Nuclear Transcription Factor NF Research Articles

FEATURES OF RELATIONS OF MELATONIN WITH THE STATE OF INTRACELLULAR REGULATORSOF THE FUNCTIONAL ACTIVITY OF WHOLE BLOOD MONONUCLEAR CELLS IN CORONARY HEART DISEASE

Introduction. Coronary heart disease (CHD), leading among the causes of death in adulthood and old age, is an urgent medical and social problem. The pathogenesis of most forms of coronary heart disease is based on stenosing atherosclerosis of the coronary arteries, which develops against the background of dyslipidemia and arterial hypertension and is accompanied by the activation of immunocompetent cells (ICCs) of the vascular wall with the development of a subclinical inflammatory reaction, as well as the production of pro-inflammatory factors such as interleukins, chemokines, growth factors and etc. In turn, ICC activity is determined by the state of their intracellular molecular cascades, which transmit signals into the cell and ensure its reactivity to various external stimuli, such as mitogens, cytokines, pathogen components, etcIt has been shown that the central nervous system plays an important role in the regulation of ICC activity due to the production of neurohumoral molecules, such as melatonin, endorphin, sero-tonin, etc., which ensure the coordination of immune responses and their control by the central nervous system. The aim of this study was to study the relationship between melatonin production and intracellular factors that regulate the pro-inflammatory activi-ty of whole blood mononuclear cells and their metabolism in patients with coronary artery disease. Material and methods. As part of the cohort study, 58 patients of both sexes with coronary artery disease aged 49 to 67 years and 20 practically healthy individuals of both sexes were examined. In accordance with the purpose of the study, the concentration of focal adhesion protein kinase (FAK), 5'AMP-activated protein kinase (AMPK), AKT1 protein kinase, signal transducers and transcription activators (STAT) was determined in nuclear cy-toplasmic lysates of whole blood mononuclear cells: STAT3, STAT5A and STAT6, c-Jun N-terminal protein kinase 1 and 2 isoforms (JNK), mitogen-activated protein kinase p38 (p38), extracellular growth kinase 1 and 2 isoforms (ERK), Janus kinase type 2 (JAK2), nuclear transcription factor NF -kB, caspase-1, cyclooxygenase-2 (COX-2), p70-S6K1 protein kinase, p53, p27, p21 proteins. In addition, the concentration of cyclic adenosine monophos-phate (cAMP) and cyclic guanosine monophosphate (cGMP) was determined in cell supernatants. Melatonin concentration was determined in blood se-rum. The material for the study was venous blood taken from the cubital vein in the morning from 6.00 to 6.15. Results. The analysis showed that in patients with coronary artery disease, in comparison with practically healthy individuals, in MNCs of whole blood, there was an increased level of protein kinases FAK, AKT, JNK, ERK, p70-S6K1, factor STAT6, protein p21, against which there was a decrease in the content of STAT3, STAT5A, JAK2, transcription factor NF-kB and caspase-1. These changes were accompanied by increased levels of cGMP and cAMP. Against this background, a decrease in the content of factors was revealed in the MNC. A high concentration of melatonin in patients with CAD was as-sociated with a decrease in the content of protein kinases AMPK, AKT, Jak2, ERK1, protein p21, caspase-1, and cAMP in MNCs, which was observed against the background of an increase in the level of protein p27 and nuclear factor NF-kB. The results of the correlation analysis indicate a different na-ture of the relationship between the level of melatonin and such factors as caspase-1, protein kinases ERK, JAK2, as well as the transcription factor NF-kB and p21 protein, depending on the characteristics of melatonin production in patients with coronary artery disease. Conclusions. In patients with coronary artery disease, melatonin exhibits a modulating effect on the energy balance of ICCs and their metabolism, helps limit pro-inflammatory activity by limiting the functional activity of MAPK/SAPK signaling pathways in MNCs.

An interview with Nobel Laureate David Baltimore, PhD.

