Sox17 Controls Emergence and Remodeling of Nestin-Expressing Coronary Vessels.

González-Hernández S, Gómez MJ, Sanchez-Cabo F, Méndez-Ferrer S, Muñoz-Cánoves P, Isern J

The molecular mechanisms underlying the formation of coronary arteries during development and during cardiac neovascularization after injury are poorly understood. However, a detailed description of the relevant signaling pathways and functional transcription factors regulating these processes is still incomplete.The goal of this study is to identify novel cardiac transcriptional mechanisms of coronary angiogenesis and vessel remodeling by defining the molecular signatures of coronary vascular endothelial cells (EC) during these complex processes.We demonstrate thatandtransgenic mouse lines are novel tools for studying the emergence of coronary endothelium and targeting sprouting coronary vessels (but not ventricular endocardium) during development. Furthermore, we identify Sox17 as a critical transcription factor upregulated during the sprouting and remodeling of coronary vessels, visualized by a specific neural enhancer from thegene that is strongly induced in developing arterioles. Functionally, genetic inducible endothelial deletion ofcauses deficient cardiac remodeling of coronary vessels, resulting in improper coronary artery formation.We demonstrated that Sox17 transcription factor regulates the transcriptional activation of Nestin\'s enhancer in developing coronary vessels while its genetic deletion leads to inadequate coronary artery formation. These findings identify Sox17 as a critical regulator for the remodeling of coronary vessels in the developing heart.



Circ Res: 13 Sep 2020; epub ahead of print

Ionic Mechanisms of Impulse Propagation Failure in the FHF2-Deficient Heart.

Park DS, Shekhar A, Santucci Iii J, Redel-Traub G, ... Goldfarb M, Fishman GI

Fibroblast growth factor homologous factors (FHFs) are key regulators of sodium channel inactivation. Mutations in these critical proteins have been implicated in human diseases including Brugada syndrome, idiopathic ventricular arrhythmias, and epileptic encephalopathy. The underlying ionic mechanisms by which reduced sodium channel availability in Fhf2 knockout mice predisposes to abnormal excitability at the tissue level are not well defined.Using animal models and theoretical multicellular linear strands, we examined how FHF2 orchestrates the interdependency of sodium, calcium, and gap junctional conductances to safeguard cardiac conduction.mice were challenged by reducing calcium conductance using verapamil or by reducing gap junctional conductance using carbenoxolone or by backcrossing into a connexin 43 heterozygous (Cx43) background. All conditions produced conduction block inmice, withshowing normal impulse propagation. To explore the ionic mechanisms of block inhearts, multicellular linear strand models incorporating FHF2-deficient sodium channel inactivation properties were constructed and faithfully recapitulated conduction abnormalities seen in mutant hearts. The mechanisms of conduction block in mutant strands with reduced calcium conductance or gap junction uncoupling are very different. Enhanced sodium channel inactivation due to FHF2 deficiency shifts dependence onto calcium current to sustain electrotonic driving force, axial current flow, and action potential generation from cell-to-cell. In the setting of gap junction uncoupling, slower charging time from upstream cells conspires with accelerated sodium channel inactivation in mutant strands to prevent sufficient downstream cell charging for action potential propagation.FHF2-dependent effects on sodium channel inactivation ensure adequate sodium current reserve to safeguard against numerous threats to reliable cardiac impulse propagation.



Circ Res: 22 Sep 2020; epub ahead of print

Arterial Stiffness Preceding Diabetes: A Longitudinal Study.

Zheng M, Zhang X, Chen S, Song Y, ... Gao X, Wu S

Previous studies on the relationship between diabetes and arterial stiffness were mostly cross-sectional. A few longitudinal studies focused on one single direction. Whether the association between arterial stiffness and diabetes is bidirectional remains unclear to date.To explore the temporal relationship between arterial stiffness and fasting blood glucose (FBG) status.Included were 14,159 participants of the Kailuan study with assessment of brachial-ankle pulse wave velocity (baPWV) from 2010 to 2015, and free of diabetes, cardiovascular and cerebrovascular diseases and chronic kidney disease at baseline. FBG and baPWV were repeatedly measured at baseline and follow-ups. Cox proportional hazard regression model was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of incident diabetes across baseline baPWV groups: <1,400 cm/s (ref), 1,400 {less than or equal to} baPWV < 1,800 cm/s, and {greater than or equal to}1,800cm/s. Path analysis was used to analyze the possible temporal causal relationship between baPWV and FBG, among 8,956 participants with repeated assessment of baPWV and FBG twice in 2010-2017. The mean baseline age of the observed population was 48.3{plus minus}12.0 years. During mean 3.72 years of follow-up, 979 incident diabetes cases were identified. After adjusting for potential confounders, the HR (95% CI) for risk of diabetes was 1.59 (1.34, 1.88) for the borderline arterial stiffness group and 2.11 (1.71, 2.61) for the elevated arterial stiffness group, compared with the normal ideal arterial stiffness group. In the path analysis, baseline baPWV was associated with follow-up FBG (the standard regression coefficient was 0.09; 95% CI: 0.05 to 0.10). In contrast, the standard regression coefficient of baseline FBG for follow-up baPWV (β = 0.00, 95% CI: -0.02 to 0.02) was not significant.Arterial stiffness, as measured by baPWV, was associated with risk of developing diabetes. Arterial stiffness appeared to precede the increase in FBG.



Circ Res: 27 Sep 2020; epub ahead of print

Platelets Can Associate with SARS-Cov-2 RNA and Are Hyperactivated in COVID-19.

Zaid Y, Puhm F, Allaeys I, Naya A, ... Guessous F, Boilard E

In addition to the overwhelming lung inflammation that prevails in COVID-19, hypercoagulation and thrombosis contribute to the lethality of subjects infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Platelets are chiefly implicated in thrombosis. Moreover, they can interact with viruses and are an important source of inflammatory mediators. While a lower platelet count is associated with severity and mortality, little is known about platelet function during COVID-19.To evaluate the contribution of platelets to inflammation and thrombosis in COVID-19 patients.Blood was collected from 115 consecutive COVID-19 patients presenting non-severe (n=71) and severe (n=44) respiratory symptoms. We document the presence of SARS-CoV-2 RNA associated with platelets of COVID-19 patients. Exhaustive assessment of cytokines in plasma and in platelets revealed the modulation of platelet-associated cytokine levels in both non-severe and severe COVID-19 patients, pointing to a direct contribution of platelets to the plasmatic cytokine load. Moreover, we demonstrate that platelets release their alpha- and dense-granule contents in both non-severe and severe forms of COVID-19. In comparison to concentrations measured in healthy volunteers, phosphatidylserine-exposing platelet extracellular vesicles were increased in non-severe, but not in severe cases of COVID-19. Levels of D-dimers, a marker of thrombosis, failed to correlate with any measured indicators of platelet activation. Functionally, platelets were hyperactivated in COVID-19 subjects presenting non-severe and severe symptoms, with aggregation occurring at suboptimal thrombin concentrations. Furthermore, platelets adhered more efficiently onto collagen-coated surfaces under flow conditions.Taken together, the data suggest that platelets are at the frontline of COVID-19 pathogenesis, as they release various sets of molecules through the different stages of the disease. Platelets may thus have the potential to contribute to the overwhelming thrombo-inflammation in COVID-19, and the inhibition of pathways related to platelet activation may improve the outcomes during COVID-19.



Circ Res: 16 Sep 2020; epub ahead of print

ApoA-I Nanoparticles Delivered Post Myocardial Infarction Moderate Inflammation.

Richart AL, Reddy M, Khalaji M, Natoli AK, ... Navdaev A, Kingwell BA

Decades of research have examined immune modulatory strategies to protect the heart after an acute myocardial infarction and prevent progression to heart failure, but have failed to translate to clinical benefit. To determine anti-inflammatory actions of apoA-I nanoparticles (n-apoA-I) that contribute to cardiac tissue recovery after myocardial infarction.Using a preclinical mouse model of myocardial infarction, we demonstrate that a single intravenous bolus of n-apoA-I (CSL111, 80mg/kg) delivered immediately after reperfusion, reduced the systemic and cardiac inflammatory response. N-apoA-I treatment lowered the number of circulating leukocytes by 30{plus minus}7% and their recruitment into the ischemic heart by 25{plus minus}10% (all p<5.0E-2). This was associated with a reduction in plasma levels of the clinical biomarker of cardiac injury, cardiac troponin-I by 52{plus minus}17% (p=1.01E-2). N-apoA-I reduced the cardiac expression of chemokines that attract neutrophils and monocytes by 60-80%, and lowered surface expression of integrin CD11b on monocytes by 20{plus minus}5% (all p<5.0E-2). Fluorescently labeled n-apoA-I entered the infarct and peri-infarct regions and co-localized with cardiomyocytes undergoing apoptosis and with leukocytes. We further demonstrate that n-apoA-I binds to neutrophils and monocytes, with preferential binding to the pro-inflammatory monocyte subtype and partially via scavenger receptor BI (SR-BI). In patients with type 2 diabetes mellitus, we also observed that intravenous infusion of the same n-apoA-I (CSL111, 80mg/kg) similarly reduced the level of circulating leukocytes by 12{plus minus}5% (all p<5.0E-2).A single intravenous bolus of n-apoA-I delivered immediately post-myocardial infarction reduced the systemic and cardiac inflammatory response through direct actions on both the ischemic myocardium and leukocytes. These data highlight the anti-inflammatory effects of n-apoA-I and provide preclinical support for investigation of its use for management of acute coronary syndromes in the setting of primary percutaneous coronary interventions.



Circ Res: 20 Sep 2020; epub ahead of print

Lgr4 Governs a Pro-Inflammatory Program in Macrophages to Antagonize Post-Infarction Cardiac Repair.

Huang CK, Dai D, Xie H, Zhu Z, ... Zhang R, Yan X
Rationale
Macrophages are critically involved in wound healing following myocardial infarction (MI). Lgr4, a member of LGR (leucine-rich repeat-containing G protein-coupled receptor) family, is emerging as a regulator of macrophage-associated immune responses. However, the contribution of Lgr4 to macrophage phenotype and function in the context of MI remains unclear.
Objective
To determine the role of macrophage Lgr4 in MI and to dissect the underlying mechanisms.
Methods and results
During early inflammatory phase of MI, infarct macrophages rather than neutrophils expressed high level of Lgr4. Macrophage-specific Lgr4 knockout mice had no baseline cardiovascular defects but manifested improved heart function, modestly reduced infarct size, decreased early mortality due to cardiac rupture, and ameliorated adverse remodeling after MI. Improved outcomes in macrophage-specific Lgr4 knockout mice subjected to MI were associated with mitigated ischemic injury and optimal infarct healing, as determined by reduction of cardiac apoptosis in the peri-infarct zone, attenuation of local myocardial inflammatory response, decrease of matrix metalloproteinase expression in the infarct, enhancement of angiogenesis, myofibroblast proliferation, and collagen I deposition in reparative granulation tissue as well as formation of collagen-rich scar. More importantly, macrophage-specific Lgr4 knockout infarcts had reduced numbers of infiltrating leukocytes and inflammatory macrophages but harbored abundant reparative macrophage subsets. Lgr4-null infarct macrophages exhibited a less inflammatory transcriptional signature. These findings were further supported by transcriptomic profiling data showing repression of multiple pathways and broad-spectrum genes associated with proinflammatory responses in macrophage-specific Lgr4 knockout infarcts. Notably, we discovered that Lgr4-mediated functional phenotype programing in infarct macrophages was at least partly attributed to regulation of AP (activator protein)-1 activity. We further demonstrated that the synergistic effects of Lgr4 on AP-1 activation in inflammatory macrophages occurred via enhancing CREB (cAMP response element-binding protein)-mediated , , andtransactivation.
Conclusions
Together, our data highlight the significance of Lgr4 in governing proinflammatory phenotype of infarct macrophages and postinfarction repair.



Circ Res: 24 Sep 2020; 127:953-973

JAM-A Acts via C/EBP-α to Promote Claudin-5 Expression and Enhance Endothelial Barrier Function.

Kakogiannos N, Ferrari L, Giampietro C, Scalise AA, ... Dejana E, Giannotta M
Rationale
Intercellular tight junctions are crucial for correct regulation of the endothelial barrier. Their composition and integrity are affected in pathological contexts, such as inflammation and tumor growth. JAM-A (junctional adhesion molecule A) is a transmembrane component of tight junctions with a role in maintenance of endothelial barrier function, although how this is accomplished remains elusive.
Objective
We aimed to understand the molecular mechanisms through which JAM-A expression regulates tight junction organization to control endothelial permeability, with potential implications under pathological conditions.
Methods and results
Genetic deletion of JAM-A in mice significantly increased vascular permeability. This was associated with significantly decreased expression of claudin-5 in the vasculature of various tissues, including brain and lung. We observed that C/EBP-α (CCAAT/enhancer-binding protein-α) can act as a transcription factor to trigger the expression of claudin-5 downstream of JAM-A, to thus enhance vascular barrier function. Accordingly, gain-of-function for C/EBP-α increased claudin-5 expression and decreased endothelial permeability, as measured by the passage of fluorescein isothiocyanate (FITC)-dextran through endothelial monolayers. Conversely, C/EBP-α loss-of-function showed the opposite effects of decreased claudin-5 levels and increased endothelial permeability. Mechanistically, JAM-A promoted C/EBP-α expression through suppression of β-catenin transcriptional activity, and also through activation of EPAC (exchange protein directly activated by cAMP). C/EBP-α then directly binds the promoter of claudin-5 to thereby promote its transcription. Finally, JAM-A-C/EBP-α-mediated regulation of claudin-5 was lost in blood vessels from tissue biopsies from patients with glioblastoma and ovarian cancer.
Conclusions
We describe here a novel role for the transcription factor C/EBP-α that is positively modulated by JAM-A, a component of tight junctions that acts through EPAC to up-regulate the expression of claudin-5, to thus decrease endothelial permeability. Overall, these data unravel a regulatory molecular pathway through which tight junctions limit vascular permeability. This will help in the identification of further therapeutic targets for diseases associated with endothelial barrier dysfunction. Graphic Abstract: An graphic abstract is available for this article.



Circ Res: 24 Sep 2020; 127:1056-1073

Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture.

Vaisar T, Hu JH, Airhart N, Fox K, ... Gharib SA, Dichek DA
Rationale
Plaque rupture is the proximate cause of most myocardial infarctions and many strokes. However, the molecular mechanisms that precipitate plaque rupture are unknown.
Objective
By applying proteomic and bioinformatic approaches in mouse models of protease-induced plaque rupture and in ruptured human plaques, we aimed to illuminate biochemical pathways through which proteolysis causes plaque rupture and identify substrates that are cleaved in ruptured plaques.
Methods and results
We performed shotgun proteomics analyses of aortas of transgenic mice with macrophage-specific overexpression of urokinase (SR-uPA mice) and of SR-uPA bone marrow transplant recipients, and we used bioinformatic tools to evaluate protein abundance and functional category enrichment in these aortas. In parallel, we performed shotgun proteomics and bioinformatics studies on extracts of ruptured and stable areas of freshly harvested human carotid plaques. We also applied a separate protein-analysis method (protein topography and migration analysis platform) to attempt to identify substrates and proteolytic fragments in mouse and human plaque extracts. Approximately 10% of extracted aortic proteins were reproducibly altered in SR-uPA aortas. Proteases, inflammatory signaling molecules, as well as proteins involved with cell adhesion, the cytoskeleton, and apoptosis, were increased. ECM (Extracellular matrix) proteins, including basement-membrane proteins, were decreased. Approximately 40% of proteins were altered in ruptured versus stable areas of human carotid plaques, including many of the same functional categories that were altered in SR-uPA aortas. Collagens were minimally altered in SR-uPA aortas and ruptured human plaques; however, several basement-membrane proteins were reduced in both SR-uPA aortas and ruptured human plaques. Protein topography and migration analysis platform did not detect robust increases in proteolytic fragments of ECM proteins in either setting.
Conclusions
Parallel studies of SR-uPA mouse aortas and human plaques identify mechanisms that connect proteolysis with plaque rupture, including inflammation, basement-membrane protein loss, and apoptosis. Basement-membrane protein loss is a prominent feature of ruptured human plaques, suggesting a major role for basement-membrane proteins in maintaining plaque stability.



Circ Res: 24 Sep 2020; 127:997-1022

CD84 Links T Cell and Platelet Activity in Cerebral Thrombo-Inflammation in Acute Stroke.

Schuhmann MK, Stoll G, Bieber M, Vögtle T, ... Nieswandt B, Stegner D
Rationale
Ischemic stroke is a leading cause of morbidity and mortality worldwide. Recanalization of the occluded vessel is essential but not sufficient to guarantee brain salvage. Experimental and clinical data suggest that infarcts often develop further due to a thromboinflammatory process critically involving platelets and T cells, but the underlying mechanisms are unknown.
Objective
We aimed to determine the role of CD (cluster of differentiation)-84 in acute ischemic stroke after recanalization and to dissect the underlying molecular thromboinflammatory mechanisms.
Methods and results
Here, we show that mice lacking CD84-a homophilic immunoreceptor of the SLAM (signaling lymphocyte activation molecule) family-on either platelets or T cells displayed reduced cerebral CD4 T-cell infiltration and thrombotic activity following experimental stroke resulting in reduced neurological damage. In vitro, platelet-derived soluble CD84 enhanced motility of wild-type but not ofCD4 T cells suggesting homophilic CD84 interactions to drive this process. Clinically, human arterial blood directly sampled from the ischemic cerebral circulation indicated local shedding of platelet CD84. Moreover, high platelet CD84 expression levels were associated with poor outcome in patients with stroke.
Conclusions
These results establish CD84 as a critical pathogenic effector and thus a potential pharmacological target in ischemic stroke.



