cav-1 (oCav-1) from a Lambda ZAP cDNA library of ovine placental artery endothelial cells. Thirty-two positive oCav-1 clones were recognized by a partial oCav-1 cDNA from this library, of which eight were sequenced. Restriction digestion of these clones revealed that the cDNAs of oCav-1 ranged from approximately 2.1 to 2.7 kb. Northern analysis of Cav-1 mRNAs in ovine uterine artery endothelial cells (UAEC) showed two transcripts of approximately 2.1 and 2.7 kb, respectively. Immunoreactive Cav-1 protein, but not caveolin-2 or caveolin-3, was detected in UAEC. Sequence analysis revealed that in addition to a 537-bp open reading frame encoding a 178 amino acid oCav-1 protein, full-length oCav-1 cDNAs apparently possess a approximately 1.6-2.1 kb 3'-untranslated region. Database searches with oCav-1 cDNA revealed that the coding region of mammalian Cav-1 genes is highly conserved. We prepared a recombinant full-length oCav-1 protein in which six consecutive histidine residues were tagged at the end of its COOH-terminus and developed a [His]6-tagged oCav-1 'pull-down assay' for studying the association of eNOS with Cav-1. Incubation of exogenous [His]6-tagged oCav-1 with resting UAEC extracts led to the formation of a [His]6-tagged oCav-1-eNOS complex. In the presence of a synthetic caveolin-scaffolding domain (CSD, aa 82-101) peptide, but not a mutated CSD peptide, [His]6-tagged oCav-1 associated eNOS was dose (0-10 microM)-dependently inhibited. eNOS association with Cav-1 in UAEC was further confirmed by the facts that eNOS co-immunoprecipitated with Cav-1 and vice versa, and that eNOS co

  12. Removal of estrogens and estrogenicity through drinking water treatment.

    PubMed

    Schenck, Kathleen; Rosenblum, Laura; Wiese, Thomas E; Wymer, Larry; Dugan, Nicholas; Williams, Daniel; Mash, Heath; Merriman, Betty; Speth, Thomas

    2012-03-01

    Estrogenic compounds have been shown to be present in surface waters, leading to concerns over their possible presence in finished drinking waters. In this work, two in vitro human cell line bioassays for estrogenicity were used to evaluate the removal of estrogens through conventional drinking water treatment using a natural water. Bench-scale studies utilizing chlorine, alum coagulation, ferric chloride coagulation, and powdered activated carbon (PAC) were conducted using Ohio River water spiked with three estrogens, 17β-estradiol, 17α-ethynylestradiol, and estriol. Treatment of the estrogens with chlorine, either alone or with coagulant, resulted in approximately 98% reductions in the concentrations of the parent estrogens, accompanied by formation of by-products. The MVLN reporter gene and MCF-7 cell proliferation assays were used to characterize the estrogenic activity of the water before and after treatment. The observed estrogenic activities of the chlorinated samples showed that estrogenicity of the water was reduced commensurate with removal of the parent estrogen. Therefore, the estrogen chlorination by-products did not contribute appreciably to the estrogenic activity of the water. Coagulation alone did not result in significant removals of the estrogens. However, addition of PAC, at a typical drinking water plant dose, resulted in removals ranging from approximately 20 to 80%. PMID:22361701

  13. Microvascular lesions by estrogen-induced ID3: its implications in cerebral & cardiorenal vascular disease

    PubMed Central

    Das, Jayanta K.; Felty, Quentin

    2014-01-01

    Severe symptoms of cerebral and cardiorenal vascular diseases can be triggered when cerebral, coronary, or glomerular arterioles grow inappropriately as a result of abnormal cell proliferation. The risk factor(s) and molecular mechanisms responsible for microvascular lesion formation are largely unknown. Although controversial, both animal and epidemiological studies have shown that estrogen increases the risk of stroke which may be due to microvascular lesions. Since microvascular diseases are characterized by excessive vessel growth, it is plausible that estrogen-induced neovascularization contributes to the growth of microvascular lesions. We present evidence for how ID3 overexpression in endothelial cells contributes to the development of an estrogen-induced neovascular phenotype with an additional focus on Pyk2 kinase. Our data showed that ID3 overexpression increased neovascularization, cell migration, and spheroid growth of human cerebral microvascular endothelial cells, hCMEC/D3. ID3 overexpressing cells showed significant estrogen-induced G2/M phase transition. Estrogen treatment increased both ID3 phosphorylation and total protein that was inhibited by tamoxifen; and Pyk2 mediated estrogen-induced ID3 mRNA expression. These findings suggest that Pyk2 signals ID3 expression and ID3 is necessary for estrogen-induced neovascularization in hCMEC/D3 cells. A better understanding of how microvascular lesions depend on ID3 may open new avenues for prevention and treatment of neurological diseases. PMID:25129100

  14. 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. PMID:26390975

  15. Soluble CD40 ligand induces endothelial dysfunction in human and porcine coronary artery endothelial cells.

    PubMed

    Chen, Changyi; Chai, Hong; Wang, Xinwen; Jiang, Jun; Jamaluddin, Md Saha; Liao, Dan; Zhang, Yuqing; Wang, Hao; Bharadwaj, Uddalak; Zhang, Sheng; Li, Min; Lin, Peter; Yao, Qizhi

    2008-10-15

    The purpose of this study was to determine the effects and mechanisms of sCD40L on endothelial dysfunction in both human coronary artery endothelial cells (HCAECs) and porcine coronary artery rings. HCAECs treated with sCD40L showed significant reductions of endothelial nitric oxide synthase (eNOS) mRNA and protein levels, eNOS mRNA stability, eNOS enzyme activity, and cellular NO levels, whereas superoxide anion (O(2)(-)) production was significantly increased. sCD40L enhanced eNOS mRNA 3'UTR binding to cytoplasmic molecules and induced a unique expression pattern of 95 microRNAs. sCD40L significantly decreased mitochondrial membrane potential, and catalase and SOD activities, whereas it increased NADPH oxidase (NOX) activity. sCD40L increased phosphorylation of MAPKs p38 and ERK1/2 as well as IkappaBalpha and enhanced NF-kappaB nuclear translocation. In porcine coronary arteries, sCD40L significantly decreased endothelium-dependent vasorelaxation and eNOS mRNA levels, whereas it increased O(2)(-) levels. Antioxidant seleno-l-methionine; chemical inhibitors of p38, ERK1/2, and mitochondrial complex II; as well as dominant negative mutant forms of IkappaBalpha and NOX4 effectively blocked sCD40L-induced eNOS down-regulation in HCAECs. Thus, sCD40L reduces eNOS levels, whereas it increases oxidative stress through the unique molecular mechanisms involving eNOS mRNA stability, 3'UTR-binding molecules, microRNAs, mitochondrial function, ROS-related enzymes, p38, ERK1/2, and NF-kappaB signal pathways in endothelial cells. PMID:18658029

  16. Removal of Estrogens and Estrogenicity through Drinking Water Treatment

    EPA Science Inventory

    Estrogenic compounds have been shown to be present in surface waters, leading to concerns over their possible presence in finished drining waters. In this work, two in vitro human cell line bioassays for estrogenicity were used to evaluate the removal of estrogens through conven...

  17. G protein-coupled estrogen receptor inhibits vascular prostanoid production and activity.

    PubMed

    Meyer, Matthias R; Fredette, Natalie C; Barton, Matthias; Prossnitz, Eric R

    2015-10-01

    Complications of atherosclerotic vascular disease, such as myocardial infarction and stroke, are the most common causes of death in postmenopausal women. Endogenous estrogens inhibit vascular inflammation-driven atherogenesis, a process that involves cyclooxygenase (COX)-derived vasoconstrictor prostanoids such as thromboxane A2. Here, we studied whether the G protein-coupled estrogen receptor (GPER) mediates estrogen-dependent inhibitory effects on prostanoid production and activity under pro-inflammatory conditions. Effects of estrogen on production of thromboxane A(2) were determined in human endothelial cells stimulated by the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-α). Moreover, Gper-deficient (Gper(-/-)) and WT mice were fed a pro-inflammatory diet and underwent ovariectomy or sham surgery to unmask the role of endogenous estrogens. Thereafter, contractions to acetylcholine-stimulated endothelial vasoconstrictor prostanoids and the thromboxane-prostanoid receptor agonist U46619 were recorded in isolated carotid arteries. In endothelial cells, TNF-α-stimulated thromboxane A2 production was inhibited by estrogen, an effect blocked by the GPER-selective antagonist G36. In ovary-intact mice, deletion of Gper increased prostanoid-dependent contractions by twofold. Ovariectomy also augmented prostanoid-dependent contractions by twofold in WT mice but had no additional effect in Gper(-/-) mice. These contractions were blocked by the COX inhibitor meclofenamate and unaffected by the nitric oxide synthase inhibitor l-N(G)-nitroarginine methyl ester. Vasoconstrictor responses to U46619 did not differ between groups, indicating intact signaling downstream of thromboxane-prostanoid receptor activation. In summary, under pro-inflammatory conditions, estrogen inhibits vasoconstrictor prostanoid production in endothelial cells and activity in intact arteries through GPER. Selective activation of GPER may therefore be considered as a novel strategy to

  18. eNOS-uncoupling in age-related erectile dysfunction

    PubMed Central

    Johnson, JM; Bivalacqua, TJ; Lagoda, GA; Burnett, AL; Musicki, B

    2011-01-01

    Aging is associated with ED. Although age-related ED is attributed largely to increased oxidative stress and endothelial dysfunction in the penis, the molecular mechanisms underlying this effect are not fully defined. We evaluated whether endothelial nitric oxide synthase (eNOS) uncoupling in the aged rat penis is a contributing mechanism. Correlatively, we evaluated the effect of replacement with eNOS cofactor tetrahydrobiopterin (BH4) on erectile function in the aged rats. Male Fischer 344 ‘young’ (4-month-old) and ‘aged’ (19-month-old) rats were treated with a BH4 precursor sepiapterin (10 mg/kg intraperitoneally) or vehicle for 4 days. After 1-day washout, erectile function was assessed in response to electrical stimulation of the cavernous nerve. Endothelial dysfunction (eNOS uncoupling) and oxidative stress (thiobarbituric acid reactive substances, TBARS) were measured by conducting western blot in penes samples. Erectile response was significantly reduced in aged rats, whereas eNOS uncoupling and TBARS production were significantly increased in the aged rat penis compared with young rats. Sepiapterin significantly improved erectile response in aged rats and prevented increase in TBARS production, but did not affect eNOS uncoupling in the penis of aged rats. These findings suggest that aging induces eNOS uncoupling in the penis, resulting in increased oxidative stress and ED. PMID:21289638

  19. Impact of Hemorheological and Endothelial Factors on Microcirculation

    NASA Astrophysics Data System (ADS)

    Turchetti, Vera; Boschi, Letizia; Donati, Giovanni; Trabalzini, Luca; Forconi, Sandro

    Previous studies showed that endothelial alterations caused by physical stress worsened the hemorheological parameters mainly in patients affected by ischemic vascular diseases: major vascular alterations have been found in patients with very high endothelial dysfunction indexes: these indexes are given by the various substances produced by the endothelium, but it is very difficult to have a value which clearly identifies the real state of the endothelial alteration. The function of the NO, an endogenous vasodilator whose synthesis is catalyzed by NOs, can be determined by the Citrulline/Arginine ratio, which represents the level of activity of the enzyme. A very good index of the endothelial dysfunction is asymmetric dimethylarginine (ADMA), a powerful endogenous inhibitor of NOs; in fact several studies have demonstrated a strong relationship between ischemic vascular disease and high levels of plasmatic ADMA. Our recent studies on heart failure and on ischemic cerebrovascular diseases evaluate endothelial dysfunctions and hemorheological parameters.

  20. Soluble endoglin, hypercholesterolemia and endothelial dysfunction.

    PubMed

    Rathouska, Jana; Jezkova, Katerina; Nemeckova, Ivana; Nachtigal, Petr

    2015-12-01

    A soluble form of endoglin (sEng) is known to be an extracellular domain of the full-length membrane endoglin, which is elevated during various pathological conditions related to vascular endothelium. In the current review, we tried to summarize a possible role of soluble endoglin in cardiovascular pathologies, focusing on its relation to endothelial dysfunction and cholesterol levels. We discussed sEng as a proposed biomarker of cardiovascular disease progression, cardiovascular disease treatment and endothelial dysfunction. We also addressed a potential interaction of sEng with TGF-β/eNOS or BMP-9 signaling. We suggest soluble endoglin levels to be monitored, because they reflect the progression/treatment efficacy of cardiovascular diseases related to endothelial dysfunction and hypercholesterolemia. A possible role of soluble endoglin as an inducer of endothelial dysfunction however remains to be elucidated. PMID:26520890

  1. The dual role of iNOS in cancer☆

    PubMed Central

    Vanini, Frederica; Kashfi, Khosrow; Nath, Niharika

    2015-01-01

    Nitric oxide (NO) is one of the 10 smallest molecules found in nature. It is a simple gaseous free radical whose predominant functions is that of a messenger through cGMP. In mammals, NO is synthesized by the enzyme nitric oxide synthase (NOS) of which there are three isoforms. Neuronal (nNOS, NOS1) and endothelial (eNOS, NOS3) are constitutive calcium-dependent forms of the enzyme that regulate neural and vascular function respectively. The third isoform (iNOS, NOS2), is calcium-independent and is inducible. In many tumors, iNOS expression is high, however, the role of iNOS during tumor development is very complex and quite perplexing, with both promoting and inhibiting actions having been described. This review will aim to summarize the dual actions of iNOS-derived NO showing that the microenvironment of the tumor is a contributing factor to these observations and ultimately to cellular outcomes. PMID:26335399

  2. Expression analysis of NOS family and HSP genes during thermal stress in goat ( Capra hircus)

    NASA Astrophysics Data System (ADS)

    Yadav, Vijay Pratap; Dangi, Satyaveer Singh; Chouhan, Vikrant Singh; Gupta, Mahesh; Dangi, Saroj K.; Singh, Gyanendra; Maurya, Vijay Prakash; Kumar, Puneet; Sarkar, Mihir

    2016-03-01

    Approximately 50 genes other than heat shock protein (HSP) expression changes during thermal stress. These genes like nitric oxide synthase (NOS) need proper attention and investigation to find out their possible role in the adaptation to thermal stress in animals. So, the present study was undertaken to demonstrate the expressions of inducible form type II NOS (iNOS), endothelial type III NOS (eNOS), constitutively expressed enzyme NOS (cNOS), HSP70, and HSP90 in peripheral blood mononuclear cells (PBMCs) during different seasons in Barbari goats. Real-time polymerase chain reaction, western blot, and immunocytochemistry were applied to investigate messenger RNA (mRNA) expression, protein expression, and immunolocalization of examined factors. The mRNA and protein expressions of iNOS, eNOS, cNOS, HSP70, and HSP90 were significantly higher ( P < 0.05) during peak summer, and iNOS and eNOS expressions were also observed to be significantly higher ( P < 0.05) during peak winter season as compared with moderate season. The iNOS, eNOS, cNOS, HSP70, and HSP90 were mainly localized in plasma membrane and cytoplasm of PBMCs. To conclude, data generated in the present study indicate the possible involvement of the NOS family genes in amelioration of thermal stress so as to maintain cellular integrity and homeostasis in goats.

