The RNA editor ADAR2 promotes immune cell trafficking by enhancing endothelial responses to interleukin-6 during sterile inflammation.

Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.

Exogenous Transforming Growth Factor-ß1 and Its Helminth-Derived Mimic Attenuate the Heart's Inflammatory Response to Ischemic Injury and Reduce Mature Scar Size.

Coronary reperfusion after acute ST-elevation myocardial infarction (STEMI) is standard therapy to salvage ischemic heart muscle. However, subsequent inflammatory responses within the infarct lead to further loss of viable myocardium. Transforming growth factor (TGF)-ß1 is a potent anti-inflammatory cytokine released in response to tissue injury. The aim of this study was to investigate the protective effects of TGF-ß1 after MI. In patients with STEMI, there was a significant correlation (P = 0.003) between higher circulating TGF-ß1 levels at 24 hours after MI and a reduction in infarct size after 3 months, suggesting a protective role of early increase in circulating TGF-ß1. A mouse model of cardiac ischemia reperfusion was used to demonstrate multiple benefits of exogenous TGF-ß1 delivered in the acute phase. It led to a significantly smaller infarct size (30% reduction, P = 0.025), reduced inflammatory infiltrate (28% reduction, P = 0.015), lower intracardiac expression of inflammatory cytokines IL-1ß and chemokine (C-C motif) ligand 2 (>50% reduction, P = 0.038 and 0.0004, respectively) at 24 hours, and reduced scar size at 4 weeks (21% reduction, P = 0.015) after reperfusion. Furthermore, a low-fibrogenic mimic of TGF-ß1, secreted by the helminth parasite Heligmosomoides polygyrus, had an almost identical protective effect on injured mouse hearts. Finally, genetic studies indicated that this benefit was mediated by TGF-ß signaling in the vascular endothelium.

Heart Disease and Ageing: The Roles of Senescence, Mitochondria, and Telomerase in Cardiovascular Disease.

During ageing molecular damage leads to the accumulation of several hallmarks of ageing including mitochondrial dysfunction, cellular senescence, genetic instability and chronic inflammation, which contribute to the development and progression of ageing-associated diseases including cardiovascular disease. Consequently, understanding how these hallmarks of biological ageing interact with the cardiovascular system and each other is fundamental to the pursuit of improving cardiovascular health globally. This review provides an overview of our current understanding of how candidate hallmarks contribute to cardiovascular diseases such as atherosclerosis, coronary artery disease and subsequent myocardial infarction, and age-related heart failure. Further, we consider the evidence that, even in the absence of chronological age, acute cellular stress leading to accelerated biological ageing expedites cardiovascular dysfunction and impacts on cardiovascular health. Finally, we consider the opportunities that modulating hallmarks of ageing offer for the development of novel cardiovascular therapeutics.

Length-independent telomere damage drives post-mitotic cardiomyocyte senescence.

Ageing is the biggest risk factor for cardiovascular disease. Cellular senescence, a process driven in part by telomere shortening, has been implicated in age-related tissue dysfunction. Here, we address the question of how senescence is induced in rarely dividing/post-mitotic cardiomyocytes and investigate whether clearance of senescent cells attenuates age-related cardiac dysfunction. During ageing, human and murine cardiomyocytes acquire a senescent-like phenotype characterised by persistent DNA damage at telomere regions that can be driven by mitochondrial dysfunction and crucially can occur independently of cell division and telomere length. Length-independent telomere damage in cardiomyocytes activates the classical senescence-inducing pathways, p21CIP and p16INK4a, and results in a non-canonical senescence-associated secretory phenotype, which is pro-fibrotic and pro-hypertrophic. Pharmacological or genetic clearance of senescent cells in mice alleviates detrimental features of cardiac ageing, including myocardial hypertrophy and fibrosis. Our data describe a mechanism by which senescence can occur and contribute to age-related myocardial dysfunction and in the wider setting to ageing in post-mitotic tissues.

