Topic: Basic Research

Abstract

Time-Restricted Salutary Effects of Blood Flow Restoration on Venous Thrombosis and Vein Wall Injury in Mouse and Human Subjects.

Li W, Kessinger CW, Orii M, Lee H, ... Henke PK, Jaffer FA
Background
Up to 50% of patients with proximal deep vein thrombosis (DVT) will develop the postthrombotic syndrome characterized by limb swelling and discomfort, hyperpigmentation, skin ulcers, and impaired quality of life. Although catheter-based interventions enabling the restoration of blood flow (RBF) have demonstrated little benefit on postthrombotic syndrome, the impact on the acuity of the thrombus and mechanisms underlying this finding remain obscure. In experimental and clinical studies, we examined whether RBF has a restricted time window for improving DVT resolution.
Methods
First, experimental stasis DVT was generated in C57/BL6 mice (n=291) by inferior vena cava ligation. To promote RBF, mice underwent mechanical deligation with or without intravenous recombinant tissue plasminogen activator administered 2 days after deligation. RBF was assessed over time by ultrasonography and intravital microscopy. Resected thrombosed inferior vena cava specimens underwent thrombus and vein wall histological and gene expression assays. Next, in a clinical study, we conducted a post hoc analysis of the ATTRACT (Acute Venous Thrombosis: Thrombus Removal with Adjunctive Catheter-Directed Thrombolysis) pharmacomechanical catheter-directed thrombolysis (PCDT) trial (NCT00790335) to assess the effects of PCDT on Venous Insufficiency Epidemiological and Economic Study quality-of-life and Villalta scores for specific symptom-onset-to-randomization timeframes.
Results
Mice that developed RBF by day 4, but not later, exhibited reduced day 8 thrombus burden parameters and reduced day 8 vein wall fibrosis and inflammation, compared with controls. In mice without RBF, recombinant tissue plasminogen activator administered at day 4, but not later, reduced day 8 thrombus burden and vein wall fibrosis. It is notable that, in mice already exhibiting RBF by day 4, recombinant tissue plasminogen activator administration did not further reduce thrombus burden or vein wall fibrosis. In the ATTRACT trial, patients receiving PCDT in an intermediate symptom-onset-to-randomization timeframe of 4 to 8 days demonstrated maximal benefits in Venous Insufficiency Epidemiological and Economic Study quality-of-life and Villalta scores (between-group difference=8.41 and 1.68, respectively, P<0.001 versus patients not receiving PCDT). PCDT did not improve postthrombotic syndrome scores for patients having a symptom-onset-to-randomization time of <4 days or >8 days.
Conclusions
Taken together, these data illustrate that, within a restricted therapeutic window, RBF improves DVT resolution, and PCDT may improve clinical outcomes. Further studies are warranted to examine the value of time-restricted RBF strategies to reduce postthrombotic syndrome in patients with DVT.



Circulation: 22 Mar 2021; 143:1224-1238
Li W, Kessinger CW, Orii M, Lee H, ... Henke PK, Jaffer FA
Circulation: 22 Mar 2021; 143:1224-1238 | PMID: 33445952
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Abstract

BMP9 and BMP10 Act Directly on Vascular Smooth Muscle Cells for Generation and Maintenance of the Contractile State.

Wang L, Rice M, Swist S, Kubin T, ... Schneider A, Braun T
Background
Vascular smooth muscle cells (VSMCs) show a remarkable phenotypic plasticity, allowing acquisition of contractile or synthetic states, but critical information is missing about the physiologic signals, promoting formation, and maintenance of contractile VSMCs in vivo. BMP9 and BMP10 (bone morphogenetic protein) are known to regulate endothelial quiescence after secretion from the liver and right atrium, whereas a direct role in the regulation of VSMCs was not investigated. We studied the role of BMP9 and BMP10 for controlling formation of contractile VSMCs.
Methods
We generated several cell type-specific loss- and gain-of-function transgenic mouse models to investigate the physiologic role of BMP9, BMP10, ALK1 (activin receptor-like kinase 1), and SMAD7 in vivo. Morphometric assessments, expression analysis, blood pressure measurements, and single molecule fluorescence in situ hybridization were performed together with analysis of isolated pulmonary VSMCs to unravel phenotypic and transcriptomic changes in response to absence or presence of BMP9 and BMP10.
Results
Concomitant genetic inactivation of Bmp9 in the germ line and Bmp10 in the right atrium led to dramatic changes in vascular tone and diminution of the VSMC layer with attenuated contractility and decreased systemic as well as right ventricular systolic pressure. On the contrary, overexpression of Bmp10 in endothelial cells of adult mice dramatically enhanced formation of contractile VSMCs and increased systemic blood pressure as well as right ventricular systolic pressure. Likewise, BMP9/10 treatment induced an ALK1-dependent phenotypic switch from synthetic to contractile in pulmonary VSMCs. Smooth muscle cell-specific overexpression of Smad7 completely suppressed differentiation and proliferation of VSMCs and reiterated defects observed in adult Bmp9/10 double mutants. Deletion of Alk1 in VSMCs recapitulated the Bmp9/10 phenotype in pulmonary but not in aortic and coronary arteries. Bulk expression analysis and single molecule RNA-fluorescence in situ hybridization uncovered vessel bed-specific, heterogeneous expression of BMP type 1 receptors, explaining phenotypic differences in different Alk1 mutant vessel beds.
Conclusions
Our study demonstrates that BMP9 and BMP10 act directly on VSMCs for induction and maintenance of their contractile state. The effects of BMP9/10 in VSMCs are mediated by different combinations of BMP type 1 receptors in a vessel bed-specific manner, offering new opportunities to manipulate blood pressure in the pulmonary circulation.



Circulation: 05 Apr 2021; 143:1394-1410
Wang L, Rice M, Swist S, Kubin T, ... Schneider A, Braun T
Circulation: 05 Apr 2021; 143:1394-1410 | PMID: 33334130
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Abstract

Isolevuglandin-Modified Cardiac Proteins Drive CD4+ T-Cell Activation in the Heart and Promote Cardiac Dysfunction.

Ngwenyama N, Kirabo A, Aronovitz M, Velázquez F, ... Harrison DG, Alcaide P
Background
Despite the well-established association between T-cell-mediated inflammation and nonischemic heart failure, the specific mechanisms triggering T-cell activation during the progression of heart failure and the antigens involved are poorly understood. We hypothesized that myocardial oxidative stress induces the formation of isolevuglandin (IsoLG)-modified proteins that function as cardiac neoantigens to elicit CD4+ T-cell receptor (TCR) activation and promote heart failure.
Methods
We used transverse aortic constriction in mice to trigger myocardial oxidative stress and T-cell infiltration. We profiled the TCR repertoire by mRNA sequencing of intramyocardial activated CD4+ T cells in Nur77GFP reporter mice, which transiently express GFP on TCR engagement. We assessed the role of antigen presentation and TCR specificity in the development of cardiac dysfunction using antigen presentation-deficient MhcII-/- mice and TCR transgenic OTII mice that lack specificity for endogenous antigens. We detected IsoLG protein adducts in failing human hearts. We also evaluated the role of reactive oxygen species and IsoLGs in eliciting T-cell immune responses in vivo by treating mice with the antioxidant TEMPOL and the IsoLG scavenger 2-hydroxybenzylamine during transverse aortic constriction, and ex vivo in mechanistic studies of CD4+ T-cell proliferation in response to IsoLG-modified cardiac proteins.
Results
We discovered that TCR antigen recognition increases in the left ventricle as cardiac dysfunction progresses and identified a limited repertoire of activated CD4+ T-cell clonotypes in the left ventricle. Antigen presentation of endogenous antigens was required to develop cardiac dysfunction because MhcII-/- mice reconstituted with CD4+ T cells and OTII mice immunized with their cognate antigen were protected from transverse aortic constriction-induced cardiac dysfunction despite the presence of left ventricle-infiltrated CD4+ T cells. Scavenging IsoLGs with 2-hydroxybenzylamine reduced TCR activation and prevented cardiac dysfunction. Mechanistically, cardiac pressure overload resulted in reactive oxygen species-dependent dendritic cell accumulation of IsoLG protein adducts, which induced robust CD4+ T-cell proliferation.
Conclusions
Our study demonstrates an important role of reactive oxygen species-induced formation of IsoLG-modified cardiac neoantigens that lead to TCR-dependent CD4+ T-cell activation within the heart.



Circulation: 22 Mar 2021; 143:1242-1255
Ngwenyama N, Kirabo A, Aronovitz M, Velázquez F, ... Harrison DG, Alcaide P
Circulation: 22 Mar 2021; 143:1242-1255 | PMID: 33463362
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Abstract

MicroRNA-21-Dependent Macrophage-to-Fibroblast Signaling Determines the Cardiac Response to Pressure Overload.

Ramanujam D, Schön AP, Beck C, Vaccarello P, ... Schulz C, Engelhardt S
Background
Cardiac macrophages (cMPs) are increasingly recognized as important regulators of myocardial homeostasis and disease, yet the role of noncoding RNA in these cells is largely unknown. Small RNA sequencing of the entire miRNomes of the major cardiac cell fractions revealed microRNA-21 (miR-21) as the single highest expressed microRNA in cMPs, both in health and disease (25% and 43% of all microRNA reads, respectively). MiR-21 has been previously reported as a key microRNA driving tissue fibrosis. Here, we aimed to determine the function of macrophage miR-21 on myocardial homeostasis and disease-associated remodeling.
Methods
Macrophage-specific ablation of miR-21 in mice driven by Cx3cr1-Cre was used to determine the function of miR-21 in this cell type. As a disease model, mice were subjected to pressure overload for 6 and 28 days. Cardiac function was assessed in vivo by echocardiography, followed by histological analyses and single-cell sequencing. Cocultures of macrophages and cardiac fibroblasts were used to study macrophage-to-fibroblast signaling.
Results
Mice with macrophage-specific genetic deletion of miR-21 were protected from interstitial fibrosis and cardiac dysfunction when subjected to pressure overload of the left ventricle. Single-cell sequencing of pressure-overloaded hearts from these mice revealed that miR-21 in macrophages is essential for their polarization toward a M1-like phenotype. Systematic quantification of intercellular communication mediated by ligand-receptor interactions across all cell types revealed that miR-21 primarily determined macrophage-fibroblast communication, promoting the transition from quiescent fibroblasts to myofibroblasts. Polarization of isolated macrophages in vitro toward a proinflammatory (M1-like) phenotype activated myofibroblast transdifferentiation of cardiac fibroblasts in a paracrine manner and was dependent on miR-21 in cMPs.
Conclusions
Our data indicate a critical role of cMPs in pressure overload-induced cardiac fibrosis and dysfunction and reveal macrophage miR-21 as a key molecule for the profibrotic role of cMPs.



Circulation: 12 Apr 2021; 143:1513-1525
Ramanujam D, Schön AP, Beck C, Vaccarello P, ... Schulz C, Engelhardt S
Circulation: 12 Apr 2021; 143:1513-1525 | PMID: 33550817
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Abstract

Antihypertrophic Memory after Regression of Exercise-induced Physiological Myocardial Hypertrophy is Mediated by the Long Noncoding RNA Mhrt779.

Lin H, Zhu Y, Zheng C, Hu D, ... Bin J, Liao Y
Background: Exercise can induce physiological myocardial hypertrophy (PMH), and former athletes can live 5-6 years longer than nonathletic controls, suggesting a benefit after regression of PMH. We previously reported that regression of pathological myocardial hypertrophy has antihypertrophic effects. Accordingly, we hypothesized that antihypertrophic memory exists even after PMH has regressed, increasing myocardial resistance to subsequent pathological hypertrophic stress.
Methods:
C57BL/6 mice were submitted to 21 days of swimming training to develop PMH. After termination of exercise, PMH regressed within 1 week. PMH regression mice (exercise hypertrophic preconditioning group, EHP) and sedentary mice (control group) then underwent transverse aortic constriction (TAC) or a sham operation for 4 weeks. Cardiac remodeling and function were evaluated using echocardiography, invasive left ventricular hemodynamic measurement and histological analysis. LncRNA sequencing, chromatin immunoprecipitation assay (ChIP), and comprehensive identification of RNA-binding proteins by mass spectrometry (CHIRP-MS) and Western blot were used to investigate the role of Mhrt779 involved in the anti-hypertrophy effect induced by EHP.
Results:
At 1 and 4 weeks after TAC, the EHP group showed less increase in myocardial hypertrophy and lower expression of the Nppa and Myh7 genes than the sedentary group. At 4 weeks after TAC, EHP mice had less pulmonary congestion, smaller left ventricular dimensions and end-diastolic pressure, and a larger left ventricular ejection fraction and maximum pressure change rate than sedentary mice. Quantitative polymerase chain reaction (qPCR) revealed that the long noncoding myosin heavy chain associated RNA transcript Mhrt779 was one of the markedly upregulated long noncoding RNAs in the EHP group. Silencing of Mhrt779 attenuated the antihypertrophic effect of EHP in mice with TAC and in cultured cardiomyocytes treated with angiotensin II, and overexpression enhanced the antihypertrophic effect. By ChIP and qPCR, we found that EHP increased histone 3 trimethylation (H3K4me3 and H3K36me3) at the a4 promoter of Mhrt779. CHIRP-MS and Western blot showed that Mhrt779 can bind Brg1 to inhibit the activation of Hdac2/Akt/GSK3β pathway induced by pressure overload. Conclusions: Myocardial hypertrophy preconditioning evoked by exercise increases resistance to pathological stress via an antihypertrophic effect mediated by a signal pathway of Mhrt779 /Brg1/Hdac2/p-Akt/p-GSK3β.




Circulation: 23 Mar 2021; epub ahead of print
Lin H, Zhu Y, Zheng C, Hu D, ... Bin J, Liao Y
Circulation: 23 Mar 2021; epub ahead of print | PMID: 33757294
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Abstract

ALDH1A3 Coordinates Metabolism with Gene Regulation in Pulmonary Arterial Hypertension.

Li D, Shao NY, Moonen JR, Zhao Z, ... Snyder MP, Rabinovitch M
Background: Metabolic alterations provide substrates that influence chromatin structure to regulate gene expression that determines cell function in health and disease. Heightened proliferation of smooth muscle cells (SMC) leading to the formation of a neointima is a feature of pulmonary arterial hypertension (PAH) and systemic vascular disease. Increased glycolysis is linked to the proliferative phenotype of these SMC.
Methods:
RNA Sequencing was applied to pulmonary arterial (PA) SMC from PAH patients with and without a BMPR2 mutation vs. control PASMC to uncover genes required for their heightened proliferation and glycolytic metabolism. Assessment of differentially expressed genes established metabolism as a major pathway, and the most highly upregulated metabolic gene in PAH PASMC was aldehyde dehydrogenase family 1 member 3 (ALDH1A3), an enzyme previously linked to glycolysis and proliferation in cancer cells and systemic vascular SMC. We determined if these functions are ALDH1A3-dependent in PAH PASMC, and if ALDH1A3 is required for the development of pulmonary hypertension in a transgenic mouse. Nuclear localization of ALDH1A3 in PAH PASMC led us to determine whether and how this enzyme coordinately regulates gene expression and metabolism in PAH PASMC. Results:ALDH1A3 mRNA and protein were increased in PAH vs control PASMC, and ALDH1A3 was required for their highly proliferative and glycolytic properties. Mice with Aldh1a3 deleted in SMC did not develop hypoxia-induced PA muscularization or pulmonary hypertension. Nuclear ALDH1A3 converted acetaldehyde to acetate to produce acetyl-CoA to acetylate H3K27, marking active enhancers. This allowed for chromatin modification at nuclear factor Y (NFY)A binding sites via the acetyltransferase KAT2B and permitted NFY mediated transcription of cell cycle and metabolic genes that is required for ALDH1A3-dependent proliferation and glycolysis. Loss of BMPR2 in PAH SMC with or without a mutation upregulated ALDH1A3, and transcription of NFYA and ALDH1A3 in PAH PASMC was β-catenin dependent. Conclusions: Our studies have uncovered a metabolic-transcriptional axis explaining how dividing cells use ALDH1A3 to coordinate their energy needs with the epigenetic and transcriptional regulation of genes required for SMC proliferation. They suggest that selectively disrupting the pivotal role of ALDH1A3 in PAH SMC, but not EC, is an important therapeutic consideration.




Circulation: 24 Mar 2021; epub ahead of print
Li D, Shao NY, Moonen JR, Zhao Z, ... Snyder MP, Rabinovitch M
Circulation: 24 Mar 2021; epub ahead of print | PMID: 33764154
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Abstract

Increased ROS-Mediated CaMKII Activation Contributes to Calcium Handling Abnormalities and Impaired Contraction in Barth Syndrome.

Liu X, Wang S, Guo X, Li Y, ... Bezzerides VJ, Pu WT
Background: Mutations in tafazzin (TAZ), a gene required for biogenesis of cardiolipin, the signature phospholipid of the inner mitochondrial membrane, causes Barth syndrome (BTHS). Cardiomyopathy and risk of sudden cardiac death are prominent features of BTHS, but the mechanisms by which impaired cardiolipin biogenesis causes cardiac muscle weakness and arrhythmia are poorly understood.
Methods:
We performed in vivo electrophysiology to define arrhythmia vulnerability in cardiac specific TAZ knockout mice. Using cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) and cardiac specific TAZ knockout mice as model systems, we investigated the effect of TAZ inactivation on Ca2+ handling. Through genome editing and pharmacology, we defined a molecular link between TAZ mutation and abnormal Ca2+ handling and contractility.
Results:
A subset of mice with cardiac-specific TAZ inactivation developed arrhythmias including bidirectional ventricular tachycardia, atrial tachycardia, and complete atrioventricular block. Compared to WT, BTHS iPSC-CMs had increased diastolic Ca2+ and decreased Ca2+ transient amplitude. BTHS iPSC-CMs had higher levels of mitochondrial and cellular ROS than WT, which activated Ca2+/calmodulin-dependent protein kinase II (CaMKII). Activated CaMKII phosphorylated the cardiac ryanodine receptor (RYR2) on serine 2814, increasing Ca2+ leak through RYR2. Inhibition of this ROS-CaMKII-RYR2 pathway through pharmacological inhibitors or genome editing normalized aberrant Ca2+ handling in BTHS iPSC-CMs and improved their contractile function. Murine Taz knockout cardiomyocytes also exhibited elevated diastolic Ca2+ and decreased Ca2+ transient amplitude. These abnormalities were ameliorated by CaMKII or ROS inhibition. Conclusions: This study identified a molecular pathway that links TAZ mutation to abnormal Ca2+ handling and decreased cardiomyocyte contractility. This pathway may offer therapeutic opportunities to treat BTHS and potentially other diseases with elevated mitochondrial ROS production.




Circulation: 31 Mar 2021; epub ahead of print
Liu X, Wang S, Guo X, Li Y, ... Bezzerides VJ, Pu WT
Circulation: 31 Mar 2021; epub ahead of print | PMID: 33793303
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Abstract

Cooperative Binding of ETS2 and NFAT Link Erk1/2 and Calcineurin Signaling in the Pathogenesis of Cardiac Hypertrophy.

Luo Y, Jiang N, May HI, Luo X, ... Gillette TG, Hill JA
Background: Cardiac hypertrophy is an independent risk factor for heart failure, a leading cause of morbidity and mortality globally. The calcineurin/NFAT (nuclear factor of activated T cells) pathway and the MAPK/Erk (extracellular signal-regulated kinase) pathway contribute to the pathogenesis of cardiac hypertrophy as an inter-dependent network of signaling cascades. However, how these pathways interact remains unclear, and specifically few direct targets responsible for the pro-hypertrophic role of NFAT have been described.
Methods:
By engineering a cardiomyocyte-specific ETS2 (a member of E26 transformationspecific sequence (ETS)-domain family) knockout mice, we investigated the role of ETS2 in cardiac hypertrophy. Primary cardiomyocytes were also used to evaluate ETS2 function in cell growth.
Results:
ETS2 is phosphorylated and activated by Erk1/2 upon hypertrophic stimulation in both mouse (n = 3) and human heart samples (n = 8-19). Conditional deletion of ETS2 in mouse cardiomyocytes protects against pressure overload-induced cardiac hypertrophy (n = 6-11). Furthermore, silencing of ETS2 in the hearts of calcineurin transgenic mice significantly attenuates hypertrophic growth and contractile dysfunction (n = 8). As a transcription factor, ETS2 is capable of binding to the promoters of hypertrophic marker genes, such as ANP, BNP and Rcan1.4 (n = 4). Additionally, we report that ETS2 forms a complex with NFAT to stimulate transcriptional activity through increased NFAT binding to the promoters of at least two hypertrophy-stimulated genes, Rcan1.4 and miR-223 (n = 4-6). Suppression of miR-223 in cardiomyocytes inhibits calcineurin-mediated cardiac hypertrophy (n = 6), revealing miR-223 as a novel pro-hypertrophic target of the calcineurin-NFAT and Erk1/2-ETS2 pathways. Conclusions: In aggregate, our findings point to a critical role for ETS2 in calcineurin-NFAT pathway-driven cardiac hypertrophy and unveil a previously unknown molecular connection between the Erk1/2 activation of ETS2 and expression of NFAT/ETS2 target genes.




Circulation: 05 Apr 2021; epub ahead of print
Luo Y, Jiang N, May HI, Luo X, ... Gillette TG, Hill JA
Circulation: 05 Apr 2021; epub ahead of print | PMID: 33821668
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Abstract

Systems Approach to Discovery of Therapeutic Targets for Vein Graft Disease PPARα Pivotally Regulates Metabolism, Activation, and Heterogeneity of Macrophages and Lesion Development.

Decano JL, Singh SA, Bueno CG, Lee LH, ... Aikawa E, Aikawa M
Background: Vein graft failure remains a common clinical challenge. We applied a systems approach in mouse experiments to discovering therapeutic targets for vein graft failure.
Methods:
Global proteomics and high-dimensional clustering on multiple vein graft tissues were used to identify potential pathogenic mechanisms. The peroxisome proliferator-activated receptors (PPARs) pathway served as an example to substantiate our discovery platform. In vivo mouse experiments with macrophage-targeted PPARα siRNA and the novel, selective activator pemafibrate demonstrate the role of PPARα in the development and inflammation of vein graft lesions. In vitro experiments further included metabolomic profiling, qPCR, flow cytometry, metabolic assays, and single-cell RNA-sequencing on primary human and mouse macrophages.
Results:
We identified changes in the vein graft proteome associated with immune responses, lipid metabolism regulated by the PPARs, fatty acid metabolism, matrix remodeling, and hematopoietic cell mobilization. PPARα agonism by pemafibrate retarded the development and inflammation of vein graft lesions in mice, while gene silencing worsened plaque formation. Pemafibrate also suppressed arteriovenous fistula lesion development. Metabolomics/lipidomics, functional metabolic assays, and single-cell analysis of cultured human macrophages revealed that PPARα modulates macrophage glycolysis, citrate metabolism, mitochondrial membrane sphingolipid metabolism, and heterogeneity. Conclusions: This study explored potential drivers of vein graft inflammation and identified PPARα as a novel potential pharmacologic treatment for this unmet medical need.




Circulation: 05 Apr 2021; epub ahead of print
Decano JL, Singh SA, Bueno CG, Lee LH, ... Aikawa E, Aikawa M
Circulation: 05 Apr 2021; epub ahead of print | PMID: 33821665
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Abstract

MicroRNA Biophysically Modulates Cardiac Action Potential by Direct Binding to Ion Channel.

