Publications by authors named "Guillermo Bodega"

26 Publications

  • Page 1 of 1

A high magnesium concentration in citrate dialysate prevents oxidative stress and damage in human monocytes .

Clin Kidney J 2021 May 30;14(5):1403-1411. Epub 2020 Aug 30.

Dpto de Biología de Sistemas, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain.

Background: The use of dialysis fluids (DFs) during haemodialysis has been associated with increased oxidative stress and reduced serum magnesium (Mg) levels, contributing to chronic inflammation. Since the role of Mg in modulating immune function and reducing oxidative stress has been demonstrated, the aim of this study was to characterize whether increasing the Mg concentration in DFs could protect immune cells from oxidative stress and damage.

Methods: The effect of citrate [citrate dialysis fluid (CDF), 1 mM] or acetate [acetate dialysis fluid (ADF), 3 mM] dialysates with low (0.5 mM; routinely used) or high (1 mM, 1.25 mM and 2 mM) Mg concentrations was assessed in THP-1 human monocytes. The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and oxidized/reduced (GSSG/GSH) glutathione were quantified under basal and inflammatory conditions (stimulation with lipopolysaccharide, LPS).

Results: The increase of Mg in CDF resulted in a significant reduction of ROS production under basal and inflammatory conditions (extremely marked in 2 mM Mg; P 0.001). These effects were not observed in ADF. Interestingly, in a dose-dependent manner, high Mg doses in CDF reduced oxidative stress in monocytes under both basal and inflammatory conditions. In fact, 2 mM Mg significantly decreased the levels of GSH, GSSG and MDA and the GSSG/GSH ratio in relation to 0.5 mM Mg.

Conclusions: CDF produces lower oxidative stress than ADF. The increase of Mg content in DFs, especially in CDF, could have a positive and protective effect in reducing oxidative stress and damage in immune cells, especially under inflammatory conditions.
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http://dx.doi.org/10.1093/ckj/sfaa131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087128PMC
May 2021

Microvesicles from indoxyl sulfate-treated endothelial cells induce vascular calcification .

Comput Struct Biotechnol J 2020 9;18:953-966. Epub 2020 Apr 9.

Departamento de Biología de Sistemas, Universidad de Alcalá (IRYCIS), Alcalá de Henares, Madrid, Spain.

Vascular calcification (VC), an unpredictable pathophysiological process and critical event in patients with cardiovascular diseases (CVDs), is the leading cause of morbi-mortality and disability in chronic kidney disease (CKD) patients worldwide. Currently, no diagnostic method is available for identifying patients at risk of VC development; the pathology is detected when the process is irreversible. Extracellular vesicles (EVs) from endothelial cells might promote VC. Therefore, their evaluation and characterization could be useful for designing new diagnostic tools. The aim of the present study is to investigate whether microvesicles (MVs) from endothelial cells damaged by uremic toxin and indoxyl sulfate (IS) could induce calcification in human vascular smooth muscle cells (VMSCs). Besides, we have also analyzed the molecular mechanisms by which these endothelial MVs can promote VC development. Endothelial damage has been evaluated according to the percentage of senescence in endothelial cells, differential microRNAs in endothelial cells, and the amount of MVs released per cell. To identify the role of MVs in VC, VSMCs were treated with MVs from IS-treated endothelial cells. Calcium, inflammatory gene expression, and procalcification mediator levels in VSMCs were determined. IS-treated endothelial cells underwent senescence and exhibited modulated microRNA expression and an increase in the release of MVs. VSMCs exposed to these MVs modulated the expression of pro-inflammatory genes and some mediators involved in calcification progression. MVs produced by IS-treated endothelial cells promoted calcification in VSMCs.
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http://dx.doi.org/10.1016/j.csbj.2020.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184105PMC
April 2020

Increasing the Magnesium Concentration in Various Dialysate Solutions Differentially Modulates Oxidative Stress in a Human Monocyte Cell Line.

Antioxidants (Basel) 2020 Apr 15;9(4). Epub 2020 Apr 15.

Departamento de Biología de Sistemas, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.

Oxidative stress is exacerbated in hemodialysis patients by several factors, including the uremic environment and the use of dialysis fluids (DFs) Since magnesium (Mg) plays a key role in modulating immune function and in reducing oxidative stress, we aimed to evaluate whether increasing the Mg concentration in different DFs could protect against oxidative stress in immunocompetent cells in vitro. Effect of ADF (acetate 3 mM), CDF (citrate 1 mM), and ACDF (citrate 0.8 mM + acetate 0.3 mM) dialysates with Mg at standard (0.5 mM) or higher (1, 1.25, and 2 mM) concentrations were assessed in THP-1 monocyte cultures. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels were quantified under basal and uremic conditions (indoxyl sulfate (IS) treatment). Under uremic conditions, the three DFs with 0.5 mM Mg promoted higher ROS production and lipid damage than the control solution. However, CDF and ACDF induced lower levels of ROS and MDA, compared to that induced by ADF. High Mg concentration (1.25 and/or 2 mM) in CDF and ACDF protected against oxidative stress, indicated by reduced ROS and MDA levels compared to respective DFs with standard concentration of Mg. Increasing Mg concentrations in ADF promoted high ROS production and MDA content. Thus, an increase in Mg content in DFs has differential effects on the oxidative stress in IS-treated THP-1 cells depending on the dialysate used.
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http://dx.doi.org/10.3390/antiox9040319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7222382PMC
April 2020

Mechanisms of Cardiovascular Disorders in Patients With Chronic Kidney Disease: A Process Related to Accelerated Senescence.

