<div><h4>An ERK5-NRF2 Axis Mediates Senescence-Associated Stemness and Atherosclerosis.</h4><i>Abe JI, Imanishi M, Li S, Zhang A, ... Le NT, Kotla S</i><br /><b>Background</b><br />ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis.<br /><b>Methods</b><br />A ERK5 S496A (dephosphorylation mimic) KI (knock in) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and WT (wild type) mice was studied using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis.<br /><b>Results</b><br />We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors.<br /><b>Conclusions</b><br />We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.<br /><br /><br /><br /><small>Circ Res: 02 Jun 2023; epub ahead of print</small></div>

<div><h4>Modulation of lncRNA links endothelial glycocalyx to vascular dysfunction of tyrosine kinase inhibitor.</h4><i>Nukala SB, Jousma J, Yan G, Han Z, ... Lee WH, Ong SG</i><br /><b>Aims</b><br />Novel cancer therapies leading to increased survivorship of cancer patients have been negated by a concomitant rise in cancer therapies-related cardiovascular toxicities. Sunitinib, a first line multi receptor tyrosine kinase inhibitor (TKI), has been reported to cause vascular dysfunction although the initiating mechanisms contributing to this side effect remain unknown. Long non-coding RNAs (lncRNAs) are emerging regulators of biological processes in endothelial cells (ECs); however, their roles in cancer therapies-related vascular toxicities remain underexplored.<br /><b>Methods and results</b><br />We performed lncRNA expression profiling to identify potential lncRNAs that are dysregulated in human induced pluripotent stem cell-derived ECs (iPSC-ECs) treated with sunitinib. We show that the lncRNA hyaluronan synthase 2 antisense 1 (HAS2-AS1) is significantly diminished in sunitinib-treated iPSC-ECs. Sunitinib was found to downregulate HAS2-AS1 by an epigenetic mechanism involving hypermethylation. Depletion of HAS2-AS1 recapitulated sunitinib-induced detrimental effects on iPSC-ECs, whereas CRISPR-mediated activation of HAS2-AS1 reversed sunitinib-induced dysfunction. We confirmed that HAS2-AS1 stabilizes the expression of its sense gene HAS2 via an RNA/mRNA heteroduplex formation. Knockdown of HAS2-AS1 led to reduced synthesis of hyaluronic acid (HA) and upregulation of ADAMTS5, an enzyme involved in extracellular matrix degradation, resulting in disruption of the endothelial glycocalyx which is critical for ECs. In vivo, sunitinib-treated mice showed reduced coronary flow reserve, accompanied by a reduction in Has2os and degradation of the endothelial glycocalyx. Finally, we identified that treatment with high molecular-weight HA can prevent the deleterious effects of sunitinib both in vitro and in vivo by preserving the endothelial glycocalyx.<br /><b>Conclusions</b><br />Our findings highlight the importance of lncRNA-mediated regulation of the endothelial glycocalyx as an important determinant of sunitinib-induced vascular toxicity and reveal potential novel therapeutic avenues to attenuate sunitinib-induced vascular dysfunction.<br /><br />© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.<br /><br /><small>Cardiovasc Res: 02 Jun 2023; epub ahead of print</small></div>

<div><h4>Piezo1 Mediates Vasodilation Induced by Acute Hyperglycemia in Mouse Renal Arteries and Microvessels.</h4><i>Fei L, Xu M, Wang H, Zhong C, ... Patzak A, Khedkar PH</i><br /><b>Background</b><br />Acute hyperglycemia is a risk factor for developing acute kidney injury and poor renal outcome in critically ill patients, whereby the role of renal vasculature remains unclear. We hypothesize that hyperglycemia-associated hyperosmolarity facilitates vasodilation through Piezo1-mediated eNOS (endothelial NO synthase) activation.<br /><b>Methods</b><br />Vasoreactivity was analyzed using wire myography in isolated mouse mesenteric arteries and renal interlobar, and using microvascular perfusion in renal afferent arterioles and efferent arterioles, and vasa recta. Immunofluorescence and Western blot were used for molecular analyses of isolated mouse blood vessels and human umbilical vein endothelial cells.<br /><b>Results</b><br />Pretreatment with hyperglycemia (44 mmol/L glucose; 4 hours) increased acetylcholine-induced relaxation in interlobar arteries and mesenteric arteries, which was prevented by eNOS inhibition using Nω-nitro-L-arginine methylester hydrochloride. Hyperosmotic mannitol solution had a similar effect. Hyperglycemia induced an immediate, Nω-nitro-L-arginine methylester hydrochloride-inhibitable dilation in afferent arterioles, efferent arterioles, and vasa recta, whereby stronger dilation in afferent arterioles compared to efferent arterioles. Hyperglycemia also increased glomerular filtration rate in mice. In human umbilical vein endothelial cells, hyperglycemia, and the Piezo1 activator Yoda-1 increased levels of Piezo1 protein, p-CaMKII (phosphorylated Ca<sup>2+</sup>/Calmodulin-dependent protein kinase type II), Akt, and p-eNOS (phosphorylated eNOS). The hyperglycemia effect could be prevented by inhibiting Piezo1 using GsMTx4 (<i>Grammostola spatulata</i> mechanotoxin 4) and CaMKII using KN93. Furthermore, in arteries and microvessels, inhibition of Piezo1 using GsMTx4 prevented the hyperglycemia -effect, while Yoda-1 caused relaxation and dilation, respectively.<br /><b>Conclusions</b><br />Results reveal that Piezo1 mediates renal vasodilation induced by hyperosmolarity in acute hyperglycemia. This mechanism may contribute to the pathogenesis of renal damage by acute hyperglycemia.<br /><br /><br /><br /><small>Hypertension: 01 Jun 2023; epub ahead of print</small></div>

<div><h4>Repetitive Antigen Responses of LDL-Reactive CD4+ T Cells Induce Tr1 Cell-Mediated Immune Tolerance.</h4><i>Mailer RK, Konrath S, Zhan L, Thode H, ... Renné T, Hansson GK</i><br /><b>Background</b><br />Inflammation triggered by the deposition of LDL (low-density lipoprotein) in the arterial wall leads to the development of atherosclerosis. Regulatory T (Treg) cells inhibit vascular inflammation through the induction of immune tolerance toward LDL-related antigens. However, tolerogenic mechanisms that promote the generation of LDL-specific Treg cells in vivo remain unclear.<br /><b>Methods</b><br />We identified LDL-specific T cells by activation-induced marker expression and analyzed expression profiles and suppressive functions of TCR (T-cell antigen receptor)-transgenic T cells upon repetitive transfer into antigen-transgenic mice via flow cytometry.<br /><b>Results</b><br />We investigated the naturally occurring Treg-cell response against human LDL in standard chow diet-fed mice that are transgenic for human ApoB100 (apolipoprotein B100). We found that IL (interleukin)-10 expression in LDL-specific T cells from spleen increases with age, albeit LDL-specific populations do not enlarge in older mice. To investigate the generation of IL-10-producing LDL-specific T cells, we transferred naive CD4+ T cells recognizing human ApoB100 from TCR-transgenic mice into human ApoB100-transgenic mice. Adoptive transfer of human ApoB100-specific T cells induced immune tolerance in recipient mice and effectively inhibited activation of subsequently transferred naive T cells of the same specificity in vivo. Moreover, repetitive transfers increased the population of Treg type 1 cells that suppress ApoB100-specific responses via IL-10. In a translational approach, LDL-specific Treg type 1 cells from blood of healthy donors suppressed the activation of monocytic THP-1 cells in an IL-10-dependent manner.<br /><b>Conclusions</b><br />We show that repetitive transfer of naive ApoB100-specific T cells and recurrent LDL-specific T-cell stimulation induces Treg type 1 cell-mediated immune tolerance against LDL in vivo. Our results provide insight into the generation of autoantigen-specific anti-inflammatory T cells under tolerogenic conditions.<br /><br /><br /><br /><small>Arterioscler Thromb Vasc Biol: 01 Jun 2023; epub ahead of print</small></div>

<div><h4>CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis.</h4><i>Singla B, Aithbathula RV, Pervaiz N, Kathuria I, ... Singh UP, Kumar S</i><br /><b>Background</b><br />TSP1 (thrombospondin-1)-a well-known angiogenesis inhibitor-mediates differential effects via interacting with cell surface receptors including CD36 and CD47. However, the role of TSP1 in regulating lymphangiogenesis is not clear. Our previous study suggested the importance of cell-specific CD47 blockade in limiting atherosclerosis. Further, our experiments revealed CD47 as a dominant TSP1 receptor in lymphatic endothelial cells (LECs). As the lymphatic vasculature is functionally linked to atherosclerosis, we aimed to investigate the effects of LEC TSP1-CD47 signaling inhibition on lymphangiogenesis and atherosclerosis.<br /><b>Methods</b><br />Murine atherosclerotic and nonatherosclerotic arteries were utilized to investigate TSP1 expression using Western blotting and immunostaining. LEC-specific knockout mice were used to determine the in vivo role of LEC <i>Cd47</i> in lymphangiogenesis and atherosclerosis. Various in vitro cell-based assays, in vivo Matrigel plug implantation, molecular biological techniques, and immunohistological approaches were used to evaluate the underlying signaling mechanisms.<br /><b>Results</b><br />Elevated TSP1 expression was observed in mouse atherosclerotic aortic tissue compared with nonatherosclerotic control tissue. TSP1 at pathological concentrations suppressed both in vitro and in vivo lymphangiogenesis. Mechanistically, TSP1 inhibited VEGF-C-induced AKT and eNOS activation in LEC and attenuated NO production. Further, <i>Cd47</i> silencing in LEC prevented the effects of TSP1 on lymphangiogenic AKT-eNOS signaling and lymphangiogenesis. Atheroprone AAV8-<i>PCSK9</i>-injected LEC-specific <i>Cd47</i> knockout mice (<i>Cd47</i><sup>ΔLEC</sup>) had reduced atherosclerosis in both aorta and aortic root compared with control mice (<i>Cd47</i><sup>ΔWT</sup>). However, no differences in metabolic parameters including body weight, plasma total cholesterol levels, and fasting blood glucose were observed. Additional immunostaining experiments performed on aortic root cross-sections indicated higher lymphatic vessel density in <i>Cd47</i><sup>ΔLEC</sup> in comparison to controls.<br /><b>Conclusions</b><br />These findings demonstrate that TSP1 inhibits lymphangiogenesis via activation of CD47 in LEC, and loss of LEC <i>Cd47</i> attenuates atherosclerotic lesion formation. Collectively, these results identify LEC CD47 as a potential therapeutic target in atherosclerosis.<br /><br /><br /><br /><small>Arterioscler Thromb Vasc Biol: 01 Jun 2023; epub ahead of print</small></div>

<div><h4>Notch3/Hes5 Induces Vascular Dysfunction in Hypoxia-Induced Pulmonary Hypertension Through ER Stress and Redox-Sensitive Pathways.</h4><i>Morris HE, Neves KB, Nilsen M, Montezano AC, MacLean MR, Touyz RM</i><br /><b>Background</b><br />Notch3 is implicated in vascular diseases, including pulmonary hypertension (PH)/pulmonary arterial hypertension. However, molecular mechanisms remain elusive. We hypothesized increased Notch3 activation induces oxidative and endoplasmic reticulum (ER) stress and downstream redox signaling, associated with procontractile pulmonary artery state, pulmonary vascular dysfunction, and PH development.<br /><b>Methods</b><br />Studies were performed in TgNotch3<sub>R169C</sub> mice (harboring gain-of-function (GOF) Notch3 mutation) exposed to chronic hypoxia to induce PH, and examined by hemodynamics. Molecular and cellular studies were performed in pulmonary artery smooth muscle cells from pulmonary arterial hypertension patients and in mouse lung. Notch3-regulated genes/proteins, ER stress, ROCK (Rho-associated kinase) expression/activity, Ca<sup>2+</sup> transients and generation of reactive oxygen species, and nitric oxide were measured. Pulmonary vascular reactivity was assessed in the presence of fasudil (ROCK inhibitor) and 4-phenylbutyric acid (ER stress inhibitor).<br /><b>Results</b><br />Hypoxia induced a more severe PH phenotype in TgNotch3<sub>R169C</sub> mice versus controls. TgNotch3<sub>R169C</sub> mice exhibited enhanced Notch3 activation and Hes5 expression (Notch3 target), with increased vascular contraction and impaired vasorelaxation that improved with fasudil/4-phenylbutyric acid. Notch3 mutation was associated with increased pulmonary vessel Ca<sup>2+</sup> transients, ROCK activation, ER stress, and increased reactive oxygen species generation, with reduced NO generation and blunted sGC/cGMP signaling. These effects were ameliorated by N-acetylcysteine. pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension recapitulated Notch3/Hes5 signaling, ER stress and redox changes observed in PH mice.<br /><b>Conclusions</b><br />Notch3 GOF amplifies vascular dysfunction in hypoxic PH. This involves oxidative and ER stress, and ROCK. We highlight a novel role for Notch3/Hes5-redox signaling and important interplay between ER and oxidative stress in PH.<br /><br /><br /><br /><small>Hypertension: 31 May 2023; epub ahead of print</small></div>

<div><h4>A rare case of extensive biventricular cardiac sarcoidosis with reversible torrential tricuspid regurgitation.</h4><i>Okafor J, Azzu A, Ahmed R, Cassimon B, ... Guha K, Khattar R</i><br /><AbstractText>Reversal of torrential tricuspid regurgitation is rarely seen. We describe a case in which effective immunosuppression alongside conventional heart failure therapies lead to reversibility of torrential tricuspid regurgitation in a patient with cardiac sarcoidosis. We also discuss the diagnostic challenge in distinguishing cardiac sarcoidosis from other myocardial diseases in a patient presenting with biventricular failure.</AbstractText><br /><br />© 2023. The Author(s).<br /><br /><small>J Nucl Cardiol: 31 May 2023; epub ahead of print</small></div>

<div><h4>Spatial Multiplexed Protein Profiling of Cardiac Ischemia-Reperfusion Injury.</h4><i>Yao L, He F, Zhao Q, Li D, ... Zhou B, Wang L</i><br /><b>Background</b><br />Reperfusion therapy is critical to myocardial salvage in the event of a myocardial infarction but is complicated by ischemia-reperfusion injury (IRI). Limited understanding of the spatial organization of cardiac cells, which governs cellular interaction and function, has hindered the search for targeted interventions minimizing the deleterious effects of IRI.<br /><b>Methods</b><br />We used imaging mass cytometry to characterize the spatial distribution and dynamics of cell phenotypes and communities in the mouse left ventricle following IRI. Heart sections were collected from 12 cardiac segments (basal, mid-cavity, apical, and apex of the anterior, lateral, and inferior wall) and 8 time points (before ischemia [I-0H], and postreperfusion [R-0H, R-2H, R-6H, R-12H, R-1D, R-3D, R-7D]), and stained with 29 metal-isotope-tagged antibodies. Cell community analysis was performed on reconstructed images, and the most disease-relevant cell type and target protein were selected for intervention of IRI.<br /><b>Results</b><br />We obtained a total of 251 multiplexed images, and identified 197 063 single cells, which were grouped into 23 distinct cell communities based on the structure of cellular neighborhoods. The cellular architecture was heterogeneous throughout the ventricular wall and exhibited swift changes following IRI. Analysis of proteins with posttranslational modifications in single cells unveiled 13 posttranslational modification intensity clusters and highlighted increased H3K9me3 (tri-methylated lysine 9 of histone H3) as a key regulatory response in endothelial cells during the middle stage of IRI. Erasing H3K9 methylation, by silencing its methyltransferase <i>Suv39h1</i> or overexpressing its demethylase <i>Kdm4d</i> in isolated endothelial cells, attenuated cardiac dysfunction and pathological remodeling following IRI. in vitro, H3K9me3 binding significantly increased at endothelial cell function-related genes upon hypoxia, suppressing tube formation, which was rescued by inhibiting H3K9me3.<br /><b>Conclusions</b><br />We mapped the spatiotemporal heterogeneity of cellular phenotypes in the adult heart upon IRI, and uncovered H3K9me3 in endothelial cells as a potential therapeutic target for alleviating pathological remodeling of the heart following myocardial IRI.<br /><br /><br /><br /><small>Circ Res: 30 May 2023; epub ahead of print</small></div>

