Metabolic and functional consequences of cytosolic 5′-nucleotidase-IA overexpression in neonatal rat cardiomyocytes

Adenosine exerts a spectrum of energy-preserving actions on the heart negative chronotropic effects. The pathways leading to adenosine formation have remained controversial. In particular, although cytosolic 5′-nucleotidases can catalyze adenosine formation in cardiomyocytes, their contribution to the actions of adenosine has not been documented previously. We recently cloned two closely related AMP-preferring cytosolic 5′-nucleotidases (cN-IA and -IB); the A form predominates in the heart. In this study, we overexpressed pigeon cN-IA in neonatal rat cardiomyocytes using an adenovirus. cN-IA overexpression increased adenosine formation and release into the medium caused by simulated hypoxia and by isoproterenol in the absence and presence of inhibitors of adenosine metabolism. Adenosine release was not affected by an ecto-5′-nucleotidase inhibitor, α,β-methylene-ADP, but was affected by a nucleoside transporter, dipyridamole. The positive chronotropic effect of isoproterenol (130 ±3 vs. 100 ±4 beats/min) was inhibited (107 ±3 vs. 94 ±3 beats/min) in cells overexpressing cN-IA, and this was reversed by the addition of the adenosine receptor antagonist 8-(p-sulfophenyl)theophilline (120 ± 3 vs. 90 ± 4 beats/min). Our results demonstrate that overexpressed cN-IA can be sufficiently active in cardiomyocytes to generate physiologically effective concentrations of adenosine at its receptors.Fil: Sala-Newby, Graciela B.. University of Bristol; Reino UnidoFil: Freeman, Nicola V. E.. University of Bristol; Reino UnidoFil: Curto, Maria de Los Angeles. University of Bristol; Reino Unido. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Newby, Andrew C.. University of Bristol; Reino Unid

Dual Role of CREB in the regulation of VSMC proliferation : mode of activation determines pro- or anti-mitogenic function

Vascular smooth muscle cell (VSMC) proliferation has been implicated in the development of restenosis after angioplasty, vein graft intimal thickening and atherogenesis. We investigated the mechanisms underlying positive and negative regulation of VSMC proliferation by the transcription factor cyclic AMP response element binding protein (CREB). Incubation with the cAMP elevating stimuli, adenosine, prostacyclin mimetics or low levels of forksolin activated CREB without changing CREB phosphorylation on serine-133 but induced nuclear translocation of the CREB co-factors CRTC-2 and CRTC-3. Overexpression of CRTC-2 or -3 significantly increased CREB activity and inhibited VSMC proliferation, whereas CRTC-2/3 silencing inhibited CREB activity and reversed the anti-mitogenic effects of adenosine A2B receptor agonists. By contrast, stimulation with serum or PDGF significantly increased CREB activity, dependent on increased CREB phosphorylation at serine-133 but not on CRTC-2/3 activation. CREB silencing significantly inhibited basal and PDGF induced proliferation. These data demonstrate that cAMP activation of CREB, which is CRTC2/3 dependent and serine-133 independent, is anti-mitogenic. Growth factor activation of CREB, which is serine-133-dependent and CRTC2/3 independent, is pro-mitogenic. Hence, CREB plays a dual role in the regulation of VSMC proliferation with the mode of activation determining its pro- or anti-mitogenic function

Constraining the initial temperature and shear viscosity in a hybrid hydrodynamic model of sNN\sqrt{s_{NN}}=200 GeV Au+Au collisions using pion spectra, elliptic flow, and femtoscopic radii

A new framework for evaluating hydrodynamic models of relativistic heavy ion collisions has been developed. This framework, a Comprehesive Heavy Ion Model Evaluation and Reporting Algorithm (CHIMERA) has been implemented by augmenting UVH 2+1D viscous hydrodynamic model with eccentricity fluctuations, pre-equilibrium flow, and the Ultra-relativistic Quantum Molecular Dynamic (UrQMD) hadronic cascade. A range of initial temperatures and shear viscosity to entropy ratios were evaluated for four initial profiles, $N_{part}$ and $N_{coll}$ scaling with and without pre-equilibrium flow. The model results were compared to pion spectra, elliptic flow, and femtoscopic radii from 200 GeV Au+Au collisions for the 0--20% centrality range.Two sets of initial density profiles, $N_{part}$ scaling with pre-equilibrium flow and $N_{coll}$ scaling without were shown to provide a consistent description of all three measurements.Comment: 21 pages, 32 figures, version 3 includes additional text for clarification, division of figures into more manageable units, and placement of chi-squared values in tables for ease of viewin

Zoledronate upregulates MMP-9 and -13 in rat vascular smooth muscle cells by inducing oxidative stress

