<div><h4>Integrin-Dependent Cell-Matrix Adhesion in Endothelial Health and Disease.</h4><i>Aman J, Margadant C</i><br /><AbstractText>The endothelium is a dynamic, semipermeable layer lining all blood vessels, regulating blood vessel formation and barrier function. Proper composition and function of the endothelial barrier are required for fluid homeostasis, and clinical conditions characterized by barrier disruption are associated with severe morbidity and high mortality rates. Endothelial barrier properties are regulated by cell-cell junctions and intracellular signaling pathways governing the cytoskeleton, but recent insights indicate an increasingly important role for integrin-mediated cell-matrix adhesion and signaling in endothelial barrier regulation. Here, we discuss diseases characterized by endothelial barrier disruption, and provide an overview of the composition of endothelial cell-matrix adhesion complexes and associated signaling pathways, their crosstalk with cell-cell junctions, and with other receptors. We further present recent insights into the role of cell-matrix adhesions in the developing and mature/adult endothelium of various vascular beds, and discuss how the dynamic regulation and turnover of cell-matrix adhesions regulates endothelial barrier function in (patho)physiological conditions like angiogenesis, inflammation and in response to hemodynamic stress. Finally, as clinical conditions associated with vascular leak still lack direct treatment, we focus on how understanding of endothelial cell-matrix adhesion may provide novel targets for treatment, and discuss current translational challenges and future perspectives.</AbstractText><br /><br /><br /><br /><small>Circ Res: 03 Feb 2023; 132:355-378</small></div>

<div><h4>Iron Metabolism in Cardiovascular Disease: Physiology, Mechanisms, and Therapeutic Targets.</h4><i>Sawicki KT, De Jesus A, Ardehali H</i><br /><AbstractText>The cardiovascular system requires iron to maintain its high energy demands and metabolic activity. Iron plays a critical role in oxygen transport and storage, mitochondrial function, and enzyme activity. However, excess iron is also cardiotoxic due to its ability to catalyze the formation of reactive oxygen species and promote oxidative damage. While mammalian cells have several redundant iron import mechanisms, they are equipped with a single iron-exporting protein, which makes the cardiovascular system particularly sensitive to iron overload. As a result, iron levels are tightly regulated at many levels to maintain homeostasis. Iron dysregulation ranges from iron deficiency to iron overload and is seen in many types of cardiovascular disease, including heart failure, myocardial infarction, anthracycline-induced cardiotoxicity, and Friedreich\'s ataxia. Recently, the use of intravenous iron therapy has been advocated in patients with heart failure and certain criteria for iron deficiency. Here, we provide an overview of systemic and cellular iron homeostasis in the context of cardiovascular physiology, iron deficiency, and iron overload in cardiovascular disease, current therapeutic strategies, and future perspectives.</AbstractText><br /><br /><br /><br /><small>Circ Res: 03 Feb 2023; 132:379-396</small></div>

<div><h4>USP25 Ameliorates Pathological Cardiac Hypertrophy by Stabilizing SERCA2a in Cardiomyocytes.</h4><i>Ye B, Zhou H, Chen Y, Luo W, ... Wang X, Liang G</i><br /><b>Background</b><br />Pathological cardiac hypertrophy can lead to heart failure and is one of the leading causes of death globally. Understanding the molecular mechanism of pathological cardiac hypertrophy will contribute to the treatment of heart failure. DUBs (deubiquitinating enzymes) are essential to cardiac pathophysiology by precisely controlling protein function, localization, and degradation. This study set out to investigate the role and molecular mechanism of a DUB, USP25 (ubiquitin-specific peptidase 25), in pathological cardiac hypertrophy.<br /><b>Methods</b><br />The role of USP25 in myocardial hypertrophy was evaluated in murine cardiomyocytes in response to Ang II (angiotensin II) and transverse aortic constriction stimulation and in hypertrophic myocardium tissues of heart failure patients. Liquid chromotography with mass spectrometry/mass spectrometry analysis combined with Co-IP was used to identify SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2A), an antihypertrophy protein, as an interacting protein of USP25. To clarify the molecular mechanism of USP25 in the regulation of SERCA2a, we constructed a series of mutant plasmids of USP25. In addition, we overexpressed USP25 and SERCA2a in the heart with adenoassociated virus serotype 9 vectors to validate the biological function of USP25 and SERCA2a interaction.<br /><b>Results</b><br />We revealed increased protein level of USP25 in murine cardiomyocytes subject to Ang II and transverse aortic constriction stimulation and in hypertrophic myocardium tissues of patients with heart failure. USP25 deficiency aggravated cardiac hypertrophy and cardiac dysfunction under Ang II and transverse aortic constriction treatment. Mechanistically, USP25 bound to SERCA2a directly via its USP (ubiquitin-specific protease) domain and cysteine at position 178 of USP25 exerts deubiquitination to maintain the stability of the SERCA2a protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby maintaining calcium handling in cardiomyocytes. Moreover, restoration of USP25 expression via adenoassociated virus serotype 9 vectors in USP25<sup>-/-</sup> mice attenuated Ang II-induced cardiac hypertrophy and cardiac dysfunction, whereas myocardial overexpression of SERCA2a could mimic the effect of USP25.<br /><b>Conclusions</b><br />We confirmed that USP25 inhibited cardiac hypertrophy by deubiquitinating and stabilizing SERCA2a.<br /><br /><br /><br /><small>Circ Res: 01 Feb 2023; epub ahead of print</small></div>

<div><h4>CNP Promotes Antiarrhythmic Effects via Phosphodiesterase 2.</h4><i>Cachorro E, Günscht M, Schubert M, Sadek MS, ... Wagner M, Kämmerer S</i><br /><b>Background</b><br />Ventricular arrhythmia and sudden cardiac death are the most common lethal complications after myocardial infarction. Antiarrhythmic pharmacotherapy remains a clinical challenge and novel concepts are highly desired. Here, we focus on the cardioprotective CNP (C-type natriuretic peptide) as a novel antiarrhythmic principle. We hypothesize that antiarrhythmic effects of CNP are mediated by PDE2 (phosphodiesterase 2), which has the unique property to be stimulated by cGMP to primarily hydrolyze cAMP. Thus, CNP might promote beneficial effects of PDE2-mediated negative crosstalk between cAMP and cGMP signaling pathways.<br /><b>Methods</b><br />To determine antiarrhythmic effects of cGMP-mediated PDE2 stimulation by CNP, we analyzed arrhythmic events and intracellular trigger mechanisms in mice in vivo, at organ level and in isolated cardiomyocytes as well as in human-induced pluripotent stem cell-derived cardiomyocytes.<br /><b>Results</b><br />In ex vivo perfused mouse hearts, CNP abrogated arrhythmia after ischemia/reperfusion injury. Upon high-dose catecholamine injections in mice, PDE2 inhibition prevented the antiarrhythmic effect of CNP. In mouse ventricular cardiomyocytes, CNP blunted the catecholamine-mediated increase in arrhythmogenic events as well as in I<sub>CaL</sub>, I<sub>NaL</sub>, and Ca<sup>2+</sup> spark frequency. Mechanistically, this was driven by reduced cellular cAMP levels and decreased phosphorylation of Ca<sup>2+</sup> handling proteins. Key experiments were confirmed in human iPSC-derived cardiomyocytes. Accordingly, the protective CNP effects were reversed by either specific pharmacological PDE2 inhibition or cardiomyocyte-specific PDE2 deletion.<br /><b>Conclusions</b><br />CNP shows strong PDE2-dependent antiarrhythmic effects. Consequently, the CNP-PDE2 axis represents a novel and attractive target for future antiarrhythmic strategies.<br /><br /><br /><br /><small>Circ Res: 30 Jan 2023; epub ahead of print</small></div>

<div><h4>Circulating Extracellular Vesicle-Propagated microRNA Signature as a Vascular Calcification Factor in Chronic Kidney Disease.</h4><i>Koide T, Mandai S, Kitaoka R, Matsuki H, ... Yokota T, Uchida S</i><br /><b>Background</b><br />Chronic kidney disease (CKD) accelerates vascular calcification via phenotypic switching of vascular smooth muscle cells (VSMCs). We investigated the roles of circulating small extracellular vesicles (sEVs) between the kidneys and VSMCs and uncovered relevant sEV-propagated microRNAs (miRNAs) and their biological signaling pathways.<br /><b>Methods and results</b><br />We established CKD models in rats and mice by adenine-induced tubulointerstitial fibrosis. Cultures of A10 embryonic rat VSMCs showed increased calcification and transcription of osterix (<i>Sp7</i>), osteocalcin (<i>Bglap</i>), and osteopontin (<i>Spp1</i>) when treated with rat CKD serum. sEVs, but not sEV-depleted serum, accelerated calcification in VSMCs. Intraperitoneal administration of a neutral sphingomyelinase and biogenesis/release inhibitor of sEVs, GW4869 (2.5 mg/kg per 2 days), inhibited thoracic aortic calcification in CKD mice under a high-phosphorus diet. GW4869 induced a nearly full recovery of calcification and transcription of osteogenic marker genes. In CKD, the miRNA transcriptome of sEVs revealed a depletion of 4 miRNAs, <i>miR-16-5p</i>, <i>miR-17~92</i> cluster-originated <i>miR-17-5p</i>/<i>miR-20a-5p</i>, and <i>miR-106b-5p</i>. Their expression decreased in sEVs from CKD patients as kidney function deteriorated. Transfection of VSMCs with each miRNA-mimic mitigated calcification. In silico analyses revealed VEGFA (vascular endothelial growth factor A) as a convergent target of these miRNAs. We found a 16-fold increase in <i>VEGFA</i> transcription in the thoracic aorta of CKD mice under a high-phosphorus diet, which GW4869 reversed. Inhibition of VEGFA-VEGFR2 signaling with sorafenib, fruquintinib, sunitinib, or <i>VEGFR2</i>-targeted siRNA mitigated calcification in VSMCs. Orally administered fruquintinib (2.5 mg/kg per day) for 4 weeks suppressed the transcription of osteogenic marker genes in the mouse aorta. The area under the curve of <i>miR-16-5p</i>, <i>miR-17-5p</i>, <i>20a-5p</i>, and <i>miR-106b-5p</i> for the prediction of abdominal aortic calcification was 0.7630, 0.7704, 0.7407, and 0.7704, respectively.<br /><b>Conclusions</b><br />The miRNA transcriptomic signature of circulating sEVs uncovered their pathologic role, devoid of the calcification-protective miRNAs that target VEGFA signaling in CKD-driven vascular calcification. These sEV-propagated miRNAs are potential biomarkers and therapeutic targets for vascular calcification.<br /><br /><br /><br /><small>Circ Res: 26 Jan 2023; epub ahead of print</small></div>