Front Cell Dev Biol 2020 20;8:185. Epub 2020 Mar 20.

Departamento Biología de Sistemas, Facultad de Medicina y Ciencias de la Salud (IRYCIS), Universidad de Alcalá, Alcalá de Henares, Madrid, Spain.

Cardiovascular diseases (CVDs), especially those involving a systemic inflammatory process such as atherosclerosis, remain the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD). CKD is a systemic condition affecting approximately 10% of the general population. The prevalence of CKD has increased over the past decades because of the aging of the population worldwide. Indeed, CVDs in patients with CKD constitute a premature form of CVD observed in the general population. Multiple studies indicate that patients with renal disease undergo accelerated aging, which precipitates the appearance of pathologies, including CVDs, usually associated with advanced age. In this review, we discuss several aspects that characterize CKD-associated CVDs, such as etiopathogenic elements that CKD patients share with the general population, changes in the cellular balance of reactive oxygen species (ROS), and the associated process of cellular senescence. Uremia-associated aging is linked with numerous changes at the cellular and molecular level. These changes are similar to those observed in the normal process of physiologic aging. We also discuss new perspectives in the study of CKD-associated CVDs and epigenetic alterations in intercellular signaling, mediated by microRNAs and/or extracellular vesicles (EVs), which promote vascular damage and subsequent development of CVD. Understanding the processes and factors involved in accelerated senescence and other abnormal intercellular signaling will identify new therapeutic targets and lead to improved methods of diagnosis and monitoring for patients with CKD-associated CVDs.
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http://dx.doi.org/10.3389/fcell.2020.00185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099607PMC
March 2020

Hypoxia-Inducible Factor-1α: The Master Regulator of Endothelial Cell Senescence in Vascular Aging.

Cells 2020 01 13;9(1). Epub 2020 Jan 13.

Departamento Biología de Sistemas, Facultad de Medicina y Ciencias de la Salud (IRYCIS), Universidad de Alcalá, Alcalá de Henares, 28805 Madrid, Spain.

Aging is one of the hottest topics in biomedical research. Advances in research and medicine have helped to preserve human health, leading to an extension of life expectancy. However, the extension of life is an irreversible process that is accompanied by the development of aging-related conditions such as weakness, slower metabolism, and stiffness of vessels. It also debated that aging can be considered an actual disease with aging-derived comorbidities, including cancer or cardiovascular disease. Currently, cardiovascular disorders, including atherosclerosis, are considered as premature aging and represent the first causes of death in developed countries, accounting for 31% of annual deaths globally. Emerging evidence has identified hypoxia-inducible factor-1α as a critical transcription factor with an essential role in aging-related pathology, in particular, regulating cellular senescence associated with cardiovascular aging. In this review, we will focus on the regulation of senescence mediated by hypoxia-inducible factor-1α in age-related pathologies, with particular emphasis on the crosstalk between endothelial and vascular cells in age-associated atherosclerotic lesions. More specifically, we will focus on the characteristics and mechanisms by which cells within the vascular wall, including endothelial and vascular cells, achieve a senescent phenotype.
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http://dx.doi.org/10.3390/cells9010195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016968PMC
January 2020

MicroRNA-126 regulates Hypoxia-Inducible Factor-1α which inhibited migration, proliferation, and angiogenesis in replicative endothelial senescence.

Sci Rep 2019 05 14;9(1):7381. Epub 2019 May 14.

Departamento Biología de Sistemas, Facultad de Medicina y Ciencias de la Salud, Universidad de Alcalá (IRYCIS), Alcalá de Henares, Madrid, Spain.

Whereas a healthy endothelium maintains physiological vascular functions, endothelial damage contributes to the development of cardiovascular diseases. Endothelial senescence is the main determinant of endothelial dysfunction and thus of age-related cardiovascular disease. The objective of this study is to test the involvement of microRNA-126 and HIF-1α in a model of replicative endothelial senescence and the interrelationship between both molecules in this in vitro model. We demonstrated that senescent endothelial cells experience impaired tube formation and delayed wound healing. Senescent endothelial cells failed to express HIF-1α, and the microvesicles released by these cells failed to carry HIF-1α. Of note, HIF-1α protein levels were restored in HIF-1α stabilizer-treated senescent endothelial cells. Finally, we show that microRNA-126 was downregulated in senescent endothelial cells and microvesicles. With regard to the interplay between microRNA-126 and HIF-1α, transfection with a microRNA-126 inhibitor downregulated HIF-1α expression in early passage endothelial cells. Moreover, while HIF-1α inhibition reduced tube formation and wound healing closure, microRNA-126 levels remained unchanged. These data indicate that HIF-1α is a target of miRNA-126 in protective and reparative functions, and suggest that their therapeutic modulation could benefit age-related vascular disease.
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http://dx.doi.org/10.1038/s41598-019-43689-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6517399PMC
May 2019

Endothelial Extracellular Vesicles Produced by Senescent Cells: Pathophysiological Role in the Cardiovascular Disease Associated with all Types of Diabetes Mellitus.

Curr Vasc Pharmacol 2019 ;17(5):447-454

Biology Systems Department, Physiology, Alcala University, Alcala de Henares, Madrid, Spain.

