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SELECTED PUBLICATIONS OF THE LAST 5 YEARS 

 

Induction of CD36 and Thrombospondin-1 in Macrophages by Hypoxia-Inducible Factor 1 and Its Relevance in the Inflammatory Process.

 

Inflammation is part of a complex biological response of vascular tissue to pathogens or damaged cells. First inflammatory cells attempt to remove the injurious stimuli and this is followed by a healing process mediated principally by phagocytosis of senescent cells. Hypoxia and p38-MAPK are associated with inflammation, and hypoxia inducible factor 1 (HIF-1) has been detected in inflamed tissues. We aimed to analyse the role of p38-MAPK and HIF-1 in the transcriptional regulation of CD36, a class B scavenger receptor, and its ligand thrombospondin (TSP-1) in macrophages and to evaluate the involvement of this pathway in phagocytosis of apoptotic neutrophils. We have also assessed HIF-1α, p38-MAPK and CD36 immunostaining in the mucosa of patients with inflammatory bowel disease. Results show that hypoxia increases neutrophil phagocytosis by macrophages and induces the expression of CD36 and TSP-1. Addition of a p38-MAPK inhibitor significantly reduced the increase in CD36 and TSP-1 expression provoked by hypoxia and decreased HIF-1α stabilization in macrophages. Transient transfection of macrophages with a miHIF-1α-targeting vector blocked the increase in mRNA expression of CD36 and TSP-1 during hypoxia and reduced phagocytosis, thus highlighting a role for the transcriptional activity of HIF-1. CD36 and TSP-1 were necessary for the phagocytosis of neutrophils induced by hypoxic macrophages, since functional blockade of these proteins undermined this process. Immunohistochemical studies revealed CD36, HIF-1α and p38-MAPK expression in the mucosa of patients with inflammatory bowel disease. A positive and significant correlation between HIF-1α and CD36 expression and CD36 and p38-MAPK expression was observed in cells of the lamina propria of the damaged mucosa. Our results demonstrate a HIF-1-dependent up-regulation of CD36 and TSP-1 that mediates the increased phagocytosis of neutrophils by macrophages during hypoxia. Moreover, they suggest that CD36 expression in the damaged mucosa of patients with inflammatory bowel disease depends on p38-MAPK and HIF-1 activity.

PMID: 23119050 [PubMed - in process] PMCID: PMC3485304

 
Differential effects of Tenofovir/Emtricitabine and Abacavir/Lamivudine on human leukocyte recruitment.

 

BACKGROUND:

The association of abacavir (ABC) with cardiovascular diseases (CVD) has led to HIV treatment guidelines favouring the combination of tenofovir/emtricitabine (TDF/FTC) over that of ABC/lamivudine (ABC/3TC). We have analysed the effects of plasma- relevant concentrations of TDF, FTC, ABC or 3TC, individually and in clinically employed combinations, on human leukocyte accumulation.The effects of ABC, 3TC, TDF and FTC on the expression of adhesion molecules were also evaluated.

METHODS:

Interactions between human leukocytes - specifically peripheral blood polymorphonuclear (PMN) or mononuclear (PBMC) cells - and human umbilical vein endothelial cells (HUVEC) were evaluated in a flow chamber reproducing in vivo conditions. The expression of adhesion molecules was analysed by flow cytometry.

RESULTS:

Concentrations of TDF, FTC or 3TC mimicking those in the plasma of patients did not have any effect on human leukocyte- endothelial cell interactions, while contrasting results were obtained with ABC. This distinct pattern was reproduced when the drugs were administered in combination; namely, ABC/3TC had a significant influence on rolling and adhesion while TDF/FTC did not.However, the effects produced by ABC alone did not differ when it was combined with 3TC, which suggests the former drug was responsible for the effects observed. ABC, 3TC, TDF and FTC did not modify the expression of endothelial adhesion molecules. Conversely, only ABC enhanced the expression of leukocyte CD11b/CD18 in neutrophils and monocytes.

CONCLUSIONS:

Our results provide evidence that the combination TDF/FTC has a better vascular profile than ABC/3TC.

