Topic: Basic Research

Abstract

VEGF-B Promotes Endocardium-Derived Coronary Vessel Development and Cardiac Regeneration.

Räsänen M, Sultan I, Paech J, Hemanthakumar KA, ... Kivelä R, Alitalo K
Background
Recent discoveries have indicated that, in the developing heart, sinus venosus and endocardium provide major sources of endothelium for coronary vessel growth that supports the expanding myocardium. Here we set out to study the origin of the coronary vessels that develop in response to vascular endothelial growth factor B (VEGF-B) in the heart and the effect of VEGF-B on recovery from myocardial infarction.
Methods
We used mice and rats expressing a VEGF-B transgene, VEGF-B-gene-deleted mice and rats, apelin-CreERT, and natriuretic peptide receptor 3-CreERT recombinase-mediated genetic cell lineage tracing and viral vector-mediated VEGF-B gene transfer in adult mice. Left anterior descending coronary vessel ligation was performed, and 5-ethynyl-2\'-deoxyuridine-mediated proliferating cell cycle labeling; flow cytometry; histological, immunohistochemical, and biochemical methods; single-cell RNA sequencing and subsequent bioinformatic analysis; microcomputed tomography; and fluorescent- and tracer-mediated vascular perfusion imaging analyses were used to study the development and function of the VEGF-B-induced vessels in the heart.
Results
We show that cardiomyocyte overexpression of VEGF-B in mice and rats during development promotes the growth of novel vessels that originate directly from the cardiac ventricles and maintain connection with the coronary vessels in subendocardial myocardium. In adult mice, endothelial proliferation induced by VEGF-B gene transfer was located predominantly in the subendocardial coronary vessels. Furthermore, VEGF-B gene transduction before or concomitantly with ligation of the left anterior descending coronary artery promoted endocardium-derived vessel development into the myocardium and improved cardiac tissue remodeling and cardiac function.
Conclusions
The myocardial VEGF-B transgene promotes the formation of endocardium-derived coronary vessels during development, endothelial proliferation in subendocardial myocardium in adult mice, and structural and functional rescue of cardiac tissue after myocardial infarction. VEGF-B could provide a new therapeutic strategy for cardiac neovascularization after coronary occlusion to rescue the most vulnerable myocardial tissue.



Circulation: 04 Jan 2021; 143:65-77
Räsänen M, Sultan I, Paech J, Hemanthakumar KA, ... Kivelä R, Alitalo K
Circulation: 04 Jan 2021; 143:65-77 | PMID: 33203221
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Abstract

Redox-Resistant SERCA [Sarco(endo)plasmic Reticulum Calcium ATPase] Attenuates Oxidant-Stimulated Mitochondrial Calcium and Apoptosis in Cardiac Myocytes and Pressure Overload-Induced Myocardial Failure in Mice.

Goodman JB, Qin F, Morgan RJ, Chambers JM, ... Cohen RA, Colucci WS
Background
SERCA [sarco(endo)plasmic reticulum calcium ATPase] is regulated by oxidative posttranslational modifications at cysteine 674 (C674). Because sarcoplasmic reticulum (SR) calcium has been shown to play a critical role in mediating mitochondrial dysfunction in response to reactive oxygen species, we hypothesized that SERCA oxidation at C674 would modulate the effects of reactive oxygen species on mitochondrial calcium and mitochondria-dependent apoptosis in cardiac myocytes.
Methods
Adult rat ventricular myocytes expressing wild-type SERCA2b or a redox-insensitive mutant in which C674 is replaced by serine (C674S) were exposed to HO (100 µmol/Lμ). Free mitochondrial calcium concentration was measured in adult rat ventricular myocytes with a genetically targeted fluorescent probe, and SR calcium content was assessed by measuring caffeine-stimulated release. Mice with heterozygous knock-in of the SERCA C674S mutation were subjected to chronic ascending aortic constriction.
Results
In adult rat ventricular myocytes expressing wild-type SERCA, HO caused a 25% increase in mitochondrial calcium concentration that was associated with a 50% decrease in SR calcium content, both of which were prevented by the ryanodine receptor inhibitor tetracaine. In cells expressing the C674S mutant, basal SR calcium content was decreased by 31% and the HO-stimulated rise in mitochondrial calcium concentration was attenuated by 40%. In wild-type cells, HO caused cytochrome c release and apoptosis, both of which were prevented in C674S-expressing cells. In myocytes from SERCA knock-in mice, basal SERCA activity and SR calcium content were decreased. To test the effect of C674 oxidation on apoptosis in vivo, SERCA knock-in mice were subjected to chronic ascending aortic constriction. In wild-type mice, ascending aortic constriction caused myocyte apoptosis, LV dilation, and systolic failure, all of which were inhibited in SERCA knock-in mice.
Conclusions
Redox activation of SERCA C674 regulates basal SR calcium content, thereby mediating the pathologic reactive oxygen species-stimulated rise in mitochondrial calcium required for myocyte apoptosis and myocardial failure.



Circulation: 21 Dec 2020; 142:2459-2469
Goodman JB, Qin F, Morgan RJ, Chambers JM, ... Cohen RA, Colucci WS
Circulation: 21 Dec 2020; 142:2459-2469 | PMID: 33076678
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Abstract

Circular RNA CircMAP3K5 Acts as a MicroRNA-22-3p Sponge to Promote Resolution of Intimal Hyperplasia via TET2-Mediated SMC Differentiation.

Zeng Z, Xia L, Fan S, Zheng J, ... Liu R, Tang WH

Aberrant expression of circular RNA (CircRNA) contributes to human diseases. CircRNAs regulate gene expression by sequestering specific microRNAs (miRNAs). In this study, we investigated whether CircMAP3K5 could act as a competing endogenous miR-22-3p sponge and regulate neointimal hyperplasia.CircRNA profiling from genome-wide RNA sequencing data was compared between human coronary artery smooth muscle cells (HCASMCs) treated with or without PDGF. Expression levels of circular MAP3K5 (CircMAP3K5) was assessed in human coronary arteries from autopsies on patients with dilated cardiomyopathy (DCM) or coronary heart disease (CHD). The role of CircMAP3K5 in intimal hyperplasia was further investigated in mice with AAV9-mediated CircMAP3K5 transfection. SMC-specific Tet2 knockout mice and global miR-22-3p knockout mice were used to delineate the mechanism by which CircMAP3K5 attenuated neointimal hyperplasia using the femoral arterial wire injury model.RNA sequencing demonstrated that treatment with PDGF-BB significantly reduced expression of CircMAP3K5 in HCASMCs. Wire-injured mouse femoral arteries and diseased arteries from CHD patients (where PDGF-BB is increased) confirmed in vivo downregulation of CircMAP3K5 associated with injury and disease. Lentivirus-mediated overexpression of CircMAP3K5 inhibited the proliferation of HCASMCs. In vivo AAV9-mediated transfection of CircMap3k5 specifically inhibited SMC proliferation in the wire-injured mouse arteries, resulting in reduced neointima formation. Using a luciferase reporter assay and RNA pull-down, CircMAP3K5 was found to sequester miR-22-3p, which in turn inhibited the expression of TET2. Both in vitro and in vivo results demonstrate that the loss of miR-22-3p recapitulated the anti-proliferative effect of CircMap3k5 on VSMCs. In SMC-specific Tet2 knockout mice, loss of Tet2 abolished the CircMap3k5-mediated anti-proliferative effect on VSMCs.We identify CircMAP3K5 as a master regulator of TET2-mediated VSMC differentiation. Targeting the CircMAP3K5/miR-22-3p/TET2 axis may provide a potential therapeutic strategy for diseases associated with intimal hyperplasia including restenosis and atherosclerosis.



Circulation: 18 Dec 2020; epub ahead of print
Zeng Z, Xia L, Fan S, Zheng J, ... Liu R, Tang WH
Circulation: 18 Dec 2020; epub ahead of print | PMID: 33207953
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Abstract

Phospholemman Phosphorylation Regulates Vascular Tone, Blood Pressure and Hypertension in Mice and Man.

Boguslavskyi A, Tokar S, Prysyazhna O, Rudyk O, ... Fuller W, Shattock MJ

Background: While it has long been recognized that smooth muscle Na/K ATPase (NKA) modulates vascular tone and blood pressure (BP), the role of its accessory protein phopholemman (PLM) has not been characterized. The aim of this study was to test the hypothesis that PLM phosphorylation regulates vascular toneand this mechanism plays an important role in modulation of vascular function and BP in experimental modelsand in man. : PLM knock-in mice (PLM), in which PLM is rendered unphosphorylatable, were used to assess the role of PLM phosphorylation in vitro in aortic and mesenteric vessels using wire myography and membrane potential measurements.BP and regional blood flow were assessed using Doppler flow and telemetry in young (14-16 weeks) and old (57-60 weeks) wild-type (WT) and transgenic mice. : We searched human genomic databases for mutations in PLM in the region of the phosphorylation sites and performed analyses within two human data cohorts (UK Biobank and GoDARTS) to assess the impact of an identified SNP on BP. This SNP was expressed in HEK cells and its effect on PLM phosphorylation determined using Western Blotting.PLM phosphorylation at Ser63 and Ser68 limited vascular constriction in response to phenylephrine. This effect was blocked by ouabain. Prevention of PLM phosphorylation in the PLM mouse profoundly enhanced vascular responses to PE bothand . In ageing WT mice PLM was hypophosphorylated and this correlated with the development of ageing-induced essential hypertension. In man we identified a non-synonymous coding variant, single nucleotide polymorphism rs61753924, which causes the substitution R70C in PLM. In HEK cells the R70C mutation prevented PLM phosphorylation at Ser68. This variant\'s rare allele is significantly associated with increased BP in middle-aged men.These studies demonstrate the importance of PLM phosphorylation in the regulation of vascular tone and BP and suggest a novel mechanism, and therapeutic target, for ageing-induced essential hypertension in man.



Circulation: 17 Dec 2020; epub ahead of print
Boguslavskyi A, Tokar S, Prysyazhna O, Rudyk O, ... Fuller W, Shattock MJ
Circulation: 17 Dec 2020; epub ahead of print | PMID: 33334125
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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

Vascular smooth muscle cells (VSMCs) show a remarkable phenotypic plasticity allowing acquisition of contractile or synthetic states but critical information is missing about the physiological signals, promoting formation and maintenance of contractile VSMCs . BMP9 and BMP10 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. Here, we studied the role of BMP9 and BMP10 for controlling formation of contractile VSMCs.We generated several cell type-specific loss- and gain-of-function transgenic mouse models to investigate the physiological role of BMP9, BMP10, ALK1 and SMAD7 . Morphometric assessments, expression analysis, blood pressure measurements, single molecule fluorescencehybridization (FISH) 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.Concomitant genetic inactivation ofin the germ line andin 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 (RVSP). , overexpression ofin endothelial cells (ECs) of adult mice dramatically enhanced formation of contractile VSMCs and increased systemic blood pressure as well as RVSP. Likewise, BMP9/10 treatment induced an ALK1-dependent phenotypic switch from synthetic to contractile in pulmonary VSMCs. SMC specific overexpression ofcompletely suppressed differentiation and proliferation of VSMCs and reiterated defects observed in adultdouble mutants. Deletion ofin VSMCs recapitulated thephenotype in pulmonary but not in aortic and coronary arteries. Bulk expression analysis and single molecule RNA-FISH uncovered vessel bed-specific, heterogeneous expression of BMP type 1 receptors, explaining phenotypic differences in differentmutant vessel beds.Our study demonstrates that BMP9 and BMP10 act directly on VSMCs for induction and maintenance of their contractile state. Surprisingly, 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: 17 Dec 2020; epub ahead of print
Wang L, Rice M, Swist S, Kubin T, ... Schneider A, Braun T
Circulation: 17 Dec 2020; epub ahead of print | PMID: 33334130
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Abstract

PCSK9 (Proprotein Convertase Subtilisin/Kexin 9) Enhances Platelet Activation, Thrombosis, and Myocardial Infarct Expansion by Binding to Platelet CD36.

Qi Z, Hu L, Zhang J, Yang W, ... Ding Z, Ge J
Background
PCSK9 (proprotein convertase subtilisin/kexin 9), mainly secreted by the liver and released into the blood, elevates plasma low-density lipoprotein cholesterol by degrading low-density lipoprotein receptor. Pleiotropic effects of PCSK9 beyond lipid metabolism have been shown. However, the direct effects of PCSK9 on platelet activation and thrombosis, and the underlying mechanisms, as well, still remain unclear.
Methods
We detected the direct effects of PCSK9 on agonist-induced platelet aggregation, dense granule ATP release, integrin αIIbβ3 activation, α-granule release, spreading, and clot retraction. These studies were complemented by in vivo analysis of FeCl-injured mouse mesenteric arteriole thrombosis. We also investigated the underlying mechanisms. Using the myocardial infarction (MI) model, we explored the effects of PCSK9 on microvascular obstruction and infarct expansion post-MI.
Results
PCSK9 directly enhances agonist-induced platelet aggregation, dense granule ATP release, integrin αIIbβ3 activation, P-selectin release from α-granules, spreading, and clot retraction. In line, PCSK9 enhances in vivo thrombosis in a FeCl-injured mesenteric arteriole thrombosis mouse model, whereas PCSK9 inhibitor evolocumab ameliorates its enhancing effects. Mechanism studies revealed that PCSK9 binds to platelet CD36 and thus activates Src kinase and MAPK (mitogen-activated protein kinase)-extracellular signal-regulated kinase 5 and c-Jun N-terminal kinase, increases the generation of reactive oxygen species, and activates the p38MAPK/cytosolic phospholipase A2/cyclooxygenase-1/thromboxane A signaling pathways downstream of CD36 to enhance platelet activation, as well. Using CD36 knockout mice, we showed that the enhancing effects of PCSK9 on platelet activation are CD36 dependent. It is important to note that aspirin consistently abolishes the enhancing effects of PCSK9 on platelet activation and in vivo thrombosis. Last, we showed that PCSK9 activating platelet CD36 aggravates microvascular obstruction and promotes MI expansion post-MI.
Conclusions
PCSK9 in plasma directly enhances platelet activation and in vivo thrombosis, and MI expansion post-MI, as well, by binding to platelet CD36 and thus activating the downstream signaling pathways. PCSK9 inhibitors or aspirin abolish the enhancing effects of PCSK9, supporting the use of aspirin in patients with high plasma PCSK9 levels in addition to PCSK9 inhibitors to prevent thrombotic complications.



Circulation: 04 Jan 2021; 143:45-61
Qi Z, Hu L, Zhang J, Yang W, ... Ding Z, Ge J
Circulation: 04 Jan 2021; 143:45-61 | PMID: 32988222
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Abstract

Cardiomyocyte Krüppel-like Factor 5 Promotes De Novo Ceramide Biosynthesis and Contributes to Eccentric Remodeling in Ischemic Cardiomyopathy.

Hoffman M, Palioura D, Kyriazis ID, Cimini M, ... Drakos SG, Drosatos K

We previously showed that cardiomyocyte Krüppel-like factor (KLF)-5 regulates cardiac fatty acid oxidation. As heart failure has been associated with altered fatty acid oxidation, we investigated the role of cardiomyocyte KLF5 in lipid metabolism and pathophysiology of ischemic heart failure.Using rtPCR and Western Blot, we investigated the KLF5 expression changes in a myocardial infarction (MI) mouse model and heart tissue from patients with ischemic heart failure. Using 2D-echocardiography, we evaluated the effect of KLF5 inhibition after MI using pharmacological KLF5 inhibitor ML264 and mice with cardiomyocyte specific KLF5 deletion (αMHC-KLF5). We identified the involvement of KLF5 in regulating lipid metabolism and ceramide accumulation after MI using liquid-chromatography-tandem-mass-spectrometry, and Western Blot and rtPCR analysis of ceramide-metabolism-related genes. We lastly evaluated the effect of cardiomyocyte-specific KLF5 overexpression (αMHC-rtTA-KLF5) on cardiac function and ceramide metabolism, and rescued the phenotype using myriocin to inhibit ceramide biosynthesis.KLF5 mRNA and protein levels were higher in human ischemic heart failure samples and in rodent models 24h, 2- and 4-weeks post-permanent left coronary artery ligation. αMHC-KLF5 mice and mice treated with ML264 had higher ejection fraction and lower ventricular volume and heart weight after MI. Lipidomic analysis showed that αMHC-KLF5 mice with MI had lower myocardial ceramide levels compared with littermate control mice with MI although basal ceramide content of αMHC-KLF5 mice was not different from control mice. KLF5 ablation suppressed the expression of serine-palmitoyl-transferase-long-chain-base-subunit (SPTLC)1 and SPTLC2, which regulate de novo ceramide biosynthesis. We confirmed our previous findings that myocardial SPTLC1 and SPTLC2 levels are increased in heart failure patients. Consistently, αMHC-rtTA-KLF5 mice showed increased SPTLC1 and SPTLC2 expression, higher myocardial ceramide levels, and systolic dysfunction beginning 2-weeks after KLF5 induction. Treatment of αMHC-rtTA-KLF5 mice with myriocin that inhibits SPT, suppressed myocardial ceramide levels and alleviated systolic dysfunction.KLF5 is induced during the development of ischemic heart failure in humans and mice and stimulates ceramide biosynthesis. Genetic or pharmacological inhibition of KLF5 in mice with MI prevents ceramide accumulation, alleviates eccentric remodeling, and increases ejection fraction. Thus, KLF5 emerges as a novel therapeutic target for the treatment of ischemic heart failure.



Circulation: 11 Jan 2021; epub ahead of print
Hoffman M, Palioura D, Kyriazis ID, Cimini M, ... Drakos SG, Drosatos K
Circulation: 11 Jan 2021; epub ahead of print | PMID: 33430631
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Abstract

Association Between Muscle Quality Measured by Abdominal Computed Tomography and Subclinical Coronary Atherosclerosis.

Jung Lee M, Kim HK, Hee Kim E, Jin Bae S, ... Kim MJ, Choe J
Objective
Low muscle mass was known to be associated with cardiovascular diseases. However, only few studies investigated the association between muscle quality and subclinical coronary atherosclerosis. Thus, we evaluated whether muscle quality measured by abdominal computed tomography is associated with the risk of coronary artery calcification. Approach and
Results:
We conducted a cross-sectional study on 4068 subjects without cardiovascular disease who underwent abdominal and coronary computed tomography between 2012 and 2013 during health examinations. The cross-sectional area of the skeletal muscle was measured at the L3 level (total abdominal muscle area, total abdominal muscle area) and segmented into normal attenuation muscle area, low attenuation muscle area, and intramuscular adipose tissue. We calculated the normal attenuation muscle area/total abdominal muscle area index, of which a higher value reflected a higher proportion of good quality muscle (normal attenuation muscle area) and a lower proportion of myosteatosis (low attenuation muscle area and intramuscular adipose tissue). In women, as the normal attenuation muscle area/total abdominal muscle area quartiles increased, the odds ratios (95% CIs) for significant coronary artery calcification (>100) consistently decreased (0.44 [0.24-0.80], 0.39 [0.19-0.81], 0.34 [0.12-0.98]; =0.003) after adjusting for cardiovascular risk factors including visceral fat area and insulin resistance. In men, the odds ratios in the Q2 group were significantly lower than those in the Q1, but the association was attenuated in Q3-4 after adjustment.
Conclusions
A higher proportion of good quality muscle was strongly associated with a lower prevalence of significant coronary artery calcification after adjustment, especially in women. Poor skeletal muscle quality may be an important risk factor for subclinical coronary atherosclerosis.



Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315054; epub ahead of print
Jung Lee M, Kim HK, Hee Kim E, Jin Bae S, ... Kim MJ, Choe J
Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315054; epub ahead of print | PMID: 33356388
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Abstract

Sexual Dimorphic Role of CD14 (Cluster of Differentiation 14) in Salt-Sensitive Hypertension and Renal Injury.

