Efectos moleculares de los productos finales de glicación avanzada en la célula endotelial humana

En la diabetes se producen alteraciones que pueden llevar a la pérdida de la función endotelial y a las distintas enfermedades cardiovasculares a través de varios mecanismos moleculares, siendo el estrés oxidativo uno de los más importantes. Durante la diabetes se producen glicaciones no enzimáticas en las proteínas y éstas se han asociado con un aumento del estrés oxidativo. La glicación más abundante en las proteínas son los productos de Amadori, tanto in vitro como in vivo. El aumento de estrés oxidativo vascular puede estar mediado por la NADPH oxidasa, principal productora de especies reactivas del oxígeno (ROS) en los vasos. Así, nuestro objetivo ha sido determinar si los productos de Amadori (albúmina glicada) pueden producir disfunción endotelial, estudiando la producción extra e intracelular de ROS inducida por estos productos, además de las rutas y mecanismos que median estos efectos. En nuestros experimentos, el tratamiento de las células endoteliales de cordón umbilical (HUVEC) con un producto de Amadori, como es la albúmina glicada (gHSA), a bajas concentraciones, aumentó la producción de aniones superóxido extracelularmente de manera dependiente de la concentración a través de la NADPH oxidasa. El aumento de la producción alcanzó un máximo a las 4 horas y se mantuvo al menos hasta las 12 horas. El aumento de ROS también se produjo a nivel intracelular. El incremento en la producción de ROS inducido por la gHSA podría deberse a un incremento en la expresión de las principales subunidades de la NADPH oxidasa en la célula endotelial humana, la Nox4 y la p22phox, observado tanto a nivel del ARN mensajero como de la proteína. La gHSA indujo la activación del NF-kB y ésta lleva al aumento de la expresión de Nox4. La inhibición de las cascadas de fosforilación mediadas por la PI3K o la MEK 1/2 no tuvo efecto en la producción de ROS por las HUVEC. Por otra parte, la inhibición de la AP-1 aumentó la producción de ROS en HUVEC tratadas con la albúmina pero no con la albúmina glicada. El tratamiento con el inhibidor de AP- 1 aumentó la expresión de las subunidades de NADPH oxidasa, Nox4 y p22phox, pero disminuyó la expresión de eNOS. El tratamiento con albúmina glicada desacopló la eNOS en las HUVEC y esta acción podría explicar el efecto diferencial en la producción de ROS en presencia del inhibidor de AP-1. Por otra parte, en las HUVEC, la gHSA indujo la expresión de ICAM-1 y VCAM-1, además de tener un papel regulador en la expresión de MCP-1. Estos datos demostraron que la gHSA puede ser un modulador importante de la actividad inflamatoria endotelial y que esta modulación puede ejercerse de forma indirecta, a través de la inducción de respuestas oxidativas. Los resultados de este estudio explican parte de los mecanismos por los que la albúmina glicada, un producto de Amadori, afecta al estrés oxidativo y al estado inflamatorio de la célula endotelial y en consecuencia, puede ser causa de la disfunción endotelial relacionada con la diabetes. Conocer estas rutas de señalización intracelular permitiría saber más sobre como se inicia la disfunción endotelial relacionada con la diabetes y como, en el futuro, se podría avanzar en el tratamiento de estos pacientes

Receptor for advanced glycation end-products expression in subcutaneous adipose tissue is related to coronary artery disease

