Lysyl oxidase-dependent extracellular matrix crosslinking modulates calcification in atherosclerosis and aortic valve disease

Atherosclerosis; Cardiovascular calcification; Lysyl oxidaseAterosclerosis; Calcificación cardiovascular; Lisil oxidasaAterosclerosi; Calcificació cardiovascular; Lisil oxidasaExtracellular matrix (ECM) is an active player in cardiovascular calcification (CVC), a major public health issue with an unmet need for effective therapies. Lysyl oxidase (LOX) conditions ECM biomechanical properties; thus, we hypothesized that LOX might impact on mineral deposition in calcific aortic valve disease (CAVD) and atherosclerosis. LOX was upregulated in calcified valves from two cohorts of CAVD patients. Strong LOX immunostaining was detected surrounding calcified foci in calcified human valves and atherosclerotic lesions colocalizing with RUNX2 on valvular interstitial cells (VICs) or vascular smooth muscle cells (VSMCs). Both LOX secretion and organized collagen deposition were enhanced in calcifying VICs exposed to osteogenic media. β-aminopropionitrile (BAPN), an inhibitor of LOX, attenuated collagen deposition and calcification. VICs seeded onto decellularized matrices from BAPN-treated VICs calcified less than cells cultured onto control scaffolds; instead, VICs exposed to conditioned media from cells over-expressing LOX or cultured onto LOX-crosslinked matrices calcified more. Atherosclerosis was induced in WT and transgenic mice that overexpress LOX in VSMC (TgLOXVSMC) by AAV-PCSK9D374Y injection and high-fat feeding. In atherosclerosis-challenged TgLOXVSMC mice both atherosclerosis burden and calcification assessed by near-infrared fluorescence (NIRF) imaging were higher than in WT mice. These animals also exhibited larger calcified areas in atherosclerotic lesions from aortic arches and brachiocephalic arteries. Moreover, LOX transgenesis exacerbated plaque inflammation, and increased VSMC cellularity, the rate of RUNX2-positive cells and both connective tissue content and collagen cross-linking. Our findings highlight the relevance of LOX in CVC and postulate this enzyme as a potential therapeutic target for CVC.We thank the technical support provided by Silvia Aguiló. This work was supported by the Spanish Ministerio de Ciencia e Innovación (RTI2018-094727-B-100 and PID2021-122509OB-I00 funded by MCIN/ AEI/10.13039/501100011033 and by “ERDF A way of making Europe”), Instituto de Salud Carlos III (ISCIII; Spain; PI21/01048), and Fundación Española de Arteriosclerosis (FEA-2022). C.B-S and L.P-U were supported by a FPU (Ministerio de Universidades; Spain), and PFIS (ISCIII) fellowships, respectively and N.L-A by a Miguel Servet contract (ISCIII). There is no financial or personal relationship with organizations that could potentially influence the described research

Rolipram Prevents the Formation of Abdominal Aortic Aneurysm (AAA) in Mice: PDE4B as a Target in AAA

Abdominal aortic aneurysm (AAA) is a common life-threatening condition characterized by exacerbated inflammation and the generation of reactive oxygen species. Pharmacological treatments to slow AAA progression or to prevent its rupture remain a challenge. Targeting phosphodiesterase 4 (PDE4) has been verified as an effective therapeutic strategy for an array of inflammatory conditions; however, no studies have assessed yet PDE4 in AAA. Here, we used angiotensin II (AngII)-infused apolipoprotein E deficient mice to study the involvement of the PDE4 subfamily in aneurysmal disease. PDE4B but not PDE4D was upregulated in inflammatory cells from both experimental and human AAA. The administration of the PDE4 selective inhibitor rolipram (3 mg/kg/day) to AngII-challenged mice (1000 ng/kg bodyweight/min) protected against AAA formation, limiting the progressive increase in the aortic diameter without affecting the blood pressure. The drug strongly attenuated the rise in vascular oxidative stress (superoxide anion) induced by AngII, and decreased the expression of inflammatory markers, as well as the recruitment of macrophages (MAC3+), lymphocytes (CD3+), and neutrophils (ELANE+) into the vessel wall. Rolipram also normalized the vascular MMP2 expression and MMP activity, preserving the elastin integrity and improving the vascular remodelling. These results point to PDE4B as a new therapeutic target for AAA.This work was supported by Instituto de Salud Carlos III (ISCIII) [PI18/0919], the Spanish Ministerio de Ciencia e Innovación (MICINN) [RTI2018-094727-B-100 and PID2019-108489RB-100], the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) [2017-SGR-00333], Sociedad Española de Arteriosclerosis [Beca FEA 2020 Investigación Básica], and Sociedad Española de Cardiología [SEC/FEC-INV-BAS 20/005]. The study was co-founded by Fondo Europeo de Desarrollo Regional (FEDER), “A way to make Europe”. L.C. was supported by a FI (AGAUR). The CNIC is supported by MICINN, ISCIII, and the Pro-CNIC Foundation

