Publications by authors named "Matilde Alique"

38 Publications

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

Statins and antiplatelet agents are associated with changes in the circulatory markers of endothelial dysfunction in chronic kidney disease.

Nefrologia (Engl Ed) 2019 May - Jun;39(3):287-293. Epub 2019 Feb 4.

Servicio de Nefrología, Hospital Universitario Puerta de Hierro Majadahonda, Majadahonda, Madrid, España; REDInREN ISCiii 016/0091009 RETYC, Madrid, España.

Backgrounds And Purposes: Patients with chronic kidney disease (CKD) have higher risk of developing cardiovascular disease. In CKD patients the mechanisms involved in, endothelial damage and the role of different drugs used on these patients are not completely understood. The aim of this work is to analyze the effect of statins and platelet antiaggregant (PA) on endothelial microvesicles (EMVs) and other markers of endothelial dysfunction.

Experimental Approach: Cross-sectional study of 41 patients with CKD 3b-4 and 8 healthy volunteers. Circulating levels of EMVs, vascular endothelial growth factor (VEGF), and advance oxidized protein products (AOPPS) were quantified and the correlation with different comorbidity variables and therapeutic strategies were evaluated.

Results: EMVs are increased in CKD patients as compared with controls (171.1 vs. 68.3/μl, P<.001). It was observed a negative correlation between age and EMVs. Statins and PA were associated with a reduction in EMVs and VEGF levels, independently of the serum total cholesterol levels (TC). The levels of AOPPS and VEGF were not different in CKD vs. controls.

Conclusion: CKD is associated with a change in EMVs, VEGF and AOPP levels. The treatment with statins and PA normalizes these values to almost the observed in controls and this effect is independently of the prevailing TC level. These findings explain the existence of the pleiotropic effects of statins and PA which deserve further studies.
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http://dx.doi.org/10.1016/j.nefro.2018.11.001DOI Listing
May 2020

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

Protein Carbamylation: A Marker Reflecting Increased Age-Related Cell Oxidation.

Int J Mol Sci 2018 May 17;19(5). Epub 2018 May 17.

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

Carbamylation is a post-translational modification of proteins that may partake in the oxidative stress-associated cell damage, and its increment has been recently proposed as a "hallmark of aging". The molecular mechanisms associated with aging are related to an increased release of free radicals. We have studied whether carbamylated proteins from the peripheral blood of healthy subjects are related to oxidative damage and aging, taking into account the gender and the immune profile of the subjects. The study was performed in healthy human volunteers. The detection of protein carbamylation and malondialdehyde (MDA) levels was evaluated using commercial kits. The immune profile was calculated using parameters of immune cell function. The results show that the individuals from the elderly group (60⁻79 years old) have increased carbamylated protein and MDA levels. When considered by gender, only men between 60 and 79 years old showed significantly increased carbamylated proteins and MDA levels. When those subjects were classified by their immune profile, the carbamylated protein levels were higher in those with an older immune profile. In conclusion, the carbamylation of proteins in peripheral blood is related to age-associated oxidative damage and to an aging functional immunological signature. Our results suggest that carbamylated proteins may play an important role at the cellular level in the aging process.
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http://dx.doi.org/10.3390/ijms19051495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983744PMC
May 2018

Residual Renal Function in Hemodialysis and Inflammation.

Ther Apher Dial 2017 Dec 3;21(6):592-598. Epub 2017 Oct 3.

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

Residual renal function (RRF) has an important effect on uremic toxin clearance, on volume control, on quality of life, and on mortality. In patients with chronic kidney disease (CKD), microinflammation with an increased percentage of CD14 /CD16 inflammatory monocytes has been reported, even with no clinical evidence of inflammation. No correlation has been established between these and RRF in hemodialysis (HD) patients. Our objective was to assess the relationship between RRF and the inflammatory parameters in HD patients. Cross-sectional observational study was carried out on 69 adult patients on chronic HD for at least 6 months, from which demographic, analytic and HD-technique data were collected and the following were measured: (i) RRF with average urea and creatinine clearance ((CCr + CU)/2) in 24-h urine (if >1 mL/min and diuresis >100 mL/day, RRF was considered); (ii) Inflammation through biochemical parameters (C-reactive protein, β microglobulin, albumin) and monocyte subpopulations in peripheral blood. The average age was 70.9 [40-88] years old; 38 (55.1%) were male; and 25 (36.2%) were diabetic. 43.5% (30/69) presented RRF, with an average of ((CCr + CU)/2): 1.8 (2.6) mL/min and diuresis: 454.5 (569) mL /24 h. Patients with RRF presented lower concentrations of C-reactive protein (6.2 vs 21.4 mg/L) (P = 0.038) and a lower percentage of non-classical CD14 /CD16 monocytes (14.6 vs. 28.3%, P = 0.02). In our study, patients with RRF present lower concentrations of inflammatory parameters, which is another reason why its preservation is an essential objective in HD.
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http://dx.doi.org/10.1111/1744-9987.12576DOI Listing
December 2017

