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SELECTED PUBLICATIONS OF THE LAST 7 YEARS/span>

2014

Lack of mitochondrial toxicity of darunavirraltegravir and rilpivirine in neurons and hepatocytes: a comparisonwith efavirenz
OBJECTIVES:
Growing evidence associates the non-nucleoside reverse transcriptase inhibitor efavirenz with several adverse events. Newer antiretrovirals, such as the integrase inhibitor raltegravir, the non-nucleoside reverse transcriptase inhibitor rilpivirine and the protease inhibitordarunavir, claim to have a better toxicological profile than efavirenz while producing similar levels of efficacy and virological suppression. The objective of this study was to determine the in vitro toxicological profile of these three new antiretrovirals by evaluating their effects on themitochondrial and cellular parameters altered by efavirenz in hepatocytes and neurons.
METHODS:
Hep3B cells and primary rat neurons were treated with clinically relevant concentrations of efavirenzdarunavirrilpivirine or raltegravir. Parameters of mitochondrial function, cytotoxicity and oxidative and endoplasmic reticulum stress were assessed using standard cell biology techniques.
RESULTS:
None of the new compounds altered the mitochondrial function of hepatic cells or neurons, while efavirenz decreased mitochondrialmembrane potential and enhanced superoxide production in both cell types, effects that are known to significantly compromise the functioning of mitochondria, cell viability and, ultimately, cell number. Of the four drugs assayed, efavirenz was the only one to alter the protein expression of LC3-II, an indicator of autophagy, and CHOP, a marker of endoplasmic reticulum stress and the unfolded protein response.
CONCLUSIONS:
Darunavirrilpivirine and raltegravir do not induce toxic effects on Hep3B cells and primary rat neurons, which suggests a safer hepatic and neurological profile than that of efavirenz.
 
Neuronal bioenergetics and acute mitochondrial dysfunction: a clue to understanding the central nervoussystem side effects of efavirenz
BACKGROUND:
Neurological pathogenesis is associated with mitochondrial dysfunction and differences in neuronal/glial handling of oxygen and glucose. The main side effects attributed to efavirenz involve the CNS, but the underlying mechanisms are unclear.
METHODS:
Human cell lines and rat primary cultures of neurons and astrocytes were treated with clinically relevant efavirenz concentration.
RESULTS:
Efavirenz alters mitochondrial respiration, enhances reactive oxygen species generation, undermines mitochondrial membrane potential, and reduces adenosine triphosphate (ATP) levels in a concentration-dependent fashion in both neurons and glial cells. However, it activates adenosine monophosphate-activated protein kinase only in glial cells, upregulating glycolysis and increasing intracellular ATP levels, which do not occur in neurons. To reproduce the conditions that often exist in human immunodeficiency virus-related neuroinflammatory disorders, the effects ofefavirenz were evaluated in the presence of exogenous nitric oxide, an inflammatory mediator and mitochondrial inhibitor. The combination potentiated the effects on mitochondrial parameters in both neurons and glial cells, but ATP generation and lactate production were enhanced only in glial cells.
CONCLUSIONS:
Efavirenz affects the bioenergetics of neurons through a mechanism involving acutemitochondrial inhibition, an action exacerbated in neuroinflammatory conditions. A similar scenario of glial cells survival and degeneration of neurons with signs of mitochondrialdysfunction and oxidative stress has been associated with neurocognitive disorders.
 
Is glycemic control modulating endoplasmic reticulum stress in leukocytes of type 2 diabetic patients?
Oxidative and endoplasmic reticulum (ER) stress is related to type 2 diabetes (T2D), but the influence of glycemic control on these parameters and its relationship with leukocyte-endothelial interactions is not known. In our study population consisting of 164 diabetic patients, (102 with glycated hemoglobin [HbA1c] <7% and 62 with HbA1c >7%) and 84 nondiabetic controls, we have verified a common anthropometric and metabolic pattern of T2D with dyslipidemia. Inflammatory parameters (high-sensitive C-reactive protein [hs-CRP] and tumor necrosis factor alpha [TNFα]) and E-selectin levels were enhanced in the HbA1c >7% group with regard to controls. O2 consumption and mitochondrial membrane potential were lower in diabeticpatients than in controls. Reactive oxygen species (ROS) production was enhanced in diabetic patients than in controls and positively correlated with HbA1c levels. GRP78 levels were higher in both diabetic groups. However, HbA1c <7% patients displayed higher levels of spliced X-box binding protein 1 (sXBP1), whereas HbA1c >7% patients exhibited preferentially enhanced levels of CHOP (CCAAT/enhancer binding protein [C/EBP] homologous protein) and activating transcription factor 6 (ATF6). Reduced leukocyte rolling velocity and increased rolling flux and adhesion were observed in diabetic patients. Our findings lead to the hypothesis of an association between poor glycemic control in T2D and increased leukocyte ROS production and chronic ER stress that could finally promote leukocyte-endothelial interactions, which, in turn, poses a risk of vascular complications for these patients.
 
