Cardiac Bmi1(+) cells contribute to myocardial renewal in the murine adult heart

Introduction: The mammalian adult heart maintains a continuous, low cardiomyocyte turnover rate throughout life. Although many cardiac stem cell populations have been studied, the natural source for homeostatic repair has not yet been defined. The Polycomb protein BMI1 is the most representative marker of mouse adult stem cell systems. We have evaluated the relevance and role of cardiac Bmi1(+) cells in cardiac physiological homeostasis. Methods: Bmi1(CreER/+); Rosa26(YFP/+) (Bmi1-YFP) mice were used for lineage tracing strategy. After tamoxifen (TM) induction, yellow fluorescent protein (YFP) is expressed under the control of Rosa26 regulatory sequences in Bmi1(+) cells. These cells and their progeny were tracked by FACS, immunofluorescence and RT-qPCR techniques from 5 days to 1 year. Results: FACS analysis of non-cardiomyocyte compartment from TM-induced Bmi1-YFP mice showed a Bmi1 (+)-expressing cardiac progenitor cell (Bmi1-CPC: B-CPC) population, SCA-1 antigen-positive (95.9 +/- 0.4 \%) that expresses some stemness-associated genes. B-CPC were also able to differentiate in vitro to the three main cardiac lineages. Pulse-chase analysis showed that B-CPC remained quite stable for extended periods (up to 1 year), which suggests that this Bmi1(+) population contains cardiac progenitors with substantial self-maintenance potential. Specific immunostaining of Bmi1-YFP hearts serial sections 5 days post-TM induction indicated broad distribution of B-CPC, which were detected in variably sized clusters, although no YFP+ cardiomyocytes (CM) were detected at this time. Between 2 to 12 months after TM induction, YFP+ CM were clearly identified (3 +/- 0.6 \% to 6.7 +/- 1.3 \%) by immunohistochemistry of serial sections and by flow cytometry of total freshly isolated CM. B-CPC also contributed to endothelial and smooth muscle (SM) lineages in vivo. Conclusions: High Bmi1 expression identifies a non-cardiomyocyte resident cardiac population (B-CPC) that contributes to the main lineages of the heart in vitro and in vivo.We wish to thank M. Torres, J.M. Perez-Pomares and B.G. Galvez for critical discussions of the manuscript, A. M. Santos for assistance with confocal microscopy and dynamic imaging, R.M. Carmona for help with the animal colony management, F.S. Cabo for bioinformatics and statistical support, J.M Ligos for the sorting strategy, and K. McCreath and C. Mark for editorial support. This study was supported by grants to A.B. from the Ministry of Science and Innovation (SAF2012-34327; PLE2009-0147 and PSE-010000-2009-3), the Research Program of the Comunidad Autonoma de Madrid (S2010/BMD-2420), the Instituto de Salud Carlos III (RETICS-RD12/0019/0018 and RETICS-RD12/0019/0023) and the European Commission (Proposal 242038). The CNB-CSIC and CNIC are supported by the Spanish Ministry of Economy and Competitiveness.S

Cell Fusion Reprogramming Leads to a Specific Hepatic Expression Pattern during Mouse Bone Marrow Derived Hepatocyte Formation In Vivo

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation

EVOLUCIÓN DE LA ACTIVIDAD DE LA BILIRRUBINA-UDP GLUCURONOSIL- TRANSFERASA (BUDP-GT) DURANTE EL DESARROLLO EN RATA

Conjugation of bilirubin and excretion by the liver is catalysed by the microsomal enzyme bilirubin-UDP- lucuronosyltransferase (bUDP-GT). It is well known that during gestation, the fetal liver in utero is incapable for capture, conjugation and clearance of bilirubin. However, we have previously demonstrated that the gene responsible for the enzyme starts to express by day 13 of gestation. Therefore, the aims of the present work are first, to carry out a procedure so as to determine the activity of the bUDP-GT in the hepatic tissue, and, second, to obtain new data about the changes during the perinatal development in the activity levels of the mentioned enzyme. On the one hand, we have analyzed both, the activation time of the microsomal fraction, which stabilizes after 30 minutes of incubation, and the changes in the enzyme activity which reach a maximum peak after 5 minutes of incubation. On the other hand, we only detect activity of the bUDP-GT at the final period of gestation, reaching adult levels during the first month of extrauterine life.La enzima microsomal bUDP-GT (bilirrubina uridindifosfo-glucuronosil transferasa) es responsable de la conjugación de la bilirrubina para ser eliminada por la bilis. Es un hecho establecido que durante la gestación la madre conjuga y elimina la bilirrubina producida por el feto, si bien hemos demostrado en nuestro laboratorio que el gen responsable de dicha enzima comienza a expresarse a partir del día 13 de gestación y por ello, nos planteamos: 1º) la puesta a punto de la técnica para determinar la actividad de la bUDP-GT en tejido hepático, y 2º) aportar nuevos datos sobre la variación de la actividad de la enzima durante el desarrollo perinatal. En cuanto al primer apartado, se han analizado el tiempo de activación de la fracción microsomal, que se estabiliza a los 30 minutos de incubación y la variación de la actividad enzimática cuyo máximo aparece a los 5 minutos de incubación de la mezcla de reacción. En cuanto a la evolución de la actividad de la bUDP-GT con la edad, hemos visto que sólo al final del periodo de gestación comienzan a aparecer valores detectables y durante el primer mes de vida ya alcanzan valores próximos al adulto