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

iNOS-Derived Nitric Oxide Induces Integrin-Linked Kinase Endocytic Lysosome-Mediated Degradation in the Vascular Endothelium.

Arterioscler Thromb Vasc Biol 2017 07 25;37(7):1272-1281. Epub 2017 May 25.

From the Biology Systems Department, Physiology, School of Medicine and Health Sciences, Universidad Alcalá (IRYCIS), Madrid, Spain (P.R., M.A., S.M., M.S.); Cardiology Department, University Francisco de Vitoria/Hospital Ramón y Cajal Research Unit (IRYCIS), Madrid, Spain (C.Z.); and Cardiology Department, School of Medicine, Cádiz University, Spain (R.T.).

Objective: ILK (integrin-linked kinase) plays a key role in controlling vasomotor tone and is decreased in atherosclerosis. The objective of this study is to test whether nitric oxide (NO) regulates ILK in vascular remodeling.

Approach And Results: We found a striking correlation between increased levels of inducible nitric oxide and decreased ILK levels in human atherosclerosis and in a mouse model of vascular remodeling (carotid artery ligation) comparing with iNOS (inducible NO synthase) knockout mice. iNOS induction produced the same result in mouse aortic endothelial cells, and these effects were mimicked by an NO donor in a time-dependent manner. We found that NO decreased ILK protein stability by promoting the dissociation of the complex ILK/Hsp90 (heat shock protein 90)/eNOS (endothelial NO synthase), leading to eNOS uncoupling. NO also destabilized ILK signaling platform and lead to decreased levels of paxillin and α-parvin. ILK phosphorylation of its downstream target GSK3-β (glycogen synthase kinase 3 beta) was decreased by NO. Mechanistically, NO increased ILK ubiquitination mediated by the E3 ubiquitin ligase CHIP (C terminus of HSC70-interacting protein), but ILK ubiquitination was not followed by proteasome degradation. Alternatively, NO drove ILK to degradation through the endocytic-lysosomal pathway. ILK colocalized with the lysosome marker LAMP-1 (lysosomal-associated membrane protein 1) in endothelial cells, and inhibition of lysosome activity with chloroquine reversed the effect of NO. Likewise, ILK colocalized with the early endosome marker EEA1 (early endosome antigen 1). ILK endocytosis proceeded via dynamin because a specific inhibitor of dynamin (Dyngo 4a) was able to reverse ILK endocytosis and its lysosome degradation.

Conclusions: Endocytosis regulates ILK signaling in vascular remodeling where there is an overload of inducible NO, and thus its inhibition may represent a novel target to fight atherosclerotic disease.
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http://dx.doi.org/10.1161/ATVBAHA.117.309560DOI Listing
July 2017

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

Susceptibility to chronic social stress increases plaque progression, vulnerability and platelet activation.

Thromb Haemost 2017 04 12;117(4):816-818. Epub 2017 Jan 12.

Juan J. Badimon, PhD, AtheroThrombosis Research Unit, Cardiovascular Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA, Tel.: +1 212 241 8484, Fax: +1 212 426 6962, E-mail:

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http://dx.doi.org/10.1160/TH16-10-0817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490439PMC
April 2017

Angiotensin receptors and β-catenin regulate brain endothelial integrity in malaria.

J Clin Invest 2016 10 19;126(10):4016-4029. Epub 2016 Sep 19.

