Topic: Basic Research

Abstract

PCSK9 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

Proprotein convertase subtilisin/kexin 9 (PCSK9), mainly secreted by the liver and released into the blood, elevates plasma low-density lipoprotein (LDL) cholesterol by degrading LDL receptor. Pleiotropic effects of PCSK9 beyond lipid-metabolism have been shown. However, the direct effects of PCSK9 on platelet activation and thrombosis, as well as the underlying mechanisms, still remain unclear.We detected the direct effects of PCSK9 on agonists-induced platelet aggregation, dense granule ATP release, integrin αIIbβ3 activation, α granule release, spreading, and clot retraction. These studies were complemented byanalysis of FeCl3-injured mouse mesenteric arteriole thrombosis. We also investigated the underlying mechanisms. Using myocardial infarct (MI) model, we explored the effects of PCSK9 on microvascular obstruction and infarct expansion post-MI.PCSK9 directly enhances agonists-induced platelet aggregation, dense granule ATP release, integrin αIIbβ3 activation, P-selection release from α granules, spreading, and clot retraction. In line, PCSK9 enhancesthrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model, while PCSK9 inhibitor evolocumab ameliorates its enhancing effects. Mechanism studies revealed that PCSK9 binds to platelet CD36 and thus activates Src kinase and mitogen-activated protein kinase (MAPK)- extracellular signal-regulated kinase 5 and c-Jun N-terminal kinase, increases the generation of reactive oxygen species, as well as activates the p38MAPK/cytosolic phospholipase A2/cyclooxygenase-1/thromboxane A signaling pathways downstream of CD36 to enhance platelet activation. Using CD36 knockout mice, we showed the enhancing effects of PCSK9 on platelet activation are CD36 dependent. Consistently and importantly, aspirin abolishes the enhancing effects of PCSK9 on platelet activation andthrombosis. Finally, we showed that PCSK9 activating platelet CD36 aggravates microvascular obstruction and promotes MI expansion post-MI.PCSK9 in plasma directly enhances platelet activation andthrombosis, as well as MI expansion post-MI, 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: 28 Sep 2020; epub ahead of print
Qi Z, Hu L, Zhang J, Yang W, ... Ding Z, Ge J
Circulation: 28 Sep 2020; epub ahead of print | PMID: 32988222
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Abstract

Development of a Cardiac Sarcomere Functional Genomics Platform to Enable Scalable Interrogation of Human Variants.

Pettinato AM, Ladha FA, Mellert DJ, Legere N, ... Chen YS, Hinson JT

Pathogenicvariants are a cause of hypertrophic (HCM) and dilated (DCM) cardiomyopathies, which promote heart failure by incompletely understood mechanisms. Additionally, the precise functional significance for 87% ofvariants remains undetermined partially due to a lack of functional genomics studies. The knowledge of which and howvariants cause HCM and DCM could improve heart failure risk determination, treatment efficacy, and therapeutic discovery, as well as provide new insights into cardiomyopathy pathogenesis.We created a toolkit of human induced pluripotent stem cell (hiPSC) models and functional assays using CRISPR/Cas9 to studyvariant pathogenicity and pathophysiology. Using hiPSC-derived cardiomyocytes (hiPSC-CMs) in cardiac microtissue and single cell assays, we functionally interrogated 51variants, including 30 pathogenic/likely pathogenic variants and 21 variants of unknown significance (VUS). We utilized RNA-sequencing to determine the transcriptomic consequences of pathogenicvariants, and adapted CRISPR/Cas9 to engineer a transcriptional reporter assay to assist prediction ofvariant pathogenicity. We also studied variant-specific pathophysiology using a thin filament-directed calcium reporter to monitor changes in myofilament calcium affinity.HCM-associatedvariants caused increased cardiac microtissue contraction, while DCM-associated variants decreased contraction.variant-dependent changes in sarcomere contractile function induced graded regulation of 101 gene transcripts, includingsignaling targets, , and . We distinguished pathogenicvariants from wildtype controls using a sarcomere functional reporter engineered by inserting tdTomato into the endogenouslocus. Based on a combination ofreporter activity and cardiac microtissue contraction, our study provides experimental support for the reclassification of 2 pathogenic/likely pathogenic variants and 2 VUSs.Our study found that HCM-associatedvariants increased cardiac microtissue contraction, while DCM-associated variants cause decreased contraction, both of which paralleled changes in myofilament calcium affinity. Transcriptomic changes, includinglevels, directly correlated with sarcomere function and can be utilized to predictvariant pathogenicity.



Circulation: 06 Oct 2020; epub ahead of print
Pettinato AM, Ladha FA, Mellert DJ, Legere N, ... Chen YS, Hinson JT
Circulation: 06 Oct 2020; epub ahead of print | PMID: 33025817
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Abstract

Dual Functional States of R406W-Desmin Assembly Complexes Cause Cardiomyopathy With Severe Intercalated Disc Derangement in Humans and in Knock-In Mice.

Herrmann H, Cabet E, Chevalier NR, Moosmann J, ... Lilienbaum A, Schröder R

Mutations in the human desmin gene cause myopathies and cardiomyopathies. Aim of this study was to elucidate molecular mechanisms initiated by the heterozygous R406W-desmin mutation in the development of a severe and early-onset cardiac phenotype.We report an adolescent patient, who underwent cardiac transplantation due to restrictive cardiomyopathy caused by a heterozygous R406W-desmin mutation. Sections of the explanted heart were analyzed with antibodies specific to 406W-desmin and to intercalated disc proteins. Effects of the R406W mutation on the molecular properties of desmin were addressed by cell transfection andassembly experiments. To prove the genuine deleterious impact of the mutation on heart tissue, we further generated and analyzed R405W-desmin knock-in mice harboring the orthologous form of the human R406W-desmin.Microscopic analysis of the explanted heart revealed desmin aggregates and the absence of desmin filaments at intercalated discs. Structural changes within intercalated discs were revealed by the abnormal organization of desmoplakin, plectin, N-cadherin, and connexin-43. Next generation sequencing confirmed thevariant c.1216C>T (p.R406W) as the sole disease-causing mutation. Cell transfection studies disclosed a dual behavior of R406W-desmin with both its integration into the endogenous intermediate filament system and segregation into protein aggregates. , R406W-desmin formed unusually thick filaments that organized into complex filament aggregates and fibrillar sheets. In contrast, assembly of equimolar mixtures of mutant and wild-type desmin generated chimeric filaments of seemingly normal morphology but with occasional prominent irregularities. Heterozygous and homozygous R405W-desmin knock-in mice develop both a myopathy and a cardiomyopathy. In particular, the main histopathological results from the patient are recapitulated in the hearts from R405W-desmin knock-in mice of both genotypes. Moreover, while heterozygous knock-in mice have a normal life span, homozygous animals die at three months of age due to a smooth muscle-related gastrointestinal phenotype.We demonstrate that R406W-desmin provokes its severe cardiotoxic potential by a novel pathomechanism, where the concurrent dual functional states of mutant desmin assembly complexes underlie the uncoupling of desmin filaments from intercalated discs and their structural disorganization.



Circulation: 06 Oct 2020; epub ahead of print
Herrmann H, Cabet E, Chevalier NR, Moosmann J, ... Lilienbaum A, Schröder R
Circulation: 06 Oct 2020; epub ahead of print | PMID: 33023321
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Abstract

Redox-Resistant SERCA Attenuates Oxidant-Stimulated Mitochondrial Calcium and Apoptosis in Cardiac Myocytes and Pressure Overload-Induced Myocardial Failure in Mice.

Goodman JB, Qin F, Morgan R, Chambers JM, ... Cohen RA, Colucci WS

Sarco(endo)plasmic reticulum calcium ATPase (SERCA) is regulated by oxidative post-translational modifications at cysteine 674 (C674). Since sarcoplasmic reticulum (SR) calcium has been shown to play a critical role in mediating mitochondrial dysfunction in response to reactive oxygen species (ROS), we hypothesized that SERCA oxidation at C674 would modulate the effects of ROS on mitochondrial calcium and mitochondria-dependent apoptosis in cardiac myocytes.Adult rat ventricular myocytes (ARVM) expressing wild-type (WT) SERCA2b or a redox-insensitive mutant in which C674 is replaced by serine (C674S) were exposed to HO (100 M). Free mitochondrial calcium concentration was measured in ARVM using 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 (SKI) were subjected to chronic ascending aortic constriction (AAC).In ARVM expressing WT 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 WT cells, HO caused cytochrome c release and apoptosis, both of which were prevented in C674S-expressing cells. In myocytes from SKI mice, basal SERCA activity and SR calcium content were decreased. To test the effect of C674 oxidation on apoptosis , SKI mice were subjected to chronic AAC. In WT mice, AAC caused myocyte apoptosis, LV dilation and systolic failure - all of which were inhibited in SKI mice.Redox activation of SERCA C674 regulates basal SR calcium content thereby mediating the pathologic ROS-stimulated rise in mitochondrial calcium required for myocyte apoptosis and myocardial failure.



Circulation: 19 Oct 2020; epub ahead of print
Goodman JB, Qin F, Morgan R, Chambers JM, ... Cohen RA, Colucci WS
Circulation: 19 Oct 2020; epub ahead of print | PMID: 33076678
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Abstract

Forty-Year Shifting Distribution of Systolic Blood Pressure With Population Hypertension Treatment and Control.

Lackland DT, Howard VJ, Cushman M, Oparil S, ... McClure LA, Howard G
Background
Hypertension awareness, treatment, and control programs were initiated in the United States during the 1960s and 1970s. Whereas blood pressure (BP) control in the population and subsequent reduced hypertension-related disease risks have improved since the implementation of these interventions, it is unclear whether these BP changes can be generalized to diverse and high-risk populations. This report describes the 4-decade change in BP levels for the population in a high disease risk southeastern region of the United States. The objective is to determine the magnitude of the shift in systolic BP (SBP) among Blacks and Whites from the Southeast between 1960 and 2005 with the assessment of the unique population cohorts.
Methods
A multicohort study design compared BPs from the CHS (Charleston Heart Study) and ECHS (Evans County Heart Study) in 1960 and the REGARDS study (Reasons for Geographic and Racial Differences in Stroke) 4 decades later. The analyses included participants ≥45 years of age from CHS (n=1323), ECHS (n=1842), and REGARDS (n=6294) with the main outcome of SBP distribution.
Results
Among Whites 45 to 54 years of age, the median SBP was 18 mm Hg (95% CI, 16-21 mm Hg) lower in 2005 than 1960. The median shift was a 45 mm Hg (95% CI, 37-51 mm Hg) decline for those ≥75 years of age. The shift was larger for Blacks, with median declines of 38 mm Hg (95% CI, 32-40 mm Hg) at 45 to 54 years of age and 50 mm Hg (95% CI, 33-60 mm Hg) for ages ≥75 years. The 95th percentile of SBP decreased 60 mm Hg for Whites and 70 mm Hg for Blacks.
Conclusions
The results of the current analyses of the unique cohorts in the Southeast confirm the improvements in population SBP levels since 1960. This assessment provides new evidence of improvement in SBP, suggesting that strategies and programs implemented to improve hypertension treatment and control have been extraordinarily successful for both Blacks and Whites residing in a high-risk region of the United States. Severe BP elevations commonly observed in the 1960s have been nearly eliminated, with the current 75th percentile of BP generally less than the 25th percentile of BP in 1960.



Circulation: 19 Oct 2020; 142:1524-1531
Lackland DT, Howard VJ, Cushman M, Oparil S, ... McClure LA, Howard G
Circulation: 19 Oct 2020; 142:1524-1531 | PMID: 33016101
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Abstract

Innate Immune Nod1/RIP2 Signaling is Essential for Cardiac Hypertrophy, but Requires Mitochondrial Antiviral Signaling Protein (MAVS) for Signal Transductions and Energy Balance.

Lin HB, Naito K, Oh Y, Farber G, ... Harper ME, Liu PP

Cardiac hypertrophy is a key biological response to injurious stresses such as pressure overload and when excessive can lead to heart failure. Innate immune activation by danger signals, through intracellular pattern recognition receptors such as nucleotide-binding oligomerization domain-containing protein 1(Nod1) and its adaptor receptor-interacting protein 2 (RIP2), might play a major role in cardiac remodeling and progression to heart failure. We hypothesize that Nod1/RIP2 are major contributors to cardiac hypertrophy, but may not be sufficient to fully express the phenotype alone.To elucidate the contribution of Nod1/RIP2 signaling to cardiac hypertrophy, we randomized Nod1, RIP2 or wild-type (WT) mice to transverse aortic constriction (TAC) or sham operations. Cardiac hypertrophy, fibrosis, and cardiac function were examined in these mice.Nod1 and RIP2 proteins were up-regulated in the heart after TAC, and this was paralleled by increased expression of mitochondrial proteins, including mitochondrial antiviral signaling protein (MAVS). Nod1 and RIP2 mice subjected to TAC exhibited better survival, improved cardiac function and decreased cardiac hypertrophy. Downstream signal transduction pathways that regulate inflammation and fibrosis including NF-κB and MAPK-GATA4/p300, were reduced in both Nod1 and RIP2 mice after TAC compared with WT mice. Co-immunoprecipitation of extracted cardiac proteins and confocal immunofluorescence microscopy showed that Nod1/ RIP2 interaction was robust and that this complex also included MAVS as an essential component. Suppression of MAVS expression attenuated the complex formation, NF-κB signalling and myocyte hypertrophy. Interrogation of mitochondrial function compared in the presence or ablation of MAVS revealed that MAVS serves to suppress mitochondrial energy output and mediate fission/fusion related dynamic changes. The latter is possibly linked to mitophagy during cardiomyocytes stress, which may provide an intriguing link between innate immune activation and mitochondrial energy balance under stress or injury conditions.We have identified that innate immune Nod1/RIP2 signaling is a major contributor to cardiac remodeling following stress. This process is critically joined by and regulated through the mitochondrial danger signal adapter MAVS. This novel complex coordinates remodeling, inflammatory response and mitochondrial energy metabolism in stressed cardiomyocytes. Thus Nod1/RIP2/MAVS signaling complex may represent an attractive new therapeutic approach toward heart failure.



Circulation: 18 Oct 2020; epub ahead of print
Lin HB, Naito K, Oh Y, Farber G, ... Harper ME, Liu PP
Circulation: 18 Oct 2020; epub ahead of print | PMID: 33070627
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Abstract

Redox Regulation of Cardiac ASK1 (Apoptosis Signal-Regulating Kinase 1) Controls p38-MAPK (Mitogen-Activated Protein Kinase) and Orchestrates Cardiac Remodeling to Hypertension.

Meijles DN, Cull JJ, Markou T, Cooper STE, ... Sugden PH, Clerk A

Systemic hypertension increases cardiac workload causing cardiomyocyte hypertrophy and increased cardiac fibrosis. An underlying feature is increased production of reactive oxygen species. Redox-sensitive ASK1 (apoptosis signal-regulating kinase 1) activates stress-regulated protein kinases (p38-MAPK [mitogen-activated protein kinases] and JNKs [c-Jun N-terminal kinases]) and promotes fibrosis in various tissues. Here, we determined the specificity of ASK1 signaling in the heart, with the hypothesis that ASK1 inhibitors may be used to manage fibrosis in hypertensive heart disease. Using immunoblotting, we established that moderate levels of HO activate ASK1 in neonatal rat cardiomyocytes and perfused rat hearts. ASK1 was activated during ischemia in adult rat hearts, but not on reperfusion, consistent with activation by moderate (not high) reactive oxygen species levels. In contrast, IL (interleukin)-1β activated an alternative kinase, TAK1 (transforming growth factor-activated kinase 1). ASK1 was not activated by IL1β in cardiomyocytes and activation in perfused hearts was due to increased reactive oxygen species. Selonsertib (ASK1 inhibitor) prevented activation of p38-MAPKs (but not JNKs) by oxidative stresses in cultured cardiomyocytes and perfused hearts. In vivo (C57Bl/6J mice with osmotic minipumps for drug delivery), selonsertib (4 mg/[kg·d]) alone did not affect cardiac function/dimensions (assessed by echocardiography). However, it suppressed hypertension-induced cardiac hypertrophy resulting from angiotensin II (0.8 mg/[kg·d], 7d), with inhibition ofmRNA upregulation, reduced cardiomyocyte hypertrophy and, notably, significant reductions in interstitial and perivascular fibrosis. Our data identify a specific reactive oxygen species→ASK1→p38-MAPK pathway in the heart and establish that ASK1 inhibitors protect the heart from hypertension-induced cardiac remodeling. Thus, targeting the ASK1→p38-MAPK nexus has potential therapeutic viability as a treatment for hypertensive heart disease.