How to compare biologic drugs [Article in English, Spanish]
This consensus document reviews the evidence on the evaluation of biological drugs. The main conclusions of the group are: a) the current evidence on biological comparisons is based on indirect comparisons and is generally unreliable and with important methodological limitations. Therefore, b) it is considered necessary to amend the regulatory directives in the sense of strongly favoring randomized non-inferiority studies comparing face to face the new biological treatment with current standards, avoiding trials versus placebo, c) A key element in this process will be determined by consensus among regulatory agencies, scientific societies, the pharmaceutical industry and health authorities regarding the clinical differences that should be considered relevant in each of the conditions tested.
 
Efavirenz induces interactions between leucocytes and endothelium through the activation of Mac-1 and gp150,95
OBJECTIVES:
The potential cardiovascular (CV) toxicity associated with combined antiretroviral therapy (cART) has been attributed mainly to the nucleoside reverse transcriptase inhibitors abacavir and didanosine. However, the other two components of cART--non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs)--may also be implicated, either directly or by influencing the action of the other drugs. This study evaluates the acute direct effects of the NNRTIs efavirenz and nevirapine and one of the most widely employed PIs, lopinavir, on leucocyte-endothelium interactions, a hallmark of CV disease.
METHODS:
Drugs were analysed in vitro in human cells (interactions of peripheral blood polymorphonuclear or mononuclear cells with human umbilical vein endothelial cells) using a flow chamber system, and in vivo in rat mesenteric vessels by means of intravital microscopy. The expression of adhesion molecules in leucocytes and endothelial cells was studied by flow cytometry, and the role of these molecules in white cell recruitment was evaluated by pre-treating human cells or rats with blocking antibodies.
RESULTS:
Efavirenz and nevirapine, but not lopinavir, increased the rolling flux and adhesion of leucocytes in vitro and in vivo while inducing emigration in rat venules. Efavirenz, but not nevirapine, augmented the levels of CD11b, CD11c and CD18 in neutrophils and monocytes. The actions of efavirenz, but not of nevirapine, were reversed by antibodies against Mac-1 (CD11b/CD18), gp150,95 (CD11c/CD18) or ICAM-1 (CD54).
CONCLUSIONS:
NNRTIs, but not PIs, interfere with leucocyte-endothelial interactions. However, differences between efavirenz and nevirapine suggest a specific CV profile for each compound.
 
Role of endothelial nitric oxide in pulmonary and systemic arteries during hypoxia
Our aim was to investigate the role played by endothelial nitric oxide (NO) during acute vascular response to hypoxia, as a modulator of both vascular tone (through guanylate cyclase (sGC) activation) and mitochondrial O2 consumption (through competitive inhibition of cytochrome-c-oxydase (CcO)). Organ bath experiments were performed and O2 consumption (Clark electrode) was determined in isolated aorta, mesenteric andpulmonary arteries of rats and eNOS-knockout mice. All pre-contracted vessels exhibited a triphasic hypoxic response consisting of an initial transient contraction (not observed in vessels from eNOS-knockout mice) followed by relaxation and subsequent sustained contraction. Removal of the endothelium, inhibition of eNOS (by L-NNA) and inhibition of sGC (by ODQ) abolished the initial contraction without altering the other two phases. The initial hypoxic contraction was observed in the presence of L-NNA+NO-donors. L-NNA and ODQ increases O2 consumption in hypoxic vessels and increases the arterial tone in normoxia but not in hypoxia. When L-NNA+mitochondrial inhibitors (cyanide, rotenone or myxothiazol) were added, the increase in tone was similar in normoxic and hypoxic vessels, which suggests that inhibition of the binding of NO to reduced CcO restored the action of NO on sGC.
CONCLUSION:
A complex equilibrium is established between NO, sGC and CcO in vessels in function of the concentration of O2: as O2 falls, NO inhibition of mitochondrial O2 consumption increases and activation of sGC decreases, thus promoting a rapid increase in tone in both pulmonaryand systemic vessels, which is followed by the triggering of NO-independent vasodilator/vasoconstrictor mechanisms.
 