Fehrenbach DJ, Abais-Battad JM, Dasinger JH, Lund H, ... Dwinell MR, Mattson DL

Genomic sequence and gene expression association studies in animals and humans have identified genes that may be integral in the pathogenesis of various diseases. CD14 (cluster of differentiation 14)-a cell surface protein involved in innate immune system activation-is one such gene associated with cardiovascular and hypertensive disease. We previously showed that this gene is upregulated in renal macrophages of Dahl salt-sensitive animals fed a high-salt diet; here we test the hypothesis that CD14 contributes to the elevated pressure and renal injury observed in salt-sensitive hypertension. Using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), we created a targeted mutation in thegene on the Dahl SS (SS/JrHSDMcwi) background and validated the absence of CD14 peptides via mass spectrometry. Radiotelemetry was used to monitor blood pressure in wild-type and CD14 animals challenged with high salt and identified infiltrating renal immune cells via flow cytometry. Germline knockout of CD14 exacerbated salt-sensitive hypertension and renal injury in female animals but not males. CD14 females demonstrated increased infiltrating macrophages but no difference in infiltrating lymphocytes. Transplant of CD14 or CD14 bone marrow was used to isolate the effects of CD14 knockout to hematopoietic cells and confirmed that the differential phenotype observed was due to knockout of CD14 in hematopoietic cells. Ovariectomy was used to remove the influence of female sex hormones, which completely abrogated the effect of CD14 knockout. These studies provide a novel treatment target and evidence of a new dichotomy in immune activation between sexes within the context of hypertensive disease where CD14 regulates immune cell activation and renal injury.



Hypertension: 30 Dec 2020; 77:228-240
Fehrenbach DJ, Abais-Battad JM, Dasinger JH, Lund H, ... Dwinell MR, Mattson DL
Hypertension: 30 Dec 2020; 77:228-240 | PMID: 33249861
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Abstract

Mendelian Randomization Analysis of Hemostatic Factors and Their Contribution to Peripheral Artery Disease-Brief Report.

Small AM, Huffman JE, Klarin D, Sabater-Lleal M, ... Damrauer SM,
Background:
and objective
Peripheral artery disease (PAD) is the third most common form of atherosclerotic vascular disease and is characterized by significant functional disability and increased cardiovascular mortality. Recent genetic data support a role for a procoagulation protein variant, the factor V Leiden mutation, in PAD. The role of other hemostatic factors in PAD remains unknown. We evaluated the role of hemostatic factors in PAD using Mendelian randomization. Approach and
Results:
Two-sample Mendelian randomization to evaluate the roles of FVII (factor VII), FVIII (factor VIII), FXI (factor XI), VWF (von Willebrand factor), and fibrinogen in PAD was performed using summary statistics from GWAS for hemostatic factors performed within the Cohorts for Heart and Aging Research in the Genome Epidemiology Consortium and from GWAS performed for PAD within the Million Veteran Program. Genetically determined FVIII and VWF, but not FVII, FXI, or fibrinogen, were associated with PAD in Mendelian randomization experiments (FVIII: odds ratio, 1.41 [95% CI, 1.23-1.62], =6.0×10, VWF: odds ratio, 1.28 [95% CI, 1.07-1.52], =0.0073). In single variant sensitivity analysis, thelocus was the strongest genetic instrument for both FVIII and VWF.
Conclusions
Our results suggest a role for hemostasis, and by extension, thrombosis in PAD. Further study is warranted to determine whether VWF and FVIII independently affect the biology of PAD.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:380-386
Small AM, Huffman JE, Klarin D, Sabater-Lleal M, ... Damrauer SM,
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:380-386 | PMID: 32847391
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Abstract

P2X7 Receptors: An Untapped Target for the Management of Cardiovascular Disease.

Shokoples BG, Paradis P, Schiffrin EL

Chronic low-grade inflammation contributes to the development of several diseases, including cardiovascular disease. Adequate strategies to target inflammation in cardiovascular disease are in their infancy and remain an avenue of great interest. The purinergic receptor P2X7 is a ubiquitously expressed receptor that predominately mediates inflammation and cellular death. P2X7 is a ligand-gated cation channel that is activated in response to high concentrations of extracellular ATP, triggering the assembly and activation of the NLRP3 (nuclear oligomerization domain like receptor family pyrin domain containing 3) inflammasome and subsequent release of proinflammatory cytokines IL (interleukin)-1β and IL-18. Increased P2X7 activation and IL-1β and IL-18 concentrations have been implicated in the development of many cardiovascular conditions including hypertension, atherosclerosis, ischemia/reperfusion injury, and heart failure. P2X7 receptor KO (knockout) mice exhibit a significant attenuation of the inflammatory response, which corresponds with reduced disease severity. P2X7 antagonism blunts blood pressure elevation in hypertension and progression of atherosclerosis in animal models. IL-1β and IL-18 inhibition has shown efficacy in clinical trials reducing major adverse cardiac events, including myocardial infarction, and heart failure. With several P2X7 antagonists available with proven safety margins, P2X7 antagonism could represent an untapped potential for therapeutic intervention in cardiovascular disorders.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:186-199
Shokoples BG, Paradis P, Schiffrin EL
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:186-199 | PMID: 32998520
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Abstract

Oxidative Stress Product, 4-Hydroxy-2-Nonenal, Induces the Release of Tissue Factor-Positive Microvesicles From Perivascular Cells Into Circulation.

Ansari SA, Keshava S, Pendurthi UR, Rao LVM
Objective
TF (Tissue factor) plays a key role in hemostasis, but an aberrant expression of TF leads to thrombosis. The objective of the present study is to investigate the effect of 4-hydroxy-2-nonenal (HNE), the most stable and major oxidant produced in various disease conditions, on the release of TF microvesicles into the circulation, identify the source of TF microvesicles origin, and assess their effect on intravascular coagulation and inflammation. Approach and
Results:
C57BL/6J mice were administered with HNE intraperitoneally, and the release of TF microvesicles into circulation was evaluated using coagulation assays and nanoparticle tracking analysis. Various cell-specific markers were used to identify the cellular source of TF microvesicles. Vascular permeability was analyzed by the extravasation of Evans blue dye or fluorescein dextran. HNE administration to mice markedly increased the levels of TF microvesicles and thrombin generation in the circulation. HNE administration also increased the number of neutrophils in the lungs and elevated the levels of inflammatory cytokines in plasma. Administration of an anti-TF antibody blocked not only HNE-induced thrombin generation but also HNE-induced inflammation. Confocal microscopy and immunoblotting studies showed that HNE does not induce TF expression either in vascular endothelium or circulating monocytes. Microvesicles harvested from HNE-administered mice stained positively with CD248 and α-smooth muscle actin, the markers that are specific to perivascular cells. HNE was found to destabilize endothelial cell barrier integrity.
Conclusions
HNE promotes the release of TF microvesicles from perivascular cells into the circulation. HNE-induced increased TF activity contributes to intravascular coagulation and inflammation.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:250-265
Ansari SA, Keshava S, Pendurthi UR, Rao LVM
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:250-265 | PMID: 33028097
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Abstract

Cholesterol-Induced Phenotypic Modulation of Smooth Muscle Cells to Macrophage/Fibroblast-like Cells Is Driven by an Unfolded Protein Response.

Chattopadhyay A, Kwartler CS, Kaw K, Li Y, ... Shen YH, Milewicz DM
Objective
Vascular smooth muscle cells (SMCs) dedifferentiate and initiate expression of macrophage markers with cholesterol exposure. This phenotypic switching is dependent on the transcription factor Klf4 (Krüppel-like factor 4). We investigated the molecular pathway by which cholesterol induces SMC phenotypic switching. Approach and
Results:
With exposure to free cholesterol, SMCs decrease expression of contractile markers, activate Klf4, and upregulate a subset of macrophage and fibroblast markers characteristic of modulated SMCs that appear with atherosclerotic plaque formation. These phenotypic changes are associated with activation of all 3 pathways of the endoplasmic reticulum unfolded protein response (UPR), Perk (protein kinase RNA-like endoplasmic reticulum kinase), Ire (inositol-requiring enzyme) 1α, and Atf (activating transcription factor) 6. Blocking the movement of cholesterol from the plasma membrane to the endoplasmic reticulum prevents free cholesterol-induced UPR, Klf4 activation, and upregulation of the majority of macrophage and fibroblast markers. Cholesterol-induced phenotypic switching is also prevented by global UPR inhibition or specific inhibition of Perk signaling. Exposure to chemical UPR inducers, tunicamycin and thapsigargin, is sufficient to induce these same phenotypic transitions. Finally, analysis of published single-cell RNA sequencing data during atherosclerotic plaque formation in hyperlipidemic mice provides preliminary in vivo evidence of a role of UPR activation in modulated SMCs.
Conclusions
Our data demonstrate that UPR is necessary and sufficient to drive phenotypic switching of SMCs to cells that resemble modulated SMCs found in atherosclerotic plaques. Preventing a UPR in hyperlipidemic mice diminishes atherosclerotic burden, and our data suggest that preventing SMC transition to dedifferentiated cells expressing macrophage and fibroblast markers contributes to this decreased plaque burden.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:302-316
Chattopadhyay A, Kwartler CS, Kaw K, Li Y, ... Shen YH, Milewicz DM
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:302-316 | PMID: 33028096
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Abstract

Stem/Progenitor Cells and Pulmonary Arterial Hypertension.

Pu X, Du L, Hu Y, Fan Y, Xu Q

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by endothelial dysfunction and vascular remodeling. Despite significant advancement in our understanding of the pathogenesis of PAH in recent years, treatment options for PAH are limited and their prognosis remains poor. PAH is now seen as a severe pulmonary arterial vasculopathy with structural changes driven by excessive vascular proliferation and inflammation. Perturbations of a number of cellular and molecular mechanisms have been described, including pathways involving growth factors, cytokines, metabolic signaling, elastases, and proteases, underscoring the complexity of the disease pathogenesis. Interestingly, emerging evidence suggests that stem/progenitor cells may have an impact on disease development and therapy. In preclinical studies, stem/progenitor cells displayed an ability to promote endothelial repair of dysfunctional arteries and induce neovascularization. The stem cell-based therapy for PAH are now under active investigation. This review article will briefly summarize the updates in the research field, with a special focus on the contribution of stem/progenitor cells to lesion formation via influencing vascular cell functions and highlight the potential clinical application of stem/progenitor cell therapy to PAH.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:167-178
Pu X, Du L, Hu Y, Fan Y, Xu Q
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:167-178 | PMID: 33028095
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Abstract

Platelet Extracellular Vesicles: Beyond the Blood.

Puhm F, Boilard E, Machlus KR

Extracellular vesicles (EVs) are a means of cell-to-cell communication and can facilitate the exchange of a broad array of molecules between adjacent or distant cells. Platelets are anucleate cells derived from megakaryocytes and are primarily known for their role in maintaining hemostasis and vascular integrity. Upon activation by a variety of agonists, platelets readily generate EVs, which were initially identified as procoagulant particles. However, as both platelets and their EVs are abundant in blood, the role of platelet EVs in hemostasis may be redundant. Moreover, findings have challenged the significance of platelet-derived EVs in coagulation. Looking beyond hemostasis, platelet EV cargo is incredibly diverse and can include lipids, proteins, nucleic acids, and organelles involved in numerous other biological processes. Furthermore, while platelets cannot cross tissue barriers, their EVs can enter lymph, bone marrow, and synovial fluid. This allows for the transfer of platelet-derived content to cellular recipients and organs inaccessible to platelets. This review highlights the importance of platelet-derived EVs in physiological and pathological conditions beyond hemostasis.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:87-96
Puhm F, Boilard E, Machlus KR
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:87-96 | PMID: 33028092
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Impact:
Abstract

Endothelial HMGB1 Is a Critical Regulator of LDL Transcytosis via an SREBP2-SR-BI Axis.

Ghaffari S, Jang E, Naderinabi F, Sanwal R, ... Ikeda J, Lee WL
Objective
LDL (low-density lipoprotein) transcytosis across the endothelium is performed by the SR-BI (scavenger receptor class B type 1) receptor and contributes to atherosclerosis. HMGB1 (high mobility group box 1) is a structural protein in the nucleus that is released by cells during inflammation; extracellular HMGB1 has been implicated in advanced disease. Whether intracellular HMGB1 regulates LDL transcytosis through its nuclear functions is unknown. Approach and
Results:
HMGB1 was depleted by siRNA in human coronary artery endothelial cells, and transcytosis of LDL was measured by total internal reflection fluorescence microscopy. Knockdown of HMGB1 attenuated LDL transcytosis without affecting albumin transcytosis. Loss of HMGB1 resulted in reduction in SR-BI levels and depletion of SREBP2 (sterol regulatory element-binding protein 2)-a transcription factor upstream of SR-BI. The effect of HMGB1 depletion on LDL transcytosis required SR-BI and SREBP2. Overexpression of HMGB1 caused an increase in LDL transcytosis that was unaffected by inhibition of extracellular HMGB1 or depletion of RAGE (receptor for advanced glycation endproducts)-a cell surface receptor for HMGB1. The effect of HMGB1 overexpression on LDL transcytosis was prevented by knockdown of SREBP2. Loss of HMGB1 caused a reduction in the half-life of SREBP2; incubation with LDL caused a significant increase in nuclear localization of HMGB1 that was dependent on SR-BI. Animals lacking endothelial HMGB1 exhibited less acute accumulation of LDL in the aorta 30 minutes after injection and when fed a high-fat diet developed fewer fatty streaks and less atherosclerosis.
Conclusions
Endothelial HMGB1 regulates LDL transcytosis by prolonging the half-life of SREBP2, enhancing SR-BI expression. Translocation of HMGB1 to the nucleus in response to LDL requires SR-BI.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:200-216
Ghaffari S, Jang E, Naderinabi F, Sanwal R, ... Ikeda J, Lee WL
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:200-216 | PMID: 33054399
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Impact:
Abstract

KLF4 (Kruppel-Like Factor 4)-Dependent Perivascular Plasticity Contributes to Adipose Tissue inflammation.

Bulut GB, Alencar GF, Owsiany KM, Nguyen AT, ... Keller SR, Owens GK
Objective
Smooth muscle cells and pericytes display remarkable plasticity during injury and disease progression. Here, we tested the hypothesis that perivascular cells give rise to -dependent macrophage-like cells that augment adipose tissue (AT) inflammation and metabolic dysfunction associated with diet-induced obesity (DIO). Approach and
Results:
UsingeYFP (enhanced yellow fluorescent protein) mice and flow cytometry of the stromovascular fraction of epididymal AT, we observed a large fraction of smooth muscle cells and pericytes lineage traced eYFP cells expressing macrophage markers. Subsequent single-cell RNA sequencing, however, showed that the majority of these cells had no detectable eYFP transcript. Further exploration revealed that intraperitoneal injection of tamoxifen in peanut oil, used for generating conditional knockout or reporter mice in thousands of previous studies, resulted in large increase in the autofluorescence and false identification of macrophages within epididymal AT as being eYFP; and unintended proinflammatory consequences. Using newly generated tdTomato mice given oral tamoxifen, we virtually eliminated the problem with autofluorescence and identified 8 perivascular cell dominated clusters, half of which were altered upon DIO. Given that perivascular cell KLF4 (kruppel-like factor 4) can have beneficial or detrimental effects, we tested its role in obesity-associated AT inflammation. While smooth muscle cells and pericytes-specificknockout (smooth muscle cells and pericytes ) mice were not protected from DIO, they displayed improved glucose tolerance upon DIO, and showed marked decreases in proinflammatory macrophages and increases in LYVE1 lymphatic endothelial cells in the epididymal AT.
Conclusions
Perivascular cells within the AT microvasculature dynamically respond to DIO and modulate tissue inflammation and metabolism in a KLF4-dependent manner.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:284-301
Bulut GB, Alencar GF, Owsiany KM, Nguyen AT, ... Keller SR, Owens GK
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:284-301 | PMID: 33054397
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Impact:
Abstract

Monosomy X in Female Mice Influences the Regional Formation and Augments the Severity of Angiotensin II-Induced Aortopathies.

AlSiraj Y, Thatcher SE, Blalock E, Saintilnord WN, ... Arnold AP, Cassis LA
Objective
Turner syndrome women (monosomy X) have high risk of aortopathies consistent with a role for sex chromosomes in disease development. We demonstrated that sex chromosomes influence regional development of Ang II (angiotensin II)-induced aortopathies in mice. In this study, we determined if the number of X chromosomes regulates regional development of Ang II-induced aortopathies. Approach and
Results:
We used females with varying numbers of X chromosomes (XX female mice [XXF] or XO female mice [XOF]) on an C57BL/6J (ascending aortopathies) or low-density lipoprotein receptor deficient () background (descending and abdominal aortopathies) compared with XY males (XYM). To induce aortopathies, mice were infused with Ang II. XOF (C57BL/6J) exhibited larger percent increases in ascending aortic lumen diameters than Ang II-infused XXF or XYM. Ang II-infused XOF () exhibited similar incidences of thoracic (XOF, 50%; XYM, 71%) and abdominal aortopathies (XOF, 83%; XYM, 71%) as XYM, which were greater than XXF (XXF, 0%). Abdominal aortic lumen diameters and maximal external diameters were similar between XOF and XYM but greater than XXF, and these effects persisted with extended Ang II infusions. Larger aortic lumen diameters, abdominal aortopathy incidence (XXF, 20%; XOF, 75%), and maximal aneurysm diameters (XXF, 1.02±0.17; XOF, 1.96±0.32 mm; =0.027) persisted in ovariectomized Ang II-infused XOF mice. Data from RNA-seq demonstrated that X chromosome genes that escape X-inactivation (histone lysine demethylasesand ) exhibited lower mRNA abundance in aortas of XOF than XXF (=0.033 and 0.024, respectively). Conversely, DNA methylation was higher in aortas of XOF than XXF (=0.038).
Conclusions
The absence of a second X chromosome promotes diffuse Ang II-induced aortopathies in females.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:269-283
AlSiraj Y, Thatcher SE, Blalock E, Saintilnord WN, ... Arnold AP, Cassis LA
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:269-283 | PMID: 33054396
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Impact:
Abstract

Targeting Transcription Factor Nrf2 (Nuclear Factor Erythroid 2-Related Factor 2) for the Intervention of Vascular Cognitive Impairment and Dementia.

Yang T, Zhang F

Vascular cognitive impairment and dementia (VCID) is an age-related, mild to severe mental disability due to a broad panel of cerebrovascular disorders. Its pathobiology involves neurovascular dysfunction, blood-brain barrier disruption, white matter damage, microRNAs, oxidative stress, neuroinflammation, and gut microbiota alterations, etc. Nrf2 (Nuclear factor erythroid 2-related factor 2) is the master regulator of redox status and controls the transcription of a panel of antioxidative and anti-inflammatory genes. By interacting with NF-κB (nuclear factor-κB), Nrf2 also fine-tunes the cellular oxidative and inflammatory balance. Aging is associated with Nrf2 dysfunction, and increasing evidence has proved the role of Nrf2 in mitigating the VCID process. Based on VCID pathobiologies and Nrf2 studies from VCID and other brain diseases, we point out several hypothetical Nrf2 targets for VCID management, including restoration of endothelial function and neurovascular coupling, preservation of blood-brain barrier integrity, reduction of amyloidopathy, promoting white matter integrity, and mitigating oxidative stress and neuroinflammation. Collectively, the Nrf2 pathway could be a promising direction for future VCID research. Targeting Nrf2 would shed light on VCID managing strategies.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:97-116
Yang T, Zhang F
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:97-116 | PMID: 33054394
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Impact:
Abstract

Novel LCAT (Lecithin:Cholesterol Acyltransferase) Activator DS-8190a Prevents the Progression of Plaque Accumulation in Atherosclerosis Models.

