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At present, the clinically most advanced strategy to treat Duchenne muscular dystrophy (DMD) is the exon-skipping strategy. Whereas antisense oligonucleotide-based clinical trials are underway for DMD, it is essential to determine the dystrophin restoration threshold needed to ensure improvement of muscle physiology at the molecular level. A preclinical trial has been conducted in golden retriever muscular dystrophy (GRMD) dogs treated in a forelimb by locoregional delivery of rAAV8-U7snRNA to promote exon skipping on the canine dystrophin messenger. Here, we exploited rAAV8-U7snRNA-transduced GRMD muscle samples, well characterized for their percentage of dystrophin-positive fibers, with the aim of defining the threshold of dystrophin rescue necessary for normalization of the status of neuronal nitric oxide synthase mu (nNOSμ), inducible nitric oxide synthase (iNOS), and ryanodine receptor-calcium release channel type 1 (RyR1), crucial actors for efficient contractile function. Results showed that restoration of dystrophin in 40% of muscle fibers is needed to decrease abnormal cytosolic nNOSμ expression and to reduce overexpression of iNOS, these two parameters leading to a reduction in the NO level in the muscle fibers. Furthermore, the same percentage of dystrophin-positive fibers of 40% was associated with the normalization of RyR1 nitrosylation status and with stabilization of the RyR1-calstabin1 complex that is required to facilitate coupled gating. We concluded that a minimal threshold of 40% of dystrophin-positive fibers is necessary for the reinstatement of central proteins needed for proper muscle contractile function, and thus identified a rate of dystrophin expression significantly improving, at the molecular level, the dystrophic muscle physiology.

The roles of neuronal and inducible nitric oxide synthases in neurones have been extensively investigated; by contrast, the biological significance of endothelial nitric oxide synthase (eNOS) overexpression that occurs in several pathological conditions has not yet been studied. We have started addressing this issue in a cell model of neurodegeneration, i.e. human SKNBE neuroblastoma cells transfected with a mutant form of alsin, a protein causing an early-onset type of amyotrophic lateral sclerosis, ALS2. We found that eNOS, which is endogenously expressed by these cells, was activated by tumour necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine that plays important roles in ALS2 and several neurodegenerative diseases. The TNF-alpha-dependent eNOS activation occurred through generation, by sphingosine-kinase-1, of sphingosine-1-phosphate, stimulation of its membrane receptors and activation of Akt, as determined using small interference RNA and dominant negative constructs specific for the enzymes and receptors. eNOS activation by TNF-alpha conferred cytoprotection from excitotoxicity and neurotoxic cues such as reactive oxygen species, endoplasmic reticulum stress, DNA damage, and mutated alsin itself. Our results suggest that overexpression of eNOS by neurones is a broad-range protective mechanism activated during damage and establish a link of pathophysiological relevance between this enzyme and inflammation accompanying neurodegenerative diseases. These findings also question the concept that high NO output in the presence of oxidative stress leads always to peroxynitrite formation contributing to neurodegeneration.

Cisplatin is one of the most effective anticancer drugs, but its severe toxic effects, including depletion of immune-competent cells, limit its efficacy. We combined the systemic treatment with cisplatin with intratumor delivery of dendritic cells (DC) previously treated ex vivo with a pulse of nitric oxide (NO) released by the NO donors (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]-diazen-1-ium-1,2-diolate or isosorbide dinitrate. We found that this chemoimmunotherapy, tested in the B16 mouse model of melanoma, was significantly more efficacious than cisplatin alone, leading to tumor regression and animal survival at low doses of cisplatin that alone had no effect. Tumor cure was not observed when combining cisplatin with DCs not exposed to NO donors, indicating the key role of the pretreatment with NO. We investigated the mechanisms responsible for the synergic effect of NO-treated DCs and cisplatin and found that NO-treated DCs were protected both in vitro and in vivo from cisplatin-induced cytotoxicity. Cisplatin triggered DC apoptosis through increased expression and activation of acid sphingomyelinase; pretreatment of DCs with NO donors prevented such activation and inhibited activation of the downstream proapoptotic events, including generation of ceramide, activation of caspases 3 and 9, and mitochondrial depolarization. The effects of NO were mediated through generation of its physiologic messenger, cyclic GMP. We conclude that NO and NO generating drugs represent promising tools to increase the efficacy of chemoimmunotherapies in vivo, promoting the survival and increasing the function of injected cells by targeting a key pathway in cisplatin-induced cytotoxicity. [Cancer Res 2007;67(16):7559–64]