Cerebral malaria is characterized by cytoadhesion of Plasmodium falciparum-infected red blood cells (Pf-iRBCs) to endothelial cells in the brain, disruption of the blood-brain barrier, and cerebral microhemorrhages. No available antimalarial drugs specifically target the endothelial disruptions underlying this complication, which is responsible for the majority of malaria-associated deaths. Here, we have demonstrated that ruptured Pf-iRBCs induce activation of β-catenin, leading to disruption of inter-endothelial cell junctions in human brain microvascular endothelial cells (HBMECs). Inhibition of β-catenin-induced TCF/LEF transcription in the nucleus of HBMECs prevented the disruption of endothelial junctions, confirming that β-catenin is a key mediator of P. falciparum adverse effects on endothelial integrity. Blockade of the angiotensin II type 1 receptor (AT1) or stimulation of the type 2 receptor (AT2) abrogated Pf-iRBC-induced activation of β-catenin and prevented the disruption of HBMEC monolayers. In a mouse model of cerebral malaria, modulation of angiotensin II receptors produced similar effects, leading to protection against cerebral malaria, reduced cerebral hemorrhages, and increased survival. In contrast, AT2-deficient mice were more susceptible to cerebral malaria. The interrelation of the β-catenin and the angiotensin II signaling pathways opens immediate host-targeted therapeutic possibilities for cerebral malaria and other diseases in which brain endothelial integrity is compromised.
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http://dx.doi.org/10.1172/JCI87306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096829PMC
October 2016

Aging-associated oxidized albumin promotes cellular senescence and endothelial damage.

Clin Interv Aging 2016 29;11:225-36. Epub 2016 Feb 29.

Department of Systems Biology, Physiology Unit, Universidad de Alcalá, Madrid, Spain.

Increased levels of oxidized proteins with aging have been considered a cardiovascular risk factor. However, it is unclear whether oxidized albumin, which is the most abundant serum protein, induces endothelial damage. The results of this study indicated that with aging processes, the levels of oxidized proteins as well as endothelial microparticles release increased, a novel marker of endothelial damage. Among these, oxidized albumin seems to play a principal role. Through in vitro studies, endothelial cells cultured with oxidized albumin exhibited an increment of endothelial damage markers such as adhesion molecules and apoptosis levels. In addition, albumin oxidation increased the amount of endothelial microparticles that were released. Moreover, endothelial cells with increased oxidative stress undergo senescence. In addition, endothelial cells cultured with oxidized albumin shown a reduction in endothelial cell migration measured by wound healing. As a result, we provide the first evidence that oxidized albumin induces endothelial injury which then contributes to the increase of cardiovascular disease in the elderly subjects.
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http://dx.doi.org/10.2147/CIA.S91453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780186PMC
September 2016

TNFα-Damaged-HUVECs Microparticles Modify Endothelial Progenitor Cell Functional Activity.

Front Physiol 2015 22;6:395. Epub 2015 Dec 22.

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

Endothelial progenitor cells (EPCs) have an important role in the maintenance of vascular integrity and homeostasis. While there are many studies that explain EPCs mechanisms action, there are few studies that demonstrate how they interact with other emerging physiological elements such as Endothelial Microparticles (EMPs). EMPs are membranous structures with a size between 100 and 1000 nm that act as molecular information transporter in biological systems and are known as an important elements in develop different pathologies; moreover a lot of works explains that are novel biomarkers. To elucidate these interactions, we proposed an in vitro model of endothelial damage mediated by TNFalpha, in which damaged EMPs and EPCs are in contact to assess EPCs functional effects. We have observed that damaged EMPs can modulate several EPCs classic factors as colony forming units (CFUs), contribution to repair a physically damaged endothelium (wound healing), binding to mature endothelium, and co-adjuvants to the formation of new vessels in vitro (angiogenesis). All of these in a dose-dependent manner. Damaged EMPs at a concentration of 10(3) MPs/ml have an activating effect of these capabilities, while at concentrations of 10(5) MPs/ml these effects are attenuated or reduced. This in vitro model helps explain that in diseases where there is an imbalance between these two elements (EPCs and damaged EMPs), the key cellular elements in the regeneration and maintenance of vascular homeostasis (EPCs) are not fully functional, and could explain, at least in part, endothelial dysfunction associated in various pathologies.
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http://dx.doi.org/10.3389/fphys.2015.00395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686689PMC
January 2016

LDL biochemical modifications: a link between atherosclerosis and aging.