Yang D, Wan X, Dennis AT, Bektik E, ... Deschênes I, Fu JD
Background
MicroRNAs (miRs) play critical roles in regulation of numerous biological events, including cardiac electrophysiology and arrhythmia, through a canonical RNA interference mechanism. It remains unknown whether endogenous miRs modulate physiologic homeostasis of the heart through noncanonical mechanisms.
Methods
We focused on the predominant miR of the heart (miR1) and investigated whether miR1 could physically bind with ion channels in cardiomyocytes by electrophoretic mobility shift assay, in situ proximity ligation assay, RNA pull down, and RNA immunoprecipitation assays. The functional modulations of cellular electrophysiology were evaluated by inside-out and whole-cell patch clamp. Mutagenesis of miR1 and the ion channel was used to understand the underlying mechanism. The effect on the heart ex vivo was demonstrated through investigating arrhythmia-associated human single nucleotide polymorphisms with miR1-deficient mice.
Results
We found that endogenous miR1 could physically bind with cardiac membrane proteins, including an inward-rectifier potassium channel Kir2.1. The miR1-Kir2.1 physical interaction was observed in mouse, guinea pig, canine, and human cardiomyocytes. miR1 quickly and significantly suppressed IK1 at sub-pmol/L concentration, which is close to endogenous miR expression level. Acute presence of miR1 depolarized resting membrane potential and prolonged final repolarization of the action potential in cardiomyocytes. We identified 3 miR1-binding residues on the C-terminus of Kir2.1. Mechanistically, miR1 binds to the pore-facing G-loop of Kir2.1 through the core sequence AAGAAG, which is outside its RNA interference seed region. This biophysical modulation is involved in the dysregulation of gain-of-function Kir2.1-M301K mutation in short QT or atrial fibrillation. We found that an arrhythmia-associated human single nucleotide polymorphism of miR1 (hSNP14A/G) specifically disrupts the biophysical modulation while retaining the RNA interference function. It is remarkable that miR1 but not hSNP14A/G relieved the hyperpolarized resting membrane potential in miR1-deficient cardiomyocytes, improved the conduction velocity, and eliminated the high inducibility of arrhythmia in miR1-deficient hearts ex vivo.
Conclusions
Our study reveals a novel evolutionarily conserved biophysical action of endogenous miRs in modulating cardiac electrophysiology. Our discovery of miRs\' biophysical modulation provides a more comprehensive understanding of ion channel dysregulation and may provide new insights into the pathogenesis of cardiac arrhythmias.



Circulation: 19 Apr 2021; 143:1597-1613
Yang D, Wan X, Dennis AT, Bektik E, ... Deschênes I, Fu JD
Circulation: 19 Apr 2021; 143:1597-1613 | PMID: 33590773
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Abstract

Long-Term Association of Air Pollution and Hospital Admissions Among Medicare Participants Using a Doubly Robust Additive Model.

Danesh Yazdi M, Wang Y, Di Q, Wei Y, ... Koutrakis P, Schwartz JD
Background
Studies examining the nonfatal health outcomes of exposure to air pollution have been limited by the number of pollutants studied and focus on short-term exposures.
Methods
We examined the relationship between long-term exposure to fine particulate matter with an aerodynamic diameter <2.5 micrometers (PM2.5), NO2, and tropospheric ozone and hospital admissions for 4 cardiovascular and respiratory outcomes (myocardial infarction, ischemic stroke, atrial fibrillation and flutter, and pneumonia) among the Medicare population of the United States. We used a doubly robust method for our statistical analysis, which relies on both inverse probability weighting and adjustment in the outcome model to account for confounding. The results from this regression are on an additive scale. We further looked at this relationship at lower pollutant concentrations, which are consistent with typical exposure levels in the United States, and among potentially susceptible subgroups.
Results
Long-term exposure to fine PM2.5 was associated with an increased risk of all outcomes with the highest effect seen for stroke with a 0.0091% (95% CI, 0.0086-0.0097) increase in the risk of stroke for each 1-µg/m3 increase in annual levels. This translated to 2536 (95% CI, 2383-2691) cases of hospital admissions with ischemic stroke per year, which can be attributed to each 1-unit increase in fine particulate matter levels among the study population. NO2 was associated with an increase in the risk of admission with stroke by 0.00059% (95% CI, 0.00039-0.00075) and atrial fibrillation by 0.00129% (95% CI, 0.00114-0.00148) per ppb and tropospheric ozone was associated with an increase in the risk of admission with pneumonia by 0.00413% (95% CI, 0.00376-0.00447) per parts per billion. At lower concentrations, all pollutants were consistently associated with an increased risk for all our studied outcomes.
Conclusions
Long-term exposure to air pollutants poses a significant risk to cardiovascular and respiratory health among the elderly population in the United States, with the greatest increase in the association per unit of exposure occurring at lower concentrations.



Circulation: 19 Apr 2021; 143:1584-1596
Danesh Yazdi M, Wang Y, Di Q, Wei Y, ... Koutrakis P, Schwartz JD
Circulation: 19 Apr 2021; 143:1584-1596 | PMID: 33611922
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Abstract

Sex-Specific Control of Human Heart Maturation by the Progesterone Receptor.

Sim CB, Phipson B, Ziemann M, Rafehi H, ... Hudson JE, Porrello ER
Background
Despite in-depth knowledge of the molecular mechanisms controlling embryonic heart development, little is known about the signals governing postnatal maturation of the human heart.
Methods
Single-nucleus RNA sequencing of 54 140 nuclei from 9 human donors was used to profile transcriptional changes in diverse cardiac cell types during maturation from fetal stages to adulthood. Bulk RNA sequencing and the Assay for Transposase-Accessible Chromatin using sequencing were used to further validate transcriptional changes and to profile alterations in the chromatin accessibility landscape in purified cardiomyocyte nuclei from 21 human donors. Functional validation studies of sex steroids implicated in cardiac maturation were performed in human pluripotent stem cell-derived cardiac organoids and mice.
Results
Our data identify the progesterone receptor as a key mediator of sex-dependent transcriptional programs during cardiomyocyte maturation. Functional validation studies in human cardiac organoids and mice demonstrate that the progesterone receptor drives sex-specific metabolic programs and maturation of cardiac contractile properties.
Conclusions
These data provide a blueprint for understanding human heart maturation in both sexes and reveal an important role for the progesterone receptor in human heart development.



Circulation: 19 Apr 2021; 143:1614-1628
Sim CB, Phipson B, Ziemann M, Rafehi H, ... Hudson JE, Porrello ER
Circulation: 19 Apr 2021; 143:1614-1628 | PMID: 33682422
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Impact:
Abstract

Cost-effectiveness of Hypertension Treatment by Pharmacists in Black Barbershops.

Bryant KB, Moran AE, Kazi DS, Zhang Y, ... Bibbins-Domingo K, Bellows BK
Background: In the Los Angeles Barbershop Blood Pressure Study (LABBPS), pharmacist-led hypertension care in Los Angeles County Black-owned barbershops significantly improved blood pressure control in non-Hispanic Black men with uncontrolled hypertension at baseline. In this analysis, 10-year health outcomes and healthcare costs of one year of the LABBPS intervention versus control are projected.
Methods:
A discrete event simulation of hypertension care processes projected blood pressure, medication-related adverse events, fatal and non-fatal cardiovascular disease events, and non-cardiovascular disease death in LABBPS participants. Program costs, total direct healthcare costs (2019 USD), and quality-adjusted life years (QALYs) were estimated for the LABBPS intervention and control arms from a healthcare sector perspective over a 10-year horizon. Future costs and QALYs were discounted 3% annually. High and intermediate cost-effectiveness thresholds were defined as <$50,000 and <$150,000 per QALY gained, respectively.
Results:
At 10 years, the intervention was projected to cost an average of $2,356 (95% uncertainty interval [UI] -$264-$4,611) more per participant than the control arm and gain 0.06 (95% UI 0.01-0.10) QALYs. The LABBPS intervention was highly cost-effective, with a mean cost of $42,717 per QALY gained (58% probability of being highly and 96% of being at least intermediately cost-effective). Exclusive use of generic drugs improved the cost-effectiveness to $17,162 per QALY gained. The LABBPS intervention would be only intermediately cost effective if pharmacists were less likely to intensify antihypertensive medications when systolic blood pressure was ≥150 mmHg or if pharmacist weekly time driving to barbershops increased. Conclusions: Hypertension care delivered by clinical pharmacists in Black barbershops is a highly cost-effective way to improve blood pressure control in Black men.




Circulation: 14 Apr 2021; epub ahead of print
Bryant KB, Moran AE, Kazi DS, Zhang Y, ... Bibbins-Domingo K, Bellows BK
Circulation: 14 Apr 2021; epub ahead of print | PMID: 33855861
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Abstract

Inducible Depletion of Calpain-2 Mitigates Abdominal Aortic Aneurysm in Mice.

Muniappan L, Okuyama M, Javidan A, Thiagarajan D, ... Saido TC, Subramanian V
Objective
Cytoskeletal structural proteins maintain cell structural integrity by bridging extracellular matrix with contractile filaments. During abdominal aortic aneurysm (AAA) development, (1) aortic medial degeneration is associated with loss of smooth muscle cell integrity and (2) fibrogenic mesenchymal cells mediate extracellular matrix remodeling. Calpains cleave cytoskeletal proteins that maintain cell structural integrity. Pharmacological inhibition of calpains exert beneficial effects on Ang II (angiotensin II)-induced AAAs in LDLR-/- (low-density receptor deficient) mice. Here, we evaluated the functional contribution of fibrogenic mesenchymal cells-derived calpain-2 on (1) cytoskeletal structural protein and extracellular matrix alterations and (2) AAA progression. Approach and
Results:
Calpain-2 protein and cytoskeletal protein (filamin and talin) fragmentation are significantly elevated in human and Ang II-induced AAAs in mice. To examine the relative contribution of calpain-2 in AAA development, calpain-2 floxed mice in an LDLr-/- background were bred to mice with a tamoxifen-inducible form of Cre under control of either the ubiquitous promoter, chicken β-actin, or fibrogenic mesenchymal cell-specific promoter, Col1α2. Ubiquitous or fibrogenic mesenchymal cell-specific depletion of calpain-2 in mice suppressed Ang II-induced AAAs, filamin/talin fragmentation, while promoting extracellular matrix protein, collagen in the aortas. Calpain-2 silencing in aortic smooth muscle cells or fibroblasts reduced Ang II-induced filamin fragmentation. In addition, silencing of filamin in aortic SMCs significantly reduced collagen protein. Furthermore, calpain-2 deficiency suppressed rupture of established Ang II-induced AAAs in mice.
Conclusions
Our studies implicate that calpain-2 deficiency prevents (1) Ang II-induced cytoskeletal structural protein fragmentation and AAA development and (2) stabilize and suppress rupture of established AAAs in mice.



Arterioscler Thromb Vasc Biol: 24 Mar 2021:ATVBAHA120315546; epub ahead of print
Muniappan L, Okuyama M, Javidan A, Thiagarajan D, ... Saido TC, Subramanian V
Arterioscler Thromb Vasc Biol: 24 Mar 2021:ATVBAHA120315546; epub ahead of print | PMID: 33761765
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Impact:
Abstract

Sexually Dimorphic Relationships Among Saa3 (Serum Amyloid A3) Inflammation, and Cholesterol Metabolism Modulate Atherosclerosis in Mice.

Chait A, Wang S, Goodspeed L, Gomes D, ... Lusis AJ, Den Hartigh LJ
Objective
Expression of the extrahepatic acute-phase protein Saa3 (serum amyloid A3) increases in response to acute and chronic inflammatory stimuli and is elevated in adipose tissue and macrophages in obese mice. A recent report suggested that Saa3 is proatherogenic in male ApoE-/- mice. Because of our previous observation that female but not male Saa3-deficient mice are protected from obesity, adipose inflammation, and hyperlipidemia, we sought to determine whether Saa3 differentially modulates atherosclerosis in mice of both sexes. Approach and
Results:
To promote atherosclerosis, Saa3+/+ and Saa3-/- male and female mice were crossed with Ldlr-/- mice. All mice consumed a diet high in saturated fat and sucrose with 0.15% added cholesterol for 16 weeks. Plasma lipids and atherosclerosis levels were assessed. Female Saa3-/-Ldlr-/- mice exhibited elevated cholesterol levels relative to Saa3+/+Ldlr-/- controls and exhibited increased atherosclerosis, while male Saa3-/-Ldlr-/- mice were protected from atherosclerosis. Data from the hybrid mouse diversity panel revealed that Saa3 associates strongly with inflammatory, Trem2-associated, and tissue remodeling genes and pathways in males but not females, an effect confirmed in liver tissue, atherosclerotic lesions, and cultured macrophages. Macrophages isolated from male and female mice showed differential inflammatory effects of Saa3 deficiency, an effect linked with sex steroid signaling. Cholesterol efflux capacity was increased in Saa3-/- males only.
Conclusions
Saa3 is proatherogenic in male but atheroprotective in female mice, effects that may be related to sex-specific relationships between Saa3, cholesterol metabolism, inflammatory genes, and Trem2 macrophages.



Arterioscler Thromb Vasc Biol: 24 Mar 2021:ATVBAHA121316066; epub ahead of print
Chait A, Wang S, Goodspeed L, Gomes D, ... Lusis AJ, Den Hartigh LJ
Arterioscler Thromb Vasc Biol: 24 Mar 2021:ATVBAHA121316066; epub ahead of print | PMID: 33761762
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Impact:
Abstract

Heme Oxygenase-1 in Macrophages Impairs the Perfusion Recovery After Hindlimb Ischemia by Suppressing Autolysosome-Dependent Degradation of NLRP3.

Ma Y, Jia L, Wang Y, Ji Y, ... Xie Y, Xiang M
Objective
Macrophage-mediated inflammatory response is closely associated with the neovascularization process following hindlimb ischemia. We previously demonstrated that HO-1 (heme oxygenase-1) in macrophages evoked proinflammatory reactions and tissue damage. Here, we evaluated the role played by macrophage-derived HO-1 and elucidated its underlying molecular mechanisms in perfusion recovery after hindlimb ischemia. Approach and
Results:
We found significant upregulation of HO-1 in mouse ischemic muscles after hindlimb ischemia surgery and with most of this expression occurring in infiltrated macrophages. Myeloid conditional HO-1-deficient mice exhibited higher perfusion recovery, evidenced by restored blood flow, motor function and attenuated tissue damage as well as increased capillary density in the gastrocnemius muscles after hindlimb ischemia, relative to littermate controls. This protective effect was accompanied by reduced nod-like receptor family, NLRP3 (pyrin domain containing 3) inflammasome activation in the infiltrated macrophages without the alteration of macrophage infiltration and polarization. Moreover, suppressing inflammasome activation with NLRP3 inhibitor MCC950 improved blood flow and capillary density in wild-type mice compared with untreated mice. Mechanistically, suppressing HO-1 abolished TNF (tumor necrosis factor)-α-induced NLRP3 protein rather than mRNA expression in bone marrow-derived macrophages, indicating that HO-1 mediated post-transcriptional regulation of NLRP3. Furthermore, HO-1 inhibition promoted autolysosome-dependent degradation of NLRP3 in bone marrow-derived macrophages. Matrigel tube formation assay revealed that HO-1 deletion abrogated the antiangiogenic effect of inflammasome-activated macrophages.
Conclusions
Taken together, these findings indicate that macrophage HO-1 deficiency promotes perfusion recovery after hindlimb ischemia by accelerating autolysosomal degradation of NLRP3. The underlying mechanism of action is a potential target for therapeutic angiogenesis in ischemic diseases.



Arterioscler Thromb Vasc Biol: 24 Mar 2021:ATVBAHA121315805; epub ahead of print
Ma Y, Jia L, Wang Y, Ji Y, ... Xie Y, Xiang M
Arterioscler Thromb Vasc Biol: 24 Mar 2021:ATVBAHA121315805; epub ahead of print | PMID: 33761761
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Impact:
Abstract

Deficiency of MMP1a (Matrix Metalloprotease 1a) Collagenase Suppresses Development of Atherosclerosis in Mice: Translational Implications for Human Coronary Artery Disease.

Fletcher EK, Wang Y, Flynn LK, Turner SE, ... Covic L, Kuliopulos A
Objective
Destruction of arterial collagen allows monocyte and macrophage infiltration leading to atherosclerotic plaque formation, but it is not clear what role the MMP1 (matrix metalloprotease 1) collagenase plays in this process in vivo. To define the specific contribution of MMP1 to atherosclerotic plaque burden and pathogenesis, we generated ApoE-/- mice deficient in the human MMP1 ortholog, MMP1a. Approach and
Results:
After 12 to 16 weeks of Western diet, genetic loss of MMP1a resulted in a significant 50% reduction in total aortic plaque burden compared with control ApoE-/- mice. MMP1a deficiency led to significant reductions in plaque monocytes/macrophages, SMCs, and necrosis, with increases in collagen content. Collagen invasion of oxidized-LDL (low-density lipoprotein) activated peripheral blood mononuclear cells from MMP1a-deficient mice was markedly attenuated and was similar to suppressive effects with pharmacological inhibitors of MMP1 and its receptor, PAR1 (protease-activated receptor 1). Patients with coronary artery disease and acute coronary syndrome undergoing cardiac catheterization in the TRIP-PCI trial were evaluated for circulating levels of all 3 major secreted collagenases, MMP1, MMP8, and MMP13 and total number of coronary lesions with ≥50% stenosis (coronary artery disease burden). MMP1 was significantly (P<0.001) higher by 19-fold and 5.7-fold relative to MMP13 and MMP8, respectively. MMP1 correlated with stenotic coronary artery disease burden, TNFα (tumor necrosis factor alpha) levels, and was co-expressed with PAR1 on monocytes. Treatment of patients with the PAR1 inhibitor, PZ-128, prevented a drop in monocytes following coronary catheterization, an acute protective effect that was reproduced in mice undergoing cardiac ischemia reperfusion.
Conclusions
These data provide evidence for an important role for the MMP1a collagenase in atherosclerotic lesion development and leukocyte behavior and validate MMP1 as a compelling target in patients with coronary artery disease/acute coronary syndrome.
Registration
URL: https://www.clinicaltrials.gov; Unique identifier: NCT02561000.



Arterioscler Thromb Vasc Biol: 24 Mar 2021:ATVBAHA120315837; epub ahead of print
Fletcher EK, Wang Y, Flynn LK, Turner SE, ... Covic L, Kuliopulos A
Arterioscler Thromb Vasc Biol: 24 Mar 2021:ATVBAHA120315837; epub ahead of print | PMID: 33761760
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Impact:
Abstract

Loss of TIMP4 (Tissue Inhibitor of Metalloproteinase 4) Promotes Atherosclerotic Plaque Deposition in the Abdominal Aorta Despite Suppressed Plasma Cholesterol Levels.

Hu M, Jana S, Kilic T, Wang F, ... Zhang DW, Kassiri Z
Objective
Atherosclerosis is accumulation of lipids and extracellular matrix in the arterial wall. TIMPs (tissue inhibitor of metalloproteinases) can impact plaque deposition by regulating ECM (extracellular matrix) turnover. TIMP4 also influences lipid metabolism and smooth muscle cell (SMC) proliferation. We investigated the role of TIMP4 in atherosclerosis. Approach and
Results:
Mice lacking low-density lipoprotein receptor (Ldlr-/-) and Timp4 (Timp4-/-/Ldlr-/-) were fed high-fat diet (HFD) or regular laboratory diet. After 3 or 6 months, HFD-fed male and female Timp4-/-/Ldlr-/- mice exhibited higher plaque density in the abdominal aorta (but not in aortic valves, arch, thoracic aorta) compared with Ldlr-/- mice. Although plasma lipid and cholesterol levels were lower in Timp4-/-/Ldlr-/--HFD, cholesterol content in the abdominal aorta was higher along with elevated inflammatory cytokines, MMP (matrix metalloproteinase) activities, CD68+/calponin+ macrophage-like SMCs in Timp4-/-/Ldlr-/--HFD compared with Ldlr-/--HFD mice. In vitro, oxidized LDL (low-density lipoprotein) markedly increased CD68 expression, reduced SMC markers, increased lipid uptake, and reduced cholesterol efflux protein ABCA1 (ATP-binding cassette transporter A1) in Timp4-/-/Ldlr-/- compared with Ldlr-/- primary SMCs from abdominal, but not thoracic aorta. TIMP4 expression in the abdominal aorta (in vivo) and its corresponding SMCs (in vitro) was ≈2-fold higher than in the thoracic aorta and SMCs; TIMP4 levels decreased following HFD. Timp4-deficiency in bone marrow-derived macrophages did not alter their foam cell formation capacity.
Conclusions
TIMP4 protects against plaque deposition in the abdominal aorta independent of plasma cholesterol levels. TIMP4 prevents proteolytic degradation of ABCA1 in SMCs, hindering cholesterol accumulation and transdifferentiation to macrophage-like foam cells, representing a novel negative regulator of atherosclerosis.



Arterioscler Thromb Vasc Biol: 31 Mar 2021:ATVBAHA120315522; epub ahead of print
Hu M, Jana S, Kilic T, Wang F, ... Zhang DW, Kassiri Z
Arterioscler Thromb Vasc Biol: 31 Mar 2021:ATVBAHA120315522; epub ahead of print | PMID: 33792349
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Impact:
Abstract

Low LAL (Lysosomal Acid Lipase) Expression by Smooth Muscle Cells Relative to Macrophages as a Mechanism for Arterial Foam Cell Formation.

Dubland JA, Allahverdian S, Besler KJ, Ortega C, ... Seidman MA, Francis GA
Objective
We previously reported smooth muscle cells (SMCs) represent ≥50% of foam cells in human coronary and ≈70% in apoE (apolipoprotein E)-deficient mouse aortic atheromas and exhibit reduced expression of the cholesterol exporter ABCA1 (ATP-binding cassette transporter A1). A major stimulus for ABCA1 expression is flux of cholesterol out of lysosomes, generated by hydrolysis of lipoprotein cholesteryl esters by LAL (lysosomal acid lipase). In this study, we investigated the potential role lysosomal dysfunction might play in foam cell formation by arterial SMCs. Approach and
Results:
Human monocyte-derived macrophages (macrophages) and arterial SMCs were treated with aggregated LDL (low-density lipoprotein) to increase intracellular cholesterol and investigated for lysosomal and postlysosomal cholesterol metabolism defects. Human and mouse atheromas were analyzed for LAL expression. Unlike macrophages, aggregated LDL uptake failed to upregulate ABCA1 expression, downregulate new cholesterol synthesis, or to significantly increase 27-hydroxycholesterol levels in SMCs. Confocal microscopy revealed retention of neutral lipids within lysosomal compartments in SMCs, while macrophages showed most lipids as cytosolic droplets. LIPA mRNA levels and LAL protein were markedly reduced in SMCs. Treatment of SMCs with medium containing LAL resulted in significantly reduced lysosomal lipid accumulation and increased cholesterol efflux to apoA-I (apolipoprotein AI). Human and mouse atheromas exhibited low LAL/Lipa expression in intimal SMCs when compared with intimal macrophages.
Conclusions
These findings indicate the inherently low level of LAL in SMCs compared with macrophages is associated with reduced capacity to catabolize atherogenic lipoproteins and is a mechanism for SMC foam cell formation in atherosclerosis.



Arterioscler Thromb Vasc Biol: 31 Mar 2021:ATVBAHA120316063; epub ahead of print
Dubland JA, Allahverdian S, Besler KJ, Ortega C, ... Seidman MA, Francis GA
Arterioscler Thromb Vasc Biol: 31 Mar 2021:ATVBAHA120316063; epub ahead of print | PMID: 33792344
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Impact:
Abstract

HSP25 Vaccination Attenuates Atherogenesis via Upregulation of LDLR Expression, Lowering of PCSK9 Levels and Curbing of Inflammation.