Loss of Endothelial Endoglin Promotes High-Output Heart Failure Through Peripheral Arteriovenous Shunting Driven by VEGF Signaling.

RATIONALE: ENG (endoglin) is a coreceptor for BMP (bone morphogenetic protein) 9/10 and is strongly expressed in endothelial cells. Mutations in ENG lead to the inherited vascular disorder hereditary hemorrhagic telangiectasia characterized by local telangiectases and larger arteriovenous malformations (AVMs); but how ENG functions to regulate the adult vasculature is not understood. OBJECTIVE: The goal of the work was to determine how ENG maintains vessel caliber in adult life to prevent AVM formation and thereby protect heart function. METHODS AND RESULTS: Genetic depletion of endothelial Eng in adult mice led to a significant reduction in mean aortic blood pressure. There was no evidence of hemorrhage, anemia, or AVMs in major organs to explain the reduced aortic pressure. However, large AVMs developed in the peripheral vasculature intimately associated with the pelvic cartilaginous symphysis-a noncapsulated cartilage with a naturally high endogenous expression of VEGF (vascular endothelial growth factor). The increased blood flow through these peripheral AVMs explained the drop in aortic blood pressure and led to increased cardiac preload, and high stroke volumes, ultimately resulting in high-output heart failure. Development of pelvic AVMs in this region of high VEGF expression occurred because loss of ENG in endothelial cells leads to increased sensitivity to VEGF and a hyperproliferative response. Development of AVMs and associated progression to high-output heart failure in the absence of endothelial ENG was attenuated by targeting VEGF signaling with an anti-VEGFR2 (VEGF receptor 2) antibody. CONCLUSIONS: ENG promotes the normal balance of VEGF signaling in quiescent endothelial cells to maintain vessel caliber-an essential function in conditions of increased VEGF expression such as local hypoxia or inflammation. In the absence of endothelial ENG, increased sensitivity to VEGF drives abnormal endothelial proliferation in local regions of high VEGF expression, leading to AVM formation and a rapid injurious impact on heart function.

Adenosine-to-inosine RNA editing contributes to type I interferon responses in systemic sclerosis.

OBJECTIVE: Adenosine deaminase acting on RNA-1 (ADAR1) enzyme is a type I interferon (IFN)-stimulated gene (ISG) catalyzing the deamination of adenosine-to-inosine, a process called A-to-I RNA editing. A-to-I RNA editing takes place mainly in Alu elements comprising a primate-specific level of post-transcriptional gene regulation. Whether RNA editing is involved in type I IFN responses in systemic sclerosis (SSc) patients remains unknown. METHODS: ISG expression was quantified in skin biopsies and peripheral blood mononuclear cells derived from SSc patients and healthy subjects. A-to-I RNA editing was examined in the ADAR1-target cathepsin S (CTSS) by an RNA editing assay. The effect of ADAR1 on interferon-α/ß-induced CTSS expression was assessed in human endothelial cells in vitro. RESULTS: Increased expression levels of the RNA editor ADAR1, and specifically the long ADAR1p150 isoform, and its target CTSS are strongly associated with type I IFN signature in skin biopsies and peripheral blood derived from SSc patients. Notably, IFN-α/ß-treated human endothelial cells show 8-10-fold increased ADAR1p150 and 23-35-fold increased CTSS expression, while silencing of ADAR1 reduces CTSS expression by 60-70%. In SSc patients, increased RNA editing rate of individual adenosines located in CTSS 3' UTR Alu elements is associated with higher CTSS expression (r = 0.36-0.6, P < 0.05 for all). Similar findings were obtained in subjects with activated type I IFN responses including SLE patients or healthy subjects after influenza vaccination. CONCLUSION: ADAR1p150-mediated A-to-I RNA editing is critically involved in type I IFN responses highlighting the importance of post-transcriptional regulation of proinflammatory gene expression in systemic autoimmunity, including SSc.

RNA epigenetics and cardiovascular diseases.