Endothelial senescence-associated with aging or induced prematurely in pathological situations, such as diabetes, is a first step in the development of Cardiovascular Disease (CVDs) and particularly inflammatory cardiovascular diseases. The main mechanism that links endothelial senescence and the progression of CVDs is the production of altered Extracellular Vesicles (EVs) by senescent endothelial cells among them, Microvesicles (MVs). MVs are recognized as intercellular signaling elements that play a key role in regulating tissue homeostasis. However, MVs produced by damage cell conveyed epigenetic signals, mainly involving microRNAs, which induce many of the injured responses in other vascular cells leading to the development of CVDs. Many studies strongly support that the quantification and characterization of the MVs released by senescent endothelial cells may be useful diagnostic tools in patients with CVDs, as well as a future therapeutic target for these diseases. In this review, we summarize the current knowledge linking senescence-associated MVs to the development of CVDs and discuss the roles of these MVs, in particular, in diabetic-associated increases the risk of CVDs.
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http://dx.doi.org/10.2174/1570161116666180820115726DOI Listing
May 2020

Senescent Microvesicles: A Novel Advance in Molecular Mechanisms of Atherosclerotic Calcification.

Int J Mol Sci 2018 Jul 9;19(7). Epub 2018 Jul 9.

Biology Systems Department, Physiology, Alcala University, Alcala de Henares, 28805 Madrid, Spain.

Atherosclerosis, a chronic inflammatory disease that causes the most heart attacks and strokes in humans, is the leading cause of death in the developing world; its principal clinical manifestation is coronary artery disease. The development of atherosclerosis is attributed to the aging process itself (biological aging) and is also associated with the development of chronic diseases (premature aging). Both aging processes produce an increase in risk factors such as oxidative stress, endothelial dysfunction and proinflammatory cytokines (oxi-inflamm-aging) that might generate endothelial senescence associated with damage in the vascular system. Cellular senescence increases microvesicle release as carriers of molecular information, which contributes to the development and calcification of atherosclerotic plaque, as a final step in advanced atherosclerotic plaque formation. Consequently, this review aims to summarize the information gleaned to date from studies investigating how the senescent extracellular vesicles, by delivering biological signalling, contribute to atherosclerotic calcification.
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http://dx.doi.org/10.3390/ijms19072003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6073566PMC
July 2018

Young and Especially Senescent Endothelial Microvesicles Produce NADPH: The Fuel for Their Antioxidant Machinery.

Oxid Med Cell Longev 2018 5;2018:3183794. Epub 2018 Apr 5.

Departamento de Biología de Sistemas, Universidad de Alcalá, Alcalá de Henares, 28805 Madrid, Spain.

In a previous study, we demonstrated that endothelial microvesicles (eMVs) have a well-developed enzymatic team involved in reactive oxygen species detoxification. In the present paper, we demonstrate that eMVs can synthesize the reducing power (NAD(P)H) that nourishes this enzymatic team, especially those eMVs derived from senescent human umbilical vein endothelial cells. Moreover, we have demonstrated that the molecules that nourish the enzymatic machinery involved in NAD(P)H synthesis are blood plasma metabolites: lactate, pyruvate, glucose, glycerol, and branched-chain amino acids. Drastic biochemical changes are observed in senescent eMVs to optimize the synthesis of reducing power. Mitochondrial activity is diminished and the glycolytic pathway is modified to increase the activity of the pentose phosphate pathway. Different dehydrogenases involved in NADPH synthesis are also increased. Functional experiments have demonstrated that eMVs can synthesize NADPH. In addition, the existence of NADPH in eMVs was confirmed by mass spectrometry. Multiphoton confocal microscopy images corroborate the synthesis of reducing power in eMVs. In conclusion, our present and previous results demonstrate that eMVs can act as autonomous reactive oxygen species scavengers: they use blood metabolites to synthesize the NADPH that fuels their antioxidant machinery. Moreover, senescent eMVs have a stronger reactive oxygen species scavenging capacity than young eMVs.
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http://dx.doi.org/10.1155/2018/3183794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5907394PMC
October 2018

Reduced TH expression and α-synuclein accumulation contribute towards nigrostriatal dysfunction in experimental hepatic encephalopathy.

Restor Neurol Neurosci 2017 ;35(5):469-481

Departamento de Biología Celular, Universidad Complutense, Madrid, Spain.

Purpose: The present work examines α-synuclein expression in the nigrostriatal system of a rat chronic hepatic encephalopathy model induced by portacaval anastomosis (PCA). There is evidence that dopaminergic dysfunction in disease conditions is strongly associated with such expression. Possible relationships among dopaminergic neurons, astroglial cells and α-synuclein expression were sought.

Methods: Brain tissue samples from rats at 1 and 6 months post-PCA, and controls, were analysed immunohistochemically using antibodies against tyrosine hydroxylase (TH), α-synuclein, glial fibrillary acidic protein (GFAP) and ubiquitin (Ub).