Mitochondria-targeted antioxidants as a therapeutic strategy for protecting endothelium in cardiovascular diseases
Endothelial dysfunction involving dysfunctional mitochondria precedes the development of cardiovascular diseases. This impairment results from an increase in reactive oxygen species, which leads to oxidative stress and a reduced bioavailability of nitric oxide. It has been demonstrated that oxidative stress and alterations in glucose and lipid homeostasis (e.g. hyperinsulinemia, hyperglycemia, insulin resistance and dyslipidemia) are linked to mitochondrial impairment and that all of them contribute to endothelial dysfunction. Anti-hyperlipidemic drugs such as statins, anti-hypertensive drugs and angiotensin receptor antagonists have been shown to exert protection through anti-oxidative stress mechanisms. Other substances with antioxidant properties, such as vitamins, are also capable of abolishing the oxidative stress associated with cardiometabolicdiseases. However, the results obtained with general antioxidants in clinical trials are contradictory, perhaps due to the unspecific nature of the targets selected. This study correlates endothelial dysfunction and mitochondrial dysfunction and examines current research for the selective targeting of specific molecules (such as ·NO donors and antioxidants) to mitochondria with the aim of protecting the endothelium against oxidative stress in cardiovascular diseases.
 
Using exomarkers to assess mitochondrial reactive species in vivo
BACKGROUND:
The ability to measure the concentrations of small damaging and signalling molecules such asreactiveoxygenspecies(ROS)in vivois essential to understanding their biological roles. While a range of methods can be applied toinvitro systems, measuring the levels and relative changesinreactivespeciesin vivois challenging.
SCOPE OF REVIEW:
One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules.
MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE:
Our laboratories have developed mitochondria-targeted probes that generate exomarkersthat can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins.
 
Perspectives and potential applications of mitochondria-targeted antioxidants in cardiometabolic diseases and type 2 diabetes
There is abundant evidence to suggest that mitochondrial dysfunction is a main cause of insulin resistance and related cardiometaboliccomorbidities. On the other hand, insulin resistance is one of the main characteristics of type 2 diabetes, obesity, and metabolic syndrome. Lipid and glucose metabolism require mitochondria to generate energy, and when O2 consumption is low due to inefficient nutrient oxidation, there is an increase in reactive oxygen species, which can impair different types of molecules, including DNA, lipids, proteins, and carbohydrates, thereby inducing proinflammatory processes. Factors which contribute to mitochondrial dysfunction, such as mitochondrial biogenesis and genetics, can also lead to insulin resistance in different insulin-target tissues, and its association with mitochondrial dysfunction can culminate in the development of cardiovascular diseases. In this context, therapies that improve mitochondrial function may also improve insulin resistance. This review explains mechanisms of mitochondrial function related to the pathological effects of insulin resistance in different tissues. The pathogenesis ofcardiometabolic diseases will be explained from a mitochondrial perspective and the potential beneficial effects of mitochondria-targeted antioxidantsas a therapy for modulating mitochondrial function in cardiometabolic diseases, especially diabetes, will also be considered.
 
