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Background. The C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with “cancer hallmarks” and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT). Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions. Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities. Methods. Structured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model. Results. We identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models. Conclusions. This study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.

The GTPase Rab14 is localized at the trans-Golgi network and at the intermediate compartment associated to sorting/recycling endosomes-like structures of the transferrin-recycling pathway: as other Rab family members, it is involved in the regulation of intracellular vesicle trafficking, though its role and functional relationship with effector/endosomal proteins is still incomplete.We have analysed whether post-translational modifications could affect Rab14 activity: the results obtained define mono-ADP-ribosylation (MARylation) as the yet-unknown Rab14 modification, catalysed by the ADP-ribosyltransferase PARP12, which specifically modifies glutamic acid residues in position 159/162. This modification is essential for the Rab14-dependent endosome progression. Accordingly, recycling of the transferrin receptor is inhibited when MARylation of Rab14 is prevented by PARP12 knocking-down or inhibition, or by overexpression of Rab14 ADP-ribosylation-defective mutant. Under these conditions, Rab14 and transferrin receptors are withheld at the cell periphery at the level of the Rab4-RUFY1-positive sorting endosomes, indicating that the interaction of Rab14 with the dual effectors RUFY and then FIP1c (which specifically binds both Rab11 and Rab14) determines the progression between the Rab4-RUFY- and Rab11-FIP1c-specific vesicles. Therefore Rab14-MARylation determines the sequential binding of this GTPase to RUFY and FIP1c, thus controlling endosome progression (i.e., transferrin receptors recycling) through the Rab4-, Rab14- and Rab11-specific vesicles. This identifies a Rab14-specific compartment of the recycling pathway and a crucial enzymatic reaction amenable to pharmacological control.

Significance Proteins are modified by many posttranslational modifications (PTMs) with crucial regulatory functions. A PTM attracting increasing interest is ADP-ribosylation, capable of altering crucial cellular targets. We show that mono-ADP-ribosylation by PARP12 of the protein Golgin-97 regulates transport to the plasma membrane of a specific group of functionally crucial cargo proteins. PARP12 is shown to be part of a regulatory cascade initiated by PKD and involving the direct phosphorylation and activation of PARP12. These events define, through Golgin-97 mono-ADP-ribosylation, a Golgi sorting mechanism for specific basolateral cargoes, including E-cadherin. Defects in this cascade impair E-cadherin transport and formation of cell–cell contacts, with potential consequences for the formation of adherens junctions, polarization of epithelial cells, and development of epithelial–mesenchymal transformation., Adenosine diphosphate (ADP)-ribosylation is a posttranslational modification involved in key regulatory events catalyzed by ADP-ribosyltransferases (ARTs). Substrate identification and localization of the mono-ADP-ribosyltransferase PARP12 at the trans-Golgi network (TGN) hinted at the involvement of ARTs in intracellular traffic. We find that Golgin-97, a TGN protein required for the formation and transport of a specific class of basolateral cargoes (e.g., E-cadherin and vesicular stomatitis virus G protein [VSVG]), is a PARP12 substrate. PARP12 targets an acidic cluster in the Golgin-97 coiled-coil domain essential for function. Its mutation or PARP12 depletion, delays E-cadherin and VSVG export and leads to a defect in carrier fission, hence in transport, with consequent accumulation of cargoes in a trans-Golgi/Rab11–positive intermediate compartment. In contrast, PARP12 does not control the Golgin-245–dependent traffic of cargoes such as tumor necrosis factor alpha (TNFα). Thus, the transport of different basolateral proteins to the plasma membrane is differentially regulated by Golgin-97 mono-ADP-ribosylation by PARP12. This identifies a selective regulatory mechanism acting on the transport of Golgin-97– vs. Golgin-245–dependent cargoes. Of note, PARP12 enzymatic activity, and consequently Golgin-97 mono-ADP-ribosylation, depends on the activation of protein kinase D (PKD) at the TGN during traffic. PARP12 is directly phosphorylated by PKD, and this is essential to stimulate PARP12 catalytic activity. PARP12 is therefore a component of the PKD-driven regulatory cascade that selectively controls a major branch of the basolateral transport pathway. We propose that through this mechanism, PARP12 contributes to the maintenance of E-cadherin–mediated cell polarity and cell–cell junctions.