<div><h4>NLRP3 inflammasome and interleukin-1 contributions to COVID-19-associated coagulopathy and immunothrombosis.</h4><i>Potere N, Garrad E, Kanthi Y, Di Nisio M, ... De Caterina R, Abbate A</i><br /><AbstractText>Immunothrombosis - immune-mediated activation of coagulation - is protective against pathogens, but excessive immunothrombosis can result in pathological thrombosis and multiorgan damage, as in severe Coronavirus Disease 2019 (COVID-19). The NACHT-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome produces major proinflammatory cytokines of the interleukin (IL)-1 family, IL-1β and IL-18, and induces pyroptotic cell death. Activation of the NLRP3 inflammasome pathway also promotes immunothrombotic programs including release of neutrophil extracellular traps and tissue factor by leukocytes, and prothrombotic responses by platelets and the vascular endothelium. NLRP3 inflammasome activation occurs in patients with COVID-19 pneumonia. In preclinical models, NLRP3 inflammasome pathway blockade restrains COVID-19-like hyperinflammation and pathology. Anakinra, recombinant human IL-1 receptor antagonist, showed safety and efficacy, and is approved for the treatment of hypoxemic COVID-19 patients with early signs of hyperinflammation. The non-selective NLRP3 inhibitor colchicine reduced hospitalization and death in a subgroup of COVID-19 outpatients, but is not approved for the treatment of COVID-19. Additional COVID-19 trials testing NLRP3 inflammasome pathway blockers are inconclusive or ongoing. We herein outline the contribution of immunothrombosis to COVID-19-associated coagulopathy, and review preclinical and clinical evidence suggesting an engagement of the NLRP3 inflammasome pathway in the immunothrombotic pathogenesis of COVID-19. We also summarize current efforts to target the NLRP3 inflammasome pathway in COVID-19, and discuss challenges, unmet gaps and the therapeutic potential that inflammasome-targeted strategies may provide for inflammation-driven thrombotic disorders including COVID-19.</AbstractText><br /><br />© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.<br /><br /><small>Cardiovasc Res: 30 May 2023; epub ahead of print</small></div>

<div><h4>HDL Function and Atherosclerosis: Reactive Dicarbonyls as Promising Targets of Therapy.</h4><i>Linton MF, Yancey PG, Tao H, Davies SS</i><br /><AbstractText>Epidemiologic studies detected an inverse relationship between HDL (high-density lipoprotein) cholesterol (HDL-C) levels and atherosclerotic cardiovascular disease (ASCVD), identifying HDL-C as a major risk factor for ASCVD and suggesting atheroprotective functions of HDL. However, the role of HDL-C as a mediator of risk for ASCVD has been called into question by the failure of HDL-C-raising drugs to reduce cardiovascular events in clinical trials. Progress in understanding the heterogeneous nature of HDL particles in terms of their protein, lipid, and small RNA composition has contributed to the realization that HDL-C levels do not necessarily reflect HDL function. The most examined atheroprotective function of HDL is reverse cholesterol transport, whereby HDL removes cholesterol from plaque macrophage foam cells and delivers it to the liver for processing and excretion into bile. Indeed, in several studies, HDL has shown inverse associations between HDL cholesterol efflux capacity and ASCVD in humans. Inflammation plays a key role in the pathogenesis of atherosclerosis and vulnerable plaque formation, and a fundamental function of HDL is suppression of inflammatory signaling in macrophages and other cells. Oxidation is also a critical process to ASCVD in promoting atherogenic oxidative modifications of LDL (low-density lipoprotein) and cellular inflammation. HDL and its proteins including apoAI (apolipoprotein AI) and PON1 (paraoxonase 1) prevent cellular oxidative stress and LDL modifications. Importantly, HDL in humans with ASCVD is oxidatively modified rendering HDL dysfunctional and proinflammatory. Modification of HDL with reactive carbonyl species, such as malondialdehyde and isolevuglandins, dramatically impairs the antiatherogenic functions of HDL. Importantly, treatment of murine models of atherosclerosis with scavengers of reactive dicarbonyls improves HDL function and reduces systemic inflammation, atherosclerosis development, and features of plaque instability. Here, we discuss the HDL antiatherogenic functions in relation to oxidative modifications and the potential of reactive dicarbonyl scavengers as a therapeutic approach for ASCVD.</AbstractText><br /><br /><br /><br /><small>Circ Res: 26 May 2023; 132:1521-1545</small></div>

<div><h4>Inflammasomes and Atherosclerosis: a Mixed Picture.</h4><i>Tall AR, Bornfeldt KE</i><br /><AbstractText>The CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcome Study) and colchicine trials suggest an important role of inflammasomes and their major product IL-1β (interleukin 1β) in human atherosclerotic cardiovascular disease. Moreover, studies in mouse models indicate a causal role of inflammasomes and IL-1β in atherosclerosis. However, recent studies have led to a more granular view of the role of inflammasomes in atherosclerosis. Studies in hyperlipidemic mouse models suggest that prominent activation of the NLRP3 inflammasome requires a second hit such as defective cholesterol efflux, defective DNA repair, clonal hematopoiesis or diabetes. Similarly in humans some mutations promoting clonal hematopoiesis increase coronary artery disease risk in part by promoting inflammasome activation. Recent studies in mice and humans point to a wider role of the AIM2 (absent in melanoma 2) inflammasome in promoting cardiovascular disease including in some forms of clonal hematopoiesis and diabetes. These developments suggest a precision medicine approach in which treatments targeting inflammasomes or IL-1β might be best employed in clinical settings involving increased inflammasome activation.</AbstractText><br /><br /><br /><br /><small>Circ Res: 26 May 2023; 132:1505-1520</small></div>

<div><h4>Distinct Roles of DRP1 in Conventional and Alternative Mitophagy in Obesity Cardiomyopathy.</h4><i>Tong M, Mukai R, Mareedu S, Zhai P, ... Babu GJ, Sadoshima J</i><br /><b>Rationale</b><br />Obesity induces cardiomyopathy characterized by hypertrophy and diastolic dysfunction. Whereas mitophagy mediated through an Atg7-dependent mechanism serves as an essential mechanism to maintain mitochondrial quality during the initial development of obesity cardiomyopathy, Rab9-dependent alternative mitophagy takes over the role during the chronic phase. Although it has been postulated that DRP1 (dynamin-related protein 1)-mediated mitochondrial fission and consequent separation of the damaged portions of mitochondria are essential for mitophagy, the involvement of DRP1 in mitophagy remains controversial.<br /><b>Objective</b><br />We investigated whether endogenous DRP1 is essential in mediating the 2 forms of mitophagy during high-fat diet (HFD)-induced obesity cardiomyopathy and, if so, what the underlying mechanisms are.<br /><b>Methods and results</b><br />Mice were fed either a normal diet or an HFD (60 kcal %fat). Mitophagy, evaluated with Mito-Keima, was increased after 3 weeks of HFD consumption. The induction of mitophagy by HFD consumption was completely abolished in tamoxifen-inducible cardiac-specific <i>Drp1</i>knockout (<i>Drp1</i> MCM) mouse hearts, in which both diastolic and systolic dysfunction were exacerbated. The increase in LC3-dependent general autophagy and colocalization between LC3 and mitochondrial proteins was abolished in <i>Drp1</i> MCM mice. Activation of alternative mitophagy was also completely abolished in <i>Drp1</i> MCM mice during the chronic phase of HFD consumption. DRP1 was phosphorylated at Ser616, localized at the mitochondria-associated membranes, and associated with Rab9 and Fis1 only during the chronic, but not acute, phase of HFD consumption.<br /><b>Conclusions</b><br />DRP1 is an essential factor in mitochondrial quality control during obesity cardiomyopathy that controls multiple forms of mitophagy. Although DRP1 regulates conventional mitophagy through a mitochondria-associated membrane-independent mechanism during the acute phase, it acts as a component of the mitophagy machinery at the mitochondria-associated membranes in alternative mitophagy during the chronic phase of HFD consumption.<br /><br /><br /><br /><small>Circ Res: 26 May 2023; epub ahead of print</small></div>

<div><h4>LTBP4 (Latent Transforming Growth Factor Beta Binding Protein 4) Protects Against Renal Fibrosis via Mitochondrial and Vascular Impacts.</h4><i>Su CT, See DHW, Huang YJ, Jao TM, ... Huang JW, Hung KY</i><br /><b>Background</b><br />As a part of natural disease progression, acute kidney injury (AKI) can develop into chronic kidney disease via renal fibrosis and inflammation. LTBP4 (latent transforming growth factor beta binding protein 4) regulates transforming growth factor beta, which plays a role in renal fibrosis pathogenesis. We previously investigated the role of LTBP4 in chronic kidney disease. Here, we examined the role of LTBP4 in AKI.<br /><b>Methods</b><br />LTBP4 expression was evaluated in human renal tissues, obtained from healthy individuals and patients with AKI, using immunohistochemistry. <i>LTBP4</i> was knocked down in both C57BL/6 mice and human renal proximal tubular cell line HK-2. AKI was induced in mice and HK-2 cells using ischemia-reperfusion injury and hypoxia, respectively. Mitochondrial division inhibitor 1, an inhibitor of DRP1 (dynamin-related protein 1), was used to reduce mitochondrial fragmentation. Gene and protein expression were then examined to assess inflammation and fibrosis. The results of bioenergetic studies for mitochondrial function, oxidative stress, and angiogenesis were assessed.<br /><b>Results</b><br />LTBP4 expression was upregulated in the renal tissues of patients with AKI. <i>Ltbp4</i>-knockdown mice showed increased renal tissue injury and mitochondrial fragmentation after ischemia-reperfusion injury, as well as increased inflammation, oxidative stress, and fibrosis, and decreased angiogenesis. in vitro studies using HK-2 cells revealed similar results. The energy profiles of Ltbp4-deficient mice and LTBP4-deficient HK-2 cells indicated decreased ATP production. LTBP4-deficient HK-2 cells exhibited decreased mitochondrial respiration and glycolysis. Human aortic endothelial cells and human umbilical vein endothelial cells exhibited decreased angiogenesis when treated with LTBP4-knockdown conditioned media. Mitochondrial division inhibitor 1 treatment ameliorated inflammation, oxidative stress, and fibrosis in mice and decreased inflammation and oxidative stress in HK-2 cells.<br /><b>Conclusions</b><br />Our study is the first to demonstrate that LTBP4 deficiency increases AKI severity, consequently leading to chronic kidney disease. Potential therapies focusing on LTBP4-associated angiogenesis and LTBP4-regulated DRP1-dependent mitochondrial division are relevant to renal injury.<br /><br /><br /><br /><small>Circ Res: 26 May 2023; epub ahead of print</small></div>

<div><h4>Immune Regulation of the Liver Through the PCSK9/CD36 Pathway During Heart Transplant Rejection.</h4><i>Zhang X, Xu H, Yu J, Cui J, ... Wu J, Xia J</i><br /><b>Background</b><br />PCSK9 (proprotein convertase subtilisin/kexin 9), which is mainly secreted by the liver, is not only a therapeutic target for hyperlipidemia and cardiovascular disease, but also has been implicated in the immune regulation of infections and tumors. However, the role of PCSK9 and the liver in heart transplant rejection (HTR) and the underlying mechanisms remain unclear.<br /><b>Methods</b><br />We assessed serum PCSK9 expression in both murine and human recipients during HTR and investigated the effect of PCSK9 ablation on HTR by using global knockout mice and a neutralizing antibody. Moreover, we performed multiorgan histological and transcriptome analyses, and multiomics and single-cell RNA-sequencing studies of the liver during HTR, as well. We further used hepatocyte-specific <i>Pcsk9</i> knockout mice to investigate whether the liver regulated HTR through PCSK9. Last, we explored the regulatory effect of the PCSK9/CD36 pathway on the phenotype and function of macrophages in vitro and in vivo.<br /><b>Results</b><br />Here, we report that murine and human recipients have high serum PCSK9 levels during HTR. PCSK9 ablation prolonged cardiac allograft survival and attenuated the infiltration of inflammatory cells in the graft and the expansion of alloreactive T cells in the spleen. Next, we demonstrated that PCSK9 was mainly produced and significantly upregulated in the recipient liver, which also showed a series of signaling changes, including changes in the TNF-α (tumor necrosis factor α) and IFN-γ (interferon γ) signaling pathways and the bile acid and fatty acid metabolism pathways. We found mechanistically that TNF-α and IFN-γ synergistically promoted PCSK9 expression in hepatocytes through the transcription factor SREBP2 (sterol regulatory element binding protein 2). Moreover, in vitro and in vivo studies indicated that PCSK9 inhibited CD36 expression and fatty acid uptake by macrophages and strengthened the proinflammatory phenotype, which facilitated their ability to promote proliferation and IFN-γ production by donor-reactive T cells. Last, we found that the protective effect of PCSK9 ablation against HTR is dependent on the CD36 pathway in the recipient.<br /><b>Conclusions</b><br />This study reveals a novel mechanism for immune regulation by the liver through the PCSK9/CD36 pathway during HTR, which influences the phenotype and function of macrophages and suggests that the modulation of this pathway may be a potential therapeutic target to prevent HTR.<br /><br /><br /><br /><small>Circulation: 26 May 2023; epub ahead of print</small></div>

<div><h4>PDE10A Inactivation Prevents Doxorubicin-Induced Cardiotoxicity and Tumor Growth.</h4><i>Chen S, Chen J, Du W, Mickelsen DM, ... Kumar S, Yan C</i><br /><b>Background</b><br />Cyclic nucleotides play critical roles in cardiovascular biology and disease. PDE10A (phosphodiesterase 10A) is able to hydrolyze both cAMP and cGMP. PDE10A expression is induced in various human tumor cell lines, and PDE10A inhibition suppresses tumor cell growth. Chemotherapy drug such as doxorubicin (DOX) is widely used in chemotherapy. However, cardiotoxicity of DOX remains to be a serious clinical complication. In the current study, we aim to determine the role of PDE10A and the effect of PDE10A inhibition on cancer growth and cardiotoxicity induced by DOX.<br /><b>Methods</b><br />We used global PDE10A KO (knockout) mice and PDE10A inhibitor TP-10 to block PDE10A function. DOX-induced cardiotoxicity was evaluated in C57Bl/6J mice and nude mice with implanted ovarian cancer xenografts. Isolated adult mouse cardiomyocytes and a human ovarian cancer cell line were used for in vitro functional and mechanistic studies.<br /><b>Results</b><br />We found that PDE10A deficiency or inhibition alleviated DOX-induced myocardial atrophy, apoptosis, and dysfunction in C57Bl/6J mice. RNA sequencing study revealed a number of PDE10A-regulated signaling pathways involved in DOX-induced cardiotoxicity. PDE10A inhibition increased the death, decreased the proliferation, and potentiated the effect of DOX on various human cancer cells. Importantly, in nude mice with implanted ovarian cancer xenografts, PDE10A inhibition attenuated tumor growth while protecting DOX-induced cardiotoxicity. In isolated cardiomyocytes, PDE10A contributed to DOX-induced cardiomyocyte death via increasing Top2β (topoisomerase 2β) expression, mitochondrial dysfunction, and DNA damage by antagonizing cGMP/PKG (protein kinase G) signaling. PDE10A contributed to cardiomyocyte atrophy via potentiating FoxO3 (forkhead box O3) signaling via both cAMP/PKA- (protein kinase A) and cGMP/PKG-dependent signaling.<br /><b>Conclusions</b><br />Taken together, our study elucidates a novel role for PDE10A in cardiotoxicity induced by DOX and cancer growth. Given that PDE10A has been already proven to be a safe drug target, PDE10A inhibition may represent a novel therapeutic strategy in cancer therapy, with effects preventing DOX-induced cardiotoxicity and simultaneously antagonizing cancer growth.<br /><br /><br /><br /><small>Circ Res: 26 May 2023; epub ahead of print</small></div>

