Nitric oxide synthetic pathway in patients with microvascular angina and its relations with oxidative stress.

A decreased nitric oxide (NO) bioavailability and an increased oxidative stress play a pivotal role in different cardiovascular pathologies. As red blood cells (RBCs) participate in NO formation in the bloodstream, the aim of this study was to outline the metabolic profile of L-arginine (Arg)/NO pathway and of oxidative stress status in RBCs and in plasma of patients with microvascular angina (MVA), investigating similarities and differences with respect to coronary artery disease (CAD) patients or healthy controls (Ctrl). Analytes involved in Arg/NO pathway and the ratio of oxidized and reduced forms of glutathione were measured by LC-MS/MS. The arginase and the NO synthase (NOS) expression were evaluated by immunofluorescence staining. RBCs from MVA patients show increased levels of NO synthesis inhibitors, parallel to that found in plasma, and a reduction of NO synthase expression. When summary scores were computed, both patient groups were associated with a positive oxidative score and a negative NO score, with the CAD group located in a more extreme position with respect to Ctrl. This finding points out to an impairment of the capacity of RBCs to produce NO in a pathological condition characterized mostly by alterations at the microvascular bed with no significant coronary stenosis.

A mass spectrometry-based workflow for the proteomic analysis of in vitro cultured cell subsets isolated by means of laser capture microdissection.

This paper describes a microproteomic workflow that is useful for simultaneously identifying and quantifying proteins from a minimal number of morphotypically heterogeneous cultured adherent cells. The analytical strategy makes use of laser capture microdissection, an effective means of harvesting pure cell populations, and label-free mass spectrometry. We optimised the workflow with particular reference to cell fixation which is crucial for successful laser-based microdissection and also downstream molecular studies. In addition, we defined the minimum number of cells to be isolated and analysed for satisfactory proteome coverage. To set up this workflow, we choose human monocyte-derived macrophages spontaneously differentiated in vitro. These cells, under our culture conditions, show distinct morphotypes, reminiscent of the heterogeneity observed in tissues in various homeostatic and pathological states, e.g. atherosclerosis. This optimised workflow may provide new insights into biology and pathology of heterogeneous cell in culture, particularly when other cell selection approaches are not suitable.

Nitric oxide synthetic pathway in red blood cells is impaired in coronary artery disease.

BACKGROUND: All the enzymatic factors/cofactors involved in nitric oxide (NO) metabolism have been recently found in red blood cells. Increased oxidative stress impairs NO bioavailability and has been described in plasma of coronary artery disease (CAD) patients. The aim of the study was to highlight a potential dysfunction of the metabolic profile of NO in red blood cells and in plasma from CAD patients compared with healthy controls. METHODS: We determined L-arginine/NO pathway by liquid-chromatography tandem mass spectrometry and high performance liquid chromatography methods. The ratio of oxidized and reduced forms of glutathione, as index of oxidative stress, was measured by liquid-chromatography tandem mass spectrometry method. NO synthase expression and activity were evaluated by immunofluorescence staining and ex-vivo experiments of L-[(15)N2]arginine conversion to L-[(15)N]citrulline respectively. RESULTS: Increased amounts of asymmetric and symmetric dimethylarginines were found both in red blood cells and in plasma of CAD patients in respect to controls. Interestingly NO synthase expression and activity were reduced in CAD red blood cells. In contrast, oxidized/reduced glutathione ratio was increased in CAD and was associated to arginase activity. CONCLUSION: Our study analyzed for the first time the whole metabolic pathway of L-arginine/NO, both in red blood cells and in plasma, highlighting an impairment of NO pathway in erythrocytes from CAD patients, associated with decreased NO synthase expression/activity and increased oxidative stress.

Human monocyte-derived macrophages spontaneously differentiated in vitro show distinct phenotypes.

