Nitric oxide favours tumour-promoting inflammation through mitochondria-dependent and -independent actions on macrophages.

Production of nitric oxide (NO) has been demonstrated in several malignancies, however its role remains not fully understood, specifically in relation to the metabolic and functional implications that it may have on immune cells participating in tumorigenesis. Here, we show that inducible NO synthase (iNOS) is expressed in cancers of the colon and the prostate, mainly by tumour cells, and NO generation is evidenced by widespread nitrotyrosine (NT) staining in tumour tissue. Furthermore, presence of NT is observed in the majority of tumour-associated macrophages (TAMs), despite low iNOS expression by these cells, suggesting that NO from the tumour microenvironment affects TAMs. Indeed, using a co-culture model, we demonstrate that NO produced by colon and prostate cancer cells is sufficient to induce NT formation in neighbouring macrophages. Moreover, exposure to exogenous NO promotes mitochondria-dependent and -independent changes in macrophages, which orientate their polarity towards an enhanced pro-inflammatory phenotype, whilst decreasing antigen-presenting function and wound healing capacity. Abrogating endogenous NO generation in murine macrophages, on the other hand, decreases their pro-inflammatory phenotype. These results suggest that the presence of NO in cancer may regulate TAM metabolism and function, favouring the persistence of inflammation, impairing healing and subverting adaptive immunity responses.

ß-cell-specific deletion of PFKFB3 restores cell fitness competition and physiological replication under diabetogenic stress.

HIF1α and PFKFB3 play a critical role in the survival of damaged ß-cells in type-2 diabetes while rendering ß-cells non-responsive to glucose stimulation. To discriminate the role of PFKFB3 from HIF1α in vivo, we generated mice with conditional ß-cell specific disruption of the Pfkfb3 gene on a human islet pancreatic polypeptide (hIAPP+/-) background and a high-fat diet (HFD) [PFKFB3ßKO + diabetogenic stress (DS)]. PFKFB3 disruption in ß-cells under DS led to selective purging of hIAPP-damaged ß-cells and the disappearance of insulin- and glucagon positive bihormonal cells. PFKFB3 disruption induced a three-fold increase in ß-cell replication as evidenced by minichromosome maintenance 2 protein (MCM2) expression. Unlike high-, lower DS or switch to restricted chow diet abolished HIF1α levels and reversed glucose intolerance of PFKFB3ßKO DS mice. Our data suggest that replication and functional recovery of ß-cells under DS depend on ß-cell competitive and selective purification of HIF1α and PFKFB3-positive ß-cells.

Inflammation, glucose, and vascular cell damage: the role of the pentose phosphate pathway.

BACKGROUND: Hyperglycemia is acknowledged as a pro-inflammatory condition and a major cause of vascular damage. Nevertheless, we have previously described that high glucose only promotes inflammation in human vascular cells previously primed with pro-inflammatory stimuli, such as the cytokine interleukin (IL)1ß. Here, we aimed to identify the cellular mechanisms by which high glucose exacerbates the vascular inflammation induced by IL1ß. METHODS: Cultured human aortic smooth muscle cells (HASMC) and isolated rat mesenteric microvessels were treated with IL1ß in medium containing 5.5-22 mmol/L glucose. Glucose uptake and consumption, lactate production, GLUT1 levels, NADPH oxidase activity and inflammatory signalling (nuclear factor-κB activation and inducible nitric oxide synthase expression) were measured in HASMC, while endothelium-dependent relaxations to acetylcholine were determined in rat microvessels. Pharmacological inhibition of IL1 receptors, NADPH oxidase and glucose-6-phosphate dehydrogenase (G6PD), as well as silencing of G6PD, were also performed. Moreover, the pentose phosphate pathway (PPP) activity and the levels of reduced glutathione were determined. RESULTS: We found that excess glucose uptake in HASMC cultured in 22 mM glucose only occurred following activation with IL1ß. However, the simple entry of glucose was not enough to be deleterious since over-expression of the glucose transporter GLUT1 or increased glucose uptake following inhibition of mitochondrial respiration by sodium azide was not sufficient to trigger inflammatory mechanisms. In fact, besides allowing glucose entry, IL1ß activated the PPP, thus permitting some of the excess glucose to be metabolized via this route. This in turn led to an over-activation NADPH oxidase, resulting in increased generation of free radicals and the subsequent downstream pro-inflammatory signalling. Moreover, in rat mesenteric microvessels high glucose incubation enhanced the endothelial dysfunction induced by IL1ß by a mechanism which was abrogated by the inhibition of the PPP. CONCLUSIONS: A pro-inflammatory stimulus like IL1ß transforms excess glucose into a vascular deleterious agent by causing an increase in glucose uptake and its subsequent diversion into the PPP, promoting the pro-oxidant conditions required for the exacerbation of pro-oxidant and pro-inflammatory pathways. We propose that over-activation of the PPP is a crucial mechanism for the vascular damage associated to hyperglycemia.