 

 

Profile of stress and toxicity gene expression in human hepatic cells treated with Efavirenz.

Hepatic toxicity and metabolic disorders are major adverse effects elicited during the pharmacological treatment of the human immunodeficiency virus (HIV) infection. Efavirenz (EFV), the most widely used non-nucleoside reverse transcriptase inhibitor (NNRTI), has been associated with these events, with recent studies implicating it in stress responses involving mitochondrial dysfunction and oxidative stress in human hepatic cells. To expand these findings, we analyzed the influence of EFV on the expression profile of selected stress and toxicity genes in these cells. Significant up-regulation was observed with Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), which indicated metabolic stress. Several genes directly related to oxidative stress and damage exhibited increased expression, including Methalothionein 2A (MT2A), Heat shock 70kDa protein 6 (HSPA6), Growth differentiation factor 15 (GDF15) and DNA-damage-inducible transcript 3 (DDIT3). In addition, Early growth response protein 1 (EGR1) was enhanced, whereas mRNA levels of the inflammatory genes Chemokine (C-X-C motif) ligand 10 (CXCL10) and Serpin peptidase inhibitor (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1) decreased and increased, respectively. This profile of gene expression supports previous data demonstrating altered mitochondrial function and presence of oxidative stress/damage in EFV-treated hepatic cells, and may be of relevance in the search for molecular targets with therapeutic potential to be employed in the prevention, diagnosis and treatment of the hepatic toxicity associated with HIV therapy.

Copyright © 2012 Elsevier B.V. All rights reserved.

 
Evidence for a relationship between mitochondrial Complex I activity and mitochondrial aldehyde dehydrogenase during nitroglycerin tolerance: Effects of mitochondrial antioxidants.

The medical use of nitroglycerin (GTN) is limited by patient tolerance. The present study evaluated the role of mitochondrial Complex I in GTN biotransformation and the therapeutic effect of mitochondrial antioxidants. The development of GTN tolerance (in rat and human vessels) produced a decrease in mitochondrial O(2) consumption. Co-incubation with the mitochondria-targeted antioxidant mitoquinone (MQ, 10(-6)mol/L) or with glutathione ester (GEE, 10(-4)mol/L) blocked GTN tolerance and the effects of GTN on mitochondrial respiration and aldehyde dehydrogenase 2 (ALDH-2) activity. Biotransformation of GTN depended on the mitochondria being functionally active, particularly mitochondrial Complex I. Tolerance induced mitochondrial ROS production and oxidative stress, though these effects were not detected in HUVECρ(0) cells or Complex I mutant cells. Experiments performed to evaluate Complex I-dependent respiration demonstrated that its inhibition by GTN was prevented by the antioxidants in control samples. These results point to a key role for mitochondrial Complex I in the adequate functioning of ALDH-2. In addition, we have identified mitochondrial Complex I as one of the targets at which the initial oxidative stress responsible for GTN tolerance takes place. Our data also suggest a role for mitochondrial-antioxidants as therapeutic tools in the control of the tolerance that accompanies chronic nitrate use.

Copyright © 2012 Elsevier B.V. All rights reserved.

 
Compromising mitochondrial function with the antiretroviral drug efavirenz induces cell survival-promoting autophagy.

Hepatotoxicity is a very common side effect associated with the pharmacological treatment of human immunodeficiency virus (HIV) infection and its pathogenesis is poorly understood. Efavirenz (EFV) is the most widely used nonnucleoside reverse transcriptase inhibitor administered for the control of HIV and some of its toxic effects in hepatic cells have been recently shown to display features of mitochondrial dysfunction. Here we studied the activation of autophagy and, in particular, mitophagy, the main mitochondrial turnover mechanism, in human hepatic cells treated with clinically relevant concentrations of this drug. EFV-treated cells had altered mitochondria, characterized by a relative increase in mitochondrial mass and defective morphology. This was followed by induction of autophagy as shown by the presence of autophagic vacuoles and the presence of the specific autophagic marker proteins microtubule-associated protein 1A/1B light chain 3 and Beclin-1. Importantly, whereas moderate levels of EFV activated autophagy, higher concentrations led to blockage in the autophagic flux, a condition that promotes "autophagic stress" and produces severe cellular damage. Finally, pharmacological inhibition of autophagy exacerbated the deleterious effect of EFV on cell survival/proliferation promoting apoptosis, which suggests that autophagy acts as an adaptive mechanism of cell survival. Conclusion: Clinical concentrations of EFV induce autophagy and, in particular, mitophagy in hepatic cells. Activation of this process promotes cell survival, but exceeding a certain threshold of mitochondrial dysfunction is associated with an autophagic overload or stress. This effect could be involved in the EFV-associated hepatotoxicity and may constitute a new mechanism implicated in the genesis of drug-induced liver damage.