Hypoxic macrophages impair autophagy in epithelial cells through Wnt1: relevance in IBD
A defective induction of epithelial autophagy may have a role in the pathogenesis of inflammatory bowel diseases. This process is regulated mainly by extracellular factors such as nutrients and growth factors and is highly induced by diverse situations of stress. We hypothesized that epithelialautophagy is regulated by the immune response that in turn is modulated by local hypoxia and inflammatory signals present in the inflamed mucosa. Our results reveal that HIF-1α and Wnt1 were co-localized with CD68 in cells of the mucosa of IBD patients. We have observed increased protein levels of β-catenin, phosphorylated mTOR, and p62 and decreased expression of LC3II in colonic epithelial crypts from damaged mucosa in which β-catenin positively correlated with phosphorylated mTOR and negatively correlated with autophagic protein markers. In cultured macrophages, HIF-1 mediated the increase in Wnt1 expression induced by hypoxia, which enhanced protein levels of β-catenin, activated mTOR, and decreasedautophagy in epithelial cells in co-culture. Our results demonstrate a HIF-1-dependent induction of Wnt1 in hypoxic macrophages that underminesautophagy in epithelial cells and suggest a role for Wnt signaling and mTOR pathways in the impaired epithelial autophagy observed in the mucosa of IBD patients.
 
The pivotal role of nitric oxide: effects on the nervous and immune systems
 
Nitric oxide (NO) has an important role in physiological and pathological processes in general, and in particular plays a homeostatic role in thenervous and immune systems. The many different physiological functions of NO include those of a mediator of blood vessel dilation, neurotransmitter, neuromodulator and inductor of mitochondrial biogenesis. In addition, NO can transform into highly reactive and harmful molecules producing an impairment of the DNA, lipids or proteins, and thus altering their function. This dual action of NO, by which it plays an important role in homeostasis and aids the development of pathological processes, makes this molecule an interesting target for medical therapies, especially with respect to the nervous and immune systems. This review describes the multiple roles of NO played out in the nervous and immune systems during different physiological and pathophysiological processes.
 

Progastrin represses the alternative activation of human macrophages and modulates their influence on coloncancer epithelial cells

 
Macrophage infiltration is a negative prognostic factor for most cancers but gastrointestinal tumors seem to be an exception. The effect ofmacrophages on cancer progression depends on their phenotype, which may vary between M1 (pro-inflammatory, defensive) to M2 (tolerogenic, pro-tumoral). Gastrointestinal cancers often become an ectopic source of gastrins and macrophages present receptors for these peptides. The aim of the present study is to analyze whether gastrins can affect the pattern of macrophage infiltration in colorectal tumors. We have evaluated the relationship between gastrin expression and the pattern of macrophage infiltration in samples from colorectal cancer and the influence of these peptides on the phenotype of macrophages differentiated from human peripheral monocytes in vitro. The total number of macrophages (CD68+ cells) was similar in tumoral and normal surrounding tissue, but the number of M2 macrophages (CD206+ cells) was significantly higher in the tumor. However, the number of these tumor-associated M2 macrophages correlated negatively with the immunoreactivity for gastrin peptides in tumor epithelial cells.Macrophages differentiated from human peripheral monocytes in the presence of progastrin showed lower levels of M2-markers (CD206, IL10) with normal amounts of M1-markers (CD86, IL12). Progastrin induced similar effects in mature macrophages treated with IL4 to obtain a M2-phenotype or with LPS plus IFNγ to generate M1-macrophages. Macrophages differentiated in the presence of progastrin presented a reduced expression of Wnt ligands and decreased the number and increased cell death of co-cultured colorectal cancer epithelial cells. Our results suggest that progastrininhibits the acquisition of a M2-phenotype in human macrophages. This effect exerted on tumor associated macrophages may modulate cancerprogression and should be taken into account when analyzing the therapeutic value of gastrin immunoneutralization.
 
Gene mutations in primary ciliary dyskinesia related to otitis media
 
Otitis media with effusion (OME) is the most common cause of conductive hearing loss in children and is strongly associated with primary ciliary dyskinesia (PCD). Approximately half of the children with PCD require otolaryngology care, posing a major problem in this population. Early diagnosis of PCD is critical in these patients to minimise the collateral damage related to OME. The current gold standard for PCD diagnosis requires determining ciliary structure defects by transmission electron microscopy (TEM) or clearly documenting ciliary dysfunction via digital high-speed video microscopy (DHSV). Although both techniques are useful for PCD diagnosis, they have limitations and need to be supported by new methodologies, including genetic analysis of genes related to PCD. In this article, we review classical and recently associated mutations related tociliary alterations leading to PCD, which can be useful for early diagnosis of the disease and subsequent early management of OME.

