scholarly journals Bradykinin stimulation of nitric oxide production is not sufficient for gamma-globin induction

2014 ◽  
Vol 142 (3-4) ◽  
pp. 189-196 ◽  
Author(s):  
Vladan Cokic ◽  
Tijana Suboticki ◽  
Bojana Beleslin-Cokic ◽  
Milos Diklic ◽  
Pavle Milenkovic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Globin Gene ◽  
No Production ◽  
Dependent Manner ◽  
Erythroid Progenitors ◽  
Globin Gene Expression ◽  
Stimulation Of

Introduction. Hydroxycarbamide, used in therapy of hemoglobinopathies, enhances nitric oxide (NO) production both in primary human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cell line (TrHBMEC). Moreover, NO increases ?-globin and fetal hemoglobin levels in human erythroid progenitors. Objective. In order to find out whether simple physiologic stimulation of NO production by components of hematopoietic microenvironment can increase ?-globin gene expression, the effects of NO-inducer bradykinin were examined in endothelial cells. Methods. The study was performed in co-cultures of human erythroid progenitors, TrHBMEC and HUVECs by ozone-based chemiluminescent determination of NO and real-time quantitative RT-PCR. Results. In accordance with previous reports, the endogenous factor bradykinin increased endothelial cell production of NO in a dose- and time-dependent manner (0.1-0.6 ?M up to 30 minutes). This induction of NO in HUVECs and TrHBMEC by bradykinin was blocked by competitive inhibitors of NO synthase (NOS), demonstrating NOS-dependence. It has been shown that bradykinin significantly reduced endothelial NOS (eNOS) mRNA level and eNOS/?-actin ratio in HUVEC (by twofold). In addition, bradykinin failed to increase ?-globin mRNA expression in erythroid progenitors only, as well as in co-culture studies of erythroid progenitors with TrHBMEC and HUVEC after 24 hours of treatment. Furthermore, bradykinin did not induce ?/? globin ratio in erythroid progenitors in co-cultures with HUVEC. Conclusion. Bradykinin mediated eNOS activation leads to short time and low NO production in endothelial cells, insufficient to induce ?-globin gene expression. These results emphasized the significance of elevated and extended NO production in augmentation of ?-globin gene expression.

EKLF Is Recruited to the γ-Globin Gene Promoter as a Co-Activator and Is Required for γ-Globin Gene Induction by Short-Chain Fatty Acids.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1771-1771
Author(s):  
Susan P. Perrine ◽  
Rishikesh Mankidy ◽  
Michael S. Boosalis ◽  
James J. Bieker ◽  
Douglas V. Faller
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Gene Induction ◽  
Therapeutic Interventions ◽  
Short Chain ◽  
Dependent Manner ◽  
Erythroid Progenitors ◽  
Globin Promoter ◽  
Globin Gene Expression

Abstract The erythroid Kruppel-like factor, EKLF, is an essential transcription factor for mammalian β-type globin gene switching, and specifically activates transcription of the adult β-globin gene through binding of its zinc finger domain to the β-globin promoter. We report now that EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives. We found that specific knockdown of EKLF levels by siRNA prevents SCFA induced-expression of an integrated γ-globin promoter in a stably-expressed mLCRβprRluc AγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. In chromatin immunoprecipitation (ChIP) assays, EKLF was found to be actively recruited to the endogenous γ-globin gene promoter with exposure of human erythroid progenitors, and hematopoietic cell lines, to SCFA derivatives. The human SWI/WNF complex is a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure in an ATP-dependent manner. We found that the SWI/SNF complex chromatin-modifying core ATPase BRG1 is also required for γ-globin gene induction by SCFA derivatives. Furthermore, BRG1 is actively recruited to the endogenous γ-globin promoter of human erythroid progenitors with exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings all demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes. Recently. we also identified a γ-globin-specific repressor complex, consisting of NCoR and HDAC3, which is displaced from the proximal γ-globin promoter by exposure to SCFA derivatives prior to activation of transcription (Blood, 108:3179–86, 2006). Collectively, these studies identify critical activating and repressing cofactors regulating γ-globin gene expression, and provide new targets for therapeutic interventions.