Chen YX, Shi C, Deng J, Diao C, ... Gerthoffer WT, O\'Brien ER
Objective
Elevated HSP27 (heat shock protein 27) levels predict relative freedom from cardiovascular events. Over-expression or twice daily subcutaneous injections of human HSP27 in ApoE -/- mice reduces blood and plaque cholesterol levels, as well as inflammation and atherosclerotic plaque burden. Antibodies to HSP27 are present in human blood, and the purpose of the current studies is to explore their role. Approach and
Results:
Blood levels of both HSP27 and anti-HSP27 IgG antibodies are elevated in healthy controls compared with patients with cardiovascular disease. ApoE -/- mice fed a high-fat diet and vaccinated with recombinant HSP25 (rHSP25, murine ortholog) show increased levels of anti-HSP25 IgG antibodies and reductions in plasma cholesterol and atherogenesis. Moreover, rHSP25 vaccination markedly lowered serum amyloid A levels as well as hepatic macrophage abundance and inflammatory cytokine expression. The effects of the HPS25 vaccination on cholesterol metabolism are divergent: increased hepatic LDLR (low-density lipoprotein receptor) mRNA and protein expression and reduced plasma PCSK9 (proprotein convertase subtilisin/kexin type 9) levels-despite no effect on PCSK9 expression. In vitro, the HSP27 immune complex upregulates hepatocyte LDLR mRNA and protein expression independent of intracellular cholesterol levels and increases LDLR promoter activity. The increase in LDLR expression by the HSP27 immune complex is dependent upon activation of the NF-κB (nuclear factor κ light chain enhancer of activated B cells) pathway. Hepatocyte PCSK9 protein levels are reduced after HSP27 immune complex treatment in vitro despite only minor transient effects on gene expression.

Conclusions
HSP27 immunotherapy represents a novel means of lowering cholesterol and PCSK9 levels, primarily due to augmentation of LDLR expression and is associated with marked reductions in inflammation.



Arterioscler Thromb Vasc Biol: 31 Mar 2021:ATVBAHA121315933; epub ahead of print
Chen YX, Shi C, Deng J, Diao C, ... Gerthoffer WT, O'Brien ER
Arterioscler Thromb Vasc Biol: 31 Mar 2021:ATVBAHA121315933; epub ahead of print | PMID: 33792343
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Impact:
Abstract

Monocyte-Chemoattractant Protein-1 Levels in Human Atherosclerotic Lesions Associate With Plaque Vulnerability.

Georgakis MK, van der Laan SW, Asare Y, Mekke JM, ... Pasterkamp G, Dichgans M
Objective
To determine whether MCP-1 (monocyte chemoattractant protein 1) levels in human atherosclerotic plaques associate with plaque vulnerability features. Approach and
Results:
We measured MCP-1 levels in human atherosclerotic plaque samples from 1199 patients in the Athero-EXPRESS Biobank who underwent endarterectomy for treatment of carotid stenosis. We explored associations with histopathologic and molecular features of plaque vulnerability, clinical plaque manifestations, and vascular events up to 3 years after endarterectomy. Following adjustments for age, sex, and vascular risk factors, MCP-1 plaque levels were associated with histopathologic markers of plaque vulnerability (large lipid core, low collagen content, high macrophage burden, low smooth muscle cell burden, intraplaque hemorrhage) and with a composite vulnerability index (range 0-5, β per SD increment in MCP-1, 0.42 [95% CI, 0.30-0.53], P=5.4×10-13). We further found significant associations with higher plaque levels of other chemokines and proinflammatory molecules and markers of neovascularization and matrix turnover. When exploring clinical plaque instability, MCP-1 plaque levels were higher among individuals with symptomatic plaques as compared with those with asymptomatic plaques (odds ratio per SD increment in MCP-1, 1.36 [95% CI, 1.09-1.69]). MCP-1 levels were further associated with a higher risk of periprocedural major adverse vascular events and strokes occurring in the first 30 days after plaque removal.
Conclusions
Higher MCP-1 plaque levels are associated with histopathologic, molecular, and clinical hallmarks of plaque vulnerability in individuals undergoing carotid endarterectomy. Our findings highlight a role of MCP-1 in clinical plaque instability in humans and complement previous epidemiological, genetic, and experimental studies supporting the translational perspective of targeting MCP-1 signaling in atherosclerosis.



Arterioscler Thromb Vasc Biol: 07 Apr 2021:ATVBAHA121316091; epub ahead of print
Georgakis MK, van der Laan SW, Asare Y, Mekke JM, ... Pasterkamp G, Dichgans M
Arterioscler Thromb Vasc Biol: 07 Apr 2021:ATVBAHA121316091; epub ahead of print | PMID: 33827260
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Impact:
Abstract

H3K4 Methyltransferase Smyd3 Mediates Vascular Smooth Muscle Cell Proliferation, Migration, and Neointima Formation.

Yang D, Su Z, Wei G, Long F, ... Liu X, Zhu YZ
Objective
Smyd3 (SET and MYND domain-containing protein 3) is an H3K4 (histone H3 lysine 4) dimethyltransferase and trimethyltransferase that activates the transcription of oncogenes and cell cycle genes in human cancer cells. We discovered its overexpression in proliferative vascular smooth muscle cells (VSMCs). However, whether Smyd3 plays a role in vascular remodeling remains unanswered. The objective of this study is to investigate the role and underlying mechanism of Smyd3 in phenotypic transition of VSMCs (such as proliferation and migration) and vascular remodeling (such as neointima formation). Approach and
Results:
We discovered upregulation of Smyd3 in both PDGF (platelet-derived growth factor) BB-induced vascular cell proliferation model and balloon injury-induced neointima formation model. Knockdown of Smyd3 or blockade of its enzymatic activity suppressed VSMCs proliferation and migration ability, whereas Smyd3 overexpression promoted VSMC migration and proliferation. Mechanistically, RNA-seq and ChIP-seq analysis revealed Smyd3 promoted neointimal formation by directly binding and increasing H3K4me3 to the promoter regions of target genes that are associated with cell proliferation and migration, cell cycle control. Furthermore, knockout of Smyd3 in mice profoundly suppressed carotid artery ligation-induced neointimal hyperplasia, consistently, local knocking down Smyd3 in rats relieved balloon injury-induced neointimal formation, while restored VSMC contractile protein expression, suggesting that Smyd3 plays a critical role in vivo.
Conclusions
Our results demonstrate that Smyd3 promotes VSMC proliferation and migration during injury-induced vascular remodeling, which provide a potential therapeutic target for preventing neointimal hyperplasia in proliferative vascular diseases.



Arterioscler Thromb Vasc Biol: 07 Apr 2021:ATVBAHA121314689; epub ahead of print
Yang D, Su Z, Wei G, Long F, ... Liu X, Zhu YZ
Arterioscler Thromb Vasc Biol: 07 Apr 2021:ATVBAHA121314689; epub ahead of print | PMID: 33827259
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Impact:
Abstract

Fetal Programming by Methyl Donor Deficiency Produces Pathological Remodeling of the Ascending Aorta.

Balint B, Hergalan S, Camadro JM, Blaise S, ... Guéant-Rodriguez RM, Guéant JL
Objective
Deficiency in vitamin B12/folate (methyl donor deficiency [MDD]) produces cardiovascular outcomes during aging and fetal programming effects in newborns of MDD mothers. Whether fetal programming provokes long-term effects on aorta remains largely unknown. Approach and
Results:
We investigated the impact of fetal programming on ascending aorta of aged rats born from mothers subjected to MDD during gestation/lactation. We performed morphological and molecular examinations of ascending aorta in 21 days- and 400 days-aged rats with initial MDD fetal programming (initial MDD) compared with control matched rats. Initial MDD induces remodeling of ascending aorta in aged rats, with collagen deposition (P=0.0008), decreased thickness of elastin (P<0.0001), and 8.7-fold increase of elastin breaks (P=0.0002). Proteomic analyses, Western blotting, and immunohistochemical examination revealed decreased expression of α-smooth muscle actin, vinculin, SM22α (smooth muscle 22α), and N-cadherin and increased expression of TGF (transforming growth factor) β1. Elastin breaks were correlated to increased neutrophil elastase (P=0.0002), cathepsin-K (P=0.0002), cathepsin-S (P<0.0001), and MMP (matrix metalloproteinase) 9, and MMP2 (P<0.0001 and P=0.02). Proximity Duolink ligation assay showed homocysteinylation of actin-associated and extracellular matrix proteins, including SM22α (P=0.01), N-cadherin (P=0.0008), and vinculin (P=0.001), which was associated with elastin breaks (P=0.002) and increased expression of MARS (methionyl-tRNA synthetase; involved in irreversible protein homocysteinylation). Furthermore, we observed an inverse relationship between elastin breaks and blood pressure (systolic, P=0.004 and diastolic, P=0.0007).
Conclusions
MDD fetal programming produced altered integrity and remodeling of ascending aorta during aging and irreversible MARS-associated homocysteinylation of key proteins of extracellular matrix and elastin homeostasis. This contributes to understanding why homocysteine-lowering vitamin B supplementation fails to relieve vascular complications in adulthood.



Arterioscler Thromb Vasc Biol: 07 Apr 2021:ATVBAHA120315587; epub ahead of print
Balint B, Hergalan S, Camadro JM, Blaise S, ... Guéant-Rodriguez RM, Guéant JL
Arterioscler Thromb Vasc Biol: 07 Apr 2021:ATVBAHA120315587; epub ahead of print | PMID: 33827257
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Impact:
Abstract

Altered Responsiveness to TGFβ and BMP and Increased CD45+ Cell Presence in Mitral Valves Are Unique Features of Ischemic Mitral Regurgitation.

Castillero E, Howsmon DP, Rego BV, Keeney S, ... Levy RJ, Ferrari G
Objective
Ischemic mitral regurgitation (IMR) often develops after an ischemic event, which results in distortion of the valvulo-ventricular complex and incomplete mitral valve (MV) leaflet coaptation. After left ventricular ischemic events, only some patients develop IMR. The susceptibility of the MV to remodel may influence whether IMR develops. We hypothesized that impaired signaling response in MV cells may contribute to IMR development by inducing maladaptive tissue remodeling. Approach and
Results:
Sheep (n=14) were subjected to ligation of the circumflex coronary artery to induce myocardial infarction. IMR was reported by echocardiography. MV leaflets and MV interstitial cells (MVICs) were collected at baseline (control, n=10), 4 and 8 weeks post-myocardial infarction. RNA sequencing highlighted differences in TGFβ (transforming growth factor beta) signaling between MV with/without IMR. SMAD6/7 and ID2 (inhibitor of DNA binding 2) were the highest increased TGFβ-signaling genes associated with IMR. MVICs from myocardial infarction sheep were less responsive to BMP (bone morphogenic protein) 4 pro-osteogenic stimulation (ID2, OPN [osteopontin], and OC [osteocalcin] mRNA) than control. MVICs from IMR sheep had a diminished COL (collagen) 1A1 mRNA response to TGFβ1 and enhanced prochondrogenic RUNX2 (runt-related transcription factor 2) and SOX9 mRNA response to BMP4 versus non-IMR MVICs. Baseline CD45 expression was detectable only in IMR MVICs. Upon TGFβ1 stimulation, CD45 expression was detected in all groups. Immunostaining confirmed increased presence of CD45+ cells in IMR MV interstitium.
Conclusions
MVs from sheep with IMR had an altered TGFβ/BMP response, associated with increased CD45+ cell presence within the tissue interstitium. Pharmacological strategies aimed to modulate TGFβ/BMP signaling after myocardial infarction may protect from pathological MV remodeling leading to IMR.



Arterioscler Thromb Vasc Biol: 07 Apr 2021:ATVBAHA121316111; epub ahead of print
Castillero E, Howsmon DP, Rego BV, Keeney S, ... Levy RJ, Ferrari G
Arterioscler Thromb Vasc Biol: 07 Apr 2021:ATVBAHA121316111; epub ahead of print | PMID: 33827255
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Impact:
Abstract

Gab2 (Grb2-Associated Binder2) Plays a Crucial Role in Inflammatory Signaling and Endothelial Dysfunction.

Kondreddy V, Magisetty J, Keshava S, Rao LVM, Pendurthi UR
Objective
In response to inflammatory insult, endothelial cells express cell adhesion molecules and TF (tissue factor), leading to increased adhesion of leukocytes to the endothelium and activation of coagulation. Enhanced coagulation could further exacerbate inflammation. Identifying key signaling molecule(s) that drive both inflammation and coagulation may help devise effective therapeutic strategies to treat inflammatory and thrombotic disorders. The aim of the current study to determine the role of Gab2 (Grb2-associated binder2), which is known to play a crucial role in the signaling evoked by growth factors and antigen receptors, in inflammatory signaling pathways and contributing to vascular dysfunction. Approach and
Results:
WT (wild type) and Gab2-silenced endothelial cells were treated with TNFα (tumor necrosis factor alpha), IL (interleukin)-1β, or lipopolysaccharide (LPS). Activation of key signaling proteins in the inflammatory signaling pathways and expression of cell adhesion molecules, TF, and inflammatory cytokines were analyzed. Gab2-/- and WT littermate mice were challenged with LPS or S pneumoniae, and parameters of inflammation and activation of coagulation were assessed. Gab2 silencing in endothelial cells markedly attenuated TNFα-induced, IL-1β-induced, and LPS-induced expression of TF, cell adhesion molecules, and inflammatory cytokines/chemokines. Gab2 silencing suppressed TNFα-induced, IL-1β-induced, and LPS-induced phosphorylation and ubiquitination of TAK1 and activation of MAPKs (mitogen-activated protein kinases) and NF-κB (nuclear factor kappa B). Immunoprecipitation studies revealed that the Src kinase Fyn phosphorylates Gab2. Gab2-/- mice are protected from LPS or S pneumoniae-induced vascular permeability, neutrophil infiltration, thrombin generation, NET formation, cytokine production, and lung injury.
Conclusions
Our studies identify, for the first time, that Gab2 integrates signaling from multiple inflammatory receptors and regulates vascular inflammation and thrombosis.



Arterioscler Thromb Vasc Biol: 07 Apr 2021:ATVBAHA121316153; epub ahead of print
Kondreddy V, Magisetty J, Keshava S, Rao LVM, Pendurthi UR
Arterioscler Thromb Vasc Biol: 07 Apr 2021:ATVBAHA121316153; epub ahead of print | PMID: 33827252
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Impact:
Abstract

Major Reservoir for Heparin-Releasable TFPIα (Tissue Factor Pathway Inhibitor α) Is Extracellular Matrix.

Peterson JA, Maroney SA, Martinez ND, Mast AE
Objective
Human endothelial cells produce 2 alternatively spliced TFPI (tissue factor pathway inhibitor) isoforms that maintain anticoagulant properties of the vasculature. TFPIβ is glycosylphosphatidylinositol anchored on the cell surface. TFPIα has a basic C terminus sharing homology with VEGF (vascular endothelial growth factor) and is a heparin-releasable protein, suggesting it binds glycosaminoglycans on the endothelium surface. However, this is unclear because TFPIα is not on the surface of cultured endothelial cells. This study identifies the source of heparin-releasable TFPIα. Approach and
Results:
ELISA assays localized heparin-releasable TFPIα to the extracellular matrix (ECM) of Ea.hy926 cells and human umbilical vein endothelial cells. Immunofluorescence microscopy for TFPIα showed punctate intracytoplasmic staining and ECM staining beneath individual cells. Flow cytometry identified TFPIβ but not TFPIα on the cell surface. TFPIα localization to ECM was confirmed with ELISA and immunohistochemistry studies of umbilical cord veins. The TFPIα C terminus interacted with Ea.hy926 ECM glycosaminoglycans, and a homologous VEGF peptide competed for this binding, suggesting these interactions modulate VEGF responses. Immobilized TFPIα C-terminal peptide bound to several ECM proteoglycans in Ea.hy926 conditioned media. Immunofluorescence studies of human kidney colocalized TFPIα with 4 of these proteoglycans surrounding the microvasculature: glypican-1, syndecan-4, thrombospondin, and laminin-5. The absence of TFPIα on the surface of endothelial cells and its co-localization with specific ECM proteins suggests TFPIα binds to unique proteoglycan structures.
Conclusions
ECM contained the primary vascular pool of heparin-releasable TFPIα. By localizing to ECM, TFPIα is positioned to inhibit the procoagulant activity of tissue factor surrounding the vasculature.



Arterioscler Thromb Vasc Biol: 07 Apr 2021:ATVBAHA120315728; epub ahead of print
Peterson JA, Maroney SA, Martinez ND, Mast AE
Arterioscler Thromb Vasc Biol: 07 Apr 2021:ATVBAHA120315728; epub ahead of print | PMID: 33827254
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Impact:
Abstract

Local Action of Neprilysin Exacerbates Pressure Overload Induced Cardiac Remodeling.

Nakagawa H, Kumazawa T, Onoue K, Nakada Y, ... Kawakami R, Saito Y
NEP (Neprilysin) degrades natriuretic peptides, and its inhibition is a clinically accepted target for heart failure treatment. NEP is widely expressed in various organs, including the heart. However, the pathophysiological significance of local cardiac NEP is not fully understood. To study the local function of NEP in the heart, we generated transgenic mice overexpressing NEP, specifically in cardiomyocytes (CM-NEP Tg). At baseline, CM-NEP Tg mice showed significantly lower levels of plasma ANP (atrial natriuretic peptide), plasma cGMP, and cardiac tissue cGMP versus wild-type (WT) mice. Blood pressure, heart weight, and cardiomyocyte diameter were greater in CM-NEP Tg than WT mice. There were no significant differences in interstitial fibrosis or ejection fraction. Transverse aortic constriction (TAC) surgery significantly increased left ventricular weight in WT and CM-NEP Tg mice 3 weeks post-op versus sham surgery; however, the cardiac hypertrophic response to TAC was higher in CM-NEP Tg than WT mice. Cardiac interstitial fibrosis was induced in TAC CM-NEP Tg mice, whereas TAC WT mice had none. TAC CM-NEP Tg, but not TAC WT, mice developed cardiac dysfunction secondary to TAC with echocardiography. Furthermore, administration of human ANP to raise plasma ANP levels comparable to those in WT mice neither improved the exacerbated cardiac hypertrophy and fibrosis nor recovered impaired cardiac function in CM-NEP Tg mice after TAC. In conclusion, overexpression of NEP in cardiomyocytes promoted degradation of natriuretic peptides in the heart and led to an exaggerated response of hypertrophy and fibrosis to pressure overload.



Hypertension: 11 Apr 2021:HYPERTENSIONAHA12016445; epub ahead of print
Nakagawa H, Kumazawa T, Onoue K, Nakada Y, ... Kawakami R, Saito Y
Hypertension: 11 Apr 2021:HYPERTENSIONAHA12016445; epub ahead of print | PMID: 33840200
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Impact:
Abstract

Dietary Magnesium Insufficiency Induces Salt-Sensitive Hypertension in Mice Associated With Reduced Kidney Catechol-O-Methyl Transferase Activity.

Kumagai A, Takeda S, Sohara E, Uchida S, ... Koya D, Kanasaki K
COMT (Catechol-O-methyl transferase), an enzyme that metabolizes catechol, requires magnesium (Mg2+) to maintain its activity. Low COMT activity causes insufficient 2-methoxyestradiol (2-ME), a biologically active metabolite from hydroxyestradiol, which leads to hypertensive disorders, including preeclampsia. Hypoestrogenism increases the risk of salt-sensitive hypertension (SSH). SSH and preeclampsia are risk factors for each other; however, the molecular mechanism of this interaction is unclear. We focused on the interactive effect of Mg2+ insufficiency and genetic COMT deficiency on SSH using 2 strains of mice with genetically distinct COMT activity. In male mice, BL6 (C57BL/6J), a high-activity COMT strain, displayed unaltered blood pressure regardless of the Mg2+ and salt levels in food; DBA (DBA/2J), a low-activity COMT strain, developed SSH under low Mg2+ and high-salt conditions. COMT inhibition in C57BL/6J strain also induced SSH. Treatment with 2-ME ameliorated SSH in both models. The ATR1 (angiotensin II type 1 receptor)-STE20-SPAK (serine-proline alanine-rich kinase)-NCC (sodium chloride cotransporter) axis, molecules associated with sodium reabsorption in distal convoluted tubules, was activated in mice that developed SSH. In female DBA mice, ovariectomized mice displayed SSH under low Mg2+ associated with activation of ATR1-SPAK-NCC axis; 2-ME inhibited all, whereas the blood pressure of sham mice was unaltered regardless of any intervention. Our findings revealed that Mg2+ insufficiency exaggerated the low COMT activity and induced SSH via the ATR1-SPAK-NCC axis due to 2-ME insufficiency, suggesting a new pathophysiological role that links COMT/2-ME deficiency with hypertensive syndrome.



Hypertension: 11 Apr 2021:HYPERTENSIONAHA12016377; epub ahead of print
Kumagai A, Takeda S, Sohara E, Uchida S, ... Koya D, Kanasaki K
Hypertension: 11 Apr 2021:HYPERTENSIONAHA12016377; epub ahead of print | PMID: 33840199
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Impact:
Abstract

Association Between Genetic Variation in Blood Pressure and Increased Lifetime Risk of Peripheral Artery Disease.

Levin MG, Klarin D, Walker VM, Gill D, ... Voight BF, Damrauer SM
Objective
We aimed to estimate the effect of blood pressure (BP) traits and BP-lowering medications (via genetic proxies) on peripheral artery disease. Approach and
Results:
Genome-wide association studies summary statistics were obtained for BP, peripheral artery disease (PAD), and coronary artery disease. Causal effects of BP on PAD were estimated by 2-sample Mendelian randomization using a range of pleiotropy-robust methods. Increased systolic BP (SBP), diastolic BP, mean arterial pressure (MAP), and pulse pressure each significantly increased risk of PAD (SBP odds ratio [OR], 1.20 [1.16-1.25] per 10 mm Hg increase, P=1×10-24; diastolic BP OR, 1.27 [1.18-1.35], P=4×10-11; MAP OR, 1.26 [1.19-1.33], P=6×10-16; pulse pressure OR, 1.31 [1.24-1.39], P=9×10-23). The effects of SBP, diastolic BP, and MAP were greater for coronary artery disease than PAD (SBP ratio of Ors, 1.06 [1.0-1.12], P=0.04; MAP ratio of OR, 1.15 [1.06-1.26], P=8.6×10-4; diastolic BP ratio of OR, 1.21 [1.08-1.35], P=6.9×10-4). Considered jointly, both pulse pressure and MAP directly increased risk of PAD (pulse pressure OR, 1.26 [1.17-1.35], P=3×10-10; MAP OR, 1.14 [1.06-1.23], P=2×10-4). The effects of antihypertensive medications were estimated using genetic instruments. SBP-lowering via β-blocker (OR, 0.74 per 10 mm Hg decrease in SBP [95% CI, 0.65-0.84]; P=5×10-6), loop diuretic (OR, 0.66 [0.48-0.91], P=0.01), and thiazide diuretic (OR, 0.57 [0.41-0.79], P=6×10-4) associated variants were protective of PAD.
Conclusions
Higher BP is likely to cause PAD. BP-lowering through β blockers, loop diuretics, and thiazide diuretics (as proxied by genetic variants) was associated with decreased risk of PAD. Future study is needed to clarify the specific mechanisms by which BP influences PAD.



Arterioscler Thromb Vasc Biol: 14 Apr 2021:ATVBAHA120315482; epub ahead of print
Levin MG, Klarin D, Walker VM, Gill D, ... Voight BF, Damrauer SM
Arterioscler Thromb Vasc Biol: 14 Apr 2021:ATVBAHA120315482; epub ahead of print | PMID: 33853351
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Impact:
Abstract

Synaptotagmin-Like Protein 2a Regulates Angiogenic Lumen Formation via Weibel-Palade Body Apical Secretion of Angiopoietin-2.