2013

Profile of leukocyte-endothelial cell interactions induced in venules and arterioles by nucleoside reverse-transcriptase inhibitors in vivo
BACKGROUND:
There is controversy regarding cardiovascular (CV) toxicity of the nucleoside reverse-transcriptase inhibitors used to treat human immunodeficiency virus infection.
METHODS:
We evaluated the effects of nucleoside reverse-transcriptase inhibitors on leukocyte-endothelium interactions, a hallmark of CV diseases, in rat mesenteric vessels using intravital microscopy and in human arterial cells using a flow chamber system.
RESULTS:
Abacavir and didanosine increased rolling, adhesion and emigration in rat vessels. These effects were reversed with antibodies against Macrophage-1 antigen (Mac-1) or intercellular adhesion molecule 1 and were reproduced in human cells. Lamivudine, zidovudine, emtricitabine, and tenofovir had no effects.
CONCLUSIONS:
Our results support the association of abacavir and didanosine with CV diseases.
 
ER stress in human hepatic cells treated with Efavirenz: mitochondria again
BACKGROUND & AIMS:
ER stress is associated with a growing number of liver diseases, including drug-induced hepatotoxicity. The non-nucleoside analogue reverse transcriptase inhibitor Efavirenz, a cornerstone of the multidrug strategy employed to treat HIV1 infection, has been related to the development of various adverse events, including metabolic disturbances and hepatic toxicity, the mechanisms of which remain elusive. Recent evidence has pinpointed a specific mitochondrial effect of Efavirenz in human hepatic cells. This study assesses the induction ofER stress by Efavirenz in the same model and the implication of mitochondria in this process.
METHODS:
Primary human hepatocytes and Hep3B were treated with clinically relevant concentrations of Efavirenz and parameters of ER stresswere studied using standard cell biology techniques.
RESULTS:
ER stress markers, including CHOP and GRP78 expression (both protein and mRNA), phosphorylation of eIF2α, and presence of the spliced form of XBP1 were upregulated. Efavirenz also enhanced cytosolic Ca(2+) content and induced morphological changes in the ER suggestive of ER stress. This response was greatly attenuated in cells with altered mitochondrial function (Rho°). The effects of Efavirenz on the ER, and particularly in regard to the mitochondrial involvement, differed from those elicited by a standard pharmacological ER stressor.
CONCLUSIONS:
This newly discovered mechanism of cellular insult involving ER stress and UPR response may help comprehend the hepatictoxicity that has been associated with the widespread and life-long use of Efavirenz. In addition, the specificity of the actions of Efavirenz observed expands our knowledge of the mechanisms that trigger ER stress and shed some light on the mitochondria/ER interplay in drug-induced hepaticchallenge.
 
M2 macrophages activate WNT signaling pathway in epithelial cells: relevance in ulcerative colitis
Macrophages, which exhibit great plasticity, are important components of the inflamed tissue and constitute an essential element of regenerative responses. Epithelial Wnt signalling is involved in mechanisms of proliferation and differentiation and expression of Wnt ligands by macrophages has been reported. We aim to determine whether the macrophage phenotype determines the expression of Wnt ligands, the influence of the macrophage phenotype in epithelial activation of Wnt signalling and the relevance of this pathway in ulcerative colitis. Human monocyte-derived macrophages and U937-derived macrophages were polarized towards M1 or M2 phenotypes and the expression of Wnt1 and Wnt3a was analyzed by qPCR. The effects of macrophages and the role of Wnt1 were analyzed on the expression of β-catenin, Tcf-4, c-Myc and markers of cell differentiation in a co-culture system with Caco-2 cells. Immunohistochemical staining of CD68, CD206, CD86, Wnt1, β-catenin and c-Myc were evaluated in the damaged and non-damaged mucosa of patients with UC. We also determined the mRNA expression of Lgr5 and c-Myc by qPCR and protein levels of β-catenin by western blot. Results show that M2, and no M1, activated the Wnt signaling pathway in co-culture epithelial cells through Wnt1 which impaired enterocyte differentiation. A significant increase in the number of CD206+ macrophages was observed in the damaged mucosa of chronic vs newly diagnosed patients. CD206 immunostaining co-localized with Wnt1 in the mucosa and these cells were associated with activation of canonical Wnt signalling pathway in epithelial cells and diminution of alkaline phosphatase activity. Our results show that M2 macrophages, and not M1, activate Wnt signalling pathways and decrease enterocyte differentiation in co-cultured epithelial cells. In the mucosa of UC patients, M2macrophages increase with chronicity and are associated with activation of epithelial Wnt signalling and diminution in enterocyte differentiation.
 