Sasaki M, Delawary M, Sakurai H, Kobayashi H, ... Terasaka N, Kubota K
Objective
Enhancement of LCAT (lecithin:cholesterol acyltransferase) activity has possibility to be beneficial for atherosclerosis. To evaluate this concept, we characterized our novel, orally administered, small-molecule LCAT activator DS-8190a, which was created from high-throughput screening and subsequent derivatization. We also focused on its mechanism of LCAT activation and the therapeutic activity with improvement of HDL (high-density lipoprotein) functionality. Approach and
Results:
DS-8190a activated human and cynomolgus monkey but not mouse LCAT enzymes in vitro. DS-8190a was orally administered to cynomolgus monkeys and dose dependently increased LCAT activity (2.0-fold in 3 mg/kg group on day 7), resulting in HDL cholesterol elevation without drastic changes of non-HDL cholesterol. Atheroprotective effects were then evaluated using × mice fed a Western diet for 8 weeks. DS-8190a treatment achieved significant reduction of atherosclerotic lesion area (48.3% reduction in 10 mg/kg treatment group). Furthermore, we conducted reverse cholesterol transport study using × mice intraperitoneally injected with J774A.1 cells loaded with [H]-cholesterol and confirmed significant increases of [H] count in plasma (1.4-fold) and feces (1.4-fold on day 2 and 1.5-fold on day3) in the DS-8190a-treated group. With regard to the molecular mechanism involved, direct binding of DS-8190a to human LCAT protein was confirmed by 2 different approaches: affinity purification by DS-8190a-immobilized beads and thermal shift assay. In addition, the candidate binding site of DS-8190a in human LCAT protein was identified by photoaffinity labeling.
Conclusions
This study demonstrates the potential of DS-8190a as a novel therapeutic for atherosclerosis. In addition, this compound proves that a small-molecule direct LCAT activator can achieve HDL-C elevation in monkey and reduction of atherosclerotic lesion area with enhanced HDL function in rodent.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:360-376
Sasaki M, Delawary M, Sakurai H, Kobayashi H, ... Terasaka N, Kubota K
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:360-376 | PMID: 33086872
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Impact:
Abstract

Local M-CSF (Macrophage Colony-Stimulating Factor) Expression Regulates Macrophage Proliferation and Apoptosis in Atherosclerosis.

Sinha SK, Miikeda A, Fouladian Z, Mehrabian M, ... Rajavashisth TB, Lusis AJ
Objective
Previous studies have shown that deficiency of M-CSF (macrophage colony-stimulating factor; or CSF1 [colony stimulating factor 1]) dramatically reduces atherosclerosis in hyperlipidemic mice. We characterize the underlying mechanism and investigate the relevant sources of CSF1 in lesions. Approach and
Results:
We quantitatively assessed the effects of CSF1 deficiency on macrophage proliferation and apoptosis in atherosclerotic lesions. Staining of aortic lesions with markers of proliferation, Ki-67 and bromodeoxyuridine, revealed around 40% reduction in CSF1 heterozygous (Csf1) as compared with WT (wild type; Csf1) mice. Similarly, staining with a marker of apoptosis, activated caspase-3, revealed a 3-fold increase in apoptotic cells in Csf1 mice. Next, we determined the cellular sources of CSF1 contributing to lesion development. Cell-specific deletions of Csf1 in smooth muscle cells using SM22α-Cre (smooth muscle protein 22-alpha-Cre) reduced lesions by about 40%, and in endothelial cells, deletions with Cdh5-Cre (VE-cadherin-Cre) reduced lesions by about 30%. Macrophage-specific deletion with LysM-Cre (lysozyme M-Cre), on the other hand, did not significantly reduce lesions size. Transplantation of Csf1 null (Csf1) mice bone marrow into Csf1 mice reduced lesions by about 35%, suggesting that CSF1 from hematopoietic cells other than macrophages contributes to atherosclerosis. None of the cell-specific knockouts affected circulating CSF1 levels, and only the smooth muscle cell deletions had any effect on the percentage monocytes in the circulation. Also, Csf1 mice did not exhibit significant differences in Ly6C/Ly6C monocytes as compared with Csf1.
Conclusions
CSF1 contributes to both macrophage proliferation and survival in lesions. Local CSF1 production by smooth muscle cell and endothelial cell rather than circulating CSF1 is the primary driver of macrophage expansion in atherosclerosis.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:220-233
Sinha SK, Miikeda A, Fouladian Z, Mehrabian M, ... Rajavashisth TB, Lusis AJ
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:220-233 | PMID: 33086870
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Impact:
Abstract

Trained Immunity: Long-Term Adaptation in Innate Immune Responses.

Tercan H, Riksen NP, Joosten LAB, Netea MG, Bekkering S

Adaptive immune responses are characterized by antigen specificity and induction of lifelong immunologic memory. Recently, it has been reported that innate immune cells can also build immune memory characteristics-a process termed trained immunity. Trained immunity describes the persistent hyperresponsive phenotype that innate immune cells can develop after brief stimulation. Pathogenic stimuli such as microorganisms, and also endogenous molecules including uric acid, oxidized LDL (low-density lipoprotein), and catecholamines, are capable of inducing memory in monocytes and macrophages. While trained immunity provides favorable cross-protection in the context of infectious diseases, the heightened immune response can be maladaptive in diseases driven by chronic systemic inflammation, such as atherosclerosis. Trained immunity is maintained by distinct epigenetic and metabolic mechanisms and persists for at least several months in vivo due to reprogramming of myeloid progenitor cells. Additionally, certain nonimmune cells are also found to exhibit trained immunity characteristics. Thus, trained immunity presents an exciting framework to develop new approaches to vaccination and also novel pharmacological targets in the treatment of inflammatory diseases.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:55-61
Tercan H, Riksen NP, Joosten LAB, Netea MG, Bekkering S
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:55-61 | PMID: 33086868
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Impact:
Abstract

Role of Platelets in Detection and Regulation of Infection.

Portier I, Campbell RA

Platelets are classically known as essential mediators of hemostasis and thrombosis. However, in recent years, platelets have gained recognition for their inflammatory functions, which modulate the immune response during infectious diseases. Platelets contain various immunoreceptors that enable them to act as sentinels to recognize intravascular pathogens. Upon activation, platelets directly limit pathogen growth through the release of AMPs (antimicrobial proteins) and ensure pathogen clearance through activation of immune cells. However, aberrant platelet activation can lead to inflammation and thrombotic events.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:70-78
Portier I, Campbell RA
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:70-78 | PMID: 33115274
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Impact:
Abstract

Transcriptional Programming in Arteriosclerotic Disease: A Multifaceted Function of the Runx2 (Runt-Related Transcription Factor 2).

Chen Y, Zhao X, Wu H

Despite successful therapeutic strategies in the prevention and treatment of arteriosclerosis, the cardiovascular complications remain a major clinical and societal issue worldwide. Increased vascular calcification promotes arterial stiffness and accelerates cardiovascular morbidity and mortality. Upregulation of the Runx2 (Runt-related transcription factor 2), an essential osteogenic transcription factor for bone formation, in the cardiovascular system has emerged as an important regulator for adverse cellular events that drive cardiovascular pathology. This review discusses the regulatory mechanisms that are critical for Runx2 expression and function and highlights the dynamic and complex cross talks of a wide variety of posttranslational modifications, including phosphorylation, acetylation, ubiquitination, and O-linked β-N-acetylglucosamine modification, in regulating Runx2 stability, cellular localization, and osteogenic transcriptional activity. How the activation of an array of signaling cascades by circulating and local microenvironmental factors upregulates Runx2 in vascular cells and promotes Runx2-mediated osteogenic transdifferentiation of vascular smooth muscle cells and expression of inflammatory cytokines that accelerate macrophage infiltration and vascular osteoclast formation is summarized. Furthermore, the increasing appreciation of a new role of Runx2 upregulation in promoting vascular smooth muscle cell phenotypic switch, and Runx2 modulated by -linked β-N-acetylglucosamine modification and Runx2-dependent repression of smooth muscle cell-specific gene expression are discussed. Further exploring the regulation of this key osteogenic transcription factor and its new perspectives in the vasculature will provide novel insights into the transcriptional regulation of vascular smooth muscle cell phenotype switch, reprograming, and vascular inflammation that promote the pathogenesis of arteriosclerosis.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:20-34
Chen Y, Zhao X, Wu H
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:20-34 | PMID: 33115268
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Impact:
Abstract

Computationally Driven Discovery in Coagulation.

Link KG, Stobb MT, Monroe DM, Fogelson AL, ... Sindi SS, Leiderman K

Bleeding frequency and severity within clinical categories of hemophilia A are highly variable and the origin of this variation is unknown. Solving this mystery in coagulation requires the generation and analysis of large data sets comprised of experimental outputs or patient samples, both of which are subject to limited availability. In this review, we describe how a computationally driven approach bypasses such limitations by generating large synthetic patient data sets. These data sets were created with a mechanistic mathematical model, by varying the model inputs, clotting factor, and inhibitor concentrations, within normal physiological ranges. Specific mathematical metrics were chosen from the model output, used as a surrogate measure for bleeding severity, and statistically analyzed for further exploration and hypothesis generation. We highlight results from our recent study that employed this computationally driven approach to identify FV (factor V) as a key modifier of thrombin generation in mild to moderate hemophilia A, which was confirmed with complementary experimental assays. The mathematical model was used further to propose a potential mechanism for these observations whereby thrombin generation is rescued in FVIII-deficient plasma due to reduced substrate competition between FV and FVIII for FXa (activated factor X).



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:79-86
Link KG, Stobb MT, Monroe DM, Fogelson AL, ... Sindi SS, Leiderman K
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:79-86 | PMID: 33115272
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Impact:
Abstract

Trained Immunity in Atherosclerotic Cardiovascular Disease.

Flores-Gomez D, Bekkering S, Netea MG, Riksen NP

Atherosclerosis is characterized by incessant inflammation in the arterial wall in which monocytes and macrophages play a crucial role. During the past few years, it has been reported that cells from the innate immune system can develop a long-lasting proinflammatory phenotype after brief stimulation not only with microbial products but also endogenous atherogenic stimuli. This persistent hyperactivation of the innate immune system is termed trained immunity and can contribute to the pathophysiology of atherosclerosis. Trained immunity is mediated via epigenetic and metabolic reprogramming and occurs both in mature innate immune cells as well as their bone marrow progenitors. In addition to monocytes, other innate immune and nonimmune cells involved in different stages of atherosclerosis can develop comparable memory characteristics. This mechanism provides exciting novel pharmacological targets that can be used to prevent or treat cardiovascular diseases.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:62-69
Flores-Gomez D, Bekkering S, Netea MG, Riksen NP
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:62-69 | PMID: 33147995
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Impact:
Abstract

Gal-1 (Galectin-1) Upregulation Contributes to Abdominal Aortic Aneurysm Progression by Enhancing Vascular Inflammation.

Chiang MT, Chen IM, Hsu FF, Chen YH, ... Chen YH, Chau LY
Objective
Abdominal aortic aneurysm (AAA) is a vascular degenerative disease causing sudden rupture of aorta and significant mortality in elders. Nevertheless, no prognostic and therapeutic target is available for disease management. Gal-1 (galectin-1) is a β-galactoside-binding lectin constitutively expressed in vasculature with roles in maintaining vascular homeostasis. This study aims to investigate the potential involvement of Gal-1 in AAA progression. Approach and
Results:
Gal-1 was significantly elevated in circulation and aortic tissues of Ang II (angiotensin II)-infused apoE-deficient mice developing AAA. Gal-1 deficiency reduced incidence and severity of AAA with lower expression of aortic MMPs (matrix metalloproteases) and proinflammatory cytokines. TNFα (tumor necrosis factor alpha) induced Gal-1 expression in cultured vascular smooth muscle cells and adventitial fibroblasts. Gal-1 deletion enhanced TNFα-induced MMP9 expression in fibroblasts but not vascular smooth muscle cells. Cysteinyl-labeling assay demonstrated that aortic Gal-1 exhibited susceptibility to oxidation in vivo. Recombinant oxidized Gal-1 induced expression of MMP9 and inflammatory cytokines to various extents in macrophages, vascular smooth muscle cells, and fibroblasts through activation of MAP (mitogen-activated protein) kinase signaling. Clinically, serum MMP9 level was significantly higher in both patients with AAA and coronary artery disease than in control subjects, whereas serum Gal-1 level was elevated in patients with AAA but not coronary artery disease when compared with controls.
Conclusions
Gal-1 is highly induced and contributes to AAA by enhancing matrix degradation activity and inflammatory responses in experimental model. The pathological link between Gal-1 and AAA is also observed in human patients. These findings support the potential of Gal-1 as a disease biomarker and therapeutic target of AAA.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:331-345
Chiang MT, Chen IM, Hsu FF, Chen YH, ... Chen YH, Chau LY
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:331-345 | PMID: 33147994
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Impact:
Abstract

Altered Macrophage Polarization Induces Experimental Pulmonary Hypertension and Is Observed in Patients With Pulmonary Arterial Hypertension.

Zawia A, Arnold ND, West L, Pickworth JA, ... Miller G, Lawrie A
Objective
To determine whether global reduction of CD68 (cluster of differentiation) macrophages impacts the development of experimental pulmonary arterial hypertension (PAH) and whether this reduction affects the balance of pro- and anti-inflammatory macrophages within the lung. Additionally, to determine whether there is evidence of an altered macrophage polarization in patients with PAH. Approach and
Results:
Macrophage reduction was induced in mice via doxycycline-induced CD68-driven cytotoxic diphtheria toxin A chain expression (macrophage low [MacLow] mice). Chimeric mice were generated using bone marrow transplant. Mice were phenotyped for PAH by echocardiography and closed chest cardiac catheterization. Murine macrophage phenotyping was performed on lungs, bone marrow-derived macrophages, and alveolar macrophages using immunohistochemical and flow cytometry. Monocyte-derived macrophages were isolated from PAH patients and healthy volunteers and polarization capacity assessed morphologically and by flow cytometry. After 6 weeks of macrophage depletion, male but not female MacLow mice developed PAH. Chimeric mice demonstrated a requirement for both MacLow bone marrow and MacLow recipient mice to cause PAH. Immunohistochemical analysis of lung sections demonstrated imbalance in M1/M2 ratio in male MacLow mice only, suggesting that this imbalance may drive the PAH phenotype. M1/M2 imbalance was also seen in male MacLow bone marrow-derived macrophages and PAH patient monocyte-derived macrophages following stimulation with doxycycline and IL (interleukin)-4, respectively. Furthermore, MacLow-derived alveolar macrophages showed characteristic differences in terms of their polarization and expression of diphtheria toxin A chain following stimulation with doxycycline.
Conclusions
These data further highlight a sex imbalance in PAH and further implicate immune cells into this paradigm. Targeting imbalance of macrophage population may offer a future therapeutic option.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:430-445
Zawia A, Arnold ND, West L, Pickworth JA, ... Miller G, Lawrie A
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:430-445 | PMID: 33147993
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Impact:
Abstract

Junctional Localization of Septin 2 Is Required for Organization of Junctional Proteins in Static Endothelial Monolayers.

Kim J, Cooper JA
Objective
Septin 2 is localized at junctions in human microvascular endothelial monolayers. The junctional localization of septin 2 is necessary for organization of cell-cell adhesion proteins of endothelial cells. Approach and
Results:
Septin 2 was depleted at junctions by suppression of expression using shRNA, treatment with inflammatory cytokine, TNF (tumor necrosis factor)-α, and ectopic overexpression of septin 2 phosphatidylinositol 4,5-bisphosphate binding mutant defect in interaction with plasma membrane. Under those conditions, organizations and expression levels of various junctional proteins were analyzed. Confocal images of immunofluorescence staining showed substantial disorganization of adherens junctional proteins, nectin-2 and afadin, TJP (tight junction protein), ZO (zonula occludens)-1, and intercellular adhesion protein, PECAM-1 (platelet-endothelial cell adhesion molecule-1). Immunoblots for those proteins did not show significant changes in expression except for nectin-2 that highly increased in expression. Significant differential gene expression profiles and biological pathway analysis by septin 2 suppression and by TNF-α treatment using RNA-seq showed common overlapping pathways. The commonalities in expression may be consistent with the similar effects on the overall organization of cell-cell adhesion proteins.
Conclusions
Localization of septin 2 at cell junctions are required for the arrangement of junctional proteins and the integrity of the barrier formed by endothelial monolayers.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:346-359
Kim J, Cooper JA
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:346-359 | PMID: 33147991
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Impact:
Abstract

Phosphoproteomic Analysis of Platelets in Severe Obesity Uncovers Platelet Reactivity and Signaling Pathways Alterations.

Barrachina MN, Hermida-Nogueira L, Moran LA, Casas V, ... Pardo M, García Á
Objective
Obesity is associated with a proinflammatory and prothrombotic state that supports atherosclerosis progression. The goal of this study was to gain insights into the phosphorylation events related to platelet reactivity in obesity and identify platelet biomarkers and altered activation pathways in this clinical condition. Approach and
Results:
We performed a comparative phosphoproteomic analysis of resting platelets from obese patients and their age- and gender-matched lean controls. The phosphoproteomic data were validated by mechanistic, functional, and biochemical assays. We identified 220 differentially regulated phosphopeptides, from at least 175 proteins; interestingly, all were up-regulated in obesity. Most of the altered phosphoproteins are involved in SFKs (Src-family kinases)-related signaling pathways, cytoskeleton reorganization, and vesicle transport, some of them validated by targeted mass spectrometry. To confirm platelet dysfunction, flow cytometry assays were performed in whole blood indicating higher surface levels of GP (glycoprotein) VI and CLEC (C-type lectin-like receptor) 2 in platelets from obese patients correlating positively with body mass index. Receiver operator characteristics curves analysis suggested a much higher sensitivity for GPVI to discriminate between obese and lean individuals. Indeed, we also found that obese platelets displayed more adhesion to collagen-coated plates. In line with the above data, soluble GPVI levels-indicative of higher GPVI signaling activation-were almost double in plasma from obese patients.
Conclusions
Our results provide novel information on platelet phosphorylation changes related to obesity, revealing the impact of this chronic pathology on platelet reactivity and pointing towards the main signaling pathways dysregulated.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:478-490
Barrachina MN, Hermida-Nogueira L, Moran LA, Casas V, ... Pardo M, García Á
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:478-490 | PMID: 33147989
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Impact:
Abstract

Calcium Ion Chelation Preserves Platelet Function During Cold Storage.

Xiang B, Zhang G, Zhang Y, Wu C, ... Whiteheart SW, Li Z
Objective
Platelet transfusion is a life-saving therapy to prevent or treat bleeding in patients with thrombocytopenia or platelet dysfunction. However, for >6 decades, safe and effective strategies for platelet storage have been an impediment to widespread use of platelet transfusion. Refrigerated platelets are cleared rapidly from circulation, precluding cold storage of platelets for transfusion. Consequently, platelets are stored at room temperature with an upper limit of 5 days due to risks of bacterial contamination and loss of platelet function. This practice severely limits platelet availability for transfusion. This study is to identify the mechanism of platelet clearance after cold storage and develop a method for platelet cold storage. Approach and
Results:
We found that rapid clearance of cold-stored platelets was largely due to integrin activation and apoptosis. Deficiency of integrin β3 or caspase-3 prolonged cold-stored platelets in circulation. Pretreatment of platelets with EGTA, a cell impermeable calcium ion chelator, reversely inhibited cold storage-induced platelet activation and consequently prolonged circulation of cold-stored platelets. Moreover, transfusion of EGTA-treated, cold-stored platelets, but not room temperature-stored platelets, into the mice deficient in glycoprotein Ibα significantly shortened tail-bleeding times and diminished blood loss.
Conclusions
Integrin activation and apoptosis is the underlying mechanism of rapid clearance of platelets after cold storage. Addition of a cell impermeable calcium ion chelator to platelet products is potentially a simple and effective method to enable cold storage of platelets for transfusion.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:234-249
Xiang B, Zhang G, Zhang Y, Wu C, ... Whiteheart SW, Li Z
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:234-249 | PMID: 33176450
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Impact:
Abstract

Nonconserved Long Intergenic Noncoding RNAs Associate With Complex Cardiometabolic Disease Traits.

