Sickle cell anemia and vascular dysfunction: The nitric oxide connection

Endothelial dysfunction and impaired nitric oxide bioavailability have been implicated in the pathogenesis of sickle cell anemia. Nitric oxide is a diatomic gas with a role in vascular homeostasis. Hemoglobin polymerization resulting from the HbS mutation produces erythrocyte deformation and hemolysis. Free hemoglobin, released into plasma by hemolysis scavenges on nitric oxide, and leads to reduced nitric oxide bioavailability. Pulmonary hypertension is a known consequence of sickle cell anemia. It occurs in 30–40% of patients with sickle cell anemia, and is associated with increased mortality. Several studies have implicated intravascular hemolysis, and impaired nitric oxide bioavailability in the pathogenesis of pulmonary hypertension. In this review, we summarize the mechanisms of altered nitric oxide bioavailability in sickle cell anemia and its possible role in the pathogenesis of pulmonary hypertension. J. Cell. Physiol. 224: 620–625, 2010. © 2010 Wiley‐Liss, Inc.     

Role of nitric oxide and reactive oxide species in disease resistance to necrotrophic pathogens

Nitric oxide (NO) and reactive oxygen species (ROS) are important signaling molecules in plant immunity. However, roles of NO and ROS in disease resistance to necrotrophic pathogens are not fully understood. We have recently demonstrated that NO plays a pivotal role in basal defense against Botrytis cinerea and the expression of the salicylic acid (SA)-responsive gene PR-1 in Nicotiana benthamiana. By contrast, ROS function negatively in resistance or positively in expansion of disease lesions during B. cinerea-N. benthamiana interaction. Here, analysis in NahG-transgenic N. benthamiana showed that SA signaling is not involved in resistance to B. cinerea in N. benthamiana. We discuss how NO and ROS participate in disease resistance to necrotrophic pathogens on the basis of recent reports.Key words: NO burst, oxidative burst, necrotrophic pathogen, salicylic acid, plant immunity, MAPKNecrotrophs are pathogens that kill host cells by means of toxic molecules and lytic enzymes, and they feed on the remains for their own growth. If the toxic molecule shows differential activity to one or a few plant species, the pathogen has a limited host range and the metabolite is referred to as a host-selective toxin (HST).¹ Several well-studied necrotrophs, in particular Cochliobolus and Alternaria spp., produce HSTs required for the pathogenicity. There are also necrotrophic fungal pathogens with a broad host range, particularly those in the order of Helotiales, including Sclerotinia sclerotiorum and Botrytis cinerea.Rapid production of nitric oxide (NO) and reactive oxygen species (ROS), called NO burst and oxidative burst, respectively, is one of the earliest responses of plants to pathogen attacks. Our recent study showed that NO and oxidative bursts accompanied by activation of the mitogen-activated protein kinase (MAPK)² are induced after inoculation with B. cinerea, and that NO plays a key role, but ROS have an opposite effect in basal defense against B. cinerea in Nicotiana benthamiana.³ NO and ROS are believed to play key roles independently or coordinately in plant innate immunity.^4,5 NO signaling comprises complex processes including increases in cytosolic Ca²⁺ concentration, cyclic GMP (cGMP), cyclic ADP ribose and activation of protein kinases. NO also modulates protein activities directly by cysteine S-nitrosylation.⁶ In addition, NO appears to act as an antioxidant of ROS, because NO can react quickly with superoxide (O₂⁻) to form peroxynitrite (ONOO⁻) and then, reduces the amount of endogenous ROS. Actually, treatment with a mammalian NO synthase inhibitor and silencing NbNOA1 decreased endogenous NO levels and increased the levels of ROS after inoculation with B. cinerea.³ The suppression of NO burst induced high susceptibility to B. cinerea, and depletion of oxidative burst by an NADPH oxidase inhibitor or silencing NbRBOHB led to reduction in disease lesions by B. cinerea,³ suggesting that the growth of B. cinerea might be determined by endogenous levels of ROS which is an important component of virulence.⁷ However, depletion of both NO and oxidative bursts by double silencing NbNOA1/NbRBOHB resulted in expansion of disease lesions compared with reduction of oxidative burst alone by silencing NbRBOHB.