OBJECTIVE: Obesity, a risk factor for coronary artery disease (CAD), is associated with inflammation and reactive oxygen species (ROS) production, while advanced glycation end-products, through their receptor (AGER or RAGE), play an important role on these processes. The aim of this study was to analyze the expression levels of RAGE, NADPH oxidase subunits, and catalase in adipose tissue in relation with CAD. DESIGN AND METHODS: Patients undergoing heart surgery were included in two groups: with and without CAD. Epicardial adipose tissue (EAT) and subcutaneous adipose tissue (SAT) biopsies were analyzed for gene expression by RT-quantitative PCR, immunohistochemistry, or western blot. RESULTS: RAGE mRNA and protein expression in SAT from patients with CAD was lower than in patients without CAD. However, there was no change in EAT from patients with or without CAD. P22-PHOX and RAGE gene expression were higher in EAT than in SAT, whereas catalase mRNA levels were lower. NADPH oxidase subunits and catalase mRNA expression were not influenced by CAD. Whereas NADPH oxidase-dependent oxidative response of SAT and EAT to lipid circulating levels could be different; glycemic levels were not related with the analyzed genes expression. CONCLUSIONS: This study demonstrates that RAGE expression in SAT, but not in EAT, is down-regulated in patients with CAD with respect to those without CAD. Although changes were not observed for NADPH oxidase subunits or catalase expression between CAD and non-CAD patients, a possible relationship between ROS production and RAGE expression in adipose tissues cannot be ruled out

Role of Corticotropin-releasing Factor in Gastrointestinal Permeability

The interface between the intestinal lumen and the mucosa is the location where the majority of ingested immunogenic particles face the scrutiny of the vast gastrointestinal immune system. Upon regular physiological conditions, the intestinal micro-flora and the epithelial barrier are well prepared to process daily a huge amount of food-derived antigens and non-immunogenic particles. Similarly, they are ready to prevent environmental toxins and microbial antigens to penetrate further and interact with the mucosal-associated immune system. These functions promote the development of proper immune responses and oral tolerance and prevent disease and inflammation. Brain-gut axis structures participate in the processing and execution of response signals to external and internal stimuli. The brain-gut axis integrates local and distant regulatory networks and super-systems that serve key housekeeping physiological functions including the balanced functioning of the intestinal barrier. Disturbance of the brain-gut axis may induce intestinal barrier dysfunction, increasing the risk of uncontrolled immunological reactions, which may indeed trigger transient mucosal inflammation and gut disease. There is a large body of evidence indicating that stress, through the brain-gut axis, may cause intestinal barrier dysfunction, mainly via the systemic and peripheral release of corticotropin-releasing factor. In this review, we describe the role of stress and corticotropin-releasing factor in the regulation of gastrointestinal permeability, and discuss the link to both health and pathological conditions

Eosinophils in the Gastrointestinal Tract: Key Contributors to Neuro-Immune Crosstalk and Potential Implications in Disorders of Brain-Gut Interaction

Disorders of brain-gut interaction; Intestinal eosinophils; Neuro-immune interactionTrastorns de la interacció cervell-intestí; Eosinòfils intestinals; Interacció neuroimmuneTrastornos de la interacción cerebro-intestino; Eosinófilos intestinales; Interacción neuroinmuneEosinophils are innate immune granulocytes actively involved in defensive responses and in local and systemic inflammatory processes. Beyond these effector roles, eosinophils are fundamental to maintaining homeostasis in the tissues they reside. Gastrointestinal eosinophils modulate barrier function and mucosal immunity and promote tissue development through their direct communication with almost every cellular component. This is possible thanks to the variety of receptors they express and the bioactive molecules they store and release, including cytotoxic proteins, cytokines, growth factors, and neuropeptides and neurotrophines. A growing body of evidence points to the eosinophil as a key neuro-immune player in the regulation of gastrointestinal function, with potential implications in pathophysiological processes. Eosinophil–neuron interactions are facilitated by chemotaxis and adhesion molecules, and the mediators released may have excitatory or inhibitory effects on each cell type, with physiological consequences dependent on the type of innervation involved. Of special interest are the disorders of the brain–gut interaction (DBGIs), mainly functional dyspepsia (FD) and irritable bowel syndrome (IBS), in which mucosal eosinophilia and eosinophil activation have been identified. In this review, we summarize the main roles of gastrointestinal eosinophils in supporting gut homeostasis and the evidence available on eosinophil–neuron interactions to bring new insights that support the fundamental role of this neuro-immune crosstalk in maintaining gut health and contributing to the pathophysiology of DBGIs.This study was funded in part by the Fondo Europeo de Desarrollo Regional (FEDER), the Fondo de Investigación Sanitaria, the Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), the Instituto de Salud Carlos III, Subdirección General de Investigación Sanitaria, and the Ministerio de Economía y Competitividad: FI12/00254 (E.S-R.), CD15/00010 (B.K.R.-J.), FI20/00256 (M.A-B.), PI19/01643 (B.L.), PI17/0190 (J.S.), CP18/00116 (C.M), CPII16/00031 and PI19/01643 (M.V.), CIBEREHD CB06/04/0021 (J.S., R.F., M.V.)

