scholarly journals Flow Cytometric Evaluation of Human Neutrophil Apoptosis During Nitric Oxide Generation In Vitro: The Role of Exogenous Antioxidants

2005 ◽  
Vol 2005 (2) ◽  
pp. 81-87 ◽  
Author(s):  
Zofia Sulowska ◽  
Ewa Majewska ◽  
Magdalena Klink ◽  
Malgorzata Banasik ◽  
Henryk Tchórzewski
Keyword(s):  
Nitric Oxide ◽  
Human Neutrophil ◽  
Flow Cytometric Analysis ◽  
Human Neutrophils ◽  
Neutrophil Apoptosis ◽  
No Donor ◽  
No Donors ◽  
Flow Cytometric

Among numerous inflammatory mediators a nitric oxide molecule is supposed to be important in the modulation of neutrophil survival in vivo and in vitro. The effect of exogenous supply of NO donors such as SNP, SIN-1, and GEA-3162 on the course of human neutrophil apoptosis and the role of extracellular antioxidants in this process was investigated. Isolated from peripheral blood, neutrophils were cultured in the presence or absence of NO donor compounds and antioxidants for 8, 12, and20hours. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V protein binding to the cell surface. Exposure of human neutrophils to GEA-3162 and SIN-1 significantly accelerates and enhances their apoptosis in vitro in a time-dependent fashion. In the presence of SNP, intensification of apoptosis has not been revealed until12hours after the culture. The inhibition of GEA-3162- and SIN-1-mediated neutrophil apoptosis by superoxide dismutase (SOD) but not by catalase (CAT) was observed. Our results show that SOD and CAT can protect neutrophils against NO-donors-induced apoptosis and suggest that the interaction of NO and oxygen metabolites signals may determine the destructive or protective role of NO donor compounds during apoptotic neutrophil death.

scholarly journals Involvement of nitric oxide donor compounds in the bactericidal activity of human neutrophils in vitro

2003 ◽  
Vol 52 (4) ◽  
pp. 303-308 ◽  
Author(s):  
Magdalena Klink ◽  
Maciej Cedzyński ◽  
Anna St Świerzko ◽  
Henryk Tchórzewski ◽  
Zofia Sulowska
Keyword(s):  
Nitric Oxide ◽  
Coronary Heart Disease ◽  
Heart Disease ◽  
Bactericidal Activity ◽  
Nitric Oxide Donor ◽  
Human Neutrophils ◽  
Bacterial Strains ◽  
No Donor ◽  
No Donors

The bactericidal activity of human neutrophils against extracellular and facultatively intracellular bacteria was studied in the presence of the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), a molsidomine metabolite. SNP and molsidomine are drugs commonly used as nitrovasodilators in coronary heart disease. It is demonstrated here that the NO donor compounds themselves did not affect the viability and survival of the bacterial strains tested. Neither SNP nor SIN-1 had any effect on the process of bacteria ingestion. In contrast, NO donors enhanced the ability of neutrophils to kill Escherichia coli, Proteus vulgaris and Salmonella Anatum. However, strains differed in their susceptibility to SNP- and SIN-1-mediated killing by neutrophils. Removal of the superoxide anion reduced the bactericidal activity of SNP- and SIN-1-treated neutrophils against E. coli and S. Anatum. This suggests that the NO derivatives formed in the reaction of NO generated from donors with the reactive oxygen species released by phagocytosed neutrophils potentiate the bactericidal activity of human neutrophils in vitro. The above original observation discussed here suggests clinical significance for the treatment of patients with nitrovasodilators in the course of coronary heart disease therapy.

scholarly journals Elevated FOXC2 Expression Promotes Invasion of HCC Cell Lines and is Associated with Poor Prognosis in Hepatocellular Carcinoma

2017 ◽  
Vol 44 (1) ◽  
pp. 99-109 ◽  
Author(s):  
Fang Yang ◽  
Lizhi Lv ◽  
Kun Zhang ◽  
Qiucheng Cai ◽  
Jianyong Liu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometric Analysis ◽  
Vital Role ◽  
Migration And Invasion ◽  
Flow Cytometric ◽  
Kaplan Meier ◽  
Proliferation Assays ◽  
The Relationship

