Identificación de nuevos biomarcadores genéticos y mecanismos moleculares de la reestenosis tras revascularización vía angioplastia: Papel del factor de transcripción NF-Y

La reestenosis es un proceso patológico consistente en la reoclusión excesiva de un vaso intervenido mediante angioplastia coronaria percutánea transluminal (ACPT), tratamiento frecuentemente utilizado para la revascularización de vasos afectados de arterioesclerosis. La reestenosis se desarrolla normalmente durante los primeros 4-12 meses tras la ACPT y tiene un elevado impacto sanitario y socio-económico, pues obliga a la revascularización del paciente, bien repitiendo la ACPT o mediante “by-pass” aortocoronario. Actualmente, el 90% de las ACPT se realiza mediante el uso de endoprótesis metálicas de soporte denominadas stents. Estos dispositivos mejoran la seguridad del proceso de intervención al reducir las tasas de reestenosis comparado con la ACPT convencional (15-30% comparado con 25-50%, respectivamente), debido principalmente a que evitan el remodelado negativo del vaso. La reciente introducción de stents liberadores de fármacos antiproliferativos (ST-FA) ha supuesto una revolución en la cardiología intervencionista al reducir un 60-80% las tasas de reestenosis en comparación con los stents convencionales. Los ST-FAs liberan de forma local drogas lipofílicas, tales como el sirolimus (rapamicina, rapamune) o el paclitaxel (taxol), que bloquean el ciclo celular, diminuyendo la proliferación celular y consecuentemente la reestenosis asociada a revascularización con stent. La reestenosis es una enfermedad de tipo hiperproliferativo iniciada por el daño mecánico que sufre el endotelio vascular que tapiza la pared arterial como consecuencia de la implantación del stent. El proceso inflamatorio iniciado posteriormente al daño endotelial desencadena la conversión de las células de músculo liso de la túnica media hacia un fenotipo caracterizado por una alta actividad proliferativa y migratoria, así como por la síntesis de componentes de la matriz extracelular, dando lugar en último término al engrosamiento de la lesión neoíntima. La correcta evolución tras la ACPT depende de la resolución de la inflamación y de la cicatrización de la herida gracias a una completa re-endotelización de la zona dañada. En este sentido, la inhibición indiscriminada de la proliferación celular por los ST-FAs afecta a la correcta re-endotelización del vaso intervenido, lo cual constituye la causa principal del elevado riesgo de sufrir trombosis tardía asociada al uso de estos stents. Así, los pacientes tratados con un ST-FA deben prolongar la terapia dual antitrombótica y antiplaquetaria para evitar la tombrosis tardía tras ACPT que se asocia con un elevado riesgo de mortalidad. Por tanto, uno de los principales objetivos de la cardiología intervencionista es la búsqueda de marcadores que permitan la identificación del riesgo individual de sufrir reestenosis tras ACPT, lo cual ayudaría en la toma de decisiones terapéuticas, por ejemplo la implantación de stent versus “by-pass” ortocoronario, o uso de stent convencional versus ST-FA dependiendo del riesgo individual de sufrir reestenosis. Sin embargo, a diferencia de la aterosclerosis nativa, no existen biomarcadores del riesgo de sufrir reestenosis. En los últimos años han ido en aumento los estudios basados en el análisis de genes candidatos que asocian polimorfismos genéticos con el riesgo individual a sufrir enfermedades complejas. Existen diversos trabajos que asocian polimorfismos de base única (SNP: single nucleotide polymorphism) en diversos genes humanos que se asocian con la susceptibilidad a desarrollar reestenosis. Es interesante destacar que Van tiel y colaboradores demostraron recientemente que el genotipo -838AA para el SNP -838(C>A) localizado en el gen supresor de tumores p27Kip1 se asocia a un riesgo disminuido de sufrir reestenosis tras ACPT debido probablemente a un aumento de la actividad promotora de este regulador negativo de la proliferación celular. El objetivo principal del proyecto es determinar si existe alguna variante genética (SNPs) en genes reguladores de ciclo celular que permita predecir el riesgo de sufrir reestenosis coronaria tras implantación de stents. El análisis de una cohorte de 284 pacientes sometidos a angioplastia con implantación de stent coronario mostró que los SNPs rs350099 (-957[T/C]), rs350104 (-475[T/C]), rs164390 (+102[G/T]) y rs875459 (+7010[G/T]) (localizados en el gen CCNB1, que codifica para la proteína ciclina B1), rs2282411 (+7733[T/C], localizado en el gen CCNA1, que codifica para la proteína ciclina A1) y el polimorfismo rs733590 (-1284[T/C]), localizado en el gen CDKN1A, que codifica para la proteína p21Cip1) se asocian con un riesgo aumentado de sufrir reestenosis tras implantación de stent coronario. Dada su localización en la región promotora de un mismo gen, CCNB1, el análisis de los polimorfismos -957[T/C], (-475[T/C]) y (+102[G/T]) en una segunda cohorte de 715 pacientes permitió validar la asociación con el riesgo de sufrir reestenosis tras implantación de stent coronario. Además, las variantes de riesgo de los polimorfismos -957[T/C], (-475[T/C]) y (+102[G/T]) se asocian con un aumento de la expresión de genes reporteros a través de la unión de los factores de transcripción NF-Y, AP-1 y SP1, respectivamente. El haplotipo formado por las variantes de riesgo de estos 3 polimorfismos se asocian, a su vez, con niveles aumentados de ciclina B1 en linfocitos circulantes y con un riesgo aumentado de sufrir reestenosis. La identificación del factor de transcripción NF-Y, conocido regulador de la transcripción de diversos genes reguladores del ciclo celular (incluida la ciclina B1), nos llevó a estudiar su papel en el contexto de la enfermedad vásculo-proliferativa. Mediante estudios moleculares y de expresión en células en cultivo, modelos animales y tejidos humanos demostramos que el PDGF-BB, conocido inductor del desarrollo de la lesión neoíntima, induce el reclutamiento de NF-Y al promotor de la ciclina B1 aumentando tanto su expresión de ARN mensajero como la proliferación celular de células de músculo liso vascular de rata y humano a través de las rutas de señalización Erk1/2 y Akt. Además, encontramos co-expresión de NF-Y y ciclina B1 en regiones proliferativas en placas ateroscleróticas y lesiones inducidas mecánicamente murinas. Por otro lado, la sobreexpresión de un mutante dominante negativo de una de las subunidades de NF-Y, NF-YA, inhibe la expresión de ciclina B1 y la proliferación celular inducida por PDGF-BB y la formación in vivo de la lesión neoíntima en un modelo de daño endoluminal de la arteria femoral común. Por último, demostramos que NF-Y se expresa y es activo en lesiones ateroscleróticas y reestenóticas humanas. En resumen, hemos identificado a NF-Y como un regulador positivo de la expresión de la ciclina B1, de la proliferación celular de las CMLVs y del desarrollo de la lesión neoíntima. En este sentido, hemos detectado la sobreexpresión y activación de NF-Y en lesiones reestenoticas y ateroscleróticas, tanto en modelos animales como en muestras humanas. Además, hemos identificado variantes polimórficas de los SNPs rs350099 (-975T/C), rs350104 (-475 T/C) y rs164390 (+102G/T) localizados en la región promotora del gen CCNB1 humano asociados a un mayor riesgo de sufrir ISR tras implantación de stent coronario que generan sitios de unión para los factores de transcripción NF-Y, AP-1 y SP1, respectivamente. De hecho, el haplotipo portador de las variantes alélicas de mayor riesgo de estos polimorfismos se asocia con niveles aumentados de ARNm de CCNB1 y con una mayor susceptibilidad a desarrollar ISR post-stent. Además, identificamos que los polimorfismos rs2282411 y rs733590 se asocian también con un riesgo aumentado de sufrir reestenosis tras implantación de stent coronario. Por lo tanto, nuestros estudios sugieren que pacientes portadores del genotipo -957TT/-475CC/+102GG presentan un reclutamiento aumentado de las proteínas NF-Y, AP-1 y SP1 que puede contribuir al mayor riesgo de sufrir ISR tras implantación de stent como resultado de un aumento de los niveles de ciclina B1 y de la proliferación de las CMLVs.Restenosis is the major limitation of atherosclerotic vessels revascularization procedure via angioplasty percutaneous coronary intervention (APCI). Restenosis, a disease that occurs during first 4-12 months after APCI, has a high economic and clinical impact due to the necessity of repeat restenotic vessel revascularization via APCI or coronary artery bypass grafting. Nowadays, APCI is performed via the implantation of a metallic prosthesis called stent. These devices improves the safety of the procedure through reducing restenosis rates compared to conventional APCI (15-30% vs 25-50%) by avoiding negative remodeling of the target vessels. The recently introduction of drug eluting stents (DES) has supposed a revolution of interventional cardiology by reducing restenosis rates (60-80%) compared to conventional stents. DES deliver lipophilic drugs (i.e sirolimus or paclitaxel) locally that block cell cycle, inhibit cell proliferation and reduce restenosis rates. Restenosis is a hiperproliferative disease initiated by mechanically-induced endothelial damage due to stent deployment. Endothelial