<div><h4>Hypoxia-Preconditioned Bone Marrow Mesenchymal Stem Cells Improved Cerebral Collateral Circulation and Stroke Outcome in Mice.</h4><i>Tian H, Yang X, Zhao J, Liu X, ... Wei L, Liu L</i><br /><b>Background</b><br />Adequate collateral circulation can remarkably improve patient prognoses for patients experiencing ischemic stroke. Hypoxic preconditioning enhances the regenerative properties of bone marrow mesenchymal stem cells (BMSCs). Rabep2 (RAB GTPase binding effector protein 2) is a key protein in collateral remodeling. We investigated whether BMSCs and hypoxia-preconditioned BMSCs (H-BMSCs) augment collateral circulation poststroke, particularly through Rabep2 regulation.<br /><b>Methods</b><br />BMSCs or H-BMSCs (1×10<sup>6</sup>) were delivered intranasally in ischemic mice with distal middle cerebral artery occlusion at 6 hours poststroke. Two-photon microscopic imaging and vessel painting methods were used to analyze collateral remodeling. Blood flow, vascular density, infarct volume, and gait analysis were assessed to evaluate poststroke outcomes. Expressions of proangiogenic marker VEGF (vascular endothelial growth factor) and Rabep2 were determined by Western blotting. Western blot, EdU incorporation, and tube formation assays were conducted on cultured endothelial cells treated with BMSCs.<br /><b>Results</b><br />BMSCs were more effectively transplanted in the ischemic brain after hypoxic preconditioning. The ipsilateral collateral diameter was increased by BMSCs and strengthened by H-BMSCs (<i>P</i><0.05). BMSCs increased peri-infarct blood flow and vascular density and reduced infarct volume, gait deficits (<i>P</i><0.05), and furthermore by H-BMSCs (<i>P</i><0.05). VEGF and Rabep2 protein expression was increased by BMSCs (<i>P</i><0.05), which was enhanced by preconditioning (<i>P</i><0.01). Additionally, BMSCs increased Rabep2 expression, proliferation, and tube formation of endothelial cells in vitro (<i>P</i><0.05). H-BMSCs enhanced these effects (<i>P</i><0.05), which were annulled by Rabep2 knockdown.<br /><b>Conclusions</b><br />BMSCs increased collateral circulation and improved poststroke outcomes, through the upregulation of Rabep2. These effects were enhanced by hypoxic preconditioning.<br /><br /><br /><br /><small>Arterioscler Thromb Vasc Biol: 25 May 2023; epub ahead of print</small></div>

<div><h4>Quartet of ApoCs and the Different Roles They Play in Diabetes.</h4><i>Hsu CC, Kanter JE, Kothari V, Bornfeldt KE</i><br /><AbstractText>APOA1 and APOB are the structural proteins of high-density lipoprotein and APOB-containing lipoproteins, such as low-density lipoprotein and very low-density lipoprotein, respectively. The 4 smaller APOCs (APOC1, APOC2, APOC3, and APOC4) are exchangeable apolipoproteins; they are readily transferred among high-density lipoproteins and APOB-containing lipoproteins. The APOCs regulate plasma triglyceride and cholesterol levels by modulating substrate availability and activities of enzymes interacting with lipoproteins and by interfering with APOB-containing lipoprotein uptake through hepatic receptors. Of the 4 APOCs, APOC3 has been best studied in relation to diabetes. Elevated serum APOC3 levels predict incident cardiovascular disease and progression of kidney disease in people with type 1 diabetes. Insulin suppresses APOC3 levels, and accordingly, elevated APOC3 levels associated with insulin deficiency and insulin resistance. Mechanistic studies in a mouse model of type 1 diabetes have demonstrated that APOC3 acts in the causal pathway of diabetes-accelerated atherosclerosis. The mechanism is likely due to the ability of APOC3 to slow the clearance of triglyceride-rich lipoproteins and their remnants, thereby causing an increased accumulation of atherogenic lipoprotein remnants in lesions of atherosclerosis. Less is known about the roles of APOC1, APOC2, and APOC4 in diabetes.</AbstractText><br /><br /><br /><br /><small>Arterioscler Thromb Vasc Biol: 25 May 2023; epub ahead of print</small></div>

<div><h4>Maresin-1 protects against pulmonary arterial hypertension by improving mitochondrial homeostasis through ALXR/HSP90α axis.</h4><i>Liu M, He H, Fan F, Qiu L, ... Yang G, Chen L</i><br /><b>Aims</b><br />Pulmonary arterial hypertension (PAH) is a progressive and lethal disease characterized by continuous proliferation of pulmonary arterial smooth muscle cell (PASMCs) and increased pulmonary vascular remodeling. Maresin-1 (MaR1) is a member of pro-resolving lipid mediators and exhibits protective effects on various inflammation-related diseases. Here we aimed to study the role of MaR1 in the development and progression of PAH and to explore the underlying mechanisms.<br /><b>Methods and results</b><br />We evaluated the effect of MaR1 treatment on PAH in both monocrotaline (MCT)-induced rat and hypoxia+SU5416 (HySu)-induced mouse models of pulmonary hypertension (PH). Plasma samples were collected from patients with PAH and rodent PH models to examine MaR1 production. Specific shRNA adenovirus or inhibitors were used to block the function of MaR1 receptors. The data showed that MaR1 significantly prevented the development and blunted the progression of PH in rodents. Blockade of the function of MaR1 receptor ALXR, but not LGR6 or RORα, with BOC-2, abolished the protective effect of MaR1 against PAH development and reduced its therapeutic potential. Mechanistically, we demonstrated that the MaR1/ALXR axis suppressed hypoxia-induced PASMCs proliferation and alleviated pulmonary vascular remodeling by inhibiting mitochondrial accumulation of heat shock protein 90α (HSP90α) and restoring mitophagy.<br /><b>Conclusion</b><br />MaR1 protects against PAH by improving mitochondrial homeostasis through ALXR/HSP90α axis and represents a promising target for PAH prevention and treatment.<br /><br />Copyright © 2023 Elsevier Ltd. All rights reserved.<br /><br /><small>J Mol Cell Cardiol: 25 May 2023; 181:15-30</small></div>

<div><h4>Macrophage angiotensin-converting enzyme (ACE) reduces atherosclerosis by increasing PPARα and fundamentally changing lipid metabolism.</h4><i>Cao D, Khan Z, Li X, Saito S, ... Okwan-Duodu D, Bernstein KE</i><br /><b>Aims</b><br />The metabolic failure of macrophages to adequately process lipid is central to the etiology of atherosclerosis. Here, we examine the role of macrophage angiotensin converting enzyme (ACE) in a mouse model of PCSK9 induced atherosclerosis.<br /><b>Methods and results</b><br />Atherosclerosis in mice was induced with AAV-PCSK9 and a high fat diet. Animals with increased macrophage ACE (ACE 10/10 mice) have a marked reduction in atherosclerosis vs. WT mice. Macrophages from both the aorta and peritoneum of ACE 10/10 express increased PPARα and have a profoundly altered phenotype to process lipids characterized by higher levels of the surface scavenger receptor CD36, increased uptake of lipid, increased capacity to transport long chain fatty acids into mitochondria, higher oxidative metabolism and lipid β-oxidation as determined using 13C isotope tracing, increased cell ATP, increased capacity for efferocytosis, increased concentrations of the lipid transporters ABCA1 and ABCG1, and increased cholesterol efflux. These effects are mostly independent of angiotensin II. Human THP-1 cells, when modified to express more ACE, increase expression of PPARα, increase cell ATP and acetyl-CoA, and increase cell efferocytosis.<br /><br /><b>Conclusion:</b><br/>and translational perspective</b><br />Increased macrophage ACE expression enhances macrophage lipid metabolism, cholesterol efflux, efferocytosis, and it reduces atherosclerosis. This has implications for the treatment of cardiovascular disease with angiotensin II receptor antagonists (ARBs) vs. ACE inhibitors.<br /><br />© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.<br /><br /><small>Cardiovasc Res: 24 May 2023; epub ahead of print</small></div>

<div><h4>Suppression of myeloid YAP antagonizes adverse cardiac remodeling during pressure overload stress.</h4><i>Francisco J, Guan J, Zhang Y, Nakada Y, ... Sadoshima J, Del Re DP</i><br /><AbstractText>Inflammation is an integral component of cardiovascular disease and is thought to contribute to cardiac dysfunction and heart failure. While ischemia-induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non-ischemic stress. Recent work has implicated a role for Yes-associated protein (YAP) in modulating inflammation in response to ischemic injury; however, whether YAP influences inflammation in the heart during non-ischemic stress is not described. We hypothesized that YAP mediates a pro-inflammatory response during pressure overload (PO)-induced non-ischemic injury, and that targeted YAP inhibition in the myeloid compartment is cardioprotective. In mice, PO elicited myeloid YAP activation, and myeloid-specific YAP knockout mice (YAP<sup>F/F</sup>;LysM<sup>Cre</sup>) subjected to PO stress had better systolic function, and attenuated pathological remodeling compared to control mice. Inflammatory indicators were also significantly attenuated, while pro-resolving genes including Vegfa were enhanced, in the myocardium, and in isolated macrophages, of myeloid YAP KO mice after PO. Experiments using bone marrow-derived macrophages (BMDMs) from YAP KO and control mice demonstrated that YAP suppression shifted polarization toward a resolving phenotype. We also observed attenuated NLRP3 inflammasome priming and function in YAP deficient BMDMs, as well as in myeloid YAP KO hearts following PO, indicating disruption of inflammasome induction. Finally, we leveraged nanoparticle-mediated delivery of the YAP inhibitor verteporfin and observed attenuated PO-induced pathological remodeling compared to DMSO nanoparticle control treatment. These data implicate myeloid YAP as an important molecular nodal point that facilitates cardiac inflammation and fibrosis during PO stress and suggest that selective inhibition of YAP may prove a novel therapeutic target in non-ischemic heart disease.</AbstractText><br /><br />Copyright © 2023 Elsevier Ltd. All rights reserved.<br /><br /><small>J Mol Cell Cardiol: 24 May 2023; 181:1-14</small></div>

<div><h4>IFN-γ deficiency in the rostral ventrolateral medulla contributes to stress-induced hypertension by impairing microglial synaptic engulfment.</h4><i>Tong L, Chen G, Liu T, Wang L, ... Zhang S, Du D</i><br /><b>Background</b><br />Dysfunctional neurons and microglia in the rostral ventrolateral medulla (RVLM) have been implicated in the pathogenesis of stress-induced hypertension (SIH). Functional perturbation of microglial synaptic engulfment can induce aberrant brain circuit activity. IFN-γ is a pleiotropic cytokine that plays a role in regulating neuronal activity. However, existing research on the exploration of the effects of microglia on synapses in the RVLM is lacking, particularly on the function of IFN-γ in microglial synaptic engulfment involved in SIH.<br /><b>Methods</b><br />A SIH rat model was established by electric foot shocks combined with noise stimulation. The underlying mechanism of IFN-γ on synaptic density and microglial synaptic engulfment was investigated through in-vivo and in-vitro experiments involving gain of function, immunofluorescence, quantitative real-time PCR, western blot, and morphometric analysis. Furthermore, the function of IFN-γ in neuronal activity, renal sympathetic nerve activity (RSNA), and blood pressure (BP) regulation was determined through in-vivo and in-vitro experiments involving Ca2+ imaging, immunofluorescence, platinum-iridium electrode recording, ELISA, the femoral artery cannulation test, and the tail-cuff method.<br /><b>Results</b><br />The BP, heart rate, RSNA, plasma norepinephrine, and the number of c-Fos-positive neurons in SIH rats increased compared with those in control rats. Pre and postsynaptic densities in the RVLM also increased in SIH rats. IFN-γ and CCL2 expression levels were significantly reduced in the RVLM of the SIH group, whose microglia also exhibited an impaired capacity for synapse engulfment. IFN-γ elevation increased CCL2 expression and microglial synaptic engulfment and decreased synaptic density in vivo and in vitro. However, CCL2 inhibition reversed these effects. Moreover, the reduction of neuronal excitability, RSNA, plasma norepinephrine, and BP by IFN-γ was abrogated through CCL2 expression.<br /><b>Conclusion</b><br />IFN-γ deficiency in the RVLM impaired the microglial engulfment of synapses by inhibiting CCL2 expression and increasing synaptic density and neuronal excitability, thereby contributing to SIH progression. Targeting IFN-γ may be considered a potential strategy to combat SIH.Graphical Abstract, http://links.lww.com/HJH/C203.<br /><br />Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.<br /><br /><small>J Hypertens: 24 May 2023; epub ahead of print</small></div>

<div><h4>Tissue factor expression in monocyte subsets during human immunothrombosis, endotoxemia and sepsis.</h4><i>Musgrave KM, Scott J, Sendama W, Gardner AI, ... Simpson AJ, Rostron AJ</i><br /><b>Introduction</b><br />Tissue factor expression on monocytes is implicated in the pathophysiology of sepsis-induced coagulopathy. How tissue factor is expressed by monocyte subsets (classical, intermediate and non-classical) is unknown.<br /><b>Methods</b><br />Monocytic tissue factor surface expression was investigated during three conditions. Primary human monocytes and microvascular endothelial cell co-cultures were used for in vitro studies. Volunteers received a bolus of lipopolysaccharide (2 ng/kg) to induce endotoxemia. Patients with sepsis, or controls with critical illness unrelated to sepsis, were recruited from four intensive care units.<br /><b>Results</b><br />Contact with endothelium and stimulation with lipopolysaccharide reduced the proportion of intermediate monocytes. Lipopolysaccharide increased tissue factor surface expression on classical and non-classical monocytes. Endotoxemia induced profound, transient monocytopenia, along with activation of coagulation pathways. In the remaining circulating monocytes, tissue factor was up-regulated in intermediate monocytes, though approximately 60 % of individuals (responders) up-regulated tissue factor across all monocyte subsets. In critically ill patients, tissue factor expression on intermediate and non-classical monocytes was significantly higher in patients with established sepsis than among non-septic patients. Upon recovery of sepsis, expression of tissue factor increased significantly in classical monocytes.<br /><b>Conclusion</b><br />Tissue factor expression in monocyte subsets varies significantly during health, endotoxemia and sepsis.<br /><br />Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.<br /><br /><small>Thromb Res: 24 May 2023; 228:10-20</small></div>

<div><h4>Observer studies of image quality of denoising reduced-count cardiac single photon emission computed tomography myocardial perfusion imaging by three-dimensional Gaussian post-reconstruction filtering and deep learning.</h4><i>Pretorius PH, Liu J, Kalluri KS, Jiang Y, ... Wernick MN, King MA</i><br /><b>Background</b><br />The aim of this research was to asses perfusion-defect detection-accuracy by human observers as a function of reduced-counts for 3D Gaussian post-reconstruction filtering vs deep learning (DL) denoising to determine if there was improved performance with DL.<br /><b>Methods</b><br />SPECT projection data of 156 normally interpreted patients were used for these studies. Half were altered to include hybrid perfusion defects with defect presence and location known. Ordered-subset expectation-maximization (OSEM) reconstruction was employed with the optional correction of attenuation (AC) and scatter (SC) in addition to distance-dependent resolution (RC). Count levels varied from full-counts (100%) to 6.25% of full-counts. The denoising strategies were previously optimized for defect detection using total perfusion deficit (TPD). Four medical physicist (PhD) and six physician (MD) observers rated the slices using a graphical user interface. Observer ratings were analyzed using the LABMRMC multi-reader, multi-case receiver-operating-characteristic (ROC) software to calculate and compare statistically the area-under-the-ROC-curves (AUCs).<br /><b>Results</b><br />For the same count-level no statistically significant increase in AUCs for DL over Gaussian denoising was determined when counts were reduced to either the 25% or 12.5% of full-counts. The average AUC for full-count OSEM with solely RC and Gaussian filtering was lower than for the strategies with AC and SC, except for a reduction to 6.25% of full-counts, thus verifying the utility of employing AC and SC with RC.<br /><b>Conclusion</b><br />We did not find any indication that at the dose levels investigated and with the DL network employed, that DL denoising was superior in AUC to optimized 3D post-reconstruction Gaussian filtering.<br /><br />© 2023. The Author(s) under exclusive licence to American Society of Nuclear Cardiology.<br /><br /><small>J Nucl Cardiol: 23 May 2023; epub ahead of print</small></div>