Circ Res: 24 Sep 2020; 127:1023-1035

Atrial Myocyte NLRP3/CaMKII Nexus Forms a Substrate for Postoperative Atrial Fibrillation.

Heijman J, Muna AP, Veleva T, Molina CE, ... Wehrens XHT, Dobrev D
Rationale
Postoperative atrial fibrillation (POAF) is a common and troublesome complication of cardiac surgery. POAF is generally believed to occur when postoperative triggers act on a preexisting vulnerable substrate, but the underlying cellular and molecular mechanisms are largely unknown.
Objective
To identify cellular POAF mechanisms in right atrial samples from patients without a history of atrial fibrillation undergoing open-heart surgery.
Methods and results
Multicellular action potentials, membrane ion-currents (perforated patch-clamp), or simultaneous membrane-current (ruptured patch-clamp) and [Ca]-recordings in atrial cardiomyocytes, along with protein-expression levels in tissue homogenates or cardiomyocytes, were assessed in 265 atrial samples from patients without or with POAF. No indices of electrical, profibrotic, or connexin remodeling were noted in POAF, but Ca-transient amplitude was smaller, although spontaneous sarcoplasmic reticulum (SR) Ca-release events and L-type Ca-current alternans occurred more frequently. CaMKII (Ca/calmodulin-dependent protein kinase-II) protein-expression, CaMKII-dependent phosphorylation of the cardiac RyR2 (ryanodine-receptor channel type-2), and RyR2 single-channel open-probability were significantly increased in POAF. SR Ca-content was unchanged in POAF despite greater SR Ca-leak, with a trend towards increased SR Ca-ATPase activity. Patients with POAF also showed stronger expression of activated components of the NLRP3 (NACHT, LRR, and PYD domains-containing protein-3)-inflammasome system in atrial whole-tissue homogenates and cardiomyocytes. Acute application of interleukin-1β caused NLRP3-signaling activation and CaMKII-dependent RyR2/phospholamban hyperphosphorylation in an immortalized mouse atrial cardiomyocyte cell-line (HL-1-cardiomyocytes) and enhanced spontaneous SR Ca-release events in both POAF cardiomyocytes and HL-1-cardiomyocytes. Computational modeling showed that RyR2 dysfunction and increased SR Ca-uptake are sufficient to reproduce the Ca-handling phenotype and indicated an increased risk of proarrhythmic delayed afterdepolarizations in POAF subjects in response to interleukin-1β.
Conclusions
Preexisting Ca-handling abnormalities and activation of NLRP3-inflammasome/CaMKII signaling are evident in atrial cardiomyocytes from patients who subsequently develop POAF. These molecular substrates sensitize cardiomyocytes to spontaneous Ca-releases and arrhythmogenic afterdepolarizations, particularly upon exposure to inflammatory mediators. Our data reveal a potential cellular and molecular substrate for this important clinical problem.



Circ Res: 24 Sep 2020; 127:1036-1055

Microanatomy of the Human Atherosclerotic Plaque by Single-Cell Transcriptomics.

Depuydt MA, Prange KH, Slenders L, Örd T, ... de Winther MP, Pasterkamp G

Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques have not been fully assessed.Using single-cell transcriptomics and chromatin accessibility we gained a better understanding of the pathophysiology underlying human atherosclerosis.We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4 and CD8 T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included two populations of pro-inflammatory macrophages showing IL1B or TNF expression as well as a foam cell-like population expressing TREM2 and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public GWAS data were particularly enriched in lesional macrophages, endothelial and smooth muscle cells.This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.



Circ Res: 27 Sep 2020; epub ahead of print

HIV Antivirals Affect Endothelial Activation and Endothelial-Platelet Crosstalk.

Khawaja AA, Taylor KA, Lovell AO, Nelson M, ... Boffito M, Emerson M

People living with human immunodeficiency virus (PLHIV) on effective antiretroviral therapy are at increased risk of cardiovascular complications, possibly due to off-target drug effects. Some studies have associated antiretroviral therapy with increased risk of myocardial infarction and endothelial dysfunction, but a link between endothelial function and antiretrovirals has not been established.To determine the effects of antiretrovirals in common clinical use upon in vitro endothelial function in order to better understand cardiovascular risk in PLHIV.Human umbilical cord vein endothelial cells (HUVEC) or human coronary artery endothelial cells (HCAEC) were pre-treated with the antiretrovirals abacavir sulphate (ABC), tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF). Expression of adhesion molecules, ectonucleotidases (CD39 and CD73), tissue factor (TF), endothelial-derived microparticle (EMP) numbers and phenotype, and platelet activation were evaluated by flow cytometry. TF and ectonucleotidase activities were measured using colourimetric plate-based assays. ABC-treated endothelial cells had higher levels of ICAM-1 and TF expression following TNF-α stimulation. In contrast, TDF and TAF treatment gave rise to greater populations of CD39+CD73+ cells. These cell surface differences were also observed within EMP repertoires. ABC-treated cells and EMP had greater TF activity, whilst TDF- and TAF-treated cells and EMP displayed higher ectonucleotidase activity. Finally, EMP isolated from ABC-treated cells enhanced collagen-evoked platelet integrin activation and α-granule release.We report differential effects of antiretrovirals used in the treatment of HIV upon endothelial function. ABC treatment led to an inflammatory, pro-thrombotic endothelial phenotype that promoted platelet activation. In contrast, TDF and TAF conferred potentially cardioprotective properties associated with ectonucleotidase activity. These observations establish a link between antiretrovirals and specific functional effects that provide insight into cardiovascular disease in PLHIV.



Circ Res: 30 Sep 2020; epub ahead of print

Dynamics of Cardiac Neutrophil Diversity in Murine Myocardial Infarction.

Vafadarnejad E, Rizzo G, Krampert L, Arampatzi P, ... Saliba AE, Cochain C
Rationale
After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they promote both tissue healing and damage.
Objective
To characterize the dynamics of circulating and cardiac neutrophil diversity after infarction.
Methods and results
We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G (lymphocyte antigen 6G) neutrophils could be delineated into 6 distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils (, , ), and putative activity of transcriptional regulators involved in hypoxic response () and emergency granulopoiesis (). At 3 and 5 days, 2 major subsets of(enriched for eg,and ) and() neutrophils were found. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis in blood and heart revealed that while circulating neutrophils undergo a process of aging characterized by loss of surface CD62L and upregulation of , heart infiltrating neutrophils acquired a unique SiglecF signature. SiglecF neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecF signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecF neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecF neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation.
Conclusions
Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse blood and ischemic heart at the single-cell level, and reveal a process of local tissue specification of neutrophils in the ischemic heart characterized by the acquisition of a SiglecF signature.



Circ Res: 08 Oct 2020; 127:e232-e249

Tamoxifen Accelerates Endothelial Healing by Targeting ERα in Smooth Muscle Cell.

Zahreddine R, Davezac M, Smirnova NF, Buscato M, ... Arnal JF, Fontaine C

Tamoxifen prevents the recurrence of breast cancer and is also beneficial against bone demineralization and arterial diseases. It acts as an Estrogen Receptor (ER) α antagonist in ER-positive breast cancers, whereas it mimics the protective action of 17β-estradiol (E2) in other tissues such as arteries. However, the mechanisms of these tissue-specific actions remain unclear. Here, we tested whether tamoxifen is able to accelerate endothelial healing and analyzed the underlying mechanisms.Using three complementary mouse models of carotid artery injury, we demonstrated that both tamoxifen and estradiol accelerated endothelial healing, but only tamoxifen required the presence of the underlying medial smooth muscle cells. Chronic treatment with E2 and tamoxifen elicited differential gene expression profiles in the carotid artery. The use of transgenic mouse models targeting either whole ERα in a cell-specific manner or ERα sub-functions (membrane/extra-nuclear versus genomic/transcriptional) demonstrated that E2-induced acceleration of endothelial healing is mediated by membrane ERα in endothelial cells, while the effect of tamoxifen is mediated by the nuclear actions of ERα in smooth muscle cells.Whereas tamoxifen acts as an anti-estrogen and ERα antagonist in breast cancer, but also on the membrane ERα of endothelial cells, it accelerates endothelial healing through activation of nuclear ERα in smooth muscle cells, inviting to revisit the mechanisms of action of selective modulation of ERα.



Circ Res: 04 Oct 2020; epub ahead of print

Extreme Acetylation of the Cardiac Mitochondrial Proteome Does Not Promote Heart Failure.

Davidson MT, Grimsrud PA, Lai L, Draper JA, ... Kelly DP, Muoio DM
Rationale
Circumstantial evidence links the development of heart failure to posttranslational modifications of mitochondrial proteins, including lysine acetylation (Kac). Nonetheless, direct evidence that Kac compromises mitochondrial performance remains sparse.
Objective
This study sought to explore the premise that mitochondrial Kac contributes to heart failure by disrupting oxidative metabolism.
Methods and results
A DKO (dual knockout) mouse line with deficiencies in CrAT (carnitine acetyltransferase) and Sirt3 (sirtuin 3)-enzymes that oppose Kac by buffering the acetyl group pool and catalyzing lysine deacetylation, respectively-was developed to model extreme mitochondrial Kac in cardiac muscle, as confirmed by quantitative acetyl-proteomics. The resulting impact on mitochondrial bioenergetics was evaluated using a respiratory diagnostics platform that permits comprehensive assessment of mitochondrial function and energy transduction. Susceptibility of DKO mice to heart failure was investigated using transaortic constriction as a model of cardiac pressure overload. The mitochondrial acetyl-lysine landscape of DKO hearts was elevated well beyond that observed in response to pressure overload or Sirt3 deficiency alone. Relative changes in the abundance of specific acetylated lysine peptides measured in DKO versus Sirt3 KO hearts were strongly correlated. A proteomics comparison across multiple settings of hyperacetylation revealed ≈86% overlap between the populations of Kac peptides affected by the DKO manipulation as compared with experimental heart failure. Despite the severity of cardiac Kac in DKO mice relative to other conditions, deep phenotyping of mitochondrial function revealed a surprisingly normal bioenergetics profile. Thus, of the >120 mitochondrial energy fluxes evaluated, including substrate-specific dehydrogenase activities, respiratory responses, redox charge, mitochondrial membrane potential, and electron leak, we found minimal evidence of oxidative insufficiencies. Similarly, DKO hearts were not more vulnerable to dysfunction caused by transaortic constriction-induced pressure overload.
Conclusions
The findings challenge the premise that hyperacetylation per se threatens metabolic resilience in the myocardium by causing broad-ranging disruption to mitochondrial oxidative machinery.



Circ Res: 24 Sep 2020; 127:1094-1108

Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells.

Aherrahrou R, Guo L, Nagraj VP, Aguhob AA, ... Berliner JA, Civelek M

Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies (GWAS) revealed 163 loci associated with CAD. However, the precise molecular mechanisms by which the majority of these loci increase CAD risk are not known. Vascular smooth muscle cells (VSMCs) are critical in the development of CAD. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes.To identify genetic variants associated with atherosclerosis-relevant phenotypes in VSMCsWe quantified twelve atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multi-ethnic heart transplant donors. After genotyping and imputation, we performed association mapping using 6.3 million genetic variants. We demonstrated significant variations in calcification, proliferation, and migration. These phenotypes were not correlated with each other. We performed GWAS for twelve atherosclerosis-relevant phenotypes and identified four genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD GWAS loci with our dataset and found nominally significant associations at 79 loci. One of them was the chromosome 1q41 locus, which harbors MIA3. The G allele of the lead risk SNP rs67180937 was associated with lower VSMC MIA3 expression and lower proliferation. Lentivirus-mediated silencing of MIA3 in VSMCs resulted in lower proliferation, consistent with human genetics findings. Further, we observed a significant reduction of MIA3 protein in VSMCs in thin fibrous caps of late-stage atherosclerotic plaques compared to early fibroatheroma with thick and protective fibrous caps in mice and humans.Our data demonstrate that genetic variants have significant influences on VSMC function relevant to the development of atherosclerosis. Further, high MIA3 expression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.



Circ Res: 11 Oct 2020; epub ahead of print

Overexpression of Activin Receptor-Like Kinase 1 in Endothelial Cells Suppresses Development of Arteriovenous Malformations in Mouse Models of Hereditary Hemorrhagic Telangiectasia.

Hwan Kim Y, Vu PN, Choe SW, Jeon CJ, ... Lee YJ, Oh SP
Rationale
Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease caused by mutations in , , or . Since proteins from all 3 HHT genes are components of signal transduction of TGF-β (transforming growth factor β) family members, it has been hypothesized that HHT is a disease caused by defects in the ENG-ALK1-SMAD4 linear signaling. However, in vivo evidence supporting this hypothesis is scarce.
Objective
We tested this hypothesis and investigated the therapeutic effects and potential risks of induced-ALK1 or -ENG overexpression (OE) for HHT.
Methods and results
We generated a novel mouse allele (ROSA26) in which HA (human influenza hemagglutinin)-tagged ALK1 and bicistronic eGFP expression are induced by Cre activity. We examined whether ALK1-OE using the ROSA26 allele could suppress the development of arteriovenous malformations (AVMs) in wounded adult skin and developing retinas of - and -inducible knockout (iKO) mice. We also used a similar approach to investigate whether ENG-OE could rescue AVMs. Biochemical and immunofluorescence analyses confirmed the Cre-dependent OE of the ALK1-HA transgene. We could not detect any pathological signs in ALK1-OE mice up to 3 months after induction. ALK1-OE prevented the development of retinal AVMs and wound-induced skin AVMs in -iKO as well as -iKO mice. ALK1-OE normalized expression of SMAD and NOTCH target genes in ENG-deficient endothelial cells (ECs) and restored the effect of BMP9 (bone morphogenetic protein 9) on suppression of phosphor-AKT levels in these endothelial cells. On the other hand, ENG-OE could not inhibit the AVM development in -iKO models.
Conclusions
These data support the notion that ENG and ALK1 form a linear signaling pathway for the formation of a proper arteriovenous network during angiogenesis. We suggest that ALK1 OE or activation can be an effective therapeutic strategy for HHT. Further research is required to study whether this therapy could be translated into treatment for humans.



Circ Res: 08 Oct 2020; 127:1122-1137

Epigenomic and Transcriptomic Dynamics During Human Heart Organogenesis.

VanOudenhove J, Yankee TN, Wilderman A, Cotney J
Rationale
There is growing evidence that common variants and rare sequence alterations in regulatory sequences can result in birth defects or predisposition to disease. Congenital heart defects are the most common birth defect and have a clear genetic component, yet only a third of cases can be attributed to structural variation in the genome or a mutation in a gene. The remaining unknown cases could be caused by alterations in regulatory sequences.
Objective
Identify regulatory sequences and gene expression networks that are active during organogenesis of the human heart. Determine whether these sites and networks are enriched for disease-relevant genes and associated genetic variation.
Methods and results
We characterized ChromHMM (chromatin state) and gene expression dynamics during human heart organogenesis. We profiled 7 histone modifications in embryonic hearts from each of 9 distinct Carnegie stages (13-14, 16-21, and 23), annotated chromatin states, and compared these maps to over 100 human tissues and cell types. We also generated RNA-sequencing data, performed differential expression, and constructed weighted gene coexpression networks. We identified 177 412 heart enhancers; 12 395 had not been previously annotated as strong enhancers. We identified 92% of all functionally validated heart-positive enhancers (n=281; 7.5× enrichment; <2.2×10). Integration of these data demonstrated novel heart enhancers are enriched near genes expressed more strongly in cardiac tissue and are enriched for variants associated with ECG measures and atrial fibrillation. Our gene expression network analysis identified gene modules strongly enriched for heart-related functions, regulatory control by heart-specific enhancers, and putative disease genes.
Conclusions
Well-connected hub genes with heart-specific expression targeted by embryonic heart-specific enhancers are likely disease candidates. Our functional annotations will allow for better interpretation of whole genome sequencing data in the large number of patients affected by congenital heart defects.



Circ Res: 08 Oct 2020; 127:e184-e209

Periostin: A Potential Therapeutic Target For Pulmonary Hypertension?