Predictive value of advanced glycation end products for the development of post-infarction heart failure: a preliminary report

Background: Since post-infarction heart failure (HF) determines a great morbidity and mortality, and given the physiopathology implications of advanced glycation end products (AGE) in the genesis of myocardial dysfunction, it was intended to analyze the prognostic value of these molecules in order to predict post-infarction HF development. Methods: A prospective clinical study in patients after first acute coronary syndrome was conducted. The follow-up period was consisted in 1 year. In 194 patients consecutively admitted in the coronary unit for myocardial infarct fluorescent AGE levels were measured. The association between glycaemic parameters and the development of post-infarction HF were analyzed in those patients. Finally, we identified the variables with independent predictor value by performing a multivariate analysis of hazard ratio for Cox regression. Results: Eleven out of 194 patients (5.6%) developed HF during follow-up (median: 1.0 years [0.8 - 1.5 years]). Even though basal glucose, fructosamine and glycated haemoglobin were significant predictive factors in the univariate analysis, after being adjusted by confounding variables and AGE they lost their statistical signification. Only AGE (Hazard Ratio 1.016, IC 95%: 1.006-1.026; p<0,001), together with NT-proBNP and the infarct extension were predictors for post-infarction HF development, where AGE levels over the median value 5-fold increased the risk of HF development during follow-up. Conclusions: AGE are an independent marker of post-infarction HF development risk

Fluorescent Advanced Glycation End Products and Their Soluble Receptor: The Birth of New Plasmatic Biomarkers for Risk Stratification of Acute Coronary Syndrome

Objective: Advanced glycation end products (AGEs) have pathophysiological implications in cardiovascular diseases. The aim of our study was to evaluate the prognostic value of fluorescent AGEs and its soluble receptor (sRAGE) in the context of acute coronary syndrome (ACS), both in-hospital phase and follow-up period. Methods: A prospective clinical study was performed in patients with debut's ACS. The endpoints were the development of cardiac events (cardiac deaths, re-infarction and new-onset heart failure) during in-hospital phase and follow-up period (366 days, inter-quartile range: 273-519 days). 215 consecutive ACS patients admitted to the coronary care unit (62.7±13.0 years, 24.2% female) were included. 47.4% had a diagnosis of ST segment elevation myocardial infarction. AGEs and sRAGE were analysed by fluorescence spectroscopy and competitive ELISA, respectively. Risk scores (GRACE, TIMI, PURSUIT) were calculated retrospectively using prospective data. The complexity of coronary artery disease was evaluated by SYNTAX score. Results: The mean fluorescent AGEs and sRAGE levels were 57.7±45.1 AU and 1045.4±850.0 pg/mL, respectively. 19 patients presented cardiac events during in-hospital phase and 29 during the follow-up. In-hospital cardiac events were significantly associated with higher sRAGE levels (p = 0.001), but not long-term cardiac events (p = 0.365). Regarding fluorescent AGE the opposite happened. After multivariate analysis correcting by gender, left ventricular ejection fraction, glucose levels, haemoglobin, GRACE and SYNTAX scores, sRAGE was significantly associated with in-hospital prognosis, whereas fluorescent AGEs was significantly associated with long-term prognosis. Conclusions: We conclude that elevated values of sRAGE are associated with worse in-hospital prognosis, whereas high fluorescent AGE levels are associated with more follow-up events