<div><h4>Homeostatic, Non-Canonical Role of Macrophage Elastase in Vascular Integrity.</h4><i>Salarian M, Ghim M, Toczek J, Han J, ... Humphrey JD, Sadeghi MM</i><br /><b>Background</b><br />Matrix metalloproteinase (MMP)-12 is highly expressed in abdominal aortic aneurysms and its elastolytic function has been implicated in the pathogenesis. This concept is challenged, however, by conflicting data. Here, we sought to revisit the role of MMP-12 in abdominal aortic aneurysm.<br /><b>Methods</b><br /><i>Apoe</i><sup>-/-</sup> and <i>Mmp12</i><sup>-/-</sup>/<i>Apoe</i><sup>-/-</sup> mice were infused with Ang II (angiotensin). Expression of neutrophil extracellular traps (NETs) markers and complement component 3 (C3) levels were evaluated by immunostaining in aortas of surviving animals. Plasma complement components were analyzed by immunoassay. The effects of a complement inhibitor, IgG-FH<sub>1-5</sub> (factor H-immunoglobulin G), and macrophage-specific MMP-12 deficiency on adverse aortic remodeling and death from rupture in Ang II-infused mice were determined.<br /><b>Results</b><br />Unexpectedly, death from aortic rupture was significantly higher in <i>Mmp12</i><sup>-/-</sup>/<i>Apoe</i><sup>-/-</sup> mice. This associated with more neutrophils, citrullinated histone H3 and neutrophil elastase, markers of NETs, and C3 levels in <i>Mmp12</i><sup><i>-/</i>-</sup> aortas. These findings were recapitulated in additional models of abdominal aortic aneurysm. MMP-12 deficiency also led to more pronounced elastic laminae degradation and reduced collagen integrity. Higher plasma C5a in <i>Mmp12</i><sup>-/-</sup> mice pointed to complement overactivation. Treatment with IgG-FH<sub>1-5</sub> decreased aortic wall NETosis and reduced adverse aortic remodeling and death from rupture in Ang II-infused <i>Mmp12</i><sup>-/-</sup> mice. Finally, macrophage-specific MMP-12 deficiency recapitulated the effects of global MMP-12 deficiency on complement deposition and NETosis, as well as adverse aortic remodeling and death from rupture in Ang II-infused mice.<br /><b>Conclusions</b><br />An MMP-12 deficiency/complement activation/NETosis pathway compromises aortic integrity, which predisposes to adverse vascular remodeling and abdominal aortic aneurysm rupture. Considering these new findings, the role of macrophage MMP-12 in vascular homeostasis demands re-evaluation of MMP-12 function in diverse settings.<br /><br /><br /><br /><small>Circ Res: 24 Jan 2023; epub ahead of print</small></div>

<div><h4>Apolipoprotein Proteomics for Residual Lipid-Related Risk in Coronary Heart Disease.</h4><i>Clarke R, Von Ende A, Schmidt L, Yin X, ... Mayr M, PROCARDIS Consortium</i><br /><b>Background</b><br />Recognition of the importance of conventional lipid measures and the advent of novel lipid-lowering medications have prompted the need for more comprehensive lipid panels to guide use of emerging treatments for the prevention of coronary heart disease (CHD). This report assessed the relevance of 13 apolipoproteins measured using a single mass-spectrometry assay for risk of CHD in the PROCARDIS case-control study of CHD (941 cases/975 controls).<br /><b>Methods</b><br />The associations of apolipoproteins with CHD were assessed after adjustment for established risk factors and correction for statin use. Apolipoproteins were grouped into 4 lipid-related classes [lipoprotein(a), low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides] and their associations with CHD were adjusted for established CHD risk factors and conventional lipids. Analyses of these apolipoproteins in a subset of the ASCOT trial (Anglo-Scandinavian Cardiac Outcomes Trial) were used to assess their within-person variability and to estimate a correction for statin use. The findings in the PROCARDIS study were compared with those for incident cardiovascular disease in the Bruneck prospective study (n=688), including new measurements of Apo(a).<br /><b>Results</b><br />Triglyceride-carrying ApoC1, ApoC3, and ApoE (apolipoproteins) were most strongly associated with the risk of CHD (2- to 3-fold higher odds ratios for top versus bottom quintile) independent of conventional lipid measures. Likewise, ApoB was independently associated with a 2-fold higher odds ratios of CHD. Lipoprotein(a) was measured using peptides from the Apo(a)-kringle repeat and Apo(a)-constant regions, but neither of these associations differed from the association with conventionally measured lipoprotein(a). Among HDL-related apolipoproteins, ApoA4 and ApoM were inversely related to CHD, independent of conventional lipid measures. The disease associations with all apolipoproteins were directionally consistent in the PROCARDIS and Bruneck studies, with the exception of ApoM.<br /><b>Conclusions</b><br />Apolipoproteins were associated with CHD independent of conventional risk factors and lipids, suggesting apolipoproteins could help to identify patients with residual lipid-related risk and guide personalized approaches to CHD risk reduction.<br /><br /><br /><br /><small>Circ Res: 24 Jan 2023; epub ahead of print</small></div>

<div><h4>ADAR1 Non-Editing Function in Macrophage Activation and Abdominal Aortic Aneurysm.</h4><i>Cai D, Sun C, Murashita T, Que X, Chen SY</i><br /><b>Background</b><br />Macrophage activation plays a critical role in abdominal aortic aneurysm (AAA) development. However, molecular mechanisms controlling macrophage activation and vascular inflammation in AAA remain largely unknown. The objective of the study was to identify novel mechanisms underlying adenosine deaminase acting on RNA (ADAR1) function in macrophage activation and AAA formation.<br /><b>Methods</b><br />Aortic transplantation was conducted to determine the importance of nonvascular ADAR1 in AAA development/dissection. Ang II (Angiotensin II) infusion of ApoE-/- mouse model combined with macrophage-specific knockout of ADAR1 was used to study ADAR1 macrophage-specific role in AAA formation/dissection. The relevance of macrophage ADAR1 to human AAA was examined using human aneurysm specimens. Moreover, a novel humanized AAA model was established to test the role of human macrophages in aneurysm formation in human arteries.<br /><b>Results</b><br />Allograft transplantation of wild-type abdominal aortas to ADAR1+/- recipient mice significantly attenuated AAA formation, suggesting that nonvascular ADAR1 is essential for AAA development. ADAR1 deficiency in hematopoietic cells decreased the prevalence and severity of AAA while inhibited macrophage infiltration and aorta wall inflammation. ADAR1 deletion blocked the classic macrophage activation, diminished NF-κB (nuclear factor kappa B) signaling, and enhanced the expression of a number of anti-inflammatory microRNAs. Mechanistically, ADAR1 interacted with Drosha to promote its degradation, which attenuated Drosha-DGCR8 (DiGeorge syndrome critical region 8) interaction, and consequently inhibited pri- to pre-microRNA processing of microRNAs targeting IKKβ, resulting in an increased IKKβ (inhibitor of nuclear factor kappa-B) expression and enhanced NF-κB signaling. Significantly, ADAR1 was induced in macrophages and interacted with Drosha in human AAA lesions. Reconstitution of ADAR1-deficient, but not the wild type, human monocytes to immunodeficient mice blocked the aneurysm formation in transplanted human arteries.<br /><b>Conclusions</b><br />Macrophage ADAR1 promotes aneurysm formation in both mouse and human arteries through a novel mechanism, that is, Drosha protein degradation, which inhibits the processing of microRNAs targeting NF-kB signaling and thus elicits macrophage-mediated vascular inflammation in AAA.<br /><br /><br /><br /><small>Circ Res: 23 Jan 2023; epub ahead of print</small></div>

<div><h4>Interdependent Nuclear Co-Trafficking of ASPP1 and p53 Aggravates Cardiac Ischemia/Reperfusion Injury.</h4><i>Yang Y, Zhang Y, Yang J, Zhang M, ... Yang B, Pan Z</i><br /><b>Objective</b><br />ASPP1 (apoptosis stimulating of p53 protein 1) is critical in regulating cell apoptosis as a cofactor of p53 to promote its transcriptional activity in the nucleus. However, whether cytoplasmic ASPP1 affects p53 nuclear trafficking and its role in cardiac diseases remains unknown. This study aims to explore the mechanism by which ASPP1 modulates p53 nuclear trafficking and the subsequent contribution to cardiac ischemia/reperfusion (I/R) injury.<br /><b>Methods and results</b><br />The immunofluorescent staining showed that under normal condition ASPP1 and p53 colocalized in the cytoplasm of neonatal mouse ventricular cardiomyocytes, while they were both upregulated and translocated to the nuclei upon hypoxia/reoxygenation treatment. The nuclear translocation of ASPP1 and p53 was interdependent, as knockdown of either ASPP1 or p53 attenuated nuclear translocation of the other one. Inhibition of importin-β1 resulted in the cytoplasmic sequestration of both p53 and ASPP1 in neonatal mouse ventricular cardiomyocytes with hypoxia/reoxygenation stimulation. Overexpression of ASPP1 potentiated, whereas knockdown of ASPP1 inhibited the expression of Bax (Bcl2-associated X), PUMA (p53 upregulated modulator of apoptosis), and Noxa, direct apoptosis-associated targets of p53. ASPP1 was also increased in the I/R myocardium. Cardiomyocyte-specific transgenic overexpression of ASPP1 aggravated I/R injury as indicated by increased infarct size and impaired cardiac function. Conversely, knockout of ASPP1 mitigated cardiac I/R injury. The same qualitative data were observed in neonatal mouse ventricular cardiomyocytes exposed to hypoxia/reoxygenation injury. Furthermore, inhibition of p53 significantly blunted the proapoptotic activity and detrimental effects of ASPP1 both in vitro and in vivo.<br /><b>Conclusions</b><br />Binding of ASPP1 to p53 triggers their nuclear cotranslocation via importin-β1 that eventually exacerbates cardiac I/R injury. The findings imply that interfering the expression of ASPP1 or the interaction between ASPP1 and p53 to block their nuclear trafficking represents an important therapeutic strategy for cardiac I/R injury.<br /><br /><br /><br /><small>Circ Res: 20 Jan 2023; 132:208-222</small></div>

