Topic: Basic Research

Abstract

Propionate attenuates atherosclerosis by immune-dependent regulation of intestinal cholesterol metabolism.

Haghikia A, Zimmermann F, Schumann P, Jasina A, ... Haghikia A, Landmesser U
Aims
Atherosclerotic cardiovascular disease (ACVD) is a major cause of mortality and morbidity worldwide, and increased low-density lipoproteins (LDLs) play a critical role in development and progression of atherosclerosis. Here, we examined for the first time gut immunomodulatory effects of the microbiota-derived metabolite propionic acid (PA) on intestinal cholesterol metabolism.
Methods and results
Using both human and animal model studies, we demonstrate that treatment with PA reduces blood total and LDL cholesterol levels. In apolipoprotein E-/- (Apoe-/-) mice fed a high-fat diet (HFD), PA reduced intestinal cholesterol absorption and aortic atherosclerotic lesion area. Further, PA increased regulatory T-cell numbers and interleukin (IL)-10 levels in the intestinal microenvironment, which in turn suppressed the expression of Niemann-Pick C1-like 1 (Npc1l1), a major intestinal cholesterol transporter. Blockade of IL-10 receptor signalling attenuated the PA-related reduction in total and LDL cholesterol and augmented atherosclerotic lesion severity in the HFD-fed Apoe-/- mice. To translate these preclinical findings to humans, we conducted a randomized, double-blinded, placebo-controlled human study (clinical trial no. NCT03590496). Oral supplementation with 500 mg of PA twice daily over the course of 8 weeks significantly reduced LDL [-15.9 mg/dL (-8.1%) vs. -1.6 mg/dL (-0.5%), P = 0.016], total [-19.6 mg/dL (-7.3%) vs. -5.3 mg/dL (-1.7%), P = 0.014] and non-high-density lipoprotein cholesterol levels [PA vs. placebo: -18.9 mg/dL (-9.1%) vs. -0.6 mg/dL (-0.5%), P = 0.002] in subjects with elevated baseline LDL cholesterol levels.
Conclusion
Our findings reveal a novel immune-mediated pathway linking the gut microbiota-derived metabolite PA with intestinal Npc1l1 expression and cholesterol homeostasis. The results highlight the gut immune system as a potential therapeutic target to control dyslipidaemia that may introduce a new avenue for prevention of ACVDs.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Eur Heart J: 30 Sep 2021; epub ahead of print
Haghikia A, Zimmermann F, Schumann P, Jasina A, ... Haghikia A, Landmesser U
Eur Heart J: 30 Sep 2021; epub ahead of print | PMID: 34597388
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Abstract

Myeloid-Derived Growth Factor Protects Against Pressure Overload-Induced Heart Failure by Preserving Sarco/Endoplasmic Reticulum Ca-ATPase Expression in Cardiomyocytes.

Korf-Klingebiel M, Reboll MR, Polten F, Weber N, ... Wang Y, Wollert KC
Background
Inflammation contributes to the pathogenesis of heart failure, but there is limited understanding of inflammation\'s potential benefits. Inflammatory cells secrete MYDGF (myeloid-derived growth factor) to promote tissue repair after acute myocardial infarction. We hypothesized that MYDGF has a role in cardiac adaptation to persistent pressure overload.
Methods
We defined the cellular sources and function of MYDGF in wild-type (WT), Mydgf-deficient (Mydgf-/-), and Mydgf bone marrow-chimeric or bone marrow-conditional transgenic mice with pressure overload-induced heart failure after transverse aortic constriction surgery. We measured MYDGF plasma concentrations by targeted liquid chromatography-mass spectrometry. We identified MYDGF signaling targets by phosphoproteomics and substrate-based kinase activity inference. We recorded Ca2+ transients and sarcomere contractions in isolated cardiomyocytes. Additionally, we explored the therapeutic potential of recombinant MYDGF.
Results
MYDGF protein abundance increased in the left ventricular myocardium and in blood plasma of pressure-overloaded mice. Patients with severe aortic stenosis also had elevated MYDGF plasma concentrations, which declined after transcatheter aortic valve implantation. Monocytes and macrophages emerged as the main MYDGF sources in the pressure-overloaded murine heart. While Mydgf-/- mice had no apparent phenotype at baseline, they developed more severe left ventricular hypertrophy and contractile dysfunction during pressure overload than WT mice. Conversely, conditional transgenic overexpression of MYDGF in bone marrow-derived inflammatory cells attenuated pressure overload-induced hypertrophy and dysfunction. Mechanistically, MYDGF inhibited G protein-coupled receptor agonist-induced hypertrophy and augmented SERCA2a (sarco/endoplasmic reticulum Ca2+-ATPase 2a) expression in cultured neonatal rat ventricular cardiomyocytes by enhancing PIM1 (Pim-1 proto-oncogene, serine/threonine kinase) expression and activity. Along this line, cardiomyocytes from pressure-overloaded Mydgf-/- mice displayed reduced PIM1 and SERCA2a expression, greater hypertrophy, and impaired Ca2+ cycling and sarcomere function compared with cardiomyocytes from pressure-overloaded WT mice. Transplanting Mydgf-/- mice with WT bone marrow cells augmented cardiac PIM1 and SERCA2a levels and ameliorated pressure overload-induced hypertrophy and dysfunction. Pressure-overloaded Mydgf-/- mice were similarly rescued by adenoviral Serca2a gene transfer. Treating pressure-overloaded WT mice subcutaneously with recombinant MYDGF enhanced SERCA2a expression, attenuated left ventricular hypertrophy and dysfunction, and improved survival.
Conclusions
These findings establish a MYDGF-based adaptive crosstalk between inflammatory cells and cardiomyocytes that protects against pressure overload-induced heart failure.



Circulation: 11 Oct 2021; 144:1227-1240
Korf-Klingebiel M, Reboll MR, Polten F, Weber N, ... Wang Y, Wollert KC
Circulation: 11 Oct 2021; 144:1227-1240 | PMID: 34372689
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Abstract

Interferon-gamma Impairs Human Coronary Artery Endothelial Glucose Metabolism via Tryptophan Catabolism and Activates Fatty Acid Oxidation.

Lee LY, Oldham WM, He H, Wang R, ... Handy DE, Loscalzo J
Background: Endothelial cells depend on glycolysis for much of their energy production. Impaired endothelial glycolysis has been associated with various vascular pathobiologies, including impaired angiogenesis and atherogenesis. Interferon-gamma (IFN-γ)-producing CD4+ and CD8+ T-lymphocytes have been identified as the predominant pathologic cell subsets in human atherosclerotic plaques. While the immunological consequences of these cells have been extensively evaluated, their IFN-γ-mediated metabolic effects on endothelial cells remain unknown. The purpose of this study was to determine the metabolic consequences of the T-lymphocyte cytokine, IFN-γ, on human coronary artery endothelial cells (HCAEC).
Methods:
The metabolic effects of IFN-γ on primary HCAEC were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux analyses and molecular mechanistic studies. Cellular phenotypic correlations were made by measuring altered endothelial intracellular cyclic guanosine monophosphate (cGMP) content, wound healing capacity, and adhesion molecule expression.
Results:
IFN-γ exposure inhibited basal glycolysis of quiescent primary HCAEC by 20% through the global transcriptional suppression of glycolytic enzymes resulting from decreased basal hypoxia inducible factor 1α (HIF1α) nuclear availability in normoxia. The decrease in HIF1α activity was a consequence of IFN-γ-induced tryptophan catabolism resulting in ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF1β sequestration by the kynurenine-activated aryl hydrocarbon receptor (AHR). Additionally, IFN-γ resulted in a 23% depletion of intracellular NAD+ in HCAEC. This altered glucose metabolism was met with concomitant activation of fatty acid oxidation, which augmented its contribution to intracellular ATP balance by over 20%. These metabolic derangements were associated with adverse endothelial phenotypic changes, including decreased basal intracellular cGMP, impaired endothelial migration, and a switch to a pro-inflammatory state. Conclusions: IFN-γ impairs endothelial glucose metabolism via altered tryptophan catabolism destabilizing HIF1, depletes NAD+, and results in a metabolic shift toward increased fatty acid oxidation. This work suggests a novel mechanistic basis for pathologic T-lymphocyte-endothelial interactions in atherosclerosis mediated by IFN-γ, linking endothelial glucose, tryptophan, and fatty acid metabolism with NAD(H) and ATP generation, and their adverse endothelial functional consequences.




Circulation: 11 Oct 2021; epub ahead of print
Lee LY, Oldham WM, He H, Wang R, ... Handy DE, Loscalzo J
Circulation: 11 Oct 2021; epub ahead of print | PMID: 34636650
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Abstract

MicroRNA-21 Controls Circadian Regulation of Apoptosis in Atherosclerotic Lesions.

Schober A, Blay RM, Saboor Maleki S, Zahedi F, ... Weber C, Nazari-Jahantigh M
Background
The necrotic core partly formed by ineffective efferocytosis increases the risk of an atherosclerotic plaque rupture. Microribonucleic acids contribute to necrotic core formation by regulating efferocytosis and macrophage apoptosis. Atherosclerotic plaque rupture occurs at increased frequency in the early morning, indicating diurnal changes in plaque vulnerability. Although circadian rhythms play a role in atherosclerosis, the molecular clock output pathways that control plaque composition and rupture susceptibility are unclear.
Methods
Circadian gene expression, necrotic core size, apoptosis, and efferocytosis in aortic lesions were investigated at different times of the day in Apoe-/-Mir21+/+ mice and Apoe-/-Mir21-/- mice after consumption of a high-fat diet for 12 weeks. Genome-wide gene expression and lesion formation were analyzed in bone marrow-transplanted mice. Diurnal changes in apoptosis and clock gene expression were determined in human atherosclerotic lesions.
Results
The expression of molecular clock genes, lesional apoptosis, and necrotic core size were diurnally regulated in Apoe-/- mice. Efferocytosis did not match the diurnal increase in apoptosis at the beginning of the active phase. However, in parallel with apoptosis, expression levels of oscillating Mir21 strands decreased in the mouse atherosclerotic aorta. Mir21 knockout abolished circadian regulation of apoptosis and reduced necrotic core size but did not affect core clock gene expression. Further, Mir21 knockout upregulated expression of proapoptotic Xaf1 (XIAP-associated factor 1) in the atherosclerotic aorta, which abolished circadian expression of Xaf1. The antiapoptotic effect of Mir21 was mediated by noncanonical targeting of Xaf1 through both Mir21 strands. Mir21 knockout in bone marrow cells also reduced atherosclerosis and necrotic core size. Circadian regulation of clock gene expression was confirmed in human atherosclerotic lesions. Apoptosis oscillated diurnally in phase with XAF1 expression, demonstrating an early morning peak antiphase to that of the Mir21 strands.
Conclusions
Our findings suggest that the molecular clock in atherosclerotic lesions induces a diurnal rhythm of apoptosis regulated by circadian Mir21 expression in macrophages that is not matched by efferocytosis, thus increasing the size of the necrotic core.



Circulation: 27 Sep 2021; 144:1059-1073
Schober A, Blay RM, Saboor Maleki S, Zahedi F, ... Weber C, Nazari-Jahantigh M
Circulation: 27 Sep 2021; 144:1059-1073 | PMID: 34233454
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Abstract

Integrated Stress Response Couples Mitochondrial Protein Translation with Oxidative Stress Control.

Zhang G, Wang X, Li C, Li Q, ... Scherer PE, Wang ZV
Background: The integrated stress response (ISR) is an evolutionarily conserved process to cope with intracellular and extracellular disturbances. Myocardial infarction is a leading cause of death worldwide. Coronary artery reperfusion is the most effective means to mitigate cardiac damage of myocardial infarction, which however causes additional reperfusion injury. This study aimed to investigate the role of the ISR in myocardial ischemia/reperfusion (I/R).
Methods:
Cardiac-specific gain- and loss-of-function approaches for the ISR were employed in vivo. Myocardial I/R was achieved by the ligation of the cardiac left anterior descending artery for 45 minutes, followed by reperfusion for different times. Cardiac function was assessed by echocardiography. Additionally, cultured H9c2 cells, primary rat cardiomyocytes, and mouse embryonic fibroblasts were used to dissect underlying molecular mechanisms. Moreover, tandem mass tag (TMT) labeling and mass spectrometry was conducted to identify protein targets of the ISR. Pharmacological means were tested to manipulate the ISR for therapeutic exploration.
Results:
We show that the PERK/eIF2α axis of the ISR is strongly induced by I/R in cardiomyocytes in vitro and in vivo. We further reveal a physiological role of PERK/eIF2α signaling by showing that acute activation of PERK in the heart confers robust cardioprotection against reperfusion injury. In contrast, cardiac-specific deletion of PERK aggravates cardiac responses to reperfusion. Mechanistically, the ISR directly targets mitochondrial complexes via translational suppression. We identify NDUFAF2, an assembly factor of mitochondrial complex I, as a selective target of PERK. Overexpression of PERK suppresses the protein expression of NDUFAF2 while PERK inhibition causes an increase of NDUFAF2. Silencing of NDUFAF2 significantly rescues cardiac cell survival from PERK knockdown under I/R. Further, we show that activation of PERK/eIF2α signaling reduces mitochondrial complex-derived reactive oxygen species and improves cardiac cell survival in response to I/R. Moreover, pharmacological stimulation of the ISR protects the heart against reperfusion damage, even after the restoration of occluded coronary artery, highlighting a clinical relevance for myocardial infarction treatment. Conclusions: These studies suggest that the ISR improves cell survival and mitigate reperfusion damage by selectively suppressing mitochondrial protein synthesis and reducing oxidative stress in the heart.




Circulation: 28 Sep 2021; epub ahead of print
Zhang G, Wang X, Li C, Li Q, ... Scherer PE, Wang ZV
Circulation: 28 Sep 2021; epub ahead of print | PMID: 34583519
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Abstract

hiPSC Modeling of Lineage-Specific Smooth Muscle Cell Defects Caused by Variant, and Its Therapeutic Implications for Loeys-Dietz Syndrome.

Zhou D, Feng H, Yang Y, Huang T, ... Mizrak D, Yang B
Background
Loeys-Dietz syndrome (LDS) is an inherited disorder predisposing individuals to thoracic aortic aneurysm and dissection. Currently, there are no medical treatments except surgical resection. Although the genetic basis of LDS is well-understood, molecular mechanisms underlying the disease remain elusive, impeding the development of a therapeutic strategy. In addition, aortic smooth muscle cells (SMCs) have heterogenous embryonic origins, depending on their spatial location, and lineage-specific effects of pathogenic variants on SMC function, likely causing regionally constrained LDS manifestations, have been unexplored.
Methods
We identified an LDS family with a dominant pathogenic variant in the TGFBR1 gene (TGFBR1A230T) causing aortic root aneurysm and dissection. To accurately model the molecular defects caused by this mutation, we used human induced pluripotent stem cells from a subject with normal aorta to generate human induced pluripotent stem cells carrying TGFBR1A230T, and corrected the mutation in patient-derived human induced pluripotent stem cells using CRISPR-Cas9 gene editing. After their lineage-specific SMC differentiation through cardiovascular progenitor cell (CPC) and neural crest stem cell lineages, we used conventional molecular techniques and single-cell RNA sequencing to characterize the molecular defects. The resulting data led to subsequent molecular and functional rescue experiments using activin A and rapamycin.
Results
Our results indicate the TGFBR1A230T mutation impairs contractile transcript and protein levels, and function in CPC-SMC, but not in neural crest stem cell-SMC. Single-cell RNA sequencing results implicate defective differentiation even in TGFBR1A230T/+ CPC-SMC including disruption of SMC contraction and extracellular matrix formation. Comparison of patient-derived and mutation-corrected cells supported the contractile phenotype observed in the mutant CPC-SMC. TGFBR1A230T selectively disrupted SMAD3 (SMAD family member 3) and AKT (AKT serine/threonine kinase) activation in CPC-SMC, and led to increased cell proliferation. Consistently, single-cell RNA sequencing revealed molecular similarities between a loss-of-function SMAD3 mutation (SMAD3c.652delA/+) and TGFBR1A230T/+. Last, combination treatment with activin A and rapamycin during or after SMC differentiation significantly improved the mutant CPC-SMC contractile gene expression and function, and rescued the mechanical properties of mutant CPC-SMC tissue constructs.
Conclusions
This study reveals that a pathogenic TGFBR1 variant causes lineage-specific SMC defects informing the etiology of LDS-associated aortic root aneurysm. As a potential pharmacological strategy, our results highlight a combination treatment with activin A and rapamycin that can rescue the SMC defects caused by the variant.



Circulation: 04 Oct 2021; 144:1145-1159
Zhou D, Feng H, Yang Y, Huang T, ... Mizrak D, Yang B
Circulation: 04 Oct 2021; 144:1145-1159 | PMID: 34346740
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Abstract

Epigenetic Regulation by in Cardiopulmonary Progenitor Cells Is Required to Prevent Pulmonary Hypertension and Chronic Obstructive Pulmonary Disease.

Qi H, Liu H, Pullamsetti SS, Günther S, ... Yuan X, Braun T
Background
The pathogenesis of life-threatening cardiopulmonary diseases such as pulmonary hypertension (PH) and chronic obstructive pulmonary disease (COPD) originates from a complex interplay of environmental factors and genetic predispositions that is not fully understood. Likewise, little is known about developmental abnormalities or epigenetic dysregulations that might predispose for PH or COPD in adult individuals.
Methods
To identify pathology-associated epigenetic alteration in diseased lung tissues, we screened a cohort of human patients with PH and COPD for changes of histone modifications by immunofluorescence staining. To analyze the function of H4K20me2/3 in lung pathogenesis, we developed a series of Suv4-20h1 knockout mouse lines targeting cardiopulmonary progenitor cells and different heart and lung cell types, followed by hemodynamic studies and morphometric assessment of tissue samples. Molecular, cellular, and biochemical techniques were applied to analyze the function of Suv4-20h1-dependent epigenetic processes in cardiopulmonary progenitor cells and their derivatives.
Results
We discovered a strong reduction of the histone modifications of H4K20me2/3 in human patients with COPD but not patients with PH that depend on the activity of the H4K20 di-methyltransferase SUV4-20H1. Loss of Suv4-20h1 in cardiopulmonary progenitor cells caused a COPD-like/PH phenotype in mice including the formation of perivascular tertiary lymphoid tissue and goblet cell hyperplasia, hyperproliferation of smooth muscle cells/myofibroblasts, impaired alveolarization and maturation defects of the microvasculature leading to massive right ventricular dilatation and premature death. Mechanistically, SUV4-20H1 binds directly to the 5\'-upstream regulatory element of the superoxide dismutase 3 (Sod3) gene to repress its expression. Increased levels of the extracellular SOD3 enzyme in Suv4-20h1 mutants increases hydrogen peroxide concentrations, causing vascular defects and impairing alveolarization.
Conclusions
Our findings reveal a pivotal role of the histone modifier SUV4-20H1 in cardiopulmonary codevelopment and uncover the developmental origins of cardiopulmonary diseases. We assume that the study will facilitate the understanding of pathogenic events causing PH and COPD and aid the development of epigenetic drugs for the treatment of cardiopulmonary diseases.



Circulation: 27 Sep 2021; 144:1042-1058
Qi H, Liu H, Pullamsetti SS, Günther S, ... Yuan X, Braun T
Circulation: 27 Sep 2021; 144:1042-1058 | PMID: 34247492
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Abstract

Role of Venous Endothelial Cells in Developmental and Pathologic Angiogenesis.

Lee HW, Xu Y, He L, Choi W, ... Jin SW, Simons M
Background
Angiogenesis is a dynamic process that involves expansion of a preexisting vascular network that can occur in a number of physiological and pathological settings. Despite its importance, the origin of the new angiogenic vasculature is poorly defined. In particular, the primary subtype of endothelial cells (capillary, venous, arterial) driving this process remains undefined.
Methods
Endothelial cells were fate-mapped with the use of genetic markers specific to arterial and capillary cells. In addition, we identified a novel venous endothelial marker gene (Gm5127) and used it to generate inducible venous endothelium-specific Cre and Dre driver mouse lines. Contributions of these various types of endothelial cells to angiogenesis were examined during normal postnatal development and in disease-specific setting.
Results
Using a comprehensive set of endothelial subtype-specific inducible reporter mice, including tip, arterial, and venous endothelial reporter lines, we showed that venous endothelial cells are the primary endothelial subtype responsible for the expansion of an angiogenic vascular network. During physiological angiogenesis, venous endothelial cells proliferate, migrating against the blood flow and differentiating into tip, capillary, and arterial endothelial cells of the new vasculature. Using intravital 2-photon imaging, we observed venous endothelial cells migrating against the blood flow to form new blood vessels. Venous endothelial cell migration also plays a key role in pathological angiogenesis. This was observed both in formation of arteriovenous malformations in mice with inducible endothelium-specific Smad4 deletion mice and in pathological vessel growth seen in oxygen-induced retinopathy.
Conclusions
Our studies establish that venous endothelial cells are the primary endothelial subtype responsible for normal expansion of vascular networks, formation of arteriovenous malformations, and pathological angiogenesis. These observations highlight the central role of the venous endothelium in normal development and disease pathogenesis.



Circulation: 18 Oct 2021; 144:1308-1322
Lee HW, Xu Y, He L, Choi W, ... Jin SW, Simons M
Circulation: 18 Oct 2021; 144:1308-1322 | PMID: 34474596
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Abstract

Long Non-coding RNA MIAT Controls Advanced Atherosclerotic Lesion Formation and Plaque Destabilization.

Fasolo F, Jin H, Winski G, Chernogubova E, ... Paloschi V, Maegdefessel L
Background: Long noncoding RNAs (lncRNAs) are important regulators of biological processes involved in vascular tissue homeostasis and disease development. The current study assessed the functional contribution of the lncRNA Myocardial Infarction Associated Transcript (MIAT) to atherosclerosis and carotid artery disease.
Methods:
We profiled differences in RNA transcript expression in patients with advanced carotid artery atherosclerotic lesions from the Biobank of Karolinska Endarterectomies (BiKE). The lncRNA MIAT was identified as the most upregulated non-coding RNA transcript in carotid plaques compared to non-atherosclerotic control arteries, which was confirmed by quantitative real time PCR (qRT-PCR) and in situ hybridization.
Results:
Experimental knockdown of MIAT, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) not only markedly decreased proliferation and migration rates of cultured human carotid artery smooth muscle cells (SMCs), but also increased their apoptosis. Mechanistically, MIAT regulated SMC proliferation via the EGR1-ELK1-ERK pathway. MIAT is further involved in SMC phenotypic transition to proinflammatory macrophage-like cells through binding to the promoter region of KLF4 and enhancing its transcription. Studies using Miat-/- and Miat-/- ApoE-/- mice as well as Yucatan LDLR-/- mini-pigs confirmed the regulatory role of this lncRNA in SMC de- and trans-differentiation and advanced atherosclerotic lesion formation. Conclusions: The lncRNA MIAT is a novel regulator of cellular processes in advanced atherosclerosis that controls proliferation, apoptosis, and phenotypic transition of SMCs as well as the pro-inflammatory properties of macrophages.




Circulation: 13 Oct 2021; epub ahead of print
Fasolo F, Jin H, Winski G, Chernogubova E, ... Paloschi V, Maegdefessel L
Circulation: 13 Oct 2021; epub ahead of print | PMID: 34647815
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Abstract

Loss of Mitochondrial Ca Uniporter Limits Inotropic Reserve and Provides Trigger and Substrate for Arrhythmias in Barth Syndrome Cardiomyopathy.