Tissue macrophages are resident phagocytes that acquire specific phenotypes according to the microenvironment. Morphological and functional heterogeneity has been evidenced in different homeostatic and pathological conditions. Indeed, the nature of macrophage subsets may have either harmful or beneficial functions in disease progression/resolution. Therefore the possibility to pharmacologically manipulate heterogeneity represents a relevant challenge. Since human tissue macrophages are not easily obtained, various in vitro models are currently used that do not adequately reflect the heterogeneity and plasticity of tissue macrophages. We had previously reported that two dominant and distinct macrophage morphotypes co-exist in the same culture of human monocytes spontaneously differentiated for 7 days in autologous serum. The present study was aimed to the phenotypic characterization of these morphotypes, that is, round- and spindle-shaped. We observed that, besides substantial differences in cytoskeleton architecture, round monocyte-derived macrophages (MDMs) showed higher lipid content, increased macropinocytosis/efferocytosis capacity, and overexpression of CD163, interleukin (IL)-10, and transforming growth factor (TGF) ß2. Conversely, spindle MDMs exhibited enhanced respiratory burst and higher expression of the chemokine (C-C motif) ligands 18 and 24 (CCL18 and CCL24). Overall, round MDMs show functional traits reminiscent of the non-inflammatory and reparative M2 phenotype, whereas spindle MDMs exhibit a pro-inflammatory profile and express genes driving lymphocyte activation and eosinophil recruitment. MDMs obtained in the culture condition herein described represent a valuable model to disentangle and manipulate the functional heterogeneity of tissue macrophages that has been disclosed in scenarios spanning from inflammatory and wounding responses to atherosclerotic lesions.

Cytoskeletal architecture regulates cyclooxygenase-2 in human endothelial cells: autocrine modulation by prostacyclin.

Endothelium is a highly dynamic tissue that controls vascular homeostasis. This requires constant rearrangements of the shape or function of endothelial cells that cannot set aside the role of the cytoskeleton. The aim of this study was to determine the mechanisms by means of which cytoskeletal alterations induce cyclooxygenase-2 (Cox-2) expression in human endothelial cells using compounds that interfere with microtubule or actin architecture. Microtubule disruption by nocodazole markedly increased Cox-2 expression and activity, and provoked paracellular gap formation, a cardinal feature of endothelial barrier dysfunction. The Cox-2 metabolite prostacyclin down-regulated Cox-2 through an autocrine receptor-mediated mechanism, and partially prevented the disassembly of endothelial monolayers. There was also an interaction between microtubules and actin filaments in nocodazole-induced Cox-2 expression. Nocodazole provoked the dissolution of the F-actin cortical ring and stress fiber formation, increased actin glutathionylation, and concomitantly lowered intracellular levels of reduced glutathione. The restoration of glutathione levels by N-acetylcysteine opposed Cox-2 expression and preserved the integrity of endothelial monolayers. Among the signaling pathways connecting microtubule disruption with Cox-2 up-regulation, crucial roles are played by Src family kinase activation, serine/threonine phosphatase 2A inhibition, and the phosphorylation of mitogen activated protein kinase p38. Our findings provide a mechanistic insight into the observation that Cox-2 is induced in endothelial cells under cytoskeleton-perturbing conditions such as those occurring in the presence of atherogenic/inflammatory stimuli and oxidative stress. In this scenario, Cox-2 up-regulation by endothelia exposed to noxious conditions can be considered protective of the vasodilatory and anti-thrombotic properties of the vessel wall.

Cyclooxygenase-2 mediates hydrogen peroxide-induced wound repair in human endothelial cells.

Cyclooxygenase-2 (Cox-2) metabolites produced by endothelial cells, particularly prostacyclin and prostaglandin E(2), profoundly affect vascular tone, regional blood flow, and angiogenesis. We have previously shown that reactive oxygen species induce Cox-2 expression in human endothelial cells (HUVEC), either on their own or as components of the signaling pathway triggered by TNFalpha, the prototypical inflammatory cytokine. Here we investigated the role of Cox-2 induced by hydrogen peroxide (H(2)O(2)), either exogenous or endogenously generated by TNFalpha, in the repair of a mechanically wounded HUVEC monolayer and probed the sources of H(2)O(2) that are involved in TNFalpha signaling and the pathways through which H(2)O(2) modulates Cox-2 expression. Results indicate that H(2)O(2)-induced Cox-2 activity participates in the repair of wounded monolayers. Both NADPH oxidase and the mitochondrial electron transport chain are involved in H(2)O(2) generation. Signaling triggered by H(2)O(2) for Cox-2 induction acts by increasing the protein tyrosine kinase phosphorylation that follows inhibition of protein phosphatase activity. The activation of p38 MAPK and its interaction in the inhibition of serine/threonine phosphatase activity are both critical steps in this event. We conclude that Cox-2 induced by H(2)O(2) plays an important role in promoting endothelial wound repair after injury, so that the cardioprotective effect of Cox-2 is due at least in part to its power of healing damaged endothelium.