Modeling the Number of People Infected With SARS-COV-2 From Wastewater Viral Load in Northwest Spain

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG[Abstract] The quantification of the SARS-CoV-2 RNA load in wastewater has emerged as a useful tool to monitor COVID–19 outbreaks in the community. This approach was implemented in the metropolitan area of A Coruña (NW Spain), where wastewater from a treatment plant was analyzed to track the epidemic dynamics in a population of 369,098 inhabitants. Viral load detected in the wastewater and the epidemiological data from A Coruña health system served as main sources for statistical models developing. Regression models described here allowed us to estimate the number of infected people (R2 = 0.9), including symptomatic and asymptomatic individuals. These models have helped to understand the real magnitude of the epidemic in a population at any given time and have been used as an effective early warning tool for predicting outbreaks in A Coruña municipality. The methodology of the present work could be used to develop a similar wastewater-based epidemiological model to track the evolution of the COVID–19 epidemic anywhere in the world where centralized water-based sanitation systems exist.This work was supported by EDAR Bens S.A., A Coruña, Spain [grant references INV04020, INV12120 and INV05921 to MP], the National Plan for Scientific Research, Development and Technological Innovation 2013-2016 funded by the ISCIII, Spain - General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe” [grant numbers PI15/00860 to GB, PI17/01482 and PI20/00413 to MP], the GAIN, Xunta de Galicia, Spain [grant number IN607A 2016/22 to GB, ED431C-2016/015 and ED431C-2020/14 to RC, ED431C 2017/58 to SL, ED431G 2019/01 to RC and SL, and ED431C 2017/66 to MCV], MINECO, Spain [grant number MTM2017-82724-R to RC], Ministerio de Ciencia e Innovación, Spain [grant number PID2020-113578RB-100 to RC], and the Spanish Network for Research in Infectious Diseases [REIPI RD16/0016/006 to GB]. The work was also supported by the European Virus Archive Global (EVA-GLOBAL) project that has received funding from the European Union's Horizon 2020 - Research and Innovation Framework Programme under grant agreement no 871029. SR-F was financially supported by REIPI RD16/0016/006, KC-P by IN607A 2016/22 and the Spanish Association against Cancer (AECC) and JAV by IN607A 2016/22. Funding for open access charge: Universidade da Coruña/CISUGEDAR Bens S.A.; INV04020EDAR Bens S.A.; INV12120EDAR Bens S.A.; INV05921Xunta de Galicia; IN607A 2016/22Xunta de Galicia; ED431C-2016/015Xunta de Galicia; ED431C-2020/14Xunta de Galicia; ED431C 2017/58Xunta de Galicia; ED431G 2019/01Xunta de Galicia; ED431C 2017/6

Acute Stress Regulates Sex-Related Molecular Responses in the Human Jejunal Mucosa: Implications for Irritable Bowel Syndrome