³ Similarly, our most recent study showed that silencing NbRibA which compromises production of both NO and ROS do not affect basal resistance against B. cinerea.⁸ These findings suggest that NO positively functions in resistance to necrotrophic pathogens in the manner other than as an antioxidant of ROS.The relationship between NO and salicylic acid (SA) has been studied.⁹ SA signaling-deficient mutants of Arabidopsis thaliana show high susceptibility to B. cinerea.^10,11 We have suggested that reduced basal defense against B. cinerea in N. benthamiana resulting from compromised endogenous NO production may be due to depletion of SA signaling, because NbNOA1-silenced plants showed suppression of the SA-responsive gene NbPR-1 expression induced by inoculation with B. cinerea.³ To confirm the possibility, we used N. benthamiana expressing NahG that converts all SA to catechol. NahG and non-NahG (WT) leaves were inoculated with B. cinerea. NahG plants showed similar susceptibility to B. cinerea compared with WT plants (Fig. 1). We also evaluated effects of silencing NbNOA1 and NbRBOHB in NahG plants on susceptibility to B. cinerea. Like NbNOA1-silenced WT plants shown previusly,³ NbNOA1-silenced NahG leaves showed high susceptibility to B. cinerea. On the other hand, NbRBOHB-silenced NahG leaves showed marked reduction of disease lesions compared with silencing-control NahG leaves. NbNOA1/NbRBOHB-silenced NahG leaves showed expansion of disease lesions compared with NbRBOHB-silenced NahG leaves (Fig. 2). These results suggest that NO-mediated basal defense against B. cinerea is not due to SA signaling, and effects of ROS on disease lesions may not depend on SA in N. benthamiana.Open in a separate windowFigure 1Effects of NahG transgene on susceptibility to B. cinerea. NahG and non-NahG (WT) leaves were inoculated with B. cinerea conidial suspension (1 × 10⁵ conidia/ml). (A) Inoculated leaves were photographed at 4 days postinoculation (dpi). (B) Average diameter of lesions formed on the leaves at 3 and 4 dpi. Data are means ± SD from fourteen experiments.Open in a separate windowFigure 2Effects of silencing NbNOA1 (N), NbRBOHB (B) or NbNOA1/NbRBOHB (N/B) in NahG plants on susceptibility to B. cinerea. Silenced NahG leaves were inoculated with B. cinerea conidial suspension (1 × 10⁵ conidia/ml). (A) Inoculated leaves were photographed at 4 dpi. (B) Average diameter of lesions formed on the leaves at 3 and 4 dpi. Data are means ± SD from four experiments. Data were subjected to Student''s t-test. *p < 0.05 versus silencing-control plants (TRV). **p < 0.05 versus NbRBOHB-silenced plants.Recently, it has been reported that NO and ROS are involved in HSTs responses.^12–15 Victorin, an HST produced by Cochliobolus victoriae, elicits generation of NO and ROS in victorin-sensitive oat leaves.¹² Cell death induced by victorin is suppressed by treatment with ROS scavengers.¹³ Similarly, treatment with ToxA, an HST produced by Pyrenophora triticirepentis, induces oxidative burst, and scavenging ROS compromises ToxA-inducible cell death in ToxA-sensitive wheatleaves.^14,15 SA-induced MAPK, which regulates both NO and ROS production,² is activated by AAL-toxin produced by Alternaria alternata f. sp. lycopersici in AAL-toxin-sensitive tobacco (Mizuno et al. unpublished data). These findings indicate requirement of ROS for the HST-inducible cell death and participation of NO in HST responses.In conclusion, NO and ROS appear to play a contrasting role in disease resistance to necrotrophic pathogens as shown in Figure 3. However, how NO signaling participates in defense responses against necrotrophic pathogens has yet to be elucidated. Recently, several targets of protein S-nitrosylation during hypersensitive response have been characterized in A. thaliana.¹⁶ Evidence is also accumulating for cGMP as an important component of NO-related signal transduction.¹⁷ Further investigations of NO signaling will lead to our understanding of interactions between plants and necrotrophic pathogens.Open in a separate windowFigure 3Model showing role of NO and oxidative bursts in disease resistance to necrotrophic pathogens. After recognition of necrotrophs, plants immediately provoke activation of MAPK which could regulate production of both NO and ROS,² and then NO and oxidative bursts. NO burst plays an important role in disease resistance to necrotrophic pathogens, whereas oxidative burst has a negative role in resistance or has a positive role in expansion of disease lesions by necrotrophs.     

