Application of a novel statistical model for quantitative proteomics by labeling to the study of VEGF-induced angiogenesis in vascular endothelial cells

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Single-reaction multi-antigen serological test for comprehensive evaluation of SARS-CoV-2 patients by flow cytometry

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19.This work was supported by: Spanish National Research Council (CSIC-202020E079, CSIC-COVID19-028); Madrid Regional Government “IMMUNOTHERCAN” [S2017/BMD-3733-2 (MVG)]; Spanish Ministry of Science and Innovation [(MCIU/AEI/FEDER, EU, RTI2018-093569-B-I00 (MVG), SAF2017-82940-R (JMRF), SAF2017-83265-R (HTR); SAF2017-82886-R (FSM)]; Health Institute Carlos III (ISCIII) [RETICS Program RD16/0012/0006; RIER (EMGC); PI19/00549 (AA)]; “La Caixa Bank Foundation” (HR17-00016), Fondo Supera COVID (CRUE-Banco de Santander), both to FSM.Peer reviewe

Bead-assisted SARS-CoV-2 multi-antigen serological test allows effective identification of patients

Many new aspects of COVID-19 disease, including different clinical manifestations, have been identified during the pandemic. The wide array of symptoms and variation in disease severity after SARS-CoV-2 infection might be related to heterogeneity in the immune responses of different patients. Here we describe a new method for a simple multi-antigen serological test that generates a full picture of seroconversion in a single reaction. The assay is based on the detection by flow cytometry of multiple immunoglobulin classes (isotypes) specific for four SARS-CoV-2 antigens: the Spike glycoprotein (one of the highly immunogenic proteins), its RBD fragment (the major target for neutralising antibodies), the nucleocapsid protein and the main cysteine-like protease. Until now, most diagnostic serological tests measured antibodies to only one antigen and some patients seemed to not make any antibody response. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. Combining all the four antigens and using machine learning techniques, it was possible to clearly discriminate between patients and healthy controls with 100% confidence. Further, combination of antigens and different immunoglobulin isotypes in this multi-antigen assay improved the classification of patients with mild and severe disease. Introduction of this method will facilitate massive screenings of patients to evaluate their immune response. It could also support vaccination campaigns both to select non-immune individuals and to distinguish infected patients from vaccine responders.This work was supported by the Spanish National Research Council (CSIC, project numbers 202020E079 and CSIC-COVID19-028) and grants from Madrid Regional Government “IMMUNOTHERCAN” [S2017/BMD-3733-2 (MVG)]; the Spanish Ministry of Science and Innovation [(MCIU/AEI/FEDER, EU): RTI2018-093569-B-I00 (MVG), SAF2017-82940-R (JMRF), SAF2017-83265-R (HTR); SAF2017-82886-R (FSM)]; Health Institute Carlos III (ISCIII) [RETICS Program RD16/0012/0006; RIER (JMRF); PI19/00549 (AA)]. The study was also funded by “La Caixa Banking Foundation” (HR17-00016 to FSM) and Fondo Supera COVID (CRUE-Banco de Santander) to FSM.N

Estudio de la modulación de la actividad transcripcional del factor inducible en hipoxia HIF-1 por el factor de transcripción C-JUN

Tesis doctoral inédita leida en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura, 17-Enero-200

New methods for disease modeling using lentiviral vectors

Lentiviral vectors (LVs) transduce quiescent cells and provide stable integration to maintain transgene expression. Several approaches have been adopted to optimize LV safety profiles. Similarly, LV targeting has been tailored through strategies including the modification of envelope components, the use of specific regulatory elements, and the selection of appropriate administration routes. Models of aortic disease based on a single injection of pleiotropic LVs have been developed that efficiently transduce the three aorta layers in wild type mice. This approach allows the dissection of pathways involved in aortic aneurysm formation and the identification of targets for gene therapy in aortic diseases. LVs provide a fast, efficient, and affordable alternative to genetically modified mice to study disease mechanisms and develop therapeutic tools.Peer reviewe

C/EBPβ and nuclear factor of activated T cells differentially regulate Adamts-1 induction by stimuli associated with vascular remodeling