Regulation of endothelial dynamics by PGC-1α relies on ROS control of VEGF-A signaling

Peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) is a regulator of mitochondrial metabolism and reactive oxygen species (ROS) that is known to play a relevant role in angiogenesis. [Aims]: This study aims to investigate the role of ROS on the regulation by PGC-1α of angiogenesis. [Methods and results]: We found that endothelial cells (ECs) from mice deleted for PGC-1α display attenuated adhesion to the extracellular matrix, together with slower and reversible spreading. Structural analysis demonstrates unstable formation of focal adhesions, defective cytoskeleton reorganization in response to cellular matrix adhesion, cell migration and cell-cell adhesion. Confluent cultures showed also a reduction of membrane bound VE-cadherin, suggesting defective inter-cellular junction formation. Functional consequences included impaired directional migration, and enhanced tip phenotype in aortic explants sprouting assays. At the molecular level, PGC-1α-deleted ECs exhibit a constitutive activation of the vascular endothelial growth factor-A (VEGF-A) signaling pathway and a defective response to VEGF-A. All these alterations are partially reversed by administration of the antioxidant EUK-189. The contribution of mitochondrial ROS and NOX activation was confirmed using a mitochondrial targeted antioxidant (MitoTEMPO) and a NOX inhibitor (VAS-2870). These results indicate that elevated production of ROS in the absence of PGC-1α is a key factor in the alteration of the VEGF-A signaling pathway and the capacity of endothelial cells to form stable interactions with other endothelial cells and with the extracellular matrix. Our findings show that PGC-1α control of ROS homeostasis plays an important role in the control of endothelial response to VEGF-A.This work was supported by grants from the Spanish ‘‘Ministerio de Economía y Competitividad’’ [Grant no. SAF2009-07599 & SAF2012-37693 to M.M and CSD 2007-00020 to M.M.]; and the ‘‘Comunidad Autónoma de Madrid’’ [Grant no. S2010/BMD-2361 to M.M.].Peer Reviewe

Oxidative stress induces loss of pericyte coverage and vascular instability in PGC-1α-deficient mice

Peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) is a regulator of mitochondrial oxidative metabolism and reactive oxygen species (ROS) homeostasis that is known to be inactivated in diabetic subjects. This study aimed to investigate the contribution of PGC-1α inactivation to the development of oxygen-induced retinopathy. We analyzed retinal vascular development in PGC-1α mice. Retinal vasculature of PGC-1α mice showed reduced pericyte coverage, a de-structured vascular plexus, and low perfusion. Exposure of PGC-1α mice to hyperoxia during retinal vascular development exacerbated these vascular abnormalities, with extensive retinal hemorrhaging and highly unstructured areas as compared with wild-type mice. Structural analysis demonstrated a reduction in membrane-bound VE-cadherin, which was suggestive of defective intercellular junctions. Interestingly, PGC-1α retinas showed a constitutive activation of the VEGF-A signaling pathway. This phenotype could be partially reversed by antioxidant administration, indicating that elevated production of ROS in the absence of PGC-1α could be a relevant factor in the alteration of the VEGF-A signaling pathway. Collectively, our findings suggest that PGC-1α control of ROS homeostasis plays an important role in the regulation of de novo angiogenesis and is required for vascular stability.This work was supported by grants from the Spanish ‘‘Ministerio de Economía y Competitividad’’ (Grant number SAF2009-07599 & SAF2012-37693 to M.M and CSD 2007-00020 to M.M.) and the ‘‘Comunidad de Madrid’’ (Grant Number S2010/BMD-2361 to M.M.).Peer Reviewe

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

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.This work was funded by the Plan Nacional Proyectos de Investigacion en Salud of Instituto de Salud Carlos III (ISCIII), EU FEDER Grant (PI14/00806). All proteomic analyses were performed at the Proteomics Facility of the Spanish National Center for Biotechnology (CNB-CSIC) (a member of ProteoRed, PRB2-ISCIII, financed via grant PT13/0001). The authors thank Jose Maria Arribas (Espectrometria de Masas y Analisis Elemental, Universidad de Alcala) for NADPH analysis and Dr. Marcos Marva (Departamento de Fisica y Matematicas, Universidad de Alcala) for statistical analysis.S