Food Nutr Res 2015 3;59:29240. Epub 2015 Dec 3.

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

Atherosclerosis is an aging disease in which increasing age is a risk factor. Modified low-density lipoprotein (LDL) is a well-known risk marker for cardiovascular disease. High-plasma LDL concentrations and modifications, such as oxidation, glycosylation, carbamylation and glycoxidation, have been shown to be proatherogenic experimentally in vitro and in vivo. Atherosclerosis results from alterations to LDL in the arterial wall by reactive oxygen species (ROS). Evidence suggests that common risk factors for atherosclerosis raise the likelihood that free ROS are produced from endothelial cells and other cells. Furthermore, oxidative stress is an important factor in the induction of endothelial senescence. Thus, endothelial damage and cellular senescence are well-established markers for atherosclerosis. This review examines LDL modifications and discusses the mechanisms of the pathology of atherosclerosis due to aging, including endothelial damage and oxidative stress, and the link between aging and atherosclerosis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670441PMC
http://dx.doi.org/10.3402/fnr.v59.29240DOI Listing
December 2015

Effects of the mas-related gene (Mrg) C receptor agonist BAM6-22 on nociceptive reflex activity in naive, monoarthritic and mononeuropathic rats after intraplantar and intrathecal administration.

Eur J Pharmacol 2016 Jan 23;770:147-53. Epub 2015 Nov 23.

Dept. Biology Systems, Unit of Physiology, Faculty of Medicine, Universidad de Alcala, Campus Universitario, 28871 Alcala de Henares, Spain.

MrgC receptors are selectively expressed on peripheral and central terminals of small calibre nociceptive fibres. Peptide agonists of the MrgC receptor were reported to modulate nociceptive transmission exerting either pro- or antinociceptive effects depending on site of action and pain model used. Here, we investigated the effect of intraplantar and intrathecal administration of the selective MrgC receptor agonist BAM6-22 on mechanically and electrically evoked nociceptive reflex activity as a uniform readout measure in naïve, monoarthritic and mononeuropathic rats. In naïve rats, intraplantar BAM6-22 enhanced, whereas intrathecal BAM6-22 did not modulate mechanically-evoked nociceptive reflex activity. In monoarthritic rats, intraplantar BAM6-22 had no effect, whereas intrathecal BAM6-22 inhibited mechanically evoked nociceptive reflex activity. In mononeuropathic rats, BAM6-22 reduced mechanically evoked nociceptive reflex activity after both intraplantar and intrathecal administration. BAM6-22 did not modulate electrically evoked nociceptive reflex activity in any condition. Thus, the results of the present investigation confirm and add to previous studies demonstrating that site of action, (patho)-physiological state and stimulus modality determine the effect quality of MrgC receptor agonists. It still needs to be explored how concurrent activation of peripheral and spinal MrgC receptors modulates nociceptive processing under conditions of both acute and chronic pain to evaluate the therapeutic potential of putative small molecule MrgC receptor agonists as innovative analgesics.
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http://dx.doi.org/10.1016/j.ejphar.2015.11.042DOI Listing
January 2016

Gremlin regulates renal inflammation via the vascular endothelial growth factor receptor 2 pathway.

J Pathol 2015 Aug 19;236(4):407-20. Epub 2015 May 19.

Cellular Biology in Renal Diseases Laboratory, IIS-Fundación Jiménez Díaz, Universidad Autónoma Madrid, REDINREN, Spain.

Inflammation is a main feature of progressive kidney disease. Gremlin binds to bone morphogenetic proteins (BMPs), acting as an antagonist and regulating nephrogenesis and fibrosis among other processes. Gremlin also binds to vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells to induce angiogenesis. In renal cells, gremlin regulates proliferation and fibrosis, but there are no data about inflammatory-related events. We have investigated the direct effects of gremlin in the kidney, evaluating whether VEGFR2 is a functional gremlin receptor. Administration of recombinant gremlin to murine kidneys induced rapid and sustained activation of VEGFR2 signalling, located in proximal tubular epithelial cells. Gremlin bound to VEGFR2 in these cells in vitro, activating this signalling pathway independently of its action as an antagonist of BMPs. In vivo, gremlin caused early renal damage, characterized by activation of the nuclear factor (NF)-κB pathway linked to up-regulation of pro-inflammatory factors and infiltration of immune inflammatory cells. VEGFR2 blockade diminished gremlin-induced renal inflammatory responses. The link between gremlin/VEGFR2 and NF-κB/inflammation was confirmed in vitro. Gremlin overexpression was associated with VEGFR2 activation in human renal disease and in the unilateral ureteral obstruction experimental model, where VEGFR2 kinase inhibition diminished renal inflammation. Our data show that a gremlin/VEGFR2 axis participates in renal inflammation and could be a novel target for kidney disease.
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http://dx.doi.org/10.1002/path.4537DOI Listing
August 2015

