scholarly journals L-sepiapterin restores SLE serum-induced markers of endothelial function in endothelial cells

2019 ◽  
Vol 6 (1) ◽  
pp. e000294 ◽  
Author(s):  
Joy N Jones Buie ◽  
Dorea Pleasant Jenkins ◽  
Robin Muise-Helmericks ◽  
Jim C Oates
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Mrna Expression ◽  
Neutrophil Migration ◽  
Basal Medium ◽  
Neutrophil Adhesion ◽  
No Production ◽  
Functional Changes ◽  
Enos Mrna ◽  
The Impact

ObjectiveSLE serves as an independent risk factor` for endothelial dysfunction (ED) not explained by Framingham risk factors. We sought to understand the development of SLE-induced ED on a cellular level in order to develop strategies aimed at reversing cellular abnormalities. This study assessed the impact of SLE patient serum on endothelial nitric oxide synthase (eNOS), nitric oxide (NO) production and functional changes in the cell.MethodsHuman umbilical vein endothelial cells (HUVECs) cultured in serum of either SLE (n=25) or healthy patients (n=14) or endothelial basal medium 2 (EBM-2) culture media supplemented with fetal bovine serum with or without L-sepiapterin were used for our studies. We applied the fluorescent probe DAF-FM diacetate for intracellular NO detection using flow cytometry. Total RNA isolates were analysed using reverse transcription PCR for eNOS mRNA expression. Oxygen consumption rate was determined using seahorse analysis. Neutrophil adhesion and migration were determined using a calcein AM microscopy assay.ResultsThe mRNA expression of eNOS was increased in SLE cultured HUVECs compared with healthy control (p<0.05). The SLE eNOS mRNA level correlated with SLE patient age (p=0.008); however, this trend was not observed with healthy patients. SLE serum reduced NO production in HUVECs compared with EBM-2 cultured cells (p<0.05). Co-treatment of endothelial cells with L-sepiapterin preserved HUVEC capacity to produce NO in SLE conditions (p<0.01). SLE serum enhanced neutrophil migration (p<0.01) but not neutrophil adhesion compared with healthy controls. The bioenergetic health index was not different.ConclusionsSLE likely causes disruption of endothelial cell eNOS function and NO modulated pathways.

Endothelial nitric oxide production during in vitro simulation of external limb compression

2002 ◽  
Vol 282 (6) ◽  
pp. H2066-H2075 ◽  
Author(s):  
Guohao Dai ◽  
Olga Tsukurov ◽  
Michael Chen ◽  
Jonathan P. Gertler ◽  
Roger D. Kamm
Keyword(s):  
Nitric Oxide ◽  
Mrna Expression ◽  
Umbilical Vein ◽  
Control Group ◽  
No Production ◽  
Vein Thrombosis ◽  
Enos Mrna ◽  
Umbilical Vein Endothelial Cells ◽  
Endothelial Nitric Oxide

External pneumatic compression (EPC) is effective in preventing deep vein thrombosis (DVT) and is thought to alter endothelial thromboresistant properties. We investigated the effect of EPC on changes in nitric oxide (NO), a critical mediator in the regulation of vasomotor and platelet function. An in vitro cell culture system was developed to simulate flow and vessel collapse conditions under EPC. Human umbilical vein endothelial cells were cultured and subjected to tube compression (C), pulsatile flow (F), or a combination of the two (FC). NO production and endothelial nitric oxide synthase (eNOS) mRNA expression were measured. The data demonstrate that in the F and FC groups, there is a rapid release of NO followed by a sustained increase. NO production levels in the F and FC groups were almost identical, whereas the C group produced the same low amount of NO as the control group. Conditions F and FC also upregulate eNOS mRNA expression by a factor of 2.08 ± 0.25 and 2.11 ± 0.21, respectively, at 6 h. Experiments with different modes of EPC show that NO production and eNOS mRNA expression respond to different time cycles of compression. These results implicate enhanced NO release as a potentially important factor in the prevention of DVT.

scholarly journals Influence of elastin-derived peptides, glucose, LDL and oxLDL on nitric oxide synthase expression in human umbilical artery endothelial cells.

