Critical Role Of Nitric Oxide Research Articles

GSNOR overexpression enhances CAR-T cell stemness and anti-tumor function by enforcing mitochondrial fitness

Chimeric antigen receptor T cell (CAR-T) has been developed as a promising agent for patients with refractory or relapsed lymphoma and leukemia, but not all the recipients could achieve a long-lasting remission. The limited capacity of in vivo expansion and memory differentiation post activation is one of the major reasons for suboptimal CAR-T therapeutic efficiency. Nitric oxide (NO) plays multifaceted roles in mitochondrial dynamics and T-cell activation, but its function on CAR-T cell persistence and anti-tumor efficacy remains unknown. Herein, we found the continuous signaling from CAR not only promotes excessive NO production, but also suppressed S-nitrosoglutathione reductase (GSNOR) expression in T cells, which collectively led to increased protein S-nitrosylation, resulting in impaired mitochondrial fitness and deficiency of T cell stemness. Intriguingly, enforced expression of GSNOR promoted memory differentiation of CAR-T cell after immune activation, rendered CAR-T better resistance to mitochondrial dysfunction, further enhanced CAR-T cell expansion and anti-tumor capacity in vitro and in mouse tumor model. Thus, we revealed a critical role of NO in restricting CAR-T cell persistence and functionality, and defined that GSNOR overexpression may provide a solution to combat NO stress and render patients with more durable protection from CAR-T therapy.

Strategies of Pathogens to Escape from NO-Based Host Defense.

Nitric oxide (NO) is an essential signaling molecule present in most living organisms including bacteria, fungi, plants, and animals. NO participates in a wide range of biological processes including vasomotor tone, neurotransmission, and immune response. However, NO is highly reactive and can give rise to reactive nitrogen and oxygen species that, in turn, can modify a broad range of biomolecules. Much evidence supports the critical role of NO in the virulence and replication of viruses, bacteria, protozoan, metazoan, and fungi, thus representing a general mechanism of host defense. However, pathogens have developed different mechanisms to elude the host NO and to protect themselves against oxidative and nitrosative stress. Here, the strategies evolved by viruses, bacteria, protozoan, metazoan, and fungi to escape from the NO-based host defense are overviewed.

Critical role of nitric oxide in impeding COVID-19 transmission and prevention: a promising possibility.

COVID-19 is a serious respiratory infection caused by a beta-coronavirus that is closely linked to SARS. Hypoxemia is a symptom of infection, which is accompanied by acute respiratory distress syndrome (ARDS). Augmenting supplementary oxygen may not always improve oxygen saturation; reversing hypoxemia in COVID-19 necessitates sophisticated means to promote oxygen transfer from alveoli to blood. Inhaled nitric oxide (iNO) has been shown to inhibit the multiplication of the respiratory coronavirus, a property that distinguishes it from other vasodilators. These findings imply that NO may have a crucial role in the therapy of COVID-19, indicating research into optimal methods to restore pulmonary physiology. According to clinical and experimental data, NO is a selective vasodilator proven to restore oxygenation by helping to normalize shunts and ventilation/perfusion mismatches. This study examines the role of NO in COVID-19 in terms of its specific physiological and biochemical properties, as well as the possibility of using inhaled NO as a standard therapy. We have also discussed how NO could be used to prevent and cure COVID-19, in addition to the limitations of NO.

The Probiotic Lactobacillus fermentum Prevents Dysbiosis and Vascular Oxidative Stress in Rats with Hypertension Induced by Chronic Nitric Oxide Blockade

Whether the probiotic Lactobacillus fermentum CECT5716 (LC40) ameliorates hypertension in rats with chronic nitric oxide (NO) synthase inhibition is tested. Rats are randomly divided into four different groups and treated for 4weeks: a) vehicle (control), b) vehicle plus NG -nitro-l-arginine methyl ester (l-NAME; 50mg100mL-1 in drinking water), c) LC40 (109 colony-forming unitsd-1 by gavage), and d) LC40 plus l-NAME. l-NAME induces gut dysbiosis, characterized mainly by an increased Fimicutes/Bacteroidetes (F/B) ratio and reduced Bifidobacterium content, increased Th17 cells and reduced Treg in mesenteric lymph nodes (MLN), increased aortic Th17 infiltration and reactive oxygen species, reduced aortic endothelium-dependent relaxant response to acetylcholine, and hypertension. LC40 prevents gut dysbiosis, alters the Th17/Treg balance in MLN, vascular oxidative stress, and inflammation, slightly improves endothelial dysfuncion but do not inhibit the development of l-NAME-induced hypertension. Chronic LC40 treatment, in this model of chronic inhibition of NO synthesis, reduces early events involved in atherosclerosis development, such as vascular oxidative stress and pro-inflammatory status, as a result of prevention of gut dysbiosis and immune changes in MLN, but not hypertension, confirming the critical role of NO in the antihypertensive effects of LC40 in genetic hypertension.

