Assessing the impact of an antigen-specific antibody response on atherosclerosis development in mice.

The antibody immune response plays a critical role in atherosclerosis. Here, we present a protocol for assessing the impact of an antigen-specific germinal center antibody response on atherosclerosis development, using a pro-atherogenic mouse model deficient for the production of germinal-center-derived antibodies. We describe steps for bone marrow transfer from donor mice into irradiated recipient mice. We then detail immunization of mouse chimeras with atheroprotective malondialdehyde low-density lipoprotein during high-fat diet feeding and atherosclerosis burden analysis. For complete details on the use and execution of this protocol, please refer to Martos-Folgado et al. (2022).1.We thank all members of the B Cell Biology Laboratory for useful discussions. A.D.M.M. is funded by ‘‘la Caixa’’ Foundation HR17-00247, I.M.-F. was a fellow of the research training program funded by Ministerio de Economía y Competitividad (SVP-2014-068216), A.R-R. is a fellow of the research training program funded by Ministerio de Ciencia, Innovacio´ n y Universidades (PRE2020-091873), and A.R. is supported by Centro Nacional de Investigaciones Cardiovasculares (CNIC). The project leading to these results has received funding from ‘‘la Caixa’’ Foundation under the project code HR17-00247 and HR22-0253 and from SAF2016-75511-R and PID2019-106773RB-I00/AEI/ 10.13039/501100011033 grants to A.R.R. (Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016 Programa Estatal de I+D+i Orientada a los Retos de la Sociedad Retos Investigación: Proyectos I + D + i 2016, Ministerio de Economía, Industria y Competitividad) and co-funding by Fondo Europeo de Desarrollo Regional (FEDER). 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 institute (CEX2020-001041-S grant funded by MCIN/AEI/ 10.13039/501100011033).S

Regulación de la expresión de lamina A/C en la célula T y su papel en la diferenciación de CD4+ “naïve” a célula efectora

La lamina A/C es una proteína nuclear con funciones estructurales y de regulación de la señalización celular y la transcripción de genes pero no se ha estudiado en detalle su papel en el sistema inmune. Los linfocitos T CD4+ “naïve” reconocen un antígeno presentado por una célula presentadora de antígeno (APC), se activan, proliferan y se diferencian a células efectoras para llevar a cabo funciones de respuesta inmune adaptativa. En la activación del linfocito T se producen eventos de señalización celular y transcripción de genes. Estudios previos en el laboratorio han mostrado que la lamina A/C se expresa en el linfocito T tras el reconocimiento de un antígeno para regular el grado de activación posterior del linfocito T. Como continuación, en este estudio se ha pretendido conocer las vías de señalización implicadas en la expresión de lamina A/C en el linfocito T así como establecer un posible papel de la lamina A/C en el proceso de diferenciación del linfocito T “naïve” a célula T efectora (Th). Para ello, con técnicas de citometría e inmunofluorescencia se ha analizado la expresión de lamina A/C en células T CD4+ de ratón estimulados a diferentes tiempos con y sin inhibidores de diferentes vías de señalización. Estos experimentos han mostrado una expresión transitoria de lamina A/C con un máximo a un día tras laestimulación así como un posible papel las vías de Src quinasas y de MAPK: ERK1/2 y p38 en esta inducción. El análisis por citometría de ensayos de activación, proliferación y diferenciación in vitro de células T CD4+ “naïve” de ratones WT y deficientes en lamina A/C ha mostrado que la lamina A/C tiene un papel fundamental en el proceso de diferenciación de la célula T CD4+ ”naïve” a Th

Disruption of the CCL1-CCR8 axis inhibits vascular Treg recruitment and function and promotes atherosclerosis in mice