Results: In the control rats, TH immunoreactivity was detected in the neuronal cell bodies and processes in the substantia nigra pars compacta (SNc). A dense TH-positive network of neurons was also seen in the striatum. In the PCA-exposed rats, however, a reduction in TH-positive neurons was seen at both 1 and 6 months in the SNc, as well as a reduction in TH-positive fibres in the striatum. This was coincident with the appearance of α-synuclein-immunoreactive neurons in the SNc; some of the TH-positive neurons also showed α-synuclein immunoreactivity. In addition, α-synuclein accumulation was seen in the SNc and striatum at both 1 and 6 months post-PCA, whereas α-synuclein was only mildly expressed in the nigrostriatal pathway of the controls. Astrogliosis was also seen following PCA, as revealed by increased GFAP expression from 1 month to 6 months post-PCA in both the SN and striatum. The astroglial activation level in the SN paralleled the reduced neuronal expression of TH throughout PCA exposure.

Conclusion: α-synuclein accumulation following PCA may induce dopaminergic dysfunction via the downregulation of TH, as well as astroglial activation.
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http://dx.doi.org/10.3233/RNN-170728DOI Listing
June 2018

The Antioxidant Machinery of Young and Senescent Human Umbilical Vein Endothelial Cells and Their Microvesicles.

Oxid Med Cell Longev 2017 31;2017:7094781. Epub 2017 May 31.

Departamento de Biología de Sistemas, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.

We examine the antioxidant role of young and senescent human umbilical vein endothelial cells (HUVECs) and their microvesicles (MVs). Proteomic and Western blot studies have shown young HUVECs to have a complete and well-developed antioxidant system. Their MVs also contain antioxidant molecules, though of a smaller and more specific range, specialized in the degradation of hydrogen peroxide and the superoxide anion via the thioredoxin-peroxiredoxin system. Senescence was shown to be associated with a large increase in the size of the antioxidant machinery in both HUVECs and their MVs. These responses might help HUVECs and their MVs deal with the more oxidising conditions found in older cells. Functional analysis confirmed the antioxidant machinery of the MVs to be active and to increase in size with senescence. No glutathione or nonpeptide antioxidant (ascorbic acid and vitamin E) activity was detected in the MVs. Endothelial cells and MVs seem to adapt to higher ROS concentrations in senescence by increasing their antioxidant machinery, although this is not enough to recover completely from the senescence-induced ROS increase. Moreover, MVs could be involved in the regulation of the blood plasma redox status by functioning as ROS scavengers.
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http://dx.doi.org/10.1155/2017/7094781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470024PMC
March 2018

Microvesicles from the plasma of elderly subjects and from senescent endothelial cells promote vascular calcification.

Aging (Albany NY) 2017 03;9(3):778-789

Departamento de Biología de Sistemas, Facultad de Medicina y Ciencias de la Salud, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain.

Vascular calcification is commonly seen in elderly people, though it can also appear in middle-aged subjects affected by premature vascular aging. The aim of this work is to test the involvement of microvesicles (MVs) produced by senescent endothelial cells (EC) and from plasma of elderly people in vascular calcification. The present work shows that MVs produced by senescent cultured ECs, plus those found in the plasma of elderly subjects, promote calcification in vascular smooth muscle cells. Only MVs from senescent ECs, and from elderly subjects' plasma, induced calcification. This ability correlated with these types of MVs' carriage of: a) increased quantities of annexins (which might act as nucleation sites for calcification), b) increased quantities of bone-morphogenic protein, and c) larger Ca contents. The MVs of senescent, cultured ECs, and those present in the plasma of elderly subjects, promote vascular calcification. The present results provide mechanistic insights into the observed increase in vascular calcification-related diseases in the elderly, and in younger patients with premature vascular aging, paving the way towards novel therapeutic strategies.
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http://dx.doi.org/10.18632/aging.101191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391231PMC
March 2017

Ammonia Affects Astroglial Proliferation in Culture.

PLoS One 2015 30;10(9):e0139619. Epub 2015 Sep 30.

Departamento de Biomedicina y Biotecnología, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.

Primary cultures of rat astroglial cells were exposed to 1, 3 and 5 mM NH4Cl for up to 10 days. Dose- and time-dependent reductions in cell numbers were seen, plus an increase in the proportion of cells in the S phase. The DNA content was reduced in the treated cells, and BrdU incorporation diminished. However, neither ammonia nor ammonia plus glutamine had any effect on DNA polymerase activity. iTRAQ analysis showed that exposure to ammonia induced a significant reduction in histone and heterochromatin protein 1 expression. A reduction in cell viability was also noted. The ammonia-induced reduction of proliferative activity in these cultured astroglial cells seems to be due to a delay in the completion of the S phase provoked by the inhibition of chromatin protein synthesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139619PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589356PMC
April 2016

Ammonia induces aquaporin-4 rearrangement in the plasma membrane of cultured astrocytes.

Neurochem Int 2012 Dec 25;61(8):1314-24. Epub 2012 Sep 25.

Departamento de Biología Celular y Genética, Facultad de Biología, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.

