Publications (16) View all
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Article: A Decoy Oligonucleotide to NF-κB Delivered Through Inhalable Particles Prevents LPS-Induced Rat Airway Inflammation.
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ABSTRACT: The inflammatory process plays a crucial role in the onset/progression of several lung pathologies including cystic fibrosis (CF), and the involvement of nuclear factor-κB (NF-κB ) is widely recognized. The specific inhibition of NF-κB by decoy oligonucleotides delivered in the lung may be beneficial although rationally designed systems are needed to optimize their pharmacological response. Prompted by this, here we have developed and tested in vivo an inhalable dry powder for prolonged delivery of a decoy oligodeoxynucleotide to NF-κB (dec-ODN), consisting of large porous particles (LPP) based on poly(lactic-co-glycolic) acid (PLGA). First, LPP containing dec-ODN (dec-ODN LPP) have been engineered to meet aerodynamic criteria crucial for pulmonary delivery, to gain an effective loading of dec-ODN, to sustain its release and to preserve its structural integrity in lung lining fluids. Then, we have investigated the effects of dec-ODN LPP in a rat model of lung inflammation induced by intra-tracheal aerosolization of lipopolysaccharide (LPS) from P. aeruginosa. The results show that a single intra-tracheal insufflation of dec-ODN LPP reduced bronchoalveolar neutrophil infiltration induced by LPS up to 72 hours, whereas naked dec-ODN was able to inhibit it only at 6 hours. The persistent inhibition of neutrophil infiltrate was associated to a reduced NF-κB/DNA binding activity as well as interleukin-6 (IL-6), interleukin-8 (IL-8) and mucin-2 (MUC2) mRNA expression in lung homogenates. It is worthy of note that the developed LPP, preventing the accumulation of neutrophils and NF-κB-related gene expression, may provide a new therapeutic option for local treatment of inflammation associated with lung diseases.American Journal of Respiratory Cell and Molecular Biology 04/2013; · 5.13 Impact Factor -
Article: Oxidative stress suppresses the cellular bioenergetic effect of the 3-mercaptopyruvate sulfurtransferase / hydrogen sulfide pathway.
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ABSTRACT: Recent data show that lower concentrations of hydrogen sulfide (H2S), as well as endogenous, intramitochondrial production of H2S by the 3-mercaptopyruvate (3-MP) / 3-mercaptopyruvate sulfurtransferase (3-MST) pathway serves as an electron donor and inorganic source of energy to support mitochondrial electron transport and ATP generation in mammalian cells by donating electrons to Complex II. The aim of our study was to investigate the role of oxidative stress on the activity of the 3-MP/3-MST/H2S pathway in vitro. Hydrogen peroxide (H2O2, 100-500 μM) caused a concentration-dependent decrease in the activity of recombinant mouse 3-MST enzyme. In mitochondria isolated from murine hepatoma cells, H2O2 (50-500 μM) caused a concentration-dependent decrease in production of H2S from 3-MP. In cultured murine hepatoma cells H2O2, (3-100 μM), did not result in overall cytotoxicity, but caused a partial decrease in basal oxygen consumption and respiratory reserve rapacity. The positive bioenergetic effect of 3-MP (100-300 nM) was completely abolished by pre-treatment of the cells with H2O2 (50 μM). The current findings demonstrate that oxidative stress inhibits 3-MST activity and interferes with the positive bioenergetic role of the 3-MP/3-MST/H2S pathway. These findings may have implications for the pathophysiology of various conditions associated with increased oxidative stress, such as various forms of critical illness, cardiovascular diseases, diabetes or physiological aging.Biochemical and Biophysical Research Communications 03/2013; · 2.48 Impact Factor -
Article: Selectivity of commonly used pharmacological inhibitors for cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE).