Angiotensin II, via angiotensin receptor type 1/nuclear factor-κB activation, causes a synergistic effect on interleukin-1-β-induced inflammatory responses in cultured mesangial cells.

J Renin Angiotensin Aldosterone Syst 2015 Mar 29;16(1):23-32. Epub 2014 Oct 29.

Cellular Biology in Renal Diseases Laboratory, IIS-Fundación Jiménez Díaz Universidad Autónoma Madrid, Spain

Introduction: The nuclear factor-κB (NF-κB) is an important regulator of the inflammatory response. Angiotensin II (Ang II) activates the NF-κB pathway linked to renal inflammation. Although both AT1 and AT2 receptors are involved in Ang II-mediated NF-κB activation, the biological processes mediated by each receptor are not fully characterized. Interleukin-1β (IL-1β) is an important macrophage-derived cytokine that regulates immune and inflammatory processes, activating intracellular pathways shared with Ang II, including the NF-κB.

Materials And Methods: In vitro studies were done in primary cultured rat mesangial cells. NF-κB pathway was evaluated by phosphorylated levels of p65/IκB and DNA binding activity. The Ang II receptor subtype was determined by pretreatment with AT1 and AT2 antagonists.

Results: In mesangial cells the simultaneous presence of Ang II and IL-1β caused a synergistic activation of the NF-κB pathway and a marked upregulation of proinflammatory factors under NF-κB control, including monocyte chemoattractant protein-1. The AT1, but not AT2, antagonist abolished the synergistic effect on NF-κB activation and proinflammatory genes caused by coincubation of Ang II and IL-1β.

Conclusions: These data indicates that Ang II, via AT1/NF-κB pathway activation, cooperates with IL-β to increase the inflammatory response in mesangial cells.
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http://dx.doi.org/10.1177/1470320314551564DOI Listing
March 2015

Targeting of Gamma-Glutamyl-Cysteine Ligase by miR-433 Reduces Glutathione Biosynthesis and Promotes TGF-β-Dependent Fibrogenesis.

Antioxid Redox Signal 2015 Nov 9;23(14):1092-105. Epub 2015 Jan 9.

1 Departamento de Biología Celular e Inmunología, Centro de Biología Molecular "Severo Ochoa, " Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid , Madrid, Spain .

Aims: Glutathione (GSH) is the main antioxidant against cell damage. Several pathological states course with reduced nucleophilic tone and perturbation of redox homeostasis due to changes in the 2GSH/GSSG ratio. Here, we investigated the regulation of the rate-limiting GSH biosynthetic heterodimeric enzyme γ-glutamyl-cysteine ligase (GCL) by microRNAs (miRNAs).

Results: "In silico" analysis of the 3'- untranslated regions (UTRs) of both catalytic (GCLc) and regulatory (GCLm) subunits of GCL enabled an identification of miR-433 as a strong candidate for the targeting of GCL. Transitory overexpression of miR-433 in human umbilical vein endothelial cells (HUVEC) showed a downregulation of both GCLc and GCLm in a nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-independent manner. Increases in pro-oxidant stimuli such as exposure to hydrogen peroxide or GSH depletion in endothelial and hepatic cells caused an expected increase in GCLc and GCLm protein expression and abrogation of miR-433 levels, thus supporting a cross-regulation of these pathways. Treatment of HUVEC with miR-433 resulted in reduced antioxidant and redox potentials, increased S-glutathionylation, and reduced endothelial nitric oxide synthase activation. In vivo models of renal and hepatic fibrosis were associated with transforming growth factor β1 (TGF-β1)-related reduction of GCLc and GCLm levels that were miR-433 dependent.