  3. Expression analysis of NOS family and HSP genes during thermal stress in goat (Capra hircus).

    PubMed

    Yadav, Vijay Pratap; Dangi, Satyaveer Singh; Chouhan, Vikrant Singh; Gupta, Mahesh; Dangi, Saroj K; Singh, Gyanendra; Maurya, Vijay Prakash; Kumar, Puneet; Sarkar, Mihir

    2016-03-01

    Approximately 50 genes other than heat shock protein (HSP) expression changes during thermal stress. These genes like nitric oxide synthase (NOS) need proper attention and investigation to find out their possible role in the adaptation to thermal stress in animals. So, the present study was undertaken to demonstrate the expressions of inducible form type II NOS (iNOS), endothelial type III NOS (eNOS), constitutively expressed enzyme NOS (cNOS), HSP70, and HSP90 in peripheral blood mononuclear cells (PBMCs) during different seasons in Barbari goats. Real-time polymerase chain reaction, western blot, and immunocytochemistry were applied to investigate messenger RNA (mRNA) expression, protein expression, and immunolocalization of examined factors. The mRNA and protein expressions of iNOS, eNOS, cNOS, HSP70, and HSP90 were significantly higher (P < 0.05) during peak summer, and iNOS and eNOS expressions were also observed to be significantly higher (P < 0.05) during peak winter season as compared with moderate season. The iNOS, eNOS, cNOS, HSP70, and HSP90 were mainly localized in plasma membrane and cytoplasm of PBMCs. To conclude, data generated in the present study indicate the possible involvement of the NOS family genes in amelioration of thermal stress so as to maintain cellular integrity and homeostasis in goats. PMID:26205811

  4. Estrogens and autoimmune diseases.

    PubMed

    Cutolo, Maurizio; Capellino, Silvia; Sulli, Alberto; Serioli, Bruno; Secchi, Maria Elena; Villaggio, Barbara; Straub, Rainer H

    2006-11-01

    Sex hormones are implicated in the immune response, with estrogens as enhancers at least of the humoral immunity and androgens and progesterone (and glucocorticoids) as natural immune-suppressors . Several physiological, pathological, and therapeutic conditions may change the serum estrogen milieu and/or peripheral conversion rate, including the menstrual cycle, pregnancy, postpartum period, menopause, being elderly, chronic stress, altered circadian rhythms, inflammatory cytokines, and use of corticosteroids, oral contraceptives, and steroid hormonal replacements, inducing altered androgen/estrogen ratios and related effects. In particular, cortisol and melatonin circadian rhythms are altered, at least in rheumatoid arthritis (RA), and partially involve sex hormone circadian synthesis and levels as well. Abnormal regulation of aromatase activity (i.e., increased activity) by inflammatory cytokine production (i.e., TNF-alpha, IL-1, and IL-6) may partially explain the abnormalities of peripheral estrogen synthesis in RA (i.e., increased availability of 17-beta estradiol and possible metabolites in synovial fluids) and in systemic lupus erythematosus, as well as the altered serum sex-hormone levels and ratio (i.e., decreased androgens and DHEAS). In the synovial fluids of RA patients, the increased estrogen concentration is observed in both sexes and is more specifically characterized by the hydroxylated forms, in particular 16alpha-hydroxyestrone, which is a mitogenic and cell proliferative endogenous hormone. Local effects of sex hormones in autoimmune rheumatic diseases seems to consist mainly in modulation of cell proliferation and cytokine production (i.e., TNF-alpha, Il-1, IL-12). In this respect, it is interesting that male patients with RA seem to profit more from anti-TNFalpha strategies than do female patients. PMID:17261796

  5. Eldecalcitol prevents endothelial dysfunction in postmenopausal osteoporosis model rats.

    PubMed

    Serizawa, Kenichi; Yogo, Kenji; Tashiro, Yoshihito; Takeda, Satoshi; Kawasaki, Ryohei; Aizawa, Ken; Endo, Koichi

    2016-02-01

    Postmenopausal women have high incidence of cardiovascular events as estrogen deficiency can cause endothelial dysfunction. Vitamin D is reported to be beneficial on endothelial function, but it remains controversial whether vitamin D is effective for endothelial dysfunction under the treatment for osteoporosis in postmenopausal women. The aim of this study was to evaluate the endothelial protective effect of eldecalcitol (ELD) in ovariectomized (OVX) rats. ELD (20  ng/kg) was orally administrated five times a week for 4 weeks from 1 day after surgery. After that, flow-mediated dilation (FMD) as an indicator of endothelial function was measured by high-resolution ultrasound in the femoral artery of living rats. ELD ameliorated the reduction of FMD in OVX rats. ELD inhibited the increase in NOX4, nitrotyrosine, and p65 and the decrease in dimer/monomer ratio of nitric oxide synthase in OVX rat femoral arteries. ELD also prevented the decrease in peroxisome proliferator-activated receptor gamma (PPARγ) in femoral arteries and cultured endothelial cells. Although PPARγ is known to inhibit osteoblastogenesis, ELD understandably increased bone mineral density of OVX rats without increase in PPARγ in bone marrow. These results suggest that ELD prevented the deterioration of endothelial function under condition of preventing bone loss in OVX rats. This endothelial protective effect of ELD might be exerted through improvement of endothelial nitric oxide synthase uncoupling, which is mediated by an antioxidative effect through normalization of vascular PPARγ/NF-κB signaling. PMID:26537128

  6. Estrogens and development of pulmonary hypertension - Interaction of estradiol metabolism and pulmonary vascular disease

    PubMed Central

    Tofovic, Stevan P.

    2010-01-01

    Severe pulmonary arterial hypertension (PAH) is characterized by clustered proliferation of endothelial cells in the lumina of small size pulmonary arteries resulting in concentric obliteration of the lumina and formation of complex vascular structures known as plexiform lesions. This debilitating disease occurs more frequently in women, yet both animal studies in classical models of PAH and limited clinical data suggest protective effects of estrogens: the estrogen paradox in pulmonary hypertension. Little is known about the role of estrogens in PAH, but one line of evidence strongly suggests that the vascular protective effects of 17β-estradiol (estradiol; E2) are mediated largely by its downstream metabolites. Estradiol is metabolized to 2-hydroxyestradiol (2HE) by CYP1A1/CYP1B1, and 2HE is converted to 2-methoxyestradiol (2ME) by catechol-O-methyl transferase. 2ME is extensively metabolized to 2-methoxyestrone, a metabolite that lacks biologic activity but which may be converted back to 2ME. 2ME has no estrogenic activity and its effects are mediated by estrogen receptors-independent mechanism(s). Notably, in systemic and pulmonary vascular endothelial cells, smooth muscle cells, and fibroblasts 2ME exerts stronger anti-mitotic effects than E2 itself. E2 and 2ME, despite having similar effects on other cardiovascular cells, have opposing effects on endothelial cells; that is, in endothelial cells, E2 is pro-mitogenic, pro-angiogenic and anti- apoptotic, whereas 2ME is antimitogenic, anti-angiogenic and pro-apoptotic. This may have significant ramifications in severe PAH that involves uncontrolled proliferation of monoclonal, apoptosis resistant endothelial cells. Based on its cellular effects, 2ME should be expected to attenuate the progression of disease and provide protection in severe PAH. In contrast, E2, due to its mitogenic, angiogenic, and anti-apoptotic effects (otherwise desirable in normal, quiescent endothelial cells), may even adversely affect

  7. Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells.

    PubMed

    Min, Jiho; Jin, Yoon-Mi; Moon, Je-Sung; Sung, Min-Sun; Jo, Sangmee Ahn; Jo, Inho

    2006-06-01

    Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression. PMID:16651461

  8. Endothelial cytosolic proteins bind to the 3' untranslated region of endothelial nitric oxide synthase mRNA: regulation by tumor necrosis factor alpha.

    PubMed Central

    Alonso, J; Sánchez de Miguel, L; Montón, M; Casado, S; López-Farré, A

    1997-01-01

    Changes in endothelial nitric oxide synthase (eNOS) expression may be involved in the endothelium-dependent vasorelaxation dysfunction associated with several vascular diseases. In the present work, we demonstrate that eNOS mRNA contains a previously undescribed cis element in the 3' untranslated region (3' UTR). A U+C-rich segment in the 3' UTR is critical in complex formation with bovine aortic endothelial cell cytosolic proteins. Tumor necrosis factor alpha (TNF-alpha), which destabilizes eNOS mRNA, increased the binding activity of the cytosolic proteins in a time-dependent manner. These data suggest that endothelial cytosolic proteins bind to the 3' UTR of eNOS mRNA. These proteins may play a role in TNF-alpha-induced eNOS mRNA destabilization. PMID:9315630

  9. Nitric Oxide Mediates Tightening of the Endothelial Barrier by Ascorbic Acid

    PubMed Central

    May, James M.; Qu, Zhi-chao

    2010-01-01

    Vitamin C, or ascorbic acid, decreases paracellular endothelial permeability in a process that requires rearrangement of the actin cytoskeleton. To define the proximal mechanism of this effect, we tested whether it might involve enhanced generation and/or sparing of nitric oxide (NO) by the vitamin. EA.hy926 endothelial cells cultured on semi-porous filter supports showed decreased endothelial barrier permeability to radiolabeled inulin in response to exogenous NO provided by the NO donor spermine NONOATE, as well as to activation of the downstream NO pathway by 8-bromo-cyclic GMP, a cell-penetrant cyclic GMP analog. Inhibition of endothelial nitric oxide synthase (eNOS) with Nω-nitro-L-arginine methyl ester increased endothelial permeability, indicating a role constitutive NO generation by eNOS in maintaining the permeability barrier. Inhibition of guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one also increased endothelial permeability and blocked barrier tightening by spermine NONOATE. Loading cells with what are likely physiologic concentrations of ascorbate decreased endothelial permeability. This effect was blocked by inhibition of either eNOS or guanylate cyclase, suggesting that it involved generation of NO by eNOS and subsequent NO-dependent activation of guanylate cyclase. These results show that endothelial permeability barrier function depends on constitutive generation of NO and that ascorbate-dependent tightening of this barrier involves maintaining NO through the eNOS/guanylate cyclase pathway. PMID:21156160

  10. Estrogens and endometrial carcinoma.

    PubMed

    Gray, L A; Christopherson, W M; Hoover, R N

    1977-04-01

    A group of 205 women with endometrial carcinoma was matched for age, parity, and year of operation with a group of 205 women who had had hysterectomies for benign disease. In the former group, 32 patients had used conjugated estrogens, while in the latter group 12 had used this hormone, yielding a relative risk of 3.1 (P = 0.0008). Users of other forms of systemic estrogens showed similar elevations in relative risk. Relative risk was related to duration of use, progressing from no evidence of risk among those using the hormone for less than 5 years to an 11.5-fold greater risk for those using it for 10 years or more. Risk was also related to the strength of the medication. The relative risk for users of the 1.25-mg tablets was 12.7 as compared to a two- to fourfold greater risk among users of lesser strength tablets. PMID:193072

  11. Translocation of Endothelial Nitric-Oxide Synthase Involves a Ternary Complex with Caveolin-1 and NOSTRIN

    PubMed Central

    Schilling, Kirstin; Opitz, Nils; Wiesenthal, Anja; Oess, Stefanie; Tikkanen, Ritva; Icking, Ann

    2006-01-01

    Recently, we characterized a novel endothelial nitric-oxide synthase (eNOS)-interacting protein, NOSTRIN (for eNOS-trafficking inducer), which decreases eNOS activity upon overexpression and induces translocation of eNOS away from the plasma membrane. Here, we show that NOSTRIN directly binds to caveolin-1, a well-established inhibitor of eNOS. Because this interaction occurs between the N terminus of caveolin (positions 1–61) and the central domain of NOSTRIN (positions 323–434), it allows for independent binding of each of the two proteins to eNOS. Consistently, we were able to demonstrate the existence of a ternary complex of NOSTRIN, eNOS, and caveolin-1 in Chinese hamster ovary (CHO)-eNOS cells. In human umbilical vein endothelial cells (HUVECs), the ternary complex assembles at the plasma membrane upon confluence or thrombin stimulation. In CHO-eNOS cells, NOSTRIN-mediated translocation of eNOS involves caveolin in a process most likely representing caveolar trafficking. Accordingly, trafficking of NOSTRIN/eNOS/caveolin is affected by altering the state of actin filaments or cholesterol levels in the plasma membrane. During caveolar trafficking, NOSTRIN functions as an adaptor to recruit mediators such as dynamin-2 essential for membrane fission. We propose that a ternary complex between NOSTRIN, caveolin-1, and eNOS mediates translocation of eNOS, with important implications for the activity and availability of eNOS in the cell. PMID:16807357

  12. Exercise, Eating, Estrogen, and Osteoporosis.

    ERIC Educational Resources Information Center

    Brown, Jim

    1986-01-01

    Osteoporosis affects millions of people, especially women. Three methods for preventing or managing osteoporosis are recommended: (1) exercise; (2) increased calcium intake; and (3) estrogen replacement therapy. (CB)

  13. Estrogen Metabolism and Breast Cancer

    PubMed Central

    Samavat, Hamed; Kurzer, Mindy S

    2015-01-01

    There is currently accumulating evidence that endogenous estrogens play a critical role in the development of breast cancer. Estrogens and their metabolites have been studied in both pre- and postmenopausal women with more consistent results shown in the latter population, in part because of large hormonal variations during the menstrual cycle and far fewer studies having been performed in premenopausal women. In this review we describe in detail estrogen metabolism and associated genetic variations, and provide a critical review of the current literature regarding the role of estrogens and their metabolites in breast cancer risk. PMID:24784887

  14. The Use of a Whole Animal Biophotonic Model as a Screen for the Angiogenic Potential of Estrogenic Compounds

    PubMed Central

    Youngblood, Ramey C; McGee, Marcus; Feugang, Jean M; Willard, Scott T; Ryan, Peter L

    2014-01-01

    Background: Vascular endothelial growth factor (VEGF) is essential for normal vascular growth and development during wound repair. VEGF is estrogen responsive and capable of regulating its own receptor, vascular endothelial growth factor receptor-2 (VEGFR-2). Several agricultural pesticides (e.g., methoxychlor) have estrogenic potential that can initiate inappropriate physiological responses in estrogenic-sensitive tissues following exposure in vivo. Thus, the current study was designed to determine whether the VEGFR-2-Luciferase (Luc) reporter transgenic mouse is a useful model for evaluating estrogenic tendencies of methoxychlor by monitoring wound healing via VEGFR-2-mediated gene expression using bioluminescence and real-time imaging technology. Results: VEGFR-2-Luc gene activity peaked by d 7 (P<0.001) in all groups but was not different (P>0.05) between control and estrogen/methoxychlor exposed mice. Conclusions: Changes in VEGFR-2-Luc gene activity associated with the dermal wound healing process were able to be measured via photonic emission. The increase in vasculature recruitment and formation is paralleled by the increase of VEGFR-2-Luc activity with a peak on day 7. However, estrogen/methoxychlor did not significantly alter wound healing mediated VEGFR-2-Luc gene expression patterns compared to controls. This suggests that the VEGFR-2-Luc transgenic mouse wound model tested in this study may not be optimal for use as a screen for the angiogenic potential of estrogenic compounds. PMID:24782643

  15. Rho GTPase/Rho Kinase Negatively Regulates Endothelial Nitric Oxide Synthase Phosphorylation through the Inhibition of Protein Kinase B/Akt in Human Endothelial Cells

    PubMed Central

    Ming, Xiu-Fen; Viswambharan, Hema; Barandier, Christine; Ruffieux, Jean; Kaibuchi, Kozo; Rusconi, Sandro; Yang, Zhihong

    2002-01-01

    Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB. PMID:12446767

  16. Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells.