Bertero E, Nickel A, Kohlhaas M, Hohl M, ... Prates Roma L, Maack C
Background: Barth syndrome (BTHS) is caused by mutations of the gene encoding tafazzin, which catalyzes maturation of mitochondrial cardiolipin and often manifests with systolic dysfunction during early infancy. Beyond the first months of life, BTHS cardiomyopathy typically transitions to a phenotype of diastolic dysfunction with preserved ejection fraction, blunted contractile reserve during exercise and arrhythmic vulnerability. Previous studies traced BTHS cardiomyopathy to mitochondrial formation of reactive oxygen species (ROS). Since mitochondrial function and ROS formation are regulated by excitation-contraction (EC) coupling, integrated analysis of mechano-energetic coupling is required to delineate the pathomechanisms of BTHS cardiomyopathy.
Methods:
We analyzed cardiac function and structure in a mouse model with global knockdown of tafazzin (Taz-KD) compared to wild-type (WT) littermates. Respiratory chain assembly and function, ROS emission, and Ca2+ uptake were determined in isolated mitochondria. EC coupling was integrated with mitochondrial redox state, ROS, and Ca2+ uptake in isolated, unloaded or preloaded cardiac myocytes, and cardiac hemodynamics analyzed in vivo.
Results:
Taz-KD mice develop heart failure with preserved ejection fraction (>50%) and age-dependent progression of diastolic dysfunction in the absence of fibrosis. Increased myofilament Ca2+ affinity and slowed cross-bridge cycling caused diastolic dysfunction, partly compensated by accelerated diastolic Ca2+ decay through preactivated sarcoplasmic reticulum Ca2+ ATPase (SERCA). Taz deficiency provoked heart-specific loss of mitochondrial Ca2+ uniporter (MCU) protein that prevented Ca2+-induced activation of the Krebs cycle during β-adrenergic stimulation, oxidizing pyridine nucleotides and triggering arrhythmias in cardiac myocytes. In vivo, Taz-KD mice displayed prolonged QRS duration as a substrate for arrhythmias, and a lack of inotropic response to β-adrenergic stimulation. Cellular arrhythmias and QRS prolongation, but not the defective inotropic reserve, were restored by inhibiting Ca2+ export via the mitochondrial Na+/Ca2+ exchanger. All alterations occurred in the absence of excess mitochondrial ROS in vitro or in vivo. Conclusions: Downregulation of MCU, increased myofilament Ca2+ affinity, and preactivated SERCA provoke mechano-energetic uncoupling that explains diastolic dysfunction and the lack of inotropic reserve in BTHS cardiomyopathy. Furthermore, defective mitochondrial Ca2+ uptake provides a trigger and a substrate for ventricular arrhythmias. These insights can guide the ongoing search for a cure of this orphaned disease.




Circulation: 13 Oct 2021; epub ahead of print
Bertero E, Nickel A, Kohlhaas M, Hohl M, ... Prates Roma L, Maack C
Circulation: 13 Oct 2021; epub ahead of print | PMID: 34648376
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Abstract

Nidogen-2 Maintains the Contractile Phenotype of Vascular Smooth Muscle Cells and Prevents Neointima Formation via Bridging Jagged1-Notch3 Signaling.

Mao C, Ma Z, Jia Y, Li W, ... Fu Y, Kong W
Background
How the extracellular matrix (ECM) microenvironment modulates the contractile phenotype of vascular smooth muscle cells (VSMCs) and confers vascular homeostasis remains elusive.
Methods
To explore the key ECM proteins in the maintenance of the contractile phenotype of VSMCs, we applied protein-protein interaction network analysis to explore novel ECM proteins associated with the VSMC phenotype. By combining in vitro and in vivo genetic mice vascular injury models, we identified nidogen-2, a basement membrane glycoprotein, as a key ECM protein for maintenance of vascular smooth muscle cell identity.
Results
We collected a VSMC phenotype-related gene dataset by using Gene Ontology annotation combined with a literature search. A computational analysis of protein-protein interactions between ECM protein genes and the genes from the VSMC phenotype-related gene dataset revealed the candidate gene nidogen-2, a basement membrane glycoprotein involved in regulation of the VSMC phenotype. Indeed, nidogen-2-deficient VSMCs exhibited loss of contractile phenotype in vitro, and compared with wild-type mice, nidogen-2-/- mice showed aggravated post-wire injury neointima formation of carotid arteries. Further bioinformatics analysis, coimmunoprecipitation assays, and luciferase assays revealed that nidogen-2 specifically interacted with Jagged1, a conventional Notch ligand. Nidogen-2 maintained the VSMC contractile phenotype via Jagged1-Notch3 signaling but not Notch1 or Notch2 signaling. Nidogen-2 enhanced Jagged1 and Notch3 interaction and subsequent Notch3 activation. Reciprocally, Jagged1 and Notch3 interaction, signaling activation, and Jagged1-triggered VSMC differentiation were significantly repressed in nidogen-2-deficient VSMCs. In accordance, the suppressive effect of Jagged1 overexpression on neointima formation was attenuated in nidogen-2-/- mice compared with wild-type mice.
Conclusions
Nidogen-2 maintains the contractile phenotype of VSMCs through Jagged1-Notch3 signaling in vitro and in vivo. Nidogen-2 is required for Jagged1-Notch3 signaling.



Circulation: 11 Oct 2021; 144:1244-1261
Mao C, Ma Z, Jia Y, Li W, ... Fu Y, Kong W
Circulation: 11 Oct 2021; 144:1244-1261 | PMID: 34315224
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Abstract

Mitochondrial Telomerase Reverse Transcriptase Protects from Myocardial Ischemia/reperfusion Injury by Improving Complex I Composition and Function.

Ale-Agha N, Jakobs P, Goy C, Zurek M, ... Altschmied J, Haendeler J
Background: The catalytic subunit of telomerase, Telomerase Reverse Transcriptase (TERT) has protective functions in the cardiovascular system. TERT is not only present in the nucleus, but also in mitochondria. However, it is unclear whether nuclear or mitochondrial TERT is responsible for the observed protection and appropriate tools are missing to dissect this.
Methods:
We generated new mouse models containing TERT exclusively in the mitochondria (mitoTERT mice) or the nucleus (nucTERT mice) to finally distinguish between the functions of nuclear and mitochondrial TERT. Outcome after ischemia/reperfusion, mitochondrial respiration in the heart as well as cellular functions of cardiomyocytes, fibroblasts, and endothelial cells were determined.
Results:
All mice were phenotypically normal. While respiration was reduced in cardiac mitochondria from TERT-deficient and nucTERT mice, it was increased in mitoTERT animals. The latter also had smaller infarcts than wildtype mice, whereas nucTERT animals had larger infarcts. The decrease in ejection fraction after one, two and four weeks of reperfusion was attenuated in mitoTERT mice. Scar size was also reduced and vascularization increased. Mitochondrial TERT protected a cardiomyocyte cell line from apoptosis. Myofibroblast differentiation, which depends on complex I activity, was abrogated in TERT-deficient and nucTERT cardiac fibroblasts and completely restored in mitoTERT cells. In endothelial cells, mitochondrial TERT enhanced migratory capacity and activation of endothelial NO synthase. Mechanistically, mitochondrial TERT improved the ratio between complex I matrix arm and membrane subunits explaining the enhanced complex I activity. In human right atrial appendages, TERT was localized in mitochondria and there increased by remote ischemic preconditioning. The Telomerase activator, TA-65 evoked a similar effect in endothelial cells, thereby increasing their migratory capacity, and enhanced myofibroblast differentiation. Conclusions: Mitochondrial, but not nuclear TERT, is critical for mitochondrial respiration and during ischemia/reperfusion injury. Mitochondrial TERT improves complex I subunit composition. TERT is present in human heart mitochondria, and remote ischemic preconditioning increases its level in those organelles. TA-65 has comparable effects ex vivo and improves migratory capacity of endothelial cells and myofibroblast differentiation. We conclude that mitochondrial TERT is responsible for cardioprotection and its increase could serve as a therapeutic strategy.




Circulation: 20 Oct 2021; epub ahead of print
Ale-Agha N, Jakobs P, Goy C, Zurek M, ... Altschmied J, Haendeler J
Circulation: 20 Oct 2021; epub ahead of print | PMID: 34672678
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Abstract

Altered Cardiac Energetics and Mitochondrial Dysfunction in Hypertrophic Cardiomyopathy.

Ranjbarvaziri S, Kooiker KB, Ellenberger M, Fajardo G, ... Contrepois K, Bernstein D
Background: Hypertrophic cardiomyopathy (HCM) is a complex disease partly explained by the effects of individual gene variants on sarcomeric protein biomechanics. At the cellular level, HCM mutations most commonly enhance force production, leading to higher energy demands. Despite significant advances in elucidating sarcomeric structure-function relationships, there is still much to be learned about the mechanisms that link altered cardiac energetics to HCM phenotypes. In this work, we test the hypothesis that changes in cardiac energetics represent a common pathophysiologic pathway in HCM.
Methods:
We performed a comprehensive multi-omics profile of the molecular (transcripts, metabolites, and complex lipids), ultrastructural, and functional components of HCM energetics using myocardial samples from 27 HCM patients and 13 normal controls (donor hearts).
Results:
Integrated omics analysis revealed alterations in a wide array of biochemical pathways with major dysregulation in fatty acid metabolism, reduction of acylcarnitines, and accumulation of free fatty acids. HCM hearts showed evidence of global energetic decompensation manifested by a decrease in high energy phosphate metabolites [ATP, ADP, and phosphocreatine (PCr)] and a reduction in mitochondrial genes involved in creatine kinase and ATP synthesis. Accompanying these metabolic derangements, electron microscopy showed an increased fraction of severely damaged mitochondria with reduced cristae density, coinciding with reduced citrate synthase (CS) activity and mitochondrial oxidative respiration. These mitochondrial abnormalities were associated with elevated reactive oxygen species (ROS) and reduced antioxidant defenses. However, despite significant mitochondrial injury, HCM hearts failed to upregulate mitophagic clearance. Conclusions: Overall, our findings suggest that perturbed metabolic signaling and mitochondrial dysfunction are common pathogenic mechanisms in patients with HCM. These results highlight potential new drug targets for attenuation of the clinical disease through improving metabolic function and reducing mitochondrial injury.




Circulation: 20 Oct 2021; epub ahead of print
Ranjbarvaziri S, Kooiker KB, Ellenberger M, Fajardo G, ... Contrepois K, Bernstein D
Circulation: 20 Oct 2021; epub ahead of print | PMID: 34672721
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Abstract

CARMN is an Evolutionarily Conserved Smooth Muscle Cell-specific LncRNA that Maintains Contractile Phenotype by Binding Myocardin.

Dong K, Shen J, He X, Hu G, ... Fulton DJR, Zhou J
Background: Vascular homeostasis is maintained by the differentiated phenotype of vascular smooth muscle cells (VSMCs). The landscape of protein coding genes comprising the transcriptome of differentiated VSMCs has been intensively investigated but many gaps remain including the emerging roles of non-coding genes.
Methods:
We re-analyzed large-scale, publicly available bulk and scRNA-seq datasets from multiple tissues and cell types to identify VSMC-enriched lncRNAs. The in vivo expression pattern of a novel SMC expressed lncRNA, Carmn (CARdiac Mesoderm Enhancer-associated Non-coding RNA) was investigated using a novel Carmn GFP knock-in reporter mouse model. Bioinformatics and qRT-PCR analysis were employed to assess CARMN expression changes during VSMC phenotypic modulation in human and murine vascular disease models. In vitro, functional assays were performed by knocking down CARMN with antisense oligonucleotides and over-expressing Carmn by adenovirus in human coronary artery SMCs. Carotid artery injury was performed in SMC-specific Carmn knockout mice to assess neointima formation and the therapeutic potential of reversing CARMN loss was tested in a rat carotid artery balloon injury model. The molecular mechanisms underlying CARMN function were investigated using RNA pull-down, RNA immunoprecipitation and luciferase reporter assays.
Results:
We identified CARMN, which was initially annotated as the host gene of the MIR143/145 cluster and recently reported to play a role in cardiac differentiation, as a highly abundant and conserved, SMC-specific lncRNA. Analysis of the Carmn GFP knock-in mouse model confirmed that Carmn is transiently expressed in embryonic cardiomyocytes and thereafter becomes restricted to SMCs. We also found that Carmn is transcribed independently of Mir143/145. CARMN expression is dramatically decreased by vascular disease in humans and murine models and regulates the contractile phenotype of VSMCs in vitro. In vivo, SMC-specific deletion of Carmn significantly exacerbated, while overexpression of Carmn markedly attenuated, injury-induced neointima formation in mouse and rat, respectively. Mechanistically, we found that Carmn physically binds to the key transcriptional cofactor myocardin, facilitating its activity and thereby maintaining the contractile phenotype of VSMCs Conclusions: CARMN is an evolutionarily conserved SMC-specific lncRNA with a previously unappreciated role in maintaining the contractile phenotype of VSMCs and is the first non-coding RNA discovered to interact with myocardin.




Circulation: 24 Oct 2021; epub ahead of print
Dong K, Shen J, He X, Hu G, ... Fulton DJR, Zhou J
Circulation: 24 Oct 2021; epub ahead of print | PMID: 34694145
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Abstract

Salt-Sensitive Hypertension of the Renal Tubular Cell-Specific NFAT5 (Nuclear Factor of Activated T-Cells 5) Knockout Mice.

Hiramatsu A, Izumi Y, Eguchi K, Matsuo N, ... Kim-Mitsuyama S, Mukoyama M
The kidney plays a crucial role in blood pressure (BP) regulation by controlling sodium reabsorption along the nephron. NFAT5 (nuclear factor of activated T-cells 5) is a transcription factor that is expressed in various tissues including the kidney and is activated at hypertonic conditions as observed in the renal medulla; the role for kidney NFAT5 in BP regulation, however, remains still obscure. In the present study, we generated inducible and renal tubular cell-specific NFAT5 knockout (KO) mice and characterized their phenotype. The NFAT5 KO mice exhibited high BP, hypernatremia, polyuria, and low urinary sodium excretion without significant alterations in the plasma renin activity or aldosterone concentration. The mice fed a high-salt diet further increased BP, revealing salt-sensitive hypertension. The KO mice ehibited the increased gene expression of the epithelial sodium channel. Protein expression of epithelial sodium channel in the membrane fraction was also significantly increased in KO mice than in wild-type mice. Treatment with amiloride, an epithelial sodium channel blocker, corrected high BP, hypernatremia, and decreased urinary sodium excretion in KO mice to the same levels of those in wild-type mice. Finally, the effects of high-salt diet and amiloride in KO mice were confirmed by the radiotelemetry method. In conclusion, these data indicate that renal tubular NFAT5 should play an important role in regulating sodium reabsorption through epithelial sodium channel under high-salt conditions, thereby preventing salt-dependent hypertension.



Hypertension: 03 Oct 2021:HYPERTENSIONAHA12117435; epub ahead of print
Hiramatsu A, Izumi Y, Eguchi K, Matsuo N, ... Kim-Mitsuyama S, Mukoyama M
Hypertension: 03 Oct 2021:HYPERTENSIONAHA12117435; epub ahead of print | PMID: 34601973
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Abstract

Turnover Rates of the Low-Density Lipoprotein Receptor and PCSK9: Added Dimension to the Cholesterol Homeostasis Model.

Dandan M, Han J, Mann S, Kim R, ... Nyangau E, Hellerstein M
Objective
We measured the turnover rates of the LDLR (low-density lipoprotein receptor) and PCSK9 (proprotein convertase subtilisin/kexin type 9) in mice by metabolic labeling with heavy water and mass spectrometry. Approach and
Results:
In liver of mice fed high-cholesterol diets, LDLR mRNA levels and synthesis rates were markedly lower with complete suppression of cholesterol synthesis and higher cholesterol content, consistent with the Brown-Goldstein model of tissue cholesterol homeostasis. We observed markedly lower PCSK9 mRNA levels and synthesis rates in liver and lower concentrations and synthesis rates in plasma. Hepatic LDLR half-life (t½) was prolonged, consistent with an effect of reduced PCSK9, and resulted in no reduction in hepatic LDLR content despite reduced mRNA levels and LDLR synthesis rates. These changes in PCSK9 synthesis complement and expand the well-established model of tissue cholesterol homeostasis in mouse liver, in that reduced synthesis and levels of PCSK9 counterbalance lower LDLR synthesis by promoting less LDLR catabolism, thereby maintaining uptake of LDL cholesterol into liver despite high intracellular cholesterol concentrations.
Conclusions
Lower hepatic synthesis and secretion of PCSK9, an SREBP2 (sterol response element binding protein) target gene, results in longer hepatic LDLR t½ in response to cholesterol feeding in mice in the face of high intracellular cholesterol content. PCSK9 modulation opposes the canonical lowering of LDLR mRNA and synthesis by cholesterol surplus and preserves LDLR levels. The physiological and therapeutic implications of these opposing control mechanisms over liver LDLR are of interest and may reflect subservience of hepatic cholesterol homeostasis to whole body cholesterol needs.



Arterioscler Thromb Vasc Biol: 06 Oct 2021:ATVBAHA121316764; epub ahead of print
Dandan M, Han J, Mann S, Kim R, ... Nyangau E, Hellerstein M
Arterioscler Thromb Vasc Biol: 06 Oct 2021:ATVBAHA121316764; epub ahead of print | PMID: 34615375
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Abstract

Inhibition of Soluble Epoxide Hydrolase Attenuates Bosutinib-Induced Blood Pressure Elevation.

Cui Z, Li B, Zhang Y, He J, ... Zhang X, Zhu Y
Endothelial cells play a critical role in maintaining homeostasis of vascular function, and endothelial activation is involved in the initial step of atherogenesis. Previously, we reported that Abl kinase mediates shear stress-induced endothelial activation. Bosutinib, a dual inhibitor of Src and Abl kinases, exerts an atheroprotective effect; however, recent studies have demonstrated an increase in the incidence of side effects associated with bosutinib, including increased arterial blood pressure (BP). To understand the effects of bosutinib on BP regulation and the mechanistic basis for novel treatment strategies against vascular dysfunction, we generated a line of mice conditionally lacking c-Abl in endothelial cells (endothelial cell-AblKO). Knockout mice and their wild-type littermates (Ablf/f) were orally administered a clinical dose of bosutinib, and their BP was monitored. Bosutinib treatment increased BP in both endothelial cell-AblKO and Ablf/f mice. Furthermore, acetylcholine-evoked endothelium-dependent relaxation of the mesenteric arteries was impaired by bosutinib treatment. RNA sequencing of mesenteric arteries revealed that the CYP (cytochrome P450)-dependent metabolic pathway was involved in regulating BP after bosutinib treatment. Additionally, bosutinib treatment led to an upregulation of soluble epoxide hydrolase in the arteries and a lower plasma content of eicosanoid metabolites in the CYP pathway in mice. Treatment with 1-Trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea, a soluble epoxide hydrolase inhibitor, reversed the bosutinib-induced changes to the eicosanoid metabolite profile, endothelium-dependent vasorelaxation, and BP. Thus, the present study demonstrates that upregulation of soluble epoxide hydrolase mediates bosutinib-induced elevation of BP, independent of c-Abl. The addition of soluble epoxide hydrolase inhibitor in patients treated with bosutinib may aid in preventing vascular side effects.



Hypertension: 03 Oct 2021:HYPERTENSIONAHA12117548; epub ahead of print
Cui Z, Li B, Zhang Y, He J, ... Zhang X, Zhu Y
Hypertension: 03 Oct 2021:HYPERTENSIONAHA12117548; epub ahead of print | PMID: 34601968
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Impact:
Abstract

Dietary Neu5Ac Intervention Protects Against Atherosclerosis Associated With Human-Like Neu5Gc Loss.

Kawanishi K, Coker JK, Grunddal KV, Dhar C, ... Varki A, Gordts PLSM
Objective
Species-specific pseudogenization of the CMAH gene during human evolution eliminated common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) biosynthesis from its precursor N-acetylneuraminic acid (Neu5Ac). With metabolic nonhuman Neu5Gc incorporation into endothelia from red meat, the major dietary source, anti-Neu5Gc antibodies appeared. Human-like Ldlr-/-Cmah-/- mice on a high-fat diet supplemented with a Neu5Gc-enriched mucin, to mimic human red meat consumption, suffered increased atherosclerosis if human-like anti-Neu5Gc antibodies were elicited. Approach and
Results:
We now ask whether interventional Neu5Ac feeding attenuates metabolically incorporated Neu5Gc-mediated inflammatory acceleration of atherogenesis in this Cmah-/-Ldlr-/- model system. Switching to a Neu5Gc-free high-fat diet or adding a 5-fold excess of Collocalia mucoid-derived Neu5Ac in high-fat diet protects against accelerated atherosclerosis. Switching completely from a Neu5Gc-rich to a Neu5Ac-rich diet further reduces severity. Remarkably, feeding Neu5Ac-enriched high-fat diet alone has a substantial intrinsic protective effect against atherosclerosis in Ldlr-/- mice even in the absence of dietary Neu5Gc but only in the human-like Cmah-null background.
Conclusions
Interventional Neu5Ac feeding can mitigate or prevent the red meat/Neu5Gc-mediated increased risk for atherosclerosis, and has an intrinsic protective effect, even in the absence of Neu5Gc feeding. These findings suggest that similar interventions should be tried in humans and that Neu5Ac-enriched diets alone should also be investigated further.



Arterioscler Thromb Vasc Biol: 29 Sep 2021:ATVBAHA120315280; epub ahead of print
Kawanishi K, Coker JK, Grunddal KV, Dhar C, ... Varki A, Gordts PLSM
Arterioscler Thromb Vasc Biol: 29 Sep 2021:ATVBAHA120315280; epub ahead of print | PMID: 34587757
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Impact:
Abstract

Vascular Smooth Muscle Cell Subpopulations and Neointimal Formation in Mouse Models of Elastin Insufficiency.

Lin CJ, Hunkins B, Roth R, Lin CY, Wagenseil JE, Mecham RP
Objective
Using a mouse model of Eln (elastin) insufficiency that spontaneously develops neointima in the ascending aorta, we sought to understand the origin and phenotypic heterogeneity of smooth muscle cells (SMCs) contributing to intimal hyperplasia. We were also interested in exploring how vascular cells adapt to the absence of Eln. Approach and
Results:
We used single-cell sequencing together with lineage-specific cell labeling to identify neointimal cell populations in a noninjury, genetic model of neointimal formation. Inactivating Eln production in vascular SMCs results in rapid intimal hyperplasia around breaks in the ascending aorta\'s internal elastic lamina. Using lineage-specific Cre drivers to both lineage mark and inactivate Eln expression in the secondary heart field and neural crest aortic SMCs, we found that cells with a secondary heart field lineage are significant contributors to neointima formation. We also identified a small population of secondary heart field-derived SMCs underneath and adjacent to the internal elastic lamina. Within the neointima of SMC-Eln knockout mice, 2 unique SMC populations were identified that are transcriptionally different from other SMCs. While these cells had a distinct gene signature, they expressed several genes identified in other studies of neointimal lesions, suggesting that some mechanisms underlying neointima formation in Eln insufficiency are shared with adult vessel injury models.
Conclusions
These results highlight the unique developmental origin and transcriptional signature of cells contributing to neointima in the ascending aorta. Our findings also show that the absence of Eln, or changes in elastic fiber integrity, influences the SMC biological niche in ways that lead to altered cell phenotypes.



Arterioscler Thromb Vasc Biol: 29 Sep 2021:ATVBAHA120315681; epub ahead of print
Lin CJ, Hunkins B, Roth R, Lin CY, Wagenseil JE, Mecham RP
Arterioscler Thromb Vasc Biol: 29 Sep 2021:ATVBAHA120315681; epub ahead of print | PMID: 34587758
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Impact:
Abstract

Myeloid Mineralocorticoid Receptor Transcriptionally Regulates P-Selectin Glycoprotein Ligand-1 and Promotes Monocyte Trafficking and Atherosclerosis.

Man JJ, Lu Q, Elizabeth Moss M, Carvajal B, ... Alcaide P, Jaffe IZ
Objective
MR (mineralocorticoid receptor) activation associates with increased risk of cardiovascular ischemia while MR inhibition reduces cardiovascular-related mortality and plaque inflammation in mouse atherosclerosis. MR in myeloid cells (My-MR) promotes inflammatory cell infiltration into injured tissues and atherosclerotic plaque inflammation by unclear mechanisms. Here, we examined the role of My-MR in leukocyte trafficking and the impact of sex. Approach and
Results:
We confirm in vivo that My-MR deletion (My-MR-KO) in ApoE-KO mice decreased plaque size. Flow cytometry revealed fewer plaque macrophages with My-MR-KO. By intravital microscopy, My-MR-KO significantly attenuated monocyte slow-rolling and adhesion to mesenteric vessels and decreased peritoneal infiltration of myeloid cells in response to inflammatory stimuli in male but not female mice. My-MR-KO mice had significantly less PSGL1 (P-selectin glycoprotein ligand 1) mRNA in peritoneal macrophages and surface PSGL1 protein on circulating monocytes in males. In vitro, MR activation with aldosterone significantly increased PSGL1 mRNA only in monocytes from MR-intact males. Similarly, aldosterone induced, and MR antagonist spironolactone inhibited, PSGL1 expression in human U937 monocytes. Mechanistically, aldosterone stimulated MR binding to a predicted MR response element in intron-1 of the PSGL1 gene by ChIP-qPCR. Reporter assays demonstrated that this PSGL1 MR response element is necessary and sufficient for aldosterone-activated, MR-dependent transcriptional activity.
Conclusions
These data identify PSGL1 as a My-MR target gene that drives leukocyte trafficking to enhance atherosclerotic plaque inflammation. These novel and sexually dimorphic findings provide insight into increased ischemia risk with MR activation, cardiovascular protection in women, and the role of MR in atherosclerosis and tissue inflammation.