Poly-ADP-ribose-polymerases (PARPs) 1 and 2 are nuclear enzymes that catalyze the poly-ADP-ribosylation of nuclear proteins transferring poly-ADP-ribose (PAR) polymers to specific residues. PARPs and PAR intervene in diverse functions, including DNA repair in the nucleus and stress granule assembly in the cytoplasm. Stress granules contribute to the regulation of translation by clustering and stabilizing mRNAs as well as several cytosolic PARPs and signaling proteins to modulate cell metabolism and survival. Our study is focused on one of these PARPs, PARP12, a Golgi-localized mono-ADP-ribosyltransferase that under stress challenge reversibly translocates from the Golgi complex to stress granules. PARP1 activation and release of nuclear PAR drive this translocation by direct PAR binding to the PARP12-WWE domain. Thus, PAR formation functionally links the activity of the nuclear and cytosolic PARPs during stress response, determining the release of PARP12 from the Golgi complex and the disassembly of the Golgi membranes, followed by a block in anterograde-membrane traffic. Notably, these functions can be rescued by reverting the stress condition (by drug wash-out). Altogether these data point at a novel, reversible nuclear signaling that senses stress to then act on cytosolic PARP12, which in turn converts the stress response into a reversible block in intracellular-membrane traffic.

Polymorphonuclear neutrophils (PMN) in atherosclerotic plaques have been identified only recently, and their contribution to plaque development is not yet fully understood. In this study, production of elastase, interleukin (IL)-8 and vascular endothelial growth factor (VEGF) by PMN was investigated in subjects with carotid stenosis undergoing carotid endarterectomy (CEA).The study enrolled 50 patients (Pts) and 10 healthy subjects (HS). Circulating PMN (cPMN) isolated from venous blood (in both Pts and HS) and from plaques (pPMN, in Pts) were cultured, alone or with 0.1 μM fMLP. Elastase, IL-8 and VEGF mRNA were analyzed by real-time PCR. In CEA specimens, PMN were localized by immunohistochemistry.In both Pts cPMN and pPMN, IL-8 mRNA was higher at rest but lower after fMLP (P0.01 vs HS), and VEGF mRNA was higher both at rest and after fMLP (P0.01 vs HS), while elastase mRNA was not significantly different. On the contrary, protein production was always higher in cPMN of HS with respect to values measured in cells of Pts. In CEA specimens, CD66b+ cells localized to areas with massive plaque formation close to neovessels. Pts with soft and mix plaques, as defined by computed tomography, did not differ in cPMN or pPMN IL-8, VEGF or elastase mRNA, or in intraplaque CD66b+ cell density. However, Pts with soft plaques had higher white blood cell count due to increased PMN.In Pts with carotid plaques, both circulating and intraplaque PMN produce IL-8, VEGF and elastase, which are crucial for plaque development and progression. These findings suggest mechanistic explanations to the reported correlation between PMN count and cardiovascular mortality in carotid ATH.

Poly-ADP-ribose-polymerases (PARPs) and the reaction product poly-ADP-ribose (PAR) intervene in diverse cellular functions, including DNA-damage repair in the nucleus and stress granule assembly in the cytoplasm. Stress granules contribute to the regulation of translation by clustering and stabilizing mRNAs as well as several cytosolic PARPs and signaling proteins to modulate cell metabolism and survival. Herein, we describe the behaviour of one of these PARPs -PARP12- and show that this enzyme contributes to cell response to oxidative stress, by translocating from the Golgi complex to the newly-formed stress granules, preventing in this way intracellular-membrane transport progression. PARP12 translocation to stress granules requires PARP1 activation and release of nuclear PAR that, in turn, is directly recognized by PARP12 through its WWE domain. Thus, PAR formation functionally links the activity of the nuclear and cytosolic PARPs during stress response, determining the release of PARP12 from the Golgi complex and, as a consequence, the disassembly of the Golgi membranes, followed by a block in the anterograde membrane traffic. Notably, Golgi complex organization and membrane traffic can be rescued by reverting the stress condition (by drug wash-out) as well as by inhibiting the nuclear signal. Altogether these data highlight a novel, reversible nuclear signaling that senses stress to then act on cytosolic PARP12, which in turn converts the stress response into a reversible block in intracellular membrane traffic.