Foulkes AS, Selvaggi C, Cao T, O\'Reilly ME, ... Xue C, Reilly MP
Objective
Transcriptome profiling of human tissues has revealed thousands of long intergenic noncoding RNAs (lincRNAs) at loci identified through large-scale genome-wide studies for complex cardiometabolic traits. This raises the question of whether genetic variation at nonconserved lincRNAs has any systematic association with complex disease, and if so, how different this pattern is from conserved lincRNAs. We evaluated whether the associations between nonconserved lincRNAs and 8 complex cardiometabolic traits resemble or differ from the pattern of association for conserved lincRNAs. Approach and
Results:
Our investigation of over 7000 lincRNA annotations from GENCODE Release 33-GRCh38.p13 for complex trait genetic associations leveraged several large, established meta-analyses genome-wide association study summary data resources, including GIANT (Genetic Investigation of Anthropometric Traits), UK Biobank, GLGC (Global Lipids Genetics Consortium), Cardiogram (Coronary Artery Disease Genome Wide Replication and Meta-Analysis), and DIAGRAM (Diabetes Genetics Replication and Meta-Analysis)/DIAMANTE (Diabetes Meta-Analysis of Trans-Ethnic Association Studies). These analyses revealed that (1) nonconserved lincRNAs associate with a range of cardiometabolic traits at a rate that is generally consistent with conserved lincRNAs; (2) these findings persist across different definitions of conservation; and (3) overall across all cardiometabolic traits, approximately one-third of genome-wide association study-associated lincRNAs are nonconserved, and this increases to about two-thirds using a more stringent definition of conservation.
Conclusions
These findings suggest that the traditional notion of conservation driving prioritization for functional and translational follow-up of complex cardiometabolic genomic discoveries may need to be revised in the context of the abundance of nonconserved long noncoding RNAs in the human genome and their apparent predilection to associate with complex cardiometabolic traits.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:501-511
Foulkes AS, Selvaggi C, Cao T, O'Reilly ME, ... Xue C, Reilly MP
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:501-511 | PMID: 33176448
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Impact:
Abstract

Sex-Specific Platelet Activation Through Protease-Activated Receptors Reverses in Myocardial Infarction.

Soo Kim B, Auerbach DS, Sadhra H, Godwin M, ... Goldenberg I, Cameron SJ
Objective
The platelet phenotype in certain patients and clinical contexts may differ from healthy conditions. We evaluated platelet activation through specific receptors in healthy men and women, comparing this to patients presenting with ST-segment-elevation myocardial infarction and non-ST-segment-elevation myocardial infarction. Approach and
Results:
We identified independent predictors of platelet activation through certain receptors and a murine MI model further explored these findings. Platelets from healthy women and female mice are more reactive through PARs (protease-activated receptors) compared with platelets from men and male mice. Multivariate regression analyses revealed male sex and non-ST-segment-elevation myocardial infarction as independent predictors of enhanced PAR1 activation in human platelets. Platelet PAR1 signaling decreased in women and increased in men during MI which was the opposite of what was observed during healthy conditions. Similarly, in mice, thrombin-mediated platelet activation was greater in healthy females compared with males, and lesser in females compared with males at the time of MI.
Conclusions
Sex-specific signaling in platelets seems to be a cross-species phenomenon. The divergent platelet phenotype in males and females at the time of MI suggests a sex-specific antiplatelet drug regimen should be prospectively evaluated.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:390-400
Soo Kim B, Auerbach DS, Sadhra H, Godwin M, ... Goldenberg I, Cameron SJ
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:390-400 | PMID: 33176447
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Impact:
Abstract

Therapeutic Potential of Tpl2 (Tumor Progression Locus 2) Inhibition on Diabetic Vasculopathy Through the Blockage of the Inflammasome Complex.

Sheu WH, Lin KH, Wang JS, Lai DW, ... Liu SH, Sheu ML
Objective
Diabetic retinopathy, one of retinal vasculopathy, is characterized by retinal inflammation, vascular leakage, blood-retinal barrier breakdown, and neovascularization. However, the molecular mechanisms that contribute to diabetic retinopathy progression remain unclear. Approach and
Results:
Tpl2 (tumor progression locus 2) is a protein kinase implicated in inflammation and pathological vascular angiogenesis. N-carboxymethyllysine (CML) and inflammatory cytokines levels in human sera and in several diabetic murine models were detected by ELISA, whereas liquid chromatography-tandem mass spectrometry analysis was used for whole eye tissues. The CML and p-Tpl2 expressions on the human retinal pigment epithelium (RPE) cells were determined by immunofluorescence. Intravitreal injection of pharmacological inhibitor or NA (neutralizing antibody) was used in a diabetic rat model. Retinal leukostasis, optical coherence tomography, and H&E staining were used to observe pathological features. Sera of diabetic retinopathy patients had significantly increased CML levels that positively correlated with diabetic retinopathy severity and foveal thickness. CML and p-Tpl2 expressions also significantly increased in the RPE of both T1DM and T2DM diabetes animal models. Mechanistic studies on RPE revealed that CML-induced Tpl2 activation and NADPH oxidase, and inflammasome complex activation were all effectively attenuated by Tpl2 inhibition. Tpl2 inhibition by NA also effectively reduced inflammatory/angiogenic factors, retinal leukostasis in streptozotocin-induced diabetic rats, and RPE secretion of inflammatory cytokines. The attenuated release of angiogenic factors led to inhibited vascular abnormalities in the diabetic animal model.
Conclusions
The inhibition of Tpl2 can block the inflammasome signaling pathway in RPE and has potential clinical and therapeutic implications in diabetes-associated retinal microvascular dysfunction.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:e46-e62
Sheu WH, Lin KH, Wang JS, Lai DW, ... Liu SH, Sheu ML
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:e46-e62 | PMID: 33176446
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Impact:
Abstract

Role of LOX-1 (Lectin-Like Oxidized Low-Density Lipoprotein Receptor 1) as a Cardiovascular Risk Predictor: Mechanistic Insight and Potential Clinical Use.

Barreto J, Karathanasis SK, Remaley A, Sposito AC

Atherosclerosis, the underlying cause of cardiovascular disease (CVD), is a worldwide cause of morbidity and mortality. Reducing ApoB-containing lipoproteins-chiefly, LDL (low-density lipoprotein)-has been the main strategy for reducing CVD risk. Although supported by large randomized clinical trials, the persistence of residual cardiovascular risk after effective LDL reduction has sparked an intense search for other novel CVD biomarkers and therapeutic targets. Recently, Lox-1 (lectin-type oxidized LDL receptor 1), an innate immune scavenger receptor, has emerged as a promising target for early diagnosis and cardiovascular risk prediction and is also being considered as a treatment target. Lox-1 was first described as a 50 kDa transmembrane protein in endothelial cells responsible for oxLDL (oxidized LDL) recognition, triggering downstream pathways that intensify atherosclerosis via endothelial dysfunction, oxLDL uptake, and apoptosis. Lox-1 is also expressed in platelets, where it enhances platelet activation, adhesion to endothelial cells, and ADP-mediated aggregation, thereby favoring thrombus formation. Lox-1 was also identified in cardiomyocytes, where it was implicated in the development of cardiac fibrosis and myocyte apoptosis, the main determinants of cardiac recovery following an ischemic insult. Together, these findings have revealed that Lox-1 is implicated in all the main steps of atherosclerosis and has encouraged the development of immunoassays for measurement of sLox-1 (serum levels of soluble Lox-1) to be used as a potential CVD biomarker. Finally, the recent development of synthetic Lox-1 inhibitors and neutralizing antibodies with promising results in animal models has made Lox-1 a target for drug development. In this review, we discuss the main findings regarding the role of Lox-1 in the development, diagnosis, and therapeutic strategies for CVD prevention and treatment.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:153-166
Barreto J, Karathanasis SK, Remaley A, Sposito AC
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:153-166 | PMID: 33176449
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Impact:
Abstract

COVID-19 and Sepsis Are Associated With Different Abnormalities in Plasma Procoagulant and Fibrinolytic Activity.

Bouck EG, Denorme F, Holle LA, Middelton EA, ... Wolberg AS, Campbell RA
Objective
Coronavirus disease 2019 (COVID-19) is associated with derangement in biomarkers of coagulation and endothelial function and has been likened to the coagulopathy of sepsis. However, clinical laboratory metrics suggest key differences in these pathologies. We sought to determine whether plasma coagulation and fibrinolytic potential in patients with COVID-19 differ compared with healthy donors and critically ill patients with sepsis. Approach and
Results:
We performed comparative studies on plasmas from a single-center, cross-sectional observational study of 99 hospitalized patients (46 with COVID-19 and 53 with sepsis) and 18 healthy donors. We measured biomarkers of endogenous coagulation and fibrinolytic activity by immunoassays, thrombin, and plasmin generation potential by fluorescence and fibrin formation and lysis by turbidity. Compared with healthy donors, patients with COVID-19 or sepsis both had elevated fibrinogen, d-dimer, soluble TM (thrombomodulin), and plasmin-antiplasmin complexes. Patients with COVID-19 had increased thrombin generation potential despite prophylactic anticoagulation, whereas patients with sepsis did not. Plasma from patients with COVID-19 also had increased endogenous plasmin potential, whereas patients with sepsis showed delayed plasmin generation. The collective perturbations in plasma thrombin and plasmin generation permitted enhanced fibrin formation in both COVID-19 and sepsis. Unexpectedly, the lag times to thrombin, plasmin, and fibrin formation were prolonged with increased disease severity in COVID-19, suggesting a loss of coagulation-initiating mechanisms accompanies severe COVID-19.
Conclusions
Both COVID-19 and sepsis are associated with endogenous activation of coagulation and fibrinolysis, but these diseases differently impact plasma procoagulant and fibrinolytic potential. Dysregulation of procoagulant and fibrinolytic pathways may uniquely contribute to the pathophysiology of COVID-19 and sepsis.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:401-414
Bouck EG, Denorme F, Holle LA, Middelton EA, ... Wolberg AS, Campbell RA
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:401-414 | PMID: 33196292
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Impact:
Abstract

Hmox1 (Heme Oxygenase-1) Protects Against Ischemia-Mediated Injury via Stabilization of HIF-1α (Hypoxia-Inducible Factor-1α).

Dunn LL, Kong SMY, Tumanov S, Chen W, ... Ng MKC, Stocker R
Objective
Hmox1 (heme oxygenase-1) is a stress-induced enzyme that catalyzes the degradation of heme to carbon monoxide, iron, and biliverdin. Induction of Hmox1 and its products protect against cardiovascular disease, including ischemic injury. Hmox1 is also a downstream target of the transcription factor HIF-1α (hypoxia-inducible factor-1α), a key regulator of the body\'s response to hypoxia. However, the mechanisms by which Hmox1 confers protection against ischemia-mediated injury remain to be fully understood. Approach and
Results:
Hmox1 deficient () mice had impaired blood flow recovery with severe tissue necrosis and autoamputation following unilateral hindlimb ischemia. Autoamputation preceded the return of blood flow, and bone marrow transfer from littermate wild-type mice failed to prevent tissue injury and autoamputation. In wild-type mice, ischemia-induced expression of Hmox1 in skeletal muscle occurred before stabilization of HIF-1α. Moreover, HIF-1α stabilization and glucose utilization were impaired inmice compared with wild-type mice. Experiments exposing dermal fibroblasts to hypoxia (1% O) recapitulated these key findings. Metabolomics analyses indicated a failure ofmice to adapt cellular energy reprogramming in response to ischemia. Prolyl-4-hydroxylase inhibition stabilized HIF-1α infibroblasts and ischemic skeletal muscle, decreased tissue necrosis and autoamputation, and restored cellular metabolism to that of wild-type mice. Mechanistic studies showed that carbon monoxide stabilized HIF-1α infibroblasts in response to hypoxia.
Conclusions
Our findings suggest that Hmox1 acts both downstream and upstream of HIF-1α, and that stabilization of HIF-1α contributes to Hmox1\'s protection against ischemic injury independent of neovascularization.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:317-330
Dunn LL, Kong SMY, Tumanov S, Chen W, ... Ng MKC, Stocker R
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:317-330 | PMID: 33207934
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Impact:
Abstract

Alternative Splicing of FN (Fibronectin) Regulates the Composition of the Arterial Wall Under Low Flow.

Murphy PA, Jailkhani N, Nicholas SA, Del Rosario AM, ... Kimble A, Hynes RO
Objective
Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered composition of the extracellular matrix at the site of lesions, and to contribute to inflammatory processes within the lesions. We have previously shown that alternative splicing of FN (fibronectin) protects against flow-induced hemorrhage. However, the impact of alternative splicing of FN on extracellular matrix composition remains unknown. Approach and
Results:
Here, we perform quantitative proteomic analysis of the matrisome of murine carotid arteries in mice deficient in the production of FN splice isoforms containing alternative exons EIIIA and EIIIB (FN-EIIIAB null) after exposure to low and disturbed flow in vivo. We also examine serum-derived and endothelial-cell contributions to the matrisome in a simplified in vitro system. We found flow-induced differences in the carotid artery matrisome that were impaired in FN-EIIIAB null mice. One of the most interesting differences was reduced recruitment of FBLN1 (fibulin-1), abundant in blood and not locally produced in the intima. This defect was validated in our in vitro assay, where FBLN1 recruitment from serum was impaired by the absence of these alternatively spliced segments.
Conclusions
Our results reveal the extent of the dynamic alterations in the matrisome in the acute response to low and disturbed flow and show how changes in the splicing of FN, a common response in vascular inflammation and remodeling, can affect matrix composition.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:e18-e32
Murphy PA, Jailkhani N, Nicholas SA, Del Rosario AM, ... Kimble A, Hynes RO
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:e18-e32 | PMID: 33207933
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Impact:
Abstract

Trained Immunity and Cardiometabolic Disease: The Role of Bone Marrow.

Mitroulis I, Hajishengallis G, Chavakis T

Until recently, immunologic memory was considered an exclusive characteristic of adaptive immunity. However, recent advances suggest that the innate arm of the immune system can also mount a type of nonspecific memory responses. Innate immune cells can elicit a robust response to subsequent inflammatory challenges after initial activation by certain stimuli, such as fungal-derived agents or vaccines. This type of memory, termed trained innate immunity (also named innate immune memory), is associated with epigenetic and metabolic alterations. Hematopoietic progenitor cells, which are the cells responsible for the generation of mature myeloid cells at steady-state and during inflammation, have a critical contribution to the induction of innate immune memory. Inflammation-triggered alterations in cellular metabolism, the epigenome and transcriptome of hematopoietic progenitor cells in the bone marrow promote long-lasting functional changes, resulting in increased myelopoiesis and consequent generation of trained innate immune cells. In the present brief review, we focus on the involvement of hematopoietic progenitors in the process of trained innate immunity and its possible role in cardiometabolic disease.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:48-54
Mitroulis I, Hajishengallis G, Chavakis T
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:48-54 | PMID: 33207931
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Impact:
Abstract

Critical Interaction Between Telomerase and Autophagy in Mediating Flow-Induced Human Arteriolar Vasodilation.

Hughes WE, Chabowski DS, Ait-Aissa K, Fetterman JL, ... Beyer AM, Gutterman DD
Objective
Coronary artery disease (CAD) is associated with a compensatory switch in mechanism of flow-mediated dilation (FMD) from nitric oxide (NO) to HO. The underlying mechanism responsible for the pathological shift is not well understood, and recent reports directly implicate telomerase and indirectly support a role for autophagy. We hypothesize that autophagy is critical for shear stress-induced release of NO and is a crucial component of for the pathway by which telomerase regulates FMD. Approach and
Results:
Human left ventricular, atrial, and adipose resistance arterioles were collected for videomicroscopy and immunoblotting. FMD and autophagic flux were measured in arterioles treated with autophagy modulators alone, and in tandem with telomerase-activity modulators. LC3B II/I was higher in left ventricular tissue from patients with CAD compared with non-CAD (2.8±0.2 versus 1.0±0.2-fold change; <0.05), although p62 was similar between groups. Shear stress increased Lysotracker fluorescence in non-CAD arterioles, with no effect in CAD arterioles. Inhibition of autophagy in non-CAD arterioles induced a switch from NO to HO, while activation of autophagy restored NO-mediated vasodilation in CAD arterioles. In the presence of an autophagy activator, telomerase inhibitor prevented the expected switch (Control: 82±4%; NG-Nitro-l-arginine methyl ester: 36±5%; polyethylene glycol catalase: 80±3). Telomerase activation was unable to restore NO-mediated FMD in the presence of autophagy inhibition in CAD arterioles (control: 72±7%; NG-Nitro-l-arginine methyl ester: 79±7%; polyethylene glycol catalase: 38±9%).
Conclusions
We provide novel evidence that autophagy is responsible for the pathological switch in dilator mechanism in CAD arterioles, demonstrating that autophagy acts downstream of telomerase as a common denominator in determining the mechanism of FMD.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:446-457
Hughes WE, Chabowski DS, Ait-Aissa K, Fetterman JL, ... Beyer AM, Gutterman DD
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:446-457 | PMID: 33232201
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Impact:
Abstract

Novel Insights From Human Studies on the Role of High-Density Lipoprotein in Mortality and Noncardiovascular Disease.

Madsen CM, Varbo A, Nordestgaard BG

The vast majority of research about HDL (high-density lipoprotein) has for decades revolved around the possible role of HDL in atherosclerosis and its therapeutic potential within cardiovascular disease prevention; however, failures with therapies aimed at increasing HDL cholesterol has left questions as to what the role and function of HDL in human health and disease is. Recent observational studies have further shown that extreme high HDL cholesterol is associated with high mortality leading to speculations that HDL could in some instances be harmful. In addition, evidence from observational, and to a lesser extent genetic studies has emerged indicating that HDL might be associated with the development of other major noncardiovascular diseases, such as infectious disease, autoimmune disease, cancer, type 2 diabetes, kidney disease, and lung disease. In this review, we discuss (1) the association between extreme high HDL cholesterol and mortality and (2) the emerging human evidence linking HDL to several major diseases outside the realm of cardiovascular disease.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:128-140
Madsen CM, Varbo A, Nordestgaard BG
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:128-140 | PMID: 33232200
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Impact:
Abstract

Oxyphospholipids in Cardiovascular Calcification.

Chignon A, Bon-Baret V, Boulanger MC, Bossé Y, Mathieu P

Mineralization of cardiovascular structures including blood vessels and heart valves is a common feature. We postulate that ectopic mineralization is a response-to-injury in which signals delivered to cells trigger a chain of events to restore and repair tissues. Maladaptive response to external or internal signals promote the expression of danger-associated molecular patterns, which, in turn, promote, when expressed chronically, a procalcifying gene program. Growing evidence suggest that danger-associated molecular patterns such as oxyphospholipids and small lipid mediators, generated by enzyme activity, are involved in the transition of vascular smooth muscle cells and valve interstitial cells to an osteoblast-like phenotype. Understanding the regulation and the molecular processes underpinning the mineralization of atherosclerotic plaques and cardiac valves are providing valuable mechanistic insights, which could lead to the development of novel therapies. Herein, we provide a focus account on the role oxyphospholipids and their mediators in the development of mineralization in plaques and calcific aortic valve disease.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:11-19
Chignon A, Bon-Baret V, Boulanger MC, Bossé Y, Mathieu P
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:11-19 | PMID: 33232199
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Impact:
Abstract

Towards the Therapeutic Use of Thrombospondin 1/CD47 Targeting TAX2 Peptide as an Antithrombotic Agent.

Jeanne A, Sarazin T, Charlé M, Kawecki C, ... Maurice P, Dedieu S
Objective
TSP-1 (thrombospondin 1) is one of the most expressed proteins in platelet α-granules and plays an important role in the regulation of hemostasis and thrombosis. Interaction of released TSP-1 with CD47 membrane receptor has been shown to regulate major events leading to thrombus formation, such as, platelet adhesion to vascular endothelium, nitric oxide/cGMP (cyclic guanosine monophosphate) signaling, platelet activation as well as aggregation. Therefore, targeting TSP-1:CD47 axis may represent a promising antithrombotic strategy. Approach and
Results:
A CD47-derived cyclic peptide was engineered, namely TAX2, that targets TSP-1 and selectively prevents TSP-1:CD47 interaction. Here, we demonstrate for the first time that TAX2 peptide strongly decreases platelet aggregation and interaction with collagen under arterial shear conditions. TAX2 also delays time for complete thrombotic occlusion in 2 mouse models of arterial thrombosis following chemical injury, whilemice recapitulate TAX2 effects. Importantly, TAX2 administration is not associated with increased bleeding risk or modification of hematologic parameters.
Conclusions
Overall, this study sheds light on the major contribution of TSP-1:CD47 interaction in platelet activation and thrombus formation while putting forward TAX2 as an innovative antithrombotic agent with high added-value.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:e1-e17
Jeanne A, Sarazin T, Charlé M, Kawecki C, ... Maurice P, Dedieu S
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:e1-e17 | PMID: 33232198
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Impact:
Abstract

Heparin-Induced Thrombocytopenia: A Focus on Thrombosis.