Nie X, Shen C, Tan J, Wu Z, ... Chen J, Bian JS
Rationale
POSTN (Periostin) is an ECM (extracellular matrix) protein involved in tissue remodeling in response to injury and a contributing factor in tumorigenesis, suggesting that POSTN plays a role in the pathogenesis of pulmonary hypertension (PH).
Objective
We aimed to gain insight into the mechanistic contribution of POSTN in experimental mouse models of PH and correlate these findings with PH in humans.
Methods and results
We used genetic epistasis approaches in human pulmonary artery endothelial cells (hPAECs), human pulmonary artery smooth muscle cells, and experimental mouse models of PH (Sugen 5416/hypoxia or chronic hypoxia) to discern the role of POSTN and its relationship to HIF (hypoxia-inducible factor)-1α signaling. We found that POSTN expression was correlated with the extent of PH in mouse models and in humans. Decreasing POSTN improved hemodynamic and cardiac responses in PH mice, blunted the release of growth factors and HIF-1α, and reversed the downregulated BMPR (bone morphogenetic protein receptor)-2 expression in hPAECs from patients with PH, whereas increasing POSTIN had the opposite effects and induced a hyperproliferative and promigratory phenotype in both hPAECs and human pulmonary artery smooth muscle cells. Overexpression of POSTN-induced activation of HIFs and increased the production of ET (endothelin)-1 and VEGF (vascular endothelial growth factor) in hPAECs. SiRNA-mediated knockdown of HIF-1α abolished the proangiogenic effect of POSTN. Blockade of TrkB (tyrosine kinase receptor B) attenuated the effect of POSTN on HIF-1α expression, while inhibition of HIF-1α reduced the expression of POSTN and TrkB. These results suggest that hPAECs produce POSTN via a HIF-1α-dependent mechanism.
Conclusions
Our study reveals that POSTN expression is increased in human and animal models of PH and fosters PH development via a positive feedback loop between HIF-1α and POSTN during hypoxia. We propose that manipulating POSTIN expression may be an efficacious therapeutic target in the treatment of PH. Our results also suggest that POSTN may serve as a biomarker to estimate the severity of PH.



Circ Res: 08 Oct 2020; 127:1138-1152

miR-18a Inhibits BMP4 and HIF-1α Normalizing Brain Arteriovenous Malformations.

Marín-Ramos NI, Thein TZ, Ghaghada KB, Chen TC, Giannotta SL, Hofman FM
Rationale
Brain arteriovenous malformations (AVMs) are abnormal tangles of vessels where arteries and veins directly connect without intervening capillary nets, increasing the risk of intracerebral hemorrhage and stroke. Current treatments are highly invasive and often not feasible. Thus, effective noninvasive treatments are needed. We previously showed that AVM-brain endothelial cells (BECs) secreted higher VEGF (vascular endothelial growth factor) and lower TSP-1 (thrombospondin-1) levels than control BEC; and that microRNA-18a (miR-18a) normalized AVM-BEC function and phenotype, although its mechanism remained unclear.
Objective
To elucidate the mechanism of action and potential clinical application of miR-18a as an effective noninvasive treatment to selectively restore the phenotype and functionality of AVM vasculature.
Methods and results
The molecular pathways affected by miR-18a in patient-derived BECs and AVM-BECs were determined by Western blot, RT-qPCR (quantitative reverse transcription polymerase chain reaction), ELISA, co-IP, immunostaining, knockdown and overexpression studies, flow cytometry, and luciferase reporter assays. miR-18a was shown to increase TSP-1 and decrease VEGF by reducing PAI-1 (plasminogen activator inhibitor-1/SERPINE1) levels. Furthermore, miR-18a decreased the expression of BMP4 (bone morphogenetic protein 4) and HIF-1α (hypoxia-inducible factor 1α), blocking the BMP4/ALK (activin-like kinase) 2/ALK1/ALK5 and Notch signaling pathways. As determined by Boyden chamber assays, miR-18a also reduced the abnormal AVM-BEC invasiveness, which correlated with a decrease in MMP2 (matrix metalloproteinase 2), MMP9, and ADAM10 (ADAM metallopeptidase domain 10) levels. In vivo pharmacokinetic studies showed that miR-18a reaches the brain following intravenous and intranasal administration. Intranasal co-delivery of miR-18a and NEO100, a good manufacturing practices-quality form of perillyl alcohol, improved the pharmacokinetic profile of miR-18a in the brain without affecting its pharmacological properties. Ultra-high-resolution computed tomography angiography and immunostaining studies in an Mgp AVM mouse model showed that miR-18a decreased abnormal cerebral vasculature and restored the functionality of the bone marrow, lungs, spleen, and liver.
Conclusions
miR-18a may have significant clinical value in preventing, reducing, and potentially reversing AVM.



Circ Res: 08 Oct 2020; 127:e210-e231

Coupling of Integrin α5 to Annexin A2 by Flow Drives Endothelial Activation.

Zhang C, Zhou T, Chen Z, Yan M, ... Zhu Y, Ai D
Rationale
Atherosclerosis preferentially occurs at specific sites of the vasculature where endothelial cells (ECs) are exposed to disturbed blood flow. Translocation of integrin α5 to lipid rafts promotes integrin activation and ligation, which is critical for oscillatory shear stress (OSS)-induced EC activation. However, the underlying mechanism of OSS promoted integrin α5 lipid raft translocation has remained largely unknown.
Objective
The objective of this study was to specify the mechanotransduction mechanism of OSS-induced integrin α5 translocation and subsequent EC activation.
Methods and results
Mass spectrometry studies identified endothelial ANXA2 (annexin A2) as a potential carrier allowing integrin α5β1 to traffic in response to OSS. Interference by siRNA ofin ECs greatly decreased OSS-induced integrin α5β1 translocation to lipid rafts, EC activation, and monocyte adhesion. Pharmacological and genetic inhibition of PTP1B (protein tyrosine phosphatase 1B) blunted OSS-induced integrin α5β1 activation, which is dependent on Piezo1-mediated calcium influx in ECs. Furthermore, ANXA2 was identified as a direct substrate of activated PTP1B by mass spectrometry. Using bioluminescence resonance energy transfer assay, PTP1B-dephosphorylated ANXA2 at Y24 was found to lead to conformational freedom of the C-terminal core domain from the N-terminal domain of ANXA2. Immunoprecipitation assays showed that this unmasked ANXA2-C-terminal core domain specifically binds to an integrin α5 nonconserved cytoplasmic domain but not β1. Importantly, ectopic lentiviral overexpression of an ANXA2 mutant increased and shRNA againstdecreased integrin α5β1 ligation, inflammatory signaling, and progression of plaques at atheroprone sites in apolipoprotein E () mice. However, the antiatherosclerotic effect ofshRNA was abolished inmice.
Conclusions
Our data elucidate a novel endothelial mechanotransduction molecular mechanism linking atheroprone flow and activation of integrin α5β1, thereby identifying a class of potential therapeutic targets for atherosclerosis. Graphic Abstract: An graphic abstract is available for this article.



Circ Res: 24 Sep 2020; 127:1074-1090

CaMKIIδC Drives Early Adaptive Ca Change and Late Eccentric Cardiac Hypertrophy.

Ljubojevic-Holzer S, Herren AW, Djalinac N, Voglhuber J, ... Bossuyt J, Bers DM
Rationale
CaMKII (Ca-Calmodulin dependent protein kinase) δC activation is implicated in pathological progression of heart failure (HF) and CaMKIIδC transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca handling and CaMKII activation in hypertrophy and HF.
Objective
To measure time- and location-dependent activation of CaMKIIδC signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKIIδC transgenic mice.
Methods and results
We used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKIIδ-knockout), CaMKIIδC transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKIIδC activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKIIδ activation caused a compensatory increase in sarcoplasmic reticulum Ca content, Ca transient amplitude, and [Ca] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKIIδ TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca transient effects reversed, but further increases in nuclear and time-averaged [Ca] and CaMKII activation occurred. CaMKIIδ TG mice lacking δB exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKIIδ expression, especially for CaMKIIδC in nuclear fractions.
Conclusions
We conclude that in early TAC perinuclear CaMKIIδC activation promotes adaptive increases in myocyte Ca transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca-CaMKIIδC axis contributes to eccentric hypertrophy and HF.



Circ Res: 08 Oct 2020; 127:1159-1178

Impact of Local Alloimmunity and Recipient Cells in Transplant Arteriosclerosis.

Cai J, Deng J, Gu W, Ni Z, ... Lu Y, Xu Q
Rationale
Transplant arteriosclerosis is the major limitation to long-term survival of solid organ transplantation. Although both immune and nonimmune cells have been suggested to contribute to this process, the complex cellular heterogeneity within the grafts, and the underlying mechanisms regulating the disease progression remain largely uncharacterized.
Objective
We aimed to delineate the cellular heterogeneity within the allografts, and to explore possible mechanisms underlying this process.
Methods and results
Here, we reported the transcriptional profiling of 11 868 cells in a mouse model of transplant arteriosclerosis by single-cell RNA sequencing. Unbiased clustering analyses identified 21 cell clusters at different stages of diseases, and focused analysis revealed several previously unknown subpopulations enriched in the allografts. Interestingly, we found evidence of the local formation of tertiary lymphoid tissues and suggested a possible local modulation of alloimmune responses within the grafts. Intercellular communication analyses uncovered a potential role of several ligands and receptors, includingand , in regulating lymphatic endothelial cell-induced early chemotaxis and infiltration of immune cells. In vivo mouse experiments confirmed the therapeutic potential of CCL21 and CXCR3 neutralizing antibodies in transplant arteriosclerosis. Combinational use of genetic lineage tracing and single-cell techniques further indicate the infiltration of host-derived c-Kit stem cells as heterogeneous populations in the allografts. Finally, we compared the immune response between mouse allograft and atherosclerosis models in single-cell RNA-seq analysis. By analyzing susceptibility genes of disease traits, we also identified several cell clusters expressing genes associated with disease risk.
Conclusions
Our study provides a transcriptional and cellular landscape of transplant arteriosclerosis, which could be fundamental to understanding the initiation and progression of this disease. CCL21/CXCR3 was also identified as important regulators of immune response and may serve as potential therapeutic targets in disease treatment.



Circ Res: 24 Sep 2020; 127:974-993

ATAC-seq Reveals an Enhancer that Regulates Sinoatrial Node Development and Function.

Galang G, Mandla R, Ruan H, Jung C, ... Black BL, Vedantham V

Cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that allows them to fire automatically and initiate the heartbeat. Although critical SAN transcription factors, including Isl1, Tbx3, and Shox2, have been identified, the cis-regulatory architecture that governs PC-specific gene expression is not understood, and discrete enhancers required for gene regulation in the SAN have not been identified.To define the epigenetic profile of PCs using comparative ATAC-seq and to identify novel enhancers involved in SAN gene regulation, development and function.We used ATAC-seq on sorted neonatal mouse SAN to compare regions of accessible chromatin in PCs and right atrial cardiomyocytes. PC-enriched ATAC-seq peaks, representing candidate SAN regulatory elements, were located near established SAN genes and were enriched for distinct sets of TF binding sites. Among several novel SAN enhancers that were experimentally validated using transgenic mice, we identified a 2.9-kb regulatory element at the Isl1 locus that was active specifically in the cardiac inflow at E8.5 and throughout later SAN development and maturation. Deletion of this enhancer from the genome of mice resulted in SAN hypoplasia and sinus arrhythmias. The mouse SAN enhancer also directed reporter activity to the inflow tract in developing zebrafish hearts, demonstrating deep conservation of its upstream regulatory network. Finally, single nucleotide polymorphisms in the human genome that occur near the region syntenic to the mouse enhancer exhibit significant associations with resting heart rate in human populations.(1) PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, (2) Cis-regulation of Isl1 specifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and (3) a corresponding human ISL1 enhancer may regulate human SAN function.



Circ Res: 11 Oct 2020; epub ahead of print

Human Pacemaker Regulatory Element Analysis Identifies TBX3 Pacemaker Enhancer.

van Eif VW, Protze S, Bosada FM, Yuan X, ... Scott IC, Christoffels VM

The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3, ISL1 and SHOX2. Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined.Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation.We performed ATAC-seq on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative regulatory DNA elements specific for either human cell type. We validated pacemaker cell-specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse orthologue of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos.We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2, TBX3 and ISL1, and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3. exercise and a variant RE with highly conserved function, driving SAN expression of TBX3.



Circ Res: 11 Oct 2020; epub ahead of print

Loss of Cardiac Ferritin H Facilitates Cardiomyopathy via Slc7a11-Mediated Ferroptosis.

Fang X, Cai Z, Wang H, Han D, ... Min J, Wang F
Rationale
Maintaining iron homeostasis is essential for proper cardiac function. Both iron deficiency and iron overload are associated with cardiomyopathy and heart failure via complex mechanisms. Although ferritin plays a central role in iron metabolism by storing excess cellular iron, the molecular function of ferritin in cardiomyocytes remains unknown.
Objective
To characterize the functional role of Fth (ferritin H) in mediating cardiac iron homeostasis and heart disease.
Methods and results
Mice expressing a conditional Fth knockout allele were crossed with 2 distinct Cre recombinase-expressing mouse lines, resulting in offspring that lackexpression specifically in myocytes (MCK-Cre) or cardiomyocytes (Myh6-Cre). Mice lacking Fth in cardiomyocytes had decreased cardiac iron levels and increased oxidative stress, resulting in mild cardiac injury upon aging. However, feeding these mice a high-iron diet caused severe cardiac injury and hypertrophic cardiomyopathy, with molecular features typical of ferroptosis, including reduced glutathione (GSH) levels and increased lipid peroxidation. Ferrostatin-1, a specific inhibitor of ferroptosis, rescued this phenotype, supporting the notion that ferroptosis plays a pathophysiological role in the heart. Finally, we found that Fth-deficient cardiomyocytes have reduced expression of the ferroptosis regulator Slc7a11, and overexpressing Slc7a11 selectively in cardiomyocytes increased GSH levels and prevented cardiac ferroptosis.
Conclusions
Our findings provide compelling evidence that ferritin plays a major role in protecting against cardiac ferroptosis and subsequent heart failure, thereby providing a possible new therapeutic target for patients at risk of developing cardiomyopathy.



Circ Res: 30 Jul 2020; 127:486-501

Sorting Nexin 10 Mediates Metabolic Reprogramming of Macrophages in Atherosclerosis Through the Lyn-Dependent TFEB Signaling Pathway.

You Y, Bao WL, Zhang SL, Li HD, ... Zheng M, Shen X
Rationale
SNX10 (sorting nexin 10) has been reported to play a critical role in regulating macrophage function and lipid metabolism.
Objective
To investigate the precise role of SNX10 in atherosclerotic diseases and the underlying mechanisms.
Methods and results
SNX10 expression was compared between human healthy vessels and carotid atherosclerotic plaques. Myeloid cell-specific SNX10 knockdown mice were crossed onto the APOE (apolipoprotein E) background and atherogenesis (high-cholesterol diet-induced) was monitored for 16 weeks. We found that SNX10 expression was increased in atherosclerotic lesions of aortic specimens from humans and APOE mice. Myeloid cell-specific SNX10 deficiency (Δ knockout [KO]) attenuated atherosclerosis progression in APOE mice. The population of anti-inflammatory monocytes/macrophages was increased in the peripheral blood and atherosclerotic lesions of ΔKO mice. In vitro experiments showed that SNX10 deficiency-inhibited foam cell formation through interrupting the internalization of CD36, which requires the interaction of SNX10 and Lyn-AKT (protein kinase B). The reduced Lyn-AKT activation by SNX10 deficiency promoted the nuclear translocation of TFEB (transcription factor EB), thereby enhanced lysosomal biogenesis and LAL (lysosomal acid lipase) activity, resulting in an increase of free fatty acids to fuel mitochondrial fatty acid oxidation. This further promoted the reprogramming of macrophages and shifted toward the anti-inflammatory phenotype.
Conclusions
Our data demonstrate for the first time that SNX10 plays a crucial role in diet-induced atherogenesis via the previously unknown link between the Lyn-Akt-TFEB signaling pathway and macrophage reprogramming, suggest that SNX10 may be a potentially promising therapeutic target for atherosclerosis treatment.



Circ Res: 30 Jul 2020; 127:534-549

The Tumor-Suppressive Human Circular RNA CircITCH Sponges miR-330-5p to Ameliorate Doxorubicin-Induced Cardiotoxicity Through Upregulating SIRT6, Survivin, and SERCA2a.

Han D, Wang Y, Wang Y, Dai X, ... Zhang J, Cao F
Rationale
Doxorubicin is one of the most potent antitumor agents available; however, its clinical use is restricted because it poses a risk of severe cardiotoxicity. Previous work has established that CircITCH (circular RNA ITCH [E3 ubiquitin-protein ligase]) is a broad-spectrum tumor-suppressive circular RNA and that its host gene, ITCH (E3 ubiquitin protein ligase), is involved in doxorubicin-induced cardiotoxicity (DOXIC). Whether CircITCH plays a role in DOXIC remains unknown.
Objective
We aimed to dissect the role of CircITCH in DOXIC and further decipher its potential mechanisms.
Methods and results
Circular RNA sequencing was performed to screen the potentially involved circRNAs in DOXI pathogenesis. Quantitative polymerase chain reaction and RNA in situ hybridization revealed that CircITCH was downregulated in doxorubicin-treated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in the autopsy specimens from cancer patients who suffered from doxorubicin-induced cardiomyopathy. Cell death/viability assays, detection of cardiomyocyte necrosis markers, microelectrode array, and cardiomyocyte functional assays revealed that CircITCH ameliorated doxorubicin-induced cardiomyocyte injury and dysfunction. Detection of cellular/mitochondrial oxidative stress and DNA damage markers verified that CircITCH alleviated cellular/mitochondrial oxidative stress and DNA damage induced by doxorubicin. RNA pull-down assays, Ago2 immunoprecipitation and double fluorescent in situ hybridization identified miR-330-5p as a direct target of CircITCH. Moreover, CircITCH was found to function by acting as an endogenous sponge that sequestered miR-330-5p. Bioinformatic analysis, luciferase reporter assays, and quantitative polymerase chain reaction showed that SIRT6 (sirtuin 6), BIRC5 (baculoviral IAP repeat containing 5, Survivin), and ATP2A2 (ATPase sarcoplasmic/endoplasmic reticulum Ca transporting 2, SERCA2a [SR Ca-ATPase 2]) were direct targets of miR-330-5p and that they were regulated by the CircITCH/miR-330-5p axis in DOXIC. Further experiments demonstrated that CircITCH-mediated alleviation of DOXIC was dependent on the interactions between miR-330-5p and the 3\'-UTRs of SIRT6, BIRC5, and ATP2A2 mRNA. Finally, AAV9 (adeno-associated virus serotype 9) vector-based overexpression of the well-conserved CircITCH partly prevented DOXIC in mice.
Conclusions
CircITCH represents a novel therapeutic target for DOXIC because it acts as a natural sponge of miR-330-5p, thereby upregulating SIRT6, Survivin and SERCA2a to alleviate doxorubicin-induced cardiomyocyte injury and dysfunction.