Francis CR, Claflin S, Kushner EJ
Objective
Vascular lumen formation requires the redistribution of intracellular proteins to instruct apicobasal polarity, thereby enforcing maturation of both luminal and basal domains. In the absence of proper apical signaling, lumen formation can be distorted leading to lumen collapse and cessation of blood flow. Slp2a (synaptotagmin-like protein-2a) has been implicated in apical membrane signaling; however, the role of Slp2a in vascular lumen formation has never been assessed. Approach and
Results:
Our results demonstrate that Slp2a is required for vascular lumen formation. Using a 3-dimensional sprouting assay, sub-cellular imaging, and zebrafish blood vessel development, we establish that Slp2a resides at the apical membrane acting as a tether for Rab27a that decorates Weibel-Palade bodies (WPBs). We show that Slp2a regulates exocytic activity of WPBs, thus regulating release of WPB contents into the luminal space during angiogenesis. Angiopoietin-2 is a Tie-2 receptor ligand that is selectively released from WPB secretory granules. We identify a critical role for angiopoietin-2 in regulating endothelial lumenization and show that in the absence of Slp2a, WPB contents cannot fuse with the apical membrane. This disrupts the release of angiopoietin-2 and blocks Tie-2 signaling necessary for proper lumen formation.
Conclusions
Our results demonstrate a novel requirement of Slp2a for vascular lumen formation. Moreover, we show that Slp2a is required for the exocytic release of WPB secretory granule cargo during vascular lumen development, and thus is a core upstream component of the WPB secretory pathway. Furthermore, we provide evidence that WPB-housed angiopoietin-2 is required for vascular lumen formation.



Arterioscler Thromb Vasc Biol: 14 Apr 2021:ATVBAHA121316113; epub ahead of print
Francis CR, Claflin S, Kushner EJ
Arterioscler Thromb Vasc Biol: 14 Apr 2021:ATVBAHA121316113; epub ahead of print | PMID: 33853352
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Impact:
Abstract

Loss of Transforming Growth Factor Beta Signaling in Aortic Smooth Muscle Cells Causes Endothelial Dysfunction and Aortic Hypercontractility.

Zhu J, Angelov S, Yildirim IA, Wei H, ... Kim F, Dichek DA
Objective
Humans and mice with loss-of-function variants of genes in the TGF-β (transforming growth factor beta) signaling pathway develop aortic aneurysms. These aneurysms could be caused by decreased aortic smooth muscle cell (SMC) contractile-protein levels and impaired aortic SMC contractile-unit function. Accordingly, we investigated whether loss of SMC TGF-β signaling in mice alters aortic contractile-protein levels and aortic contractility. Approach and
Results:
We used immunoblotting, wire myography, histological analyses, and measurements of aortic nitric oxide and superoxide levels to assess aortic contractile-protein levels and vasomotor function in mice with SMC-specific deletion of the type 2 TGF-β receptor (TBR2SMΔ mice). Aortic contractile-protein levels were not altered in TBR2SMΔ mice. Surprisingly, TBR2SMΔ mice had increased aortic contractility and severe endothelial dysfunction. Endothelial dysfunction was manifested as decreased relaxation to acetylcholine (Emax 37% versus 97%; P<0.0001), decreased aortic nitric oxide (50%; P=0.005), decreased endothelial nitric oxide synthase activation (31%; P=0.002), and lower aortic levels of phosphorylated vasodilator-stimulated phosphoprotein (an indicator of nitric oxide bioavailability: 65%; P<0.0001). Aortic hypercontractility was reduced by mechanical denudation of endothelium and was eliminated by pretreatment of TBR2SMΔ and control aortas with a nitric oxide synthase inhibitor, revealing a significant positive interaction between aortic hypercontractility and absence of endothelium-derived nitric oxide (P<0.05 for both denudation and nitric oxide inhibition).
Conclusions
Aortic aneurysms that develop in TBR2SMΔ mice are not caused by decreased SMC contractility. Loss of physiological SMC TGF-β signaling causes endothelial dysfunction leading to aortic hypercontractility. Endothelial dysfunction may contribute to vascular pathologies associated with abnormal TGF-β signaling.



Arterioscler Thromb Vasc Biol: 14 Apr 2021:ATVBAHA121315878; epub ahead of print
Zhu J, Angelov S, Yildirim IA, Wei H, ... Kim F, Dichek DA
Arterioscler Thromb Vasc Biol: 14 Apr 2021:ATVBAHA121315878; epub ahead of print | PMID: 33853348
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Impact:
Abstract

Carbon Monoxide Suppresses Neointima Formation in Transplant Arteriosclerosis by Inhibiting Vascular Progenitor Cell Differentiation.

Sakihama H, Lee GR, Chin BY, Csizmadia E, ... Bach FH, Otterbein LE
Objective
Evidence indicates that bone marrow progenitor cells (BMPC) are a major contributor to neointima formation in transplant arteriosclerosis. HO-1 (heme oxygenase 1, Hmox1) and carbon monoxide (CO), a product of heme degradation by HO-1, ameliorate neointima formation by inhibiting proliferation of smooth muscle cells. We investigated the mechanism whereby HO-1 and CO modulate BMPC and mitigates neointima formation in transplant arteriosclerosis. Approach and
Results:
Using a murine model of aortic transplantation, bone marrow chimeric mice, and in vitro experiments, we report that CO does not inhibit mobilization of BMPC into the circulation or their homing to the vessel adventitia, but instead suppresses differentiation of BMPC into smooth muscle cells after they arrive in the adventitia. Specifically, the effect of CO on differentiation of BMPC into smooth muscle cell is mediated in part, by limiting PDGFR-β (platelet derived growth factor receptor-β) signaling. Hmox1 -/- BMPC exhibit a greater propensity to differentiate into smooth muscle cell in vitro, in part by regulating PDGFR-β+ expression. Furthermore, wild-type mice transplanted with Hmox1 -/- bone marrow cells show augmented neointima formation after allografting versus control. CO exposure significantly ameliorated neointima formation, which remains more severe with Hmox1 -/- bone marrow cell versus air-treated mice receiving HO-1-expressing bone marrow cell, highlighting the importance of endogenous HO-1 in neointima formation.

Conclusions
Host BMPC contribute to neointima formation in transplant arteriosclerosis and the protective effect afforded by HO-1/CO against neointima formation is mediated in part through the regulation of PDGFR-β expression. We propose that suppressing differentiation of BMPC is a major mechanism by which HO-1 and CO prevent neointima expansion after transplant.



Arterioscler Thromb Vasc Biol: 14 Apr 2021:ATVBAHA120315558; epub ahead of print
Sakihama H, Lee GR, Chin BY, Csizmadia E, ... Bach FH, Otterbein LE
Arterioscler Thromb Vasc Biol: 14 Apr 2021:ATVBAHA120315558; epub ahead of print | PMID: 33853347
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Impact:
Abstract

Anxa1 in smooth muscle cells protects against acute aortic dissection.

Zhou C, Lin Z, Cao H, Chen Y, ... Pan B, Zheng L
Aims
Acute aortic dissection (AAD) is a life-threatening disease with high morbidity and mortality. Previous studies have showed that vascular smooth muscle cell (VSMC) phenotype switching modulates vascular function and AAD progression. However, whether an endogenous signaling system that protects AAD progression exists, remains unknown. Our aim is to investigate the role of Anxa1 in VSMC phenotype switching and the pathogenesis of AAD.
Methods and results
We first assessed Anxa1 expression levels by immunohistochemical staining in control aorta and AAD tissue from mice. A strong increase of Anxa1 expression was seen in the mouse AAD tissues. In line with these findings, micro-CT scan results indicated that Anxa1 plays a role in the development of AAD in our murine model, with systemic deficiency of Anxa1 markedly progressing AAD. Conversely, administration of Anxa1 mimetic peptide, Ac2-26, rescued the AAD phenotype in Anxa1-/- mice. Transcriptomic studies revealed a novel role for Anxa1 in VSMC phenotype switching, with Anxa1 deficiency triggering the synthetic phenotype of VSMCs via down-regulation of the JunB/MYL9 pathway. The resultant VSMC synthetic phenotype rendered elevated inflammation and enhanced matrix metalloproteinases (MMPs) production, leading to augmented elastin degradation. VSMC-restricted deficiency of Anxa1 in mice phenocopied VSMC phenotype switching and the consequent exacerbation of AAD. Finally, our studies in human AAD aortic specimens recapitulated key findings in murine AAD, specifically that the decrease of Anxa1 is associated with VSMC phenotype switch, heightened inflammation, and enhanced MMP production in human aortas.
Conclusions
Our findings demonstrated that Anxa1 is a novel endogenous defender that prevents acute aortic dissection by inhibiting vascular smooth muscle cell phenotype switching, suggesting that Anxa1 signaling may be a potential target for AAD pharmacological therapy.
Translational perspective
Our studies herein may lead to a paradigm shift for pharmacologic therapy towards acute aortic dissection. Through careful examination of the pathological changes that occur during AAD onset in experimental animal models, we demonstrated that VSMC phenotype switching plays a critical role in the development of AAD. Inhibition of VSMC phenotype switching and its attendant impacts on aortic function may be a viable approach for future treatment. Toward that end, our studies highlighted the protective benefit of Anxa1 and its mimetic peptide Ac2-26 in AAD through prevention of the switching of VSMC to a synthetic phenotype.

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

Cardiovasc Res: 22 Mar 2021; epub ahead of print
Zhou C, Lin Z, Cao H, Chen Y, ... Pan B, Zheng L
Cardiovasc Res: 22 Mar 2021; epub ahead of print | PMID: 33757117
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Impact:
Abstract

Treatment and prevention of lipoprotein(a)-mediated cardiovascular disease: the emerging potential of RNA interference therapeutics.

Swerdlow DI, Rider DA, Yavari A, Lindholm MW, Campion GV, Nissen SE
Lipid- and lipoprotein-modifying therapies have expanded substantially in the last 25 years, resulting in reduction in the incidence of major adverse cardiovascular events. However, no specific lipoprotein (a) Lp(a)]-targeting therapy has yet been shown to reduce cardiovascular disease risk. Many epidemiological and genetic studies have demonstrated that lipoprotein(a) is an important genetically-determined causal risk factor for coronary heart disease, aortic valve disease, stroke, heart failure and peripheral vascular disease. Accordingly, the need for specific lipoprotein(a)-lowering therapy has become a major public health priority. Approximately 20% of the global population (1.4 billion people) have elevated levels of Lp(a) associated with higher cardiovascular risk, though the threshold for determining \'high risk\' is debated. Traditional lifestyle approaches to cardiovascular risk reduction are ineffective at lowering Lp(a). To address a lifelong risk factor unmodifiable by non-pharmacological means, Lp(a)-lowering therapy needs to be safe, highly effective, and tolerable for a patient population who will likely require several decades of treatment. N-acetylgalactosamine (GalNAc)-conjugated gene silencing therapeutics such as small interfering RNA (siRNA) and antisense oligonucleotide targeting LPA are ideally suited for this application, offering a highly tissue- and target transcript-specific approach with the potential for safe and durable lipoprotein(a) lowering with as few as three or four doses per year. In this review, we evaluate the causal role of lipoprotein(a) across the cardiovascular disease spectrum, examine the role of established lipid modifying therapies in lowering lipoprotein(a), and focus on the anticipated role for siRNA therapeutics in treating and preventing lipoprotein(a)-related disease.

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

Cardiovasc Res: 24 Mar 2021; epub ahead of print
Swerdlow DI, Rider DA, Yavari A, Lindholm MW, Campion GV, Nissen SE
Cardiovasc Res: 24 Mar 2021; epub ahead of print | PMID: 33769464
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Impact:
Abstract

Guideline-Driven Management of Hypertension: An Evidence-Based Update.

Carey RM, Wright JT, Taler SJ, Whelton PK
Several important findings bearing on the prevention, detection, and management of hypertension have been reported since publication of the 2017 American College of Cardiology/American Heart Association Blood Pressure Guideline. This review summarizes and places in context the results of relevant observational studies, randomized clinical trials, and meta-analyses published between January 2018 and March 2021. Topics covered include blood pressure measurement, patient evaluation for secondary hypertension, cardiovascular disease risk assessment and blood pressure threshold for drug therapy, lifestyle and pharmacological management, treatment target blood pressure goal, management of hypertension in older adults, diabetes, chronic kidney disease, resistant hypertension, and optimization of care using patient, provider, and health system approaches. Presenting new information in each of these areas has the potential to increase hypertension awareness, treatment, and control which remain essential for the prevention of cardiovascular disease and mortality in the future.



Circ Res: 01 Apr 2021; 128:827-846
Carey RM, Wright JT, Taler SJ, Whelton PK
Circ Res: 01 Apr 2021; 128:827-846 | PMID: 33793326
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Impact:
Abstract

Gut microbiota dysbiosis promotes age-related atrial fibrillation by lipopolysaccharide and glucose-induced activation of NLRP3-inflammasome.

Zhang Y, Zhang S, Li B, Luo Y, ... Wu Y, Li Y
Aims
Aging is the most significant contributor to the increasing prevalence of atrial fibrillation (AF). The gut microbiota dysbiosis is involved in age-related diseases. However, whether the aged-associated dysbiosis contributes to age-related AF is still unknown. Direct demonstration that the aged gut microbiota is sufficient to transmit the enhanced AF susceptibility in a young host via microbiota-intestinal barrier-atria axis has not yet been reported. This study aimed to determine whether gut microbiota dysbiosis affects age-related AF.
Methods and results
Herein, by using a fecal microbiota transplantation (FMT) rat model, we demonstrated that the high AF susceptibility of aged rats could be transmitted to a young host. Specially, we found the dramatically increased levels of circulating lipopolysaccharide (LPS) and glucose led to the up-regulated expression of NLR family pyrin domain containing 3 (NLRP3)-inflammasome, promoting the development of AF which depended on the enhanced atrial fibrosis in recipient host. Inhibition of inflammasome by a potent and selective inhibitor of the NLRP3 inflammasome, MCC950, resulted in a lower atrial fibrosis and AF susceptibility. Then we conducted cross-sectional clinical studies to explore the effect of aging on the altering trends with glucose levels and circulating LPS among clinical individuals in two China hospitals. We found that both of serum LPS and glucose levels were progressively increased in elderly patients as compared with those young. Furthermore, the aging phenotype of circulating LPS and glucose levels, intestinal structure and atrial NLRP3-inflammasome of rats were also confirmed in clinical AF patients. Finally, aged rats colonized with youthful microbiota restored intestinal structure and atrial NLRP3-inflammasome activity, which suppressed the development of aged-related AF.
Conclusions
Collectively, these studies described a novel causal role of aberrant gut microbiota in the pathogenesis of age-related AF, which indicates that the microbiota-intestinal barrier-atrial NLRP3 inflammasome axis may be a rational molecular target for the treatment of aged-related arrhythmia disease.
Translational perspective
The current study demonstrates that aged-associated microbiota dysbiosis promotes AF in part through a microbiota-gut-atria axis. Increased AF susceptibility due to enhanced atrial NLRP3-inflammasome activity by LPS and high glucose was found in an aged FMT rat model, and also confirmed within elderly clinical individuals. In a long-term FMT rat study, the AF susceptibility was ameliorated by treatment with youthful microbiota. The present findings can further increase our understanding of aged-related AF and address a promising therapeutic strategy that involves modulation of gut microbiota composition.

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

Cardiovasc Res: 22 Mar 2021; epub ahead of print
Zhang Y, Zhang S, Li B, Luo Y, ... Wu Y, Li Y
Cardiovasc Res: 22 Mar 2021; epub ahead of print | PMID: 33757127
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Impact:
Abstract

SPARC-related modular calcium binding 1 regulates aortic valve calcification by disrupting BMPR-II/p-p38 signalling.

Wang Y, Gu J, Du A, Zhang S, ... Sun W, Kong X
Aims
Aortic valve calcification is more prevalent in chronic kidney disease accompanied by hypercalcemia. SPARC (Secreted Protein Acidic and Rich in Cysteine)-related modular calcium binding 1 (SMOC1) is a regulator of BMP2 signalling, but the role of SMOC1 in aortic valve calcification under different conditions has not been studied. This study aimed to investigate the roles of SMOC1 in aortic valve calcification under normal and high calcium conditions, focusing on the effects on aortic valve interstitial cells (AVICs).
Methods and results
SMOC1 was expressed by aortic valve endothelial cells and secreted into the extracellular matrix in non-calcific valves and downregulated in calcific aortic valves. In vitro studies demonstrated that HUVEC secreted SMOC1 could enter the cytoplasm of AVICs. Overexpression of SMOC1 attenuated warfarin-induced AVIC calcification but promoted high calcium/phosphate or vitamin D-induced AVIC and aortic valve calcification by regulating BMP2 signalling both in vitro and in vivo. Co-immunoprecipitation revealed that SMOC1 binds to BMP receptor II (BMPR-II) and inhibits BMP2-induced phosphorylation of p38 (p-p38) via amino acids 372-383 of its EF-hand calcium-binding domain. Inhibition of p-p38 by the p38 inhibitor SB203580 blocked the effects of SMOC1 on BMP2 signalling and AVIC calcification induced by high calcium/phosphate medium. In high-calcium-treated AVICs, SMOC1 lost its ability to bind to BMPR-II, but not to caveolin-1, promoting p-p38 and cell apoptosis due to increased expression of BMPR-II and enhanced endocytosis.
Conclusions
These observations support that SMOC1 works as a dual-directional modulator of AVIC calcification by regulating p38-dependent BMP2 signalling transduction according to different extracellular calcium concentrations.

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

Cardiovasc Res: 22 Mar 2021; epub ahead of print
Wang Y, Gu J, Du A, Zhang S, ... Sun W, Kong X
Cardiovasc Res: 22 Mar 2021; epub ahead of print | PMID: 33757126
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Impact:
Abstract

The subfornical organ drives hypertension in polycystic kidney disease via the hypothalamic paraventricular nucleus.

Underwood CF, Mcmullan S, Goodchild AK, Phillips JK, Hildreth CM
Aims
Hypertension is a prevalent yet poorly understood feature of polycystic kidney disease. Previously we demonstrated that increased glutamatergic neurotransmission within the hypothalamic paraventricular nucleus produces hypertension in the Lewis Polycystic Kidney rat model of polycystic kidney disease. Here we tested the hypothesis that augmented glutamatergic drive to the paraventricular nucleus in Lewis Polycystic Kidney rats originates from the forebrain lamina terminalis, a sensory structure that relays blood-borne information throughout the brain.
Methods and results
Anatomical experiments revealed that 38% of paraventricular nucleus-projecting neurons in the subfornical organ of the lamina terminalis expressed Fos/Fra, an activation marker, in Lewis Polycystic Kidney rats while <1% of neurons were Fos/Fra+ in Lewis control rats (P = 0.01, n = 8). In anaesthetised rats, subfornical organ neuronal inhibition using isoguvacine produced a greater reduction in systolic blood pressure in the Lewis Polycystic Kidney versus Lewis rats (-21 ± 4 vs. -7 ± 2 mmHg, P < 0.01; n = 10), which could be prevented by prior blockade of paraventricular nucleus ionotropic glutamate receptors using kynurenic acid. Blockade of ionotropic glutamate receptors in the paraventricular nucleus produced an exaggerated depressor response in Lewis Polycystic Kidney relative to Lewis rats (-23 ± 4 vs. -2 ± 3 mmHg, P < 0.001; n = 13), which was corrected by prior inhibition of the subfornical organ with muscimol but unaffected by chronic systemic angiotensin II type I receptor antagonism or lowering of plasma hyperosmolality through high-water intake (P > 0.05); treatments that both nevertheless lowered blood pressure in Lewis Polycystic Kidney rats (P < 0.0001).
Conclusion
Our data reveal multiple independent mechanisms contribute to hypertension in polycystic kidney disease, and identify high plasma osmolality, angiotensin II type I receptor activation and, importantly, a hyperactive subfornical organ to paraventricular nucleus glutamatergic pathway as potential therapeutic targets.
Translational perspective
Hypertension is a significant comorbidity for all forms of chronic kidney disease and for individuals with polycystic kidney disease, often an early presenting feature. Nevertheless, the cause(s) of hypertension in polycystic kidney disease are poorly defined. Here we define the contribution of a neural pathway that contributes to hypertension in polycystic kidney disease. Critically, targeting this pathway may provide an additional antihypertensive effect beyond that achieved with current conventional antihypertensive therapies. Future work identifying the drivers of this neural pathway will aid in the development of newer generation antihypertensive medication.

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 Mar 2021; epub ahead of print
Underwood CF, Mcmullan S, Goodchild AK, Phillips JK, Hildreth CM
Cardiovasc Res: 27 Mar 2021; epub ahead of print | PMID: 33774660
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Impact:
Abstract

Zebrafish scube1 and scube2 cooperate in promoting Vegfa signaling during embryonic vascularization.

Tsao KC, Lin YC, Chen YT, Lai SL, Yang RB
Aims
The secreted and membrane-anchored SCUBE (signal peptide-CUB-EGF domain-containing proteins) gene family composed of 3 members was originally identified from endothelial cells (ECs). We recently showed that membrane SCUBE2 binds vascular endothelial growth factor A (VEGFA) and acts as a co-receptor for VEGF receptor 2 (VEGFR2) to modulate EC migration, proliferation and tube formation during postnatal and tumor angiogenesis. However, whether these SCUBE genes cooperate in modulating VEGF signaling during embryonic vascular development remains unknown.
Methods and results
To further dissect the genetic interactions of these scube genes, transcription activator-like effector nuclease-mediated genome editing was used to generate knockout (KO) alleles of each scube gene. No overt vascular phenotypes were seen in any single scube KO mutants because of compensation by other scube genes during zebrafish development. However, scube1 and scube2 double KO (DKO) severely impaired EC filopodia extensions, migration, and proliferation, thus disrupting proper vascular lumen formation during vasculogenesis and angiogenesis as well as development of the organ-specific intestinal vasculature. Further genetic, biochemical, and molecular analyses revealed that Scube1 and Scube2 might act cooperatively at the cell-surface receptor level to facilitate Vegfa signaling during zebrafish embryonic vascularization.
Conclusions
We showed for the first time that cooperation between scube1 and scube2 is critical for proper regulation of angiogenic cell behaviors and formation of functional vessels during zebrafish embryonic development.
Translational perspective
Our studies indicate that targeting SCUBE1 and/or SCUBE2 on modulating VEGF signaling might provide potential therapeutic treatments or VEGF-mediated proliferative pathological vascular 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: 30 Mar 2021; epub ahead of print
Tsao KC, Lin YC, Chen YT, Lai SL, Yang RB
Cardiovasc Res: 30 Mar 2021; epub ahead of print | PMID: 33788916
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Impact:
Abstract

SERCA2a stimulation by istaroxime improves intracellular Ca2+ handling and diastolic dysfunction in a model of diabetic cardiomyopathy.

Torre E, Arici M, Lodrini AM, Ferrandi M, ... Bianchi G, Rocchetti M
Aims
Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes.
Methods and results
Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed 1) marked DD not associated to cardiac fibrosis, 2) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, 3) reduced LV SERCA2 protein level and activity and 4) slower SR Ca2+ uptake rate, 5) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, 6) increased diastolic Ca2+, and 7) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent.
Conclusions
SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment.
Translational perspective
Deficient sarcoplasmic reticulum (SR) Ca2+ uptake has been identified in cardiomyocytes from failing human hearts with impaired diastolic relaxation (e.g. diabetic hearts) and has been associated with a decreased SERCA2a expression and activity and/or with a higher SERCA2a inhibition by phospholamban. Thus, SERCA2a may represent a pharmacological target for interventions aimed at improving cytosolic Ca2+ compartmentalization into the SR to limit diastolic dysfunction pathologies. In this context, istaroxime is the first-in-class luso-inotropic agent targeting SERCA2a that has already demonstrated its efficacy in clinical trials and may be useful to clarify the relevance of SERCA2a stimulation in controlling cytosolic Ca2+ level.