Cardiovascular disease (CVD) remains the leading cause of death in the Western world. Despite advances in the prevention and in the management of CVD, the role of RNA epigenetics in the cardiovascular system has been until recently unexplored. The rapidly expanding research field of RNA modifications has introduced a novel layer of gene regulation in mammalian cells. RNA modifications may control all aspects of RNA metabolism, and their study reveals previously unrecognized regulatory pathways that may determine gene expression at a post-transcriptional level. Understanding the role of RNA modifications in CVD may lead towards a better understanding of disease mechanisms and the development of novel biomarkers or therapeutic strategies. In this review, we highlight the most recent and major reports in the field of RNA methylation and adenosine to inosine RNA editing related to the cardiovascular field and we discuss how this breakthrough will advance the field of precision medicine.

Decreased Expression of Vascular Endothelial Growth Factor Receptor 1 Contributes to the Pathogenesis of Hereditary Hemorrhagic Telangiectasia Type 2.

BACKGROUND: Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is an inherited genetic disorder characterized by vascular malformations and hemorrhage. HHT2 results from ACVRL1 haploinsufficiency, the remaining wild-type allele being unable to contribute sufficient protein to sustain endothelial cell function. Blood vessels function normally but are prone to respond to angiogenic stimuli, leading to the development of telangiectasic lesions that can bleed. How ACVRL1 haploinsufficiency leads to pathological angiogenesis is unknown. METHODS: We took advantage of Acvrl1+/- mutant mice that exhibit HHT2 vascular lesions and focused on the neonatal retina and the airway system after Mycoplasma pulmonis infection, as physiological and pathological models of angiogenesis, respectively. We elucidated underlying disease mechanisms in vitro by generating Acvrl1+/- mouse embryonic stem cell lines that underwent sprouting angiogenesis and performed genetic complementation experiments. Finally, HHT2 plasma samples and skin biopsies were analyzed to determine whether the mechanisms evident in mice are conserved in humans. RESULTS: Acvrl1+/- retinas at postnatal day 7 showed excessive angiogenesis and numerous endothelial "tip cells" at the vascular front that displayed migratory defects. Vascular endothelial growth factor receptor 1 (VEGFR1; Flt-1) levels were reduced in Acvrl1+/- mice and HHT2 patients, suggesting similar mechanisms in humans. In sprouting angiogenesis, VEGFR1 is expressed in stalk cells to inhibit VEGFR2 (Flk-1, KDR) signaling and thus limit tip cell formation. Soluble VEGFR1 (sVEGFR1) is also secreted, creating a VEGF gradient that promotes orientated sprout migration. Acvrl1+/- embryonic stem cell lines recapitulated the vascular anomalies in Acvrl1+/- (HHT2) mice. Genetic insertion of either the membrane or soluble form of VEGFR1 into the ROSA26 locus of Acvrl1+/- embryonic stem cell lines prevented the vascular anomalies, suggesting that high VEGFR2 activity in Acvrl1+/- endothelial cells induces HHT2 vascular anomalies. To confirm our hypothesis, Acvrl1+/- mice were infected by Mycoplasma pulmonis to induce sustained airway inflammation. Infected Acvrl1+/- tracheas showed excessive angiogenesis with the formation of multiple telangiectases, vascular defects that were prevented by VEGFR2 blocking antibodies. CONCLUSIONS: Our findings demonstrate a key role of VEGFR1 in HHT2 pathogenesis and provide mechanisms explaining why HHT2 blood vessels respond abnormally to angiogenic signals. This supports the case for using anti-VEGF therapy in HHT2.

Circulating microparticles from obstructive sleep apnea syndrome patients induce endothelin-mediated angiogenesis.