Background/Aims: Increasing evidence has indicated that Forkhead box protein C2 (FOXC2) plays an important role in carcinogenesis. However, the expression and the role of FOXC2 in hepatocellular carcinoma (HCC) have not been extensively studied. Methods: FOXC2 expression was analyzed by quantitative real-time polymerase chain reaction, Western blot analysis and immunohistochemistry in HCC tissue and cells. The relationship between FOXC2 expression and patient clinical significance and survival were assessed by Pearson’s correlation and Kaplan-Meier analysis, respectively. Cell proliferation assays, colony formation assays, flow cytometric analysis and Transwell assays were employed to measure the effects of FOXC2 on HCC cells in vitro. Results: The expression of FOXC2 was increased in HCC tissue, and high FOXC2 expression was associated with worse patient survival. Knockdown of FOXC2 inhibited HCC cell growth, migration, and invasion in vitro, as well as tumor growth. Furthermore, we found that activation of AKT-mediated MMP-2 and MMP-9 was involved in FOXC2 promoting an aggressive phenotype. Conclusions: Taken together, these findings demonstrate that FOXC2 is upregulated in HCC tissue and is associated with tumor size, vascular invasion and advanced TNM stage. Further investigation suggested that FOXC2 may play a vital role in promoting proliferation and invasion in HCC and serves as a novel therapeutic target in HCC.

scholarly journals Nitric oxide synthase acutely regulates progesterone production by in vitro cultured rabbit corpora lutea

1999 ◽  
Vol 160 (2) ◽  
pp. 275-283 ◽  
Author(s):  
A Gobbetti ◽  
C Boiti ◽  
C Canali ◽  
M Zerani
Keyword(s):  
Nitric Oxide ◽  
In Vitro Release ◽  
Inhibitory Effect ◽  
Corpora Lutea ◽  
No Donor ◽  
Nos Inhibitor ◽  
Oxide Synthase ◽  
Vitro Release

We examined the presence and the regulation of nitric oxide (NO) synthase (NOS) using in vitro cultured corpora lutea (CL) obtained from rabbits at days 4 and 9 of pseudopregnancy. The role of NO and NOS on steroidogenesis was also investigated using the same CL preparations after short-term incubations (30 min and 2 h) with the NO donor, sodium nitroprusside (NP), the NOS inhibitor, Nomega-nitro-l-arginine methyl ester (l-NAME) and prostaglandin (PG) F-2alpha. The basal NOS activity was greater in CL at day 4 than at day 9, and was also differently modulated by PGF-2alpha, depending on the age of the CL. The addition of PGF-2alpha to day 4 CL had no effect, but PGF-2alpha on day 9 caused a threefold increase in NOS activity. NP caused a two- to fivefold decrease in release of progesterone from CL of both ages, and this inhibitory effect on steroidogenesis was reversed by l-NAME. All treatments failed to modify basal androgens and 17beta-oestradiol was not detectable in either control or treated CL. These results suggest that NO is effectively involved in the regulation process of steroidogenesis, independently of 17beta-oestradiol. PGF-2alpha had no effect on day 4, but induced luteolysis on day 9, by reducing progesterone (P</=0. 01) to about 18% of control. The luteolytic action of PGF-2alpha was completely reversed by co-incubation with l-NAME, thus supporting the hypothesis that luteolysis is mediated by NO. The addition of NP or l-NAME did not modify the in vitro release of PGF-2alpha. We hypothesised that PGF-2alpha upregulates NOS activity and, consequently, the production of NO, which acutely inhibits progesterone release from day 9 CL of pseudopregnant rabbits.

Restoring Oxygen Carrying Capacity of Sickle Erythrocytes with Nitric Oxide Donors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3785-3785
Author(s):  
Borys Hrinczenko
Keyword(s):  
Nitric Oxide ◽  
Oxygen Affinity ◽  
Red Cell ◽  
No Donors ◽  
Sickle Erythrocytes ◽  
Low Levels ◽  
Pre Treatment ◽  
Normal Controls