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

Cardiovasc Res: 31 Mar 2021; epub ahead of print
Torre E, Arici M, Lodrini AM, Ferrandi M, ... Bianchi G, Rocchetti M
Cardiovasc Res: 31 Mar 2021; epub ahead of print | PMID: 33792692
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Impact:
Abstract

Novel insights into the electrophysiology of murine cardiac macrophages: relevance of voltage-gated potassium channels.

Simon-Chica A, Fernández MC, Wülfers EM, Lother A, ... Kohl P, Schneider-Warme F
Aims
Macrophages (MΦ), known for immunological roles such as phagocytosis and antigen presentation, have been found to electrotonically couple to cardiomyocytes (CM) of the atrio-ventricular node via Cx43, affecting cardiac conduction in isolated mouse hearts. Here, we characterise passive and active electrophysiological properties of murine cardiac resident MΦ, and model their potential electrophysiological relevance for CM.
Methods and results
We combined classic electrophysiological approaches with 3 D florescence imaging, RNA-sequencing, pharmacological interventions and computer simulations. We used Cx3cr1eYFP/+ mice wherein cardiac MΦ were fluorescently labelled. FACS-purified fluorescent MΦ from mouse hearts were studied by whole-cell patch-clamp. MΦ electrophysiological properties include: membrane resistance 2.2 ± 0.1 GΩ (all data mean±SEM), capacitance 18.3 ± 0.1 pF, resting membrane potential -39.6 ± 0.3 mV, and several voltage-activated, outward or inwardly-rectifying potassium currents. Using ion channel blockers (barium, TEA, 4-AP, margatoxin, XEN-D0103, DIDS), flow cytometry, immuno-staining and RNA-sequencing, we identified Kv1.3, Kv1.5 and Kir2.1 as channels contributing to observed ion currents. MΦ displayed four patterns for outward and two for inward-rectifier potassium currents. Additionally, MΦ showed surface expression of Cx43, a prerequisite for homo- and/or heterotypic electrotonic coupling. Experimental results fed into development of an original computational model to describe cardiac MΦ electrophysiology. Computer simulations to quantitatively assess plausible effects of MΦ on electrotonically coupled CM showed that MΦ can depolarise resting CM, shorten early and prolong late action potential duration, with effects depending on coupling strength and individual MΦ electrophysiological properties, in particular resting membrane potential and presence/absence of Kir2.1.
Conclusions
Our results provide a first electrophysiological characterisation of cardiac resident MΦ, and a computational model to quantitatively explore their relevance in the heterocellular heart. Future work will be focussed at distinguishing electrophysiological effects of MΦ-CM coupling on both cell types during steady-state and in patho-physiological remodelling, when immune cells change their phenotype, proliferate, and/or invade from external sources.
Translational perspective
Cardiac tissue contains resident macrophages (MΦ) which, beyond immunological and housekeeping roles, have been found to electrotonically couple via connexins to cardiomyocytes (CM), stabilising atrio-ventricular conduction at high excitation rates. Here, we characterise structure and electrophysiological function of murine cardiac MΦ and provide a computational model to quantitatively probe the potential relevance of MΦ-CM coupling for cardiac electrophysiology. We find that MΦ are unlikely to have major electrophysiological effects in normal tissue, where they would hasten early and slow late CM-repolarisation. Further work will address potential arrhythmogenicity of MΦ in patho-physiologically remodelled tissue containing elevated MΦ-numbers, incl. non-resident recruited cells.

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

Cardiovasc Res: 05 Apr 2021; epub ahead of print
Simon-Chica A, Fernández MC, Wülfers EM, Lother A, ... Kohl P, Schneider-Warme F
Cardiovasc Res: 05 Apr 2021; epub ahead of print | PMID: 33823533
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Impact:
Abstract

Intracerebral Injection of Heme Induces Lipid Peroxidation, Neuroinflammation, and Sensorimotor Deficits.

Vasconcellos LRC, Martimiano L, Dantas DP, Fonseca FM, ... Bozza MT, Pimentel-Coelho PM
Background:
and purpose
Heme is a red blood cell component released in the brain parenchyma following intracerebral hemorrhage. However, the study of the pathophysiological mechanisms triggered by heme in the brain is hampered by the lack of well-established in vivo models of intracerebral heme injection. This study aims to optimize and characterize a protocol of intrastriatal heme injection in mice, with a focus on the induction of lipid peroxidation, neuroinflammation and, ultimately, sensorimotor deficits. We also evaluated the involvement of NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), an inflammasome sensor, in the behavior deficits induced by heme in this model.
Methods
Mice were injected with heme in the striatum for the evaluation of neuroinflammation and brain damage through histological and biochemical techniques. Immunoblot was used to evaluate the expression of proteins involved in heme/iron metabolism and antioxidant responses and the activation of the MAPK (mitogen-activated protein kinase) signaling pathway. For the assessment of neurological function, we followed-up heme-injected mice for 2 weeks using the rotarod, elevated body swing, and cylinder tests. Mice injected with the vehicle (sham), or autologous blood were used as controls.
Results
Heme induced lipid peroxidation and inflammation in the brain. Moreover, heme increased the expression of HO-1 (heme oxygenase-1), ferritin, p62, and superoxide dismutase 2, and activated the MAPK signaling pathway promoting pro-IL (interleukin)-1β production and its cleavage to the active form. Heme-injected mice exhibited signs of brain damage and reactive astrogliosis around the injection site. Behavior deficits were observed after heme or autologous blood injection in comparison to sham-operated controls. In addition, behavior deficits and IL-1β production were reduced in Nlrp3 knockout mice in comparison to wild-type mice.
Conclusions
Our results show that intracerebral heme injection induces neuroinflammation, and neurological deficits, in an NLRP3-dependent manner, suggesting that this is a feasible model to evaluate the role of heme in neurological disorders.



Stroke: 07 Apr 2021:STROKEAHA120031911; epub ahead of print
Vasconcellos LRC, Martimiano L, Dantas DP, Fonseca FM, ... Bozza MT, Pimentel-Coelho PM
Stroke: 07 Apr 2021:STROKEAHA120031911; epub ahead of print | PMID: 33827248
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Impact:
Abstract

Effects of canagliflozin on myocardial infarction: a post hoc analysis of the CANVAS Program and CREDENCE trial.

Yu J, Li J, Leaver PJ, Arnott C, ... Neal B, Figtree GA
Aims
Given the benefits of sodium glucose co-transporter 2 inhibition (SGLT2i) in protecting against heart failure in diabetic patients, we sought to explore the potential impact of SGLT2i on the clinical features of patients presenting with myocardial infarction (MI) through a post-hoc analysis of CANVAS Program and CREDENCE trial.
Methods and results
Individuals with type 2 diabetes and history or high risk of cardiovascular disease (CANVAS Program) or type 2 diabetes and chronic kidney disease (CREDENCE) were included. The intervention was Canagliflozin 100 or 300 mg (combined in the analysis) or placebo. MI events were adjudicated as ST-elevation myocardial infarction (STEMI), non-STEMI as well as type 1 MI or type 2 MI. 421 first MI events in the CANVAS Program and 178 first MI events in the CREDENCE trial were recorded (83 fatal, 128 STEMI, 431 non-STEMI, and 40 unknown). No benefit of canagliflozin compared with placebo on time to first MI event was observed (HR 0.89; 95% CI 0.75, 1.05). Canagliflozin was associated with lower risk for non-STEMI (HR 0.78; 95% CI 0.65, 0.95) but suggested a possible increase in STEMI (HR 1.55; 95% CI 1.06, 2.27), with no difference in risk of type 1 or type 2 MI. There was no change in fatal MI (HR 1.22, 95% CI 0.78, 1.93).
Conclusions
Canagliflozin was not associated with a reduction in overall MI in the pooled CANVAS Program and CREDENCE trial population. The possible differential effect on STEMI and Non-STEMI observed in the CANVAS cohort warrants further investigation.

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

Cardiovasc Res: 06 Apr 2021; epub ahead of print
Yu J, Li J, Leaver PJ, Arnott C, ... Neal B, Figtree GA
Cardiovasc Res: 06 Apr 2021; epub ahead of print | PMID: 33826709
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Impact:
Abstract

Single cell dual-omics reveals the transcriptomic and epigenomic diversity of cardiac non-myocytes.

Wang L, Yang Y, Ma H, Xie Y, ... Liu J, Qian L
Aims
The precise cellular identity and molecular features of non-myocytes (nonCM) in a mammalian heart at a single cell level remain elusive. Depiction of epigenetic landscape with transcriptomic signatures using the latest single-cell multi-omics has the potential to unravel the molecular programs underlying the cellular diversity of cardiac non-myocytes. Here, we characterized the molecular and cellular features of cardiac nonCM populations in the adult murine heart at the single cell level.
Methods and results
Through single-cell dual omics analysis, we mapped the epigenetic landscapes, characterized the transcriptomic profiles and delineated the molecular signatures of cardiac nonCMs in the adult murine heart. Distinct cis-regulatory elements and trans-acting factors for the individual major nonCM cell types (endothelial cells, fibroblast, pericytes and immune cells) were identified. In particular, unbiased sub-clustering and functional annotation of cardiac fibroblasts (FB) revealed extensive FB heterogeneity and identified FB subtypes with functional states related to cellular response to stimuli, cytoskeleton organization and immune regulation, respectively. We further explored the function of marker genes Hsd11b1 and Gfpt2 that label major FB sub-populations and determined the distribution of Hsd11b1+ and Gfp2+ FBs in murine healthy and diseased hearts.
Conclusions
In summary, we characterized the nonCM cellular identity at the transcriptome and epigenome levels using single-cell omics approaches and discovered previously unrecognized cardiac fibroblast subpopulations with unique functional states.
Translational perspective
Our research identified discrete cell types of nonCM in the heart and differentially expressed genes with regulatory factors. Unveiling the heterogeneity of nonCMs and molecular signatures of each cell type or subtypes allows for study, precise capture and manipulation of specific cell type(s) in heart and will provide insights into the development of therapeutics for 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: 10 Apr 2021; epub ahead of print
Wang L, Yang Y, Ma H, Xie Y, ... Liu J, Qian L
Cardiovasc Res: 10 Apr 2021; epub ahead of print | PMID: 33839759
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Impact:
Abstract

FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

Jeong K, Murphy JM, Erin Ahn EY, Steve Lim ST
Aim
Vascular smooth muscle cells (VSMCs) normally exhibit a very low proliferative rate. Vessel injury triggers VSMC proliferation, in part, through focal adhesion kinase (FAK) activation, which increases transcription of cyclin D1, a key activator for cell cycle-dependent kinases (CDKs). At the same time, we also observe that FAK regulates the expression of the CDK inhibitors (CDKIs) p27 and p21. However, the mechanism of how FAK controls CDKIs in cell cycle progression is not fully understood.
Methods and results
We found that pharmacological and genetic FAK inhibition increased p27 and p21 by reducing stability of S-phase kinase-associated protein 2 (Skp2), which targets the CDKIs for degradation. FAK N-terminal domain interacts with Skp2 and an APC/C E3 ligase activator, fizzy-related 1 (Fzr1) in the nucleus, which promotes ubiquitination and degradation of both Skp2 and Fzr1. Notably, overexpression of cyclin D1 alone failed to promote proliferation of genetic FAK kinase-dead (KD) VSMCs, suggesting that the FAK-Skp2-CDKI signaling axis is distinct from the FAK-cyclin D1 pathway. However, overexpression of both cyclin D1 and Skp2 enables proliferation of FAK-KD VSMCs, implicating that FAK ought to control both activating and inhibitory switches for CDKs. In vivo, wire injury activates FAK in the cytosol and increased Skp2 and decreased p27 and p21 levels.
Conclusions
Both pharmacological FAK and genetic FAK inhibition reduced Skp2 expression in VSMCs upon injury, which significantly reduced intimal hyperplasia through elevated expression of p27 and p21. This study revealed that nuclear FAK-Skp2-CDKI signaling negatively regulates CDK activity in VSMC proliferation.
Translational perspective
Increased VSMC proliferation contributes to pathological vessel narrowing in atherosclerosisand following vascular interventions. Blocking VSMC proliferation will reduce atherosclerosisprogression and increase patency of vascular interventions. We found that forced nuclear FAKlocalization by FAK inhibition reduced VSMC proliferation upon vessel injury. Nuclear FAKdecreased Skp2 protein expression by proteasomal degradation, thereby increasing theexpression of cell cycle inhibitors p27 and p21 and blocking cell cycle progression. This studyhas demonstrated the potential for FAK inhibitors in blocking VSMC proliferation to treat vessel narrowing 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: 10 Apr 2021; epub ahead of print
Jeong K, Murphy JM, Erin Ahn EY, Steve Lim ST
Cardiovasc Res: 10 Apr 2021; epub ahead of print | PMID: 33839758
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Impact:
Abstract

Apolipoprotein genetic variants and hereditary amyloidosis.

Jeraj N, Hegele RA, Berberich AJ
Purpose of review
Amyloidosis is caused by the deposition of misfolded aggregated proteins called amyloid fibrils that in turn cause organ damage and dysfunction. In this review, we aim to summarize the genetic, clinical, and histological findings in apolipoprotein-associated hereditary amyloidosis and the growing list of mutations and apolipoproteins associated with this disorder. We also endeavor to summarize the features of apolipoproteins that have led them to be overrepresented among amyloidogenic proteins. Additionally, we aim to distinguish mutations leading to amyloidosis from those that lead to inherited dyslipidemias.
Recent findings
Apolipoproteins are becoming increasingly recognized in hereditary forms of amyloidosis. Although mutations in APOA1 and APOA2 have been well established in hereditary amyloidosis, new mutations are still being detected, providing further insight into the pathogenesis of apolipoprotein-related amyloidosis. Furthermore, amyloidogenic mutations in APOC2 and APOC3 have more recently been described. Although no hereditary mutations in APOE or APOA4 have been described to date, both protein products are amyloidogenic and frequently found within amyloid deposits.
Summary
Understanding the underlying apolipoprotein mutations that contribute to hereditary amyloidosis may help improve understanding of this rare but serious disorder and could open the door for targeted therapies and the potential development of new treatment options.

Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

Curr Opin Lipidol: 31 Mar 2021; 32:132-140
Jeraj N, Hegele RA, Berberich AJ
Curr Opin Lipidol: 31 Mar 2021; 32:132-140 | PMID: 33395107
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Impact:
Abstract

Polygenic scores for dyslipidemia: the emerging genomic model of plasma lipoprotein trait inheritance.

Trinder M, Brunham LR
Purpose of review
Contemporary polygenic scores, which summarize the cumulative contribution of millions of common single-nucleotide variants to a phenotypic trait, can have effects comparable to monogenic mutations. This review focuses on the emerging use of \'genome-wide\' polygenic scores for plasma lipoproteins to define the etiology of clinical dyslipidemia, modify the severity of monogenic disease, and inform therapeutic options.
Recent findings
Polygenic scores for low-density lipoprotein cholesterol (LDL-C), triglycerides, and high-density lipoprotein cholesterol are associated with severe hypercholesterolemia, hypertriglyceridemia, or hypoalphalipoproteinemia, respectively. These polygenic scores for LDL-C or triglycerides associate with risk of incident coronary artery disease (CAD) independent of polygenic scores designed specifically for CAD and may identify individuals that benefit most from lipid-lowering medication. Additionally, the severity of hypercholesterolemia and CAD associated with familial hypercholesterolemia-a common monogenic disorder-is modified by these polygenic factors. The current focus of polygenic scores for dyslipidemia is to design predictive polygenic scores for diverse populations and determining how these polygenic scores could be implemented and standardized for use in the clinic.
Summary
Polygenic scores have shown early promise for the management of dyslipidemias, but several challenges need to be addressed before widespread clinical implementation to ensure that potential benefits are robust and reproducible, equitable, and cost-effective.

Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

Curr Opin Lipidol: 31 Mar 2021; 32:103-111
Trinder M, Brunham LR
Curr Opin Lipidol: 31 Mar 2021; 32:103-111 | PMID: 33395106
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Impact:
Abstract

Recent advances in ABCG5 and ABCG8 variants.

Fong V, Patel SB
Purpose of review
In this review, we summarize the genetics and mechanisms of sitosterolemia and sterol trafficking, and provide an update on the understanding of the prevalence of ABCG5 and ABCG8 variants and their role in human disease.
Recent findings
Defects in ABCG5/G8 result in the accumulation of xenosterols. It had been previously thought that near total LoF of one of the proteins was required to cause pathology. However, recently there was the first report of a patient with Sitosterolemia who was heterozygous for mutations in both genes. Moreover, large population studies have demonstrated the even simple heterozygous carriers are associated with altered lipid profiles and cardiovascular risk. Broader screening has added to the rapidly growing list of gene variants indicating that the prevalence of ABCG5/G8 variants is higher than previous thought, especially in patients with hypercholesterolemia.
Summary
These findings support a strategy of measuring xenosterol levels in patients with hypercholesterolemia to screen for ABCG5/G8 variants, and then tailoring treatment with a sterol absorption inhibitor, like ezetimibe, where indicated. Xenosterol trafficking affects remnant clearance and maybe pathogenically linked to the increased risk of atherosclerosis.

Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

Curr Opin Lipidol: 31 Mar 2021; 32:117-122
Fong V, Patel SB
Curr Opin Lipidol: 31 Mar 2021; 32:117-122 | PMID: 33395105
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Impact:
Abstract

Pharmacogenomics of statins and familial hypercholesterolemia.

Chora JR, Bourbon M
Purpose of review
To collect evidence on statin pharmacogenomics, and review what is known in this field for familial hypercholesterolemia (FH) patients.
Recent findings
There are well-known associations between specific single nucleotide polymorphisms involved in statin transport and metabolism and either adverse effects or altered lipid-lowering efficacy. However, the applicability of this knowledge is uncertain, especially in high-risk populations. There are alternative approaches to study plasma concentrations of statins and new insights on why some association studies fail to be replicated.
Summary
Statin therapy recommendations are not always followed in primary and secondary prevention and, even when followed, patients often fail to reach therapeutic target values. Considering the stringent 2019 European Atherosclerosis Society and European Society of Cardiology recommended target lipid levels, as well as the persistently high cost for alternative lipid-lowering therapies such as PCSK9 inhibitors, the variability in low-density lipoprotein cholesterol reductions on statin therapy is still an important factor that needs to be addressed to ensure better cardiovascular disease risk management, especially for FH patients, who have not been well studied historically in this context.

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

Curr Opin Lipidol: 31 Mar 2021; 32:96-102
Chora JR, Bourbon M
Curr Opin Lipidol: 31 Mar 2021; 32:96-102 | PMID: 33591029
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Impact:
Abstract

Update on cerebrotendinous xanthomatosis.

DeBarber AE, Duell PB
Purpose of review
Cerebrotendinous xanthomatosis (CTX) is a rare genetic lipid storage disorder with highly pleomorphic clinical phenotype. Complications of this disease can be devastating and may include severe cognitive impairment and dementia in later stages. Disease progression can be prevented or stabilized by bile acid replacement therapy, although a subset of patients with advanced disease continue to deteriorate despite therapy.
Recent findings
Delayed diagnosis of CTX continues to impede effective treatment. A clinical diagnostic algorithm for CTX was developed that can decrease the age of diagnosis of CTX. The strategy of screening children with bilateral juvenile cataracts for CTX also improved diagnosis, as this group had a 500-fold higher-rate of CTX than the general population. Improved diagnosis of CTX is critical, as patients treated early in the course of the disease have significantly better outcomes compared with those treated later. More sensitive and specific biochemical testing for CTX has been developed that is potentially more informative than blood cholestanol to assess treatment efficacy and medication compliance in CTX.
Summary
Because we are recognizing more severe presentations of CTX in infants and children, and delayed diagnosis and treatment worsens the prognosis, CTX is an excellent candidate disorder for newborn screening using recently reported methods for newborn dried bloodspot analysis.

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

Curr Opin Lipidol: 31 Mar 2021; 32:123-131
DeBarber AE, Duell PB
Curr Opin Lipidol: 31 Mar 2021; 32:123-131 | PMID: 33630770
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Impact:
Abstract

Cross-species data integration to prioritize causal genes in lipid metabolism.

Votava JA, Parks BW
Purpose of review
More than one hundred loci have been identified from human genome-wide association studies (GWAS) for blood lipids. Despite the success of GWAS in identifying loci, subsequent prioritization of causal genes related to these loci remains a challenge. To address this challenge, recent work suggests that candidate causal genes within loci can be prioritized through cross-species integration using genome-wide data from the mouse.
Recent findings
Mouse model systems provide unparalleled access to primary tissues, like the liver, that are not readily available for human studies. Given the key role the liver plays in controlling blood lipid levels and the wealth of liver genome-wide transcript and protein data available in the mouse, these data can be leveraged. Using coexpression network analysis approaches with mouse genome-wide data, coupled with cross-species analysis of human lipid GWAS, causal genes within lipid loci can be prioritized. Prioritization through both mouse and human along with biochemical validation provide a systematic and valuable method to discover lipid metabolism genes.
Summary
The prioritization of causal lipid genes within GWAS loci is a challenging process requiring a multidisciplinary approach. Integration of data types across species, such as the mouse, can aid in causal gene prioritization.

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

Curr Opin Lipidol: 31 Mar 2021; 32:141-146
Votava JA, Parks BW
Curr Opin Lipidol: 31 Mar 2021; 32:141-146 | PMID: 33651746
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Impact:
Abstract

Role of the Peroxisome Proliferator Activated Receptors in Hypertension.

Fang S, Livergood MC, Nakagawa P, Wu J, Sigmund CD
Nuclear receptors represent a large family of ligand-activated transcription factors which sense the physiological environment and make long-term adaptations by mediating changes in gene expression. In this review, we will first discuss the fundamental mechanisms by which nuclear receptors mediate their transcriptional responses. We will focus on the PPAR (peroxisome proliferator-activated receptor) family of adopted orphan receptors paying special attention to PPARγ, the isoform with the most compelling evidence as an important regulator of arterial blood pressure. We will review genetic data showing that rare mutations in PPARγ cause severe hypertension and clinical trial data which show that PPARγ activators have beneficial effects on blood pressure. We will detail the tissue- and cell-specific molecular mechanisms by which PPARs in the brain, kidney, vasculature, and immune system modulate blood pressure and related phenotypes, such as endothelial function. Finally, we will discuss the role of placental PPARs in preeclampsia, a life threatening form of hypertension during pregnancy. We will close with a viewpoint on future research directions and implications for developing novel therapies.



Circ Res: 01 Apr 2021; 128:1021-1039
Fang S, Livergood MC, Nakagawa P, Wu J, Sigmund CD
Circ Res: 01 Apr 2021; 128:1021-1039 | PMID: 33793338
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Impact:
Abstract

Hypertension: Do Inflammation and Immunity Hold the Key to Solving this Epidemic?