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ABSTRACT: BACKGROUND AND PURPOSE: Hydrogen sulfide (H2 S) is a signaling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H2 S are cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE). In the present study we examined the selectivity of commonly used pharmacological inhibitors of H2 S biosynthesis towards CSE and CBS. EXPERIMENTAL APPROACH: To address this question human CSE or CBS enzymes were expressed and purified from E. coli as fusion proteins with glutathione-S-transferase. After purification the activity of the recombinant enzymes was tested using the methylene blue method. KEY RESULTS: β-cyanoalanine (BCA), a CSE inhibitor, was more potent in inhibiting CSE than propargylglycine (PAG) (IC50 14+0.2 μM vs 40+8 μM, respectively). Similarly to PAG, L-aminoethoxyvinylglycine only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared to BCA and PAG (IC50 1.1+0.1μM); the IC50 for AOAA for inhibiting CBS was 8.5+0.7 μM. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H2 S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a four-fold lower IC50 for CBS vs CSE, while hydroxylamine was 60-fold more selective against CSE. CONCLUSIONS AND IMPLICATIONS: In conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE vs. CBS, no selective pharmacological CBS inhibitor is currently available.British Journal of Pharmacology 03/2013; · 4.41 Impact Factor -
Article: Potential Role of Hydrogen Sulfide in the Pathogenesis of Vascular Dysfunction in Septic Shock.
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ABSTRACT: Various forms of circulatory shock (including septic shock) lead to an impairment of vascular function, which importantly contributes to the development of multiple organ failure and mortality. Such dysfunction of blood vessels consists of two principal components: vascular smooth muscle (VSM) dysfunction, and endothelial dysfunction. The VSM dysfunction (progressive, therapy-resistant loss of VSM responsiveness to vasoconstrictor catecholamines, such as noradrenaline) leads to a progressive deterioration of blood pressure in patients with circulatory shock. The endothelial dysfunction (loss of the ability of the endothelium to produce nitric oxide and other endothelium-derived factors) contributes to the impairment of microvascular blood flow, to the enhanced adhesion and activation of neutrophils and platelets, to coagulation problems, and perfusion/metabolism mismatch in the affected organs. Here we overview the vascular regulatory functions of the novel gasotransmitter hydrogen sulfide (H2S), with an emphasis on its potential role in the pathogenesis of vascular dysfunction in circulatory shock. We first review the roles of endogenously produced or exogenously administered H2S on vascular function. Next, we review the results of published studies using shock models induced by bacterial lipopolysaccharide, and by cecal ligation and puncture, a polymicrobial model of sepsis showing overproduction of H2S. Finally, we summarize the potential mechanisms by which H2S may contribute to vascular dysfunction in shock and show an example of how the vascular response to H2S is altered in a rat model of endotoxemia. In addition, we outline the potential means by which modulation of H2S (pharmacological inhibition of its biosynthesis or therapeutic donation) may affect the outcome in circulatory shock.Current Vascular Pharmacology 03/2013; 11(2):208-221. · 2.90 Impact Factor -
Article: Cell-Based Screening Identifies Paroxetine as an Inhibitor of Diabetic Endothelial Dysfunction.
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ABSTRACT: We have conducted a phenotypic screening in endothelial cells exposed to elevated extracellular glucose (an in vitro model of hyperglycemia) to identify compounds that prevent hyperglycemia-induced reactive oxygen species (ROS) formation without adversely affecting cell viability. From a focused library of >6,000 clinically used drug-like and pharmacologically active compounds, several classes of active compounds emerged, with a confirmed hit rate of <0.5%. Follow-up studies focused on paroxetine, a clinically used antidepressant compound that has not been previously implicated in the context of hyperglycemia or diabetes. Paroxetine reduced hyperglycemia-induced mitochondrial ROS formation, mitochondrial protein oxidation, and mitochondrial and nuclear DNA damage, without interfering with mitochondrial electron transport or cellular bioenergetics. The ability of paroxetine to improve hyperglycemic endothelial cell injury was unique among serotonin reuptake blockers and can be attributed to its antioxidant effect, which primarily resides within its sesamol moiety. Paroxetine maintained the ability of vascular rings to respond to the endothelium-dependent relaxant acetylcholine, both during in vitro hyperglycemia and ex vivo, in a rat model of streptozotocin-induced diabetes. Thus, the current work identifies a novel pharmacological action of paroxetine as a protector of endothelial cells against hyperglycemic injury and raises the potential of repurposing of this drug for the experimental therapy of diabetic cardiovascular complications.Diabetes 12/2012; · 8.29 Impact Factor