Intraprotein electron transfer in a two-domain construct of neuronal nitric oxide synthase: the output state in nitric oxide formation

Intersubunit intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation, which serves as the input state for reduction of FMN by electrons from NADPH and flavin adenine dinucleotide (FAD) in the reductase domain. To favor the formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct of rat neuronal NOS (nNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of IET between the FMN and heme domains in the nNOS oxyFMN construct in the presence and absence of added calmodulin (CaM) were directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single-domain heme oxygenase constructs. The IET rate constant in the presence of CaM (262 s(-)(1)) was increased approximately 10-fold compared to that in the absence of CaM (22 s(-)(1)). The effect of CaM on interdomain interactions was further evidenced by electron paramagnetic resonance (EPR) spectra. This work provides the first direct evidence of the CaM control of electron transfer (ET) between FMN and heme domains through facilitation of the FMN/heme interactions in the output state. Therefore, CaM controls IET between heme and FMN domains by a conformational gated mechanism. This is essential in coupling ET in the reductase domain in NOS with NO synthesis in the oxygenase domain.     

Sickle cell disease and malaria morbidity: a tale with two tails

More than 230,000 children are born in Africa with sickle cell disease (SCD) each year: approximately 85% of all affected births worldwide. Although malaria is commonly viewed as a major problem for African patients with this condition, questions still remain about its relative importance as a cause of ill heath and death. In the absence of definitive studies investigating the contribution of malaria to morbidity and mortality in African children with SCD, policy makers will continue to lack the evidence on which to base appropriate management guidelines.     

Examination of bacterial resistance to exogenous nitric oxide

While much research has been directed to harnessing the antimicrobial properties of exogenous NO, the possibility of bacteria developing resistance to such therapy has not been thoroughly studied. Herein, we evaluate potential NO resistance using spontaneous and serial passage mutagenesis assays. Specifically, Staphylococcus aureus, Methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa were systematically exposed to NO-releasing 75mol% MPTMS-TEOS nitrosothiol particles at or below minimum inhibitory concentration (MIC) levels. In the spontaneous mutagenesis assay, bacteria that survived exposure to lethal concentrations of NO showed no increase in MIC. Similarly, no increase in MIC was observed in the serial passage mutagenesis assay after exposure of these species to sub-inhibitory concentrations of NO through 20 d.     

Sickle cell disease as an accelerated aging syndrome

Sickle cell disease (SCD) is characterized by vaso-occlusion, hemolysis, and systemic manifestations that form the hallmark of the disease. Apart from morbidity, SCD is also associated with increased mortality and decreased quality of life. Aging is a natural phenomenon that is associated with changes at cellular, tissue, and organ levels, in addition to the loss of physical fitness, increased susceptibility to diseases, and a higher likelihood of mortality. Some of the cellular mechanisms involved in normal (or physiological) aging include abnormalities of sphingolipids (ceramides) and reduced length of the telomere. These changes have also been documented in SCD. Cellular, organs, and physical manifestations of SCD resemble an accelerated aging syndrome. Sickle erythrocytes also acquire morphological features similar to that of aged normal erythrocytes and are thus picked up early by the macrophages for destruction. Brain, kidney, heart, innate and adaptive immune system, and musculoskeletal system of patients with SCD exhibit morphological and functional changes that are ordinarily seen in the elderly in the general population. Stroke, silent cerebral infarcts, cardiomegaly, heart failure, pulmonary hypertension, nephropathy with proteinuria, osteopenia, osteoporosis, osteonecrosis, gout, and infections are exceedingly common in SCD. In this review, we have attempted to draw parallels between SCD and accelerated aging syndromes.     

Src kinase activation by nitric oxide promotes resistance to anoikis in tumour cell lines

Tumour progression involves the establishment of tumour metastases at distant sites. Resistance to anoikis, a form of cell death that occurs when cells lose contact with the extracellular matrix and with neighbouring cells, is essential for metastases. NO has been associated with anoikis. NO treated HeLa cells and murine melanoma cells in suspension triggered a nitric oxide (NO)-Src kinase signalling circuitry that enabled resistance to anoikis. Two NO donors, sodium nitroprusside (SNP) (500 µM) and DETANO (125 µM), protected against cell death derived from detachment of a growth permissive surface (experimental anoikis). Under conditions of NO-mediated Src activation the following were observed: (a) down-regulation of the pro-apoptotic proteins Bim and cleaved caspase-3 and the cell surface protein, E-cadherin, (b) up-regulation of caveolin-1, and (c) the dissociation of cell aggregates formed when cells are detached from a growth permissive surface. Efficiency of reattachment of tumour cells in suspension and treated with different concentrations of an NO donor, was dependent on the NO concentration. These findings indicate that NO-activated Src kinase triggers a signalling circuitry that provides resistance to anoikis, and allows for metastases.     