Madhur MS, Elijovich F, Alexander MR, Pitzer A, ... Laffer CL, Kirabo A
Elevated cardiovascular risk including stroke, heart failure, and heart attack is present even after normalization of blood pressure in patients with hypertension. Underlying immune cell activation is a likely culprit. Although immune cells are important for protection against invading pathogens, their chronic overactivation may lead to tissue damage and high blood pressure. Triggers that may initiate immune activation include viral infections, autoimmunity, and lifestyle factors such as excess dietary salt. These conditions activate the immune system either directly or through their impact on the gut microbiome, which ultimately produces chronic inflammation and hypertension. T cells are central to the immune responses contributing to hypertension. They are activated in part by binding specific antigens that are presented in major histocompatibility complex molecules on professional antigen-presenting cells, and they generate repertoires of rearranged T-cell receptors. Activated T cells infiltrate tissues and produce cytokines including interleukin 17A, which promote renal and vascular dysfunction and end-organ damage leading to hypertension. In this comprehensive review, we highlight environmental, genetic, and microbial associated mechanisms contributing to both innate and adaptive immune cell activation leading to hypertension. Targeting the underlying chronic immune cell activation in hypertension has the potential to mitigate the excess cardiovascular risk associated with this common and deadly disease.



Circ Res: 01 Apr 2021; 128:908-933
Madhur MS, Elijovich F, Alexander MR, Pitzer A, ... Laffer CL, Kirabo A
Circ Res: 01 Apr 2021; 128:908-933 | PMID: 33793336
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Impact:
Abstract

Oxidative Stress and Hypertension.

Griendling KK, Camargo LL, Rios FJ, Alves-Lopes R, Montezano AC, Touyz RM
A link between oxidative stress and hypertension has been firmly established in multiple animal models of hypertension but remains elusive in humans. While initial studies focused on inactivation of nitric oxide by superoxide, our understanding of relevant reactive oxygen species (superoxide, hydrogen peroxide, and peroxynitrite) and how they modify complex signaling pathways to promote hypertension has expanded significantly. In this review, we summarize recent advances in delineating the primary and secondary sources of reactive oxygen species (nicotinamide adenine dinucleotide phosphate oxidases, uncoupled endothelial nitric oxide synthase, endoplasmic reticulum, and mitochondria), the posttranslational oxidative modifications they induce on protein targets important for redox signaling, their interplay with endogenous antioxidant systems, and the role of inflammasome activation and endoplasmic reticular stress in the development of hypertension. We highlight how oxidative stress in different organ systems contributes to hypertension, describe new animal models that have clarified the importance of specific proteins, and discuss clinical studies that shed light on how these processes and pathways are altered in human hypertension. Finally, we focus on the promise of redox proteomics and systems biology to help us fully understand the relationship between ROS and hypertension and their potential for designing and evaluating novel antihypertensive therapies.



Circ Res: 01 Apr 2021; 128:993-1020
Griendling KK, Camargo LL, Rios FJ, Alves-Lopes R, Montezano AC, Touyz RM
Circ Res: 01 Apr 2021; 128:993-1020 | PMID: 33793335
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Impact:
Abstract

Vascular Stress Signaling in Hypertension.

Cicalese SM, da Silva JF, Priviero F, Webb RC, Eguchi S, Tostes RC
Cells respond to stress by activating a variety of defense signaling pathways, including cell survival and cell death pathways. Although cell survival signaling helps the cell to recover from acute insults, cell death or senescence pathways induced by chronic insults can lead to unresolved pathologies. Arterial hypertension results from chronic physiological maladaptation against various stressors represented by abnormal circulating or local neurohormonal factors, mechanical stress, intracellular accumulation of toxic molecules, and dysfunctional organelles. Hypertension and aging share common mechanisms that mediate or prolong chronic cell stress, such as endoplasmic reticulum stress and accumulation of protein aggregates, oxidative stress, metabolic mitochondrial stress, DNA damage, stress-induced senescence, and proinflammatory processes. This review discusses common adaptive signaling mechanisms against these stresses including unfolded protein responses, antioxidant response element signaling, autophagy, mitophagy, and mitochondrial fission/fusion, STING (signaling effector stimulator of interferon genes)-mediated responses, and activation of pattern recognition receptors. The main molecular mechanisms by which the vasculature copes with hypertensive and aging stressors are presented and recent advancements in stress-adaptive signaling mechanisms as well as potential therapeutic targets are discussed.



Circ Res: 01 Apr 2021; 128:969-992
Cicalese SM, da Silva JF, Priviero F, Webb RC, Eguchi S, Tostes RC
Circ Res: 01 Apr 2021; 128:969-992 | PMID: 33793333
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Impact:
Abstract

Device Therapy of Hypertension.

Mahfoud F, Schlaich MP, Lobo MD
In the past decade, efforts to improve blood pressure control have looked beyond conventional approaches of lifestyle modification and drug therapy to embrace interventional therapies. Based upon animal and human studies clearly demonstrating a key role for the sympathetic nervous system in the etiology of hypertension, the newer technologies that have emerged are predominantly aimed at neuromodulation of peripheral nervous system targets. These include renal denervation, baroreflex activation therapy, endovascular baroreflex amplification therapy, carotid body ablation, and pacemaker-mediated programmable hypertension control. Of these, renal denervation is the most mature, and with a recent series of proof-of-concept trials demonstrating the safety and efficacy of radiofrequency and more recently ultrasound-based renal denervation, this technology is poised to become available as a viable treatment option for hypertension in the foreseeable future. With regard to baroreflex activation therapy, endovascular baroreflex amplification, carotid body ablation, and programmable hypertension control, these are developing technologies for which more human data are required. Importantly, central nervous system control of the circulation remains a poorly understood yet vital component of the hypertension pathway and mandates further investigation. Technology to improve blood pressure control through deep brain stimulation of key cardiovascular control territories is, therefore, of interest. Furthermore, alternative nonsympathomodulatory intervention targeting the hemodynamics of the circulation may also be worth exploring for patients in whom sympathetic drive is less relevant to hypertension perpetuation. Herein, we review the aforementioned technologies with an emphasis on the preclinical data that underpin their rationale and the human evidence that supports their use.



Circ Res: 01 Apr 2021; 128:1080-1099
Mahfoud F, Schlaich MP, Lobo MD
Circ Res: 01 Apr 2021; 128:1080-1099 | PMID: 33793330
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Impact:
Abstract

Obesity, Adipose Tissue and Vascular Dysfunction.

Koenen M, Hill MA, Cohen P, Sowers JR
Cardiovascular diseases are the leading cause of death worldwide. Overweight and obesity are strongly associated with comorbidities such as hypertension and insulin resistance, which collectively contribute to the development of cardiovascular diseases and resultant morbidity and mortality. Forty-two percent of adults in the United States are obese, and a total of 1.9 billion adults worldwide are overweight or obese. These alarming numbers, which continue to climb, represent a major health and economic burden. Adipose tissue is a highly dynamic organ that can be classified based on the cellular composition of different depots and their distinct anatomical localization. Massive expansion and remodeling of adipose tissue during obesity differentially affects specific adipose tissue depots and significantly contributes to vascular dysfunction and cardiovascular diseases. Visceral adipose tissue accumulation results in increased immune cell infiltration and secretion of vasoconstrictor mediators, whereas expansion of subcutaneous adipose tissue is less harmful. Therefore, fat distribution more than overall body weight is a key determinant of the risk for cardiovascular diseases. Thermogenic brown and beige adipose tissue, in contrast to white adipose tissue, is associated with beneficial effects on the vasculature. The relationship between the type of adipose tissue and its influence on vascular function becomes particularly evident in the context of the heterogenous phenotype of perivascular adipose tissue that is strongly location dependent. In this review, we address the abnormal remodeling of specific adipose tissue depots during obesity and how this critically contributes to the development of hypertension, endothelial dysfunction, and vascular stiffness. We also discuss the local and systemic roles of adipose tissue derived secreted factors and increased systemic inflammation during obesity and highlight their detrimental impact on cardiovascular health.



Circ Res: 01 Apr 2021; 128:951-968
Koenen M, Hill MA, Cohen P, Sowers JR
Circ Res: 01 Apr 2021; 128:951-968 | PMID: 33793327
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Impact:
Abstract

Pathophysiology of Hypertension: The Mosaic Theory and Beyond.

Harrison DG, Coffman TM, Wilcox CS
Dr Irvine Page proposed the Mosaic Theory of Hypertension in the 1940s advocating that hypertension is the result of many factors that interact to raise blood pressure and cause end-organ damage. Over the years, Dr Page modified his paradigm, and new concepts regarding oxidative stress, inflammation, genetics, sodium homeostasis, and the microbiome have arisen that allow further refinements of the Mosaic Theory. A constant feature of this approach to understanding hypertension is that the various nodes are interdependent and that these almost certainly vary between experimental models and between individuals with hypertension. This review discusses these new concepts and provides an introduction to other reviews in this compendium of Circulation Research.



Circ Res: 01 Apr 2021; 128:847-863
Harrison DG, Coffman TM, Wilcox CS
Circ Res: 01 Apr 2021; 128:847-863 | PMID: 33793328
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Impact:
Abstract

Prevention of Fibrosis and Pathological Cardiac Remodeling by Salinomycin.

Burke RM, Dirkx RA, Quijada P, Lighthouse JK, ... Ashton J, Small EM
Rationale: Cardiomyopathy is characterized by the deposition of extracellular matrix by activated resident cardiac fibroblasts, called myofibroblasts. There are currently no therapeutic approaches to blunt the development of pathological fibrosis and ventricle chamber stiffening that ultimately leads to heart failure. Objective: We undertook a high-throughput screen to identify small molecule inhibitors of myofibroblast activation that might limit the progression of heart failure. We evaluated the therapeutic efficacy of the polyether ionophore salinomycin in patient derived cardiac fibroblasts and pre-clinical mouse models of ischemic and non-ischemic heart failure.
Methods and results:
Here, we demonstrate that salinomycin displays potent anti-fibrotic activity in cardiac fibroblasts obtained from heart failure patients. In pre-clinical studies, salinomycin prevents cardiac fibrosis and functional decline in mouse models of ischemic and non-ischemic heart disease. Remarkably, interventional treatment with salinomycin attenuates pre-established pathological cardiac remodeling secondary to hypertension, and limits scar expansion when administered after a severe myocardial infarction. Mechanistically, salinomycin inhibits cardiac fibroblast activation by preventing p38/MAPK and Rho signaling. Salinomycin also promotes cardiomyocyte survival and improves coronary vessel density, suggesting that cardioprotection conferred by salinomycin occurs via the integration of multiple mechanisms in multiple relevant cardiac cell types. Conclusions: These data establish salinomycin as an anti-fibrotic agent that targets multiple cardioprotection pathways, thereby holding promise for the treatment of heart failure patients.




Circ Res: 06 Apr 2021; epub ahead of print
Burke RM, Dirkx RA, Quijada P, Lighthouse JK, ... Ashton J, Small EM
Circ Res: 06 Apr 2021; epub ahead of print | PMID: 33825488
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Impact:
Abstract

Over-Expression of Intestinal Alkaline Phosphatase Attenuates Atherosclerosis.

Ghosh SS, Wang J, Yannie PJ, Cooper RC, ... Korzun W, Ghosh S
Rationale: Intestinal Alkaline Phosphatase (IAP) is secreted by enterocytes and is present on the apical surface. It not only detoxifies bacterial endotoxin lipopolysaccharide (LPS) in the gut lumen and limits intestinal inflammation but also restricts translocation of LPS into systemic circulation. Diet-induced intestinal barrier dysfunction and subsequent development of metabolic endotoxemia seen in diabetes and heart disease is associated with reduced IAP levels. To examine the direct effects of increased IAP expression on barrier function and development of metabolic diseases, we developed intestine-specific IAP transgenic mice (IAPTg) over-expressing human chimeric IAP. Objective:The aim of this study was to evaluate the effects of intestine-specific IAP overexpression on Western-type diet (WD)-induced atherosclerosis in Ldlr-/- mice.
Methods and results:
IAPTg mice crossed into Ldlr-/- background (Ldlr-/-IAPTg) and Ldlr-/- littermates were fed WD for 16 weeks. Intestinal barrier dysfunction was assessed by monitoring plasma LPS levels and histological examination of colon. Over-expression of IAP attenuated WD-induced disruption of the colonic mucous layer, reducing intestinal barrier dysfunction and plasma LPS levels. Significant reduction in body, liver and adipose tissue weight was also seen in WD-fed Ldlr-/-IAPTg mice. Plasma and hepatic lipids were also significantly reduced in WD-fed Ldlr-/-IAPTg mice. Consistently, intestinal lipid absorption was attenuated in Ldlr-/-IAPTg mice with reduced expression of apical lipid transporters (CD36, FATP4 and NPC1L1) and intracellular lipid transport proteins (FABP1/2, SCP2). Attenuation of WD-induced atherosclerosis in Ldlr-/-IAPTg mice was demonstrated by significant reduction in arch and total aortic lesions as seen by enface analyses as well as significantly reduced atherosclerotic lesions in the ascending aorta of these mice.Conclusions: IAP overexpression improves intestinal barrier function by maintaining the integrity of the mucin layer in WD fed Ldlr-/-IAPTg mice and attenuates intestinal lipid absorption. Thus, by limiting translocation of gut-derived LPS and/or reducing plasma lipids, over-expression of IAP attenuates development of WD-induced atherosclerosis.




Circ Res: 08 Apr 2021; epub ahead of print
Ghosh SS, Wang J, Yannie PJ, Cooper RC, ... Korzun W, Ghosh S
Circ Res: 08 Apr 2021; epub ahead of print | PMID: 33834851
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Impact:
Abstract

An Immuno-Cardiac Model for Macrophage-Mediated Inflammation in COVID-19 Hearts.

Yang L, Han Y, Jaffre F, Nilsson-Payant BE, ... Chen Z, Chen S
Rationale: While respiratory failure is a frequent and clinically significant outcome of COVID-19, cardiac complications are a common feature in hospitalized COVID-19 patients and are associated with worse patient outcomes. The cause of cardiac injury in COVID-19 patients is not yet known. Case reports of COVID-19 autopsy heart samples have demonstrated abnormal inflammatory infiltration of macrophages in heart tissues.Objective: Generate an immuno-cardiac co-culture platform to model macrophage-mediated hyper-inflammation in COVID-19 hearts and screen for drugs that can block the macrophage-mediated inflammation.
Methods and results:
We systematically compared autopsy samples from non-COVID-19 donors and COVID-19 patients using RNA-seq and immunohistochemistry. We observed strikingly increased expression levels of CCL2 as well as macrophage infiltration in heart tissues of COVID-19 patients. We generated an immuno-cardiac co-culture platform containing human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) and macrophages. We found that macrophages induce increased reactive oxygen species (ROS) and apoptosis in CMs by secreting IL-6 and TNF-α after SARS-CoV-2 exposure. Using this immuno-cardiac co-culture platform, we performed a high content screen and identified ranolazine and tofacitinib as compounds that protect CMs from macrophage-induced cardiotoxicity. Conclusions: We established an immuno-host co-culture system to study macrophage-induced host cell damage following SARS-CoV-2 infection and identified FDA-approved drug candidates that alleviate the macrophage-mediated hyper-inflammation and cellular injury.




Circ Res: 14 Apr 2021; epub ahead of print
Yang L, Han Y, Jaffre F, Nilsson-Payant BE, ... Chen Z, Chen S
Circ Res: 14 Apr 2021; epub ahead of print | PMID: 33853355
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Impact:
Abstract

Intrinsic Electrical Remodeling Underlies Atrioventricular Block in Athletes.

Mesirca P, Nakao S, Nissen SD, Forte G, ... Boyett MR, D\'Souza A
Rationale: Athletes present with atrioventricular node (AV node) dysfunction manifesting as AV block. This can necessitate electronic pacemaker implantation, known to be more frequent in athletes with a long training history. Objective: AV block in athletes is attributed to high vagal tone. Here we investigated the alternative hypothesis that electrical remodeling of the AV node is responsible.
Methods and results:
Radio-telemetry ECG data and AV node biopsies were collected in sedentary and trained Standardbred racehorses, a large-animal model of the athlete\'s heart. Trained horses presented with longer PR intervals (that persisted under complete autonomic block) versus sedentary horses, concomitant with reduced expression of key ion channels involved in AV node conduction: L-type Ca2+ channel subunit CaV1.2 and the hyperpolarization-activated cyclic nucleotide gated channel 4 (HCN4). AV node electrophysiology was explored further in mice; prolongation of the PR interval (in vivo and ex vivo), Wenckebach cycle length and AV node refractory period was observed in mice trained by swimming versus sedentary mice. Transcriptional profiling in laser-capture microdissected AV node revealed striking reduction in pacemaking ion channels in trained mice, translating into protein downregulation of CaV1.2 and HCN4. Correspondingly, patch clamp recordings in isolated AV node myocytes demonstrated a training-induced reduction in ICa,L and If density that likely contributed to the observed lower frequency of action potential firing in trained cohorts. microRNA (miR) profiling and in vitro studies revealed miR-211-5p and miR-432 as direct regulators of CaV1.2 and HCN4. In vivo miRs suppression or detraining restored training-induced PR prolongation and ion channel remodeling. Conclusions: Training-induced AV node dysfunction is underscored by likely miR-mediated transcriptional remodeling that translates into reduced current density of key ionic currents involved in impulse generation and conduction. We conclude that electrical remodeling is a key mechanism underlying AV block in athletes.




Circ Res: 13 Apr 2021; epub ahead of print
Mesirca P, Nakao S, Nissen SD, Forte G, ... Boyett MR, D'Souza A
Circ Res: 13 Apr 2021; epub ahead of print | PMID: 33849278
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Impact:
Abstract

Update on apolipoprotein B.

Sniderman A, Langlois M, Cobbaert C
Purpose of review
The 2019 European Society of Cardiology/European Atherosclerosis Society Guidelines concluded that apolipoprotein B (apoB) was a more accurate measure of cardiovascular risk and a better guide to the adequacy of lipid lowering than low-density lipoprotein cholesterol (LDL-C) or non-high-density lipoprotein cholesterol (HDL-C). Also, they stated that apoB can be measured more accurately than LDL-C or non-HDL-C. This strong endorsement of the central role of apoB contrasts with the limited endorsement of apoB by the 2018 American College of Cardiology/American Heart Association Multisociety Guidelines. Nevertheless, both retained LDL-C as the primary metric to guide statin/ezetimibe/Proprotein convertase subtilisin/kexin type 9 (PCSK9) therapy.
Recent findings
This essay will review the most important recent advances in knowledge about apoB with particular emphasis on the results of Mendelian randomization studies and a new discordance analysis in subjects on statin therapy. We will also lay out why using LDL-C to guide the adequacy of lipid lowering therapy represents an interpretive error of the results of the statin/ezetimibe/PCSK9 inhibitor randomized clinical trials and therefore why apoB should be the primary metric to guide statin/ezetimibe/PCSK9 therapy.
Summary
There is now a robust body of evidence demonstrating the superiority of apoB over LDL-C and non-HDL-C as a clinical marker of cardiovascular risk. LDL-C is not the appropriate marker to assess the benefits of statin/ezetimibe/PCSK9 therapy.

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

Curr Opin Lipidol: 15 Apr 2021; epub ahead of print
Sniderman A, Langlois M, Cobbaert C
Curr Opin Lipidol: 15 Apr 2021; epub ahead of print | PMID: 33870931
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Impact:
Abstract

Lipid effects of sodium-glucose cotransporter 2 inhibitors.

Lazarte J, Kanagalingam T, Hegele RA
Purpose of review
Sodium-glucose cotransporter 2 (SGLT2) inhibitors are widely used antihyperglycemic drugs that show remarkable cardiorenal protective effects in patients with or without type 2 diabetes. Furthermore, they are effective among patients across a wide range of baseline renal and cardiac function. Numerous mechanisms have been evaluated to understand these remarkable clinical benefits. From an early stage, these agents were noted to affect the plasma lipid profile. Here we review lipid profile alterations attributable to SGLT2 inhibitors and also some mechanisms explored in model systems and human studies.
Recent findings
SGLT2 inhibitors given to patients with diabetes as monotherapy shift substrate utilization from carbohydrates to lipids, and have mild effects on the lipid profile. Increased LDL cholesterol appears to be associated with increased hepatic production and decreased catabolism. Increased HDL cholesterol and decreased triglycerides appear to be associated with improved insulin sensitivity and increased lipolysis. Lipid effects of SGLT2 inhibitors are further modulated by background therapy with other diabetes medications and statins.
Summary
The minor lipid profile alterations observed in patients treated with SGLT2 inhibitors are offset by the staggering range of beneficial pleiotropic mechanisms that likely explain the marked cardiorenal benefits of these agents.

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

Curr Opin Lipidol: 15 Apr 2021; epub ahead of print
Lazarte J, Kanagalingam T, Hegele RA
Curr Opin Lipidol: 15 Apr 2021; epub ahead of print | PMID: 33870930
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Impact:
Abstract

Circulating senescent angiogenic T cells are linked with endothelial dysfunction and systemic inflammation in hypertension.

Zhang G, Liu Y, Qiu Y, Zhang J, ... Tao J, He J
Objective
Angiogenic T cells (Tang cells), a recently discovered T-cell subset, have been reported involved in the repair of endothelial injury. The purpose of this study was to explore the correlation of immunologic senescence and pro-inflammatory capacity of Tang cells with endothelial dysfunction in hypertensive patients.
Methods
Immunological characteristics of Tang cells (CD3+CD31+CXCR4+) from hypertensive patients with or without endothelial dysfunction were elucidated by surface immunophenotyping and intracellular cytokine staining. Endothelial function was measured by flow-mediated dilation (FMD).
Results
The frequency of CD28null subset in CD4+ Tang cells was notably elevated in hypertensive patients with endothelial dysfunction, which was negatively associated with FMD. The high frequency of CD28nullCD4+ Tang cells was an independent risk factor of endothelial dysfunction with good diagnostic performance in ROC curve analysis. Immunophenotyping revealed that this specific subset of Tang cells exhibited senescent profile and has low hTERT expression. CD28nullCD4+ Tang cells produced high levels of inflammatory cytokines, IL-6, IFN-γ and TNF-α, and significantly correlated with the systemic inflammation in hypertensive patients with endothelial dysfunction.
Conclusion
Collectively, our findings demonstrate for the first time that CD28null subset in CD4+ Tang cells with senescent and pro-inflammatory phenotype is dependently correlated with impaired FMD and systemic inflammation, which might contribute to the immunopathologic mechanism of endothelial dysfunction. Identification of a pathogenic CD4+ Tang-cell subset lacking CD28 may offer opportunities for the evaluation and management of endothelial dysfunction in hypertension.

Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc.

J Hypertens: 30 Apr 2021; 39:970-978
Zhang G, Liu Y, Qiu Y, Zhang J, ... Tao J, He J
J Hypertens: 30 Apr 2021; 39:970-978 | PMID: 33196557
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Impact:
Abstract

Cardiac and renal protective effects of 2,5-dimethylcelecoxib in angiotensin II and high-salt-induced hypertension model mice.

Yamamoto M, Takahashi-Yanaga F, Arioka M, Igawa K, ... Yamaura K, Sasaguri T
Background
We reported that 2,5-dimethylcelecoxib (DM-celecoxib), a celecoxib derivative that is unable to inhibit cyclooxygenase-2, prevented cardiac remodeling induced by sarcomeric gene mutation, left ventricular pressure overload, or β-adrenergic receptor stimulation. This effect seemed to be mediated by the inhibition of the canonical Wnt/β-catenin signaling pathway, which has been suggested to play a key role in the development of chronic kidney disease and chronic heart failure.
Method
We investigated the effect of DM-celecoxib on cardiac remodeling and kidney injury in hypertension model mice induced by angiotensin II infusion in the absence or presence of high-salt load.
Results
DM-celecoxib prevented cardiac remodeling and markedly reduced urinary albumin excretion without altering blood pressure in those mice. Moreover, DM-celecoxib prevented podocyte injury, glomerulosclerosis, and interstitial fibrosis in the kidney of mice loaded with angiotensin II and high-salt load. DM-celecoxib reduced the phosphorylation level of Akt and activated glycogen synthase kinase-3, which led to the suppression of the Wnt/β-catenin signal in the heart and kidney. DM-celecoxib also reduced the expression level of snail, a key transcription factor for the epithelial-mesenchymal transition and of which gene is a target of the Wnt/β-catenin signal.
Conclusion
Results of the current study suggested that DM-celecoxib could be beneficial for patients with hypertensive heart and kidney diseases.

Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

J Hypertens: 30 Apr 2021; 39:892-903
Yamamoto M, Takahashi-Yanaga F, Arioka M, Igawa K, ... Yamaura K, Sasaguri T
J Hypertens: 30 Apr 2021; 39:892-903 | PMID: 33252422
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Impact:
Abstract

Effects of inverted photoperiods on the blood pressure and carotid artery of spontaneously hypertensive rats and Wistar-Kyoto rats.

Wang Y, Zhang T, Zhang Y, Yu Y, ... Zhang H, Chi Y
Objective
The objective of this study was to investigate the effects of inverted photoperiods on the blood pressure and carotid arteries in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats (homologous control group).
Methods and results
This study used two inverted photoperiods [inverted light:dark (ILD)16 : 8 and ILD12 : 12] to create the model. A total of 27 male SHR and 27 male WKY rats were randomly divided into six groups (nine rats per group): SHR (LD12 : 12), SHR (ILD16 : 8), SHR (ILD12 : 12), WKY (LD12 : 12), WKY (ILD16 : 8) and WKY (ILD12 : 12). We recorded the trajectory of the activity rhythm of the rats and performed carotid vascular ultrasound examination, MRI (arterial spin labelling) analysis and carotid biopsy. The results showed that inverted photoperiods increased the blood pressure, carotid intima-media thickness, resistance index and blood flow velocity. In addition, inverted photoperiods led to the development of carotid arterial thrombosis, significantly reduced cerebral blood flow and increased the number of collagen fibres. Moreover, it increased the expression of angiotensin receptor and low-density lipoprotein receptor in the carotid arteries, leading to decreased expression of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase and nitric oxide synthase. Inverted photoperiods induced the formation of atherosclerotic plaque. Multiple results of SHR were worse than those of WKY rats.
Conclusion
Taken together, inverted photoperiods can produce a series of adverse consequences on blood pressure and carotid arteries. Hypertension can aggravate the adverse effects of inverted photoperiods.

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

J Hypertens: 30 Apr 2021; 39:871-879
Wang Y, Zhang T, Zhang Y, Yu Y, ... Zhang H, Chi Y
J Hypertens: 30 Apr 2021; 39:871-879 | PMID: 33824259
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Impact:
Abstract

Associations of plasma PAPP-A2 and genetic variations with salt sensitivity, blood pressure changes and hypertension incidence in Chinese adults.

Wang Y, Jia H, Gao WH, Zou T, ... Yang TL, Mu JJ
Objective
Pregnancy-associated plasma protein-A2 (PAPP-A2) is the homolog of PAPP-A in the vertebrate genome and its role in protecting against salt-induced hypertension in salt-sensitive rats has been confirmed. We sought to examine the associations of plasma PAPP-A2 levels and its genetic variants with salt sensitivity, blood pressure (BP) changes and hypertension incidence in humans.
Methods
Eighty participants (18-65 years old) sequentially consuming a usual diet, a 7-day low-salt diet (3.0 g/day) and a 7-day high-salt diet (18 g/day). In addition, we studied participants of the original Baoji Salt-Sensitive Study, recruited from 124 families in Northern China in 2004 who received the same salt intake intervention, and evaluated them for the development of hypertension over 14 years.
Results
The plasma PAPPA2 levels significantly decreased with the change from baseline to a low-salt diet and decreased further when converting from the low-salt to high-salt diet. SNP rs12042763 in the PAPP-A2 gene was significantly associated with systolic BP responses to both low-salt and high-salt diet while SNP rs2861813 showed a significant association with the changes in SBP and pulse pressure at 14-year follow-up. Additionally, SNPs rs2294654 and rs718067 demonstrated a significant association with the incidence of hypertension over the 14-year follow-up. Finally, the gene-based analysis found that Pappa2 was significantly associated with longitudinal SBP changes and the incidence of hypertension over the 14-year follow-up.
Conclusions
This study shows that dietary salt intake affects plasma PAPP-A2 levels and that PAPP-A2 may play a role in salt sensitivity, BP progression and development of hypertension in the Chinese populations.

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

J Hypertens: 25 Mar 2021; epub ahead of print
Wang Y, Jia H, Gao WH, Zou T, ... Yang TL, Mu JJ
J Hypertens: 25 Mar 2021; epub ahead of print | PMID: 33783375
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Impact:
Abstract

Aging-induced isoDGR-modified fibronectin activates monocytic and endothelial cells to promote atherosclerosis.

Park JE, JebaMercy G, Pazhanchamy K, Guo X, ... McCarthy NE, Sze SK
Background:
and aims
Aging is the primary risk factor for cardiovascular disease (CVD), but the mechanisms underlying age-linked atherosclerosis remain unclear. We previously observed that long-lived vascular matrix proteins can acquire \'gain-of-function\' isoDGR motifs that might play a role in atherosclerotic pathology.
Methods
IsoDGR-specific mAb were generated and used for ELISA-based measurement of motif levels in plasma samples from patients with coronary artery diseases (CAD) and non-CAD controls. Functional consequences of isoDGR accumulation in age-damaged fibronectin were determined by bioassay for capacity to activate monocytes, macrophages, and endothelial cells (signalling activity, pro-inflammatory cytokine expression, and recruitment/adhesion potential). Mice deficient in the isoDGR repair enzyme PCMT1 were used to assess motif distribution and macrophage localisation in vivo.
Results
IsoDGR-modified fibronectin and fibrinogen levels in patient plasma were significantly enhanced in CAD and further associated with smoking status. Functional assays demonstrated that isoDGR-modified fibronectin activated both monocytes and macrophages via integrin receptor \'outside in\' signalling, triggering an ERK:AP-1 cascade and expression of pro-inflammatory cytokines MCP-1 and TNFα to drive additional recruitment of circulating leukocytes. IsoDGR-modified fibronectin also induced endothelial cell expression of integrin β1 to further enhance cellular adhesion and matrix deposition. Analysis of murine aortic tissues confirmed accumulation of isoDGR-modified proteins co-localised with CD68+ macrophages in vivo.
Conclusions
Age-damaged fibronectin features isoDGR motifs that increase binding to integrins on the surface of monocytes, macrophages, and endothelial cells. Subsequent activation of \'outside-in\' signalling elicits a range of potent cytokines and chemokines that drive additional leukocyte recruitment to the developing atherosclerotic matrix.

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

Atherosclerosis: 24 Mar 2021; 324:58-68
Park JE, JebaMercy G, Pazhanchamy K, Guo X, ... McCarthy NE, Sze SK
Atherosclerosis: 24 Mar 2021; 324:58-68 | PMID: 33831670
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Impact:
Abstract

Flecainide-Induced QRS Complex Widening Correlates with Negative Inotropy.

Rabelo Evangelista AB, Monteiro FR, Nearing BD, Belardinelli L, Verrier RL
Background
The negative inotropic effect of Class IC antiarrhythmic drugs limits their use for acute cardioversion of atrial fibrillation (AF).
Objective
In an intact porcine model, we examined effects of pulmonary and intravenous (IV) administration of flecainide on left ventricular (LV) contractility and QRS complex width at doses that are effective in converting new-onset AF to sinus rhythm.
Methods
Flecainide (1.5 mg/kg bolus) was delivered by intratracheal administration and compared to 2.0 mg/kg 10-min IV administration (ESC guideline) and to 0.5 and 1.0 mg/kg 2-min IV doses in 40 closed-chest, anesthetized Yorkshire pigs. Catheters were fluoroscopically positioned in the LV to monitor QRS complex width and contractility and at the bifurcation of the main bronchi to deliver intratracheal flecainide.
Results
Peak flecainide plasma concentrations (Cmax) were similar but the 30-min AUC of plasma levels was 1.4- to 2.9-fold greater for 2.0 mg/kg 10-min IV infusion than for the lower, more rapidly delivered intratracheal and IV doses. The AUC for LV contractility, i.e., negative inotropic burden, was 2.2- to 3.6-fold greater for 2.0 mg/kg 10-min IV dose than for the lower, more rapidly delivered doses. QRS complex widening by flecainide was highly correlated with the decrease in LV contractility (r2=0.890, p<0.0001, for all IV doses; r2=0.812, p=0.014, for intratracheal flecainide).
Conclusions
QRS complex widening in response to flecainide is strongly correlated with the decrease in LV contractility. Rapid pulmonary or IV flecainide delivery reduces the negative inotropic burden while quickly achieving Cmax levels associated with conversion of AF.

Copyright © 2021. Published by Elsevier Inc.

Heart Rhythm: 09 Apr 2021; epub ahead of print
Rabelo Evangelista AB, Monteiro FR, Nearing BD, Belardinelli L, Verrier RL
Heart Rhythm: 09 Apr 2021; epub ahead of print | PMID: 33848647
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Impact:
Abstract

A NOVEL PATHOGENIC ROLE FOR GALECTIN-3 IN EARLY DISEASE STAGES OF ARRHYTHMOGENIC CARDIOMYOPATHY.

Cason M, Celeghin R, Marinas MB, Beffagna G, ... Basso C, Pilichou K
Background
Arrhythmogenic cardiomyopathy (AC) is a myocardial disease due to desmosomal mutations, whose pathogenesis remains incompletely understood.
Objective
To identify molecular pathways underlying early AC by gene expression profiling in both humans and animal models.
Methods
RNA sequencing for differentially expressed genes (DEGs) was performed on the myocardium of transgenic mice over-expressing the Desmoglein2-N271S mutation before phenotype onset. Zebrafish signaling reporters were used for in vivo validation. Whole exome sequencing was undertaken in 10 genotype-negative AC patients and subsequent direct sequencing in 140 AC index cases.
Results
Among 29 DEGs identified at early disease stages, Lgals3/GAL3 (lectin, galactoside-binding, soluble, 3) showed reduced cardiac expression in transgenic mice and in 3 AC patients who suffered sudden cardiac death without overt structural remodeling. Four rare missense variants of LGALS3 were identified in 5 human AC probands. Pharmacological inhibition of Lgals3 in zebrafish reduced Wnt and TGFβ signaling, increased Hippo/YAP-TAZ signaling, and induced alterations in desmoplakin membrane localization, desmosome integrity and stability. Increased LGALS3 plasma expression in genotype-positive AC patients and CD98 activation supported the GAL3 release by circulating macrophages pointing toward the stabilization of desmosomal assembly at the injured regions.
Conclusions
GAL3 plays a crucial role in early AC onset through regulation of Wnt/β-catenin signaling and intercellular adhesion.

Copyright © 2021. Published by Elsevier Inc.

Heart Rhythm: 11 Apr 2021; epub ahead of print
Cason M, Celeghin R, Marinas MB, Beffagna G, ... Basso C, Pilichou K
Heart Rhythm: 11 Apr 2021; epub ahead of print | PMID: 33857645
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Impact:
Abstract

Cardiac Potassium Inward Rectifier Kir2: Review of Structure, Regulation, Pharmacology and Arrhythmogenesis.

Reilly L, Eckhardt L
Potassium inward rectifier channel Kir2 is an important component of terminal cardiac repolarization and resting membrane stability. This functionality is part of balanced cardiac excitability and is a defining feature of excitable cardiac membranes. \"Gain\"- or \"loss-of-function\" mutations in KCNJ2, the gene encoding Kir2.1, cause genetic sudden cardiac death syndromes, and loss of Kir2 current, IK1, is a major contributing factor to arrhythmogenesis in failing human hearts. Here we provide a contemporary review of the Kir2 channels\' functional structure, physiology and pharmacology. Beyond the structure and functional relationships, we will focus on the elements of clinically used drugs that block the channel and the implications for treatment of atrial fibrillation with IK1 blocking agents. We will also review the clinical disease entities associated with KCNJ2 mutations and the growing area of research into associated arrhythmia mechanisms. Lastly, Kir2 channels\' presence has become a tipping point for electrical maturity in induced pluripotent stem cell derived cardiomyocytes (iPS-CMs) and highlights the significance of understanding why Kir2 in iPS-CMs is important to consider for CIPA and drug safety testing.

Copyright © 2021. Published by Elsevier Inc.

Heart Rhythm: 11 Apr 2021; epub ahead of print
Reilly L, Eckhardt L
Heart Rhythm: 11 Apr 2021; epub ahead of print | PMID: 33857643
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Impact:
Abstract

Triglyceride-induced cardiac lipotoxicity is mitigated by Silybum marianum.

Vilahur G, Sutelman P, Mendieta G, Ben-Aicha S, ... Casaní L, Badimon L
Background:
and aims
Silybum marianum (SM) is an herbal product with cytoprotective and antioxidant properties. We have previously demonstrated that SM ameliorates ventricular remodeling and improves cardiac performance. Here, we evaluated whether SM could exert beneficial effects against cardiac lipotoxicity in a pig model of closed-chest myocardial infarction (MI).
Methods
Study 1 investigated the effect of SM administration on lipid profile and any potential SM-related adverse effects. Animals received SM or placebo during 10 days and were afterward sacrificed. Study 2 evaluated the effectiveness of SM daily administration in reducing cardiac lipotoxicity in animals subjected to a 1.5h myocardial infarction (MI), who were subsequently reperfused for 2.5h and euthanized or kept under study for three weeks and then sacrificed.
Results
Animals administered a 10-day SM regime presented a sharp decline in plasma triglyceride levels vs. controls, with no other modifications in lipid profile. The decrease in triglyceride concentration was accompanied by a marked reduction in triglyceride intestinal absorption and glycoprotein-P expression. Three weeks post-MI the triglyceride content in the ischemic myocardium of the SM-treated animals was significantly lower than in the ischemic myocardium of placebo-controls. This effect was associated with an enhanced cardiac expression of PPARγ and triglyceride clearance receptors. This long-term SM-administration induced a lower expression of lipid receptors in subcutaneous adipose tissue. No SM-related side-effects were registered.
Conclusion
SM administration reduces plasma triglyceride levels through attenuation of triglyceride intestinal absorption and modulates cardiac lipotoxicity in the ischemic myocardium, likely contributing to improve ventricular remodeling.

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

Atherosclerosis: 26 Mar 2021; 324:91-101
Vilahur G, Sutelman P, Mendieta G, Ben-Aicha S, ... Casaní L, Badimon L
Atherosclerosis: 26 Mar 2021; 324:91-101 | PMID: 33857761
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Impact:
Abstract

Treatment of atherosclerosis through transplantation of endothelial progenitor cells overexpressing dimethylarginine dimethylaminohydrolase (DDAH) in rabbits.

Shoeibi S, Mahdipour E, Mohammadi S, Moohebati M, Ghayour-Mobarhan M
Background
Endothelial dysfunction is a key event in the development of vascular diseases, including atherosclerosis. Endothelial progenitor cells (EPCs) play an important role in vascular repair. Decreased dimethylarginine dimethylaminohydrolase (DDAH) activity is observed in several pathological conditions, and it is associated with an increased risk of vascular disease. We hypothesized that bone marrow-derived EPCs and combination therapy with DDAH2-EPCs could reduce plaque size and ameliorate endothelial dysfunction in an atherosclerosis rabbit model.
Method
Four groups of rabbits (n = 8 per group) were subjected to a hyperlipidemic diet for a month. After establishing the atherosclerosis model, rabbits received 4 × 106 EPC, EPCs expressing DDAH2, through femoral vein injection, or saline (the control group with basic food and the untreated group). One month after transplantation, plaque thickness, endothelial function, oxidative stress, and inflammatory mRNAs, DDAH, and eNOS function were assessed.
Results
DDAH2-EPCs transplantation (p < 0.05) and EPCs transplantation (p < 0.05) were both associated with a reduction in plaque size compared to the control saline injection. The antiproliferative and antiatherogenic effects of EPCs were further enhanced by the overexpression of DDAH2 (p < 0.05, DDAH2-EPCs vs. EPCs). Furthermore, DDAH2-EPCs transplantation significantly increased endothelium integrity compared to the EPCs transplantation.
Conclusion
Transplantation of EPCs overexpressing DDAH2 may enhance the repair of injured endothelium by reducing inflammation and restoring endothelial function. Therefore, pCMV6-mediated DDAH2 gene-transfected EPCs are a potentially valuable tool for the treatment of atherosclerosis.

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

Int J Cardiol: 14 May 2021; 331:189-198
Shoeibi S, Mahdipour E, Mohammadi S, Moohebati M, Ghayour-Mobarhan M
Int J Cardiol: 14 May 2021; 331:189-198 | PMID: 33535073
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Impact:
Abstract

Characterizing modifier genes of cardiac fibrosis phenotype in hypertrophic cardiomyopathy.

Xu F, Chen Y, Tillman KA, Cui Y, ... Lu L, Sun Y
Background
Clinical phenotypes of hypertrophic cardiomyopathy (HCM) vary greatly even among patients with the same gene mutations. This variability is largely regulated by unidentified modifier loci. The purpose of the study is to identify modifier genes for cardiac fibrosis-a major phenotype of HCM-using the BXD family, a murine cohort.
Methods
The relative severity of cardiac fibrosis was estimated by quantitation of cardiac collagen volume fraction (CCVF) across 66 members of the BXD family. Quantitative trait locus (QTL) mapping for cardiac fibrosis was done using GeneNetwork. Candidate modifier loci and genes associated with fibrosis were prioritized based on an explicit scoring system. Networks of correlation between fibrosis and cardiac transcriptomes were evaluated to generate causal models of disease susceptibility.
Results
CCVF levels varied greatly within this family. Interval mapping identified a significant CCVF-related QTL on chromosome (Chr) 2 in males, and a significant QTL on Chr 4 Mb in females. The scoring system highlighted two strong candidate genes in the Chr 2 locus-Nek6 and Nr6a1. Both genes are highly expressed in the heart. Cardiac Nek6 mRNA levels are significantly correlated with CCVF. Nipsnap3b and Fktn are lead candidate genes for the Chr 4 locus, and both are also highly expressed in heart. Cardiac Nipsnap3b gene expression correlates well with CCVF.
Conclusion
Our study demonstrated that candidate modifier genes of cardiac fibrosis phenotype in HCM are different in males and females. Nek6 and Nr6a1 are strong candidates in males, while Nipsnap3b and Fktn are top candidates in females.

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

Int J Cardiol: 30 Apr 2021; 330:135-141
Xu F, Chen Y, Tillman KA, Cui Y, ... Lu L, Sun Y
Int J Cardiol: 30 Apr 2021; 330:135-141 | PMID: 33529666
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Impact:
Abstract

Empagliflozin increases plasma levels of campesterol, a marker of cholesterol absorption, in patients with type 2 diabetes: Association with a slight increase in high-density lipoprotein cholesterol.

Jojima T, Sakurai S, Wakamatsu S, Iijima T, ... Usui I, Aso Y
Background:
and aims
Sodium/glucose cotransporter 2 (SGLT2) inhibitors decrease plasma triglyceride levels and slightly increase low-density lipoprotein (LDL-c) and high-density lipoprotein cholesterol (HDL-c). However, the mechanisms underlying such changes in the blood lipid profile remain to be determined. We investigated how empagliflozin affects plasma markers of cholesterol absorption and synthesis, and evaluated the relationship between changes in these markers and blood lipids in patients with type 2 diabetes.
Methods and results
In a randomized, active-controlled, open-label trial, 51 patients were randomly allocated in 2:1 ratio to receive empagliflozin 10 mg/day (n = 32) or standard therapy (n = 19) for 12 weeks. We measured plasma levels of lathosterol as a marker of cholesterol synthesis, and campesterol and sitosterol as markers of cholesterol absorption, at baseline and 12 weeks after treatment. In the empagliflozin group, serum HDL-c, but not LDL-c, significantly increased between baseline and 12 weeks (54.4 ± 16.3 vs. 58.8 ± 19.6 mg/dl; p = 0.0006), whereas in the standard therapy group, HDL-c and LDL-c remained unchanged. In the empagliflozin group, plasma campesterol also increased significantly (4.14 ± 1.88 vs. 4.90 ± 2.26 μg/ml, p = 0.0008), whereas no change in plasma campesterol or sitosterol was found in the control group. Although plasma lathosterol showed no change in the whole empagliflozin group, it decreased significantly in patients who were not taking statins. In statin non-users, plasma lathosterol decreased significantly after treatment with empagliflozin (2.71 ± 0.99 vs. 1.91 ± 0.99 μg/ml, p < 0.05). In the empagliflozin group, changes in plasma campesterol correlated positively with changes in HDL-c.
Conclusion
Empagliflozin increases serum campesterol, a marker of cholesterol absorption, in patients with type 2 diabetes. This increase may be associated with SGLT2 inhibitor-induced increases in HDL cholesterol.

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

Int J Cardiol: 14 May 2021; 331:243-248
Jojima T, Sakurai S, Wakamatsu S, Iijima T, ... Usui I, Aso Y
Int J Cardiol: 14 May 2021; 331:243-248 | PMID: 33556413
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Impact:
Abstract

Adenosine A3 agonists reverse neuropathic pain via T cell-mediated production of IL-10.

Durante M, Squillace S, Lauro F, Giancotti LA, ... Jacobson KA, Salvemini D
The A3 adenosine receptor (A3AR) has emerged as a therapeutic target with A3AR agonists to tackle the global challenge of neuropathic pain, and investigation into its mode of action is essential for ongoing clinical development. Immune cell A3ARs, and their activation during pathology, modulate cytokine release. Thus, the use of immune cells as a cellular substrate for the pharmacological action of A3AR agonists is enticing, but unknown. The present study discovered that Rag-KO mice lacking T and B cells, as compared with WT mice, are insensitive to the anti-allodynic effects of A3AR agonists. Similar findings were observed in interleukin-10 and interleukin-10 receptor knockout mice. Adoptive transfer of CD4+ T cells from WT mice infiltrated the dorsal root ganglion (DRG) and restored A3AR agonist-mediated anti-allodynia in Rag-KO mice. CD4+ T cells from Adora3-KO or Il10-KO mice did not. Transfer of CD4+ T cells from WT mice, but not Il10-KO mice, into Il10-KO mice or Adora3-KO mice fully reinstated the anti-allodynic effects of A3AR activation. Notably, A3AR agonism reduced DRG neuron excitability when cocultured with CD4+ T cells in an IL-10-dependent manner. A3AR action on CD4+ T cells infiltrated in the DRG decreased phosphorylation of GluN2B-containing N-methyl-D-aspartate receptors at Tyr1472, a modification associated with regulating neuronal hypersensitivity. Our findings establish that activation of A3AR on CD4+ T cells to release IL-10 is required and sufficient evidence for the use of A3AR agonists as therapeutics.



J Clin Invest: 31 Mar 2021; 131
Durante M, Squillace S, Lauro F, Giancotti LA, ... Jacobson KA, Salvemini D
J Clin Invest: 31 Mar 2021; 131 | PMID: 33621215
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Impact:
Abstract

Blocking podoplanin inhibits platelet activation and decreases cancer-associated venous thrombosis.