damage triggers an inflammatory response that activates vascular smooth muscle cells (VSMC) to a “synthetic” phenotype characterized by high proliferative and migratory response and elevated extracellular matrix components production. These events end up with the neointimal lesion development and vessel re-occlusion. The proper process development requires inflammation resolution and complete re-endothelization of affected lesion. In this sense, unselective inhibition of cell proliferation by DES dampers re-endothelization causing an increase in the risk to suffer late thrombosis (a feature of these stents). Therefore, considering the socioeconomic impact of restenosis and drawbacks associated to use of DES respect to BMS appears to be necessary identify biomarkers of the risk of restenosis in order to stratify patients to develop a patient-tailored therapy. During last years, increasing evidence suggest the association of genetic polymorphisms with the risk to suffer complex diseases. Severals studies demonstrate that single nucleotide polymorphisms (SNPS) located in different human genes are associated with increased in-stent restenosis (ISR). Interestingly, Van tiel and colleagues demonstrated that -838AA individuals (SNP -838(C>A) within the p27Kip1 gene) show reduced ISR risk probably due to reduced promoter activity. The goal of this work is to identify SNPs in cell cycle regulatory genes that predict ISR risk after coronary stent deployment. Our analysis of a cohort of 284 patients undergoing coronary angioplasty and stent deployment showed that SNPs rs350099 (-957[T/C]), rs350104 (-475[T/C]), rs164390 (+102[G/T]) y rs875459 (+7010[G/T]) (located in CCNB1 gene, codifying cyclin B1 protein), rs2282411 (+7733[T/C], located in CCNA1 gen, codifying cyclin A1) and rs733590 polymorphism (-1284[T/C]), located in CDKN1A gene, codifying p21Cip1 protein) are associated with increased ISR risk. Based on its location in CCNB1 promoter region, analysis of -957[T/C], (-475[T/C]) y (+102[G/T]) polymorphisms in additional 715 patients cohort also showed significant association with increased ISR risk. Moreover, risk allelic variants of -957[T/C], (-475[T/C]) y (+102[G/T]) polymorphisms are associated with increased reporter gene expression through NF-Y, AP-1 and SP1 transcription factors, respectively. High risk alleles containing haplotype is associated with augmented cyclin mRNA levels in peripheral blood lymphocytes and increased ISR risk. The identification of NF-Y transcription factor, known controller of cell cycle regulation gene transcription (cyclin B1 included), led us to investigate its role in the context of vasculo-proliferative diseases. By using molecular and expression studies in cultured cells, animal models and human tissues we demonstrate that platelet-derived growth factor-BB (PDGF-BB), a main inductor of neointimal lesion development, induces in rat and human VSMCs the recruitment of NF-Y to the CCNB1 promoter and augments both CCNB1 mRNA expression and cell proliferation through extracellular signal-regulated kinase 1/2 (Erk1/2) and Akt activation. We find co-expression of NF-Y and cyclin B1 in proliferative regions of murine atherosclerotic plaques and mechanically-induced lesions, which correlates with higher binding of NF-Y to target sequences in the CCNB1 promoter. Moreover, adenovirus-mediated overexpression of a NF-YA dominant-negative mutant inhibits PDGF-BB-induced CCNB1 expression and VSMC proliferation in vitro and neointimal lesion formation in a mouse model of femoral artery injury. We also detect NF-Y expression and DNA-binding activity in human neointimal lesions. In summary, we have identified NF-Y as a key positive regulator of cyclin B1 expression, VSMC proliferation and neointimal lesion formation. We have also found overexpression and activation of NF-Y in restenotic and atherosclerotic lesions, both in animal models and human samples. Moreover, we have identify polymorphic variants of rs350099 (-975T/C), rs350104 (-475 T/C) y rs164390 (+102G/T) within human CCNB1 promoter region that are associated with increased risk of ISR and generates novel NF-Y, AP-1 and SP1 DNA-binding sites, respectively. In fact, high risk haplotype is associated with increased cyclin B1 mRNA levels and augmented ISR risk. In addition, we also found significant association of rs2282411 and rs733590 polymorphisms with increased ISR risk. Therefore, our studies suggest that individuals carrying -957TT/-475CC/+102GG genotype show increased NF-Y, AP-1 and SP1 recruitment contributing through higher cyclin B1 levels and VSMC proliferation to increased ISR risk after coronary stent deployment