<div><h4>Distinct effects of cardiac mitochondrial calcium uniporter inactivation via EMRE deletion in the short and long term.</h4><i>Villanueva HC, Sung JH, Stevens JA, Zhang MJ, ... Townsend D, Liu JC</i><br /><AbstractText>Transport of Ca<sup>2+</sup> into mitochondria is thought to stimulate the production of ATP, a critical process in the heart\'s fight or flight response, but excess Ca<sup>2+</sup> can trigger cell death. The mitochondrial Ca<sup>2+</sup> uniporter complex is the primary route of Ca<sup>2+</sup> transport into mitochondria, in which the channel-forming protein MCU and the regulatory protein EMRE are essential for activity. In previous studies, chronic Mcu or Emre deletion differ from acute cardiac Mcu deletion in response to adrenergic stimulation and ischemia/reperfusion (I/R) injury, despite equivalent inactivation of rapid mitochondrial Ca<sup>2+</sup> uptake. To explore this discrepancy between chronic and acute loss of uniporter activity, we compared short-term and long-term Emre deletion using a novel conditional cardiac-specific, tamoxifen-inducible mouse model. After short-term Emre deletion (3 weeks post-tamoxifen) in adult mice, cardiac mitochondria were unable to take up Ca<sup>2+</sup>, had lower basal mitochondrial Ca<sup>2+</sup> levels, and displayed attenuated Ca<sup>2+</sup>-induced ATP production and mPTP opening. Moreover, short-term EMRE loss blunted cardiac response to adrenergic stimulation and improved maintenance of cardiac function in an ex vivo I/R model. We then tested whether the long-term absence of EMRE (3 months post-tamoxifen) in adulthood would lead to distinct outcomes. After long-term Emre deletion, mitochondrial Ca<sup>2+</sup> handling and function, as well as cardiac response to adrenergic stimulation, were similarly impaired as in short-term deletion. Interestingly, however, protection from I/R injury was lost in the long-term. These data suggest that several months without uniporter function are insufficient to restore bioenergetic response but are sufficient to restore susceptibility to I/R.</AbstractText><br /><br />Copyright © 2023. Published by Elsevier Ltd.<br /><br /><small>J Mol Cell Cardiol: 23 May 2023; epub ahead of print</small></div>

<div><h4>Myeloid CD40 deficiency reduces atherosclerosis by impairing macrophages\' transition into a pro-inflammatory state.</h4><i>Bosmans LA, van Tiel CM, Aarts SABM, Willemsen L, ... Shami A, Lutgens E</i><br /><b>Aims</b><br />CD40 and its ligand, CD40L, play a critical role in driving atherosclerotic plaque development. Disrupted CD40-signalling reduces experimental atherosclerosis and induces a favourable stable plaque phenotype. We recently showed that small molecule-based inhibition of CD40-tumour necrosis factor receptor associated factor-6 interactions attenuates atherosclerosis in hyperlipidaemic mice via macrophage-driven mechanisms. The present study aims to detail the function of myeloid CD40 in atherosclerosis using myeloid-specific CD40-deficient mice.<br /><b>Method and results</b><br />Cd40flox/flox and LysM-cre Cd40flox/flox mice on an Apoe-/- background were generated (CD40wt and CD40mac-/-, respectively). Atherosclerotic lesion size, as well as plaque macrophage content, was reduced in CD40mac-/- compared to CD40wt mice, and their plaques displayed a reduction in necrotic core size. Transcriptomics analysis of the CD40mac-/- atherosclerotic aorta revealed downregulated pathways of immune pathways and inflammatory responses. Loss of CD40 in macrophages changed the representation of aortic macrophage subsets. Mass cytometry analysis revealed a higher content of a subset of alternative or resident-like CD206+CD209b- macrophages in the atherosclerotic aorta of CD40mac-/- compared to CD40wt mice. RNA-sequencing of bone marrow-derived macrophages of CD40mac-/- mice demonstrated upregulation of genes associated with alternatively activated macrophages (including Folr2, Thbs1, Sdc1, and Tns1).<br /><b>Conclusions</b><br />We here show that absence of CD40 signalling in myeloid cells reduces atherosclerosis and limits systemic inflammation by preventing a shift in macrophage polarization towards pro-inflammatory states. Our study confirms the merit of macrophage-targeted inhibition of CD40 as a valuable therapeutic strategy to combat atherosclerosis.<br /><br />© The Author(s) 2022. Published by Oxford University Press on behalf of the European Society of Cardiology.<br /><br /><small>Cardiovasc Res: 22 May 2023; 119:1146-1160</small></div>

<div><h4>Inhibition of the extracellular enzyme ADAMTS4 prevents cardiac fibrosis and dysfunction.</h4><i>Vistnes M, Erusappan PM, Sasi A, Nordén ES, ... Sjaastad I, Christensen G</i><br /><b>Aims</b><br />Heart failure is a condition with high mortality rates, and there is a lack of therapies that directly target maladaptive changes in the extracellular matrix (ECM), such as fibrosis. We investigated whether the ECM enzyme known as A disintegrin and metalloprotease with thrombospondin motif (ADAMTS) 4 might serve as a therapeutic target in treatment of heart failure and cardiac fibrosis.<br /><b>Methods and results</b><br />The effects of pharmacological ADAMTS4 inhibition on cardiac function and fibrosis were examined in rats exposed to cardiac pressure overload. Disease mechanisms affected by the treatment were identified based on changes in the myocardial transcriptome. Following aortic banding (AB), rats receiving an ADAMTS inhibitor, with high inhibitory capacity for ADAMTS4, showed substantially better cardiac function than vehicle-treated rats, including ∼30 % reduction in E/e\' and left atrial diameter, indicating an improvement in diastolic function. ADAMTS inhibition also resulted in a marked reduction in myocardial collagen content and a downregulation of transforming growth factor (TGF) β target genes. The mechanism for the beneficial effects of ADAMTS inhibition was further studied in cultured human cardiac fibroblasts producing mature ECM. ADAMTS4 caused a 50% increase in the TGF-β levels in the medium. Simultaneously, ADAMTS4 elicited a not previously known cleavage of TGF-β-binding proteins, i.e. latent binding protein of TGF-β (LTBP1) and extra domain A (EDA)-fibronectin. These effects were abolished by the ADAMTS inhibitor. In failing human hearts, we observed a marked increase in ADAMTS4 expression and cleavage activity.<br /><b>Conclusion</b><br />Inhibition of ADAMTS4 improves cardiac function and reduces collagen accumulation in rats with cardiac pressure overload, possibly through a not previously known cleavage of molecules that control TGF-β availability. Targeting ADAMTS4 may serve as a novel strategy in heart failure treatment, in particular in heart failure with fibrosis and diastolic dysfunction.<br /><br />© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.<br /><br /><small>Cardiovasc Res: 22 May 2023; epub ahead of print</small></div>

<div><h4>Hepatic Cdkal1 deletion regulates HDL catabolism and promotes reverse cholesterol transport.</h4><i>An DB, Ann SJ, Seok S, Kang Y, Lee SH</i><br /><b>Background:</b><br/>and aims</b><br />Associations between CDKAL1 variants and cholesterol efflux capacity (CEC) have been reported. This study aimed to investigate the effects of Cdkal1 deficiency on high-density lipoprotein (HDL) metabolism, atherosclerosis, and related pathways.<br /><b>Methods</b><br />Lipid and glucose metabolic profiles, CEC, and in vivo reverse cholesterol transport (RCT) were compared in liver-specific Alb-Cre:Cdkal1<sup>fl/fl</sup> and Cdkal1<sup>fl/fl</sup> mice. Aortic atherosclerosis was compared in Apoe<sup>-/-</sup>Alb-Cre:Cdkal1<sup>fl/fl</sup> and Apoe<sup>-/-</sup> mice fed high-fat diets. HDL subclasses and mediators of HDL metabolism from Alb-Cre:Cdkal1<sup>fl/fl</sup> mice were examined.<br /><b>Results</b><br />HDL-cholesterol level tended to be higher in the Alb-Cre:Cdkal1<sup>fl/fl</sup> mice (p = 0.050). Glucose and other lipid profiles were similar in the two groups of mice, irrespective of diet. The mean CEC was 27% higher (p = 0.007) in the Alb-Cre:Cdkal1<sup>fl/fl</sup> mice, as were the radioactivities of bile acids (mean difference 17%; p = 0.035) and cholesterol (mean difference 42%; p = 0.036) from faeces. The radioactivity tendency was largely similar in mice fed a high-fat diet. Atherosclerotic lesion area tended to be smaller in the Apoe<sup>-/-</sup>Alb-Cre:Cdkal1<sup>fl/fl</sup> mice than in the Apoe<sup>-/-</sup> mice (p = 0.067). Cholesterol concentrations in large HDLs were higher in the Alb-Cre:Cdkal1<sup>fl/fl</sup> mice (p = 0.024), whereas in small HDLs, they were lower (p = 0.024). Endothelial lipase (mean difference 39%; p = 0.002) and hepatic lipase expression levels (mean difference 34%; p < 0.001) were reduced in the Alb-Cre:Cdkal1<sup>fl/fl</sup> mice, whereas SR-B1 expression was elevated (mean difference 35%; p = 0.007).<br /><b>Conclusions</b><br />The promotion of CEC and RCT in Alb-Cre:Cdkal1<sup>fl/fl</sup> mice verified the effect of CDKAL1 seen in human genetic data. These phenotypes were related to regulation of HDL catabolism. This study suggests that CDKAL1 and associated molecules could be targets for improving RCT and vascular pathology.<br /><br />Copyright © 2023 Elsevier B.V. All rights reserved.<br /><br /><small>Atherosclerosis: 22 May 2023; 375:21-29</small></div>

<div><h4>Novel mechanisms and therapeutic targets in atherosclerosis: inflammation and beyond.</h4><i>Weber C, Habenicht AJR, von Hundelshausen P</i><br /><AbstractText>This review based on the ESC William Harvey Lecture in Basic Science 2022 highlights recent experimental and translational progress on the therapeutic targeting of the inflammatory components in atherosclerosis, introducing novel strategies to limit side effects and to increase efficacy. Since the validation of the inflammatory paradigm in CANTOS and COLCOT, efforts to control the residual risk conferred by inflammation have centred on the NLRP3 inflammasome-driven IL-1β-IL6 axis. Interference with the co-stimulatory dyad CD40L-CD40 and selective targeting of tumour necrosis factor-receptor associated factors (TRAFs), namely the TRAF6-CD40 interaction in macrophages by small molecule inhibitors, harbour intriguing options to reduce established atherosclerosis and plaque instability without immune side effects. The chemokine system crucial for shaping immune cell recruitment and homoeostasis can be fine-tuned and modulated by its heterodimer interactome. Structure-function analysis enabled the design of cyclic, helical, or linked peptides specifically targeting or mimicking these interactions to limit atherosclerosis or thrombosis by blunting myeloid recruitment, boosting regulatory T cells, inhibiting platelet activity, or specifically blocking the atypical chemokine MIF without notable side effects. Finally, adventitial neuroimmune cardiovascular interfaces in advanced atherosclerosis show robust restructuring of innervation from perivascular ganglia and employ sensory neurons of dorsal root ganglia to enter the central nervous system and to establish an atherosclerosis-brain circuit sensor, while sympathetic and vagal efferents project to the celiac ganglion to create an atherosclerosis-brain circuit effector. Disrupting this circuitry by surgical or chemical sympathectomy limited disease progression and enhanced plaque stability, opening exciting perspectives for selective and tailored intervention beyond anti-inflammatory strategies.</AbstractText><br /><br />© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.<br /><br /><small>Eur Heart J: 20 May 2023; epub ahead of print</small></div>

<div><h4>Bestrophin3 Deficiency in Vascular Smooth Muscle Cells Activates MEKK2/3-MAPK Signaling to Trigger Spontaneous Aortic Dissection.</h4><i>Zhang TT, Lei QQ, He J, Guan X, ... Huang H, Zhou JG</i><br /><b>Background</b><br />Aortic dissection (AD) is a fatal cardiovascular disorder without effective medications due to unclear pathogenic mechanisms. Bestrophin3 (Best3), the predominant isoform of bestrophin family in vessels, has emerged as critical for vascular pathological processes. However, the contribution of Best3 to vascular diseases remains elusive.<br /><b>Methods</b><br />Smooth muscle cell-specific and endothelial cell-specific Best3 knockout mice (Best3<sup>SMKO</sup> and Best3<sup>ECKO</sup>, respectively) were engineered to investigate the role of Best3 in vascular pathophysiology. Functional studies, single-cell RNA sequencing, proteomics analysis, and coimmunoprecipitation coupled with mass spectrometry were performed to evaluate the function of Best3 in vessels.<br /><b>Results</b><br />Best3 expression in aortas of human AD samples and mouse AD models was decreased. Best3<sup>SMKO</sup> but not Best3<sup>ECKO</sup> mice spontaneously developed AD with age, and the incidence reached 48% at 72 weeks of age. Reanalysis of single-cell transcriptome data revealed that reduction of fibromyocytes, a fibroblast-like smooth muscle cell cluster, was a typical feature of human ascending AD and aneurysm. Consistently, Best3 deficiency in smooth muscle cells decreased the number of fibromyocytes. Mechanistically, Best3 interacted with both MEKK2 and MEKK3, and this interaction inhibited phosphorylation of MEKK2 at serine153 and MEKK3 at serine61. Best3 deficiency induced phosphorylation-dependent inhibition of ubiquitination and protein turnover of MEKK2/3, thereby activating the downstream mitogen-activated protein kinase signaling cascade. Furthermore, restoration of Best3 or inhibition of MEKK2/3 prevented AD progression in angiotensin II-infused Best3<sup>SMKO</sup> and ApoE<sup>-/-</sup> mice.<br /><b>Conclusions</b><br />These findings unveil a critical role of Best3 in regulating smooth muscle cell phenotypic switch and aortic structural integrity through controlling MEKK2/3 degradation. Best3-MEKK2/3 signaling represents a novel therapeutic target for AD.<br /><br /><br /><br /><small>Circulation: 19 May 2023; epub ahead of print</small></div>

<div><h4>Endothelial PHACTR1 Promotes Endothelial Activation and Atherosclerosis by Repressing PPARγ Activity Under Disturbed Flow in Mice.</h4><i>Jiang D, Liu H, Zhu G, Li X, ... Xu Y, Pang J</i><br /><b>Background</b><br />Numerous genome-wide association studies revealed that SNPs at the <i>PHACTR1</i> (phosphatase and actin regulator 1) locus strongly correlate with coronary artery disease. However, the biological function of PHACTR1 remains poorly understood. Here, we identified the proatherosclerotic effect of endothelial PHACTR1, contrary to macrophage PHACTR1.<br /><b>Methods</b><br />We generated global (<i>Phactr1</i><sup><i>-/</i></sup><sup><i>-</i></sup>) and endothelial cell (EC)-specific (<i>Phactr1</i><sup><i>ECKO</i></sup>) <i>Phactr1</i> KO (knockout) mice and crossed these mice with apolipoprotein E-deficient (<i>Apoe</i><sup><i>-/-</i></sup>) mice. Atherosclerosis was induced by feeding the high-fat/high-cholesterol diet for 12 weeks or partially ligating carotid arteries combined with a 2-week high-fat/high-cholesterol diet. PHACTR1 localization was identified by immunostaining of overexpressed PHACTR1 in human umbilical vein ECs exposed to different types of flow. The molecular function of endothelial PHACTR1 was explored by RNA sequencing using EC-enriched mRNA from global or EC-specific <i>Phactr1</i> KO mice. Endothelial activation was evaluated in human umbilical vein ECs transfected with siRNA targeting <i>PHACTR1</i> and in <i>Phactr1</i><sup><i>ECKO</i></sup> mice after partial carotid ligation.<br /><b>Results</b><br />Global or EC-specific <i>Phactr1</i> deficiency significantly inhibited atherosclerosis in regions of disturbed flow. PHACTR1 was enriched in ECs and located in the nucleus of disturbed flow areas but shuttled to cytoplasm under laminar flow in vitro. RNA sequencing showed that endothelial <i>Phactr1</i> depletion affected vascular function, and PPARγ (peroxisome proliferator-activated receptor gamma) was the top transcription factor regulating differentially expressed genes. PHACTR1 functioned as a PPARγ transcriptional corepressor by binding to PPARγ through the corepressor motifs. PPARγ activation protects against atherosclerosis by inhibiting endothelial activation. Consistently, <i>PHACTR1</i> deficiency remarkably reduced endothelial activation induced by disturbed flow in vivo and in vitro. PPARγ antagonist GW9662 abolished the protective effects of <i>Phactr1</i> KO on EC activation and atherosclerosis in vivo.<br /><b>Conclusions</b><br />Our results identified endothelial PHACTR1 as a novel PPARγ corepressor to promote atherosclerosis in disturbed flow regions. Endothelial PHACTR1 is a potential therapeutic target for atherosclerosis treatment.<br /><br /><br /><br /><small>Arterioscler Thromb Vasc Biol: 18 May 2023; epub ahead of print</small></div>