Wang X, Liu B, Xu M, Jiang Y, ... Wu J, Zhao Y
Background
Patients with cancer are at a high risk of venous thromboembolism (VTE), studies have shown that high expression of podoplanin (PDPN) in tumors is associated with increased risk of VTE.
Methods
Two human malignant cell lines (NCI-H226 and C8161) expressing high levels of PDPN were selected to explore the role of platelet in cancer-associated venous thrombosis in vitro and in vivo. Immunohistochemical staining using anti-PDPN antibody was performed in the pulmonary carcinoma patients.
Results
Both NCI-H226 and C8161 cells expressing high PDPN triggered platelet activation via CLEC-2 in vitro, which was abrogated by an anti-PDPN antibody SZ-168. Furthermore, the in vivo study revealed that injection of CHO-PDPN or C8161 in two mouse model of venous thrombosis activated platelets, increased platelet counts and enhanced thrombosis. More importantly, PDPN-enhanced thrombosis was reduced in mice treated with SZ168. A total of 63.3% tumor specimens stained positive for PDPN. High PDPN expression was associated with an increased risk of VTE and poor prognosis.
Conclusions
PDPN expression in tumors induced platelet activation and was related to a high risk of VTE via platelet activation. SZ168 inhibited PDPN-induced platelet activation in vitro and decreased the incidence of VTE in mice.

Copyright © 2021 Elsevier Ltd. All rights reserved.

Thromb Res: 30 Mar 2021; 200:72-80
Wang X, Liu B, Xu M, Jiang Y, ... Wu J, Zhao Y
Thromb Res: 30 Mar 2021; 200:72-80 | PMID: 33548843
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Impact:
Abstract

Sulfated non-anticoagulant low molecular weight heparin in the prevention of cancer and non-cancer associated thrombosis without compromising hemostasis.

Darwish NHE, Godugu K, Mousa SA
Introduction
Cancer-associated thrombosis (CAT) accounts for about 20% of all cases of Venous Thromboembolism (VTE). Tissue factor (TF) is documented to be highly expressed on cancer cells and pathological angiogenic endothelial cells. Here, we used a novel oxidized sulfated ultra-LMWH, S-NACH, which is devoid of anti-factor Xa and IIa activities with limited to no systemic anticoagulant effects. This sulfated form has enhanced binding to vascular endothelial cells (EC) and releases and potentiates the action of tissue factor pathway inhibitor (TFPI). S-NACH binds with high affinity to EC, releases and binds to EC TFPI, and promotes vascular antithrombotic effect with limited to no risk of bleeding complications.
Materials and methods
We investigated the effects of S-NACH on clot kinetics in vitro and in vivo. Also, we investigated the effects of S-NACH on CAT mediated by human acute leukemia cells (K562) and human pancreatic cancer cells (SUIT2).
Results
S-NACH was associated with ~3-fold increase of TFPI 2 levels within 3 h. Also, S-NACH reversed the hypercoagulability state that is associated with cancer cells in vitro. In vivo, S-NACH at 20 mg/kg subcutaneously (SC) had no effect on bleeding time compared to both tinzaparin and enoxaparin at 5 mg/kg SC. S-NACH did not show any anti-IIa or anti-Xa activities in comparison to tinzaparin and enoxaparin (p < 0.001).
Conclusion
Data suggest the importance of S-NACH through its EC binding, EC TFPI release and its interaction with TFPI in enhancing its activity in the prevention of cancer and non-cancer associated thrombosis with limited to no bleeding complications.

Copyright © 2021 Elsevier Ltd. All rights reserved.

Thromb Res: 30 Mar 2021; 200:109-114
Darwish NHE, Godugu K, Mousa SA
Thromb Res: 30 Mar 2021; 200:109-114 | PMID: 33582600
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Impact:
Abstract

Selatogrel, a reversible P2Y12 receptor antagonist, has reduced off-target interference with haemostatic factors in a mouse thrombosis model.

Crescence L, Darbousset R, Caroff E, Hubler F, ... Panicot-Dubois L, Dubois C
Introduction
Selatogrel is a reversible antagonist of the P2Y12 receptor. In rat thrombosis/haemostasis models, selatogrel was associated with lower blood loss than clopidogrel or ticagrelor at equivalent anti-thrombotic effect.
Material and methods
We sought to elucidate the mechanism underlying the observed differences in blood loss, using real-time intravital microscopy in mouse.
Results
Selatogrel, ticagrelor and clopidogrel dose-dependently inhibited laser-induced platelet thrombus formation. At maximal antithrombotic effect, only small mural platelets aggregates, corresponding to hemostatic seals, were present. The phenotype of these hemostatic seals was dependent on the type of P2Y12 receptor antagonist. In the presence of clopidogrel and ticagrelor, detachment of platelets from the hemostatic seals was increased, indicative of reduced stability. In contrast, in the presence of selatogrel, platelet detachment was not increased. Moreover, equivalent antithrombotic dosing regimens of ticagrelor and clopidogrel reduced laser-induced calcium mobilization in the endothelium, restricted neutrophil adhesion and subsequent fibrin formation and thus reduced fibrin-mediated stabilization of the hemostatic seals. The effects of ticagrelor were also observed in P2Y12 receptor deficient mice, indicating that the effects are off-target and independent of the P2Y12 receptor. In contrast, selatogrel did not interfere with these elements of haemostasis in wild-type or in P2Y12 receptor deficient mice.
Conclusion
In the presence of selatogrel the stability of hemostatic seals was unperturbed, translating to an improved blood loss profile. Our data suggest that the mechanism underlying the differences in blood loss profiles of P2Y12 receptor antagonists is by off-target interference with endothelial activation, neutrophil function and thus, fibrin-mediated stabilization of haemostatic seals.

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

Thromb Res: 30 Mar 2021; 200:133-140
Crescence L, Darbousset R, Caroff E, Hubler F, ... Panicot-Dubois L, Dubois C
Thromb Res: 30 Mar 2021; 200:133-140 | PMID: 33610885
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Impact:
Abstract

The role of β-adrenergic system remodeling in human heart failure: A mechanistic investigation.

Mora MT, Gong JQX, Sobie EA, Trenor B
β-adrenergic receptor antagonists (β-blockers) are extensively used to improve cardiac performance in heart failure (HF), but the electrical improvements with these clinical treatments are not fully understood. The aim of this study was to analyze the electrophysiological effects of β-adrenergic system remodeling in heart failure with reduced ejection fraction and the underlying mechanisms. We used a combined mathematical model that integrated β-adrenergic signaling with electrophysiology and calcium cycling in human ventricular myocytes. HF remodeling, both in the electrophysiological and signaling systems, was introduced to quantitatively analyze changes in electrophysiological properties due to the stimulation of β-adrenergic receptors in failing myocytes. We found that the inotropic effect of β-adrenergic stimulation was reduced in HF due to the altered Ca2+ dynamics resulting from the combination of structural, electrophysiological and signaling remodeling. Isolated cells showed proarrhythmic risk after sympathetic stimulation because early afterdepolarizations appeared, and the vulnerability was greater in failing myocytes. When analyzing coupled cells, β-adrenergic stimulation reduced transmural repolarization gradients between endocardium and epicardium in normal tissue, but was less effective at reducing these gradients after HF remodeling. The comparison of the selective activation of β-adrenergic isoforms revealed that the response to β2-adrenergic receptors stimulation was blunted in HF while β1-adrenergic receptors downstream effectors regulated most of the changes observed after sympathetic stimulation. In conclusion, this study was able to reproduce an altered β-adrenergic activity on failing myocytes and to explain the mechanisms involved. The derived predictions could help in the treatment of HF and guide in the design of future experiments.

Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:14-25
Mora MT, Gong JQX, Sobie EA, Trenor B
J Mol Cell Cardiol: 30 Mar 2021; 153:14-25 | PMID: 33326834
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Impact:
Abstract

Ubiquitin-like protein FAT10 suppresses SIRT1-mediated autophagy to protect against ischemic myocardial injury.

Wan R, Yuan P, Guo L, Shao J, ... Jiang X, Hong K
Autophagy plays a deleterious role in ischemic myocardial injury. The deacetylase SIRT1 is a well-established regulator of autophagy that can be modified by the ubiquitin-like protein SUMO1. Our previous work demonstrated that another ubiquitin-like protein, FAT10, exerts cardioprotective effects against myocardial ischemia by stabilizing the caveolin-3 protein; however, the effects of FAT10 on autophagy through SIRT1 are unclear. Here, we constructed a Fat10-knockout rat model to evaluate the role of FAT10 in autophagy. In vivo and in vitro assays confirmed that FAT10 suppressed autophagy to protect the heart from ischemic myocardial injury. Mechanistically, FAT10 was mainly involved in the regulation of the autophagosome formation process. FAT10 affected autophagy through modulating SIRT1 degradation, which resulted in reduced SIRT1 nuclear translocation and inhibited SIRT1 activity via its C-terminal glycine residues. Notably, FAT10 competed with SUMO1 at the K734 modification site of SIRT1, which further reduced LC3 deacetylation and suppressed autophagy. Our findings suggest that FAT10 inhibits autophagy by antagonizing SIRT1 SUMOylation to protect the heart from ischemic myocardial injury. This is a novel mechanism through which FAT10 regulates autophagy as a cardiac protector.

Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:1-13
Wan R, Yuan P, Guo L, Shao J, ... Jiang X, Hong K
J Mol Cell Cardiol: 30 Mar 2021; 153:1-13 | PMID: 33307094
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Impact:
Abstract

Elevated EZH2 in ischemic heart disease epigenetically mediates suppression of Na1.5 expression.

Zhao L, You T, Lu Y, Lin S, Li F, Xu H
Suppression of the cardiac sodium channel NaV1.5 leads to fatal arrhythmias in ischemic heart disease (IHD). However, the transcriptional regulation of NaV1.5 in cardiac ischemia is still unclear. Our studies are aimed to investigate the expression of enhancer of zeste homolog 2 (EZH2) in IHD and regulation of cardiac NaV1.5 expression by EZH2. Human heart tissue was obtained from IHD and non-failing heart (NFH) patients; mouse heart tissue was obtained from the peri-infarct zone of hearts with myocardial infarction (MI) and hearts with a sham procedure. Protein and mRNA expression were measured by immunoblotting, immunostaining, and qRT-PCR. Protein-DNA binding and promoter activity were analyzed by ChIP-qPCR and luciferase assays, respectively. Na+ channel activity was assessed by whole-cell patch clamp recordings. EZH2 and H3K27me3 were increased while NaV1.5 expression was reduced in IHD hearts and in mouse MI hearts compared to the controls. Reduced NaV1.5 and increased EZH2 mRNA levels were observed in mouse MI hearts. A selective EZH2 inhibitor, GSK126 decreased H3K27me3 and elevated NaV1.5 in HL-1 cells. Silencing of EZH2 expression decreased H3K27me3 and increased NaV1.5 in these cells. EZH2 and H3K27me3 were enriched in the promoter regions of Scn5a and were decreased by treatment with EZH2 siRNA. GSK126 inhibited the enrichment of H3K27me3 in the Scn5a promoter and enhanced Scn5a transcriptional activity. GSK126 significantly increased Na+ channel activity. Taken together, EZH2 is increased in ischemic hearts and epigenetically suppresses Scn5a transcription by H3K27me3, leading to decreased NaV1.5 expression and Na+ channel activity underlying the pathogenesis of arrhythmias.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:95-103
Zhao L, You T, Lu Y, Lin S, Li F, Xu H
J Mol Cell Cardiol: 30 Mar 2021; 153:95-103 | PMID: 33370552
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Impact:
Abstract

Suppression of canonical TGF-β signaling enables GATA4 to interact with H3K27me3 demethylase JMJD3 to promote cardiomyogenesis.

Riching AS, Danis E, Zhao Y, Cao Y, ... Buttrick PM, Song K
Direct reprogramming of fibroblasts into cardiomyocytes (CMs) represents a promising strategy to regenerate CMs lost after ischemic heart injury. Overexpression of GATA4, HAND2, MEF2C, TBX5, miR-1, and miR-133 (GHMT2m) along with transforming growth factor beta (TGF-β) inhibition efficiently promote reprogramming. However, the mechanisms by which TGF-β blockade promotes cardiac reprogramming remain unknown. Here, we identify interactions between the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3, the SWI/SNF remodeling complex subunit BRG1, and cardiac transcription factors. Furthermore, canonical TGF-β signaling regulates the interaction between GATA4 and JMJD3. TGF-β activation impairs the ability of GATA4 to bind target genes and prevents demethylation of H3K27 at cardiac gene promoters during cardiac reprogramming. Finally, a mutation in GATA4 (V267M) that is associated with congenital heart disease exhibits reduced binding to JMJD3 and impairs cardiomyogenesis. Thus, we have identified an epigenetic mechanism wherein canonical TGF-β pathway activation impairs cardiac gene programming, in part by interfering with GATA4-JMJD3 interactions.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:44-59
Riching AS, Danis E, Zhao Y, Cao Y, ... Buttrick PM, Song K
J Mol Cell Cardiol: 30 Mar 2021; 153:44-59 | PMID: 33359755
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Impact:
Abstract

Considerations for using isolated cell systems to understand cardiac metabolism and biology.

McNally LA, Altamimi TR, Fulghum K, Hill BG
Changes in myocardial metabolic activity are fundamentally linked to cardiac health and remodeling. Primary cardiomyocytes, induced pluripotent stem cell-derived cardiomyocytes, and transformed cardiomyocyte cell lines are common models used to understand how (patho)physiological conditions or stimuli contribute to changes in cardiac metabolism. These cell models are helpful also for defining metabolic mechanisms of cardiac dysfunction and remodeling. Although technical advances have improved our capacity to measure cardiomyocyte metabolism, there is often heterogeneity in metabolic assay protocols and cell models, which could hinder data interpretation and discernment of the mechanisms of cardiac (patho)physiology. In this review, we discuss considerations for integrating cardiomyocyte cell models with techniques that have become relatively common in the field, such as respirometry and extracellular flux analysis. Furthermore, we provide overviews of metabolic assays that complement XF analyses and that provide information on not only catabolic pathway activity, but biosynthetic pathway activity and redox status as well. Cultivating a more widespread understanding of the advantages and limitations of metabolic measurements in cardiomyocyte cell models will continue to be essential for the development of coherent metabolic mechanisms of cardiac health and pathophysiology.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:26-41
McNally LA, Altamimi TR, Fulghum K, Hill BG
J Mol Cell Cardiol: 30 Mar 2021; 153:26-41 | PMID: 33359038
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Impact:
Abstract

Nano-scale morphology of cardiomyocyte t-tubule/sarcoplasmic reticulum junctions revealed by ultra-rapid high-pressure freezing and electron tomography.

Rog-Zielinska EA, Moss R, Kaltenbacher W, Greiner J, ... Kohl P, Cannell MB
Detailed knowledge of the ultrastructure of intracellular compartments is a prerequisite for our understanding of how cells function. In cardiac muscle cells, close apposition of transverse (t)-tubule (TT) and sarcoplasmic reticulum (SR) membranes supports stable high-gain excitation-contraction coupling. Here, the fine structure of this key intracellular element is examined in rabbit and mouse ventricular cardiomyocytes, using ultra-rapid high-pressure freezing (HPF, omitting aldehyde fixation) and electron microscopy. 3D electron tomograms were used to quantify the dimensions of TT, terminal cisternae of the SR, and the space between SR and TT membranes (dyadic cleft). In comparison to conventional aldehyde-based chemical sample fixation, HPF-preserved samples of both species show considerably more voluminous SR terminal cisternae, both in absolute dimensions and in terms of junctional SR to TT volume ratio. In rabbit cardiomyocytes, the average dyadic cleft surface area of HPF and chemically fixed myocytes did not differ, but cleft volume was significantly smaller in HPF samples than in conventionally fixed tissue; in murine cardiomyocytes, the dyadic cleft surface area was higher in HPF samples with no difference in cleft volume. In both species, the apposition of the TT and SR membranes in the dyad was more likely to be closer than 10 nm in HPF samples compared to CFD, presumably resulting from avoidance of sample shrinkage associated with conventional fixation techniques. Overall, we provide a note of caution regarding quantitative interpretation of chemically-fixed ultrastructures, and offer novel insight into cardiac TT and SR ultrastructure with relevance for our understanding of cardiac physiology.

Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:86-92
Rog-Zielinska EA, Moss R, Kaltenbacher W, Greiner J, ... Kohl P, Cannell MB
J Mol Cell Cardiol: 30 Mar 2021; 153:86-92 | PMID: 33359037
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Impact:
Abstract

Blockade of sodium‑calcium exchanger via ORM-10962 attenuates cardiac alternans.

Szlovák J, Tomek J, Zhou X, Tóth N, ... Rodriguez B, Nagy N
Repolarization alternans, a periodic oscillation of long-short action potential duration, is an important source of arrhythmogenic substrate, although the mechanisms driving it are insufficiently understood. Despite its relevance as an arrhythmia precursor, there are no successful therapies able to target it specifically. We hypothesized that blockade of the sodium‑calcium exchanger (NCX) could inhibit alternans. The effects of the selective NCX blocker ORM-10962 were evaluated on action potentials measured with microelectrodes from canine papillary muscle preparations, and calcium transients measured using Fluo4-AM from isolated ventricular myocytes paced to evoke alternans. Computer simulations were used to obtain insight into the drug\'s mechanisms of action. ORM-10962 attenuated cardiac alternans, both in action potential duration and calcium transient amplitude. Three morphological types of alternans were observed, with differential response to ORM-10962 with regards to APD alternans attenuation. Analysis of APD restitution indicates that calcium oscillations underlie alternans formation. Furthermore, ORM-10962 did not markedly alter APD restitution, but increased post-repolarization refractoriness, which may be mediated by indirectly reduced L-type calcium current. Computer simulations reproduced alternans attenuation via ORM-10962, suggesting that it is acts by reducing sarcoplasmic reticulum release refractoriness. This results from the ORM-10962-induced sodium‑calcium exchanger block accompanied by an indirect reduction in L-type calcium current. Using a computer model of a heart failure cell, we furthermore demonstrate that the anti-alternans effect holds also for this disease, in which the risk of alternans is elevated. Targeting NCX may therefore be a useful anti-arrhythmic strategy to specifically prevent calcium driven alternans.

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

J Mol Cell Cardiol: 30 Mar 2021; 153:111-122
Szlovák J, Tomek J, Zhou X, Tóth N, ... Rodriguez B, Nagy N
J Mol Cell Cardiol: 30 Mar 2021; 153:111-122 | PMID: 33383036
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Impact:
Abstract

Mechanisms underlying age-associated manifestation of cardiac sodium channel gain-of-function.

Nowak MB, Poelzing S, Weinberg SH
Cardiac action potentials are initiated by sodium ion (Na+) influx through voltage-gated Na+ channels. Na+ channel gain-of-function (GOF) can arise in inherited conditions due to mutations in the gene encoding the cardiac Na+ channel, such as Long QT syndrome type 3 (LQT3). LQT3 can be a \"concealed\" disease, as patients with LQT3-associated mutations can remain asymptomatic until later in life; however, arrhythmias can also arise early in life in LQT3 patients, demonstrating a complex age-associated manifestation. We and others recently demonstrated that cardiac Na+ channels preferentially localize at the intercalated disc (ID) in adult cardiac tissue, which facilitates ephaptic coupling and formation of intercellular Na+ nanodomains that regulate pro-arrhythmic early afterdepolarization (EAD) formation in tissue with Na+ channel GOF. Several properties related to ephaptic coupling vary with age, such as cell size and Na+ channel and gap junction (GJ) expression and distribution: neonatal cells have immature IDs, with Na+ channels and GJs primarily diffusively distributed, while adult myocytes have mature IDs with preferentially localized Na+ channels and GJs. Here, we perform an in silico study varying critical age-dependent parameters to investigate mechanisms underlying age-associated manifestation of Na+ channel GOF in a model of guinea pig cardiac tissue. Simulations predict that total Na+ current conductance is a critical factor in action potential duration (APD) prolongation. We find a complex cell size/ Na+ channel expression relationship: increases in cell size (without concurrent increases in Na+ channel expression) suppress EAD formation, while increases in Na+ channel expression (without concurrent increases in cell size) promotes EAD formation. Finally, simulations with neonatal and early age-associated parameters predict normal APD with minimal dependence on intercellular cleft width; however, variability in cellular properties can lead to EADs presenting in early developmental stages. In contrast, for adult-associated parameters, EAD formation is highly dependent on cleft width, consistent with a mechanism underlying the age-associated manifestation of the Na+ channel GOF.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:60-71
Nowak MB, Poelzing S, Weinberg SH
J Mol Cell Cardiol: 30 Mar 2021; 153:60-71 | PMID: 33373643
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Impact:
Abstract

Recombinant Soluble Corin Improves Cardiac Function in Mouse Models of Heart Failure.

Niu Y, Zhang S, Gu X, Zhou T, ... Wu Q, Dong N

Background:
Corin is a transmembrane protease that activates ANP and BNP (atrial and B-type natriuretic peptides). Impaired corin expression and function are associated with heart failure. In this study, we characterized a soluble form of corin (sCorin) and examined its effects on cardiac morphology and function in mouse heart failure models. Methods and Results sCorin, consisting of the full-length extracellular fragment of human corin with an engineered activation site, was expressed in Chinese hamster ovary cells, purified from the conditioned medium with affinity chromatography, and characterized in pro-ANP processing assays in vitro and pharmacokinetic studies in mice. Effects of sCorin on mouse models of heart failure induced by left coronary artery ligation and transverse aortic constriction were assessed by ELISA analysis of plasma markers, histologic examination, and echocardiography. We showed that purified and activated sCorin converted pro-ANP to ANP that stimulated cGMP production in cultured cells. In mice, intravenously and intraperitoneally administered sCorin had plasma half-lives of 3.5±0.1 and 8.3±0.3 hour, respectively. In the mouse heart failure models, intraperitoneal injection of sCorin increased plasma ANP, BNP, and cGMP levels; lowered plasma levels of NT-proANP (N-terminal-pro-ANP), angiotensin II, and aldosterone; reduced cardiac hypertrophy and fibrosis; and improved cardiac function.
Conclusions:
We show that sCorin treatment enhanced natriuretic peptide processing and activity, suppressed the renin-angiotensin-aldosterone system, and improved cardiac morphology and function in mice with failing hearts.




J Am Heart Assoc: 05 Apr 2021; 10:e019961
Niu Y, Zhang S, Gu X, Zhou T, ... Wu Q, Dong N
J Am Heart Assoc: 05 Apr 2021; 10:e019961 | PMID: 33759549
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Impact:
Abstract

Creld1 regulates myocardial development and function.

Beckert V, Rassmann S, Kayvanjoo AH, Klausen C, ... Mass E, Wachten D
CRELD1 (Cysteine-Rich with EGF-Like Domains 1) is a risk gene for non-syndromic atrioventricular septal defects in human patients. In a mouse model, Creld1 has been shown to be essential for heart development, particularly in septum and valve formation. However, due to the embryonic lethality of global Creld1 knockout (KO) mice, its cell type-specific function during peri- and postnatal stages remains unknown. Here, we generated conditional Creld1 KO mice lacking Creld1 either in the endocardium (KOTie2) or the myocardium (KOMyHC). Using a combination of cardiac phenotyping, histology, immunohistochemistry, RNA-sequencing, and flow cytometry, we demonstrate that Creld1 function in the endocardium is dispensable for heart development. Lack of myocardial Creld1 causes extracellular matrix remodeling and trabeculation defects by modulation of the Notch1 signaling pathway. Hence, KOMyHC mice die early postnatally due to myocardial hypoplasia. Our results reveal that Creld1 not only controls the formation of septa and valves at an early stage during heart development, but also cardiac maturation and function at a later stage. These findings underline the central role of Creld1 in mammalian heart development and function.

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

J Mol Cell Cardiol: 23 Mar 2021; 156:45-56
Beckert V, Rassmann S, Kayvanjoo AH, Klausen C, ... Mass E, Wachten D
J Mol Cell Cardiol: 23 Mar 2021; 156:45-56 | PMID: 33773996
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Impact:
Older ...

This program is still in alpha version.