Hypertension: 29 Sep 2020; 76:1208-1218
Meijles DN, Cull JJ, Markou T, Cooper STE, ... Sugden PH, Clerk A
Hypertension: 29 Sep 2020; 76:1208-1218 | PMID: 32903101
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Abstract

VWF (von Willebrand Factor) Is a Critical Mediator of Deep Vein Thrombosis in a Mouse Model of Diet-Induced Obesity.

Michels A, Dwyer CN, Mewburn J, Nesbitt K, ... Swystun LL, Lillicrap D
Objective
Obesity is characterized by chronic low-grade inflammation and consequentially a hypercoagulable state, associating with an increased incidence of venous thromboembolism. Increased VWF (von Willebrand factor) plasma concentration and procoagulant function are independent risk factors for venous thromboembolism and are elevated in obese patients. Here, we explore the pathobiological role of VWF in obesity-associated venous thrombosis using murine models. Approach and Results: We first showed that diet-induced obese mice have increased VWF plasma levels and FVIII (factor VIII) activity compared with littermate controls. Elevated VWF levels appeared to be because of both increased synthesis and impaired clearance. Diet-induced obesity-associated venous thrombosis was assessed using the inferior vena cava-stenosis model of deep vein thrombosis. Diet-induced obese mice developed larger venous thrombi that were rich in VWF, erythrocytes, and leukocytes. Administering a polyclonal anti-VWF antibody or an anti-VWF A1 domain nanobody was protective against obesity-mediated thrombogenicity. Delayed administration (3 hours post-inferior vena cava stenosis) similarly reduced thrombus weight in diet-induced obese mice.
Conclusions
This study demonstrates the critical role of VWF in the complex, thrombo-inflammatory state of obesity. It adds to the growing rationale for targeting VWF-specific interactions in thrombotic disease.



Arterioscler Thromb Vasc Biol: 23 Sep 2020:ATVBAHA120314690; epub ahead of print
Michels A, Dwyer CN, Mewburn J, Nesbitt K, ... Swystun LL, Lillicrap D
Arterioscler Thromb Vasc Biol: 23 Sep 2020:ATVBAHA120314690; epub ahead of print | PMID: 32967458
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Abstract

Antenatal Dexamethasone Exposure Impairs the High-Conductance Ca-Activated K Channels via Epigenetic Alteration at Gene Promoter in Male Offspring.

Xu T, Zhao M, Li H, Zhou X, ... Xu Z, Gao Q
Objective
Antenatal exposure to glucocorticoids increases cardiovascular risks related to vascular dysfunctions in offspring, although underlying mechanisms are still unknown. As an important vascular mediator, high-conductance Ca-activated K channels (BK) plays an essential role in determining vascular tone. Long-term effects of antenatal glucocorticoids on BK in offspring are largely unknown. This study examined the effects and mechanisms of antenatal exposure to clinically relevant doses of glucocorticoids on vascular BK in offspring. Approach and Results: Pregnant Sprague-Dawley rats received synthetic glucocorticoids dexamethasone or vehicle during the last week of pregnancy. Vascular functions, cellular electrophysiology, target gene expression, and promoter methylation were examined in mesenteric arteries of male offspring (gestational day 21 [fetus] and postnatal day 120 [adult offspring]). Antenatal dexamethasone exposure impaired BK activators-mediated relaxation and reduced whole-cell BK currents in mesenteric arteries. Antenatal dexamethasone exposure did not alter Ca/voltage-sensitivity of BK but downregulated the expressions of BK α and β1 subunits in both fetal and adult mesenteric arteries. In addition, increased promoter methylations withinandwere compatible with reduced expressions of the 2 genes.
Conclusions
Our findings showed a profound and long-term impact of antenatal dexamethasone exposure on vascular BK via an altered epigenetic pattern from fetal stage to adulthood, advancing understanding of prolonged adverse effects and mechanisms of antenatal glucocorticoids exposure on vascular health in offspring.



Arterioscler Thromb Vasc Biol: 23 Sep 2020:ATVBAHA120314905; epub ahead of print
Xu T, Zhao M, Li H, Zhou X, ... Xu Z, Gao Q
Arterioscler Thromb Vasc Biol: 23 Sep 2020:ATVBAHA120314905; epub ahead of print | PMID: 32967457
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Abstract

Role of the Carotid Body in an Ovine Model of Renovascular Hypertension.

Chang JW, Tromp TR, Joles JA, McBryde FD, Paton JFR, Ramchandra R

The carotid body is implicated as an important mediator and potential treatment target for hypertension. The mechanisms driving increased carotid body tonicity in hypertension are incompletely understood. Using a large preclinical animal model, which is crucial for translation, we hypothesized that carotid sinus nerve denervation would chronically decrease blood pressure in a renovascular ovine model of hypertension in which hypertonicity of the carotid body is associated with reduced common carotid artery blood flow. Adult ewes underwent either unilateral renal artery clipping or sham surgery. Two weeks later, flow probes were placed around the contralateral renal and common carotid arteries. Hypertension was accompanied by a significant reduction in common carotid blood flow but no change in renal blood flow. Carotid sinus nerve denervation significantly reduced blood pressure compared with sham. In both hypertensive and normotensive animals, carotid body stimulation using potassium cyanide caused dose-dependent increases in mean arterial pressure and common carotid conductance but a reduction in renal vascular conductance. These responses were not different between the animal groups. Taken together, our findings indicate that (1) the carotid body is activated in renovascular hypertension, and this is associated with reduced blood flow (decreased vascular conductance) in the common carotid artery and (2) the carotid body can differentially regulate blood flow to the common carotid and renal arteries. We suggest that in the ovine renovascular model, carotid body hypertonicity may be a product of reduced common carotid artery blood flow and plays an amplifying role with the kidney in the development of hypertension.



Hypertension: 27 Sep 2020:HYPERTENSIONAHA12015676; epub ahead of print
Chang JW, Tromp TR, Joles JA, McBryde FD, Paton JFR, Ramchandra R
Hypertension: 27 Sep 2020:HYPERTENSIONAHA12015676; epub ahead of print | PMID: 32981362
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Abstract

P2X7: 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 Sep 2020:ATVBAHA120315116; epub ahead of print
Shokoples BG, Paradis P, Schiffrin EL
Arterioscler Thromb Vasc Biol: 30 Sep 2020:ATVBAHA120315116; epub ahead of print | PMID: 32998520
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Abstract

Spontaneous Atherosclerosis in Aged LCAT-Deficient Hamsters With Enhanced Oxidative Stress.

Guo M, Liu Z, Xu Y, Ma P, ... Liu G, Xian X
Objective
LCAT (lecithin cholesterol acyltransferase) deficiency results in severe low HDL (high-density lipoprotein). Although whether LCAT is pro- or antiatherosclerosis was in debate in mouse studies, our previous study clearly shows that LCAT deficiency (LCAT) in hamster accelerates atherosclerotic development on high-fat diet. However, unlike in hypercholesterolemia and hypertriglyceridemia, whether LCAT deficiency could lead to spontaneous atherosclerosis has not been studied yet in animal models. We, therefore, sought to investigate the atherosclerosis in LCAT hamsters on standard laboratory diet and explore the potential underlying mechanisms. Approach and Results: Young (<8 months) and aged (>16 months) male and female wild-type and LCAT hamsters on standard laboratory diet were used. Compared with age- and sex-matched wild-type hamsters, LCAT hamsters showed a complete loss of plasma HDL and an increase in triglyceride by 2- to 8-folds at different stages of age. In aged LCAT hamsters, the lesion areas at the aortic roots were ≈40×10 μm in males and 18×10 μm in females, respectively, which were consistent with the en face plaques observed in male (1.2%) and (1.5%) female groups, respectively. The results of plasma malondialdehyde measurement showed that malondialdehyde concentrations were markedly elevated to 54.4 μmol/L in males and 30 μmol/L in females, which are significantly associated with the atherosclerotic lesions.
Conclusions
Our study demonstrates the development of spontaneous atherosclerotic lesions in aged male and female LCAT hamsters with higher plasma oxidative lipid levels independent of plasma total cholesterol levels, further confirming the antiatherosclerotic role of LCAT.



Arterioscler Thromb Vasc Biol: 30 Sep 2020:ATVBAHA120315265; epub ahead of print
Guo M, Liu Z, Xu Y, Ma P, ... Liu G, Xian X
Arterioscler Thromb Vasc Biol: 30 Sep 2020:ATVBAHA120315265; epub ahead of print | PMID: 32998519
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Abstract

Aging-Associated miR-217 Aggravates Atherosclerosis and Promotes Cardiovascular Dysfunction.

de Yébenes VG, Briones AM, Martos-Folgado I, Mur SM, ... Redondo JM, Ramiro AR
Objective
microRNAs are master regulators of gene expression with essential roles in virtually all biological processes. miR-217 has been associated with aging and cellular senescence, but its role in vascular disease is not understood. Approach and Results: We have used an inducible endothelium-specific knock-in mouse model to address the role of miR-217 in vascular function and atherosclerosis. miR-217 reduced NO production and promoted endothelial dysfunction, increased blood pressure, and exacerbated atherosclerosis in proatherogenic apoE mice. Moreover, increased endothelial miR-217 expression led to the development of coronary artery disease and altered left ventricular heart function, inducing diastolic and systolic dysfunction. Conversely, inhibition of endogenous vascular miR-217 in apoE mice improved vascular contractility and diminished atherosclerosis. Transcriptome analysis revealed that miR-217 regulates an endothelial signaling hub and downregulates a network of eNOS (endothelial NO synthase) activators, including VEGF (vascular endothelial growth factor) and apelin receptor pathways, resulting in diminished eNOS expression. Further analysis revealed that human plasma miR-217 is a biomarker of vascular aging and cardiovascular risk.
Conclusions
Our results highlight the therapeutic potential of miR-217 inhibitors in aging-related cardiovascular disease.



Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2408-2424
de Yébenes VG, Briones AM, Martos-Folgado I, Mur SM, ... Redondo JM, Ramiro AR
Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2408-2424 | PMID: 32847388
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Abstract

Arterial Structure and Stiffness Are Altered in Young Adults Born Preterm.

Flahault A, Oliveira Fernandes R, De Meulemeester J, Ravizzoni Dartora D, ... Luu TM, Nuyt AM
Objective
Preterm birth has been associated with changes in arterial structure and function. Association with complications occurring during the neonatal period, including bronchopulmonary dysplasia, on vascular outcomes in adulthood is unknown. Approach and Results: We evaluated a cohort of 86 adults born preterm (below 30 weeks of gestation), compared to 85 adults born term, at a mean age of 23 years. We performed ultrasonographic assessment of the dimensions of the ascending aorta, carotid and brachial arteries, and estimated flow-mediated dilation, carotid-femoral pulse wave velocity, augmentation index corrected for heart rate, and carotid intima-media thickness. All analyses were performed with and without adjustment for potential confounding variables, including height, sex, and body mass index. Ascending aorta diameter in diastole was smaller in the preterm group, but carotid and brachial arteries were similar. Carotid and brachial strain, a marker of arterial distensibility, was smaller in the preterm group, while carotid-femoral pulse wave velocity, was similar between groups, indicating similar aortic stiffness. Carotid intima-media thickness, endothelial function flow-mediated dilation, blood nitrite, and nitrate levels were similar between groups. Individuals with bronchopulmonary dysplasia had lower brachial artery strain suggesting long-term association of this neonatal complication with vascular structure. Diastolic blood pressure was higher in the preterm group and was associated with decreased brachial and carotid distensibility.
Conclusions
Young adults born preterm display alterations in arterial distensibility that are associated with a history of bronchopulmonary dysplasia.



Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2548-2556
Flahault A, Oliveira Fernandes R, De Meulemeester J, Ravizzoni Dartora D, ... Luu TM, Nuyt AM
Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2548-2556 | PMID: 32847389
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Abstract

Internalization of HMGB1 (High Mobility Group Box 1) Promotes Angiogenesis in Endothelial Cells.

Lan J, Luo H, Wu R, Wang J, ... Jiang Y, Xu J
Objective
In patients with peripheral artery disease, blockages in arterioles <1 mm cannot be treated surgically, and there are currently few effective medicines. Studies have shown that inflammation in ischemic tissue is related to injury recovery and angiogenesis, but insufficient attention has been paid to this area. Studies have suggested that HMGB1 (high mobility group protein 1), which is released by ischemic tissue, promotes angiogenesis, but the mechanism is not entirely clear. In this study, we tested the internalization of HMGB1 in endothelial cells and investigated a novel proangiogenic pathway. Approach and Results: Using green fluorescent protein-tagged HMGB1 to stimulate endothelial cells, we demonstrated HMGB1 internalization via dynamin and RAGE (receptor for advanced glycation end products)-dependent signaling. Using a fluorescence assay, we detected internalized protein fusion to lysosomes, followed by activation of CatB (cathepsin B) and CatL (cathepsin L). The latter promoted the release of VEGF (vascular endothelial growth factor)-A and endoglin and upregulated the capacities of cell migration, proliferation, and tube formation in endothelial cells. We identified that the cytokine-induced fragment-a key functional domain in HMGB1-mediates the internalization and angiogenic function of HMGB1. We further confirmed that HMGB1 internalization also occurs in vivo in endothelial cells and promotes angiogenesis in mouse femoral artery ligation.
Conclusions
In this study, we identified a novel pathway of HMGB1 internalization-induced angiogenesis in endothelial cells. This finding sheds light on the regulatory role of inflammatory factors in angiogenesis through cell internalization and opens a new door to understand the relationship between inflammation and angiogenesis in ischemic diseases.



Arterioscler Thromb Vasc Biol: 30 Sep 2020:ATVBAHA120315151; epub ahead of print
Lan J, Luo H, Wu R, Wang J, ... Jiang Y, Xu J
Arterioscler Thromb Vasc Biol: 30 Sep 2020:ATVBAHA120315151; epub ahead of print | PMID: 32998518
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Impact:
Abstract

Comparison of the Postprandial Metabolic Fate of U-C Stearic Acid and U-C Oleic Acid in Postmenopausal Women.

Rodríguez-Morató J, Galluccio J, Dolnikowski GG, Lichtenstein AH, Matthan NR
Objective
Compare the postprandial fatty acid metabolism of isotopically labeled stearate [U-C18:0] and oleate [U-C18:1]. Approach and Results: In conjunction with a randomized-controlled crossover trial, 6 hypercholesterolemic postmenopausal women (≥50 years; body mass index: 25.6±3.0 kg/m; LDL [low-density lipoprotein]-cholesterol ≥110 mg/dL) consumed isocaloric diets enriched in 18:0 or 18:1 (10%-15% E) for 5 weeks each. On day 1 of week 5, following a 12-hour fast, participants receive their experimental diet divided into 13 hourly meals beginning at 8 am. U-C18:0 or U-C18:1 was incorporated into the 1:00 pm meal (1.0 mg/kg body weight). Serial blood and breath samples were collected over 12 hours and fasting samples at 24 and 48 hours. Plasma and lipid subfraction fatty acid profiles were assessed by gas chromatography-flame ionization detector, isotope-enrichment by liquid chromatography time-of-flight mass spectrometry, and fatty acid oxidation rate (expired 13CO) by isotope ratio mass spectrometry. Both diets resulted in similar plasma LDL-cholesterol concentrations. Kinetic curves showed that U-C18:0 had a higher plasma area under the curve (66%), lower plasma clearance rate (-46%), and a lower cumulative oxidation rate (-34%) than U-C18:1. Three labeled plasma metabolites of U-C18:0 were detected: 13C16:0, C16:1, and C18:1. No plasma metabolites of U-C18:1 were detected within the study time-frame. Higher incorporation of 18:0 in cholesteryl ester and triglyceride fractions was observed on the 18:0 compared with the 18:1 diet.
Conclusions
The neutrality of 18:0 on plasma LDL-cholesterol concentrations is not attributable to a single factor. Compared with 18:1, 18:0 had higher plasma area under the curve because of lower clearance and oxidation rates, underwent both a direct and a multistage conversion to 18:1, and was preferentially incorporated into cholesteryl esters and triglycerides.



Arterioscler Thromb Vasc Biol: 30 Sep 2020:ATVBAHA120315260; epub ahead of print
Rodríguez-Morató J, Galluccio J, Dolnikowski GG, Lichtenstein AH, Matthan NR
Arterioscler Thromb Vasc Biol: 30 Sep 2020:ATVBAHA120315260; epub ahead of print | PMID: 32998517
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Abstract

Endothelial Loss Drives a Proliferative Response to BMP (Bone Morphogenetic Protein) 9 via Prolonged Canonical Signaling.