<div><h4>ROR2 (Receptor Tyrosine Kinase-Like Orphan Receptor 2)/Planar Cell Polarity a New Pathway Controlling Endothelial Cell Polarity Under Flow Conditions.</h4><i>Bougaran P, Bats ML, Delobel V, Rubin S, ... Duplàa C, Dufourcq P</i><br /><b>Background</b><br />Endothelial cells (ECs) are sensitive to physical forces created by blood flow, especially to laminar shear stress. Among the cell responses to laminar flow, EC polarization against the flow direction emerges as a key event, particularly during the development and remodeling of the vascular network. EC adopt an elongated planar cell shape with an asymmetrical distribution of intracellular organelles along the axis of blood flow. This study aimed to investigate the involvement of planar cell polarity via the receptor ROR2 (receptor tyrosine kinase-like orphan receptor 2) in endothelial responses to laminar shear stress.<br /><b>Methods</b><br />We generated a genetic mouse model with EC-specific deletion of <i>Ror2</i>, in combination with in vitro approaches involving loss- and gain-of-function experiments.<br /><b>Results</b><br />During the first 2 weeks of life, the endothelium of the mouse aorta undergoes a rapid remodeling associated with a loss of EC polarization against the flow direction. Notably, we found a correlation between ROR2 expression and endothelial polarization levels. Our findings demonstrate that deletion of <i>Ror2</i> in murine ECs impaired their polarization during the postnatal development of the aorta. in vitro experiments further validated the essential role of ROR2 in both EC collective polarization and directed migration under laminar flow conditions. Exposure to laminar shear stress triggered the relocalization of ROR2 to cell-cell junctions where it formed a complex with VE-Cadherin and β-catenin, thereby regulating adherens junctions remodeling at the rear and front poles of ECs. Finally, we showed that adherens junctions remodeling and cell polarity induced by ROR2 were dependent on the activation of the small GTPase Cdc42.<br /><b>Conclusions</b><br />This study identified ROR2/planar cell polarity pathway as a new mechanism controlling and coordinating collective polarity patterns of EC during shear stress response.<br /><br /><br /><br /><small>Arterioscler Thromb Vasc Biol: 18 May 2023; epub ahead of print</small></div>

<div><h4>Label-Free Characterization of Atherosclerotic Plaques Via High-Resolution Multispectral Fluorescence Lifetime Imaging Microscopy.</h4><i>Han J, Kim S, Jung Kim H, Soo Nam H, ... Kim JW, Yoo H</i><br /><b>Background</b><br />Autofluorescence lifetime (AFL) imaging, a robust technique that enables label-free molecular investigation of biological tissues, is being introduced into the field of cardiovascular diagnostics. However, detailed AFL characteristics of coronary arteries remain elusive and there is a lack of methodology enabling such characterization.<br /><b>Methods</b><br />We developed multispectral fluorescence lifetime imaging microscopy (FLIM) based on analog-mean-delay. Freshly sectioned coronary arteries and atheromas, harvested from 5 swine models, were imaged using FLIM and stained to label lipids, macrophages, collagen, and smooth muscle cells. The components were quantitated from digitized histological images and compared with the corresponding FLIM. Multispectral AFL parameters derived from 2 different spectral bands (390 nm and 450 nm) were analyzed.<br /><b>Results</b><br />FLIM provided a wide field-of-view, high-resolution AFL imaging of frozen sections. Principal compositions of coronary arteries, such as tunica media, tunica adventitia, elastic laminas, smooth muscle cell-enriched fibrous plaque, lipid-rich core, and foamy macrophages, were well visualized in FLIM images and were found to have each different AFL spectra. In particular, proatherogenic components including lipids and foamy macrophages exhibited significantly different AFL values compared with plaque-stabilizing collagen- or smooth muscle cell-enriched tissues (<i>P</i><0.0001). Pairwise comparisons showed that each composition was distinguishable from another by the difference in multispectral AFL parameters. Pixel-level analysis based on coregistered FLIM-histology dataset showed that each component of atherosclerosis (lipids, macrophages, collagen, and smooth muscle cells) had distinct correlation pattern with AFL parameters. Random forest regressors trained with the dataset allowed automated, simultaneous visualization of the key atherosclerotic components with high accuracy (r>0.87).<br /><b>Conclusions</b><br />FLIM provided detailed pixel-level AFL investigation of the complex composition of coronary artery and atheroma. Our FLIM strategy enabling an automated, comprehensive visualization of multiple plaque components from unlabeled sections will be highly useful to efficiently evaluate ex vivo samples without the need for histological staining and analysis.<br /><br /><br /><br /><small>Arterioscler Thromb Vasc Biol: 18 May 2023; epub ahead of print</small></div>

<div><h4> is a Novel Long Noncoding RNA Promoting Vascular Smooth Muscle Inflammation via Scaffolding MKL1 and USP10.</h4><i>Zhang W, Zhao J, Deng L, Ishimwe N, ... Baker AH, Long X</i><br /><b>Background</b><br />Activation of vascular smooth muscle cell (VSMC) inflammation is vital to initiate vascular disease. The role of human-specific long noncoding RNAs in VSMC inflammation is poorly understood.<br /><b>Methods</b><br />Bulk RNA sequencing in differentiated human VSMCs revealed a novel human-specific long noncoding RNA called inflammatory MKL1 (megakaryoblastic leukemia 1) interacting long noncoding RNA (<i>INKILN</i>). <i>INKILN</i> expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation as well as human atherosclerosis and abdominal aortic aneurysm. The transcriptional regulation of <i>INKILN</i> was verified through luciferase reporter and chromatin immunoprecipitation assays. Loss-of-function and gain-of-function studies and multiple RNA-protein and protein-protein interaction assays were used to uncover a mechanistic role of <i>INKILN</i> in the VSMC proinflammatory gene program. Bacterial artificial chromosome transgenic mice were used to study <i>INKIL</i><i>N</i> expression and function in ligation injury-induced neointimal formation.<br /><b>Results</b><br /><i>INKILN</i> expression is downregulated in contractile VSMCs and induced in human atherosclerosis and abdominal aortic aneurysm. <i>INKILN</i> is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB (nuclear factor kappa B) site within its proximal promoter. <i>INKILN</i> activates proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. <i>INKILN</i> physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. <i>INKILN</i> depletion blocks interleukin-1β-induced nuclear localization of both p65 and MKL1. Knockdown of <i>INKILN</i> abolishes the physical interaction between p65 and MKL1 and the luciferase activity of an NF-κB reporter. Furthermore, <i>INKILN</i> knockdown enhances MKL1 ubiquitination through reduced physical interaction with the deubiquitinating enzyme USP10 (ubiquitin-specific peptidase 10). <i>INKILN</i> is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in bacterial artificial chromosome transgenic mice.<br /><b>Conclusions</b><br />These findings elucidate an important pathway of VSMC inflammation involving an <i>INKILN</i>/MKL1/USP10 regulatory axis. Human bacterial artificial chromosome transgenic mice offer a novel and physiologically relevant approach for investigating human-specific long noncoding RNAs under vascular disease conditions.<br /><br /><br /><br /><small>Circulation: 18 May 2023; epub ahead of print</small></div>

<div><h4>PRMT5-mediated arginine methylation stabilizes KLF4 to accelerate neointimal formation.</h4><i>Liu H, Dong X, Jia K, Yuan B, ... Sun J, Pan LL</i><br /><b>Aims</b><br />Accumulating evidence supports an indispensable role of protein arginine methyltransferase 5 (PRMT5) in the pathological progression of several human cancers. As an important enzyme regulating protein methylation, how PRMT5 participates in vascular remodeling remains unknown. To investigate the role and underlying mechanism of PRMT5 in neointimal formation and to evaluate its potential as an effective therapeutic target for the condition.<br /><b>Methods and results</b><br />Aberrant PRMT5 overexpression was positively correlated with clinical carotid arterial stenosis. Vascular smooth muscle cells (SMC)-specific PRMT5 knockout inhibited intimal hyperplasia with enhanced expression of contractile markers in mice. Conversely, PRMT5 overexpression inhibited SMC contractile markers and promoted intimal hyperplasia. Furthermore, we showed that PRMT5 promoted SMC phenotypic switching through stabilizing Kruppel-like factor 4 (KLF4). Mechanistically, PRMT5-mediated KLF4 methylation inhibited ubiquitin-dependent proteolysis of KLF4, leading to a disruption of myocardin (MYOCD) -serum response factor (SRF) interaction and MYOCD-SRF-mediated the transcription of SMC contractile markers.<br /><b>Conclusions</b><br />Our data demonstrated that PRMT5 critically mediated vascular remodeling by promoting KLF4-mediated SMC phenotypic conversion and consequently the progression of intimal hyperplasia. Therefore, PRMT5 may represent a potential therapeutic target for intimal hyperplasia-associated vascular diseases.<br /><br />© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.<br /><br /><small>Cardiovasc Res: 18 May 2023; epub ahead of print</small></div>

<div><h4>Circadian PER1 Controls Daily Fat Absorption with the Regulation of PER1-PKA on Phosphorylation of Bile Acid Synthetase.</h4><i>Ge W, Sun Q, Yang Y, Ding Z, Liu J, Zhang J</i><br /><AbstractText>Several epidemiological studies suggest a correlation between eating time and obesity. Night-eating syndrome characterized by a time-delayed eating pattern is positively associated with obesity in humans as well as in experimental animals. Here we show that oil intake at night significantly makes more fat than that at day in wild type (WT) mice, and circadian Period 1 (Per1) contributes to this day-night difference. Per1-knockout mice are protected from high-fat diet (HFD)-induced obesity, which is accompanied by a reduction in the size of the bile acid pool, and the oral administration of bile acids restores fat absorption and accumulation. We identify that PER1 directly binds to the major hepatic enzymes involved in bile acid synthesis such as cholesterol 7alpha-hydroxylase (CYP7A1) and sterol 12alpha-hydroxylase (CYP8B1). A biosynthesis rhythm of bile acids is accompanied by the activity and instability of bile acid synthases with PER1/PKA-mediated phosphorylation pathways. Both fasting and high fat stress enhance Per1 expression, increasing the fat absorption and accumulation. Our findings reveal that Per1 is an energy regulator and controls daily fat absorption and accumulation.</AbstractText><br /><br />Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.<br /><br /><small>J Lipid Res: 18 May 2023:100390; epub ahead of print</small></div>

<div><h4>Retained Metabolic Flexibility of the Failing Human Heart.</h4><i>Watson WD, Green PG, Lewis AJM, Arvidsson P, ... Herring N, Rider OJ</i><br /><b>Background</b><br />The failing heart is traditionally described as metabolically inflexible and oxygen starved, causing energetic deficit and contractile dysfunction. Current metabolic modulator therapies aim to increase glucose oxidation by increasing oxygen efficiency of adenosine triphosphate production, with mixed results.<br /><b>Methods</b><br />To investigate metabolic flexibility and oxygen delivery in the failing heart, 20 patients with nonischemic heart failure with reduced ejection fraction (left ventricular ejection fraction 34.9±9.1) underwent separate infusions of insulin+glucose infusion (I+G) or Intralipid infusion. We used cardiovascular magnetic resonance to assess cardiac function and measured energetics using phosphorus-31 magnetic resonance spectroscopy. To investigate the effects of these infusions on cardiac substrate use, function, and myocardial oxygen uptake (MVo<sub>2</sub>), invasive arteriovenous sampling and pressure-volume loops were performed (n=9).<br /><b>Results</b><br />At rest, we found that the heart had considerable metabolic flexibility. During I+G, cardiac glucose uptake and oxidation were predominant (70±14% total energy substrate for adenosine triphosphate production versus 17±16% for Intralipid; <i>P</i>=0.002); however, no change in cardiac function was seen relative to basal conditions. In contrast, during Intralipid infusion, cardiac long-chain fatty acid (LCFA) delivery, uptake, LCFA acylcarnitine production, and fatty acid oxidation were all increased (LCFA 73±17% of total substrate versus 19±26% total during I+G; <i>P</i>=0.009).Myocardial energetics were better with Intralipid compared with I+G (phosphocreatine/adenosine triphosphate 1.86±0.25 versus 2.01±0.33; <i>P</i>=0.02), and systolic and diastolic function were improved (LVEF 34.9±9.1 baseline, 33.7±8.2 I+G, 39.9±9.3 Intralipid; <i>P</i><0.001). During increased cardiac workload, LCFA uptake and oxidation were again increased during both infusions. There was no evidence of systolic dysfunction or lactate efflux at 65% maximal heart rate, suggesting that a metabolic switch to fat did not cause clinically meaningful ischemic metabolism.<br /><b>Conclusions</b><br />Our findings show that even in nonischemic heart failure with reduced ejection fraction with severely impaired systolic function, significant cardiac metabolic flexibility is retained, including the ability to alter substrate use to match both arterial supply and changes in workload. Increasing LCFA uptake and oxidation is associated with improved myocardial energetics and contractility. Together, these findings challenge aspects of the rationale underlying existing metabolic therapies for heart failure and suggest that strategies promoting fatty acid oxidation may form the basis for future therapies.<br /><br /><br /><br /><small>Circulation: 18 May 2023; epub ahead of print</small></div>

<div><h4>Attenuation of ST-segment elevation by ischemic preconditioning: Reflection of cardioprotection in Göttingen but not in Ossabaw minipigs.</h4><i>Lieder HR, Adam V, Skyschally A, Sturek M, Kleinbongard P, Heusch G</i><br /><b>Background</b><br />Ischemic preconditioning (IPC; brief cycles of coronary occlusion/ reperfusion) reduces myocardial infarct size. The ST-segment elevation during coronary occlusion is progressively attenuated with increasing number of IPC cycles. Progressive attenuation of ST-segment elevation is considered a result of sarcolemmal K<sub>ATP</sub> channel activation and has been considered to reflect and predict IPC\'s cardioprotection. We have recently demonstrated that IPC failed to reduce infarct size in minipigs of a particular strain (Ossabaw), which had a genetic predisposition to develop, but not yet established a metabolic syndrome. To determine whether or not Ossabaw minipigs nevertheless had attenuated ST-segment elevation over repetitive IPC cycles, we compared Göttingen vs. Ossabaw minipigs in which IPC reduces infarct size.<br /><b>Methods and results</b><br />We analyzed surface chest electrocardiographic (ECG) recordings of anesthetized open-chest contemporary Göttingen (n = 43) and Ossabaw minipigs (n = 53). Both minipig strains were subjected to 60 min coronary occlusion and 180 min reperfusion without or with IPC (3 × 5 min/ 10 min coronary occlusion/ reperfusion). ST-segment elevations during the repetitive coronary occlusions were analyzed. In both minipig strains, IPC attenuated ST-segment elevation with increasing number of coronary occlusions. IPC reduced infarct size in Göttingen minipigs (45 ± 10% without vs. 25 ± 13% of area at risk with IPC), whereas such cardioprotection was absent in Ossabaw minipigs (54 ± 11% vs. 50 ± 11%).<br /><b>Conclusion</b><br />Apparently, the block of signal transduction of IPC in Ossabaw minipigs occurs distal to the sarcolemma, where K<sub>ATP</sub> channel activation still attenuates ST-segment elevation as it does in Göttingen minipigs.<br /><br />Copyright © 2023. Published by Elsevier B.V.<br /><br /><small>Int J Cardiol: 17 May 2023; epub ahead of print</small></div>