In an online interview, Nobel Laureate David Baltimore, Ph.D., reflected on his contributions to biomedical science that have had a major influence on the fields of molecular biology, virology, cancer, and immunology. Dr. Baltimore is President Emeritus and Distinguished Professor of Biology at the California Institute of Technology. Among other notable works, he discovered the critical nuclear transcription factor NF Kappa B and the Rag1 and Rag2 proteins that rearrange adaptive immune cell receptors.
 His career path, he says, evolved naturally, as math and science came easily to him. As a high school student, he participated in a summer program at the Jackson Lab in Bar Harbor, Maine, where he says he came away feeling that experimental biology was exciting and rewarding.
 “That's where I discovered that the frontiers of science, were not so distant; that I could actually make a discovery that nobody else in the world knew about,” he says. 
 And that he did. Independently, he and Howard Temin discovered the viral enzyme reverse transcriptase revising the canon of cellular information transfer. They published back-to-back papers in Nature demonstrating that this enzyme in virus particles could transcribe RNA to DNA. Both received a Nobel Prize for this work.
 In reflecting on his early experience evaluating how to work with recombinant DNA and how we should scientifically and safely approach gain of function research, he says, “We have to be very honest with ourselves about what might hold danger, and we have to control our instinct … to do anything we can to generate progress and understanding of life. …At the same time, we don’t want to hold back progress, and so there is a balancing.”
 Dr. Baltimore also discussed his optimism about vectored immunoprophylaxis as a strategy for prevention of HIV and his doubt that scalable strategies will be able to cure HIV. He also reflected on his philosophy for the training of young scientists and the successful training program that he developed at the Whitehead Institute.

DNA methylation of a NF-\u03baB binding site in the aquaporin 5 promoter impacts on mortality in sepsis

Altered aquaporin 5 (AQP5) expression in immune cells impacts on key mechanisms of inflammation and is associated with sepsis survival. Since epigenetic regulation via DNA methylation might contribute to a differential AQP5 expression in sepsis, we tested the hypotheses that DNA methylation of the AQP5 promotor (1) influences AQP5 expression, (2) is associated with the 30-day survival of septic patients, and (3) alters the nuclear transcription factor NF-κB binding. AQP5 mRNA expression was quantified by real-time PCR in whole blood samples of 135 septic patients. In silico computer analysis of the AQP5 promoter (nt-567 to nt-975) revealed seven putative inflammatory transcription factor binding sites and methylation of these sites was analyzed. Electrophoretic mobility shift assays were performed to assess the binding of nuclear NF-κB to the AQP5 promoter region nt-937. After adjustment for multiple testing, a greater methylation rate was found at cytosine site nt-937 in the AQP5 promoter linked to NF-κB binding in non-survivors compared to survivors (p = 0.002, padj = 0.014). This was associated with greater AQP5 mRNA expression in non-survivors (p = 0.037). Greater (≥16%) promoter methylation at nt-937 was also associated with an independently increased risk of death within 30 days (HR: 3.31; 95% CI: 1.54–6.23; p = 0.002). We detected a functionally important AQP5 promoter cytosine site (nt-937) linked to the binding of the inflammatorily acting nuclear transcription factor NF-κB, with increased methylation in sepsis non-survivors. Thus, nt-937 APQ5 promoter methylation, presumably related to NF-κB binding, is prognostically relevant in sepsis and demonstrates that epigenetic changes impact on sepsis outcome.

PATHOGENIC MECHANISMS OF GENERALIZED PERIODONTITIS FROM THE POSITION OF POLYMORPHISM OF NUCLEAR TRANSCRIPTION FACTOR NF- κВ1

Background. Periodontal diseases are a topical issue of contemporary dentistry because they are accompanied by severe morphological and functional disorders of maxillodental system; and are characterized by polyetiology and a number of metabolic disorders.The purpose of this study was to substantiate the pathogenic mechanisms of generalized periodontitis in relation to polymorphism of nuclear transcription factor NF- κB1.Objective. The aim of the study was to determine the genetic factors in the development of generalized periodontitis and the relationship of this parameter with immunohistochemical affiliation for cellular infiltrate of the lamina propria of gum at this nosology in young people. Hence, 2 groups were formed: І – control and II – observational.Methods. Polymorphic gene section NF-κB1 was determined using the cells of buccal epithelium of the examined people by means of polymerase chain reaction. Collection of material was performed with sterile disposable dental brush, followed by the introduction of a reagent in ependorph with DNA Express reagent (LyTeh NPF, Russia). Genome deoxyribonucleic acid was isolated by DNA Express set (LyTeh, Moscow).Results. The lack of correlation in this case indicates that no matter how parameters change, relatively major genotype (Del/Del) in this case is unchanged and the determining factor causes the development of generalized periodontitis, clinical picture of which is rapidly progressing.Conclusions. Results of correlation analysis proved that genotype (Del/Del), as polymorphic variant of gene transcription factor NF-κB1, was significantly associated with the emergence of rapidly progressive periodontitis in young people.

