Nuevos modelos genéticos murinos para el estudio del endotelio vascular sanguíneo: análisis de la relevancia de c-Met durante la neovascularización en el ratón

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-07-201

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

In vivo imaging of lymphatic vessels in development, wound healing, inflammation, and tumor metastasis

Lymphatic vessel growth or lymphangiogenesis occurs during embryonic development and wound healing and plays an important role in tumor metastasis and inflammatory diseases. However, the possibility of noninvasive detection and quantification of lymphangiogenesis has been lacking. Here, we present the Vegfr3EGFPLuc mouse model, where an EGFP-luciferase fusion protein, expressed under the endogenous transcriptional control of the Vegfr3 gene, allows the monitoring of physiological and pathological lymphangiogenesis in vivo. We show tracking of lymphatic vessel development during embryogenesis as well as lymphangiogenesis induced by specific growth factors, during wound healing and in contact hypersensitivity (CHS) - induced inflammation where we also monitor down-regulation of lymphangiogenesis by the glucocorticoid dexamethasone. Importantly, the Vegfr3-reporter allowed us to tracking tumor-induced lymphangiogenesis at the tumor periphery and in lymph nodes in association with the metastatic process. This is the first reporter mouse model for luminescence imaging of lymphangiogenesis. It should provide an important tool for studying the involvement of lymphangiogenesis in pathological processes.The support of the Spanish National Cancer Research Centre (CNIO) Transgenesis, Comparative Pathology, Confocal Microscopy, Molecular Imaging, and Animal Facility Core Units is greatly acknowledged. This work was supported by the Ministry of Science and Innovation of Spain Grants BIO2009-09488 and BIO2006-03213 (to S.O.) and by the CNIO.Peer reviewe

Pericytes regulate VEGF-induced endothelial sprouting through VEGFR1.

Pericytes adhere to the abluminal surface of endothelial tubules and are required for the formation of stable vascular networks. Defective endothelial cell-pericyte interactions are frequently observed in diseases characterized by compromised vascular integrity such as diabetic retinopathy. Many functional properties of pericytes and their exact role in the regulation of angiogenic blood vessel growth remain elusive. Here we show that pericytes promote endothelial sprouting in the postnatal retinal vasculature. Using genetic and pharmacological approaches, we show that the expression of vascular endothelial growth factor receptor 1 (VEGFR1) by pericytes spatially restricts VEGF signalling. Angiogenic defects caused by pericyte depletion are phenocopied by intraocular injection of VEGF-A or pericyte-specific inactivation of the murine gene encoding VEGFR1. Our findings establish that pericytes promote endothelial sprouting, which results in the loss of side branches and the enlargement of vessels when pericyte function is impaired or lost

Wnt/β-catenin signaling regulates VE-cadherin-mediated anastomosis of brain capillaries by counteracting S1pr1 signaling.

Canonical Wnt signaling is crucial for vascularization of the central nervous system and blood-brain barrier (BBB) formation. BBB formation and modulation are not only important for development, but also relevant for vascular and neurodegenerative diseases. However, there is little understanding of how Wnt signaling contributes to brain angiogenesis and BBB formation. Here we show, using high resolution in vivo imaging and temporal and spatial manipulation of Wnt signaling, different requirements for Wnt signaling during brain angiogenesis and BBB formation. In the absence of Wnt signaling, premature Sphingosine-1-phosphate receptor (S1pr) signaling reduces VE-cadherin and Esama at cell-cell junctions. We suggest that Wnt signaling suppresses S1pr signaling during angiogenesis to enable the dynamic junction formation during anastomosis, whereas later S1pr signaling regulates BBB maturation and VE-cadherin stabilization. Our data provides a link between brain angiogenesis and BBB formation and identifies Wnt signaling as coordinator of the timing and as regulator of anastomosis.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