Arepally GM, Padmanabhan A

Heparin-induced thrombocytopenia is an immune-mediated disorder caused by antibodies that recognize complexes of platelet factor 4 and heparin. Thrombosis is a central and unpredictable feature of this syndrome. Despite optimal management, disease morbidity and mortality from thrombosis remain high. The hypercoagulable state in heparin-induced thrombocytopenia is biologically distinct from other thrombophilic disorders in that clinical complications are directly attributable to circulating ultra-large immune complexes. In some individuals, ultra-large immune complexes elicit unchecked cellular procoagulant responses that culminate in thrombosis. To date, the clinical and biologic risk factors associated with thrombotic risk in heparin-induced thrombocytopenia remain elusive. This review will summarize our current understanding of thrombosis in heparin-induced thrombocytopenia with attention to its clinical features, cellular mechanisms, and its management.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:141-152
Arepally GM, Padmanabhan A
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:141-152 | PMID: 33267665
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Impact:
Abstract

Lipid Receptor GPR31 (G-Protein-Coupled Receptor 31) Regulates Platelet Reactivity and Thrombosis Without Affecting Hemostasis.

Van Doren L, Nguyen N, Garzia C, Fletcher EK, ... Kuliopulos A, Covic L
Objective
12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein-coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity. Approach and
Results:
Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4-mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 (Ras-related protein 1) and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop-derived pepducin, GPR310 (G-protein-coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection (=0.0018) against ferric chloride-induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE-mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems.
Conclusions
The 12-LOX product 12(S)-HETE stimulates GPR31-Gi-signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:e33-e45
Van Doren L, Nguyen N, Garzia C, Fletcher EK, ... Kuliopulos A, Covic L
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:e33-e45 | PMID: 33267659
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Impact:
Abstract

Arterial Thrombotic Complications of Tyrosine Kinase Inhibitors.

Wu MD, Moslehi JJ, Lindner JR

Abnormal expression or function of several classes of kinases contribute to the development of many types of solid and hematologic malignancies. TKs (tyrosine kinases) in particular play a role in tumor growth, metastasis, neovascularization, suppression of immune surveillance, and drug resistance. TKIs (tyrosine kinase inhibitors) targeted to TKs such as BCR-ABL1, VEGF receptors, PDGF receptors, have transformed therapy of certain forms of cancer by providing excellent efficacy with relatively low adverse event rates. Yet some of these agents have been associated with high rates of vascular events, presumably from prothrombotic complications that result in myocardial infarction, stroke, and critical limb ischemia. This review describes the scope of the problem evidenced by clinical experience with some of the most commonly used TKIs, with a focus on TKIs targeted to the BCR-ABL1 (breakpoint cluster region-Abelson 1) translocation. We also discuss the potential mechanisms responsible for arterial thrombotic complications that could lead to mitigation strategies or unique TK targeting strategies to reduce adverse event rates without compromising efficacy.



Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:3-10
Wu MD, Moslehi JJ, Lindner JR
Arterioscler Thromb Vasc Biol: 30 Dec 2020; 41:3-10 | PMID: 33275447
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Impact:
Abstract

Magnesium Supplementation Attenuates Pulmonary Hypertension via Regulation of Magnesium Transporters.

Wang D, Zhu ZL, Lin DC, Zheng SY, ... Sham JSK, Lin MJ

Pulmonary hypertension (PH) is characterized by profound vascular remodeling and altered Ca homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Magnesium ion (Mg), a natural Ca antagonist and a cofactor for numerous enzymes, is crucial for regulating diverse cellular functions, but its roles in PH remains unclear. Here, we examined the roles of Mg and its transporters in PH development. Chronic hypoxia and monocrotaline induced significant PH in adult male rats. It was associated with a reduction of [Mg] in PASMCs, a significant increase in gene expressions of , , , , , , , , ,and ; upregulation of SLC41A1, SLC41A2, CNNM2, and TRPM7 proteins; and downregulation of SLC41A3 mRNA and protein. Mg supplement attenuated pulmonary arterial pressure, right heart hypertrophy, and medial wall thickening of pulmonary arteries, and reversed the changes in the expression of Mg transporters. Incubation of PASMCs with a high concentration of Mg markedly inhibited PASMC proliferation and migration, and increased apoptosis, whereas a low level of Mg produced the opposite effects. siRNA targetingattenuated PASMC proliferation and migration, but promoted apoptosis; andoverexpression also caused similar effects. Moreover, siRNA targetingor high [Mg] incubation inhibited hypoxia-induced upregulation and nuclear translocation of NFATc3 in PASMCs. The results, for the first time, provide the supportive evidence that Mg transporters participate in the development of PH by modulating PASMC proliferation, migration, and apoptosis; and Mg supplementation attenuates PH through regulation of Mg transporters involving the NFATc3 signaling pathway.



Hypertension: 27 Dec 2020:HYPERTENSIONAHA12014909; epub ahead of print
Wang D, Zhu ZL, Lin DC, Zheng SY, ... Sham JSK, Lin MJ
Hypertension: 27 Dec 2020:HYPERTENSIONAHA12014909; epub ahead of print | PMID: 33356397
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Impact:
Abstract

CROT (Carnitine O-Octanoyltransferase) Is a Novel Contributing Factor in Vascular Calcification via Promoting Fatty Acid Metabolism and Mitochondrial Dysfunction.

Okui T, Iwashita M, Rogers MA, Halu A, ... Singh SA, Aikawa E
Objective
Vascular calcification is a critical pathology associated with increased cardiovascular event risk, but there are no Food and Drug Administration-approved anticalcific therapies. We hypothesized and validated that an unbiased screening approach would identify novel mediators of human vascular calcification. Approach and
Results:
We performed an unbiased quantitative proteomics and pathway network analysis that identified increased CROT (carnitine O-octanoyltransferase) in calcifying primary human coronary artery smooth muscle cells (SMCs). Additionally, human carotid artery atherosclerotic plaques contained increased immunoreactive CROT near calcified regions.siRNA reduced fibrocalcific response in calcifying SMCs. In agreement, histidine 327 to alanine point mutation inactivated human CROT fatty acid metabolism enzymatic activity and suppressed SMC calcification.siRNA suppressed type 1 collagen secretion, and restored mitochondrial proteome alterations, and suppressed mitochondrial fragmentation in calcifying SMCs. Lipidomics analysis of SMCs incubated withsiRNA revealed increased eicosapentaenoic acid, a vascular calcification inhibitor. CRISPR/Cas9-mediateddeficiency in LDL (low-density lipoprotein) receptor-deficient mice reduced aortic and carotid artery calcification without altering bone density or liver and plasma cholesterol and triglyceride concentrations.
Conclusions
CROT is a novel contributing factor in vascular calcification via promoting fatty acid metabolism and mitochondrial dysfunction, as such CROT inhibition has strong potential as an antifibrocalcific therapy.



Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315007; epub ahead of print
Okui T, Iwashita M, Rogers MA, Halu A, ... Singh SA, Aikawa E
Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315007; epub ahead of print | PMID: 33356393
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Impact:
Abstract

Effects of Evolocumab on the Postprandial Kinetics of Apo (Apolipoprotein) B100- and B48-Containing Lipoproteins in Subjects With Type 2 Diabetes.

Taskinen MR, Björnson E, Kahri J, Söderlund S, ... Packard CJ, Borén J
Objective
Increased risk of atherosclerotic cardiovascular disease in subjects with type 2 diabetes is linked to elevated levels of triglyceride-rich lipoproteins and their remnants. The metabolic effects of PCSK9 (proprotein convertase subtilisin/kexin 9) inhibitors on this dyslipidemia were investigated using stable-isotope-labeled tracers. Approach and
Results:
Triglyceride transport and the metabolism of apos (apolipoproteins) B48, B100, C-III, and E after a fat-rich meal were investigated before and on evolocumab treatment in 13 subjects with type 2 diabetes. Kinetic parameters were determined for the following: apoB48 in chylomicrons; triglyceride in VLDL (very low-density lipoprotein) and VLDL; and apoB100 in VLDL, VLDL, IDL (intermediate-density lipoprotein), and LDL (low-density lipoprotein). Evolocumab did not alter the kinetics of apoB48 in chylomicrons or apoB100 or triglyceride in VLDL. In contrast, the fractional catabolic rates of VLDL-apoB100 and VLDL-triglyceride were both increased by about 45%, which led to a 28% fall in the VLDL plasma level. LDL-apoB100 was markedly reduced by evolocumab, which was linked to metabolic heterogeneity in this fraction. Evolocumab increased clearance of the more rapidly metabolized LDL by 61% and decreased production of the more slowly cleared LDL by 75%. ApoC-III kinetics were not altered by evolocumab, but the apoE fractional catabolic rates increased by 45% and the apoE plasma level fell by 33%. The apoE fractional catabolic rates was associated with the decrease in VLDL- and IDL-apoB100 concentrations.
Conclusions
Evolocumab had only minor effects on lipoproteins that are involved in triglyceride transport (chylomicrons and VLDL) but, in contrast, had a profound impact on lipoproteins that carry cholesterol (VLDL, IDL, LDL). Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02948777.



Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315446; epub ahead of print
Taskinen MR, Björnson E, Kahri J, Söderlund S, ... Packard CJ, Borén J
Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315446; epub ahead of print | PMID: 33356392
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Impact:
Abstract

Reduction of Connexin 43 Attenuates Angiogenic Effects of Human Smooth Muscle Progenitor Cells via Inactivation of Akt and NF-κB Pathway.

Tien TY, Wu YJ, Su CH, Wang HH, ... Su Y, Yeh HI
Objective
Circulating progenitor cells possess vasculogenesis property and participate in repair of vascular injury. Cx (connexin) 43-a transmembrane protein constituting gap junctions-is involved in vascular pathology. However, the role of Cx43 in smooth muscle progenitor cells (SPCs) remained unclear. Approach and
Results:
Human SPCs cultured from CD34 peripheral blood mononuclear cells expressed smooth muscle cell markers, such as smooth muscle MHC (myosin heavy chain), nonmuscle MHC, calponin, and CD140B, and Cx43 was the most abundant Cx isoform. To evaluate the role of Cx43 in SPCs, short interference RNA was used to knock down Cx43 expression. Cellular activities of SPCs were reduced by Cx43 downregulation. In addition, Cx43 downregulation attenuated angiogenic potential of SPCs in hind limb ischemia mice. Protein array and ELISA of the supernatant from SPCs showed that IL (interleukin)-6, IL-8, and HGF (hepatocyte growth factor) were reduced by Cx43 downregulation. Simultaneously, Cx43 downregulation reduced the phosphorylation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Akt (protein kinase B) pathway and reactivation of NF-κB and Akt using betulinic acid, and SC79 could restore the secretion of growth factors and cytokines. Moreover, FAK (focal adhesion kinase)-Src (proto-oncogene tyrosine-protein kinase Src) activation was increased by Cx43 downregulation, and inactivation of Akt-NF-κB could be restored by Src inhibitor (PP2), indicating that Akt-NF-κB inactivated by Cx43 downregulation arose from FAK-Src activation. Finally, the depressed cellular activities and secretion of SPCs after Cx43 downregulation were restored by FAK inhibitor PF-562271 or PP2.
Conclusions
SPCs possess angiogenic potential to repair ischemic tissue mainly through paracrine effects. Gap junction protein Cx43 plays an important role in regulating cellular function and paracrine effects of SPCs through FAK-Src axis.



Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315650; epub ahead of print
Tien TY, Wu YJ, Su CH, Wang HH, ... Su Y, Yeh HI
Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315650; epub ahead of print | PMID: 33356390
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Impact:
Abstract

Intracellular AIBP (Apolipoprotein A-I Binding Protein) Regulates Oxidized LDL (Low-Density Lipoprotein)-Induced Mitophagy in Macrophages.

Choi SH, Agatisa-Boyle C, Gonen A, Kim A, ... Tsimikas S, Miller YI
Objective
Atherosclerotic lesions are often characterized by accumulation of OxLDL (oxidized low-density lipoprotein), which is associated with vascular inflammation and lesion vulnerability to rupture. Extracellular AIBP (apolipoprotein A-I binding protein; encoded bygene), when secreted, promotes cholesterol efflux and regulates lipid rafts dynamics, but its role as an intracellular protein in mammalian cells remains unknown. The aim of this work was to determine the function of intracellular AIBP in macrophages exposed to OxLDL and in atherosclerotic lesions. Approach and
Results:
Using a novel monoclonal antibody against human and mouse AIBP, which are highly homologous, we demonstrated robust AIBP expression in human and mouse atherosclerotic lesions. We observed significantly reduced autophagy in bone marrow-derived macrophages, isolated fromcompared with wild-type mice, which were exposed to OxLDL. In atherosclerotic lesions frommice subjected toknockdown and fed a Western diet, autophagy was reduced, whereas apoptosis was increased, when compared with that in wild-type mice. AIBP expression was necessary for efficient control of reactive oxygen species and cell death and for mitochondria quality control in macrophages exposed to OxLDL. Mitochondria-localized AIBP, via its N-terminal domain, associated with E3 ubiquitin-protein ligase PARK2 (Parkin), MFN (mitofusin)1, and MFN2, but not BNIP3 (Bcl2/adenovirus E1B 19-kDa-interacting protein-3), and regulated ubiquitination of MFN1 and MFN2, key components of mitophagy.
Conclusions
These data suggest that intracellular AIBP is a new regulator of autophagy in macrophages. Mitochondria-localized AIBP augments mitophagy and participates in mitochondria quality control, protecting macrophages against cell death in the context of atherosclerosis.



Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315485; epub ahead of print
Choi SH, Agatisa-Boyle C, Gonen A, Kim A, ... Tsimikas S, Miller YI
Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315485; epub ahead of print | PMID: 33356389
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Impact:
Abstract

Novel Dual Reporter Mouse Model Provides Definitive Labeling and Identification of Smooth Muscle Cells.

Ruan J, Zhang L, Hu D, Qu X, ... Zhou J, Cai CL
Objective
encodes a myosin heavy chain protein that is specifically expressed in smooth muscle cells (SMCs) and is important for maintaining vascular wall stability. The goal of this study is to generate adual reporter mouse line for definitive visualization of MYH11 SMCs in vivo. Approach and
Results:
We generated aknock-in mouse model by insertingreporter cassette into thegene locus. The nuclear (n)cassette is flanked by 2sites followed by H2B-GFP (histone 2B fused green fluorescent protein). Upon Cre-mediated recombination,cassette is removed thereby permitting nucleus localized H2B-GFP expression. Expression of the nuclear localized lacZ or H2B-GFP is under control of the endogenouspromoter. Nuclear lacZ was expressed specifically in SMCs at embryonic and adult stages. Following germline Cre-mediated deletion of nuclear lacZ, H2B-GFP was specifically expressed in the nuclei of SMCs. Comparison of nuclear lacZ expression withandmediated-H2B-GFP expression revealed heterogenous origins of SMCs from neural crest and second heart field in the great arteries and coronary vessels adjacent to aortic root.
Conclusions
Theknock-in dual reporter mouse model offers an exceptional genetic tool to visualize and trace the origins of SMCs in mice.



Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315107; epub ahead of print
Ruan J, Zhang L, Hu D, Qu X, ... Zhou J, Cai CL
Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315107; epub ahead of print | PMID: 33356387
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Impact:
Abstract

Runx2 (Runt-Related Transcription Factor 2) Links the DNA Damage Response to Osteogenic Reprogramming and Apoptosis of Vascular Smooth Muscle Cells.

Cobb AM, Yusoff S, Hayward R, Ahmad S, ... D\'Haese PC, Shanahan CM
Objective
The development of ectopic vascular calcification is strongly linked with organismal aging, which is primarily caused by the accumulation of DNA damage over time. As Runx2 (Runt-related transcription factor 2) has been identified as a regulator of vascular smooth muscle cell osteogenic transition, a key component of vascular calcification, we examined the relationship between DNA damage and Runx2 activation. Approach and
Results:
We found genotoxic stress-stimulated Runx2 accumulation and transactivation of its osteogenic target genes, leading to enhanced calcification. Inhibition of DNA damage signaling attenuated this response. Runx2 localized to sites of DNA damage and participated in DNA repair by regulating phosphorylation events on histone H2AX, with exogenous expression of Runx2 resulting in unrepaired DNA damage and increased apoptosis. Mechanistically, Runx2 was PARylated in response to genotoxic stress, and inhibition of this modification disrupted its localization at DNA lesions and reduced its binding to osteogenic gene promoters.
Conclusions
These data identify Runx2 as a novel component of the DNA damage response, coupling DNA damage signaling to both osteogenic gene transcription and apoptosis and providing a mechanism for accelerated mineralization in aging and chronic disease.



Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315206; epub ahead of print
Cobb AM, Yusoff S, Hayward R, Ahmad S, ... D'Haese PC, Shanahan CM
Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315206; epub ahead of print | PMID: 33356386
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Impact:
Abstract

Targeted Molecular Imaging of Cardiovascular Diseases by Iron Oxide Nanoparticles.

Vazquez-Prada KX, Lam J, Kamato D, Ping Xu Z, Little PJ, Ta HT

Cardiovascular disease is one of the major contributors to global disease burden. Atherosclerosis is an inflammatory process that involves the accumulation of lipids and fibrous elements in the large arteries, forming an atherosclerotic plaque. Rupture of unstable plaques leads to thrombosis that triggers life-threatening complications such as myocardial infarction. Current diagnostic methods are invasive as they require insertion of a catheter into the coronary artery. Molecular imaging techniques, such as magnetic resonance imaging, have been developed to image atherosclerotic plaques and thrombosis due to its high spatial resolution and safety. The sensitivity of magnetic resonance imaging can be improved with contrast agents, such as iron oxide nanoparticles. This review presents the most recent advances in atherosclerosis, thrombosis, and myocardial infarction molecular imaging using iron oxide-based nanoparticles. While some studies have shown their effectiveness, many are yet to undertake comprehensive testing of biocompatibility. There are still potential hazards to address and complications to diagnosis, therefore strategies for overcoming these challenges are required.



Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315404; epub ahead of print
Vazquez-Prada KX, Lam J, Kamato D, Ping Xu Z, Little PJ, Ta HT
Arterioscler Thromb Vasc Biol: 23 Dec 2020:ATVBAHA120315404; epub ahead of print | PMID: 33356385
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Impact:
Abstract

Electronic Cigarettes Induce Mitochondrial DNA Damage and Trigger TLR 9 (Toll-Like Receptor 9)-Mediated Atherosclerosis.

Li J, Huynh L, Cornwell WD, Tang MS, ... Liu C, Wang H
Objective
Electronic cigarette (e-cig) use has recently been implicated in promoting atherosclerosis. In this study, we aimed to investigate the mechanism of e-cig exposure accelerated atherosclerotic lesion development. Approach and
Results:
Eight-week-old ApoE mice fed normal laboratory diet were exposed to e-cig vapor (ECV) for 2 hours/day, 5 days/week for 16 weeks. We found that ECV exposure significantly induced atherosclerotic lesions as examined by Oil Red O staining and greatly upregulated TLR9 (toll-like receptor 9) expression in classical monocytes and in the atherosclerotic plaques, which the latter was corroborated by enhanced TLR9 expression in human femoral artery atherosclerotic plaques from e-cig smokers. Intriguingly, we found a significant increase of oxidative mitochondria DNA lesion in the plasma of ECV-exposed mice. Administration of TLR9 antagonist before ECV exposure not only alleviated atherosclerosis and the upregulation of TLR9 in plaques but also attenuated the increase of plasma levels of inflammatory cytokines, reduced the plaque accumulation of lipid and macrophages, and decreased the frequency of blood CCR2 classical monocytes. Surprisingly, we found that cytoplasmic mitochondrial DNA isolated from ECV extract-treated macrophages can enhance TLR9 activation in reporter cells and the induction of inflammatory cytokine could be suppressed by TLR9 inhibitor in macrophages.
Conclusions
E-cig increases level of damaged mitochondrial DNA in circulating blood and induces the expression of TLR9, which elevate the expression of proinflammatory cytokines in monocyte/macrophage and consequently lead to atherosclerosis. Our results raise the possibility that intervention of TLR9 activation is a potential pharmacological target of ECV-related inflammation and cardiovascular diseases.