    PubMed

    Jin, Benjamin Y; Sartoretto, Juliano L; Gladyshev, Vadim N; Michel, Thomas

    2009-10-13

    Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H(2)O(2) is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H(2)O(2) treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC(50) for H(2)O(2)-promoted phosphorylation of AMPK is 65 + or - 15 microM, within the physiological range of cellular H(2)O(2) concentrations. The Ca(2+)/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) inhibitor STO-609 abolishes H(2)O(2)-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKbeta abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H(2)O(2) biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H(2)O(2) generation, which is blocked by PEG-catalase. eNOS(-/-) mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS(-/-) mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKbeta is critically involved in mediating the phosphorylation of AMPK promoted by H(2)O(2) in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H(2)O(2) generation in endothelial cells. PMID:19805165

  17. 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. PMID:25510468

  18. Regulation of endothelial nitric oxide synthase activation in endothelial cells by S1P1 and S1P3.

    PubMed

    Tölle, M; Klöckl, L; Wiedon, A; Zidek, W; van der Giet, M; Schuchardt, M

    2016-08-01

    Endothelial nitric oxide synthase (eNOS) plays a crucial role in vascular homeostasis. Lysophospholipid interaction with sphingosine 1-phosphat (S1P) receptors results in eNOS activation in different cells. In endothelial cells, eNOS activation via S1P1 or S1P3 was shown controversially. The aim of this study is to investigate the meaning of both S1P receptors for eNOS activation in human endothelial cells. Therefore, several S1P1 and S1P3 agonists in combination with antagonists and specific RNAi approach were used. eNOS activation was measured in human umbilical vein endothelial cells (HUVEC) via DAF2-DA-based fluorescence microscopy. For investigation of the signaling pathway, agonists/antagonist studies, RNAi approach, Luminex™ multiplex, and Western Blot were used. In HUVEC, both the S1P1 agonist AUY954 as well as the S1P1,3 agonist FTY720P induced eNOS activation in a time- and dose-dependent manner. Other S1P1 agonists activated eNOS to a lesser extent. The AUY954-induced eNOS activation was blocked by the S1P1 antagonist W146, the combination of W146 and the S1P3 antagonist CAY10444 and the S1P1,3 antagonist VPC23019, but not by CAY10444 indicating the meaning of S1P1 for the AUY954-induced eNOS activation. The FTY720P-induced eNOS activation was blocked only by the combination of W146 and CAY10444 and the combined S1P1,3 antagonist VPC23019, but not by W146 or CAY10444 indicating the importance of both S1P1 and S1P3 for FTY720-induced eNOS activation. These results were confirmed using specific siRNA against S1P1 and S1P3. The S1P1,3 activation results in Akt phosphorylation and subsequent activation of eNOS via phosphorylation at serine(1177) and dephosphorylation at threonine(495). Beside former investigations with rather unspecific S1P receptor activation these data show potent selective S1P1 activation by using AUY954 and with selective S1P receptor inhibition evidence was provided that both S1P1 and S1P3 lead to downstream activation of eNOS in

  19. Islet Endothelial Cells Derived From Mouse Embryonic Stem Cells.

    PubMed

    Jain, Neha; Lee, Eun Jung

    2016-01-01

    The islet endothelium comprises a specialized population of islet endothelial cells (IECs) expressing unique markers such as nephrin and α-1 antitrypsin (AAT) that are not found in endothelial cells in surrounding tissues. However, due to difficulties in isolating and maintaining a pure population of these cells, the information on these islet-specific cells is currently very limited. Interestingly, we have identified a large subpopulation of endothelial cells exhibiting IEC phenotype, while deriving insulin-producing cells from mouse embryonic stem cells (mESCs). These cells were identified by the uptake of low-density lipoprotein (LDL) and were successfully isolated and subsequently expanded in endothelial cell culture medium. Further analysis demonstrated that the mouse embryonic stem cell-derived endothelial cells (mESC-ECs) not only express classical endothelial markers, such as platelet endothelial cell adhesion molecule (PECAM1), thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), and endothelial nitric oxide synthase (eNOS) but also IEC-specific markers such as nephrin and AAT. Moreover, mESC-ECs secrete basement membrane proteins such as collagen type IV, laminin, and fibronectin in culture and form tubular networks on a layer of Matrigel, demonstrating angiogenic activity. Further, mESC-ECs not only express eNOS, but also its eNOS expression is glucose dependent, which is another characteristic phenotype of IECs. With the ability to obtain highly purified IECs derived from pluripotent stem cells, it is possible to closely examine the function of these cells and their interaction with pancreatic β-cells during development and maturation in vitro. Further characterization of tissue-specific endothelial cell properties may enhance our ability to formulate new therapeutic angiogenic approaches for diabetes. PMID:25751085

  20. Role of estrogen receptor β selective agonist in ameliorating portal hypertension in rats with CCl4-induced liver cirrhosis

    PubMed Central

    Zhang, Cheng-Gang; Zhang, Bin; Deng, Wen-Sheng; Duan, Ming; Chen, Wei; Wu, Zhi-Yong

    2016-01-01

    AIM: To investigate the role of diarylpropionitrile (DPN), a selective agonist of estrogen receptor β (ERβ), in liver cirrhosis with portal hypertension (PHT) and isolated hepatic stellate cells (HSCs). METHODS: Female Sprague-Dawley rats were ovariectomized (OVX), and liver cirrhosis with PHT was induced by CCl4 injection. DPN and PHTPP, the selective ERβ agonist and antagonist, were used as drug interventions. Liver fibrosis was assessed by hematoxylin and eosin (HE) and Masson’s trichrome staining and by analyzing smooth muscle actin expression. Hemodynamic parameters were determined in vivo using colored microspheres technique. Protein expression and phosphorylation were determined by immunohistochemical staining and Western blot analysis. Messenger RNA levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Collagen gel contraction assay was performed using gel lattices containing HSCs treated with DPN, PHTPP, or Y-27632 prior to ET-1 addition. RESULTS: Treatment with DPN in vivo greatly lowered portal pressure and improved hemodynamic parameters without affecting mean arterial pressure, which was associated with the attenuation of liver fibrosis and intrahepatic vascular resistance (IHVR). In CCl4-treated rat livers, DPN significantly decreased the expression of RhoA and ROCK II, and even suppressed ROCK II activity. Moreover, DPN remarkedly increased the levels of endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS, and promoted the activities of protein kinase G (PKG), which is an NO effector in the liver. Furthermore, DPN reduced the contractility of activated HSCs in the 3-dimensional stress-relaxed collagen lattices, and decreased the ROCK II activity in activated HSCs. Finally, in vivo/in vitro experiments demonstrated that MLC activity was inhibited by DPN. CONCLUSION: For OVX rats with liver cirrhosis, DPN suppressed liver RhoA/ROCK signal, facilitated NO/PKG pathways, and decreased IHVR, giving rise to

  1. Estrogen receptor scintigraphy.

    PubMed

    Scheidhauer, K; Scharl, A; Schicha, H

    1998-03-01

    Radio-labeled estrogen receptor ligands are tracers that can be used for functional receptor diagnosis. Their specificity towards receptors, together with the fact that only 50-70% of mammary carcinomas are receptor positive, renders them unsuitable for detection of primary tumors or metastases, and this means that estrogen receptor scintigraphy can be used neither for tumor screening nor for staging. However, both 18F-labeled and 123I-labeled estradiol derivatives are suitable for in vivo imaging of estrogen receptors. Their high specificity, established in animal experiments and in vitro studies has been reproduced in in vivo applications in humans. Tracers with positron radiation emitters are, however, hardly suitable for broad application owing to the short half-life of 18F, which would mean that users would need to be situated close to a cyclotron and a correspondingly equipped radiochemical laboratory. The number of available PET scanners, on the other hand, has increased over the last few years, especially in Germany, so that this, at least, does not present a limiting factor. All the same, 123I-labeled estradiol derivatives will find more widespread application, since the number of gamma-cameras incorporating modern multi-head systems is several times greater. The results of studies with 123I-E2-scintigraphy published to date are very promising, even given the initial technical problems mentioned above. As a method of examination, it could be optimised by using improved tracers with a higher tumor contrast and less disturbance from overlapping in diagnostically relevant locations, for instance, by selecting tracers with higher activities whose excretion is more renal than hepatobiliary. The use of modern multi-head camera systems can also be expected to improve the photon yield. PMID:9646642

  2. Vascular endothelial-cadherin downregulation as a feature of endothelial transdifferentiation in monocrotaline-induced pulmonary hypertension.

    PubMed

    Nikitopoulou, Ioanna; Orfanos, Stylianos E; Kotanidou, Anastasia; Maltabe, Violetta; Manitsopoulos, Nikolaos; Karras, Panagiotis; Kouklis, Panos; Armaganidis, Apostolos; Maniatis, Nikolaos A

    2016-08-01

    Increased pulmonary vascular resistance in pulmonary hypertension (PH) is caused by vasoconstriction and obstruction of small pulmonary arteries by proliferating vascular cells. In analogy to cancer, subsets of proliferating cells may be derived from endothelial cells transitioning into a mesenchymal phenotype. To understand phenotypic shifts transpiring within endothelial cells in PH, we injected rats with alkaloid monocrotaline to induce PH and measured lung tissue levels of endothelial-specific protein and critical differentiation marker vascular endothelial (VE)-cadherin. VE-cadherin expression by immonoblotting declined significantly 24 h and 15 days postinjection to rebound to baseline at 30 days. There was a concomitant increase in transcriptional repressors Snail and Slug, along with a reduction in VE-cadherin mRNA. Mesenchymal markers α-smooth muscle actin and vimentin were upregulated by immunohistochemistry and immunoblotting, and α-smooth muscle actin was colocalized with endothelial marker platelet endothelial cell adhesion molecule-1 by confocal microscopy. Apoptosis was limited in this model, especially in the 24-h time point. In addition, monocrotaline resulted in activation of protein kinase B/Akt, endothelial nitric oxide synthase (eNOS), nuclear factor (NF)-κB, and increased lung tissue nitrotyrosine staining. To understand the etiological relationship between nitrosative stress and VE-cadherin suppression, we incubated cultured rat lung endothelial cells with endothelin-1, a vasoconstrictor and pro-proliferative agent in pulmonary arterial hypertension. This resulted in activation of eNOS, NF-κB, and Akt, in addition to induction of Snail, downregulation of VE-cadherin, and synthesis of vimentin. These effects were blocked by eNOS inhibitor N(ω)-nitro-l-arginine methyl ester. We propose that transcriptional repression of VE-cadherin by nitrosative stress is involved in endothelial-mesenchymal transdifferentiation in experimental PH. PMID

  3. A Scallop Nitric Oxide Synthase (NOS) with Structure Similar to Neuronal NOS and Its Involvement in the Immune Defense

    PubMed Central

    Jiang, Qiufen; Zhou, Zhi; Wang, Leilei; Wang, Lingling; Yue, Feng; Wang, Jingjing; Song, Linsheng

    2013-01-01

    Background Nitric oxide synthase (NOS) is responsible for synthesizing nitric oxide (NO) from L-arginine, and involved in multiple physiological functions. However, its immunological role in mollusc was seldom reported. Methodology In the present study, an NOS (CfNOS) gene was identified from the scallop Chlamys farreri encoding a polypeptide of 1486 amino acids. Its amino acid sequence shared 50.0~54.7, 40.7~47.0 and 42.5~44.5% similarities with vertebrate neuronal (n), endothelial (e) and inducible (i) NOSs, respectively. CfNOS contained PDZ, oxygenase and reductase domains, which resembled those in nNOS. The CfNOS mRNA transcripts expressed in all embryos and larvae after the 2-cell embryo stage, and were detectable in all tested tissues with the highest level in the gonad, and with the immune tissues hepatopancreas and haemocytes included. Moreover, the immunoreactive area of CfNOS distributed over the haemocyte cytoplasm and cell membrane. After LPS, β-glucan and PGN stimulation, the expression level of CfNOS mRNA in haemocytes increased significantly at 3 h (4.0-, 4.8- and 2.7-fold, respectively, P < 0.01), and reached the peak at 12 h (15.3- and 27.6-fold for LPS and β-glucan respectively, P < 0.01) and 24 h (17.3-fold for PGN, P < 0.01). In addition, TNF-α also induced the expression of CfNOS, which started to increase at 1 h (5.2-fold, P < 0.05) and peaked at 6 h (19.9-fold, P < 0.01). The catalytic activity of the native CfNOS protein was 30.3 ± 0.3 U mgprot-1, and it decreased significantly after the addition of the selective inhibitors of nNOS and iNOS (26.9 ± 0.4 and 29.3 ± 0.1 U mgprot-1, respectively, P < 0.01). Conclusions These results suggested that CfNOS, with identical structure with nNOS and similar enzymatic characteristics to nNOS and iNOS, played the immunological role of iNOS to be involved in the scallop immune defense against PAMPs and TNF-α. PMID:23922688

  4. 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. PMID:26976926

  5. Caveolin-1 mediates endotoxin inhibition of endothelin-1-induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells.

    PubMed

    Kwok, Willson; Lee, Sang Ho; Culberson, Cathy; Korneszczuk, Katarzyna; Clemens, Mark G

    2009-11-01

    Endothelin-1 (ET-1) plays a key role in the regulation of endothelial nitric oxide synthase (eNOS) activation in liver sinusoidal endothelial cells (LSECs). In the presence of endotoxin, an increase in caveolin-1 (Cav-1) expression impairs ET-1/eNOS signaling; however, the molecular mechanism is unknown. The objective of this study was to investigate the molecular mechanism of Cav-1 in the regulation of LPS suppression of ET-1-mediated eNOS activation in LSECs by examining the effect of caveolae disruption using methyl-beta-cyclodextrin (CD) and filipin. Treatment with 5 mM CD for 30 min increased eNOS activity (+255%, P < 0.05). A dose (0.25 microg/ml) of filipin for 30 min produced a similar effect (+111%, P < 0.05). CD induced the perinuclear localization of Cav-1 and eNOS and stimulated NO production in the same region. Readdition of 0.5 mM cholesterol to saturate CD reversed these effects. Both the combined treatment with CD and ET-1 (CD + ET-1) and with filipin and ET-1 stimulated eNOS activity; however, pretreatment with endotoxin (LPS) abrogated these effects. Following LPS pretreatment, CD + ET-1 failed to stimulate eNOS activity (+51%, P > 0.05), which contributed to the reduced levels of eNOS-Ser1177 phosphorylation and eNOS-Thr495 dephosphorylation, the LPS/CD-induced overexpression and translocation of Cav-1 in the perinuclear region, and the increased perinuclear colocalization of eNOS with Cav-1. These results supported the hypothesis that Cav-1 mediates the action of endotoxin in suppressing ET-1-mediated eNOS activation and demonstrated that the manipulation of caveolae produces significant effects on ET-1-mediated eNOS activity in LSECs. PMID:20501440

  6. Selective Estrogen Receptor Modulators

    PubMed Central

    2016-01-01

    Selective estrogen receptor modulators (SERMs) are now being used as a treatment for breast cancer, osteoporosis and postmenopausal symptoms, as these drugs have features that can act as an estrogen agonist and an antagonist, depending on the target tissue. After tamoxifen, raloxifene, lasofoxifene and bazedoxifene SERMs have been developed and used for treatment. The clinically decisive difference among these drugs (i.e., the key difference) is their endometrial safety. Compared to bisphosphonate drug formulations for osteoporosis, SERMs are to be used primarily in postmenopausal women of younger age and are particularly recommended if there is a family history of invasive breast cancer, as their use greatly reduces the incidence of this type of cancer in women. Among the above mentioned SERMs, raloxifene has been widely used in prevention and treatment of postmenopausal osteoporosis and vertebral compression fractures, and clinical studies are now underway to test the comparative advantages of raloxifene with those of bazedoxifene, a more recently developed SERM. Research on a number of adverse side effects of SERM agents is being performed to determine the long-term safety of this class of compouds for treatment of osteoporosis. PMID:27559463

  7. Estrogen Receptor Agonists and Antagonists in the Yeast Estrogen Bioassay.

    PubMed

    Wang, Si; Bovee, Toine F H

    2016-01-01

    Cell-based bioassays can be used to predict the eventual biological activity of a substance on a living organism. In vitro reporter gene bioassays are based on recombinant vertebrate cell lines or yeast strains and especially the latter are easy-to-handle, cheap, and fast. Moreover, yeast cells do not express estrogen, androgen, progesterone or glucocorticoid receptors, and are thus powerful tools in the development of specific reporter gene systems that are devoid of crosstalk from other hormone pathways. This chapter describes our experience with an in-house developed RIKILT yeast estrogen bioassay for testing estrogen receptor agonists and antagonists, focusing on the applicability of the latter. PMID:26585147

  8. NOSTRIN: A protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase

    PubMed Central

    Zimmermann, Kirstin; Opitz, Nils; Dedio, Jürgen; Renné, Christoph; Müller-Esterl, Werner; Oess, Stefanie

    2002-01-01

    Activity and localization of endothelial nitric oxide synthase (eNOS) is regulated in a remarkably complex fashion, yet the complex molecular machinery mastering stimulus-induced eNOS translocation and trafficking is poorly understood. In a search by the yeast two-hybrid system using the eNOS oxygenase domain as bait, we have identified a previously uncharacterized eNOS-interacting protein, dubbed NOSTRIN (for eNOS traffic inducer). NOSTRIN contains a single polypeptide chain of 506-aa residues of 58 kDa with an N-terminal cdc15 domain and a C-terminal SH3 domain. NOSTRIN mRNA is abundant in highly vascularized tissues such as placenta, kidney, lung, and heart, and NOSTRIN protein is expressed in vascular endothelial cells. Coimmunoprecipitation experiments demonstrated the eNOS–NOSTRIN interaction in vitro and in vivo, and NOSTRIN's SH3 domain was essential and sufficient for eNOS binding. NOSTRIN colocalized extensively with eNOS at the plasma membrane of confluent human umbilical venous endothelial cells and in punctate cytosolic structures of CHO-eNOS cells. NOSTRIN overexpression induced a profound redistribution of eNOS from the plasma membrane to vesicle-like structures matching the NOSTRIN pattern and at the same time led to a significant inhibition of NO release. We conclude that NOSTRIN contributes to the intricate protein network controlling activity, trafficking, and targeting of eNOS. PMID:12446846

  9. Nitric oxide modulates lipopolysaccharide-induced endothelial platelet endothelial cell adhesion molecule expression via interleukin-10.