Copyright © 2011 American Association for the Study of Liver Diseases.

 
Autophagy as a rescue mechanism in efavirenz-induced mitochondrial dysfunction: a lesson from hepatic cells.

Efavirenz (EFV) is the most widely used non-nucleoside reverse transcriptase inhibitor applied in highly active antiretroviral therapy (HAART), the combined pharmacological treatment of the human immunodeficiency virus infection. Its use has been associated with the development of several adverse events including hepatotoxicity. The molecular pathogenesis of this effect is poorly understood but recent reports have highlighted features of mitochondrial dysfunction in hepatic cells exposed to clinically relevant concentrations of EFV. In this study, we investigated the activation of autophagy and, in particular, mitophagy, in human hepatic cells exposed to EFV. We detected the presence of altered mitochondria with abnormal morphology and relative increase in mitochondrial mass. Several autophagic markers reveal specific induction of autophagy. Of special note, while moderate levels of EFV activate autophagy, higher concentrations exceeding the threshold of mitochondrial dysfunction, lead to a blockage in the autophagic flux, thus promoting "autophagic stress". Pharmacological inhibition of autophagy exacerbates the deleterious effect of EFV on cell survival/proliferation thereby promoting apoptosis, a finding which points to the fact that autophagy is triggered as a rescue mechanism enabling cell survival. The effect described in this study could be involved in the EFV-associated hepatotoxicity. It may constitute a new mechanism implicated in the genesis of pharmacological liver damage and in the recovery of hepatic homeostasis upon a drug-induced cellular insult.

Twenty years of HIV-1 non-nucleoside reverse transcriptase inhibitors: time to reevaluate their toxicity.

Twenty years of effective clinical application have consolidated non-nucleoside reverse transcriptase inhibitors (NNRTI) as essential components of the Highly Active Antiretroviral Therapy (HAART) employed in the treatment of Human Immunodeficiency Virus (HIV). However, as the disease has come under control, there has been growing emphasis on the long-term adverse effects induced by this chronic pharmacological therapy. Although traditionally considered to be safe and well-tolerated drugs, there is mounting evidence that associates NNRTI with the onset of cutaneous reactions, neuropsychiatric symptoms, hepatotoxicity, metabolic disturbances and gastrointestinal toxicity. Though the clinical manifestations of these detrimental events are increasingly recognised, the cellular and molecular mechanisms underlying them have received little attention. This review revaluates the toxicities associated with the use of NNRTI by analysing data from both clinical trials and recent in vitro studies. Particular emphasis is placed on the specific characteristics of each of the compounds that comprise this class of anti-HIV drugs, including some that are currently in clinical development. A deeper understanding of the causes of NNRTI-induced side effects would greatly help to improve existing anti-HIV-1 therapies and to develop safer and better tolerated drugs in the future, thus increasing the long term efficacy of NNRTI-containing regimens.

 
Nano-jewels in biology. Gold and platinum on diamond nanoparticles as antioxidant systems against cellular oxidative stress.

Diamond nanoparticles (DNPs) obtained by explosive detonation have become commercially available. These commercial DNPs can be treated under Fenton conditions (FeSO(4) and H(2)O(2) at acidic pH) to obtain purer DNP samples with a small average particle size (4 nm) and a large population of surface OH groups (HO-DNPs). These Fenton-treated HO-DNPs have been used as a support of gold and platinum nanoparticles (≤2 nm average size). The resulting materials (Au/HO-DNP and Pt/HO-DNP) exhibit a high antioxidant activity against reactive oxygen species induced in a hepatoma cell line. In addition to presenting good biocompatibility, Au/HO- and Pt/HO-DNP exhibit about a two-fold higher antioxidant activity than glutathione, one of the reference antioxidant systems. The most active material against cellular oxidative stress was Au/HO-DNP.