Innovation And Conclusion: We describe for the first time an miRNA, miR-433, capable of directly targeting GCL and promoting functional consequences in endothelial physiology and fibrotic processes by decreasing GSH levels.
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http://dx.doi.org/10.1089/ars.2014.6025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4657521PMC
November 2015

Alternatively spliced tissue factor promotes plaque angiogenesis through the activation of hypoxia-inducible factor-1α and vascular endothelial growth factor signaling.

Circulation 2014 Oct 12;130(15):1274-86. Epub 2014 Aug 12.

From the AtheroThrombosis Research Unit (C.G., M.A., D.T.R., J.J.B.), Cardiovascular Research Institute (C.G., D.K.Y., D.J., J.C.K., T.W., R.J.H., V.F.), Translational and Molecular Imaging Institute (C.C., A.M., Z.A.F.), Department of Radiology (C.C., A.M., Z.A.F.), and Vascular Surgery (P.L.F.), Icahn School of Medicine at Mount Sinai, New York, NY; Memorial Sloan-Kettering, New York, NY (G.A.S.); and CNIC, Madrid, Spain (V.F.).

Background: Alternatively spliced tissue factor (asTF) is a novel isoform of full-length tissue factor, which exhibits angiogenic activity. Although asTF has been detected in human plaques, it is unknown whether its expression in atherosclerosis causes increased neovascularization and an advanced plaque phenotype.

Methods And Results: Carotid (n=10) and coronary (n=8) specimens from patients with stable or unstable angina were classified as complicated or uncomplicated on the basis of plaque morphology. Analysis of asTF expression and cell type-specific expression revealed a strong expression and colocalization of asTF with macrophages and neovessels within complicated, but not uncomplicated, human plaques. Our results showed that the angiogenic activity of asTF is mediated via hypoxia-inducible factor-1α upregulation through integrins and activation of phosphatidylinositol-3-kinase/Akt and mitogen-activated protein kinase pathways. Hypoxia-inducible factor-1α upregulation by asTF also was associated with increased vascular endothelial growth factor expression in primary human endothelial cells, and vascular endothelial growth factor-Trap significantly reduced the angiogenic effect of asTF in vivo. Furthermore, asTF gene transfer significantly increased neointima formation and neovascularization after carotid wire injury in ApoE(-/-) mice.

Conclusions: The results of this study provide strong evidence that asTF promotes neointima formation and angiogenesis in an experimental model of accelerated atherosclerosis. Here, we demonstrate that the angiogenic effect of asTF is mediated via the activation of the hypoxia-inducible factor-1/vascular endothelial growth factor signaling. This mechanism may be relevant to neovascularization and the progression and associated complications of human atherosclerosis as suggested by the increased expression of asTF in complicated versus uncomplicated human carotid and coronary plaques.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.114.006614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4190117PMC
October 2014

Integrin-linked kinase plays a key role in the regulation of angiotensin II-induced renal inflammation.

Clin Sci (Lond) 2014 Jul;127(1):19-31

*Cellular Biology in Renal Diseases Laboratory, IIS-Fundación Jiménez Díaz, Universidad Autónoma Madrid, Madrid, Spain.

ILK (integrin-linked kinase) is an intracellular serine/threonine kinase involved in cell-matrix interactions. ILK dysregulation has been described in chronic renal disease and modulates podocyte function and fibrosis, whereas data about its role in inflammation are scarce. AngII (angiotensin II) is a pro-inflammatory cytokine that promotes renal inflammation. AngII blockers are renoprotective and down-regulate ILK in experimental kidney disease, but the involvement of ILK in the actions of AngII in the kidney has not been addressed. Therefore we have investigated whether ILK signalling modulates the kidney response to systemic AngII infusion in wild-type and ILK-conditional knockout mice. In wild-type mice, AngII induced an inflammatory response, characterized by infiltration of monocytes/macrophages and lymphocytes, and up-regulation of pro-inflammatory factors (chemokines, adhesion molecules and cytokines). AngII activated several intracellular signalling mechanisms, such as the NF-κB (nuclear factor κB) transcription factor, Akt and production of ROS (reactive oxygen species). All these responses were prevented in AngII-infused ILK-deficient mice. In vitro studies characterized further the mechanisms regulating the inflammatory response modulated by ILK. In cultured tubular epithelial cells ILK blockade, by siRNA, inhibited AngII-induced NF-κB subunit p65 phosphorylation and its nuclear translocation. Moreover, ILK gene silencing prevented NF-κB-related pro-inflammatory gene up-regulation. The results of the present study demonstrate that ILK plays a key role in the regulation of renal inflammation by modulating the canonical NF-κB pathway, and suggest a potential therapeutic target for inflammatory renal diseases.
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http://dx.doi.org/10.1042/CS20130412DOI Listing
July 2014

Connective tissue growth factor is a new ligand of epidermal growth factor receptor.