2011 ◽  
Vol 58 (3) ◽  
Author(s):  
Wojciech Garczorz ◽  
Tomasz Francuz ◽  
Jan Gmiński ◽  
Wirginia Likus ◽  
Krzysztof Siemianowicz ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Mrna Expression ◽  
Umbilical Artery ◽  
Oxidized Ldl ◽  
Enos Gene ◽  
Human Umbilical Artery ◽  
Enos Mrna ◽  
Oxide Synthase ◽  
Enos Protein

Endothelial dysfunction plays an important role in the development of atherosclerosis. Elastin-derived peptides (EDP), hyperglycemia, hypercholesterolemia and oxidized LDL have a proven proatherosclerotic potential. Nitric oxide generated by endothelial nitric oxide synthase (eNOS; EC 1.14.13.39) is an important vasorelaxant. Here we studied the influence of those proatherosclerotic factors on eNOS gene and protein expression in artery-derived endothelial cells. Human umbilical artery endothelial cells (HUAEC) were incubated with or without: glucose (270 mg/dl), LDL (200 mg/dl), oxidized LDL (oxLDL 25 mg/dl) or κ-elastin (0.5 and 2.5 µg/ml). Gene expression was assessed by real time RT-PCR, whilst the eNOS protein by ELISA. In cells incubated with 2.5 µg/ml of κ-elastin, a 31 % increase of eNOS mRNA expression was observed, but the protein level remained unchanged. OxLDL, LDL and glucose decreased the eNOS protein level by 74 %, 37 % and 29 %, respectively. OxLDL decreased eNOS mRNA by 42 %. LDL non-significantly decreased eNOS mRNA expression. An elevated glucose level did not affect the eNOS mRNA expression. Hyperglycemia and an elevated level of LDL, particularly oxLDL, decreased the level of eNOS protein in endothelial cells. As κ-elastin did not decrease the expression of eNOS gene in HUAEC, the proatherogenic properties of elastin-derived peptides are unlikely to be due to their influence on eNOS.

scholarly journals Repeated and long-term treatment with physiological concentrations of resveratrol promotes NO production in vascular endothelial cells

2011 ◽  
Vol 107 (6) ◽  
pp. 774-780 ◽  
Author(s):  
Satoru Takahashi ◽  
Yukiko Nakashima
Keyword(s):  
Endothelial Cells ◽  
Mrna Expression ◽  
Term Treatment ◽  
No Synthase ◽  
No Production ◽  
Dependent Manner ◽  
Long Term Treatment ◽  
Enos Mrna ◽  
Enos Protein

In the present study, we examined the effect of repeated and long-term treatment with resveratrol on NO production in endothelial cells as a model of routine wine consumption. Repeated treatment with resveratrol for 5 d resulted in an increase in endothelial NO synthase (eNOS) protein content and NO production in human umbilical vein endothelial cell (HUVEC) in a concentration-dependent manner. A significant increase in functional eNOS protein content was observed with resveratrol, even at 50 nm. In contrast, eNOS phosphorylation was not stimulated and inducible NO synthase (iNOS) was not detected after resveratrol treatment. Both eNOS protein and mRNA expression were promoted by 50 nm-resveratrol in a time-dependent manner. Increased eNOS mRNA expression in response to resveratrol was not decreased by an oestrogen receptor (ER) antagonist ICI182780, a PPARα inhibitor MK886 or a sirtuin inhibitor Salermide. However, a combination of ICI182780 and MK886 significantly inhibited resveratrol-induced eNOS mRNA expression. Salermide had no effect even in the presence of ICI182780 or MK886. These results demonstrate that resveratrol within the physiological range increases eNOS mRNA and protein expression through ER and PPARα activation, thereby promoting NO production in endothelial cells. eNOS induction might result from the accumulative effect of nanomolar concentrations of resveratrol. The present study results can account in part for the observation that cardiovascular benefits of red wine are experienced with routine consumption, but not with acute consumption.