 
Abacavir and didanosine induce the interaction between human leukocytes and endothelial cells through Mac-1 upregulation

OBJECTIVE:

Abacavir and didanosine are nucleoside reverse transcriptase inhibitors (NRTI) widely used in therapy for HIV-infection but which have been linked to cardiovascular complications. The objective of this study was to analyze the effects of clinically relevant doses of abacavir and didanosine on human leukocyte-endothelium interactions and to compare them with those of other NRTIs.

DESIGN AND METHODS:

The interactions between human leukocytes - specifically peripheral blood polymorphonuclear (PMN) or mononuclear (PBMC) cells - and human umbilical vein endothelial cells were evaluated in a flow chamber system that reproduces conditions in vivo. The expression of adhesion molecules was analyzed by flow cytometry.

RESULTS:

Abacavir induced a dose-dependent increase in PMN and PBMC rolling and adhesion. This was reproduced by didanosine but not by lamivudine or zidovudine. Both abacavir and didanosine increased Mac-1 expression in neutrophils and monocytes, but produced no effects on either lymphocytes or the expression of endothelial adhesion molecules. The PMN/PBMC rolling and adhesion induced by abacavir or didanosine did not occur when antibodies against Mac-1 or its ligand ICAM-1 were blocked.

CONCLUSION:

Abacavir induces significant human leukocyte accumulation through the activation of Mac-1, which in turn interacts with its endothelial ligand ICAM-1. The fact that didanosine exhibits similar effects and that lamivudine and zidovudine do not points to a relationship between the chemical structure of NRTIs and the induction of leukocyte/endothelial cell interactions. This mechanism may be especially relevant to the progression of the vascular damage associated with atherosclerosis and myocardial infarction in abacavir and didanosine-treated patients.

 

 
Enhanced oxidative stress and increased mitochondrial mass during efavirenz-induced apoptosis in human hepatic cells.

BACKGROUND AND PURPOSE:

Efavirenz (EFV) is widely used in the treatment of HIV-1 infection. Though highly efficient, there is growing concern about EFV-related side effects, the molecular basis of which remains elusive.

EXPERIMENTAL APPROACH:

In vitro studies were performed to address the effect of clinically relevant concentrations of EFV (10, 25 and 50 microM) on human hepatic cells.

KEY RESULTS:

Cellular proliferation and viability were reduced in a concentration-dependent manner. Analyses of the cell cycle and several cell death parameters (chromatin condensation, phosphatidylserine exteriorization, mitochondrial proapoptotic protein translocation and caspase activation) revealed that EFV triggered apoptosis via the intrinsic pathway. In addition, EFV directly affected mitochondrial function in a reversible manner, inducing a decrease in mitochondrial membrane potential and an increase in mitochondrial superoxide production, followed by a reduction in cellular glutathione content. The rapidity of these actions rules out any involvement of mitochondrial DNA replication, which, until now, was thought to be the main mechanism of mitochondrial toxicity of antiretroviral drugs. Importantly, we also observed an increase in mitochondrial mass, manifested as an elevated cardiolipin content and enhanced expression of mitochondrial proteins, which was not paralleled by an increase in the mtDNA/nuclear DNA copy number ratio. The toxic effect of EFV was partially reversed by antioxidant pretreatment, which suggests ROS generation is involved in this effect.

CONCLUSION AND IMPLICATIONS:

Clinically relevant concentrations of EFV were shown to be mitotoxic in human hepatic cells in vitro, which may be pertinent to the understanding of the hepatotoxicity associated with this drug.


Gastrin: an acid-releasing, proliferative and immunomodulatory peptide?