Synergy between medicinal chemistry and biological research.

Salvador Moncada studied medicine at the University of El Salvador (El Salvador) before coming to the UK in 1971 to work on a PhD with Professor John Vane at the Institute of Basic Medical Sciences, Royal College of Surgeons (UK). After a short period of research at the University of Honduras (Honduras), he joined the Wellcome Research Laboratories (UK) where he became Head of the Department of Prostaglandin Research and later, Director of Research. He returned to academic life in 1996 as founder and director of the Wolfson Institute for Biomedical Research at University College London (UK). Moncada played a role in the discovery of the mechanism of action of aspirin-like drugs and later led the teams which discover prostacyclin and identified nitric oxide as a biological mediator. In his role as a Director of Research of the Wellcome Laboratories, he oversaw the discovery and development of medicines for epilepsy, migraine, malaria and cancer. Currently, he is working on the regulation of cell proliferation as Director of the Institute of Cancer Sciences at the University of Manchester (UK). Moncada has won numerous awards from the international scientific community and in 2010, he received a knighthood from Her Majesty Queen Elizabeth II for his services to science.

El endotelio vascular / The vascular endothelium

La investigación sobre el endotelio vascular en los últimos 40 años ha provisto ideas para entender la enfermedad vascular. Este nuevo conocimiento ha encontrado su camino en la medicina clínica. En esta revisión nos ocupamos de ciertas áreas de la investigación en las que se ha obtenido avances significativos en la prevención y el tratamiento cardiovascular, así como algunas interrogantes que aún permanecen sin respuesta...

Over the last 40 years, research on the vascular endothelium has provided important clues for the understanding of vascular disease. This new knowledge is finding its way into clinical medicine. In this review we deal with some areas where significant advances in the prevention and treatment of cardiovascular research has been achieved and with some of the remaining questions...

Oncogenic transformation of mesenchymal stem cells decreases Nrf2 expression favoring in vivo tumor growth and poorer survival.

BACKGROUND: The transcription factor Nrf2 is a key regulator of the cellular antioxidant response, and its activation by chemoprotective agents has been proposed as a potential strategy to prevent cancer. However, activating mutations in the Nrf2 pathway have been found to promote tumorigenesis in certain models. Therefore, the role of Nrf2 in cancer remains contentious. METHODS: We employed a well-characterized model of stepwise human mesenchymal stem cell (MSC) transformation and breast cancer cell lines to investigate oxidative stress and the role of Nrf2 during tumorigenesis. The Nrf2 pathway was studied by microarray analyses, qRT-PCR, and western-blotting. To assess the contribution of Nrf2 to transformation, we established tumor xenografts with transformed MSC expressing Nrf2 (n = 6 mice per group). Expression and survival data for Nrf2 in different cancers were obtained from GEO and TCGA databases. All statistical tests were two-sided. RESULTS: We found an accumulation of reactive oxygen species during MSC transformation that correlated with the transcriptional down-regulation of antioxidants and Nrf2-downstream genes. Nrf2 was repressed in transformed MSC and in breast cancer cells via oncogene-induced activation of the RAS/RAF/ERK pathway. Furthermore, restoration of Nrf2 function in transformed cells decreased reactive oxygen species and impaired in vivo tumor growth (P = 0.001) by mechanisms that included sensitization to apoptosis, and a decreased hypoxic/angiogenic response through HIF-1α destabilization and VEGFA repression. Microarray analyses showed down-regulation of Nrf2 in a panel of human tumors and, strikingly, low Nrf2 expression correlated with poorer survival in patients with melanoma (P = 0.0341), kidney (P = 0.0203) and prostate (P = 0.00279) cancers. CONCLUSIONS: Our data indicate that oncogene-induced Nrf2 repression is an adaptive response for certain cancers to acquire a pro-oxidant state that favors cell survival and in vivo tumor growth.