Aquaporin-4 (AQP4) is a water channel protein mainly located in the astroglial plasma membrane, the precise function of which in the brain edema that accompanies hepatic encephalopathy (HE) is unclear. Since ammonia is the main pathogenic agent in HE, its effect on AQP4 expression and distribution in confluent primary astroglial cultures was examined via their exposure to ammonium chloride (1, 3 and 5 mM) for 5 and 10 days. Ammonia induced the general inhibition of AQP4 mRNA synthesis except in the 1 mM/5 day treatment. However, the AQP4 protein content measured was dependent on the method of analysis; an apparent increase was recorded in treated cells in in-cell Western assays, while an apparent reduction was seen with the classic Western blot method, perhaps due to differences in AQP4 aggregation. Ammonia might therefore induce the formation of insoluble AQP4 aggregates in the astroglial plasma membrane. The finding of AQP4 in the pellet of classic Western blot samples, plus data obtained via confocal microscopy, atomic force microscopy (using immunolabeled cells with gold nanoparticles) and scanning electron microscopy, all corroborate this hypothesis. The effect of ammonia on AQP4 seems not to be due to any osmotic effect; identical osmotic stress induced by glutamine and salt had no significant effect on the AQP4 content. AQP4 functional analysis (subjecting astrocytes to a hypo-osmotic medium and using flow cytometry to measure cell size) demonstrated a smaller water influx in ammonia-treated astrocytes suggesting that AQP4 aggregates are representative of an inactive status; however, more confirmatory studies are required to fully understand the functional status of AQP4 aggregates. The present results suggest that ammonia affects AQP4 expression and distribution, and that astrocytes change their expression of AQP4 mRNA as well as the aggregation status of the ensuing protein depending on the ammonia concentration and duration of exposure.
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http://dx.doi.org/10.1016/j.neuint.2012.09.008DOI Listing
December 2012

Motor-evoked potentials in awake rats are a valid method of assessing hepatic encephalopathy and of studying its pathogenesis.

Hepatology 2010 Dec 1;52(6):2077-85. Epub 2010 Oct 1.

Servei de Medicina Interna-Hepatologia, Hospital Vall d'Hebron, Barcelona, Spain.

Unlabelled: Experimental models of hepatic encephalopathy (HE) are limited by difficulties in objectively monitoring neuronal function. There are few models that examine a well-defined neuronal pathway and lack the confounding effects of anesthetics. Motor-evoked potentials (MEPs) assess the function of the motor tract, which has been shown to be impaired in patients with cirrhosis. MEPs were elicited by cranial stimulation (central) and compound motor action potential by sciatic nerve stimulation (peripheral) in several models of HE in the rat. The experiments were performed using subcutaneous electrodes without anesthetics. Brain water content was assessed by gravimetry, brain metabolites were measured by magnetic resonance spectroscopy, and amino acids in microdialysates from the frontal cortex were analyzed by high-performance liquid chromatography. Abnormalities of MEP were observed in acute liver failure (ALF) induced by hepatic devascularization in relation to the progression of neurological manifestations. Similar disturbances were seen in rats with portocaval anastomosis after the administration of blood or lipopolysaccharide, but were absent in rats with biliary duct ligation. Hypothermia (≤35°C) and mannitol prevented the development of brain edema in acute liver failure, but only hypothermia avoided the decrease in the amplitude of MEP. Disturbances of MEP caused by the administration of blood into the gastrointestinal tract in rats with portocaval anastomosis were associated with an increase in ammonia, glutamine, and glutamate in brain microdialysate.

Conclusion: Assessment of MEP in awake rats is a valid method to monitor HE in models of ALF and precipitated HE. This method shows the lack of efficacy of mannitol, a therapy that decreases brain edema, and relates disturbances of the function of the motor tract to ammonia and its metabolites.
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http://dx.doi.org/10.1002/hep.23938DOI Listing
December 2010

Astrocytes express Mxi2, a splice isoform of p38MAPK.

J Mol Histol 2009 Oct 31;40(5-6):325-9. Epub 2009 Dec 31.

Departamento de Biología Celular y Genética, Facultad de Biología, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain.

Mitogen-activated protein kinases (MAPKs) are a superfamily of cytoplasmic serine/threonine kinases that transduce many types of extracellular stimuli into cellular responses. p38MAPK is a member of this family with its active form in a diphosphorylated state (p38MAPKdiP). Two strong anti-p38MAPKdiP immunoreactive bands (apparent molecular weight 38 and 34 kDa) were detected by Western blotting in cultured astrocytes. Using a specific antibody and employing immunoprecipitation procedures and SELDI-TOF analysis, the 34 kDa band was found to correspond to Mxi2, a splice variant of p38MAPK; cultured astrocytes therefore express Mxi2. Separate protein extractions of different subcellular fractions, and fluorescent immunovisualisation employing confocal microscopy, showed Mxi2 to have a non-nuclear, cytosolic distribution in the studied cells. ERK1/2, protein whose intracellular distribution is influenced by Mxi2, showed the same cytoplasmic pattern than Mxi2.
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http://dx.doi.org/10.1007/s10735-009-9248-8DOI Listing
October 2009

Induction of NOS and nitrotyrosine expression in the rat striatum following experimental hepatic encephalopathy.

Metab Brain Dis 2009 Sep;24(3):395-408

Departamento de Biología Celular y Genética, Facultad de Biología, Universidad de Alcalá, 28871 Madrid, Spain.