Arterioscler Thromb Vasc Biol: 30 Dec 2020:ATVBAHA120315556; epub ahead of print
Li J, Huynh L, Cornwell WD, Tang MS, ... Liu C, Wang H
Arterioscler Thromb Vasc Biol: 30 Dec 2020:ATVBAHA120315556; epub ahead of print | PMID: 33380174
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Impact:
Abstract

Helix-Loop-Helix Factor Id3 (Inhibitor of Differentiation 3): A Novel Regulator of Hyaluronan-Mediated Adipose Tissue Inflammation.

Misiou A, Garmey JC, Hensien JM, Harmon DB, ... Grandoch M, McNamara CA
Objective
The aim of this study was to unravel mechanisms whereby deficiency of the transcription factor Id3 (inhibitor of differentiation 3) leads to metabolic dysfunction in visceral obesity. We investigated the impact of loss of Id3 on hyaluronic acid (HA) production by the 3 HAS (HA synthases; -1, -2, and -3) and on obesity-induced adipose tissue (AT) accumulation of proinflammatory B cells. Approach and
Results:
Malemice and respective wild-type littermate controls were fed a 60% high-fat diet for 4 weeks. An increase in inflammatory B2 cells was detected inepididymal AT. HA accumulated in epididymal AT of high-fat diet-fedmice and circulating levels of HA were elevated.mRNA expression was increased in epididymal AT ofmice. Luciferase promoter assays showed that Id3 suppressedpromoter activity, while loss ofstimulatedpromoter activity. Functionally, HA strongly promoted B2 cell adhesion in the AT and on cultured vascular smooth muscle cells ofmice, an effect sensitive to hyaluronidase.
Conclusions
Our data demonstrate that loss ofincreasesexpression in the epididymal AT, thereby promoting HA accumulation. In turn, elevated HA content promotes HA-dependent binding of B2 cells and an increase in the B2 cells in the AT, which contributes to AT inflammation.



Arterioscler Thromb Vasc Biol: 30 Dec 2020:ATVBAHA120315588; epub ahead of print
Misiou A, Garmey JC, Hensien JM, Harmon DB, ... Grandoch M, McNamara CA
Arterioscler Thromb Vasc Biol: 30 Dec 2020:ATVBAHA120315588; epub ahead of print | PMID: 33380173
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Impact:
Abstract

Impact of Age, Menopause, and Obesity on Oxylipins Linked to Vascular Health.

Pauls SD, Du Y, Clair L, Winter T, ... Taylor CG, Zahradka P
Objective
Cardiovascular disease, a major cause of mortality and morbidity, exhibits sexual dimorphism since the onset of cardiovascular disease occurs later in women than in men. The loss of cardioprotection in older women may be due to an increase in arterial stiffness after menopause. Free fatty acid metabolites of polyunsaturated fatty acids, called oxylipins, are known to impact vessel function and may be responsible for the vascular benefits of polyunsaturated fatty acids. The objectives of this study were to compare the plasma oxylipin profiles of young females (20-55 years), older females (55), and older males (55) and to identify associations between oxylipins and cardiovascular disease risk factors, such as obesity and arterial stiffness. Approach and
Results:
We quantified plasma oxylipins by high-performance liquid chromatography-tandem mass spectrometry in archived samples taken from completed clinical trials. We identified 3 major 12-lipoxygenase products, 12-hydroxy-eicosatetraenoic acid, 12-hydroxy-eicosapentaenoic acid, and 14-hydroxy-docosahexaenoic acid, that are present at high levels in young females compared with older females and males. These oxylipins also decreased with obesity and displayed robust negative associations with arterial stiffness as assessed by brachial-ankle pulse wave velocity. According to multiple linear regression modeling, these associations were maintained even after correcting for body mass index category combined with either age, menopausal status, or estradiol levels. Using linear discriminant analysis, the combination of these 3 oxylipins effectively distinguished participants according to both brachial-ankle pulse wave velocity risk group and age.
Conclusions
Higher 12-lipoxygenase oxylipin plasma concentrations associated with lower arterial stiffness in premenopausal females may be an important contributing factor to sex differences in cardiovascular disease.
Registration
URL: https://www.clinicaltrials.gov; Unique identifiers: NCT01661543, NCT01562171, NCT01890330, NCT02571114 and NCT02317588.



Arterioscler Thromb Vasc Biol: 30 Dec 2020:ATVBAHA120315133; epub ahead of print
Pauls SD, Du Y, Clair L, Winter T, ... Taylor CG, Zahradka P
Arterioscler Thromb Vasc Biol: 30 Dec 2020:ATVBAHA120315133; epub ahead of print | PMID: 33380172
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Impact:
Abstract

Trained Immunity and Reactivity of Macrophages and Endothelial Cells.

Drummer C, Saaoud F, Shao Y, Sun Y, ... Wang H, Yang X

Innate immune cells can develop exacerbated immunologic response and long-term inflammatory phenotype following brief exposure to endogenous or exogenous insults, which leads to an altered response towards a second challenge after the return to a nonactivated state. This phenomenon is known as trained immunity (TI). TI is not only important for host defense and vaccine response but also for chronic inflammations such as cardiovascular and metabolic diseases such as atherosclerosis. TI can occur in innate immune cells such as monocytes/macrophages, natural killer cells, endothelial cells (ECs), and nonimmune cells, such as fibroblast. In this brief review, we analyze the significance of TI in ECs, which are also considered as innate immune cells in addition to macrophages. TI can be induced by a variety of stimuli, including lipopolysaccharides, bacillus Calmette-Guerin, and oxLDL (oxidized low-density lipoprotein), which are defined as risk factors for cardiovascular and metabolic diseases. Furthermore, TI in ECs is functional for inflammation effectiveness and transition to chronic inflammation. Rewiring of cellular metabolism of the trained cells takes place during induction of TI, including increased glycolysis, glutaminolysis, increased accumulation of tricarboxylic acid cycle metabolites and acetyl-coenzyme A production, as well as increased mevalonate synthesis. Subsequently, this leads to epigenetic remodeling, resulting in important changes in chromatin architecture that enables increased gene transcription and enhanced proinflammatory immune response. However, TI pathways and inflammatory pathways are separated to ensure memory stays when inflammation undergoes resolution. Additionally, reactive oxygen species play context-dependent roles in TI. Therefore, TI plays significant roles in EC and macrophage pathology and chronic inflammation. However, further characterization of TI in ECs and macrophages would provide novel insights into cardiovascular disease pathogenesis and new therapeutic targets.



Arterioscler Thromb Vasc Biol: 30 Dec 2020:ATVBAHA120315452; epub ahead of print
Drummer C, Saaoud F, Shao Y, Sun Y, ... Wang H, Yang X
Arterioscler Thromb Vasc Biol: 30 Dec 2020:ATVBAHA120315452; epub ahead of print | PMID: 33380171
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Impact:
Abstract

Loss of Chloride Channel 6 (CLC-6) Affects Vascular Smooth Muscle Contractility and Arterial Stiffness via Alterations to Golgi Calcium Stores.

Klemens CA, Chulkov EG, Wu J, Khan MAH, ... Palygin O, Staruschenko A

Genome-wide association studies have found a number of potential genes involved in blood pressure regulation; however, the functional role of many of these candidates has yet to be established. One such candidate gene is , which encodes the transmembrane protein, chloride channel 6 (ClC-6). Although thelocus has been widely associated with human blood pressure regulation, the mechanistic role of ClC-6 in blood pressure homeostasis at the molecular, cellular, and physiological levels is completely unknown. In this study, we demonstrate that rats with a functional knockout of ClC-6 on the Dahl Salt-Sensitive rat background (SS-) have lower diastolic but not systolic blood pressures. The effect of diastolic blood pressure attenuation was independent of dietary salt exposure in knockout animals. Moreover, SS- rats are protected from hypertension-induced cardiac hypertrophy and arterial stiffening; however, they have impaired vasodilation and dysregulated intracellular calcium handling. ClC-6 is highly expressed in vascular smooth muscle cells where it is targeted to the Golgi apparatus. Using bilayer electrophysiology, we provide evidence that recombinant human ClC-6 protein can function as a channel. Last, we demonstrate that loss of ClC-6 function reduces Golgi calcium stores, which may play a previously unidentified role in vascular contraction and relaxation signaling in vascular smooth muscle cells. Collectively, these data indicate that ClC-6 may modulate blood pressure by regulating Golgi calcium reserves, which in turn contribute to vascular smooth muscle function.



Hypertension: 03 Jan 2021:HYPERTENSIONAHA12016589; epub ahead of print
Klemens CA, Chulkov EG, Wu J, Khan MAH, ... Palygin O, Staruschenko A
Hypertension: 03 Jan 2021:HYPERTENSIONAHA12016589; epub ahead of print | PMID: 33390052
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Impact:
Abstract

Direct Actions of AT (Type 1 Angiotensin) Receptors in Cardiomyocytes Do Not Contribute to Cardiac Hypertrophy.

Sparks MA, Rianto F, Diaz E, Revoori R, ... Crowley SD, Coffman TM

Activation of AT (type 1 Ang) receptors stimulates cardiomyocyte hypertrophy in vitro. Accordingly, it has been suggested that regression of cardiac hypertrophy associated with renin-Ang system blockade is due to inhibition of cellular actions of Ang II in the heart, above and beyond their effects to reduce pressure overload. We generated 2 distinct mouse lines with cell-specific deletion of AT receptors, from cardiomyocytes. In the first line (C-SMKO), elimination of AT receptors was achieved using a heterologoustransgene under control of thepromoter, which expresses in cells of smooth muscle lineage including cardiomyocytes and vascular smooth muscle cells of conduit but not resistance vessels. The second line (R-SMKO) utilized atransgene knocked-in to thelocus, which drives expression in cardiac myocytes and vascular smooth muscle cells in both conduit and resistance arteries. Thus, although both groups lack AT receptors in the cardiomyocytes, they are distinguished by presence (C-SMKO) or absence (R-SMKO) of peripheral vascular responses to Ang II. Similar to wild-types, chronic Ang II infusion caused hypertension and cardiac hypertrophy in C-SMKO mice, whereas both hypertension and cardiac hypertrophy were reduced in R-SMKOs. Thus, despite the absence of AT receptors in cardiomyocytes, C-SMKOs develop robust cardiac hypertrophy. By contrast, R-SMKOs developed identical levels of hypertrophy in response to pressure overload-induced by transverse aortic banding. Our findings suggest that direct activation of AT receptors in cardiac myocytes has minimal influence on cardiac hypertrophy induced by renin-Ang system activation or pressure overload.



Hypertension: 03 Jan 2021:HYPERTENSIONAHA11914079; epub ahead of print
Sparks MA, Rianto F, Diaz E, Revoori R, ... Crowley SD, Coffman TM
Hypertension: 03 Jan 2021:HYPERTENSIONAHA11914079; epub ahead of print | PMID: 33390039
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Impact:
Abstract

The Long Noncoding RNA RP11-728F11.4 Promotes Atherosclerosis.

Dong XH, Lu ZF, Kang CM, Li XH, ... Ye S, Hu YW
Objective
Noncoding RNAs are emerging as important players in gene regulation and cardiovascular diseases. Their roles in the pathogenesis of atherosclerosis are not fully understood. The purpose of this study was to determine the role played by a previously uncharacterized long noncoding RNA, RP11-728F11.4, in the development of atherosclerosis and the mechanisms by which it acts. Approach and
Results:
Expression microarray analysis revealed that atherosclerotic plaques had increased expression of RP11-728F11.4 as well as the cognate gene(FXYD domain containing ion transport regulator 6), which encodes a modulator of Na/K-ATPase. In vitro experiments showed that RP11-728F11.4 interacted with the RNA-binding protein EWSR1 (Ewings sarcoma RNA binding protein-1) and upregulatedexpression. Lentivirus-induced overexpression of RP11-728F11.4 in cultured monocytes-derived macrophages resulted in higher Na/K-ATPase activity, intracellular cholesterol accumulation, and increased proinflammatory cytokine production. The effects of RP11-728F11.4 were enhanced by siRNA-mediated knockdown of EWSR1 and reduced by downregulation of FXYD domain containing ion transport regulator 6. In vivo experiments in apoE knockout mice fed a Western diet demonstrated that RP11-728F11.4 increased proinflammatory cytokine production and augmented atherosclerotic lesions.
Conclusions
RP11-728F11.4 promotes atherosclerosis, with an influence on cholesterol homeostasis and proinflammatory molecule production, thus representing a potential therapeutic target.



Arterioscler Thromb Vasc Biol: 05 Jan 2021:ATVBAHA120315114; epub ahead of print
Dong XH, Lu ZF, Kang CM, Li XH, ... Ye S, Hu YW
Arterioscler Thromb Vasc Biol: 05 Jan 2021:ATVBAHA120315114; epub ahead of print | PMID: 33406853
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Impact:
Abstract

MEF2 (Myocyte Enhancer Factor 2) Is Essential for Endothelial Homeostasis and the Atheroprotective Gene Expression Program.

Lu YW, Martino N, Gerlach BD, Lamar JM, ... Adam AP, Schwarz JJ
Objective
Atherosclerosis predominantly forms in regions of oscillatory shear stress while regions of laminar shear stress are protected. This protection is partly through the endothelium in laminar flow regions expressing an anti-inflammatory and antithrombotic gene expression program. Several molecular pathways transmitting these distinct flow patterns to the endothelium have been defined. Our objective is to define the role of the MEF2 (myocyte enhancer factor 2) family of transcription factors in promoting an atheroprotective endothelium. Approach and
Results:
Here, we show through endothelial-specific deletion of the 3 MEF2 factors in the endothelium, Mef2a, -c, and -d, that MEF2 is a critical regulator of vascular homeostasis. MEF2 deficiency results in systemic inflammation, hemorrhage, thrombocytopenia, leukocytosis, and rapid lethality. Transcriptome analysis reveals that MEF2 is required for normal regulation of 3 pathways implicated in determining the flow responsiveness of the endothelium. Specifically, MEF2 is required for expression of Klf2 and Klf4, 2 partially redundant factors essential for promoting an anti-inflammatory and antithrombotic endothelium. This critical requirement results in phenotypic similarities between endothelial-specific deletions of Mef2a/c/d and Klf2/4. In addition, MEF2 regulates the expression of Notch family genes, Notch1, Dll1, and Jag1, which also promote an atheroprotective endothelium. In contrast to these atheroprotective pathways, MEF2 deficiency upregulates an atherosclerosis promoting pathway through increasing the amount of TAZ (transcriptional coactivator with PDZ-binding motif).
Conclusions
Our results implicate MEF2 as a critical upstream regulator of several transcription factors responsible for gene expression programs that affect development of atherosclerosis and promote an anti-inflammatory and antithrombotic endothelium.



Arterioscler Thromb Vasc Biol: 31 Dec 2020:ATVBAHA120314978; epub ahead of print
Lu YW, Martino N, Gerlach BD, Lamar JM, ... Adam AP, Schwarz JJ
Arterioscler Thromb Vasc Biol: 31 Dec 2020:ATVBAHA120314978; epub ahead of print | PMID: 33406884
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Impact:
Abstract

ODC (Ornithine Decarboxylase)-Dependent Putrescine Synthesis Maintains MerTK (MER Tyrosine-Protein Kinase) Expression to Drive Resolution.

Yurdagul A, Kong N, Gerlach BD, Wang X, ... Shi J, Tabas I
Objective
ODC (ornithine decarboxylase)-dependent putrescine synthesis promotes the successive clearance of apoptotic cells (ACs) by macrophages, contributing to inflammation resolution. However, it remains unknown whether ODC is required for other arms of the resolution program. Approach and
Results:
RNA sequencing of ODC-deficient macrophages exposed to ACs showed increases in mRNAs associated with heightened inflammation and decreases in mRNAs related to resolution and repair compared with WT (wild type) macrophages. In zymosan peritonitis, myeloid ODC deletion led to delayed clearance of neutrophils and a decrease in the proresolving cytokine, IL (interleukin)-10. Nanoparticle-mediated silencing of macrophage ODC in a model of atherosclerosis regression lowered IL-10 expression, decreased efferocytosis, enhanced necrotic core area, and reduced fibrous cap thickness. Mechanistically, ODC deletion lowered basal expression of MerTK (MER tyrosine-protein kinase)-an AC receptor-via a histone methylation-dependent transcriptional mechanism. Owing to lower basal MerTK, subsequent exposure to ACs resulted in lower MerTK-Erk (extracellular signal-regulated kinase) 1/2-dependent IL-10 production. Putrescine treatment of ODC-deficient macrophages restored the expression of both MerTK and AC-induced IL-10.
Conclusions
These findings demonstrate that ODC-dependent putrescine synthesis in macrophages maintains a basal level of MerTK expression needed to optimally resolve inflammation upon subsequent AC exposure.



Arterioscler Thromb Vasc Biol: 05 Jan 2021:ATVBAHA120315622; epub ahead of print
Yurdagul A, Kong N, Gerlach BD, Wang X, ... Shi J, Tabas I
Arterioscler Thromb Vasc Biol: 05 Jan 2021:ATVBAHA120315622; epub ahead of print | PMID: 33406854
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Impact:
Abstract

Role of Klotho in the Development of Essential Hypertension.

Kanbay M, Demiray A, Afsar B, Covic A, ... Ureche C, Ortiz A

Klotho has antiaging properties, and serum levels decrease with physiological aging and aging-related diseases, such as hypertension, cardiovascular, and chronic kidney disease. Klotho deficiency in mice results in accelerated aging and cardiovascular injury, whereas Klotho supplementation slows down the progression of aging-related diseases. The pleiotropic functions of Klotho include, but are not limited to, inhibition of insulin/IGF-1 (insulin-like growth factor 1) and WNT (wingless-related integration site) signaling pathways, suppression of oxidative stress and aldosterone secretion, regulation of calcium-phosphate homeostasis, and modulation of autophagy with inhibition of apoptosis, fibrosis, and cell senescence. Accumulating evidence shows an interconnection between Klotho deficiency and hypertension, and Klotho gene polymorphisms are associated with hypertension in humans. In this review, we critically review the current understanding of the role of Klotho in the development of essential hypertension and the most important underlying pathways involved, such as the FGF23 (fibroblast growth factor 23)/Klotho axis, aldosterone, Wnt5a/RhoA, and SIRT1 (Sirtuin1). Based on this critical review, we suggest avenues for further research.



Hypertension: 10 Jan 2021:HYPERTENSIONAHA12016635; epub ahead of print
Kanbay M, Demiray A, Afsar B, Covic A, ... Ureche C, Ortiz A
Hypertension: 10 Jan 2021:HYPERTENSIONAHA12016635; epub ahead of print | PMID: 33423524
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Impact:
Abstract

Apoptotic Ablation of Platelets Reduces Atherosclerosis in Mice With Diabetes.

Lee MKS, Kraakman MJ, Dragoljevic D, Hanssen NMJ, ... Nagareddy PR, Murphy AJ
Objective
People with diabetes are at a significantly higher risk of cardiovascular disease, in part, due to accelerated atherosclerosis. Diabetic subjects have increased number of platelets that are activated, more reactive, and respond suboptimally to antiplatelet therapies. We hypothesized that reducing platelet numbers by inducing their premature apoptotic death would decrease atherosclerosis. Approach and
Results:
This was achieved by targeting the antiapoptotic protein Bcl-x (B-cell lymphoma-extra large; which is essential for platelet viability) via distinct genetic and pharmacological approaches. In the former, we transplanted bone marrow from mice carrying the Tyr15 to Cys loss of function allele of Bcl-x (known as ) or wild-type littermate controls into atherosclerotic-pronemice made diabetic with streptozotocin and fed a Western diet. Reduced Bcl-x function in hematopoietic cells significantly decreased platelet numbers, exclusive of other hematologic changes. This led to a significant reduction in atherosclerotic lesion formation inbone marrow transplantedmice. To assess the potential therapeutic relevance of reducing platelets in atherosclerosis, we next targeted Bcl-x with a pharmacological strategy. This was achieved by low-dose administration of the BH3 (B-cell lymphoma-2 homology domain 3) mimetic, ABT-737 triweekly, in diabeticmice for the final 6 weeks of a 12-week study. ABT-737 normalized platelet numbers along with platelet and leukocyte activation to that of nondiabetic controls, significantly reducing atherosclerosis while promoting a more stable plaque phenotype.
Conclusions
These studies suggest that selectively reducing circulating platelets, by targeting Bcl-x to promote platelet apoptosis, can reduce atherosclerosis and lower cardiovascular disease risk in diabetes.