CNS Pericytes Modulate Local T Cell Infiltration in EAE

Pericytes at the blood–brain barrier (BBB) are located between the tight endothelial cell layer of the blood vessels and astrocytic endfeet. They contribute to central nervous system (CNS) homeostasis by regulating BBB development and maintenance. Loss of pericytes results in increased numbers of infiltrating immune cells in the CNS in experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis (MS). However, little is known about their competence to modulate immune cell activation or function in CNS autoimmunity. To evaluate the capacity of pericytes to directly interact with T cells in an antigen-specific fashion and potentially (re)shape their function, we depleted major histocompatibility complex (MHC) class II from pericytes in a cell type-specific fashion and performed T cell-pericyte cocultures and EAE experiments. We found that pericytes present antigen in vitro to induce T cell activation and proliferation. In an adoptive transfer EAE experiment, pericyte-specific MHC II KO resulted in locally enhanced T cell infiltration in the CNS; even though, overall disease course of mice was not affected. Thus, pericytes may serve as non-professional antigen-presenting cells affecting states of T cell activation, thereby locally shaping lesion formation in CNS inflammation but without modulating disease severity

Phosphoinositide 3-Kinase-Regulated pericyte maturation governs vascular remodeling

© 2020 American Heart Association, Inc.Background: Pericytes regulate vessel stabilization and function, and their loss is associated with diseases such as diabetic retinopathy or cancer. Despite their physiological importance, pericyte function and molecular regulation during angiogenesis remain poorly understood. Methods: To decipher the transcriptomic programs of pericytes during angiogenesis, we crossed Pdgfrb(BAC)-CreERT2 mice into RiboTagflox/flox mice. Pericyte morphological changes were assessed in mural cell-specific R26-mTmG reporter mice, in which low doses of tamoxifen allowed labeling of single-cell pericytes at high resolution. To study the role of phosphoinositide 3-kinase (PI3K) signaling in pericyte biology during angiogenesis, we used genetic mouse models that allow selective inactivation of PI3Kα and PI3Kβ isoforms and their negative regulator phosphate and tensin homolog deleted on chromosome 10 (PTEN) in mural cells. Results: At the onset of angiogenesis, pericytes exhibit molecular traits of cell proliferation and activated PI3K signaling, whereas during vascular remodeling, pericytes upregulate genes involved in mature pericyte cell function, together with a remarkable decrease in PI3K signaling. Immature pericytes showed stellate shape and high proliferation, and mature pericytes were quiescent and elongated. Unexpectedly, we demonstrate that PI3Kβ, but not PI3Kα, regulates pericyte proliferation and maturation during vessel formation. Genetic PI3Kβ inactivation in pericytes triggered early pericyte maturation. Conversely, unleashing PI3K signaling by means of PTEN deletion delayed pericyte maturation. Pericyte maturation was necessary to undergo vessel remodeling during angiogenesis. Conclusions: Our results identify new molecular and morphological traits associated with pericyte maturation and uncover PI3Kβ activity as a checkpoint to ensure appropriate vessel formation. In turn, our results may open new therapeutic opportunities to regulate angiogenesis in pathological processes through the manipulation of pericyte PI3Kβ activity.Dr Graupera’s laboratory is supported by the research grants SAF2017-89116R-P from Ministerio de Ciencia (Spain) cofunded by European Regional Developmental Fund (ERDF), a Way to Build Europe; by the Catalan government through the project 2017-SGR; by La Caixa Foundation (HR18-00120); by la Asociación Española contra el Cancer (AECC)-Grupos Traslacionales (GCTRA18006CARR); by la Fundación BBVA (Beca Leonardo a Investigadores y Creadores Culturales 2017); and by the People Program (Marie Curie Actions; grant agreement 317250) of the European Union’s Seventh Framework Program FP7/2007 to 2013/, and the Marie Skłodowska-Curie (grant agreement 675392) of the European Union’s Horizon 2020 research. Dr Carracedo’s laboratory is supported by the Basque Department of Industry, Tourism and Trade (Elkartek) and the Department of Education (IKERTALDE IT1106-16), the Ministerio de Ciencia (SAF2016-79381-R [FEDER/EU], Severo Ochoa Excellence Accreditation SEV-2016-0644; Excellence Networks SAF2016-81975-REDT), European Training Networks Project (H2020-MSCA-ITN-308 2016 721532), the AECC (IDEAS175CARR, GCTRA18006CARR), La Caixa Foundation (HR17-00094), and the European Research Council (StG 336343, PoC 754627, CoG 819242). Centro de Investigación Biomédica en Red Cáncer (CIBERONC) was cofunded with FEDER funds and funded by Instituto de Salud Carlos III. Dr Aransay’s laboratory is supported by the Basque Department of Industry, Tourism and Trade (Elkartek) and the Severo Ochoa Excellence Accreditation SEV-2016-0644. Dr Franco was supported by European Research Council (StG 679368), the H2020-Twinning grant (692322), the Fundação para a Ciência e a Tecnologia funding (grants IF/00412/2012; EXPL-BEX-BCM-2258-2013; PRECISE-LISBOA-01-0145-FEDER-016394), and a grant from the Fondation Leducq (17CVD03). Personal support was from Marie-Curie ITN Actions (Dr Figueiredo and Kobialka), Juan de la Cierva (IJCI-2015-23455, Dr Villacampa), and CIBERONC (A. Martinez-Romero).info:eu-repo/semantics/publishedVersio