Arterioscler Thromb Vasc Biol: 06 Oct 2021:ATVBAHA121316929; epub ahead of print
Man JJ, Lu Q, Elizabeth Moss M, Carvajal B, ... Alcaide P, Jaffe IZ
Arterioscler Thromb Vasc Biol: 06 Oct 2021:ATVBAHA121316929; epub ahead of print | PMID: 34615372
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Impact:
Abstract

Molecular and Cellular Dynamics of Aortic Aneurysms Revealed by Single-Cell Transcriptomics.

Li Y, LeMaire SA, Shen YH
The aorta is highly heterogeneous, containing many different types of cells that perform sophisticated functions to maintain aortic homeostasis. Recently, single-cell RNA sequencing studies have provided substantial new insight into the heterogeneity of vascular cell types, the comprehensive molecular features of each cell type, and the phenotypic interrelationship between these cell populations. This new information has significantly improved our understanding of aortic biology and aneurysms at the molecular and cellular level. Here, we summarize these findings, with a focus on what single-cell RNA sequencing analysis has revealed about cellular heterogeneity, cellular transitions, communications among cell populations, and critical transcription factors in the vascular wall. We also review the information learned from single-cell RNA sequencing that has contributed to our understanding of the pathogenesis of vascular disease, such as the identification of cell types in which aneurysm-related genes and genetic variants function. Finally, we discuss the challenges and future directions of single-cell RNA sequencing applications in studies of aortic biology and diseases.



Arterioscler Thromb Vasc Biol: 06 Oct 2021:ATVBAHA121315852; epub ahead of print
Li Y, LeMaire SA, Shen YH
Arterioscler Thromb Vasc Biol: 06 Oct 2021:ATVBAHA121315852; epub ahead of print | PMID: 34615376
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Impact:
Abstract

2-Methoxyestradiol Ameliorates Angiotensin II-Induced Hypertension by Inhibiting Cytosolic Phospholipase Aα Activity in Female Mice.

Song CY, Singh P, Motiwala M, Shin JS, ... Bonventre JV, Malik KU
We tested the hypothesis that CYP1B1 (cytochrome P450 1B1)-17β-estradiol metabolite 2-methoxyestradiol protects against Ang II (angiotensin II)-induced hypertension by inhibiting group IV cPLA2α (cytosolic phospholipase A2α) activity and production of prohypertensive eicosanoids in female mice. Ang II (700 ng/kg per minute, SC) increased mean arterial blood pressure (BP), systolic and diastolic BP measured by radiotelemetry, renal fibrosis, and reactive oxygen species production in wild-type mice (cPLA2α+/+/Cyp1b1+/+) that were enhanced by ovariectomy and abolished in intact and ovariectomized-cPLA2α-/-/Cyp1b1+/+ mice. Ang II-induced increase in SBP measured by tail-cuff, renal fibrosis, reactive oxygen species production, and cPLA2α activity measured by its phosphorylation in the kidney, and urinary excretion of prostaglandin E2 and thromboxane A2 metabolites were enhanced in ovariectomized-cPLA2α+/+/Cyp1b1+/+ and intact cPLA2α+/+/Cyp1b1-/- mice. 2-Methoxyestradiol and arachidonic acid metabolism inhibitor 5,8,11,14-eicosatetraynoic acid attenuated the Ang II-induced increase in SBP, renal fibrosis, reactive oxygen species production, and urinary excretion of prostaglandin E2, and thromboxane A2 metabolites in ovariectomized-cPLA2α+/+/Cyp1b1+/+ and intact cPLA2α+/+/Cyp1b1-/- mice. Antagonists of prostaglandin E2 and thromboxane A2 receptors EP1 and EP3 and TP, respectively, inhibited Ang II-induced increases in SBP and reactive oxygen species production and renal fibrosis in ovariectomized-cPLA2α+/+/Cyp1b1+/+ and intact cPLA2α+/+/Cyp1b1-/- mice. These data suggest that CYP1B1-generated metabolite 2-methoxyestradiol mitigates Ang II-induced hypertension and renal fibrosis by inhibiting cPLA2α activity, reducing prostaglandin E2, and thromboxane A2 production and stimulating EP1 and EP3 and TP receptors, respectively. Thus, 2-methoxyestradiol and the drugs that selectively block EP1 and EP3 and TP receptors could be useful in treating hypertension and its pathogenesis in females.



Hypertension: 10 Oct 2021:HYPERTENSIONAHA12118181; epub ahead of print
Song CY, Singh P, Motiwala M, Shin JS, ... Bonventre JV, Malik KU
Hypertension: 10 Oct 2021:HYPERTENSIONAHA12118181; epub ahead of print | PMID: 34628937
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Abstract

Platelet Heterogeneity in Myeloproliferative Neoplasms.

Thomas S, Krishnan A
Myeloproliferative neoplasms (MPNs) are a group of malignant disorders of the bone marrow where a dysregulated balance between proliferation and differentiation gives rise to abnormal numbers of mature blood cells. MPNs encompass a spectrum of disease entities with progressively more severe clinical features, including complications with thrombosis and hemostasis and an increased propensity for transformation to acute myeloid leukemia. There is an unmet clinical need for markers of disease progression. Our understanding of the precise mechanisms that influence pathogenesis and disease progression has been limited by access to disease-specific cells as biosources. Here, we review the landscape of MPN pathology and present blood platelets as potential candidates for disease-specific understanding. We conclude with our recent work discovering progressive platelet heterogeneity by subtype in a large clinical cohort of patients with MPN.



Arterioscler Thromb Vasc Biol: 06 Oct 2021:ATVBAHA121316373; epub ahead of print
Thomas S, Krishnan A
Arterioscler Thromb Vasc Biol: 06 Oct 2021:ATVBAHA121316373; epub ahead of print | PMID: 34615371
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Impact:
Abstract

Cardiotoxicity of Cancer Treatments: Focus on Anthracycline Cardiomyopathy.

Nishi M, Wang PY, Hwang PM
Significant progress has been made in developing new treatments and refining the use of preexisting ones against cancer. Their successful use and the longer survival of cancer patients have been associated with reports of new cardiotoxicities and the better characterization of the previously known cardiac complications. Immunotherapies with monoclonal antibodies against specific cancer-promoting genes, chimeric antigen receptor T cells, and immune checkpoint inhibitors have been developed to fight cancer cells, but they can also show off-target effects on the heart. Some of these cardiotoxicities are thought to be due to nonspecific immune activation and inflammatory damage. Unlike immunotherapy-associated cardiotoxicities which are relatively new entities, there is extensive literature on anthracycline-induced cardiomyopathy. Here, we provide a brief overview of the cardiotoxicities of immunotherapies for the purpose of distinguishing them from anthracycline cardiomyopathy. This is especially relevant as the expansion of oncological treatments presents greater diagnostic challenges in determining the cause of cardiac dysfunction in cancer survivors with a history of multiple cancer treatments including anthracyclines and immunotherapies administered concurrently or serially over time. We then provide a focused review of the mechanisms proposed to underlie the development of anthracycline cardiomyopathy based on experimental data mostly in mouse models. Insights into its pathogenesis may stimulate the development of new strategies to identify patients who are susceptible to anthracycline cardiomyopathy while permitting low cardiac risk patients to receive optimal treatment for their cancer.



Arterioscler Thromb Vasc Biol: 29 Sep 2021:ATVBAHA121316697; epub ahead of print
Nishi M, Wang PY, Hwang PM
Arterioscler Thromb Vasc Biol: 29 Sep 2021:ATVBAHA121316697; epub ahead of print | PMID: 34587760
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Impact:
Abstract

Inhibition of Vascular Growth by Modulation of the Anandamide/Fatty Acid Amide Hydrolase Axis.

Rieck S, Kilgus S, Meyer JH, Huang H, ... Fleischmann BK, Wenzel D
Objective
Pathological angiogenesis is a hallmark of various diseases characterized by local hypoxia and inflammation. These disorders can be treated with inhibitors of angiogenesis, but current compounds display a variety of side effects and lose efficacy over time. This makes the identification of novel signaling pathways and pharmacological targets involved in angiogenesis a top priority. Approach and
Results:
Here, we show that inactivation of FAAH (fatty acid amide hydrolase), the enzyme responsible for degradation of the endocannabinoid anandamide, strongly impairs angiogenesis in vitro and in vivo. Both, the pharmacological FAAH inhibitor URB597 and anandamide induce downregulation of gene sets for cell cycle progression and DNA replication in endothelial cells. This is underscored by cell biological experiments, in which both compounds inhibit proliferation and migration and evoke cell cycle exit of endothelial cells. This prominent antiangiogenic effect is also of pathophysiological relevance in vivo, as laser-induced choroidal neovascularization in the eye of FAAH-/- mice is strongly reduced.
Conclusions
Thus, elevation of endogenous anandamide levels by FAAH inhibition represents a novel antiangiogenic mechanism.



Arterioscler Thromb Vasc Biol: 06 Oct 2021:ATVBAHA121316973; epub ahead of print
Rieck S, Kilgus S, Meyer JH, Huang H, ... Fleischmann BK, Wenzel D
Arterioscler Thromb Vasc Biol: 06 Oct 2021:ATVBAHA121316973; epub ahead of print | PMID: 34615374
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Impact:
Abstract

Disruption of the Blood-Brain Barrier by Extracellular Vesicles From Preeclampsia Plasma and Hypoxic Placentae: Attenuation by Magnesium Sulfate.

León J, Acurio J, Bergman L, López J, ... Vatish M, Escudero C
Preeclampsia, a pregnancy-related endothelial disorder, is associated with both cardiovascular and cerebrovascular complications. Preeclampsia requires the presence of a placenta as part of its pathophysiology, yet the role of this organ in the cerebrovascular complications remains unclear. Research has shown that circulating small extracellular vesicles (also known as exosomes) present in preeclampsia plasma can generate endothelial dysfunction, but it is unclear whether the impairment of function of brain endothelial cells at the blood-brain barrier is secondary to plasma-derived or placental-derived exosomes. In this study, we evaluated the effect of small extracellular vesicles isolated from plasma samples of women with preeclampsia (n=12) and women with normal pregnancy (n=11) as well as from human placental explants from normotensive pregnancies (n=6) subjected to hypoxia (1% oxygen) on the integrity of the blood-brain barrier, using both in vitro and animal models. Exposure of human-derived brain endothelial cell monolayers to plasma and plasma-derived small extracellular vesicles from preeclamptic pregnancies increased the permeability and reduced the transendothelial electrical resistance. A similar outcome was observed with hypoxic placental-derived small extracellular vesicles, which also increased the permeability to Evan\'s blue in the brain of C57BL6 nonpregnant mice. Cotreatment with magnesium sulfate reversed the effects elicited by plasma, plasma-derived, and hypoxic placental-derived small extracellular vesicles in the employed models. Thus, circulating small extracellular vesicles in plasma from women with preeclampsia or from hypoxic placentae disrupt the blood-brain barrier, which can be prevented using magnesium sulfate. These findings provide new insights into the pathophysiology of cerebral complications associated with preeclampsia.



Hypertension: 03 Oct 2021:HYPERTENSIONAHA12117744; epub ahead of print
León J, Acurio J, Bergman L, López J, ... Vatish M, Escudero C
Hypertension: 03 Oct 2021:HYPERTENSIONAHA12117744; epub ahead of print | PMID: 34601964
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Impact:
Abstract

Tgfβ1-Cthrc1 Signaling Axis Plays an Important Role in the Short-Term Reparative Response to Heart Valve Endothelial Injury.

Nordquist EM, Dutta P, Kodigepalli KM, Mattern C, ... Lindner V, Lincoln J
Objective
Aortic valve disease is a common worldwide health burden with limited treatment options. Studies have shown that the valve endothelium is critical for structure-function relationships, and disease is associated with its dysfunction, damage, or injury. Therefore, therapeutic targets to maintain a healthy endothelium or repair damaged endothelial cells could hold promise. In this current study, we utilize a surgical mouse model of heart valve endothelial cell injury to study the short-term response to injury at molecular and cellular levels. The goal is to determine if the native heart valve exhibits a reparative response and identify the mechanisms underlying this process. Approach and
Results:
Mild aortic valve endothelial injury and abrogated function was evoked by inserting a guidewire down the carotid artery of young (3 months) and aging (16-18 months) wild-type mice. Short-term cellular responses were examined at 6 hours, 48 hours, and 4 weeks following injury, whereas molecular profiles were determined after 48 hours by RNA-sequencing. Within 48 hours following endothelial injury, young wild-type mice restore endothelial barrier function in association with increased cell proliferation, and upregulation of transforming growth factor beta 1 (Tgfβ1) and the glycoprotein, collagen triple helix repeat containing 1 (Cthrc1). Interestingly, this beneficial response to injury was not observed in aging mice with known underlying endothelial dysfunction.
Conclusions
Data from this study suggests that the healthy valve has the capacity to respond to mild endothelial injury, which in short term has beneficial effects on restoring endothelial barrier function through acute activation of the Tgfβ1-Cthrc1 signaling axis and cell proliferation.



Arterioscler Thromb Vasc Biol: 13 Oct 2021:ATVBAHA121316450; epub ahead of print
Nordquist EM, Dutta P, Kodigepalli KM, Mattern C, ... Lindner V, Lincoln J
Arterioscler Thromb Vasc Biol: 13 Oct 2021:ATVBAHA121316450; epub ahead of print | PMID: 34645278
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Impact:
Abstract

Endothelial METTL3 (Methyltransferase-Like 3) Inhibits Fibrinolysis by Promoting PAI-1 (Plasminogen Activator Inhibitor-1) Expression Through Enhancing Jun Proto-Oncogene N6-Methyladenosine Modification.

Bai Q, Lu Y, Chen Y, Zhang H, ... Wu H, Wen A
Objective
METTL3 (methyltransferase-like protein 3)-mediated N6-methyladenosine modification is the most abundant RNA modification on eukaryote mRNAs and plays a crucial role in diverse physiological and pathological processes. However, whether N6-methyladenosine modification has function in thrombosis is unknown. This study aims to determine the role of METTL3 in the endothelial cells-mediated thrombosis. Approach and
Results:
RNA-sequencing and real-time quantitative PCR revealed that the expression of PAI-1 (plasminogen activator inhibitor-1) was downregulated in METTL3 knockdown human umbilical vein endothelial cells. In vitro experiments showed that METTL3 suppressed fibrinolysis. Mechanically, RNA methylation sequencing and meRIP-quantitative real-time PCR showed that METTL3 catalyzed N6-methyladenosine modification on 3\' UTR of JUN mRNA. Western blotting analysis showed that METTL3 promoted JUN protein expression. Chromatin immunoprecipitation analysis demonstrated that JUN bound to the PAI-1 promoter in human umbilical vein endothelial cells. Furthermore, mice challenged with lipopolysaccharide resulted in higher METTL3 expression in vessels. Endothelial-specific knockdown of Mettl3 decreased expression of active PAI-1 in plasma and attenuated fibrin deposition in livers and lungs during endotoxemia.
Conclusions
Our study reveals that METTL3-mediated N6-methyladenosine modification plays a crucial role in fibrinolysis and is an underlying target for the therapy of thrombotic disorders.



Arterioscler Thromb Vasc Biol: 13 Oct 2021:ATVBAHA121316414; epub ahead of print
Bai Q, Lu Y, Chen Y, Zhang H, ... Wu H, Wen A
Arterioscler Thromb Vasc Biol: 13 Oct 2021:ATVBAHA121316414; epub ahead of print | PMID: 34645279
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Impact:
Abstract

Endothelial GTPCH (GTP Cyclohydrolase 1) and Tetrahydrobiopterin Regulate Gestational Blood Pressure, Uteroplacental Remodeling, and Fetal Growth.

Chuaiphichai S, Yu GZ, Tan CMJ, Whiteman C, ... Leeson P, Channon KM
Abnormal uteroplacental remodeling leads to placental hypoperfusion, causing fetal growth restriction and pregnancy-related hypertension, which are associated with endothelial dysfunction and markers of reduced vascular NO bioavailability and oxidative stress. Tetrahydrobiopterin (BH4) is a redox cofactor for eNOS (endothelial NO synthase) with a required role in NO generation. Using mice models and human samples, we investigated the physiological requirement for endothelial cell BH4 in uteroplacental vascular adaptation and blood pressure regulation to pregnancy. In pregnant mice, selective maternal endothelial BH4 deficiency resulting from targeted deletion of Gch1 caused progressive hypertension during pregnancy and fetal growth restriction. Maternal endothelial cell Gch1 deletion caused defective functional and structural remodeling in uterine arteries and in spiral arteries, leading to placental insufficiency. Using primary endothelial cells isolated from either normal or hypertensive pregnancies, we found that hypertensive pregnancies are associated with reduced endothelial cell BH4 levels, impaired eNOS activity, and reduced endothelial cell proliferation, mediated by reduced GTPCH (GTP cyclohydrolase 1) protein. In rescue experiments, high blood pressure and fetal growth restriction in pregnant endothelial cell Gch1 deficient mice was not rescued by oral BH4 supplementation, due to systemic oxidation of BH4 to dihydrobiopterin. However, the fully reduced folate, 5-methyltetrahydrofolate prevented BH4 oxidation, reduced blood pressure to normal levels, and normalized fetal growth. We identify a critical requirement for maternal endothelial cell BH4 biosynthesis in uteroplacental vascular remodeling in pregnancy. Restoration of endothelial cell BH4 with reduced folates identifies a novel therapeutic target for the prevention and treatment of pregnancy-related hypertension such as preeclampsia.



Hypertension: 24 Oct 2021:HYPERTENSIONAHA12017646; epub ahead of print
Chuaiphichai S, Yu GZ, Tan CMJ, Whiteman C, ... Leeson P, Channon KM
Hypertension: 24 Oct 2021:HYPERTENSIONAHA12017646; epub ahead of print | PMID: 34689592
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Impact:
Abstract

Resolvin E1 Attenuates Pulmonary Hypertension by Suppressing Wnt7a/β-Catenin Signaling.

Liu G, Wan N, Liu Q, Chen Y, ... Shen Y, Wang J
Pulmonary arterial hypertension (PAH) is a devastating disease characterized by severe pulmonary vascular wall remodeling and perivascular inflammation. Resolvin E1 (RvE1), a proresolving lipid mediator, has protective effects against various inflammatory diseases. However, the effect of RvE1 on PAH development remains to be determined. We aimed to investigate whether RvE1 has a therapeutic effect on PAH and, if so, to elucidate the molecular mechanisms underlying its effects. A hypoxia+SU5416-induced mouse model of pulmonary hypertension (PH) and an monocrotaline-induced rat model of PH were used to test therapeutic effect of RvE1. Lung tissues and plasma samples were collected from patients with PAH and rodent models to examine RvE1 production and its receptor chemerin chemokine-like receptor 1 (ChemR23) expression. We observed that RvE1 generation was reduced in the plasma of patients with idiopathic PAH and in lungs from experimental rodent models of PH. ChemR23 expression was markedly downregulated in hypoxia-exposed mouse pulmonary artery smooth muscle cells (PASMCs) and pulmonary arteries from PH rodents and patients with idiopathic PAH. RvE1 treatment alleviated experimental PH in both male and female rodents by inhibiting PASMC proliferation. Deletion of ChemR23 in vascular SMCs abolished the protective effect of RvE1 against hypoxia+SU5416-induced PAH in mice. Mechanistically, the RvE1/ChemR23 axis suppressed hypoxia-induced PASMC proliferation by inhibiting proliferative wingless-type MMTV integration site family member 7a/β-catenin signaling. Activation of ChemR23 by RvE1 diminished wingless-type MMTV integration site family member 7a expression in PASMCs by inhibiting protein kinase A-mediated Egr2 (early growth response 2) phosphorylation at Ser349. Thus, the RvE1/ChemR23 axis represses experimental PAH by modulating wingless-type MMTV integration site family member 7a/β-catenin signaling in PASMCs and may serve as a therapeutic target for the management of PAH.



Hypertension: 24 Oct 2021:HYPERTENSIONAHA12117809; epub ahead of print
Liu G, Wan N, Liu Q, Chen Y, ... Shen Y, Wang J
Hypertension: 24 Oct 2021:HYPERTENSIONAHA12117809; epub ahead of print | PMID: 34689593
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Abstract

PD-L1 (Programmed Death Ligand 1) Regulates T-Cell Differentiation to Control Adaptive Venous Remodeling.

Matsubara Y, Gonzalez L, Kiwan G, Liu J, ... Mori M, Dardik A
Objective
Patients with end-stage renal disease depend on hemodialysis for survival. Although arteriovenous fistulae (AVF) are the preferred vascular access for hemodialysis, the primary success rate of AVF is only 30% to 50% within 6 months, showing an urgent need for improvement. PD-L1 (programmed death ligand 1) is a ligand that regulates T-cell activity. Since T cells have an important role during AVF maturation, we hypothesized that PD-L1 regulates T cells to control venous remodeling that occurs during AVF maturation. Approach and results: In the mouse aortocaval fistula model, anti-PD-L1 antibody (200 mg, 3×/wk intraperitoneal) was given to inhibit PD-L1 activity during AVF maturation. Inhibition of PD-L1 increased T-helper type 1 cells and T-helper type 2 cells but reduced regulatory T cells to increase M1-type macrophages and reduce M2-type macrophages; these changes were associated with reduced vascular wall thickening and reduced AVF patency. Inhibition of PD-L1 also inhibited smooth muscle cell proliferation and increased endothelial dysfunction. The effects of anti-PD-L1 antibody on adaptive venous remodeling were diminished in nude mice; however, they were restored after T-cell transfer into nude mice, indicating the effects of anti-PD-L1 antibody on venous remodeling were dependent on T cells.
Conclusions
Regulation of PD-L1 activity may be a potential therapeutic target for clinical translation to improve AVF maturation.



Arterioscler Thromb Vasc Biol: 20 Oct 2021:ATVBAHA121316380; epub ahead of print
Matsubara Y, Gonzalez L, Kiwan G, Liu J, ... Mori M, Dardik A
Arterioscler Thromb Vasc Biol: 20 Oct 2021:ATVBAHA121316380; epub ahead of print | PMID: 34670406
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Abstract

CCM3 Loss-Induced Lymphatic Defect Is Mediated by the Augmented VEGFR3-ERK1/2 Signaling.

Qin L, Zhang H, Li B, Jiang Q, ... Min W, Zhou JH
Objective
Cerebral cavernous malformations (CCMs) can happen anywhere in the body, although they most commonly produce symptoms in the brain. The role of CCM genes in other vascular beds outside the brain and retina is not well-examined, although the 3 CCM-associated genes (CCM1, CCM2, and CCM3) are ubiquitously expressed in all tissues. We aimed to determine the role of CCM gene in lymphatics. Approach and
Results:
Mice with an inducible pan-endothelial cell (EC) or lymphatic EC deletion of Ccm3 (Pdcd10ECKO or Pdcd10LECKO) exhibit dilated lymphatic capillaries and collecting vessels with abnormal valve structure. Morphological alterations were correlated with lymphatic dysfunction in Pdcd10LECKO mice as determined by Evans blue dye and fluorescein isothiocyanate(FITC)-dextran transport assays. Pdcd10LECKO lymphatics had increased VEGFR3 (vascular endothelial growth factor receptor-3)-ERK1/2 signaling with lymphatic hyperplasia. Mechanistic studies suggested that VEGFR3 is primarily regulated at a transcriptional level in Ccm3-deficient lymphatic ECs, in an NF-κB (nuclear factor κB)-dependent manner. CCM3 binds to importin alpha 2/KPNA2 (karyopherin subunit alpha 2), and a CCM3 deletion releases KPNA2 to activate NF-κB P65 by facilitating its nuclear translocation and P65-dependent VEGFR3 transcription. Moreover, increased VEGFR3 in lymphatic EC preferentially activates ERK1/2 signaling, which is critical for lymphatic EC proliferation. Importantly, inhibition of VEGFR3 or ERK1/2 rescued the lymphatic defects in structure and function.
Conclusions
Our data demonstrate that CCM3 deletion augments the VEGFR3-ERK1/2 signaling in lymphatic EC that drives lymphatic hyperplasia and malformation and warrant further investigation on the potential clinical relevance of lymphatic dysfunction in patients with CCM.



Arterioscler Thromb Vasc Biol: 20 Oct 2021:ATVBAHA121316707; epub ahead of print
Qin L, Zhang H, Li B, Jiang Q, ... Min W, Zhou JH
Arterioscler Thromb Vasc Biol: 20 Oct 2021:ATVBAHA121316707; epub ahead of print | PMID: 34670407
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Abstract

Endothelial p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels.