The CC chemokine 1 (CCL1, also called I-309 or TCA3) is a potent chemoattractant for leukocytes that plays an important role in inflammatory processes and diseases through binding to its receptor CCR8. Here, we investigated the role of the CCL1-CCR8 axis in atherosclerosis. We found increased expression of CCL1 in the aortas of atherosclerosis-prone fat-fed apolipoprotein E (Apoe)-null mice; moreover, in vitro flow chamber assays and in vivo intravital microscopy demonstrated an essential role for CCL1 in leukocyte recruitment. Mice doubly deficient for CCL1 and Apoe exhibited enhanced atherosclerosis in aorta, which was associated with reduced plasma levels of the anti-inflammatory interleukin 10, an increased splenocyte Th1/Th2 ratio, and a reduced regulatory T cell (Treg) content in aorta and spleen. Reduced Treg recruitment and aggravated atherosclerosis were also detected in the aortas of fat-fed low-density lipoprotein receptor-null mice treated with CCR8 blocking antibodies. These findings demonstrate that disruption of the CCL1-CCR8 axis promotes atherosclerosis by inhibiting interleukin 10 production and Treg recruitment and function.This study was supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (MCIU, grants SAF2016-79490-R and SAF2014-57845-R) and the Instituto de Salud Carlos III (ISCIII, grants PI14/00526, PI17/01395, CP11/00145, and CPII16/00022) with co-funding from the European Regional Development Fund (ERDF, “Una manera de hacer Europa”), the Fundación Ramón Areces, European Union (EuroCellNet COST Action CA15214) and the INSERM. VZG is supported by the ISCIII, JMG-G by the ISCIII Miguel Servet Program and the Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), AdMM by the MCIU (predoctoral contract BES-2014-06779), and ZM by a British Heart Foundation Professorship. The CNIC is supported by the MCIU and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

A survey of the clinicopathological and molecular characteristics of patients with suspected Lynch syndrome in Latin America

Background: Genetic counselling and testing for Lynch syndrome (LS) have recently been introduced in several Latin America countries. We aimed to characterize the clinical, molecular and mismatch repair (MMR) variants spectrum of patients with suspected LS in Latin America. Methods: Eleven LS hereditary cancer registries and 34 published LS databases were used to identify unrelated families that fulfilled the Amsterdam II (AMSII) criteria and/or the Bethesda guidelines or suggestive of a dominant colorectal (CRC) inheritance syndrome. Results: We performed a thorough investigation of 15 countries and identified 6 countries where germline genetic testing for LS is available and 3 countries where tumor testing is used in the LS diagnosis. The spectrum of pathogenic MMR variants included MLH1 up to 54%, MSH2 up to 43%, MSH6 up to 10%, PMS2 up to 3% and EPCAM up to 0.8%. The Latin America MMR spectrum is broad with a total of 220 different variants which 80% were private and 20% were recurrent. Frequent regions included exons 11 of MLH1 (15%), exon 3 and 7 of MSH2 (17 and 15%, respectively), exon 4 of MSH6 (65%), exons 11 and 13 of PMS2 (31% and 23%, respectively). Sixteen international founder variants in MLH1, MSH2 and MSH6 were identified and 41 (19%) variants have not previously been reported, thus representing novel genetic variants in the MMR genes. The AMSII criteria was the most used clinical criteria to identify pathogenic MMR carriers although microsatellite instability, immunohistochemistry and family history are still the primary methods in several countries where no genetic testing for LS is available yet. Conclusion: The Latin America LS pathogenic MMR variants spectrum included new variants, frequently altered genetic regions and potential founder effects, emphasizing the relevance implementing Lynch syndrome genetic testing and counseling in all of Latin America countries.Radium Hospital Foundation (Oslo, Norway) in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript, Helse Sør-Øst (Norway) in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript, the French Association Recherche contre le Cancer (ARC) in the analysis, and interpretation of data, the Groupement des Entreprises Françaises dans la Lutte contre le Cancer (Gefluc) in the analysis, and interpretation of data, the Association Nationale de la Recherche et de la Technologie (ANRT, CIFRE PhD fellowship to H.T.) in the analysis, and interpretation of data and by the OpenHealth Institute in the analysis, and interpretation of data. Barretos Cancer Hospital received financial support by FINEP-CT-INFRA (02/2010)info:eu-repo/semantics/publishedVersio

Papel de la Lamina A/C en la enfermedad cardiovascular asociada al envejecimiento.