NR4A3: A Key Nuclear Receptor in Vascular Biology, Cardiovascular Remodeling, and Beyond

Aneurisma aórtico abdominal; Aterosclerosis; Remodelación cardiovascularAneurisma aòrtic abdominal; Aterosclerosi; Remodelació cardiovascularAbdominal aortic aneurysm; Atherosclerosis; Cardiovascular remodelingThe mechanisms committed in the activation and response of vascular and inflammatory immune cells play a major role in tissue remodeling in cardiovascular diseases (CVDs) such as atherosclerosis, pulmonary arterial hypertension, and abdominal aortic aneurysm. Cardiovascular remodeling entails interrelated cellular processes (proliferation, survival/apoptosis, inflammation, extracellular matrix (ECM) synthesis/degradation, redox homeostasis, etc.) coordinately regulated by a reduced number of transcription factors. Nuclear receptors of the subfamily 4 group A (NR4A) have recently emerged as key master genes in multiple cellular processes and vital functions of different organs, and have been involved in a variety of high-incidence human pathologies including atherosclerosis and other CVDs. This paper reviews the major findings involving NR4A3 (Neuron-derived Orphan Receptor 1, NOR-1) in the cardiovascular remodeling operating in these diseases.This research was funded by the Spanish Ministerio de Ciencia e Innovación (RTI2018-094727-B-100), Instituto de Salud Carlos III (ISCIII; PI18/0919 and PI20/01649), the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR; 2017-SGR-00333). The study was cofounded by Fondo Europeo de Desarrollo Regional (FEDER), a way to make Europe. C.B.-S. is supported by a FPU fellowship (Ministerio de Ciencia, Innovación y Universidades)

CD69-oxLDL ligand engagement induces Programmed Cell Death 1 (PD-1) expression in human CD4 + T lymphocytes

The mechanisms that control the inflammatory–immune response play a key role in tissue remodelling in cardiovascular diseases. T cell activation receptor CD69 binds to oxidized low-density lipoprotein (oxLDL), inducing the expression of anti-inflammatory NR4A nuclear receptors and modulating inflammation in atherosclerosis. To understand the downstream T cell responses triggered by the CD69-oxLDL binding, we incubated CD69-expressing Jurkat T cells with oxLDL. RNA sequencing revealed a differential gene expression profile dependent on the presence of CD69 and the degree of LDL oxidation. CD69-oxLDL binding induced the expression of NR4A receptors (NR4A1 and NR4A3), but also of PD-1. These results were confirmed using oxLDL and a monoclonal antibody against CD69 in CD69-expressing Jurkat and primary CD4 + lymphocytes. CD69-mediated induction of PD-1 and NR4A3 was dependent on NFAT activation. Silencing NR4A3 slightly increased PD-1 levels, suggesting a potential regulation of PD-1 by this receptor. Moreover, expression of PD-1, CD69 and NR4A3 was increased in human arteries with chronic inflammation compared to healthy controls, with a strong correlation between PD-1 and CD69 mRNA expression (r = 0.655 P < 0.0001). Moreover, PD-1 was expressed in areas enriched in CD3 infiltrating T cells. Our results underscore a novel mechanism of PD-1 induction independent of TCR signalling that might contribute to the role of CD69 in the modulation of inflammation and vascular remodelling in cardiovascular diseasesOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by grant S2017/BMD-3671-INFLAMUNE-CM from the Comunidad de Madrid, a grant from the Ramón Areces Foundation “Ciencias de la Vida y la Salud”, “La Caixa” Banking Foundation (HR17-00016) to FSM; grants PDC2021-121719-I00 and PDI-2020-120412RBI00 to FSM, and RTI2018-094727-B-100 to JMG funded by MCIN/ AEI/10.13039/501100011033 and by “ERDF A way of making Europe”; the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) (2017-SGR-00333) to JMG; and a grant from the Instituto de Salud Carlos III (PI18/0919) to CR. M. Jiménez-Fernández is supported by a FPI-Severo Ochoa-CNIC (PRE2019-087941); C. Ballester-Servera is supported by a FPU fellowship (Ministerio de Universidades). Data availability The data underlying this article are available in the article and in its online Supplementary material. Declarations Conflict of interest The authors have no confict of interest to declare. Ethical approval Written consent was obtained from all participating subjects. The procedure was approved by the Ethics Committee of the Hospital de la Santa Creu i Sant Pau (Barcelona, Spain) and was conducted in accordance with the Declaration of Helsinki. Consent for publication Consent to publish has been received from all participant