Circ Res: 30 Jul 2020; 127:e108-e125

Gut Microbiota-Derived Short-Chain Fatty Acids Promote Poststroke Recovery in Aged Mice.

Lee J, d\'Aigle J, Atadja L, Quaicoe V, ... McCullough LD, Venna VR
Rationale
The elderly experience profound systemic responses after stroke, which contribute to higher mortality and more severe long-term disability. Recent studies have revealed that stroke outcomes can be influenced by the composition of gut microbiome. However, the potential benefits of manipulating the gut microbiome after injury is unknown.
Objective
To determine if restoring youthful gut microbiota after stroke aids in recovery in aged subjects, we altered the gut microbiome through young fecal transplant gavage in aged mice after experimental stroke. Further, the effect of direct enrichment of selective bacteria producing short-chain fatty acids (SCFAs) was tested as a more targeted and refined microbiome therapy.
Methods and results
Aged male mice (18-20 months) were subjected to ischemic stroke by middle cerebral artery occlusion. We performed fecal transplant gavage 3 days after middle cerebral artery occlusion using young donor biome (2-3 months) or aged biome (18-20 months). At day 14 after stroke, aged stroke mice receiving young fecal transplant gavage had less behavioral impairment, and reduced brain and gut inflammation. Based on data from microbial sequencing and metabolomics analysis demonstrating that young fecal transplants contained much higher SCFA levels and related bacterial strains, we selected 4 SCFA-producers (, , , and ) for transplantation. These SCFA-producers alleviated poststroke neurological deficits and inflammation, and elevated gut, brain and plasma SCFA concentrations in aged stroke mice.
Conclusions
This is the first study suggesting that the poor stroke recovery in aged mice can be reversed via poststroke bacteriotherapy following the replenishment of youthful gut microbiome via modulation of immunologic, microbial, and metabolomic profiles in the host.



Circ Res: 30 Jul 2020; 127:453-465

The Calcineurin-TFEB-p62 Pathway Mediates the Activation of Cardiac Macroautophagy by Proteasomal Malfunction.

Pan B, Li J, Parajuli N, Tian Z, ... Su H, Wang X
Rationale
The ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway are pivotal to proteostasis. Targeting these pathways is emerging as an attractive strategy for treating cancer. However, a significant proportion of patients who receive a proteasome inhibitor-containing regime show cardiotoxicity. Moreover, UPS and autophagic-lysosomal pathway defects are implicated in cardiac pathogenesis. Hence, a better understanding of the cross-talk between the 2 catabolic pathways will help advance cardiac pathophysiology and medicine.
Objective
Systemic proteasome inhibition (PSMI) was shown to increase p62/SQSTM1 expression and induce myocardial macroautophagy. Here we investigate how proteasome malfunction activates cardiac autophagic-lysosomal pathway.
Methods and results
Myocardial macroautophagy, TFEB (transcription factor EB) expression and activity, and p62 expression were markedly increased in mice with either cardiomyocyte-restricted ablation of(an essential proteasome subunit gene) or pharmacological PSMI. In cultured cardiomyocytes, PSMI-induced increases in TFEB activation and p62 expression were blunted by pharmacological and genetic calcineurin inhibition and by siRNA-mediatedsilencing. PSMI induced remarkable increases in myocardial autophagic flux in wild type mice but not p62 null (p62-KO) mice. Bortezomib-induced left ventricular wall thickening and diastolic malfunction was exacerbated by p62 deficiency. In cultured cardiomyocytes from wild type mice but not p62-KO mice, PSMI induced increases in LC3-II flux and the lysosomal removal of ubiquitinated proteins. Myocardial TFEB activation by PSMI as reflected by TFEB nuclear localization and target gene expression was strikingly less in p62-KO mice compared with wild type mice.
Conclusions
(1) The activation of cardiac macroautophagy by proteasomal malfunction is mediated by the Mocln1-calcineurin-TFEB-p62 pathway; (2) p62 unexpectedly exerts a feed-forward effect on TFEB activation by proteasome malfunction; and (3) targeting the Mcoln1 (mucolipin1)-calcineurin-TFEB-p62 pathway may provide new means to intervene cardiac autophagic-lysosomal pathway activation during proteasome malfunction.



Circ Res: 30 Jul 2020; 127:502-518

PKG1α Cysteine-42 Redox State Controls mTORC1 Activation in Pathological Cardiac Hypertrophy.

Oeing CU, Nakamura T, Pan S, Mishra S, ... Kass DA, Ranek MJ
Rationale
Stimulated PKG1α (protein kinase G-1α) phosphorylates TSC2 (tuberous sclerosis complex 2) at serine 1365, potently suppressing mTORC1 (mechanistic [mammalian] target of rapamycin complex 1) activation by neurohormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine-42 is also induced by these stressors, which blunts its cardioprotective effects.
Objective
We tested the dependence of mTORC1 activation on PKG1α C42 oxidation and its capacity to suppress such activation by soluble GC-1 (guanylyl cyclase 1) activation.
Methods and results
Cardiomyocytes expressing wild-type (WT) PKG1α (PKG1α) or cysteine-42 to serine mutation redox-dead (PKG1α) were exposed to ET-1 (endothelin 1). Cells expressing PKG1α exhibited substantial mTORC1 activation (p70 S6K [p70 S6 kinase], 4EBP1 [elF4E binding protein-1], and Ulk1 [Unc-51-like kinase 1] phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1α expression. Mice with global knock-in of PKG1α subjected to pressure overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1α mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2-S1365 phosphorylation increased in PKG1α more than PKG1α myocardium following PO. TSC2 (TSC2 S1365 phospho-null, created by a serine to alanine mutation) knock-in mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1α mice, protection against PO-induced mTORC1 activation, cardiodepression, and mortality in PKG1α mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1α and PKG1α after PO and blocked ET-1 stimulated mTORC1 in TSC2-expressing myocytes.
Conclusions
Oxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.



Circ Res: 30 Jul 2020; 127:522-533

Role of the GLUT1 Glucose Transporter in Postnatal CNS Angiogenesis and Blood-Brain Barrier Integrity.

Veys K, Fan Z, Ghobrial M, Bouché A, ... Carmeliet P, De Bock K
Rationale
Endothelial cells (ECs) are highly glycolytic and generate the majority of their energy via the breakdown of glucose to lactate. At the same time, a main role of ECs is to allow the transport of glucose to the surrounding tissues. GLUT1 (glucose transporter isoform 1/) is highly expressed in ECs of the central nervous system (CNS) and is often implicated in blood-brain barrier (BBB) dysfunction, but whether and how GLUT1 controls EC metabolism and function is poorly understood.
Objective
We evaluated the role of GLUT1 in endothelial metabolism and function during postnatal CNS development as well as at the adult BBB.
Methods and results
Inhibition of GLUT1 decreases EC glucose uptake and glycolysis, leading to energy depletion and the activation of the cellular energy sensor AMPK (AMP-activated protein kinase), and decreases EC proliferation without affecting migration. Deletion of GLUT1 from the developing postnatal retinal endothelium reduces retinal EC proliferation and lowers vascular outgrowth, without affecting the number of tip cells. In contrast, in the brain, we observed a lower number of tip cells in addition to reduced brain EC proliferation, indicating that within the CNS, organotypic differences in EC metabolism exist. Interestingly, when ECs become quiescent, endothelial glycolysis is repressed, and GLUT1 expression increases in a Notch-dependent fashion. GLUT1 deletion from quiescent adult ECs leads to severe seizures, accompanied by neuronal loss and CNS inflammation. Strikingly, this does not coincide with BBB leakiness, altered expression of genes crucial for BBB barrier functioning nor reduced vascular function. Instead, we found a selective activation of inflammatory and extracellular matrix related gene sets.
Conclusions
GLUT1 is the main glucose transporter in ECs and becomes uncoupled from glycolysis during quiescence in a Notch-dependent manner. It is crucial for developmental CNS angiogenesis and adult CNS homeostasis but does not affect BBB barrier function.



Circ Res: 30 Jul 2020; 127:466-482

Critical Role of Neprilysin in Kidney Angiotensin Metabolism.

Kaltenecker CC, Domenig O, Kopecky C, Antlanger M, ... Säemann MD, Kovarik JJ
Rationale
Kidney homeostasis is critically determined by the coordinated activity of the renin-angiotensin system (RAS), including the balanced synthesis of its main effector peptides Ang (angiotensin) II and Ang (1-7). The condition of enzymatic overproduction of Ang II relative to Ang (1-7) is termed RAS dysregulation and leads to cellular signals, which promote hypertension and organ damage, and ultimately progressive kidney failure. ACE2 (angiotensin-converting enzyme 2) and NEP (neprilysin) induce the alternative, and potentially reno-protective axis by enhancing Ang (1-7) production. However, their individual contribution to baseline RAS balance and whether their activities change in chronic kidney disease (CKD) has not yet been elucidated.
Objective
To examine whether NEP-mediated Ang (1-7) generation exceeds Ang II formation in the healthy kidney compared with diseased kidney.
Methods and results
In this exploratory study, we used liquid chromatography-tandem mass spectrometry to measure Ang II and Ang (1-7) synthesis rates of ACE, chymase and NEP, ACE2, PEP (prolyl-endopeptidase), PCP (prolyl-carboxypeptidase) in kidney biopsy homogenates in 11 healthy living kidney donors, and 12 patients with CKD. The spatial expression of RAS enzymes was determined by immunohistochemistry. Healthy kidneys showed higher NEP-mediated Ang (1-7) synthesis than Ang II formation, thus displaying a strong preference towards the reno-protective alternative RAS axis. In contrast, in CKD kidneys higher levels of Ang II were recorded, which originated from mast cell chymase activity.
Conclusions
Ang (1-7) is the dominant RAS peptide in healthy human kidneys with NEP rather than ACE2 being essential for its generation. Severe RAS dysregulation is present in CKD dictated by high chymase-mediated Ang II formation. Kidney RAS enzyme analysis might lead to novel therapeutic approaches for CKD.



Circ Res: 13 Aug 2020; 127:593-606

CITED4 Protects Against Adverse Remodeling in Response to Physiological and Pathological Stress.

Lerchenmüller C, Rabolli CP, Yeri A, Kitchen R, ... Das S, Rosenzweig A
Rationale
Cardiac CITED4 (CBP/p300-interacting transactivators with E [glutamic acid]/D [aspartic acid]-rich-carboxylterminal domain4) is induced by exercise and is sufficient to cause physiological hypertrophy and mitigate adverse ventricular remodeling after ischemic injury. However, the role of endogenous CITED4 in response to physiological or pathological stress is unknown.
Objective
To investigate the role of CITED4 in murine models of exercise and pressure overload.
Methods and results
We generated cardiomyocyte-specific CITED4 knockout mice (C4KO) and subjected them to an intensive swim exercise protocol as well as transverse aortic constriction (TAC). Echocardiography, Western blotting, qPCR, immunohistochemistry, immunofluorescence, and transcriptional profiling for mRNA and miRNA (microRNA) expression were performed. Cellular crosstalk was investigated in vitro. CITED4 deletion in cardiomyocytes did not affect baseline cardiac size or function in young adult mice. C4KO mice developed modest cardiac dysfunction and dilation in response to exercise. After TAC, C4KOs developed severe heart failure with left ventricular dilation, impaired cardiomyocyte growth accompanied by reduced mTOR (mammalian target of rapamycin) activity and maladaptive cardiac remodeling with increased apoptosis, autophagy, and impaired mitochondrial signaling. Interstitial fibrosis was markedly increased in C4KO hearts after TAC. RNAseq revealed induction of a profibrotic miRNA network. miR30d was decreased in C4KO hearts after TAC and mediated crosstalk between cardiomyocytes and fibroblasts to modulate fibrosis. miR30d inhibition was sufficient to increase cardiac dysfunction and fibrosis after TAC.
Conclusions
CITED4 protects against pathological cardiac remodeling by regulating mTOR activity and a network of miRNAs mediating cardiomyocyte to fibroblast crosstalk. Our findings highlight the importance of CITED4 in response to both physiological and pathological stimuli.



Circ Res: 13 Aug 2020; 127:631-646

S100A9 Links Inflammation and Repair in Myocardial Infarction.

Marinković G, Koenis DS, de Camp L, Jablonowski R, ... Jovinge S, Schiopu A
Rationale
The alarmin S100A9 has been identified as a potential therapeutic target in myocardial infarction. Short-term S100A9 blockade during the inflammatory phase post-myocardial infarction inhibits systemic and cardiac inflammation and improves cardiac function long term.
Objective
To evaluate the impact of S100A9 blockade on postischemic cardiac repair.
Methods and results
We assessed cardiac function, hematopoietic response, and myeloid phagocyte dynamics in WT (wild type) C57BL/6 mice with permanent coronary artery ligation, treated with the specific S100A9 blocker ABR-238901 for 7 or 21 days. In contrast to the beneficial effects of short-term therapy, extended S100A9 blockade led to progressive deterioration of cardiac function and left ventricle dilation. The treatment reduced the proliferation of LinSca-1c-Kit hematopoietic stem and progenitor cells in the bone marrow and the production of proreparatory CD150CD48CCR2 hematopoietic stem cells. Monocyte trafficking from the spleen to the myocardium and subsequent phenotype switching to reparatory Ly6CMerTK macrophages was also impaired, leading to inefficient efferocytosis, accumulation of apoptotic cardiomyocytes, and a larger myocardial scar. The transcription factor Nur77 (Nr4a1 [nuclear receptor subfamily 4 group A member 1]) mediates the transition from inflammatory Ly6C monocytes to reparatory Ly6C macrophages. S100A9 upregulated the levels and activity of Nur77 in monocytes and macrophages in vitro and in Ly6C monocytes in vivo, and S100A9 blockade antagonized these effects. Finally, the presence of reparatory macrophages in the myocardium was also impaired in S100A9 mice with permanent myocardial ischemia, leading to depressed cardiac function long term.
Conclusions
We show that S100A9 plays an important role in both the inflammatory and the reparatory immune responses to myocardial infarction. Long-term S100A9 blockade negatively impacts cardiac recovery and counterbalances the beneficial effects of short-term therapy. These results define a therapeutic window targeting the inflammatory phase for optimal effects of S100A9 blockade as potential immunomodulatory treatment in acute myocardial infarction.



Circ Res: 13 Aug 2020; 127:664-676

Modulation of Mammalian Cardiomyocyte Cytokinesis by the Extracellular Matrix.

Wu CC, Jeratsch S, Graumann J, Stainier DYR
Rationale
After birth, cycling mammalian CMs (cardiomyocytes) progressively lose the ability to undergo cytokinesis and hence they become binucleated, which leads to cell cycle exit and loss of regenerative capacity. During late embryonic and early postnatal heart growth, CM development is accompanied by an expansion of the cardiac fibroblast (cFb) population and compositional changes in the ECM (extracellular matrix). Whether and how these changes influence cardiomyocyte cytokinesis is currently unknown.
Objective
To elucidate the role of postnatal cFbs and the ECM in cardiomyocyte cytokinesis and identify ECM proteins that promote cardiomyocyte cytokinesis.
Methods and results
Using primary rat cardiomyocyte cultures, we found that a proportion of postnatal, but not embryonic, cycling cardiomyocytes fail to progress through cytokinesis and subsequently binucleate, consistent with published reports of in vitro and in vivo observations. Direct coculture with postnatal cFbs increased cardiomyocyte binucleation, which could be inhibited by RGD peptide treatment. In contrast, cFb-conditioned medium or transwell coculture did not significantly increase cardiomyocyte binucleation, suggesting that cFbs inhibit cardiomyocyte cytokinesis through ECM modulation rather than by secreting diffusible factors. Furthermore, we found that both embryonic and postnatal CMs binucleate at a significantly higher rate when cultured on postnatal cFb-derived ECM compared with embryonic cFb-derived ECM. These cytokinetic defects correlate with cardiomyocyte inefficiency in mitotic rounding, a process which is key to successful cytokinesis. To identify ECM proteins that modulate cardiomyocyte cytokinesis, we compared the composition of embryonic and postnatal cFb-derived ECM by mass spectrometry followed by functional assessment. We found that 2 embryonically enriched ECM proteins, SLIT2 and NPNT (nephronectin), promote cytokinesis of postnatal CMs in vitro and in vivo.
Conclusions
We identified the postnatal cardiac ECM as a nonpermissive environment for cardiomyocyte cytokinesis and uncovered novel functions for the embryonic ECM proteins SLIT2 and NPNT (nephronectin) in promoting postnatal cardiomyocyte cytokinesis. Graphic Abstract: A graphic abstract is available for this article.