Fulfilling the metabolic requirements for cell proliferation.

The activity of key metabolic enzymes is regulated by the ubiquitin ligases that control the function of the cyclins; therefore the activity of these ubiquitin ligases explains the coordination of cell-cycle progression with the supply of substrates necessary for cell duplication. APC/C (anaphase-promoting complex/cyclosome)-Cdh1, the ubiquitin ligase that controls G(1)- to S-phase transition by targeting specific degradation motifs in cell-cycle proteins, also regulates the glycolysis-promoting enzyme PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3) and GLS1 (glutaminase 1), a critical enzyme in glutaminolysis. A decrease in the activity of APC/C-Cdh1 in mid-to-late G(1) releases both proteins, thus explaining the simultaneous increase in the utilization of glucose and glutamine during cell proliferation. This occurs at a time consistent with the point in G(1) that has been described as the nutrient-sensitive restriction point and is responsible for the transition from G(1) to S. PFKFB3 is also a substrate at the onset of S-phase for the ubiquitin ligase SCF (Skp1/cullin/F-box)-ß-TrCP (ß-transducin repeat-containing protein), so that the activity of PFKFB3 is short-lasting, coinciding with a peak in glycolysis in mid-to-late G(1), whereas the activity of GLS1 remains high throughout S-phase. The differential regulation of the activity of these proteins indicates that a finely-tuned set of mechanisms is activated to fulfil specific metabolic demands at different stages of the cell cycle. These findings have implications for the understanding of cell proliferation in general and, in particular, of cancer, its prevention and treatment.

E3 ubiquitin ligase APC/C-Cdh1 accounts for the Warburg effect by linking glycolysis to cell proliferation.

Cell proliferation is known to be accompanied by activation of glycolysis. We have recently discovered that the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is degraded by the E3 ubiquitin ligase APC/C-Cdh1, which also degrades cell-cycle proteins. We now show in two different cell types (neoplastic and nonneoplastic) that both proliferation and aerobic glycolysis are prevented by overexpression of Cdh1 and enhanced by its silencing. Furthermore, we have coexpressed Cdh1 with PFKFB3--either wild-type or a mutant form resistant to ubiquitylation by APC/C-Cdh1--or with the glycolytic enzyme 6-phosphofructo-1-kinase and demonstrated that whereas glycolysis is essential for cell proliferation, its initiation in the presence of active Cdh1 does not result in proliferation. Our experiments indicate that the proliferative response, regardless of whether it occurs in normal or neoplastic cells, is dependent on a decrease in the activity of APC/C-Cdh1, which activates both proliferation and glycolysis. These observations have implications for cell proliferation, neoplastic transformation, and the prevention and treatment of cancer.

Nitric oxide, cytochrome C oxidase, and the cellular response to hypoxia.

Cytochrome c oxidase (CcO; complex IV of the mitochondrial electron transport chain) is the primary site of cellular oxygen consumption and, as such, is central to oxidative phosphorylation and the generation of adenosine-triphosphate. Nitric oxide (NO), an endogenously-generated gas, modulates the activity of CcO. Depending on the intracellular oxygen concentration and the resultant dominant redox state of CcO, the interaction between CcO and NO can have a range of signaling consequences for cells in the perception of changes in oxygen concentration and the initiation of adaptive responses. At higher oxygen concentrations, when CcO is predominantly in an oxidized state, it consumes NO. At lower oxygen concentrations, when CcO is predominantly reduced, NO is not consumed and accumulates in the microenvironment, with implications for both the respiratory rate of cells and the local vascular tone. Changes in the availability of intracellular oxygen and in the generation of reactive oxygen species that accompany these interactions result in cell signaling and in regulation of oxygen-sensitive pathways that ultimately determine the nature of the cellular response to hypoxia.

Lack of oxygen deactivates mitochondrial complex I: implications for ischemic injury?