Abstract Sickle cell anemia (SCA) is an inherited blood disorder of hemoglobin function. A genetic mutation results in the substitution of a valine for glutamic acid residue at position 6 of the beta-globin chain yielding the mutant hemoglobin S (HbS). HbS polymerizes within erythrocytes during deoxygenation resulting in altered affinity of oxygen binding. The slightly different P50 (PO2 at which Hb is half-saturated with oxygen) values of sickle erythrocytes obtained during either oxygenation or deoxygenation (hysteresis) demonstrate HbS polymerization induced inhibition of oxygen affinity. Nitric oxide (NO) has been found to be an important signaling molecule in the circulatory system. NO derivatives of Hb provide insights into the physiological role of Hb. NO can bind to Hb at either the heme moiety forming nitrosylhemoglobin (HbNO) or to the conserved beta-93 cysteine yielding S-nitrosohemoglobin (SNO-Hb). In deoxygenated venous blood NO preferentially binds to the hemes of Hb forming HbNO while in oxygenated arterial blood NO binds to the beta-93 cysteine residues forming SNO-Hb. Increased oxygen affinity is seen in both SNO-Hb (Bonaventura C, et al, 1999) and also with HbNO. Decreasing the HbS P50 inhibits intra-erythrocyte HbS polymerization that may be an effective strategy to treat SCA. Clinical trials of NO breathing effects on oxygen affinity are conflicting. One study found an increased oxygen affinity of blood from SCA patients breathing 80 ppm NO with no effect seen in normal controls (Head A, et al, 1997). Another study found that levels of NO bound to Hb are too low to affect overall oxygen affinity (Gladwin M, et al, 1999). The purpose of this in vitro study was to determine the oxygen affinity of deoxygenated sickle erythrocytes pre-treated with exogenous NO donors. Blood from SCA (HbSS) and normal controls (HbAA) were collected and suspended in PBS buffer and deoxygenated with argon gas. The Hb concentration of each sample was calculated and then was either left untreated (control) or treated with varying concentrations of NO donors. The NO donors included: 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO), S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside (SNP), an aqueous solution of NO, and sodium trioxodinitrate (Angeli’s salt, AS). Methemoglobin and protein degradation were negligible. Samples were then transferred via airtight syringes into a stirred and temperature controlled (37°C) chamber of PBS solution at ambient oxygen pressure fitted with a very sensitive oxygen electrode. Oxygen levels were measured in real time. The amount of oxygen extracted from the PBS medium followed first order kinetics. Studies with HbSS red cell suspensions showed that the largest increment in oxygen extraction from the medium was obtained with DEANO pre-treatment. Calculations indicated that low levels of NO treatment, at approximately a 1:1000 ratio of [NO]/[heme], yielded the largest oxygen consumption. The effects of pre-treatment with the other NO donors on sickle erythrocytes (HbSS) were not as pronounced. DEANO is an NO donor yielding a “pure” NO radical as opposed to other redox forms. Similar studies with HbAA and HbSC did not show increases in oxygen extraction. Taken together the data suggest that low levels of NO perturb the quaternary structure of intraerythrocyte HbS polymer allowing depolymerization and increased oxygen affinity. The hope is that these in vitro studies will better characterize the role of NO in its interactions with Hb and the red cell and to use this knowledge for potential therapies in diseases such as SCA.

Role of BKCa Channels in Cephalic Vasodilation Induced by CGRP, NO and Transcranial Electrical Stimulation In The Rat

Cephalalgia ◽  
2007 ◽  
Vol 27 (10) ◽  
pp. 1120-1127 ◽  
Author(s):  
A Gozalov ◽  
I Jansen-Olesen ◽  
D Klaerke ◽  
J Olesen
Keyword(s):  
Nitric Oxide ◽  
Electrical Stimulation ◽  
Selective Inhibitor ◽  
Transcranial Electrical Stimulation ◽  
Bkca Channels ◽  
No Donor ◽  
Calcitonin Gene

Both calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are potent vasodilators that have been shown to induce headache in migraine patients. Their antagonists are effective in the treatment of migraine attacks. In the present study, we hypothesize that vasodilation induced by the NO donor glyceryltrinitrate (GTN) or by CGRP is partially mediated via large conductance calcium-activated potassium (BKCa) channels. The effects of the BKCa channel selective inhibitor iberiotoxin on dural and pial vasodilation induced by CGRP, GTN and endogenously released CGRP by transcranial electrical stimulation (TES) were examined. Iberiotoxin significantly attenuated GTN-induced dural and pial artery dilation in vivo and in vitro, but had no effect on vasodilation induced by CGRP and TES. Our results show that GTN- but not CGRP-induced dural and pial vasodilation involves opening of BKCa channels in rat.