Endothelial C-Type Natriuretic Peptide Acts on Pericytes to Regulate Microcirculatory Flow and Blood Pressure

Background: Peripheral vascular resistance has a major impact on arterial blood pressure levels. Endothelial C-type natriuretic peptide (CNP) participates in the local regulation of vascular tone, but the target cells remain controversial. The cGMP-producing guanylyl cyclase-B (GC-B) receptor for CNP is expressed in vascular smooth muscle cells (SMCs). However, whereas endothelial cell-specific CNP knockout mice are hypertensive, mice with deletion of GC-B in vascular SMCs have unaltered blood pressure. Methods: We analyzed whether the vasodilating response to CNP changes along the vascular tree, ie, whether the GC-B receptor is expressed in microvascular types of cells. Mice with a floxed GC-B (Npr2) gene were interbred with Tie2-Cre or PDGF-R-Cre(ERT2) lines to develop mice lacking GC-B in endothelial cells or in precapillary arteriolar SMCs and capillary pericytes. Intravital microscopy, invasive and noninvasive hemodynamics, fluorescence energy transfer studies of pericyte cAMP levels in situ, and renal physiology were combined to dissect whether and how CNP/GC-B/cGMP signaling modulates microcirculatory tone and blood pressure. Results: Intravital microscopy studies revealed that the vasodilatatory effect of CNP increases toward small-diameter arterioles and capillaries. CNP consistently did not prevent endothelin-1-induced acute constrictions of proximal arterioles, but fully reversed endothelin effects in precapillary arterioles and capillaries. Here, the GC-B receptor is expressed both in endothelial and mural cells, ie, in pericytes. It is notable that the vasodilatatory effects of CNP were preserved in mice with endothelial GC-B deletion, but abolished in mice lacking GC-B in microcirculatory SMCs and pericytes. CNP, via GC-B/cGMP signaling, modulates 2 signaling cascades in pericytes: it activates cGMP-dependent protein kinase I to phosphorylate downstream targets such as the cytoskeleton-associated vasodilator-activated phosphoprotein, and it inhibits phosphodiesterase 3A, thereby enhancing pericyte cAMP levels. These pathways ultimately prevent endothelin-induced increases of pericyte calcium levels and pericyte contraction. Mice with deletion of GC-B in microcirculatory SMCs and pericytes have elevated peripheral resistance and chronic arterial hypertension without a change in renal function. Conclusions: Our studies indicate that endothelial CNP regulates distal arteriolar and capillary blood flow. CNP-induced GC-B/cGMP signaling in microvascular SMCs and pericytes is essential for the maintenance of normal microvascular resistance and blood pressure

Iron accumulation drives fibrosis, senescence and the senescence-associated secretory phenotype.