For S-nitrosothiols and peroxynitrite to interfere with the activity of mitochondrial complex I, prior transition of the enzyme from its active (A) to its deactive, dormant (D) state is necessary. We now demonstrate accumulation of the D-form of complex I in human epithelial kidney cells after prolonged hypoxia. Upon reoxygenation after hypoxia there was an initial delay in the return of the respiration rate to normal. This was due to the accumulation of the D-form and its slow, substrate-dependent reconversion to the A-form. Reconversion to the A-form could be prevented by prolonged incubation with endogenously generated NO. We propose that the hypoxic transition from the A-form to the D-form of complex I may be protective, because it would act to reduce the electron burst and the formation of free radicals during reoxygenation. However, this may become an early pathophysiological event when NO-dependent formation of S-nitrosothiols or peroxynitrite structurally modifies complex I in its D-form and impedes its return to the active state. These observations provide a mechanism to account for the severe cell injury that follows hypoxia and reoxygenation when accompanied by NO generation.

Mitochondrial dysfunction and HIF1alpha stabilization in inflammation.

Activation of murine-derived J774.A1 macrophages with interferon gamma and lipopolysaccharide leads to a progressive mitochondrial defect characterized by inhibition of oxygen consumption and a decrease in the generation of ATP by oxidative phosphorylation. These changes are dependent on the generation of nitric oxide (NO) by an inducible NO synthase that becomes a significant consumer of oxygen. Furthermore, in these activated cells there is a biphasic stabilization of the hypoxia-inducible factor HIF1alpha, the second phase of which is also dependent on the presence of NO. The mitochondrial defect and stabilization of HIF1alpha synergize to activate glycolysis, which, at its maximum, generates quantities of ATP greater than those produced by non-activated cells. Nevertheless, the amount of ATP generated is not sufficient to fulfil the energy requirements of the activated cells, probably leading to a progressive energy deficit with the consequent inhibition of cell proliferation and death.

Transformation of human mesenchymal stem cells increases their dependency on oxidative phosphorylation for energy production.

An increased dependency on glycolysis for ATP production is considered to be a hallmark of tumor cells. Whether this increase in glycolytic activity is due mainly to inherent metabolic alterations or to the hypoxic microenvironment remains controversial. Here we have transformed human adult mesenchymal stem cells (MSC) using genetic alterations as described for differentiated cells. Our data suggest that MSC require disruption of the same pathways as have been shown for differentiated cells to confer a fully transformed phenotype. Furthermore, we found that MSC are more glycolytic than primary human fibroblasts and, in contrast to differentiated cells, do not depend on increased aerobic glycolysis for ATP production during transformation. These data indicate that aerobic glycolysis (the Warburg effect) is not an intrinsic component of the transformation of adult stem cells, and that oncogenic adaptation to bioenergetic requirements, in some circumstances, may also rely on increases in oxidative phosphorylation. We did find, however, a reversible increase in the transcription of glycolytic enzymes in tumors generated by transformed MSC, indicating this is a secondary phenomenon resulting from adaptation of the tumor to its microenvironment.

TNF-alpha downregulates eNOS expression and mitochondrial biogenesis in fat and muscle of obese rodents.

Obesity is associated with chronic low-grade inflammation. Thus, at metabolically relevant sites, including adipose tissue and muscle, there is abnormal production of proinflammatory cytokines such as TNF-alpha. Here we demonstrate that eNOS expression was reduced, with a concomitant reduction of mitochondrial biogenesis and function, in white and brown adipose tissue and in the soleus muscle of 3 different animal models of obesity. The genetic deletion of TNF receptor 1 in obese mice restored eNOS expression and mitochondrial biogenesis in fat and muscle; this was associated with less body weight gain than in obese wild-type controls. Furthermore, TNF-alpha downregulated eNOS expression and mitochondrial biogenesis in cultured white and brown adipocytes and muscle satellite cells of mice. The NO donors DETA-NO and SNAP prevented the reduction of mitochondrial biogenesis observed with TNF-alpha. Our findings demonstrate that TNF-alpha impairs mitochondrial biogenesis and function in different tissues of obese rodents by downregulating eNOS expression and suggest a novel pathophysiological process that sustains obesity.

Nitric oxide is a factor in the stabilization of hypoxia-inducible factor-1alpha in cancer: role of free radical formation.