The Role and Mechanism of VCAM-1 in 2-Methoxyestradiol Inhibiting Monocyte-Endothelial Cell Adhesions

2-Methoxyestradiol (2-ME) is a major endogenous metabolite of estrogen, which formed by catalysis of cytochrome P450 (CYP450) and catechins-O-methoxy transferase (COMT), has an important role in atherosclerosis (AS), but the underlying mechanisms remain obscure. In our work, we investigated the role and mechanism of 2-ME inhibiting monocyte-endothelial cell adhesion. In vitro studies using monocyte-endothelial cell adhesion assay and cell immunofluorescence method showed that lipopolysaccharide (LPS) promoted monocyte-endothelial cell adhesion, these effects were blocked by 2-ME through a dose- and time-dependent manner. Furthermore, Vascular cell adhesion molecular-1 (VCAM-1) specific antibody (anti- VCAM-1, 1:1000) were pre-incubated the cells for 48 h, and by monocyte-endothelial cell adhesion experiment, results showed that anti-VCAM-1 can significantly inhibit the LPS-induced monocyte-endothelial cell adhesion, indicating VCAM-1 may be the key molecule of the LPS-nduced cell adhesion. Moreover, through Western Blot and monocyte-endothelial cell adhesion assay observing, results showed that 2-ME (10^(-5) M) pretreatment for 48 h was strongest inhibited the expression of VCAM-1, NF-_κBp65 nucleus protein and nuclear translocation on LPS-induced endothelial cell. In conclusion, 2-ME has the effect on monocytes-endothelial cell adhesion, and the mechanism is that inhibiting the role of the nuclear transcription factor NF-_κBp65 and down regulation the expression of VCAM-1. This study will be helpful for further clarify the mechanism of 2-ME anti-AS, and can be used as a new type of clinical drug development.

Effect of Ganoderma lucidum powder on liver cell apoptosis and nuclear transcription factor NF - κB/65 expression in type 2 diabetes rat

Objective To observe the effect of Ganoderma lucidum powder on liver cell apoptosis and nuclear transcription factor NF - κB/65 expression in type 2 diabetes rat. Methods 40 Wistar big mice were divided into three groups randomly: control group, diabetes group and ganoderma lucidum group. The apoptosis rate of liver cell was detected by Giemsa staining and the expression of nuclear transcription factor NF - κB/65 was detected by immunohistochemical staining. Results The apoptosis rate and the expression of NF - κB/65in diabetes group was obviously higher than that of control group and ganoderma lucidum group ( P ＜ 0.05 ).Conclusion Ganoderma lucidum powder can protect the liver organization of diabetes rats and restraint the liver cell apoptosis rate, which may be the result of reducing the oxidative stress and reducing the NF - κB/65 overexpression of diabetic liver. Key words: Type 2 diabetes mellitus; Nuclear transcription factor( NF-κB) ; Apoptosis; Ganoderma lucidum

Type IVB piliated Salmonella typhi enhance IL-6 and NF- κB production in human monocytic THP-1 cells through activation of protein kinase C

Salmonella typhi is an important human pathogen responsible for typhoid fever. Type IVB pili, encoded by the S. typhi pil operon located in the major pathogenicity island, are used to facilitate bacterial entry into human intestinal cells in vitro and may be important in the mediation of enteric fever in humans. However, possible involvement of the type IVB pili of S. typhi in signal transduction in infected immune cells has not been examined previously. In this study, we have compared the effect of piliated and nonpiliated S. typhi on the activities of protein kinase C (PKC), the production of interleukin-6 (IL-6) and nuclear transcription factor NF- κB in human monocytic THP-1 cells. We find that piliated S. typhi can stimulate significantly higher activities of PKC, the production of IL-6 and NF- κB than a nonpiliated strain based on substrate phosphorolysis kinase assay, Western blot, RT-PCR, and luciferase reporter gene assay. In time course experiments, PKC activity increased in a time-dependent fashion after stimulation by the piliated bacteria. The PKC inhibitor Dequalinium chloride (DECA) remarkably reduced the production of IL-6, NF- κB and the activity of PKC induced by the piliated S. typhi. These results suggest that the induction of IL-6 and NF- κB depend on the PKC signal pathway. Our report demonstrates that the type IVB pili of S. typhi play important roles in the production of NF- κB and the proinflammatory cytokine IL-6, and in the stimulation of PKC activity and therefore, may have effects on the development of fever and other inflammatory responses.