Intracellular membrane trafficking among organelles occurs through vesicular or tubular intermediates that selectively deliver proteins and lipids from one compartment to another. The Rab proteins are small GTPases that function as master regulators of this process. All Rabs localize at specific subcellular compartments and their distribution is crucial for the specificity and directionality of membrane traffic (i.e. secretion, endocytosis, recycling etc.). In particular, Rab14, one of the recently discovered members of the Rab family is localized at the Golgi/trans-Golgi network (TGN) and endosomes and has been associated with different steps of the endosomal recycling pathway. Here we have identified Rab14 as a novel substrate of PARP12, a mono-ADP-ribosyl transferase mainly localized at the Golgi complex. The physiological functions of PARP12 are not yet completely elucidated. With this study we demonstrate that PARP12 is able to interact and modify Rab14 and that its depletion impairs Rab14 functions. Inhibition of ADP-ribosylation alters Rab14 localization, which in turn affects membrane flow through the recycling endosomes. Using bioinformatics and mutagenesis approaches, we have generated a Rab14 mutant that cannot be ADP-ribosylated by PARP12. The analysis of the mutant suggests that Rab14 participates in a cascade of events controlling the maturation of recycling endosomes and that the ADP-ribosylation of Rab14 is essential for this step. Overall our data identify mono-ADP-ribosylation as a novel post-translation modification of the small GTPase Rab14 that may be used to modulate the spatial-temporal regulation of the Rab14-GTPase activation cycle. In addition our data highlight a novel role of PARP12 in the progression of intracellular membrane traffic.

FeCl4− heterogenized on a Dowex 2-X8 anion exchange resin catalyzes the photooxidation of ethanol to acetaldehyde under visible and near-UV irradiation (>345 nm). The rate of reaction is proportional to the oxygen partial pressure up to 1 atm. Oxidation is suggested to occur through the formation of 1-hydroxyethylhydroperoxide, initiated by the photodissociation of a chlorine atom. The hydroperoxide re-oxidizes the iron(II) species, both the oxidation and reduction steps producing acetaldehyde. This mechanism is consistent with the increases in yield with ethanol concentration in ethanol–toluene mixtures towards an asymptotic limit. Copyright © 2014 John Wiley & Sons, Ltd.

Atherosclerosis is an inflammatory disease characterized by immunological activity, in which endothelial dysfunction represents an early event leading to subsequent inflammatory vascular damage. We investigated gene expression of the adhesion molecules (AMs) ICAM-1, VCAM-1, andβ1-integrin in endothelial cells (ECs) isolated from venous blood (circulating EC, cEC) and purified from femoral plaques (pEC) obtained from 9 patients with peripheral artery disease (PAD) submitted to femoral artery thrombendarterectomy (FEA). In addition, in peripheral blood mononuclear cells (PBMCs) of the same subjects, we investigated gene expression of IFN-γ, IL-4, TGF-β, and IL-10. Patients were longitudinally evaluated 1 month before surgery, when statin treatment was established, at the time of surgery, and after 2 and 5 months. All AM mRNA levels, measured by means of real-time PCR, in cEC diminished during the study, up to 41–50% of initial levels at followup. AM mRNA expression was significantly higher in pEC than in cEC. During the study, in PBMCs, TGF-βand IL-10 mRNA levels remained unchanged while IFN-γand IL-4 levels increased; however, the ratio IFN-γ/IL-4 showed no significant modification. In PAD patients, FEA and statin treatment induce a profound reduction of AM expression in cEC and affect cytokine mRNA expression in PBMCs.

Objective Sample manipulation to obtain isolated granulocytes represents a key, and often necessary, step in the in vitro studies. We investigated by the means of flow cytometry and microscopic techniques (both optical microscopy [OM] and scanning electron microscopy [SEM]), the granulocyte-endothelium adhesion and the role of sample manipulation. Methods By means of a co-culture method, we have analysed the adhesion of human leukocytes, originated from two different blood samples (fresh venous blood [FB] and buffy coat [BC]), to the human umbilical venous endothelial cell (HUVEC) monolayer. Cultured HUVEC were analysed for adhesion molecule expression by means of flow cytometry, while the morphological changes were evaluated by means of SEM. Cell adhesion was evaluated by means of flow cytometry and both OM and SEM. Results HUVEC expressed under resting conditions the adhesion molecules ICAM-1, VCAM-1 and E-selectin and their expression was upregulated by stimulation with TNF-α (0.1–10 ng/ml) as well as with LPS (1 μg/ml). SEM analysis showed that stimulation with both stimuli profoundly affect cell morphology. Flow cytometric evaluation of cell adhesion showed that the ability of cells to adhere to HUVEC monolayer was quite different in the two preparations, with the lowest adhesion for FB in all the cell subsets analysed. Finally, isolated granulocytes were able to adhere to HUVEC monolayer more than cells identified in FB or BC and the adhesion was increased during activation of HUVEC with 10 ng/ml of TNF-α. Conclusion Our data showed that cell manipulation necessary for the isolation of specific immune cells from whole blood profoundly affect the ability of these cells to adhere to the HUVEC monolayer although their functional properties remain unchanged.