Hepatic encephalopathy (HE) is a neurologic disease associated with hepatic dysfunction. Astroglial and neuronal alterations have been described in the basal ganglia in HE. Our study was performed to determine whether such alterations are mediated by nitric oxide (NO), by using an experimental model of HE (portacaval anastomosis [PCA]). The expression of the NO synthases (nNOS and iNOS) and the production of nitrotyrosine (NT) were evaluated in the striatum of rats exposed to PCA for 1 and 6 months. The expression of nNOS in the striatal neurons of PCA rats was increased compared to controls. nNOS expression was also detectable in astrocytes after 6 months of exposure to PCA. Whereas astroglial cells in the normal striatum showed no iNOS expression, iNOS was expressed in the astrocytes of PCA brains, mainly in perivascular processes at 6 months PCA exposure (demonstrated by colocalization with GFAP). The increased expression of both the nNOS and iNOS isoforms in PCA rats might indicate a critical role for NO in the pathomechanism of HE. To study the potential cell damage caused by NO, the deposition of NT in PCA-rats was analysed. Nitrotyrosine was detected in neurons although it was mainly seen in the astrocytes of PCA brains, in which double immunolabelling showed NT to be colocalized with GFAP. Thus, the present study shows the induction of iNOS and NT in astrocytes, which increases with the duration of PCA exposure. This suggests that the induced astroglial production of NO during PCA might be one of the main factors contributing to HE.
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http://dx.doi.org/10.1007/s11011-009-9154-5DOI Listing
September 2009

Effect of ammonia, glutamine, and serum on calcineurin, p38MAPK-diP, GADD153/CHOP10, and CNTF in primary rat astrocyte cultures.

Brain Res 2007 Oct 8;1175:126-33. Epub 2007 Aug 8.

Departamento de Biología Celular y Genética, Facultad de Biología, Universidad de Alcalá, E-28871 Alcalá de Henares, Madrid, Spain.

Primary astrocyte cultures were subjected to different experimental schedules using several concentrations of ammonia (1, 3, and 5 mM ammonium chloride), serum (2.5%, 5%, and 12%), and glutamine (0.5, 1, and 3 mM) to analyze the involvement of calcineurin (CaN) in hyperammonemia and its relation with p38MAPK-diP and ciliary neurotrophic factor (CNTF). We demonstrated that exposure to ammonia affects CaN content, and confirmed the ammonia-induced reduction of CNTF expression; however, the involvement of CaN and p38MAPK-diP in CNTF reduction could not be confirmed. On the contrary, an inverse relationship between CaN and p38MAPK-diP contents was clearly demonstrated. GADD153/CHOP10 content was always higher under hyperammonemic conditions as well as under glutamine exposure, probably due to the osmotic stress provoked by glutamine accumulation, which was induced after exposure to ammonia. Statistical analysis demonstrated significant interactions of ammonia and serum for CaN, GADD153/CHOP10 and CNTF contents. The exposure to glutamine also induced changes in GADD153/CHOP10 and CaN; however, CNTF content was not affected. In conclusion, CaN content was affected by exposure to ammonia and glutamine; the serum content of the culture medium had a strong influence on the astroglial response to ammonium chloride, and glutamine exposure only reproduced some of the ammonia effects.
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http://dx.doi.org/10.1016/j.brainres.2007.07.058DOI Listing
October 2007

Possible implication of ciliary neurotrophic factor (CNTF) and beta-synuclein in the ammonia effect on cultured rat astroglial cells: a study using DNA and protein microarrays.

Neurochem Int 2006 Jun 17;48(8):729-38. Epub 2006 Feb 17.

Departamento de Biología Celular y Genética, Facultad de Biología, Universidad de Alcalá, E-28871 Alcalá de Henares, Madrid, Spain.

Astrocytes are considered the key cell in hepatic encephalopathy; although their precise role in the disease has not yet been determined, exposure to ammonia appears to have an important pathogenic effect. We exposed confluent cultures of rat astroglial cells to ammonia (5mM NH(4)Cl) for 1, 3, 5 and 7 days, and determined astroglial levels of actin, glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), GLAST glutamate transporter, 25kDa heat-shock protein (HSP25), HSP60 and HSP70 by Western blot; the glutamine content in culture medium was measured by mass spectrometry. Significant increases were observed for GS, HSP60 and glutamine, and significant reductions for actin and GFAP. Astrocytes exposed to ammonia for 4 days were used to analyze the effect of ammonia in protein and DNA microarrays. After protein microarray data filtration by signal intensity, x-fold change and z-score, 11 proteins were selected, among which the significant increase in beta-synuclein was confirmed by Western blot. DNA microarray data filtration by intensity signal, x-fold change and p-value selected almost 600 genes. The significant increase in alpha-synuclein mRNA was confirmed by quantitative RT-PCR, but no change was observed in alpha-synuclein protein levels. A notable decrease in ciliary neurotrophic factor (CNTF) was demonstrated by Western blot after ammonia treatment, concurring with the reduction in CNTF mRNA observed in DNA microarrays. We discuss the possibility of a pathogenic role for CNTF and a protective role for beta-synuclein in experimental hyperammonemia. This study demonstrates the use of microarrays as tools to ascertain the possible implication of previously unidentified proteins in the pathogenesis of hepatic encephalopathy.
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http://dx.doi.org/10.1016/j.neuint.2005.12.014DOI Listing
June 2006

Pituitary adenylate cyclase-activating peptide/vasoactive intestinal peptide receptors in human normal mammary gland and breast cancer tissue.