Gastrin release is affected by gastric inflammatory conditions. Antral G cells respond to inflammatory mediators by increasing gastrin secretion. Accumulating experimental evidence suggests that gastrin exerts immunomodulatory and proinflammatory effects. Gastrin could be a contributing factor to these pathologies, which may constitute a new justification for pharmacological blockade of gastrin action.

 

 
Inhibition of mitochondrial function by efavirenz increases lipid content in hepatic cells.

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in human immunodeficiency virus (HIV) infection therapy. It has been associated with hepatotoxic effects and alterations in lipid and body fat composition. Given the importance of the liver in lipid regulation, we have evaluated the effects of clinically used concentrations of EFV on the mitochondria and lipid metabolism of human hepatic cells in vitro. Mitochondrial function was rapidly undermined by EFV to an extent that varied with the concentration employed; in particular, respiration and intracellular adenosine triphosphate (ATP) levels were reduced whereas reactive oxygen species (ROS) production increased. Results in isolated mitochondria suggest that the mechanism responsible for these actions was a specific inhibition of complex I of the respiratory chain. The reduction in energy production triggered a compensatory mechanism mediated by the enzyme adenosine monophosphate-activated protein kinase (AMPK), the master switch of cellular bioenergetics. Fluorescence and nuclear magnetic resonance demonstrated a rapid intracellular increase of neutral lipids, usually in the form of droplets. This was prevented by the AMPK inhibitor compound C and by removal of fatty acids from the culture medium. These effects were not reproduced by Nevirapine, another NNRTI. EFV is clinically coadministered with two nucleoside reverse transcriptase inhibitors. Evaluation of one of the most common combination, EFV/Lamivudine/Abacavir, revealed that the effects of EFV on ROS production were enhanced. CONCLUSION: Clinical concentrations of EFV induce bioenergetic stress in hepatic cells by acutely inhibiting mitochondrial function. This new mechanism of mitochondrial interference leads to an accumulation of lipids in the cytoplasm that is mediated by activation of AMPK.

 

 
Nitric oxide, derived from inducible nitric oxide synthase, decreases hypoxia inducible factor-1alpha in macrophages during aspirin-induced mesenteric inflammation.

BACKGROUND AND PURPOSE:

Nitric oxide (NO) modulates expression of hypoxia inducible factor-1 (HIF-1), a transcription factor regulating function of myeloid cells. Here, we have assessed the role played by NO, formed by inducible NOS (iNOS), in the inflammation induced by aspirin in the gut, by modulating HIF-1 activity.

EXPERIMENTAL APPROACH:

The role of iNOS-derived NO on leucocyte-endothelial interactions induced by aspirin was evaluated by intravital microscopy in mesenteric venules of rats pretreated with selective iNOS inhibitors, 1400W or l-N6-(1-iminoethyl)-lysine. NO was localized by fluorescence microscopy, using DAF-FM. iNOS, HIF-1alpha and CD36 were localized by immunohistochemistry.

KEY RESULTS:

Leucocyte-endothelial interactions increased at 6 h and returned to normal levels 24 h after aspirin administration. Numbers of migrated leucocytes were similar between 6 and 24 h after aspirin. iNOS expression and iNOS-derived NO synthesis were observed in leucocytes of the mesentery of aspirin-treated rats. Blockade of iNOS activity in aspirin-treated rats: (i) did not modify leucocyte infiltration at 6 h, but reduced the number of polymorphonuclear leucocyte and increased that of macrophages at 24 h; (ii) increased HIF-1alpha immunostaining in macrophages of the mesentery; and (iii) prevented the decrease in CD36 immunostaining induced by aspirin in these cells.

CONCLUSIONS AND IMPLICATIONS:

NO, associated with acute gut inflammation induced by aspirin, diminished HIF-1alpha stabilization in macrophages. Early inhibition of iNOS-derived NO synthesis, by increasing the activity of HIF-1 in these cells, may accelerate the clearance of leucocytes.

 

 
iNOS-derived nitric oxide mediates the increase in TFF2 expression associated with gastric damage: role of HIF-1.