<div><h4>Immunology of Giant Cell Arteritis.</h4><i>Weyand CM, Goronzy JJ</i><br /><AbstractText>Giant cell arteritis is an autoimmune disease of medium and large arteries, characterized by granulomatous inflammation of the three-layered vessel wall that results in vaso-occlusion, wall dissection, and aneurysm formation. The immunopathogenesis of giant cell arteritis is an accumulative process in which a prolonged asymptomatic period is followed by uncontrolled innate immunity, a breakdown in self-tolerance, the transition of autoimmunity from the periphery into the vessel wall and, eventually, the progressive evolution of vessel wall inflammation. Each of the steps in pathogenesis corresponds to specific immuno-phenotypes that provide mechanistic insights into how the immune system attacks and damages blood vessels. Clinically evident disease begins with inappropriate activation of myeloid cells triggering the release of hepatic acute phase proteins and inducing extravascular manifestations, such as muscle pains and stiffness diagnosed as polymyalgia rheumatica. Loss of self-tolerance in the adaptive immune system is linked to aberrant signaling in the NOTCH pathway, leading to expansion of NOTCH1<sup>+</sup>CD4<sup>+</sup> T cells and the functional decline of NOTCH4<sup>+</sup> T regulatory cells (Checkpoint 1). A defect in the endothelial cell barrier of adventitial vasa vasorum networks marks Checkpoint 2; the invasion of monocytes, macrophages and T cells into the arterial wall. Due to the failure of the immuno-inhibitory PD-1 (programmed cell death protein 1)/PD-L1 (programmed cell death ligand 1) pathway, wall-infiltrating immune cells arrive in a permissive tissues microenvironment, where multiple T cell effector lineages thrive, shift toward high glycolytic activity, and support the development of tissue-damaging macrophages, including multinucleated giant cells (Checkpoint 3). Eventually, the vascular lesions are occupied by self-renewing T cells that provide autonomy to the disease process and limit the therapeutic effectiveness of currently used immunosuppressants. The multi-step process deviating protective to pathogenic immunity offers an array of interception points that provide opportunities for the prevention and therapeutic management of this devastating autoimmune disease.</AbstractText><br /><br /><br /><br /><small>Circ Res: 20 Jan 2023; 132:238-250</small></div>

<div><h4>The Circadian Biology of Heart Failure.</h4><i>El Jamal N, Lordan R, Teegarden SL, Grosser T, FitzGerald G</i><br /><AbstractText>Driven by autonomous molecular clocks that are synchronized by a master pacemaker in the suprachiasmatic nucleus, cardiac physiology fluctuates in diurnal rhythms that can be partly or entirely circadian. Cardiac contractility, metabolism, and electrophysiology, all have diurnal rhythms, as does the neurohumoral control of cardiac and kidney function. In this review, we discuss the evidence that circadian biology regulates cardiac function, how molecular clocks may relate to the pathogenesis of heart failure, and how chronotherapeutics might be applied in heart failure. Disrupting molecular clocks can lead to heart failure in animal models, and the myocardial response to injury seems to be conditioned by the time of day. Human studies are consistent with these findings, and they implicate the clock and circadian rhythms in the pathogenesis of heart failure. Certain circadian rhythms are maintained in patients with heart failure, a factor that can guide optimal timing of therapy. Pharmacologic and nonpharmacologic manipulation of circadian rhythms and molecular clocks show promise in the prevention and treatment of heart failure.</AbstractText><br /><br /><br /><br /><small>Circ Res: 20 Jan 2023; 132:223-237</small></div>

<div><h4>Mechanistic Insights of the LEMD2 p.L13R Mutation and Its Role in Cardiomyopathy.</h4><i>Chen R, Buchmann S, Kroth A, Arias-Loza AP, ... Frantz S, Gerull B</i><br /><b>Background</b><br />Nuclear envelope proteins play an important role in the pathogenesis of hereditary cardiomyopathies. Recently, a new form of arrhythmic cardiomyopathy caused by a homozygous mutation (p.L13R) in the inner nuclear membrane protein LEMD2 was discovered. The aim was to unravel the molecular mechanisms of mutant LEMD2 in the pathogenesis of cardiomyopathy.<br /><b>Methods</b><br />We generated a Lemd2 p.L13R knock-in mouse model and a corresponding cell model via CRISPR/Cas9 technology and investigated the cardiac phenotype as well as cellular and subcellular mechanisms of nuclear membrane rupture and repair.<br /><b>Results</b><br />Knock-in mice developed a cardiomyopathy with predominantly endocardial fibrosis, left ventricular dilatation, and systolic dysfunction. Electrocardiograms displayed pronounced ventricular arrhythmias and conduction disease. A key finding of knock-in cardiomyocytes on ultrastructural level was a significant increase in nuclear membrane invaginations and decreased nuclear circularity. Furthermore, increased DNA damage and premature senescence were detected as the underlying cause of fibrotic and inflammatory remodeling. As the p.L13R mutation is located in the Lap2/Emerin/Man1 (LEM)-domain, we observed a disrupted interaction between mutant LEMD2 and BAF (barrier-to-autointegration factor), which is required to initiate the nuclear envelope rupture repair process. To mimic increased mechanical stress with subsequent nuclear envelope ruptures, we investigated mutant HeLa-cells upon electrical stimulation and increased stiffness. Here, we demonstrated impaired nuclear envelope rupture repair capacity, subsequent cytoplasmic leakage of the DNA repair factor KU80 along with increased DNA damage, and recruitment of the cGAS (cyclic GMP-AMP synthase) to the nuclear membrane and micronuclei.<br /><b>Conclusions</b><br />We show for the first time that the Lemd2 p.L13R mutation in mice recapitulates human dilated cardiomyopathy with fibrosis and severe ventricular arrhythmias. Impaired nuclear envelope rupture repair capacity resulted in increased DNA damage and activation of the cGAS/STING/IFN pathway, promoting premature senescence. Hence, LEMD2 is a new player inthe disease group of laminopathies.<br /><br /><br /><br /><small>Circ Res: 20 Jan 2023; 132:e43-e58</small></div>

<div><h4>Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection.</h4><i>Carnevale R, Cammisotto V, Bartimoccia S, Nocella C, ... Pignatelli P, Violi F</i><br /><b>Background</b><br />SARS-CoV-2 is associated with an increased risk of venous and arterial thrombosis, but the underlying mechanism is still unclear.<br /><b>Methods</b><br />We performed a cross-sectional analysis of platelet function in 25 SARS-CoV-2 and 10 healthy subjects by measuring Nox2 (NADPH oxidase 2)-derived oxidative stress and thromboxane B<sub>2</sub>, and investigated if administration of monoclonal antibodies against the S protein (Spike protein) of SARS-CoV-2 affects platelet activation. Furthermore, we investigated in vitro if the S protein of SARS-CoV-2 or plasma from SARS-CoV-2 enhanced platelet activation.<br /><b>Results</b><br />Ex vivo studies showed enhanced platelet Nox2-derived oxidative stress and thromboxane B<sub>2</sub> biosynthesis and under laminar flow platelet-dependent thrombus growth in SARS-CoV-2 compared with controls; both effects were lowered by Nox2 and TLR4 (Toll-like receptor 4) inhibitors. Two hours after administration of monoclonal antibodies, a significant inhibition of platelet activation was observed in patients with SARS-CoV-2 compared with untreated ones. In vitro study showed that S protein per se did not elicit platelet activation but amplified the platelet response to subthreshold concentrations of agonists and functionally interacted with platelet TLR4. A docking simulation analysis suggested that TLR4 binds to S protein via three receptor-binding domains; furthermore, immunoprecipitation and immunofluorescence showed S protein-TLR4 colocalization in platelets from SARS-CoV-2. Plasma from patients with SARS-CoV-2 enhanced platelet activation and Nox2-related oxidative stress, an effect blunted by TNF (tumor necrosis factor) α inhibitor; this effect was recapitulated by an in vitro study documenting that TNFα alone promoted platelet activation and amplified the platelet response to S protein via p47phox upregulation.<br /><b>Conclusions</b><br />The study identifies 2 TLR4-dependent and independent pathways promoting platelet-dependent thrombus growth and suggests inhibition of TLR4. or p47phox as a tool to counteract thrombosis in SARS-CoV-2.<br /><br /><br /><br /><small>Circ Res: 13 Jan 2023; epub ahead of print</small></div>

<div><h4>Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation.</h4><i>Tousif S, Singh AP, Umbarkar P, Galindo C, ... Prabhu SD, Lal H</i><br /><b>Background</b><br />The tyrosine kinase inhibitor ponatinib is the only treatment option for chronic myelogenous leukemia patients with T315I (gatekeeper) mutation. Pharmacovigilance analysis of Food and Drug Administration and World Health Organization datasets has revealed that ponatinib is the most cardiotoxic agent among all Food and Drug Administration-approved tyrosine kinase inhibitors in a real-world scenario. However, the mechanism of ponatinib-induced cardiotoxicity is unknown.<br /><b>Methods</b><br />The lack of well-optimized mouse models has hampered the in vivo cardio-oncology studies. Here, we show that cardiovascular comorbidity mouse models evidence a robust cardiac pathological phenotype upon ponatinib treatment. A combination of multiple in vitro and in vivo models was employed to delineate the underlying molecular mechanisms.<br /><b>Results</b><br />An unbiased RNA sequencing analysis identified the enrichment of dysregulated inflammatory genes, including a multifold upregulation of alarmins S100A8/A9, as a top hit in ponatinib-treated hearts. Mechanistically, we demonstrate that ponatinib activates the S100A8/9-TLR4 (Toll-like receptor 4)-NLRP3 (NLR family pyrin domain-containing 3)-IL (interleukin)-1β signaling pathway in cardiac and systemic myeloid cells, in vitro and in vivo, thereby leading to excessive myocardial and systemic inflammation. Excessive inflammation was central to the cardiac pathology because interventions with broad-spectrum immunosuppressive glucocorticoid dexamethasone or specific inhibitors of NLRP3 (CY-09) or S100A9 (paquinimod) nearly abolished the ponatinib-induced cardiac dysfunction.<br /><b>Conclusions</b><br />Taken together, these findings uncover a novel mechanism of ponatinib-induced cardiac inflammation leading to cardiac dysfunction. From a translational perspective, our results provide critical preclinical data and rationale for a clinical investigation into immunosuppressive interventions for managing ponatinib-induced cardiotoxicity.<br /><br /><br /><br /><small>Circ Res: 10 Jan 2023; epub ahead of print</small></div>