Regulation of endothelial dynamics by PGC-1α relies on ROS control of VEGF-A signaling

Peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) is a regulator of mitochondrial metabolism and reactive oxygen species (ROS) that is known to play a relevant role in angiogenesis. Aims: This study aims to investigate the role of ROS on the regulation by PGC-1α of angiogenesis. Methods and results: We found that endothelial cells (ECs) from mice deleted for PGC-1α display attenuated adhesion to the extracellular matrix, together with slower and reversible spreading. Structural analysis demonstrates unstable formation of focal adhesions, defective cytoskeleton reorganization in response to cellular matrix adhesion, cell migration and cell-cell adhesion. Confluent cultures showed also a reduction of membrane bound VE-cadherin, suggesting defective inter-cellular junction formation. Functional consequences included impaired directional migration, and enhanced tip phenotype in aortic explants sprouting assays. At the molecular level, PGC-1α-deleted ECs exhibit a constitutive activation of the vascular endothelial growth factor-A (VEGF-A) signaling pathway and a defective response to VEGF-A. All these alterations are partially reversed by administration of the antioxidant EUK-189. The contribution of mitochondrial ROS and NOX activation was confirmed using a mitochondrial targeted antioxidant (MitoTEMPO) and a NOX inhibitor (VAS-2870). These results indicate that elevated production of ROS in the absence of PGC-1α is a key factor in the alteration of the VEGF-A signaling pathway and the capacity of endothelial cells to form stable interactions with other endothelial cells and with the extracellular matrix. Our findings show that PGC-1α control of ROS homeostasis plays an important role in the control of endothelial response to VEGF-A.Ministerio de Economía y Competitividad (España)Comunidad de MadridDepto. de Biología CelularFac. de Ciencias BiológicasTRUEpu

Chromatin dynamics through mouse preimplantation development revealed by single molecule localisation microscopy.

Most studies addressing chromatin behaviour during preimplantation development are based on biochemical assays that lack spatial and cell-specific information, crucial during early development. Here, we describe the changes in chromatin taking place at the transition from totipotency to lineage specification, by using direct stochastical optical reconstruction microscopy (dSTORM) in whole-mount embryos during the first stages of mouse development. Through the study of two post-translational modifications of Histone 3 related to active and repressed chromatin, H3K4me3 and H3K9me3 respectively, we obtained a time-course of chromatin states, showing spatial differences between cell types, related to their differentiation state. This analysis adds a new layer of information to previous biochemical studies and provides novel insight to current models of chromatin organisation during the first stages of development.This work was supported by grants BFU2017-84914-P and PID2020-115755GB-I00 from the Spanish State Research Agency (AEI). M.P. was supported by fellowships from the Comunidad de Madrid (PEJD-2018-PRE/BMD-9079) and the Ministerio de Ciencia e Innovación (PRE2018-083477). The CBMSO is supported by an Institutional grant from the Fundación Ramon Areces, and the the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence grant (CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). Deposited in PMC for immediate release.S

Number and brightness analysis reveals that NCAM and FGF2 elicit different assembly and dynamics of FGFR1 in live cells

Both fibroblast growth factor-2 (FGF2) and neural cell adhesion molecule (NCAM) trigger FGF receptor 1 (FGFR1) signaling; however, they induce remarkably distinct receptor trafficking and cellular responses. The molecular basis of such a dichotomy and the role of distinct types of ligand-receptor interaction remain elusive. Number of molecules and brightness (N&B) analysis revealed that FGF2 and NCAM promote different FGFR1 assembly and dynamics at the plasma membrane. NCAM stimulation elicits long-lasting cycles of short-lived FGFR1 monomers and multimers, a behavior that might reflect a rapid FGFR1 internalization and recycling. FGF2, instead, induces stable dimerization at the dose that stimulates cell proliferation. Reducing the occupancy of FGFR1 in response to low FGF2 doses causes a switch towards cyclically exposed and unstable receptor dimers, consistently with previously reported biphasic response to FGF2 and with the divergent signaling elicited by different ligand concentrations. Similar instability was observed upon altering the endocytic pathway. Thus, FGF2 and NCAM induce differential FGFR1 clustering at the cell surface, which might account for the distinct intracellular fate of the receptor and, hence, for the different signaling cascades and cellular responses.U.C. and V.R.C. acknowledge the support of Fondazione CARIPLO, Milano (Grant 2375 2009-2012). U.C. also acknowledges the support from the Associazione Italiana Ricerca sul Cancro, the Association for International Cancer Research (now known as Worldwide Cancer Research), and the Italian Ministry of Health. A. T. acknowledges the 'Fondazione Banca del Monte di Lombardia' for partly supporting his work with the PV Fellowship 'Progetto Professionalita Ivano Becchi' (20112012). M.G. was supported by a fellowship from Fondazione IEO-CCM. The CNIC is supported by the Ministry of Ciencia, Innovaciòn y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S