Theilmann AL, Hawke LG, Hilton LR, Whitford MKM, ... Archer SL, Ormiston ML
Objective
Pulmonary arterial hypertension is a disease of proliferative vascular occlusion that is strongly linked to mutations in -the gene encoding the BMPR-II (BMP [bone morphogenetic protein] type II receptor). The endothelial-selective BMPR-II ligand, BMP9, reverses disease in animal models of pulmonary arterial hypertension and suppresses the proliferation of healthy endothelial cells. However, the impact ofloss on the antiproliferative actions of BMP9 has yet to be assessed. Approach and Results: BMP9 suppressed proliferation in blood outgrowth endothelial cells from healthy control subjects but increased proliferation in blood outgrowth endothelial cells from pulmonary arterial hypertension patients withmutations. This shift from growth suppression to enhanced proliferation was recapitulated in control human pulmonary artery endothelial cells following siRNA-mediatedsilencing, as well as in mouse pulmonary endothelial cells isolated from endothelial-conditionalknockout mice (). BMP9-induced proliferation was not attributable to altered metabolic activity or elevated TGFβ (transforming growth factor beta) signaling but was linked to the prolonged induction of the canonical BMP targetin the context ofloss. In vivo, daily BMP9 administration to neonatal mice impaired both retinal and lung vascular patterning in control mice () but had no measurable effect on mice bearing a heterozygous endothelialdeletion () and caused excessive angiogenesis in both vascular beds formice.
Conclusions
loss reverses the endothelial response to BMP9, causing enhanced proliferation. This finding has potential implications for the proposed translation of BMP9 as a treatment for pulmonary arterial hypertension and suggests the need for focused patient selection in clinical trials.



Arterioscler Thromb Vasc Biol: 30 Sep 2020:ATVBAHA119313357; epub ahead of print
Theilmann AL, Hawke LG, Hilton LR, Whitford MKM, ... Archer SL, Ormiston ML
Arterioscler Thromb Vasc Biol: 30 Sep 2020:ATVBAHA119313357; epub ahead of print | PMID: 32998516
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Abstract

STEAP3 (Six-Transmembrane Epithelial Antigen of Prostate 3) Inhibits Pathological Cardiac Hypertrophy.

Li PL, Liu H, Chen GP, Li L, ... Huang Z, Zhu L

Pathological cardiac hypertrophy is one of the major predictors and inducers of heart failure, the end stage of various cardiovascular diseases. However, the molecular mechanisms underlying pathogenesis of pathological cardiac hypertrophy remain largely unknown. Here, we provided the first evidence that STEAP3 (Six-Transmembrane Epithelial Antigen of Prostate 3) is a key negative regulator of this disease. We found that the expression of STEAP3 was reduced in pressure overload-induced hypertrophic hearts and phenylephrine-induced hypertrophic cardiomyocytes. In a transverse aortic constriction-triggered mouse cardiac hypertrophy model, STEAP3 deficiency remarkably deteriorated cardiac hypertrophy and fibrosis, whereas the opposite phenotype was observed in the cardiomyocyte-specific STEAP3 overexpressing mice. Accordingly, STEAP3 significantly mitigated phenylephrine-induced cell enlargement in primary neonatal rat cardiomyocytes. Mechanistically, via RNA-seq and immunoprecipitation-mass screening, we demonstrated that STEAP3 directly bond to Rho family small GTPase 1 and suppressed the activation of downstream mitogen-activated protein kinase-extracellular signal-regulated kinase signaling cascade. Remarkably, the antihypertrophic effect of STEAP3 was largely blocked by overexpression of constitutively active mutant Rac1 (G12V). Our study indicates that STEAP3 serves as a novel negative regulator of pathological cardiac hypertrophy by blocking the activation of the Rac1-dependent signaling cascade and may contribute to exploring effective therapeutic strategies of pathological cardiac hypertrophy treatment.



Hypertension: 29 Sep 2020; 76:1219-1230
Li PL, Liu H, Chen GP, Li L, ... Huang Z, Zhu L
Hypertension: 29 Sep 2020; 76:1219-1230 | PMID: 32862709
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Abstract

TRAF3IP2 (TRAF3 Interacting Protein 2) Mediates Obesity-Associated Vascular Insulin Resistance and Dysfunction in Male Mice.

Grunewald ZI, Ramirez-Perez FI, Woodford ML, Morales-Quinones M, ... Chandrasekar B, Padilla J

Insulin resistance in the vasculature is a characteristic feature of obesity and contributes to the pathogenesis of vascular dysfunction and disease. However, the molecular mechanisms underlying obesity-associated vascular insulin resistance and dysfunction remain poorly understood. We hypothesized that TRAF3IP2 (TRAF3 interacting protein 2), a proinflammatory adaptor molecule known to activate pathological stress pathways and implicated in cardiovascular diseases, plays a causal role in obesity-associated vascular insulin resistance and dysfunction. We tested this hypothesis by employing genetic-manipulation in endothelial cells in vitro, in isolated arteries ex vivo, and diet-induced obesity in a mouse model ofablation in vivo. We show that ectopic expression of TRAF3IP2 blunts insulin signaling in endothelial cells and diminishes endothelium-dependent vasorelaxation in isolated aortic rings. Further, 16 weeks of high fat/high sucrose feeding impaired glucose tolerance, aortic insulin-induced vasorelaxation, and hindlimb postocclusive reactive hyperemia, while increasing blood pressure and arterial stiffness in wild-type male mice. Notably,ablation protected mice from such high fat/high sucrose feeding-induced metabolic and vascular defects. Interestingly, wild-type female mice expressed markedly reduced levels of TRAF3IP2 mRNA independent of diet and were protected against high fat/high sucrose diet-induced vascular dysfunction. These data indicate that TRAF3IP2 plays a causal role in vascular insulin resistance and dysfunction. Specifically, the present findings highlight a sexual dimorphic role of TRAF3IP2 in vascular control and identify it as a promising therapeutic target in vasculometabolic derangements associated with obesity, particularly in males.



Hypertension: 29 Sep 2020; 76:1319-1329
Grunewald ZI, Ramirez-Perez FI, Woodford ML, Morales-Quinones M, ... Chandrasekar B, Padilla J
Hypertension: 29 Sep 2020; 76:1319-1329 | PMID: 32829657
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Abstract

Platelet Dysfunction and Thrombosis in JAK2-Mutated Primary Myelofibrotic Mice.

Matsuura S, Thompson CR, Belghasem ME, Bekendam RH, ... Flaumenhaft R, Ravid K
Objective
The risk of thrombosis in myeloproliferative neoplasms, such as primary myelofibrosis varies depending on the type of key driving mutation (JAK2 [janus kinase 2], CALR [calreticulin], and MPL [myeloproliferative leukemia protein or thrombopoietin receptor]) and the accompanying mutations in other genes. In the current study, we sought to examine the propensity for thrombosis, as well as platelet activation properties in a mouse model of primary myelofibrosis induced by JAK2 (janus kinase 2 with valine to phenylalanine substitution on codon 617) mutation. Approach and Results: Vav1-hJAK2 transgenic mice show hallmarks of primary myelofibrosis, including significant megakaryocytosis and bone marrow fibrosis, with a moderate increase in red blood cells and platelet number. This mouse model was used to study responses to 2 models of vascular injury and to investigate platelet properties. Platelets derived from the mutated mice have reduced aggregation in response to collagen, reduced thrombus formation and thrombus size, as demonstrated using laser-induced or FeCl-induced vascular injury models, and increased bleeding time. Strikingly, the mutated platelets had a significantly reduced number of dense granules, which could explain impaired ADP secretion upon platelet activation, and a diminished second wave of activation.
Conclusions
Together, our study highlights for the first time the influence of a hyperactive JAK2 on platelet activation-induced ADP secretion and dense granule homeostasis, with consequent effects on platelet activation properties.



Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:e262-e272
Matsuura S, Thompson CR, Belghasem ME, Bekendam RH, ... Flaumenhaft R, Ravid K
Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:e262-e272 | PMID: 32814440
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Abstract

Role of Kv1.3 Channels in Platelet Functions and Thrombus Formation.

Fan C, Yang X, Wang WW, Wang J, ... Wang Z, Liu K
Objective
Platelet activation by stimulatory factors leads to an increase in intracellular calcium concentration ([Ca]), which is essential for almost all platelet functions. Modulation of Ca influx and [Ca] in platelets has been emerging as a possible strategy for preventing and treating platelet-dependent thrombosis. Voltage-gated potassium 1.3 channels (Kv1.3) are highly expressed in platelets and able to regulate agonist-evoked [Ca] increase. However, the role of Kv1.3 channels in regulating platelet functions and thrombosis has not yet been elucidated. In addition, it is difficult to obtain a specific blocker for this channel, since Kv1.3 shares identical drug-binding sites with other K channels. Here, we investigate whether specific blockade of Kv1.3 channels by monoclonal antibodies affects platelet functions and thrombosis. Approach and Results: In this study, we produced the anti-Kv1.3 monoclonal antibody 6E12#15, which could specifically recognize both human and mouse Kv1.3 proteins and sufficiently block Kv1.3 channel currents. We found Kv1.3 blockade by 6E12#15 inhibited platelet aggregation, adhesion, and activation upon agonist stimulation. In vivo treatment with 6E12#15 alleviated thrombus formation in a mesenteric arteriole thrombosis mouse model and protected mice from collagen/epinephrine-induced pulmonary thromboembolism. Furthermore, we observed Kv1.3 regulated platelet functions by modulating Ca influx and [Ca] elevation, and that this is mediated in part by P2X. Interestingly,mice showed impaired platelet aggregation while displayed no abnormalities in in vivo thrombus formation. This phenomenon was related to more megakaryocytes and platelets produced inmice compared with wild-type mice.
Conclusions
We showed specific inhibition of Kv1.3 by the novel monoclonal antibody 6E12#15 suppressed platelet functions and platelet-dependent thrombosis through modulating platelet [Ca] elevation. These results indicate that Kv1.3 could act as a promising therapeutic target for antiplatelet therapies.



Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2360-2375
Fan C, Yang X, Wang WW, Wang J, ... Wang Z, Liu K
Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2360-2375 | PMID: 32787516
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Abstract

SMIT (Sodium-Myo-Inositol Transporter) 1 Regulates Arterial Contractility Through the Modulation of Vascular Kv7 Channels.

Barrese V, Stott JB, Baldwin SN, Mondejar-Parreño G, Greenwood IA
Objective
The SMIT1 (sodium:myo-inositol transporter 1) regulates myo-inositol movement into cells and responses to hypertonic stimuli. Alteration of myo-inositol levels has been associated with several diseases, including hypertension, but there is no evidence of a functional role of SMIT1 in the vasculature. Recent evidence showed that in the nervous system SMIT1 interacted and modulated the function of members of the Kv7 family of voltage-gated potassium channels, which are also expressed in the vasculature where they regulate arterial contractility. Therefore, in this study, we evaluated whether SMIT1 was functionally relevant in arterial smooth muscle. Approach and Results: Immunofluorescence and polymerase chain reaction experiments revealed that SMIT1 was expressed in rat renal and mesenteric vascular smooth muscle cells. Isometric tension recordings showed that incubation of renal arteries with raffinose and myo-inositol (which increases SMIT1 expression) reduced the contractile responses to methoxamine, an effect that was abolished by preincubation with the pan-Kv7 blocker linopirdine and by molecular knockdown of Kv7.4 and Kv7.5. Knockdown of SMIT1 increased the contraction of renal arteries induced by methoxamine, impaired the response to the Kv7.2-Kv7.5 activator ML213 but did not interfere with the relaxant responses induced by openers of other potassium channels. Proximity ligation assay showed that SMIT1 interacted with heteromeric channels formed by Kv7.4 and Kv7.5 proteins in both renal and mesenteric vascular smooth muscle cells. Patch-clamp experiments showed that incubation with raffinose plus myo-inositol increased Kv7 currents in vascular smooth muscle cells.
Conclusions
SMIT1 protein is expressed in vascular smooth muscle cells where it modulates arterial contractility through an association with Kv7.4/Kv7.5 heteromers.



Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2468-2480
Barrese V, Stott JB, Baldwin SN, Mondejar-Parreño G, Greenwood IA
Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2468-2480 | PMID: 32787517
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Abstract

Aberrant Exon 8/8a Splicing by Downregulated PTBP (Polypyrimidine Tract-Binding Protein) 1 Increases Ca1.2 Dihydropyridine Resistance to Attenuate Vasodilation.

Lei J, Liu X, Song M, Zhou Y, ... Zhu G, Wang J
Objective
Calcium channel blockers, such as dihydropyridines, are commonly used to inhibit enhanced activity of vascular Ca1.2 channels in hypertension. However, patients who are insensitive to such treatments develop calcium channel blocker-resistant hypertension. The function of Ca1.2 channel is diversified by alternative splicing, and the splicing factor PTBP (polypyrimidine tract-binding protein) 1 influences the utilization of mutually exclusive exon 8/8a of the Ca1.2 channel during neuronal development. Nevertheless, whether and how PTBP1 makes a role in the calcium channel blocker sensitivity of vascular Ca1.2 channels, and calcium channel blocker-induced vasodilation remains unknown. Approach and Results: We detected high expression of PTBP1 and, inversely, low expression of exon 8a in Ca1.2 channels (Ca1.2) in rat arteries. In contrast, the opposite expression patterns were observed in brain and heart tissues. In comparison to normotensive rats, the expressions of PTBP1 and Ca1.2 channels were dysregulated in mesenteric arteries of hypertensive rats. Notably, PTBP1 expression was significantly downregulated, and Ca1.2 channels were aberrantly increased in dihydropyridine-resistant arteries compared with dihydropyridine-sensitive arteries of rats and human. In rat vascular smooth muscle cells, PTBP1 knockdown resulted in shifting of Ca1.2 exon 8 to 8a. Using patch-clamp recordings, we demonstrated a concomitant reduction of sensitivity of Ca1.2 channels to nifedipine, due to the higher expression of Ca1.2 isoform. In vascular myography experiments, small interfering RNA-mediated knockdown of PTBP1 attenuated nifedipine-induced vasodilation of rat mesenteric arteries.
Conclusions
PTBP1 finely modulates the sensitivities of Ca1.2 channels to dihydropyridine by shifting the utilization of exon 8/8a and resulting in changes of responses in dihydropyridine-induced vasodilation.



Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2440-2453
Lei J, Liu X, Song M, Zhou Y, ... Zhu G, Wang J
Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2440-2453 | PMID: 32787518
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Impact:
Abstract

Antiplatelet Effect of Carbon Monoxide Is Mediated by NAD and ATP Depletion.

Kaczara P, Sitek B, Przyborowski K, Kurpinska A, ... Stojak M, Chlopicki S
Objectives
Carbon monoxide (CO) produced by haem oxygenases or released by CO-releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO-induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results: To analyze the effects of CORM-A1 on human platelet aggregation and bioenergetics, a light transmission aggregometry, Seahorse XFe technique and liquid chromatography tandem-mass spectrometry-based metabolomics were used. CORM-A1-induced inhibition of platelet aggregation was accompanied by the inhibition of mitochondrial respiration and glycolysis. Interestingly, specific inhibitors of these processes applied individually, in contrast to combined treatment, did not inhibit platelet aggregation considerably. A CORM-A1-induced delay of tricarboxylic acid cycle was associated with oxidized nicotinamide adenine dinucleotide (NAD) depletion, compatible with the inhibition of oxidative phosphorylation. CORM-A1 provoked an increase in concentrations of proximal (before GAPDH [glyceraldehyde 3-phosphate dehydrogenase]), but not distal glycolysis metabolites, suggesting that CO delayed glycolysis at the level of NAD-dependent GAPDH; however, GAPDH activity was directly not inhibited. In the presence of exogenous pyruvate, CORM-A1-induced inhibition of platelet aggregation and glycolysis were lost, but were restored by the inhibition of lactate dehydrogenase, involved in cytosolic NAD regeneration, pointing out to the key role of NAD depletion in the inhibition of platelet bioenergetics by CORM-A1.
Conclusions
The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration-attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis-ascribed to cytosolic NAD depletion.



Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2376-2390
Kaczara P, Sitek B, Przyborowski K, Kurpinska A, ... Stojak M, Chlopicki S
Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2376-2390 | PMID: 32787519
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Abstract

Loss of Down Syndrome Critical Region-1 Mediated-Hypercholesterolemia Accelerates Corneal Opacity Via Pathological Neovessel Formation.