Microparticles are deemed true biomarkers and vectors of biological information between cells. Depending on their origin, the composition of microparticles varies and the subsequent message transported by them, such as proteins, mRNA, or miRNA, can differ. In obstructive sleep apnea syndrome (OSAS), circulating microparticles are associated with endothelial dysfunction by reducing endothelial-derived nitric oxide production. Here, we have analyzed the potential role of circulating microparticles from OSAS patients on the regulation of angiogenesis and the involved pathway. VEGF content carried by circulating microparticles from OSAS patients was increased when compared with microparticles from non-OSAS patients. Circulating microparticles from OSAS patients induced an increase of angiogenesis that was abolished in the presence of the antagonist of endothelin-1 receptor type B. In addition, endothelin-1 secretion was increased in human endothelial cells treated by OSAS microparticles. We highlight that circulating microparticles from OSAS patients can modify the secretome of endothelial cells leading to angiogenesis.

Interaction in endothelium of non-muscular myosin light-chain kinase and the NF-κB pathway is critical to lipopolysaccharide-induced vascular hyporeactivity.

During sepsis, endothelial barrier dysfunction contributes to cardiovascular failure, mainly through the release of oxidative metabolites by penetrant leukocytes. We reported the non-muscular isoform of myosin light chain kinase (nmMLCK) playing a pivotal role in endotoxin shock injury associated with oxidative and nitrative stresses, and vascular hyporeactivity. The present study was aimed at understanding the molecular mechanism of lipopolysaccharide (LPS)-induced vascular alterations as well as studying a probable functional association of nmMLCK with nuclear factor κ-light-chain enhancer of activated B cells (NF-κB). Aortic rings from mice were exposed in vitro to LPS and, then, vascular reactivity was measured. Human aortic endothelial cells (HAoECs) were incubated with LPS, and interaction of nmMLCK with NF-κB was analysed. We provide evidence that nmMLCK deletion prevents vascular hyporeactivity induced by in vitro LPS treatment but not endothelial dysfunction in the aorta. Deletion of nmMLCK inhibits LPS-induced NF-κB activation and increases nitric oxide (NO) release via induction of inducible NO synthase (iNOS) within the vascular wall. Also, removal of endothelium prevented both NF-κB and iNOS expression in aortic rings. Among the proinflammatory factors released by LPS-treated endothelial cells, interleukin-6 accounts for the induction of iNOS on smooth muscle cells in response to LPS. Of particular interest is the demonstration that, in HAoECs, LPS-induced NF-κB activation occurs via increased MLCK activity sensitive to the MLCK inhibitor, ML-7, and physical interactions between nmMLCK and NF-κB. We report for the first time on NF-κB as a novel partner of nmMLCK within endothelial cells. The present study demonstrates a pivotal role of nmMLCK in vascular inflammatory pathologies.

Amyloid beta is associated with carotid wall echolucency and atherosclerotic plaque composition.

Circulating amyloid-beta 1-40 (Αb40) has pro-atherogenic properties and could serve as a biomarker in atherosclerotic cardiovascular disease (ASCVD). However, the association of Ab40 levels with morphological characteristics reflecting atherosclerotic plaque echolucency and composition is not available. Carotid atherosclerosis was assessed in consecutively recruited individuals without ASCVD (n = 342) by ultrasonography. The primary endpoint was grey scale median (GSM) of intima-media complex (IMC) and plaques, analysed using dedicated software. Vascular markers were assessed at two time-points (median follow-up 35.5 months). In n = 56 patients undergoing carotid endarterectomy, histological plaque features were analysed. Plasma Αb40 levels were measured at baseline. Ab40 was associated with lower IMC GSM and plaque GSM and higher plaque area at baseline after multivariable adjustment. Increased Ab40 levels were also longitudinally associated with decreasing or persistently low IMC and plaque GSM after multivariable adjustment (p < 0.05). In the histological analysis, Ab40 levels were associated with lower incidence of calcified plaques and plaques without high-risk features. Ab40 levels are associated with ultrasonographic and histological markers of carotid wall composition both in the non-stenotic arterial wall and in severely stenotic plaques. These findings support experimental evidence linking Ab40 with plaque vulnerability, possibly mediating its established association with major adverse cardiovascular events.