Trefoil (TFF) peptides are involved in gastrointestinal mucosal restitution. An hypoxia inducible factor 1 (HIF-1)-dependent induction of TFF genes has been reported in gastric epithelial cells. Nitric oxide (NO) is associated with mucosal damage and modulates HIF-1 activity. The aim of the present study was to analyze the role of iNOS-derived NO in HIF-1alpha stabilization and TFF gene expression in damaged gastric mucosa. Aspirin caused gastric injury that peaked 6 h after dosing and returned to normality at 24 h. iNOS mRNA expression occurs in the corpus in parallel with damage. Blockade of iNOS activity did not modify gastric lesions induced by aspirin but delayed mucosal healing. Aspirin induced HIF-1alpha stabilization and TFF2 mRNA up-regulation in the mucosa, but these effects were diminished when iNOS activity was inhibited. Results obtained using a coculture setup showed that iNOS-derived NO from activated macrophages induced HIF-1alpha stabilization, TFF gene expression, and accelerated wound healing in cultured epithelial cells. Finally, transient silencing of endogenous HIF-1alpha in epithelial cells significantly undermined activated macrophage-induced TFF gene expression. Evidence suggests that the iNOS-derived NO associated with NSAID-induced gastric injury is implicated in mucosal restitution via the HIF-1-mediated induction of TFF genes.

 

 
Inhibition of mitochondrial function by efavirenz increases lipid content in hepatic cells.

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in human immunodeficiency virus (HIV) infection therapy. It has been associated with hepatotoxic effects and alterations in lipid and body fat composition. Given the importance of the liver in lipid regulation, we have evaluated the effects of clinically used concentrations of EFV on the mitochondria and lipid metabolism of human hepatic cells in vitro. Mitochondrial function was rapidly undermined by EFV to an extent that varied with the concentration employed; in particular, respiration and intracellular adenosine triphosphate (ATP) levels were reduced whereas reactive oxygen species (ROS) production increased. Results in isolated mitochondria suggest that the mechanism responsible for these actions was a specific inhibition of complex I of the respiratory chain. The reduction in energy production triggered a compensatory mechanism mediated by the enzyme adenosine monophosphate-activated protein kinase (AMPK), the master switch of cellular bioenergetics. Fluorescence and nuclear magnetic resonance demonstrated a rapid intracellular increase of neutral lipids, usually in the form of droplets. This was prevented by the AMPK inhibitor compound C and by removal of fatty acids from the culture medium. These effects were not reproduced by Nevirapine, another NNRTI. EFV is clinically coadministered with two nucleoside reverse transcriptase inhibitors. Evaluation of one of the most common combination, EFV/Lamivudine/Abacavir, revealed that the effects of EFV on ROS production were enhanced. CONCLUSION: Clinical concentrations of EFV induce bioenergetic stress in hepatic cells by acutely inhibiting mitochondrial function. This new mechanism of mitochondrial interference leads to an accumulation of lipids in the cytoplasm that is mediated by activation of AMPK.

 

 
Regulation of homocysteine homeostasis through the transcriptional coactivator PGC-1alpha

Plasma homocysteine (Hcy) is an independent risk factor for cardiovascular disease. Hcy is a nonprotein amino acid derivative that is generated from the methionine cycle, which provides the methyl group for essentially all biological methylation reactions. Although plasma Hcy levels are elevated in patients with cardiovascular disease, the mechanisms that regulate Hcy homeostasis remain poorly defined. In this study, we found that the expression of key enzymes involved in Hcy metabolism is induced in the liver in response to fasting. This induction coincides with increased expression of peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha, a transcriptional coactivator that regulates hepatic gluconeogenesis and mitochondrial function. PGC-1alpha stimulates the expression of genes involved in Hcy metabolism in cultured primary hepatocytes as well as in the liver. Adenoviral-mediated expression of PGC-1alpha in vivo leads to elevated plasma Hcy levels. In contrast, mice deficient in PGC-1alpha have lower plasma Hcy concentrations. These results define a novel role for the PGC-1alpha coactivator pathway in the regulation of Hcy homeostasis and suggest a potential pathogenic mechanism that contributes to hyperhomocysteinemia.