Muramatsu M, Nakagawa S, Osawa T, Toyono T, ... Ryeom S, Minami T
Objective
The calcineurin-NFAT (nuclear factor for activated T cells)-DSCR (Down syndrome critical region)-1 pathway plays a crucial role as the downstream effector of VEGF (vascular endothelial growth factor)-mediated tumor angiogenesis in endothelial cells. A role for DSCR-1 in different organ microenvironment such as the cornea and its role in ocular diseases is not well understood. Corneal changes can be indicators of various disease states and are easily detected through ocular examinations. Approach and Results: The presentation of a corneal arcus or a corneal opacity due to lipid deposition in the cornea often indicates hyperlipidemia and in most cases, hypercholesterolemia. Although the loss of Apo (apolipoprotein) E has been well characterized and is known to lead to elevated serum cholesterol levels, there are few corneal changes observed inmice. In this study, we show that the combined loss of ApoE and DSCR-1 leads to a dramatic increase in serum cholesterol levels and severe corneal opacity with complete penetrance. The cornea is normally maintained in an avascular state; however, loss ofis sufficient to induce hyper-inflammatory and -oxidative condition, increased corneal neovascularization, and lymphangiogenesis. Furthermore, immunohistological analysis and genome-wide screening revealed that loss ofin mice triggers increased immune cell infiltration and upregulation of SDF (stromal derived factor)-1 and its receptor, CXCR4 (C-X-C motif chemokine ligand receptor-4), potentiating this signaling axis in the cornea, thereby contributing to pathological corneal angiogenesis and opacity.
Conclusions
This study is the first demonstration of the critical role for the endogenous inhibitor of calcineurin, DSCR-1, and pathological corneal angiogenesis in hypercholesterolemia induced corneal opacity.



Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2425-2439
Muramatsu M, Nakagawa S, Osawa T, Toyono T, ... Ryeom S, Minami T
Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2425-2439 | PMID: 32787520
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Impact:
Abstract

Reelin Amplifies Glycoprotein VI Activation and AlphaIIb Beta3 Integrin Outside-In Signaling via PLC Gamma 2 and Rho GTPases.

Krueger I, Gremer L, Mangels L, Klier M, ... Bock HH, Elvers M
Objective
Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice.
Conclusions
Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.



Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2391-2403
Krueger I, Gremer L, Mangels L, Klier M, ... Bock HH, Elvers M
Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2391-2403 | PMID: 32787521
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Abstract

Cholesterol Efflux-Independent Modification of Lipid Rafts by AIBP (Apolipoprotein A-I Binding Protein).

Low H, Mukhamedova N, Capettini LDSA, Xia Y, ... Miller YI, Sviridov D
Objective
AIBP (apolipoprotein A-I binding protein) is an effective and selective regulator of lipid rafts modulating many metabolic pathways originating from the rafts, including inflammation. The mechanism of action was suggested to involve stimulation by AIBP of cholesterol efflux, depleting rafts of cholesterol, which is essential for lipid raft integrity. Here we describe a different mechanism contributing to the regulation of lipid rafts by AIBP. Approach and Results: We demonstrate that modulation of rafts by AIBP may not exclusively depend on the rate of cholesterol efflux or presence of the key regulator of the efflux, ABCA1 (ATP-binding cassette transporter A-I). AIBP interacted with phosphatidylinositol 3-phosphate, which was associated with increased abundance and activation of Cdc42 and rearrangement of the actin cytoskeleton. Cytoskeleton rearrangement was accompanied with reduction of the abundance of lipid rafts, without significant changes in the lipid composition of the rafts. The interaction of AIBP with phosphatidylinositol 3-phosphate was blocked by AIBP substrate, NADPH (nicotinamide adenine dinucleotide phosphate), and both NADPH and silencing of Cdc42 interfered with the ability of AIBP to regulate lipid rafts and cholesterol efflux.
Conclusions
Our findings indicate that an underlying mechanism of regulation of lipid rafts by AIBP involves PIP-dependent rearrangement of the cytoskeleton.



Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2346-2359
Low H, Mukhamedova N, Capettini LDSA, Xia Y, ... Miller YI, Sviridov D
Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2346-2359 | PMID: 32787522
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Abstract

BAF60a Deficiency in Vascular Smooth Muscle Cells Prevents Abdominal Aortic Aneurysm by Reducing Inflammation and Extracellular Matrix Degradation.

Chang Z, Zhao G, Zhao Y, Lu H, ... Chen YE, Zhang J
Objective
Currently, there are no approved drugs for abdominal aortic aneurysm (AAA) treatment, likely due to limited understanding of the primary molecular mechanisms underlying AAA development and progression. BAF60a-a unique subunit of the SWI/SNF (switch/sucrose nonfermentable) chromatin remodeling complex-is a novel regulator of metabolic homeostasis, yet little is known about its function in the vasculature and pathogenesis of AAA. In this study, we sought to investigate the role and underlying mechanisms of vascular smooth muscle cell (VSMC)-specific BAF60a in AAA formation. Approach and Results: BAF60a is upregulated in human and experimental murine AAA lesions. In vivo studies revealed that VSMC-specific knockout of BAF60a protected mice from both Ang II (angiotensin II)-induced and elastase-induced AAA formation with significant suppression of vascular inflammation, monocyte infiltration, and elastin fragmentation. Through RNA sequencing and pathway analysis, we found that the expression of inflammatory response genes in cultured human aortic smooth muscle cells was significantly downregulated by small interfering RNA-mediated BAF60a knockdown while upregulated upon adenovirus-mediated BAF60a overexpression. BAF60a regulates VSMC inflammation by recruiting BRG1 (Brahma-related gene-1)-a catalytic subunit of the SWI/SNF complex-to the promoter region of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) target genes. Furthermore, loss of BAF60a in VSMCs prevented the upregulation of the proteolytic enzyme cysteine protease CTSS (cathepsin S), thus ameliorating ECM (extracellular matrix) degradation within the vascular wall in AAA.
Conclusions
Our study demonstrated that BAF60a is required to recruit the SWI/SNF complex to facilitate the epigenetic regulation of VSMC inflammation, which may serve as a potential therapeutic target in preventing and treating AAA.



Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2494-2507
Chang Z, Zhao G, Zhao Y, Lu H, ... Chen YE, Zhang J
Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2494-2507 | PMID: 32787523
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Abstract

Systematic Interrogation of Angiogenesis in the Ischemic Mouse Hind Limb: Vulnerabilities and Quality Assurance.

Lee JJ, Arpino JM, Yin H, Nong Z, ... O\'Neil C, Pickering JG
Objective
There has been little success in translating preclinical studies of mouse hind limb ischemia into benefit for patients with peripheral artery disease. Using systematic strategies, we sought to define the injury and angiogenesis landscapes in mice subjected to hind limb ischemia and ascertain whether published studies to date have used an analysis strategy concordant with these data. Approach and Results: Maps of ischemic injury were generated from 22 different hind limb muscles and 33 muscle territories in 12-week-old C57BL/6 mice, based on loss or centralization of myofiber nuclei. Angiogenesis was similarly mapped based on CD (cluster of differentiation) 31-positive capillary content. Only 10 of 33 muscle territories displayed consistent muscle injury, with the distal anterior hind limb muscles most reliably injured. Angiogenesis was patchy and exclusively associated with zones of regenerated muscle (central nuclei). Angiogenesis was not observed in normal appearing muscle, necrotic muscle, or injury border zones. Systematic review of mouse hind limb angiogenesis studies identified 5147 unique publications, of which 509 met eligibility criteria for analysis. Only 7% of these analyzed manuscripts evaluated angiogenesis in distal anterior hind limb muscles and only 15% consistently examined for angiogenesis in zones of muscle regeneration.
Conclusions
In 12-week C57BL/6 mice, angiogenesis postfemoral artery excision proceeds exclusively in zones of muscle regeneration. Only a minority of studies to date have analyzed angiogenesis in regions of demonstrably regenerating muscle or in high-likelihood territories. Quality assurance standards, informed by the atlas and mapping data herein, could augment data reliability and potentially help translate mouse hind limb ischemia studies to patient care.



Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2454-2467
Lee JJ, Arpino JM, Yin H, Nong Z, ... O'Neil C, Pickering JG
Arterioscler Thromb Vasc Biol: 29 Sep 2020; 40:2454-2467 | PMID: 32787524
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Abstract

Long Noncoding RNA Rps4l Mediates the Proliferation of Hypoxic Pulmonary Artery Smooth Muscle Cells.

Liu Y, Zhang H, Li Y, Yan L, ... Li G, Zhu D

Pulmonary hypertension (PH) is a rare and fatal disorder involving the vascular remodeling of pulmonary arteries mediated by the enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs). Long noncoding RNAs are a subclass of regulatory molecules with diverse cellular functions, but their role in PH remains largely unexplored. We aimed to identify and determine the functions of long noncoding RNAs involved in hypoxia-induced PH and PASMC proliferation. RNA sequencing in a hypoxic mouse model identified hypoxia-regulated long noncoding RNAs, including Rps4l. Rps4l expression was significantly reduced in PH-model mice and hypoxic PASMCs. The subcellular localization of Rps4l was detected by RNA fluorescence in situ hybridization and quantification of nuclear/cytoplasmic RNA. Rps4l overexpression rescued pulmonary arterial hypertension features, as demonstrated by right ventricle hypertrophy, right ventricular systolic pressure, hemodynamics, cardiac function, and vascular remodeling. At the cellular level, Rps4l overexpression weakened cell viability and proliferation and suppressed cell cycle progression. Potential Rps4l-binding proteins were identified via RNA pull-down followed by mass spectrometry, RNA immunoprecipitation, and microscale thermophoresis. These results indicated that Rps4l is associated with and affects the stabilization of ILF3 (interleukin enhancer-binding factor 3). Rps41 further regulates the levels of HIF-1α and consequently leads to hypoxia-induced PASMC proliferation and migration. Our results showed that in hypoxic PASMCs, Rps4l expression decreases due to regulation by hypoxia. This decrease affects the proliferation, migration, and cell cycle progression of PASMCs through ILF3/HIF-1α. These results provide a theoretical basis for further investigations into the pathological mechanism of hypoxic PH and may provide insight for the development of novel treatments.



Hypertension: 29 Sep 2020; 76:1124-1133
Liu Y, Zhang H, Li Y, Yan L, ... Li G, Zhu D
Hypertension: 29 Sep 2020; 76:1124-1133 | PMID: 32772647
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Abstract

Impaired Hedgehog-Gli1 Pathway Activity Underlies the Vascular Phenotype of Polycystic Kidney Disease.

Franchi F, Peterson KM, Quandt K, Domnick D, ... Fernandez-Zapico ME, Rodriguez-Porcel MG

Polycystic kidney disease (PKD) has been linked to abnormal structure/function of ciliary proteins, leading to renal dysfunction. Recently, attention has been focused in the significant vascular abnormalities associated with PKD, but the mechanisms underlying this phenomenon remain elusive. Here, we seek to define the molecular events regulating the angiogenic imbalance observed in PKD. Using micro computed tomography (n=7) and protein expression analysis (n=5), we assessed the vascular density and the angiogenic profile of noncystic organs in a well-established PKD rat model (Polycystic Kidney-PCK rat). Heart and lungs of PCK rats have reduced vascular density and decreased expression of angiogenic factors compared with wild type. Similarly, PCK-vascular smooth muscle cells (VSMCs; n=4) exhibited lower levels of vascular markers. Then, using small interfering RNA (n=4), we determined the role of the ciliary protein fibrocystin in wild type-VSMCs, a critical component/regulator of vascular structure and function. Reduction of fibrocystin in wild type-VSMCs (n=4) led to an abnormal angiogenic potential similar to that observed in PCK-VSMCs. Furthermore, we investigated the involvement of the hedgehog signaling, a pathway closely linked to the primary cilium and associated with vascular development, in PKD. Mechanistically, we demonstrated that impairment of the hedgehog signaling mediates, in part, this abnormal angiogenic phenotype. Lastly, overexpression of Gli1 in PCK-VSMCs (n=4) restored the expression levels of proangiogenic molecules. Our data support a critical role of fibrocystin in the abnormal vascular phenotype of PKD and indicate that a dysregulation of hedgehog may be responsible, at least in part, for these vascular deficiencies.



Hypertension: 04 Oct 2020:HYPERTENSIONAHA12015483; epub ahead of print
Franchi F, Peterson KM, Quandt K, Domnick D, ... Fernandez-Zapico ME, Rodriguez-Porcel MG
Hypertension: 04 Oct 2020:HYPERTENSIONAHA12015483; epub ahead of print | PMID: 33012205
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Abstract

Myeloid-Derived TSP1 (Thrombospondin-1) Contributes to Abdominal Aortic Aneurysm Through Suppressing Tissue Inhibitor of Metalloproteinases-1.

Yang H, Zhou T, Sorenson CM, Sheibani N, Liu B
Objective
Abdominal aortic aneurysm is characterized by the progressive loss of aortic integrity and accumulation of inflammatory cells primarily macrophages. We previously reported that global deletion of matricellular protein TSP1 (thrombospondin-1) protects mice from aneurysm formation. The objective of the current study is to investigate the cellular and molecular mechanisms underlying TSP1\'s action in aneurysm. Approach and Results: Using RNA fluorescent in situ hybridization, we identified macrophages being the major source of TSP1 in human and mouse aneurysmal tissues, accounting for over 70% of cells that actively expressedmRNA. Lack of TSP1 in macrophages decreased solution-based gelatinase activities by elevating TIMP1 (tissue inhibitor of metalloproteinases-1) without affecting the major MMPs (matrix metalloproteinases). Knocking downrestored the ability ofmacrophages to invade matrix. Finally, we generatedmice and crossed them withmice. In the CaCl-induced model of abdominal aortic aneurysm, lacking TSP1 in myeloid cells was sufficient to protect mice from aneurysm by reducing macrophage accumulation and preserving aortic integrity.
Conclusions
TSP1 contributes to aneurysm pathogenesis, at least in part, by suppressing TIMP1 expression, which subsequently enables inflammatory macrophages to infiltrate vascular tissues.



Arterioscler Thromb Vasc Biol: 07 Oct 2020:ATVBAHA120314913; epub ahead of print
Yang H, Zhou T, Sorenson CM, Sheibani N, Liu B
Arterioscler Thromb Vasc Biol: 07 Oct 2020:ATVBAHA120314913; epub ahead of print | PMID: 33028100
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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: 07 Oct 2020:ATVBAHA120315187; epub ahead of print
Ansari SA, Keshava S, Pendurthi UR, Rao LVM
Arterioscler Thromb Vasc Biol: 07 Oct 2020:ATVBAHA120315187; epub ahead of print | 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 methyl-β-cyclodextrin 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 methyl-β-cyclodextrin 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: 07 Oct 2020:ATVBAHA120315164; epub ahead of print
Chattopadhyay A, Kwartler CS, Kaw K, Li Y, ... Shen YH, Milewicz DM
Arterioscler Thromb Vasc Biol: 07 Oct 2020:ATVBAHA120315164; epub ahead of print | 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 suggest 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: 07 Oct 2020:ATVBAHA120315052; epub ahead of print
Pu X, Du L, Hu Y, Fan Y, Xu Q
Arterioscler Thromb Vasc Biol: 07 Oct 2020:ATVBAHA120315052; epub ahead of print | PMID: 33028095
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Abstract

Desert Hedgehog-Driven Endothelium Integrity Is Enhanced by Gas1 (Growth Arrest-Specific 1) but Negatively Regulated by Cdon (Cell Adhesion Molecule-Related/Downregulated by Oncogenes).

Chapouly C, Hollier PL, Guimbal S, Cornuault L, Gadeau AP, Renault MA
Objective
Evidences accumulated within the past decades identified hedgehog signaling as a new regulator of endothelium integrity. More specifically, we recently identified desert hedgehog (Dhh) as a downstream effector of Klf2 (Kruppel-like factor 2) in endothelial cells (ECs). The purpose of this study is to investigate whether hedgehog coreceptors Gas1 (growth arrest-specific 1) and Cdon (cell adhesion molecule-related/downregulated by oncogenes) may be used as therapeutic targets to modulate Dhh signaling in ECs. Approach and Results: We demonstrated that both Gas1 and Cdon are expressed in adult ECs and relied on either siRNAs- or EC-specific conditional knockout mice to investigate their role. We found that Gas1 deficiency mainly phenocopies Dhh deficiency especially by inducing VCAM (vascular cell adhesion molecule)-1 and ICAM (intercellular adhesion molecule)-1 overexpression while Cdon deficiency has opposite effects by promoting endothelial junction integrity. At a molecular level, Cdon prevents Dhh binding to Ptch1 (patched-1) and thus acts as a decoy receptor for Dhh, while Gas1 promotes Dhh binding to Smo (smoothened) and as a result potentiates Dhh effects. Since Cdon is upregulated in ECs treated by inflammatory cytokines, including TNF (tumor necrosis factor)-α and Il (interleukin)-1β, we then tested whether Cdon inhibition would promote endothelium integrity in acute inflammatory conditions and found that both fibrinogen and IgG extravasation were decreased in association with an increased Cdh5 (cadherin-5) expression in the brain cortex of EC-specific Cdon knockout mice administered locally with Il-1β.
Conclusions
Altogether, these results demonstrate that Gas1 is a positive regulator of Dhh in ECs while Cdon is a negative regulator. Interestingly, Cdon blocking molecules may then be used to promote endothelium integrity, at least in inflammatory conditions.



Arterioscler Thromb Vasc Biol: 07 Oct 2020:ATVBAHA120314441; epub ahead of print
Chapouly C, Hollier PL, Guimbal S, Cornuault L, Gadeau AP, Renault MA
Arterioscler Thromb Vasc Biol: 07 Oct 2020:ATVBAHA120314441; epub ahead of print | PMID: 33028094
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Abstract

Endogenous THBD (Thrombomodulin) Mediates Angiogenesis in the Ischemic Brain.

Wenzel J, Spyropoulos D, Assmann JC, Khan MA, ... Isermann B, Schwaninger M
Objective
THBD (thrombomodulin) is part of the anticoagulant protein C-system that acts at the endothelium and is involved in anti-inflammatory and barrier-stabilizing processes. A recombinant soluble form of THBD was shown to have protective effects in different organs, but how the endogenous THBD is regulated during ischemia, particularly in the brain is not known to date. The aim of this study was to investigate the role of THBD, especially in brain endothelial cells, during ischemic stroke. Approach and Results: To induce ischemic brain damage, we occluded the middle cerebral artery occlusion of mice. We found an increased endothelial expression ofin the peri-infarct area, whereas in the core of the ischemic tissueexpression was decreased compared with the contralateral side. We generated a novel Cre/loxP-based mouse line that allows for the inducible deletion ofspecifically in brain endothelial cells, which worsened stroke outcome 48 hours after middle cerebral artery occlusion. Unexpectedly, we found no signs of increased coagulation, thrombosis, or inflammation in the brain but decreased vessel diameters and impaired angiogenesis in the peri-infarct area that led to a reduced overall vessel length 1 week after stroke induction.
Conclusions
Endogenous THBD acts as a protective factor in the brain during ischemic stroke and enhances vessel diameter and proliferation. These previously unknown properties of THBD could offer new opportunities to affect vessel function after ischemia and thereby improve stroke outcome.



Arterioscler Thromb Vasc Biol: 07 Oct 2020:ATVBAHA120315061; epub ahead of print
Wenzel J, Spyropoulos D, Assmann JC, Khan MA, ... Isermann B, Schwaninger M
Arterioscler Thromb Vasc Biol: 07 Oct 2020:ATVBAHA120315061; epub ahead of print | PMID: 33028093
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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: 07 Oct 2020:ATVBAHA120314644; epub ahead of print
Puhm F, Boilard E, Machlus KR
Arterioscler Thromb Vasc Biol: 07 Oct 2020:ATVBAHA120314644; epub ahead of print | PMID: 33028092
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Abstract

Role of Endothelial G Protein-Coupled Receptor Kinase 2 in Angioedema.

Gambardella J, Sorriento D, Bova M, Rusciano M, ... Santulli G, Ciccarelli M

Excessive BK (bradykinin) stimulation is responsible for the exaggerated permeabilization of the endothelium in angioedema. However, the molecular mechanisms underlying these responses have not been investigated. BK receptors are Gq-protein-coupled receptors phosphorylated by GRK2 (G protein-coupled receptor kinase 2) with a hitherto unknown biological and pathophysiological significance. In the present study, we sought to identify the functional role of GRK2 in angioedema through the regulation of BK signaling. We found that the accumulation of cytosolic Ca in endothelial cells induced by BK was sensitive to GRK2 activity, as it was significantly augmented by inhibiting the kinase. Accordingly, permeabilization and NO production induced by BK were enhanced, as well. In vivo, mice with reduced GRK2 levels in the endothelium (Tie2-CRE/GRK2) exhibited an increased response to BK in terms of vascular permeability and extravasation. Finally, patients with reduced GRK2 levels displayed a severe phenotype of angioedema. Taken together, these findings establish GRK2 as a novel pivotal regulator of BK signaling with an essential role in the pathophysiology of vascular permeability and angioedema.



Hypertension: 30 Oct 2020; 76:1625-1636
Gambardella J, Sorriento D, Bova M, Rusciano M, ... Santulli G, Ciccarelli M
Hypertension: 30 Oct 2020; 76:1625-1636 | PMID: 32895019
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Abstract

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

Ghaffari S, Jang E, Nabi FN, 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: 14 Oct 2020:ATVBAHA120314557; epub ahead of print
Ghaffari S, Jang E, Nabi FN, Sanwal R, ... Ikeda J, Lee WL
Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA120314557; epub ahead of print | PMID: 33054399
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Abstract

Naive CD8 T Cells Expressing CD95 Increase Human Cardiovascular Disease Severity.

Padgett LE, Dinh HQ, Wu R, Gaddis DE, ... McNamara CA, Hedrick CC
Objective
Cardiovascular disease (CVD) remains a significant global health concern with a high degree of mortality. While CD4 T cells have been extensively studied in CVD, the importance of CD8 T cells in this disease, despite their abundance and increased activation in human atherosclerotic plaques, remains largely unknown. To compare the peripheral T-cell signatures between humans with a high (severe) risk of CVD (including myocardial infarction or stroke) to those with a low risk of CVD. Approach and Results: Using mass cytometry, we uncovered a naive CD8 T (T) cell population expressing CD95 (termed CD95CD8 stem cell memory T [CD8 T] cells) that was enriched in patients with high compared with low CVD. This T-cell subset enrichment within individuals with high CVD was a relative increase and resulted from the loss of CD95 cells within the T compartment. We found that CD8 T cells positively correlated with CVD risk in humans, while CD8 T cells were inversely correlated. Atherosclerotic apolipoprotein E-deficient (ApoE) mice also displayed respective 7- and 2-fold increases in CD8 T frequencies within the peripheral blood and aorta-draining paraaortic lymph nodes compared with C57BL/6J mice. CD8 T cells were 1.7-fold increased in aortas from western diet fed ApoE mice compared with normal laboratory diet-fed ApoE mice. Importantly, transfer of T cells into immune-deficientrecipient mice that lacked T cells increased atherosclerosis, illustrating the importance of these cells in atherogenesis.
Conclusions
CD8 T cells are increased in humans with high CVD. As these T cells promote atherosclerosis, targeting them may attenuate atherosclerotic plaque progression.



Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA120315106; epub ahead of print
Padgett LE, Dinh HQ, Wu R, Gaddis DE, ... McNamara CA, Hedrick CC
Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA120315106; epub ahead of print | PMID: 33054398
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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: 14 Oct 2020:ATVBAHA120314703; epub ahead of print
Bulut GB, Alencar GF, Owsiany KM, Nguyen AT, ... Keller SR, Owens GK
Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA120314703; epub ahead of print | PMID: 33054397
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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: 14 Oct 2020:ATVBAHA120314407; epub ahead of print
AlSiraj Y, Thatcher SE, Blalock E, Saintilnord WN, ... Arnold AP, Cassis LA
Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA120314407; epub ahead of print | PMID: 33054396
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Abstract

Fibroblast Nox2 (NADPH Oxidase-2) Regulates ANG II (Angiotensin II)-Induced Vascular Remodeling and Hypertension via Paracrine Signaling to Vascular Smooth Muscle Cells.

Harrison CB, Trevelin SC, Richards DA, Santos CXC, ... Mayr M, Shah AM
Objective
The superoxide-generating Nox2 (NADPH oxidase-2) is expressed in multiple cell types. Previous studies demonstrated distinct roles for cardiomyocyte, endothelial cell, and leukocyte cell Nox2 in ANG II (angiotensin II)-induced cardiovascular remodeling. However, the in vivo role of fibroblast Nox2 remains unclear. Approach and Results: We developed a novel mouse model with inducible fibroblast-specific deficiency of Nox2 (Fibro-Nox2KO mice) and investigated the responses to chronic ANG II stimulation. Fibro-Nox2KO mice showed no differences in basal blood pressure or vessel wall morphology, but the hypertensive response to ANG II infusion (1.1 mg/[kg·day] for 14 days) was substantially reduced as compared to control Nox2-Flox littermates. This was accompanied by a significant attenuation of aortic and resistance vessel remodeling. The conditioned medium of ANG II-stimulated primary fibroblasts induced a significant increase in vascular smooth muscle cell, which was inhibited by the shRNA-mediated knockdown of fibroblast Nox2. Mass spectrometric analysis of the secretome of ANG II-treated primary fibroblasts identified GDF6 (growth differentiation factor 6) as a potential growth factor that may be involved in these effects. Recombinant GDF6 induced a concentration-dependent increase in vascular smooth muscle cell growth while chronic ANG II infusion in vivo significantly increased aortic GDF6 protein levels in control mice but not Fibro-Nox2KO animals. Finally, silencing GDF6 in fibroblasts prevented the induction of vascular smooth muscle cell growth by fibroblast-conditioned media in vitro.
Conclusions
These results indicate that fibroblast Nox2 plays a crucial role in the development of ANG II-induced vascular remodeling and hypertension in vivo. Mechanistically, fibroblast Nox2 may regulate paracrine signaling to medial vascular smooth muscle cells via factors, such as GDF6.



Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA120315322; epub ahead of print
Harrison CB, Trevelin SC, Richards DA, Santos CXC, ... Mayr M, Shah AM
Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA120315322; epub ahead of print | PMID: 33054395
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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: 14 Oct 2020:ATVBAHA120314804; epub ahead of print
Yang T, Zhang F
Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA120314804; epub ahead of print | PMID: 33054394
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Abstract

Recent Advances in Vascular Imaging.

Nishimiya K, Matsumoto Y, Shimokawa H

Recent advances in vascular imaging have enabled us to uncover the underlying mechanisms of vascular diseases both ex vivo and in vivo. In the past decade, efforts have been made to establish various methodologies for evaluation of atherosclerotic plaque progression and vascular inflammatory changes in addition to biomarkers and clinical manifestations. Several recent publications inhighlighted the essential roles of in vivo and ex vivo vascular imaging, including magnetic resonance image, computed tomography, positron emission tomography/scintigraphy, ultrasonography, intravascular ultrasound, and most recently, optical coherence tomography, all of which can be used in bench and clinical studies at relative ease. With new methods proposed in several landmark studies, these clinically available imaging modalities will be used in the near future. Moreover, future development of intravascular imaging modalities, such as optical coherence tomography-intravascular ultrasound, optical coherence tomography-near-infrared autofluorescence, polarized-sensitive optical coherence tomography, and micro-optical coherence tomography, are anticipated for better management of patients with cardiovascular disease. In this review article, we will overview recent advances in vascular imaging and ongoing works for future developments.



Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA120313609; epub ahead of print
Nishimiya K, Matsumoto Y, Shimokawa H
Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA120313609; epub ahead of print | PMID: 33054393
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Abstract

Macrophage Biology in Cardiovascular Diseases.

Khoury MK, Yang H, Liu B

Macrophages have key functional role in the pathogenesis are various cardiovascular diseases, such as atherosclerosis and aortic aneurysm. Their accumulation within the vessel wall leads to sustained local inflammatory responses characterized by secretion of chemokines, cytokines, and matrix protein degrading enzymes. Here, we summarize recent findings on macrophage contribution to cardiovascular disease published in . In this issue, we focus on the origin, survival/death, and phenotypic switching of macrophages within vessel walls.



Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA120313584; epub ahead of print
Khoury MK, Yang H, Liu B
Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA120313584; epub ahead of print | PMID: 33054391
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Impact:
Abstract

Epac1 (Exchange Protein Directly Activated by cAMP 1) Upregulates LOX-1 (Oxidized Low-Density Lipoprotein Receptor 1) to Promote Foam Cell Formation and Atherosclerosis Development.

Robichaux WG, Mei FC, Yang W, Wang H, ... Teng BB, Cheng X
Objective
The cAMP second messenger system, a major stress-response pathway, plays essential roles in normal cardiovascular functions and in pathogenesis of heart diseases. Here, we test the hypothesis that the Epac1 (exchange protein directly activated by cAMP 1) acts as a major downstream effector of cAMP signaling to promote atherogenesis and represents a novel therapeutic target. Approach and Results: To ascertain Epac1\'s function in atherosclerosis development, a triple knockout mouse model () was generated by crossingmice with atherosclerosis-pronemice lacking bothand . Deletion of Epac1 led to a significant reduction of atherosclerotic lesion formation as measured by postmortem staining, accompanied by attenuated macrophage/foam cell infiltrations within atherosclerotic plaques as determined by immunofluorescence staining inanimals compared withlittermates. Primary bone marrow-derived macrophages were isolated from Epac1-null and wild-type mice to investigate the role of Epac1 in lipid uptake and foam cell formation. ox-LDLs (oxidized low-density lipoproteins) stimulation of bone marrow-derived macrophages led to elevated intracellular cAMP and Epac1 levels, whereas an Epac-specific agonist, increased lipid accumulation in wild-type, but not Epac1-null, bone marrow-derived macrophages. Mechanistically, Epac1 acts through PKC (protein kinase C) to upregulate LOX-1 (ox-LDL receptor 1), a major scavenger receptor for ox-LDL uptake, exerting a feedforward mechanism with ox-LDL to increase lipid uptake and propel foam cell formation and atherogenesis.
Conclusions
Our study demonstrates a fundamental role of cAMP/Epac1 signaling in vascular remodeling by promoting ox-LDL uptake and foam cell formation during atherosclerosis lesion development. Therefore, Epac1 represents a promising, unexplored therapeutic target for atherosclerosis.



Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA119314238; epub ahead of print
Robichaux WG, Mei FC, Yang W, Wang H, ... Teng BB, Cheng X
Arterioscler Thromb Vasc Biol: 14 Oct 2020:ATVBAHA119314238; epub ahead of print | PMID: 33054390
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Impact:
Abstract

Platelets Promote Thromboinflammation in SARS-CoV-2 Pneumonia.

Taus F, Salvagno G, Canè S, Fava C, ... Bronte V, Minuz P
Objective
Pulmonary thrombosis is observed in severe acute respiratory syndrome coronavirus 2 pneumonia. Aim was to investigate whether subpopulations of platelets were programmed to procoagulant and inflammatory activities in coronavirus disease 2019 (COVID-19) patients with pneumonia, without comorbidities predisposing to thromboembolism. Approach and Results: Overall, 37 patients and 28 healthy subjects were studied. Platelet-leukocyte aggregates, platelet-derived microvesicles, the expression of P-selectin, and active fibrinogen receptor on platelets were quantified by flow cytometry. The profile of 45 cytokines, chemokines, and growth factors released by platelets was defined by immunoassay. The contribution of platelets to coagulation factor activity was selectively measured. Numerous platelet-monocyte (mean±SE, 67.9±4.9%, n=17 versus 19.4±3.0%, n=22; <0.0001) and platelet-granulocyte conjugates (34.2±4.04% versus 8.6±0.7%; <0.0001) were detected in patients. Resting patient platelets had similar levels of P-selectin (10.9±2.6%, n=12) to collagen-activated control platelets (8.7±1.5%), which was not further increased by collagen activation on patient platelets (12.4±2.5%, =nonsignificant). The agonist-stimulated expression of the active fibrinogen receptor was reduced by 60% in patients (<0.0001 versus controls). Cytokines (IL [interleukin]-1α, IL-1β, IL-1RA, IL-4, IL-10, IL-13, IL, 17, IL-27, IFN [interferon]-α, and IFN-γ), chemokines (MCP-1/CCL2), and growth factors (VEGF [vascular endothelial growth factor]-A/D) were released in significantly larger amounts upon stimulation of COVID-19 platelets. Platelets contributed to increased fibrinogen, VWF (von Willebrand factor), and factor XII in COVID-19 patients. Patients (28.5±0.7 s, n=32), unlike controls (31.6±0.5 s, n=28; <0.001), showed accelerated factor XII-dependent coagulation.
Conclusions
Platelets in COVID-19 pneumonia are primed to spread proinflammatory and procoagulant activities in systemic circulation.



Arterioscler Thromb Vasc Biol: 13 Oct 2020:ATVBAHA120315175; epub ahead of print
Taus F, Salvagno G, Canè S, Fava C, ... Bronte V, Minuz P
Arterioscler Thromb Vasc Biol: 13 Oct 2020:ATVBAHA120315175; epub ahead of print | PMID: 33052054
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Impact:
Abstract

Mas Receptor Activation Contributes to the Improvement of Nitric Oxide Bioavailability and Vascular Remodeling During Chronic AT1R (Angiotensin Type-1 Receptor) Blockade in Experimental Hypertension.

Savoia C, Arrabito E, Parente R, Nicoletti C, ... Touyz RM, Volpe M

Angiotensin (1-7) production increases during AT1R (angiotensin type-1 receptor) blockade. The contribution of Ang (1-7) (angiotensin [1-7]) and its receptor (MasR) to the favorable effect of angiotensin receptor blockers on remodeling and function of resistance arteries remains unclear. We sought to determine whether MasR contributes to the improvement of vascular structure and function during chronic AT1R blockade. Spontaneously hypertensive rats were treated with Ang (1-7) or olmesartan ± MasR antagonist A-779, or vehicle, for 14 days. Blood pressure was measured by tail cuff methodology. Mesenteric arteries were dissected and mounted on a pressurized micromyograph to evaluate media-to-lumen ratio (M/L) and endothelial function. Expression of MasR and eNOS (endothelial nitric oxide synthase) was evaluated by immunoblotting, plasma nitrate by colorimetric assay, and reactive oxygen species production by dihydroethidium staining. Independently of blood pressure, olmesartan significantly reduced M/L and improved NO bioavailability, A-779 prevented these effects. Likewise, Ang (1-7) significantly reduced M/L and NO bioavailability. MasR expression was significantly increased by Ang (1-7) as well as by olmesartan, and it was blunted in the presence of A-779. Both Ang (1-7) and olmesartan increased eNOS expression and plasma nitrite which were reduced by A-779. Superoxide generation was attenuated by olmesartan and Ang (1-7) and was blunted in the presence of A-779. These MasR-mediated actions were independent of AT2R activation since olmesartan and Ang (1-7) increased MasR expression and reduced M/L in Ang II (angiotensin II)-infused AT2R knockout mice, independently of blood pressure control. A-779 prevented these effects. Hence, MasR activation may contribute to the favorable effects of AT1R antagonism on NO bioavailability and microvascular remodeling, independently of AT2R activation and blood pressure control.



Hypertension: 18 Oct 2020:HYPERTENSIONAHA12015527; epub ahead of print
Savoia C, Arrabito E, Parente R, Nicoletti C, ... Touyz RM, Volpe M
Hypertension: 18 Oct 2020:HYPERTENSIONAHA12015527; epub ahead of print | PMID: 33070664
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Impact:
Abstract

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

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



Circ Res: 24 Sep 2020; 127:1023-1035
Schuhmann MK, Stoll G, Bieber M, Vögtle T, ... Nieswandt B, Stegner D
Circ Res: 24 Sep 2020; 127:1023-1035 | PMID: 32762491
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Abstract

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

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



Circ Res: 24 Sep 2020; 127:997-1022
Vaisar T, Hu JH, Airhart N, Fox K, ... Gharib SA, Dichek DA
Circ Res: 24 Sep 2020; 127:997-1022 | PMID: 32762496
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Impact:
Abstract

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

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



Circ Res: 24 Sep 2020; 127:1056-1073
Kakogiannos N, Ferrari L, Giampietro C, Scalise AA, ... Dejana E, Giannotta M
Circ Res: 24 Sep 2020; 127:1056-1073 | PMID: 32673519
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Impact:
Abstract

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

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



Circ Res: 24 Sep 2020; 127:953-973
Huang CK, Dai D, Xie H, Zhu Z, ... Zhang R, Yan X
Circ Res: 24 Sep 2020; 127:953-973 | PMID: 32600176
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Impact:
Abstract

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

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

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



Circ Res: 27 Sep 2020; epub ahead of print
Depuydt MA, Prange KH, Slenders L, Örd T, ... de Winther MP, Pasterkamp G
Circ Res: 27 Sep 2020; epub ahead of print | PMID: 32981416
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Impact:
Abstract

Recent Advances in the Role of the Adenosinergic System in Coronary Artery Disease.

Paganelli F, Gaudry M, Ruf J, Guieu R

Adenosine is an endogenous nucleoside that plays a major role in the physiology and physiopathology of the coronary artery system, mainly by activating its A2A receptors (A2AR). Adenosine is released by myocardial, endothelial and immune cells during hypoxia, ischaemia or inflammation, each condition being present in coronary artery disease (CAD). While activation of A2AR improves coronary blood circulation and leads to anti-inflammatory effects, downregulation of A2AR has many deleterious effects during CAD. A decrease in the level and/or activity of A2AR leads to: i) lack of vasodilation, which decreases blood flow, leading to a decrease in myocardial oxygenation and tissue hypoxia; ii) an increase in the immune response, favouring inflammation; and iii) platelet aggregation, which therefore participates, in part, in the formation of a fibrin-platelet thrombus after the rupture or erosion of the plaque, leading to the occurrence of acute coronary syndrome. Inflammation contributes to the development of atherosclerosis, leading to myocardial ischaemia, which in turn leads to tissue hypoxia. Therefore, a vicious circle is created that maintains and aggravates CAD. In some cases, studying the adenosinergic profile can help assess the severity of CAD. In fact, inducible ischaemia in CAD patients, as assessed by exercise stress test or fractional flow reserve, is associated with the presence of a reserve of A2AR called spare receptors. The purpose of this review is to present emerging experimental evidence supporting the existence of this adaptive adenosinergic response to ischaemia or inflammation in CAD. We believe that we have achieved a breakthrough in the understanding and modeling of spare A2AR, based upon a new concept allowing for a new and non-invasive CAD management.

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

Cardiovasc Res: 28 Sep 2020; epub ahead of print
Paganelli F, Gaudry M, Ruf J, Guieu R
Cardiovasc Res: 28 Sep 2020; epub ahead of print | PMID: 32991685
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Abstract

PCSK9 and LRP5 in macrophage lipid internalization and inflammation.

Badimon L, Luquero A, Crespo J, Peña E, Borrell-Pages M
Aims
Atherosclerosis, the leading cause of cardiovascular diseases, is driven by high blood cholesterol levels and chronic inflammation. Low-Density Lipoprotein Receptor (LDLR) play a critical role in regulating blood cholesterol levels by binding to and clearing LDLs from the circulation. The disruption of the interaction between Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) and LDLR reduces blood cholesterol levels. It is not well known whether other members of the LDLR superfamily may be targets of PCSK9. The aim of this work was to determine if LDLR-related protein 5 (LRP5) is a PCSK9 target, and to study the role of PCSK9 and LRP5 in foam cell formation and lipid accumulation.
Methods and results
Primary cultures of human inflammatory cells (monocytes and macrophages) were silenced for LRP5 or PCSK9 and challenged with LDLs. We first show that LRP5 is needed for macrophage lipid uptake since LRP5-silenced macrophages show less intracellular CE accumulation. In macrophages, internalization of LRP5-bound LDL is already highly evident after 5 hours of LDL incubation and lasts up to 24hours; however in the absence of both LRP5 and PCSK9 there is a strong reduction of CE accumulation indicating a role for both proteins in lipid uptake. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in lipid-loaded macrophages. Finally PCSK9 participates in TLR4/NFkB signaling; a decreased TLR4 protein expression levels and a decreased nuclear translocation of NFκB was detected in PCSK9 silenced cells after lipid loading, indicating a down-regulation of the TLR4/NFκB pathway.
Conclusion
Our results show that both LRP5 and PCSK9 participate in lipid uptake in macrophages. In the absence of LRP5 there is a reduced release of PCSK9 indicating that LRP5 also participates in the mechanism of release of soluble PCSK9. Furthermore, PCSK9 up-regulates TLR4/NFκB favoring inflammation.
Translational perspective
We demonstrate that PCSK9 and LRP5 contribute to lipid uptake. We also show that LRP5 participates in PCSK9 transport to the plasma membrane and that PCSK9 inhibition protects against agLDL-induced inflammation associated to the TLR4/NFκB pathway. These results offer new targets to prevent the progression of inflammation and hypercholesterolemia and their increased risk of cardiovascular events.

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

Cardiovasc Res: 28 Sep 2020; epub ahead of print
Badimon L, Luquero A, Crespo J, Peña E, Borrell-Pages M
Cardiovasc Res: 28 Sep 2020; epub ahead of print | PMID: 32991689
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Impact:
Abstract

Pathobiology of pulmonary artery hypertension: role of long non-coding RNAs.

Zahid KR, Raza U, Chen J, Raj UJ, Gou D

Pulmonary arterial hypertension (PAH) is a disease with complex pathobiology, significant morbidity and mortality, and remains without a cure. It is characterized by vascular remodelling associated with uncontrolled proliferation of pulmonary artery smooth muscle cells, endothelial cell proliferation and dysfunction, and endothelial-to-mesenchymal transition, leading to narrowing of the vascular lumen, increased vascular resistance and pulmonary arterial pressure, which inevitably results in right heart failure and death. There are multiple molecules and signalling pathways that are involved in the vascular remodelling, including non-coding RNAs, i.e. microRNAs and long non-coding RNAs (lncRNAs). It is only in recent years that the role of lncRNAs in the pathobiology of pulmonary vascular remodelling and right ventricular dysfunction is being vigorously investigated. In this review, we have summarized the current state of knowledge about the role of lncRNAs as key drivers and gatekeepers in regulating major cellular and molecular trafficking involved in the pathogenesis of PAH. In addition, we have discussed the limitations and challenges in translating lncRNA research in vivo and in therapeutic applications of lncRNAs in PAH.

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

Cardiovasc Res: 30 Sep 2020; 116:1937-1947
Zahid KR, Raza U, Chen J, Raj UJ, Gou D
Cardiovasc Res: 30 Sep 2020; 116:1937-1947 | PMID: 32109276
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Abstract

The LINC00961 transcript and its encoded micropeptide, small regulatory polypeptide of amino acid response, regulate endothelial cell function.

Spencer HL, Sanders R, Boulberdaa M, Meloni M, ... Rodor J, Baker AH
Aims
Long non-coding RNAs (lncRNAs) play functional roles in physiology and disease, yet understanding of their contribution to endothelial cell (EC) function is incomplete. We identified lncRNAs regulated during EC differentiation and investigated the role of LINC00961 and its encoded micropeptide, small regulatory polypeptide of amino acid response (SPAAR), in EC function.
Methods and results
Deep sequencing of human embryonic stem cell differentiation to ECs was combined with Encyclopedia of DNA Elements (ENCODE) RNA-seq data from vascular cells, identifying 278 endothelial enriched genes, including 6 lncRNAs. Expression of LINC00961, first annotated as an lncRNA but reassigned as a protein-coding gene for the SPAAR micropeptide, was increased during the differentiation and was EC enriched. LINC00961 transcript depletion significantly reduced EC adhesion, tube formation, migration, proliferation, and barrier integrity in primary ECs. Overexpression of the SPAAR open reading frame increased tubule formation; however, overexpression of the full-length transcript did not, despite production of SPAAR. Furthermore, overexpression of an ATG mutant of the full-length transcript reduced network formation, suggesting a bona fide non-coding RNA function of the transcript with opposing effects to SPAAR. As the LINC00961 locus is conserved in mouse, we generated an LINC00961 locus knockout (KO) mouse that underwent hind limb ischaemia (HLI) to investigate the angiogenic role of this locus in vivo. In agreement with in vitro data, KO animals had a reduced capillary density in the ischaemic adductor muscle after 7 days. Finally, to characterize LINC00961 and SPAAR independent functions in ECs, we performed pull-downs of both molecules and identified protein-binding partners. LINC00961 RNA binds the G-actin sequestering protein thymosin beta-4x (Tβ4) and Tβ4 depletion phenocopied the overexpression of the ATG mutant. SPAAR binding partners included the actin-binding protein, SYNE1.
Conclusion
The LINC00961 locus regulates EC function in vitro and in vivo. The gene produces two molecules with opposing effects on angiogenesis: SPAAR and LINC00961.

© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 30 Sep 2020; 116:1981-1994
Spencer HL, Sanders R, Boulberdaa M, Meloni M, ... Rodor J, Baker AH
Cardiovasc Res: 30 Sep 2020; 116:1981-1994 | PMID: 31990292
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Abstract

Mechanistic definition of the cardiovascular mPGES-1/COX-2/ADMA axis.

Kirkby NS, Raouf J, Ahmetaj-Shala B, Liu B, ... Jakobsson PJ, Mitchell JA
Aims
Cardiovascular side effects caused by non-steroidal anti-inflammatory drugs (NSAIDs), which all inhibit cyclooxygenase (COX)-2, have prevented development of new drugs that target prostaglandins to treat inflammation and cancer. Microsomal prostaglandin E synthase-1 (mPGES-1) inhibitors have efficacy in the NSAID arena but their cardiovascular safety is not known. Our previous work identified asymmetric dimethylarginine (ADMA), an inhibitor of endothelial nitric oxide synthase, as a potential biomarker of cardiovascular toxicity associated with blockade of COX-2. Here, we have used pharmacological tools and genetically modified mice to delineate mPGES-1 and COX-2 in the regulation of ADMA.
Methods and results
Inhibition of COX-2 but not mPGES-1 deletion resulted in increased plasma ADMA levels. mPGES-1 deletion but not COX-2 inhibition resulted in increased plasma prostacyclin levels. These differences were explained by distinct compartmentalization of COX-2 and mPGES-1 in the kidney. Data from prostanoid synthase/receptor knockout mice showed that the COX-2/ADMA axis is controlled by prostacyclin receptors (IP and PPARβ/δ) and the inhibitory PGE2 receptor EP4, but not other PGE2 receptors.
Conclusion
These data demonstrate that inhibition of mPGES-1 spares the renal COX-2/ADMA pathway and define mechanistically how COX-2 regulates ADMA.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 30 Sep 2020; 116:1972-1980
Kirkby NS, Raouf J, Ahmetaj-Shala B, Liu B, ... Jakobsson PJ, Mitchell JA
Cardiovasc Res: 30 Sep 2020; 116:1972-1980 | PMID: 31688905
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Abstract

HIV Antivirals Affect Endothelial Activation and Endothelial-Platelet Crosstalk.

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

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



Circ Res: 30 Sep 2020; epub ahead of print
Khawaja AA, Taylor KA, Lovell AO, Nelson M, ... Boffito M, Emerson M
Circ Res: 30 Sep 2020; epub ahead of print | PMID: 32998637
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Abstract

Co-expression of calcium and hERG potassium channels reduces the incidence of proarrhythmic events.

Ballouz S, Mangala MM, Perry MD, Heitmann S, ... Hill AP, Vandenberg JI
Aims
Cardiac electrical activity is extraordinarily robust. However, when it goes wrong it can have fatal consequences. Electrical activity in the heart is controlled by the carefully orchestrated activity of more than a dozen different ion conductances. Whilst there is considerable variability in cardiac ion channel expression levels between individuals, studies in rodents have indicated that there are modules of ion channels whose expression co-vary. The aim of this study was to investigate whether meta-analytic co-expression analysis of large-scale gene expression data sets could identify modules of co-expressed cardiac ion channel genes in human hearts that are of functional importance.
Methods and results
Meta-analysis of 3653 public human RNA-seq datasets identified a strong correlation between expression of CACNA1C (L-type calcium current, ICaL) and KCNH2 (rapid delayed rectifier K+ current, IKr), which was also observed in human adult heart tissue samples. In silico modeling suggested that co-expression of CACNA1C and KCNH2 would limit the variability in action potential duration seen with variations in expression of ion channel genes and reduce susceptibility to early afterdepolarizations, a surrogate marker for pro-arrhythmia. We also found that levels of KCNH2 and CACNA1C expression are correlated in human induced pluripotent stem cell derived cardiac myocytes and the levels of CACNA1C and KCNH2 expression were inversely correlated with the magnitude of changes in repolarization duration following inhibition of IKr.
Conclusions
Meta-analytic approaches of multiple independent human gene expression datasets can be used to identify gene modules that are important for regulating heart function. Specifically, we have verified that there is co-expression of CACNA1C and KCNH2 ion channel genes in human heart tissue, and in silico analyses suggest that CACNA1C-KCNH2 co-expression increases the robustness of cardiac electrical activity.
Translational perspective
Here, we show, using meta-analysis of multiple independent human gene expression datasets, that there is co-expression of KCNH2-CACNA1C in human heart tissue which was then confirmed in human cardiac myocytes derived from induced pluripotent stem cells. Both in silico and functional studies show that the co-expression of CACNA1C and KCNH2 increases the robustness of cardiac electrical signalling. Our data also suggest that those patients who express higher levels of CACNA1C and KCNH2 are likely to be more susceptible to arrhythmias when exposed to drugs that block IKr, the major cause of drug-induced cardiac arrhythmias.

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

Cardiovasc Res: 30 Sep 2020; epub ahead of print
Ballouz S, Mangala MM, Perry MD, Heitmann S, ... Hill AP, Vandenberg JI
Cardiovasc Res: 30 Sep 2020; epub ahead of print | PMID: 33002116
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Abstract

Transcriptome Analysis of Non-Human Primate Induced Pluripotent Stem Cell-Derived Cardiomyocytes in 2D Monolayer Culture versus 3D Engineered Heart Tissue.

Yang H, Shao N, Holmström A, Zhao X, ... Abilez OJ, Wu JC
Aims
Stem cell therapy has shown promise for treating myocardial infarction (MI) via re-muscularization and paracrine signaling in both small and large animals. Non-human primates (NHPs), such as rhesus macaques (Macaca mulatta), are primarily utilized in preclinical trials due to their similarity to humans, both genetically and physiologically. Currently, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are delivered into the infarcted myocardium by either direct cell injection or an engineered tissue patch. Although both approaches have advantages in terms of sample preparation, cell-host interaction, and engraftment, how the iPSC-CMs respond to ischemic conditions in the infarcted heart under these two different delivery approaches remain unclear. Here we aim to gain a better understanding of the effects of hypoxia on iPSC-CMs at the transcriptome level.
Methods and results
NHP iPSC-CMs in both monolayer culture (2 D) and engineered heart tissue (EHT) (3 D) format were exposed to hypoxic conditions to serve as surrogates of direct cell injection and tissue implantation in vivo, respectively. Outcomes were compared at the transcriptome level. We found the 3 D EHT model was more sensitive to ischemic conditions and similar to the native in vivo myocardium in terms of cell-extracellular matrix/cell-cell interactions, energy metabolism, and paracrine signaling.
Conclusions
By exposing NHP iPSC-CMs to different culture conditions, transcriptome profiling improves our understanding of the mechanism of ischemic injury.
Translational perspective
Stem cell therapy has shown promise for treating ischemic heart tissue. However, how stem cells respond following different delivery method is unclear. Here hypoxic conditioning was applied to non-human primate iPSC-CMs in 2 D monolayer culture and 3 D engineered heart tissue to model cell injection versus patch implantation, respectively, in an ischemic milieu. The differential transcriptome of hypoxic effects on iPSC-CMs show upregulation of ECM-cell/cell-cell interactions (COL9A1, ITGB6, CTSV, and EPHA1), energy metabolism/hypoxia (ALDOC, ENO2, PFKFB4, CA3, and CA9), and paracrine signaling (WNT, PDGF, FGFR, EGFR, PI3K, and VEGF) in the 3 D format, which suggest engineered heart tissue as more suitable model for evaluating cardiac regenerative therapy.

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

Cardiovasc Res: 30 Sep 2020; epub ahead of print
Yang H, Shao N, Holmström A, Zhao X, ... Abilez OJ, Wu JC
Cardiovasc Res: 30 Sep 2020; epub ahead of print | PMID: 33002105
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Abstract

3-Hydroxyanthralinic acid metabolism controls the hepatic SREBP/lipoprotein axis, inhibits inflammasome activation in macrophages, and decreases atherosclerosis in Ldlr-/- mice.

Berg M, Polyzos KA, Agardh H, Baumgartner R, ... Hansson GK, Ketelhuth DFJ
Aims
Atherosclerosis is a chronic inflammatory disease involving immunological and metabolic processes. Metabolism of tryptophan (Trp) via the kynurenine pathway has shown immunomodulatory properties and the ability to modulate atherosclerosis. We identified 3-hydroxyanthranilic acid (3-HAA) as a key metabolite of Trp modulating vascular inflammation and lipid metabolism. The molecular mechanisms driven by 3-HAA in atherosclerosis have not been completely elucidated. In this study, we investigated whether two major signalling pathways, activation of SREBPs and inflammasome, are associated with the 3-HAA-dependent regulation of lipoprotein synthesis and inflammation in the atherogenesis process. Moreover, we examined whether inhibition of endogenous 3-HAA degradation affects hyperlipidaemia and plaque formation.
Methods and results
In vitro, we showed that 3-HAA reduces SREBP-2 expression and nuclear translocation and apolipoprotein B secretion in HepG2 cell cultures, and inhibits inflammasome activation and IL-1β production by macrophages. Using Ldlr-/- mice, we showed that inhibition of 3-HAA 3,4-dioxygenase (HAAO), which increases the endogenous levels of 3-HAA, decreases plasma lipids and atherosclerosis. Notably, HAAO inhibition led to decreased hepatic SREBP-2 mRNA levels and lipid accumulation, and improved liver pathology scores.
Conclusions
We show that the activity of SREBP-2 and the inflammasome can be regulated by 3-HAA metabolism. Moreover, our study highlights that targeting HAAO is a promising strategy to prevent and treat hypercholesterolaemia and atherosclerosis.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 30 Sep 2020; 116:1948-1957
Berg M, Polyzos KA, Agardh H, Baumgartner R, ... Hansson GK, Ketelhuth DFJ
Cardiovasc Res: 30 Sep 2020; 116:1948-1957 | PMID: 31589306
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Abstract

Abnormal neurovascular coupling as a cause of excess cerebral vasodilation in familial migraine.

Staehr C, Rajanathan R, Postnov DD, Hangaard L, ... Aalkjaer C, Matchkov VV
Aims
Acute migraine attack in familial hemiplegic migraine type 2 (FHM2) patients is characterized by sequential hypo- and hyperperfusion. FHM2 is associated with mutations in the Na, K-ATPase α2 isoform. Heterozygous mice bearing one of these mutations (α2+/G301R mice) were shown to have elevated cerebrovascular tone and, thus, hypoperfusion that might lead to elevated concentrations of local metabolites. We hypothesize that these α2+/G301R mice also have increased cerebrovascular hyperaemic responses to these local metabolites leading to hyperperfusion in the affected part of the brain.
Methods and results
Neurovascular coupling was compared in α2+/G301R and matching wild-type (WT) mice using Laser Speckle Contrast Imaging. In brain slices, parenchymal arteriole diameter and intracellular calcium changes in neuronal tissue, astrocytic endfeet, and smooth muscle cells in response to neuronal excitation were assessed. Wall tension and smooth muscle membrane potential were measured in isolated middle cerebral arteries. Quantitative polymerase chain reaction, western blot, and immunohistochemistry were used to assess the molecular background underlying the functional changes. Whisker stimulation induced larger increase in blood perfusion, i.e. hyperaemic response, of the somatosensory cortex of α2+/G301R than WT mice. Neuronal excitation was associated with larger parenchymal arteriole dilation in brain slices from α2+/G301R than WT mice. These hyperaemic responses in vivo and ex vivo were inhibited by BaCl2, suggesting involvement of inward-rectifying K+ channels (Kir). Relaxation to elevated bath K+ was larger in arteries from α2+/G301R compared to WT mice. This difference was endothelium-dependent. Endothelial Kir2.1 channel expression was higher in arteries from α2+/G301R mice. No sex difference in functional responses and Kir2.1 expression was found.
Conclusion
This study suggests that an abnormally high cerebrovascular hyperaemic response in α2+/G301R mice is a result of increased endothelial Kir2.1 channel expression. This may be initiated by vasospasm-induced accumulation of local metabolites and underlie the hyperperfusion seen in FHM2 patients during migraine attack.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 30 Sep 2020; 116:2009-2020
Staehr C, Rajanathan R, Postnov DD, Hangaard L, ... Aalkjaer C, Matchkov VV
Cardiovasc Res: 30 Sep 2020; 116:2009-2020 | PMID: 31710670
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Abstract

Interstitial macrophage-derived thrombospondin-1 contributes to hypoxia-induced pulmonary hypertension.

Kumar R, Mickael C, Kassa B, Sanders L, ... Tuder RM, Graham BB
Aims
Transforming growth factor-β (TGF-β) signalling is required for chronic hypoxia-induced pulmonary hypertension (PH). The activation of TGF-β by thrombospondin-1 (TSP-1) contributes to the pathogenesis of hypoxia-induced PH. However, neither the cellular source of pathologic TSP-1 nor the downstream signalling pathway that link activated TGF-β to PH have been determined. In this study, we hypothesized that circulating monocytes, which are recruited to become interstitial macrophages (IMs), are the major source of TSP-1 in hypoxia-exposed mice, and TSP-1 activates TGF-β with increased Rho-kinase signalling, causing vasoconstriction.
Methods and results
Flow cytometry revealed that a specific subset of IMs is the major source of pathologic TSP-1 in hypoxia. Intravenous depletion and parabiosis experiments demonstrated that these cells are circulating prior to recruitment into the interstitium. Rho-kinase-mediated vasoconstriction was a major downstream target of active TGF-β. Thbs1 deficient bone marrow (BM) protected against hypoxic-PH by blocking TGF-β activation and Rho-kinase-mediated vasoconstriction.
Conclusion
In hypoxia-challenged mice, BM derived and circulating monocytes are recruited to become IMs which express TSP-1, resulting in TGF-β activation and Rho-kinase-mediated vasoconstriction.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 30 Sep 2020; 116:2021-2030
Kumar R, Mickael C, Kassa B, Sanders L, ... Tuder RM, Graham BB
Cardiovasc Res: 30 Sep 2020; 116:2021-2030 | PMID: 31710666
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Abstract

Nitric oxide modulates cardiomyocyte pH control through a biphasic effect on sodium/hydrogen exchanger-1.

Richards MA, Simon JN, Ma R, Loonat AA, ... Fliegel L, Swietach P
Aims
When activated, Na+/H+ exchanger-1 (NHE1) produces some of the largest ionic fluxes in the heart. NHE1-dependent H+ extrusion and Na+ entry strongly modulate cardiac physiology through the direct effects of pH on proteins and by influencing intracellular Ca2+ handling. To attain an appropriate level of activation, cardiac NHE1 must respond to myocyte-derived cues. Among physiologically important cues is nitric oxide (NO), which regulates a myriad of cardiac functions, but its actions on NHE1 are unclear.
Methods and results
NHE1 activity was measured using pH-sensitive cSNARF1 fluorescence after acid-loading adult ventricular myocytes by an ammonium prepulse solution manoeuvre. NO signalling was manipulated by knockout of its major constitutive synthase nNOS, adenoviral nNOS gene delivery, nNOS inhibition, and application of NO-donors. NHE1 flux was found to be activated by low [NO], but inhibited at high [NO]. These responses involved cGMP-dependent signalling, rather than S-nitros(yl)ation. Stronger cGMP signals, that can inhibit phosphodiesterase enzymes, allowed [cAMP] to rise, as demonstrated by a FRET-based sensor. Inferring from the actions of membrane-permeant analogues, cGMP was determined to activate NHE1, whereas cAMP was inhibitory, which explains the biphasic regulation by NO. Activation of NHE1-dependent Na+ influx by low [NO] also increased the frequency of spontaneous Ca2+ waves, whereas high [NO] suppressed these aberrant forms of Ca2+ signalling.
Conclusions
Physiological levels of NO stimulation increase NHE1 activity, which boosts pH control during acid-disturbances and results in Na+-driven cellular Ca2+ loading. These responses are positively inotropic but also increase the likelihood of aberrant Ca2+ signals, and hence arrhythmia. Stronger NO signals inhibit NHE1, leading to a reversal of the aforementioned effects, ostensibly as a potential cardioprotective intervention to curtail NHE1 overdrive.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 30 Sep 2020; 116:1958-1971
Richards MA, Simon JN, Ma R, Loonat AA, ... Fliegel L, Swietach P
Cardiovasc Res: 30 Sep 2020; 116:1958-1971 | PMID: 31742355
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Abstract

Mas receptor is translocated to the nucleus upon agonist stimulation in brainstem neurons from spontaneously hypertensive rats but not normotensive rats.

Cerniello FM, Silva MG, Carretero OA, Gironacci MM
Aims 
Activation of the angiotensin (Ang)-(1-7)/Mas receptor (R) axis protects from sympathetic overactivity. Endocytic trafficking is an essential process that regulates receptor (R) function and its ultimate cellular responses. We investigated whether the blunted responses to Ang-(1-7) in hypertensive rats are associated to an alteration in MasR trafficking.
Methods and results 
Brainstem neurons from Wistar-Kyoto (WKY) or spontaneously hypertensive rats (SHRs) were investigated for (i) Ang-(1-7) levels and binding and MasR expression, (ii) Ang-(1-7) responses (arachidonic acid and nitric oxide release and Akt and ERK1/2 phosphorylation), and (iii) MasR trafficking. Ang-(1-7) was determined by radioimmunoassay. MasR expression and functionality were evaluated by western blot and binding assays. MasR trafficking was evaluated by immunofluorescence. Ang-(1-7) treatment induced an increase in nitric oxide and arachidonic acid release and ERK1/2 and Akt phosphorylation in WKY neurons but did not have an effect in SHR neurons. Although SHR neurons showed greater MasR expression, Ang-(1-7)-elicited responses were substantially diminished presumably due to decreased Ang-(1-7) endogenous levels concomitant with impaired binding to its receptor. Through immunocolocalization studies, we evidenced that upon Ang-(1-7) stimulation MasRs were internalized through clathrin-coated pits and caveolae into early endosomes and slowly recycled back to the plasma membrane. However, the fraction of internalized MasRs into early endosomes was larger and the fraction of MasRs recycled back to the plasma membrane was smaller in SHR than in WKY neurons. Surprisingly, in SHR neurons but not in WKY neurons, Ang-(1-7) induced MasR translocation to the nucleus. Nuclear MasR expression and Ang-(1-7) levels were significantly greater in the nuclei of Ang-(1-7)-stimulated SHR neurons, indicating that the MasR is translocated with its ligand bound to it.
Conclusion 
MasRs display differential trafficking in brainstem neurons from SHRs, which may contribute to the impaired responses to Ang-(1-7).

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 30 Sep 2020; 116:1995-2008
Cerniello FM, Silva MG, Carretero OA, Gironacci MM
Cardiovasc Res: 30 Sep 2020; 116:1995-2008 | PMID: 31825460
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Abstract

Dynamics of Cardiac Neutrophil Diversity in Murine Myocardial Infarction.

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



Circ Res: 08 Oct 2020; 127:e232-e249
Vafadarnejad E, Rizzo G, Krampert L, Arampatzi P, ... Saliba AE, Cochain C
Circ Res: 08 Oct 2020; 127:e232-e249 | PMID: 32811295
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Abstract

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

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

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



Circ Res: 04 Oct 2020; epub ahead of print
Zahreddine R, Davezac M, Smirnova NF, Buscato M, ... Arnal JF, Fontaine C
Circ Res: 04 Oct 2020; epub ahead of print | PMID: 33012251
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Abstract

Fibroblasts in atherosclerosis: heterogeneous and plastic participants.

Tillie RJHA, van Kuijk K, Sluimer JC
Purpose of review
Fibroblasts are very heterogeneous and plastic cells in the vasculature. A growing interest in fibroblasts in healthy and atherosclerotic vasculature is observed, next to macrophages, endothelial cells, and smooth muscle cells (SMCs). In this review, we discuss fibroblast presence, heterogeneity, origin, and plasticity in health and atherosclerosis based on latest literature.
Recent findings
With help of single cell sequencing (SCS) techniques, we have gained more insight into presence and functions of fibroblasts in atherosclerosis. Next to SMCs, fibroblasts are extracellular matrix-producing cells abundant in the vasculature and involved in atherogenesis. Fibroblasts encompass a heterogeneous population and SCS data reveal several fibroblast clusters in healthy and atherosclerotic tissue with varying gene expression and function. Moreover, recent findings indicate interesting similarities between adventitial stem and/or progenitor cells and fibroblasts. Also, communication with inflammatory cells opens up a new therapeutic avenue.
Summary
Because of their highly plastic and heterogeneous nature, modulating fibroblast cell function and communication in the atherosclerotic vessel might be useful in battling atherosclerosis from within the plaque.



Curr Opin Lipidol: 29 Sep 2020; 31:273-278
Tillie RJHA, van Kuijk K, Sluimer JC
Curr Opin Lipidol: 29 Sep 2020; 31:273-278 | PMID: 32773464
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Abstract

Transport of LDLs into the arterial wall: impact in atherosclerosis.

Zhang X, Fernández-Hernando C
Purpose of review
Atherosclerosis is a complicated cardiovascular disease characterized by unbalanced lipid metabolism and unresolved inflammation that occurred inside of arteries. The transcytosis of LDL across the endothelium and its accumulation in the arterial wall is the initial step of atherosclerosis. Here, we summarize recent research into the understanding of the regulatory mechanisms of endothelial LDL transcytosis and its relevance in the development of atherosclerosis.
Recent findings
A number of recent studies have revealed the contribution of caveolae, activin-like kinase 1 (ALK1) or scavenger receptor B1 (SR-B1) in endothelial LDL transcytosis and the progression of atherosclerosis. Caveolin-1 (Cav-1), the major protein component in caveolae, is required for the formation of caveolae and caveolae-mediated LDL uptake and transcytosis across the endothelium. SR-B1 and ALK1 directly bind LDL and facilitate the transport of LDL through the endothelial cells. The change in expression of caveolae-associated proteins and SR-B1 regulates endothelial LDL transcytosis and the pathogenesis of atherosclerosis.
Summary
Caveolae, ALK1 and SR-B1 are identified as key regulators in the LDL transcytosis across the endothelium. Endothelial LDL transcytosis might be a potential therapeutic approach to limit the initiation of early atherosclerosis and treat the atherosclerotic vascular diseases.



Curr Opin Lipidol: 29 Sep 2020; 31:279-285
Zhang X, Fernández-Hernando C
Curr Opin Lipidol: 29 Sep 2020; 31:279-285 | PMID: 32773465
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Abstract

Age-associated arterial calcification: the current pursuit of aggravating and mitigating factors.

Chakrabarti A, Goldstein DR, Sutton NR
Purpose of review
The incidence of arterial calcification increases with age, can occur independently of atherosclerosis and hyperlipidemia, contributes to vessel stiffening, and is associated with adverse cardiovascular outcomes. Here, we provide an up-to-date review of how aging leads to arterial calcification and discuss potential therapies.
Recent findings
Recent research suggests that mitochondrial dysfunction (impaired efficiency of the respiratory chain, increased reactive oxygen species production, and a high mutation rate of mitochondrial DNA), cellular senescence, ectonucleotidases, and extrinsic factors such as hyperglycemia promote age-determined calcification. We discuss the future potential impact of antilipidemics, senolytics, and poly(ADP-ribose)polymerases inhibitors on age-associated arterial calcification.
Summary
Understanding how mechanisms of aging lead to arterial calcification will allow us to pinpoint prospective strategies to mitigate arterial calcification, even after the effects of aging have already begun to occur.



Curr Opin Lipidol: 29 Sep 2020; 31:265-272
Chakrabarti A, Goldstein DR, Sutton NR
Curr Opin Lipidol: 29 Sep 2020; 31:265-272 | PMID: 32773466
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Impact:
Abstract

Atherosclerosis: cell biology and lipoproteins.

Getz GS, Reardon CA
Purpose of review
Lipoproteins have significant role in both the promotion and prevention of atherosclerosis. This brief review will focus on recent reports on relationship between HDL and HDL subclasses and their composition and function, the role of apoC-III in metabolism of triglyceride-rich lipoproteins, the impact of Lipoprotein (a) (Lp(a)) on endothelial cells, and the mechanism of uptake of aggregated LDL by macrophages.
Recent findings
The complexity of the protein and lipid content of murine and human HDL and their relationship to its cholesterol efflux capacity have been examined. HDL has also been shown to have both antiatherogenic and proatherogenic properties. The relationship between apoC-III and LPL activity, apoprotein E mediated clearance of triglyceride-rich lipoproteins and the potential importance of apoC-III in the increased risk of cardiovascular disease in type 1 diabetics has been investigated. Oxidized phospholipid in Lp(a) promotes endothelial cells inflammatory and glycolytic responses. TLR4 participates in the uptake of aggregated LDL to contribute to foam cell formation.
Summary
These studies contribute to our mechanistic understanding of how lipoproteins contribute to atherogenesis and identify potential therapeutic targets.



Curr Opin Lipidol: 29 Sep 2020; 31:286-290
Getz GS, Reardon CA
Curr Opin Lipidol: 29 Sep 2020; 31:286-290 | PMID: 32773467
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Impact:
Abstract

Measurement of carotid plaque burden.

Spence JD
Purpose of review
To describe the uses of measurement of carotid plaque burden, as total plaque area (TPA), total plaque volume (TPV), and vessel wall volume (VWV), which includes plaque burden and wall volume.
Recent findings
Measurement of plaque burden is useful for risk stratification, research into the genetics and biology of atherosclerosis, for measuring effects of new therapies for atherosclerosis, and for treatment of high-risk patients with severe atherosclerosis.
Summary
Measurement of plaque burden is far superior to measurement of carotid intima-media thickness (IMT) in many ways, and should replace it. Vessel wall volume can be measured in persons with no plaque as an alternative to IMT.



Curr Opin Lipidol: 29 Sep 2020; 31:291-298
Spence JD
Curr Opin Lipidol: 29 Sep 2020; 31:291-298 | PMID: 32773468
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Impact:
Abstract

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

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



Circ Res: 24 Sep 2020; 127:1094-1108
Davidson MT, Grimsrud PA, Lai L, Draper JA, ... Kelly DP, Muoio DM
Circ Res: 24 Sep 2020; 127:1094-1108 | PMID: 32660330
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Impact:
Abstract

Mast Cell Promotes the Development of Intracranial Aneurysm Rupture.

Furukawa H, Wada K, Tada Y, Kuwabara A, ... Lawton MT, Hashimoto T
Background and purpose
Inflammation has emerged as a key component of the pathophysiology of intracranial aneurysms. Mast cells have been detected in human intracranial aneurysm tissues, and their presence was associated with intramural microhemorrhage and wall degeneration. We hypothesized that mast cells play a critical role in the development of aneurysmal rupture, and that mast cells can be used as a therapeutic target for the prevention of aneurysm rupture.
Methods
Intracranial aneurysms were induced in adult mice using a combination of induced systemic hypertension and a single injection of elastase into the cerebrospinal fluid. Aneurysm formation and rupture were assessed over 3 weeks. Roles of mast cells were assessed using a mast cell stabilizer (cromolyn), a mast cell activator (C48/80), and mice that are genetically lacking mature mast cells (Kit mice).
Results
Pharmacological stabilization of mast cells with cromolyn markedly decreased the rupture rate of aneurysms (80% versus 19%, n=10 versus n =16) without affecting the aneurysm formation. The activation of mast cells with C48/80 significantly increased the rupture rate of aneurysms (25% versus 100%, n=4 versus n=5) without affecting the overall rate of aneurysm formation. Furthermore, the genetic deficiency of mast cells significantly prevented aneurysm rupture (80% versus 25%, n=10 versus n=8, wild-type versus Kit mice).
Conclusions
These results suggest that mast cells play a key role in promoting aneurysm rupture but not formation. Stabilizers of mast cells may have a potential therapeutic value in preventing intracranial aneurysm rupture in patients.



Stroke: 05 Oct 2020:STROKEAHA120030834; epub ahead of print
Furukawa H, Wada K, Tada Y, Kuwabara A, ... Lawton MT, Hashimoto T
Stroke: 05 Oct 2020:STROKEAHA120030834; epub ahead of print | PMID: 33019897
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Impact:
Abstract

The fish-oil paradox.

Nicholls SJ, Nelson AJ
Purpose of review
Increasing interest has focused on the potential cardioprotective effects of the omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the basis of findings from epidemiology and cohort studies. This review will summarize the findings of contemporary clinical trials of omega-3 fatty acids.
Recent findings
Although a large clinical trial performed prior to the widespread use of statins demonstrated cardiovascular benefit with fish oils, subsequent studies have failed to reproduce this result. More recent studies have demonstrated a reduction in cardiovascular risk with administration of high-dose EPA, but not a carboxylic acid formulation containing both EPA and DHA or with lower doses of omega-3 fatty acids.
Summary
Administration of omega-3 fatty acids differing in either composition or dose produce variable effects on cardiovascular outcomes. This has implications for both the public health and pharmacological approach to cardiovascular prevention.



Curr Opin Lipidol: 28 Sep 2020; epub ahead of print
Nicholls SJ, Nelson AJ
Curr Opin Lipidol: 28 Sep 2020; epub ahead of print | PMID: 33027227
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Abstract

Targeting epigenetics as atherosclerosis treatment: an updated view.

Neele AE, Willemsen L, Chen HJ, Dzobo KE, de Winther MPJ
Purpose of review
This review discusses the current developments on epigenetic inhibition as treatment for atherosclerosis.
Recent findings
The first phase III clinical trial targeting epigenetics in cardiovascular disease (CVD), BETonMACE, using the bromodomain inhibitor apabetalone (RVX-208) showed no significant effect on major adverse cardiovascular events (MACE) in patients with type II diabetes, low HDL-c and a recent acute coronary artery event compared with its placebo arm.
Summary
Preclinical and clinical studies suggest that targeting epigenetics in atherosclerosis is a promising novel therapeutic strategy against CVD. Interfering with histone acetylation by targeting histone deacetylates (HDACs) and bromodomain and extraterminal domain (BET) proteins demonstrated encouraging results in modulating disease progression in model systems. Although the first phase III clinical trial targeting BET in CVD showed no effect on MACE, we suggest that there is sufficient potential for future clinical usage based on the outcomes in specific subgroups and the fact that the study was slightly underpowered. Lastly, we propose that there is future window for targeting repressive histone modifications in atherosclerosis.



Curr Opin Lipidol: 28 Sep 2020; epub ahead of print
Neele AE, Willemsen L, Chen HJ, Dzobo KE, de Winther MPJ
Curr Opin Lipidol: 28 Sep 2020; epub ahead of print | PMID: 33027226
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Abstract

Hypertriglyceridemia: new approaches in management and treatment.

Wolska A, Yang ZH, Remaley AT
Purpose of review
Hypertriglyceridemia (HTG), a form of dyslipidemia characterized by elevated plasma of triglycerides (TG), is associated with an increased risk for acute pancreatitis. Moreover, HTG has recently been shown to be linked to the development of atherosclerotic cardiovascular disease (ASCVD); therefore, there is a great interest in better understanding the pathophysiology of HTG and improving its clinical management. In this review, we briefly describe TG metabolism, recent guidelines for the clinical management of HTG and provide an overview of the current and potential new therapies for HTG.
Recent findings
Screening patients for HTG is valuable for not only identifying patients with extreme TG elevations, who are at risk for pancreatitis, but also for managing ASCVD risk in patients with more moderate forms of HTG. Therefore, the most recent USA guidelines for cardiovascular diseases recommend using TG as a risk enhancer test, leading to a more aggressive treatment of patients with intermediate risk. Currently, there are several available approaches for reducing plasma TG, which include lifestyle changes, fibrates and omega-3 fatty acid treatment. The addition of eicosapentaenoic acid (EPA) on top of statins has recently been shown to significantly reduce ASCVD events. Nevertheless, there is an unmet need for more effective treatment options. Several new therapies based on newly identified targets in TG metabolism, such as apolipoprotein C-III and angiopoietin-like 3 protein, are currently under development.
Summary
The clinical management of HTG is important in the prevention and treatment of acute pancreatitis and also impacts on how ASCVD risk is managed. More work needs to be done to establish the mechanism for the ability of how EPA lowers ASCVD and how to best integrate it with other lipid-lowering therapies. The efficacy and safety of the novel therapies for HTG should be established soon in the ongoing late-stage clinical trials.



Curr Opin Lipidol: 28 Sep 2020; epub ahead of print
Wolska A, Yang ZH, Remaley AT
Curr Opin Lipidol: 28 Sep 2020; epub ahead of print | PMID: 33027225
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Abstract

Changing dietary approaches to prevent cardiovascular disease.

Nestel PJ, Beilin LJ, Mori TA
Purpose of review
We have focused on recent research relevant to effects of dietary patterns and major food groups on cardiovascular outcomes, taking into account guidelines and position statements from expert authorities, with an emphasis on important changes in recommendations, some of which remain controversial.
Recent findings
Major findings include: refocusing on qualitative patterns of food consumption replacing quantitative prescriptive advice on nutrients; increasing intake of plant foods; substituting saturated fats with polyunsaturated and monounsaturated oils; reducing salt intake; regular consumption of fish with a focus on omega-3 enrichment; not restricting dairy foods, other than butter and cream, with encouragement of some fermented products; reducing cholesterol intake for those at increased cardiovascular risk and diabetes, allowing 7-eggs weekly; restricting processed meats and allowing moderate lean meat consumption; preference for fiber-rich complex carbohydrates and reduced sugar intake; maintaining healthy bodyweight; and although water is the preferred beverage, allowing moderate alcohol consumption to national guidelines and avoiding alcohol in specific cardiovascular disorders.
Summary
The new approach that focuses on healthier patterns of food intake is more readily understood by health practitioners and translatable to consumers and patients.



Curr Opin Lipidol: 28 Sep 2020; epub ahead of print
Nestel PJ, Beilin LJ, Mori TA
Curr Opin Lipidol: 28 Sep 2020; epub ahead of print | PMID: 33027224
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Abstract

The interconnection between lipoprotein(a), lipoprotein(a) cholesterol and true LDL-cholesterol in the diagnosis of familial hypercholesterolemia.

Yeang C, Willeit P, Tsimikas S
Purpose of review
Elevated levels of lipoprotein(a) [Lp(a)] are present in 30-50% of patients with familial hypercholesterolemia. The contribution of Lp(a) towards risk stratification of patients with familial hypercholesterolemia has been recently recognized, with studies showing a significantly worse prognosis if Lp(a) is elevated. However, the role of elevated Lp(a) in diagnosis of familial hypercholesterolemia is less well defined or accepted.
Recent findings
An important confounder in the diagnosis of familial hypercholesterolemia is the significant contribution of the cholesterol content on Lp(a) (Lp(a)-C) in individuals with elevated Lp(a). Because Lp(a)-C is incorporated into all clinical LDL-C measurements, it can contribute significantly to the cholesterol threshold diagnostic criteria for familial hypercholesterolemia used in most clinical algorithms.
Summary
In this review, we discuss the interrelationship of Lp(a), Lp(a)-C and correct LDL-C in the diagnosis and prognosis of familial hypercholesterolemia. Future studies of accurately measuring correct LDL-C or in using apoB-100 and Lp(a) criteria may overcome the limitations of using estimated LDL-C in the diagnosis of familial hypercholesterolemia in individuals with concomitant elevation of Lp(a).



Curr Opin Lipidol: 05 Oct 2020; epub ahead of print
Yeang C, Willeit P, Tsimikas S
Curr Opin Lipidol: 05 Oct 2020; epub ahead of print | PMID: 33027223
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Abstract

Advances, gaps and opportunities in the detection of familial hypercholesterolemia: overview of current and future screening and detection methods.

Ibrahim S, Reeskamp LF, Stroes ESG, Watts GF
Purpose of review
Studies reaffirm that familial hypercholesterolemia is more prevalent than initially considered, with a population frequency of approximately one in 300. The majority of patients remains unidentified. This warrants critical evaluation of existing screening methods and exploration of novel methods of detection.
Recent findings
New public policy recommendations on the detection of familial hypercholesterolemia have been made by a global community of experts and advocates. Phenotypic tools for diagnosing index cases remain inaccurate. Genetic testing is the gold standard for familial hypercholesterolemia and a new international position statement has been published. Correction of LDL cholesterol (LDL-C) for the cholesterol content of lipoprotein(a) [Lp(a)] may increase the precision of the phenotypic diagnosis of familial hypercholesterolemia. Cascade cotesting for familial hypercholesterolemia and elevated Lp(a) levels provides a new opportunity to stratify risk in families. Digital technology and machine learning methods, coupled with clinical alert and decision support systems, lead the way in more efficient approaches for detecting and managing index cases. Universal screening of children, combined with child-parent cascade testing, appears to be the most effective method for underpinning a population strategy for maximizing the detection of familial hypercholesterolemia.
Summary
Detection of familial hypercholesterolemia can be enhanced by optimizing current diagnostic algorithms, probing electronic health records with novel information technologies and integrating universal screening of children with cascade testing of parents and other relatives.



Curr Opin Lipidol: 05 Oct 2020; epub ahead of print
Ibrahim S, Reeskamp LF, Stroes ESG, Watts GF
Curr Opin Lipidol: 05 Oct 2020; epub ahead of print | PMID: 33027222
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Abstract

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

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

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



Circ Res: 11 Oct 2020; epub ahead of print
Aherrahrou R, Guo L, Nagraj VP, Aguhob AA, ... Berliner JA, Civelek M
Circ Res: 11 Oct 2020; epub ahead of print | PMID: 33040646
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Abstract

Epigenomic and Transcriptomic Dynamics During Human Heart Organogenesis.

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



Circ Res: 08 Oct 2020; 127:e184-e209
VanOudenhove J, Yankee TN, Wilderman A, Cotney J
Circ Res: 08 Oct 2020; 127:e184-e209 | PMID: 32772801
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Abstract

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

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



Circ Res: 08 Oct 2020; 127:1122-1137
Hwan Kim Y, Vu PN, Choe SW, Jeon CJ, ... Lee YJ, Oh SP
Circ Res: 08 Oct 2020; 127:1122-1137 | PMID: 32762495
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Impact:
Abstract

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

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



Circ Res: 08 Oct 2020; 127:e210-e231
Marín-Ramos NI, Thein TZ, Ghaghada KB, Chen TC, Giannotta SL, Hofman FM
Circ Res: 08 Oct 2020; 127:e210-e231 | PMID: 32755283
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Abstract

Periostin: A Potential Therapeutic Target For Pulmonary Hypertension?

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



Circ Res: 08 Oct 2020; 127:1138-1152
Nie X, Shen C, Tan J, Wu Z, ... Chen J, Bian JS
Circ Res: 08 Oct 2020; 127:1138-1152 | PMID: 32752980
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Impact:
Abstract

Impact of Local Alloimmunity and Recipient Cells in Transplant Arteriosclerosis.

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



Circ Res: 24 Sep 2020; 127:974-993
Cai J, Deng J, Gu W, Ni Z, ... Lu Y, Xu Q
Circ Res: 24 Sep 2020; 127:974-993 | PMID: 32689904
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Impact:
Abstract

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

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



Circ Res: 08 Oct 2020; 127:1159-1178
Ljubojevic-Holzer S, Herren AW, Djalinac N, Voglhuber J, ... Bossuyt J, Bers DM
Circ Res: 08 Oct 2020; 127:1159-1178 | PMID: 32821022
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Impact:
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