Expression and secretion of chemotactic cytokines IL-8 and MCP-1 by human endothelial cells after Rickettsia rickettsii infection: Regulation by nuclear transcription factor NF- κB

Infection of endothelial cells (EC) with Rickettsia rickettsii results in Rocky Mountain spotted fever, an acute illness characterized by systemic inflammation. Interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are important chemokines for activating neutrophils and monocytes, respectively, and recruiting these circulating immune cells to the sites of inflammation. In this study, we have measured the expression and secretion of these chemokines during R. rickettsii infection of cultured human EC. In comparison to uninfected controls, increased mRNA expression of IL-8 and MCP-1 in R. rickettsii-infected EC was evident as early as 3 h and was sustained up to 21 h. Subsequent analysis of culture supernatants revealed significantly enhanced secretion of both chemokines at 3, 8, and 18 h post-infection (5–28-fold increase in IL-8 and 4–16-fold increase in MCP-1). The presence of peptide-aldehyde compound MG132 to inhibit proteasome-mediated degradation of the inhibitory protein I κB α and synthetic peptide SN-50 to inhibit the nuclear translocation of nuclear factor-kappa B (NF- κB) resulted in significant inhibition of the chemokine response. Also, T24 cells expressing a super-repressor mutant of I κB α (to render NF- κB inactivatable) secreted significantly lower quantities of IL-8 than mock-transfected cells. A neutralizing antibody against IL-1 α or an IL-1 specific receptor antagonist had no effect on the early phase of R. rickettsii-induced NF- κB activation and IL-8/MCP-1 secretion at 3 h. Both of these treatments, however, diminished late-phase NF- κB activation by about 33% and only partially suppressed the infection-induced chemokine release at 21 h. Thus, while chemokine response early during the infection likely depends on the direct activation of NF- κB, subtle autocrine effects of newly synthesized IL-1 α may contribute, in part, to the control of NF- κB activation and chemokine production at later times. These findings implicate a prominent role for host EC in recruiting immune cells to the site of inflammation during Rickettsia infection and provide important insights to further our understanding of the pathogenesis of spotted fever group rickettsioses.

Thalidomide as an Immunotherapeutic Agent: The Effects on Neutrophil- Mediated Inflammation

Thalidomide was developed in the 1950s as a sedative drug and withdrawn in 1961 because of its teratogenic effects, but has been rediscovered as an immuno-modifying drug. It has been administered successfully for the treatment of erythema nodosum leprosum, aphthous ulceration in HIV disease, inflammatory bowel diseases, and multiple myeloma. So far, investigations into the mode of action of thalidomide have focused on lymphocytes and vascular endothelial cells and have shown that this agent inhibits the production of tumor necrosis factor (TNF)-alpha and is an inhibitor of tumor angiogenesis. Recently, other immunological effects of this drug have been gaining attention, including attenuation of neutrophil activation and inhibition of myelo-proliferative responses. In autoimmune diseases, inflammation is characterized by an influx of granulocytes, and the association of granulocytes with gastrointestinal ulcer formation or rheumatic arthritis has been well documented. The suppressive effect of thalidomide on the activation of the nuclear transcription factor NF-(kappa)B may explain these effects of thalidomide. NF-(kappa)B is retained in the cytoplasm with I(kappa)B(alpha), and is activated by a wide variety of inflammatory stimuli including TNF, IL-1 and endotoxin followed by its translocation to the nucleus. Constitutive activation of NF-(kappa)B has been detected in various inflammatory diseases, while angiogenesis and organogenesis also require NF-(kappa)B activation. Thalidomide, on the other hand, has been shown to selectively suppress NF-(kappa)B activation induced by inflammatory mediators. NF-(kappa)B is known to be located downstream of proliferative and/or survival signaling induced by growth factors, which regulate anti-apoptotic genes. Myeloid cells in vitro, however, have been found to proceed to apoptosis as the result of the treatment with thalidomide and subsequent inactivation of NF-(kappa)B. These findings are consistent with clinical symptoms that showed the recovery from leukocytosis and/or neutrophilia after the administration of thalidomide. These findings shed new light on the anti-inflammatory properties of thalidomide and suggested that they may inhibit granulocyte-mediated tissue injury.