An In Vitro Co-culture Mouse Model Demonstrates Efficient Vaccine-Mediated Control of Francisella tularensis SCHU S4 and Identifies Nitric Oxide as a Predictor of Efficacy.

Francisella tularensis is a highly virulent intracellular bacterium and cell-mediated immunity is critical for protection, but mechanisms of protection against highly virulent variants, such as the prototypic strain F. tularensis strain SCHU S4, are poorly understood. To this end, we established a co-culture system, based on splenocytes from naïve, or immunized mice and in vitro infected bone marrow-derived macrophages that allowed assessment of mechanisms controlling infection with F. tularensis. We utilized the system to understand why the clpB gene deletion mutant, ΔclpB, of SCHU S4 shows superior efficacy as a vaccine in the mouse model as compared to the existing human vaccine, the live vaccine strain (LVS). Compared to naïve splenocytes, ΔclpB-, or LVS-immune splenocytes conferred very significant control of a SCHU S4 infection and the ΔclpB-immune splenocytes were superior to the LVS-immune splenocytes. Cultures with the ΔclpB-immune splenocytes also contained higher levels of IFN-γ, IL-17, and GM-CSF and nitric oxide, and T cells expressing combinations of IFN-γ, TNF-α, and IL-17, than did cultures with LVS-immune splenocytes. There was strong inverse correlation between bacterial replication and levels of nitrite, an end product of nitric oxide, and essentially no control was observed when BMDM from iNOS−/− mice were infected. Collectively, the co-culture model identified a critical role of nitric oxide for protection against a highly virulent strain of F. tularensis.

Macrophage-derived nitric oxide initiates T-cell diapedesis and tumor rejection

ABSTRACTIn tumor biology, nitric oxide (NO) is generally regarded as an immunosuppressive molecule that impedes T-cell functions and activation of endothelial cells. Contrasting with this view, we here describe a critical role for NO derived from inducible nitric oxide (iNOS)-expressing tumor macrophages in T-cell infiltration and tumor rejection as shown by iNOS gene deletion, inhibition of iNOS, or NO donors. Specifically, macrophage-derived NO was found to induce on tumor vessels adhesion molecules that were required for T-cell extravasation. Experiments with human endothelial cells revealed a bimodal dose-dependent effect of NO. High doses of NO donors were indeed suppressive but lower, more physiological concentrations, induced adhesion molecules in an NFkB-dependent pathway and preferentially activated transcription of genes involved in lymphocyte diapedesis. iNOS+ macrophages in tumors appear to generate precisely the amount of NO that promotes endothelial activation and T-cell infiltration. These results will be valuable for the development of strategies designed to overcome the paucity of T-cell infiltration into tumors that is a major obstacle in clinical cancer immunotherapy.

Melatonin and nitric oxide modulate glutathione content and glutathione reductase activity in sunflower seedling cotyledons accompanying salt stress