 

 
Regulation of oxygen distribution in tissues by endothelial nitric oxide.

Nitric oxide (NO) decreases cellular oxygen (O(2)) consumption by competitively inhibiting cytochrome c oxidase. Here, we show that endogenously released endothelial NO, either basal or stimulated, can modulate O(2) consumption both throughout the thickness of conductance vessels and in the microcirculation. Furthermore, we have shown that such modulation regulates O(2) distribution to the surrounding tissues. We have demonstrated these effects by measuring O(2) consumption in blood vessels in a hypoxic chamber and O(2) distribution in the microcirculation using the fluorescent oxygen-probe Ru(phen)(3)(2+). Removal of NO by physical or pharmacological means, or in eNOS(-/-) mice, abolishes this regulatory mechanism. Our results indicate that, in addition to its well-known effect on the regulation of vascular tone, endothelial NO plays a major role in facilitating the distribution of O(2), an action which is crucial for the adaptation of tissues, including the vessel wall itself, to hypoxia. It is possible that changes in the distribution of O(2) throughout the vessel wall may be implicated in the origin of vascular pathologies such as atherosclerosis.

 

 
Gastrin induces the interaction between human mononuclear leukocytes and endothelial cells through the endothelial expression of P-selectin and VCAM-1.

Gastric mucosal inflammation is frequently associated with hypergastrinemia, and a correlation exists between the level of gastrin and degree of gastritis. We have previously observed that gastrin promotes leukocyte-endothelial interactions and contributes to Helicobacter-induced inflammation in the rat mesentery. In the present study, we aimed to evaluate a possible proinflammatory activity of gastrin in humans. The interaction between human leukocytes [U-937 cells, peripheral blood polymorphonuclear (PMN), and peripheral blood mononuclear (PBMC) cells] and human umbilical vein endothelial cells (HUVEC) was analyzed in static and dynamic conditions. The endothelial expression of adhesion molecules [P-selectin, E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule (VCAM)-1] was analyzed by flow cytometry and fluorescent microscopy screening. Gastrin increased the static adhesion of U-937 cells to HUVEC (1 h; 10(-9) M: 122 +/- 9%; 10(-8) M: 143 +/- 17%; 10(-7) M: 162 +/- 14% vs. control, all P < 0.05). Incubation of HUVEC with gastrin (4 h) also increased PBMC rolling (vehicle: 63 +/- 12; 10(-9) M: 109 +/- 29; 10(-8) M: 141 +/- 24; 10(-7) M: 261 +/- 16 leukocytes/min, P < 0.05) and adhesion (vehicle: 3 +/- 2, 10(-9) M: 11 +/- 4, 10(-8) M: 17 +/- 5, 10(-7) M: 15 +/- 5 leukocytes/mm(2), all P < 0.05) in the parallel-plate flow chamber. Treatment of PBMC with gastrin had no effects. The cholecystokinin (CCK)-2 receptor antagonist (L-365,260, 10(-7) M) prevented the effects of gastrin. P-selectin and VCAM-1 expression were enhanced by gastrin, and neutralizing antibodies against these molecules prevented PBMC rolling and adhesion. Gastrin did not affect the interactions between HUVEC and PMN. Gastrin induces interactions between human mononuclear leukocytes and endothelial cells through the activation of CCK-2 receptors and the enhancement of endothelial P-selectin and VCAM-1.

 

 
Mitochondrial-targeted antioxidants and oxidative stress: a proteomic prospective study.

Mitochondria produce large amounts of free radicals and play an important role in the life and death of a cell, regulating the signalling, metabolism, and energy production needed for cellular function. In this way, mitochondrial oxidative damage and dysfunction contribute to a number of cell pathologies that are manifested through a range of conditions that include cardiovascular diseases (CVD). Although the molecular mechanisms responsible for mitochondria-mediated disease processes are not yet completely understood, oxidative stress definitely seems to play an important role. When examined at the protein level, both expression levels and protein modifications are altered by oxidative stress. While these effects have been studied in the past by classic biochemical methods, recent developments in proteomics have allowed the oxidative stress response to be studied in more depth. The focus of this work is the mitochondrial proteome/genome interplay that is currently believed to be implicated in a range of human diseases. Particular attention is given to the current knowledge of the role of mitochondria in the development of oxidative-stress-based diseases; e.g. CVD is highlighted together with the prospective proteomics perspective as an alternative prognostic and diagnostic tool for interpreting many mitochondria-related anomalies. Accordingly, strategies for the targeted delivery of antioxidants to mitochondria are being developed. The insight provided by recent proteomic research and the effects of mitochondrial-antioxidants on possible interventions are also discussed.