J Mol Cell Biol 2013 Oct 8;5(5):323-35. Epub 2013 Aug 8.

Cellular Biology in Renal Diseases Laboratory, Universidad Autónoma de Madrid, 28040 Madrid, Spain.

Chronic kidney disease is reaching epidemic proportions worldwide and there is no effective treatment. Connective tissue growth factor (CCN2) has been suggested as a risk biomarker and a potential therapeutic target for renal diseases, but its specific receptor has not been identified. Epidermal growth factor receptor (EGFR) participates in kidney damage, but whether CCN2 activates the EGFR pathway is unknown. Here, we show that CCN2 is a novel EGFR ligand. CCN2 binding to EGFR extracellular domain was demonstrated by surface plasmon resonance. CCN2 contains four distinct structural modules. The carboxyl-terminal module (CCN2(IV)) showed a clear interaction with soluble EGFR, suggesting that EGFR-binding site is located in this module. Injection of CCN2(IV) in mice increased EGFR phosphorylation in the kidney, mainly in tubular epithelial cells. EGFR kinase inhibition decreased CCN2(IV)-induced renal changes (ERK activation and inflammation). Studies in cultured tubular epithelial cells showed that CCN2(IV) binds to EGFR leading to ERK activation and proinflammatory factors overexpression. CCN2 interacts with the neurotrophin receptor TrkA, and EGFR/TrkA receptor crosstalk was found in response to CCN2(IV) stimulation. Moreover, endogenous CCN2 blockade inhibited TGF-β-induced EGFR activation. These findings indicate that CCN2 is a novel EGFR ligand that contributes to renal damage through EGFR signalling.
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http://dx.doi.org/10.1093/jmcb/mjt030DOI Listing
October 2013

The C-terminal module IV of connective tissue growth factor is a novel immune modulator of the Th17 response.

Lab Invest 2013 Jul 6;93(7):812-24. Epub 2013 May 6.

Cellular Biology in Renal Diseases Laboratory, Instituto de Investigacion Sanitaria Fundación Jiménez Díaz, Universidad Autónoma Madrid, Madrid, Spain.

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein susceptible to proteolytic degradation. CCN2 levels have been suggested as a potential risk biomarker in several chronic diseases. In body fluids, CCN2 full-length and its degradation fragments can be found; however, their in vivo effects are far from being elucidated. CCN2 was described as a profibrotic mediator, but this concept is changing to a proinflammatory cytokine. In vitro, CCN2 full-length and its C-terminal module IV (CCN2(IV)) exert proinflammatory properties. Emerging evidence suggest that Th17 cells, and its effector cytokine IL-17A, participate in chronic inflammatory diseases. Our aim was to explore whether CCN2(IV) could regulate the Th17 response. In vitro, stimulation of human naive CD4+ T lymphocytes with CCN2(IV) resulted in differentiation to Th17 phenotype. The in vivo effects of CCN2(IV) were studied in C57BL/6 mice. Intraperitoneal administration of recombinant CCN2(IV) did not change serum IL-17A levels, but caused an activation of the Th17 response in the kidney, characterized by interstitial infiltration of Th17 (IL17A+/CD4+) cells and upregulation of proinflammatory mediators. In CCN2(IV)-injected mice, elevated renal levels of Th17-related factors (IL-17A, IL-6, STAT3 and RORγt) were found, whereas Th1/Th2 cytokines or Treg-related factors (TGF-β and Foxp-3) were not modified. Treatment with an anti-IL-17A neutralizing antibody diminished CCN2(IV)-induced renal inflammation. Our findings unveil that the C-terminal module of CCN2 induces the Th17 differentiation of human Th17 cells and causes a renal Th17 inflammatory response. Furthermore, these data bear out that IL-17A targeting is a promising tool for chronic inflammatory diseases, including renal pathologies.
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http://dx.doi.org/10.1038/labinvest.2013.67DOI Listing
July 2013

Angiotensin II contributes to renal fibrosis independently of Notch pathway activation.