Estrés agudo; Barrera intestinal; SexoEstrès agut; Barrera intestinal; SexeCute stress; Intestinal barrier; SexIrritable bowel syndrome (IBS) is a prevalent gastrointestinal disorder linked to intestinal barrier dysfunction and life stress. We have previously reported that female sex per se determines an increased susceptibility to intestinal barrier dysfunction after cold pain stress (CPS). We aimed to identify sex-related molecular differences in response to CPS in healthy subjects to understand the origin of sex bias predominance in IBS. In 13 healthy males and 21 females, two consecutive jejunal biopsies were obtained using Watson’s capsule, at baseline, and ninety minutes after CPS. Total mucosal RNA and protein were isolated from jejunal biopsies. Expression of genes related to epithelial barrier (CLDN1, CLDN2, OCLN, ZO-1, and ZO-3), mast cell (MC) activation (TPSAB1, SERPINA1), and the glucocorticoid receptor (NR3C1) were analyzed using RT-qPCR. NR3C1, ZO-1 and OCLN protein expression were evaluated through immunohistochemistry and western blot, and mucosal inflammation through MC, lymphocyte, and eosinophil numbering. Autonomic, hormonal, and psychological responses to CPS were monitored. We found an increase in jejunal MCs, a reduced CLDN1 and OCLN expression, and an increased CLDN2 and SERPINA1 expression 90 min after CPS. We also found a significant decrease in ZO-1, OCLN, and NR3C1 gene expression, and a decrease in OCLN protein expression only in females, when compared to males. CPS induced a significant increase in blood pressure, plasma cortisol and ACTH, and subjective stress perception in all participants. Specific and independent sex-related molecular responses in epithelial barrier regulation are unraveled by acute stress in the jejunum of healthy subjects and may partially explain female predominance in IBS.Supported in part by Fondo Europeo de Desarrollo Regional (FEDER), Fondo de Investigación Sanitaria and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Instituto de Salud Carlos III, Subdirección General de Investigación Sanitaria, Ministerio de Economiá y Competitividad: CM08/00229 (BL); CM10/00155 (MP); EII2011-0035, CD15/00010, and MV17-00043 (BKRJ.); FI12/00254 (ESR.), PI17/0190 (JS), PI12/00314 and PI15/00301 (CAC), CIBEREHD CB06/04/0021 (JS, CAC.); Vall d’Hebron Institut de Recerca, Programa de becas predoctorales Amics de Vall d’Hebron: PRED-VHIR-2014-018 (MF), PRED-VHIR-2016-53 34 (CPC.)

miR-16 and miR-125b are involved in barrier function dysregulation through the modulation of claudin-2 and cingulin expression in the jejunum in IBS with diarrhoea

Micro-RNAs (miRNAs) play a crucial role in controlling intestinal epithelial barrier function partly by modulating the expression of tight junction (TJ) proteins. We have previously shown differential messenger RNA (mRNA) expression correlated with ultrastructural abnormalities of the epithelial barrier in patients with diarrhoea-predominant IBS (IBS-D). However, the participation of miRNAs in these differential mRNA-associated findings remains to be established. Our aims were (1) to identify miRNAs differentially expressed in the small bowel mucosa of patients with IBS-D and (2) to explore putative target genes specifically involved in epithelial barrier function that are controlled by specific dysregulated IBS-D miRNAs. Healthy controls and patients meeting Rome III IBS-D criteria were studied. Intestinal tissue samples were analysed to identify potential candidates by: (a) miRNA-mRNA profiling; (b) miRNA-mRNA pairing analysis to assess the co-expression profile of miRNA-mRNA pairs; (c) pathway analysis and upstream regulator identification; (d) miRNA and target mRNA validation. Candidate miRNA-mRNA pairs were functionally assessed in intestinal epithelial cells. IBS-D samples showed distinct miRNA and mRNA profiles compared with healthy controls. TJ signalling was associated with the IBS-D transcriptional profile. Further validation of selected genes showed consistent upregulation in 75% of genes involved in epithelial barrier function. Bioinformatic analysis of putative miRNA binding sites identified hsa-miR-125b-5p and hsa-miR-16 as regulating expression of the TJ genes CGN (cingulin) and CLDN2 (claudin-2), respectively. Consistently, protein expression of CGN and CLDN2 was upregulated in IBS-D, while the respective targeting miRNAs were downregulated. In addition, bowel dysfunction, perceived stress and depression and number of mast cells correlated with the expression of hsa-miR-125b-5p and hsa-miR-16 and their respective target proteins. Modulation of the intestinal epithelial barrier function in IBS-D involves both transcriptional and post-transcriptional mechanisms. These molecular mechanisms include miRNAs as master regulators in controlling the expression of TJ proteins and are associated with major clinical symptoms