IL-4 stimulates the expression of CXCL-8, E-selectin, VEGF, and inducible nitric oxide synthase mRNA by equine pulmonary artery endothelial cells

2007 ◽  
Vol 292 (5) ◽  
pp. L1147-L1154 ◽  
Author(s):  
Hong Huang ◽  
Anouk Lavoie-Lamoureux ◽  
Kantuta Moran ◽  
Jean-Pierre Lavoie
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Mrna Expression ◽  
Inducible Nitric Oxide Synthase ◽  
Neutrophil Migration ◽  
Pulmonary Arteries ◽  
Dependent Manner ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Little is known concerning the possible contribution of T helper 2 (Th2)-type cytokines to the recruitment of neutrophils into the lung tissue. In the present study, endothelial cells from equine pulmonary arteries were cultured in the presence of recombinant equine (re) IL-4 and reIL-5, and the cytokine mRNA expression of molecules implicated in the chemotaxis and migration of neutrophils was studied using real-time RT-PCR. The functional response of reIL-4-induced endothelial cell stimulation on neutrophil migration was also studied using a chemotaxis chamber. ReIL-4 either increased the expression of CXCL-8, E-selectin, vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS), or potentiated the coeffects of lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α) on CXCL-8. Supernatants collected from cultured endothelial cells stimulated with reIL-4 significantly promoted neutrophil migration in a dose-dependent manner. Dexamethasone (DXM) decreased the expression of CXCL-8, VEGF, and iNOS induced by reIL-4, while 1400W dihydrochloride (1400W), a selective inhibitor of iNOS, decreased the expression of E-selectin, VEGF, and iNOS. DXM and 1400W attenuated the mRNA expression of E-selectin and iNOS induced by the costimulation of reIL-4, reTNF-α, and LPS. Neither equine nor human recombinant IL-5 influenced the mRNA expression of CXCL-8, E-selectin, or VEGF. These findings suggest that Th2-type cytokines may contribute to pulmonary neutrophilia during allergic inflammation by the increased expression of neutrophil chemokines and adhesion molecules by endothelial cells. DXM and the iNOS inhibitors may decrease pulmonary neutrophilia due, in part, to a direct inhibition of some of these factors.

scholarly journals Thyroid hormones stimulate L-arginine transport in human endothelial cells

2018 ◽  
Vol 239 (1) ◽  
pp. 49-62 ◽  
Author(s):  
Marta Toral ◽  
Rosario Jimenez ◽  
Sebastián Montoro-Molina ◽  
Miguel Romero ◽  
Rosemary Wangensteen ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Thyroid Hormones ◽  
Intracellular Calcium ◽  
Signaling Pathways ◽  
No Production ◽  
Αvβ3 Integrin ◽  
Arginine Uptake ◽  
Intracellular Ca ◽  
Enos Mrna

Thyroid hormone activity is associated with L-arginine metabolism and nitric oxide (NO) production, which participate in the cardiovascular manifestations of thyroid disorders. L-arginine transporters play an important role in activating L-arginine uptake and NO production. However, the effects of thyroid hormones on L-arginine transporters in endothelial cells have not yet been evaluated. The following methods were used. We measured L-arginine uptake, mRNA expression of L-arginine transporters, endothelial nitric oxide synthase (eNOS) mRNA and NO generation after the administration of T3, T4 and the T3 analog, 3,3′,5-triiodothyroacetic acid TRIAC in human umbilical vein endothelial cells (HUVECs). We also analyzed the role of αvβ3 integrin and of phosphatidyl-inositol-3 kinase (PI3K), mitogen-activated protein kinases (MAPKs: ERK1/2, p38 and SAPK-JNK) and intracellular calcium signaling pathways as underlying mechanisms. To this end, αvβ3 integrin was pharmacologically inhibited by tetraiodothyroacetic acid (TETRAC) or genetically blocked by silencing αv mRNA and PI3K, MAPKs and intracellular calcium by selective inhibitors. The following results were obtained. Thyroid hormones and the T3 analog TRIAC increased L-arginine uptake in HUVECs, the sodium-independent y+/CAT isoforms, except CAT2b, sodium-dependent y+L system and sodium-independent system b0,+L-arginine transporters, eNOS mRNA and NO production. These effects were suppressed by αvβ3 integrin inhibition with TETRAC or αv integrin downregulation or by PI3K, MAPK or intracellular Ca2+ signaling inhibitors. In conclusion, we report for the first time that activation of L-arginine uptake by thyroid hormones is related to an upregulation of L-arginine transporters. This effect seems to be mediated by activation of αvβ3 integrin receptor and subsequent PI3K, MAPK and intracellular Ca2+ signaling pathways.