Mechanisms of Vascular Inflammation and Potential Therapeutic Targets: A Position Paper From the ESH Working Group on Small Arteries.

Inflammatory responses in small vessels play an important role in the development of cardiovascular diseases, including hypertension, stroke, and small vessel disease. This involves various complex molecular processes including oxidative stress, inflammasome activation, immune-mediated responses, and protein misfolding, which together contribute to microvascular damage. In addition, epigenetic factors, including DNA methylation, histone modifications, and microRNAs influence vascular inflammation and injury. These phenomena may be acquired during the aging process or due to environmental factors. Activation of proinflammatory signaling pathways and molecular events induce low-grade and chronic inflammation with consequent cardiovascular damage. Identifying mechanism-specific targets might provide opportunities in the development of novel therapeutic approaches. Monoclonal antibodies targeting inflammatory cytokines and epigenetic drugs, show promise in reducing microvascular inflammation and associated cardiovascular diseases. In this article, we provide a comprehensive discussion of the complex mechanisms underlying microvascular inflammation and offer insights into innovative therapeutic strategies that may ameliorate vascular injury in cardiovascular disease.

Circulating microparticles from patients with obstructive sleep apnea enhance vascular contraction: mandatory role of the endothelium.

Obstructive sleep apnea (OSA) is characterized by repetitive apnea-hypopnea cycles during sleep associated with oxygen desaturation and sleep disruption. We evaluated the role of circulating microparticles (MPs) from patients with OSA in the regulation of vascular function. MPs from whole blood from patients with OSA or control subjects were injected i.v. into mice. Injection of MPs from patients with OSA induced ex vivo vascular hyperreactivity in aortas with functional endothelium but, in contrast, hyporeactivity in vessels without functional endothelium. Vascular hyperreactivity was blunted in the presence of a nitric oxide synthase inhibitor alone or combined with the cyclooxygenase inhibitor indomethacin. MPs from patients with OSA reduced endothelial nitric oxide synthase activity and nitric oxide production, increased aortic cyclooxygenase-1 and cyclooxygenase-2 expression, and increased thromboxane A(2) and prostacyclin production. Blockade of thromboxane A(2) receptor did not affect the serotonin response in arteries from OSA MP-treated mice. A superoxide dismutase mimetic reduced the vascular hyperreactivity induced by MPs from patients with OSA but had no effect on contraction in vessels from control and non-OSA MP-treated mice. These data provide evidence that circulating MPs from patients with OSA induce ex vivo vascular hyperreactivity with the obligatory role of the endothelium and subtle interactions between the nitric oxide and cyclooxygenase pathways and metabolites. These results highlight the participation of MPs in vascular dysfunction associated with OSA.

RNA-binding proteins in vascular inflammation and atherosclerosis.

Atherosclerotic cardiovascular disease (ASCVD) remains the major cause of premature death and disability worldwide, even when patients with an established manifestation of atherosclerotic heart disease are optimally treated according to the clinical guidelines. Apart from the epigenetic control of transcription of the genetic information to messenger RNAs (mRNAs), gene expression is tightly controlled at the post-transcriptional level before the initiation of translation. Although mRNAs are traditionally perceived as the messenger molecules that bring genetic information from the nuclear DNA to the cytoplasmic ribosomes for protein synthesis, emerging evidence suggests that processes controlling RNA metabolism, driven by RNA-binding proteins (RBPs), affect cellular function in health and disease. Over the recent years, vascular endothelial cell, smooth muscle cell and immune cell RBPs have emerged as key co- or post-transcriptional regulators of several genes related to vascular inflammation and atherosclerosis. In this review, we provide an overview of cell-specific function of RNA-binding proteins involved in all stages of ASCVD and how this knowledge may be used for the development of novel precision medicine therapeutics.

Endothelial dysfunction and immunothrombosis in sepsis.