 

 
Oxidative stress and endothelial dysfunction in cardiovascular disease: mitochondria-targeted therapeutics.

Functional impairment of endothelial activity (endothelial dysfunction) precedes the development of cardiovascular diseases (CVD). This condition is a result of a reduced bioavailability of nitric oxide (NO), a well known vasodilator, which is mainly due to increased NO degradation caused by its reaction with reactive oxygen species (ROS). Although there are several conditions that contribute independently to endothelial dysfunction, such as hyperglycemia, insulin resistance, hyperinsulinemia and dyslipidemia, increased oxidative stress seems to play a key role. In addition to their original pharmacological properties, drugs used clinically at present, including anti-hypertension reagents, angiotensin receptor blockers and anti-hyperlipidemic reagents such as statins, protect various organs via anti-oxidative stress mechanisms. Moreover, some substances with antioxidant properties, such as vitamin C or vitamin E, have been used to eradicate the oxidative stress associated with CVD. The results of the clinical trials employing anti-oxidative stress reagents in patients with CVD are contradictory, which could be a result of inadequate study design or selected targets. This review considers the process of endothelial dysfunction and CVD from a mitochondrial perspective and evaluates strategies currently under development for the targeted delivery of antioxidants or NO to mitochondria. It endorses the idea that selectively targeting specific antioxidants and NO donors to mitochondria is an effective strategy for modulating mitochondrial respiration and ROS production and protecting mitochondria against oxidative stress.

 

 

Induction of trefoil factor (TFF)1, TFF2 and TFF3 by hypoxia is mediated by hypoxia inducible factor-1: implications for gastric mucosal healing.


BACKGROUND AND PURPOSE:

Mucosal microcirculation is compromised during gastric damage induced by non-steroidal anti-inflammatory drugs, such as aspirin. Consequently, oxygen supply to epithelial cells is decreased. The trefoil factor (TFF) peptides are involved in mechanisms of defence and repair in the gastrointestinal tract but their regulation at sites of gastric injury is unknown.

EXPERIMENTAL APPROACH:

Hypoxia and expression of TFF genes and peptides were measured in the damaged stomach of aspirin-treated rats. In a human gastric cell line (AGS cells), the effects of hypoxia and of hypoxia inducible factor (HIF)-1 (through transient transfection of HIF-1alpha siRNA or over-expression of HIF-1alpha) on TFF gene expression were evaluated.

KEY RESULTS:

Hypoxyprobe immunostaining, up-regulation of TFF2 (1.9-fold) and TFF3 (1.8-fold) and a non-significant increase of TFF1 (1.5-fold) mRNA were observed in the damaged stomach of aspirin-treated rats, compared with control animals. Hypoxia (3% O(2), 16 h) induced mRNA for TFF1 (5.8-fold), TTF2 (9.1-fold) and TFF3 (9.3-fold) in AGS cells, an effect mediated by HIF-1, as transient transfection of HIF-1alpha siRNA reduced the effects of hypoxia. Over-expression of HIF-1alpha by transfection in non-hypoxic epithelial cells produced a similar pattern of TFF induction to that observed with hypoxia and transactivated a TFF1 reporter construct.

CONCLUSIONS AND IMPLICATIONS:

Hypoxia inducible factor-1 mediated the induction of TFF gene expression by hypoxia in gastric epithelial cells. Low oxygen levels and up-regulation of TFF gene expression in the damaged stomach of aspirin-treated rats suggest that hypoxia induced expression of TFF genes at sites of gastric injury.

 

 
Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells.

Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys(118), suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys(118) contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments.

 

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