High dietary fat induces NADPH oxidase-associated oxidative stress and inflammation in rat cerebral cortex

Epidemiological and experimental studies have suggested that high dietary intake of fats is associated with cognitive decline and a significantly increased risk of dementia. Since oxidative stress and inflammation have been speculated to be critical mechanisms underlying neurodegenerative diseases, we hypothesized that a high fat (HF) diet might induce cerebral oxidative stress or neural inflammation and subsequently contribute to the high risk of dementia. To test this hypothesis, male rats were placed on either a HF diet or a low fat (LF) diet starting at 1 month of age and lasting for 5 months. Intracellular reactive oxidative species (ROS) generation in the cerebral cortex was measured by the oxidant-sensitive dye 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H 2DCFDA). Cortical tissue concentration of prostaglandin E 2 (PGE 2) was determined using an enzymatic immunoassay. Expression of NADPH oxidase subunits, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), nuclear transcription factor NF kappa-B (NF-κB) p65 subunit, Ikappa B(IκB), and phospho-Ikappa B(phospho-IκB) was evaluated by Western blot analysis. The HF diet significantly increased ROS generation and expression of gp91 phox, p22 phox, p47 phox, and p67 phox NADPH oxidase subunits in cerebral cortex. Elevated PGE 2 levels and markedly increased COX-2 expression suggested a neural inflammatory response in response to excessive fat intake. These findings were further supported by significantly increased phospho-IκB and nuclear NF-κB expression that suggested a role of IκB phosphorylation in HF diet-induced NF-κB translocation. The present study revealed that HF diet induced neural oxidative stress, inflammation, and NF-κB activation in rat cerebral cortex, and provided novel evidence regarding the link between high dietary fat and increased risk of dementia.

In vivo cytokine secretion and NF- κB activation around titanium and copper implants

The early biological response at titanium (Ti), copper (Cu)-coated Ti and sham sites was evaluated in an in vivo rat model. Material surface chemical and topographical properties were characterized using Auger electron spectroscopy, energy dispersive X-ray spectroscopy and interferometry, respectively. The number of leukocytes, cell types and cell viability (release of lactate dehydrogenase) were determined in the implant-interface exudate. The contents of activated nuclear transcription factor NF- κB, interleukin-6 (IL-6) and interleukin-10 (IL-10) were determined by enzyme linked immunosorbent assay. An increase in the number of leukocytes, in particular, polymorphonuclear leukocytes, was observed between 12 and 48 h around Cu. A marked decrease of exudate cell viability was found around Cu after 48 h. The total amounts of activated NF- κB after 12 h was highest in Ti exudates whereas after 48 h the highest amount of NF- κB was detected around Cu. The levels of cytokine IL-6 were consistently high around Cu at both time periods. No differences in IL-10 contents were detected, irrespective of material/sham and time. The results show that materials with different toxicity grades (titanium with low and copper with high toxicity) exhibit early differences in the activation of NF- κB, extracellular expression and secretion of mediators, causing major differences in inflammatory cell accumulation and death in vivo.

Tumor necrosis factor-α mediates polymethylmethacrylate particle-induced NF- κB activation in osteoclast precursor cells

Tumor necrosis factor-α (TNF) is a potent osteoclastogenic cytokine that has a fundamental role in the pathogenesis of implant particle-induced osteolysis. The nuclear transcription factor NF- κB mediates TNF signaling and this transcription complex is necessary for osteoclastogenesis. Because polymethylmethacrylate (PMMA) particles cause osteolysis, we reasoned the PMMA would induce NF- κB activation. In fact, we find that exposure of osteoclast precursors, in the form of colony stimulating factor-1 (CSF-1) dependent murine bone marrow macrophages, to PMMA particles prompts nuclear translocation and activation of NF- κB. Supershift assays confirm the presence of the p50 and p65 NF- κB subunits in the activated transcription factor. Particle-induced NF- κB activation is equal in both wild type and LPS- hyporesponsive cells indicating that the phenomenon does not represent endotoxin contamination. A soluble, competitive inhibitor of TNF (huTNFr:Fc) dampens particle-directed NF- κB activation and this response is also abrogated in TNF −/− osteoclast precursors. Thus, PMMA particle activation of NF- κB is a secondary event resulting from enhanced TNF expression and is independent of LPS contamination.