<div><h4>Platelet-Mimicking Nanosponges for Functional Reversal of Antiplatelet Agents.</h4><i>Xu J, Yan N, Wang C, Gao C, ... Zhang Y, Nie G</i><br /><AbstractText>During long-term antiplatelet agents (APAs) administration, patients with thrombotic diseases take a fairly high risk of life-threatening bleeding, especially when in need of urgent surgery. Rapid functional reversal of APAs remains an issue yet to be efficiently resolved by far due to the lack of any specific reversal agent in the clinic, which greatly restricts the use of APAs. Although platelet transfusion has become a common practice to counteract the function of APAs, personalized dosage of platelet transfusion is difficult to be judged by assessing the trade-off between its effectiveness and additional thrombotic risk. Here we reported a platelet-mimicking perfluorocarbon-based nanosponge, which potently reversed the antiplatelet effect of APAs by competitively binding with APAs. Platelet-mimicking perfluorocarbon-based nanosponges showed high binding affinity comparable to fresh platelets in vitro with first-line APAs, ticagrelor and tirofiban, and efficiently reversed their function in both tail bleeding and postischemic-reperfusion models. Moreover, the deficiency of platelet intrinsic thrombotic activity diminished the risk of thrombogenesis. In summary, our results demonstrated the safety and effectiveness of the platelet-mimicking nanosponge in ameliorating the bleeding risk of different APAs, which offers a promising strategy for the management of bleeding complications induced by antiplatelet therapy.</AbstractText><br /><br /><br /><br /><small>Circ Res: 10 Jan 2023; epub ahead of print</small></div>

<div><h4> Mutation and Metabolic Reprogramming in Pulmonary Arterial Hypertension.</h4><i>Cuthbertson I, Morrell NW, Caruso P</i><br /><AbstractText>Pulmonary arterial hypertension forms the first and most severe of the 5 categories of pulmonary hypertension. Disease pathogenesis is driven by progressive remodeling of peripheral pulmonary arteries, caused by the excessive proliferation of vascular wall cells, including endothelial cells, smooth muscle cells and fibroblasts, and perivascular inflammation. Compelling evidence from animal models suggests endothelial cell dysfunction is a key initial trigger of pulmonary vascular remodeling, which is characterised by hyperproliferation and early apoptosis followed by enrichment of apoptosis-resistant populations. Dysfunctional pulmonary arterial endothelial cells lose their ability to produce vasodilatory mediators, together leading to augmented pulmonary arterial smooth muscle cell responses, increased pulmonary vascular pressures and right ventricular afterload, and progressive right ventricular hypertrophy and heart failure. It is recognized that a range of abnormal cellular molecular signatures underpin the pathophysiology of pulmonary arterial hypertension and are enhanced by loss-of-function mutations in the <i>BMPR2</i> gene, the most common genetic cause of pulmonary arterial hypertension and associated with worse disease prognosis. Widespread metabolic abnormalities are observed in the heart, pulmonary vasculature, and systemic tissues, and may underpin heterogeneity in responsivity to treatment. Metabolic abnormalities include hyperglycolytic reprogramming, mitochondrial dysfunction, aberrant polyamine and sphingosine metabolism, reduced insulin sensitivity, and defective iron handling. This review critically discusses published mechanisms linking metabolic abnormalities with dysfunctional BMPR2 (bone morphogenetic protein receptor 2) signaling; hypothesized mechanistic links requiring further validation; and their relevance to pulmonary arterial hypertension pathogenesis and the development of potential therapeutic strategies.</AbstractText><br /><br /><br /><br /><small>Circ Res: 06 Jan 2023; 132:109-126</small></div>

<div><h4>Cardiac Alternans: From Bedside to Bench and Back.</h4><i>Qu Z, Weiss JN</i><br /><AbstractText>Cardiac alternans arises from dynamical instabilities in the electrical and calcium cycling systems of the heart, and often precedes ventricular arrhythmias and sudden cardiac death. In this review, we integrate clinical observations with theory and experiment to paint a holistic portrait of cardiac alternans: the underlying mechanisms, arrhythmic manifestations and electrocardiographic signatures. We first summarize the cellular and tissue mechanisms of alternans that have been demonstrated both theoretically and experimentally, including 3 voltage-driven and 2 calcium-driven alternans mechanisms. Based on experimental and simulation results, we describe their relevance to mechanisms of arrhythmogenesis under different disease conditions, and their link to electrocardiographic characteristics of alternans observed in patients. Our major conclusion is that alternans is not only a predictor, but also a causal mechanism of potentially lethal ventricular and atrial arrhythmias across the full spectrum of arrhythmia mechanisms that culminate in functional reentry, although less important for anatomic reentry and focal arrhythmias.</AbstractText><br /><br /><br /><br /><small>Circ Res: 06 Jan 2023; 132:127-149</small></div>

<div><h4>Genetic Regulation of Gene Expression and Splicing Predict Causal Genes.</h4><i>Aherrahrou R, Lue D, Perry RN, Aberra YT, ... Kaikkonen MU, Civelek M</i><br /><b>Background</b><br />Coronary artery disease (CAD) is the leading cause of death worldwide. Recent meta-analyses of genome-wide association studies have identified over 175 loci associated with CAD. The majority of these loci are in noncoding regions and are predicted to regulate gene expression. Given that vascular smooth muscle cells (SMCs) play critical roles in the development and progression of CAD, we aimed to identify the subset of the CAD genome-wide association studies risk loci associated with the regulation of transcription in distinct SMC phenotypes.<br /><b>Methods</b><br />Here, we measured gene expression in SMCs isolated from the ascending aortas of 151 heart transplant donors of various genetic ancestries in quiescent or proliferative conditions and calculated the association of their expression and splicing with ~6.3 million imputed single-nucleotide polymorphism markers across the genome.<br /><b>Results</b><br />We identified 4910 expression and 4412 splice quantitative trait loci representing regions of the genome associated with transcript abundance and splicing. A total of 3660 expression quantitative trait locus (eQTLs) had not been observed in the publicly available Genotype-Tissue Expression dataset. Further, 29 and 880 eQTLs were SMC- and sex-specific, respectively. We made these results available for public query on a user-friendly website. To identify the effector transcript(s) regulated by CAD genome-wide association studies loci, we used 4 distinct colocalization approaches. We identified 84 eQTL and 164 splice quantitative trait loci that colocalized with CAD loci, highlighting the importance of genetic regulation of mRNA splicing as a molecular mechanism for CAD genetic risk. Notably, 20% and 35% of the eQTLs were unique to quiescent or proliferative SMCs, respectively. One CAD locus colocalized with an SMC sex-specific eQTL (<i>TERF2IP</i>), and another locus colocalized with SMC-specific eQTL (<i>ALKBH8</i>). The most significantly associated CAD locus, 9p21, was an splice quantitative trait loci for the long noncoding RNA <i>CDKN2B-AS1</i>, also known as <i>ANRIL</i>, in proliferative SMCs.<br /><b>Conclusions</b><br />Collectively, our results provide evidence for the molecular mechanisms of genetic susceptibility to CAD in distinct SMC phenotypes.<br /><br /><br /><br /><small>Circ Res: 04 Jan 2023; epub ahead of print</small></div>

<div><h4>Metabolomic Signatures Associated With Pulmonary Arterial Hypertension Outcomes.</h4><i>Pi H, Xia L, Ralph DD, Rayner SG, ... Leary PJ, Gharib SA</i><br /><b>Background</b><br />Pulmonary arterial hypertension (PAH) is a complex disease characterized by progressive right ventricular (RV) failure leading to significant morbidity and mortality. Investigating metabolic features and pathways associated with RV dilation, mortality, and measures of disease severity can provide insight into molecular mechanisms, identify subphenotypes, and suggest potential therapeutic targets.<br /><b>Methods</b><br />We collected data from a prospective cohort of PAH participants and performed untargeted metabolomic profiling on 1045 metabolites from circulating blood. Analyses were intended to identify metabolomic differences across a range of common metrics in PAH (eg, dilated versus nondilated RV). Partial least squares discriminant analysis was first applied to assess the distinguishability of relevant outcomes. Significantly altered metabolites were then identified using linear regression, and Cox regression models (as appropriate for the specific outcome) with adjustments for age, sex, body mass index, and PAH cause. Models exploring RV maladaptation were further adjusted for pulmonary vascular resistance. Pathway enrichment analysis was performed to identify significantly dysregulated processes.<br /><b>Results</b><br />A total of 117 participants with PAH were included. Partial least squares discriminant analysis showed cluster differentiation between participants with dilated versus nondilated RVs, survivors versus nonsurvivors, and across a range of NT-proBNP (N-terminal probrain natriuretic peptide) levels, REVEAL 2.0 composite scores, and 6-minute-walk distances. Polyamine and histidine pathways were associated with differences in RV dilation, mortality, NT-proBNP, REVEAL score, and 6-minute walk distance. Acylcarnitine pathways were associated with NT-proBNP, REVEAL score, and 6-minute walk distance. Sphingomyelin pathways were associated with RV dilation and NT-proBNP after adjustment for pulmonary vascular resistance.<br /><b>Conclusions</b><br />Distinct plasma metabolomic profiles are associated with RV dilation, mortality, and measures of disease severity in PAH. Polyamine, histidine, and sphingomyelin metabolic pathways represent promising candidates for identifying patients at high risk for poor outcomes and investigation into their roles as markers or mediators of disease progression and RV adaptation.<br /><br /><br /><br /><small>Circ Res: 04 Jan 2023; epub ahead of print</small></div>