Calorie restriction extends life span in organisms ranging from yeast to mammals. Here, we report that calorie restriction for either 3 or 12 months induced endothelial nitric oxide synthase (eNOS) expression and 3′,5′-cyclic guanosine monophosphate formation in various tissues of male mice. This was accompanied by mitochondrial biogenesis, with increased oxygen consumption and adenosine triphosphate production, and an enhanced expression of sirtuin 1. These effects were strongly attenuated in eNOS null-mutant mice. Thus, nitric oxide plays a fundamental role in the processes induced by calorie restriction and may be involved in the extension of life span in mammals.

The gaseous messenger nitric oxide plays a role in a variety of biological functions. Evidence accumulated over the last 7 years indicates that functions of nitric oxide in apoptosis growth and differentiation may originate in part from an interplay with signalling members of the sphingolipid family. Interactions between nitric oxide and sphingolipids have been shown to be multiple, to involve regulation of activity and expression of the enzymes responsible for the synthesis of nitric oxide and of those involved in the sphingolipid metabolic pathways. Recent evidence indicates that one of these interactions, namely the cross-talk of sphingomyelinases and their product ceramide with nitric oxide and its generating enzyme endothelial nitric oxide synthase, plays prominent roles during key patho-physiological processes such as inflammation, proliferation, death and differentiation.

We recently found that long-term exposure to nitric oxide (NO) triggers mitochondrial biogenesis in mammalian cells and tissues by activation of guanylate cyclase and generation of cGMP. Here, we report that the NO/cGMP-dependent mitochondrial biogenesis is associated with enhanced coupled respiration and content of ATP in U937, L6, and PC12 cells. The observed increase in ATP content depended entirely on oxidative phosphorylation, because ATP formation by glycolysis was unchanged. Brain, kidney, liver, heart, and gastrocnemius muscle from endothelial NO synthase null mutant mice displayed markedly reduced mitochondrial content associated with significantly lower oxygen consumption and ATP content. In these tissues, ultrastructural analyses revealed significantly smaller mitochondria. Furthermore, a significant reduction in the number of mitochondria was observed in the subsarcolemmal region of the gastrocnemius muscle. We conclude that NO/cGMP stimulates mitochondrial biogenesis, both in vitro and in vivo , and that this stimulation is associated with increased mitochondrial function, resulting in enhanced formation of ATP.

Susceptibility of dendritic cells (DCs) to tumor-induced apoptosis reduces their efficacy in cancer therapy. Here we show that delivery within exponentially growing B16 melanomas of DCs treated ex vivo with nitric oxide (NO), released by the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), significantly reduced tumor growth, with cure of 37% of animals. DETA-NO-treated DCs became resistant to tumor-induced apoptosis because DETA-NO prevented tumor-induced changes in the expression of Bcl-2, Bax, and Bcl-xL; activation of caspase-9; and a reduction in the mitochondrial membrane potential. DETA-NO also increased DC cytotoxic activity against tumor cells and DC ability to trigger T-lymphocyte proliferation. All of the effects of DETA-NO were mediated through cGMP generation. NO and NO-generating drugs may therefore be used to increase the anticancer efficacy of DCs.

The gaseous messenger nitric oxide (NO) contributes to biological effects of oestrogen in target tissues, including reproductive organs, bone, cardiovascular and central nervous systems. Vasodilation and anti-atherosclerotic properties of NO have been shown to play a role in these effects. The possibility that NO acts also through regulation of the signal transduction cascade triggered by oestrogen, instead, has never been investigated. To study this we have used the MCF-7 human breast cancer cell line, an established model for oestrogen signalling. Exposure of these cells to 17-β-oestradiol (E2) in the presence of NO gave rise to activation of signalling events additional to those triggered by E2 alone, namely tyrosine phosphorylation of specific proteins, including the insulin receptor substrate-1, with recruitment to this adapter of the phosphatidylinositol 3′-kinase and persistent activation of Akt (protein kinase B). Active Akt, in turn, prevented E2 from activating p42/44 extracellular signal-regulated kinases (ERK 1/2). These effects of NO, which were mediated through generation of cyclic GMP and activation of the cGMP-dependent protein kinase I, initiated in the first minutes after administration of oestrogen. The consequences, however, were long lasting, as modulation of Akt and ERK 1/2 activities by NO was responsible for inhibition of E2-triggered cell growth and regulation of oestrogen responsive-element dependent gene transcription. Generation of NO is stimulated by both E2 and growth factors known to contribute to the complex network of intracellular events regulating the biological actions of oestrogen. It is conceivable, therefore, that modulation by NO of E2 early signalling, here described for the first time, has broad significance in regulating cellular responses to the hormone.