    PubMed

    Hebeda, C B; Teixeira, S A; Tamura, E K; Muscará, M N; de Mello, S B V; Markus, R P; Farsky, S H P

    2011-08-01

    We have shown previously that nitric oxide (NO) controls platelet endothelial cell adhesion molecule (PECAM-1) expression on both neutrophils and endothelial cells under physiological conditions. Here, the molecular mechanism by which NO regulates lipopolysaccharide (LPS)-induced endothelial PECAM-1 expression and the role of interleukin (IL)-10 on this control was investigated. For this purpose, N-(G)-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg/day for 14 days dissolved in drinking water) was used to inhibit both constitutive (cNOS) and inducible nitric oxide (iNOS) synthase activities in LPS-stimulated Wistar rats (5 mg/kg, intraperitoneally). This treatment resulted in reduced levels of serum NO. Under this condition, circulating levels of IL-10 was enhanced, secreted mainly by circulating lymphocytes, dependent on transcriptional activation, and endothelial PECAM-1 expression was reduced independently on reduced gene synthesis. The connection between NO, IL-10 and PECAM-1 expression was examined by incubating LPS-stimulated (1 µg/ml) cultured endothelial cells obtained from naive rats with supernatant of LPS-stimulated lymphocytes, which were obtained from blood of control or L-NAME-treated rats. Supernatant of LPS-stimulated lymphocytes obtained from L-NAME-treated rats, which contained higher levels of IL-10, reduced LPS-induced PECAM-1 expression by endothelial cells, and this reduction was reversed by adding the anti-IL-10 monoclonal antibody. Therefore, an association between NO, IL-10 and PECAM-1 was found and may represent a novel mechanism by which NO controls endothelial cell functions. PMID:21564091

  10. 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

  11. Estrogen receptor signaling during vertebrate development

    PubMed Central

    Bondesson, Maria; Hao, Ruixin; Lin, Chin-Yo; Williams, Cecilia; Gustafsson, Jan-Åke

    2014-01-01

    Estrogen receptors are expressed and their cognate ligands produced in all vertebrates, indicative of important and conserved functions. Through evolution estrogen has been involved in controlling reproduction, affecting both the development of reproductive organs and reproductive behavior. This review broadly describes the synthesis of estrogens and the expression patterns of aromatase and the estrogen receptors, in relation to estrogen functions in the developing fetus and child. We focus on the role of estrogens for development of reproductive tissues, as well as non-reproductive effects on the developing brain. We collate data from human, rodent, bird and fish studies and highlight common and species-specific effects of estrogen signaling on fetal development. Morphological malformations originating from perturbed estrogen signaling in estrogen receptor and aromatase knockout mice are discussed, as well as the clinical manifestations of rare estrogen receptor alpha and aromatase gene mutations in humans. PMID:24954179

  12. Endothelial cell expression of haemoglobin α regulates nitric oxide signalling.

    PubMed

    Straub, Adam C; Lohman, Alexander W; Billaud, Marie; Johnstone, Scott R; Dwyer, Scott T; Lee, Monica Y; Bortz, Pamela Schoppee; Best, Angela K; Columbus, Linda; Gaston, Benjamin; Isakson, Brant E

    2012-11-15

    Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signalling in many cell systems. For example, endothelial NOS controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone, but the regulation of these processes is not adequately explained by simple NO diffusion from endothelium to smooth muscle. Here we report a new model for the regulation of NO signalling by demonstrating that haemoglobin (Hb) α (encoded by the HBA1 and HBA2 genes in humans) is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to Hb α and is abrogated by its genetic depletion. Mechanistically, endothelial Hb α haem iron in the Fe(3+) state permits NO signalling, and this signalling is shut off when Hb α is reduced to the Fe(2+) state by endothelial cytochrome b5 reductase 3 (CYB5R3, also known as diaphorase 1). Genetic and pharmacological inhibition of CYB5R3 increases NO bioactivity in small arteries. These data reveal a new mechanism by which the regulation of the intracellular Hb α oxidation state controls NOS signalling in non-erythroid cells. This model may be relevant to haem-containing globins in a broad range of NOS-containing somatic cells. PMID:23123858

  13. Aging and vascular endothelial function in humans

    PubMed Central

    SEALS, Douglas R.; JABLONSKI, Kristen L.; DONATO, Anthony J.

    2012-01-01

    Advancing age is the major risk factor for the development of CVD (cardiovascular diseases). This is attributable, in part, to the development of vascular endothelial dysfunction, as indicated by reduced peripheral artery EDD (endothelium-dependent dilation) in response to chemical [typically ACh (acetylcholine)] or mechanical (intravascular shear) stimuli. Reduced bioavailability of the endothelium-synthesized dilating molecule NO (nitric oxide) as a result of oxidative stress is the key mechanism mediating reduced EDD with aging. Vascular oxidative stress increases with age as a consequence of greater production of reactive oxygen species (e.g. superoxide) without a compensatory increase in antioxidant defences. Sources of increased superoxide production include up-regulation of the oxidant enzyme NADPH oxidase, uncoupling of the normally NO-producing enzyme, eNOS (endothelial NO synthase) (due to reduced availability of the cofactor tetrahydrobiopterin) and increased mitochondrial synthesis during oxidative phosphorylation. Increased bioactivity of the potent endothelial-derived constricting factor ET-1 (endothelin-1), reduced endothelial production of/responsiveness to dilatory prostaglandins, the development of vascular inflammation, formation of AGEs (advanced glycation end-products), an increased rate of endothelial apoptosis and reduced expression of oestrogen receptor α (in postmenopausal females) also probably contribute to impaired EDD with aging. Several lifestyle and biological factors modulate vascular endothelial function with aging, including regular aerobic exercise, dietary factors (e.g. processed compared with non-processed foods), body weight/fatness, vitamin D status, menopause/oestrogen deficiency and a number of conventional and non-conventional risk factors for CVD. Given the number of older adults now and in the future, more information is needed on effective strategies for the prevention and treatment of vascular endothelial aging. PMID

  14. Endothelial cell nitric oxide production in acute chest syndrome.

    PubMed

    Hammerman, S I; Klings, E S; Hendra, K P; Upchurch, G R; Rishikof, D C; Loscalzo, J; Farber, H W

    1999-10-01

    Acute chest syndrome (ACS) is the most common form of acute pulmonary disease associated with sickle cell disease. To investigate the possibility that alterations in endothelial cell (EC) production and metabolism of nitric oxide (NO) products might be contributory, we measured NO products from cultured pulmonary EC exposed to red blood cells and/or plasma from sickle cell patients during crisis. Exposure to plasma from patients with ACS caused a 5- to 10-fold increase in S-nitrosothiol (RSNO) and a 7- to 14-fold increase in total nitrogen oxide (NO(x)) production by both pulmonary arterial and microvascular EC. Increases occurred within 2 h of exposure to plasma in a concentration-dependent manner and were associated with increases in endothelial nitric oxide synthase (eNOS) protein and eNOS enzymatic activity, but not with changes in nitric oxide synthase (NOS) III or NOS II transcripts, inducible NOS (iNOS) protein nor iNOS enzymatic activity. RSNO and NO(x) increased whether plasma was obtained from patients with ACS or other forms of vasoocclusive crisis. Furthermore, an oxidative state occurred and oxidative metabolites of NO, particularly peroxynitrite, were produced. These findings suggest that altered NO production and metabolism to damaging oxidative molecules contribute to the pathogenesis of ACS. PMID:10516198

  15. Expression, localization, and regulation of NOS in human mast cell lines: effects on leukotriene production.

    PubMed

    Gilchrist, Mark; McCauley, Scott D; Befus, A Dean

    2004-07-15

    Nitric oxide (NO) is a potent radical produced by nitric oxide synthase (NOS) and has pleiotrophic activities in health and disease. As mast cells (MCs) play a central role in both homeostasis and pathology, we investigated NOS expression and NO production in human MC populations. Endothelial NOS (eNOS) was ubiquitously expressed in both human MC lines and skin-derived MCs, while neuronal NOS (nNOS) was variably expressed in the MC populations studied. The inducible (iNOS) isoform was not detected in human MCs. Both growth factor-independent (HMC-1) and -dependent (LAD 2) MC lines showed predominant nuclear eNOS protein localization, with weaker cytoplasmic expression. nNOS showed exclusive cytoplasmic localization in HMC-1. Activation with Ca(2+) ionophore (A23187) or IgE-anti-IgE induced eNOS phosphorylation and translocation to the nucleus and nuclear and cytoplasmic NO formation. eNOS colocalizes with the leukotriene (LT)-initiating enzyme 5-lipoxygenase (5-LO) in the MC nucleus. The NO donor, S-nitrosoglutathione (SNOG), inhibited, whereas the NOS inhibitor, N(G)-nitro-l-arginine methyl ester (L-NAME), potentiated LT release in a dose-dependent manner. Thus, human MC lines produce NO in both cytoplasmic and nuclear compartments, and endogenously produced NO can regulate LT production by MCs. PMID:15044250

  16. Targeted overexpression of endothelial nitric oxide synthase in endothelial cells improves cerebrovascular reactivity in Ins2Akita-type-1 diabetic mice.

    PubMed

    Chandra, Saurav B; Mohan, Sumathy; Ford, Bridget M; Huang, Lei; Janardhanan, Preethi; Deo, Kaiwalya S; Cong, Linlin; Muir, Eric R; Duong, Timothy Q

    2016-06-01

    Reduced bioavailability of nitric oxide due to impaired endothelial nitric oxide synthase (eNOS) activity is a leading cause of endothelial dysfunction in diabetes. Enhancing eNOS activity in diabetes is a potential therapeutic target. This study investigated basal cerebral blood flow and cerebrovascular reactivity in wild-type mice, diabetic mice (Ins2(Akita+/-)), nondiabetic eNOS-overexpressing mice (TgeNOS), and the cross of two transgenic mice (TgeNOS-Ins2(Akita+/-)) at six months of age. The cross was aimed at improving eNOS expression in diabetic mice. The major findings were: (i) Body weights of Ins2(Akita+/-) and TgeNOS-Ins2(Akita+/-) were significantly different from wild-type and TgeNOS mice. Blood pressure of TgeNOS mice was lower than wild-type. (ii) Basal cerebral blood flow of the TgeNOS group was significantly higher than cerebral blood flow of the other three groups. (iii) The cerebrovascular reactivity in the Ins2(Akita+/-) mice was significantly lower compared with wild-type, whereas that in the TgeNOS-Ins2(Akita+/-) was significantly higher compared with the Ins2(Akita+/-) and TgeNOS groups. Overexpression of eNOS rescued cerebrovascular dysfunction in diabetic animals, resulting in improved cerebrovascular reactivity. These results underscore the possible role of eNOS in vascular dysfunction in the brain of diabetic mice and support the notion that enhancing eNOS activity in diabetes is a potential therapeutic target. PMID:26661212

  17. Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling.

    PubMed Central

    García-Cardeña, G; Oh, P; Liu, J; Schnitzer, J E; Sessa, W C

    1996-01-01

    The membrane association of endothelial nitric oxide synthase (eNOS) plays an important role in the biosynthesis of nitric oxide (NO) in vascular endothelium. Previously, we have shown that in cultured endothelial cells and in intact blood vessels, eNOS is found primarily in the perinuclear region of the cells and in discrete regions of the plasma membrane, suggesting trafficking of the protein from the Golgi to specialized plasma membrane structures. Here, we show that eNOS is found in Triton X-100-insoluble membranes prepared from cultured bovine aortic endothelial cells and colocalizes with caveolin, a coat protein of caveolae, in cultured bovine lung microvascular endothelial cells as determined by confocal microscopy. To examine if eNOS is indeed in caveolae, we purified luminal endothelial cell plasma membranes and their caveolae directly from intact, perfused rat lungs. eNOS is found in the luminal plasma membranes and is markedly enriched in the purified caveolae. Because palmitoylation of eNOS does not significantly influence its membrane association, we next examined whether this modification can affect eNOS targeting to caveolae. Wild-type eNOS, but not the palmitoylation mutant form of the enzyme, colocalizes with caveolin on the cell surface in transfected NIH 3T3 cells, demonstrating that palmitoylation of eNOS is necessary for its targeting into caveolae. These data suggest that the subcellular targeting of eNOS to caveolae can restrict NO signaling to specific targets within a limited microenvironment at the cell surface and may influence signal transduction through caveolae. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692835

  18. Phosphorylation inactivation of endothelial nitric oxide synthesis in pulmonary arterial hypertension.

    PubMed

    Ghosh, Sudakshina; Gupta, Manveen; Xu, Weiling; Mavrakis, Deloris A; Janocha, Allison J; Comhair, Suzy A A; Haque, Mohammad Mahfuzul; Stuehr, Dennis J; Yu, Jun; Polgar, Peter; Naga Prasad, Sathyamangla V; Erzurum, Serpil C

    2016-06-01

    The impairment of vasodilator nitric oxide (NO) production is well accepted as a typical marker of endothelial dysfunction in vascular diseases, including in the pathophysiology of pulmonary arterial hypertension (PAH), but the molecular mechanisms accounting for loss of NO production are unknown. We hypothesized that low NO production by pulmonary arterial endothelial cells in PAH is due to inactivation of NO synthase (eNOS) by aberrant phosphorylation of the protein. To test the hypothesis, we evaluated eNOS levels, dimerization, and phosphorylation in the vascular endothelial cells and lungs of patients with PAH compared with controls. In mechanistic studies, eNOS activity in endothelial cells in PAH lungs was found to be inhibited due to phosphorylation at T495. Evidence pointed to greater phosphorylation/activation of protein kinase C (PKC) α and its greater association with eNOS as the source of greater phosphorylation at T495. The presence of greater amounts of pT495-eNOS in plexiform lesions in lungs of patients with PAH confirmed the pathobiological mechanism in vivo. Transfection of the activating mutation of eNOS (T495A/S1177D) restored NO production in PAH cells. Pharmacological blockade of PKC activity by β-blocker also restored NO formation by PAH cells, identifying one mechanism by which β-blockers may benefit PAH and cardiovascular diseases through recovery of endothelial functions. PMID:27130529

  19. Estrogen Signaling in Metabolic Inflammation

    PubMed Central

    Monteiro, Rosário; Teixeira, Diana; Calhau, Conceição

    2014-01-01

    There is extensive evidence supporting the interference of inflammatory activation with metabolism. Obesity, mainly visceral obesity, is associated with a low-grade inflammatory state, triggered by metabolic surplus where specialized metabolic cells such as adipocytes activate cellular stress initiating and sustaining the inflammatory program. The increasing prevalence of obesity, resulting in increased cardiometabolic risk and precipitating illness such as cardiovascular disease, type 2 diabetes, fatty liver, cirrhosis, and certain types of cancer, constitutes a good example of this association. The metabolic actions of estrogens have been studied extensively and there is also accumulating evidence that estrogens influence immune processes. However, the connection between these two fields of estrogen actions has been underacknowledged since little attention has been drawn towards the possible action of estrogens on the modulation of metabolism through their anti-inflammatory properties. In the present paper, we summarize knowledge on the modification inflammatory processes by estrogens with impact on metabolism and highlight major research questions on the field. Understanding the regulation of metabolic inflammation by estrogens may provide the basis for the development of therapeutic strategies to the management of metabolic dysfunctions. PMID:25400333