Blockade of NF- κ B Activation and Donation of Nitric Oxide: New Treatment Options in Inflammatory Bowel Disease?

Background : Inhibition of NF- s B activation has been suggested as an anti-inflammatory treatment strategy in inflammatory bowel disease (IBD). However, NF- s B regulated genes like inducible nitric oxide synthase (iNOS) are also involved in cell survival mechanisms. Methods : Review of the literature on NF- s B activation and iNOS induction in IBD. Results : In patients with IBD the mucosal immune response is derailed. The nuclear transcription factor NF- s B is a key regulator of the inducible expression of many genes involved in immune and inflammatory responses in the gut. Stimuli like oxidative stress, cytokines (IL-1, IL-6, TNF- α ), bacteria and viruses can release NF- s B from their inactive cytoplasmatic form to the nucleus. Drugs like corticosteroids, sulphasalazine, mesalazine and inhibitory cytokines (e.g. IL-10, IL-11) can prevent the activation of NF- s B. New, more potent and selective treatment strategies with antisense p65, proteasome inhibitors and viral I s B α expression vectors aim at the prevention of NF- s B activation in mucosal macrophages and T lymphocytes. However, NF- s B regulated genes are also involved in survival responses of epithelial cells. For example, inhibition of the NF- s B mediated induction of iNOS in epithelial cells could block important anti-apoptotic and anti-microbial survival mechanisms. Nitric oxide may also serve in a negative feedback loop to antagonize prolonged activation of NF- s B, thereby limiting chronic inflammation. Conclusion : Luminal donation of nitric oxide could block NF- s B activation. Selective inhibition of NF- s B activation in inflammatory cells could be a treatment option in IBD.

Oxygen free radical signaling in ischemic preconditioning.

This review will focus on the free radical signaling mechanism of preconditioning. The results from our laboratory as well as studies from other laboratories suggest that reactive oxygen species function as second messenger during myocardial adaptation to ischemia. This review provides evidence for the first time that tyrosine kinase and MAP kinases are the targets for reactive oxygen species generated in the preconditioned myocardium. The finding that p38 MAP kinase might be upstream of NF kappa B further supports our previous reports that MAPKAP kinase 2 could be the most likely link between the preconditioning and adaptation mediated by gene expression. p38 activation appears to be an important step in the translocation and activation of the nuclear transcription factor NF kappa B, which in turn may be involved in the induction of the expression of a variety of stress-inducible genes.

Differential regulation of apoptosis by ischemia-reperfusion and ischemic adaptation.

Ischemia and reperfusion injure the heart, as manifested by myocardial infarction, postischemic ventricular functional dysfunctions, arrhythmias, and cardiomyocyte apoptosis. Hearts can be adapted to ischemic-reperfusion injury by subjecting them to non-lethal cyclic episodes of short-term ischemia and reperfusion. The adapted myocardium becomes resistant to subsequent lethal ischemic injury. Reactive oxygen species and oxidative stress play crucial roles in the pathophysiology of ischemic-reperfusion injury. The adapted hearts, when subjected to subsequent ischemia and reperfusion, generate a reduced amount of oxygen free radicals compared to the nonadapted hearts. The number of cardiomyocytes undergoing apoptotic cell death is reduced in the adapted hearts subjected to ischemia and reperfusion. In concert, the adapted myocardium is associated with increased antioxidant gene Bcl-2, increased binding activity of the nuclear transcription factor NF kappa B, and reduced binding activity of AP-1 compared to nonadapted hearts. Yet when nonadapted hearts are subjected to ischemia and reperfusion, Bcl-2 is down-regulated while NF kappa B is moderately upregulated and AP-1 is significantly upregulated.

The effects of L-arginine on atherosclerosis and heart disease.