<div><h4>RyR2 Serine-2030 PKA Site Governs Ca Release Termination and Ca Alternans.</h4><i>Wei J, Guo W, Wang R, Paul Estillore J, ... Hove-Madsen L, Chen SRW</i><br /><b>Background</b><br />PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca<sup>2+</sup> release and induction of cardiac Ca<sup>2+</sup> alternans. To determine the role of the PKA phosphorylation site RyR2-S2030 in Ca<sup>2+</sup> release termination in vitro and cardiac Ca<sup>2+</sup> alternans in intact hearts.<br /><b>Methods and results</b><br />We performed single-cell endoplasmic reticulum Ca<sup>2+</sup> imaging to assess the impact of S2030 mutations on Ca<sup>2+</sup> release termination in HEK293 cells. We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca<sup>2+</sup> level at which Ca<sup>2+</sup> release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca<sup>2+</sup> binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca<sup>2+</sup>/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca<sup>2+</sup> release termination. To determine the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation. Using confocal Ca<sup>2+</sup> imaging, we found that isoproterenol and CaM-M suppressed pacing-induced Ca<sup>2+</sup> alternans and accelerated Ca<sup>2+</sup> transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor H89 and the CaMKII inhibitor KN93 individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and H89-sensitive component, but not the KN93-sensitive component, of isoproterenol.<br /><b>Conclusions</b><br />These data demonstrate, for the first time, that the PKA phosphorylation site RyR-S2030 is an important determinant of PKA-regulated, CaM-dependent Ca<sup>2+</sup> release termination, and Ca<sup>2+</sup> alternans.<br /><br /><br /><br /><small>Circ Res: 30 Dec 2022; epub ahead of print</small></div>

<div><h4>Impaired Human Cardiac Cell Development due to NOTCH1 Deficiency.</h4><i>Ye S, Wang C, Xu Z, Lin H, ... Wu JC, Zhao MT</i><br /><b>Background</b><br /><i>NOTCH1</i> pathogenic variants are implicated in multiple types of congenital heart defects including hypoplastic left heart syndrome, where the left ventricle is underdeveloped. It is unknown how <i>NOTCH1</i> regulates human cardiac cell lineage determination and cardiomyocyte proliferation. In addition, mechanisms by which <i>NOTCH1</i> pathogenic variants lead to ventricular hypoplasia in hypoplastic left heart syndrome remain elusive.<br /><b>Methods</b><br />CRISPR/Cas9 genome editing was utilized to delete <i>NOTCH1</i> in human induced pluripotent stem cells. Cardiac differentiation was carried out by sequential modulation of WNT signaling, and <i>NOTCH1</i> knockout and wild-type differentiating cells were collected at day 0, 2, 5, 10, 14, and 30 for single-cell RNA-seq.<br /><b>Results</b><br />Human <i>NOTCH1</i> knockout induced pluripotent stem cells are able to generate functional cardiomyocytes and endothelial cells, suggesting that NOTCH1 is not required for mesoderm differentiation and cardiovascular development in vitro. However, disruption of NOTCH1 blocks human ventricular-like cardiomyocyte differentiation but promotes atrial-like cardiomyocyte generation through shortening the action potential duration. <i>NOTCH1</i> deficiency leads to defective proliferation of early human cardiomyocytes, and transcriptomic analysis indicates that pathways involved in cell cycle progression and mitosis are downregulated in <i>NOTCH1</i> knockout cardiomyocytes. Single-cell transcriptomic analysis reveals abnormal cell lineage determination of cardiac mesoderm, which is manifested by the biased differentiation toward epicardial and second heart field progenitors at the expense of first heart field progenitors in <i>NOTCH1</i> knockout cell populations.<br /><b>Conclusions</b><br /><i>NOTCH1</i> is essential for human ventricular-like cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm and modulating cell cycle progression. Because first heart field progenitors primarily contribute to the left ventricle, we speculate that pathogenic <i>NOTCH1</i> variants lead to biased differentiation of first heart field progenitors, blocked ventricular-like cardiomyocyte differentiation, and defective cardiomyocyte proliferation, which collaboratively contribute to left ventricular hypoplasia in hypoplastic left heart syndrome.<br /><br /><br /><br /><small>Circ Res: 30 Dec 2022; epub ahead of print</small></div>

<div><h4> Protects Against Elevated Arterial Stiffness.</h4><i>Luo S, Zhao Y, Zhu S, Liu L, ... Fan J, Xia M</i><br /><b>Background</b><br />Dysbiosis of gut microbiota plays a pivotal role in vascular dysfunction and microbial diversity was reported to be inversely correlated with arterial stiffness. However, the causal role of gut microbiota in the progression of arterial stiffness and the specific species along with the molecular mechanisms underlying this change remain largely unknown.<br /><b>Methods</b><br />Participants with elevated arterial stiffness and normal controls free of medication were matched for age and sex. The microbial composition and metabolic capacities between the 2 groups were compared with the integration of metagenomics and metabolomics. Subsequently, AngII (angiotensin II)-induced and humanized mouse model were employed to evaluate the protective effect of <i>Flavonifractor plautii</i> (<i>F. plautii</i>) and its main effector cis-aconitic acid.<br /><b>Results</b><br />Human fecal metagenomic sequencing revealed a significantly high abundance and centrality of <i>F. plautii</i> in normal controls, which was absent in the microbial community of subjects with elevated arterial stiffness. Moreover, blood pressure only mediated part of the effect of <i>F. plautii</i> on lower arterial stiffness. The microbiome of normal controls exhibited an enhanced capacity for glycolysis and polysaccharide degradation, whereas, those of subjects with increased arterial stiffness were characterized by increased biosynthesis of fatty acids and aromatic amino acids. Integrative analysis with metabolomics profiling further suggested that increased cis-aconitic acid served as the main effector for the protective effect of <i>F. plautii</i> against arterial stiffness. Replenishment with <i>F. plautii</i> and cis-aconitic acid improved elastic fiber network and reversed increased pulse wave velocity through the suppression of MMP-2 (matrix metalloproteinase-2) and inhibition of MCP-1 (monocyte chemoattractant protein-1) and NF-κB (nuclear factor kappa-B) activation in both AngII-induced and humanized model of arterial stiffness.<br /><b>Conclusions</b><br />Our translational study identifies a novel link between <i>F. plautii</i> and arterial function and raises the possibility of sustaining vascular health by targeting gut microbiota.<br /><br /><br /><br /><small>Circ Res: 28 Dec 2022; epub ahead of print</small></div>

<div><h4>Hydrogen Sulfide Modulates Endothelial-Mesenchymal Transition in Heart Failure.</h4><i>Li Z, Xia H, Sharp TE, LaPenna KB, ... Papapetropoulos A, Lefer DJ</i><br /><b>Background</b><br />Hydrogen sulfide is a critical endogenous signaling molecule that exerts protective effects in the setting of heart failure. Cystathionine γ-lyase (CSE), 1 of 3 hydrogen-sulfide-producing enzyme, is predominantly localized in the vascular endothelium. The interaction between the endothelial CSE-hydrogen sulfide axis and endothelial-mesenchymal transition, an important pathological process contributing to the formation of fibrosis, has yet to be investigated.<br /><b>Methods</b><br />Endothelial-cell-specific CSE knockout and Endothelial cell-CSE overexpressing mice were subjected to transverse aortic constriction to induce heart failure with reduced ejection fraction. Cardiac function, vascular reactivity, and treadmill exercise capacity after transverse aortic constriction were measured to determine the severity of heart failure. Histological and gene expression analyses were performed to investigate changes in cardiac fibrosis and endothelial-mesenchymal transition activation.<br /><b>Results</b><br />Endothelial-cell-specific CSE knockout mice exhibited increased endothelial-mesenchymal transition and reduced nitric oxide bioavailability in the myocardium, which was associated with increased cardiac fibrosis, impaired cardiac and vascular function, and worsened exercise performance. In contrast, genetic overexpression of CSE in endothelial cells led to increased myocardial nitric oxide, decreased endothelial-mesenchymal transition and cardiac fibrosis, preserved cardiac and endothelial function, and improved exercise capacity.<br /><b>Conclusions</b><br />Our data demonstrate that endothelial CSE modulates endothelial-mesenchymal transition and ameliorate the severity of pressure-overload-induced heart failure, in part, through nitric oxide-related mechanisms. These data further suggest that endothelium-derived hydrogen sulfide is a potential therapeutic for the treatment of heart failure with reduced ejection fraction.<br /><br /><br /><br /><small>Circ Res: 28 Dec 2022; epub ahead of print</small></div>

<div><h4>NDRG1 Signaling is Essential for Endothelial Inflammation and Vascular Remodeling.</h4><i>Zhang G, Qin Q, Zhang C, Sun X, ... Guo ZF, Sun J</i><br /><b>Background</b><br />NDRG-1 (N-myc downstream-regulated gene 1) is a member of NDRG family that plays essential roles in cell differentiation, proliferation, and stress responses. Although the expression of NDRG1 is regulated by fluid shear stress, its roles in vascular biology remain poorly understood. The purpose of the study is to determine the functional significance of NDRG1 in vascular inflammation and remodeling.<br /><b>Methods and results</b><br />By using quantitative polymerase chain reaction, western blot, and immunohistochemistry, we demonstrate that the expression of NDRG1 is markedly increased in cytokine-stimulated endothelial cells and in human and mouse atherosclerotic lesions. To determine the role of NDRG1 in endothelial activation, we performed loss-of-function studies using NDRG1 short hairpin RNA. Our results demonstrate that NDRG1 knockdown by lentivirus bearing NDRG1 short hairpin RNA substantially attenuates both IL-1β and TNF-α (tumor necrosis factor-α)-induced expression of cytokines/chemokines and adhesion molecules. Intriguingly, inhibition of NDRG1 also significantly attenuates the expression of procoagulant molecules, such as PAI-1 (plasminogen activator inhibitor type 1) and TF (tissue factor), and increases the expression of TM (thrombomodulin) and t-PA (tissue-type plasminogen activator), thus exerting potent antithrombotic effects in endothelial cells. Mechanistically, we showed that NDRG1 interacts with orphan Nur77 (nuclear receptor) and functionally inhibits the transcriptional activity of Nur77 and NF-κB (nuclear factor Kappa B) in endothelial cells. Moreover, in NDRG1 knockdown cells, both cytokine-induced mitogen-activated protein kinase activation, c-Jun phosphorylation, and AP-1 (activator protein 1) transcriptional activity are substantially inhibited. Neointima and atherosclerosis formation induced by carotid artery ligation and arterial thrombosis were markedly attenuated in endothelial cell-specific NDRG1 knockout mice compared with their wild-type littermates.<br /><b>Conclusions</b><br />Our results for the first time identify NDRG1 as a critical mediator implicated in regulating endothelial inflammation, thrombotic responses, and vascular remodeling, and suggest that inhibition of NDRG1 may represent a novel therapeutic strategy for inflammatory vascular diseases, such as atherothrombosis and restenosis.<br /><br /><br /><br /><small>Circ Res: 23 Dec 2022; epub ahead of print</small></div>