Fibrogenesis is part of a normal protective response to tissue injury that can become irreversible and progressive, leading to fatal diseases. Senescent cells are a main driver of fibrotic diseases through their secretome, known as senescence-associated secretory phenotype (SASP). Here, we report that cellular senescence, and multiple types of fibrotic diseases in mice and humans are characterized by the accumulation of iron. We show that vascular and hemolytic injuries are efficient in triggering iron accumulation, which in turn can cause senescence and promote fibrosis. Notably, we find that senescent cells persistently accumulate iron, even when the surge of extracellular iron has subdued. Indeed, under normal conditions of extracellular iron, cells exposed to different types of senescence-inducing insults accumulate abundant ferritin-bound iron, mostly within lysosomes, and present high levels of labile iron, which fuels the generation of reactive oxygen species and the SASP. Finally, we demonstrate that detection of iron by magnetic resonance imaging might allow non-invasive assessment of fibrotic burden in the kidneys of mice and in patients with renal fibrosis. Our findings suggest that iron accumulation plays a central role in senescence and fibrosis, even when the initiating events may be independent of iron, and identify iron metabolism as a potential therapeutic target for senescence-associated diseases.Acknowledgements: We are grateful to K. Raj (Altos Laboratories) for his help with the experiments on replicative senescence. We thank D. Muñoz Espin (University of Cambridge) for sending us the IMR90 cells stably transduced with tamoxifen inducible Ras-G12V. We thank R. Mendez (IRB) for the H5V and HUVEC cells. We thank staf at the TEM-SEM Electron Microscopy Unit from Scientific and Technological Centers (CCiTUB), Universitat de Barcelona for their support and advice on TEM techniques. We are thankful to the Magnetic Resonance Imaging Core Facility of the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) for the scientific and technical support in MRI acquisition and analysis. M.M. received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement (no. 794744) and from the Spanish Ministry of Science and Innovation (MCIN) (RYC2020-030652-I/AEI/10.13039/501100011033). V.L.P. was a recipient of a predoctoral contract from the Spanish Ministry of Education (FPU-18/05917). K.M. was a recipient of fellowships from the German Cardiac, the German Research Foundation and a postdoctoral contract Juan de la Cierva from the MCIN. F.H.G. was supported by the PhD4MD Collaborative Research Training Program for Medical Doctors (IRB Barcelona/Hospital Clinic/IDIBAPS). M. Sanchez was funded by grants PID2021- 122436OB-I00 from MCIN/AEI/10.13039/501100011033/FEDER, UE, RETOS COLABORACION RTC2019-007074-1 from MCIN/ AEI/10.13039/501100011033. C.L.-M. was a recipient of a predoctoral contract from the Spanish Ministry of Education (FPU-18/02965). G.A. was funded by the Instituto de Salud Carlos III through project PI 20/01360, FEDER funds. J.M.C. was funded by the Instituto de Salud Carlos III through projects PI18/00910 and PI21/00931 (co-funded by European Regional Development Fund, a way to build Europe) and thanks the CERCA Programme/ Generalitat de Catalunya for institutional support. Work in the laboratory of M. Serrano was funded by the IRB and ‘laCaixa’ Foundation and by grants from the Spanish Ministry of Science co-funded by the European Regional Development Fund (SAF2017- 82613-R), European Research Council (ERC-2014-AdG/669622) and grant RETOS COLABORACION RTC2019-007125-1 from MCIN/AEI and Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement of Catalonia (Grup de Recerca consolidat 2017 SGR 282