Huang L, Bichsel C, Norris A, Thorpe J, ... Greene AK, Bischoff J
Objective
Capillary malformation (CM) occurs sporadically and is associated with Sturge-Weber syndrome. The somatic mosaic mutation in GNAQ (c.548G>A, p.R183Q) is enriched in endothelial cells (ECs) in skin CM and Sturge-Weber syndrome brain CM. Our goal was to investigate how the mutant Gαq (G-protein αq subunit) alters EC signaling and disrupts capillary morphogenesis. Approach and
Results:
We used lentiviral constructs to express p.R183Q or wild-type GNAQ in normal human endothelial colony forming cells (EC-R183Q and EC-WT, respectively). EC-R183Q constitutively activated PLC (phospholipase C) β3, a downstream effector of Gαq. Activated PLCβ3 was also detected in human CM tissue sections. Bulk RNA sequencing analyses of mutant versus wild-type EC indicated constitutive activation of PKC (protein kinase C), NF-κB (nuclear factor kappa B) and calcineurin signaling in EC-R183Q. Increased expression of downstream targets in these pathways, ANGPT2 (angiopoietin-2) and DSCR (Down syndrome critical region protein) 1.4 were confirmed by qPCR and immunostaining of human CM tissue sections. The Gαq inhibitor YM-254890 as well as siRNA targeted to PLCβ3 reduced mRNA expression levels of these targets in EC-R183Q while the pan-PKC inhibitor AEB071 reduced ANGPT2 but not DSCR1.4. EC-R183Q formed enlarged blood vessels in mice, reminiscent of those found in human CM. shRNA knockdown of ANGPT2 in EC-R183Q normalized the enlarged vessels to sizes comparable those formed by EC-WT.
Conclusions
Gαq-R183Q, when expressed in ECs, establishes constitutively active PLCβ3 signaling that leads to increased ANGPT2 and a proangiogenic, proinflammatory phenotype. EC-R183Q are sufficient to form enlarged CM-like vessels in mice, and suppression of ANGPT2 prevents the enlargement. Our study provides the first evidence that endothelial Gαq-R183Q is causative for CM and identifies ANGPT2 as a contributor to CM vascular phenotype.



Arterioscler Thromb Vasc Biol: 20 Oct 2021:ATVBAHA121316651; epub ahead of print
Huang L, Bichsel C, Norris A, Thorpe J, ... Greene AK, Bischoff J
Arterioscler Thromb Vasc Biol: 20 Oct 2021:ATVBAHA121316651; epub ahead of print | PMID: 34670408
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Abstract

CircSOD2: A Novel Regulator for Smooth Muscle Proliferation and Neointima Formation.

Mei X, Cui XB, Li Y, Chen SY
Objective
Vascular smooth muscle cell (SMC) proliferation contributes to neointima formation following vascular injury. Circular RNA-a novel type of noncoding RNA with closed-loop structure-exhibits cell- and tissue-specific expression patterns. However, the role of circular RNA in SMC proliferation and neointima formation is largely unknown. The objective of this study is to investigate the role and mechanism of circSOD2 in SMC proliferation and neointima formation. Approach and
Results:
Circular RNA profiling of human aortic SMCs revealed that PDGF (platelet-derived growth factor)-BB up- and downregulated numerous circular RNAs. Among them, circSOD2, derived from back-splicing event of SOD2 (superoxide dismutase 2), was significantly enriched. Knockdown of circSOD2 by short hairpin RNA blocked PDGF-BB-induced SMC proliferation. Inversely, circSOD2 ectopic expression promoted SMC proliferation. Mechanistically, circSOD2 acted as a sponge for miR-206, leading to upregulation of NOTCH3 and NOTCH3 signaling, which regulates cyclin D1 and CDK (cyclin-dependent kinase) 4/6. In vivo studies showed that circSOD2 was induced in neointima SMCs in balloon-injured rat carotid arteries. Importantly, knockdown of circSOD2 attenuated injury-induced neointima formation along with decreased neointimal SMC proliferation.
Conclusions
CircSOD2 is a novel regulator mediating SMC proliferation and neointima formation following vascular injury. Therefore, circSOD2 could be a potential therapeutic target for inhibiting the development of proliferative vascular diseases.



Arterioscler Thromb Vasc Biol: 20 Oct 2021:ATVBAHA121316911; epub ahead of print
Mei X, Cui XB, Li Y, Chen SY
Arterioscler Thromb Vasc Biol: 20 Oct 2021:ATVBAHA121316911; epub ahead of print | PMID: 34670409
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Impact:
Abstract

Knockout of Macula Densa Neuronal Nitric Oxide Synthase Increases Blood Pressure in db/db Mice.

Zhang J, Wang X, Cui Y, Jiang S, ... Vallon V, Liu R
Hypertension is a common comorbid condition in patients with diabetes. The pathogenesis of hypertension in diabetes has not been fully clarified. Primary tubular hyperreabsorption may contribute, which may be counteracted by glomerular hyperfiltration in the early diabetic kidney. In this study, we hypothesize that in early diabetes, the macula densa neuronal nitric oxide synthase (NOS1)-derived nitric oxide (NO) production is enhanced, which blunts tubuloglomerular feedback (TGF) response, promotes glomerular hyperfiltration, and maintains normal blood pressure; conversely, insufficient NO generation by the macula densa induces hypertension by lowering glomerular filtration rate and thus inhibiting natriuresis. To test this hypothesis, we examined the changes of macula densa NOS1 expression and phosphorylation as well as NO production, TGF response, glomerular filtration rate, sodium excretion, and blood pressure in a murine model of leptin receptor-deficient (db/db) diabetes with or without macula densa-specific NOS1 deletion. We found that db/db mice presented reduced fractional renal sodium excretion and only a small increase in blood pressure, associated with upregulated expression and activity of macula densa NOS1, inhibited TGF response, and glomerular hyperfiltration. Genetic knockout of macula densa NOS1 restored the TGF response and attenuated glomerular hyperfiltration in db/db mice but also further reduced fractional renal sodium excretion and substantially increased blood pressure. In conclusion, the present study demonstrates that in the early stage of leptin receptor-deficient diabetes, the upregulation of macula densa NOS1 inhibits TGF and increases glomerular filtration rate, which counteracts renal sodium retention and limits the rise in blood pressure.



Hypertension: 17 Oct 2021:HYPERTENSIONAHA12117643; epub ahead of print
Zhang J, Wang X, Cui Y, Jiang S, ... Vallon V, Liu R
Hypertension: 17 Oct 2021:HYPERTENSIONAHA12117643; epub ahead of print | PMID: 34657443
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Impact:
Abstract

The iterative lipid impact on inflammation in atherosclerosis.

Kraaijenhof JM, Hovingh GK, Stroes ESG, Kroon J
Purpose of review
Lipid-mediated atherogenesis is hallmarked by a chronic inflammatory state. Low-density lipoprotein cholesterol (LDL-C), triglyceride rich lipoproteins (TRLs), and lipoprotein(a) [Lp(a)] are causally related to atherosclerosis. Within the paradigm of endothelial activation and subendothelial lipid deposition, these lipoproteins induce numerous pro-inflammatory pathways. In this review, we will outline the effects of lipoproteins on systemic inflammatory pathways in atherosclerosis.
Recent findings
Apolipoprotein B-containing lipoproteins exert a variety of pro-inflammatory effects, ranging from the local artery to systemic immune cell activation. LDL-C, TRLs, and Lp(a) induce endothelial dysfunction with concomitant activation of circulating monocytes through enhanced lipid accumulation. The process of trained immunity of the innate immune system, predominantly induced by LDL-C particles, hallmarks the propagation of the low-grade inflammatory response. In concert, bone marrow activation induces myeloid skewing, further contributing to immune cell mobilization and plaque progression.
Summary
Lipoproteins and inflammation are intertwined in atherogenesis. Elucidating the inflammatory pathways will provide new opportunities for therapeutic agents.

Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.

Curr Opin Lipidol: 30 Sep 2021; 32:286-292
Kraaijenhof JM, Hovingh GK, Stroes ESG, Kroon J
Curr Opin Lipidol: 30 Sep 2021; 32:286-292 | PMID: 34392272
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Impact:
Abstract

Metabolic regulation of macrophage proliferation and function in atherosclerosis.

Patterson MT, Williams JW
Purpose of review
Macrophage accumulation within atherosclerotic plaque is a primary driver of disease progression. However, recent advances in both phenotypic and functional heterogeneity of these cells have allowed for improved insight into potential regulation of macrophage function within lesions. In this review, we will discuss recent insights on macrophage heterogeneity, lipid processing, metabolism, and proliferation in atherosclerosis. Furthermore, we will identify outstanding questions in the field that are pertinent to future studies.
Recent findings
With the recent development of single-cell RNA sequencing, several studies have highlighted the diverse macrophage populations within plaques, including pro-inflammatory, anti-inflammatory, lipid loaded and tissue resident macrophages. Furthermore, new data has suggested that differential activation of metabolic pathways, including glycolysis and fatty acid oxidation, may play a key role in determining function. Recent works have highlighted that different populations retain varying capacity to undergo proliferation; regulating the proliferation pathway may be highly effective in reducing plaque in advanced lesions.
Summary
Macrophage populations within atherosclerosis are highly heterogeneous; differences in cytokine production, lipid handling, metabolism, and proliferation are seen between subpopulations. Understanding the basic cellular mechanisms that drive this heterogeneity will allow for the development of highly specific disease modulating agents to combat atherosclerosis.

Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.

Curr Opin Lipidol: 30 Sep 2021; 32:293-300
Patterson MT, Williams JW
Curr Opin Lipidol: 30 Sep 2021; 32:293-300 | PMID: 34334628
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Impact:
Abstract

Extracellular matrix: paving the way to the newest trends in atherosclerosis.

Gialeli C, Shami A, Gonçalves I
Purpose of review
The extracellular matrix (ECM) is critical for all aspects of vascular pathobiology. In vascular disease the balance of its structural components is shifted. In atherosclerotic plaques there is in fact a dynamic battle between stabilizing and proinflammatory responses. This review explores the most recent strides that have been made to detail the active role of the ECM - and its main binding partners - in driving atherosclerotic plaque development and destabilization.
Recent findings
Proteoglycans-glycosaminoglycans (PGs-GAGs) synthesis and remodelling, as well as elastin synthesis, cross-linking, degradation and its elastokines potentially affect disease progression, providing multiple steps for potential therapeutic intervention and diagnostic targeted imaging. Of note, GAGs biosynthetic enzymes modulate the phenotype of vascular resident and infiltrating cells. In addition, while plaque collagen structure exerts very palpable effects on its immediate surroundings, a new role for collagen is also emerging on a more systemic level as a biomarker for cardiovascular disease as well as a target for selective drug-delivery.
Summary
The importance of studying the ECM in atherosclerosis is more and more acknowledged and various systems are being developed to visualize, target and mimic it.

Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.

Curr Opin Lipidol: 30 Sep 2021; 32:277-285
Gialeli C, Shami A, Gonçalves I
Curr Opin Lipidol: 30 Sep 2021; 32:277-285 | PMID: 34320563
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Impact:
Abstract

Lipids and cardiovascular calcification: contributions to plaque vulnerability.

Hsu JJ, Tintut Y, Demer LL
Purpose of review
Cardiovascular calcification, a common feature of atherosclerotic lesions, has long been known to associate with cardiovascular risk. The roles of lipoproteins in atherosclerosis are also established, and lipid-modifying therapies have shown capacity for plaque regression. However, the association of lipid-modifying therapies with calcification is more complex, and currently no medical therapies have been found to reverse or attenuate calcification in patients. In this review, we summarize recent developments in our understanding of the interplay between lipids and cardiovascular calcification, as well as new imaging modalities for assessing calcified atherosclerotic plaque vulnerability.
Recent findings
Recent clinical studies have highlighted the associations of lipoprotein subtypes, such as low-density and high-density lipoprotein particles, as well as lipoprotein (a) [Lp(a)], with coronary calcification and calcific aortic valve disease. Further, evidence continues to emerge for the utility of fused 18F-sodium fluoride positron-emission tomographic and computed tomographic (18F-NaF PET/CT) imaging in characterizing the microarchitecture and vulnerability of atherosclerotic plaque, in both humans and animal models.
Summary
The relationship between lipids and cardiovascular calcification is complex, and new imaging techniques, such as 18F-NaF PET/CT imaging, may allow for better identification of disease-modifying therapies and prediction of calcified plaque progression and stability to help guide clinical management.

Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.

Curr Opin Lipidol: 30 Sep 2021; 32:308-314
Hsu JJ, Tintut Y, Demer LL
Curr Opin Lipidol: 30 Sep 2021; 32:308-314 | PMID: 34320564
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Impact:
Abstract

HDL and persistent inflammation immunosuppression and catabolism syndrome.

Barker G, Weiner JR, Guirgis FW, Reddy S
Purpose of review
This study reviews the mechanisms of HDL cholesterol immunomodulation in the context of the mechanisms of chronic inflammation and immunosuppression causing persistent inflammation, immunosuppression and catabolism syndrome (PICS) and describes potential therapies and gaps in current research.
Recent findings
Low HDL cholesterol is predictive of acute sepsis severity and outcome. Recent research has indicated apolipoprotein is a prognostic indicator of long-term outcomes. The pathobiologic mechanisms of PICS have been elucidated in the past several years. Recent research of the interaction of HDL pathways in related chronic inflammatory diseases may provide insights into further mechanisms and therapeutic targets.
Summary
HDL significantly influences innate and adaptive immune pathways relating to chronic disease and inflammation. Further research is needed to better characterize these interactions in the setting of PICS.

Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.

Curr Opin Lipidol: 30 Sep 2021; 32:315-322
Barker G, Weiner JR, Guirgis FW, Reddy S
Curr Opin Lipidol: 30 Sep 2021; 32:315-322 | PMID: 34374677
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Impact:
Abstract

LDL receptor-related protein 1 and its interacting partners in tissue homeostasis.

Jaeschke A, Hui DY
Purpose of review
LDL receptor-related protein 1 (LRP1) is a multifunctional protein with endocytic and signal transduction properties due to its interaction with numerous extracellular ligands and intracellular proteins. This brief review highlights key developments in identifying novel functions of LRP1 in liver, lung, and the central nervous system in disease pathogenesis.
Recent findings
In hepatocytes, LRP1 complexes with phosphatidylinositol 4-phosphate 5-kinase-1 and its related protein to maintain intracellular levels of phosphatidylinositol (4,5) bisphosphate and preserve lysosome and mitochondria integrity. In contrast, in smooth muscle cells, macrophages, and endothelial cells, LRP1 interacts with various different extracellular ligands and intracellular proteins in a tissue-dependent and microenvironment-dependent manner to either enhance or suppress inflammation, disease progression or resolution. Similarly, LRP1 expression in astrocytes and oligodendrocyte progenitor cells regulates cell differentiation and maturation in a developmental-dependent manner to modulate neurogenesis, gliogenesis, and white matter repair after injury.
Summary
LRP1 modulates metabolic disease manifestation, inflammation, and differentiation in a cell-dependent, time-dependent, and tissue-dependent manner. Whether LRP1 expression is protective or pathogenic is dependent on its interaction with specific ligands and intracellular proteins, which in turn is dependent on the cell type and the microenvironment where these cells reside.

Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.

Curr Opin Lipidol: 30 Sep 2021; 32:301-307
Jaeschke A, Hui DY
Curr Opin Lipidol: 30 Sep 2021; 32:301-307 | PMID: 34310383
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Impact:
Abstract

Matricellular Protein Cilp1 Promotes Myocardial Fibrosis in Response to Myocardial Infarction.

Zhang QJ, He Y, Li Y, Shen H, ... Yu Y, Liu ZP
Rationale: Cartilage intermediate layer protein 1 (Cilp1) is a secreted extracellular matrix (ECM) protein normally associated with bone and cartilage development. Its function and mechanism of action in adult heart disease remain elusive.Objective: To establish the function and mechanism of action of Cilp1 in post-myocardial infarction (MI) cardiac remodeling.
Methods and results:
We investigated the expression of Cilp1 in mouse models of pathological cardiac remodeling and human heart failure patients. Cilp1 was expressed predominantly in cardiac fibroblasts and upregulated in response to cardiac injury and in the heart and blood of heart failure patients. We generated Cilp1 knock out (KO) and transgenic (Tg) mice with N-terminal half of the protein (NCilp1) overexpressed in myofibroblasts. Cilp1 KO mice had better cardiac function, reduced number of immune cells and myofibroblasts, and enhanced microvascular survival after MI compared to wild-type (WT) littermates. Conversely, NCilp1-Tg mice had augmented loss of cardiac function, increased number of myofibroblasts and infarct size after the MI injury. RNA-seq and gene ontology analysis indicated that cell proliferation and mTORC1 signaling were downregulated in KO hearts compared to WT hearts. In vivo BrdU labeling and immunofluorescence staining showed that myofibroblast proliferation in the Cilp1 KO heart was downregulated. Biaxial mechanical testing and ECM gene expression analysis indicated that while MI caused significant stiffness in WT hearts it had little effect on KO hearts. Upregulation of collagen expression after MI injury was attenuated in KO hearts. Recombinant CILP1 protein or NCilp1-conditioned medium promoted proliferation of neonatal rat ventricular cardiac fibroblasts via the mTORC1 signaling pathway. Conclusions: Our studies established a pathological role of Cilp1 in promoting post-MI remodeling, identified a novel function of Cilp1 in promoting myofibroblast proliferation, and suggested that Cilp1 may serve as a potential biomarker for pathological cardiac remodeling and target for fibrotic heart disease.




Circ Res: 05 Oct 2021; epub ahead of print
Zhang QJ, He Y, Li Y, Shen H, ... Yu Y, Liu ZP
Circ Res: 05 Oct 2021; epub ahead of print | PMID: 34610755
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Impact:
Abstract

ANKRD36 Is Involved in Hypertension by Altering ENaC Genes Expression.

Yan Y, Wang J, Yu L, Cui B, ... Hui R, Wang Y
Rationale: Hypertension is the most important risk factor for cardiovascular and cerebrovascular diseases. Getting deep insight into the pathogenesis of hypertension is necessary. Objective: To investigate the role of ANKRD36 in hypertension.
Methods and results:
We firstly recruited an essential hypertension cohort, and then performed genome-wide transcriptome analysis with peripheral blood mRNA. ANKRD36 (ankyrin repeat domain 36) was found to be significantly lower expressed in hypertension. The anchorin repeat domain mediates a variety of protein-protein interactions. The ENaC genes expression was found up-regulated in HUVECs with ANKRD36 knockdown by using Affymetrix expression profile chip. In HKC and HEK293T cells, ANKRD36 overexpression significantly down-regulated ENaC genes expression, and ANKRD36 knockdown up-regulated their expression. The ChIP assay and YY1 knockdown showed the expression of ENaC was regulated by ANKRD36 via YY1, a dual function transcription factor ubiquitously expressed in human tissues. CO-IP and fluorescence resonance energy transfer assay confirmed the interaction between ANKRD36 and YY1. The nucleo-cytoplasmic ratio of YY1 decreased when ANKRD36 was overexpressed, and also increased when ANKRD36 was knocked down. ANK2 domain of ANKRD36 was critical to its interacting with YY1. Ankrd36 knockout mice showed higher blood pressure levels and Na+ reabsorption, especially when fed with high-salt diet. Higher ENaC genes expression was observed in renal tubular epithelial cells from the knockout mice, and Yy1 knockdown mitigated the alteration. Ankrd36 knockout mice also showed more sensitive response to ENaC inhibitor amiloride treatment. Conclusions: We identified that ANKRD36 was involved in blood pressure regulation by interacting with YY1 and then altering ENaC genes expression. Lower expressed ANKRD36 in hypertension might be a potential therapeutic target, and the application of ENaC inhibitors on hypertension treatment might be extended when serum K+ levels are closely monitored.




Circ Res: 06 Oct 2021; epub ahead of print
Yan Y, Wang J, Yu L, Cui B, ... Hui R, Wang Y
Circ Res: 06 Oct 2021; epub ahead of print | PMID: 34615377
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Impact:
Abstract

Remdesivir induces persistent mitochondrial and structural damage in human induced pluripotent stem cell derived cardiomyocytes.

Kwok M, Lee C, Li HS, Deng R, ... Yan BP, Poon EN
Aims
Remdesivir is a prodrug of an adenosine triphosphate analogue and is currently the only drug formally approved for the treatment of hospitalised COVID-19 patients. Nucleoside/nucleotide analogues have been shown to induce mitochondrial damage and cardiotoxicity, and this may be exacerbated by hypoxia, which frequently occurs in severe COVID-19 patients. Although there have been few reports of adverse cardiovascular events associated with remdesivir, clinical data are limited. Here, we investigated whether remdesivir induced cardiotoxicity using an in vitro human cardiac model.
Methods and results
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were exposed to remdesivir under normoxic and hypoxic conditions to simulate mild and severe COVID-19 respectively. Remdesivir induced mitochondrial fragmentation, reduced redox potential and suppressed mitochondrial respiration at levels below the estimated plasma concentration under both normoxic and hypoxic conditions. Non-mitochondrial damage such as electrophysiological alterations and sarcomere disarray were also observed. Importantly, some of these changes persisted after the cessation of treatment, culminating in increased cell death. Mechanistically, we found that inhibition of DRP1, a regulator of mitochondrial fission, ameliorated the cardiotoxic effects of remdesivir, showing that remdesivir-induced cardiotoxicity was preventable and excessive mitochondrial fission might contribute to this phenotype.
Conclusions
Using an in vitro model, we demonstrated that remdesivir can induce cardiotoxicity in hiPSC-CMs at clinically relevant concentrations. These results reveal previously unknown potential side-effects of remdesivir and highlight the importance of further investigations with in vivo animal models and active clinical monitoring to prevent lasting cardiac damage to patients.
Translational perspective
Adult cardiomyocytes have limited ability to regenerate, thus treatment-induced cardiotoxicity can potentially cause irreparable harm. Remdesivir is currently the only FDA approved treatment for COVID-19 but clinical safety data are limited. Using human pluripotent stem cell-derived cardiomyocytes, we revealed that remdesivir induced persistent mitochondrial and structural abnormalities at clinically relevant concentrations. We advise confirmatory experiments in in vivo animal models, investigations of cardioprotective strategies, and closer patient monitoring such that treatment-induced cardiotoxicity does not contribute to the long term sequelae of COVID-19 patients.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 04 Oct 2021; epub ahead of print
Kwok M, Lee C, Li HS, Deng R, ... Yan BP, Poon EN
Cardiovasc Res: 04 Oct 2021; epub ahead of print | PMID: 34609482
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Impact:
Abstract

Atherosclerosis is a Major Human Killer and Non-resolving Inflammation is a Prime Suspect.

Fredman G, MacNamara KC
The resolution of inflammation (or inflammation-resolution) is an active and highly coordinated process. Inflammation-resolution is governed by several endogenous factors, and specialized pro-resolving mediators (SPMs) are one such class of molecules that have robust biological function. Non-resolving inflammation is associated with a variety of human diseases, including atherosclerosis. Moreover, non-resolving inflammation is a hallmark of aging, an inevitable process associated with increased risk for cardiovascular disease. Uncovering mechanisms as to why inflammation-resolution is impaired in aging and in disease and identifying useful biomarkers for non-resolving inflammation are unmet needs. Recent work has pointed to a critical role for balanced ratios of SPMs and pro-inflammatory lipids (i.e., leukotrienes and/or specific prostaglandins) as a key determinant of timely inflammation resolution. This review will focus on the accumulating findings that support the role of non-resolving inflammation and imbalanced pro-resolving and pro-inflammatory mediators in atherosclerosis. We aim to provide insight as to why these imbalances occur, the importance of aging in disease progression, and how hematopoietic function impacts inflammation-resolution and atherosclerosis. We highlight open questions regarding therapeutic strategies and mechanisms of disease to provide a framework for future studies that aim to tackle this important human disease.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 04 Oct 2021; epub ahead of print
Fredman G, MacNamara KC
Cardiovasc Res: 04 Oct 2021; epub ahead of print | PMID: 34609505
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Impact:
Abstract

Dyslipidemia and aortic valve disease.