Adaptive resistance to nitric oxide in motor neurons

Nitric oxide (NO) is a free radical produced actively by mammalian cells, including neurons. Low levels of NO can function in intercellular signaling, but high levels are cytotoxic. This cytotoxic potential suggests that cells at risk for NO damage, such as neurons, might have NO resistance mechanisms to prevent cell death, and adaptive resistance to NO-releasing compounds has been reported for some non-neuronal cell types. Here we show that immortalized mouse motor neurons (NSC34 cells) respond to sub-lethal fluxes of pure NO by activating adaptive resistance mechanisms that counteract cytotoxic NO exposure. This adaptive NO resistance is reversible and is paralleled by the induction of the oxidative stress enzyme heme oxygenase 1 (HO-1). An inhibitor of both HO-1 and heme-dependent guanylate cyclase (tin-protoporphyrin IX) greatly sensitized NO-pretreated NSC34 cells to the NO challenge. However, readdition of cyclic GMP (in the form of the 8-bromo derivative) restored rather little resistance, and a more selective guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxaline-1-one (at 10 microM), did not have the sensitizing effect. Therefore, the inducible HO-1 pathway contributes substantially to adaptive NO resistance, while cyclic GMP seems to play at most a small role. A similar adaptive resistance to NO was observed in primary rat spinal chord motor neurons. The activation of NO resistance in motor neurons may counteract age- or disease-related neurodegeneration.     

Podokinesis in endothelial cell migration: role of nitric oxide

Previously, we demonstrated the role of nitric oxide (NO) intransforming epithelial cells from a stationary to locomoting phenotype[E. Noiri, T. Peresleni, N. Srivastava, P. Weber, W. F. Bahou, N. Peunova, and M. S. Goligorsky. Am. J. Physiol. 270 (CellPhysiol. 39): C794-C802, 1996] and itspermissive function in endothelin-1-stimulated endothelial cellmigration (E. Noiri, Y. Hu, W. F. Bahou, C. Keese, I. Giaever, and M. S. Goligorsky. J. Biol. Chem. 272:1747-1753, 1997). In the present study, the role of functional NOsynthase in executing the vascular endothelial growth factor(VEGF)-guided program of endothelial cell migration and angiogenesiswas studied in two independent experimental settings. First, VEGF,shown to stimulate NO release from simian virus 40-immortalized microvascular endothelial cells, induced endothelial cell transwell migration, whereasN^G-nitro-L-arginine methyl ester(L-NAME) or antisenseoligonucleotides to endothelial NO synthase suppressed this effect ofVEGF. Second, in a series of experiments on endothelial cell woundhealing, the rate of VEGF-stimulated cell migration was significantlyblunted by the inhibition of NO synthesis. To gain insight into thepossible mode of NO action, we next addressed the possibility that NOmodulates cell matrix adhesion by performing impedance analysis ofendothelial cell monolayers subjected to NO. The data showed thepresence of spontaneous fluctuations of the resistance in ostensiblystationary endothelial cells. Spontaneous oscillations were induced byNO, which also inhibited cell matrix adhesion. This process we propose to term "podokinesis" to emphasize a scalar form ofmicromotion that, in the presence of guidance cues, e.g., VEGF, istransformed to a vectorial movement. In conclusion, execution of theprogram for directional endothelial cell migration requires twocoexisting messages: NO-induced podokinesis (scalar motion) andguidance cues, e.g., VEGF, which imparts a vectorial component to themovement. Such a requirement for the dual signaling may explain amismatch in the demand and supply with newly formed vessels indifferent pathological states accompanied by the inhibition of NOsynthase.