Clinically isolated syndrome (CIS) is a first, usually recovering, episode of neurological disturbance(s) suggestive of multiple sclerosis (MS). CIS subjects might benefit from early disease-modifying drugs, provided that those at high risk of developing MS can be identified. Gene expression for dopaminergic receptors (DR) and adrenoceptors (AR) is dysregulated in lymphocytes of MS patients and is affected by treatment with interferon (IFN)-β. In particular, lymphocyte DR D5 mRNA might be a marker of IFN-β response in MS patients. No information exists so far in CIS subjects. We investigated DR and AR gene expression in peripheral blood mononuclear cells (PBMC) and in CD4+ T effector (Teff) and regulatory (Treg) cells from CIS subjects, and assessed their relationship with MS progression after 12months. Expression of several DR and AR are upregulated in PBMC, Teff and Treg from CIS subjects. DR D3 and α2A-AR mRNA in PBMC, and DR D5 mRNA in Treg correlate with the risk of MS at 12months. Results show the involvement of dopaminergic and adrenergic pathways in CIS as well as in MS pathogenesis, supporting the evaluation of dopaminergic and adrenergic agents in MS.

Objective: To determine the effectiveness of nutrition education on improving nutrition-related health outcomes in Aboriginal and Torres Strait Islander people. Methods: Databases Medline, Cinahl, Scopus, ProQuest and ATSI Health were searched in September 2013 to identify nutrition education intervention studies in Indigenous Australian populations. Peer-reviewed and grey literature with nutrition-related biochemical or anthropometrical health outcomes were included in a qualitative comparative analysis. Results: Of 1,162 studies identified from the search, six met inclusion criteria. Three studies were from a remote setting and three from an urban setting. Four of the six education interventions were shown to improve body mass index (BMI) and/or nutritional biochemical indicators. Components of the nutrition education interventions showing greatest effect included cooking skills workshops, group education sessions and store interventions. Community involvement in the program design was most strongly associated with a positive effect on BMI. Conclusion: Nutrition education had some effect in reducing biochemical and anthropometric risk factors for chronic disease in Indigenous Australians. Implications: Nutrition education can be considered as part of a range of strategies to improve nutrition-related health for Aboriginal and Torres Strait Islander people. Further evidence is needed to strengthen this recommendation.

Objective Angiotensin II, through the activation of angiotensin II type 1 receptors, plays a crucial role in atherosclerosis. Statins may interfere with the effects of angiotensin II. Methods We have investigated the expression of angiotensin II type 1 receptor, angiotensin II type 2 receptor and angiotensinogen on circulating monocytes and T-lymphocytes from subjects at high risk for vascular events before and during simvastatin treatment, and healthy controls. In-vitro experiments were also performed to assess the ability of simvastatin to interfere with angiotensin II signalling. Results In comparison with controls, high-risk subjects had similar angiotensin II type 1 receptor expression on the cell membranes but significantly higher angiotensin II type 1 receptor mRNA levels at least in monocyte subsets whereas their expression on T cells was similar. Angiotensin II type 2 receptor mRNA expression was higher than controls in both monocytes and T lymphocytes. No differences were observed in angiotensinogen expression on monocytes while T lymphocytes of high-risk subjects show higher expression. One-month treatment of high-risk subjects with simvastatin resulted in a reduction of angiotensin II type 1 receptor mRNA without affecting angiotensin II type 2 receptor whereas angiotensinogen mRNA expression was reduced at least in monocytes. Incubation in vitro with simvastatin reduces the expression of angiotensin II type 1 receptor mRNA levels on monocytes from untreated subjects. Conclusion Simvastatin induces down-regulation of the angiotensin II type 1 receptor, interferes with angiotensin II activity in immune cells and contributes to the anti-inflammatory profile of statins that can explain the therapeutic effects of these drugs.