Neutrophil extracellular traps: from physiology to pathology.

At the frontline of the host defence response, neutrophil antimicrobial functions have adapted to combat infections and injuries of different origins and magnitude. The release of web-like DNA structures named neutrophil extracellular traps (NETs) constitutes an important mechanism by which neutrophils prevent pathogen dissemination or deal with microorganisms of a bigger size. At the same time, nuclear and granule proteins with microbicidal activity bind to these DNA structures promoting the elimination of entrapped pathogens. However, these toxic properties may produce unwanted effects in the host, when neutrophils uncontrollably release NETs upon persistent inflammation. As a consequence, NET accumulation can produce vessel occlusion, tissue damage, and prolonged inflammation associated with the progression and exacerbation of multiple pathologic conditions. This review outlines recent advances in understanding the mechanisms of NET release and functions in sterile disease. We also discuss mechanisms of physiological regulation and the importance of neutrophil heterogeneity in NET formation and composition.C.S.-R. receives funding from the Deutsche Forschungsgemeinschaft (SFB1123 TP A6). O.S. receives funding from the Deutsche Forschungsgemeinschaft (SFB914 TP B8, SFB1123 TP A6, TP B5, SFB1009 TP A13), the Vetenskapsra˚det (2017-01762), the Else-Kro¨ner-Fresenius Stiftung (2017_A13), the Swedish Heart–Lung Foundation (20190317), and the Leducq foundation (TNE-18CVD04). P.L. receives funding support from the National Heart, Lung, and Blood Institute (1R01HL134892), the American Heart Association (18CSA34080399), the RRM Charitable Fund, and the Simard Fund. V.P. receives core funding from the Francis Crick institute funded by UK Medical Research Council, Cancer Research UK and the Wellcome Trust (FC0010129, FC001134). I.V.A was funded by an EMBO LTF (ALTF 113-2019). A.H. is funded by Ministerio de Ciencia e Innovacion (RTI2018-095497-B-I00), La Caixa Foundation (HR17_00527), and the European Commision (FET-OPEN 861878).S