<div><h4>Inhibition of Eicosanoid Degradation Mitigates Fibrosis of the Heart.</h4><i>Rubino M, Travers JG, Headrick AL, Enyart BT, ... Koch KA, McKinsey TA</i><br /><b>Background</b><br />Organ fibrosis due to excessive production of extracellular matrix by resident fibroblasts is estimated to contribute to >45% of deaths in the Western world, including those due to cardiovascular diseases such as heart failure. Here, we screened for small molecule inhibitors with a common ability to suppress activation of fibroblasts across organ systems.<br /><b>Methods</b><br />High-content imaging of cultured cardiac, pulmonary, and renal fibroblasts was used to identify nontoxic compounds that blocked induction of markers of activation in response to the profibrotic stimulus, transforming growth factor-β1. SW033291, which inhibits the eicosanoid-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase, was chosen for follow-up studies with cultured adult rat ventricular fibroblasts and human cardiac fibroblasts (CFs), and for evaluation in mouse models of cardiac fibrosis and diastolic dysfunction. Additional mechanistic studies were performed with CFs treated with exogenous eicosanoids.<br /><b>Results</b><br />Nine compounds, including SW033291, shared a common ability to suppress transforming growth factor-β1-mediated activation of cardiac, pulmonary, and renal fibroblasts. SW033291 dose-dependently inhibited transforming growth factor-β1-induced expression of activation markers (eg, α-smooth muscle actin and periostin) in adult rat ventricular fibroblasts and normal human CFs, and reduced contractile capacity of the cells. Remarkably, the 15-hydroxyprostaglandin dehydrogenase inhibitor also reversed constitutive activation of fibroblasts obtained from explanted hearts from patients with heart failure. SW033291 blocked cardiac fibrosis induced by angiotensin II infusion and ameliorated diastolic dysfunction in an alternative model of systemic hypertension driven by combined uninephrectomy and deoxycorticosterone acetate administration. Mechanistically, SW033291-mediated stimulation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase signaling was required for SW033291 to block CF activation. Of the 12 exogenous eicosanoids that were tested, only 12-hydroxyeicosatetraenoic acid, which signals through the G protein-coupled receptor, GPR31, recapitulated the suppressive effects of SW033291 on CF activation.<br /><b>Conclusions</b><br />Inhibition of degradation of eicosanoids, arachidonic acid-derived fatty acids that signal through G protein-coupled receptors, is a potential therapeutic strategy for suppression of pathological organ fibrosis. In the heart, we propose that 15-hydroxyprostaglandin dehydrogenase inhibition triggers CF-derived autocrine/paracrine signaling by eicosanoids, including 12-hydroxyeicosatetraenoic acid, to stimulate extracellular signal-regulated kinase 1/2 and block conversion of fibroblasts into activated cells that secrete excessive amounts of extracellular matrix and contribute of heart failure pathogenesis.<br /><br /><br /><br /><small>Circ Res: 08 Dec 2022; epub ahead of print</small></div>

<div><h4>LLPS of DDR1 Counteracts the Hippo Pathway to Orchestrate Arterial Stiffening.</h4><i>Liu J, Wang J, Liu Y, Xie SA, ... Wang X, Zhou J</i><br /><b>Background</b><br />The Hippo-YAP (yes-associated protein) signaling pathway is modulated in response to various environmental cues. Activation of YAP in vascular smooth muscle cells conveys the extracellular matrix stiffness-induced changes in vascular smooth muscle cells phenotype and behavior. Recent studies have established a mechanoreceptive role of receptor tyrosine kinase DDR1 (discoidin domain receptor 1) in vascular smooth muscle cells.<br /><b>Methods</b><br />We conduced 5/6 nephrectomy in vascular smooth muscle cells-specific Ddr1-knockout mice, accompanied by pharmacological inhibition of the Hippo pathway kinase large tumor suppressor 1 (LATS1), to investigate DDR1 in YAP activation. We utilized polyacrylamide gels of varying stiffness or the DDR1 ligand, type I collagen, to stimulate the cells. We employed multiple molecular biological techniques to explore the role of DDR1 in controlling the Hippo pathway and to determine the mechanistic basis by which DDR1 exerts this effect.<br /><b>Results</b><br />We identified the requirement for DDR1 in stiffness/collagen-induced YAP activation. We uncovered that DDR1 underwent stiffness/collagen binding-stimulated liquid-liquid phase separation and co-condensed with LATS1 to inactivate LATS1. Mutagenesis experiments revealed that the transmembrane domain is responsible for DDR1 droplet formation. Purified DDR1 N-terminal and transmembrane domain was sufficient to drive its reversible condensation. Depletion of the DDR1 C-terminus led to failure in co-condensation with LATS1. Interaction between the DDR1 C-terminus and LATS1 competitively inhibited binding of MOB1 (Mps one binder 1) to LATS1 and thus the subsequent phosphorylation of LATS1. <br /><b>Introduction:</b><br/>of the single-point mutants, histidine-745-proline and histidine-902-proline, to DDR1 on the C-terminus abolished the co-condensation. In mouse models, YAP activity was positively correlated with collagen I expression and arterial stiffness. LATS1 inhibition reactivated the YAP signaling in Ddr1-deficient vessels and abrogated the arterial softening effect of Ddr1 deficiency.<br /><b>Conclusions</b><br />These findings identify DDR1 as a mediator of YAP activation by mechanical and chemical stimuli and demonstrate that DDR1 regulates LATS1 phosphorylation in an liquid-liquid phase separation-dependent manner.<br /><br /><br /><br /><small>Circ Res: 08 Dec 2022; epub ahead of print</small></div>

<div><h4>Tropoelastin Improves Post-Infarct Cardiac Function by Increasing Scar Elastin.</h4><i>Hume RD, Kanagalingam S, Deshmukh T, Chen S, ... Weiss AS, Chong JJH</i><br /><b>Background</b><br />Myocardial infarction (MI) is among the leading causes of death worldwide. Following MI, necrotic cardiomyocytes are replaced by a stiff collagen-rich scar. Compared to collagen, the extracellular matrix protein elastin has high elasticity and may have more favorable properties within the cardiac scar. We sought to improve post-MI healing by introducing tropoelastin, the soluble subunit of elastin, to alter scar mechanics early after MI.<br /><b>Methods and results</b><br />We developed an ultrasound-guided direct intramyocardial injection method to administer tropoelastin directly into the left ventricular anterior wall of rats subjected to induced MI. Experimental groups included shams and infarcted rats injected with either PBS vehicle control or tropoelastin. Compared to vehicle treated controls, echocardiography assessments showed tropoelastin significantly improved left ventricular ejection fraction (64.7±4.4% versus 46.0±3.1% control) and reduced left ventricular dyssynchrony (11.4±3.5 ms versus 31.1±5.8 ms control) 28 days post-MI. Additionally, tropoelastin reduced post-MI scar size (8.9±1.5% versus 20.9±2.7% control) and increased scar elastin (22±5.8% versus 6.2±1.5% control) as determined by histological assessments. Ribonucleic acid sequencing analyses of rat infarcts showed that tropoelastin injection increased genes associated with elastic fiber formation 7 days post-MI and reduced genes associated with immune response 11 days post-MI. To show translational relevance, we performed immunohistochemical analyses on human ischemic heart disease cardiac samples and showed an increase in tropoelastin within fibrotic areas. Using ribonucleic acid sequencing we also demonstrated the tropoelastin gene <i>ELN</i> is upregulated in human ischemic heart disease and during human cardiac fibroblast-myofibroblast differentiation. Furthermore, we showed by immunocytochemistry that human cardiac fibroblast synthesize increased elastin in direct response to tropoelastin treatment.<br /><b>Conclusions</b><br />We demonstrate for the first time that purified human tropoelastin can significantly repair the infarcted heart in a rodent model of MI and that human cardiac fibroblast synthesize elastin. Since human cardiac fibroblasts are primarily responsible for post-MI scar synthesis, our findings suggest exciting future clinical translation options designed to therapeutically manipulate this synthesis.<br /><br /><br /><br /><small>Circ Res: 01 Dec 2022; epub ahead of print</small></div>

<div><h4>Praliciguat Promotes Ischemic Leg Reperfusion on Leptin Receptor-Deficient Mice.</h4><i>Foussard N, Rouault P, Cornuault L, Reynaud A, ... Mohammedi K, Renault MA</i><br /><b>Background</b><br />Lower-limb peripheral artery disease is one of the major complications of diabetes. Peripheral artery disease is associated with poor limb and cardiovascular prognoses, along with a dramatic decrease in life expectancy. Despite major medical advances in the treatment of diabetes, a substantial therapeutic gap remains in the peripheral artery disease population. Praliciguat is an orally available sGC (soluble guanylate cyclase) stimulator that has been reported both preclinically and in early stage clinical trials to have favorable effects in metabolic and hemodynamic outcomes, suggesting that it may have a potential beneficial effect in peripheral artery disease.<br /><b>Objective</b><br />We evaluated the effect of praliciguat on hind limb ischemia recovery in a mouse model of type 2 diabetes.<br /><b>Methods</b><br />HLI was induced in leptin receptor-deficient (Lepr<sup><i>db/db</i></sup>) mice by ligation and excision of the left femoral artery. Praliciguat (10 mg/kg/day) was administered in the diet starting 3 days before surgery.<br /><b>Results</b><br />Twenty-eight days after surgery, ischemic foot perfusion and function parameters were better in praliciguat-treated mice than in vehicle controls. Improved ischemic foot perfusion was not associated with either improved traditional cardiovascular risk factors (ie, weight, glycemia) or increased angiogenesis. However, treatment with praliciguat significantly increased arteriole diameter, decreased ICAM1 (intercellular adhesion molecule 1) expression, and prevented the accumulation of oxidative proangiogenic and proinflammatory muscle fibers. While investigating the mechanism underlying the beneficial effects of praliciguat therapy, we found that praliciguat significantly downregulated <i>Myh2</i> and <i>Cxcl12</i> mRNA expression in cultured myoblasts and that conditioned medium form praliciguat-treated myoblast decreased <i>ICAM1</i> mRNA expression in endothelial cells. These results suggest that praliciguat therapy may decrease <i>ICAM1</i> expression in endothelial cells by downregulating <i>Cxcl12</i> in myocytes.<br /><b>Conclusions</b><br />Our results demonstrated that praliciguat promotes blood flow recovery in the ischemic muscle of mice with type 2 diabetes, at least in part by increasing arteriole diameter and by downregulating ICAM1 expression.<br /><br /><br /><br /><small>Circ Res: 30 Nov 2022; epub ahead of print</small></div>