<div><h4>Takotsubo syndrome: getting closer to its causes.</h4><i>Akhtar MM, Cammann VL, Templin C, Ghadri JR, Lüscher TF</i><br /><AbstractText>Takotsubo syndrome (TTS) accounts for between 1 and 4% of cases presenting clinically as an acute coronary syndrome. It typically presents as a transient cardiac phenotype of left ventricular dysfunction with spontaneous recovery. More dramatic presentations may include cardiogenic shock or cardiac arrest. Despite progress in the understanding of the condition since its first description in 1990, considerable questions remain into understanding underlying pathomechanisms. In this review article, we describe the current published data on potential underlying mechanisms associated with the onset of TTS including sympathetic nervous system over-stimulation, structural and functional alterations in the central nervous system, catecholamine secretion, alterations in the balance and distribution of adrenergic receptors, the additive impact of hormones including oestrogen, epicardial coronary or microvascular spasm, endothelial dysfunction, and genetics as potentially contributing to the cascade of events leading to the onset. These pathomechanisms provide suggestions for novel potential therapeutic strategies in patients with TTS including the role of cognitive behavioural therapy, beta-blockers, and endothelin-A antagonists. The underlying mechanism of TTS remains elusive. In reality, physical or emotional stressors likely trigger through the amygdala and hippocampus a central neurohumoral activation with the local and systemic secretion of excess catecholamine and other neurohormones, which exert its effect on the myocardium through a metabolic switch, altered cellular signalling, and endothelial dysfunction. These complex pathways exert a regional activation in the myocardium through the altered distribution of adrenoceptors and density of autonomic innervation as a protective mechanism from myocardial apoptosis. More research is needed to understand how these different complex mechanisms interact with each other to bring on the TTS phenotype.</AbstractText><br /><br />© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.<br /><br /><small>Cardiovasc Res: 15 May 2023; epub ahead of print</small></div>

<div><h4>Human antigen R regulates autophagic flux by stabilizing autophagy-associated mRNA in calcific aortic valve disease.</h4><i>Fang J, Qian Y, Chen J, Xu D, ... Wang J, Liu X</i><br /><b>Aims</b><br />The incidence of calcific aortic valve disease (CAVD) has risen over the last decade and is expected to continue rising; however, pharmacological approaches have proven ineffective. In this study, we evaluated the role and underlying mechanisms of human antigen R (HuR)-mediated post-transcriptional regulation in CAVD.<br /><b>Methods and results</b><br />We found that HuR was significantly upregulated in human calcified aortic valves and primary aortic valvular interstitial cells (VICs) following osteogenic stimulation. Subsequent functional studies revealed that HuR silencing ameliorated calcification both in vitro and in vivo. For the first time, we demonstrated that HuR directly interacted with the transcript of phosphatidylinositol-5-phosphate 4-kinase, type II, alpha (PIP4K2A), which mediates phosphatidylinositol signaling, facilitates autophagy, and act as an mRNA stabilizer. HuR positively modulated PIP4K2A expression at the post-transcriptional level, and consequently influenced the AKT/mTOR/ATG13 pathway to regulate autophagy and CAVD progression.<br /><b>Conclusion</b><br />Our study provides new insights into the post-transcriptional regulatory role of HuR in modulating autophagy-positive factors to regulate the pathogenesis of CAVD. Our findings highlight the potential of HuR as an innovative therapeutic target in CAVD treatment.<br /><b>Translational perspective</b><br />As the incidence of CAVD has been rising over the past decade with only invasive, expensive, and risky interventions available, it is imperative to explore novel approaches to halt aortic valve calcification. Currently, regulation of CAVD pathogenesis is not fully understood. HuR, an RNA-binding protein, and autophagy are thought to play key roles in CAVD; however, the precise underlying mechanisms have not been elucidated. Therefore, we believe that the findings of this study are relevant to the field because they provide direct evidence of a critical role for HuR, hVIC phenotypic transition, and autophagy in CAVD and suggest a novel target for hindering CAVD progression.<br /><br />© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.<br /><br /><small>Cardiovasc Res: 15 May 2023; epub ahead of print</small></div>

<div><h4>Prediction of Kv11.1 potassium channel PAS-domain variants trafficking via machine learning.</h4><i>Fang X, Immadisetty K, Ramon GS, Hartle CM, ... Delisle BP, Kekenes-Huskey PM</i><br /><AbstractText>Congenital long QT syndrome (LQTS) is characterized by a prolonged QT-interval on an electrocardiogram (ECG). An abnormal prolongation in the QT-interval increases the risk for fatal arrhythmias. Genetic variants in several different cardiac ion channel genes, including KCNH2, are known to cause LQTS. Here, we evaluated whether structure-based molecular dynamics (MD) simulations and machine learning (ML) could improve the identification of missense variants in LQTS-linked genes. To do this, we investigated KCNH2 missense variants in the Kv11.1 channel protein shown to have wild type (WT) like or class II (trafficking-deficient) phenotypes in vitro. We focused on KCNH2 missense variants that disrupt normal Kv11.1 channel protein trafficking, as it is the most common phenotype for LQTS-associated variants. Specifically, we used computational techniques to correlate structural and dynamic changes in the Kv11.1 channel protein PAS domain (PASD) with Kv11.1 channel protein trafficking phenotypes. These simulations unveiled several molecular features, including the numbers of hydrating waters and hydrogen bonding pairs, as well as folding free energy scores, that are predictive of trafficking. We then used statistical and machine learning (ML) (Decision tree (DT), Random forest (RF), and Support vector machine (SVM)) techniques to classify variants using these simulation-derived features. Together with bioinformatics data, such as sequence conservation and folding energies, we were able to predict with reasonable accuracy (≈75%) which KCNH2 variants do not traffic normally. We conclude that structure-based simulations of KCNH2 variants localized to the Kv11.1 channel PASD led to an improvement in classification accuracy. Therefore, this approach should be considered to complement the classification of variant of unknown significance (VUS) in the Kv11.1 channel PASD.</AbstractText><br /><br />Copyright © 2023. Published by Elsevier Ltd.<br /><br /><small>J Mol Cell Cardiol: 13 May 2023; epub ahead of print</small></div>

<div><h4>A Post-Pandemic Enigma: The Cardiovascular Impact of Post-Acute Sequelae of SARS-CoV-2.</h4><i>Singh TK, Zidar DA, McCrae K, Highland KB, ... Cameron SJ, Chung MK</i><br /><AbstractText>COVID-19 has become the first modern-day pandemic of historic proportion, affecting >600 million individuals worldwide and causing >6.5 million deaths. While acute infection has had devastating consequences, postacute sequelae of SARS-CoV-2 infection appears to be a pandemic of its own, impacting up to one-third of survivors and often causing symptoms suggestive of cardiovascular phenomena. This review will highlight the suspected pathophysiology of postacute sequelae of SARS-CoV-2, its influence on the cardiovascular system, and potential treatment strategies.</AbstractText><br /><br /><br /><br /><small>Circ Res: 12 May 2023; 132:1358-1373</small></div>

<div><h4>Platelets and SARS-CoV-2 During COVID-19: Immunity, Thrombosis, and Beyond.</h4><i>Sciaudone A, Corkrey H, Humphries F, Koupenova M</i><br /><AbstractText>COVID-19 is characterized by dysregulated thrombosis and coagulation that can increase mortality in patients. Platelets are fast responders to pathogen presence, alerting the surrounding immune cells and contributing to thrombosis and intravascular coagulation. The SARS-CoV-2 genome has been found in platelets from patients with COVID-19, and its coverage varies according to the method of detection, suggesting direct interaction of the virus with these cells. Antibodies against Spike and Nucleocapsid have confirmed this platelet-viral interaction. This review discusses the immune, prothrombotic, and procoagulant characteristics of platelets observed in patients with COVID-19. We outline the direct and indirect interaction of platelets with SARS-CoV-2, the contribution of the virus to programmed cell death pathway activation in platelets and the consequent extracellular vesicle release. We discuss platelet activation and immunothrombosis in patients with COVID-19, the effect of Spike on platelets, and possible activation of platelets by classical platelet activation triggers as well as contribution of platelets to complement activation. As COVID-19-mediated thrombosis and coagulation are still not well understood in vivo, we discuss available murine models and mouse adaptable strains.</AbstractText><br /><br /><br /><br /><small>Circ Res: 12 May 2023; 132:1272-1289</small></div>

<div><h4>Renal medullary overexpression of sphingosine-1-phosphate receptor 1 transgene attenuates deoxycorticosterone acetate (DOCA)-salt hypertension.</h4><i>Hu G, Xie D, Chen C, Wang W, ... Ritter JK, Li N</i><br /><b>Objective</b><br />Our previous studies showed that renal medullary sphingosine-1-phosphate receptor 1 (S1PR1) mediated sodium excretion, high salt intake increased S1PR1 level, deoxycorticosterone acetate (DOCA) blocked high salt-induced S1PR1 in the renal medulla, and that conditional knockout of S1PR1 in the collecting duct aggravated DOCA-salt hypertension. The present study tested the hypothesis that overexpression of S1PR1 transgene in the renal medulla attenuates the sodium retention and hypertension in DOCA-salt mouse model.<br /><b>Methods</b><br />Male C57BL/6J mice received renal medullary transfection of control or S1PR1-expressing plasmids and then DOCA-salt treatment. Renal sodium excretion and arterial pressure were compared between control and S1PR1-overexpressed mice in response to high salt loading or pressure natriuresis.<br /><b>Results</b><br />S1PR1-transfected mice showed significantly enhanced urinary sodium excretion in response to acute sodium loading (0.93±0.27 in control vs. 4.72±1.12 µmol/min/gKW in S1PR1-overexpressed mice, p<0.05) and the pressure natriuresis (3.58±1.77 vs. 9.52±1.38, p<0.05), less positive sodium balance in response to chronic high-salt intake (3.05±0.39 vs. 1.65±0.39mmol/72hr, p<0.05), and consequently, the attenuation of DOCA-salt hypertension (134.2±6.79 vs. 109.8±3.54 mmHg, p<0.05). The αENaC protein amount in the renal medulla was not changed, however, the βENaC was significantly decreased and the γENaC was significantly increased in S1PR1-overexpressed mice. The immunostaining showed apical membrane translocation of γENaC, while no change of αENaC and βENaC in control mice, and that the apical membrane translocation of γENaC was blocked in S1PR1-treasffected mice.<br /><b>Conclusion</b><br />These results suggested that activation of S1PR1 in the renal medulla attenuates DOCA-induced sodium retention and salt-sensitive hypertension associated with inhibition of ENaC.<br /><br />© The Author(s) 2023. Published by Oxford University Press on behalf of American Journal of Hypertension, Ltd. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.<br /><br /><small>Am J Hypertens: 12 May 2023; epub ahead of print</small></div>

<div><h4>R-spondin 3/LGR4 (Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 4) Axis Is a Novel Inflammatory and Neurite Outgrowth Signaling System in the Ischemic Brain in Mice.</h4><i>Shimamura M, Hayashi H, Ju N, Yoshida S, ... Morishita R, Nakagami H</i><br /><b>Background</b><br />Although stimulation of Wnt/β-catenin signaling is an important strategy to treat ischemic stroke, its signaling pathway has not been fully clarified yet. Recently, RSPO3 (R-spondin 3)/LGR4 (leucine-rich repeat-containing G protein-coupled receptor 4) signaling has resolved TLR4 (toll-like receptor 4)-induced inflammation in lung injury; however, whether this signal is critical in the ischemic brain remains unknown. Therefore, we investigated the role of RSPO3/LGR4 signaling in the ischemic brain.<br /><b>Methods</b><br />BALB/c mice were exposed to permanent distal middle cerebral artery and common carotid artery occlusion. Temporal RSPO3 and LGR4 expressions were examined, and the mice were randomly assigned to receive vehicle or recombinant RSPO3. The underlying mechanisms were investigated using microglial cell lines and primary mixed glia-endothelia-neuron and primary neuronal cultures.<br /><b>Results</b><br />In the ischemic brain, RSPO3 and LGR4 were expressed in endothelial cells and microglia/macrophages and neurons, respectively. Stimulation of RSPO3/LGR4 signaling by recombinant RSPO3 recovered neurological deficits with decreased <i>Il1β</i> and <i>iNOS</i> mRNA on day 3 and increased <i>Gap43</i> on day 9. In cultured cells, LGR4 was expressed in neuron and microglia, whereas RSPO3 promoted nuclear translocation of β-catenin. Neuroprotective effects with reduced expression of inflammatory cytokines were observed in lipopolysaccharide-stimulated glia-endothelium-neuron cultures but not in glutamate-, CoCl<sub>2</sub>-, H<sub>2</sub>O<sub>2</sub>-, or oxygen glucose deprivation-stimulated neuronal cultures, indicating that RSPO3/LGR4 can protect neurons by regulating inflammatory cytokines. LGR4-Fc chimera, which was used to block endogenous RSPO3/LGR4 signaling, increased LPS-induced production of inflammatory cytokines, suggesting that endogenous RSPO3 suppresses inflammation. RSPO3 decreased TLR4-related inflammatory cytokine expression by decreasing TLR4 expression without affecting the M1/M2 phenotype. RSPO3 also inhibited TLR2- and TLR9-induced inflammation but not TLR7-induced inflammation, and promoted neurite outgrowth.<br /><b>Conclusions</b><br />RSPO3/LGR4 signaling plays a critical role in regulating TLR-induced inflammation and neurite outgrowth in the ischemic brain. Enhancing this signal will be a promising approach for treating ischemic stroke.<br /><br /><br /><br /><small>Stroke: 11 May 2023; epub ahead of print</small></div>

<div><h4>Impairment of Angiogenesis-Driven Clot Resolution Is a Key Event in the Progression to Chronic Thromboembolic Pulmonary Hypertension: Validation in a Novel Rabbit Model.</h4><i>Quarck R, Willems L, Tielemans B, Stoian L, ... Jacquemin M, Delcroix M</i><br /><b>Background</b><br />Chronic thromboembolic pulmonary hypertension (CTEPH) is a life-threatening condition and rare complication of acute pulmonary embolism. Mechanisms underlying impaired clot resolution and in sustained fibrothrombotic obstruction of the pulmonary arterial bed remain poorly understood. Since defective angiogenesis correlated to defective clot resolution based on observations in surgical material from patients with CTEPH, we aimed to validate its crucial pathogenic role by intrathrombus inhibition of angiogenesis in a novel CTEPH rabbit model.<br /><b>Methods</b><br />We aimed to compare whether intrathrombus administration of an antifibrinolytic agent, tranexamic acid, or an inhibitor of angiogenesis, SU5416, would contribute to CTEPH progression. Both products were administered on a weekly basis by autologous clot embolization in rabbits. Right ventricular pressure was monitored by telemetry, right ventricular function by transthoracic echocardiography, and a complete pulmonary hemodynamic evaluation was obtained through right heart catheterization. Markers of inflammation, endothelial dysfunction, heart failure, and fibrinolysis were measured in plasma. Pulmonary vessel remodeling was analyzed by immunohistochemistry.<br /><b>Results</b><br />Impairing intrathrombus angiogenesis by repeatedly embolizing autologous blood clots containing SU5416 resulted in elevated mean pulmonary arterial pressure (38 mm Hg), increased indexed pulmonary vascular resistance, and enhanced right ventricular hypertrophy (80%, 1.9-fold, 36%, respectively, compared with rabbits embolized with clots containing an antifibrinolytic agent). This was caused by both obstruction of large pulmonary arteries with fibrothrombotic material and muscularization of pulmonary microvessels, and accompanied by inflammatory cell infiltration and increased circulating endothelin-1.<br /><b>Conclusions</b><br />The key role of angiogenesis-driven clot resolution was validated in a reliable small-animal model reproducing the major pathophysiological hallmarks of CTEPH.<br /><br /><br /><br /><small>Arterioscler Thromb Vasc Biol: 11 May 2023; epub ahead of print</small></div>