     

Constitutive expression of mammalian nitric oxide synthase in tobacco plants triggers disease resistance to pathogens

Nitric oxide (NO) is known for its role in the activation of plant defense responses. To examine the involvement and mode of action of NO in plant defense responses, we introduced calmodulin-dependent mammalian neuronal nitric oxide synthase (nNOS), which controls the CaMV35S promoter, into wild-type and NahG tobacco plants. Constitutive expression of nNOS led to NO production and triggered spontaneous induction of leaf lesions. Transgenic plants accumulated high amounts of H₂O₂, with catalase activity lower than that in the wild type. nNOS transgenic plants contained high levels of salicylic acid (SA), and they induced an array of SA-, jasmonic acid (JA)-, and/or ethylene (ET)-related genes. Consequently, NahG co-expression blocked the induction of systemic acquired resistance (SAR)-associated genes in transgenic plants, implying SA is involved in NO-mediated induction of SAR genes. The transgenic plants exhibited enhanced resistance to a spectrum of pathogens, including bacteria, fungi, and viruses. Our results suggest a highly ranked regulatory role for NO in SA-, JA-, and/or ET-dependent pathways that lead to disease resistance.     

Regulation of obesity and insulin resistance by nitric oxide

Obesity is a risk factor for developing type 2 diabetes and cardiovascular disease and has quickly become a worldwide pandemic with few tangible and safe treatment options. Although it is generally accepted that the primary cause of obesity is energy imbalance, i.e., the calories consumed are greater than are utilized, understanding how caloric balance is regulated has proven a challenge. Many “distal” causes of obesity, such as the structural environment, occupation, and social influences, are exceedingly difficult to change or manipulate. Hence, molecular processes and pathways more proximal to the origins of obesity—those that directly regulate energy metabolism or caloric intake—seem to be more feasible targets for therapy. In particular, nitric oxide (NO) is emerging as a central regulator of energy metabolism and body composition. NO bioavailability is decreased in animal models of diet-induced obesity and in obese and insulin-resistant patients, and increasing NO output has remarkable effects on obesity and insulin resistance. This review discusses the role of NO in regulating adiposity and insulin sensitivity and places its modes of action into context with the known causes and consequences of metabolic disease.     

Myristoylation of endothelial cell nitric oxide synthase is important for extracellular release of nitric oxide

Endothelial cell nitric oxide synthase (NOS) is known to have a N-myristoylation consensus sequence. Such a consensus sequence is not evident in the macrophage, smooth muscle and neuronal NOS. A functional role for this N-terminal myristoylation is not clear yet. In the present study, we examined the effect of N-terminal myristoylation on the NOS activity determined by the conversion of L-[³H]arginine to L-[³H]citrulline and extracellular NO release determined by nitrite production in the conditioned medium from the COS-7 cells transfected with wild type bovine aortic endothelial cell (BAEC) NOS cDNA or nonmyristoylated BAEC-NOS mutant cDNA. NOS activity of wild type BAEC-NOS in COS-7 cells was localized in the particulate fraction and that of mutant NOS was in the cytosolic fraction. In contrast, nitrite production from COS-7 cells transfected with wild type BAEC-NOS cDNA was greater than that of mutant cDNA in a time dependent and a concentration dependent manner. These results suggest that membrane localization of NOS with myristoylation facilitates extracellular transport of NO and leads to enhanced NO signaling on the vascular smooth muscle cells and the intravascular blood cells including neutrophils, macrophages and platelets.     

Analysis of nitric oxide donor effectiveness in resistance vessels

Decreased nitric oxide (NO) bioavailability is associated with a number of pathological conditions. Administration of a supplemental source of NO can counter the pathological effects arising from decreased NO bioavailability. A class of NO-nucleophile adducts that spontaneously release NO (NONOates) has been developed, and its members show promise as therapeutic sources of NO. Because the NONOates release NO spontaneously, a significant portion of the NO may be consumed by the myriad of NO reactive species present in the body. Here we develop a model to analyze the efficacy of NO delivery, by membrane-impermeable NONOates, in the resistance arterioles. Our model identifies three features of blood vessels that will enhance NONOate efficacy: 1) the amount of NO delivered to the abluminal region increases with lumen radius; 2) the presence of a flow-induced red blood cell-free zone will augment NO delivery; and 3) extravasation of the NONOate into the interstitial space will increase abluminal NO delivery. These results suggest that NONOates may be more effective in larger vessels and that NONOate efficacy can be altered by modifying permeability to the interstitial space.