L-arginine, a semi-essential amino acid, can enhance the synthesis of nitric oxide (NO) via the nitric oxide synthase pathway. Enhanced bioavailability of NO may prevent activation of pro-inflammatory endothelial genes by the inhibition of nuclear transcription factor NF 3B, thus preventing the expression of adhesion molecules on endothelial surfaces. Animal studies have demonstrated that the chronic administration of L-arginine reduces the extent of atherosclerosis and prevents xanthoma development in LDL receptor knockout mice. Human studies have demonstrated improvement in endothelium vasodilator function both in coronary arteries and forearm flow responses. In addition oral L-arginine reverses an increased monocyte-endothelial adhesion in men with coronary artery disease and normalizes platelet aggregation in hypercholesterolemic humans. L-arginine may be a promising drug in the therapy of atherosclerosis. (Int J Cardiovasc Interventions

Ethanol Inhibits the Potentiation of Endotoxin by Dibutyryl cAMP and 2‐Methylthio ATP In Vivo

Ethanol (ETOH) selectively suppressed Escherichia coli endotoxin lipopolysaccharide (LPS)-stimulated, but not dibutyryl cAMP (db-cAMP)-stimulated upregulation of inducible nitric oxide synthase (iNOS) in rat alveolar macrophages (AMs) in vivo (Zhao et al., Alcohol. Clin. Exp. Res. 21:1062-1074, 1997). LPS-induced stimulation of iNOS is inhibited in vitro by db-cAMP and purine-2-Y (P2Y) receptor-mediated agonists. We examined the effect of ETOH on this interaction in rat lung AMs in vivo. Two hours after co-administration of LPS [0.6 mg/kg, intratracheal (i.t.)] with db-cAMP (0.1 mg/kg, i.t.) or 2-methylthio-adenosine-triphosphate (2-mes-ATP) to Sprague-Dawley rats (225 to 250 g) (n = 10 to 24/gp) iNOS messenger ribonucleic acid (mRNA), iNOS protein, and nitrate and nitrite anions [reactive nitrogen intermediates (RNIs)] in bronchoalveolar lavage fluid (BALf), and the ex vivo incubates of AMs were increased more than when these compounds were given individually to the rats. Co-administration of LPS with the autacoids did not affect LPS-stimulated increases of tumor necrosis factor-alpha (TNF alpha) mRNA, but inhibited LPS-stimulated BALf TNF alpha protein and attenuated LPS-mediated decreases of cell-associated TNF alpha. Pretreatment of rats with ETOH (4.5 g/kg, i.p.) or diethyidithiocarbamate (DETC; 5 mg/kg, i.t.), an inhibitor of the nuclear transcription factor NF kappa B (NF-kappaB), 30 min before co-administration of LPS with the autacoids restored iNOS mRNA levels to that obtained with the autacoid alone. Pretreatment of rats with ETOH decreased iNOS protein and RNI below that produced by either compound given individually to the rats. Although pretreatment of rats with DETC also decreased iNOS protein and RNI levels produced by LPS, the levels of both substances were elevated above that of the autacoid alone, LPS-induced upregulation of iNOS mRNA was associated with elevated levels of p65/p50 NF-kappaB in the nucleus of AMs. Neither db-cAMP or 2-mes-ATP affected LPS-stimulated NF-kappaB-DNA binding. Moreover, ETOH and DETC inhibited LPS-stimulated NF-kappaB-DNA binding. We conclude that in rat AMs in vivo: (1) both db-cAMP and P2Y-receptor stimulation summate with, rather than inhibit, LPS-induced upregulation of the iNOS mRNA, protein, and RNI; (2) ETOH and DETC inhibit LPS-induced upregulation of iNOS mRNA only when stimulated through the NF-kappaB pathway; and (3) ETOH inhibits both autacoid and LPS-stimulated formation of iNOS protein by a mechanism independent of its ability to suppress iNOS transcription.

On the possible role of reactive oxygen species in angiogenesis.