Circ Res: 10 Sep 2020; 127:896-907

Iron-Deficiency and Estrogen Are Associated With Ischemic Stroke by Up-Regulating Transferrin to Induce Hypercoagulability.

Tang X, Fang M, Cheng R, Zhang Z, ... Long C, Lai R
Rationale
Epidemiological studies have identified an associate between iron deficiency (ID) and the use of oral contraceptives (CC) and ischemic stroke (IS). To date, however, the underlying mechanism remains poorly understood. Both ID and CC have been demonstrated to upregulate the level and iron-binding ability of Tf (transferrin), with our recent study showing that this upregulation can induce hypercoagulability by potentiating FXIIa/thrombin and blocking antithrombin-coagulation proteases interactions.
Objective
To investigate whether Tf mediates IS associated with ID or CC and the underlying mechanisms.
Methods and results
Tf levels were assayed in the plasma of IS patients with a history of ID anemia, ID anemia patients, venous thromboembolism patients using CC, and ID mice, and in the cerebrospinal fluid of some IS patients. Effects of ID and estrogen administration on Tf expression and coagulability and the underlying mechanisms were studied in vivo and in vitro. High levels of Tf and Tf-thrombin/FXIIa complexes were found in patients and ID mice. Both ID and estrogen upregulated Tf through hypoxia and estrogen response elements located in the Tf gene enhancer and promoter regions, respectively. In addition, ID, administration of exogenous Tf or estrogen, and Tf overexpression promoted platelet-based thrombin generation and hypercoagulability and thus aggravated IS. In contrast, anti-Tf antibodies, Tf knockdown, and peptide inhibitors of Tf-thrombin/FXIIa interaction exerted anti-IS effects in vivo.
Conclusions
Our findings revealed that certain factors (ie, ID and CC) upregulating Tf are risk factors of thromboembolic diseases decipher a previously unrecognized mechanistic association among ID, CC, and IS and provide a novel strategy for the development of anti-IS medicine by interfering with Tf-thrombin/FXIIa interactions.



Circ Res: 13 Aug 2020; 127:651-663

Novel Methods for Quantification of Vasodepression and Cardioinhibition During Tilt-Induced Vasovagal Syncope.

van Dijk JG, Ghariq M, Kerkhof FI, Reijntjes R, ... Thijs RD, Benditt DG
Rationale
Assessing the relative contributions of cardioinhibition and vasodepression to the blood pressure (BP) decrease in tilt-induced vasovagal syncope requires methods that reflect BP physiology accurately.
Objective
To assess the relative contributions of cardioinhibition and vasodepression to tilt-induced vasovagal syncope using novel methods.
Methods and results
We studied the parameters determining BP, that is, stroke volume (SV), heart rate (HR), and total peripheral resistance (TPR), in 163 patients with tilt-induced vasovagal syncope documented by continuous ECG and video EEG monitoring. We defined the beginning of cardioinhibition as the start of an HR decrease (HR) before syncope and used logarithms of SV, HR, and TPR ratios to quantify the multiplicative relation BP=SV·HR·TPR. We defined 3 stages before syncope and 2 after it based on direction changes of these parameters. The earliest BP decrease occurred 9 minutes before syncope. Cardioinhibition was observed in 91% of patients at a median time of 58 seconds before syncope. At that time, SV had a strong negative effect on BP, TPR a lesser negative effect, while HR had increased (all <0.001). At the onset of cardioinhibition, the median HR was at 98 bpm higher than baseline. Cardioinhibition thus initially only represented a reduction of the corrective HR increase but was nonetheless accompanied by an immediate acceleration of the ongoing BP decrease. At syncope, SV and HR contributed similarly to the BP decrease (<0.001), while TPR did not affect BP.
Conclusions
The novel methods allowed the relative effects of SV, HR, and TPR on BP to be assessed separately, although all act together. The 2 major factors lowering BP in tilt-induced vasovagal syncope were reduced SV and cardioinhibition. We suggest that the term vasodepression in reflex syncope should not be limited to reduced arterial vasoconstriction, reflected in TPR, but should also encompass venous pooling, reflected in SV.



Circ Res: 13 Aug 2020; 127:e126-e138

Self-Maintenance of Cardiac Resident Reparative Macrophages Attenuates Doxorubicin-Induced Cardiomyopathy Through the SR-A1-c-Myc Axis.

Zhang H, Xu A, Sun X, Yang Y, ... Xu Y, Chen Q
Rationale
Doxorubicin-induced cardiomyopathy (DiCM) is a primary cause of heart failure and mortality in cancer patients, in which macrophage-orchestrated inflammation serves as an essential pathological mechanism. However, the specific roles of tissue-resident and monocyte-derived macrophages in DiCM remain poorly understood.
Objective
Uncovering the origins, phenotypes, and functions of proliferative cardiac resident macrophages and mechanistic insights into the self-maintenance of cardiac macrophage during DiCM progression.
Methods and results
Mice were administrated with doxorubicin to induce cardiomyopathy. Dynamic changes of resident and monocyte-derived macrophages were examined by lineage tracing, parabiosis, and bone marrow transplantation. We found that the monocyte-derived macrophages primarily exhibited a proinflammatory phenotype that dominated the whole DiCM pathological process and impaired cardiac function. In contrast, cardiac resident macrophages were vulnerable to doxorubicin insult. The survived resident macrophages exhibited enhanced proliferation and conferred a reparative role. Global or myeloid specifically ablation of SR-A1 (class A1 scavenger receptor) inhibited proliferation of cardiac resident reparative macrophages and, therefore, exacerbated cardiomyopathy in DiCM mice. Importantly, the detrimental effect of macrophage SR-A1 deficiency was confirmed by transplantation of bone marrow. At the mechanistic level, we show that c-Myc (Avian myelocytomatosis virus oncogene cellular homolog), a key transcriptional factor for the SR-A1-P38-SIRT1 (Sirtuin 1) pathway, mediated the effect of SR-A1 in reparative macrophage proliferation in DiCM.
Conclusions
The SR-A1-c-Myc axis may represent a promising target to treat DiCM through augmentation of cardiac resident reparative macrophage proliferation.



Circ Res: 13 Aug 2020; 127:610-627

LDL Receptor Regulates the Reverse Transport of Macrophage-Derived Unesterified Cholesterol via Concerted Action of the HDL-LDL Axis: Insight From Mouse Models.

Cedó L, Metso J, Santos D, García-León A, ... Blanco-Vaca F, Escolà-Gil JC
Rationale
The HDL (high-density lipoprotein)-mediated stimulation of cellular cholesterol efflux initiates macrophage-specific reverse cholesterol transport (m-RCT), which ends in the fecal excretion of macrophage-derived unesterified cholesterol (UC). Early studies established that LDL (low-density lipoprotein) particles could act as efficient intermediate acceptors of cellular-derived UC, thereby preventing the saturation of HDL particles and facilitating their cholesterol efflux capacity. However, the capacity of LDL to act as a plasma cholesterol reservoir and its potential impact in supporting the m-RCT pathway in vivo both remain unknown.
Objective
We investigated LDL contributions to the m-RCT pathway in hypercholesterolemic mice.
Methods and results
Macrophage cholesterol efflux induced in vitro by LDL added to the culture media either alone or together with HDL or ex vivo by plasma derived from subjects with familial hypercholesterolemia was assessed. In vivo, m-RCT was evaluated in mouse models of hypercholesterolemia that were naturally deficient in CETP (cholesteryl ester transfer protein) and fed a Western-type diet. LDL induced the efflux of radiolabeled UC from cultured macrophages, and, in the simultaneous presence of HDL, a rapid transfer of the radiolabeled UC from HDL to LDL occurred. However, LDL did not exert a synergistic effect on HDL cholesterol efflux capacity in the familial hypercholesterolemia plasma. The m-RCT rates of the LDLr (LDL receptor)-KO (knockout), LDLr-KO/APOB100, and PCSK9 (proprotein convertase subtilisin/kexin type 9)-overexpressing mice were all significantly reduced relative to the wild-type mice. In contrast, m-RCT remained unchanged in HAPOB100 Tg (human APOB100 transgenic) mice with fully functional LDLr, despite increased levels of plasma APO (apolipoprotein)-B-containing lipoproteins.
Conclusions
Hepatic LDLr plays a critical role in the flow of macrophage-derived UC to feces, while the plasma increase of APOB-containing lipoproteins is unable to stimulate m-RCT. The results indicate that, besides the major HDL-dependent m-RCT pathway via SR-BI (scavenger receptor class B type 1) to the liver, a CETP-independent m-RCT path exists, in which LDL mediates the transfer of cholesterol from macrophages to feces. Graphical Abstract: A graphical abstract is available for this article.



Circ Res: 27 Aug 2020; 127:778-792

Long Range Endocrine Delivery of Circulating miR-210 to Endothelium Promotes Pulmonary Hypertension.

Zhao J, Florentin J, Tai YY, Torrino S, ... Dutta P, Chan SY
Rationale
Unproven theories abound regarding the long-range uptake and endocrine activity of extracellular blood-borne microRNAs into tissue. In pulmonary hypertension (PH), microRNA-210 (miR-210) in pulmonary endothelial cells promotes disease, but its activity as an extracellular molecule is incompletely defined.
Objective
We investigated whether chronic and endogenous endocrine delivery of extracellular miR-210 to pulmonary vascular endothelial cells promotes PH.
Methods and results
Usingreplete (wild-type [WT]) and knockout mice, we tracked blood-borne miR-210 using bone marrow transplantation and parabiosis (conjoining of circulatory systems). With bone marrow transplantation, circulating miR-210 was derived predominantly from bone marrow. Via parabiosis during chronic hypoxia to induce miR-210 production and PH, miR-210 was undetectable in knockout-knockout mice pairs. However, in plasma and lung endothelium, but not smooth muscle or adventitia, miR-210 was observed in knockout mice of WT-knockout pairs. This was accompanied by downregulation of miR-210 targets ISCU (iron-sulfur assembly proteins)1/2 and COX10 (cytochrome c oxidase assembly protein-10), indicating endothelial import of functional miR-210. Via hemodynamic and histological indices, knockout-knockout pairs were protected from PH, whereas knockout mice in WT-knockout pairs developed PH. In particular, pulmonary vascular engraftment of miR-210-positive interstitial lung macrophages was observed in knockout mice of WT-knockout pairs. To address whether engrafted miR-210-positive myeloid or lymphoid cells contribute to paracrine miR-210 delivery, we studied miR-210 knockout mice parabiosed with miR-210 WT;knockout mice (deficient in myeloid recruitment) or miR-210 WT;knockout mice (deficient in lymphocytes). In both pairs, miR-210 knockout mice still displayed miR-210 delivery and PH, thus demonstrating a pathogenic endocrine delivery of extracellular miR-210.
Conclusions
Endogenous blood-borne transport of miR-210 into pulmonary vascular endothelial cells promotes PH, offering fundamental insight into the systemic physiology of microRNA activity. These results also describe a platform for RNA-mediated crosstalk in PH, providing an impetus for developing blood-based miR-210 technologies for diagnosis and therapy in this disease.



Circ Res: 13 Aug 2020; 127:677-692

GRK5 Controls SAP97-Dependent Cardiotoxic β Adrenergic Receptor-CaMKII Signaling in Heart Failure.

Xu B, Li M, Wang Y, Zhao M, ... Nieves-Cintron M, Xiang YK
Rationale
Cardiotoxic β adrenergic receptor (βAR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of βAR and organizes a receptor signalosome.
Objective
We aim to elucidate the dynamics of βAR-SAP97 signalosome and its potential role in chronic cardiotoxic βAR-CaMKII signaling that contributes to development of heart failure.
Methods and results
The integrity of cardiac βAR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine βAR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the βAR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of βAR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from βAR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of βAR-SAP97 complex and increases in CaMKII activity in hearts.
Conclusions
These data reveal a critical role of SAP97 in maintaining the integrity of cardiac βAR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.



Circ Res: 27 Aug 2020; 127:796-810

Histone Deacetylase 9 Activates IKK to Regulate Atherosclerotic Plaque Vulnerability.

Asare Y, Campbell-James TA, Bokov Y, Yu LL, ... Bernhagen J, Dichgans M
Rationale
Arterial inflammation manifested as atherosclerosis is the leading cause of mortality worldwide. Genome-wide association studies have identified a prominent role of HDAC (histone deacetylase)-9 in atherosclerosis and its clinical complications including stroke and myocardial infarction.
Objective
To determine the mechanisms linking HDAC9 to these vascular pathologies and explore its therapeutic potential for atheroprotection.
Methods and results
We studied the effects ofon features of plaque vulnerability using bone marrow reconstitution experiments and pharmacological targeting with a small molecule inhibitor in hyperlipidemic mice. We further used 2-photon and intravital microscopy to study endothelial activation and leukocyte-endothelial interactions. We show that hematopoieticdeficiency reduces lesional macrophage content while increasing fibrous cap thickness thus conferring plaque stability. We demonstrate that HDAC9 binds to IKK (inhibitory kappa B kinase)-α and β, resulting in their deacetylation and subsequent activation, which drives inflammatory responses in both macrophages and endothelial cells. Pharmacological inhibition of HDAC9 with the class IIa HDAC inhibitor TMP195 attenuates lesion formation by reducing endothelial activation and leukocyte recruitment along with limiting proinflammatory responses in macrophages. Transcriptional profiling using RNA sequencing revealed that TMP195 downregulates key inflammatory pathways consistent with inhibitory effects on IKKβ. TMP195 mitigates the progression of established lesions and inhibits the infiltration of inflammatory cells. Moreover, TMP195 diminishes features of plaque vulnerability and thereby enhances plaque stability in advanced lesions. Ex vivo treatment of monocytes from patients with established atherosclerosis reduced the production of inflammatory cytokines including IL (interleukin)-1β and IL-6.
Conclusions
Our findings identify HDAC9 as a regulator of atherosclerotic plaque stability and IKK activation thus providing a mechanistic explanation for the prominence of HDAC9 as a vascular risk locus in genome-wide association studies. Its therapeutic inhibition may provide a potent lever to alleviate vascular inflammation. Graphical Abstract: A graphical abstract is available for this article.



Circ Res: 27 Aug 2020; 127:811-823

Epigenomes of Human Hearts Reveal New Genetic Variants Relevant for Cardiac Disease and Phenotype.

Tan WLW, Anene-Nzelu CG, Wong E, Lee CJM, ... Foo RSY,
Rationale
Identifying genetic markers for heterogeneous complex diseases such as heart failure is challenging and requires prohibitively large cohort sizes in genome-wide association studies to meet the stringent threshold of genome-wide statistical significance. On the other hand, chromatin quantitative trait loci, elucidated by direct epigenetic profiling of specific human tissues, may contribute toward prioritizing subthreshold variants for disease association.
Objective
Here, we captured noncoding genetic variants by performing epigenetic profiling for enhancer H3K27ac chromatin immunoprecipitation followed by sequencing in 70 human control and end-stage failing hearts.
Methods and results
We have mapped a comprehensive catalog of 47 321 putative human heart enhancers and promoters. Three thousand eight hundred ninety-seven differential acetylation peaks (FDR [false discovery rate], 5%) pointed to pathways altered in heart failure. To identify cardiac histone acetylation quantitative trait loci (haQTLs), we regressed out confounding factors including heart failure disease status and used the G-SCI (Genotype-independent Signal Correlation and Imbalance) test to call out 1680 haQTLs (FDR, 10%). RNA sequencing performed on the same heart samples proved a subset of haQTLs to have significant association also to gene expression (expression quantitative trait loci), either in(180) or through long-range interactions (81), identified by Hi-C (high-throughput chromatin conformation assay) and HiChIP (high-throughput protein centric chromatin) performed on a subset of hearts. Furthermore, a concordant relationship between the gain or disruption of TF (transcription factor)-binding motifs, inferred from alternative alleles at the haQTLs, implied a surprising direct association between these specific TF and local histone acetylation in human hearts. Finally, 62 unique loci were identified by colocalization of haQTLs with the subthreshold loci of heart-related genome-wide association studies datasets.
Conclusions
Disease and phenotype association for 62 unique loci are now implicated. These loci may indeed mediate their effect through modification of enhancer H3K27 acetylation enrichment and their corresponding gene expression differences (bioRxiv: https://doi.org/10.1101/536763). Graphical Abstract: A graphical abstract is available for this article.



Circ Res: 27 Aug 2020; 127:761-777

B55α/PP2A Limits Endothelial Cell Apoptosis During Vascular Remodeling: A Complementary Approach To Disrupt Pathological Vessels?