Wastewater early warning system for SARS-CoV-2 outbreaks and variants in a Coruña, Spain

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG[Abstract]: Wastewater-based epidemiology has been widely used as a cost-effective method for tracking the COVID-19 pandemic at the community level. Here we describe COVIDBENS, a wastewater surveillance program running from June 2020 to March 2022 in the wastewater treatment plant of Bens in A Coruña (Spain). The main goal of this work was to provide an effective early warning tool based in wastewater epidemiology to help in decision-making at both the social and public health levels. RT-qPCR procedures and Illumina sequencing were used to weekly monitor the viral load and to detect SARS-CoV-2 mutations in wastewater, respectively. In addition, own statistical models were applied to estimate the real number of infected people and the frequency of each emerging variant circulating in the community, which considerable improved the surveillance strategy. Our analysis detected 6 viral load waves in A Coruña with concentrations between 103 and 106 SARS-CoV-2 RNA copies/L. Our system was able to anticipate community outbreaks during the pandemic with 8-36 days in advance with respect to clinical reports and, to detect the emergence of new SARS-CoV-2 variants in A Coruña such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529 and BA.2) in wastewater with 42, 30, and 27 days, respectively, before the health system did. Data generated here helped local authorities and health managers to give a faster and more efficient response to the pandemic situation, and also allowed important industrial companies to adapt their production to each situation. The wastewater-based epidemiology program developed in our metropolitan area of A Coruña (Spain) during the SARS-CoV-2 pandemic served as a powerful early warning system combining statistical models with mutations and viral load monitoring in wastewater over time.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Funding for open access charge: Universidade da Coruña/CISUG. This work was supported by EDAR Bens S.A., A Coruña, Spain [grant references INV04020, INV12120, INV05921, and INV148721 to MP], by the National Plan for Scientific Research, Development and Technological Innovation funded by the Institute of Health Carlos III (ISCIII), Spain—General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe” [grant references PI15/00860 to GB, PI17/01482, and PI20/00413 to MP], by the Galician Innovation Agency (GAIN) (Xunta de Galicia, Spain) [grant references IN607A 2016/22 to GB, ED431C-2016/015 and ED431C-2020/14 to RC, ED431C 2021/53 to SL and ED431G 2019/01 and COV20/00604 to RC and SL, by Ministry of Economic Affairs and Digital Transformation (MINECO), Spain [grant references MTM2017-82724-R to RC], by the Spanish Network for Research in Infectious Diseases [REIPI RD16/0016/0006 to GB], by the “Innova Saúde” Program, (INNOVAMICROLAB project) co-founded by the Galician Healthcare Service (SERGAS) and the Spanish Ministry of Science and Innovation, and by the Spanish Network of Research in Infectious Diseases (CIBERINFEC, ISCIII), and by the European Virus Archive Global (EVA-GLOBAL) project that has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 871029. SR-F was financially supported by REIPI RD16/0016/006, KC-P by IN607A 2016/22 and the Spanish Association against Cancer (AECC) and JAV by IN607A 2016/22. DP was funded by grant EPICOVIGAL FONDO SUPERA-COVID19 from Banco Santander-CSIC-CRUE, Spain, and grant CT850A-2 from (Health Knowledge Agency) ACIS SERGAS from the Consellería de Sanidade of Xunta de Galicia, Spain.EDAR Bens S.A.; INV04020EDAR Bens S.A.; INV12120EDAR Bens S.A.; INV05921EDAR Bens S.A.; INV148721Xunta de Galicia; IN607A 2016/22Xunta de Galicia; ED431C-2016/015Xunta de Galicia; ED431C-2020/14Xunta de Galicia; ED431C 2021/53Xunta de Galicia; ED431G 2019/01Xunta de Galicia; COV20/0060