Adenosine enhances nitric oxide production by vascular endothelial cells

1995 ◽  
Vol 269 (2) ◽  
pp. C519-C523 ◽  
Author(s):  
J. M. Li ◽  
R. A. Fenton ◽  
B. S. Cutler ◽  
J. G. Dobson
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Vascular Tone ◽  
Vascular Endothelial Cells ◽  
Competitive Inhibitor ◽  
No Production ◽  
Dependent Manner ◽  
Bathing Medium ◽  
Adenosine Receptor Antagonist ◽  
Potent Vasodilator

Adenosine per se is a potent vasodilator of vascular smooth muscle. Endothelial cells modulate vascular tone via the release of nitric oxide (NO), which also elicits vasodilation. This study was undertaken to determine whether adenosine could directly stimulate endothelial cells to enhance NO production, which could subsequently reduce vascular tone. NO production was evaluated in porcine carotid artery endothelial cells (PCAEC) and human saphenous vein endothelial cells (HSVEC) seeded on multiwell plates, grown to confluence, and treated with adenosine for 1 h. The bathing medium was collected, and the NO production was determined as reflected by the formation of NO2- and NO3-. NO production by PCAEC was significantly increased by adenosine in a dose-dependent manner, whereas there was only an insignificant tendency for an increase by HSVEC. The addition of the NO synthase competitive inhibitor, NG-monomethyl-L-arginine (NMMA), or the adenosine receptor antagonist, theophylline, prevented the increase in NO production by adenosine. The results suggest that adenosine stimulates, by a receptor-mediated mechanism, the production of NO by arterial, but not by venous, endothelial cells.

Gene Expression Profiles Involved in γ to β Globin Gene Switching during Erythroid Maturation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 820-820
Author(s):  
Wei Li ◽  
Betty S. Pace
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Globin Gene ◽  
Gene Expression Profiles ◽  
Phase System ◽  
Mrna Levels ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Gene Switching ◽  
Globin Gene Expression

Abstract The design and evaluation of therapies for sickle cell disease (SCD) rely on our understanding of hemoglobin accumulation during erythropoiesis and sequential globin gene expression (ε → Gγ → Aγ → δ → β) during development. To gain insights into globin gene switching, we completed time course micorarray analyses of erythroid progenitors to identify trans-factors involved in γ gene activation. Studies were completed to map the pattern of γ and β globin gene expression in progenitors grown from normal peripheral blood mononuclear cells. We compared cells grown in a 2-phase (phase 1, d0-6: SCF, IL-3, IL-6, and GM-CSF and phase 2, d7-25: SCF and EPO) vs. 1-phase (d0-34: SCF, IL-3, and EPO) liquid culture system. From day 0 to 34 in either system cell viability remained >99%. Total RNA was isolated using Trizol and column cleanup (Qiagen). Globin mRNA levels were measured at 2–3 day intervals by quantitative PCR (qPCR). In the 2-phase system γ-globin mRNA>β-globin mRNA up to d14, 4 days of approximately equal expression then β mRNA > γ mRNA by d20. By contrast, in 1-phase studies there was a rapid switch around d20(see graph). We speculate that this difference may be due to the early addition of EPO on d0 therefore we continued our detailed analysis in this system. To confirm that our in vitro system recapitulates in vivo gene expression patterns, we completed studies to ascertain Gγ - vs. Aγ globin mRNA levels. The normalized Gγ:Aγ ratio decreased from ~3:1 on d7 to ~1:1 by d34; These findings were confirmed using two sets of Gγ and Aγ globin primers. We concluded that the 1-phase system recapitulated normal γ/β globin switching and that gene profiling studies to identify the trans-factor involved in switching mechanisms were feasible. We used Discover oligo chips (ArrayIt, Sunnyvale, CA) containing 380 human genes selected from 30 major functional groups including hematopoiesis. To aide interpretation of chip data, cell populations were rated morphologically using Giemsa stained cytospin preps. From d16 on we observed an increase in late erythroid progenitors (normoblasts) from 1% to 71% by d31. After verifying RNA quality by gel inspection of ribosomal molecules, we prepared Cy3 and Cy5 probes for early and late time-point RNA samples respectively. Chip analysis was performed at several time points but d0/21, d7/21, and d21/28 were most informative. Based on Axon GenePixPro 6.0 and Acuity 4.0 software analysis we found the following genes with >1.5-fold change in expression profile (shown as down-regulated/up-regulated genes): d0/21: 33/73, d7/21: 13/25, and d21/28:35/26. Principal component analysis (PCA), hierarchical clusters and self organizing maps were constructed. Gene profiles were correlated with the γ/β switching curve using d7 (γ >β), d21 (γ ~ β), and d28 (γ <β) data. Hematopoietic dataset analysis at d21 revealed 4 candidate γ-globin gene activators including v-myb, upsteam binding transfactor -RNApol1 and 2 zinc finger proteins. Analysis of a d28 dataset revealed 12 proteins involved in γ-globin gene silencing including IL-3, SCF, MAPKKK3, v-raf-1, ATF-2, and glucocorticoid receptor DNA binding factor 1 among others. Gene expression profiles will be validated using qPCR and promising candidates will be tested by forced expression in transient and stable reporter systems. Figure Figure