Inhibition of NET Release Fails to Reduce Adipose Tissue Inflammation in Mice

Obesity-associated diseases such as Type 2 diabetes, liver disease and cardiovascular diseases are profoundly mediated by low-grade chronic inflammation of the adipose tissue. Recently, the importance of neutrophils and neutrophil-derived myeloperoxidase and neutrophil elastase on the induction of insulin resistance has been established. Since neutrophil elastase and myeloperoxidase are critically involved in the release of neutrophil extracellular traps (NETs), we here hypothesized that NETs may be relevant to early adipose tissue inflammation. Thus, we tested the effect of the Peptidyl Arginine Deiminase 4 inhibitor Cl-amidine, a compound preventing histone citrullination and subsequent NET release, in a mouse model of adipose tissue inflammation. C57BL6 mice received a 60% high fat diet for 10 weeks and were treated with either Cl-amidine or vehicle. Flow cytometry of adipose tissue and liver, immunohistological analysis and glucose and insulin tolerance tests were performed to determine the effect of the treatment and diet. Although high fat diet feeding induced insulin resistance no significant effect was observed between the treatment groups. In addition no effect was found in leukocyte infiltration and activation in the adipose tissue and liver. Therefore we concluded that inhibition of neutrophil extracellular trap formation may have no clinical relevance for early obesity-mediated pathogenesis of the adipose tissue and liver

Resolving lipid mediators maresin 1 and resolvin D2 prevent atheroprogression in mice

RATIONALE: Atheroprogression is a consequence of non-resolved inflammation and currently a comprehensive overview of the mechanisms preventing resolution is missing. However, in acute inflammation, resolution is known to be orchestrated by a switch from inflammatory to resolving lipid mediators. Therefore we hypothesized that lesional lipid mediator imbalance favors atheroprogression. OBJECTIVE: To understand the lipid mediator balance during atheroprogression and to establish an interventional strategy based on delivery of resolving lipid mediators. METHODS AND RESULTS: Aortic lipid mediator profiling of aortas from Apoe(-/-) mice fed a high fat diet for four weeks, eight weeks, or four months revealed an expansion of inflammatory lipid mediators, Leukotriene B4 (LTB4) and Prostaglandin E2 (PGE2), and a concomitant decrease of resolving lipid mediators, Resolvin D2 (RvD2) and Maresin 1 (MaR1), during advanced atherosclerosis. Functionally, aortic LTB4 and PGE2 levels correlated with traits of plaque instability while RvD2 and MaR1 levels correlated with signs of plaque stability. In a therapeutic context, repetitive RvD2 and MaR1 delivery prevented atheroprogression as characterized by halted expansion of the necrotic core and accumulation of macrophages along with increased fibrous cap thickness and smooth muscle cell numbers. Mechanistically, RvD2 and MaR1 induced a shift in macrophage profile towards a reparative phenotype which secondarily stimulated collagen synthesis in smooth muscle cells. CONCLUSIONS: We present evidence for the imbalance between inflammatory and resolving lipid mediators during atheroprogression. Delivery of RvD2 and MaR1 successfully prevented atheroprogression suggesting that resolving lipid mediators potentially represent an innovative strategy to resolve arterial inflammation

Atherosclerotic plaque destabilization in Mice: A comparative study

Atherosclerosis-Associated diseases are the main cause ofmortality and morbidity in western societies. The progression of atherosclerosis is a dynamic process evolving from early to advanced lesions thatmay become rupture-prone vulnerable plaques. Acute coronary syndromes are the clinical manifestation of life-Threatening thrombotic events associated with high-risk vulnerable plaques. Hyperlipidemic mouse models have been extensively used in studying the mechanisms controlling initiation and progression of atherosclerosis. However, the understanding of mechanisms leading to atherosclerotic plaque destabilization has been hampered by the lack of proper animalmodelsmimicking this process. Although various mouse models generate atherosclerotic plaques with histological features of human advanced lesions, a consensus model to study atherosclerotic plaque destabilization is still lacking. Hence, we studied the degree and features of plaque vulnerability in different mouse models of atherosclerotic plaque destabilization and find that the model based on the placement of a shear stress modifier in combination with hypercholesterolemia represent with high incidence the most human like lesions compared to the other models