Lysyl oxidase-dependent extracellular matrix crosslinking modulates calcification in atherosclerosis and aortic valve disease

Extracellular matrix (ECM) is an active player in cardiovascular calcification (CVC), a major public health issue with an unmet need for effective therapies. Lysyl oxidase (LOX) conditions ECM biomechanical properties; thus, we hypothesized that LOX might impact on mineral deposition in calcific aortic valve disease (CAVD) and atherosclerosis. LOX was upregulated in calcified valves from two cohorts of CAVD patients. Strong LOX immunostaining was detected surrounding calcified foci in calcified human valves and atherosclerotic lesions colocalizing with RUNX2 on valvular interstitial cells (VICs) or vascular smooth muscle cells (VSMCs). Both LOX secretion and organized collagen deposition were enhanced in calcifying VICs exposed to osteogenic media. β-aminopropionitrile (BAPN), an inhibitor of LOX, attenuated collagen deposition and calcification. VICs seeded onto decellularized matrices from BAPN-treated VICs calcified less than cells cultured onto control scaffolds; instead, VICs exposed to conditioned media from cells over-expressing LOX or cultured onto LOX-crosslinked matrices calcified more. Atherosclerosis was induced in WT and transgenic mice that overexpress LOX in VSMC (TgLOXVSMC) by AAV-PCSK9D374Y injection and high-fat feeding. In atherosclerosis-challenged TgLOXVSMC mice both atherosclerosis burden and calcification assessed by near-infrared fluorescence (NIRF) imaging were higher than in WT mice. These animals also exhibited larger calcified areas in atherosclerotic lesions from aortic arches and brachiocephalic arteries. Moreover, LOX transgenesis exacerbated plaque inflammation, and increased VSMC cellularity, the rate of RUNX2-positive cells and both connective tissue content and collagen cross-linking. Our findings highlight the relevance of LOX in CVC and postulate this enzyme as a potential therapeutic target for CVC.This work was supported by the Spanish Ministerio de Ciencia e Innovación (RTI2018-094727-B-100 and PID2021-122509OB-I00 funded by MCIN/ AEI/10.13039/501100011033 and by “ERDF A way of making Europe”), Instituto de Salud Carlos III (ISCIII; Spain; PI21/01048), and Fundación Española de Arteriosclerosis (FEA-2022). C.B-S and L.P-U were supported by a FPU (Ministerio de Universidades; Spain), and PFIS (ISCIII) fellowships, respectively and N.L-A by a Miguel Servet contract (ISCIII). There is no financial or personal relationship with organizations that could potentially influence the described research.Peer reviewe

El receptor NOR-1 en el remodelado cardiovascular: análisis de mecanismos fisiopatológicos y validación de nuevos modelos animales de utilidad preclínica