Cucurbitacin D Upregulates Fetal Hemoglobin Expression in K562 Cells and Human Erythroid Progenitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3833-3833
Author(s):  
Hongtao Xing ◽  
Siwei Zhang ◽  
H. Phillip Koeffler ◽  
Ming Chiu Fung
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Negative Control ◽  
Optimal Dosage ◽  
Erythroid Progenitors ◽  
Cucurbitacin D ◽  
Globin Gene Expression ◽  
Sodium Phenylbutyrate

Abstract The search for novel therapeutic candidates causing reactivation of fetal hemoglobin (a2g2; HbF) to reduce the imbalance of globin gene expression is important in order to develop effective approach for the clinical management of sickle cell anemia and b-thalassemia. For the first time, we have identified cucurbitacin D (CuD), a naturally occurring oxygenated tetracyclic triterpenoid, as a molecular entity inducing g-globin gene expression and HbF synthesis in K562 cells and human erythroid progenitors from either peripheral blood or bone marrow. The upregulation of HbF induced by CuD was dose- and time- dependent. CuD was compared to hydroxyurea (HU), 5-azacytidine, amifostine, recombinant human erythropoietin (rhEPO), and sodium phenylbutyrate. At their optimal dosage, CuD (12.5 ng/mL) and HU (25.0 μg/mL) induced nearly 70% K562 cells to express total hemoglobin after 6 days culture, which was higher than the induction by Amifostine (30%), 5-azacytidine (36%), rhEPO (16%), sodium phenylbutyrate (23%) at their optimal concentrations and negative control (11%). Fetal hemoglobin ELISA showed that CuD (12.5 ng/mL) and 5-azacytidine (400 ng/mL) induced higher levels of fetal hemoglobin in K562 cells (15.4 ng/μL and 29.3 ng/μL, respectively), compared to HU (10.3 ng/μL), amifostine (7.8 ng/μL), rhEPO (10.9 ng/μL), sodium phenylbutyrate (9.9 ng/μL) at their optimal concentrations and negative control (5.3 ng/μL). CuD induced a significantly higher fetal cell percentage than HU in K562 cells (65% vs 37% maximum) and primary erythroid progenitors (36% vs 21% maximum) based on the immunofluorescence imaging and flow cytometry analysis. Real-time PCR results showed that the amount of γ-globin mRNA increased from 2.5-fold in CuD-optimal-treated cells (12.5 ng/mL, 48 hours) compared with 1.5-fold in HU-optimal-treated cells (25.0 μg/mL, 48 hours). Growth inhibition assay (MTT) demonstrated that CuD at its optimal γ-globin inducing dosage (12.5 ng/mL) inhibited proliferation of K562 by less than 10% of untreated control cells; while hydroxyurea at its optimal dosage (25.0 μg/mL) inhibited 80% of cell division. The in vitro therapeutic index (calculated by dividing the dose inhibiting 50% cell growth (IC50) by dose inducing 50% maximal HbF production (ED50)) of CuD was 40-fold greater than HU. Taken together, the results suggest that CuD has the potential to be a therapeutic agent for treatment of sickle cell anemia and b-thalassemia.