et al.Emerging evidence indicates that the metalloproteinase Adamts-1 plays a significant role in the pathophysiology of vessel remodeling, but little is known about the signaling pathways that control Adamts-1 expression. We show that vascular endothelial growth factor (VEGF), angiotensin-II, interleukin-1β, and tumor necrosis factor α, stimuli implicated in pathological vascular remodeling, increase Adamts-1 expression in endothelial and vascular smooth muscle cells. Analysis of the intracellular signaling pathways implicated in this process revealed that VEGF and angiotensin-II upregulate Adamts-1 expression via activation of differential signaling pathways that ultimately promote functional binding of the NFAT or C/EBPβ transcription factors, respectively, to the Adamts-1 promoter. Infusion of mice with angiotensin-II triggered phosphorylation and nuclear translocation of C/EBPβ proteins in aortic cells concomitantly with an increase in the expression of Adamts-1, further underscoring the importance of C/EBPβ signaling in angiotensin-II-induced upregulation of Adamts-1. Similarly, VEGF promoted NFAT activation and subsequent Adamts-1 induction in aortic wall in a calcineurin-dependent manner. Our results demonstrate that Adamts-1 upregulation by inducers of pathological vascular remodeling is mediated by specific signal transduction pathways involving NFAT or C/EBPβ transcription factors. Targeting of these pathways may prove useful in the treatment of vascular disease.This work was funded by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad; SAF2012-34296 to J.M.R. and SAF2013-45258-P to M.R.C.), the Fundación La Marató TV3 (264/C/2012 to J.M.R.), the Spanish Ministry of Health (Ministerio de Sanidad y Consumo) Red de Investigación Cardiovascular (RIC) cofounded by FEDER (grants RD06/0042/0022 to J.M.R. and RD12/0042/0023 to A.G.A), and the Spanish Council for Scientific Research (CSIC; to M.R.C.). The Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness and the Pro-CNIC Foundation. J.O. is the holder of an FPI fellowship from the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad; BES 2010-034552). The cost of this publication has been paid in part by FEDER funds.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

A major role for RCAN1 in atherosclerosis progression

This is an open access article under the terms of the Creative Commons Attribution License.-- et al.Atherosclerosis is a complex inflammatory disease involving extensive vascular vessel remodelling and migration of vascular cells. As RCAN1 is implicated in cell migration, we investigated its contribution to atherosclerosis. We show RCAN1 induction in atherosclerotic human and mouse tissues. Rcan1 was expressed in lesional macrophages, endothelial cells and vascular smooth muscle cells and was induced by treatment of these cells with oxidized LDLs (oxLDLs). Rcan1 regulates CD36 expression and its genetic inactivation reduced atherosclerosis extension and severity in Apoe-/- mice. This effect was mechanistically linked to diminished oxLDL uptake, resistance to oxLDL-mediated inhibition of macrophage migration and increased lesional IL-10 and mannose receptor expression. Moreover, Apoe-/-Rcan1-/- macrophages expressed higher-than-Apoe-/- levels of anti-inflammatory markers. We previously showed that Rcan1 mediates aneurysm development and that its expression is not required in haematopoietic cells for this process. However, transplantation of Apoe-/-Rcan1-/- bone-marrow (BM) cells into Apoe-/- recipients confers atherosclerosis resistance. Our data define a major role for haematopoietic Rcan1 in atherosclerosis and suggest that therapies aimed at inhibiting RCAN1 expression or function might significantly reduce atherosclerosis burden. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.The Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad) supports MRC, JMR and JM‐G with grants SAF2010‐15126, SAF2009‐10708 and SAF2012‐40127, respectively; MRC is also supported by the Spanish Council for Scientific Research (CSIC); JMR is also supported by Fundación La Marató TV3 (080731), and Fundación Genoma España (GENOMA). The Red de Investigacion Cardiovascular (RIC) of the Spanish Ministry of Health (Ministerio de Sanidad) supports the research of JMR, VA and JM‐G with grants RD12/0042/0022, RD12/0042/0028 and RD12/0042/0053, respectively. The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness and the Pro‐CNIC Foundation. TM was supported in part by the Leading‐edge Research Promotion Fund (LS038, TM). VE was an inve stigator of the Sara Borrell Program (CD06/00232), and NM‐B holds an FPU fellowship (FPU2008‐1500).Peer Reviewe

c-Jun and Hypoxia-Inducible Factor 1 Functionally Cooperate in Hypoxia-Induced Gene Transcription

Under low-oxygen conditions, cells develop an adaptive program that leads to the induction of several genes, which are transcriptionally regulated by hypoxia-inducible factor 1 (HIF-1). On the other hand, there are other factors which modulate the HIF-1-mediated induction of some genes by binding to cis-acting motifs present in their promoters. Here, we show that c-Jun functionally cooperates with HIF-1 transcriptional activity in different cell types. Interestingly, a dominant-negative mutant of c-Jun which lacks its transactivation domain partially inhibits HIF-1-mediated transcription. This cooperative effect is not due to an increase in the nuclear amount of the HIF-1α subunit, nor does it require direct binding of c-Jun to DNA. c-Jun and HIF-1α are able to associate in vivo but not in vitro, suggesting that this interaction involves the participation of additional proteins and/or a posttranslational modification of these factors. In this context, hypoxia induces phosphorylation of c-Jun at Ser(63) in endothelial cells. This process is involved in its cooperative effect, since specific blockade of the JNK pathway and mutation of c-Jun at Ser(63) and Ser(73) impair its functional cooperation with HIF-1. The functional interplay between c-Jun and HIF-1 provides a novel insight into the regulation of some genes, such as the one for VEGF, which is a key regulator of tumor angiogenesis