Mata P, Alonso R, Pérez de Isla L, Badimón L
Purpose of review
Degenerative aortic stenosis (AS) is one of the most prevalent heart valve diseases in the adult population. The understanding of AS pathophysiology and involved risk factors have recently undergone a great advance, with low-density lipoprotein cholesterol (LDL-C), lipoprotein (a) [Lp(a)] and other clinical conditions taking on a relevant role. Although little is known about the prevention of AS, we can progressively find more evidence of the possible use of drugs to control risk factors as tools that may delay the progression to severe AS and aortic valve replacement.
Recent findings
Several factors have shown to be solid predictors of the development of AS. Mendelian randomization and observational studies on risk factors specifically lipid factors, such as hypercholesterolemia, Lp(a), proprotein convertase subtilisin/kexin type 9 and hypertension have provided meaningful new information. The SAFEHEART study has significantly contributed to define the role of LDL-C and Lp(a) in AS.
Summary
In this review we discuss the interrelationship of dyslipidemia, especially hypercholesterolemia and Lp(a) in the development and prognosis of valvular AS. New imaging tools may contribute to its early detection. Future studies with proprotein convertase subtilisin/kexin type 9 inhibitors and specific therapies to lower Lp(a) might contribute to delay AS development.

Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.

Curr Opin Lipidol: 07 Oct 2021; epub ahead of print
Mata P, Alonso R, Pérez de Isla L, Badimón L
Curr Opin Lipidol: 07 Oct 2021; epub ahead of print | PMID: 34629429
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Abstract

Management of familial hypercholesterolemia in pregnancy.

Graham DF, Raal FJ
Purpose of review
To highlight quandaries and review options for the management of familial hypercholesterolemia (FH) during pregnancy.
Recent findings
Women with FH face barriers to effective care and consequently face significant disease related long term morbidity and mortality.Pregnancy includes major maternal physiological changes resulting in exacerbation of maternal hypercholesterolemia compounded by the current practice of cessation or reduction in the dose of lipid-lowering therapy during pregnancy and lactation that may impact short and long term cardiac morbidity and mortality. Although lipoprotein apheresis is the treatment of choice for high- risk FH patients, reassuring safety evidence for the use of statins during pregnancy is mounting rapidly. However, it will be some time before subtle effects on the development of the offspring can be definitively excluded. Women with homozygous FH or with an established atherosclerotic vessel or aortic disease should be offered therapy with statins during pregnancy if lipoprotein apheresis is not readily available. Pregnancy outcomes tend to be favourable in women with FH. We have reviewed the currently available evidence regarding the risks and benefits of treatment options for FH during pregnancy.

Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.

Curr Opin Lipidol: 05 Oct 2021; epub ahead of print
Graham DF, Raal FJ
Curr Opin Lipidol: 05 Oct 2021; epub ahead of print | PMID: 34619689
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Abstract

Haploinsufficiency of Tmem43 in cardiac myocytes activates the DNA damage response pathway leading to a late-onset senescence-associated pro-fibrotic cardiomyopathy.

Rouhi L, Cheedipudi SM, Chen SN, Fan S, ... Gurha P, Marian AJ
Aims
Arrhythmogenic cardiomyopathy (ACM) encompasses a genetically heterogeneous group of myocardial diseases whose manifestations are sudden cardiac death, cardiac arrhythmias, heart failure, and in a subset fibro-adipogenic infiltration of the myocardium. Mutations in the TMEM43 gene, encoding transmembrane protein 43 (TMEM43) are known to cause ACM. The purpose of the study was to gain insights into the molecular pathogenesis of ACM caused by TMEM43 haploinsufficiency.
Methods and results
The Tmem43 gene was specifically deleted in cardiac myocytes by crossing the Myh6-Cre and floxed Tmem43 mice. Myh6-Cre:Tmem43W/F mice showed an age-dependent phenotype characterized by an increased mortality, cardiac dilatation and dysfunction, myocardial fibrosis, adipogenesis, and apoptosis. Sequencing of cardiac myocyte transcripts prior to and after the onset of cardiac phenotype predicted early activation of the TP53 pathway. Increased TP53 activity was associated with increased levels of markers of DNA damage response (DDR), and a subset of senescence-associated secretary phenotype (SASP). Activation of DDR, TP53, SASP, and their selected downstream effectors, including phospho-SMAD2 and phospho-SMAD3 were validated by alternative methods, including immunoblotting. Expression of SASP was associated with epithelial-mesenchymal transition and age-dependent expression of myocardial fibrosis and apoptosis in the Myh6-Cre:Tmem43W/F mice.
Conclusion
TMEM43 haploinsufficiency is associated with activation of the DDR and the TP53 pathways, which lead to increased expression of SASP and an age-dependent expression of a pro-fibrotic cardiomyopathy. Given that TMEM43 is a nuclear envelope protein and our previous data showing deficiency of another nuclear envelope protein, namely lamin A/C, activates the DDR/TP53 pathway, we surmise that DNA damage is a shared mechanism in the pathogenesis of cardiomyopathies caused by mutations involving nuclear envelope proteins.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 27 Sep 2021; 117:2377-2394
Rouhi L, Cheedipudi SM, Chen SN, Fan S, ... Gurha P, Marian AJ
Cardiovasc Res: 27 Sep 2021; 117:2377-2394 | PMID: 33070193
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Abstract

SMC Derived Hyaluronan Modulates Vascular SMC-Phenotype in Murine Atherosclerosis.

Hartmann F, Gorski DJ, Newman AA, Homann S, ... Owens GK, Fischer JW
Rationale: Plaque instability remains poorly understood and new therapeutic approaches to reduce plaque rupture and subsequent clinical events are of great interest. Recent studies revealed an important role of phenotypic switching of smooth muscle cells (SMC) in controlling plaque stability, including extracellular matrix (ECM) deposition. Objective: The aim of this study was to elucidate the role of hyaluronan (HA) derived from SMC-HA synthase 3 (Has3), in phenotypic switching and plaque stability in an animal model of atherosclerosis.
Methods and results:
A mouse line with SMC-specific deletion of Has3 and simultaneous SMC lineage tracing (eYFP) on an Apoe-/- background was used. Lineage tracing of SMC with eYFP revealed that SMC-specific deletion of Has3 significantly increased the number of galectin-3 (LGALS3+) \"transition-state\" SMC and decreased alpha-smooth muscle actin (ACTA2+) SMC. Notably, SMC-Has3 deletion led to significantly increased collagen deposition and maturation within the fibrous cap (FC) and the whole lesion, as evidenced by Picrosirius red staining and LC-PolScope analysis. Single-cell RNA sequencing (scRNA-seq) of brachiocephalic artery (BCA) lesions demonstrated that the loss of SMC-Has3 enhanced the transition of SMC to an Lgals3+, ECM-producing phenotype with elevated acute-phase response gene expression. Experiments using cultured murine aortic SMC revealed that blocking cluster of differentiation-44 (CD44), an important HA binding receptor, recapitulated the enhanced acute-phase response and synthesis of fibrous ECM. Conclusions: These studies provide evidence that the deletion of SMC-Has3 results in an ECM-producing \"transition state\" SMC phenotype (characterized by LGALS3+ expression), likely via reduced CD44 signaling, resulting in increased collagen formation and maturation, an index consistent with increased plaque stability.




Circ Res: 06 Oct 2021; epub ahead of print
Hartmann F, Gorski DJ, Newman AA, Homann S, ... Owens GK, Fischer JW
Circ Res: 06 Oct 2021; epub ahead of print | PMID: 34615369
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Abstract

Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity.

Basehore S, Bohlman S, Weber C, Swaminathan S, ... Arany Z, Clyne AM
Rationale: In diabetic animals as well as high glucose cell culture conditions, endothelial nitric oxide synthase (eNOS) is heavily O-GlcNAcylated, which inhibits its phosphorylation and nitric oxide (NO) production. It is unknown, however, whether varied blood flow conditions, which affect eNOS phosphorylation, modulate eNOS activity via O-GlcNAcylation-dependent mechanisms. Objective: The goal of this study was to test if steady laminar flow, but not oscillating disturbed flow, decreases eNOS O-GlcNAcylation, thereby elevating eNOS phosphorylation and NO production.
Methods and results:
Human umbilical vein endothelial cells (HUVEC) were exposed to either laminar flow (20 dynes/cm2 shear stress) or oscillating disturbed flow (4{plus minus}6 dynes/cm2 shear stress) for 24 hours in a cone-and-plate device. eNOS O-GlcNAcylation was almost completely abolished in cells exposed to steady laminar but not oscillating disturbed flow. Interestingly, there was no change in protein level or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway (HBP) activity, thereby reducing UDP-GlcNAc pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation, thereby increasing eNOS phosphorylation and NO production. Finally, we detected significantly higher O-GlcNAcylated proteins in endothelium of the inner aortic arch in mice, suggesting that disturbed flow increases protein O-GlcNAcylation in vivo. Conclusions: Our data demonstrate that steady laminar but not oscillating disturbed flow decreases eNOS O-GlcNAcylation by limiting glycolysis and UDP-GlcNAc substrate availability, thus enhancing eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli, relates flow-induced glycolytic reductions to macrovascular disease, and highlights targeting HBP metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent EC dysfunction in cardiovascular disease.




Circ Res: 03 Oct 2021; epub ahead of print
Basehore S, Bohlman S, Weber C, Swaminathan S, ... Arany Z, Clyne AM
Circ Res: 03 Oct 2021; epub ahead of print | PMID: 34605247
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Abstract

Approaches to treat pulmonary arterial hypertension by targeting BMPR2: from cell membrane to nucleus.

Dunmore BJ, Jones RJ, Toshner MR, Upton PD, Morrell NW
Pulmonary arterial hypertension (PAH) is estimated to affect between 10 and 50 people per million worldwide. The lack of cure and devastating nature of the disease means that treatment is crucial to arrest rapid clinical worsening. Current therapies are limited by their focus on inhibiting residual vasoconstriction rather than targeting key regulators of the cellular pathology. Potential disease-modifying therapies may come from research directed towards causal pathways involved in the cellular and molecular mechanisms of disease. It is widely acknowledged that targeting reduced expression of the critical bone morphogenetic protein type-2 receptor and its associated signalling pathways is a compelling therapeutic avenue to explore. In this review, we highlight the advances that have been made in understanding this pathway and the therapeutics that are being tested in clinical trials and the clinic to treat PAH.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 27 Sep 2021; 117:2309-2325
Dunmore BJ, Jones RJ, Toshner MR, Upton PD, Morrell NW
Cardiovasc Res: 27 Sep 2021; 117:2309-2325 | PMID: 33399862
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Abstract

Dynamin-related protein 1 inhibition reduces hepatic PCSK9 secretion.

Rogers MA, Hutcheson JD, Okui T, Goettsch C, ... Aikawa M, Aikawa E
Aims
Proteostasis maintains protein homeostasis and participates in regulating critical cardiometabolic disease risk factors including proprotein convertase subtilisin/kexin type 9 (PCSK9). Endoplasmic reticulum (ER) remodeling through release and incorporation of trafficking vesicles mediates protein secretion and degradation. We hypothesized that ER remodeling that drives mitochondrial fission participates in cardiometabolic proteostasis.
Methods and results
We used in vitro and in vivo hepatocyte inhibition of a protein involved in mitochondrial fission, dynamin-related protein 1 (DRP1). Here, we show that DRP1 promotes remodeling of select ER microdomains by tethering vesicles at ER. A DRP1 inhibitor, mitochondrial division inhibitor 1 (mdivi-1) reduced ER localization of a DRP1 receptor, mitochondrial fission factor, suppressing ER remodeling-driven mitochondrial fission, autophagy, and increased mitochondrial calcium buffering and PCSK9 proteasomal degradation. DRP1 inhibition by CRISPR/Cas9 deletion or mdivi-1 alone or in combination with statin incubation in human hepatocytes and hepatocyte-specific Drp1-deficiency in mice reduced PCSK9 secretion (-78.5%). In HepG2 cells, mdivi-1 increased low-density lipoprotein receptor via c-Jun transcription and reduced PCSK9 mRNA levels via suppressed sterol regulatory binding protein-1c. Additionally, mdivi-1 reduced macrophage burden, oxidative stress, and advanced calcified atherosclerotic plaque in aortic roots of diabetic Apoe-deficient mice and inflammatory cytokine production in human macrophages.
Conclusions
We propose a novel tethering function of DRP1 beyond its established fission function, with DRP1-mediated ER remodeling likely contributing to ER constriction of mitochondria that drives mitochondrial fission. We report that DRP1-driven remodeling of select ER micro-domains may critically regulate hepatic proteostasis and identify mdivi-1 as a novel small molecule PCSK9 inhibitor.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 27 Sep 2021; 117:2340-2353
Rogers MA, Hutcheson JD, Okui T, Goettsch C, ... Aikawa M, Aikawa E
Cardiovasc Res: 27 Sep 2021; 117:2340-2353 | PMID: 33523181
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Abstract

Inducibility, but not stability, of atrial fibrillation is increased by NOX2 overexpression in mice.

Mighiu AS, Recalde A, Ziberna K, Carnicer R, ... Simon JN, Casadei B
Aims
Gp91-containing NADPH oxidases (NOX2) are a significant source of myocardial superoxide production. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, a direct causal role for NOX2 in AF has not been demonstrated. Accordingly, we investigated whether myocardial NOX2 overexpression in mice (NOX2-Tg) is sufficient to generate a favourable substrate for AF and further assessed the effects of atorvastatin, an inhibitor of NOX2, on atrial superoxide production and AF susceptibility.
Methods and results
NOX2-Tg mice showed a 2- to 2.5-fold higher atrial protein content of NOX2 compared with wild-type (WT) controls, which was associated with a significant (twofold) increase in NADPH-stimulated superoxide production (2-hydroxyethidium by HPLC) in left and right atrial tissue homogenates (P = 0.004 and P = 0.019, respectively). AF susceptibility assessed in vivo by transoesophageal atrial burst stimulation was modestly increased in NOX2-Tg compared with WT (probability of AF induction: 88% vs. 69%, respectively; P = 0.037), in the absence of significant alterations in AF duration, surface ECG parameters, and LV mass or function. Mechanistic studies did not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of atrial tissues showed no differences in action potential duration and conduction velocity between genotypes. In addition, we did not observe any genotype difference in markers of fibrosis and inflammation, including atrial collagen content and Col1a1, Il-1β, Il-6, and Mcp-1 mRNA. Similarly, NOX2 overexpression did not have consistent effects on RyR2 Ca2+ leak nor did it affect PKA or CaMKII-mediated RyR2 phosphorylation. Finally, treatment with atorvastatin significantly inhibited atrial superoxide production in NOX2-Tg but had no effect on AF induction in either genotype.
Conclusion
Together, these data indicate that while atrial NOX2 overexpression may contribute to atrial arrhythmogenesis, NOX2-derived superoxide production does not affect the electrical and structural properties of the atrial myocardium.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 27 Sep 2021; 117:2354-2364
Mighiu AS, Recalde A, Ziberna K, Carnicer R, ... Simon JN, Casadei B
Cardiovasc Res: 27 Sep 2021; 117:2354-2364 | PMID: 33483749
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Abstract

Dietary carbohydrates restriction inhibits the development of cardiac hypertrophy and heart failure.

Nakamura M, Odanovic N, Nakada Y, Dohi S, ... Abdellatif M, Sadoshima J
Aims
A diet with modified components, such as a ketogenic low-carbohydrate (LC) diet, potentially extends longevity and healthspan. However, how an LC diet impacts on cardiac pathology during haemodynamic stress remains elusive. This study evaluated the effects of an LC diet high in either fat (Fat-LC) or protein (Pro-LC) in a mouse model of chronic hypertensive cardiac remodelling.
Methods and results
Wild-type mice were subjected to transverse aortic constriction, followed by feeding with the Fat-LC, the Pro-LC, or a high-carbohydrate control diet. After 4 weeks, echocardiographic, haemodynamic, histological, and biochemical analyses were performed. LC diet consumption after pressure overload inhibited the development of pathological hypertrophy and systolic dysfunction compared to the control diet. An anti-hypertrophic serine/threonine kinase, GSK-3β, was re-activated by both LC diets; however, the Fat-LC, but not the Pro-LC, diet exerted cardioprotection in GSK-3β cardiac-specific knockout mice. β-hydroxybutyrate, a major ketone body in mammals, was increased in the hearts of mice fed the Fat-LC, but not the Pro-LC, diet. In cardiomyocytes, ketone body supplementation inhibited phenylephrine-induced hypertrophy, in part by suppressing mTOR signalling.
Conclusion
Strict carbohydrate restriction suppresses pathological cardiac growth and heart failure after pressure overload through distinct anti-hypertrophic mechanisms elicited by supplemented macronutrients.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 27 Sep 2021; 117:2365-2376
Nakamura M, Odanovic N, Nakada Y, Dohi S, ... Abdellatif M, Sadoshima J
Cardiovasc Res: 27 Sep 2021; 117:2365-2376 | PMID: 33070172
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Abstract

Cardiac Resident Macrophages Prevent Fibrosis and Stimulate Angiogenesis.

Revelo X, Parthiban P, Chen C, Barrow F, ... Herman A, van Berlo JH
Rationale: The initial hypertrophy response to cardiac pressure overload is considered compensatory, but with sustained stress, it eventually leads to heart failure. Recently, a role for recruited macrophages (mψs) in determining the transition from compensated to decompensated hypertrophy has been established. However, whether cardiac-resident immune cells influence the early phase of hypertrophy development has not been established.Objective:To assess the role of cardiac immune cells in the early hypertrophy response to cardiac pressure overload-induced by transverse aortic constriction (TAC).
Methods and results:
We performed cytometry-by-time-of-flight to determine the identity and abundance of immune cells in the heart at 1 and 4 weeks after TAC. We observed a substantial increase in cardiac mψs 1 week after TAC. We then conducted Cite-Seq single-cell RNA sequencing of cardiac immune cells isolated from 4 sham and 6 TAC hearts. We identified 12 clusters of monocytes and mψs, categorized as either resident or recruited mψs, that showed remarkable changes in their abundance between sham and TAC conditions. To determine the role of cardiac-resident mψs early in the response to a hypertrophic stimulus, we used a blocking antibody against macrophage colony-stimulating factor 1 receptor (CD115). As blocking CD115 initially depletes all macrophages, we allowed the replenishment of recruited mψs by monocytes before performing TAC. This preferential depletion of resident mψs resulted in enhanced fibrosis and a blunted angiogenesis response to TAC. Mψ-depletion in CCR2 knockout mice showed that aggravated fibrosis was primarily caused by the recruitment of monocyte-derived mψs. Finally, 6 weeks after TAC these early events lead to depressed cardiac function and enhanced fibrosis, despite complete restoration of cardiac immune cells.<Conclusions: Cardiac resident mψs are a heterogeneous population of immune cells with key roles in stimulating angiogenesis and inhibiting fibrosis in response to cardiac pressure overload.




Circ Res: 13 Oct 2021; epub ahead of print
Revelo X, Parthiban P, Chen C, Barrow F, ... Herman A, van Berlo JH
Circ Res: 13 Oct 2021; epub ahead of print | PMID: 34645281
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Abstract

Cardiac SARS-CoV-2 infection is associated with pro-inflammatory transcriptomic alterations within the heart.

Bräuninger H, Stoffers B, Fitzek ADE, Meißner K, ... Westermann D, Lindner D
Aims
Cardiac involvement in COVID-19 is associated with adverse outcome. However, it is unclear whether cell specific consequences are associated with cardiac SARS-CoV-2 infection. Therefore, we investigated heart tissue utilizing in situ hybridization, immunohistochemistry and RNA-sequencing in consecutive autopsy cases to quantify virus load and characterize cardiac involvement in COVID-19.
Methods and results
In this study, 95 SARS-CoV-2-positive autopsy cases were included. A relevant SARS-CoV-2 virus load in the cardiac tissue was detected in 41/95 deceased (43%). MACE-RNA-sequencing was performed to identify molecular pathomechanisms caused by the infection of the heart. A signature matrix was generated based on the single-cell dataset \"Heart Cell Atlas\" and used for digital cytometry on the MACE-RNA-sequencing data. Thus, immune cell fractions were estimated and revealed no difference in immune cell numbers in cases with and without cardiac infection. This result was confirmed by quantitative immunohistological diagnosis.MACE-RNA-sequencing revealed 19 differentially expressed genes (DEGs) with a q-value <0.05 (e.g. up: IFI44L, IFT3, TRIM25; down: NPPB, MB, MYPN). The upregulated DEGs were linked to interferon pathways and originate predominantly from endothelial cells. In contrast, the downregulated DEGs originate predominately from cardiomyocytes. Immunofluorescent staining showed viral protein in cells positive for the endothelial marker ICAM1 but rarely in cardiomyocytes. The GO term analysis revealed that downregulated GO terms were linked to cardiomyocyte structure, whereas upregulated GO terms were linked to anti-virus immune response.
Conclusion
This study reveals, that cardiac infection induced transcriptomic alterations mainly linked to immune response and destruction of cardiomyocytes. While endothelial cells are primarily targeted by the virus, we suggest cardiomyocyte-destruction by paracrine effects. Increased pro-inflammatory gene expression was detected in SARS-CoV-2-infected cardiac tissue but no increased SARS-CoV-2 associated immune cell infiltration was observed.
Translational perspective
Cardiac injury can be documented in COVID-19, regardless the direct cardiac virus infection and is known to be associated with outcome. However, the direct virus infection of the myocardium leads to transcriptomic alterations and might therefore additionally contribute to pathophysiological processes in COVID-19. Therefore, consequences of cardiac virus infection need to be investigated in future studies, since they might also contribute to long-term effects in case of survival.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 13 Oct 2021; epub ahead of print
Bräuninger H, Stoffers B, Fitzek ADE, Meißner K, ... Westermann D, Lindner D
Cardiovasc Res: 13 Oct 2021; epub ahead of print | PMID: 34647998
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Abstract

MMP-2 knockdown blunts age-dependent carotid stiffness by decreasing elastin degradation and augmenting eNOS activation.

Diaz-Canestro C, Puspitasari YM, Liberale L, Guzik TJ, ... Sudano I, Camici GG
Aims
Arterial stiffness is a hallmark of vascular aging that precedes and strongly predicts the development of cardiovascular diseases. Age-dependent stiffening of large elastic arteries is primarily attributed to increased levels of matrix metalloproteinase-2 (MMP-2). However, the mechanistic link between age-dependent arterial stiffness and MMP-2 remains unclear. Thus, we aimed to investigate the efficacy of MMP-2 knockdown using small interfering RNA (siRNA) on age-dependent arterial stiffness.
Methods and results
Pulse wave velocity (PWV) was assessed in right carotid artery of wild type (WT) mice from different age groups. MMP-2 levels in the carotid artery and plasma of young (3 months) and old (20-25 months) WT mice were determined. Carotid PWV as well as vascular and circulating MMP-2 were elevated with increasing age in mice. Old WT mice (18-21-month-old) were treated for 4 weeks with either MMP-2 or scrambled (Scr) siRNA via tail vein injection. Carotid PWV was assessed at baseline, 2 and 4 weeks after start of the treatment. MMP-2 knockdown reduced vascular MMP-2 levels and attenuated age-dependent carotid stiffness. siMMP-2 treated mice showed increased elastin to collagen ratio, lower plasma desmosine (DES), enhanced phosphorylation of endothelial nitric oxide synthase (eNOS) and higher levels of vascular cyclic guanosine monophosphate (cGMP). An age-dependent increase in direct protein-protein interaction between MMP-2 and eNOS was also observed. Lastly, DES, an elastin breakdown product, was measured in a patient cohort (n = 64, 23-86 years old), where carotid-femoral PWV was also assessed; here, plasma levels of DES directly correlated with age and arterial stiffness.
Conclusion
MMP-2 knockdown attenuates age-dependent carotid stiffness by blunting elastin degradation and augmenting eNOS bioavailability. Given the increasing clinical use of siRNA technology, MMP2 knockdown should be investigated further as a possible strategy to mitigate age-dependent arterial stiffness and related CV diseases.
Translational perspective
Arterial stiffness is a hallmark of vascular aging that precedes and strongly predicts the development of cardiovascular diseases. This study provides translational evidence to support a key role for MMP-2 on the development of age-associated arterial stiffness. Silencing of MMP-2 using siRNA technology shows an effect on aged mice where it attenuates age-dependent carotid stiffness by reducing elastin degradation and increasing eNOS bioavailability. Additionally, in humans we show that elastin breakdown increases with age and increased PWV. These findings indicate MMP-2 knockdown as a promising novel strategy to attenuate age-dependent arterial stiffness and cardiovascular diseases.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 28 Sep 2021; epub ahead of print
Diaz-Canestro C, Puspitasari YM, Liberale L, Guzik TJ, ... Sudano I, Camici GG
Cardiovasc Res: 28 Sep 2021; epub ahead of print | PMID: 34586381
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Abstract

Genetic inhibition of Nuclear Factor of Activated T-cell c2 (NFATc2) prevents atrial fibrillation in CREM transgenic mice.