PLoS One 2012 9;7(7):e40490. Epub 2012 Jul 9.

Cellular Biology in Renal Diseases Laboratory, Universidad Autónoma, Madrid, Spain.

Recent studies have described that the Notch signaling pathway is activated in a wide range of renal diseases. Angiotensin II (AngII) plays a key role in the progression of kidney diseases. AngII contributes to renal fibrosis by upregulation of profibrotic factors, induction of epithelial mesenchymal transition and accumulation of extracellular matrix proteins. In cultured human tubular epithelial cells the Notch activation by transforming growth factor-β1 (TGF-β1) has been involved in epithelial mesenchymal transition. AngII mimics many profibrotic actions of TGF-β1. For these reasons, our aim was to investigate whether AngII could regulate the Notch/Jagged system in the kidney, and its potential role in AngII-induced responses. In cultured human tubular epithelial cells, TGF-β1, but not AngII, increased the Notch pathway-related gene expression, Jagged-1 synthesis, and caused nuclear translocation of the activated Notch. In podocytes and renal fibroblasts, AngII did not modulate the Notch pathway. In tubular epithelial cells, pharmacological Notch inhibition did not modify AngII-induced changes in epithelial mesenchymal markers, profibrotic factors and extracellular matrix proteins. Systemic infusion of AngII into rats for 2 weeks caused tubulointerstitial fibrosis, but did not upregulate renal expression of activated Notch-1 or Jagged-1, as observed in spontaneously hypertensive rats. Moreover, the Notch/Jagged system was not modulated by AngII type I receptor blockade in the model of unilateral ureteral obstruction in mice. These data clearly indicate that AngII does not regulate the Notch/Jagged signaling system in the kidney, in vivo and in vitro. Our findings showing that the Notch pathway is not involved in AngII-induced fibrosis could provide important information to understand the complex role of Notch system in the regulation of renal regeneration vs damage progression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040490PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3392235PMC
January 2013

GSK3, snail, and adhesion molecule regulation by cyclosporine A in renal tubular cells.

Toxicol Sci 2012 Jun 12;127(2):425-37. Epub 2012 Mar 12.

Division of Nephrology and Hypertension, Laboratory of Nephrology and Vascular Pathology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz, Universidad Autónoma de Madrid, 28040 Madrid, Spain.

Tubular cell injury and fibrosis are key features of calcineurin inhibitor nephrotoxicity, but the molecular processes involved are not fully understood. In cultured murine MCT and human kidney 2 proximal tubular cells, gene expression and protein levels were studied by real-time polymerase chain reaction, Western blot, and confocal microscopy. Protein function was evaluated by pharmacological inhibitors and confirmed by small interfering RNA (siRNA) gene targeting. In renal tubular cells, cytotoxic concentrations of cyclosporine A (CsA) inhibited both gene and protein expression of adherent and tight junction proteins (E-cadherin, ZO-1, claudin-1, and β-catenin) and increased vimentin expression, without involvement of transforming growth factor β1 or caspase activity. CsA upregulated transcriptional repressors (Snail, Slug, and Twist) of the adherent and tight junction proteins were studied. Snail siRNA targeting prevented the downregulation of E-cadherin by CsA. CsA promoted glycogen synthase kinase 3 (GSK3) phosphorylation and increased Snail half-life. The GSK3 inhibitor lithium upregulated Snail and decreased E-cadherin expression in a Snail-dependent manner. Moreover, targeting GSK3 activity by siRNA also upregulated Snail. Furthermore, GSK3 siRNA had a negative impact on CsA-induced upregulation of Snail. Tacrolimus also inhibited GSK3 and mimicked CsA responses in tubular cells. We conclude that calcineurin inhibitors may directly decrease the expression of epithelial adhesion molecules by repressing GSK3 and stabilizing Snail. This offers potential pharmacological targets for prevention of nephrotoxicity.
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http://dx.doi.org/10.1093/toxsci/kfs108DOI Listing
June 2012