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 23-10-2020Esta tesis tiene embargado el acceso al texto completo hasta el 23-04-2022Las enfermedades cardiovasculares (ECVs) son la primera causa de mortalidad en el mundo. El envejecimiento y la hipercolesterolemia son dos factores de riesgo cardiovascular muy importantes. La lamina A/C es una proteína nuclear codificada por el gen LMNA que juega un papel estructural y regulador de diversas funciones celulares. El objetivo de este trabajo de Tesis Doctoral ha sido estudiar el papel del envejecimiento y la hipercolesterolemia sobre la expresión de lamina A/C y su posible efecto sobre la ECV asociada. Por un lado, observamos un descenso en los niveles de lamina A/C en el sistema inmune humano y de ratón durante el envejecimiento. Además, demostramos que la hipercolesterolemia está asociada a una disminución de la expresión de lamina A/C en leucocitos de ratón. Experimentos funcionales de trasplante de ratones deficientes en Ldlr (Ldlr-/-, del inglés Low-density lipoprotein receptor) utilizando médula ósea (MO) Lmna-/- y silvestre (WT: Wild-type) (MO Lmna-/- �� Ldlr-/- y MO WT �� Ldlr-/-, respectivamente) demostraron un aumento en el desarrollo de arteriosclerosis en la arteria aorta y en arterias carótidas de los ratones MO Lmna-/- �� Ldlr-/- alimentados con dieta grasa (DG) en comparación con los controles MO WT �� Ldlr-/-, sin que los parámetros fisiológicos básicos estuvieran alterados. El aumento de arteriosclerosis en ratones hipercolesterolémicos carentes de lamina A/C en las células del sistema inmune estaba asociado a un aumento de la extravasación de leucocitos y a una menor velocidad de movimiento de los leucocitos extravasados. De acuerdo con los resultados anteriores, demostramos mediante experimentos in vitro que los monocitos proinflamatorios Lmna-/- migraban con mayor facilidad a través de constricciones de pequeño tamaño, pero eran más lentos al desplazarse en ausencia de constricciones. Por otra parte, observamos un descenso en los niveles de lamina A/C durante el envejecimiento en tejido arterial humano y de ratón debido a una disminución de la expresión en células de músculo liso vascular (CMLVs) y células endoteliales. Para profundizar en el papel de lamina A/C en el desarrollo de ECV, desarrollamos el modelo de ratón Lmnaflox/flox SM22αCre, deficiente en lamina A/C en CMLVs, cardiomiocitos, y fibroblastos cardíacos. En comparación con los controles, los ratones Lmnaflox/flox SM22αCre morían prematuramente (vida media ≈ 5 semanas) y mostraban una pérdida grave de función cardíaca en ambos ventrículos, así como problemas de conducción eléctrica en el corazón. Además, los ratones Lmnaflox/flox SM22αCre mostraron fibrosis cardíaca asociada a la expresión exacerbada de marcadores de miofibroblastos (FSP- 1 y SMA). En resumen, nuestros resultados sugieren que el descenso de la lamina A/C contribuye al desarrollo de ECVs asociadas al envejecimiento e hipercolesterolemia. Serán necesarios más estudios para determinar los mecanismos moleculares que subyacen a esta nueva función reguladora de la lamina A/C en el envejecimiento y la ECVCardiovascular disease (CVD) is the main cause of mortality worldwide. Aging and hypercholesterolemia are critical cardiovascular risk factors. Lamin A/C, encoded by the LMNA gene, are nuclear proteins that play important structural roles and regulate a broad range of cellular function. In this Doctoral Thesis, we studied the regulation of lamin A/C by aging and hypercholesterolemia, and its possible role in CVD. We observed an age dependent decrease in lamin A/C expression in human and mouse immune cells. Likewise, hypercholesterolemia is associated with lower lamin A/C expression in mouse leukocytes. We next performed functional studies by reconstituting lethally-irradiated low-density lipoprotein receptor mice (Ldlr-/-) with wild-type or Lmna-null (Lmna-/-) bone marrow (BM-WT Ldlr-/-, BM-Lmna-/- Ldlr-/-, respectively). We found higher atherosclerosis burden in aorta and carotid arteries of BM-Lmna-/- Ldlr-/-mice fed high fat diet for 6 weeks compared with BM-WT Ldlr-/- controls, without significant changes in circulating blood cell populations, body weight and lipid profile. Augmented atherosclerosis in BM- Lmna-/- Ldlr-/- mice was associated with increased leukocyte migration, although their migration velocity within the tissue was lower. Consistent with these findings, Lmna-/- proimmflamatory monocytes migrated in vitro more easily through small constrictions but exhibited slower velocity when passing through straight channels without constrictions. We also found a significant downregulation of lamin A/C in aged mouse and human arterial tissue, due to diminished expression in both smooth muscle cells (SMCs) and endothelial cells. To further investigate the role of A-type lamins in CVD, we generated a mouse model with genetic disruption of Lmna specifically in SMCs, cardiomyocytes and cardiac fibroblasts (Lmnaflox/flox SM22αCre). Compared with controls, Lmnaflox/flox SM22αCre mice died prematurely (average life span: 5 weeks) and exhibited cardiac alterations, including defective conduction and repolarization, severe left ventricle and right ventricle dysfunction. Remarkably, Lmnaflox/flox SM22αCre developed prominent interstitial cardiac fibrosis associated with augmented expression of FSP-1 and SMA. Collectively, our results suggest that lamin A/C downregulation contributes to CVD during aging and in the setting of hypercholesterolemia. Future studies will examine the molecular mechanisms underlying these new regulatory functions of lamin A/C in aging and CV