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

    PubMed

    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

  1. 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

  2. Estrogens, breast cancer, and intestinal flora.

    PubMed

    Gorbach, S L

    1984-01-01

    Epidemiologic evidence has linked diet to breast cancer, with the highest cancer rates observed in women who eat a high fat-low fiber diet. There is also substantial information, both clinical and experimental, that implicates estrogens in the etiology of breast cancer. A recent study from our laboratory has shown that diet influences levels of estrogens, and the main mechanism is metabolism of estrogens in the intestine. The intestinal microflora plays a key role in the enterohepatic circulation of estrogens by deconjugating bound estrogens that appear in the bile, thereby permitting the free hormones to be reabsorbed. By suppressing the microflora with antibiotic therapy, fecal estrogens increase and urinary estrogens decrease, changes indicating diminished intestinal reabsorption. A low fat-high fiber diet is associated with similar findings-high fecal estrogens and low urinary estrogens. It appears that the microflora plays a key role in the metabolism of female sex hormones. PMID:6326245

  3. Resveratrol Ameliorates High Glucose and High-Fat/Sucrose Diet-Induced Vascular Hyperpermeability Involving Cav-1/eNOS Regulation

    PubMed Central

    Peng, Xiao lin; Qu, Wei; Wang, Lin zhi; Huang, Bin qing; Ying, Chen jiang; Sun, Xiu fa; Hao, Li ping

    2014-01-01

    Vascular endothelial hyperpermeability is one of the manifestations of endothelial dysfunction. Resveratrol (Res) is considered to be beneficial in protecting endothelial function. However, currently, the exact protective effect and involved mechanisms of Res on endothelial dysfunction-hyperpermeability have not been completely clarified. The aim of present study is to investigate the effects of Res on amelioration of endothelial hyperpermeability and the role of caveolin-1 (Cav-1)/endothelial nitric oxide synthase (eNOS) pathway. Adult male Wistar rats were treated with a normal or high-fat/sucrose diet (HFS) with or without Res for 13 weeks. HFS and in vitro treatment with high glucose increased hyperpermeability in rat aorta, heart, liver and kidney and cultured bovine aortic endothelial cells (BAECs), respectively, which was attenuated by Res treatment. Application of Res reversed the changes in eNOS and Cav-1 expressions in aorta and heart of rats fed HFS and in BAECs incubated with high glucose. Res stimulated the formation of NO inhibited by high glucose in BAECs. Beta-Cyclodextrin (β-CD), caveolae inhibitor, showed the better beneficial effect than Res alone to up-regulate eNOS phosphorylative levels, while NG-Nitro-77 L-arginine methyl ester (L-NAME), eNOS inhibitor, had no effect on Cav-1 expression. Our studies suggested that HFS and in vitro treatment with high glucose caused endothelial hyperpermeability, which were ameliorated by Res at least involving Cav-1/eNOS regulation. PMID:25419974

  4. Baicalin attenuates angiotensin II-induced endothelial dysfunction.

    PubMed

    Wei, Xiling; Zhu, Xingyu; Hu, Nan; Zhang, Xiuqin; Sun, Tianjiao; Xu, Jiyang; Bian, Xiaohong

    2015-09-11

    Angiotensin II (Ang II) has been shown to activate multiple downstream pathways resulting in endothelial dysfunction and oxidative stress. Baicalin, a natural flavone, exerts anti-oxidant and anti-apoptotic effects in cardiovascular diseases. In the present study, we hypothesized that baicalin has beneficial effects in Ang II-induced endothelial cells injury. Here, we shown that baicalin improved endothelial fuction impaired by Ang II through promoting endothelial-dependent vasodilation and suppressing the apoptosis of HUVECs in which baicalin decreased the expression of bax and cleaved caspase-3, and increased bcl-2 expression. Additionally, baicalin significantly conversed Ang II to angiotensin-1-7 [Ang-(1-7)] by activating angiotensin-converting enzyme 2 (ACE2) and Mas receptor mRNA expression and protein expression. Moreover, treatment with baicalin significantly reduced cell oxidative damage induced by Ang II through MDA/ROS decrease and NO/T-AOC increase. This antioxidant capacity was related to the increases of PI3K, phosphor-AKT (Ser-473) and phosphor-eNOS (Ser-1177). In conclusion, our results implicate that baicalin could protect endothelial cells from Ang II-induced endothelial dysfunction and oxidative stress via modulating the expression of bax, bcl-2 and cleaved caspase-3, activating ACE2/Ang-(1-7)/Mas axis and up-regulating PI3K/AKT/eNOS pathway. PMID:26239661

  5. Diverse Functions of Endothelial NO Synthases System: NO and EDH

    PubMed Central

    Godo, Shigeo

    2016-01-01

    Abstract: Endothelium-dependent relaxations are predominantly regulated by nitric oxide (NO) in large conduit arteries and by endothelium-dependent hyperpolarization (EDH) in small resistance vessels. Although the nature of EDH factors varies depending on species and vascular beds, we have previously demonstrated that endothelial NO synthases (eNOS)-derived hydrogen peroxide (H2O2) is an EDH factor in animals and humans. This vessel size-dependent contribution of NO and EDH is, at least in part, attributable to the diverse roles of endothelial NOSs system; in large conduit arteries, eNOS mainly serves as a NO-generating system to elicit soluble guanylate cyclase–cyclic guanosine monophosphate-mediated relaxations, whereas in small resistance vessels, it serves as a superoxide-generating system to cause EDH/H2O2-mediated relaxations. Endothelial caveolin-1 may play an important role for the diverse roles of NOSs. Although reactive oxygen species are generally regarded harmful, the physiological roles of H2O2 have attracted much attention as accumulating evidence has shown that endothelium-derived H2O2 contributes to cardiovascular homeostasis. The diverse functions of endothelial NOSs system with NO and EDH/H2O2 could account for a compensatory mechanism in the setting of endothelial dysfunction. In this review, we will briefly summarize the current knowledge on the diverse functions of endothelial NOSs system: NO and EDH/H2O2. PMID:26647119

  6. Diverse Functions of Endothelial NO Synthases System: NO and EDH.

    PubMed

    Shimokawa, Hiroaki; Godo, Shigeo

    2016-05-01

    Endothelium-dependent relaxations are predominantly regulated by nitric oxide (NO) in large conduit arteries and by endothelium-dependent hyperpolarization (EDH) in small resistance vessels. Although the nature of EDH factors varies depending on species and vascular beds, we have previously demonstrated that endothelial NO synthases (eNOS)-derived hydrogen peroxide (H2O2) is an EDH factor in animals and humans. This vessel size-dependent contribution of NO and EDH is, at least in part, attributable to the diverse roles of endothelial NOSs system; in large conduit arteries, eNOS mainly serves as a NO-generating system to elicit soluble guanylate cyclase-cyclic guanosine monophosphate-mediated relaxations, whereas in small resistance vessels, it serves as a superoxide-generating system to cause EDH/H2O2-mediated relaxations. Endothelial caveolin-1 may play an important role for the diverse roles of NOSs. Although reactive oxygen species are generally regarded harmful, the physiological roles of H2O2 have attracted much attention as accumulating evidence has shown that endothelium-derived H2O2 contributes to cardiovascular homeostasis. The diverse functions of endothelial NOSs system with NO and EDH/H2O2 could account for a compensatory mechanism in the setting of endothelial dysfunction. In this review, we will briefly summarize the current knowledge on the diverse functions of endothelial NOSs system: NO and EDH/H2O2. PMID:26647119

  7. The NOS Challenge

    ERIC Educational Resources Information Center

    Quigley, Cassie; Buck, Gayle; Akerson, Valarie

    2011-01-01

    "The picture of a scientist is me!" exclaims first grader Kendra during a nature of science (NOS) lesson. She drew a picture of a scientist and explained that she was going to be a scientist when she grew up because she "loved to observe like a scientist." Kendra's experience was a part of a 30-day unit designed specifically for first graders.…

  8. [Progress of non-genomic action of estrogen and its impact on female reproduction].

    PubMed

    Yu, Lin-Lin; Yuan, Dong-Zhi; Zhang, Shi-Mao; Yue, Li-Min

    2016-08-25

    Estrogen is one of the steroid hormones. Besides the genomic action mediated by its intracellular receptor on target cells, there is now increasing body of evidence indicating that estrogen also has non-genomic action. For the non-genomic action, estrogen binds to its receptor on cell membrane, subsequently rapidly activates various intracellular signaling pathways, such as PLC/Ca(2+), ERK/MAPK, cAMP-PKA, PI3K-AKT-NOS, and finally induces biological effects. The non-genomic effects of estrogen on physiologic and pathologic processes have been found in many tissues within the reproductive, nervous and cardiovascular systems and bone etc. In reproductive system, it has been demonstrated that estrogen plays important roles in follicle development, fertilization and embryo implantation, and it is involved in the genesis and development of genital tract tumors and breast cancer. In this review, we focus on the general characteristics of non-genomic action of estrogen, its main nonnuclear signaling pathways and physiological and pathological significance, especially its influences in female reproductive functions. PMID:27546514

  9. Effective treatment of vascular endothelial growth factor refractory hindlimb ischemia by a mutant endothelial nitric oxide synthase gene.

    PubMed

    Qian, H S; Liu, P; Huw, L-Y; Orme, A; Halks-Miller, M; Hill, S M; Jin, F; Kretschmer, P; Blasko, E; Cashion, L; Szymanski, P; Vergona, R; Harkins, R; Yu, J; Sessa, W C; Dole, W P; Rubanyi, G M; Kauser, K

    2006-09-01

    Gene delivery of angiogenic growth factors is a promising approach for the treatment of ischemic cardiovascular diseases. However, success of this new therapeutic principle is hindered by the lack of critical understanding as to how disease pathology affects the efficiency of gene delivery and/or the downstream signaling pathways of angiogenesis. Critical limb ischemia occurs in patients with advanced atherosclerosis often exhibiting deficiency in endothelial nitric oxide production. Similar to these patients, segmental femoral artery resection progresses into severe ischemic necrosis in mice deficient in endothelial nitric oxide synthase (ecNOS-KO) as well as in balb/c mice. We used these models to evaluate the influence of severe ischemia on transfection efficiency and duration of transgene expression in the skeletal muscle following plasmid injection in combination with electroporation. Subsequently, we also explored the potential therapeutic effect of the phosphomimetic mutant of ecNOS gene (NOS1177D) using optimized delivery parameters, and found significant benefit both in ecNOS-KO and balb/c mice. Our results indicate that NOS1177D gene delivery to the ischemic skeletal muscle can be efficient to reverse critical limb ischemia in pathological settings, which are refractory to treatments with a single growth factor, such as vascular endothelial growth factor. PMID:16642030

  10. S-glutathionylation uncouples eNOS and regulates its cellular and vascular function

    PubMed Central

    Chen, Chun-An; Wang, Tse-Yao; Varadharaj, Saradhadevi; Reyes, Levy A.; Hemann, Craig; Hassan Talukder, M. A.; Chen, Yeong-Renn; Druhan, Lawrence J.; Zweier, Jay L.

    2012-01-01

    Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular function, and can generate both nitric oxide (NO) and superoxide (O2•−), which are key mediators of cellular signalling. In the presence of Ca2+/calmodulin, eNOS produces NO, endothelial-derived relaxing factor, from L-arginine (L-Arg) by means of electron transfer from NADPH through a flavin containing reductase domain to oxygen bound at the haem of an oxygenase domain, which also contains binding sites for tetrahydrobiopterin (BH4) and L-Arg1–3. In the absence of BH4, NO synthesis is abrogated and instead O2•− is generated4–7. While NOS dysfunction occurs in diseases with redox stress, BH4 repletion only partly restores NOS activity and NOS-dependent vasodilation7. This suggests that there is an as yet unidentified redox-regulated mechanism controlling NOS function. Protein thiols can undergo S-glutathionylation, a reversible protein modification involved in cellular signalling and adaptation8,9. Under oxidative stress, S-glutathionylation occurs through thiol–disulphide exchange with oxidized glutathione or reaction of oxidant-induced protein thiyl radicals with reduced glutathione10,11. Cysteine residues are critical for the maintenance of eNOS function12,13; we therefore speculated that oxidative stress could alter eNOS activity through S-glutathionylation. Here we show that S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in O2•− generation primarily from the reductase, in which two highly conserved cysteine residues are identified as sites of S-glutathionylation and found to be critical for redox-regulation of eNOS function. We show that eNOS S-glutathionylation in endothelial cells, with loss of NO and gain of O2•− generation, is associated with impaired endothelium-dependent vasodilation. In hypertensive vessels, eNOS S-glutathionylation is increased with impaired endothelium-dependent vasodilation that is restored by thiol

  11. Asymmetric Dimethylarginine, Endothelial Dysfunction and Renal Disease

    PubMed Central

    Aldámiz-Echevarría, Luis; Andrade, Fernando

    2012-01-01

    l-Arginine (Arg) is oxidized to l-citrulline and nitric oxide (NO) by the action of endothelial nitric oxide synthase (NOS). In contrast, protein-incorporated Arg residues can be methylated with subsequent proteolysis giving rise to methylarginine compounds, such as asymmetric dimethylarginine (ADMA) that competes with Arg for binding to NOS. Most ADMA is degraded by dimethylarginine dimethyaminohydrolase (DDAH), distributed widely throughout the body and regulates ADMA levels and, therefore, NO synthesis. In recent years, several studies have suggested that increased ADMA levels are a marker of atherosclerotic change, and can be used to assess cardiovascular risk, consistent with ADMA being predominantly absorbed by endothelial cells. NO is an important messenger molecule involved in numerous biological processes, and its activity is essential to understand both pathogenic and therapeutic mechanisms in kidney disease and renal transplantation. NO production is reduced in renal patients because of their elevated ADMA levels with associated reduced DDAH activity. These factors contribute to endothelial dysfunction, oxidative stress and the progression of renal damage, but there are treatments that may effectively reduce ADMA levels in patients with kidney disease. Available data on ADMA levels in controls and renal patients, both in adults and children, also are summarized in this review. PMID:23109853

  12. 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

  13. Relationship between estrogen receptor-binding and estrogenic activities of environmental estrogens and suppression by flavonoids.