A dual-action ligand targeting both integrin alpha(V)beta(3) and vascular endothelial growth factor receptors (VEGFRs), was synthesized via conjugation of a cyclic peptidomimetic alpha(V)beta(3) Arg-Gly-Asp (RGD) ligand with a decapentapeptide. The latter was obtained from a known VEGFR antagonist by acetylation at the Lys13 side chain. Functionalization of the precursor ligands was carried out in solution and in the solid phase, affording two fragments: an alkyne VEGFR ligand and the azide integrin alpha(V)beta(3) ligand, which were conjugated by click chemistry. Circular dichroism studies confirmed that both the RGD and VEGFR ligand portions of the dual-action compound substantially adopt the biologically active conformation. In vitro binding assays on isolated integrin alpha(V)beta(3) and VEGFR-1 showed that the dual-action conjugate retains a good level of affinity for both its target receptors, although with one order of magnitude (10/20 times) decrease in potency. The dual-action ligand strongly inhibited the VEGF-induced morphogenesis in Human Umbilical Vein Endothelial Cells (HUVECs). Remarkably, its efficiency in preventing the formation of new blood vessels was similar to that of the original individual ligands, despite the worse affinity towards integrin alpha(V)beta(3) and VEGFR-1.

Adrenergic modulation of immunity has been extensively characterized, however, few information exist regarding polymorphonuclear leukocytes (PMN), despite their key role in immunity and inflammation. We investigated the effect of adrenergic agents on human PMN migration, CD11b and CD18 expression, reactive oxygen species (ROS) and interleukin (IL)-8 production, and on adrenoceptor (AR) expression.Migration was measured by the Boyden chamber assay, CD11b/CD18 expression was assessed by flow cytometry, intracellular ROS were detected by spectrofluorimetry, and IL-8 was quantitated by standard ELISA assay. AR mRNA levels were measured by real-time PCR and PMN morphology was studied by scanning electron microscopy.Adrenaline(A), noradrenaline and the β-AR agonist isoprenaline reduced N-formyl-Met-Leu-Phe (fMLP)-induced migration, CD11b/CD18 expression, and ROS production, without affecting IL-8. The effect of A on CD11b was antagonized by yohimbine and propranolol, and increased by prazosin. The effect on ROS production was completely abolished by propranolol. PMN expressed α(1A)-, α(1B)-, α(1D)-, α(2A)-, α(2C)-, β(1)-, β(2)-, and β(3)-AR mRNA. A prevented fMLP-induced morphological changes of PMN.Adrenergic agents reduced PMN responses mainly through β-AR, although α-AR may contribute at least to CD11b expression. AR-operated pathways in PMN should be investigated in disease conditions and in the response to therapeutic agents.

Outline: Anterior Knee Pain (AKP) is the most prevalent injury in running. Growing evidence suggests that hip muscle imbalance plays a role in the development of AKP. Gluteus Medius (GMed) is the main abductor of the hip and also stabilises the pelvis during gait. Studies show that hip-abduction strength is decreased in populations with AKP. This relates to increased hip-adduction, hip internal rotation and knee abduction, which are associated with higher patellofemoral contact pressures. Objective: The main aim of this study is to determine whether a standing exercise programme will improve hip-abductor strength. The second aim of the study is to determine whether there will be a decrease in running-related pain after completing a six-week standing exercise programme. Design: Within-participant pre-test/post-test comparative trial.Setting: The Sport’s Clinic at the Malta Football Association. Ta Qali National Stadium (Malta).Main outcome measures: GMed torque was measured using a Biodex® System 3 isokinetic dynamometer as demonstrated by Brent et al (2013). The Numerical Rating Scale was used to measure pain related to running. Results: Pre-test torque scores were higher on the affected side. Scores on the affected side improved by 22.81% from a mean baseline average of 96.00Nm/kg to 117.86Nm/kg, (p-value 0.013). Scores on the unaffected side improved by 27.97% from a mean baseline average of 90.14Nm/kg to 115.29Nm/kg (p-value 0.031). Post-test scores were more balanced between limbs. Pain scores decreased from 5.29 as measured on the NRS to 1.00 (p-value 0.000). Conclusion: Standing exercises aimed at strengthening the hip-abductors may prove to be a valid intervention for prevention and rehabilitation of AKP. However, better quality studies are needed in order to validate this.