scholarly journals Androgen Receptor-Dependent Activation of Endothelial Nitric Oxide Synthase in Vascular Endothelial Cells: Role of Phosphatidylinositol 3-Kinase/Akt Pathway

Endocrinology ◽  
2010 ◽  
Vol 151 (4) ◽  
pp. 1822-1828 ◽  
Author(s):  
Jing Yu ◽  
Masahiro Akishita ◽  
Masato Eto ◽  
Sumito Ogawa ◽  
Bo-Kyung Son ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Androgen Receptor ◽  
Pi3 Kinase ◽  
Maximal Response ◽  
No Production ◽  
Dependent Manner ◽  
Small Interference Rna ◽  
Enos Phosphorylation ◽  
Interference Rna

The mechanisms of testosterone-induced vasodilatation are not fully understood. This study investigated the effect of testosterone on nitric oxide (NO) synthesis and its molecular mechanism using human aortic endothelial cells (HAEC). Testosterone at physiological concentrations (1–100 nm) induced a rapid (15–30 min) increase in NO production, which was associated with phosphorylation and activation of endothelial NO synthase (eNOS). Then, the involvement of the androgen receptor (AR), which is abundantly expressed in HAEC, was examined. The effect of testosterone on eNOS activation and NO production were abolished by pretreatment with an AR antagonist nilutamide and by transfection with AR small interference RNA. In contrast, testosterone-induced eNOS phosphorylation was unchanged by pretreatment with an aromatase inhibitor or by transfection with ERα small interference RNA. 5α-Dihydrotestosterone, a nonaromatizable androgen, also stimulated eNOS phosphorylation. Next, the signaling cascade that leads to eNOS phosphorylation was explored. Testosterone stimulated rapid phosphorylation of Akt in a time- and dose-dependent manner, with maximal response at 15–60 min. The rapid phosphorylation of eNOS or NO production induced by testosterone was inhibited by Akt inhibitor SH-5 or by phosphatidylinositol (PI) 3-kinase inhibitor wortmannin. Co-immunoprecipitation assays revealed a testosterone-dependent interaction between AR and the p85α subunit of PI3-kinase. In conclusion, testosterone rapidly induces NO production via AR-dependent activation of eNOS in HAEC. Activation of PI3-kinase/Akt signaling and the direct interaction of AR with p85α are involved, at least in part, in eNOS phosphorylation.

scholarly journals Nitric oxide-dependent stimulation of endothelial cell proliferation by sustained high flow

2008 ◽  
Vol 295 (2) ◽  
pp. H736-H742 ◽  
Author(s):  
Eleni Metaxa ◽  
Hui Meng ◽  
Shashikanth R. Kaluvala ◽  
Michael P. Szymanski ◽  
Rocco A. Paluch ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Proliferation ◽  
Endothelial Cell ◽  
High Flow ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Brdu Incorporation ◽  
Endothelial Cell Proliferation ◽  
Tunel Staining ◽  
No Production ◽  
Stimulation Of

Little is understood about endothelial cell (EC) responses to high flow, which mediate adaptive outward remodeling as well as cerebral aneurysm development. Opposite EC behaviors have been reported in vivo including cell loss during aneurysm initiation and cell proliferation during adaptive outward remodeling. This study aims at elucidating the EC growth response to elevated wall shear stress (WSS) and determining if nitric oxide (NO) is involved. A confluent EC monolayer was subjected to steady-state, laminar flow with WSS ranging from 15 to 100 dyn/cm2 for 24 and 48 h. Cells oriented to the direction of the flow with a time course that varied with WSS. At 48 h, all cells were aligned with the flow. EC proliferation was examined using bromodeoxyuridine (BrdU) incorporation. The percentage of proliferating ECs rose linearly from 15 to 50 dyn/cm2 to more than sixfold at 50–100 dyn/cm2 compared with the accepted physiological baseline of 15–20 dyn/cm2. In addition, terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) staining revealed that apoptosis decreased with increasing WSS. These results demonstrate that high WSS stimulates EC proliferation and suppresses apoptosis. Furthermore, immunostaining revealed increased endothelial nitric oxide synthase (eNOS) production with increasing WSS. NOS inhibition with Nω-nitro-l-arginine methyl ester (l-NAME) drastically reduced the WSS-stimulated proliferation, indicating a critical role of NO production in the stimulation of EC proliferation by high WSS.