The present findings demonstrate significant modulation of total glutathione content, reduced glutathione (GSH) content, oxidized glutathione (GSSG) content, GSH/GSSG ratio and glutathione reductase (GR; EC 1.6.4.2) activity in dark-grown seedling cotyledons in response to salt-stress (120 mM NaCl) in sunflower (Helianthus annuus L.) seedlings. A differential spatial distribution of GR activity (monitored by confocal laser scanning microscopic (CLSM) imaging) is also evident. Melatonin and nitric oxide (NO) differentially ameliorate salt stress effect by modulating GR activity and GSH content in seedling cotyledons. Total glutathione content (GSH + GSSG) exhibit a seedling age-dependent increase in the cotyledons, more so in salt-stressed conditions and when subjected to melatonin treatment. Seedlings raised in presence of 15 μM of melatonin exhibit significant increase in GR activity in cotyledon homogenates (10,000 g supernatant) coinciding with significant increase in GSH content. GSSG content and GSH/GSSG ratio also increased due to melatonin treatment. A correlation is thus evident in NaCl–sensitized modulation of GSH content and GR activity by melatonin. GSH content is down regulated by NO provided as 250 μM of sodium nitroprusside (SNP) although total glutathione content remained in similar range. A reversal of response (enhanced total glutathione accumulation) by NO scavenger (cPTIO) highlights the critical role of NO in modulating glutathione homeostasis. SNP lowers the activity of hydroxyindole-O-methyltransferase (HIOMT) - a regulatory enzyme in melatonin biosynthesis in control seedlings whereas its activity is upregulated in salt-stressed seedling cotyledons. Melatonin content of seedling cotyledons is also modulated by NO. NO and melatonin thus seem to modulate GR activity and GSH content during seedling growth under salt stress.

Plasma nitric oxide and oxidized LDL levels in systemic lupus erythematosus (SLE)

Abstract Background: Systemic lupus erythematosus (SLE) is an autoimmune disease with different clinical manifestations. The inflammatory and oxidative modification reactions are the most important events associated with cardiovascular complications of SLE patients. The aim of this study was to investigate the nitric oxide (NO) and the oxidized low-density lipoprotein (Ox-LDL) levels in order to explain the role of oxidized particles in development of clinical manifestations. Methods: A total of 80 subjects, SLE patients (n=40) and healthy controls (n=40), were recruited and matched regarding to age, gender, and body mass index (BMI). The biochemical parameters were measured using routine laboratory methods. The plasma Ox-LDL and NO levels were assayed with ELISA and colorimetric techniques, respectively. Results: The plasma NO level was significantly high in SLE patients (33.03±18.09 μmol/mL) in comparison to healthy controls (15.25±11.54 μmol/mL). In contrast, the total and normalized (Ox-LDL/LDL) plasma Ox-LDL values were low in SLE patients (p=0.2 and p<0.05, respectively). A linear negative correlation was also observed between the plasma Ox-LDL and NO levels (r=0.25, p<0.05). Multiple logistic regression analysis showed the critical role of NO on Ox-LDL level. Moreover, the disease activity and medications did not relate to the plasma Ox-LDL level (p>0.5). Conclusions: The results showed that the excess NO prevents the oxidation of LDL particles so that the inflammatory events in comparison to oxidative modifications may be most involved in clinical complications of SLE patients.

Critical role of nitric oxide in the modulation of prepulse inhibition in Swiss mice

Nitric oxide (NO) modulates the dopamine uptake and release processes and appears to be implicated in dopamine-related pathologies, such as schizophrenia. However, it is unclear whether there is excess or deficient NO synthesis in schizophrenia pathophysiology. Analyses of the intracellular pathways downstream of NO system activation have identified the cyclic nucleotide cyclic guanosine monophosphate (cGMP) as a possible target for drug development. Defects in the sensorimotor gating of the neural mechanism underlying the integration and processing of sensory information have been detected across species through prepulse inhibition (PPI). The aim of this study was to investigate the effects of NO/cGMP increase on sensorimotor gating modulation during dopamine hyperfunction. Mice were treated with NO donors and subjected to the PPI test. Treatment with the NO donor sodium nitroprusside was preceded by pretreatment with a soluble guanylate cyclase (sGC) inhibitor. Additionally, the mice were treated with NO donors and phosphodiesterases inhibitors prior to amphetamine treatment. Pretreatment with the NO donors enhanced the PPI response and attenuated the amphetamine-disruptive effects on the PPI. The sGC inhibitor did not modify the sodium nitroprusside effects. Additionally, the cGMP increase induced by a specific phosphodiesterase inhibitor did not modify the amphetamine-disruptive effect. This study provides the first demonstration that an increase in NO can improve the PPI response and block the amphetamine-disruptive effects on the PPI response. Our data are consistent with recent clinical results. However, these effects do not appear to be related to an increase in cGMP levels, and further investigation is thus required.