Widespread expression of the alpha-subunit of hypoxia-inducible factor (HIF-1alpha) was observed in samples of human oral squamous cell carcinoma. In all the cases, this was accompanied by a widespread distribution of nitric oxide (NO) synthases (NOS). Furthermore, in three human cell lines derived from human oral squamous cell carcinoma, the accumulation of HIF-1alpha was prevented either by inhibition of NOS activity with the nonspecific NOS inhibitor N(G)-monomethyl-L-arginine or by the antioxidants N-acetyl-L-cysteine and ascorbic acid. We suggest that, in certain forms of cancer, NO might be responsible for the accumulation of HIF-1alpha by a mechanism dependent on free radicals.

Calorie restriction promotes mitochondrial biogenesis by inducing the expression of eNOS.

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.

Therapeutic potential of nitric oxide donors in the prevention and treatment of atherosclerosis.

Well-known risk factors for atherosclerosis include hypercholesterolaemia, hypertension, diabetes, and smoking. These conditions are associated with endothelial dysfunction, which itself is associated with reduced endothelial generation of nitric oxide (NO). This is an overview of the implications of NO generation in atherosclerosis and of the potential therapeutic benefit of drugs which donate NO, such as organic nitrates, nicorandil, and sydnonimines, or those which increase the availability of endogenous NO, such as statins, angiotensin-converting enzyme inhibitors, L-arginine, and tetrahydrobiopterin.

Hypoxia accelerates nitric oxide-dependent inhibition of mitochondrial complex I in activated macrophages.

Excess production of nitric oxide (NO) is implicated in the development of multiple organ failure, with a putative mechanism involving direct mitochondrial inhibition, predominantly affecting complex I. The persistent effects of NO on complex I may be mediated through S-nitrosylation and/or nitration. The temporal contribution of these chemical modifications to the inhibition of respiration and the influence of concurrent hypoxia have not been previously examined. We therefore addressed these questions using J774 macrophages activated by endotoxin and interferon-gamma over a 24-h period, incubated at 21% and 1% oxygen. Oxygen consumption and complex I activity fell progressively over time in the activated cells. This was largely prevented by coincubation with the nonspecific NO synthase inhibitor L-N5-(1-iminoethyl)-ornithine. Addition of glutathione ethyl ester reversed the inhibition at initial time points, suggesting an early mechanism involving nitrosylation. Thereafter, the inhibition of complex I became more persistent, coinciding with a progressive increase in mitochondrial nitration. Hypoxia accelerated the persistent inhibition of complex I, despite a reduction in the total amount of NO generated. Our results suggest that hypoxia amplified the mitochondrial inhibition induced by NO generated during inflammatory disease states.

Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals.

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.

Inhibition of mitochondrial respiration by the anticancer agent 2-methoxyestradiol.

2-Methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol, is known to have antiproliferative, antiangiogenic, and proapoptotic activity. Mechanistically, 2ME2 has been shown to downregulate hypoxia-inducible factor 1alpha (HIF1alpha) and to induce apoptosis in tumour cells by generating reactive oxygen species (ROS). In this study we report that 2ME2 inhibits mitochondrial respiration in both intact cells and submitochondrial particles, and that this effect is due to inhibition of complex I of the mitochondrial electron transport chain (ETC). The prevention by 2ME2 of hypoxia-induced stabilisation of HIF1alpha in HEK293 cells was found not to be due to an effect on HIF1alpha synthesis but rather to an effect on protein degradation. This is in agreement with our recent observation using other inhibitors of mitochondrial respiration which bring about rapid degradation of HIF1alpha in hypoxia due to increased availability of oxygen and reactivation of prolyl hydroxylases. The concentrations of 2ME2 that inhibited complex I also induced the generation of ROS. 2ME2 did not, however, cause generation of ROS in 143B rho(-) cells, which lack a functional mitochondrial ETC. We conclude that inhibition of mitochondrial respiration explains, at least in part, the effect of 2ME2 on hypoxia-dependent HIF1alpha stabilisation and cellular ROS production. Since these actions of 2ME2 occur at higher concentrations than those known to inhibit cell proliferation, it remains to be established whether they contribute to its therapeutic effect.