Background Neutrophil (PMN) leukocytes participate to the initial phases of atherosclerosis through the release of Interleukin 8 (CxCL8; IL-8) that contribute to amplification of inflammation. Aim of the study is to investigate the production of IL-8 by PMN leukocytes from dyslipidemic patients treated with simvastatin. Methods In 15 dyslipidemic subjects with moderately increased cardiovascular risk, assessed by Framingham Risk Score, blood samples were obtain to investigate PMNs IL-8 production [at baseline and after N-formyl-Met-Leu-Phe (fMLP) stimulation] before and after long-term (1-year) simvastatin treatment. Results The resting release of IL-8 was higher in dyslipidemic patients at baseline when compared with control subjects (p

Background In diabetes, a variety of pro-inflammatory cellular changes has been found in various cell types, including monocytes which are known to be involved in all the phases of atherogenesis. Angiotensin II (Ang II) type 1 receptor (AT1R) mediates the pro-atherogenic effects of Ang II whereas the type 2 receptor (AT2R) seems associated with atheroprotection. We sought to investigate the potential changes of AT1R-AT2R expression in human monocytes of type 2 diabetic- hypercholesterolemic patients and in hypercholesterolemic subjects, upon clinical treatment with rosuvastatin. Methods The AT1R membrane protein and mRNA AT1R and AT2R expression in monocytes were investigated in 10 type 2 diabetic-hypercholesterolemic patients and in 10 hypercholesterolemic subjects, before and after 3-month rosuvastatin treatment. Moreover, the serum cytokine levels of interferon-γ (IFN-γ) and interleukin-4 (IL-4) were detected. Results As expected, rosuvastatin was associated with a change in the lipid profile in the two groups. Both the membrane protein (P = 0.008) and the AT1R mRNA expression (P = 0.038) were significantly reduced during treatment in the absence of AT2R expression change in diabetic-hypercholesterolemic patients whereas no significant difference was observed in hypercholesterolemic subjects. The serum IL-4 levels were increased during treatment whereas no change was observed in IFN-γ in diabetic-hypercholesterolemic patients. No cytokine change was observed in hypercholesterolemic subjects. Conclusions Our study on monocytes of diabetic-hypercholesterolemic patients, showing a reduced AT1R but not AT2R expression during rosuvastatin treatment, suggests that statin therapy may modulate favorably the AT1-AT2 receptor balance in subjects with coexistent type 2 diabetes.

Atherosclerotic calcifications are related to poor prognosis and all-cause mortality in large population studies.1 Moreover, vascular calcifications are inversely associated with the potential reduction of plaque volume in regression studies during statin treatment, which suggests that more calcified lesions are less likely to undergo positive remodeling.2 Recently, it has been suggested that calcification is a tightly regulated process of mineralization akin to bone formation.3 In plaques, the deposits consist of a nonhomogeneous composite that contains hydroxyapatite mineral nanocrystals embedded in a collagenous organic matrix, whereas at a nanoscale level, apatite crystals interact with cholesterol crystallites. In this context, mineralization may result from a basic template pattern generated by the organic …

Recombinant fluorescent probes allow the detection of molecular events inside living cells. Many of them exploit the intracellular space to provide positional signals and, thus, require detection by single cell imaging. We describe here a novel strategy based on probes capable of encoding the spatial dimension of intracellular signals into "all-or-none" fluorescence intensity changes (differential anchorage probes, DAPs). The resulting signals can be acquired in single cells at high throughput by automated flow cytometry, (i) bypassing image acquisition and analysis, (ii) providing a direct quantitative readout, and (iii) allowing the exploration of large experimental series. We illustrate our purpose with DAPs for Bax and the effector caspases 3 and 7, which are keys players in apoptotic cell death, and show applications in basic research, high content multiplexed library screening, compound characterization, and drug profiling.

In recent years, experimental and clinical evidence has been provided regarding endothelial dysfunction and its contribution to the inflammatory process leading to atherosclerotic plaque formation. All the techniques employed direct to the study of endothelial dysfunction, however usually require isolation of endothelial cells and therefore tissue manipulation and destruction, with subsequent loss of information regarding morphology and topographical distribution of the lesions. By means of scanning electron microscopy (SEM), we have investigated the characteristics of the endothelial layer in carotid specimens obtained from subjects undergoing carotid endarterectomy. Carotid specimens obtained from 6 patients undergoing carotid endarterectomy for stenosis> or =70% were fixed, prepared and examined by use of SEM in a direct mode. All the plaque specimens showed increased thickness of subendothelial connective tissue with respect to post-mortem tissue obtained from an healthy subject. Endothelial layers were typically detached from the basal lamina surface and infiltrating cells (mainly erythrocytes and, possibly, monocytes) could be identified. Endothelial cells in proximity of the plaques had irregular shape, with prominent nuclei. In several areas, the endothelial layer was completely absent and basal lamina completely uncovered. In the present study, by using SEM analysis, the morphological features of dysfunctional endothelium in human carotid plaques were extensively documented at the ultrastructural level. SEM is a powerful investigational technique which allows tridimensional examination of specimens without disruption of the originary morphology.