Rationalizing the use of PPIs: an unresolved matter
The abnormally high use of proton-pump inhibitors (PPIs) has been repeatedly denounced for many years (1), and attention has been drawn towards the economic burden it represents (2,3). Multiple scientific publications have reported this problem in many countries. In the present issue of the Journal an observational, prospective study based on a survey to analyze the inappropriate (not recommended by clinical guidelines) indication of chronic PPI use (daily for over one year) in outpatients seen at a specialty hospital in Mexico is reported (4). The authors find an overall rate of inappropriateness of 35.3 %, a figure within the lower range of those reported, which exceed 60 % in many countries including Australia (5,6), United Kingdom (7), and Greece (8), and in four studies performed in Spain (9,10-12). All these studies discuss oral therapy with PPIs, but inappropriateness is also very high (above 75 %) in terms of dosage and length of intravenous therapy (13).
 
2012
 
Induction of CD36 and Thrombospondin-1 in Macrophages by Hypo xia-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.

 
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.

 
Guidelines for the use and interpretation of assays for monitoring autophagy
In 2008 we published the first set ofguidelines for standardizing research inautophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update theseguidelines formonitoringautophagy in different organisms. Various reviews have described the range ofassays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measureautophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through theautophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increasedautophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with moreautophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set ofguidelines for the selection andinterpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. Theseguidelines are not meant to be a formulaic set of rules, because the appropriateassays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multipleassays to monitorautophagy. In theseguidelines, we consider these various methods of assessingautophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particularautophagyassays, we hope to encourage technical innovation in the field.

 

Gold nanoparticles supported on nanoparticulate ceria as a powerful agent against intracellular oxidative stress
Ceria-supported gold nanoparticles are prepared exhibiting peroxidase activity and acting as radical traps. Au/CeO(2) shows a remarkable biocompatibility as demonstrated by measuring cellular viability, proliferation, and lack of apoptosis for two human cell lines (Hep3B and HeLa). The antioxidant activity of Au/CeO(2) against reactive oxygen species (ROS) is demonstrated by studying the cellular behavior of Hep3B and HeLa in a model of cellular oxidative stress. It is determined that Au/CeO(2) exhibits higher antioxidant activity than glutathione, the main cytosolic antioxidant compound, and its CeO(2) carrier. Overall the result presented here shows the potential of implementing well-established nanoparticulated gold catalysts with remarkable biocompatibility in cellular biology.

 

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.

 

Metabolomics of the effect of AMPK activation by AICAR on humanumbilical vein endothelial cells

AMP-activated protein kinase (AMPK) is a metabolic master switch expressed in a great number ofcells and tissues. AMPK is thought to modulate the cellular response to different stresses that increase cellular AMP concentration. The adenosine analog, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) is an AMPK activator used in many studies to assess the effects ofAMPK activation on cellular metabolism and function. However, the effect of AICAR on cell metabolism reaches many different pathways and metabolites, some of which do not seem to be fully related to AMPK activation. We have now for the first time used NMR metabolomics onhuman umbilical vein endothelial cells (HUVEC) for the study of the global metabolic impact ofAMPK activation by AICAR. In our study, incubation with AICAR activates AMPK and is associated with, among others, broad metabolic alterations in energy metabolism and phospholipid biosynthesis. Using NMR spectroscopy and metabolic network tools, we analyzed the connections between the different metabolic switches activated by AICAR. Our approach reveals a strong interconnection between different phospholipid precursors and oxidation by-products. Metabolomicsprofiling is a useful tool for detecting major metabolic alterations, generating new hypotheses and provides some insight about the different molecular correlations in a complex system. The present study shows that AICAR induces metabolic effects in cell metabolism well beyond energy production pathways.

2011
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.

 
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.

 
2010
 
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.

 
 
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.

 

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.

 

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.

 

2009

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.

 

 
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.

 

2008

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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