Arterioscler Thromb Vasc Biol: 13 Jan 2021:ATVBAHA120315369; epub ahead of print
Lee MKS, Kraakman MJ, Dragoljevic D, Hanssen NMJ, ... Nagareddy PR, Murphy AJ
Arterioscler Thromb Vasc Biol: 13 Jan 2021:ATVBAHA120315369; epub ahead of print | PMID: 33441028
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Impact:
Abstract

Platelet Proteomes, Pathways, and Phenotypes as Informants of Vascular Wellness and Disease.

Aslan JE

Platelets rapidly undergo responsive transitions in form and function to repair vascular endothelium and mediate hemostasis. In contrast, heterogeneous platelet subpopulations with a range of primed or refractory phenotypes gradually arise in chronic inflammatory and other conditions in a manner that may indicate or support disease. Qualitatively distinguishable platelet phenotypes are increasingly associated with a variety of physiological and pathological circumstances; however, the origins and significance of platelet phenotypic variation remain unclear and conceptually vague. As changes in platelet function in disease exhibit many similarities to platelets following the activation of platelet agonist receptors, the intracellular responses of platelets common to hemostasis and inflammation may provide insights to the molecular basis of platelet phenotype. Here, we review concepts around how protein-level relations-from platelet receptors through intracellular signaling events-may help to define platelet phenotypes in inflammation, immune responses, aging, and other conditions. We further discuss how representing systems-wide platelet proteomics data profiles as circuit-like networks of causally related intracellular events, or, pathway maps, may inform molecular definitions of platelet phenotype. In addition to offering insights into platelets as druggable targets, maps of causally arranged intracellular relations underlying platelet function can also advance precision and interceptive medicine efforts by leveraging platelets as accessible, dynamic, endogenous, circulating biomarkers of vascular wellness and disease.



Arterioscler Thromb Vasc Biol: 13 Jan 2021:ATVBAHA120314647; epub ahead of print
Aslan JE
Arterioscler Thromb Vasc Biol: 13 Jan 2021:ATVBAHA120314647; epub ahead of print | PMID: 33441027
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Abstract

E17241 as a Novel ABCA1 (ATP-Binding Cassette Transporter A1) Upregulator Ameliorates Atherosclerosis in Mice.

Xu Y, Liu C, Han X, Jia X, ... Hong B, Si S
Objective
Reverse cholesterol transport, removing excess cholesterol from peripheral tissues, is an important therapeutic target for atherosclerosis treatment. In this study, we propose a new small molecule, E17241, that may be used to treat atherosclerosis by promoting reverse cholesterol transport via ABCA1 (ATP-binding cassette transporter A1) upregulation. Approach and
Results:
E17241 (4-(1,3-dithiolan-2-yl)-N-(3-hydroxypyridin-2-yl)benzamide) was first identified as an ABCA1 upregulator using a cell-based reporter assay. E17241 significantly increases the mRNA and protein expression levels of ABCA1 in both hepatic cells and macrophages. It promotes cholesterol efflux to apo AI in macrophage cells, and this effect depends on ABCA1. It also decreases total cholesterol content in Ox-LDL (oxidized low-density lipoprotein) loading macrophage cells. E17241 treatment increases the content of H-labeled cholesterol in the feces of male C57BL/6J mice intraperitoneally injected with 3H-cholesterol-labeled macrophage J774 cells, indicating that it could promote in vivo macrophage reverse cholesterol transport. Compared with the western diet group (western diet-fed male ApoE mice), the E17241 group (western diet+E17241 treatment) shows decreased plasma cholesterol, liver cholesterol, and triglyceride levels, with increased fecal cholesterol content. Importantly, E17241 reduces atherosclerotic lesion areas in the en face aorta and aortic sinus while increasing ABCA1 protein levels in both liver and macrophages. Human proteome microarray, coimmunoprecipitation, and other assays demonstrate that PKCζ (protein kinase C zeta) is a binding target of E17241, and this small molecule increases ABCA1 expression in macrophages via the PKCζ-NR (nuclear receptor) pathway.
Conclusions
E17241 may be developed as a new lead or drug candidate for the treatment of atherosclerosis by upregulating ABCA1.



Arterioscler Thromb Vasc Biol: 13 Jan 2021:ATVBAHA120314156; epub ahead of print
Xu Y, Liu C, Han X, Jia X, ... Hong B, Si S
Arterioscler Thromb Vasc Biol: 13 Jan 2021:ATVBAHA120314156; epub ahead of print | PMID: 33441025
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Abstract

Single-Cell RNA Sequencing to Disentangle the Blood System.

Acosta J, Ssozi D, van Galen P

The blood system is often represented as a tree-like structure with stem cells that give rise to mature blood cell types through a series of demarcated steps. Although this representation has served as a model of hierarchical tissue organization for decades, single-cell technologies are shedding new light on the abundance of cell type intermediates and the molecular mechanisms that ensure balanced replenishment of differentiated cells. In this Brief Review, we exemplify new insights into blood cell differentiation generated by single-cell RNA sequencing, summarize considerations for the application of this technology, and highlight innovations that are leading the way to understand hematopoiesis at the resolution of single cells.



Arterioscler Thromb Vasc Biol: 13 Jan 2021:ATVBAHA120314654; epub ahead of print
Acosta J, Ssozi D, van Galen P
Arterioscler Thromb Vasc Biol: 13 Jan 2021:ATVBAHA120314654; epub ahead of print | PMID: 33441024
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Abstract

Different DOACs Control Inflammation in Cardiac Ischemia-Reperfusion Differently.

Gadi I, Fatima S, Elwakiel A, Nazir S, ... Isermann B, Shahzad K

While thrombin is the key protease in thrombus formation, other coagulation proteases, such as fXa or activated protein C (aPC), independently modulate intracellular signaling via partially distinct receptors.To study the differential effects of fXa or fIIa inhibition on gene expression and inflammation in myocardial ischemia-reperfusion injury (IRI).Mice were treated with a direct fIIa inhibitor (fIIai) or direct fXa inhibitor (fXai) at doses that induced comparable anticoagulant effects ex vivo and in vivo (tail bleeding assay and FeCl3-induced thrombosis). Myocardial IRI was induced via LAD ligation. We determined infarct size and in vivo aPC generation, analyzed gene expression by RNAseq, and performed immunoblotting and ELISA. The signaling-only 3K3A-aPC variant and inhibitory antibodies that blocked all or only the anticoagulant function of aPC were used to determine the role of aPC. Doses of fIIai and fXai that induced comparable anticoagulant effects resulted in a comparable reduction in infarct size. However, unbiased gene expression analyses revealed marked differences, including pathways related to sterile inflammation and inflammasome regulation. fXai but not fIIai inhibited sterile inflammation by reducing the expression of proinflammatory cytokines (IL-1beta, IL-6, and TNFalpha) as well as NF-κB and inflammasome activation. This anti-inflammatory effect was associated with reduced myocardial fibrosis 28 days post myocardial IRI. Mechanistically, in vivo aPC generation was higher with fXai than with fIIai. Inhibition of the anticoagulant and signaling properties of aPC abolished the anti-inflammatory effect associated with fXai, while inhibiting only the anticoagulant function of aPC had no effect. Combining 3K3A-aPC with fIIai reduced the inflammatory response, mimicking the fXai-associated effect.We showed that specific inhibition of coagulation via DOACs had differential effects on gene expression and inflammation, despite comparable anticoagulant effects and infarct sizes. Targeting individual coagulation proteases induces specific cellular responses unrelated to their anticoagulant effect.



Circ Res: 22 Dec 2020; epub ahead of print
Gadi I, Fatima S, Elwakiel A, Nazir S, ... Isermann B, Shahzad K
Circ Res: 22 Dec 2020; epub ahead of print | PMID: 33353373
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Abstract

Klotho Deficiency Causes Heart Aging via Impairing the Nrf2-GR Pathway.

Chen K, Wang S, Sun QW, Zhang B, Ullah MF, Sun Z

Cardiac aging is an important contributing factor for heart failure which affects a large population but remains poorly understood.The purpose of this study is to investigate whether Klotho plays a role in cardiac aging.Heart function declined in old mice (24 months), as evidenced by decreases in fractional shortening, ejection fraction, and cardiac output. Heart size and weight, cardiomyocyte size and cardiac fibrosis were increased in old mice, indicating that aging causes cardiac hypertrophy and remodeling. Circulating Klotho levels were dramatically decreased in old mice, which prompted us to investigate whether the Klotho decline may cause heart aging. We found that Klotho gene mutation (KL-/-) largely decreased serum klotho levels and impaired heart function. Interestingly, supplement of exogenous secreted Klotho prevented heart failure, hypertrophy, and remodeling in both old mice and KL (-/-) mice. Secreted Klotho treatment inhibited excessive cardiac oxidative stress, senescence and apoptosis in old mice and KL (-/-) mice. Serum phosphate levels in KL (-/-) mice were kept in the normal range, suggesting that Klotho deficiency-induced heart aging is independent of phosphate metabolism. Mechanistically, Klotho deficiency suppressed glutathione reductase (GR) expression and activity in the heart via inhibition of transcription factor Nrf2. Furthermore, cardiac-specific overexpression of GR prevented excessive oxidative stress, apoptosis, and heart failure in both old and KL (-/-) mice.Klotho deficiency causes cardiac aging via impairing the Nrf2-GR pathway. Supplement of exogenous secreted Klotho represents a promising therapeutic strategy for aging-associated cardiomyopathy and heart failure.



Circ Res: 17 Dec 2020; epub ahead of print
Chen K, Wang S, Sun QW, Zhang B, Ullah MF, Sun Z
Circ Res: 17 Dec 2020; epub ahead of print | PMID: 33334122
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Abstract

Structural and Functional Characterization of A Nav1.5-Mitochondrial Couplon.

Pérez-Hernández Duran M, Leo-Macias A, Keegan S, Jouni M, ... Rothenberg E, Delmar M

The cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined.To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes, and the subjacent subsarcolemmal mitochondria (SSM). Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to SSM; we further found that SSM preferentially host the mitochondrial Na+/Ca2+ exchanger (NCLX). This anatomical proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased ROS production when compared to interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome dataset (GTEx) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A.We describe an anatomical hub (a couplon) formed by sodium channel clusters and SSM. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.



Circ Res: 20 Dec 2020; epub ahead of print
Pérez-Hernández Duran M, Leo-Macias A, Keegan S, Jouni M, ... Rothenberg E, Delmar M
Circ Res: 20 Dec 2020; epub ahead of print | PMID: 33342222
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Abstract

SIRT6 Protects Smooth Muscle Cells from Senescence and Reduces Atherosclerosis.

Grootaert M, Finigan A, Figg N, Uryga AK, Bennett M

Vascular smooth muscle cell (VSMC) senescence promotes atherosclerosis and features of plaque instability, in part through lipid-mediated oxidative DNA damage and telomere dysfunction. Sirtuin 6 (SIRT6) is a nuclear deacetylase involved in DNA damage response signaling, inflammation and metabolism; however, its role in regulating VSMC senescence and atherosclerosis is unclear.We examined SIRT6 expression in human VSMCs (hVSMCs), the role, regulation and downstream pathways activated by SIRT6, and how VSMC SIRT6 regulates atherogenesis.SIRT6 protein, but not mRNA, expression was markedly reduced in VSMCs in human and mouse atherosclerotic plaques, and in hVSMCs derived from plaques or undergoing replicative or palmitate-induced senescence vs. healthy aortic VSMCs. The ubiquitin ligase CHIP promoted SIRT6 stability, but CHIP expression was reduced in human and mouse plaque VSMCs and by palmitate in a p38- and c-Jun N-terminal kinase-dependent manner. SIRT6 bound to telomeres, while SIRT6 inhibition using shRNA or a deacetylase-inactive mutant (SIRT6) shortened hVSMC lifespan and induced senescence, associated with telomeric H3K9 hyperacetylation and 53BP1 binding, indicative of telomere damage. In contrast, SIRT6 overexpression preserved telomere integrity, delayed cellular senescence, and reduced inflammatory cytokine expression and changes in VSMC metabolism associated with senescence. SIRT6, but not SIRT6, promoted proliferation and lifespan of mouse VSMCs, and prevented senescence-associated metabolic changes. ApoE mice were generated that overexpress SIRT6 or SIRT6H133Y in VSMCs only. SM22alpha-hSIRT6/ApoE mice had reduced atherosclerosis, markers of senescence and inflammation compared to littermate controls, while plaques of SM22alpha-hSIRT6/ApoE mice showed increased features of plaque instability.SIRT6 protein expression is reduced in human and mouse plaque VSMCs and is positively regulated by CHIP. SIRT6 regulates telomere maintenance and VSMC lifespan, and inhibits atherogenesis, all dependent on its deacetylase activity. Our data shows that endogenous SIRT6 deacetylase is an important and unrecognized inhibitor of VSMC senescence and atherosclerosis.



Circ Res: 21 Dec 2020; epub ahead of print
Grootaert M, Finigan A, Figg N, Uryga AK, Bennett M
Circ Res: 21 Dec 2020; epub ahead of print | PMID: 33353368
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Abstract

Single-Cell Analysis of Blood-Brain Barrier Response to Pericyte Loss.

Andaloussi Mäe M, He L, Nordling S, Vazquez-Liebanas E, ... Keller A, Betsholtz C

Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain disorders, and genetically induced pericyte deficiency in the brain leads to increased macromolecular leakage across the blood-brain barrier (BBB). However, the molecular details of the endothelial response to pericyte deficiency remain elusive.To map the transcriptional changes in brain endothelial cells resulting from lack of pericyte contact at single-cell level, and to correlate them with regional heterogeneities in BBB function and vascular phenotype.We reveal transcriptional, morphological and functional consequences of pericyte absence for brain endothelial cells using a combination of methodologies, including single-cell RNA sequencing, tracer analyses and immunofluorescent detection of protein expression in pericyte-deficient adult Pdgfbret/ret mice. We find that endothelial cells without pericyte contact retain a general BBB-specific gene expression profile, however, they acquire a venous-shifted molecular pattern and become transformed regarding the expression of numerous growth factors and regulatory proteins. Adult Pdgfbret/ret brains display ongoing angiogenic sprouting without concomitant cell proliferation providing unique insights into the endothelial tip cell transcriptome. We also reveal heterogeneous modes of pericyte-deficient BBB impairment, where hotspot leakage sites display arteriolar-shifted identity and pinpoint putative BBB regulators. By testing the causal involvement of some of these using reverse genetics, we uncover a reinforcing role for angiopoietin 2 at the BBB.By elucidating the complexity of endothelial response to pericyte deficiency at cellular resolution, our study provides insight into the importance of brain pericytes for endothelial arterio-venous zonation, angiogenic quiescence and a limited set of BBB functions. The BBB-reinforcing role of ANGPT2 is paradoxical given its wider role as TIE2 receptor antagonist and may suggest a unique and context-dependent function of ANGPT2 in the brain.



Circ Res: 29 Dec 2020; epub ahead of print
Andaloussi Mäe M, He L, Nordling S, Vazquez-Liebanas E, ... Keller A, Betsholtz C
Circ Res: 29 Dec 2020; epub ahead of print | PMID: 33375813
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Abstract

Ca-CaM Dependent Inactivation of RyR2 Underlies Ca Alternans in Intact Heart.

Wei J, Yao J, Belke D, Guo W, ... Echebarria B, Chen SW

Ca alternans plays an essential role in cardiac alternans that can lead to ventricular fibrillation, but the mechanism underlying Ca alternans remains undefined. Increasing evidence suggests that Ca alternans results from alternations in the inactivation of cardiac ryanodine receptor (RyR2). However, what inactivates RyR2 and how RyR2 inactivation leads to Ca2+ alternans are unknown.To determine the role of calmodulin (CaM) on Ca alternans in intact working mouse hearts. We used an in vivo local gene delivery approach to alter CaM function by directly injecting adenoviruses expressing CaM-wild type (CaM-WT), a loss-of-function CaM mutation, CaM (1-4), and a gain-of-function mutation, CaM-M37Q, into the anterior wall of the left ventricle of RyR2 WT or mutant mouse hearts. We monitored Ca transients in ventricular myocytes near the adenovirus injection sites in Langendorff-perfused intact working hearts using confocal Ca imaging. We found that CaM-WT and CaM-M37Q promoted Ca alternans and prolonged Ca transient recovery in intact RyR2 WT and mutant hearts, whereas, CaM (1-4) exerted opposite effects. Altered CaM function also affected the recovery from inactivation of the L-type Ca current, but had no significant impact on sarcoplasmic reticulum Ca content. Further, we developed a novel numerical myocyte model of Ca alternans that incorporates Ca-CaM-dependent regulation of RyR2 and the L-type Ca channel. Remarkably, the new model recapitulates the impact on Ca alternans of altered CaM and RyR2 functions under 9 different experimental conditions. Our simulations reveal that diastolic cytosolic Ca elevation as a result of rapid pacing triggers Ca-CaM dependent inactivation of RyR2. The resultant RyR2 inactivation diminishes SR Carelease, which in turn reduces diastolic cytosolic Ca, leading to alternations in diastolic cytosolic Ca, RyR2 inactivation, and SR Ca release (i.e. Ca alternans).Our results demonstrate that inactivation of RyR2 by Ca-CaM is a major determinant of Ca alternans, making Ca-CaM dependent regulation of RyR2 an important therapeutic target for cardiac alternans.



Circ Res: 29 Dec 2020; epub ahead of print
Wei J, Yao J, Belke D, Guo W, ... Echebarria B, Chen SW
Circ Res: 29 Dec 2020; epub ahead of print | PMID: 33375811
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Abstract

Hydralazine protects the heart against acute ischemia/reperfusion injury by inhibiting Drp1-mediated mitochondrial fission.

Kalkhoran SB, Kriston-Vizi J, Hernandez-Resendiz S, Crespo-Avilan GE, ... Lim SY, Hausenloy DJ
Aims
Genetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission.
Methods and results
Pre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6 ± 1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated ischaemia/reperfusion injury (IRI), hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7 ± 2.5% vs control 34.1 ± 1.5%, P = 0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6 ± 6.5% vs vehicle control 54.1 ± 4.9%, P = 0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9 ± 3.0% vs vehicle control 58.2 ± 3.8%, P < 0.001).
Conclusion
We show that, in addition to its anti-oxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.
Translational perspective
Hydralazine is used clinically as a treatment for patients with hypertension and chronic heart failure, and experimental studies have known it to have anti-oxidant and anti-apoptotic effects. In our study, we show that administration of hydralazine immediately prior to reperfusion inhibited ischaemia/reperfusion injury-induced mitochondrial fission, and reduced myocardial infarct size. These findings raise the possibility of repurposing hydralazine as a novel potential cardioprotective therapy, which can be administered to acute myocardial infarction patients immediately prior to reperfusion by primary percutaneous coronary intervention, to reduce myocardial infarct size and prevent heart failure.

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

Cardiovasc Res: 01 Jan 2021; epub ahead of print
Kalkhoran SB, Kriston-Vizi J, Hernandez-Resendiz S, Crespo-Avilan GE, ... Lim SY, Hausenloy DJ
Cardiovasc Res: 01 Jan 2021; epub ahead of print | PMID: 33386841
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Abstract

Approaches to treat pulmonary arterial hypertension by targeting bmpr2 - from cell membrane to nucleus.

Dunmore BJ, Jones RJ, Toshner MR, Upton PD, Morrell NW

Pulmonary arterial hypertension (PAH) is estimated to affect between 10-50 people per million worldwide. The lack of cure and devastating nature of the disease means that treatment is crucial to arrest rapid clinical worsening. Current therapies are limited by their focus on inhibiting residual vasoconstriction rather than targeting key regulators of the cellular pathology. Potential disease-modifying therapies may come from research directed towards causal pathways involved in the cellular and molecular mechanisms of disease. It is widely acknowledged, that targeting reduced expression of the critical bone morphogenetic protein type-2 receptor (BMPR2) and its associated signalling pathways is a compelling therapeutic avenue to explore. In this review we highlight the advances that have been made in understanding this pathway and the therapeutics that are being tested in clinical trials and the clinic to treat PAH.