WOS: 000374502300001PubMed ID: 27143852Background: Bisphosphonates, including zoledronate, target osteoclasts and are widely used in the treatment of osteoporosis and other bone resorption diseases, despite side effects that include damaging the stomach epithelium. Beneficial and adverse effects on other organ systems, including the cardiovascular system, have also been described and could impact on the use of bisphosphonates as therapeutic agents. Vascular smooth muscle cells (VSMCs) are major constituents of the normal vascular wall and have a key role in intimal thickening and atherosclerosis, in part by secreting MMPs that remodel the extracellular matrix and cleave cell surface proteins or secreted mediators. In this study, we investigated the effects of zoledronate on MMP expression. Methods: Rat VSMCs were stimulated by PDGF (50 ng/mL) plus TNF-alpha (10 ng/mL) or left unstimulated for a further 24 hours in serum-free medium. In other series of experiments, cells were pre-treated either with SC-514 (50 mu M) or with apocynin (20 nM) for 2 hours, then zoledronate (100 mu M) was added into 2% fetal calf serum containing medium for 24 hours. Results and discussion: Using isolated rat VSMCs in culture, zoledronate (100 mu M) increased MMP-9 and -13 mRNA expressions but inhibited MMP-2 expression. MMP-9 and MMP-13 up-regulation was shown to depend on the NF-kappa B pathway; and this was activated by zoledronate. Furthermore, zoledronate elevated the levels of reactive oxygen species detected by either dichlorofluorescein in isolated VSMCs or lucigenin enhanced chemiluminescence in rat aortic rings in vitro. Apocynin, an inhibitor of NADPH oxidase, reversed NF-kappa B activation and MMP-9 and MMP-13 up-regulation by zoledronate. Conclusion: We conclude that zoledronate increases MMP-9 and MMP-13 expressions in rat VSMCs dependent upon stimulation of the NF-kappa B pathway by reactive oxygen species. Effects on MMP expression may contribute to the pharmacologic profile of bisphosphonates.British Heart FoundationBritish Heart Foundation [CH95/001]; British Heart FoundationBritish Heart Foundation [RG/09/006/27918]The authors would like to thank Dr Goksel Gokce, Ege University Faculty of Pharmacy and Dr Steve White, University of Bristol for valuable help and expertise on oxidative stress measurements. MZA would also like to thank Prof Levent Ustunes for kind help and encouragement. This study was supported by the British Heart Foundation grant CH95/001

cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1

AbstractElevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1. In cultured rat VSMC, CCN1-silencing significantly inhibited BrdU incorporation and migration in a wound healing assay. Recombinant CCN1 enhanced chemotaxis in a Boyden chamber. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Forskolin also inhibited CCN1 expression in balloon injured rat carotid arteries in vivo. Inhibiting RhoA activity, which regulates actin-polymerisation, by cAMP-elevation or pharmacologically with C3-transferase, or inhibiting its downstream kinase, ROCK, with Y27632, significantly inhibited CCN1 expression. Conversely, expression of constitutively active RhoA reversed the inhibitory effects of forskolin on CCN1 mRNA. Furthermore, CCN1 mRNA levels were significantly decreased by inhibiting actin-polymerisation with latrunculin B or increased by stimulating actin-polymerisation with Jasplakinolide. We next tested the role of the actin-dependent SRF co-factor, MKL1, in CCN1 expression. Forskolin inhibited nuclear translocation of MKL1 and binding of MKL1 to the CCN1 promoter. Constitutively-active MKL1 enhanced basal promoter activity of wild-type but not SRE-mutated CCN1; and prevented forskolin inhibition. Furthermore, pharmacological MKL-inhibition with CCG-1423 significantly inhibited CCN1 promoter activity as well as mRNA and protein expression. Our data demonstrates that cAMP-induced actin-cytoskeleton remodelling regulates expression of CCN1 through MKL1: it highlights a novel cAMP-dependent mechanism controlling VSMC behaviour

QT interval prolongation and torsades de pointes in a patient undergoing treatment with vorinostat: A case report and review of the literature

Vorinostat is a histone deacetylase inhibitor used in the treatment of recurrent or persistent cases of cutaneous T-cell lymphoma (CTCL). A retrospective review of 116 patients from phase I and II clinical trials who had a baseline and at least one subsequent ECG revealed that four patients had Grade 2 and one patient had Grade 3 QTc interval prolongation; however, a MEDLINE search found no reported cases of torsades de pointes (TdP) in patients treated with vorinostat. We describe the case of a 49 year-old male with a history of CTCL actively undergoing treatment with vorinostat. During day 1 of hospitalization, he developed a pulseless polymorphic ventricular tachycardia requiring resuscitation. He was found to have a QTc of 826 ms. Following correction of potassium and magnesium, QTc gradually decreased and no further ventricular arrhythmia was noted. Other factors implicated in this case included concurrent sertraline and doxepin therapy (both drugs have been associated with the development of TdP in overdose). The mechanism of development of TdP in this patient is postulated to be related to vorinostat use in combination with hypokalemia and concomitant treatment with medications associated with QTc prolongation. This case highlights the importance of post-market surveillance

UVA irradiation of human skin vasodilates arterial vasculature and lowers blood pressure independently of nitric oxide synthase

The incidence of hypertension and cardiovascular disease correlates with latitude and rises in winter. The molecular basis for this remains obscure. As nitric oxide (NO) metabolites are abundant in human skin we hypothesised that exposure to UVA may mobilise NO bioactivity into the circulation to exert beneficial cardiovascular effects independently of vitamin D. In 24 healthy volunteers irradiation of the skin with 2 Standard Erythemal Doses of UVA lowered BP, with concomitant decreases in circulating nitrate and rises in nitrite concentrations. Unexpectedly, acute dietary intervention aimed at modulating systemic nitrate availability had no effect on UV-induced hemodynamic changes, indicating that cardiovascular effects were not mediated via direct utilization of circulating nitrate. UVA irradiation of the forearm caused increased blood flow independently of NO-synthase activity, suggesting involvement of pre-formed cutaneous NO stores. Confocal fluorescence microscopy studies of human skin pre-labelled with the NO-imaging probe DAF2-DA revealed that UVA-induced NO release occurs in a NOS-independent, dose-dependent fashion, with the majority of the light-sensitive NO pool in the upper epidermis. Collectively, our data provide mechanistic insights into an important function of the skin in modulating systemic NO bioavailability which may account for the latitudinal and seasonal variations of BP and cardiovascular disease.Journal of Investigative Dermatology accepted article preview online, 20 January 2014