Ni L, Lahiri SK, Nie J, Pan X, ... Dobrev D, Wehrens XHT
Aims
Abnormal intracellular calcium handling contributes to the progressive nature of atrial fibrillation (AF), the most common sustained cardiac arrhythmia. Evidence in mouse models suggests that activation of the nuclear factor of activated T-cell (NFAT) signaling pathway contributes to atrial remodeling. Our aim was to determine the role of NFATc2 in AF in humans and mouse models.
Methods and results
Expression levels of NFATc1-c4 isoforms were assessed by quantitative reverse transcription-polymerase chain reaction in right atrial appendages from patients with chronic AF. NFATc1 and NFATc2 mRNA levels were elevated in chronic AF (cAF) patients compared with those in sinus rhythm (SR). Western blotting revealed increased cytosolic and nuclear levels of NFATc2 in AF patients. Similar findings were obtained in CREM-IbΔC-X transgenic (CREM) mice, a model of progressive AF. Telemetry ECG recordings revealed age-dependent spontaneous AF in CREM mice, which was prevented by NFATc2 knockout in CREM: NFATc2-/- mice. Programmed electrical stimulation revealed that CREM: NFATc2-/- mice lacked an AF substrate. Morphometric analysis and histology revealed increased atrial weight and atrial fibrosis in CREM mice compared with WT controls, which was reversed in CREM: NFATc2-/- mice. Confocal microscopy showed an increased Ca2+ spark frequency despite a reduced sarcoplasmic reticulum (SR) Ca2+ load in CREM mice compared with controls, whereas these abnormalities were normalized in CREM: NFATc2-/- mice. Western blotting revealed that genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of S2814 on RyR2 in CREM: RyR2-S2814A mice suppressed NFATc2 activation observed in CREM mice, suggesting that NFATc2 is activated by excessive SR Ca2+ leak via RyR2. Finally, chromatin immunoprecipitation sequencing from AF patients identified Ras And EF-Hand Domain-Containing Protein (RASEF) as a direct target of NFATc2 mediated transcription.
Conclusion
Our findings reveal activation of the NFAT signaling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model.
Translational perspective
Atrial fibrillation (AF) is a progressive disease characterized by electrical and structural remodeling which promotes atrial arrhythmias. This study provides evidence for increased \'nuclear factor of activated T-cell\' (NFAT) signaling in patients with chronic AF. Studies in the CREM transgenic model of progressive AF revealed that the NFATc2 isoform mediates atrial remodeling associated with AF substrate development. Chromatin immunoprecipitation sequencing of atrial biopsies from AF patients identified \'Ras And EF-Hand Domain-Containing Protein\' (RASEF) as a downstream target of NFATc2-mediated transcription, suggesting that targeting these factors might be beneficial for curtailing AF progression.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 13 Oct 2021; epub ahead of print
Ni L, Lahiri SK, Nie J, Pan X, ... Dobrev D, Wehrens XHT
Cardiovasc Res: 13 Oct 2021; epub ahead of print | PMID: 34648001
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Abstract

Recent dynamic studies of the metabolism of atherogenic lipoproteins: elucidating the mode of action of new therapies.

Chan DC, Ying Q, Watts GF
Purpose of review
LDL, triglyceride-rich lipoprotein (TRL) and lipoprotein(a) [Lp(a)] particles are the key atherogenic lipoproteins. Deranged metabolism of these lipoproteins accounts for a spectrum of clinically important dyslipidemias, such as FH, elevated Lp(a) and diabetic dyslipidemia. We review the findings from recent dynamic and tracer studies that have contributed to expanding knowledge in this field.
Recent findings
Deficiency in LDL receptor activity does not only impair the catabolism of LDL-apoB-100 in FH, but also induces hepatic overproduction and decreases catabolism of TRLs. Patients with elevated Lp(a) are characterized by increased hepatic secretion of Lp(a) particles. Elevation of TRLs in diabetes is partly mediated by increased production of apoB-48 and apoC-III, and impaired clearance of apoB-48 in the postprandial state. Tracer kinetic studies show that proprotein convertase subtilisin/kexin type 9 mAbs alone or in combination with statin can increase the catabolism and decrease production of LDL and Lp(a) particles. By contrast, angiopoietin-like protein 3 inhibitors (e.g. evinacumab) reduce VLDL production and increase LDL clearance in FH. Glucagon-like peptide-1 receptor agonists can improve diabetic dyslipidemia by increasing the catabolism of apoB-48 and decreasing the production of apoB-48 and apoC-III.
Summary
Dynamic studies of the metabolism of atherogenic lipoproteins provide new insight into the nature of dyslipidemias and point to how new therapies with complementary modes of action may have maximal clinical impact.

Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.

Curr Opin Lipidol: 10 Oct 2021; epub ahead of print
Chan DC, Ying Q, Watts GF
Curr Opin Lipidol: 10 Oct 2021; epub ahead of print | PMID: 34636776
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Abstract

Truncated YY1 interacts with BASP1 through a 339KLK341 motif in YY1 and suppresses vascular smooth muscle cell growth and intimal hyperplasia after vascular injury.

Santiago FS, Li Y, Zhong L, Raftery MJ, Lins L, Khachigian LM
Aims
In-stent restenosis and late stent thrombosis are complications associated with the use of metallic and drug-coated stents. Strategies that inhibit vascular smooth muscle cell (SMC) proliferation without affecting endothelial cell (EC) growth would be helpful in reducing complications arising from percutaneous interventions. SMC hyperplasia is also a pathologic feature of graft stenosis and fistula failure. Our group previously showed that forced expression of the injury-inducible zinc finger (ZNF) transcription factor, yin yang-1 (YY1), comprising 414 residues inhibits neointima formation in carotid arteries of rabbits and rats. YY1 inhibits SMC proliferation without affecting EC growth in vitro. Identifying a shorter version of YY1 retaining cell-selective inhibition would make it more amenable for potential use as a gene therapeutic agent.
Methods and results
We dissected YY1 into a range of shorter fragments (YY1A-D, YY1Δ) and found that the first two ZNFs in YY1 (construct YY1B, spanning 52 residues) repressed SMC proliferation. Receptor binding domain analysis predicts a three-residue (339KLK341) interaction domain. Mutation of 339KLK341 to 339AAA341 in YY1B (called YY1Bm) abrogated YY1B\'s ability to inhibit SMC but not EC proliferation and migration. Incubation of recombinant GST-YY1B and GST-YY1Bm with SMC lysates followed by precipitation with glutathione-agarose beads and mass spectrometric analysis identified a novel interaction between YY1B and BASP1. Overexpression of BASP1, like YY1, inhibited SMC but not EC proliferation and migration. BASP1 siRNA partially rescued SMC from growth inhibition by YY1B. In the rat carotid balloon injury model, adenoviral overexpression of YY1B, like full-length YY1, reduced neointima formation, whereas YY1Bm had no such effect. CD31+ immunostaining suggested YY1B could increase re-endothelialization in a 339KLK341-dependent manner.
Conclusion
These studies identify a truncated form of YY1 (YY1B) that can interact with BASP1 and inhibit SMC proliferation, migration, and intimal hyperplasia after balloon injury of rat carotid arteries as effectively as full length YY1. We demonstrate the therapeutic potential of YY1B in vascular proliferative disease.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 27 Sep 2021; 117:2395-2406
Santiago FS, Li Y, Zhong L, Raftery MJ, Lins L, Khachigian LM
Cardiovasc Res: 27 Sep 2021; 117:2395-2406 | PMID: 33508088
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Abstract

Vascular smooth muscle cells in atherosclerosis: time for a re-assessment.

Grootaert MOJ, Bennett MR
Vascular smooth muscle cells (VSMCs) are key participants in both early and late-stage atherosclerosis. VSMCs invade the early atherosclerotic lesion from the media, expanding lesions, but also forming a protective fibrous cap rich in extracellular matrix to cover the \'necrotic\' core. Hence, VSMCs have been viewed as plaque-stabilizing, and decreased VSMC plaque content-often measured by expression of contractile markers-associated with increased plaque vulnerability. However, the emergence of lineage-tracing and transcriptomic studies has demonstrated that VSMCs comprise a much larger proportion of atherosclerotic plaques than originally thought, demonstrate multiple different phenotypes in vivo, and have roles that might be detrimental. VSMCs down-regulate contractile markers during atherosclerosis whilst adopting alternative phenotypes, including macrophage-like, foam cell-like, osteochondrogenic-like, myofibroblast-like, and mesenchymal stem cell-like. VSMC phenotypic switching can be studied in tissue culture, but also now in the media, fibrous cap and deep-core region, and markedly affects plaque formation and markers of stability. In this review, we describe the different VSMC plaque phenotypes and their presumed cellular and paracrine functions, the regulatory mechanisms that control VSMC plasticity, and their impact on atherogenesis and plaque stability.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 27 Sep 2021; 117:2326-2339
Grootaert MOJ, Bennett MR
Cardiovasc Res: 27 Sep 2021; 117:2326-2339 | PMID: 33576407
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Impact:
Abstract

Novel Mechanisms of Exosome-Mediated Phagocytosis of Dead Cells in Injured Heart.

Patil M, Saheera S, Dubey PK, Kahn-Krell A, ... Qin G, Krishnamurthy P
Rationale: After myocardial ischemic injury, improper phagocytic clearance of dying cardiac cells and the ensuing lack of inflammation resolution results in adverse cardiac remodeling and dysfunction that might lead to heart failure. Therefore, therapeutic strategies to ameliorate immune cell phagocytic function is critical for augmenting cardiac repair after injury. Objective: To determine if mesenchymal stem cell-derived exosomes (MSC-Exo) act as opsonin for apoptotic cells and/or trigger \"eat me\" phagocytic signaling in resident/recruited phagocytes after myocardial ischemic injury.
Methods and results:
We evaluated MSC-Exo-mediated opsonization of apoptotic cardiomyocytes; and invitro and invivo effects of milk fat globule- epidermal growth factor-factor VIII (MFGE8)-deficient mouse MSC-Exo on macrophage engulfment of apoptotic cardiomyocytes and its implications on cardiac remodeling, repair and function. Microscopy and FACS analyses show that opsonization of apoptotic cardiomyocytes with MSC-Exo enhances their engulfment by macrophages. Furthermore, pre-incubation of macrophages with MSC-Exo reprogrammed the signaling pathways involved in phagocytosis and expression of pro-reparative cytokines. Protein analysis of MSC-Exo reveals expression of MFGE8, a glycoprotein which bridges externalized phosphatidylserine (PS) on the apoptotic cell surface to alphaVbeta3 or alphaVbeta5 integrins on the phagocyte. Most intriguingly, siRNA inhibition of MFGE8 significantly reduced the MSC-Exo-mediated augmentation of dead cell engulfment, associated signaling and pro-reparative phenotype. After myocardial ischemic injury, intramyocardial administration of MSC-Exo increases macrophage uptake of apoptotic bodies in the border zone of infarct and is associated with reduced proinflammatory response, increase in neovascularization, lower infarct size and an improvement in cardiac function and MFGE8-deficient MSC-Exo administration failed to protect mice against MI. Conclusions: Our data demonstrates that exosome-associated MFGE8 on one hand enhances opsonization of dead cells and on the other activates phagocytic signaling thus augmenting removal of apoptotic cells, resolution of inflammation and therefore efficient cardiac recovery after injury.




Circ Res: 07 Oct 2021; epub ahead of print
Patil M, Saheera S, Dubey PK, Kahn-Krell A, ... Qin G, Krishnamurthy P
Circ Res: 07 Oct 2021; epub ahead of print | PMID: 34623174
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Abstract

Temporal relationship between systemic endothelial dysfunction and alterations in erythrocyte function in a murine model of chronic heart failure.

Mohaissen T, Proniewski B, Targosz-Korecka M, Bar A, ... Marzec KM, Chlopicki S
Aims
Endothelial dysfunction (ED) and red blood cell distribution width (RDW) are both prognostic factors in heart failure (HF), but the relationship between them is not clear. In this study, we used a unique mouse model of chronic HF driven by cardiomyocyte-specific overexpression of activated Gαq protein (Tgαq*44 mice) to characterise the relationship between the development of peripheral ED and the occurrence of structural nanomechanical and biochemical changes in red blood cells (RBCs).
Methods and results
Systemic ED was detected in vivo in 8-month-old Tgαq*44 mice, as evidenced by impaired acetylcholine-induced vasodilation in the aorta and increased endothelial permeability in the brachiocephalic artery. ED in the aorta was associated with impaired nitric oxide (NO) production in the aorta and diminished systemic NO bioavailability. ED in the aorta was also characterised by increased superoxide and eicosanoid production. In 4- to 6-month-old Tgαq*44 mice, RBC size and membrane composition displayed alterations that did not result in significant changes in their nanomechanical and functional properties. However, 8-month-old Tgαq*44 mice presented greatly accentuated structural and size changes and increased RBC stiffness. In 12-month-old Tgαq*44 mice, the erythropathy was featured by severely altered RBC shape and elasticity, increased RDW, impaired RBC deformability, and increased oxidative stress (GSH/GSSH ratio). Moreover, RBCs taken from 12-month-old Tgαq*44 mice, but not from 12-month-old FVB mice, co-incubated with aortic rings from FVB mice, induced impaired endothelium-dependent vasodilation and this effect was partially reversed by an arginase inhibitor (ABH, 2(S)-amino-6-boronohexanoic acid).
Conclusion
In the Tgαq*44 murine model of HF, systemic endothelial dysfunction accelerates erythropathy and, conversely, erythropathy may contribute to endothelial dysfunction. These results suggest that erythropathy may be regarded as a marker and a mediator of systemic endothelial dysfunction in HF. In particular, targeting RBC arginase may represent a novel treatment strategy for systemic endothelial dysfunction in HF. RBC arginase and possibly other RBC-mediated mechanisms may represent novel therapeutic targets for systemic endothelial dysfunction in HF.
Translational perspective
Endothelial dysfunction (ED) and red blood cell distribution width (RDW) both have prognostic value for heart failure (HF), but it is not known whether these pathologies are related. We comprehensively characterized endothelial and RBC functional status in a unique murine model of chronic heart failure with a prolonged time course of HF progression. Our results suggest that ED accelerates erythropathy and, conversely, erythropathy may contribute to ED. Accordingly, erythropathy in HF reflects ED and involves various changes (in functional, structural, nanomechanical, and biochemical levels) that could have diagnostic and therapeutic significance for HF.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 06 Oct 2021; epub ahead of print
Mohaissen T, Proniewski B, Targosz-Korecka M, Bar A, ... Marzec KM, Chlopicki S
Cardiovasc Res: 06 Oct 2021; epub ahead of print | PMID: 34617995
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Impact:
Abstract

Integration of multiple imaging platforms to uncover cardiovascular defects in adult zebrafish.

Bensimon-Brito A, Boezio GLM, Cardeira-da-Silva J, Wietelmann A, ... Mueller C, Stainier DYR
Aims
Mammalian models have been instrumental in investigating adult heart function and human disease. However, electrophysiological differences with human hearts and high costs motivate the need for non-mammalian models. The zebrafish is a well-established genetic model to study cardiovascular development and function; however, analysis of cardiovascular phenotypes in adult specimens is particularly challenging as they are opaque.
Methods and results
Here, we optimized and combined multiple imaging techniques including echocardiography, magnetic resonance imaging, and micro-computed tomography to identify and analyze cardiovascular phenotypes in adult zebrafish. Using alk5a/tgfbr1a mutants as a case study, we observed morphological and functional cardiovascular defects that were undetected with conventional approaches. Correlation analysis of multiple parameters revealed an association between hemodynamic defects and structural alterations of the heart, as observed clinically.
Conclusion
We report a new, comprehensive, and sensitive platform to identify otherwise indiscernible cardiovascular phenotypes in adult zebrafish.
Translational perspective
This study further illustrates the importance of the zebrafish model to investigate cardiovascular phenotypes including morphological and functional alterations as observed in human disease settings.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 04 Oct 2021; epub ahead of print
Bensimon-Brito A, Boezio GLM, Cardeira-da-Silva J, Wietelmann A, ... Mueller C, Stainier DYR
Cardiovasc Res: 04 Oct 2021; epub ahead of print | PMID: 34609500
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Impact:
Abstract

Cardiotoxicities of novel cancer immunotherapies.

Stein-Merlob AF, Rothberg MV, Ribas A, Yang EH
Immunotherapy revolutionised oncology by harnessing the native immune system to effectively treat a wide variety of malignancies even at advanced stages. Off-target immune activation leads to immune-related adverse events affecting multiple organ systems, including the cardiovascular system. In this review, we discuss the current literature describing the epidemiology, mechanisms and proposed management of cardiotoxicities related to immune checkpoint inhibitors (ICIs), chimeric antigen receptor (CAR) T-cell therapies and bispecific T-cell engagers. ICIs are monoclonal antibody antagonists that block a co-inhibitory pathway used by tumour cells to evade a T cell-mediated immune response. ICI-associated cardiotoxicities include myocarditis, pericarditis, atherosclerosis, arrhythmias and vasculitis. ICI-associated myocarditis is the most recognised and potentially fatal cardiotoxicity with mortality approaching 50%. Recently, ICI-associated dysregulation of the atherosclerotic plaque immune response with prolonged use has been linked to early progression of atherosclerosis and myocardial infarction. Treatment strategies include immunosuppression with corticosteroids and supportive care. In CAR T-cell therapy, autologous T cells are genetically engineered to express receptors targeted to cancer cells. While stimulating an effective tumour response, they also elicit a profound immune reaction called cytokine release syndrome (CRS). High-grade CRS causes significant systemic abnormalities, including cardiovascular effects such as arrhythmias, haemodynamic compromise and cardiomyopathy. Treatment with interleukin-6 inhibitors and corticosteroids is associated with improved outcomes. The evidence shows that, although uncommon, immunotherapy-related cardiovascular toxicities confer significant risk of morbidity and mortality and benefit from rapid immunosuppressive treatment. As new immunotherapies are developed and adopted, it will be imperative to closely monitor for cardiotoxicity.

© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.

Heart: 30 Oct 2021; 107:1694-1703
Stein-Merlob AF, Rothberg MV, Ribas A, Yang EH
Heart: 30 Oct 2021; 107:1694-1703 | PMID: 33722826
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Impact:
Abstract

Familial combined hyperlipidemia is a polygenic trait.

Gill PK, Hegele RA
Purpose of review
: Familial combined hyperlipidemia (FCH), defined by concurrently elevated plasma triglyceride (TG) and low-density lipoprotein (LDL) cholesterol, has long been investigated to characterize its genetic basis. Despite almost half a century of searching, a single gene cause for the phenotype has not yet been identified.
Recent findings
: Recent studies using next-generation genetic analytic methods confirm that FCH has a polygenic basis, with a clear large contribution from the accumulation of small-to-moderate effect common single nucleotide polymorphisms (SNPs) throughout the genome that is associated with raising TG, and probably also those raising LDL cholesterol. On the other hand, rare monogenic variants, such as those causing familial hypercholesterolemia, play a negligible role, if any. Genetic profiling suggests that patients with FCH and hypertriglyceridemia share a strong polygenic basis and show a similar profile of multiple TG-raising common SNPs.
Summary
: Recent progress in genomics has shown that most if not all of the genetic susceptibility to FCH is polygenic in nature. Future research should include larger cohort studies, with wider ancestral diversity, ancestry-specific polygenic scores, and investigation of epigenetic and lifestyle factors to help further elucidate the causative agents at play in cases where the genetic etiology remains to be defined.

Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.

Curr Opin Lipidol: 21 Oct 2021; epub ahead of print
Gill PK, Hegele RA
Curr Opin Lipidol: 21 Oct 2021; epub ahead of print | PMID: 34690300
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Impact:
Abstract

Interferon stimulated gene 15 pathway is a novel mediator of endothelial dysfunction and aneurysms development in angiotensin II infused mice through increased oxidative stress.

González-Amor M, García-Redondo AB, Jorge I, Zalba G, ... Salaices M, Briones AM
Aims
Interferon-stimulated gene 15 (ISG15) encodes an ubiquitin-like protein that induces a reversible post-translational modification (ISGylation) and can also be secreted as a free form. ISG15 plays an essential role as host-defense response to microbial infection; however, its contribution to vascular damage associated to hypertension is unknown.
Methods and results
Bioinformatics identified ISG15 as a mediator of hypertension-associated vascular damage. ISG15 expression positively correlated with systolic and diastolic blood pressure and carotid intima-media thickness in human peripheral blood mononuclear cells. Consistently, Isg15 expression was enhanced in aorta from hypertension models and in angiotensinII (AngII)-treated vascular cells and macrophages. Proteomics revealed differential expression of proteins implicated in cardiovascular function, extracellular matrix and remodeling, and vascular redox state in aorta from AngII-infused ISG15-/- mice. Moreover, ISG15-/- mice were protected against AngII-induced hypertension, vascular stiffness, elastin remodeling, endothelial dysfunction, and expression of inflammatory and oxidative stress markers. Conversely, mice with excessive ISGylation (USP18C61A) show enhanced AngII-induced hypertension, vascular fibrosis, inflammation and reactive oxygen species (ROS) generation along with elastin breaks, aortic dilation and rupture. Accordingly, human and murine abdominal aortic aneurysms showed augmented ISG15 expression. Mechanistically, ISG15 induces vascular ROS production, while antioxidant treatment prevented ISG15-induced endothelial dysfunction and vascular remodeling.
Conclusion
ISG15 is a novel mediator of vascular damage in hypertension through oxidative stress and inflammation.
Translational perspective
Recent evidence from randomized clinical trials demonstrate the effectiveness of specific anti-inflammatory treatments in cardiovascular prevention. In this study we have identified a new inflammatory mediator involved in vascular damage in experimental and human hypertension and aneurysms. We found that interferon stimulated gene 15 (ISG15) is increased at the vascular level in animal models of hypertension and aneurysms. More importantly, ISG15 correlates with human hypertension, vascular remodeling, and aneurysms presence. Underlying mechanisms responsible for vascular damage induced by ISG15 include oxidative and inflammation. Our results further support the role of inflammation in vascular damage in different cardiovascular pathologies.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 20 Oct 2021; epub ahead of print
González-Amor M, García-Redondo AB, Jorge I, Zalba G, ... Salaices M, Briones AM
Cardiovasc Res: 20 Oct 2021; epub ahead of print | PMID: 34672341
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Impact:
Abstract

SR-mitochondria crosstalk shapes Ca signaling to impact pathophenotype in disease models marked by dysregulated intracellular Ca release.

Tow BD, Deb A, Neupane S, Patel S, ... Györke S, Liu B
Aims
Diastolic Ca release (DCR) from sarcoplasmic reticulum (SR) Ca release channel ryanodine receptor (RyR2) has been linked to multiple cardiac pathologies, but its exact role in shaping divergent cardiac pathologies remains unclear. We hypothesize that the SR-mitochondria interplay contributes to disease phenotypes by shaping Ca signaling.
Methods and results
A genetic model of catecholaminergic polymorphic ventricular tachycardia (CPVT2 model of CASQ2 knockout) and a pre-diabetic cardiomyopathy model of fructose fed mice (FFD), both marked by DCR, are employed in this study. Mitochondria Ca (mCa) is modulated by pharmacologically targeting mitochondria Ca uniporter (MCU) or permeability transition pore (mPTP), mCa uptake and extrusion mechanisms, respectively. An MCU activator abolished Ca waves in CPVT2 but exacerbated waves in FFD cells. Mechanistically this is ascribed to mitochondria\'s function as a Ca buffer or source of reactive oxygen species (mtROS) to exacerbate RyR2 functionality, respectively. Enhancing mCa uptake reduced and elevated mtROS production in CPVT2 and FFD respectively. In CPVT2, mitochondria took up more Ca in permeabilized cells, and had higher level of mCa content in intact cells vs FFD. Conditional ablation of MCU in the CPVT2 model caused lethality and cardiac remodeling, but reduced arrhythmias in the FFD model. In parallel, CPVT2 mitochondria also employ upregulated mPTP-mediated Ca efflux to avoid mCa overload, as seen by elevated incidence of MitoWinks (an indicator of mPTP-mediated Ca efflux) vs FFD. Both pharmacological and genetic inhibition of mPTP promoted mtROS production and exacerbation of myocyte Ca handling in CPVT2. Further, genetic inhibition of mPTP exacerbated arrhythmias in CPVT2.
Conclusion
In contrast to FFD, which is more susceptible to mtROS-dependent RyR2 leak, in CPVT2 mitochondria buffer SR-derived DCR to mitigate Ca-dependent pathological remodeling and rely on mPTP-mediated Ca efflux to avoid mCa overload. SR-mitochondria interplay contributes to the divergent pathologies by disparately shaping intracellular Ca signaling.
Translational perspective
It is well-established that RyR2 dysfunction is involved in a spectrum of pathological conditions including cardiac arrhythmias. In this study, two disease models marked by RyR2 dysfunction were employed to explore how the interplay between SR and mitochondria contributes to divergent cardiac pathologies. We found mitochondria act as essential Ca buffer to absorb SR-derived Ca to mitigate pathological remodeling in the genetic arrhythmic syndrome CPVT, but they are more susceptible to Ca overload or ROS-related exacerbation of RyR2 dysfunction in pre-diabetic cardiomyopathy. Thus, tailored therapies should be developed to target SR-mitochondria interplay in the aims of treating these diseases.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 21 Oct 2021; epub ahead of print
Tow BD, Deb A, Neupane S, Patel S, ... Györke S, Liu B
Cardiovasc Res: 21 Oct 2021; epub ahead of print | PMID: 34677619
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Impact:
Abstract

Ogfod1 deletion increases cardiac beta-alanine levels and protects mice against ischemia-reperfusion injury.