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

Cardiovasc Res: 04 Jan 2021; epub ahead of print
Dunmore BJ, Jones RJ, Toshner MR, Upton PD, Morrell NW
Cardiovasc Res: 04 Jan 2021; epub ahead of print | PMID: 33399862
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Abstract

Flow-Mediated Susceptibility and Molecular Response of Cerebral Endothelia to SARS-CoV-2 Infection.

Kaneko N, Satta S, Komuro Y, Muthukrishnan SD, ... Hsiai T, Hinman JD
Background:
and purpose
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with an increased rate of cerebrovascular events including ischemic stroke and intracerebral hemorrhage. The mechanisms underlying cerebral endothelial susceptibility and response to SARS-CoV-2 are unknown yet critical to understanding the association of SARS-CoV-2 infection with cerebrovascular events.
Methods
Endothelial cells were isolated from human brain and analyzed by RNA sequencing. Human umbilical vein and human brain microvascular cells were used in both monolayer culture and endothelialized within a 3-dimensional printed vascular model of the middle cerebral artery. Gene expression levels were measured by quantitative polymerase chain reaction and direct RNA hybridization. Recombinant SARS-CoV-2 S protein and S protein-containing liposomes were used to measure endothelial binding by immunocytochemistry.
Results
(angiotensin-converting enzyme-2) mRNA levels were low in human brain and monolayer endothelial cell culture. Within the 3-dimensional printed vascular model,gene expression and protein levels were progressively increased by vessel size and flow rates. SARS-CoV-2 S protein-containing liposomes were detected in human umbilical vein endothelial cells and human brain microvascular endothelial cells in 3-dimensional middle cerebral artery models but not in monolayer culture consistent with flow dependency of ACE2 expression. Binding of SARS-CoV-2 S protein triggered 83 unique genes in human brain endothelial cells including upregulation of complement component C3.
Conclusions
Brain endothelial cells are susceptible to direct SARS-CoV-2 infection through flow-dependent expression of ACE2. Viral S protein binding triggers a unique gene expression profile in brain endothelia that may explain the association of SARS-CoV-2 infection with cerebrovascular events.



Stroke: 30 Dec 2020; 52:260-270
Kaneko N, Satta S, Komuro Y, Muthukrishnan SD, ... Hsiai T, Hinman JD
Stroke: 30 Dec 2020; 52:260-270 | PMID: 33161843
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Abstract

Liver fat storage pathways: methodologies and dietary effects.

Roumans KHM, Basset Sagarminaga J, Peters HPF, Schrauwen P, Schrauwen-Hinderling VB
Purpose of review
Nonalcoholic fatty liver is the result of an imbalance between lipid storage [from meal, de novo lipogenesis (DNL) and fatty acid (FA) uptake] and disposal (oxidation and VLDL output). Knowledge on the contribution of each of these pathways to liver fat content in humans is essential to develop tailored strategies to prevent and treat nonalcoholic fatty liver. Here, we review the techniques available to study the different storage pathways and review dietary modulation of these pathways.
Recent findings
The type of carbohydrate and fat could be of importance in modulating DNL, as complex carbohydrates and omega-3 FAs have been shown to reduce DNL. No effects were found on the other pathways, however studies investigating this are scarce.
Summary
Techniques used to assess storage pathways are predominantly stable isotope techniques, which require specific expertise and are costly. Validated biomarkers are often lacking. These methodological limitations also translate into a limited number of studies investigating to what extent storage pathways can be modulated by diet. Further research is needed to elucidate in more detail the impact that fat and carbohydrate type can have on liver fat storage pathways and content.

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

Curr Opin Lipidol: 31 Jan 2021; 32:9-15
Roumans KHM, Basset Sagarminaga J, Peters HPF, Schrauwen P, Schrauwen-Hinderling VB
Curr Opin Lipidol: 31 Jan 2021; 32:9-15 | PMID: 33234776
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Abstract

Impact of metabolic dysfunction on cognition in humans.

Juul Rasmussen I, Qvist Thomassen J, Frikke-Schmidt R
Purpose of review
The current review evaluates the recent literature on the impact of metabolic dysfunction in human cognition, focusing on epidemiological studies and meta-analyses of these.
Recent findings
Worldwide around 50 million people live with dementia, a number projected to triple by 2050. Recent reports from the Lancet Commission suggest that 40% of dementia cases may be preventable primarily by focusing on well established metabolic dysfunction components and cardiovascular risk factors.
Summary
There is robust evidence that type 2 diabetes and midlife hypertension increase risk of dementia in late life. Obesity and elevated levels of LDL cholesterol in midlife probably increase risk of dementia, but further research is needed in these areas. Physical activity, diet, alcohol, and smoking might also influence the risk of dementia through their effect on metabolic dysfunction. A key recommendation is to be ambitious about prevention, focusing on interventions to promote healthier lifestyles combating metabolic dysfunction. Only comprehensive multidomain and staff-requiring interventions are however efficient to maintain or improve cognition in at-risk individuals and will be unrealistic economic burdens for most societies to implement. Therefore, a risk score that identifies high-risk individuals will enable a targeted early intensive intervention toward those high-risk individuals that will benefit the most from a prevention against cardiovascular risk factors and metabolic dysfunction.

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

Curr Opin Lipidol: 31 Jan 2021; 32:55-61
Juul Rasmussen I, Qvist Thomassen J, Frikke-Schmidt R
Curr Opin Lipidol: 31 Jan 2021; 32:55-61 | PMID: 33278082
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Impact:
Abstract

Mendelian randomization as a tool for causal inference in human nutrition and metabolism.

Larsson SC
Purpose of review
The current review describes the fundamentals of the Mendelian randomization framework and its current application for causal inference in human nutrition and metabolism.
Recent findings
In the Mendelian randomization framework, genetic variants that are strongly associated with the potential risk factor are used as instrumental variables to determine whether the risk factor is a cause of the disease. Mendelian randomization studies are less susceptible to confounding and reverse causality compared with traditional observational studies. The Mendelian randomization study design has been increasingly used in recent years to appraise the causal associations of various nutritional factors, such as milk and alcohol intake, circulating levels of micronutrients and metabolites, and obesity with risk of different health outcomes. Mendelian randomization studies have confirmed some but challenged other nutrition-disease associations recognized by traditional observational studies. Yet, the causal role of many nutritional factors and intermediate metabolic changes for health and disease remains unresolved.
Summary
Mendelian randomization can be used as a tool to improve causal inference in observational studies assessing the role of nutritional factors and metabolites in health and disease. There is a need for more large-scale genome-wide association studies to identify more genetic variants for nutritional factors that can be utilized for Mendelian randomization analyses.

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

Curr Opin Lipidol: 31 Jan 2021; 32:1-8
Larsson SC
Curr Opin Lipidol: 31 Jan 2021; 32:1-8 | PMID: 33278081
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Impact:
Abstract

The role of the molecular circadian clock in human energy homeostasis.

Dollet L, Pendergrast LA, Zierath JR
Purpose of review
The aim of this review is to present the latest findings on the role of the circadian clock in the control of metabolism, and the therapeutic potential of chronotherapy to regulate energy homeostasis in humans.
Recent findings
We summarized the recent advances related to circadian clock regulation of food intake and energy expenditure. In peripheral organs, mitochondrial oxidative capacity and lipolysis show circadian pattern in humans, and rhythms disruption may be involved in the pathogenesis of metabolic diseases. Indeed, circadian desynchrony affects food intake, insulin sensitivity, and increases the risk of developing metabolic disease. Time-targeted strategies, which aim to synchronize external cues with the molecular clock to improve metabolic outcomes, have positive effects on metabolism in humans, with several studies showing that time-targeted feeding improves body weight loss and glucose tolerance.
Summary
The interest in time-targeted strategies to prevent or manage metabolic disturbances has grown this past year with encouraging health benefits. To maximize the therapeutic effect of these strategies, further research is warranted to delineate the molecular regulation of metabolic processes controlled by the clock and especially its modulation in contexts such as aging, sex differences, or metabolic diseases.

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

Curr Opin Lipidol: 31 Jan 2021; 32:16-23
Dollet L, Pendergrast LA, Zierath JR
Curr Opin Lipidol: 31 Jan 2021; 32:16-23 | PMID: 33278080
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Impact:
Abstract

Radiation Impacts Early Atherosclerosis by Suppressing Intimal LDL Accumulation.

Ikeda J, Scipione CA, Hyduk S, Althagafi MG, ... MacParland SA, Cybulsky MI

Bone marrow transplantation (BMT) is used frequently to study the role of hematopoietic cells in atherosclerosis, but aortic arch lesions are smaller in mice after BMT.To identify the earliest stage of atherosclerosis inhibited by BMT and elucidate potential mechanisms.mice underwent total body γ-irradiation, bone marrow reconstitution and 6-week recovery. Atherosclerosis was studied in the ascending aortic arch and compared to mice without BMT. In BMT mice neutral lipid and myeloid cell topography were lower in lesions after feeding a cholesterol-rich diet (CRD) for 3, 6 and 12 weeks. Lesion coalescence and height were suppressed dramatically in mice post-BMT, whereas lateral growth was inhibited minimally. Targeted radiation to the upper thorax alone reproduced the BMT phenotype. Classical monocyte recruitment, intimal myeloid cell proliferation and apoptosis did not account for the post-BMT phenotype. Neutral lipid accumulation was reduced in 5-day lesions, thus we developed quantitative assays for LDL accumulation and paracellular leakage using DiI-labeled human LDL and rhodamine B-labeled 70kD dextran. LDL accumulation was dramatically higher in the intima ofrelative tomice, and was inhibited by injection of HDL mimics, suggesting a regulated process. LDL, but not dextran, accumulation was lower in mice post-BMT both at baseline and in 5-day lesions. Since the transcript abundance of molecules implicated in LDL transcytosis was not significantly different in the post-BMT intima, transcriptomics from whole aortic arch intima, and at single cell resolution, was performed to give insights into pathways modulated by BMT.Radiation exposure inhibits LDL entry into the aortic intima at baseline and the earliest stages of atherosclerosis. Single cell transcriptomic analysis suggests that LDL uptake by endothelial cells is diverted to lysosomal degradation and reverse cholesterol transport pathways. This reduces intimal accumulation of lipid and impacts lesion initiation and growth.



Circ Res: 04 Jan 2021; epub ahead of print
Ikeda J, Scipione CA, Hyduk S, Althagafi MG, ... MacParland SA, Cybulsky MI
Circ Res: 04 Jan 2021; epub ahead of print | PMID: 33397122
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Abstract

MTORC1-Regulated Metabolism Controlled by TSC2 Limits Cardiac Reperfusion Injury.

Oeing CU, Jun S, Mishra S, Dunkerly-Eyring B, ... Ranek MJ, Kass DA

The mechanistic target of rapamycin complex-1 (mTORC1) controls metabolism and protein homeostasis, and is activated following ischemic reperfusion (IR) injury and by ischemic preconditioning (IPC). However, studies vary as to whether this activation is beneficial or detrimental, and its influence on metabolism after IR is little studied. A limitation of prior investigations is their use of broad gain/loss of mTORC1 function, mostly applied prior to ischemic stress. This can be circumvented by regulating one serine (S1365) on tuberous sclerosis complex (TSC2) to achieve bi-directional mTORC1 modulation but only with TCS2-regulated co-stimulation.We tested the hypothesis that reduced TSC2 S1365 phosphorylation protects the myocardium against IR and IPC by amplifying mTORC1 activity to favor glycolytic metabolism.Mice with either S1365A (TSC2; phospho-null) or S1365E (TSC2; phosphomimetic) knock-in mutations were studied ex vivo and in vivo. In response to IR, hearts from TSC2 mice had amplified mTORC1 activation and improved heart function compared to WT and TSC2 hearts. The magnitude of protection matched IPC. IPC requited less S1365 phosphorylation, as TSC2 hearts gained no benefit and failed to activate mTORC1 with IPC. IR metabolism was altered in TSC2, with increased mitochondrial oxygen consumption rate and glycolytic capacity (stressed/maximal extracellular acidification) after myocyte hypoxia-reperfusion. In whole heart, lactate increased and long-chain acyl-carnitine levels declined during ischemia. The relative IR protection in TSC2 was lost by lowering glucose in the perfusate by 36%. Adding fatty acid (palmitate) compensated for reduced glucose in WT and TSC2 but not TSC2 which had the worst post-IR function under these conditions.TSC2-S1365 phosphorylation status regulates myocardial substrate utilization, and its decline activates mTORC1 biasing metabolism away from fatty acid oxidation to glycolysis to confer protection against IR. This pathway is also engaged and reduced TSC2 S1365 phosphorylation required for effective IPC.



Circ Res: 05 Jan 2021; epub ahead of print
Oeing CU, Jun S, Mishra S, Dunkerly-Eyring B, ... Ranek MJ, Kass DA
Circ Res: 05 Jan 2021; epub ahead of print | PMID: 33401933
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Abstract

Deep dive to the secrets of the PREDIMED trial.

Fernández-Lázaro CI, Ruiz-Canela M, Martínez-González MÁ
Purpose of review
The aim of this study was to briefly summarize the contribution of the PREDIMED (PREvención con DIeta MEDiterránea) trial on cardiovascular evidence and examine in depth its groundbreaking trajectory.PREDIMED was conducted during 2003-2010 and represented the largest primary prevention trial ever testing the effects of changes in a complete food pattern (namely, the Mediterranean diet) on cardiovascular disease (CVD). Major contributions relied on the relevant changes in the food pattern attained by the behavioural intervention and their robust effect in reducing hard clinical end-points. Given some potential concerns, which were appropriately addressed with supporting analyses, this review is timely and relevant.
Recent findings
PREDIMED has continued contributing to the existing literature with extensive, robust and abundant new evidence on the benefits of the Mediterranean diet, particularly on cardiovascular health, including recent studies using high-throughput metabolomic techniques. After robustly addressing some controversies, the conclusions of the original trial remained unaltered.
Summary
The Mediterranean diet represents an effective and robust nutritional strategy against CVD in high cardiovascular risk populations. Recent findings from the PREDIMED have identified a metabolic signature of the Mediterranean diet that can objectively determine dietary adherence and predict CVD risk. This metabolomic signature opens up a new era for nutritional epidemiology and personalized nutrition.

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

Curr Opin Lipidol: 31 Jan 2021; 32:62-69
Fernández-Lázaro CI, Ruiz-Canela M, Martínez-González MÁ
Curr Opin Lipidol: 31 Jan 2021; 32:62-69 | PMID: 33315620
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Abstract

Aberrant Activation of Notch1 Signaling in Glomerular Endothelium Induces Albuminuria.

Li L, Liu Q, Shang T, Song W, ... Lobe CG, Liu J

Glomerular capillaries are lined with a highly specialized fenestrated endothelium and contribute to the glomerular filtration barrier (GFB). The Notch signaling pathway is involved in regulation of GFB, but its role in glomerular endothelium has not been investigated due to the embryonic lethality of animal models with genetic modification of Notch pathway components in the endothelium.To determine the effects of aberrant activation of the Notch signaling in glomerular endothelium and the underlying molecular mechanisms.We established the ZEG-Notch1 intracellular domain (NICD1)/Tie2-tTA/Tet-O-Cre transgenic mouse model to constitutively activate Notch1 signaling in endothelial cells of adult mice. The triple transgenic mice developed severe albuminuria with significantly decreased VE-cadherin expression in the glomerular endothelium. In vitro studies showed that either NICD1 lentiviral infection or treatment with Notch ligand DLL4 markedly reduced VE-cadherin expression and increased monolayer permeability of human renal glomerular endothelial cells (HRGECs). In addition, Notch1 activation or gene knockdown of VE-cadherin reduced the glomerular endothelial glycocalyx. Further investigation demonstrated that activated Notch1 suppression of VE-cadherin was through the transcription factors SNAI1 and ERG, which bind to the -373 E-box and the -134/-118 ETS element of the VE-cadherin promoter, respectively. Our results reveal novel regulatory mechanisms whereby endothelial Notch1 signaling dictates the level of VE-cadherin through the transcription factors SNAI1 and ERG, leading to dysfunction of GFB and induction of albuminuria.



Circ Res: 12 Jan 2021; epub ahead of print
Li L, Liu Q, Shang T, Song W, ... Lobe CG, Liu J
Circ Res: 12 Jan 2021; epub ahead of print | PMID: 33435713
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Abstract

Impact of ultra-processed food consumption on metabolic health.

Costa de Miranda R, Rauber F, Levy RB
Purpose of review
Ultra-processed foods (UPF) have been associated with poor diet quality and adverse health outcomes. Our aim in this review is to summarize recent research assessing the impact of UPF consumption, classified according to the NOVA system, on outcomes related to metabolic health.
Recent findings
Thirty recent studies with different design, quality and target population have investigated the impact of UPF consumption on parameters related to metabolic health, which were organized into: metabolic syndrome; body weight change and obesity indicators; blood pressure and hypertension; glucose profile, insulin resistance and type 2 diabetes; other metabolic risks and cardiovascular diseases and mortality. Most of the studies demonstrated adverse associations between high UPF consumption and metabolic health, mainly those with robust design and involving adults.
Summary
Most of the latest findings have revealed an adverse impact of high UPF consumption on metabolic health, including cardiovascular diseases and mortality. Scientific evidence is accumulating towards the necessity of curbing UPF consumption worldwide at different life stages. Nevertheless, other studies are needed to confirm the causality between UPF consumption and metabolic health in diverse scenarios and to better elucidate all likely mechanisms involved in this relationship.

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

Curr Opin Lipidol: 31 Jan 2021; 32:24-37
Costa de Miranda R, Rauber F, Levy RB
Curr Opin Lipidol: 31 Jan 2021; 32:24-37 | PMID: 33315618
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Abstract

Impact drugs targeting cardiometabolic risk on the gut microbiota.

Balvers M, van den Born BH, Levin E, Nieuwdorp M
Purpose of review
Alterations in the gut microbiome composition or function are associated with risk factors for cardiometabolic diseases, including hypertension, hyperlipidemia and hyperglycemia. Based on recent evidence that also oral medications used to treat these conditions could alter the gut microbiome composition and function and, vice versa, that the gut microbiome could affect the efficacy of these treatments, we reviewed the literature on these observed interactions.
Recent findings
While the interaction of metformin with the gut microbiome has been studied most, other drugs that target cardiometabolic risk are gaining attention and often showed associations with alterations in microbiome-related features, including alterations in specific microbial taxa or pathways, microbiome composition or microbiome-derived metabolites, while the gut microbiome was also involved in drug metabolism and drug efficacy. As for metformin, for some of them even a potential therapeutic effect via the gut microbiome is postulated. However, exact mechanisms remain to be elucidated.
Summary
There is growing interest in clarifying the interactions between the gut microbiome and drugs to treat hypertension, hyperlipidemia and hyperglycemia as well as the first pass effect of microbiome on drug efficacy. While mostly analysed in animal models, also human studies are gaining more and more traction. Improving the understanding of the gut microbiome drug interaction can provide clinical directions for therapy by optimizing drug efficacy or providing new targets for drug development.

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

Curr Opin Lipidol: 31 Jan 2021; 32:38-54
Balvers M, van den Born BH, Levin E, Nieuwdorp M
Curr Opin Lipidol: 31 Jan 2021; 32:38-54 | PMID: 33332920
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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: 30 Dec 2020; epub ahead of print
Jeraj N, Hegele RA, Berberich AJ
Curr Opin Lipidol: 30 Dec 2020; epub ahead of print | PMID: 33395107
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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: 30 Dec 2020; epub ahead of print
Trinder M, Brunham LR
Curr Opin Lipidol: 30 Dec 2020; epub ahead of print | PMID: 33395106
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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: 30 Dec 2020; epub ahead of print
Fong V, Patel SB
Curr Opin Lipidol: 30 Dec 2020; epub ahead of print | PMID: 33395105
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Impact:
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