scholarly journals Effects of chitosan on nitric oxide production and inducible nitric oxide synthase activity and mRNA expression in weaned piglets

2018 ◽  
Vol 60 (No. 8) ◽  
pp. 359-366
Author(s):  
J. Li ◽  
B. Shi ◽  
S. Yan ◽  
L. Jin ◽  
Y. Guo ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Mrna Expression ◽  
Inducible Nitric Oxide Synthase ◽  
No Production ◽  
Weaned Piglets ◽  
Inos Activity ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

The effects of chitosan on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) activity and gene expression in vivo or vitro were investigated in weaned piglets. In vivo, 180 weaned piglets were assigned to five dietary treatments with six replicates. The piglets were fed on a basal diet supplemented with 0 (control), 100, 500, 1000, and 2000 mg chitosan/kg feed, respectively. In vitro, the peripheral blood mononuclear cells (PBMCs) from a weaned piglet were cultured respectively with 0 (control), 40, 80, 160, and 320 &micro;g chitosan/ml medium. Results showed that serum NO concentrations on days 14 and 28 and iNOS activity on day 28 were quadratically improved with increasing chitosan dose (P &lt; 0.05). The iNOS mRNA expressions were linearly or quadratically enhanced in the duodenum on day 28, and were improved quadratically in the jejunum on days 14 and 28 and in the ileum on day 28 (P &lt; 0.01). In vitro, the NO concentrations, iNOS activity, and mRNA expression in unstimulated PBMCs were quadratically enhanced by chitosan, but the improvement of NO concentrations and iNOS activity by chitosan were markedly inhibited by N-(3-[aminomethyl] benzyl) acetamidine (1400w) (P&nbsp;&lt; 0.05). Moreover, the increase of NO concentrations, iNOS activity, and mRNA expression in PBMCs induced by lipopolysaccharide (LPS) were suppressed significantly by chitosan (P &lt; 0.05). The results indicated that the NO concentrations, iNOS activity, and mRNA expression in piglets were increased by feeding chitosan in a dose-dependent manner. In addition, chitosan improved the NO production in unstimulated PBMCs but inhibited its production in LPS-induced cells, which exerted bidirectional regulatory effects on the NO production via modulated iNOS activity and mRNA expression.

scholarly journals Hydrogen Sulfide Increases Nitric Oxide Production and Subsequent S-Nitrosylation in Endothelial Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Ping-Ho Chen ◽  
Yaw-Syan Fu ◽  
Yun-Ming Wang ◽  
Kun-Han Yang ◽  
Danny Ling Wang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Hydrogen Sulfide ◽  
Protein Kinase ◽  
Fluorescent Probe ◽  
Acid Methyl Ester ◽  
High Specificity ◽  
Protein S ◽  
No Production ◽  
No Donor

Hydrogen sulfide (H2S) and nitric oxide (NO), two endogenous gaseous molecules in endothelial cells, got increased attention with respect to their protective roles in the cardiovascular system. However, the details of the signaling pathways between H2S and NO in endothelia cells remain unclear. In this study, a treatment with NaHS profoundly increased the expression and the activity of endothelial nitric oxide synthase. Elevated gaseous NO levels were observed by a novel and specific fluorescent probe, 5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid methyl ester (FA-OMe), and quantified by flow cytometry. Further study indicated an increase of upstream regulator for eNOS activation, AMP-activated protein kinase (AMPK), and protein kinase B (Akt). By using a biotin switch, the level of NO-mediated protein S-nitrosylation was also enhanced. However, with the addition of the NO donor, NOC-18, the expressions of cystathionine-γ-lyase, cystathionine-β-synthase, and 3-mercaptopyruvate sulfurtransferase were not changed. The level of H2S was also monitored by a new designed fluorescent probe, 4-nitro-7-thiocyanatobenz-2-oxa-1,3-diazole (NBD-SCN) with high specificity. Therefore, NO did not reciprocally increase the expression of H2S-generating enzymes and the H2S level. The present study provides an integrated insight of cellular responses to H2S and NO from protein expression to gaseous molecule generation, which indicates the upstream role of H2S in modulating NO production and protein S-nitrosylation.