Harris M, Sun J, Keeran K, Aponte A, ... Murphy E, Kennedy LM
Aims
Prolyl hydroxylation is a post-translational modification that regulates protein stability, turnover, and activity. The proteins that catalyze prolyl hydroxylation belong to the 2-oxoglutarate- and iron-dependent oxygenase family of proteins. 2-oxoglutarate- and iron-dependent oxygenase domain-containing protein 1 (Ogfod1), which hydroxylates a proline in ribosomal protein s23 is a newly-described member of this family. The aims of this study were to investigate roles for Ogfod1 in the heart, and in the heart\'s response to stress.
Methods and results
We isolated hearts from wild type (WT) and Ogfod1 knockout (KO) mice and performed quantitative proteomics using Tandem Mass Tag labelling coupled to Liquid Chromatography and tandem Mass Spectrometry (LC-MS/MS) to identify protein changes. Ingenuity Pathway Analysis identified \"Urate Biosynthesis/Inosine 5\'-phosphate Degradation\" and \"Purine Nucleotides Degradation II (Aerobic)\" as the most significantly-enriched pathways. We performed metabolomics analysis and found that both purine and pyrimidine pathways were altered with the purine nucleotide inosine 5\'-monophosphate (IMP) showing a 3.5-fold enrichment in KO hearts (P = 0.011) and the pyrimidine catabolism product beta-alanine showing a 1.7-fold enrichment in KO hearts (P = 0.014). As changes in these pathways have been shown to contribute to cardioprotection, we subjected isolated perfused hearts to ischemia and reperfusion (I/R). KO hearts showed a 41.4% decrease in infarct size and a 34% improvement in cardiac function compared to WT hearts. This protection was also evident in an in vivo I/R model. Additionally, our data show that treating isolated perfused WT hearts with carnosine, a metabolite of beta-alanine, improved protection in the context of I/R injury, whereas treating KO hearts with carnosine had no impact on recovery of function or infarct size.
Conclusions
Taken together, these data show that Ogfod1 deletion alters the myocardial proteome and metabolome to confer protection against I/R injury.
Translational perspective
Heart disease is the leading cause of death in the US. In characterizing the cardiovascular effects of deleting the prolyl hydroxylase Ogfod1 and investigating its role in disease pathology, we found that deleting Ogfod1 protected hearts against ex vivo and in vivo I/R injury. Ogfod1-KO hearts showed significant metabolomic and proteomic changes that supported altered purine and pyrimidine nucleotide synthesis and turnover. Beta-alanine, a precursor of the anti-oxidant carnosine and a product of pyrimidine degradation, accumulated in KO hearts to help confer cardioprotection. Altogether, these data suggest a role for Ogfod1 downregulation as a therapeutic strategy for heart disease.

Published on behalf of the European Society of Cardiology 2021.

Cardiovasc Res: 19 Oct 2021; epub ahead of print
Harris M, Sun J, Keeran K, Aponte A, ... Murphy E, Kennedy LM
Cardiovasc Res: 19 Oct 2021; epub ahead of print | PMID: 34668514
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Impact:
Abstract

Pannexin 1-a novel regulator of acute hypoxic pulmonary vasoconstriction.

Grimmer B, Krauszman A, Hu X, Kabir G, ... Isakson BE, Kuebler WM
Aims
Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to alveolar hypoxia that diverts blood flow from poorly ventilated to better aerated lung areas to optimize ventilation-perfusion matching. Yet, the exact sensory and signaling mechanisms by which hypoxia triggers pulmonary vasoconstriction remain incompletely understood. Recently, ATP release via pannexin 1 (Panx1) and subsequent signaling via purinergic P2Y receptors has been identified as regulator of vasoconstriction in systemic arterioles. Here, we probed for the role of Panx1-mediated ATP release in HPV and chronic hypoxic pulmonary hypertension (PH).
Methods and results
Pharmacological inhibition of Panx1 by probenecid, spironolactone, the Panx1 specific inhibitory peptide (10Panx1) and genetic deletion of Panx1 specifically in smooth muscle attenuated HPV in isolated perfused mouse lungs. In pulmonary artery smooth muscle cells (PASMC), both spironolactone and 10Panx1 attenuated the increase in intracellular Ca2+ concentration ([Ca2+]i) in response to hypoxia. Yet, genetic deletion of Panx1 in either endothelial or smooth muscle cells did not prevent the development of PH in mice. Unexpectedly, ATP release in response to hypoxia was not detectable in PASMC, and inhibition of purinergic receptors or ATP degradation by ATPase failed to attenuate HPV. Rather, transient receptor potential vanilloid 4 (TRPV4) antagonism and Panx1 inhibition inhibited the hypoxia-induced [Ca2+]i increase in PASMC in an additive manner, suggesting that Panx1 regulates [Ca2+]i independently of the ATP-P2Y-TRPV4 pathway. In line with this notion, Panx1 overexpression increased the [Ca2+]i response to hypoxia in HeLa cells.
Conclusion
In the present study we identify Panx1 as novel regulator of HPV. Yet, the role of Panx1 in HPV was not attributable to ATP release and downstream signaling via P2Y receptors or TRPV4 activation, but relates to a role of Panx1 as direct or indirect modulator of the PASMC Ca2+ response to hypoxia. Panx1 did not affect the development of chronic hypoxic PH.
Translational perspective
Hypoxic pulmonary vasoconstriction (HPV) optimizes lung ventilation-perfusion matching, but also contributes to pulmonary pathologies including high altitude pulmonary edema (HAPE) or chronic hypoxic pulmonary hypertension. Here, we demonstrate that pharmaceutical inhibition as well as genetic deletion of the hemichannel pannexin-1 (Panx1) in pulmonary artery smooth muscle cells attenuates the physiological HPV response. Panx1 deficiency did, however, not prevent the development of chronic hypoxic pulmonary hypertension in mice. Panx1 inhibitors such as the mineralocorticoid receptor antagonist spironolactone may thus present a putative strategy for the prevention or treatment of HAPE, yet not for chronic hypoxic lung disease.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 19 Oct 2021; epub ahead of print
Grimmer B, Krauszman A, Hu X, Kabir G, ... Isakson BE, Kuebler WM
Cardiovasc Res: 19 Oct 2021; epub ahead of print | PMID: 34668529
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Impact:
Abstract

Association of central arterial stiffness with hippocampal blood flow and N-acetyl aspartate concentration in hypertensive adult Dahl salt sensitive rats.

Ajamu SO, Fenner RC, Grigorova YN, Cezayirli D, ... Fedorova OV, Fishbein KW
Background
Central arterial stiffness (CAS) is associated with elevated arterial blood pressure (BP) and is likely associated with stiffening of cerebral artery walls, with attendant cerebral hypoperfusion, neuronal density loss and cognitive decline. Dahl salt-sensitive (Dahl-S) rats exhibit age-associated hypertension and memory loss, even on a normal salt intake.
Method
We sought to explore whether central arterial pulse wave velocity (PWV), a marker of CAS, is associated with hippocampal cerebral blood flow (CBF) and neuronal density in hypertensive Dahl-S rats. We measured systolic BP (by tail-cuff plethysmography), aortic PWV (by echocardiography) and CBF and N-acetyl aspartate (NAA) (by magnetic resonance imaging) in 6 month-old male Dahl-S rats (n = 12).
Results
Greater PWV was significantly associated with lower CBF and lower NAA concentration in the hippocampus, supporting a role of CAS in cerebrovascular dysfunction and decline in cognitive performance with aging.
Conclusion
These findings implicate increased CAS in cerebral hypoperfusion and loss of neuronal density and function in the Dahl-S model of age-associated cardiovascular dysfunction.



J Hypertens: 30 Sep 2021; 39:2113-2121
Ajamu SO, Fenner RC, Grigorova YN, Cezayirli D, ... Fedorova OV, Fishbein KW
J Hypertens: 30 Sep 2021; 39:2113-2121 | PMID: 34001812
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Impact:
Abstract

Tachypacing-induced CREB/CD44 signaling contributes to the suppression of L-type calcium channel expression and the development of atrial remodeling.

Chang SH, Chan YH, Chen WJ, Chang GJ, Lee JL, Yeh YH
Background
Atrial fibrillation (AF), a common arrhythmia in clinics, is characterized as downregulation of L-type calcium channel (LTCC) and shortening of atrial action potential duration (APD). Our prior studies have shown the association of CD44 with AF genesis.
Objective
The purpose of this study was to explore the potential role of CD44 and its related signaling in tachypacing-induced downregulation of LTCC.
Methods and results
In vitro, tachypacing in atrium-derived myocytes (HL-1 cell line) induced activation (phosphorylation) of cyclic adenosine monophosphate response element-binding protein (CREB). Furthermore, tachypacing promoted an association between CREB and CD44 in HL-1 myocytes, which was documented in atrial tissues from patients with AF. Deletion and mutational analysis of the LTCC promoter along with chromatin immunoprecipitation revealed that cyclic adenosine monophosphate response element is essential for tachypacing-inhibited LTCC transcription. Tachypacing also hindered the binding of p-CREB to the promoter of LTCC. Blockade of CREB/CD44 signaling in HL-1 cells attenuated tachypacing-triggered downregulation of LTCC and shortening of APD. Atrial myocytes isolated from CD44-/- mice exhibited higher LTCC current and longer APD than did those from wild-type mice. Ex vivo, tachypacing caused less activation of CREB in CD44-/- mice than in wild-type mice. In vivo, burst atrial pacing stimulated less inducibility of AF in CREB inhibitor-treated mice than in controls.
Conclusion
Tachypacing-induced CREB/CD44 signaling contributes to the suppression of LTCC, which provides valuable information about the pathogenesis of atrial modeling and AF.

Copyright © 2021 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Heart Rhythm: 29 Sep 2021; 18:1760-1771
Chang SH, Chan YH, Chen WJ, Chang GJ, Lee JL, Yeh YH
Heart Rhythm: 29 Sep 2021; 18:1760-1771 | PMID: 34023501
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Impact:
Abstract

Simple electrophysiological predictor of QRS change induced by cardiac resynchronization therapy: A novel marker of complete left bundle branch block.

Sedláček K, Jansová H, Vančura V, Grieco D, Kautzner J, Wichterle D
Background
QRS complex shortening by cardiac resynchronization therapy (CRT) has been associated with improved outcomes.
Objective
We hypothesized that the absence of QRS duration (QRSd) prolongation by right ventricular mid-septal pacing (RVP) may indicate complete left bundle branch block (cLBBB).
Methods
We prospectively collected 12-lead surface electrocardiograms (ECGs) and intracardiac electrograms during CRT implant procedures. Digital recordings were edited and manually measured. The outcome measure was a change in QRSd induced by CRT (delta CRT). Several outcome predictors were investigated: native QRSd, cLBBB (by using Strauss criteria), interval between the onset of the QRS complex and the local left ventricular electrogram (Q-LV), and a newly proposed index defined by the difference between RVP and native QRSd (delta RVP).
Results
One hundred thirty-three consecutive patients were included in the study. Delta RVP was 27 ± 25 ms, and delta CRT was -14 ± 28 ms. Delta CRT correlated with native QRSd (r = -0.65), with the presence of ECG-based cLBBB (r = -0.40), with Q-LV (r = -0.68), and with delta RVP (r = 0.72) (P < .00001 for all correlations). In multivariable analysis, delta CRT was most strongly associated with delta RVP (P < .00001), followed by native QRSd and Q-LV, while ECG-based cLBBB became a nonsignificant factor.
Conclusion
Baseline QRSd, delta RVP, and LV electrical lead position (Q-LV) represent strong independent predictors of ECG response to CRT. The absence of QRSd prolongation by RVP may serve as an alternative and more specific marker of cLBBB. Delta RVP correlates strongly with the CRT effect on QRSd and outperforms the predictive value of ECG-based cLBBB.

Copyright © 2021 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Heart Rhythm: 29 Sep 2021; 18:1717-1723
Sedláček K, Jansová H, Vančura V, Grieco D, Kautzner J, Wichterle D
Heart Rhythm: 29 Sep 2021; 18:1717-1723 | PMID: 34098086
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Impact:
Abstract

Deficiency of CXXC finger protein 1 leads to small changes in heart rate but moderate epigenetic alterations and significant protein downregulation of hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) ion channels in mice.

Shi L, Shen J, Jin X, Li Z, ... Yang B, Pan Z
Background
The normal cardiac rhythm is generated in the sinoatrial node (SAN). Changes in ionic currents of the SAN may cause sinus arrhythmia. CXXC finger protein 1 (Cfp1) is an epigenetic regulator that is involved in transcriptional regulation of multiple genes.
Objective
The purpose of this study was to explore whether Cfp1 controls SAN function through regulation of ion channel-related genes.
Methods
Electrophysiological study, patch clamp recording, reverse transcriptase polymerase chain reaction, optical mapping, chromatin immunoprecipitation, and immunofluorescence staining were performed to evaluate the function of SAN and underlying mechanism on Cfp1 heterozygous knockout (Cfp1+/-) mice.
Results
Heart rate was slower slightly and SAN recovery time was longer in Cfp1+/- mice than controls. Whole-cell patch-clamp recording showed that the firing rate of action potentials was reduced in Cfp1+/- mice. The density of If current was reduced by 66% in SAN cells of Cfp1+/- mice but the densities of ICa, ICa-L, and ICa-T were not changed. The hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) mRNA level in SAN tissue of Cfp1+/- mice was reduced. The HCN4 protein was significantly decreased in SAN cells and tissues after heterozygous deletion of Cfp1. Chromatin immunoprecipitation assay on cultured HL-1 cells demonstrated that Cfp1 was enriched in the promoter regions of HCN4. Knockdown of Cfp1 reduced H3K4 trimethylation, H3K9 acetylation, and H3K27 acetylation of HCN4 promoter region.
Conclusion
Deficiency of Cfp1 leads to small changes in heart rate by moderate epigenetic modification alterations and significant protein downregulation of HCN4 ion channels in mice.

Copyright © 2021 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Heart Rhythm: 29 Sep 2021; 18:1780-1789
Shi L, Shen J, Jin X, Li Z, ... Yang B, Pan Z
Heart Rhythm: 29 Sep 2021; 18:1780-1789 | PMID: 34182171
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Impact:
Abstract

The regulation of Ero1-alpha in homocysteine-induced macrophage apoptosis and vulnerable plaque formation in atherosclerosis.

Zhang N, Zhu L, Wu X, Yan R, ... Yang Z, Jia S
Background:
and aims
Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis and plaque vulnerability. Macrophage apoptosis mediated by endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of HHcy-aggravated atherosclerosis. Endoplasmic reticulum oxidoreductase 1α (Ero1α) is critical for ER stress-induced apoptosis. We hypothesized that Ero1α may contribute to ER-stress induced macrophage apoptosis and plaque stability in advanced atherosclerotic lesions by HHcy.
Methods
Apoe-/- mice were maintained on drinking water containing homocysteine (Hcy, 1.8 g/L) to establish HHcy atherosclerotic models. The role of Ero1α in atherosclerotic plaque stability, macrophage apoptosis and ER stress were monitored in the plaque of aortic roots in HHcy Apoe-/- mice with or without silence or overexpression of Ero1α through lentivirus. Mouse peritoneal macrophages were used to confirm the regulation of Ero1α on ER stress dependent apoptosis in the presence of HHcy.
Results
Atherosclerotic plaque vulnerability and macrophage apoptosis were promoted in Apoe-/- mice by high Hcy diet, accompanied by the upregulation of Ero1α expression and ER stress. Inhibition of Ero1α prevented macrophage apoptosis and atherosclerotic plaque vulnerability, and vice versa. Consistently, in mouse peritoneal macrophages, ER stress and apoptosis were attenuated by Ero1α deficiency, but enhanced by Ero1α overexpression.
Conclusions
Hcy, via upregulation of Ero1α expression, activates ER stress-dependent macrophage apoptosis to promote vulnerable plaque formation in atherosclerosis. Ero1α may be a potential therapeutic target for atherosclerosis induced by Hcy.

Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.

Atherosclerosis: 29 Sep 2021; 334:39-47
Zhang N, Zhu L, Wu X, Yan R, ... Yang Z, Jia S
Atherosclerosis: 29 Sep 2021; 334:39-47 | PMID: 34478920
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Impact:
Abstract

Identification of a SCN5A founder mutation causing sudden death, Brugada syndrome, and conduction blocks in Southern Italy.

Curcio A, Malovini A, Mazzanti A, Memmi M, ... Bellazzi R, Napolitano C
Background
The genetic architecture of Brugada syndrome (BrS) is emerging as an increasingly complex area of investigation. The identification of genetically homogeneous populations can provide mechanistic insights and improve genotype-phenotype correlation.
Objective
To characterize and define the clinical implications of a novel BrS founder mutation. Using a haplotype-based approach we investigated whether 2 SCN5A genetic variants could derive from founder events.
Methods
Single nucleotide polymorphisms were genotyped in 201 subjects, haplotypes reconstructed, and mutational age estimated. Clinical phenotypes and historical records were collected.
Results
A SCN5A variant (c.3352C>T; p.Gln1118Ter) was identified in 3 probands with BrS originating from south Italy. The same mutation was identified in a proband from central Italy and in 1 U.S. resident subject with Italian ancestry. The 5 individuals carried a common core haplotype, whose frequency was extremely low in local noncarrier probands and in population controls (0%-6.06%). The clinical presentation included multigenerational dominant transmission of Brugada electrocardiographic pattern, high incidence of sudden cardiac death (SCD), and cardiac conduction defects (CCD). We reconstructed 7-generation pedigrees with common geographic origin. Variant\'s age estimates suggested that origin of the p.Gln1118Ter dates back 76 generations (95% confidence interval: 28-200). A second SCN5A variant (c.5350G>A; p.Glu1784Lys) identified in the region did not show similar founder signal.
Conclusion
p.Gln1118Ter is a novel BrS/CCD/SCD founder mutation. We illustrate how these findings provide insights on the inheritance patterns and phenotypes associated with SCN5A mutation.

Copyright © 2021 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Heart Rhythm: 29 Sep 2021; 18:1698-1706
Curcio A, Malovini A, Mazzanti A, Memmi M, ... Bellazzi R, Napolitano C
Heart Rhythm: 29 Sep 2021; 18:1698-1706 | PMID: 34245912
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Impact:
Abstract

Disruption of Protein Quality Control of Human Ether-à-go-go Related Gene K Channel Results in Profound Long QT Syndrome.

Ledford HA, Ren L, Thai PN, Park S, ... Zhang XD, Chiamvimonvat N
Background
Long QT syndrome (LQTS) is a hereditary disease that predisposes patients to life-threatening cardiac arrhythmias and sudden cardiac death. Our previously study of human ether-à-go-go related gene (hERG)-encoded K+ channel (Kv11.1) supports an association between hERG and RING Finger Protein 207 (RNF207) variants in aggravating the onset and severity of LQTS, specifically T613M hERG (hERGT613M) and RNF207 frameshift (RNF207G603fs) mutations. However, the underlying mechanistic underpinning remains unknown.
Objective
The purpose of the current study is to test the role of RNF207 on the function of hERG-encoded K+ channel subunits.
Methods and results
Here, we demonstrate that RNF207 serves as an E3 ubiquitin ligase and targets misfolded hERGT613M proteins for degradation. RNF207G603fs exhibits decreased activity and hinders the normal degradation pathway; this increases the levels of hERGT613M subunits and their dominant-negative effect on the wild-type (WT) subunits, ultimately resulting in decreased current density. Similar findings are shown for hERGA614V, a known dominant-negative mutant subunit. Finally, the presence of RNF207G603fs with hERGT613M results in significantly prolonged action potential durations and reduced hERG current in human pluripotent stem cell-derived cardiomyocytes.
Conclusions
Our study establishes RNF207 as an interacting protein serving as a ubiquitin ligase for hERG-encoded K+ channel subunits. Normal function of RNF207 is critical for the quality control of hERG subunits and, consequently, cardiac repolarization. Moreover, our study provides evidence for protein quality control as a new paradigm in life-threatening cardiac arrhythmias in LQTS patients.

Copyright © 2021. Published by Elsevier Inc.

Heart Rhythm: 07 Oct 2021; epub ahead of print
Ledford HA, Ren L, Thai PN, Park S, ... Zhang XD, Chiamvimonvat N
Heart Rhythm: 07 Oct 2021; epub ahead of print | PMID: 34634443
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Impact:
Abstract

Targeting cytokines and immune checkpoints in atherosclerosis with monoclonal antibodies.

Lutgens E, Joffre J, van Os B, Ait-Oufella H
Over the past fifteen years, treatments using monoclonal antibodies specifically targeting cytokines have been developed to treat chronic inflammatory diseases, including rheumatoid arthritis or psoriasis, both associated with increased cardiovascular risk. The cardiovascular impact of these therapies allows us to validate the clinical relevance of the knowledge acquired from experimental studies about the role of cytokines in atherosclerosis. Several clinical studies have confirmed the protective effects of anti-TNFα and anti-IL-6R monoclonal antibodies against athero-thrombotic cardiovascular risk in patients with chronic inflammatory diseases. Yet, caution is needed since anti-TNFα treatment can aggravate chronic heart failure. More recently, the CANTOS study showed for the first time that an anti-inflammatory treatment using anti-IL-1β monoclonal antibody in coronary artery disease patients significantly reduced cardiovascular events. The effects of IL-23/IL-17 axis blockade on cardiovascular risk in patients with psoriasis or arthritis remain controversial. Several monoclonal antibodies targeting costimulatory molecules have also been developed, a direct way to confirm their involvement in atherothrombotic cardiovascular diseases. Blocking the CD28-CD80/86 axis with Abatacept has been shown to reduce cardiovascular risk. In contrast, the treatment of cancer patients with antibodies blocking immune checkpoint inhibitory receptors, such as CTLA-4, PD1, or PDL1, could worsen the risk of atherothrombotic events. In the future, cardiologists will be increasingly solicited to assess the cardiovascular risk of patients suffering from chronic inflammatory diseases or cancer and participate in choosing the most appropriate treatment. At the same time, immunomodulatory approaches directly targeting cardiovascular diseases will be developed as a complement to the usual treatment strategies.

Copyright © 2021 Elsevier B.V. All rights reserved.

Atherosclerosis: 29 Sep 2021; 335:98-109
Lutgens E, Joffre J, van Os B, Ait-Oufella H
Atherosclerosis: 29 Sep 2021; 335:98-109 | PMID: 34593238
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Impact:
Abstract

Promoting athero-protective immunity by vaccination with low density lipoprotein-derived antigens.

Nilsson J, Shah PK
Immune responses activated by LDL particles that have been trapped and oxidized in the arterial wall play an important role in atherosclerosis. Some of these immune responses are protective by facilitating the removal of pro-inflammatory and toxic lipid species formed as result of LDL oxidation. However, should these protective immune responses be insufficient, other more potent pro-inflammatory immune responses instead contributing to disease progression will gradually become dominant. The importance of the balance between protective and pathogenic immunity is particularly apparent when it comes to the adaptive immune system where pro-inflammatory T helper 1 (Th1) type T cells aggravate atherosclerosis, while regulatory T cells (Tregs) have an opposing role. As oxidized LDL is a key autoantigen in atherosclerosis, it has become an interesting possibility that immune-modulatory therapy that favors the activity of apolipoprotein B peptide-specific Tregs could be developed into a novel treatment strategy for prevention/stabilization of atherosclerosis and ischemic cardiovascular events. Indeed, several such oxidized LDL tolerance vaccines have shown promising results in animal models of atherosclerosis. This review will discuss the experimental background for development of atherosclerosis vaccines based on LDL-derived antigens as well as the challenges involved in translating these findings into clinical application.

Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.

Atherosclerosis: 29 Sep 2021; 335:89-97
Nilsson J, Shah PK
Atherosclerosis: 29 Sep 2021; 335:89-97 | PMID: 34462127
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Impact:
Abstract

ATP-binding cassette transporters mediate differential biosynthesis of glycosphingolipid species.

Budani M, Auray-Blais C, Lingwood C
The cytosolic-oriented glucosylceramide (GlcCer) synthase is enigmatic, requiring nascent GlcCer translocation to the luminal Golgi membrane to access glycosphingolipid (GSL) anabolic glycosyltransferases. The mechanism by which GlcCer is flipped remains unclear. To investigate the role of GlcCer binding partners in this process, we previously made cleavable, biotinylated, photoreactive GlcCer analogs in which the reactive nitrene was closely apposed to the GlcCer head group, while maintaining a C16-acyl chain. GlcCer binding protein specificity was validated for both photoprobes. Using one probe, XLB, here we identified ATP-binding cassette (ABC) transporters ABCA3, ABCB4, and ABCB10 as unfractionated microsomal GlcCer-binding proteins in DU-145 prostate tumor cells. siRNA knockdown (KD) of these transporters differentially blocked GSL synthesis assessed in toto and via metabolic labelling. KD of ABCA3 reduced acid/neutral GSL levels, but increased those of LacCer, while KD of ABCB4 preferentially reduced neutral GSL levels, and KD of ABCB10 reduced levels of both neutral and acidic GSLs. Depletion of ABCA12, implicated in GlcCer transport, preferentially decreased neutral GSL levels, while ABCB1 KD preferentially reduced gangliosides, but increased neutral GSL Gb3. These results imply that multiple ABC transporters may provide distinct but overlapping GlcCer and LacCer pools within the Golgi lumen for anabolism of different GSL series by metabolic channeling. Differential ABC family member usage may fine tune GSL biosynthesis depending on cell/tissue type. We conclude that ABC transporters provide a new tool for the regulation of GSL biosynthesis and serve as potential targets to reduce selected GSL species/subsets in diseases in which GSLs are dysregulated.

Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

J Lipid Res: 27 Sep 2021:100128; epub ahead of print
Budani M, Auray-Blais C, Lingwood C
J Lipid Res: 27 Sep 2021:100128; epub ahead of print | PMID: 34597626
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Impact:
Abstract

Low production of 12α-hydroxylated bile acids prevents hepatic steatosis in Cyp2c70 mice by reducing fat absorption.

Li R, Palmiotti A, de Vries HD, Hovingh MV, ... de Boer JF, Kuipers F
Bile acids (BAs) play important roles in lipid homeostasis and BA signaling pathways serve as therapeutic targets for non-alcoholic fatty liver disease (NAFLD). Recently, we generated Cyp2c70-/- mice with a human-like BA composition lacking mouse/rat-specific muricholic acids (MCAs) to accelerate translation from mice to humans. We employed this model to assess the consequences of a human-like BA pool on diet-induced obesity and NAFLD development. Male and female Cyp2c70-/- mice and wild-type (WT) littermates were challenged with a 12-week Western-type high-fat diet (WTD) supplemented with 0.25% cholesterol. Cyp2c70-deficiency induced a hydrophobic BA pool with high abundances of chenodeoxycholic acid, particularly in females, due to sex-dependent suppression of sterol 12α-hydroxylase (Cyp8b1). Plasma transaminases were elevated and hepatic fibrosis was present in Cyp2c70-/- mice, especially in females. Surprisingly, female Cyp2c70-/- mice were resistant to WTD-induced obesity and hepatic steatosis while male Cyp2c70-/- mice showed similar adiposity and moderately reduced steatosis compared to WT controls. Both intestinal cholesterol and fatty acid absorption were reduced in Cyp2c70-/- mice, the latter more strongly in females, despite unaffected biliary BA secretion rates. Intriguingly, the biliary ratio 12α-/non-12α-hydroxylated BAs significantly correlated with fatty acid absorption and hepatic triglyceride content as well as with specific changes in gut microbiome composition. The hydrophobic human-like BA pool in Cyp2c70-/- mice prevents WTD-induced obesity in female mice and NAFLD development in both genders, primarily due to impaired intestinal fat absorption. Our data point to a key role for 12α-hydroxylated BAs in control of intestinal fat absorption and modulation of gut microbiome composition.

Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

J Lipid Res: 05 Oct 2021:100134; epub ahead of print
Li R, Palmiotti A, de Vries HD, Hovingh MV, ... de Boer JF, Kuipers F
J Lipid Res: 05 Oct 2021:100134; epub ahead of print | PMID: 34626589
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Impact:
Abstract

Rotors anchored by refractory islands drive Torsades de Pointes in an experimental model of electrical storm.

Yamazaki M, Tomii N, Tsuneyama K, Takanari H, ... Nattel S, Tsuji Y
Background
Electrical storm (ES) is a life-threatening emergency in patients at high risk of ventricular tachycardia/fibrillation (VT/VF), but the pathophysiology and molecular basis are poorly understood.
Objective
To explore the electrophysiological substrate for experimental ES.
Methods
A model was created by inducing chronic complete atrioventricular-block in defibrillator-implanted rabbits, which recapitulates QT-prolongation, Torsades-des-Pointes (TdP) and VF-episodes.
Results
Optical mapping revealed island-like regions with action potential duration (APD) prolongation in the left ventricle (LV), leading to increased spatial APD-dispersion, in rabbits with ES (defined as ≥3 VF-episodes/24-h). The maximum APD and its dispersion correlated with the total number of VF-episodes in-vivo. TdP was initiated by an ectopic beat that failed to enter the island and formed a reentrant wave, and perpetuated by rotors whose centers swirled in the periphery of the island. Epinephrine exacerbated the island by prolonging APD and enhancing APD-dispersion, which was less evident after late Na+-current (INa-L) blockade with 10 μM ranolazine. Non-sustained VT in a non-ES rabbit heart with homogeneous APD prolongation resulted from multiple foci with an electrocardiographic morphology different from TdP driven by drifting rotors in ES-rabbit hearts. The neuronal Na+-channel subunit NaV1.8 was upregulated in ES-rabbit LV-tissues and expressed within myocardium corresponding to the island location in optically mapped ES-rabbit hearts. The NaV1.8-blocker A-803467 (10 mg/kg, i.v.) attenuated QT-prolongation and suppressed epinephrine-evoked TdP.
Conclusion
A tissue-island with enhanced refractoriness contributes to the generation of drifting rotors that underlies ES in this model. NaV1.8-mediated INa-L merits further investigation as a contributor to the substrate for ES.

Copyright © 2021. Published by Elsevier Inc.

Heart Rhythm: 18 Oct 2021; epub ahead of print
Yamazaki M, Tomii N, Tsuneyama K, Takanari H, ... Nattel S, Tsuji Y
Heart Rhythm: 18 Oct 2021; epub ahead of print | PMID: 34678525
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Impact:
Abstract

Modest decrease in severity of obesity in adolescence associates with low arterial stiffness.

Saner C, Laitinen TT, Nuotio J, Arnup SJ, ... Juonala M, Burgner DP
Background:
and aims
Childhood obesity is associated with cardiovascular risk factors (CVRF), subclinical cardiovascular phenotypes (carotid intima-media thickness, cIMT; pulse-wave velocity, PWV; and carotid elasticity), and adult cardiovascular disease (CVD) mortality. In youth with obesity (body mass index, BMI ≥95th centile), we investigated associations between changes in adiposity and CVRF in early adolescence and subclinical cardiovascular phenotypes in late adolescence.
Methods
Participants had adiposity measures (the severity of obesity in percentage >95th BMI-centile (%>95th BMI-centile)), waist circumference (WC), percentage total body fat (%BF) and CVRF (systolic blood pressure, SBP; glycoprotein acetyls, GlycA; and low-density lipoprotein cholesterol) assessed in early (mean age 10.2 ± 3.5y) and late (15.7 ± 3.7y) adolescence. Subclinical cardiovascular phenotypes were assessed in late adolescence. Multivariable regression analysis was performed.
Results
Decreasing the %>95th BMI-centile was associated with carotid elasticity (0.945%/10 mmHg, p = 0.002) in females, and with PWV in males (-0.75 m/s, p < 0.001). Changes in all adiposity measures (per 1-unit increase) were associated with carotid elasticity (-0.020 to -0.063%/10 mmHg, p < 0.005), and PWV (0.011-0.045 m/s, p < 0.005). Changes in GlycA (per 50μmol-increase) were associated with elasticity (-0.162%/10 mmHg, p = 0.042), and changes in SBP (per 10 mmHg-increase) were associated with PWV (0.260 m/s, p < 0.001). Adjusted for change in BMI, the coefficient for GlycA was reduced by 46% and for SBP by 12%. Only male sex was associated with cIMT (+34 μm, p = 0.006).
Conclusions
In youth with obesity, decreasing or maintaining the severity of obesity, and decreasing the levels of SBP and GlycA from early to late adolescence was associated with low arterial stiffness.

Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.

Atherosclerosis: 29 Sep 2021; 335:23-30
Saner C, Laitinen TT, Nuotio J, Arnup SJ, ... Juonala M, Burgner DP
Atherosclerosis: 29 Sep 2021; 335:23-30 | PMID: 34543877
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Impact:
Abstract

LncRNA HCP5 in hBMSC-derived exosomes alleviates myocardial ischemia reperfusion injury by sponging miR-497 to activate IGF1/PI3K/AKT pathway.

Li KS, Bai Y, Li J, Li SL, ... Zhou Q, Wang DJ
Ischemia/reperfusion (I/R) injury is an inevitable process during heart transplant and suppressing I/R injury could greatly improve the survival rate of recipients. Mesenchymal stem cells (MSCs) have positive effects on I/R. We aimed to investigate the mechanisms underlying the protective roles of MSCs in I/R. Both cell model and rat model of myocardial I/R were used. MTT assay and flow cytometry were used to measure cell viability and apoptosis, respectively. QRT-PCR and western blotting were employed to measure levels of lncRNA HCP5 (HLA complex P5), miR-497, apoptosis-related proteins, and insulin-like growth factor (IGF1)/PI3K/AKT pathway. Dual luciferase assay was used to validate interactions of HCP5 and miR-497, miR-497 and IGF1. Echocardiography was performed to evaluate cardiac function of rats. Serum levels of CK-MB and LDH were measured. H&E and Masson staining were used to examine morphology of myocardial tissues. hBMSC-derived exosomes (hBMSC-Exos) increased the viability of cardiomyocytes following hypoxia/reperfusion (H/R) and decreased apoptosis. H/R diminished HCP5 expression in cardiomyocytes while hBMSC-Exos recovered the level. Overexpression of HCP5 in hBMSC-Exos further enhanced the protective effects in H/R while HCP5 knockdown suppressed. HCP5 directly bound miR-497 and miR-497 targeted IGF1. miR-497 mimics or si-IGF1 blocked the effects of HCP5 overexpression. Further, hBMSC-Exos alleviated I/R injury in vivo and knockdown of HCP5 in hBMSC-Exos decreased the beneficial effects. AntagomiR-497 blocked the effects of HCP5 knockdown. HCP5 from hBMSC-Exos protects cardiomyocytes against I/R injury via sponging miR-497 to disinhibit IGF1/PI3K/AKT pathway. These results shed light on mechanisms underlying the protective role of hBMSC-Exos in I/R.

Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.

Int J Cardiol: 31 Oct 2021; 342:72-81
Li KS, Bai Y, Li J, Li SL, ... Zhou Q, Wang DJ
Int J Cardiol: 31 Oct 2021; 342:72-81 | PMID: 34311013
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Impact:
Abstract

Neuropeptide Y promotes adipogenesis of human cardiac mesenchymal stromal cells in arrhythmogenic cardiomyopathy.

Stadiotti I, Di Bona A, Pilato CA, Scalco A, ... Mongillo M, Zaglia T
Background
Arrhythmogenic Cardiomyopathy (AC) is a familial cardiac disease, mainly caused by mutations in desmosomal genes. AC hearts show fibro-fatty myocardial replacement, which favors stress-related life-threatening arrhythmias, predominantly in the young and athletes. AC lacks effective therapies, as its pathogenesis is poorly understood. Recently, we showed that cardiac Mesenchymal Stromal Cells (cMSCs) contribute to adipose tissue in human AC hearts, although the underlying mechanisms are still unclear.
Purpose
We hypothesize that the sympathetic neurotransmitter, Neuropeptide Y (NPY), participates to cMSC adipogenesis in human AC.
Methods
For translation of our findings, we combined in vitro cytochemical, molecular and pharmacologic assays on human cMSCs, from myocardial biopsies of healthy controls and AC patients, with the use of existing drugs to interfere with the predicted AC mechanisms. Sympathetic innervation was inspected in human autoptic heart samples, and NPY plasma levels measured in healthy and AC subjects.
Results
AC cMSCs expressed higher levels of pro-adipogenic isotypes of NPY-receptors (i.e. Y1-R, Y5-R). Consistently, NPY enhanced adipogenesis in AC cMSCs, which was blocked by FDA-approved Y1-R and Y5-R antagonists. AC-associated PKP2 reduction directly caused NPY-dependent adipogenesis in cMSCs. In support of the involvement of sympathetic neurons (SNs) and NPY in AC myocardial remodeling, patients had elevated NPY plasma levels and, in human AC hearts, SNs accumulated in fatty areas and were close to cMSCs.
Conclusions
Independently from the disease origin, AC causes in cMSCs a targetable gain of responsiveness to NPY, which leads to increased adipogenesis, thus playing a role in AC myocardial remodeling.

Copyright © 2021 Elsevier B.V. All rights reserved.

Int J Cardiol: 31 Oct 2021; 342:94-102
Stadiotti I, Di Bona A, Pilato CA, Scalco A, ... Mongillo M, Zaglia T
Int J Cardiol: 31 Oct 2021; 342:94-102 | PMID: 34400166
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Impact:
Abstract

Protection of the enhanced Nrf2 deacetylation and its downstream transcriptional activity by SIRT1 in myocardial ischemia/reperfusion injury.

Xu JJ, Cui J, Lin Q, Chen XY, ... Wei B, Zhao W
Nrf2, the master gene transcriptor of antioxidant proteins, and SIRT1, the unique Class III histone deacetylase of sirtuins, have been involved in protecting myocardial ischemia/reperfusion (MI/R) injury. However, whether the protective effect of SIRT1 is directly related to the deacetylation of Nrf2 in the pathology of MI/R remains to be investigated. The current study was designed to evaluate the regulation of Nrf2 deacetylation and transcriptional activity by SIRT1 in MI/R. Hypoxia/reoxygenation (H/R) cardiomyocytes and MI/R mice were used to assess the role of SIRT1 in Nrf2 activation. Oxidative stress, cardiac function, LDH release, ROS and infarct size were also evaluated. We found that Nrf2 physically interacted with SIRT1 not only in normal and H/R cardiomyocytes in vitro, but also in Sham or I/R hearts in vivo. Adenovirus induced SIRT1 overexpression resulted in protected H/R induced cell death, accompanied by declined LDH release. Through MI/R in vivo, cardiac overexpression of SIRT1 led to ameliorated cardiac function and infarct size, as well as the decreased cardiac oxidative stress. Notably, such beneficial actions of SIRT1 were blocked by the Nrf2 silence. Mechanically, acetylation of Nrf2 was significantly decreased by SIRT1 overexpression in cardiomyocytes or in whole hearts, which upregulated the downstream signaling pathway of Nrf2. Taken together, we uncovered a clue, for the first time that SIRT1 physically interacts with Nrf2. The cardioprotective effect of SIRT1 overexpression against MI/R is associated with the increased Nrf2 deacetylation and activity. These findings have offered a direct proof and new perspective of post-translational modification in the understanding of oxidative stress and MI/R treatment.

Copyright © 2021 Elsevier B.V. All rights reserved.

Int J Cardiol: 31 Oct 2021; 342:82-93
Xu JJ, Cui J, Lin Q, Chen XY, ... Wei B, Zhao W
Int J Cardiol: 31 Oct 2021; 342:82-93 | PMID: 34403762
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Impact:
Abstract

NKTR-255 is a polymer-conjugated IL-15 with unique mechanisms of action on T and natural killer cells.

Robinson TO, Hegde SM, Chang A, Gangadharan A, ... Miyazaki T, Schluns KS
NKTR-255 is a PEG conjugate of recombinant human IL-15 (rhIL-15) being examined as a potential cancer immunotherapeutic. Since IL-15 responses can be mediated by trans or cis presentation via IL-15Rα or soluble IL-15/IL-15Rα complexes, we investigated the role of IL-15Rα in driving NKTR-255 responses using defined naive and memory OVA-specific CD8+ T cells (OT-I) and NK cells in mice. NKTR-255 induced a 2.5- and 2.0-fold expansion of CD8+ T and NK cells, respectively, in WT mice. In adoptive transfer studies, proliferation of naive and memory WT OT-I T cells in response to NKTR-255 was not impaired in IL-15Rα-/- mice, suggesting trans presentation was not utilized by NKTR-255. Interestingly, naive IL-15Rα-/- OT-I cells had deficient responses to NKTR-255, while memory IL-15Rα-/- OT-I cell responses were partially impaired, suggesting that naive CD8+ T cells are more dependent on cis presentation of NKTR-255 than memory CD8+ T cells. In bone marrow chimera studies, IL-15Rα-/- and WT NK cells present in WT recipients had similar responses to NKTR-255, suggesting that cis presentation is not utilized by NK cells. NKTR-255 could form soluble complexes with IL-15Rα; binding to murine IL-15Rα generated superagonists that preferentially stimulated NK cells, showing that conversion to IL-15Rβ agonist biases the response toward NK cells. These findings highlight the ability of NKTR-255 to utilize IL-15Rα for cis presentation and act as an IL-15Rαβ agonist on CD8+ T cells.



J Clin Invest: 30 Sep 2021; 131
Robinson TO, Hegde SM, Chang A, Gangadharan A, ... Miyazaki T, Schluns KS
J Clin Invest: 30 Sep 2021; 131 | PMID: 34375310
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Impact:
Abstract

Exercise-induced late preconditioning in mice is triggered by eNOS-dependent generation of nitric oxide and activation of PKCε and is mediated by increased iNOS activity.

Guo Y, Li Q, Xuan YT, Wu WJ, ... Tomlin A, Bolli R
The purpose of this study was to assess whether short-term, mild exercise induces protection against myocardial infarction and, if so, what role the eNOS-PKCε-iNOS axis plays. Mice were subjected to 2 bouts/day of treadmill exercise (60 min at 15 m/min) for 2 consecutive days. At 24 h after the last bout of exercise, mice were subjected to a 30-min coronary artery occlusion and 24 h of reperfusion. In the exercise group (group III, wild-type mice), infarct size (25.5 ± 8.8% of risk region) was significantly (P < 0.05) reduced compared with the control groups (sham exercise, group II [63.4 ± 7.8%] and acute myocardial infarction, group I [58.6 ± 7.0%]). This effect was abolished by pretreatment with the NOS inhibitor L-NA (group VI, 56.1 ± 16.2%) and the PKC inhibitor chelerythrine (group VIII, 57.9 ± 12.5%). Moreover, the late PC effect of exercise was completely abrogated in eNOS-/- mice (group XIII, 61.0 ± 11.2%). The myocardial phosphorylated eNOS at Ser-1177 was significantly increased at 30 min after treadmill training (exercise group) compared with sham-exercised hearts. PKCε translocation was significantly increased at 30 min after exercise in WT mice but not in eNOS-/- mice. At 24 h after exercise, iNOS protein was upregulated compared with sham-exercised hearts. The protection of late PC was abrogated in iNOS-/- mice (group XVI, 56.4 ± 12.9%) and in wildtype mice given the selective iNOS inhibitor 1400 W prior to ischemia (group X 62.0 ± 8.8% of risk region). We conclude that 1) even short, mild exercise induces a delayed PC effect that affords powerful protection against infarction; 2) this cardioprotective effect is dependent on activation of eNOS, eNOS-derived NO generation, and subsequent PKCε activation during PC; 3) the translocation of PKCε is dependent on eNOS; 4) the protection 24 h later is dependent on iNOS activity. Thus, eNOS is the trigger and iNOS the mediator of PC induced by mild exercise.

Copyright © 2021 Elsevier B.V. All rights reserved.

Int J Cardiol: 30 Sep 2021; 340:68-78
Guo Y, Li Q, Xuan YT, Wu WJ, ... Tomlin A, Bolli R
Int J Cardiol: 30 Sep 2021; 340:68-78 | PMID: 34400167
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Impact:
Abstract

Loss-of-function mutations in cardiac ryanodine receptor channel cause various types of arrhythmias including long QT syndrome.

Hirose S, Murayama T, Tetsuo N, Hoshiai M, ... Kurebayashi N, Ohno S
Aims
Gain-of-function mutations in RYR2, encoding the cardiac ryanodine receptor channel (RyR2), cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Whereas, genotype-phenotype correlations of loss-of-function mutations remains unknown, due to a small number of analysed mutations. In this study, we aimed to investigate their genotype-phenotype correlations in patients with loss-of-function RYR2 mutations.
Methods and results
We performed targeted gene sequencing for 710 probands younger than 16-year-old with inherited primary arrhythmia syndromes (IPAS). RYR2 mutations were identified in 63 probands, and 3 probands displayed clinical features different from CPVT. A proband with p.E4146D developed ventricular fibrillation (VF) and QT prolongation whereas that with p.S4168P showed QT prolongation and bradycardia. Another proband with p.S4938F showed short-coupled variant of torsade de pointes (scTdP). To evaluate the functional alterations in these three mutant RyR2s and p.K4594Q previously reported in a long QT syndrome (LQTS), we measured Ca2+ signals in HEK293 cells and HL-1 cardiomyocytes as well as Ca2+-dependent [3H]ryanodine binding. All mutant RyR2s demonstrated a reduced Ca2+ release, an increased endoplasmic reticulum Ca2+, and a reduced [3H]ryanodine binding, indicating loss-of-functions. In HL-1 cells, the exogenous expression of S4168P and K4594Q reduced amplitude of Ca2+ transients without inducing Ca2+ waves, whereas that of E4146D and S4938F evoked frequent localized Ca2+ waves.
Conclusion
Loss-of-function RYR2 mutations may be implicated in various types of arrhythmias including LQTS, VF, and scTdP, depending on alteration of the channel activity. Search of RYR2 mutations in IPAS patients clinically different from CPVT will be a useful strategy to effectively discover loss-of-function RYR2 mutations.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Europace: 17 Oct 2021; epub ahead of print
Hirose S, Murayama T, Tetsuo N, Hoshiai M, ... Kurebayashi N, Ohno S
Europace: 17 Oct 2021; epub ahead of print | PMID: 34661651
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Abstract

Effect of leukopenia induced by cyclophosphamide on the initial stage of arterial thrombosis in mice.

Lee KO, Kwon I, Nam HS, Park Y, ... Song JW, Heo JH
Introduction
Leukocytes are found in organizing thrombi and are associated with thrombus growth. However, their role in the initial stage of thrombus formation is not well known. We investigated the role of leukocytes in the early stage of arterial thrombosis by inducing leukopenia.
Methods
In this double-blind, randomized, placebo-controlled study, 72 Institute of Cancer Research mice were randomly treated with intraperitoneal 100 mg/kg cyclophosphamide or normal saline. The primary outcome was time to occlusion after FeCl3 treatment. We also compared thrombus size, histological composition, and association with peripheral blood cell counts between cyclophosphamide and control groups.
Results
Cyclophosphamide treatment significantly decreased leukocyte counts by 82.8% compared to placebo (P < 0.001). The time to occlusion was significantly longer in the cyclophosphamide group (3.31 ± 1.59 min) than in the control group (2.30 ± 1.14 min; P = 0.003). The immunoreactivity for Ly6G-positive cells, intracellular histone H3, and released histone H3 in thrombi was significantly reduced in the cyclophosphamide group by 92.8%, 50.2%, and 34.3%, respectively. Time to occlusion had a moderate negative correlation with leukocyte count in peripheral blood (r = -0.326, P = 0.022) in the entire group.
Conclusions
Cyclophosphamide-induced leukopenia attenuated thrombus formation during the early stage of arterial thrombosis. Our findings suggest the potential role of leukocytes in the initial stage of arterial thrombosis.

Copyright © 2021 Elsevier Ltd. All rights reserved.

Thromb Res: 29 Sep 2021; 206:111-119
Lee KO, Kwon I, Nam HS, Park Y, ... Song JW, Heo JH
Thromb Res: 29 Sep 2021; 206:111-119 | PMID: 34455128
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