<div><h4>New Loss-of-Function Mutations in PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) Reduce Plasma LDL (Low-Density Lipoprotein) Cholesterol.</h4><i>Meng FH, Liu S, Xiao J, Zhou YX, ... Fu ZY, Luo J</i><br /><b>Background</b><br />Lower plasma levels of LDL (low-density lipoprotein) cholesterol (LDL-C) can reduce the risk of atherosclerotic cardiovascular disease. The loss-of-function mutations in <i>PCSK9</i> (proprotein convertase subtilisin/kexin type 9) have been known to associate with low LDL-C in many human populations. PCSK9 genetic variants in Chinese Uyghurs who are at high risk of atherosclerotic cardiovascular disease due to their dietary habits have not been reported.<br /><b>Methods</b><br />The study involved the whole-exome and target sequencing of college students from Uyghur and other ethnic groups in Xinjiang, China, for the association of PCSK9 loss-of-function mutations with low plasma levels of LDL-C. The mechanisms by which the identified mutations affect the function of PCSK9 were investigated in cultured cells using biochemical and cell assays. The causal effects of the identified PCSK9 mutations on LDL-C levels were verified in mice injected with adeno-associated virus expressing different forms of PCSK9 and fed a high-cholesterol diet.<br /><b>Results</b><br />We identified 2 PCSK9 mutations-E144K and C378W-in Chinese Uyghurs with low plasma levels of LDL-C. The E144K and C378W mutations impaired the maturation and secretion of the PCSK9 protein, respectively. Adeno-associated virus-mediated expression of E144K and C378W mutants in <i>Pcsk9</i> KO (knockout) mice fed a high-cholesterol diet also hampered PCSK9 secretion into the serum, resulting in elevated levels of LDL receptor in the liver and reduced levels of LDL-C in the serum.<br /><b>Conclusions</b><br />Our study shows that E144K and C378W are PCSK9 loss-of-function mutations causing low LDL-C levels in mice and probably in humans as well.<br /><br /><br /><br /><small>Arterioscler Thromb Vasc Biol: 11 May 2023; epub ahead of print</small></div>

<div><h4>Rhodiola wallichiana var.cholaensis protects against myocardial ischemia-reperfusion injury by attenuating oxidative stress-mediated apoptosis via enhancing Nrf2 signaling.</h4><i>Yan T, Li X, Wang X, Zhang Y, ... Jia Y, Xiao W</i><br /><AbstractText>The present study aimed to explore the cardioprotective effects of Rhodiola wallichiana var.cholaensis (RW) against hypoxia/reoxygenation (H/R)-induced H9c2 cell injury and ischemia/reperfusion (I/R)-induced myocardial injury. Following treatment with RW, H9c2 cells were subjected to 4 h of hypoxia/3 h of reoxygenation. MTT assay, LDH assay, and flow cytometry were employed to detect cell viability and changes of ROS and mitochondrial membrane potential. Moreover, after RW treatment, rats underwent 30 min of ischemia, followed by 120 min of reperfusion. Masson and TUNEL staining were performed to measure myocardial damage and apoptosis, respectively. The changes in the levels of proteins were detected by ELISA and western blot. The results showed that RW attenuated the H/R-induced increase in LDH release and loss of the mitochondrial membrane potential, as well as the apoptosis in H9c2 cells. Meanwhile, RW significantly reduces the ST-segment elevation and improves cardiomyocytes\' injury, inhibit the apoptosis induced by I/R in rats. Furthermore, RW could decrease the levels of MDA and increase the levels of SOD, T-AOC. GSH-Px and GSH both in vivo and in vitro. Besides, RW increased the expressions of Nrf2, HO-1, ARE and NQO1, and decreased the expressions of Keap1, activating the Nrf2 signaling pathway. Taken together, these results suggested that RW exerts cardioprotection on H/R injury in H9c2 cells and I/R injury in rats by attenuating oxidative stress-mediated apoptosis via enhancing Nrf2 signaling.</AbstractText><br /><br />Copyright © 2023. Published by Elsevier B.V.<br /><br /><small>Int J Cardiol: 11 May 2023; epub ahead of print</small></div>

<div><h4>Exercise-induced endothelial Mecp2 lactylation suppresses atherosclerosis via the Ereg/MAPK signalling pathway.</h4><i>Wang Y, Chen L, Zhang M, Li X, ... Wu J, Yu B</i><br /><b>Background:</b><br/>and aims</b><br />Lactylation, a recently identified post-translational modification (PTM), plays a central role in the regulation of multiple physiological and pathological processes. Exercise is known to provide protection against cardiovascular disease. However, whether exercise-generated lactate changes lactylation and is involved in the exercise-induced attenuation of atherosclerotic cardiovascular disease (ASCVD) remains unclear. The purpose of this study was to investigate the effects and mechanisms of exercise-induced lactylation on ASCVD.<br /><b>Methods and results</b><br />Using the high-fat diet-induced apolipoprotein-deficient mouse model of ASCVD, we found that exercise training promoted Mecp2 lysine lactylation (Mecp2k271la); it also decreased the expression of vascular cell adhesion molecule 1 (Vcam-1), intercellular adhesion molecule 1 (Icam-1), monocyte chemoattractant protein 1 (Mcp-1), interleukin (IL)-1β, IL-6, and increased the level of endothelial nitric oxide synthase (Enos) in the aortic tissue of mice. To explore the underlying mechanisms, mouse aortic endothelial cells (MAECs) were subjected to RNA-sequencing and CHIP-qPCR, which confirmed that Mecp2k271la repressed the expression of epiregulin (Ereg) by binding to its chromatin, demonstrating Ereg as a key downstream molecule for Mecp2k271la. Furthermore, Ereg altered the mitogen-activated protein kinase (MAPK) signalling pathway through regulating the phosphorylation level of epidermal growth factor receptor, thereby affecting the expression of Vcam-1, Icam-1, Mcp-1, IL-1β, IL-6, and Enos in ECs, which in turn promoted the regression of atherosclerosis. In addition, increasing the level of Mecp2k271la by exogenous lactate administration in vivo also inhibits the expression of Ereg and the MAPK activity in ECs, resulting in repressed atherosclerotic progression.<br /><b>Conclusions</b><br />In summary, this study provides a mechanistic link between exercise and lactylation modification, offering new insight into the anti-atherosclerotic effects of exercise-induced PTM.<br /><br />Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.<br /><br /><small>Atherosclerosis: 11 May 2023; 375:45-58</small></div>

<div><h4>Monocyte Heterogeneity in Cardiovascular Disease.</h4><i>Ruder AV, Wetzels SMW, Temmerman L, Biessen EAL, Goossens P</i><br /><AbstractText>Monocytes circulate the vasculature at steady state and are recruited to sites of inflammation where they differentiate into macrophages (MФ) to replenish tissue-resident MФ populations and engage in the development of cardiovascular disease (CVD). Monocytes display considerable heterogeneity, currently reflected by a nomenclature based on their expression of cluster of differentiation (CD) 14 and CD16, distinguishing CD14++CD16- classical (cMo), CD14++CD16+ intermediate (intMo) and CD14 + CD16++ non-classical (ncMo) monocytes. Several reports point to shifted subset distributions in the context of CVD, with significant association of intMo numbers with atherosclerosis, myocardial infarction, and heart failure. However, clear indications of their causal involvement as well as their predictive value for CVD are lacking. As recent high-parameter cytometry and single-cell RNA sequencing (scRNA-Seq) studies suggest an even higher degree of heterogeneity, better understanding of the functionalities of these subsets is pivotal. Considering their high heterogeneity, surprisingly little is known about functional differences between MФ originating from monocytes belonging to different subsets, and implications thereof for CVD pathogenesis. This paper provides an overview of recent findings on monocyte heterogeneity in the context of homeostasis and disease as well as functional differences between the subsets and their potential to differentiate into MФ, focusing on their role in vessels and the heart. The emerging paradigm of monocyte heterogeneity transcending the current tripartite subset division argues for an updated nomenclature and functional studies to substantiate marker-based subdivision and to clarify subset-specific implications for CVD.</AbstractText><br /><br />© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.<br /><br /><small>Cardiovasc Res: 10 May 2023; epub ahead of print</small></div>

<div><h4>Nedd4-2 upregulation is associated with ACE2 ubiquitination in hypertension.</h4><i>Mohammed M, Ogunlade B, Elgazzaz M, Berdasco C, ... Lazartigues E, Filipeanu CM</i><br /><b>Background</b><br />ACE2 is a critical component of the compensatory renin-angiotensin system that is downregulated during the development of hypertension, possibly via ubiquitination. However, little is known about the mechanisms involved in ACE2 ubiquitination in neurogenic hypertension. This study aimed at identifying ACE2 ubiquitination partners, establish causal relationships, clinical relevance and test a gene therapy strategy to mitigate ACE2 ubiquitination in neurogenic hypertension.<br /><b>Methods and results</b><br />Bioinformatics and proteomics were combined to identify E3 ubiquitin ligases associated with ACE2 ubiquitination in chronically hypertensive mice. In vitro gain/loss of function experiments assessed ACE2 expression and activity to validate the interaction between ACE2 and the identified E3 ligase. Mutation experiments were further used to generate a ubiquitination-resistant ACE2 mutant (ACE2-5R). Optogenetics, blood pressure telemetry, pharmacological blockade of GABAA receptors in mice expressing ACE2-5R in the bed nucleus of the stria terminalis (BNST) and capillary Western were used to assess the role of ACE2 ubiquitination in neurogenic hypertension.Ubiquitination was first validated as leading to ACE2 downregulation and Nedd4-2 identified as a E3 ligase up-regulated in hypertension and promoting ACE2 ubiquitination. Mutation of lysine residues in the C-terminal of ACE2 was associated with increased activity and resistance to Ang-II-mediated degradation. Mice transfected with ACE2-5R in the BNST exhibited enhanced GABAergic input to the paraventricular nucleus (PVN) and reduction of hypertension. ACE2-5R expression was associated with reduced Nedd4-2 levels in the BNST.<br /><b>Conclusions</b><br />Our data identify Nedd4-2 as the first E3 ubiquitin ligase involved in ACE2 ubiquitination in Ang-II-mediated hypertension. We demonstrate the pivotal role of ACE2 on GABAergic neurons in the maintenance of a tonic inhibitory tone to the PVN, and the regulation of pre-sympathetic activity. These findings provide a new working model where Nedd4-2 could contribute to ACE2 ubiquitination leading to the development of neurogenic hypertension and highlight potential novel therapeutic strategies.<br /><b>Translational perspective</b><br />While ACE2 conversion of Ang-II to Ang-(1-7) is supposed to limit the overactivity of the renin-angiotensin system (RAS), the enzyme is downregulated during the development of hypertension. As antihypertensive RAS blockers on the market only provide limited control of BP among hypertensive patients, understanding the mechanisms responsible for this blunted compensation provides new possible targets for the treatment of hypertension. In this study we show that Nedd4-2 upregulation is associated with ACE2 ubiquitination while prevention of this post-translational modification prevents the development of hypertension. Accordingly, targeting of ACE2 ubiquitination provides a new treatment strategy to reduce hypertension.<br /><br />Published by Oxford University Press on behalf of the European Society of Cardiology 2023.<br /><br /><small>Cardiovasc Res: 10 May 2023; epub ahead of print</small></div>

<div><h4>Endothelial mechanobiology in atherosclerosis.</h4><i>Wang X, Shen Y, Shang M, Liu X, Munn LL</i><br /><AbstractText>Cardiovascular disease (CVD) is a serious health challenge, causing more deaths world-wide than cancer. The vascular endothelium, which forms the inner lining of blood vessels, plays a central role in maintaining vascular integrity and homeostasis and is in direct contact with the blood flow. Research over the past century has shown that mechanical perturbations of the vascular wall contribute to formation and progression of atherosclerosis. While the straight part of the artery is exposed to sustained laminar flow and physiological high shear stress, flow near branch points or in curved vessels can exhibit \"disturbed\" flow. Clinical studies as well as carefully controlled in vitro analyses have confirmed that these regions of disturbed flow, which can include low shear stress, recirculation, oscillation, or lateral flow, are preferential sites of atherosclerotic lesion formation. Because of their critical role in blood flow homeostasis, vascular endothelial cells (ECs) have mechanosensory mechanisms that allow them to react rapidly to changes in mechanical forces, and to execute context-specific adaptive responses to modulate EC functions. This review summarizes the current understanding of endothelial mechanobiology, which can guide the identification of new therapeutic targets to slow or reverse the progression of atherosclerosis.</AbstractText><br /><br />© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.<br /><br /><small>Cardiovasc Res: 10 May 2023; epub ahead of print</small></div>

<div><h4>Sympathetic tone dictates the impact of lipolysis on FABP4 secretion.</h4><i>Prentice KJ, Lee A, Cedillo P, Inouye KE, ... Lee GY, Hotamisligil GS</i><br /><AbstractText>Levels of circulating FABP4 protein are strongly associated with obesity and metabolic disease in both mice and humans, and secretion is stimulated by ß-adrenergic stimulation both in vivo and in vitro. Previously, lipolysis-induced FABP4 secretion was found to be significantly reduced upon pharmacological inhibition of adipose triglyceride lipase (ATGL), and was absent from adipose tissue explants from mice specifically lacking ATGL in their adipocytes (ATGL<sup>AdpKO</sup>). Here we find that upon activation of ß-adrenergic receptors in vivo, ATGL<sup>AdpKO</sup> mice unexpectedly exhibited significantly higher levels of circulating FABP4 as compared to ATGL<sup>fl/fl</sup> controls, despite no corresponding induction of lipolysis. We generated an additional model with adipocyte-specific deletion of both FABP4 and ATGL (ATGL/FABP4<sup>AdpKO</sup>) to evaluate the cellular source of this circulating FABP4. In these animals, there was no evidence of lipolysis-induced FABP4 secretion, indicating that the source of elevated FABP4 levels in ATGL<sup>AdpKO</sup> mice was indeed from the adipocytes. ATGL<sup>AdpKO</sup> mice exhibited significantly elevated corticosterone levels, which positively correlated with plasma FABP4 levels. Pharmacological inhibition of sympathetic signaling during lipolysis using hexamethonium, or housing mice at thermoneutrality to chronically reduce sympathetic tone significantly reduced FABP4 secretion in ATGL<sup>AdpKO</sup> mice compared to controls. Therefore, activity of a key enzymatic step of lipolysis mediated by ATGL, per se, is not required for in vivo stimulation of FABP4 secretion from adipocytes, which can be induced through sympathetic signaling.</AbstractText><br /><br />Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.<br /><br /><small>J Lipid Res: 10 May 2023:100386; epub ahead of print</small></div>

<div><h4>NLRP3 inflammasome-driven IL-1β and IL-18 contribute to lipopolysaccharide-induced septic cardiomyopathy.</h4><i>Fujimura K, Karasawa T, Komada T, Yamada N, ... Kario K, Takahashi M</i><br /><AbstractText>Sepsis is a life-threatening syndrome, and its associated mortality is increased when cardiac dysfunction and damage (septic cardiomyopathy [SCM]) occur. Although inflammation is involved in the pathophysiology of SCM, the mechanism of how inflammation induces SCM in vivo has remained obscure. NLRP3 inflammasome is a critical component of the innate immune system that activates caspase-1 (Casp1) and causes the maturation of IL-1β and IL-18 as well as the processing of gasdermin D (GSDMD). Here, we investigated the role of the NLRP3 inflammasome in a murine model of lipopolysaccharide (LPS)-induced SCM. LPS injection induced cardiac dysfunction, damage, and lethality, which was significantly prevented in NLRP3<sup>-/-</sup> mice, compared to wild-type (WT) mice. LPS injection upregulated mRNA levels of inflammatory cytokines (Il6, Tnfa, and Ifng) in the heart, liver, and spleen of WT mice, and this upregulation was prevented in NLRP3<sup>-/-</sup> mice. LPS injection increased plasma levels of inflammatory cytokines (IL-1β, IL-18, and TNF-α) in WT mice, and this increase was markedly inhibited in NLRP3<sup>-/-</sup> mice. LPS-induced SCM was also prevented in Casp1/11<sup>-/-</sup> mice, but not in Casp11<sup>mt</sup>, IL-1β<sup>-/-</sup>, IL-1α<sup>-/-</sup>, or GSDMD<sup>-/-</sup> mice. Notably, LPS-induced SCM was apparently prevented in IL-1β<sup>-/-</sup> mice transduced with adeno-associated virus vector expressing IL-18 binding protein (IL-18BP). Furthermore, splenectomy, irradiation, or macrophage depletion alleviated LPS-induced SCM. Our findings demonstrate that the cross-regulation of NLRP3 inflammasome-driven IL-1β and IL-18 contributes to the pathophysiology of SCM and provide new insights into the mechanism underlying the pathogenesis of SCM.</AbstractText><br /><br />Copyright © 2023. Published by Elsevier Ltd.<br /><br /><small>J Mol Cell Cardiol: 10 May 2023; epub ahead of print</small></div>