    PubMed

    Han, Dal-Ho; Denison, Michael S; Tachibana, Hirofumi; Yamada, Koji

    2002-07-01

    In this study, we investigated the estrogenic activity of environmental estrogens by a competition binding assay using a human recombinant estrogens receptor (hERbeta) and by a proliferation assay using MCF-7 cells and a sulforhodamine-B assay. In the binding assay, pharmaceuticals had a stronger binding activity to hERbeta than that of some phytoestrogens (coumestrol, daidzein, genistein, luteolin, chrysin, flavone, and naringenin) or industrial chemicals, but phytoestrogens such as coumestrol had a binding activity as strong as pharmaceuticals such as 17alpha-ethynylestradiol (EE), tamoxifen (Tam), and mestranol. In the proliferation assay, pharmaceuticals such as diethylstilbestrol, EE, Tam, and clomiphene, and industrial chemicals such as 4-nonylphenol, bisphenol A, and 4-dihydroxybiphenyl had a proliferation-stimulating activity as strong as 17beta-estradiol (ES). In addition, we found that phytoestrogens such as coumestrol, daidzein, luteolin, and quercetin exerted a proliferation stimulating activity as strong as ES. Furthermore, we examined the suppression of proliferation-stimulating activity, induced by environmental estrogen, by flavonoids, such as daidzein, genistein, quercetin, and luteolin, and found that these flavonoids suppressed the induction of the proliferation-stimulating activity of environmental estrogens. The suppressive effect of flavonoids suggests that these compounds have anti-estrogenic and anti-cancer activities. PMID:12224631

  14. Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells

    PubMed Central

    Jin, Benjamin Y.; Sartoretto, Juliano L.; Gladyshev, Vadim N.; Michel, Thomas

    2009-01-01

    Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H2O2 is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H2O2 treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC50 for H2O2-promoted phosphorylation of AMPK is 65 ± 15 μM, within the physiological range of cellular H2O2 concentrations. The Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKβ) inhibitor STO-609 abolishes H2O2-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKβ abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H2O2 biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H2O2 generation, which is blocked by PEG-catalase. eNOS−/− mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS−/− mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKβ is critically involved in mediating the phosphorylation of AMPK promoted by H2O2 in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H2O2 generation in endothelial cells. PMID:19805165

  15. Modulation of endothelial nitric oxide by plant-derived products.

    PubMed

    Schmitt, Christoph A; Dirsch, Verena M

    2009-09-01

    Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is recognised as a central anti-inflammatory and anti-atherogenic principle in the vasculature. Decreased availability of NO in the vasculature promotes the progression of cardiovascular diseases. Epidemiological and clinical studies have demonstrated that a growing list of natural products, as components of the daily diet or phytomedical preparations, may improve vascular function by enhancing NO bioavailability. In this article we first outline common pathways modulating endothelial NO production or bioavailability to provide a basis for subsequent mechanistic discussions. Then we comprehensively review natural products and plant extracts known to positively influence eNOS activity and/or endothelial function in vitro or in vivo. We will discuss red wine, highlighting polyphenols, oligomeric procyanidins (OPC) and resveratrol as modulators of endothelial NO production. Other dietary products and their active components known to activate eNOS include cocoa (OPC and its monomer (-)-epicatechin), pomegranates (polyphenols), black and green tea (flavanoids, especially epigallocatechin gallate), olive oil (oleic acid and polyphenols), soy (genistein), and quercetin, one of the most abundant flavonoids in plants. In addition, phytomedical preparations made from ginkgo, hawthorn and ginseng, as well as formulations used in traditional Chinese Medicine, have been shown to affect endothelial NO production. Recurring phytochemical patterns among active fractions and purified compounds are discussed. In summary, there is increasing evidence that several single natural products and plant extracts influence endothelial NO production. Identification of such compounds and characterisation of their cellular actions may increase our knowledge of the regulation of endothelial NO production and could provide valuable clues for the prevention or treatment of cardiovascular diseases. PMID:19497380

  16. Glucocorticoids and estrogens modulate the NF-κB pathway differently in the micro- and macrovasculature.

    PubMed

    Edgar, Abarca-Rojano; Judith, Pacheco-Yépez; Elisa, Drago-Serrano Maria; Rafael, Campos-Rodríguez

    2013-12-01

    Estrogens and glucocorticoids have synergistic effects in the micro and macrovasculature of endothelial cells (ECs), having pro-inflammatory effects in the former and inhibiting the expression of adhesion molecules in the latter. The molecular basis of these effects in the endothelium has not yet been clarified. We postulate that the ECs of the micro- and macrovasculature have different non-genomic mechanisms that regulate levels of preexisting complexes of glucocorticoids and estrogens with their respective receptors. Since these receptors are regulated by NF-κB, their expression could be critical to the activation of a pro- or anti-inflammatory response. In the macrovasculature the synergistic effects of estrogens and glucocorticoids on ECs may be through the inhibition of NF-κB, leading to the inhibition of the expression of inflammatory molecules. It seems likely that glucocorticoid-receptor and estrogen-receptor complexes directly bind to NF-κB proteins in the macrovasculature, resulting in the inhibition of an excessive proinflammatory response. Further insights into these processes may help clarify the role of the endothelial cells of different vascular beds during the inflammatory response and chronic inflammation, and thus contribute to the design of more effective therapeutic strategies for the prevention of diseases related to inflammation, including atherosclerosis, systemic lupus erythematosus and rheumatoid arthritis. PMID:24199951

  17. Estrogenic profile on a water-soluble estrogen, estrazinol hydrobromide.

    PubMed

    Rassaert, C L; DiPasquale, G; Giannina, T; Manning, J P; Meli, A

    1973-01-01

    The estrogenic properties of estrazinol hydrobromide (EZ), a water-soluble estrogen, were compared with those of Premarin (PR), another water-soluble estrogen preparation consisting of conjugated equine estrogens. Estradiol-17beta, estra-1,3,5(10)-triene-3,17beta-diol (E), and ethinyl estradiol, 17alpha-ethinyl-1,3,5 (10)-estratriene-3,17beta-diol (EE) were used as reference standards. Subcutaneous progesterone (400 mcg) given to rabbits primed with comparable subcutaneous doses of either E or EE produced full secretory changes of the endometrium, while such a transformation could not be elicited in orally primed animals regardless of the estrogen used. The biological profile or orally administered EZ was very similar to that of oral EE and different from oral PR. Howerver, the oral EZ-induced morphological changes of the rabbit endometrium appeared somewhat different from those produced by oral EE. The findings indicated that following oral administration, EZ-induced endometrial transformation is more "normal" and/or adequate than the changes produced by either EE or PR. PMID:4368700

  18. Formononetin promotes angiogenesis through the estrogen receptor alpha-enhanced ROCK pathway

    PubMed Central

    Li, Shang; Dang, Yuanye; Zhou, Xuelin; Huang, Bin; Huang, Xiaohui; Zhang, Zherui; Kwan, Yiu Wa; Chan, Shun Wan; Leung, George Pak Heng; Lee, Simon Ming Yuen; Hoi, Maggie Pui Man

    2015-01-01

    Formononetin is an isoflavone that has been shown to display estrogenic properties and induce angiogenesis activities. However, the interrelationship between the estrogenic properties and angiogenesis activities of formononetin are not well defined. In the present study, docking and enzymatic assay demonstrated that formononetin displayed direct binding to the ligand-binding domain (LBD) of estrogen receptor alpha (ERα) with an agonistic property. Results from Human Umbilical Vein Endothelial Cells (HUVEC) by using real-time migration xCELLigence system, immunofluorescence and western blotting provided strong evidences of formononetin induced endothelial cell migration and dramatic actin cytoskeleton spatial modification through ERα-enhanced-ROCK-II/MMP2/9 signaling pathways. In addition, results from co-immunoprecipitation suggested formononetin induced cell migration via recruiting of ERα/ROCK-II activated complex formation. More interestingly, in zebrafish embryo we observed that formononetin significantly promoted angiogenic sproutings in the subintestinal vessels (SIVs) that could be completely abolished by ROCK inhibitor. In this study, we elucidated the underlying mechanisms that formononetin produced proangiogenesis effects through an ERα-enhanced ROCK-II signaling pathways. Results from the present study also expand our knowledge about the enigmatic underlying mechanisms of phytoestrogenic compounds in the promotion of angiogenesis in relation to ERα and ROCK interaction in endothelial cells and their relationship with actin assembly and cell migration. PMID:26568398

  19. Transcriptional regulation of endothelial nitric oxide synthase expression in uterine artery endothelial cells by c-Jun/AP-1

    PubMed Central

    Qian, Xiao-Xian; Mata-Greenwood, Eugenia; Liao, Wu Xiang; Zhang, Honghai; Zheng, Jing; Chen, Dong-bao

    2007-01-01

    Despite extensive studies have shown that increased endothelial nitric oxide synthase (NOS3) expression in the uterine artery endothelial cells (UAEC) plays a key role in uterine vasodilatation, the molecular mechanism controlling NOS3 expression in UAEC is unknown. According to the sheep NOS3 promoter sequence isolated in our laboratory, we hypothesize that the activator protein-1 (AP-1) site in the proximal sheep NOS3 promoter (TGAGTCA, -682 to -676) is important for NOS3 expression. We developed a c-Jun adenoviral expression system to overexpress c-Jun protein into UAEC to investigate the effects of c-Jun/AP-1 on NOS3 expression. Basal levels of c-Jun protein and mRNA were detected in UAEC. C-Jun protein was overexpressed in a concentration and time-dependent fashion in UAEC infected with sense c-Jun (S-c-Jun), but not sham and antisense c-Jun (A-c-Jun) adenoviruses. Infection with S-c-Jun adenovirus (25 MOI, multiplicity of infection) resulted in efficient c-Jun protein overexpression in UAEC up to 3 days. In S-c-Jun, but not sham and A-c-Jun adenovirus infected UAEC, NOS3 mRNA and protein levels were increased (P<0.05) compared to noninfected controls. Increased NOS3 expression was associated with increased total NOS activity. Transient transfections showed that c-Jun overexpression augmented the transactivation of the sheep NOS3 promoter-driven luciferase/reporter constructs with the AP-1 site but not of deletion constructs without the AP-1 site. When the AP-1 site was mutated, c-Jun failed to trans-activate the sheep NOS3 promoter. AP-1 DNA binding activity also increased in c-Jun overexpressed UAEC. Lastly, the pharmacological AP-1 activator phorbol myristate acetate increased AP-1 binding, trans-activated the wild-type but not the AP-1 mutant NOS3 promoter and dose-dependently stimulated UAEC NOS3 and c-Jun protein expression. Hence, our data show that c-Jun/AP-1 regulates NOS3 transcription involving the proximal AP-1 site in the 5′-regulatory region of

  20. Effects of pinostrobin on estrogen metabolism and estrogen receptor transactivation.

    PubMed

    Le Bail, J C; Aubourg, L; Habrioux, G

    2000-08-01

    The interaction between the estrogen receptor and 5-hydroxy-7-methoxyflavanone (pinostrobin) was studied in the presence or absence of estradiol or dehydroepiandrosterone sulfate (DHEAS), respectively, using a stably transfected human breast cancer cell line (MVLN). We also evaluated its action on the proliferation in estrogen-dependent (MCF-7) human breast cancer cells in the same conditions than the estrogen receptor assay. On the other hand pinostrobin was evaluated for their effects on the human placental aromatase, 3beta-hydroxysteroid dehydrogenase Delta(4)/Delta(5) isomerase and 17beta-hydroxysteroid dehydrogenase activities. Pinostrobin did not possess antiestrogenic activity but presented anti-aromatase activity and decreased the growth of MCF-7 cells induced by DHEAS and E(2). This study provides particularly evidence of the potential biological interest of pinostrobin among the flavonoids. PMID:10840157

  1. Bidirectional cross-regulation between the endothelial nitric oxide synthase and β-catenin signalling pathways

    PubMed Central

    Warboys, Christina M.; Chen, Nan; Zhang, Qiuping; Shaifta, Yasin; Vanderslott, Genevieve; Passacquale, Gabriella; Hu, Yanhua; Xu, Qingbo; Ward, Jeremy P.T.; Ferro, Albert

    2014-01-01

    Aims β-catenin has been shown to be regulated by inducible nitric oxide synthase (NOS) in endothelial cells. We investigated here whether β-catenin interacts with and regulates endothelial NOS (eNOS) and whether eNOS activation promotes β-catenin signalling. Methods and results We identified β-catenin as a novel eNOS binding protein in human umbilical vein endothelial cells (HUVECs) by mass spectroscopy and western blot analyses of β-catenin and eNOS immunoprecipitates. This was confirmed by in situ proximity ligation assay. eNOS activity, assessed by cGMP production and eNOS phosphorylation (Ser1177), was enhanced in β-catenin−/− mouse pulmonary endothelial cells (MPECs) relative to wild-type MPECs. eNOS activation (using adenosine, salbutamol, thrombin, or histamine), or application of an NO donor (spermine NONOate) or cGMP-analogue (8-bromo-cGMP) caused nuclear translocation of β-catenin in HUVEC as shown by western blotting of nuclear extracts. Exposure to spermine NONOate, 8-bromo-cGMP, or sildenafil (a phosphodiesterase type 5 inhibitor) also increased the expression of β-catenin-dependent transcripts, IL-8, and cyclin D1. Stimulation of wild-type MPECs with basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), spermine NONOate, 8-bromo-cGMP, or sildenafil increased tube length relative to controls in an angiogenesis assay. These responses were abrogated in β-catenin−/− MPECs, with the exception of that to bFGF which is NO-independent. In C57BL/6 mice, subcutaneous VEGF-supplemented Matrigel plugs containing β-catenin−/− MPECs exhibited reduced angiogenesis compared with plugs containing wild-type MPECs. Angiogenesis was not altered in bFGF-supplemented Matrigel. Conclusion These data reveal bidirectional cross-talk and regulation between the NO-cGMP and β-catenin signalling pathways. PMID:25062958

  2. A protective role for endothelial nitric oxide synthase in glomerulonephritis.

    PubMed

    Heeringa, Peter; Steenbergen, Eric; van Goor, Harry

    2002-03-01

    In acute glomerulonephritis (GN), increased nitric oxide (NO) production occurs, suggesting a pathophysiological role for NO in the disease process. Although NO potentially could have both toxic as well as protective effects, its exact role in the pathophysiology of GN is unclear and may depend on the NOS isoform generating NO. The protective effects of NO such as prevention of leukocyte and platelet activation and adhesion have been attributed to NO generated by endothelial nitric oxide synthase (eNOS). Evidence for a beneficial role for eNOS includes the demonstration of reduced eNOS expression in experimental models of GN as well as human biopsy specimens that is mostly likely due to endothelial cell necrosis. Reduced NO production in GN also may occur through reaction of NO with superoxide anions or the myeloperoxidase (MPO)/hypochlorous acid (HOCL) system. Further evidence has been provided by the observation that in several experimental models of GN, glomerular injury is exacerbated following treatment with non-selective NO inhibitors. Finally, the development of GN is severely aggravated in mice lacking a functional gene for eNOS as compared to wild-type mice, providing direct support for a protective role of eNOS-derived NO in acute GN. PMID:11849432

  3. ERGDB: Estrogen Responsive Genes Database.

    PubMed

    Tang, Suisheng; Han, Hao; Bajic, Vladimir B

    2004-01-01

    ERGDB is an integrated knowledge database dedicated to genes responsive to estrogen. Genes included in ERGDB are those whose expression levels are experimentally proven to be either up-regulated or down-regulated by estrogen. Genes included are identified based on publications from the PubMed database and each record has been manually examined, evaluated and selected for inclusion by biologists. ERGDB aims to be a unified gateway to store, search, retrieve and update information about estrogen responsive genes. Each record contains links to relevant databases, such as GenBank, LocusLink, Refseq, PubMed and ATCC. The unique feature of ERGDB is that it contains information on the dependence of gene reactions on experimental conditions. In addition to basic information about the genes, information for each record includes gene functional description, experimental methods used, tissue or cell type, gene reaction, estrogen exposure time and the summary of putative estrogen response elements if the gene's promoter sequence was available. Through a web interface at http://sdmc.i2r.a-star.edu.sg/ergdb/ cgi-bin/explore.pl users can either browse or query ERGDB. Access is free for academic and non-profit users. PMID:14681475

  4. Fenofibrate activates AMPK and increases eNOS phosphorylation in HUVEC

    SciTech Connect

    Murakami, Hisashi; Murakami, Ryuichiro . E-mail: ryuichi@med.nagoya-u.ac.jp; Kambe, Fukushi; Cao, Xia; Takahashi, Ryotaro; Asai, Toru; Hirai, Toshihisa; Numaguchi, Yasushi; Okumura, Kenji; Seo, Hisao; Murohara, Toyoaki

    2006-03-24

    Fenofibrate improves endothelial function by lipid-lowering and anti-inflammatory effects. Additionally, fenofibrate has been demonstrated to upregulate endothelial nitric oxide synthase (eNOS). AMP-activated protein kinase (AMPK) has been reported to phosphorylate eNOS at Ser-1177 and stimulate vascular endothelium-derived nitric oxide (NO) production. We report here that fenofibrate activates AMPK and increases eNOS phosphorylation and NO production in human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with fenofibrate increased the phosphorylation of AMPK and acetyl-CoA carboxylase. Fenofibrate simultaneously increased eNOS phosphorylation and NO production. Inhibitors of protein kinase A and phosphatidylinositol 3-kinase failed to suppress the fenofibrate-induced eNOS phosphorylation. Neither bezafibrate nor WY-14643 activated AMPK in HUVEC. Furthermore, fenofibrate activated AMPK without requiring any transcriptional activities. These results indicate that fenofibrate stimulates eNOS phosphorylation and NO production through AMPK activation, which is suggested to be a novel characteristic of this agonist and unrelated to its effects on peroxisome proliferator-activated receptor {alpha}.