Gynecol Endocrinol 2005 Jun;20(6):327-33

Department of Biochemistry and Molecular Biology, University of Alcalá, E-28871 Alcalá de Henares, Spain.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) bind similarly to VPAC1 and VPAC2 receptors, whereas PACAP binds with higher affinity than VIP to PAC1 receptors. Here we demonstrate by different approaches the expression of the three subclasses of PACAP/VIP receptors in human normal and malignant breast tissue. At the mRNA level, reverse transcription-polymerase chain reaction experiments showed VPAC1 and VPAC2 receptors as well as various isoforms (null, hip/hop) of PAC1 receptors due to alternative splicing. At the protein level, Western blot experiments revealed the three subclasses of receptor although no conclusive differences could be established when comparing control, peritumoral and tumoral tissue samples. Immunohistochemistry showed the distribution of these receptors: they were located at epithelial cells in normal and cancer conditions but also in leukocytes at the stromal level in carcinomatous tissue. A weaker immunostaining of PAC1 receptors in normal tissue and a strong density of the three PACAP/VIP receptor subclasses in cancer tissue may be related to differential expression patterns during breast tumor progression but more samples need to be studied to validate this hypothesis. PAC1, VPAC1 and VPAC2 receptors were functional, as shown by their coupling to adenylate cyclase stimulation: VIP, PACAP-27 and PACAP-38 behaved similarly at this level, whereas both VPAC receptors acted alike as shown by means of specific peptide agonists and antagonists. The present results together with the known presence of PACAP and VIP in the mammary gland support a paracrine/autocrine involvement of both peptides at this level in physiological and pathological conditions, i.e. during malignant transformation.
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http://dx.doi.org/10.1080/09513590500098240DOI Listing
June 2005

Down-regulation of the AMPA glutamate receptor subunits GluR1 and GluR2/3 in the rat cerebellum following pre- and perinatal delta9-tetrahydrocannabinol exposure.

Cerebellum 2004 ;3(2):66-74

Departamento Biología Celular y Genética, Universidad de Alcalá, Madrid, Spain.

This paper reports the effects of pre- and perinatal exposure to delta9-tetrahydrocannabinol (THC) on expression levels of specific AMPA glutamate receptor subunits (GluR1 and GluR2/3) in the cerebellum of male and female rats. Pregnant rats were administered saline or THC from gestational day 5 (ED5) to postnatal day 20 (PD20). Expression of the GluR1 and GluR2/3 subunits of AMPA glutamate receptors was analyzed by immunohistochemistry in THC-exposed rats at three postnatal ages: PD20 (still exposed to THC) to study the direct effect of drug exposure, and PD30 and PD70 (10 and 50 days following THC withdrawal) to analyze the long-term effects of prenatal exposure. Compared to controls, pre- and perinatal THC exposure decreased the immunoreactivity levels of the GluR1 subunit in Bergmann glial cells, as well as levels of the GluR2/3 subunit in Purkinje neurons at PD20. These changes in AMPA receptor subunit levels may correlate with the decreased excitatory neurotransmission described in the cerebellum after cannabinoid treatment, which could play a significant role in the biochemical effects of THC. In addition, the reduced glutamate receptor expression observed at PD20 did not return to normal even after THC withdrawal (PD30 and PD70). The results support the idea that THC exposure during critical stages of cerebellar development may alter the glutamatergic system, not only during the drug exposure period itself but also in adults following THC withdrawal. The decreased expressions of glutamate receptors induced by developmental THC exposure could lead to functional alterations through the inhibition of glutamatergic neurotransmission, and clearly demonstrate an interaction between cannabinoids and the glutamatergic system.
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http://dx.doi.org/10.1080/14734220310017230DOI Listing
August 2004

PACAP expression and distribution in human breast cancer and healthy tissue.

Cancer Lett 2004 Mar;205(2):189-95

Department of Biochemistry and Molecular Biology, University of Alcalá, 28871 Alcalá de Henares, Spain.

Pituitary adenylate cyclase-activating peptide (PACAP) is involved in various biological processes including cell growth, proliferation and differentiation. Here, we demonstrate for the first time the presence of both PACAP mRNA and PACAP immunoreactivity in human normal breast and breast carcinoma. Control, peritumoral and tumoral tissue pieces expressed preproPACAP mRNA since reverse transcription-polymerase chain reaction analysis gave an amplification product of the expected size (226 bp), which was further identified by analysis of the sequence. A main 19.9-kDa product (preproPACAP protein) was identified by Western blot in the three classes of breast tissue together with a small amount of a 14.6-kDa product (conceivably a result of partial processing by proprotein convertases). However, the mature peptide PACAP-38 was absent. The levels of both PACAP mRNA and PACAP immunoreactivity increased from normal to peritumoral and tumoral breast tissues but more patients must be considered to reinforce this feature. Immunohistochemistry showed the localization of PACAP immunoreactivity in alveolar epithelial cells in normal and carcinomatous tissues but also, at high density, in duct-like structures and in leucocytes in the connective tissue from breast cancer pieces. The results suggest that PACAP may play a role by autocrine/paracrine mechanisms in both normal human breast physiology and breast tumorigenesis.
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http://dx.doi.org/10.1016/j.canlet.2003.10.008DOI Listing
March 2004

Expression of functional PACAP/VIP receptors in human prostate cancer and healthy tissue.

Peptides 2003 Jun;24(6):893-902

Department of Biochemistry and Molecular Biology, University of Alcalá, E-28871 Alcalá de Henares, Spain.