Ehling M, Celus W, Martín-Pérez R, Alba-Rovira R, ... Di Conza G, Mazzone M
Rationale
How endothelial cells (ECs) migrate and form an immature vascular plexus has been extensively studied. Yet, mechanisms underlying vascular remodeling remain poorly established. A better understanding of these processes may lead to the design of novel therapeutic strategies complementary to current angiogenesis inhibitors.
Objective
Starting from our previous observations that PP2A (protein phosphatase 2) regulates the HIF (hypoxia-inducible factor)/PHD-2 (prolyl hydroxylase 2)-constituted oxygen machinery, we hypothesized that this axis could play an important role during blood vessel formation, tissue perfusion, and oxygen restoration.
Methods and results
We show that the PP2A regulatory subunit B55α is at the crossroad between vessel pruning and vessel maturation. Blood vessels with high B55α counter cell stress conditions and thrive for stabilization and maturation. When B55α is inhibited, ECs cannot cope with cell stress and undergo apoptosis, leading to massive pruning of nascent blood vessels. Mechanistically, we found that the B55α/PP2A complex restrains PHD-2 activity, promoting EC survival in a HIF-dependent manner, and furthermore dephosphorylates p38, altogether protecting ECs against cell stress occurring, for example, during the onset of blood flow. In tumors, EC-specific B55α deficiency induces pruning of immature-like tumor blood vessels resulting in delayed tumor growth and metastasis, without affecting nonpathological vessels. Consistently, systemic administration of a pan-PP2A inhibitor disrupts vascular network formation and tumor progression in vivo without additional effects on B55α-deficient vessels.
Conclusions
Our data underline a unique role of the B55α/PP2A phosphatase complex in vessel remodeling and suggest the use of PP2A-inhibitors as potent antiangiogenic drugs targeting specifically nascent blood vessels with a mode-of-action complementary to VEGF-R (vascular endothelial growth factor receptor)-targeted therapies. Graphical Abstract: A graphical abstract is available for this article.



Circ Res: 27 Aug 2020; 127:707-723

Large Extracellular Vesicle-Associated Rap1 Accumulates in Atherosclerotic Plaques, Correlates With Vascular Risks and Is Involved in Atherosclerosis.

Perdomo L, Vidal-Gómez X, Soleti R, Vergori L, ... Martínez MC,
Rationale
Metabolic syndrome (MetS) is a cluster of interrelated risk factors for cardiovascular diseases and atherosclerosis. Circulating levels of large extracellular vesicles (lEVs), submicrometer-sized vesicles released from plasma membrane, from MetS patients were shown to induce endothelial dysfunction, but their role in early stage of atherosclerosis and on vascular smooth muscle cells (SMC) remain to be fully elucidated.
Objective
To determine the mechanisms by which lEVs lead to the progression of atherosclerosis in the setting of MetS.
Methods and results
Proteomic analysis revealed that the small GTPase, Rap1 was overexpressed in lEVs from MetS patients compared with those from non-MetS subjects. Rap1 was in GTP-associated active state in both types of lEVs, and Rap1-lEVs levels correlated with increased cardiovascular risks, including stenosis. MetS-lEVs, but not non-MetS-lEVs, increased Rap1-dependent endothelial cell permeability. MetS-lEVs significantly promoted migration and proliferation of human aortic SMC and increased expression of proinflammatory molecules and activation of ERK (extracellular signal-regulated kinase) 5/p38 pathways. Neutralization of Rap1 by specific antibody or pharmacological inhibition of Rap1 completely prevented the effects of lEVs from MetS patients. High-fat diet-fed ApoE mice displayed an increased expression of Rap1 both in aortas and circulating lEVs. lEVs accumulated in plaque atherosclerotic lesions depending on the progression of atherosclerosis. lEVs from high-fat diet-fed ApoE mice, but not those from mice fed with a standard diet, enhanced SMC proliferation. Human atherosclerotic lesions were enriched in lEVs expressing Rap1.
Conclusions
These data demonstrate that Rap1 carried by MetS-lEVs participates in the enhanced SMC proliferation, migration, proinflammatory profile, and activation of ERK5/p38 pathways leading to vascular inflammation and remodeling, and atherosclerosis. These results highlight that Rap1 carried by MetS-lEVs may be a novel determinant of diagnostic value for cardiometabolic risk factors and suggest Rap1 as a promising therapeutic target against the development of atherosclerosis. Graphical Abstract: A graphical abstract is available for this article.



Circ Res: 27 Aug 2020; 127:747-760

Somatic Gain of KRAS Function in the Endothelium Is Sufficient to Cause Vascular Malformations That Require MEK but Not PI3K Signaling.

Fish JE, Flores Suarez CP, Boudreau E, Herman AM, ... Radovanovic I, Wythe JD
Rationale
We previously identified somatic activating mutations in the() gene in the endothelium of the majority of human sporadic brain arteriovenous malformations; a disorder characterized by direct connections between arteries and veins. However, whether this genetic abnormality alone is sufficient for lesion formation, as well as how active KRAS signaling contributes to arteriovenous malformations, remains unknown.
Objective
To establish the first in vivo models of somatic KRAS gain of function in the endothelium in both mice and zebrafish to directly observe the phenotypic consequences of constitutive KRAS activity at a cellular level in vivo, and to test potential therapeutic interventions for arteriovenous malformations.
Methods and results
Using both postnatal and adult mice, as well as embryonic zebrafish, we demonstrate that endothelial-specific gain of function mutations in(G12D or G12V) are sufficient to induce brain arteriovenous malformations. Active KRAS signaling leads to altered endothelial cell morphogenesis and increased cell size, ectopic sprouting, expanded vessel lumen diameter, and direct connections between arteries and veins. Furthermore, we show that these lesions are not associated with altered endothelial growth dynamics or a lack of proper arteriovenous identity but instead seem to feature exuberant angiogenic signaling. Finally, we demonstrate that KRAS-dependent arteriovenous malformations in zebrafish are refractory to inhibition of the downstream effector PI3K but instead require active MEK (mitogen-activated protein kinase kinase 1) signaling.
Conclusions
We demonstrate that active KRAS expression in the endothelium is sufficient for brain arteriovenous malformations, even in the setting of uninjured adult vasculature. Furthermore, the finding that KRAS-dependent lesions are reversible in zebrafish suggests that MEK inhibition may represent a promising therapeutic treatment for arteriovenous malformation patients. Graphical Abstract: A graphical abstract is available for this article.



Circ Res: 27 Aug 2020; 127:727-743

Gut Microbiota and Cardiovascular Disease.

Witkowski M, Weeks TL, Hazen SL

Fecal microbial community changes are associated with numerous disease states, including cardiovascular disease (CVD). However, such data are merely associative. A causal contribution for gut microbiota in CVD has been further supported by a multitude of more direct experimental evidence. Indeed, gut microbiota transplantation studies, specific gut microbiota-dependent pathways, and downstream metabolites have all been shown to influence host metabolism and CVD, sometimes through specific identified host receptors. Multiple metaorganismal pathways (involving both microbe and host) both impact CVD in animal models and show striking clinical associations in human studies. For example, trimethylamine N-oxide and, more recently, phenylacetylglutamine are gut microbiota-dependent metabolites whose blood levels are associated with incident CVD risks in large-scale clinical studies. Importantly, a causal link to CVD for these and other specific gut microbial metabolites/pathways has been shown through numerous mechanistic animal model studies. Phenylacetylglutamine, for example, was recently shown to promote adverse cardiovascular phenotypes in the host via interaction with multiple ARs (adrenergic receptors)-a class of key receptors that regulate cardiovascular homeostasis. In this review, we summarize recent advances of microbiome research in CVD and related cardiometabolic phenotypes that have helped to move the field forward from associative to causative results. We focus on microbiota and metaorganismal compounds/pathways, with specific attention paid to short-chain fatty acids, secondary bile acids, trimethylamine N-oxide, and phenylacetylglutamine. We also discuss novel therapeutic strategies for directly targeting the gut microbiome to improve cardiovascular outcomes.



Circ Res: 30 Jul 2020; 127:553-570

Transient Intermittent Hyperglycemia Accelerates Atherosclerosis by Promoting Myelopoiesis.

Flynn MC, Kraakman MJ, Tikellis C, Lee MKS, ... Thomas MC, Murphy AJ
Rationale
Treatment efficacy for diabetes mellitus is largely determined by assessment of HbA1c (glycated hemoglobin A1c) levels, which poorly reflects direct glucose variation. People with prediabetes and diabetes mellitus spend >50% of their time outside the optimal glucose range. These glucose variations, termed transient intermittent hyperglycemia (TIH), appear to be an independent risk factor for cardiovascular disease, but the pathological basis for this association is unclear.
Objective
To determine whether TIH per se promotes myelopoiesis to produce more monocytes and consequently adversely affects atherosclerosis.
Methods and results
To create a mouse model of TIH, we administered 4 bolus doses of glucose at 2-hour intervals intraperitoneally once to WT (wild type) or once weekly to atherosclerotic prone mice. TIH accelerated atherogenesis without an increase in plasma cholesterol, seen in traditional models of diabetes mellitus. TIH promoted myelopoiesis in the bone marrow, resulting in increased circulating monocytes, particularly the inflammatory Ly6-C subset, and neutrophils. Hematopoietic-restricted deletion of , , or its cognate receptorprevented monocytosis. Mechanistically, glucose uptake via GLUT (glucose transporter)-1 and enhanced glycolysis in neutrophils promoted the production of S100A8/A9. Myeloid-restricted deletion of(GLUT-1) or pharmacological inhibition of S100A8/A9 reduced TIH-induced myelopoiesis and atherosclerosis.
Conclusions
Together, these data provide a mechanism as to how TIH, prevalent in people with impaired glucose metabolism, contributes to cardiovascular disease. These findings provide a rationale for continual glucose control in these patients and may also suggest that strategies aimed at targeting the S100A8/A9-RAGE (receptor for advanced glycation end products) axis could represent a viable approach to protect the vulnerable blood vessels in diabetes mellitus. Graphic Abstract: A graphic abstract is available for this article.



Circ Res: 10 Sep 2020; 127:877-892

Reactive Oxygen-Forming Nox5 Links Vascular Smooth Muscle Cell Phenotypic Switching and Extracellular Vesicle-Mediated Vascular Calcification.

Furmanik M, Chatrou M, van Gorp R, Akbulut A, ... Reutelingsperger C, Schurgers L
Rationale
Vascular calcification, the formation of calcium phosphate crystals in the vessel wall, is mediated by vascular smooth muscle cells (VSMCs). However, the underlying molecular mechanisms remain elusive, precluding mechanism-based therapies.
Objective
Phenotypic switching denotes a loss of contractile proteins and an increase in migration and proliferation, whereby VSMCs are termed synthetic. We examined how VSMC phenotypic switching influences vascular calcification and the possible role of the uniquely calcium-dependent reactive oxygen species (ROS)-forming Nox5 (NADPH oxidase 5).
Methods and results
In vitro cultures of synthetic VSMCs showed decreased expression of contractile markers CNN-1 (calponin 1), α-SMA (α-smooth muscle actin), and SM22-α (smooth muscle protein 22α) and an increase in synthetic marker S100A4 (S100 calcium binding protein A4) compared with contractile VSMCs. This was associated with increased calcification of synthetic cells in response to high extracellular Ca. Phenotypic switching was accompanied by increased levels of ROS and Ca-dependent Nox5 in synthetic VSMCs. Nox5 itself regulated VSMC phenotype as siRNA knockdown of Nox5 increased contractile marker expression and decreased calcification, while overexpression of Nox5 decreased contractile marker expression. ROS production in synthetic VSMCs was cytosolic Ca-dependent, in line with it being mediated by Nox5. Treatment of VSMCs with Ca loaded extracellular vesicles (EVs) lead to an increase in cytosolic Ca. Inhibiting EV endocytosis with dynasore blocked the increase in cytosolic Ca and VSMC calcification. Increased ROS production resulted in increased EV release and decreased phagocytosis by VSMCs.
Conclusions
We show here that contractile VSMCs are resistant to calcification and identify Nox5 as a key regulator of VSMC phenotypic switching. Additionally, we describe a new mechanism of Ca uptake via EVs and show that Ca induces ROS production in VSMCs via Nox5. ROS production is required for release of EVs, which promote calcification. Identifying molecular pathways that control Nox5 and VSMC-derived EVs provides potential targets to modulate vascular remodeling and calcification in the context of mineral imbalance. Graphic Abstract: A graphic abstract is available for this article.



Circ Res: 10 Sep 2020; 127:911-927

NAADP/SERCA3-Dependent Ca Stores Pathway Specifically Controls Early Autocrine ADP Secretion Potentiating Platelet Activation.

Feng M, Elaïb Z, Borgel D, Denis CV, ... Rosa JP, Bobe R
Rationale
Ca signaling is a key and ubiquitous actor of cell organization and its modulation controls many cellular responses. SERCAs (sarco-endoplasmic reticulum Ca-ATPases) pump Ca into internal stores that play a major role in the cytosolic Ca concentration rise upon cell activation. Platelets exhibit 2 types of SERCAs, SERCA2b and SERCA3 (SERCA3 deficient mice), which may exert specific roles, yet ill-defined. We have recently shown that Ca mobilization from SERCA3-dependent stores was required for full platelet activation in weak stimulation conditions.
Objective
To uncover the signaling mechanisms associated with Ca mobilization from SERCA3-dependent stores leading to ADP secretion.
Methods and results
Using platelets from wild-type or -deficient mice, we demonstrated that an early (within 5-10 s following stimulation) secretion of ADP specifically dependent on SERCA3 stored Ca is exclusively mobilized by nicotinic acid adenosine dinucleotide-phosphate (NAADP): both Ca mobilization from SERCA3-dependent stores and primary ADP secretion are blocked by the NAADP receptor antagonist Ned-19, and reciprocally both are stimulated by permeant NAADP. In contrast, Ca mobilization from SERCA3-dependent stores and primary ADP secretion were unaffected by inhibition of the production of IP3 (inositol-1,4,5-trisphosphate) by phospholipase-C and accordingly were not stimulated by permeant IP3.
Conclusions
Upon activation, an NAADP/SERCA3 Ca mobilization pathway initiates an early ADP secretion, potentiating platelet activation, and a secondary wave of ADP secretion driven by both an IP3/SERCA2b-dependent Ca stores pathway and the NAADP/SERCA3 pathway. This does not exclude that Ca mobilized from SERCA3 stores may also enhance platelet global reactivity to agonists. Because of its modulating effect on platelet activation, this NAADP-SERCA3 pathway may be a relevant target for anti-thrombotic therapy. Graphic Abstract: A graphic abstract is available for this article.



Circ Res: 10 Sep 2020; 127:e166-e183

Reduced Platelet miR-223 Induction in Kawasaki Disease Leads to Severe Coronary Artery Pathology Through a miR-223/PDGFRβ Vascular Smooth Muscle Cell Axis.

Zhang Y, Wang Y, Zhang L, Xia L, ... Hwa J, Tang WH
Rationale
Kawasaki disease (KD) is an acute vasculitis of early childhood that can result in permanent coronary artery structural damage. The cause for this arterial vulnerability in up to 15% of patients with KD is unknown. Vascular smooth muscle cell dedifferentiation play a key role in the pathophysiology of medial damage and aneurysm formation, recognized arterial pathology in KD. Platelet hyperreactivity is also a hallmark of KD. We recently demonstrated that uptake of platelets and platelet-derived miRNAs influences vascular smooth muscle cell phenotype in vivo.
Objective
We set out to explore whether platelet/vascular smooth muscle cell (VSMC) interactions contribute to coronary pathology in KD.
Methods and results
We prospectively recruited and studied 242 patients with KD, 75 of whom had documented coronary artery pathology. Genome-wide miRNA sequencing and droplet digital PCR demonstrated that patient with KD platelets have significant induction of miR-223 compared with healthy controls (HCs). Platelet-derived miR-223 has recently been shown to promote vascular smooth muscle quiescence and resolution of wound healing after vessel injury. Paradoxically, patients with KD with the most severe coronary pathology (giant coronary artery aneurysms) exhibited a lack of miR-223 induction. Hyperactive platelets isolated from patients with KD are readily taken up by VSMCs, delivering functional miR-223 into the VSMCs promoting VSMC differentiation via downregulation of PDGFRβ (platelet-derived growth factor receptor β). The lack of miR-223 induction in patients with severe coronary pathology leads to persistent VSMC dedifferentiation. In a mouse model of KD ( cell wall extract injection), miR-223 knockout mice exhibited increased medial thickening, loss of contractile VSMCs in the media, and fragmentation of medial elastic fibers compared with WT mice, which demonstrated significant miR-223 induction uponcell wall extract challenge. The excessive arterial damage in the miR-223 knockout could be rescued by adoptive transfer of platelet, administration of miR-223 mimics, or the PDGFRβ inhibitor imatinib mesylate. Interestingly, miR-223 levels progressively increase with age, with the lowest levels found in <5-year-old. This provides a basis for coronary pathology susceptibility in this very young cohort.
Conclusions
Platelet-derived miR-223 (through PDGFRβ inhibition) promotes VSMC differentiation and resolution of KD induced vascular injury. Lack of miR-223 induction leads to severe coronary pathology characterized by VSMC dedifferentiation and medial damage. Detection of platelet-derived miR-223 in patients with KD (at the time of diagnosis) may identify patients at greatest risk of coronary artery pathology. Moreover, targeting platelet miR-223 or VSMC PDGFRβ represents potential therapeutic strategies to alleviate coronary pathology in KD. Graphic Abstract: A graphic abstract is available for this article.



Circ Res: 10 Sep 2020; 127:855-873

Glutamyl-Prolyl-tRNA Synthetase Regulates Proline-Rich Pro-Fibrotic Protein Synthesis During Cardiac Fibrosis.