Inactivation of nuclear factor-Y inhibits vascular smooth muscle cell proliferation and neointima formation

OBJECTIVE: Atherosclerosis and restenosis are multifactorial diseases associated with abnormal vascular smooth muscle cell (VSMC) proliferation. Nuclear factor-Y (NF-Y) plays a major role in transcriptional activation of the CYCLIN B1 gene (CCNB1), a key positive regulator of cell proliferation and neointimal thickening. Here, we investigated the role of NF-Y in occlusive vascular disease. APPROACH AND RESULTS: We performed molecular and expression studies in cultured cells, animal models, and human tissues. We find upregulation of NF-Y and cyclin B1 expression in proliferative regions of murine atherosclerotic plaques and mechanically induced lesions, which correlates with higher binding of NF-Y to target sequences in the CCNB1 promoter. NF-YA expression in neointimal lesions is detected in VSMCs, macrophages, and endothelial cells. Platelet-derived growth factor-BB, a main inductor of VSMC growth and neointima development, induces the recruitment of NF-Y to the CCNB1 promoter and augments both CCNB1 mRNA expression and cell proliferation through extracellular signal-regulated kinase 1/2 and Akt activation in rat and human VSMCs. Moreover, adenovirus-mediated overexpression of a NF-YA-dominant negative mutant inhibits platelet-derived growth factor-BB-induced CCNB1 expression and VSMC proliferation in vitro and neointimal lesion formation in a mouse model of femoral artery injury. We also detect NF-Y expression and DNA-binding activity in human neointimal lesions. CONCLUSIONS: Our results identify NF-Y as a key downstream effector of the platelet-derived growth factor-BB-dependent mitogenic pathway that is activated in experimental and human vasculoproliferative diseases. They also identify NF-Y inhibition as a novel and attractive strategy for the local treatment of neointimal formation induced by vessel denudation.This study was funded by the Spanish Ministry of Economy and Competiveness (MINECO; grants SAF2010-16044, SAF200911949), Instituto de Salud Carlos III (ISCIII; grants RD12/0042/0021, RD12/0042/0028, RD12/0042/0053), and the Dr Léon Dumont Prize 2010 by the Belgian Society of Cardiology (to Vicente Andrés). Patricia Fernández received salary support from ISCIII and Carlos Silvestre-Roig from Fundación Mario Losantos del Campo and Fundación Ferrer para la Investigación. Óscar M. Pello and Ricardo Rodríguez-Calvo hold a Juan de la Cierva contract from MINECO. Vanesa Esteban is an investigator of the Sara Borell program from ISCIII (CD06/00232). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by MINECO and Pro-CNIC Foundation.S

Macrophage Inflammation, Erythrophagocytosis, and Accelerated Atherosclerosis in Jak2(V617F) Mice