scholarly journals Platelet CXCL4 mediates neutrophil extracellular traps formation in ANCA-associated vasculitis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kotaro Matsumoto ◽  
Hidekata Yasuoka ◽  
Keiko Yoshimoto ◽  
Katsuya Suzuki ◽  
Tsutomu Takeuchi
Keyword(s):  
Flow Cytometric Analysis ◽  
Neutrophil Extracellular Traps ◽  
Platelet Factor ◽  
Extracellular Traps ◽  
Flow Cytometric ◽  
Anca Associated Vasculitis ◽  
Tlr Agonist ◽  
Tlr9 Signaling

AbstractNeutrophils form neutrophil extracellular traps (NETs), which are involved in the pathogenesis of ANCA-associated vasculitis (AAV). Recent reports suggest that platelets stimulated via toll-like receptor (TLR) pathways can induce NETs formation. However, the mechanism underlying the involvement of platelets in NETs formation in AAV is unknown. We investigated the role of platelets in the pathogenesis of AAV. Platelets from AAV patients and healthy controls (HCs) were co-cultured with peripheral neutrophils, and NETs formation was visualized and quantified. The expression levels of TLRs on platelets were examined by flow cytometry. Platelets were treated with a TLR agonist, platelet-derived humoral factor, CXCL4 (platelet factor 4: PF4), and/or anti-CXCL4 antibody to investigate the effects of TLR–CXCL4 signaling on NETs formation. Platelets from AAV significantly upregulated NETs formation in vitro. Flow cytometric analysis revealed that the proportion of TLR9 positive platelets was significantly higher in AAV than HCs. CXCL4 released from TLR9 agonist-stimulated platelets was significantly enhanced in AAV, which subsequently increased NETs formation. Further, neutralizing anti-CXCL4 antibody significantly inhibited NETs formation enhanced by platelets from AAV. TLR9 signaling and CXCL4 release underlie the key role that platelets play in NETs formation in the pathogenesis of AAV.

scholarly journals Biphasic roles for soluble guanylyl cyclase (sGC) in platelet activation

Blood ◽  
2011 ◽  
Vol 118 (13) ◽  
pp. 3670-3679 ◽  
Author(s):  
Guoying Zhang ◽  
Binggang Xiang ◽  
Anping Dong ◽  
Radek C. Skoda ◽  
Alan Daugherty ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Guanylyl Cyclase ◽  
Soluble Guanylyl Cyclase ◽  
Whole Body ◽  
No Donor ◽  
No Donors ◽  
Deficient Mice

AbstractNitric oxide (NO) stimulates cGMP synthesis by activating its intracellular receptor, soluble guanylyl cyclase (sGC). It is a currently prevailing concept that No and cGMP inhibits platelet function. However, the data supporting the inhibitory role of NO/sGC/cGMP in platelets have been obtained either in vitro or using whole body gene deletion that affects vessel wall function. Here we have generated mice with sGC gene deleted only in megakaryocytes and platelets. Using the megakaryocyte- and platelet-specific sGC-deficient mice, we identify a stimulatory role of sGC in platelet activation and in thrombosis in vivo. Deletion of sGC in platelets abolished cGMP production induced by either NO donors or platelet agonists, caused a marked defect in aggregation and attenuated secretion in response to low doses of collagen or thrombin. Importantly, megakaryocyte- and platelet-specific sGC deficient mice showed prolonged tail-bleeding times and impaired FeCl3-induced carotid artery thrombosis in vivo. Interestingly, the inhibitory effect of the NO donor SNP on platelet activation was sGC-dependent only at micromolar concentrations, but sGC-independent at millimolar concentrations. Together, our data demonstrate important roles of sGC in stimulating platelet activation and in vivo thrombosis and hemostasis, and sGC-dependent and -independent inhibition of platelets by NO donors.