Wu J, Subbaiah KCV, Xie LH, Jiang F, ... Tang WHW, Yao P
Rationale
Increased protein synthesis of profibrotic genes is a common feature in cardiac fibrosis and heart failure. Despite this observation, critical factors and molecular mechanisms for translational control of profibrotic genes during cardiac fibrosis remain unclear.
Objective
To investigate the role of a bifunctional ARS (aminoacyl-tRNA synthetase), EPRS (glutamyl-prolyl-tRNA synthetase) in translational control of cardiac fibrosis.
Methods and results
Results from reanalyses of multiple publicly available data sets of human and mouse heart failure, demonstrated that EPRS acted as an integrated node among the ARSs in various cardiac pathogenic processes. We confirmed that EPRS was induced at mRNA and protein levels (≈1.5-2.5-fold increase) in failing hearts compared with nonfailing hearts using our cohort of human and mouse heart samples. Genetic knockout of one allele ofglobally () using CRISPR-Cas9 technology or in a Postn-Cre-dependent manner (; ) strongly reduces cardiac fibrosis (≈50% reduction) in isoproterenol-, transverse aortic constriction-, and myocardial infarction (MI)-induced heart failure mouse models. Inhibition of EPRS using a PRS (prolyl-tRNA synthetase)-specific inhibitor, halofuginone, significantly decreases translation efficiency (TE) of proline-rich collagens in cardiac fibroblasts as well as TGF-β (transforming growth factor-β)-activated myofibroblasts. Overexpression of EPRS increases collagen protein expression in primary cardiac fibroblasts under TGF-β stimulation. Using transcriptome-wide RNA-Seq and polysome profiling-Seq in halofuginone-treated fibroblasts, we identified multiple novel Pro-rich genes in addition to collagens, such as Ltbp2 (latent TGF-β-binding protein 2) and Sulf1 (sulfatase 1), which are translationally regulated by EPRS. SULF1 is highly enriched in human and mouse myofibroblasts. In the primary cardiac fibroblast culture system, siRNA-mediated knockdown of SULF1 attenuates cardiac myofibroblast activation and collagen deposition. Overexpression of SULF1 promotes TGF-β-induced myofibroblast activation and partially antagonizes anti-fibrotic effects of halofuginone treatment.
Conclusions
Our results indicate that EPRS preferentially controls translational activation of proline codon rich profibrotic genes in cardiac fibroblasts and augments pathological cardiac remodeling. Graphical Abstract: A graphical abstract is available for this article.



Circ Res: 27 Aug 2020; 127:827-846

Hematoma Resolution In Vivo Is Directed by Activating Transcription Factor 1.

Seneviratne A, Han Y, Wong E, Walter ERH, ... Haskard DO, Boyle JJ
Rationale
The efficient resolution of tissue hemorrhage is an important homeostatic function. In human macrophages in vitro, heme activates an AMPK (AMP-activated protein kinase)/ATF1 (activating transcription factor-1) pathway that directs Mhem macrophages through coregulation of HO-1 (heme oxygenase-1; ) and lipid homeostasis genes.
Objective
We asked whether this pathway had an in vivo role in mice.
Methods and results
Perifemoral hematomas were used as a model of hematoma resolution. In mouse bone marrow-derived macrophages, heme induced HO-1, lipid regulatory genes including LXR (lipid X receptor), the growth factor IGF1 (insulin-like growth factor-1), and the splenic red pulp macrophage gene . This response was lost in bone marrow-derived macrophages from mice deficient in AMPK () or ATF1 (). In vivo, femoral hematomas resolved completely between days 8 and 9 in littermate control mice (n=12), but were still present at day 9 in mice deficient in either AMPK () or ATF1 (; n=6 each). Residual hematomas were accompanied by increased macrophage infiltration, inflammatory activation and oxidative stress. We also found that fluorescent lipids and a fluorescent iron-analog were trafficked to lipid-laden and iron-laden macrophages respectively. Moreover erythrocyte iron and lipid abnormally colocalized in the same macrophages inmice. Therefore, iron-lipid separation was -dependent.
Conclusions
Taken together, these data demonstrate that both AMPK and ATF1 are required for normal hematoma resolution. Graphic Abstract: An online graphic abstract is available for this article.



Circ Res: 10 Sep 2020; 127:928-944

Integrative Genomic Analysis Reveals Four Protein Biomarkers for Platelet Traits.

Lee DH, Yao C, Bhan A, Schlaeger T, ... Levy D, Johnson AD
Rationale
Mean platelet volume (MPV) and platelet count (PLT) are platelet measures that have been linked to cardiovascular disease (CVD) and mortality risk. Identifying protein biomarkers for these measures may yield insights into CVD mechanisms.
Objective
We aimed to identify causal protein biomarkers for MPV and PLT among 71 CVD-related plasma proteins measured in FHS (Framingham Heart Study) participants.
Methods and results
We conducted integrative analyses of genetic variants associated with PLT/MPV with protein quantitative trait locus variants associated with plasma proteins followed by Mendelian randomization to infer causal relations of proteins for PLT/MPV. We also tested protein-PLT/MPV association in FHS participants. Using induced pluripotent stem cell-derived megakaryocyte clones that produce functional platelets, we conducted RNA-sequencing and analyzed expression differences between low- and high-platelet producing clones. We then performed small interfering RNA gene knockdown experiments targeting genes encoding proteins with putatively causal platelet effects in megakaryocyte clones to examine effects on platelet production. In protein-trait association analyses, ten proteins were associated with MPV and 31 with PLT. Mendelian randomization identified 4 putatively causal proteins for MPV and 4 for PLT. GP-5 (Glycoprotein V), GRN (granulin), and MCAM (melanoma cell adhesion molecule) were associated with PLT, while MPO (myeloperoxidase) showed significant association with MPV in both analyses. RNA-sequencing analysis results were directionally concordant with observed and Mendelian randomization-inferred associations for GP-5, GRN, and MCAM. In siRNA gene knockdown experiments, silencing GP-5, GRN, and MPO decreased PLTs. Genome-wide association study results suggest several of these may be linked to CVD risk.
Conclusions
We identified 4 proteins that are causally linked to PLTs. These proteins may also have roles in the pathogenesis of CVD via a platelet/blood coagulation-based mechanism.



Circ Res: 08 Oct 2020; 127:1182-1194

Downregulation of LAPTM4B Contributes to the Impairment of the Autophagic Flux via Unopposed Activation of mTORC1 Signaling During Myocardial Ischemia/Reperfusion Injury.

Gu S, Tan J, Li Q, Liu S, ... Cao N, Yang HT
Rationale
Impaired autophagic flux contributes to ischemia/reperfusion (I/R)-induced cardiomyocyte death, but the underlying molecular mechanisms remain largely unexplored.
Objective
To determine the role of LAPTM4B (lysosomal-associated transmembrane protein 4B) in the regulation of autophagic flux and myocardial I/R injury.
Methods and results
LAPTM4B was expressed in murine hearts but downregulated in hearts with I/R (30 minutes/2 hours) injury and neonatal rat cardiomyocytes with hypoxia/reoxygenation (6 hours/2 hours) injury. During myocardial reperfusion, LAPTM4B-knockout (LAPTM4B) mice had a significantly increased infarct size and lactate dehydrogenase release, whereas adenovirus-mediated LAPTM4B-overexpression was cardioprotective. Concomitantly, LAPTM4B mice showed higher accumulation of the autophagy markers LC3-II (microtubule-associated protein 1A/1B-light chain 3), but not P62, in the I/R heart, whereas they did not alter chloroquine-induced further increases of LC3-II and P62 in both sham and I/R hearts. Conversely, LAPTM4B-overexpression had opposite effects. The hypoxia/reoxygenation-reduced viability of neonatal rat cardiomyocytes, ratio of autolysosomes/autophagosomes, and function of lysosomes were further decreased by LAPTM4B-knockdown but reversed by LAPTM4B-overexpression. Moreover, the LAPTM4B-overexpression-mediated benefits were abolished by knockdown of lysosome-associated membrane protein-2 (an autophagosome-lysosome fusion protein) in vivo and by the autophagy inhibitor bafilomycin A1 in vivo. In contrast, rapamycin (Rapa) successfully restored the impaired autophagic flux in LAPTM4B mice and the subsequent myocardial I/R injury. Mechanistically, LAPTM4B regulated the activity of mTORC1 (mammalian target of rapamycin complex 1) via interacting with mTOR through its EC3 (extracelluar) domain. Thus, mTORC1 was overactivated in LAPTM4B mice, leading to the repression of TFEB (transcription factor EB), a master regulator of lysosomal and autophagic genes, during myocardial I/R. The mTORC1 inhibition or TFEB-overexpression rescued the LAPTM4B-induced impairment in autophagic flux and I/R injury, whereas TFEB-knockdown abolished the LAPTM4B-overexpression-mediated recovery of autophagic flux and cardioprotection.
Conclusions
The downregulation of LAPTM4B contributes to myocardial I/R-induced impairment of autophagic flux via modulation of the mTORC1/TFEB pathway. Graphic Abstract: A graphic abstract is available for this article.



Circ Res: 10 Sep 2020; 127:e148-e165

No Evidence for Erythro-Myeloid Progenitor-Derived Vascular Endothelial Cells in Multiple Organs.

Feng T, Gao Z, Kou S, Huang X, ... Lin CP, Zhang H

Endothelial cells are thought to emerge de novo from the mesoderm to form the entire circulatory system. Recently, erythro-myeloid progenitors (EMPs) have been proposed to be another remarkable developmental origin for blood vessels in multiple organs, including the hindbrain, liver, lung and heart, as demonstrated by lineage tracing studies using different genetic tools. These observations challenge the current consensus that intraembryonic vessels are thought to expand solely by the proliferation of pre-existing endothelial cells. Resolution of this controversy over the developmental origin of endothelial cells is crucial for developing future therapeutics for vessel-dependent organ repair and regeneration.To examine the contribution of EMPs to intraembryonic endothelial cells.We first employed a transgenic mouse expressing a tamoxifen-inducible Mer-iCre fusion protein driven by the colony stimulating factor 1 receptor (Csf1r) promoter. Genetic lineage tracing based on Csf1r-Mer-iCre-Mer showed no contribution of EMPs to brain endothelial cells identified by several markers. We also generated a knock-in mouse line by inserting an internal ribosome entry site (ires)-iCre cassette into the 3\' untranslated region of Csf1r gene to further investigate the cellular fates of EMPs. Similarly, we did not find any Csf1r-ires-iCre traced endothelial cells in brain, liver, lung or heart in development either. Additionally, we found that KIT proto-oncogene receptor tyrosine kinase (Kit) was expressed not only in EMPs but also in embryonic hindbrain endothelial cells. Therefore, Kit promoter driven recombinase, such as Kit-CreER, is a flawed tool for lineage tracing when examining the contribution of EMPs to hindbrain endothelial cells. We also traced protein tyrosine phosphatase receptor type C (Ptprc/CD45)circulating EMPs and did not find any CD45 lineage-derived endothelial cells during development.Our study suggested that EMPs are not the origin of intraembryonic endothelial cells.



Circ Res: 13 Aug 2020; epub ahead of print

Diabetes Impairs Cellular Cholesterol Efflux From ABCA1 to Small HDL Particles.

He Y, Ronsein GE, Tang C, Jarvik GP, ... Bornfeldt KE, Heinecke JW
Rationale
HDL (high-density lipoprotein) may be cardioprotective because it accepts cholesterol from macrophages via the cholesterol transport proteins ABCA1 (ATP-binding cassette transporter A1) and ABCG1 (ATP-binding cassette transporter G1). The ABCA1-specific cellular cholesterol efflux capacity (ABCA1 CEC) of HDL strongly and negatively associates with cardiovascular disease risk, but how diabetes mellitus impacts that step is unclear.
Objective
To test the hypothesis that HDL\'s cholesterol efflux capacity is impaired in subjects with type 2 diabetes mellitus.
Methods and results
We performed a case-control study with 19 subjects with type 2 diabetes mellitus and 20 control subjects. Three sizes of HDL particles, small HDL, medium HDL, and large HDL, were isolated by high-resolution size exclusion chromatography from study subjects. Then we assessed the ABCA1 CEC of equimolar concentrations of particles. Small HDL accounted for almost all of ABCA1 CEC activity of HDL. ABCA1 CEC-but not ABCG1 CEC-of small HDL was lower in the subjects with type 2 diabetes mellitus than the control subjects. Isotope dilution tandem mass spectrometry demonstrated that the concentration of SERPINA1 (serpin family A member 1) in small HDL was also lower in subjects with diabetes mellitus. Enriching small HDL with SERPINA1 enhanced ABCA1 CEC. Structural analysis of SERPINA1 identified 3 amphipathic α-helices clustered in the N-terminal domain of the protein; biochemical analyses demonstrated that SERPINA1 binds phospholipid vesicles.
Conclusions
The ABCA1 CEC of small HDL is selectively impaired in type 2 diabetes mellitus, likely because of lower levels of SERPINA1. SERPINA1 contains a cluster of amphipathic α-helices that enable apolipoproteins to bind phospholipid and promote ABCA1 activity. Thus, impaired ABCA1 activity of small HDL particles deficient in SERPINA1 could increase cardiovascular disease risk in subjects with diabetes mellitus.



Circ Res: 08 Oct 2020; 127:1198-1210

Hyperglycemia Induces Myocardial Dysfunction via Epigenetic Regulation of JunD.

Hussain S, Khan AW, Akhmedov A, Suades R, ... Lüscher TF, Cosentino F

Hyperglycemia-induced reactive oxygen species (ROS) are key mediators of cardiac dysfunction. JunD, a member of the Activated Protein-1 (AP-1) family of transcription factors, is emerging as a major gatekeeper against oxidative stress. However, its contribution to redox state and inflammation in the diabetic heart remains to be elucidated.The present study investigates the role of JunD in hyperglycemia-induced and ROS-driven myocardial dysfunction.JunD mRNA and protein expression were reduced in the myocardium of mice with streptozotocin-induced diabetes as compared to controls. JunD downregulation was associated with oxidative stress and left ventricular dysfunction assessed by electron spin resonance spectroscopy as well as conventional and 2D speckle-tracking echocardiography. Furthermore, myocardial expression of free radical scavenger superoxide dismutase 1 and aldehyde dehydrogenase 2 was reduced, whereas the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits NOX2 and NOX4 were upregulated. The redox changes were associated with increased NF-kappaB binding activity and expression of inflammatory mediators. Interestingly, mice with cardiac-specific overexpression of JunD via the α-myosin heavy chain promoter (α-MHC-) were protected against hyperglycemia-induced cardiac dysfunction. We also showed that JunD was epigenetically regulated by promoter hypermethylation, posttranslational modification of histone marks and translational repression by miRNA-673/menin. Reduced JunD mRNA and protein expression were confirmed in left ventricular specimens obtained from patients with type 2 diabetes and heart failure as compared to non-diabetic subjects.Here we show that a complex epigenetic machinery involving DNA methylation, histone modifications and microRNAs mediates hyperglycemia-induced JunD downregulation and myocardial dysfunction in experimental and human diabetes. Our results pave the way for tissue-specific therapeutic modulation of JunD to prevent diabetic cardiomyopathy.



Circ Res: 19 Aug 2020; epub ahead of print

Gut Microbiota-Associated Activation of TLR5 Induces Apolipoprotein A1 Production in the Liver.

Yiu JHC, Chan KS, Cheung J, Li J, ... Xu A, Woo CWH

Dysbiosis of gut microbiota plays an important role in cardiovascular diseases but the molecular mechanisms are complex. An association between gut microbiome and the variance in HDL-cholesterol (HDL-C) level was suggested in a human study. Besides, dietary fat was shown to increase both HDL-C and LDL-C levels. We speculate that certain types of gut bacteria responding to dietary fat may help to regulate HDL-C level and potentially affect atherosclerotic development.We aimed to investigate whether and how high-fat diet (HFD)-associated gut microbiota regulated HDL level.We found that HFD increased gut flagellated bacteria population in mice. The increase in HDL-C levels was adopted by mice receiving fecal microbiome transplantion from HFD-fed mouse donors. HFD led to increased hepatic but not circulating flagellin, and deletion of TLR5, a receptor sensing flagellin, suppressed HFD-stimulated HDL-C and apolipoprotein A1 (ApoA1) levels. Overexpression of TLR5 in the liver of TLR5-knockout mice was able to partially restore the production of ApoA1 and HDL-C levels. Mechanistically, TLR5 activation by flagellin in primary hepatocytes stimulated ApoA1 production through the transcriptional activation responding to the binding of NFkappaB on Apoa1 promoter region. Furthermore, oral supplementation of flagellin was able to stimulate hepatic ApoA1 production and HDL-C level, and decrease atherosclerotic lesion size in apolipoprotein E-deficient (Apoe-/-) mice without triggering hepatic and systemic inflammation. The stimulation of ApoA1 production was also seen in human ApoA1 transgenic mice treated with oral flagellin.Our finding suggests that commensal flagellated bacteria in gut can facilitate ApoA1 and HDL productions in liver through activation of TLR5 in hepatocytes. Hepatic TLR5 may be a potential drug target to increase ApoA1.



Circ Res: 20 Aug 2020; epub ahead of print

High-Density Lipoprotein Carries Markers that Track with Recovery from Stroke.