  5. 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

  6. Endothelial nitric oxide synthase regulates microvascular hyperpermeability in vivo

    PubMed Central

    Hatakeyama, Takuya; Pappas, Peter J; Hobson, Robert W; Boric, Mauricio P; Sessa, William C; Durán, Walter N

    2006-01-01

    Nitric oxide (NO) is an important regulator of blood flow, but its role in permeability is still challenged. We tested in vivo the hypotheses that: (a) endothelial nitric oxide synthase (eNOS) is not essential for regulation of baseline permeability; (b) eNOS is essential for hyperpermeability responses in inflammation; and (c) molecular inhibition of eNOS with caveolin-1 scaffolding domain (AP-Cav) reduces eNOS-regulated hyperpermeability. We used eNOS-deficient (eNOS−/−) mice and their wild-type control as experimental animals, platelet-activating factor (PAF) at 10−7 m as the test pro-inflammatory agent, and integrated optical intensity (IOI) as an index of microvascular permeability. PAF increased permeability in wild-type cremaster muscle from a baseline of 2.4 ± 2.2 to a peak net value of 84.4 ± 2.7 units, while the corresponding values in cremaster muscle of eNOS−/− mice were 1.0 ± 0.3 and 15.6 ± 7.7 units (P < 0.05). Similarly, PAF increased IOI in the mesentery of wild-type mice but much less in the mesentery of eNOS−/− mice. PAF increased IOI to comparable values in the mesenteries of wild-type mice and those lacking the gene for inducible NOS (iNOS). Administration of AP-Cav blocked the microvascular hyperpermeability responses to 10−7 m PAF. We conclude that: (1) baseline permeability does not depend on eNOS; (2) eNOS and NO are integral elements of the signalling pathway for the hyperpermeability response to PAF; (3) iNOS does not affect either baseline permeability or hyperpermeability responses to PAF; and (4) caveolin-1 inhibits eNOS regulation of microvascular permeability in vivo. Our results establish eNOS as an important regulator of microvascular permeability in inflammation. PMID:16675496

  7. Mixture interactions of xenoestrogens with endogenous estrogens.

    EPA Science Inventory

    There is growing concern of exposure to fish, wildlife, and humans to water sources contaminated with estrogens and the potential impact on reproductive health. These environmental estrogens originate from various sources including concentrated animal feedlot operations (CAFO), m...

  8. Novel mechanisms of endothelial mechano-transduction

    PubMed Central

    Abe, Jun-ichi; Berk, Bradford C

    2014-01-01

    Atherosclerosis is a focal disease that develops preferentially where non-laminar, disturbed blood flow (d-flow) occurs such as branches, bifurcations, and curvatures of large arteries. Endothelial cells sense and respond differently to d-flow compared to steady laminar flow (s-flow). D-flow that occurs in so-called athero-prone areas activates pro-inflammatory and apoptotic signaling, and this results in endothelial dysfunction and leads to subsequent development of atherosclerosis. In contrast, s-flow as “athero-protective flow” promotes expression of many anti-inflammatory genes such as Kruppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) and inhibits endothelial inflammation and athrogenesis. Here we will discuss that d-flow and s-flow induce pro- and anti-atherogenic events via flow type-specific “mechanotransduction” pathways. We will focus on five mechano-sensitive pathways: MEK5 (MAPK/ERK kinase 5)-ERK5-KLF2 signaling, ERK5-PPAR (peroxisome proliferator-activated receptor) signaling, and mechano-signaling pathways involving SUMOylation, protein kinase C-ζ, (PKCζ), and p90 ribosomal S6 kinase (p90RSK). We believe that clarifying regulation mechanisms between these two flow types will provide new insights into therapeutic approaches for the prevention and treatment of atherosclerosis. PMID:25301843

  9. 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

  10. Functional interaction of endothelial nitric oxide synthase with a voltage-dependent anion channel

    PubMed Central

    Sun, Jianxin; Liao, James K.

    2002-01-01

    Endothelium-derived nitric oxide (NO) is an important regulator of vascular function. NO is produced by endothelial NO synthase (eNOS) whose function is modulated, in part, by specific protein interactions. By coimmunoprecipitation experiments followed by MS analyses, we identified a human voltage-dependent anion/cation channel or porin as a binding partner of eNOS. The interaction between porin and eNOS was demonstrated by coimmunoprecipitation studies in nontransfected human endothelial cells and Cos-7 cells transiently transfected with eNOS and porin cDNAs. In vitro binding studies with glutathione S-transferase–porin indicated that porin binds directly to eNOS and that this interaction augmented eNOS activity. The calcium ionophore, A23187, and bradykinin, which are known to activate eNOS, markedly increased porin–eNOS interaction, suggesting a potential role of intracellular Ca2+ in mediating this interaction. Theses results indicate that the interaction between a voltage-dependent membrane channel and eNOS may be important for regulating eNOS activity. PMID:12228731

  11. Vascular nitric oxide: Beyond eNOS.

    PubMed

    Zhao, Yingzi; Vanhoutte, Paul M; Leung, Susan W S

    2015-10-01

    As the first discovered gaseous signaling molecule, nitric oxide (NO) affects a number of cellular processes, including those involving vascular cells. This brief review summarizes the contribution of NO to the regulation of vascular tone and its sources in the blood vessel wall. NO regulates the degree of contraction of vascular smooth muscle cells mainly by stimulating soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP), although cGMP-independent signaling [S-nitrosylation of target proteins, activation of sarco/endoplasmic reticulum calcium ATPase (SERCA) or production of cyclic inosine monophosphate (cIMP)] also can be involved. In the blood vessel wall, NO is produced mainly from l-arginine by the enzyme endothelial nitric oxide synthase (eNOS) but it can also be released non-enzymatically from S-nitrosothiols or from nitrate/nitrite. Dysfunction in the production and/or the bioavailability of NO characterizes endothelial dysfunction, which is associated with cardiovascular diseases such as hypertension and atherosclerosis. PMID:26499181

  12. Not lost in translation: Emerging clinical importance of the G protein-coupled estrogen receptor GPER.

    PubMed

    Barton, Matthias

    2016-07-01

    It has been 20years that the G protein-coupled estrogen receptor (GPER) was cloned as the orphan receptor GPR30 from multiple cellular sources, including vascular endothelial cells. Here, I will provide an overview of estrogen biology and the historical background leading to the discovery of rapid vascular estrogen signaling. I will also review the recent advances in the understanding of the mechanisms underlying GPER function, its role in physiology and disease, some of the currently available GPER-targeting drugs approved for clinical use such as SERMs (selective estrogen receptor modulators) and SERDs (selective estrogen receptor downregulators). Many of currently used drugs such as tamoxifen, raloxifene, or faslodex™/fulvestrant were discovered targeting GPER many years after they had been introduced to the clinics for entirely different purposes. This has important implications for the clinical use of these drugs and their modes of action, which I have termed 'reverse translational medicine'. In addition, environmental pollutants known as 'endocrine disruptors' have been found to bind to GPER. This article also discusses recent evidence in these areas as well as opportunities in translational clinical medicine and GPER research, including medical genetics, personalized medicine, prevention, and its theranostic use. PMID:26921679

  13. Design of pathway preferential estrogens that provide beneficial metabolic and vascular effects without stimulating reproductive tissues.

    PubMed

    Madak-Erdogan, Zeynep; Kim, Sung Hoon; Gong, Ping; Zhao, Yiru C; Zhang, Hui; Chambliss, Ken L; Carlson, Kathryn E; Mayne, Christopher G; Shaul, Philip W; Korach, Kenneth S; Katzenellenbogen, John A; Katzenellenbogen, Benita S

    2016-01-01

    There is great medical need for estrogens with favorable pharmacological profiles that support desirable activities for menopausal women, such as metabolic and vascular protection, but that lack stimulatory activities on the breast and uterus. We report the development of structurally novel estrogens that preferentially activate a subset of estrogen receptor (ER) signaling pathways and result in favorable target tissue-selective activity. Through a process of structural alteration of estrogenic ligands that was designed to preserve their essential chemical and physical features but greatly reduced their binding affinity for ERs, we obtained "pathway preferential estrogens" (PaPEs), which interacted with ERs to activate the extranuclear-initiated signaling pathway preferentially over the nuclear-initiated pathway. PaPEs elicited a pattern of gene regulation and cellular and biological processes that did not stimulate reproductive and mammary tissues or breast cancer cells. However, in ovariectomized mice, PaPEs triggered beneficial responses both in metabolic tissues (adipose tissue and liver) that reduced body weight gain and fat accumulation and in the vasculature that accelerated repair of endothelial damage. This process of designed ligand structure alteration represents a novel approach to develop ligands that shift the balance in ER-mediated extranuclear and nuclear pathways to obtain tissue-selective, non-nuclear PaPEs, which may be beneficial for postmenopausal hormone replacement. The approach may also have broad applicability for other members of the nuclear hormone receptor superfamily. PMID:27221711

  14. Effects of estrogen on beta-amyloid-induced cholinergic cell death in the nucleus basalis magnocellularis.

    PubMed

    Szego, Eva M; Csorba, Attila; Janáky, Tamás; Kékesi, Katalin A; Abrahám, István M; Mórotz, Gábor M; Penke, Botond; Palkovits, Miklós; Murvai, Unige; Kellermayer, Miklós S Z; Kardos, József; Juhász, Gábor D

    2011-01-01

    Alzheimer disease is characterized by accumulation of β-amyloid (Aβ) and cognitive dysfunctions linked to early loss of cholinergic neurons. As estrogen-based hormone replacement therapy has beneficial effects on cognition of demented patients, and it may prevent memory impairments, we investigated the effect of estrogen-pretreatment on Aβ-induced cholinergic neurodegeneration in the nucleus basalis magnocellularis (NBM). We tested which Aβ species induces the more pronounced cholinotoxic effect in vivo. We injected different Aβ assemblies in the NBM of mice, and measured cholinergic cell and cortical fiber loss. Spherical Aβ oligomers had the most toxic effect. Pretreatment of ovariectomized mice with estrogen before Aβ injection decreased cholinergic neuron loss and partly prevented fiber degeneration. By using proteomics, we searched for proteins involved in estrogen-mediated protection and in Aβ toxicity 24 h following injection. The change in expression of, e.g., DJ-1, NADH ubiquinone oxidoreductase, ATP synthase, phosphatidylethanolamine-binding protein 1, protein phosphatase 2A and dimethylarginine dimethylaminohydrolase 1 support our hypothesis that Aβ induces mitochondrial dysfunction, decreases MAPK signaling, and increases NOS activation in NBM. On the other hand, altered expression of, e.g., MAP kinase kinase 1 and 2, protein phosphatase 1 and 2A by Aβ might increase MAPK suppression and NOS signaling in the cortical target area. Estrogen pretreatment reversed most of the changes in the proteome in both areas. Our experiments suggest that regulation of the MAPK pathway, mitochondrial pH and NO production may all contribute to Aβ toxicity, and their regulation can be prevented partly by estrogen pretreatment. PMID:20938166

  15. Estradiol coupling to human monocyte nitric oxide release is dependent on intracellular calcium transients: evidence for an estrogen surface receptor.

    PubMed

    Stefano, G B; Prevot, V; Beauvillain, J C; Fimiani, C; Welters, I; Cadet, P; Breton, C; Pestel, J; Salzet, M; Bilfinger, T V

    1999-10-01

    We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase (cNOS) activity in human peripheral monocytes by acting on an estrogen surface receptor. NO release was measured in real time with an amperometric probe. 17beta-estradiol exposure to monocytes stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol had no effect. 17beta-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17beta-estradiol and E2-BSA, whereas ICI 182,780, a selective inhibitor of the nuclear estrogen receptor, had no effect. We further showed, using a dual emission microfluorometry in a calcium-free medium, that the 17beta-estradiol-stimulated release of monocyte NO was dependent on the initial stimulation of intracellular calcium transients in a tamoxifen-sensitive process. Leeching out the intracellular calcium stores abolished the effect of 17beta-estradiol on NO release. RT-PCR analysis of RNA obtained from the cells revealed a strong estrogen receptor-alpha amplification signal and a weak beta signal. Taken together, a physiological dose of estrogen acutely stimulates NO release from human monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium. PMID:10490972

  16. Estrogen Treatment in Multiple Sclerosis

    PubMed Central

    Gold, Stefan M; Voskuhl, Rhonda R

    2009-01-01

    Currently available treatments for multiple sclerosis reduce inflammatory lesions on MRI and decrease clinical relapses but have limited effects on disability. Novel treatment options that target both the inflammatory as well as the neurodegenerative component of the disease are therefore needed. A growing body of evidence from basic science and clinical studies supports the therapeutic potential of estrogens in MS. Mechanisms of action include both immunomodulatory and directly neuroprotective pathways. A first pilot trial of oral estriol treatment showed encouraging results. There are now several phase II trials underway to further determine the efficacy of estrogen treatment in MS. PMID:19539954

  17. 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

  18. 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

  19. Shear-Induced Nitric Oxide Production by Endothelial Cells.

    PubMed

    Sriram, Krishna; Laughlin, Justin G; Rangamani, Padmini; Tartakovsky, Daniel M

    2016-07-12

    We present a biochemical model of the wall shear stress-induced activation of endothelial nitric oxide synthase (eNOS) in an endothelial cell. The model includes three key mechanotransducers: mechanosensing ion channels, integrins, and G protein-coupled receptors. The reaction cascade consists of two interconnected parts. The first is rapid activation of calcium, which results in formation of calcium-calmodulin complexes, followed by recruitment of eNOS from caveolae. The second is phosphorylation of eNOS by protein kinases PKC and AKT. The model also includes a negative feedback loop due to inhibition of calcium influx into the cell by cyclic guanosine monophosphate (cGMP). In this feedback, increased nitric oxide (NO) levels cause an increase in cGMP levels, so that cGMP inhibition of calcium influx can limit NO production. The model was used to predict the dynamics of NO production by an endothelial cell subjected to a step increase of wall shear stress from zero to a finite physiologically relevant value. Among several experimentally observed features, the model predicts a highly nonlinear, biphasic transient behavior of eNOS activation and NO production: a rapid initial activation due to the very rapid influx of calcium into the cytosol (occurring within 1-5 min) is followed by a sustained period of activation due to protein kinases. PMID:27410748

  20. ATP-Binding Cassette Transporter G1 and High-Density Lipoprotein Promote Endothelial NO Synthesis Through a Decrease in the Interaction of Caveolin-1 and Endothelial NO Synthase

    PubMed Central

    Terasaka, Naoki; Westerterp, Marit; Koetsveld, Joris; Fernández-Hernando, Carlos; Yvan-Charvet, Laurent; Wang, Nan; Sessa, William C.; Tall, Alan R.

    2016-01-01

    Objective To investigate whether cholesterol efflux to high-density lipoprotein (HDL) via ATP-binding cassette transporter G1 (ABCG1) modulates the interaction of caveolin (Cav) 1 and endothelial NO synthase (eNOS). Methods and Results ABCG1 promotes cholesterol and 7-oxysterol efflux from endothelial cells (ECs) to HDL. It was previously reported that ABCG1 protects against dietary cholesterol-induced endothelial dysfunction by promoting the efflux of 7-oxysterols to HDL. Increased cholesterol loading in ECs is known to cause an inhibitory interaction between Cav-1 and eNOS and impaired NO release. In human aortic ECs, free cholesterol loading promoted the interaction of Cav-1 with eNOS, reducing eNOS activity. These effects of cholesterol loading were reversed by HDL in an ABCG1-dependent manner. HDL also reversed the inhibition of eNOS by cholesterol loading in murine lung ECs, but this effect of HDL was abolished in Cav-1–deficient murine lung ECs. Increased interaction of Cav-1 with eNOS was also detected in aortic homogenates of high-cholesterol diet–fed Abcg1−/− mice, paralleling a decrease in eNOS activity and impaired endothelial function. Conclusion The promotion of cholesterol efflux via ABCG1 results in a reduced inhibitory interaction of eNOS with Cav-1. PMID:20798376

  1. Regulation of Placental Villous Angiopoietin-1 and -2 Expression by Estrogen During Baboon Pregnancy1

    PubMed Central

    Albrecht, Eugene D.; Babischkin, Jeffery S.; Pepe, Gerald J.