Role of the Hypoxic Bone Marrow Microenvironment in Multiple Myeloma Tumor Progression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2348-2348
Author(s):  
Kewal Asosingh ◽  
Hendrik De Raeve ◽  
Mark de Ridder ◽  
Guy A. Storme ◽  
Angelo Willems ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Mouse Model ◽  
Tumor Progression ◽  
Flow Cytometric Analysis ◽  
Surrogate Marker ◽  
Hypoxic Conditions ◽  
Flow Cytometric

Abstract Recently we reported that pre-clinical myeloma disease progression in the 5T2MM mouse model is characterized by predominant CD45+ MM-cells in the early, pre-angiogenic stage stage of slow tumor progression, followed by expansion of CD45− MM-cells during the subsequent angiogenic stage of progressive tumor growth. Unlike other cancer cells, multiple myeloma (MM) cells have to survive and to grow in a microenvironment which is already hypoxic by nature. This hypoxic bone marrow (BM) microenvironment is essential for normal hematopoiesis. However, the role of BM hypoxia in myeloma tumor progression is not known. Herein we addressed this topic in the 5T2MM mouse model. Flow cytometric analysis of control mice and 5T2MM diseased mice injected with pimonidazole hypoxyprobe indicated that both normal BM and myeloma infiltrated BM are hypoxic. However, in myelomatous BM the hypoxia was significantly decreased. Analysis of HIF-1a expression, a surrogate marker of hypoxia, by flow cytometry also demonstrated significantly lower levels of hypoxia in myeloma infiltrated BM. HIF-1a expression was found in 5T2MM-cells and was significantly higher compared to the non-tumor cell fraction. In vitro culturing of 5T2MM cells under hypoxic conditions, indicated increased activation of apoptosis inducing caspase-3 in the CD45− MM-fraction, but not in the CD45+ 5T2MM-cells, suggesting that native BM hypoxia selects the tumor population for tumor initiating CD45+ 5T2MM-cells. Although angiogeneic switch and angiogeneic heterogeneity has been reported in MM, the role of myeloma associated angiogensis is remains unclear. The decreased hypoxia in myeloma infiltrated BM adds strength to the hypothesis that myeloma associated neovascularization is functional by increasing BM oxygenation. The data also suggest that the angiogenesis allows expansion of CD45− 5T2MM-cells by decreasing BM hypoxia. All together, these findings suggest an important role of BM hypoxia in myeloma tumor progression.

Morphine Inhibits NF-κB Nuclear Binding in Human Neutrophils and Monocytes by a Nitric Oxide–dependent Mechanism

2000 ◽  
Vol 92 (6) ◽  
pp. 1677-1684 ◽  
Author(s):  
Ingeborg D. Welters ◽  
Axel Menzebach ◽  
Yannick Goumon ◽  
Patrick Cadet ◽  
Thilo Menges ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Transcriptional Activation ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
No Donor ◽  
Nuclear Binding ◽  
Flow Cytometric ◽  
No Release ◽  
Blood Neutrophils ◽  
Nf Kappab

Background The transcription factor NF-kappaB plays a pivotal role in gene expression of inflammatory mediators such as cytokines or adhesion molecules. NF-kappaB-mediated transcriptional activation of these genes is inhibited by nitric oxide (NO) in a variety of cells, including monocytes. Morphine mediates NO release in a naloxone antagonizable manner in monocytes and neutrophils. Methods The influence of morphine on NF-kappaB activation was investigated in a whole-blood flow cytometric assay. A specific antibody against the p65 subunit of NF-kappaB was used and detected by fluoresceine-isothiocyanate-labeled anti-immunoglobulin G. Nuclei were stained with propidium iodide. Leukocyte subpopulations were evaluated by gating on neutrophils and monocytes. The median fluorescence channel was determined. Different morphine concentrations (50 nm, 50 microm, 1 mm) and incubation intervals (10-150 min) were used. Results Morphine inhibits lipopolysaccharide-induced NF-kappaB nuclear binding in human blood neutrophils and monocytes in a time-, concentration-, and naloxone-sensitive-dependent manner. Similar effects were achieved with the NO donor S-nitroso-N-acetyl-pencillamine and the antioxidant N-acetyl-cysteine. The NO synthase inhibitors Nomega-nitro-l-arginine-methyl-esther and Nomega-nitro-l-arginine completely abolished the morphine-induced attenuation of NF-kappaB nuclear binding, demonstrating that the inhibitory action is mediated by NO release. Conclusion Morphine causes immunosuppression, at least in part, via the NO-stimulated depression of NF-kappaB nuclear binding.

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