During myogenic differentiation the short mitochondria of myoblasts change into the extensively elongated network observed in myotubes. The functional relevance and the molecular mechanisms driving the formation of this mitochondrial network are unknown. We now show that mitochondrial elongation is required for myogenesis to occur and that this event depends on the cellular generation of nitric oxide (NO). Inhibition of NO synthesis in myogenic precursor cells leads to inhibition of mitochondrial elongation and of myogenic differentiation. This is due to the enhanced activity, translocation and docking of the pro-fission GTPase dynamin-related protein-1 (Drp1) to mitochondria, leading also to a latent mitochondrial dysfunction that increased sensitivity to apoptotic stimuli. These effects of NO inhibition were not observed in myogenic precursor cells containing a dominant-negative form of Drp1. Both NO-dependent repression of Drp1 action and maintenance of mitochondrial integrity and function were mediated through the soluble guanylate cyclase. These data uncover a novel level of regulation of differentiation linking mitochondrial morphology and function to myogenic differentiation. © 2010 Macmillan Publishers Limited All rights reserved.

Nitric oxide (NO), generated by phagocytes at inflammation sites, contributes to regulate immune responses through autocrine and paracrine actions on bystander cells. Among the latter are dendritic cells (DCs). Little is known about regulation of DC function by NO, especially in the human system. We exposed human monocyte-derived DCs to the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2 diolate (DETA-NO) during their maturation process induced by treatment with tumor necrosis factor α or lipopolysaccharide or by CD40 activation. We report here that after exposure to DETA-NO, DCs exhibit a significantly increased ability to activate T lymphocytes stimulated by mycobacterial antigens, Staphylococcus aureus Cowen strain B, allo-antigens, or cross-linking of the CD3–T cell receptor complex. This effect persists after removal of DETA-NO, depends on the generation of cyclic guanosine 5′-monophosphate, and is a result of enhanced release by DCs of soluble factors, in particular interleukin (IL)-12. This modulation of DC function is a result of a synergism between NO and the various maturation stimuli, as neither enhanced T cell activation nor IL-12 release was observed after DC exposure to DETA-NO only. These results provide the first evidence that NO acts as a cosignaling molecule regulating human DC response to maturation stimuli.

Depletion of dendritic cells (DCs) via apoptosis contributes to sepsis-induced immune suppression. The mechanisms leading to DC apoptosis during sepsis are not known. In this study we report that immature DCs undergo apoptosis when treated with high numbers of Escherichia coli. This effect was mimicked by high concentrations of LPS. Apoptosis was accompanied by generation of ceramide through activation of acid sphingomyelinase (A-SMase), was prevented by inhibitors of this enzyme, and was restored by exogenous ceramide. Compared with immature DCs, mature DCs expressed significantly reduced levels of A-SMase, did not generate ceramide in response to E. coli or LPS, and were insensitive to E. coli- and LPS-triggered apoptosis. However, sensitivity to apoptosis was restored by addition of exogenous A-SMase or ceramide. Furthermore, inhibition of A-SMase activation and ceramide generation was found to be the mechanism through which the immune-modulating messenger NO protects immature DCs from the apoptogenic effects of E. coli and LPS. NO acted through formation of cGMP and stimulation of the cGMP-dependent protein kinase. The relevance of A-SMase and its inhibition by NO/cGMP were confirmed in a mouse model of LPS-induced sepsis. DC apoptosis was significantly higher in inducible NO synthase-deficient mice than in wild-type animals and was significantly reduced by treatment ex vivo with NO, cGMP, or the A-SMase inhibitor imipramine. Thus, A-SMase plays a central role in E. coli/LPS-induced DC apoptosis and its inhibition by NO, and it might be a target of new therapeutic approaches to sepsis.