Human microvascular endothelial cells grown on a 3-D reconstituted extracellular matrix (Matrigel) spontaneously and rapidly form a capillary network of tubular structures, thus modeling part of the angiogenic cascade. Exposure of the cells at the time of plating onto Matrigel to a brief episode of hypoxia (40-60) min and subsequent reoxygenation, significantly accelerated (up to 3-fold) the rate of tubular morphogenesis, as determined by computer-aided morphometry. This effect was not dependent on activation of PKC or upregulation/release of angiogenic growth factors. Rather, hypoxia/reoxygenation (H/R), but not hypoxia alone, caused the formation of reactive oxygen species (ROS) and the activation of the nuclear transcription factor NF kappa B, both of which were inhibited by ROS-scavengers, such as pyrollidine dithiocarbamate. Tube formation was inhibited, also under normoxic conditions, by diverse ROS antagonists in a dose-dependent fashion. Our results indicate that angiogenesis is accompanied by and/or requires generation of ROS. We hypothesize that in the clinical setting of hypoxia/reoxygenation during ischemic pre-conditioning, enhanced activation of ROS-dependent intracellular signaling may accelerate the rate of neovascularization also in vivo, thus contributing to the alleviation of certain ischemic lesions.

Transforming growth factor beta 1 prevents cytokine-mediated inhibitory effects and induction of nitric oxide synthase in the RINm5F insulin-containing beta-cell line.

The aim of this study was to examine if the growth factor, transforming growth factor beta 1 (TGF beta 1), could prevent induction of nitric oxide synthase and cytokine-mediated inhibitory effects in the insulin-containing, clonal beta cell line RINm5F. Treatment of RINm5F cells for 24 h with interleukin-1 beta (IL-1 beta) (100 pM) induced expression of nitric oxide synthase and inhibited glyceraldehyde-stimulated insulin secretion. Combinations of IL-1 beta (100 pM), tumour necrosis factor-alpha (100 pM) and interferon-gamma (100 pM) reduced RINm5F cell viability (determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium (MTT) reduction assay) and de novo protein synthesis, as measured by incorporation of radiolabelled amino acids into perchloric acid-precipitable protein. Pretreatment of RINm5F cells with TGF beta 1 (10 pM) for 18 or 24 h, prior to the addition of either IL-1 beta or combined cytokines, prevented cytokine-induced inhibition of insulin secretion, protein synthesis and the loss of cell viability. TGF beta 1 pretreatment inhibited cytokine-induced expression and activity of nitric oxide synthase in RINm5F cells as determined by Western blotting and by cytosolic conversion of radiolabelled arginine into labelled citrulline and nitric oxide. Chemically generated superoxide also induced expression of nitric oxide synthase possibly due to direct activation of the nuclear transcription factor NF kappa B, an effect prevented by both an antioxidant and TGF beta 1 pretreatment. In conclusion, the mechanism of action of TGF beta 1 in blocking cytokine inhibitory effects was by preventing induction of nitric oxide synthase.

Induction of endothelial cell surface adhesion molecules by tumor necrosis factor is blocked by protein tyrosine phosphatase inhibitors: role of the nuclear transcription factor NF-kappa B.

Recent studies from our laboratory have indicated that protein tyrosine phosphatase (PTPase) inhibitors can down-modulate the tumor necrosis factor (TNF)-mediated activation of the nuclear transcription factor NF-kappa B in ML-1a, a monocytic cell line (Singh and Aggarwal, J. Biol. Chem. 1995: 270: 10631). Since TNF is one of the major inducers of various adhesion molecules in human endothelial cells and their expression is known to require the activation of NF-kappa B, we examined the effect of PTPase inhibitors on the TNF-mediated induction of intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and endothelial leukocyte adhesion molecule (ELAM)-1. Like ML-1a, human dermal microvessel endothelial cells (MVEC) treated with TNF rapidly activated (within 30 min) NF-kappa B; this effect was completely abolished by co-treatment with phenylarsine oxide (PAO), a specific inhibitor of PTPase. The induction of ICAM-1, VCAM-1, and ELAM-1 by TNF in MVEC occurred within 6 h and was also completely down-regulated by PAO in a dose-dependent manner. PAO was found to be effective even when added 3 h after TNF, suggesting a rapid mode of action of this inhibitor. Besides PAO, other inhibitors of PTPase, including pervanadate and diamide, also blocked TNF-dependent NF-kappa B activation and induction of all the three adhesion proteins. Consistent with these results, the attachment of monocytes to MVEC was also blocked by the PTPase inhibitors. Thus, overall, our results demonstrate that a PTPase is involved either directly or indirectly in the pathway leading to the induction of endothelial cell adhesion molecules by TNF. Because of their role in cell adhesion, PTPase may provide a novel target of drug development for treatment of inflammation, atherogenesis, and tumor metastasis.