    2009-01-01

    We recently showed an increase in vascular endothelial growth factor (VEGF), decrease in angiopoietin-1 (Ang-1) and unaltered Ang-2 expression by the villous placenta with advancing baboon pregnancy. Moreover, placental VEGF expression was increased by estrogen in early pregnancy. In the present study, we determined whether placental Ang-1 and Ang-2 are regulated by estrogen. Ang-1 and Ang-2 mRNA and protein were determined by RT-PCR and immunocytochemistry in the placenta of baboons on day 60 of gestation (term is 184 days) after administration of estrogen precursor androstenedione on days 25-59 or on day 54 after acute estradiol administration. Chronic androstenedione treatment increased serum estradiol levels 3-fold (P<0.001) and decreased (P<0.05) villous cytotrophoblast Ang-1 mRNA to a level (0.36 ± 0.08 relative to 18S rRNA) that was one-third of that in untreated animals (0.98 ± 0.26). Within 2 h of estradiol administration, cytotrophoblast Ang-1 mRNA was decreased to a level (0.24 ± 0.05) one-fifth (P<0.05) of that in untreated animals (1.14 ± 0.23). However, Ang-2 mRNA levels were unaltered. Ang-1, Ang-2 and estrogen receptors α and β protein were localized within villous cytotrophoblasts providing a mechanism for estrogen action at this site. In summary, estrogen increased VEGF, decreased Ang-1, and had no effect on Ang-2 expression within placental cytotrophoblasts during early baboon pregnancy. We propose that the estrogen-dependent differential regulation of these angioregulatory factors underpins the unique pattern of neovascularization established within the villous placenta during primate pregnancy. PMID:18022824

  2. Effect of Excessive Potassium Iodide on Rat Aorta Endothelial Cells.

    PubMed

    Zhang, Man; Zou, Xiaoyan; Lin, Xinying; Bian, Jianchao; Meng, Huicui; Liu, Dan

    2015-08-01

    The aim of the current study was to investigate the effect of excess iodine on rat aorta endothelial cells and the potential underlying mechanisms. Rat aorta endothelial cells were cultured with iodide ion (3506, 4076, 4647, 5218, 5789, 6360, 6931, and 7512 mg/L) for 48 h. Morphological changes of cells were observed with microscope after Wright-Giemsa staining and acridine orange staining. Cell proliferation was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell apoptosis was assessed with flow cytometry. The activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), endothelial nitric oxide synthase (eNOS), induced nitric oxide synthase (iNOS), and concentrations of malondialdehyde (MDA), glutathione (GSH), and protein carbonyl in culture medium were determined with colorimetric method. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was detected by enzyme linked immunosorbent assay. The results showed that excess iodine induced abnormal morphologic changes of cells, inhibited cell proliferation, and increased apoptosis rate. Iodine also reduced the activity of SOD, GSH-Px, and concentrations of GSH and increased the concentrations of MDA and protein carbonyl in a dose-dependent manner. Moreover, excess iodine decreased the activity of eNOS and increased the activity of iNOS and the expression of ICAM-1 and VCAM-1 in culture medium. Our results suggested that excess iodine exposure increased oxidative stress, caused damage of vascular endothelial cells, and altered the expression of adhesion factors and the activity of NOS. These changes may explain the mechanisms underlying excess iodine-induced vascular injury. PMID:25690517

  3. Endostatin induces acute endothelial nitric oxide and prostacyclin release

    SciTech Connect

    Li Chunying; Harris, M. Brennan; Venema, Virginia J.; Venema, Richard C. . E-mail: rvenema@mcg.edu

    2005-04-15

    Chronic exposure to endostatin (ES) blocks endothelial cell (EC) proliferation, and migration and induces EC apoptosis thereby inhibiting angiogenesis. Nitric oxide (NO) and prostacyclin (PGI{sub 2}), in contrast, play important roles in promoting angiogenesis. In this study, we examined the acute effects of ES on endothelial NO and PGI{sub 2} production. Unexpectedly, a cGMP reporter cell assay showed that ES-induced acute endothelial NO release in cultured bovine aortic endothelial cells (BAECs). Enzyme immunoassay showed that ES also induced an acute increase in PGI{sub 2} production in BAECs. These results were confirmed by ex vivo vascular ring studies that showed vascular relaxation in response to ES. Immunoblot analysis showed that ES stimulated acute phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser116, Ser617, Ser635, and Ser1179, and dephosphorylation at Thr497 in BAECs, events associated with eNOS activation. Short-term exposure of EC to ES, therefore, unlike long-term exposure which is anti-angiogenic, may be pro-angiogenic.

  4. Influence of coronary artery diameter on eNOS protein content

    NASA Technical Reports Server (NTRS)

    Laughlin, M. H.; Turk, J. R.; Schrage, W. G.; Woodman, C. R.; Price, E. M.

    2003-01-01

    The purpose of this study was to test the hypothesis that the content of endothelial nitric oxide synthase (eNOS) protein (eNOS protein/g total artery protein) increases with decreasing artery diameter in the coronary arterial tree. Content of eNOS protein was determined in porcine coronary arteries with immunoblot analysis. Arteries were isolated in six size categories from each heart: large arteries [301- to 2,500-microm internal diameter (ID)], small arteries (201- to 300-microm ID), resistance arteries (151- to 200-microm ID), large arterioles (101- to 150-microm ID), intermediate arterioles (51- to 100-microm ID), and small arterioles(<50-microm ID). To obtain sufficient protein for analysis from small- and intermediate-sized arterioles, five to seven arterioles 1-2 mm in length were pooled into one sample for each animal. Results establish that the number of smooth muscle cells per endothelial cell decreases from a number of 10 to 15 in large coronary arteries to 1 in the smallest arterioles. Immunohistochemistry revealed that eNOS is located only in endothelial cells in all sizes of coronary artery and in coronary capillaries. Contrary to our hypothesis, eNOS protein content did not increase with decreasing size of coronary artery. Indeed, the smallest coronary arterioles had less eNOS protein per gram of total protein than the large coronary arteries. These results indicate that eNOS protein content is greater in the endothelial cells of conduit arteries, resistance arteries, and large arterioles than in small coronary arterioles.

  5. Estrogens are needed for the improvement in endothelium-mediated dilation induced by a chronic increase in blood flow in rat mesenteric arteries.

    PubMed

    Tarhouni, K; Guihot, A L; Vessieres, E; Procaccio, V; Grimaud, L; Abraham, P; Lenfant, F; Arnal, J F; Favre, J; Loufrani, L; Henrion, D

    2016-05-01

    Resistance arteries play a key role in the control of local blood flow. They undergo outward remodeling in response to a chronic increase in blood flow as seen in collateral artery growth in ischemic disorders. We have previously shown that mesenteric artery outward remodeling depends on the endothelial estrogen receptor alpha. As outward arterial remodeling is associated with improved endothelium-dependent dilation, we hypothesized that estrogens might also play a role in flow-mediated improvement of endothelium-dependent dilation. Local increase in blood flow in first order mesenteric arteries was obtained after ligation of adjacent arteries in three-month old ovariectomized female rats treated with 17-beta-estradiol (OVX+E2) or vehicle (OVX). After 2 weeks, diameter was equivalent in high flow (HF) than in normal flow (NF) arteries with a greater wall to lumen ratio in HF vessels in OVX rats. Acetylcholine-mediated relaxation was lower in HF than in NF vessels. eNOS and caveolin-1 expression level was equivalent in HF and NF arteries. By contrast, arterial diameter was 30% greater in HF than in NF arteries and the wall to lumen ratio was not changed in OVX+E2 rats. Acetylcholine-mediated relaxation was higher in HF than in NF arteries. The expression level of eNOS was higher and that of caveolin-1 was lower in HF than in NF arteries. Acetylcholine (NO-dependent)-mediated relaxation was partly inhibited by the NO-synthesis blocker L-NAME in OVX rats whereas L-NAME blocked totally the relaxation in OVX+E2 rats. Endothelium-independent relaxation (sodium nitroprusside) was equivalent in OXV and OVX+E2 rats. Similarly, serotonin- and phenylephrine-mediated contractions were higher in HF than in NF arteries in both OVX and OVX+E2 rats in association with high ratio of phosphorylated ERK1/2 to ERK1/2. Thus, we demonstrated the essential role of endogenous E2 in flow-mediated improvement of endothelium (NO)-mediated dilatation in rat mesenteric arteries. PMID:26471832

  6. Protein kinase Cδ regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation

    PubMed Central

    Sud, Neetu; Wedgwood, Stephen; Black, Stephen M.

    2008-01-01

    In this study, we explore the roles of the delta isoform of PKC (PKCδ) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKCδ with either rottlerin or with the peptide, δV1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKCδ inhibition using either rottlerin or the overexpression of a dominant negative PKCδ mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKCδ inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKCδ is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKCδ-mediated Akt activation and NO generation in maintaining eNOS expression. PMID:18192589

  7. Cellular and molecular biology of aging endothelial cells.

    PubMed

    Donato, Anthony J; Morgan, R Garrett; Walker, Ashley E; Lesniewski, Lisa A

    2015-12-01

    Cardiovascular disease (CVD) is the leading cause of death in the United States and aging is a major risk factor for CVD development. One of the major age-related arterial phenotypes thought to be responsible for the development of CVD in older adults is endothelial dysfunction. Endothelial function is modulated by traditional CVD risk factors in young adults, but advancing age is independently associated with the development of vascular endothelial dysfunction. This endothelial dysfunction results from a reduction in nitric oxide bioavailability downstream of endothelial oxidative stress and inflammation that can be further modulated by traditional CVD risk factors in older adults. Greater endothelial oxidative stress with aging is a result of augmented production from the intracellular enzymes NADPH oxidase and uncoupled eNOS, as well as from mitochondrial respiration in the absence of appropriate increases in antioxidant defenses as regulated by relevant transcription factors, such as FOXO. Interestingly, it appears that NFkB, a critical inflammatory transcription factor, is sensitive to this age-related endothelial redox change and its activation induces transcription of pro-inflammatory cytokines that can further suppress endothelial function, thus creating a vicious feed-forward cycle. This review will discuss the two macro-mechanistic processes, oxidative stress and inflammation, that contribute to endothelial dysfunction with advancing age as well as the cellular and molecular events that lead to the vicious cycle of inflammation and oxidative stress in the aged endothelium. Other potential mediators of this pro-inflammatory endothelial phenotype are increases in immune or senescent cells in the vasculature. Of note, genomic instability, telomere dysfunction or DNA damage has been shown to trigger cell senescence via the p53/p21 pathway and result in increased inflammatory signaling in arteries from older adults. This review will discuss the current state

  8. The role of Hath6, a newly identified shear-stress-responsive transcription factor, in endothelial cell differentiation and function

    PubMed Central

    Fang, Fang; Wasserman, Scott M.; Torres-Vazquez, Jesus; Weinstein, Brant; Cao, Feng; Li, Zongjin; Wilson, Kitchener D.; Yue, Wen; Wu, Joseph C.; Xie, Xiaoyan; Pei, Xuetao

    2014-01-01

    ABSTRACT The key regulators of endothelial differentiation that is induced by shear stress are mostly unclear. Human atonal homolog 6 (Hath6 or ATOH8) is an endothelial-selective and shear-stress-responsive transcription factor. In this study, we sought to elucidate the role of Hath6 in the endothelial specification of embryonic stem cells. In a stepwise human embryonic stem cell to endothelial cell (hESC-EC) induction system, Hath6 mRNA was upregulated synchronously with endothelial determination. Subsequently, gain-of-function and loss-of-function studies of Hath6 were performed using the hESC-EC induction model and endothelial cell lines. The overexpression of Hath6, which mimics shear stress treatment, resulted in an increased CD45−CD31+KDR+ population, a higher tubular-structure-formation capacity and increased endothelial-specific gene expression. By contrast, the knockdown of Hath6 mRNA markedly decreased endothelial differentiation. Hath6 also facilitated the maturation of endothelial cells in terms of endothelial gene expression, tubular-structure formation and cell migration. We further demonstrated that the gene encoding eNOS is a direct target of Hath6 through a reporter system assay and western blot analysis, and that the inhibition of eNOS diminishes hESC-EC differentiation. These results suggest that eNOS plays a key role in linking Hath6 to the endothelial phenotype. Further in situ hybridization studies in zebrafish and mouse embryos indicated that homologs of Hath6 are involved in vasculogenesis and angiogenesis. This study provides the first confirmation of the positive impact of Hath6 on human embryonic endothelial differentiation and function. Moreover, we present a potential signaling pathway through which shear stress stimulates endothelial differentiation. PMID:24463812

  9. The role of Hath6, a newly identified shear-stress-responsive transcription factor, in endothelial cell differentiation and function.

    PubMed

    Fang, Fang; Wasserman, Scott M; Torres-Vazquez, Jesus; Weinstein, Brant; Cao, Feng; Li, Zongjin; Wilson, Kitchener D; Yue, Wen; Wu, Joseph C; Xie, Xiaoyan; Pei, Xuetao

    2014-04-01

    The key regulators of endothelial differentiation that is induced by shear stress are mostly unclear. Human atonal homolog 6 (Hath6 or ATOH8) is an endothelial-selective and shear-stress-responsive transcription factor. In this study, we sought to elucidate the role of Hath6 in the endothelial specification of embryonic stem cells. In a stepwise human embryonic stem cell to endothelial cell (hESC-EC) induction system, Hath6 mRNA was upregulated synchronously with endothelial determination. Subsequently, gain-of-function and loss-of-function studies of Hath6 were performed using the hESC-EC induction model and endothelial cell lines. The overexpression of Hath6, which mimics shear stress treatment, resulted in an increased CD45(-)CD31(+)KDR(+) population, a higher tubular-structure-formation capacity and increased endothelial-specific gene expression. By contrast, the knockdown of Hath6 mRNA markedly decreased endothelial differentiation. Hath6 also facilitated the maturation of endothelial cells in terms of endothelial gene expression, tubular-structure formation and cell migration. We further demonstrated that the gene encoding eNOS is a direct target of Hath6 through a reporter system assay and western blot analysis, and that the inhibition of eNOS diminishes hESC-EC differentiation. These results suggest that eNOS plays a key role in linking Hath6 to the endothelial phenotype. Further in situ hybridization studies in zebrafish and mouse embryos indicated that homologs of Hath6 are involved in vasculogenesis and angiogenesis. This study provides the first confirmation of the positive impact of Hath6 on human embryonic endothelial differentiation and function. Moreover, we present a potential signaling pathway through which shear stress stimulates endothelial differentiation. PMID:24463812

  10. Association of NOS2 and NOS3 gene polymorphisms with susceptibility to type 2 diabetes mellitus and diabetic nephropathy in the Chinese Han population.

    PubMed

    Chen, Feng; Li, Yu-Mei; Yang, Lin-Qing; Zhong, Cai-Gao; Zhuang, Zhi-Xiong

    2016-07-01

    Inducible nitric oxide synthase (NOS2) and endothelial nitric oxide synthase (NOS3) gene play important roles in the susceptibility to type 2 diabetes mellitus (T2DM). The present study aims to detect the potential association of NOS2 and NOS3 gene polymorphisms with the susceptibility toT2DM and diabetic nephropathy (DN) in the Chinese Han population. Four hundred and ninety T2DM patients and 485 healthy controls were enrolled in this case-control study. The genotypes of NOS2 and NOS3 gene polymorphisms were analyzed by the polymerase chain reaction (PCR)-ligase detection reaction (LDR) method. Our data demonstrated that the NOS2 rs2779248 and NOS2 rs1137933 genetic polymorphisms were significantly associated with the increased susceptibility to T2DM in the heterozygote comparison, dominant model, and allele contrast; and NOS3 rs3918188 genetic polymorphism was significantly associated with the increased susceptibility to T2DM in the homozygote comparison and recessive model. The allele-C and genotype-TC of NOS2 rs2779248, allele-A and genotype-GA of NOS2 rs1137933 and genotype-AA of NOS3 rs3918188 genetic polymorphisms might be the risk factors for increasing the susceptibility to T2DM. And a significant haplotype effect of NOS2 rs10459953/C- rs1137933/G- rs2779248/T was found between T2DM cases and controls. Moreover, NOS3 rs1800783 polymorphism was significantly associated with the increased susceptibility to DN in the heterozygote comparison, recessive model and allele contrast. At last, a positive correlation of family history of diabetes with NOS3 rs11771443 polymorphism was found in DN. These preliminary findings indicate that the NOS2 rs2779248, NOS2 rs1137933, and NOS3 rs3918188 genetic polymorphisms are potentially related to the susceptibility to T2DM, and the rs1800783 polymorphism might be considered as genetic risk factors for diabetic nephropathy, and family history of diabetes was closely associated with rs11771443 polymorphism in DN, and the