Papel de la lamina A/C en la enfermedad cardiovascular asociada al envejecimiento

Las enfermedades cardiovasculares (ECVs) son la primera causa de mortalidad en el mundo. El envejecimiento y la hipercolesterolemia son dos factores de riesgo cardiovascular muy importantes. La lamina A/C es una proteína nuclear codificada por el gen LMNA que juega un papel estructural y regulador de diversas funciones celulares. El objetivo de este trabajo de Tesis Doctoral ha sido estudiar el papel del envejecimiento y la hipercolesterolemia sobre la expresión de lamina A/C y su posible efecto sobre la ECV asociada. Por un lado, observamos un descenso en los niveles de lamina A/C en el sistema inmune humano y de ratón durante el envejecimiento. Además, demostramos que la hipercolesterolemia está asociada a una disminución de la expresión de lamina A/C en leucocitos de ratón. Experimentos funcionales de trasplante de ratones deficientes en Ldlr (Ldlr-/-, del inglés Low-density lipoprotein receptor) utilizando médula ósea (MO) Lmna-/- y silvestre (WT: Wild-type) (MO Lmna-/- Ldlr-/- y MO WT Ldlr-/-, respectivamente) demostraron un aumento en el desarrollo de arteriosclerosis en la arteria aorta y en arterias carótidas de los ratones MO Lmna-/- Ldlr-/- alimentados con dieta grasa (DG) en comparación con los controles MO WT Ldlr-/-, sin que los parámetros fisiológicos básicos estuvieran alterados. El aumento de arteriosclerosis en ratones hipercolesterolémicos carentes de lamina A/C en las células del sistema inmune estaba asociado a un aumento de la extravasación de leucocitos y a una menor velocidad de movimiento de los leucocitos extravasados. De acuerdo con los resultados anteriores, demostramos mediante experimentos in vitro que los monocitos proinflamatorios Lmna-/- migraban con mayor facilidad a través de constricciones de pequeño tamaño, pero eran más lentos al desplazarse en ausencia de constricciones. Por otra parte, observamos un descenso en los niveles de lamina A/C durante el envejecimiento en tejido arterial humano y de ratón debido a una disminución de la expresión en células de músculo liso vascular (CMLVs) y células endoteliales. Para profundizar en el papel de lamina A/C en el desarrollo de ECV, desarrollamos el modelo de ratón Lmnaflox/flox SM22αCre, deficiente en lamina A/C en CMLVs, cardiomiocitos, y fibroblastos cardíacos. En comparación con los controles, los ratones Lmnaflox/flox SM22αCre morían prematuramente (vida media ≈ 5 semanas) y mostraban una pérdida grave de función cardíaca en ambos ventrículos, así como problemas de conducción eléctrica en el corazón. Además, los ratones Lmnaflox/flox SM22αCre mostraron fibrosis cardíaca asociada a la expresión exacerbada de marcadores de miofibroblastos (FSP- 1 y SMA). En resumen, nuestros resultados sugieren que el descenso de la lamina A/C contribuye al desarrollo de ECVs asociadas al envejecimiento e hipercolesterolemia. Serán necesarios más estudios para determinar los mecanismos moleculares que subyacen a esta nueva función reguladora de la lamina A/C en el envejecimiento y la ECVS

Lamin A/C Ablation Restricted to Vascular Smooth Muscle Cells, Cardiomyocytes, and Cardiac Fibroblasts Causes Cardiac and Vascular Dysfunction.

Mutations in the LMNA gene (encoding lamin A/C proteins) cause several human cardiac diseases, including dilated cardiomyopathies (LMNA-DCM). The main clinical risks in LMNA-DCM patients are sudden cardiac death and progressive left ventricular ejection fraction deterioration, and therefore most human and animal studies have sought to define the mechanisms through which LMNA mutations provoke cardiac alterations, with a particular focus on cardiomyocytes. To investigate if LMNA mutations also cause vascular alterations that might contribute to the etiopathogenesis of LMNA-DCM, we generated and characterized Lmnaflox/floxSM22αCre mice, which constitutively lack lamin A/C in vascular smooth muscle cells (VSMCs), cardiac fibroblasts, and cardiomyocytes. Like mice with whole body or cardiomyocyte-specific lamin A/C ablation, Lmnaflox/floxSM22αCre mice recapitulated the main hallmarks of human LMNA-DCM, including ventricular systolic dysfunction, cardiac conduction defects, cardiac fibrosis, and premature death. These alterations were associated with elevated expression of total and phosphorylated (active) Smad3 and cleaved (active) caspase 3 in the heart. Lmnaflox/floxSM22αCre mice also exhibited perivascular fibrosis in the coronary arteries and a switch of aortic VSMCs from the 'contractile' to the 'synthetic' phenotype. Ex vivo wire myography in isolated aortic rings revealed impaired maximum contraction capacity and an altered response to vasoconstrictor and vasodilator agents in Lmnaflox/floxSM22αCre mice. To our knowledge, our results provide the first evidence of phenotypic alterations in VSMCs that might contribute significantly to the pathophysiology of some forms of LMNA-DCM. Future work addressing the mechanisms underlying vascular defects in LMNA-DCM may open new therapeutic avenues for these diseases.This study was supported by grants SAF2016-79490-R and PID2019-108489RB-I00 from the Spanish Ministerio de Ciencia e Innovación (MCIN)/Agencia Estatal de Investigación (AEI)/10.13039/ 501100011033, with co-funding from the European Social Fund (“The ESF invests in your future”). Microscopy was conducted at the Microscopy and Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/10.13039/501100011033. A.D.M.-M. was supported by the MCIN (predoctoral contract BES-2014-067791), C.E.-E. and V.F. by the Fundación “la Caixa” (predoctoral contracts LCF/BQ/DR19/1170012 and LCF/BQ/DE14/10320024, respectively), Í.R.-P.d.L. by MCIN/AEI/ 10.13039/501100011033 and the European Social Fund (“The ESF invests in your future”) (predoctoral contract PRE2020-092264), and M.G.-A. by MCIN (post-doctoral contract FJC 2021-047576-I). The CNIC is supported by the MCIN, the Instituto de Salud Carlos III, and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (grant number CEX2020-001041-S funded by MCIN/AEI/10.13039/501100011033).S

Endothelial NOD1 directs myeloid cell recruitment in atherosclerosis through VCAM-1