Plubell DL, Fenton AM, Rosario S, Bergstrom P, ... Fazio S, Pamir N

Prospective cohort studies question the value of HDL-C for stroke risk prediction.Investigate the relationship between long-term functional recovery and HDL proteome and function.Changes in HDL protein composition and function (cholesterol efflux capacity, or CEC) in patients after acute ischemic stroke at two time points (24 h, 35 patients; 96 h, 20 patients) and in 35 control subjects were measured. The recovery from stroke was assessed by 3 month The National Institute of Health Stroke Scale (NIHSS) and Modified Rankin scale (mRS) scores. When compared to control subject after adjustments for sex and HDL-C levels, twelve proteins some of which participate in acute phase response and platelet activation (APMAP, GPLD1, APOE, IHH, ITIH4, SAA2, APOA4, CLU, ANTRX2, PON1, SERPINA1, and APOF) were significantly (adj. p<0.05) altered in stroke HDL at 96h. The first eight of these proteins were also significantly altered at 24h. Consistent with inflammatory remodeling, CEC was reduced by 32% (P<0.001) at both time points. Baseline stroke severity adjusted regression model showed that changes within 96 hours post stroke in APOF, APOL1, APMAP, APOC4, APOM, PCYOX1, PON1, and APOE correlate with stroke recovery scores (R2=0.38-0.73, adjusted p<0.05). APOF (R2=0.73), and APOL1 (R2=0.60) continued to significantly correlate with recovery scores after accounting for tPA treatment. Changes in HDL proteins during early acute phase of stroke associate with recovery. Monitoring HDL proteins may provide clinical biomarkers that inform on stroke recuperation .



Circ Res: 25 Aug 2020; epub ahead of print

Association of Habitual Physical Activity with Cardiovascular Disease Risk.

Lin H, Sardana M, Zhang Y, Liu C, ... Murabito JM, McManus DD

A sedentary lifestyle is associated with increased risk for cardiovascular disease (CVD). Smartwatches enable accurate daily activity monitoring for physical activity measurement and intervention. Few studies, however, have examined physical activity measures from smartwatches in relation to traditional risk factors associated with future risk for CVD.To investigate the association of habitual physical activity measured by smartwatch with predicted CVD risk in adults. We enrolled consenting Framingham Heart Study (FHS) participants in an ongoing electronic Framingham Heart Study (eFHS) at the time of their FHS research center examination. We provided participants with a smartwatch (Apple Watch Series 0) and instructed them to wear it daily, which measured their habitual physical activity as the average daily step count. We estimated the 10-year predicted risk of CVD using the ACC/AHA 2013 pooled cohort risk equation. We estimated the association between physical activity and predicted risk of CVD using linear mixed effects models adjusting for age, sex, wear time, and familial structure. Our study included 903 eFHS participants (mean age 53{plus minus}9 years, 61% women, 9% non-white) who wore the smartwatch greater than 5 hours per day for more than 30 days. Median daily step count was similar among men (7,202 with interquartile range [IQR] 3,619) and women (7,260 with IQR 3,068, P=0.52). Average 10-year predicted CVD risk was 4.5% (IQR 6.1%) for men and 1.2% (IQR 2.2%) for women (P=1.3x10-26). Every 1,000 steps higher habitual physical activity was associated with 0.18% lower predicted CVD risk (P=3.2x10-4). The association was attenuated but remained significant after further adjustment for body mass index (P=0.01).In this community-based sample of adults, higher daily physical activity measured by a study smartwatch was associated with lower predicted risk of CVD. Future research should examine the longitudinal association of prospectively measured daily activity and incident CVD.



Circ Res: 25 Aug 2020; epub ahead of print

Infarct Collagen Topography Regulates Fibroblast Fate Via p38-Yap-TEAD Signals.

Bugg D, Bretherton RC, Kim P, Olszewski E, ... Kim DH, Davis JM

Myocardial infarction (MI) causes spatial variation in collagen organization and phenotypic diversity in fibroblasts, which regulate the heart\'s extracellular matrix (ECM). The relationship between collagen structure and fibroblast phenotype is poorly understood but could provide insights regarding the mechanistic basis for myofibroblast heterogeneity in the injured heart.To investigate the role of collagen organization in cardiac fibroblast fate determination.Biomimetic topographies were nanofabricated to recapitulate differential collagen organization in the infarcted mouse heart. Here adult cardiac fibroblasts were freshly isolated and cultured on ECM topographical mimetics for 72 hours. Aligned mimetics caused cardiac fibroblasts to elongate while randomly organized topographies induced circular morphology similar to the disparate myofibroblast morphologies measured in vivo. Alignment cues also induced myofibroblast differentiation, as more than 60% of fibroblasts formed -smooth muscle actin (alphaSMA) stress fibers and expressed myofibroblast-specific ECM genes like periostin. By contrast, random organization caused 38% of cardiac fibroblasts to express alphaSMA albeit with down-regulated myofibroblast-specific ECM genes. Coupling topographical cues with the profibrotic agonist, TGFb, additively upregulated myofibroblast-specific ECM genes independent of topography, but only fibroblasts on flat and randomly oriented mimetics had increased percentages of fibroblasts with alphaSMA stress fibers. Increased tension sensation at focal adhesions induced myofibroblast differentiation on aligned mimetics. These signals were transduced by p38-YAP-TEAD interactions, in which both p38 and YAP-TEAD binding were required for myofibroblast differentiation. By contrast randomly oriented mimetics did not change focal adhesion tension sensation or enrich for p38-YAP-TEAD interactions, which explains the topography-dependent diversity in fibroblast phenotypes observed here.Spatial variations in collagen organization regulate cardiac fibroblast phenotype through mechanical activation of p38-YAP-TEAD signaling, which likely contribute to myofibroblast heterogeneity in the infarcted myocardium.



Circ Res: 03 Sep 2020; epub ahead of print

Shaping Waves of Bone Morphogenetic Protein Inhibition During Vascular Growth.

Guihard P, Guo Y, Wu X, Yao J, ... Bostrom KI, Zhang L

The bone morphogenetic proteins (BMPs) are essential morphogens in angiogenesis and vascular development. Disruption of BMP signaling can trigger cardiovascular disease such as arteriovenous malformations.A computational model predicted that BMP4 and BMP9 and their inhibitors matrix Gla protein (MGP) and Crossveinless-2 (CV2) would form a regulatory system consisting of negative feedback loops with time delays, and that BMP9 would trigger oscillatory expression of the two inhibitors. The goal was to investigate this regulatory system in endothelial differentiation and vascular growth.Oscillations in the expression of MGP and CV2 were detected in endothelial cells (ECs) in vitro, using qPCR and immunoblotting. These organized temporally downstream BMP-related activities including expression of stalk cell markers and cell proliferation, consistent with an integral role of BMP9 in vessel maturation. In vivo, the inhibitors were located in distinct zones in relation to the front of the expanding retinal network, as determined by immunofluorescence. Time-dependent changes of the CV2 location in the retina, and the existence of an endothelial population with signs of oscillatory MGP expression in developing vasculature supported the in vitro findings. Loss of MGP or its BMP4-binding capacity disrupted the retinal vasculature, resulting in poorly formed networks, especially in the venous drainage areas, and arteriovenous malformations as determined by increased cell coverage and functional testing.Our results suggest a previously unknown mechanism of temporal orchestration of BMP4 and BMP9 activities that utilizes the tandem actions of the extracellular antagonists MGP and CV2. Disruption of this mechanism may contribute to vascular malformations and disease.



Circ Res: 27 Aug 2020; epub ahead of print

Niacin Attenuates Pulmonary Hypertension Through H-PGDS in Macrophages.

Jia D, Bai P, Wan N, Liu J, ... Zhang J, Shen Y

Pulmonary arterial hypertension (PAH) is characterized by progressive pulmonary vascular remodeling, accompanied by varying degrees of perivascular inflammation. Niacin, a commonly used lipid-lowering drug, possesses vasodilating and pro-resolution effects by promoting the release of prostaglandin D2 (PGD2). However, whether or not niacin confers protection against PAH pathogenesis is still unknown.This study aimed to determine whether or not niacin attenuates the development of PAH and, if so, to elucidate the molecular mechanisms underlying its effects.Vascular endothelial growth factor receptor inhibitor SU5416 and hypoxic exposure were used to induce pulmonary hypertension (PH) in rodents. We found that niacin attenuated the development of this hypoxia/SU5416 (HySu) -induced PH in mice and suppressed progression of monocrotaline- and HySu -induced PH in rats through the reduction of pulmonary artery remodeling. Niacin boosted PGD2 generation in lung tissue, mainly through hematopoietic PGD2 synthases (H-PGDS). Deletion of H-PGDS, but not lipocalin-type PGDS, exacerbated the HySu -induced PH in mice and abolished the protective effects of niacin against PAH. Moreover, H-PGDS was expressed dominantly in infiltrated macrophages in lungs of PH mice and idiopathic PAH patients. Macrophage-specific deletion of H-PGDS markedly decreased PGD2 generation in lungs, aggravated HySu -induced PH in mice and attenuated the therapeutic effect of niacin on PAH.Niacin treatment ameliorates the progression of PAH through the suppression of vascular remodeling by stimulating H-PGDS-derived PGD2 release from macrophages.



Circ Res: 10 Sep 2020; epub ahead of print

ILRUN, a Human Plasma Lipid GWAS Locus, Regulates Lipoprotein Metabolism in Mice.

Bi X, Kuwano T, Lee PC, Millar JS, ... Hand NJ, Rader DJ

Single nucleotide polymorphisms (SNPs) near the ILRUN gene are genome-wide significantly associated with plasma lipid traits and coronary artery disease (CAD) but the biological basis of this association is unknown.To investigate the role of ILRUN in plasma lipid and lipoprotein metabolism.ILRUN encodes a protein that contains a ubiquitin-associated (UBA)-like domain, suggesting that it may interact with ubiquitinylated proteins. We generated mice globally deficient for Ilrun (IlrunKO) and found they had significantly lower plasma cholesterol levels resulting from reduced liver lipoprotein production. Liver transcriptome analysis uncovered altered transcription of genes downstream of lipid-related transcription factors, particularly PPARα, and livers from IlrunKO mice had increased PPARα protein. Human ILRUN was shown to bind to ubiquitinylated proteins including PPARα, and the UBA-like domain of ILRUN was found to be required for its interaction with PPARα.These findings establish ILRUN as a novel regulator of lipid metabolism that promotes hepatic lipoprotein production. Our results also provide functional evidence that ILRUN may be the casual gene underlying the observed genetic associations with plasma lipids at 6p21 in human.



Circ Res: 10 Sep 2020; epub ahead of print

Exercise Regulates microRNAs to Preserve Coronary and Cardiac Function in the Diabetic Heart.

Lew JK, Pearson JT, Saw E, Tsuchimochi H, ... Katare R, Schwenke DO

Diabetic heart disease (DHD) is a debilitating manifestation of type 2 diabetes. Exercise has been proposed as a potential therapy for DHD, although the effectiveness of exercise in preventing or reversing the progression of DHD remains controversial. Cardiac function is critically dependent on the preservation of coronary vascular function.We aimed to elucidate the effectiveness and mechanisms by which exercise facilitates coronary and cardiac-protection during the onset and progression of DHD.Diabeticand non-diabetic mice, with or without underlying cardiac dysfunction (16 and 8 weeks old, respectively) were subjected to either moderate-intensity exercise (MIE) or high-IE (HIE) for eight weeks. Subsequently, synchrotron microangiography, immunohistochemistry, Western blot, and RT-PCR were used to assess time-dependent changes in cardiac and coronary structure and function associated with diabetes and exercise, and determine whether these changes reflect the observed changes in cardiac-enriched and vascular-enriched microRNAs (miRNAs). We show that, if exercise is initiated from 8 weeks of age, both MIE and HIE prevented the onset of coronary and cardiac dysfunction, apoptosis, fibrosis, microvascular rarefaction, and disruption of miRNA signaling, as seen in the non-exercised diabetic mice. Conversely, the cardiovascular benefits of MIE were absent if the exercise was initiated after the diabetic mice had already established cardiac dysfunction (i.e. from 16 weeks of age). The experimental silencing or upregulation of miRNA-126 activity suggests the mechanism underpinning the cardiovascular benefits of exercise were mediated, at least in part, through tissue-specific miRNAs.Our findings provide the first experimental evidence for the critical importance of early exercise intervention in ameliorating the onset and progression of DHD. Our results also suggest that the beneficial effects of exercise are mediated through the normalization of cardiovascular-enriched miRNAs, which are dysregulated in DHD.



Circ Res: 09 Sep 2020; epub ahead of print

Abstract 466: Vascular Claudin-11 Plays a Protective Role for Human Atherosclerosis Progression.

Kim EH, Jung MH, Park MS, Choi KH, Lee SJ, Kim HS

The expression and the role of tight junction protein, Claudin-11 (CLDN11) in vasculat smooth muscle cell (vSMC) is unknown.To understand the role of CLDN11 in the vSMC, we transfected siRNA-CLDN11 into human coronary vascular smooth muscle cell line (hCSMC). cDNA microarray using Agilent Human mRNA arrays, immunocytochemistry, tube formation assay, FACS, and cell cycle analysis were performed 7 days after CLDN11 knock-down. To know the role of CLDN11 in the human cardiovascular system, we obtained vascular smooth muscle layer from autopsied left anterior descending artery and CLDN11 mRNA expression was evaluated following modified AHA Consensus Classification Based on Morphologic Descriptions (three groups, total n=45).CLDN11 was well expressed in vascular smooth mucscle layer in immunohistochemisty and western blot analysis. We observed the angiogenesis (CXCL8, SOX17, HEY1), cell proliferation (EGR3, ITGB2), and extracellular matrix (BMPER, WNT1) associated gene expression. Following CLDN11-siRNA transfection, the tube formation assay and proliferating cellular phase was markedly increased in siRNA treatment group (p<0.01, respectively). In the human sample, CLDN11 expression was inversely correlated with the progression of coronary atherosclerosis (p=0.0026), and the sudden cardiac death with complicated coronary atherosclerosis (p<0.05).These results indicate that CLDN11 could tightly regulate the vascular smooth muscle physiology. And CLDN11 might play a certain role for atherosclerosis propagation to regulate the vSMCs plasticity and possible new etiology for cardiovascular events.



Circ Res: 30 Jul 2020; 127:A466

tPA Mobilizes Immune Cells that Exacerbate Hemorrhagic Transformation in Stroke.

Shi K, Zou M, Jia DM, Shi S, ... Wang X, Shi FD

Hemorrhagic complications represent a major limitation of intravenous thrombolysis using tissue plasminogen activator (tPA) in patients with ischemic stroke. The expression of tPA receptors on immune cells raises the question of what effects tPA exerts on these cells and whether these effects contribute to thrombolysis-related hemorrhagic transformation.We aim to determine the impact of tPA on immune cells and investigate the association between observed immune alteration with hemorrhagic transformation in ischemic stroke patients and in a rat model of embolic stroke.Paired blood samples were collected before and 1 hour after tPA infusion from 71 ischemic stroke patients. Control blood samples were collected from 27 ischemic stroke patients without tPA treatment. A rat embolic middle cerebral artery occlusion model was adopted to investigate the underlying mechanisms of hemorrhagic transformation. We report that tPA induces a swift surge of circulating neutrophils and T cells with profoundly altered molecular features in ischemic stroke patients and a rat model of focal embolic stroke. tPA exacerbates endothelial injury, increases adhesion and migration of neutrophils and T cells, which are associated with brain hemorrhage in rats subjected to embolic stroke. Genetic ablation of annexin A2 in neutrophils and T cells diminishes the effect of tPA on these cells. Decoupling the interaction between mobilized neutrophils/T cells and the neurovascular unit, achieved via a sphingosine-1-phosphate receptor 1 modulator RP101075 and a CCL2 synthesis inhibitor bindarit, which block lymphocyte egress and myeloid cell recruitment, respectively, attenuates hemorrhagic transformation and improves neurological function after tPA thrombolysis.Our findings suggest that immune invasion of the neurovascular unit represents a previously unrecognized mechanism underlying tPA-mediated brain hemorrhage, which can be overcome by precise immune modulation during thrombolytic therapy.



Circ Res: 18 Oct 2020; epub ahead of print

Adrenergic Ca1.2 Activation via Rad Phosphorylation Converges at α I-II Loop.

Papa A, Kushner JS, Hennessey JA, Katchman AN, ... Johny MB, Marx SO

Changing activity of cardiac Ca1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently up-regulated via activation of protein kinase A, essential molecular details remained stubbornly enigmatic.The primary goal of this study was to determine how various factors converging at the Ca1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure.We generated transgenic mice with expression of Ca1.2 α subunits with: 1) mutations ablating interaction between α and β subunits; 2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α); or 3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α up-regulated in the hypertrophied heart. Introducing three glycine residues that disrupt a rigid IS6-AID helix markedly reduced basal open probability despite intact binding of Caβ to α I-II loop, and eliminated β-adrenergic agonist stimulation of Ca1.2 current. In contrast, introduction of the exon 9* splice variant in α I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β and Rad or transgenically expressed in cardiomyocytes.Ca channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α I-II loop. Caβ binding to α stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β binding to α.



Circ Res: 21 Oct 2020; epub ahead of print