Vasoactive intestinal peptide (VIP) is involved in prostate cell proliferation and function. VIP and pituitary adenylate cyclase-activating peptide (PACAP) are similarly recognized by VPAC(1)/VPAC(2) receptors whereas PACAP binds with higher affinity than VIP to PAC(1) receptor. Here we systematically studied the presence and distribution of functional PAC(1), VPAC(1) and VPAC(2) receptors in human normal and malignant prostate tissue. Functional PACAP/VIP receptors were detected in normal and malignant prostate by adenylyl cyclase stimulation with PACAP-27/38 and VIP. RT-PCR experiments showed PAC(1) (various isoforms due to alternative splicing), VPAC(1) and VPAC(2) receptor expression at the mRNA level, whereas Western blots found the three receptor protein classes in normal and pathological conditions. No conclusive differences could be established when comparing control and cancer tissue samples. Immunohistochemistry showed a weaker immunostaining in tumoral than in normal epithelial cells for the three receptor subtypes. In conclusion, we demonstrate the expression of functional PAC(1), VPAC(1) and VPAC(2) receptors in human prostate as well as its maintenance after malignant transformation.
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http://dx.doi.org/10.1016/s0196-9781(03)00162-1DOI Listing
June 2003

VIP and PACAP receptors coupled to adenylyl cyclase in human lung cancer: a study in biopsy specimens.

Peptides 2003 Mar;24(3):429-36

Department of Biochemistry and Molecular Biology, University of Alcalá, E-28871 Alcalá de Henares, Spain.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are important neuropeptides in the control of lung physiology. Both of these commonly bind to specific G protein coupled receptors named VPAC(1)-R and VPAC(2)-R, and PAC(1)-R (with higher affinity for PACAP). VIP and PACAP have been implicated in the control of cell proliferation and tumor growth. This study examined the presence of VIP and PACAP receptors in human lung cancer samples, as well as the functionality of adenylyl cyclase (AC) stimulated by both peptides. Results from RT-PCR and immunoblot experiments showed the expression of VPAC(1)-, VPAC(2)- and PAC(1)-R in lung cancer samples. Immunohistochemical studies showed the expression of VPAC(1) and VPAC(2) receptors. These receptors were positively coupled to AC, but the enzyme activity was impaired as compared to normal lung. There were no changes in Galpha(s) or Galpha(i) levels. Present results contribute to a better knowledge of VIP/PACAP actions in lung cancer and support the interest for the development of VIP/PACAP analogues with therapeutic roles.
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http://dx.doi.org/10.1016/s0196-9781(03)00058-5DOI Listing
March 2003

Expression and distribution of pituitary adenylate cyclase-activating peptide in human prostate and prostate cancer tissues.

Regul Pept 2002 Dec;110(1):9-15

Molecular Neuroendocrinology Unit, Department of Biochemistry and Molecular Biology, University of Alcalá, 28871 Alcalá de Henares, Spain.

The presence, expression and distribution of pituitary adenylate cyclase-activating peptide (PACAP) in human prostate cancer and healthy tissue were investigated by means of biochemical and morphological procedures. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated the presence of its precursor encoding mRNA in both normal and pathological conditions (amplification products with 577 or 226 bp were identified). Immunochemistry using an appropriate antibody served to detect in both classes of tissues a 19.9-kDa product corresponding to the PACAP preproprotein and another protein of 14.6 kDa that may represent a product partially processed by convertases. However, a 5-kDa band characteristic of PACAP-38 peptide was not observed. Immunohistochemistry on tissue sections indicated the location of PACAP in the epithelial layer of prostate glands (and in some scarce leucocytes) but not in the stroma, either in normal or carcinomatous tissues. No clear differences could be established when comparing samples from patients with different tumor Gleason grades. These results are the first demonstration of the localization of PACAP or its precursors and its mRNA in the human prostate gland and their presence during the progression of prostate carcinoma.
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http://dx.doi.org/10.1016/s0167-0115(02)00108-8DOI Listing
December 2002

HSP70 constitutive expression in rat central nervous system from postnatal development to maturity.

J Histochem Cytochem 2002 Sep;50(9):1161-8

Departamento de Biología Celular y Genética, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain.

We studied the level of the basal (constitutive) HSP70 expression (inducible and constitutive forms) in the central nervous system (CNS) of male and female rats from the postnatal period to maturity. HSP70 levels were analyzed by immunoblotting in five different areas (cortex, hippocampus, hypothalamus, cerebellum, and spinal cord). The highest levels of HSP70 were found in juvenile rats and decreased progressively until reaching baseline levels between 2 and 4 months. A slight and nonsignificant increase in aged (2-year-old) rats compared with adult subjects was observed in some cerebral areas (cerebral cortex, hippocampus, and cerebellum). In the first weeks of postnatal development, HSP70 immunoreactivity was distributed throughout CNS sections and no specific immunopositive cells could be clearly determined. In adult animals, strong immunostaining was observed in some large neurons (Purkinje neurons and mesencephalic and spinal cord motor neurons), some perivascular and subpial astrocytes, and ependymocytes. Immunoelectron microscopy revealed that HSP70 in these cells is located in the perinuclear area and in mitochondria, rough endoplasmic reticulum, and microtubules. In neurons, strong immunolabeling was also observed in synaptic membranes. The postnatal time course of HSP70 levels and the location and size of HSP70-immunopositive cells suggest that HSP70 constitutively expressed in the rat CNS may be mainly determined by the degree of development and metabolic activity of the neural cells.
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http://dx.doi.org/10.1177/002215540205000902DOI Listing
September 2002