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

2008 ◽  
Vol 294 (1) ◽  
pp. C295-C305 ◽  
Author(s):  
James White ◽  
Theresa Guerin ◽  
Hollie Swanson ◽  
Steven Post ◽  
Haining Zhu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Myristic Acid ◽  
Acetylcholine Receptors ◽  
Diabetic Mouse ◽  
No Synthase ◽  
Diabetic Patients ◽  
No Production ◽  
Dependent Manner ◽  
Endothelial No Synthase ◽  
Stimulation Of

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

Role of G proteins in shear stress-mediated nitric oxide production by endothelial cells

1994 ◽  
Vol 267 (3) ◽  
pp. C753-C758 ◽  
Author(s):  
M. J. Kuchan ◽  
H. Jo ◽  
J. A. Frangos
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
G Protein ◽  
G Proteins ◽  
No Production ◽  
Dependent Manner ◽  
Protein Activation ◽  
Synthase Inhibitor

Exposure of cultured endothelial cells to shear stress resulting from well-defined fluid flow stimulates the production of nitric oxide (NO). We have established that an initial burst in production is followed by sustained steady-state NO production. The signal transduction events leading to this stimulation are not well understood. In the present study, we examined the role of regulatory guanine nucleotide binding proteins (G proteins) in shear stress-mediated NO production. In endothelial cells not exposed to shear stress, AIF4-, a general activator of G proteins, markedly elevated the production of guanosine 3',5'-cyclic monophosphate (cGMP). Pretreatment with NO synthase inhibitor N omega-nitro-L-arginine completely blocked this stimulation. Incubation with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a general G protein inhibitor, blocked the flow-mediated burst in cGMP production in a dose-dependent manner. Likewise, GDP beta S inhibited NOx (NO2 + NO3) production for the 1st h. However, inhibition was not detectable between 1 and 3 h. Pertussis toxin (PTx) had no effect on the shear response at any time point. The burst in NO production caused by a change in shear stress appears to be dependent on a PTx-refractory G protein. Sustained shear-mediated production is independent of G protein activation.

VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells

1998 ◽  
Vol 274 (3) ◽  
pp. H1054-H1058 ◽  
Author(s):  
John D. Hood ◽  
Cynthia J. Meininger ◽  
Marina Ziche ◽  
Harris J. Granger
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Northern Blotting ◽  
No Production ◽  
Vascular Endothelial ◽  
Umbilical Vein Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Growth Factor ◽  
Stimulation Of

Vascular endothelial growth factor (VEGF) is an endothelium-specific secreted protein that potently stimulates vasodilation, microvascular hyperpermeability, and angiogenesis. Nitric oxide (NO) is also reported to modulate vascular tone, permeability, and capillary growth. Therefore, we hypothesized that VEGF might regulate endothelial production of NO. The production of nitrogen oxides by human umbilical vein endothelial cells (HUVECs) was measured after 1, 12, 24, and 48 h of incubation with VEGF. VEGF treatment resulted in both an acute (1 h) and chronic (>24 h) stimulation of NO production. Furthermore, Western and Northern blotting revealed a VEGF-elicited, dose-dependent increase in the cellular content of endothelial cell nitric oxide synthase (ecNOS) message and protein that may account for the chronic upregulation of NO production elicited by VEGF. Finally, endothelial cells pretreated with VEGF for 24 h and subsequently exposed to A-23187 for 1 h produced NO at approximately twice the rate of cells that were not pretreated with VEGF. We conclude that VEGF upregulates ecNOS enzyme and elicits a biphasic stimulation of endothelial NO production.

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