Rationale: The mechanisms driving atherothrombotic risk in individuals with JAK2(V617F) (Jak2(VF)) positive clonal hematopoiesis or myeloproliferative neoplasms are poorly understood. Objective: The goal of this study was to assess atherosclerosis and underlying mechanisms in hypercholesterolemic mice with hematopoietic Jak2(VF) expression. Methods and Results: Irradiated low-density lipoprotein receptor knockout (Ldlr(-/-)) mice were transplanted with bone marrow from wild-type or Jak2(VF) mice and fed a high-fat high-cholesterol Western diet. Hematopoietic functions and atherosclerosis were characterized. After 7 weeks of Western diet, Jak2(VF) mice showed increased atherosclerosis. Early atherosclerotic lesions showed increased neutrophil adhesion and content, correlating with lesion size. After 12 weeks of Western diet, Jak2(VF) lesions showed increased complexity, with larger necrotic cores, defective efferocytosis, prominent iron deposition, and costaining of erythrocytes and macrophages, suggesting erythrophagocytosis. Jak2(VF) erythrocytes were more susceptible to phagocytosis by wild-type macrophages and showed decreased surface expression of CD47, a "don't-eat-me" signal. Human JAK2VF erythrocytes were also more susceptible to erythrophagocytosis. Jak2(VF) macrophages displayed increased expression and production of proinflammatory cytokines and chemokines, prominent inflammasome activation, increased p38 MAPK (mitogen-activated protein kinase) signaling, and reduced levels of MerTK (c-Mer tyrosine kinase), a key molecule mediating efferocytosis. Increased erythrophagocytosis also suppressed efferocytosis. Conclusions: Hematopoietic Jak2(VF) expression promotes early lesion formation and increased complexity in advanced atherosclerosis. In addition to increasing hematopoiesis and neutrophil infiltration in early lesions, Jak2(VF) caused cellular defects in erythrocytes and macrophages, leading to increased erythrophagocytosis but defective efferocytosis. These changes promote accumulation of iron in plaques and increased necrotic core formation which, together with exacerbated proinflammatory responses, likely contribute to plaque instability

Histone Deacetylase 9 Activates IKK to Regulate Atherosclerotic Plaque Vulnerability

Rationale: Arterial inflammation manifested as atherosclerosis is the leading cause of mortality worldwide. Genome-wide association studies have identified a prominent role of histone deacetylase 9 (HDAC9) in atherosclerosis and its clinical complications including stroke and myocardial infarction. Objective: To determine the mechanisms linking HDAC9 to these vascular pathologies and explore its therapeutic potential for atheroprotection. Methods and Results: We studied the effects of Hdac9 on features of plaque vulnerability using bone marrow reconstitution experiments and pharmacological targeting with a small molecule inhibitor in hyperlipidemic mice. We further employed two-photon and intravital microscopy to study endothelial activation and leukocyte-endothelial interactions. We show that hematopoietic Hdac9 deficiency reduces lesional macrophage content whilst increasing fibrous cap thickness thus conferring plaque stability. We demonstrate that HDAC9 binds to IKKα and β resulting in their deacetylation and subsequent activation, which drives inflammatory responses in both macrophages and endothelial cells. Pharmacological inhibition of HDAC9 with the class IIa HDAC inhibitor TMP195 attenuates lesion formation by reducing endothelial activation and leukocyte recruitment along with limiting pro-inflammatory responses in macrophages. Transcriptional profiling using RNA-Seq revealed that TMP195 downregulates key inflammatory pathways consistent with inhibitory effects on IKKβ. TMP195 mitigates the progression of established lesions and inhibits the infiltration of inflammatory cells. Moreover, TMP195 diminishes features of plaque vulnerability and thereby enhances plaque stability in advanced lesions. Ex vivo treatment of monocytes from patients with established atherosclerosis reduced the production of inflammatory cytokines including IL-1β and IL-6. Conclusions: Our findings identify HDAC9 as a regulator of atherosclerotic plaque stability and IKK activation thus providing a mechanistic explanation for the prominence of HDAC9 as a vascular risk locus in genome-wide association studies. Its therapeutic inhibition may provide a potent lever to alleviate vascular inflammation

The AIM2 inflammasome exacerbates atherosclerosis in clonal haematopoiesis

Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors(1). Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2(V617F) (JAK2(VF)) mutation, which increases JAK-STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease(1,2). Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2(VF) selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2(VF) lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2(VF) lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1 beta reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2(VF) macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1 beta or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk