Funciones de Calcineurina y Rcan1 en el remodelado patológico de la pared vascular

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 18-07-2014El remodelado de la pared vascular es un distintivo común que se observa en patologías y procesos como hipertensión, restenosis, aterosclerosis y aneurismas. Este remodelado que implica cambios en la masa de células de la capa media y afecta al tamaño del lumen del vaso, se caracteriza por la proliferación y migración de células de músculo liso vascular (CMLVs), la acumulación de elementos de matriz extracelular, y la presencia de células inflamatorias. Aunque la angiotensina II (AngII) desempeña un papel central en el remodelado vascular, en procesos como la formación de aneurismas y de neoíntima, aún se conoce poco de los mecanismos implicados en su participación. La identificación de moléculas efectoras de AngII implicadas en estos procesos podría ayudar al desarrollo de terapias que mejorasen el tratamiento de estas enfermedades. En este trabajo mostramos que AngII induce la migración de CMLVs y el desarrollo de aneurisma abdominal aórtico (AAA) y restenosis y que estos efectos son bloqueados por el tratamiento farmacológico con inhibidores de calcineurina (CN) o por la administración de péptidos inhibidores de CN mediante partículas lentivirales. El análisis transcriptómico de genoma completo en CMLVs reveló que >1500 genes son regulados por AngII, y que solo 11 de ellos requieren de la activación de CN para su regulación. De todos estos genes, el denominado regulador de CN 1 (Rcan1) es el gen activado por AngII más dependiente de CN. Rcan1 se induce tanto in vitro como in vivo por AngII, y es necesario para la migración inducida por AngII en las CMLVs. Observamos que los ratones Rcan1-/- son resistentes al desarrollo de AAA inducidos por infusión de AngII, y a la formación de neoíntima producida por daño femoral. También mostramos la inducción de la expresión de RCAN1 en lesiones ateroscleróticas humanas y de ratón, y que se induce en CMLVs, macrófagos y células endoteliales por su tratamiento con lipoproteínas de baja densidad oxidadas (LDLox). Mostramos que los ratones Rcan1-/- presentan menos área de lesión y ateromas en estadios menos avanzados. Describimos que Rcan1 regula en macrófagos la expresión de CD36, la captación de LDLox, la migración, y sus características inflamatorias. Nuestros resultados indican que aunque Rcan1 está implicado en el desarrollo de aneurismas, su expresión en células hematopoyéticas no es esencial para este proceso. También, mostramos, sin embargo, que el trasplante de médula ósea Rcan1-/- en ratones Rcan1+/+ confiere resistencia al desarrollo de placas de ateroma, definiendo así una función esencial de Rcan1 en células hematopoyéticas en esta patología. Todos estos resultados aportan una nueva visión de Rcan1 como diana terapéutica en enfermedades de remodelado vascular como los AAA, la restenosis y la aterosclerosis.Artery wall remodeling, a major feature of diseases such as hypertension, restenosis, atherosclerosis, and aneurysm, involves cellular processes including altered vascular smooth muscle cell (VSMCs) growth, migration and differentiation, increased extracellular matrix deposition and inflammation. Pathological vessel remodeling triggers changes in the tunica media mass that reduce or increase the vessel lumen. Angiotensin II (AngII) is a key effector of aortic wall remodeling that contributes to aneurysm formation and restenosis through incompletely defined signaling pathways. The identification of molecules involved in vessel remodeling could aid the development of improved treatments for these pathologies. Here we have showed that AngII induces VSMC migration and vessel remodeling in mouse models of restenosis and aneurysm. These effects were prevented by pharmacological inhibition of calcineurin (CN) or lentiviral delivery of CN-inhibitory peptides. Whole-genome analysis revealed >1,500 AngII-regulated genes in VSMCs, with just 11 of them requiring CN activation. Of these, the most sensitive to CN activation was regulator of CN 1 (Rcan1). Rcan1 was strongly activated by AngII in vitro and in vivo and was required for AngII-induced VSMC migration. Remarkably, Rcan1-/- mice were resistant to AngII-induced aneurysm and neoíntima formation after femoral injury. Atherosclerosis, a complex inflammatory disease, also involves extensive vascular vessel remodeling and migration of vascular cells. We also show that RCAN1 is induced, in human and mouse atheroma lesion. Indeed Rcan1 is induced in lesional macrophages, endothelial cells and VSMCs by treatment of these cells with oxidized LDLs (oxLDLs). Rcan1 genetic inactivation diminished atherosclerosis lesion severity and extension in Apoe-/- mice. We show that this effect was mechanistically linked to CD36 downregulation, diminished oxLDL uptake resistance to oxLDL-mediated inhibition of macrophage migration and to induction of antiinflammatory properties in macrophages. By using bone marrow transplantation, we show that Rcan1 expression is not required in hematopoietic cells for aneurysm development. In contrast, our data indicate that transplantation of Apoe-/-Rcan1-/- bone marrow cells into Apoe-/- recipients confers atherosclerosis resistance, thus defining a major role for Rcan1 in hematopoietic cells in atherosclerosis progression. Together, our findings provide a new vision of Rcan1 as an essential target in pathological vessel remodeling, and support that therapies aimed at inhibiting RCAN1 expression or function might significantly reduce aneurysm, restenosis and atherosclerosis burde

A major role of TWEAK/Fn14 axis as a therapeutic target for post-angioplasty restenosis

Background: Tumor necrosis factor-like weak inducer of apoptosis (Tnfsf12; TWEAK) and its receptor Fibroblast growth factor-inducible 14 (Tnfrsf12a; Fn14) participate in the inflammatory response associated with vascular remodeling.However, the functional effect ofTWEAK on vascular smoothmuscle cells (VSMCs) is not completely elucidated. Methods: Next generation sequencing-based methodswere performed to identify genes and pathways regulated by TWEAK in VSMCs. Flow-citometry, wound-healing scratch experiments and transwellmigration assays were used to analyze VSMCs proliferation and migration. Mouse wire injury model was done to evaluate the role of TWEAK/Fn14 during neointimal hyperplasia. Findings: TWEAK up-regulated 1611 and down-regulated 1091 genes in VSMCs. Using a gene-set enrichment method,we found a functionalmodule involved in cell proliferation defined as the minimal network connecting top TWEAK up-regulated genes. In vitro experiments in wild-type or Tnfrsf12a deficient VSMCs demonstrated that TWEAK increased cell proliferation, VSMCs motility and migration. Mechanistically, TWEAK increased cyclins (cyclinD1), cyclin-dependent kinases (CDK4, CDK6) and decreased cyclin-dependent kinase inhibitors (p15lNK4B) mRNA and protein expression. Downregulation of p15INK4B induced by TWEAK was mediated by mitogen-activated protein kinase ERK and Akt activation. Tnfrsf12a or Tnfsf12 genetic depletion and pharmacological intervention with TWEAK blocking antibody reduced neointimal formation, decreasing cell proliferation, cyclin D1 and CDK4/6 expression, and increasing p15INK4B expression compared with wild type or IgG-treated mice in wire-injured femoral arteries. Finally, immunohistochemistry in human coronary arteries with stenosis or in-stent restenosis revealed high levels of Fn14, TWEAK and PCNA in VSMCs enriched areas of the neointima as compared with healthy coronary arteries. Interpretation: Our data define a major role of TWEAK/Fn14 in the control of VSMCs proliferation and migration during neointimal hyperplasia after wire injury in mice, and identify TWEAK/Fn14 as a potential target for treating in-stent restenosis.This work was supported by Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias ISCiii/FEDER PI13/00395; PI16/01419; PI17/ 01495) and Spanish Biomedical Research Centre in Cardiovascular Disease (CIBERCV) and Metabolic Diseases and Diabetes (CIBERDEM). PM was supported by ISCIII Miguel Servet Program (CP16/00116). CGM was supported by Fundación Conchita Rábago. NMB and VE were supported by the Spanish Ministry of Economy and Competitiveness (Juan de la Cierva IJCI-2016-29630 and Ramón y Ramón Cajal Program RyC-2013-12880, respectively). JMM has been supported a postdoctoral fellowship fromthe American Diabetes Association (Grant 1-15-MI-03) and a postdoctoral fellowship fromthe American Heart Association

The impact of type 2 immunity and allergic diseases in atherosclerosis.

Allergic diseases are allergen-induced immunological disorders characterized by the development of type 2 immunity and IgE responses. The prevalence of allergic diseases has been on the rise alike cardiovascular disease (CVD), which affects arteries of different organs such as the heart, the kidney and the brain. The underlying cause of CVD is often atherosclerosis, a disease distinguished by endothelial dysfunction, fibrofatty material accumulation in the intima of the artery wall, smooth muscle cell proliferation, and Th1 inflammation. The opposed T-cell identity of allergy and atherosclerosis implies an atheroprotective role for Th2 cells by counteracting Th1 responses. Yet, the clinical association between allergic disease and CVD argues against it. Within, we review different phases of allergic pathology, basic immunological mechanisms of atherosclerosis and the clinical association between allergic diseases (particularly asthma, atopic dermatitis, allergic rhinitis and food allergy) and CVD. Then, we discuss putative atherogenic mechanisms of type 2 immunity and allergic inflammation including acute allergic reactions (IgE, IgG1, mast cells, macrophages and allergic mediators such as vasoactive components, growth factors and those derived from the complement, contact and coagulation systems) and late phase inflammation (Th2 cells, eosinophils, type 2 innate-like lymphoid cells, alarmins, IL-4, IL-5, IL-9, IL-13 and IL-17).Severo Ochoa Center of Excellence, Grant/Award Number: CEX2020-001041- S; Pro CNIC Foundation; Ministerio de Ciencia e Innovación; Ministry of Science and Innovation, Grant/ Award Number: PID2019-110369RB- I00; European Commission, Grant/Award Number: ERC-CoG 819775 and H2020-HEALTH 945118; Spanish Ministry of Universities; Ayudas Margarita Salas para la Formación de Jóvenes Doctores—Universidad Autónoma de Madrid, Grant/ Award Number: CA1/RSUE/2021–00577; Formación de Profesorado Universitario, Grant/Award Number: FPU16/03953; Sociedad Española de Alergología e Inmunología Clínica (SEAIC), Grant/ Award Number: BECA20A9; New Frontiers in Research Fund, Grant/ Award Number: NFRFE-2019- 00083; The Nutricia Research Foundation, Grant/Award Number: NRF-2021- 13; Instituto de Salud Carlos III, Grant/Award Number: PI21/00158, PI21/01126, CP20/00043, PI18/01467, PI19/00044, RD16/0006/0015 and RD21/0002/0008; Severo Ochoa Program, Grant/Award Number: AEI/SEV-2017- 0712S

Mcp-1 predicts recurrent cardiovascular events in patients with persistent inflammation

Clinical data indicate that patients with C-reactive protein (CRP) levels higher than 2 mg per liter suffer from persistent inflammation, which is associated with high risk of cardiovascular disease (CVD). We determined whether a panel of biomarkers associated with CVD could predict recurrent events in patients with low or persistent inflammation and coronary artery disease (CAD). We followed 917 patients with CAD (median 4.59 ± 2.39 years), assessing CRP, galectin-3, monocyte chemoattractant protein-1 (MCP-1), N-terminal fragment of brain natriuretic peptide (NT-proBNP) and troponin-I plasma levels. The primary outcome was the combination of cardiovascular events (acute coronary syndrome, stroke or transient ischemic event, heart failure or death). Patients with persistent inflammation (n = 343) showed higher NT-proBNP and MCP-1 plasma levels compared to patients with CRP < 2 mg/L. Neither MCP-1 nor NT-proBNP was associated with primary outcome in patients with CRP < 2 mg/L. However, NT-proBNP and MCP-1 plasma levels were associated with increased risk of the primary outcome in patients with persistent inflammation. When patients were divided by type of event, MCP-1 was associated with an increased risk of acute ischemic events. A significant interaction between MCP-1 and persistent inflammation was found (synergy index: 6.17 (4.39–7.95)). In conclusion, MCP-1 plasma concentration is associated with recurrent cardiovascular events in patients with persistent inflammation.This research was funded by grants from Fondo de Investigaciones Sanitarias (PI14/1567, PI05/0451, PI16/01419, PI17/01615, PI17/01495, PI19/00128); RETOS-Colaboración (RTC2019-006826-1); Spanish Society of Arteriosclerosis; and Instituto de Salud Carlos III FEDER (FJD biobank: RD09/0076/00101

The impact of type 2 immunity and allergic diseases in atherosclerosis.

Allergic diseases are allergen-induced immunological disorders characterized by the development of type 2 immunity and IgE responses. The prevalence of allergic diseases has been on the rise alike cardiovascular disease (CVD), which affects arteries of different organs such as the heart, the kidney and the brain. The underlying cause of CVD is often atherosclerosis, a disease distinguished by endothelial dysfunction, fibrofatty material accumulation in the intima of the artery wall, smooth muscle cell proliferation, and Th1 inflammation. The opposed T-cell identity of allergy and atherosclerosis implies an atheroprotective role for Th2 cells by counteracting Th1 responses. Yet, the clinical association between allergic disease and CVD argues against it. Within, we review different phases of allergic pathology, basic immunological mechanisms of atherosclerosis and the clinical association between allergic diseases (particularly asthma, atopic dermatitis, allergic rhinitis and food allergy) and CVD. Then, we discuss putative atherogenic mechanisms of type 2 immunity and allergic inflammation including acute allergic reactions (IgE, IgG1, mast cells, macrophages and allergic mediators such as vasoactive components, growth factors and those derived from the complement, contact and coagulation systems) and late phase inflammation (Th2 cells, eosinophils, type 2 innate-like lymphoid cells, alarmins, IL-4, IL-5, IL-9, IL-13 and IL-17).N

Regulator of calcineurin 1 mediates pathological vascular wall remodeling

Angiotensin-II–driven calcineurin activation and regulator of calcineurin-1 (Rcan-1) expression is required for pathological vascular remodeling in mice

Procede in vitro d'identification d'anevrismesd l'aorte thoracique (AAT) chez un sujet

The present invention refers to an In vitro method for screening for subjects at risk of developing thoracic aortic aneurysm (TAA) or a disease causing TAA comprising: (a) measuring the expression pattern or level of at least A Disintegrin And Metalloproteinase with Thrombospondin Motifs 1 (ADAMTS1) obtained from an isolated biological sample of the subjects to be screened; and (b) comparing said expression pattern or level of at least ADAMTS1 of the subjects to be screened with an already established expression pattern or level, wherein reduced expression of at least ADAMTS1 is indicative of a thoracic aortic aneurysm (TAA).CNIC Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Consejo Superior de Investigaciones Científicas (España), Universidad Autónoma de MadridA1 Solicitud de patente con informe sobre el estado de la técnic