Tesis doctoral presentada para lograr el título de Doctor por la Universidad de Barcelona, Facultad de Farmacia y Ciencias de la Alimentación.--Calificación: Sobresaliente cum laudeActualmente no se dispone de fármacos eficaces ni de modelos animales adecuados para estudiar el aneurisma de la aorta abdominal (AAA) ni la hipertrofia cardiaca, patologías con una elevada morbi-mortalidad. La expresión del receptor nuclear NOR-1 es elevada en el miocardio y aumenta en la aorta de pacientes con AAA. Animales modificados genéticamente para este receptor podrían ser útiles para estudiar los mecanismos de estas enfermedades y para realizar ensayos preclínicos de potenciales terapias. Estudios ecocardiográficos de nuestro grupo habían mostrado que la angiotensina II (AngII) induce dilatación aórtica tanto en un ratón que sobreexpresa NOR-1 humano (hNOR-1) preferentemente en la pared vascular y el miocardio (TgNOR-1) como en el que la expresión de hNOR-1 se dirige células musculares (TgNOR1CMLV). También se observó que los ratones TgNOR-1 desarrollan hipertrofia cardíaca en respuesta a sobrecarga de presión, y que ambos podrían ser útiles como modelos preclínicos ya que la doxiciclina prevenía la formación de aneurismas inducidos por AngII. En el presente trabajo hemos profundizado en los mecanismos celulares y moleculares por los que la transgénesis de NOR-1 predispone a la formación de AAA. En respuesta a AngII en la pared vascular de los animales transgénicos se producen más roturas de fibras elásticas y un mayor aumento de la actividad MMP, del infiltrado inflamatorio y del estrés oxidativo que en los animales control. En estos animales la doxiciclina previno la formación de aneurismas inducidos por AngII a través de la reducción del remodelado vascular, la actividad MMP, la inflamación y el estrés oxidativo. El análisis del patrón de expresión diferencial en respuesta a AngII en la aorta abdominal de ratones TgNOR1CMLV y controles mediante microarrays identificó 1512 genes regulados diferencialmente y el análisis de vías (GSEA; Gene Set Enrichment Analysis) determinó la regulación diferencial de procesos biológicos, relacionados con inflamación, ciclo celular, citoesqueleto, diferenciación de célula muscular y activación simpática. Confirmamos que en respuesta a la AngII la transgénesis de NOR-1 se asocia a la inducción de genes implicados en la síntesis y transporte de catecolaminas, entre ellos la tirosina hidroxilasa (TH), enzima limitante de esta vía. Su expresión se incrementó de manera significativa en el AAA humano en el que la TH se localiza no sólo en las terminaciones nerviosas, sino también en células inflamatorias y en menor medida en CMLV.Un perfil similar se detectó en el ratón TgNOR1CMLV y en el modelo clásico de AAA (ratón deficiente en apolipoproteína E (ApoE-/-) infundido con AngII). Destacar que la inhibición específica de la TH mediante α-Metil-DL-tirosina (AMPT), previno el desarrollo de AAA en ambos modelos reduciendo el remodelado vascular, la inflamación y el estrés oxidativo. Por tanto, se propone a la TH como una nueva diana farmacológica en el AAA. En relación con la hipertrofia cardíaca. Los cardiomiocitos de los ratones TgNOR-1 son de mayor tamaño que los controles y cuando se estimulan eléctricamente experimentan mayor acortamiento que éstos. A su vez, la transgénesis de NOR-1 en cardiofibroblastos aumentó la expresión de marcadores del cambio fenotípico fibroblasto/miofibroblasto. Estos animales presentaban un agravamiento de la hipertrofia cardiaca asociada a la edad sin alteración de la función sistólica. Análogamente, confirmamos la mayor predisposición de los ratones transgénicos a la hipertrofia cardiaca inducida por AngII, con una hipertrofia concéntrica más acentuada que los ratones control, un aumento compensatorio de la función sistólica y una exacerbada respuesta inflamatoria y fibrótica. De hecho, la sobreexpresión de NOR-1 incrementó la deposición y el entrecruzamiento de las fibras de colágeno y la expresión de marcadores fibróticos, como la lisil oxidasa like 2 (LOXL2). Asimismo, la transgénesis de NOR-1 potenció la inducción de marcadores de hipertrofia cardíaca causada por la AngII. Nuestros resultados indican que en los ratones transgénicos existe una mayor expresión basal de Myh7 y de Loxl2 y mediante ensayos de actividad transcripcional demostramos que NOR-1 regula directamente la expresión de estos genes. Estos resultados sugieren que NOR-1 está implicado en el complejo programa transcripcional que lleva a la hipertrofia hipertensiva

Altamira: From cave to tourism (1921-1996)

Máster en Prehistoria y Arqueologí