Atherosclerosis is a chronic disease characterized by vascular lipid retention and inflammation, and pattern recognition receptors (PRRs) are important contributors in early stages of the disease. Given the implication of the intracellular PRR nucleotide-binding oligomerization domain 1 (NOD1) in cardiovascular diseases, we investigated its contribution to early atherosclerosis. We evidenced NOD1 induction in atherosclerotic human and mouse tissues, predominantly in vascular endothelial cells. Accordingly, NOD1 genetic inactivation in Apoe-/- mice reduced not only atherosclerosis burden, but also monocyte and neutrophil accumulation in atheromata. Of note, in the presence of either peptidoglycan or oxidized LDLs, endothelial NOD1 triggered VCAM-1 up-regulation through the RIP2-NF-¿B axis in an autocrine manner, enhancing firm adhesion of both sets of myeloid cells to the inflamed micro- and macrovasculature in vivo. Our data define a major proatherogenic role for endothelial NOD1 in early leukocyte recruitment to the athero-prone vasculature, thus introducing NOD1 as an innovative therapeutic target and potential prognostic moleculeThis work was supported bythe the Ministry of Science, Innovation and Universities(Grants SAF2015-64767-R, SAF2016-79490-R, SAF2015-70747-R, SAF2016-75004-R, SAF2017-82436-R/RTC2017-6283-1), the Center for Biomedical Research Network Consortium in Cardiovascular Diseases (Grants CB16/11/00257, CB16/11/00405, CB/11/00222), Community of Madrid Grant BMD/3686, the German Research Foundation (Grants SFB914 TPA02/B08/B09/Z03, SFB1123 TP A06/B05/B08/Z01), and the Netherlands Organization for Scientific Research (VIDI proj-ect 91712303). This project was also financed, in part, by theEuropean Regional Development Fund. O.S. and L.B. con-tributed equally as senior authors

MDA-LDL vaccination induces athero-protective germinal-center-derived antibody responses.

Atherosclerosis is a chronic inflammatory disease of the arteries that can lead to thrombosis, infarction, and stroke and is the leading cause of mortality worldwide. Immunization of pro-atherogenic mice with malondialdehyde-modified low-density lipoprotein (MDA-LDL) neo-antigen is athero-protective. However, the immune response to MDA-LDL and the mechanisms responsible for this athero-protection are not completely understood. Here, we find that immunization of mice with MDA-LDL elicits memory B cells, plasma cells, and switched anti-MDA-LDL antibodies as well as clonal expansion and affinity maturation, indicating that MDA-LDL triggers a bona fide germinal center antibody response. Further, Prdm1fl/flAicda-Cre+/kiLdlr-/- pro-atherogenic chimeras, which lack germinal center-derived plasma cells, show accelerated atherosclerosis. Finally, we show that MDA-LDL immunization is not athero-protective in mice lacking germinal-center-derived plasma cells. Our findings give further support to the development of MDA-LDL-based vaccines for the prevention or treatment of atherosclerosis.We thank all members of the B Cell Biology Laboratory for useful discussions, A. Rodriguez-Ronchel for the design of the graphical abstract, V.G. de Ye´benes for critical reading of the manuscript, and V. Labrador for help with microscopy and image analysis. I.M.-F. and C.L. were fellows of the research training program funded by Ministerio de Economı´a y Competitividad (SVP-2014-068216 and SVP-2014-068289, respectively), A.d.M.M. is funded by ‘‘la Caixa’’ Foundation HR17–00247, and A.R.R. was supported by Centro Nacional de Investigaciones Cardiovasculares (CNIC). The project leading to these results has received funding from la Caixa Banking Foundation under the project code HR17-00247 and from SAF2016-75511-R and PID2019-106773RB-I00/AEI/10.13039/501100 011033 grants to A.R.R. (Plan Estatal de Investigacio´ n Cientı´fica y Te´ cnica y de Innovacio´ n 2013–2016 Programa Estatal de I+D+i Orientada a los Retos de la Sociedad Retos Investigacio´ n: Proyectos I + D + i 2016, Ministerio de Economı´a,Industria y Competitividad) and co-funding by Fondo Europeo de Desarrollo Regional (FEDER). CIBERCV (J.L.M.-V.) and CIBERDEM (J.C.E.-G.) are Instituto de Salud Carlos III projects. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovacio´ n (MCIN), and the Pro CNIC Foundation and is a Severo Ochoa institute (CEX2020-001041-S grant funded by MCIN/AEI/10.13039/501100011033).S