<div><h4>TXNIP Suppresses the Osteochondrogenic Switch of VSMCs in Atherosclerosis.</h4><i>Woo SH, Kyung D, Hyun Lee S, Seong Park K, ... Choi JH, Kim DY</i><br /><b>Background</b><br />The osteochondrogenic switch of vascular smooth muscle cells (VSMCs) is a pivotal cellular process in atherosclerotic calcification. However, the exact molecular mechanism of the osteochondrogenic transition of VSMCs remains to be elucidated. Here, we explore the regulatory role of thioredoxin-interacting protein (TXNIP) in the phenotypical transitioning of VSMCs toward osteochondrogenic cells responsible for atherosclerotic calcification.<br /><b>Methods</b><br />The atherosclerotic phenotypes of <i>Txnip</i><sup>-/-</sup> mice were analyzed in combination with single-cell RNA-sequencing. The atherosclerotic phenotypes of <i>Tagln</i>-Cre; <i>Txnip</i><sup>flox/flox</sup> mice (smooth muscle cell-specific <i>Txnip</i> ablation model), and the mice transplanted with the bone marrow of <i>Txnip</i><sup>-/-</sup> mice were analyzed. Public single-cell RNA-sequencing dataset (GSE159677) was reanalyzed to define the gene expression of TXNIP in human calcified atherosclerotic plaques. The effect of TXNIP suppression on the osteochondrogenic phenotypic changes in primary aortic VSMCs was analyzed.<br /><b>Results</b><br />Atherosclerotic lesions of <i>Txnip</i><sup>-/-</sup> mice presented significantly increased calcification and deposition of collagen content. Subsequent single-cell RNA-sequencing analysis identified the modulated VSMC and osteochondrogenic clusters, which were VSMC-derived populations. The osteochondrogenic cluster was markedly expanded in <i>Txnip</i><sup>-/-</sup> mice. The pathway analysis of the VSMC-derived cells revealed enrichment of bone- and cartilage-formation-related pathways and bone morphogenetic protein signaling in <i>Txnip</i><sup>-/-</sup> mice. Reanalyzing public single-cell RNA-sequencing dataset revealed that TXNIP was downregulated in the modulated VSMC and osteochondrogenic clusters of human calcified atherosclerotic lesions. <i>Tagln</i>-Cre; <i>Txnip</i><sup>flox/flox</sup> mice recapitulated the calcification and collagen-rich atherosclerotic phenotypes of <i>Txnip</i><sup>-/-</sup> mice, whereas the hematopoietic deficiency of TXNIP did not affect the lesion phenotype. Suppression of TXNIP in cultured VSMCs accelerates osteodifferentiation and upregulates bone morphogenetic protein signaling. Treatment with the bone morphogenetic protein signaling inhibitor K02288 abrogated the effect of TXNIP suppression on osteodifferentiation.<br /><b>Conclusions</b><br />Our results suggest that TXNIP is a novel regulator of atherosclerotic calcification by suppressing bone morphogenetic protein signaling to inhibit the transition of VSMCs toward an osteochondrogenic phenotype.<br /><br /><br /><br /><small>Circ Res: 30 Nov 2022; epub ahead of print</small></div>

<div><h4>Maintenance of Enteral ACE2 Prevents Diabetic Retinopathy in Type 1 Diabetes.</h4><i>Prasad R, Floyd JL, Dupont M, Harbour A, ... Li Q, Grant MB</i><br /><b>Background</b><br />We examined components of systemic and intestinal renin-angiotensin system on gut barrier permeability, glucose homeostasis, systemic inflammation, and progression of diabetic retinopathy (DR) in human subjects and mice with type 1 diabetes.<br /><b>Methods</b><br />Type 1 diabetes individual with (n=18) and without (n=20) DR and controls (n=34) were examined for changes in gut-regulated components of the immune system, gut leakage markers (FABP2 [fatty acid binding protein 2] and PGN [peptidoglycan]), and Ang II (angiotensin II); <i>Akita</i> mice were orally administered a <i>Lactobacillus paracasei</i> (LP) probiotic expressing humanized ACE2 (angiotensin-converting enzyme 2) protein (LP-ACE2) as either a prevention or an intervention. <i>Akita</i> mice with genetic overexpression of <i>humanAce2</i> by small intestine epithelial cells (<i>Vil-Cre.hAce2KI-Akita</i>) were similarly examined. After 9 months of type 1 diabetes, circulatory, enteral, and ocular end points were assessed.<br /><b>Results</b><br />Type 1 diabetes subjects exhibit elevations in gut-derived circulating immune cells (Th17 and ILC1 cells) and higher gut leakage markers, which were positively correlated with plasma Ang II and DR severity. The LP-ACE2 prevention cohort and genetic overexpression of intestinal ACE2 preserved barrier integrity, reduced inflammatory response, improved hyperglycemia, and delayed development of DR. Improvements in glucose homeostasis were due to intestinal MasR activation, resulting in a GSK-3β/c-Myc-mediated decrease in intestinal glucose transporter expression. In the LP-ACE2 intervention cohort, gut barrier integrity was improved and DR reversed, but no improvement in hyperglycemia was observed. These data support that the beneficial effects of LP-ACE2 on DR are due to the action of ACE2, not improved glucose homeostasis.<br /><b>Conclusions</b><br />Dysregulated systemic and intestinal renin-angiotensin system was associated with worsening gut barrier permeability, gut-derived immune cell activation, systemic inflammation, and progression of DR in human subjects. In <i>Akita</i> mice, maintaining intestinal ACE2 expression prevented and reversed DR, emphasizing the multifaceted role of the intestinal renin-angiotensin system in diabetes and DR.<br /><br /><br /><br /><small>Circ Res: 30 Nov 2022; epub ahead of print</small></div>

<div><h4>Epsin Nanotherapy Regulates Cholesterol Transport to Fortify Atheroma Regression.</h4><i>Cui K, Gao X, Wang B, Wu H, ... Chen K, Chen H</i><br /><b>Background</b><br />Excess cholesterol accumulation in lesional macrophages elicits complex responses in atherosclerosis. Epsins, a family of endocytic adaptors, fuel the progression of atherosclerosis; however, the underlying mechanism and therapeutic potential of targeting Epsins remains unknown. In this study, we determined the role of Epsins in macrophage-mediated metabolic regulation. We then developed an innovative method to therapeutically target macrophage Epsins with specially designed S2P-conjugated lipid nanoparticles, which encapsulate small-interfering RNAs to suppress Epsins.<br /><b>Methods</b><br />We used single-cell RNA sequencing with our newly developed algorithm MEBOCOST to study cell-cell communications mediated by metabolites from sender cells and sensor proteins on receiver cells. Biomedical, cellular, and molecular approaches were utilized to investigate the role of macrophage Epsins in regulating lipid metabolism and transport. We performed this study using myeloid-specific Epsin double knockout (LysM-DKO) mice and mice with a genetic reduction of ABCG1 (ATP-binding cassette subfamily G member 1; LysM-DKO-ABCG1<sup>fl/+</sup>). The nanoparticles targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis.<br /><b>Results</b><br />We revealed that Epsins regulate lipid metabolism and transport in atherosclerotic macrophages. Inhibiting Epsins by nanotherapy halts inflammation and accelerates atheroma resolution. Harnessing lesional macrophage-specific nanoparticle delivery of Epsin small-interfering RNAs, we showed that silencing of macrophage Epsins diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, we demonstrated that Epsins bound to CD36 to facilitate lipid uptake by enhancing CD36 endocytosis and recycling. Conversely, Epsins promoted ABCG1 degradation via lysosomes and hampered ABCG1-mediated cholesterol efflux and reverse cholesterol transport. In a LysM-DKO-ABCG1<sup>fl/+</sup> mouse model, enhanced cholesterol efflux and reverse transport due to Epsin deficiency was suppressed by the reduction of ABCG1.<br /><b>Conclusions</b><br />Our findings suggest that targeting Epsins in lesional macrophages may offer therapeutic benefits for advanced atherosclerosis by reducing CD36-mediated lipid uptake and increasing ABCG1-mediated cholesterol efflux.<br /><br /><br /><br /><small>Circ Res: 29 Nov 2022; epub ahead of print</small></div>

<div><h4>RBPMS2 Is a Myocardial-Enriched Splicing Regulator Required for Cardiac Function.</h4><i>Akerberg AA, Trembley M, Butty V, Schwertner A, ... Burns CE, Burns CG</i><br /><b>Background</b><br />RBPs (RNA-binding proteins) perform indispensable functions in the post-transcriptional regulation of gene expression. Numerous RBPs have been implicated in cardiac development or physiology based on gene knockout studies and the identification of pathogenic RBP gene mutations in monogenic heart disorders. The discovery and characterization of additional RBPs performing indispensable functions in the heart will advance basic and translational cardiovascular research. <u>Methods:</u> We performed a differential expression screen in zebrafish embryos to identify genes enriched in <i>nkx2.5</i>-positive cardiomyocytes or cardiopharyngeal progenitors compared to <i>nkx2.5</i>-negative cells from the same embryos. We investigated the myocardial-enriched gene RNA-binding protein with multiple splicing (variants) 2 [<i>RBPMS2</i>)] by generating and characterizing <i>rbpms2</i> knockout zebrafish and human cardiomyocytes derived from <i>RBPMS2</i>-deficient induced pluripotent stem cells.<br /><b>Results</b><br />We identified 1848 genes enriched in <i>nkx2.5</i>-positive population. Among the most highly enriched genes, most with well-established functions in the heart, we discovered the ohnologs <i>rbpms2a</i> and <i>rbpms2b</i>, which encode an evolutionarily conserved RBP. Rbpms2 localizes selectively to cardiomyocytes during zebrafish heart development and strong cardiomyocyte expression persists into adulthood. Rbpms2-deficient embryos suffer from early cardiac dysfunction characterized by reduced ejection fraction. The functional deficit is accompanied by myofibril disarray, altered calcium handling, and differential alternative splicing events in mutant cardiomyocytes. These phenotypes are also observed in <i>RBPMS2</i>-deficient human cardiomyocytes, indicative of conserved molecular and cellular function. RNA-sequencing and comparative analysis of genes mis-spliced in <i>RBPMS2</i>-deficient zebrafish and human cardiomyocytes uncovered a conserved network of 29 ortholog pairs that require <i>RBPMS2</i> for alternative splicing regulation, including <i>RBFOX2</i>, <i>SLC8A1</i>, and <i>MYBPC3</i>.<br /><b>Conclusions</b><br />Our study identifies <i>RBPMS2</i> as a conserved regulator of alternative splicing, myofibrillar organization, and calcium handling in zebrafish and human cardiomyocytes.<br /><br /><br /><br /><small>Circ Res: 11 Nov 2022; epub ahead of print</small></div>