Sepsis is a life-threatening clinical syndrome characterized by multiorgan dysfunction caused by a dysregulated or over-reactive host response to infection. During sepsis, the coagulation cascade is triggered by activated cells of the innate immune system, such as neutrophils and monocytes, resulting in clot formation mainly in the microcirculation, a process known as immunothrombosis. Although this process aims to protect the host through inhibition of the pathogen's dissemination and survival, endothelial dysfunction and microthrombotic complications can rapidly lead to multiple organ dysfunction. The development of treatments targeting endothelial innate immune responses and immunothrombosis could be of great significance for reducing morbidity and mortality in patients with sepsis. Medications modifying cell-specific immune responses or inhibiting platelet-endothelial interaction or platelet activation have been proposed. Herein, we discuss the underlying mechanisms of organ-specific endothelial dysfunction and immunothrombosis in sepsis and its complications, while highlighting the recent advances in the development of new therapeutic approaches aiming at improving the short- or long-term prognosis in sepsis.

Senescent cardiomyocytes contribute to cardiac dysfunction following myocardial infarction.

Myocardial infarction is a leading cause of morbidity and mortality. While reperfusion is now standard therapy, pathological remodeling leading to heart failure remains a clinical problem. Cellular senescence has been shown to contribute to disease pathophysiology and treatment with the senolytic navitoclax attenuates inflammation, reduces adverse myocardial remodeling and results in improved functional recovery. However, it remains unclear which senescent cell populations contribute to these processes. To identify whether senescent cardiomyocytes contribute to disease pathophysiology post-myocardial infarction, we established a transgenic model in which p16 (CDKN2A) expression was specifically knocked-out in the cardiomyocyte population. Following myocardial infarction, mice lacking cardiomyocyte p16 expression demonstrated no difference in cardiomyocyte hypertrophy but exhibited improved cardiac function and significantly reduced scar size in comparison to control animals. This data demonstrates that senescent cardiomyocytes participate in pathological myocardial remodeling. Importantly, inhibition of cardiomyocyte senescence led to reduced senescence-associated inflammation and decreased senescence-associated markers within other myocardial lineages, consistent with the hypothesis that cardiomyocytes promote pathological remodeling by spreading senescence to other cell-types. Collectively this study presents a novel demonstration that senescent cardiomyocytes are major contributors to myocardial remodeling and dysfunction following a myocardial infarction. Therefore, to maximize the potential for clinical translation, it is important to further understand the mechanisms underlying cardiomyocyte senescence and how to optimize senolytic strategies to target this cell lineage.

Senescent cardiomyocytes contribute to cardiac dysfunction following myocardial infarction.

Myocardial infarction is a leading cause of morbidity and mortality. While reperfusion is now standard therapy, pathological remodelling leading to heart failure remains a clinical problem. Cellular senescence has been shown to contribute to disease pathophysiology and treatment with the senolytic navitoclax attenuates inflammation, reduces adverse myocardial remodelling and results in improved functional recovery. However, it remains unclear which senescent cell populations contribute to these processes. To identify whether senescent cardiomyocytes contribute to disease pathophysiology post-myocardial infarction, we established a transgenic model in which p16 (CDKN2A) expression was specifically knocked-out in the cardiomyocyte population. Following myocardial infarction, mice lacking cardiomyocyte p16 expression demonstrated no difference in cardiomyocyte hypertrophy but exhibited improved cardiac function and significantly reduced scar size in comparison to control animals. This data demonstrates that senescent cardiomyocytes participate in pathological myocardial remodelling. Importantly, inhibition of cardiomyocyte senescence led to reduced senescence-associated inflammation and decreased senescence-associated markers within other myocardial lineages, consistent with the hypothesis that cardiomyocytes promote pathological remodelling by spreading senescence to other cell-types. Collectively this study presents the demonstration that senescent cardiomyocytes are major contributors to myocardial remodelling and dysfunction following a myocardial infarction. Therefore, to maximise the potential for clinical translation, it is important to further understand the mechanisms underlying cardiomyocyte senescence and how to optimise senolytic strategies to target this cell lineage.