Host Immune Response to Intestinal Amebiasis

Entamoeba histolytica is an invasive enteric protozoan parasite that causes amebiasis. Globally, diarrheal disease is second only to pneumonia as a leading cause of death in children under five, and intestinal amebiasis is one of the top ten causes of severe diarrhea in the developing world. Amebiasis is more common in malnourished children, a state that afflicts approximately one-third of children in the developing world. In the critical first year of life, 11% of Bangladeshi infants living in poverty suffer from E. histolytica diarrhea [1], [2]. There is currently no vaccine for this devastating disease, thus an understanding of the human immune response toward the parasite would greatly enhance the ability to develop effective immunotherapies. The host deploys a series of immune defenses against the parasite as it invades the colon. The ameba, however, has developed complex strategies to evade host defenses and promote its own survival. Here, we summarize the dynamics of the interaction of parasite with host and its importance in the pathogenesis of amebiasis (Figure 1). Figure 1 A. Host Immune Response to Intestinal Amebiasis. Innate Immunity Stomach acid serves as an important first line of defense against enteropathogens through its ability to kill acid-sensitive microorganisms. However, infectious amebic cysts are highly resistant and survive passage through the acidic environment of the stomach. In the intestine, the next layer of innate defense may be the mucus layer, which is thought to act as a protective barrier, preventing E. histolytica from invading intestinal epithelial cells (IECs). Mucin, a major constituent of the intestinal mucus layer, is a glycoprotein secreted by goblet cells and submucosal glands. Mucin glycoproteins bind to and inhibit the Gal/GalNAc adherence lectin of the parasite, preventing in vitro adherence and killing of CHO cells [3]. Trophozoites, however, can disrupt the mucus layer and intestinal barrier by secreting cysteine proteases (CPs) and glycosidases to allow for penetration of the colonic mucosa. Specifically, E. histolytica cysteine protease-A5 (EhCP-A5) degrades mucin-2 (MUC2) and extracellular matrix (ECM) proteins [4]. The importance of cysteine proteases was demonstrated by an ex vivo human intestinal model, where EhCP-A5–silenced parasites failed to penetrate into the colonic lamina propria [5]. IECs exposed to E. histolytica trophozoites secrete potent chemokines, such as IL-8, resulting in immune cell recruitment and infiltration of the lamina propria and intestinal epithelium [6]. Neutrophils are one of the first immune cells to respond to amebic invasion. Neutrophils activated by interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), or lipopolysaccharides (LPS) carry out amebicidal activity in vitro by releasing reactive oxygen species (ROS) [7], [8]. Depletion of neutrophils with anti-Gr-1 antibodies resulted in exacerbated intestinal disease in murine models, supporting the protective role of neutrophils in amebiasis [9]. It should be noted, however, that anti-Gr-1 antibodies can deplete other granulocytes such as eosinophils. Macrophages also play a crucial role in the host response against intestinal amebiasis. Macrophages are amebicidal after stimulation with IFN-γ or TNF-α [10], [11]. Several amebic antigens are known to activate these cells via pattern recognition receptors. Toll-like receptor (TLR)-2 expression in macrophages is upregulated when exposed to the Gal/GalNAc lectin of E. histolytica, triggering pro-inflammatory cytokine production via NF-κB activation [12]. Macrophages that lack TLR-2 and TLR-4 displayed impaired response to E. histolytica lipopeptidophosphoglycan (LPPG), suggesting that pattern recognition is essential to the immune response [13]. Additionally, E. histolytica DNA can activate macrophages through interacting with TLR-9 [14]. Amebicidal activity of macrophages is contributed to by the production of nitric oxide (NO) from L-arginine, which is mediated by macrophage nitric oxide synthase. Inducible nitric oxide synthase (iNOS)–deficient mice were more susceptible to amebic liver abscess and to E. histolytica–induced hepatocytic apoptosis, implicating a critical role for NO in the host defense against amebiasis [15].

Effect of a free radical scavenger on nitric oxide release in microvessels

BackgroundSuperoxides impair nitric oxide (NO) bioactivity; however, the dynamics of NO release in the peripheral microcirculation remain unknown. We investigated the effect of a free-radical scavenger (edaravone) on dynamic NO release and the expression of eNOS and iNOS in microvessels. Methods and resultsAn electrochemical microsensor was positioned at the iliac artery bifurcation of the rat abdominal aorta, and NO release was measured in response to edaravone. A bio-imaging model was also used to obtain images of NO release from microvessels. Moreover, eNOS expression and iNOS expression were investigated in inflammatory and non-inflammatory models. NO was observed in association with microvessels in the mesentery. NO release in the aorta was significantly greater with edaravone than with placebo in the non-inflammatory model (P<0.05). Acetylcholine-induced NO release with edaravone was greater than with placebo in both models. Bio-imaging showed greater NO release from arterioles than from venules. eNOS expression with edaravone was greater than with placebo with or without inflammation. iNOS expression was increased by inflammation, but edaravone inhibited this increase. ConclusionThese results support the critical role of NO in the microcirculation and suggest that free-radical scavenging increases the bioavailability of NO in the microcirculation via eNOS upregulation.

Nitric oxide synthesis and biological functions of nitric oxide released from ruthenium compounds

During three decades, an enormous number of studies have demonstrated the critical role of nitric oxide (NO) as a second messenger engaged in the activation of many systems including vascular smooth muscle relaxation. The underlying cellular mechanisms involved in vasodilatation are essentially due to soluble guanylyl-cyclase (sGC) modulation in the cytoplasm of vascular smooth cells. sGC activation culminates in cyclic GMP (cGMP) production, which in turn leads to protein kinase G (PKG) activation. NO binds to the sGC heme moiety, thereby activating this enzyme. Activation of the NO-sGC-cGMP-PKG pathway entails Ca(2+) signaling reduction and vasodilatation. Endothelium dysfunction leads to decreased production or bioavailability of endogenous NO that could contribute to vascular diseases. Nitrosyl ruthenium complexes have been studied as a new class of NO donors with potential therapeutic use in order to supply the NO deficiency. In this context, this article shall provide a brief review of the effects exerted by the NO that is enzymatically produced via endothelial NO-synthase (eNOS) activation and by the NO released from NO donor compounds in the vascular smooth muscle cells on both conduit and resistance arteries, as well as veins. In addition, the involvement of the nitrite molecule as an endogenous NO reservoir engaged in vasodilatation will be described.

Nitric Oxide Signaling as a Common Target of Organohalogens and Other Neuroendocrine Disruptors

Organohalogen compounds such as polychlorinated biphenyls (PCB) and polybrominated diphenyl ethers (PBDE) are global environmental pollutants and highly persistent, bioaccumulative chemicals that produce adverse effects in humans and wildlife. Because of the widespread use of these organohalogens in household items and consumer products, indoor contamination is a significant source of human exposure, especially for children. One significant concern with regard to health effects associated with exposure to organohalogens is endocrine disruption. Toxicological studies on organohalogen pollutants primarily focused on sex steroid and thyroid hormone actions, and findings have largely shaped the way one envisions their disruptive effects occurring. Organohalogens exert additional effects on other systems including other complex endocrine systems that may be disregulated at various levels of organization. Over the last 20 years evidence has mounted in favor of a critical role of nitric oxide (NO) in numerous functions ranging from neuroendocrine functions to learning and memory. With its participation in multiple systems and action at several levels of integration, NO signaling has a pervasive influence on nervous and endocrine functions. Like blockers of NO synthesis, PCBs and PBDEs produce multifaceted effects on physiological systems. Based on this unique set of converging information it is proposed that organohalogen actions occur, in part, by hijacking processes associated with this ubiquitous bioactive molecule. The current review examines the emerging evidence for NO involvement in selected organohalogen actions and includes recent progress from our laboratory that adds to our current understanding of the actions of organohalogens within hypothalamic neuroendocrine circuits. The thyroid, vasopressin, and reproductive systems as well as processes associated with long-term potentiation were selected as sample targets of organohalogens that rely on regulation by NO. Information is provided about other toxicants with demonstrated interference of NO signaling. Our focus on the convergence between NO system and organohalogen toxicity offers a novel approach to understanding endocrine and neuroendocrine disruption that is particularly problematic for developing organisms. This new working model is proposed as a way to encourage future study in elucidating common mechanisms of action that are selected with a better operational understanding of the systems affected.

Critical role of Nitric Oxide on Nicotine‐Induced Hyperactivation of Dopaminergic Nigrostriatal System: Electrophysiological and Neurochemical evidence in Rats

Nicotine, the main psychoactive ingredient in tobacco, stimulates dopamine (DA) function, increasing DA neuronal activity and DA release. DA is involved in both motor control and in the rewarding and reinforcing effects of nicotine; however, the complete understanding of its molecular mechanisms is yet to be attained. Substantial evidence indicates that the reinforcing properties of drugs of abuse, including nicotine, can be affected by the nitric oxide (NO) system, which may act by modulating central dopaminergic function. In this study, using single cell recordings in vivo coupled with microiontophoresis and microdialysis in freely moving animals, the role of NO signaling on the hyperactivation elicited by nicotine of the nigrostriatal system was investigated in rats. Nicotine induced a dose-dependent increase of the firing activity of the substantia nigra pars compacta (SNc) DA neurons and DA and 3,4-dihydroxyphenylacetic acid (DOPAC) release in the striatum. Pharmacological manipulation of the NO system did not produce any change under basal condition in terms of neuronal discharge and DA release. In contrast, pretreatments with two NO synthase (NOS) inhibitors, N-omega-nitro-l-arginine methyl ester (l-NAME) and 7-nitroindazole (7-NI) were both capable of blocking the nicotine-induced increase of SNc DA neuron activity and DA striatal levels. The effects of nicotine in l-NAME and 7-NI-pretreated rats were partially restored when rats were pretreated with the NO donor molsidomine. These results further support the evidence of an important role played by NO on modulation of dopaminergic function and drug addiction, thus revealing new pharmacological possibilities in the treatment of nicotine dependence and other DA dysfunctions.

PII: S1043-4666(09)00292-0

PII: S1043-4666(09)00292-0

Induction of NOS and nitrotyrosine expression in the rat striatum following experimental hepatic encephalopathy

Hepatic encephalopathy (HE) is a neurologic disease associated with hepatic dysfunction. Astroglial and neuronal alterations have been described in the basal ganglia in HE. Our study was performed to determine whether such alterations are mediated by nitric oxide (NO), by using an experimental model of HE (portacaval anastomosis [PCA]). The expression of the NO synthases (nNOS and iNOS) and the production of nitrotyrosine (NT) were evaluated in the striatum of rats exposed to PCA for 1 and 6 months. The expression of nNOS in the striatal neurons of PCA rats was increased compared to controls. nNOS expression was also detectable in astrocytes after 6 months of exposure to PCA. Whereas astroglial cells in the normal striatum showed no iNOS expression, iNOS was expressed in the astrocytes of PCA brains, mainly in perivascular processes at 6 months PCA exposure (demonstrated by colocalization with GFAP). The increased expression of both the nNOS and iNOS isoforms in PCA rats might indicate a critical role for NO in the pathomechanism of HE. To study the potential cell damage caused by NO, the deposition of NT in PCA-rats was analysed. Nitrotyrosine was detected in neurons although it was mainly seen in the astrocytes of PCA brains, in which double immunolabelling showed NT to be colocalized with GFAP. Thus, the present study shows the induction of iNOS and NT in astrocytes, which increases with the duration of PCA exposure. This suggests that the induced astroglial production of NO during PCA might be one of the main factors contributing to HE.

Role of nitric oxide (NO) in the hyperbaric oxygen (HBO2)‐induced long‐lasting activation of endogenous opioid systems in mice

Exposure to HBO2 causes a long‐lasting analgesic effect in patients with chronic pain (Kiralp et al., J. Int. Med. Res. 32:258–262, 2004). To determine the duration of antinociceptive effect in an animal model following exposure to HBO2, male NIH Swiss mice (20–30 g) were exposed to HBO2 at 2.5 ATA for 60 min then removed to room air for varying durations of time (5, 15, 30, 60, 90, 120, 150 or 180 min) prior to antinociceptive testing. The antinociceptive effect of HBO2 remained robust up to 90 min but subsided by 120 min. By contrast, mice exposed to 70% nitrous oxide for 60 min exhibited antinociception only through the 15‐min interval. When administered prior to HBO2 exposure, both naltrexone and L‐NAME antagonized the antinociceptive effect seen 90 min later. When administered prior to antinociceptive testing, naltrexone but not L‐NAME antagonized the antinociceptive effect of HBO2. These results suggest that there is a critical role of NO during HBO2 exposure that is required for expression of the opioid‐mediated antinociceptive effect 90 min later. (Supported in part by NIH Grant GM‐77153, ASPET SURF Program and the Chico Hyperbaric Center).

A negative feedback mechanism involving nitric oxide and nuclear factor kappa-B modulates endothelial nitric oxide synthase transcription

Nuclear factor kappa B (NFκB), commonly a proinflammatory transcription factor, is responsible for increasing transcription of the endothelial cell nitric oxide synthase (eNOS) in response to laminar shear stress. Nitric oxide (NO) production can be stimulated by shear, and NO is known to inhibit NFκB activation. We hypothesized that this inhibitory action of NO on NFκB activation serves as a negative feedback to inhibit NFκB activity and eNOS transcription. Exposure of bovine aortic endothelial cells to laminar shear stimulated steady state eNOS mRNA expression and eNOS promoter activity as measured using an eNOS promoter/CAT construct. These effects of shear were enhanced by the NOS inhibitor l-NAME and decreased by the NO-donor DPTA-NO by 30–50%. The NFκB inhibitor panepoxydone prevented the increase in eNOS mRNA caused by shear confirming a role of NFκB in this response. Shear stress stimulated a transient (30 min) nuclear translocation of the NFκB subunit p50. Treatment with l-NAME increased binding of the NFκB subunit p50 to consensus oligonucleotide-coated microtiter plates, while having only minimal effect on binding of p65, strongly suggesting that nitric oxide mainly inhibits p50 activation. Using the biotin switch method, we found that shear stress stimulates p50 nitrosylation and this was prevented by l-NAME. Moreover, transfection of endothelial cells with a vector encoding the C62S p50, a variant with a point mutation of the nitrosylation site C62, markedly increased nuclear translocation of p50 and doubled eNOS mRNA expression under shear stress compared to that observed in cells transfected with wild-type p50. We conclude that this interaction between shear, NFκB activation, NO production and NFκB inhibition represents a classical negative feedback loop, which prevents sustained activation of NFκB. In the absence of NO, shear stimulation of NFκB and eNOS transcription are enhanced. Our findings emphasize the critical role of NO in modulation of the endothelial cell inflammatory state. Several common diseases, including hypercholesteremia, hypertension and diabetes, are associated with eNOS dysfunction. Under these conditions, decreased NO availability may result in sustained activation of NFκB in response to shear and unrestrained endothelial inflammation.

Nitric oxide-based molecular strategies for restenosis therapy

Arterial renarrowing (restenosis) limits balloon angioplasty’s therapeutic potential, despite the benefits of intravascular prosthetic devices called stents. Nitric oxide (NO) mediates/regulates diverse aspects of blood vessel function, and NO insufficiency contributes to many occlusive vascular diseases. Numerous preclinical and more restricted human studies suggest that NO supplementation may help solve the restenosis problem, although the extant data do not conclusively demonstrate that pharmacologic NO prevents clinical restenosis. This article reviews 20 patents from the past three years that claim methods for treating restenosis with NO by using NO-releasing polymers, small-molecule NO donors, and nitric oxide synthase (NOS)-based molecular approaches. The authors suggest that a controlled-release delivery platform capable of extending the intrinsically short half-life of NO, and comprised of a stent coated with a stable, biocompatible polymer containing a low-molecular-weight NO donor represents a most promising, state-of-the-art approach for treating restenosis with NO. The expanding clinical applications of angioplasty, the persistence of the restenosis problem, and the critical role of NO in maintaining arterial patency and homeostasis will undoubtedly elicit further inventions for NO-based restenosis therapy.

Arteriogenesis: role of nitric oxide.

Arteriogenesis is an important process for adapting the pre-existing circuit of vessels into functional collateral conduits for delivery of oxygen enriched blood to tissue distal to occlusion of a large, peripheral conduit artery. Recent evidence has shown that arteriogenesis is regulated by nitric oxide (NO), angiogenic factors and shear stress. NO significantly impacts vasomotor tone to enhance conductance of the newly recruited collateral arteries, and this effect is augmented by exercise training prior to arterial occlusion. NO-mediated increases in vascular conductance allows for greater collateral dependent blood flow to the tissue distal to occlusion. NO production is also critical to the efficacy of therapeutic arteriogenesis achieved by delivery of exogenous angiogenic growth factors (VEGF, FGF-2) or by exercise training. The critical role of NO in therapeutic arteriogenesis is independent of NO-mediated changes in vascular conductance and implies a central role in arteriogenic signaling events. Maintenance, or improvement, of NO production and signaling, such as with regular exercise, may improve endothelial cell function and thus may help preserve the arteriogenic potential of preexisting collateral networks.