<div><h4>Nidogen-2 is a Novel Endogenous Ligand of LGR4 to Inhibit Vascular Calcification.</h4><i>Chen Y, Mao C, Gu R, Zhao R, ... Sun J, Kong W</i><br /><AbstractText><br /><b>Background:</b><br/>Vascular calcification is closely related to the all-cause mortality of cardiovascular events. Basement membrane protein nidogen-2 is a key component of the vascular extracellular matrix microenvironment and we recently found it is pivotal for the maintenance of contractile phenotype in vascular smooth muscle cell (VSMCs). However, whether nidogen-2 is involved in VSMCs osteochondrogenic transition and vascular calcification remains unclear.</AbstractText><br /><b>Methods</b><br />VSMCs was treated with high-phosphate to study VSMCs calcification in vitro. Three different mice models (5/6 nephrectomy-induced chronic renal failure, cholecalciferol-overload, and periadventitially administered with CaCl<sub>2</sub>) were used to study vascular calcification in vivo. Membrane protein interactome, coimmunoprecipitation, flow cytometric binding assay, surface plasmon resonance, G protein signaling , VSMCs calcium assays were performed to clarify the phenotype and elucidate the molecular mechanisms.<br /><b>Results</b><br />Nidogen-2 protein levels were significantly reduced in calcified VSMCs and aortas from mice in different vascular calcification model. Nidogen-2 deficiency exacerbated high-phosphate-induced VSMC calcification, whereas the addition of purified nidogen-2 protein markedly alleviated VSMC calcification in vitro. <i>Nidogen-2</i><sup>-/-</sup> mice exhibited aggravated aorta calcification compared to wild-type (WT) mice in response to 5/6 nephrectomy, cholecalciferol-overload, and CaCl<sub>2</sub> administration. Further unbiased coimmunoprecipitation and interactome analysis of purified nidogen-2 and membrane protein in VSMCs revealed that nidogen-2 directly binds to LGR4 (leucine-rich repeat G-protein-coupled receptor 4) with <i>K</i><sub>D</sub> value 26.77 nM. LGR4 deficiency in VSMCs in vitro or in vivo abolished the protective effect of nidogen-2 on vascular calcification. Of interest, nidogen-2 biased activated LGR4-Gαq-PKCα (protein kinase Cα)-AMPKα1 (AMP-activated protein kinase α1) signaling to counteract VSMCs osteogenic transition and mineralization.<br /><b>Conclusions</b><br />Nidogen-2 is a novel endogenous ligand of LGR4 that biased activated Gαq- PKCα-AMPKα1 signaling and inhibited vascular calcification.<br /><br /><br /><br /><small>Circ Res: 10 Nov 2022; epub ahead of print</small></div>

<div><h4>The Effect of a Neuronal Nitric Oxide Synthase Inhibitor on Neurovascular Regulation in Humans.</h4><i>O\'Gallagher K, Rosentreter RE, Elaine Soriano J, Roomi A, ... Shah AM, Phillips AA</i><br /><b>Background</b><br />Neurovascular coupling (NVC) is a key process in cerebral blood flow regulation. NVC ensures adequate brain perfusion to changes in local metabolic demands. Neuronal nitric oxide synthase (nNOS) is suspected to be involved in NVC; however, this has not been tested in humans. Our objective was to investigate the effects of nNOS inhibition on NVC in humans.<br /><b>Methods</b><br />We performed a 3-visit partially randomized, double-blinded, placebo-controlled, crossover study in 12 healthy subjects. On each visit, subjects received an intravenous infusion of either S-methyl-L-thiocitrulline (a selective nNOS-inhibitor), 0.9% saline (placebo control), or phenylephrine (pressor control). The NVC assessment involved eliciting posterior circulation hyperemia through visual stimulation while measuring posterior and middle cerebral arteries blood velocity.<br /><b>Results</b><br />nNOS inhibition blunted the rapidity of the NVC response versus pressor control, evidenced by a reduced initial rise in mean posterior cerebral artery velocity (-3.3% [-6.5, -0.01], <i>P</i>=0.049), and a reduced rate of increase (ie, acceleration) in posterior cerebral artery velocity (slope reduced -4.3% [-8.5, -0.1], <i>P</i>=0.045). The overall magnitude of posterior cerebral artery response relative to placebo control or pressor control was not affected. Changes in BP parameters were well-matched between the S-methyl-L-thiocitrulline and pressor control arms.<br /><b>Conclusions</b><br />Neuronal NOS plays a role in dynamic cerebral blood flow control in healthy adults, particularly the rapidity of the NVC response to visual stimulation. This work opens the way to further investigation of the role of nNOS in conditions of impaired NVC, potentially revealing a therapeutic target.<br /><br /><br /><br /><small>Circ Res: 09 Nov 2022; epub ahead of print</small></div>

<div><h4>Spatiotemporal Control of Vascular Ca1.2 by α1 S1928 Phosphorylation.</h4><i>Martín-Aragón Baudel M, Flores-Tamez VA, Hong J, Reddy GR, ... Nieves-Cintrón M, Navedo MF</i><br /><b>Background</b><br />L-type Ca<sub>V</sub>1.2 channels undergo cooperative gating to regulate cell function, although mechanisms are unclear. This study tests the hypothesis that phosphorylation of the Ca<sub>V</sub>1.2 pore-forming subunit α1<sub>C</sub> at S1928 mediates vascular Ca<sub>V</sub>1.2 cooperativity during diabetic hyperglycemia.<br /><b>Methods</b><br />A multiscale approach including patch-clamp electrophysiology, super-resolution nanoscopy, proximity ligation assay, pressure myography, and Laser Speckle imaging was implemented to examine Ca<sub>V</sub>1.2 cooperativity, α1<sub>C</sub> clustering, myogenic tone, and blood flow in human and mouse arterial myocytes/vessels.<br /><b>Results</b><br />Ca<sub>V</sub>1.2 activity and cooperative gating increase in arterial myocytes from patients with type 2 diabetes and type 1 diabetic mice, and in wild-type mouse arterial myocytes after elevating extracellular glucose. These changes were prevented in wild-type cells pre-exposed to a PKA inhibitor or cells from knock-in S1928A but not S1700A mice. In addition, α1<sub>C</sub> clustering at the surface membrane of wild-type, but not wild-type cells pre-exposed to PKA or P2Y<sub>11</sub> inhibitors and S1928A arterial myocytes, was elevated upon hyperglycemia and diabetes. Ca<sub>V</sub>1.2 spatial and gating remodeling correlated with enhanced arterial myocyte Ca<sup>2+</sup> influx and contractility and <i>in vivo</i> reduction in arterial diameter and blood flow upon hyperglycemia and diabetes in wild-type but not S1928A cells/mice.<br /><b>Conclusions</b><br />These results suggest that PKA-dependent pS1928 promotes the spatial reorganization of vascular α1<sub>C</sub> into \"superclusters\" upon hyperglycemia and diabetes. This triggers Ca<sub>V</sub>1.2 activity and cooperativity, directly impacting vascular reactivity. The results may lay the foundation for developing therapeutics to correct Ca<sub>V</sub>1.2 and arterial function during diabetic hyperglycemia.<br /><br /><br /><br /><small>Circ Res: 08 Nov 2022; epub ahead of print</small></div>

<div><h4>Blocking MG53 Phosphorylation Protects Diabetic Heart From Ischemic Injury.</h4><i>Lv F, Wang Y, Shan D, Guo S, ... Hu X, Xiao RP</i><br /><b>Background</b><br />As an integral component of cell membrane repair machinery, MG53 (mitsugumin 53) is important for cardioprotection induced by ischemia preconditioning and postconditioning. However, it also impairs insulin signaling via its E3 ligase activity-mediated ubiquitination-dependent degradation of IR (insulin receptor) and IRS1 (insulin receptor substrate 1) and its myokine function-induced allosteric blockage of IR. Here, we sought to develop MG53 into a cardioprotection therapy by separating its detrimental metabolic effects from beneficial actions.<br /><b>Methods</b><br />Using immunoprecipitation-mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we investigated the role of MG53 phosphorylation at serine 255 (S255). In particular, utilizing recombinant proteins and gene knock-in approaches, we evaluated the potential therapeutic effect of MG53-S255A mutant in treating cardiac ischemia/reperfusion injury in diabetic mice.<br /><b>Results</b><br />We identified S255 phosphorylation as a prerequisite for MG53 E3 ligase activity. Furthermore, MG53<sup>S255</sup> phosphorylation was mediated by GSK3β (glycogen synthase kinase 3 beta) and markedly elevated in the animal models with metabolic disorders. Thus, IR-IRS1-GSK3β-MG53 formed a vicious cycle in the pathogenesis of metabolic disorders where aberrant insulin signaling led to hyper-activation of GSK3β, which in turn, phosphorylated MG53 and enhanced its E3 ligase activity, and further impaired insulin sensitivity. Importantly, S255A mutant eliminated the E3 ligase activity while retained cell protective function of MG53. Consequently, the S255A mutant, but not the wild type MG53, protected the heart against ischemia/reperfusion injury in <i>db/db</i> mice with advanced diabetes, although both elicited cardioprotection in normal mice. Moreover, in S255A knock-in mice, S255A mutant also mitigated ischemia/reperfusion-induced myocardial damage in the diabetic setting.<br /><b>Conclusions</b><br />S255 phosphorylation is a biased regulation of MG53 E3 ligase activity. The MG53-S255A mutant provides a promising approach for the treatment of acute myocardial injury, especially in patients with metabolic disorders.<br /><br /><br /><br /><small>Circ Res: 07 Nov 2022; epub ahead of print</small></div>