We investigated angiotensin type 1 receptor (AT1R) expression and interleukin-8 (IL-8) productions in polymorphonuclear leukocytes obtained from patients with peripheral arterial disease (PAD) undergoing femoral endarterectomy. Subjects at high cardiovascular risk (high-risk subjects, HRS) and healthy controls (HC) were also enrolled. To this end, patients with PAD were studied 1 month before surgery, at the time of surgery, and 3 and 6 months after surgery. Polymorphonuclear leukocytes were obtained from venous blood and evaluated for AT1R expression at messenger RNA (mRNA) and protein level and IL-8 production (by means of enzyme-linked immunosorbent assay). At baseline, AT1R membrane expression was similar in cells from patients with PAD, HRS, and HC, whereas AT1R mRNA was similar in patients with PAD and HC and higher in HRS. During the follow-up period, AT1R expression progressively decreased both on the cell membrane and at the mRNA level. Both resting and stimulated production of IL-8 was lower in patients with PAD in comparison to HC and HRS and did not change during the follow up period. In PAD patients, femoral endarterectomy is associated with reduction of AT1R expression however with no apparent effect on IL-8 production. The relevance of such effects for cardiovascular protection deserves consideration.

Voltage-dependent anion channel (VDAC)1 is the main channel of the mitochondrial outer membrane (MOM) and it has been proposed to be part of the permeability transition pore (PTP), a putative multiprotein complex candidate agent of the mitochondrial permeability transition (MPT). Working at the single live cell level, we found that overexpression of VDAC1 triggers MPT at the mitochondrial inner membrane (MIM). Conversely, silencing VDAC1 expression results in the inhibition of MPT caused by selenite-induced oxidative stress. This MOM-MIM crosstalk was modulated by Cyclosporin A and mitochondrial Cyclophilin D, but not by Bcl-2 and Bcl-X(L), indicative of PTP operation. VDAC1-dependent MPT engages a positive feedback loop involving reactive oxygen species and p38-MAPK, and secondarily triggers a canonical apoptotic response including Bax activation, cytochrome c release and caspase 3 activation. Our data thus support a model of the PTP complex involving VDAC1 at the MOM, and indicate that VDAC1-dependent MPT is an upstream mechanism playing a causal role in oxidative stress-induced apoptosis.

Bax is considered to be pivotal in inducing cytochrome c release (CCR) from mitochondria during apoptosis. Indeed, Bax redistributes to the mitochondrial outer membrane (MOM) upon activation and forms homo-multimers that are capable of permeabilizing the MOM. Our attempts to image this sequence of events in single live cells resulted in unexpected observations. Bax redistribution exhibited two distinct components: an early minor redistribution that was silent in terms of homo-multimerization and a major late redistribution that was synchronous with the formation of Bax multimers, but that proceeded belatedly, i.e. only after caspase 3/7 (C3/7) had already been activated. Intriguingly, neither of these two components of redistribution correlated with CCR, which turned out to be spatially organized, propagating as a traveling wave at constant velocity. Strikingly, propagation of the CCR wave (1) preceded signs of in situ Bax conformational activation; (2) appeared to be independent of autocatalytic loops involving a positive feedback of either C3/7, Ca2+ mobilization or mitochondrial permeability transition; and (3) was triggered by diffuse stimulation with the synthetic Bak activator BH3I-1 but then proceeded independently of Bak activation. Thus, the CCR wave not only questions the exact role of Bax redistribution in cell death, but also indicates the existence of yet unidentified positive-feedback loops that ensure a spatiotemporal control of apoptosis at the subcellular scale.