Arginase inhibition increases nitric oxide production in bovine pulmonary arterial endothelial cells

2004 ◽  
Vol 287 (1) ◽  
pp. L60-L68 ◽  
Author(s):  
Louis G. Chicoine ◽  
Michael L. Paffett ◽  
Tamara L. Young ◽  
Leif D. Nelin
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
No Synthase ◽  
No Production ◽  
Urea Production ◽  
Pulmonary Arterial ◽  
Factor Α ◽  
Arterial Endothelial Cells ◽  
Regular Medium ◽  
Necrosis Factor

Nitric oxide (NO) is produced by NO synthase (NOS) from l-arginine (l-Arg). Alternatively, l-Arg can be metabolized by arginase to produce l-ornithine and urea. Arginase (AR) exists in two isoforms, ARI and ARII. We hypothesized that inhibiting AR with l-valine (l-Val) would increase NO production in bovine pulmonary arterial endothelial cells (bPAEC). bPAEC were grown to confluence in either regular medium (EGM; control) or EGM with lipopolysaccharide and tumor necrosis factor-α (L/T) added. Treatment of bPAEC with L/T resulted in greater ARI protein expression and ARII mRNA expression than in control bPAEC. Addition of l-Val to the medium led to a concentration-dependent decrease in urea production and a concentration-dependent increase in NO production in both control and L/T-treated bPAEC. In a second set of experiments, control and L/T bPAEC were grown in EGM, EGM with 30 mM l-Val, EGM with 10 mM l-Arg, or EGM with both 10 mM l-Arg and 30 mM l-Val. In both control and L/T bPAEC, treatment with l-Val decreased urea production and increased NO production. Treatment with l-Arg increased both urea and NO production. The addition of the combination l-Arg and l-Val decreased urea production compared with the addition of l-Arg alone and increased NO production compared with l-Val alone. These data suggest that competition for intracellular l-Arg by AR may be involved in the regulation of NOS activity in control bPAEC and in response to L/T treatment.

scholarly journals Decreased endothelial nitric oxide, systemic oxidative stress, and increased sympathetic modulation contribute to hypertension in obese rats

2014 ◽  
Vol 306 (10) ◽  
pp. H1472-H1480 ◽  
Author(s):  
Natalia Veronez da Cunha ◽  
Phileno Pinge-Filho ◽  
Carolina Panis ◽  
Bruno Rodrigues Silva ◽  
Laena Pernomian ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Lipid Peroxidation ◽  
Endothelial Cells ◽  
Arterial Pressure ◽  
Vascular Reactivity ◽  
Monosodium Glutamate ◽  
Saline Control ◽  
Pulse Interval ◽  
No Production ◽  
Obese Rats

We investigated the involvement of nitric oxide (NO) and reactive oxygen species (ROS) on autonomic cardiovascular parameters, vascular reactivity, and endothelial cells isolated from aorta of monosodium glutamate (MSG) obese rats. Obesity was induced by administration of 4 mg/g body wt of MSG or equimolar saline [control (CTR)] to newborn rats. At the 60th day, the treatment was started with NG-nitro-l-arginine methyl ester (l-NAME, 20 mg/kg) or 0.9% saline. At the 90th day, after artery catheterization, mean arterial pressure (MAP) and heart rate were recorded. Plasma was collected to assess lipid peroxidation. Endothelial cells isolated from aorta were evaluated by flow cytometry and fluorescence intensity (FI) emitted by NO-sensitive dye [4,5-diaminofluoresceindiacetate (DAF-2DA)] and by ROS-sensitive dye [dihydroethidium (DHE)]. Vascular reactivity was made by concentration-response curves of acetylcholine. MSG showed hypertension compared with CTR. Treatment with l-NAME increased MAP only in CTR. The MSG induced an increase in the low-frequency (LF) band and a decrease in the high-frequency band of pulse interval. l-NAME treatment increased the LF band of systolic arterial pressure only in CTR without changes in MSG. Lipid peroxidation levels were higher in MSG and were attenuated after l-NAME. In endothelial cells, basal FI to DAF was higher in CTR than in MSG. In both groups, acetylcholine increased FI for DAF from basal. The FI baseline to DHE was higher in MSG than in CTR. Acetylcholine increased FI to DHE in the CTR group, but decreased in MSG animals. We suggest that reduced NO production and increased production of ROS may contribute to hypertension in obese MSG animals.

Sign in / Sign up

Export Citation Format

Share Document