Atherosclerosis is a chronic progressive inflammatory disease of the arterial wall, in which calcification is a critical determinant of plaque stability and has major consequences for the overall clinical burden of the atherosclerotic process.1 Calcification of the vascular wall occurs through osteoblast-mediated ectopic mineral deposition, resembling the process of orthotopic (skeletal) osteogenesis.2,3 Energy dispersive x-ray spectroscopy (EDAX) is a potent analytical tool that allows the elemental analysis and characterization of biological specimens.4 Coupling EDAX to scanning electron …

ObjectivesPolymorphonuclear neutrophils (PMN) in atherosclerotic plaques have been identified only recently, and their contribution to plaque development is not yet fully understood. In this study, production of elastase, interleukin (IL)-8 and vascular endothelial growth factor (VEGF) by PMN was investigated in subjects with carotid stenosis undergoing carotid endarterectomy (CEA).MethodsThe study enrolled 50 patients (Pts) and 10 healthy subjects (HS). Circulating PMN (cPMN) isolated from venous blood (in both Pts and HS) and from plaques (pPMN, in Pts) were cultured, alone or with 0.1 μM fMLP. Elastase, IL-8 and VEGF mRNA were analyzed by real-time PCR. In CEA specimens, PMN were localized by immunohistochemistry.ResultsIn both Pts cPMN and pPMN, IL-8 mRNA was higher at rest but lower after fMLP (PConclusionsIn Pts with carotid plaques, both circulating and intraplaque PMN produce IL-8, VEGF and elastase, which are crucial for plaque development and progression. These findings suggest mechanistic explanations to the reported correlation between PMN count and cardiovascular mortality in carotid ATH.

Background The CD4+ T-lymphocytes and their subtype CD4 + CD25highFoxP3+ regulatory T cells are receiving growing interest as major regulators of atherogenesis. We sought to investigate 1) whether the CD4 + cell subsets were expressed differently in dyslipidemic patients (Pts) and healthy subjects (HS) and 2) whether atorvastatin treatment could be associated in-vivo and in-vitro with cell changes in expression and functional response. Methods CD4+ subsets frequency (CD4 + CD25highFoxP3+, CD4 + CD25-FoxP3+) and mRNA expression for FoxP3, IL-10 and TGF-β were evaluated in 30 consecutive Pts at baseline and after a 3-month atorvastatin therapy, and in 17 HS. Results The % of CD4 + cells did not differ between HS and Pts. The % of CD4 + CD25highFoxP3+ was higher in Pts than HS and did not change during treatment. The CD4 + CD25-FoxP3+ cells were similar between the two groups and were lower in Pts at visit 2. Cytokine expression and FoxP3 did not differ in HS and Pts and no substantial change was observed during treatment. At visit 1, CD4 + CD25highFoxP3+ cells were significantly correlated with both total-cholesterol (r = 0.570, P = 0.0002), LDL-cholesterol (r = 0.715, P = 0.0001), Apolipoprotein B (r = 0.590, P = 0.0001). In-vitro atorvastatin (up to 5 μM) failed to induce any significant modulation of cell functions. Conclusion CD4 + CD25highFoxP3+ regulatory cells seem to be over-stimulated in the early pre-clinical phase of atherosclerosis and a relationship exists between their frequency and circulating lipids. A potential immuno-modulation by statin treatment is not achieved through a normalization in peripheral CD4 + cell subsets.

Background Cyclic RGD peptidomimetics containing a bifunctional diketopiperazine scaffold are a novel class of high-affinity ligands for the integrins αVβ3 and αVβ5. Since integrins are a promising target for the modulation of normal and pathological angiogenesis, the present study aimed at characterizing the ability of the RGD peptidomimetic cyclo[DKP-RGD] 1 proliferation, migration and network formation in human umbilical vein endothelial cells (HUVEC). Methods Cell viability was assessed by flow cytometry and annexin V (ANX)/propidium iodide (PI) staining. Cell proliferation was evaluated by the ELISA measurement of bromodeoxyuridine (BrdU) incorporation. Network formation by HUVEC cultured in Matrigel-coated plates was evaluated by optical microscopy and image analysis. Integrin subunit mRNA expression was assessed by real time-PCR and Akt phosphorylation by western blot analysis. Results Cyclo[DKP-RGD] 1 does not affect cell viability and proliferation either in resting conditions or in the presence of the pro-angiogenic growth factors VEGF, EGF, FGF, and IGF-I. Addition of cyclo[DKP-RGD] 1 however significantly decreased network formation induced by pro-angiogenic growth factors or by IL-8. Cyclo[DKP-RGD] 1 did not affect mRNA levels of αV, β3 or β5 integrin subunits, however it significantly reduced the phosphorylation of Akt. Conclusions Cyclo[DKP-RGD] 1 can be a potential modulator of angiogenesis induced by different growth factors, possibly devoid of the adverse effects of cytotoxic RGD peptidomimetic analogues.