<div><h4>Polycomb Group Protein CBX7 Represses Cardiomyocyte Proliferation Through Modulation of the TARDBP/RBM38 Axis.</h4><i>Cho KW, Andrade M, Bae S, Kim S, ... Park C, Yoon YS</i><br /><b>Background</b><br />Shortly after birth, cardiomyocytes exit the cell cycle and cease proliferation. At present, the regulatory mechanisms for this loss of proliferative capacity are poorly understood. CBX7 (chromobox 7), a polycomb group (PcG) protein, regulates the cell cycle, but its role in cardiomyocyte proliferation is unknown.<br /><b>Methods</b><br />We profiled CBX7 expression in the mouse hearts through quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. We overexpressed CBX7 in neonatal mouse cardiomyocytes through adenoviral transduction. We knocked down CBX7 by using constitutive and inducible conditional knockout mice (<i>Tnnt2-Cre;Cbx7</i><sup><i>fl/+</i></sup> and <i>Myh6-MCM;Cbx7</i><sup><i>fl/fl</i></sup>, respectively). We measured cardiomyocyte proliferation by immunostaining of proliferation markers such as Ki67, phospho-histone 3, and cyclin B1. To examine the role of CBX7 in cardiac regeneration, we used neonatal cardiac apical resection and adult myocardial infarction models. We examined the mechanism of CBX7-mediated repression of cardiomyocyte proliferation through coimmunoprecipitation, mass spectrometry, and other molecular techniques.<br /><b>Result</b><br />We explored <i>Cbx7</i> expression in the heart and found that mRNA expression abruptly increased after birth and was sustained throughout adulthood. Overexpression of CBX7 through adenoviral transduction reduced proliferation of neonatal cardiomyocytes and promoted their multinucleation. On the other hand, genetic inactivation of <i>Cbx7</i> increased proliferation of cardiomyocytes and impeded cardiac maturation during postnatal heart growth. Genetic ablation of <i>Cbx7</i> promoted regeneration of neonatal and adult injured hearts. Mechanistically, CBX7 interacted with TARDBP (TAR DNA-binding protein 43) and positively regulated its downstream target, RBM38 (RNA Binding Motif Protein 38), in a TARDBP-dependent manner. Overexpression of RBM38 inhibited the proliferation of CBX7-depleted neonatal cardiomyocytes.<br /><b>Conclusions</b><br />Our results demonstrate that CBX7 directs the cell cycle exit of cardiomyocytes during the postnatal period by regulating its downstream targets TARDBP and RBM38. This is the first study to demonstrate the role of CBX7 in regulation of cardiomyocyte proliferation, and CBX7 could be an important target for cardiac regeneration.<br /><br /><br /><br /><small>Circulation: 09 May 2023; epub ahead of print</small></div>

<div><h4>Downregulation of FKBP5 Promotes Atrial Arrhythmogenesis.</h4><i>Wang X, Song J, Yuan Y, Li L, ... Dobrev D, Li N</i><br /><b>Background</b><br />Atrial fibrillation (AF), the most common arrhythmia, is associated with the downregulation of <i>FKBP5</i> (encoding FKBP5 [FK506 binding protein 5]). However, the function of FKBP5 in the heart remains unknown. Here, we elucidate the consequences of cardiomyocyte-restricted loss of FKBP5 on cardiac function and AF development and study the underlying mechanisms.<br /><b>Methods</b><br />Right atrial samples from patients with AF were used to assess the protein levels of FKBP5. A cardiomyocyte-specific FKBP5 knockdown mouse model was established by crossbreeding <i>Fkbp5</i><sup><i>flox/flox</i></sup> mice with <i>Myh6</i><sup><i>MerCreMer/+</i></sup> mice. Cardiac function and AF inducibility were assessed by echocardiography and programmed intracardiac stimulation. Histology, optical mapping, cellular electrophysiology, and biochemistry were employed to elucidate the proarrhythmic mechanisms due to loss of cardiomyocyte FKBP5.<br /><b>Results</b><br />FKBP5 protein levels were lower in the atrial lysates of patients with paroxysmal AF or long-lasting persistent chronic AF. Cardiomyocyte-specific knockdown mice exhibited increased AF inducibility and duration compared with control mice. Enhanced AF susceptibility in cardiomyocyte-specific knockdown mice was associated with the development of action potential alternans and spontaneous Ca<sup>2+</sup> waves, and increased protein levels and activity of the NCX1 (Na<sup>+</sup>/Ca<sup>2+</sup>-exchanger 1), mimicking the cellular phenotype of chronic AF patients. FKBP5-deficiency enhanced transcription of <i>Slc8a1</i> (encoding NCX1) via transcription factor hypoxia-inducible factor 1α. In vitro studies revealed that FKBP5 negatively modulated the protein levels of hypoxia-inducible factor 1α by competitively interacting with heat-shock protein 90. Injections of the heat-shock protein 90 inhibitor 17-AAG normalized protein levels of hypoxia-inducible factor 1α and NCX1 and reduced AF susceptibility in cardiomyocyte-specific knockdown mice. Furthermore, the atrial cardiomyocyte-selective knockdown of FKBP5 was sufficient to enhance AF arrhythmogenesis.<br /><b>Conclusions</b><br />This is the first study to demonstrate a role for the FKBP5-deficiency in atrial arrhythmogenesis and to establish FKBP5 as a negative regulator of hypoxia-inducible factor 1α in cardiomyocytes. Our results identify a potential molecular mechanism for the proarrhythmic NCX1 upregulation in chronic AF patients.<br /><br /><br /><br /><small>Circ Res: 08 May 2023; epub ahead of print</small></div>

<div><h4>Transcriptomic and Proteomic of Gastrocnemius Muscle in Peripheral Artery Disease.</h4><i>Ferrucci L, Candia J, Ubaida-Mohien C, Lyaskov A, ... Peterson CA, McDermott MM</i><br /><b>Background</b><br />Few effective therapies exist to improve lower extremity muscle pathology and mobility loss due to peripheral artery disease (PAD), in part because mechanisms associated with functional impairment remain unclear.<br /><b>Methods</b><br />To better understand mechanisms of muscle impairment in PAD, we performed in-depth transcriptomic and proteomic analyses on gastrocnemius muscle biopsies from 31 PAD participants (mean age, 69.9 years) and 29 age- and sex-matched non-PAD controls (mean age, 70.0 years) free of diabetes or limb-threatening ischemia.<br /><b>Results</b><br />Transcriptomic and proteomic analyses suggested activation of hypoxia-compensatory mechanisms in PAD muscle, including inflammation, fibrosis, apoptosis, angiogenesis, unfolded protein response, and nerve and muscle repair. Stoichiometric proportions of mitochondrial respiratory proteins were aberrant in PAD compared to non-PAD, suggesting that respiratory proteins not in complete functional units are not removed by mitophagy, likely contributing to abnormal mitochondrial activity. Supporting this hypothesis, greater mitochondrial respiratory protein abundance was significantly associated with greater complex II and complex IV respiratory activity in non-PAD but not in PAD. Rate-limiting glycolytic enzymes, such as hexokinase and pyruvate kinase, were less abundant in muscle of people with PAD compared with non-PAD participants, suggesting diminished glucose metabolism.<br /><b>Conclusions</b><br />In PAD muscle, hypoxia induces accumulation of mitochondria respiratory proteins, reduced activity of rate-limiting glycolytic enzymes, and an enhanced integrated stress response that modulates protein translation. These mechanisms may serve as targets for disease modification.<br /><br /><br /><br /><small>Circ Res: 08 May 2023; epub ahead of print</small></div>

<div><h4>Critical Role of the cGAS-STING Pathway in Doxorubicin-Induced Cardiotoxicity.</h4><i>Luo W, Zou X, Wang Y, Dong Z, ... Sun A, Ge J</i><br /><b>Background</b><br />Doxorubicin is an effective chemotherapy drug for treating various types of cancer. However, lethal cardiotoxicity severely limits its clinical use. Recent evidence has indicated that aberrant activation of the cytosolic DNA-sensing cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-STING (stimulator of interferon genes) pathway plays a critical role in cardiovascular destruction. Here, we investigate the involvement of this mechanism in doxorubicin-induced cardiotoxicity (DIC).<br /><b>Methods</b><br />Mice were treated with low-dose doxorubicin to induce chronic DIC. The role of the cGAS-STING pathway in DIC was evaluated in <i>cGAS</i>-deficiency (c<i>GAS</i><sup>-/-</sup>), <i>Sting</i> deficiency (<i>Sting</i><sup>-/-</sup>), and interferon regulatory factor 3 (<i>Irf3</i>)-deficiency (<i>Irf3</i><sup>-/-</sup>) mice. Endothelial cell (EC)-specific conditional <i>Sting</i> deficiency (<i>Sting</i><sup>flox/flox</sup>/Cdh5-Cre<sup>ERT</sup>) mice were used to assess the importance of this pathway in ECs during DIC. We also examined the direct effects of the cGAS-STING pathway on nicotinamide adenine dinucleotide (NAD) homeostasis in vitro and in vivo.<br /><b>Results</b><br />In the chronic DIC model, we observed significant activation of the cGAS-STING pathway in cardiac ECs. Global <i>cGAS</i>, <i>Sting,</i> and <i>Irf3</i> deficiency all markedly ameliorated DIC. EC-specific <i>Sting</i> deficiency significantly prevented DIC and endothelial dysfunction. Mechanistically, doxorubicin activated the cardiac EC cGAS-STING pathway and its target, IRF3, which directly induced CD38 expression. In cardiac ECs, the cGAS-STING pathway caused a reduction in NAD levels and subsequent mitochondrial dysfunction via the intracellular NAD glycohydrolase (NADase) activity of CD38. Furthermore, the cardiac EC cGAS-STING pathway also regulates NAD homeostasis and mitochondrial bioenergetics in cardiomyocytes through the ecto-NADase activity of CD38. We also demonstrated that pharmacological inhibition of TANK-binding kinase 1 or CD38 effectively ameliorated DIC without compromising the anticancer effects of doxorubicin.<br /><b>Conclusions</b><br />Our findings indicate a critical role of the cardiac EC cGAS-STING pathway in DIC. The cGAS-STING pathway may represent a novel therapeutic target for preventing DIC.<br /><br /><br /><br /><small>Circ Res: 08 May 2023; epub ahead of print</small></div>

<div><h4>Down-regulation of WWP2 aggravates Type 2 diabetes mellitus-induced vascular endothelial injury through modulating ubiquitination and degradation of DDX3X.</h4><i>You S, Xu J, Yin Z, Wu B, ... Sun Y, Zhang N</i><br /><b>Background</b><br />Endothelial injury caused by Type 2 diabetes mellitus (T2DM) is considered as a mainstay in the pathophysiology of diabetic vascular complications (DVCs). However, the molecular mechanism of T2DM-induced endothelial injury remains largely unknown. Here, we found that endothelial WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) act as a novel regulator for T2DM-induced vascular endothelial injury through modulating ubiquitination and degradation of DEAD-box helicase 3 X-linked (DDX3X).<br /><b>Methods</b><br />Single-cell transcriptome analysis was used to evaluate WWP2 expression in vascular endothelial cells of T2DM patients and healthy controls. Endothelial-specific Wwp2 knockout mice were used to investigate the effect of WWP2 on T2DM-induced vascular endothelial injury. In vitro loss- and gain-of-function studies were performed to assess the function of WWP2 on cell proliferation and apoptosis of human umbilical vein endothelial cells. The substrate protein of WWP2 was verified using mass spectrometry, coimmunoprecipitation assays and immunofluorescence assays. The mechanism of WWP2 regulation on substrate protein was investigated by pulse-chase assay and ubiquitination assay.<br /><b>Results</b><br />The expression of WWP2 was significantly down-regulated in vascular endothelial cells during T2DM. Endothelial-specific Wwp2 knockout in mice significantly aggravated T2DM-induced vascular endothelial injury and vascular remodeling after endothelial injury. Our in vitro experiments showed that WWP2 protected against endothelial injury by promoting cell proliferation and inhibiting apoptosis in ECs. Mechanically, we found that WWP2 is down-regulated in high glucose and palmitic acid (HG/PA)-induced ECs due to c-Jun N-terminal kinase (JNK) activation, and uncovered that WWP2 suppresses HG/PA-induced endothelial injury by catalyzing K63-linked polyubiquitination of DDX3X and targeting it for proteasomal degradation.<br /><b>Conclusion</b><br />Our studies revealed the key role of endothelial WWP2 and the fundamental importance of the JNK-WWP2-DDX3X regulatory axis in T2DM-induced vascular endothelial injury, suggesting that WWP2 may serve as a new therapeutic target for DVCs.<br /><br />© 2023. The Author(s).<br /><br /><small>Cardiovasc Diabetol: 06 May 2023; 22:107</small></div>

<div><h4>Role of Dickkopf-3 in Blood Pressure Regulation in Mice and Hypertensive Rats.</h4><i>Busceti CL, Carrizzo A, Bianchi F, De Lucia M, ... Nicoletti F, Vecchione C</i><br /><b>Background</b><br />Dkk3 (Dickkopf-3) is a secreted glycoprotein known for its proapoptotic and angiogenic activity. The role of Dkk3 in cardiovascular homeostasis is largely unknown. Remarkably, the Dkk3 gene maps within a chromosome segment linked to the hypertensive phenotype in spontaneously hypertensive rats (SHR).<br /><b>Methods</b><br />We used Dkk3<sup>-/-</sup> mice or stroke-resistant (sr) and stroke-prone (sp) SHR to examine the role of Dkk3 in the central and peripheral regulation of blood pressure (BP). We used lentiviral expression vector to rescue Dkk3 in knockout mice or to induce Dkk3 overexpression or silencing in SHR.<br /><b>Results</b><br />Genetic deletion of Dkk3 in mice enhanced BP and impaired endothelium-dependent acetylcholine-induced relaxation of resistance arteries. These alterations were rescued by restoring Dkk3 expression either in the periphery or in the CNS. Dkk3 was required for the constitutive expression of VEGF (vascular endothelium growth factor), and the action of Dkk3 on BP and endothelium-dependent vasorelaxation was mediated by VEGF-stimulated phosphatidylinositol-3-kinase pathway, leading to eNOS (endothelial NO synthase) activation both in resistance arteries and the CNS. The regulatory function of Dkk3 on BP was confirmed in SHR stroke-resistant and SHR stroke-prone in which was blunted in both resistance arteries and brainstem. In SHR stroke-resistant, lentiviral expression vector-induced Dkk3 expression in the CNS largely reduced BP, whereas Dkk3 knock-down further enhanced BP. In SHR stroke-prone challenged with a hypersodic diet, lentiviral expression vector-induced Dkk3 expression in the CNS displayed a substantial antihypertensive effect and delayed the occurrence of stroke.<br /><b>Conclusions</b><br />These findings demonstrate that Dkk3 acts as peripheral and central regulator of BP by promoting VEGF expression and activating a VEGF/Akt/eNOS hypotensive axis.<br /><br /><br /><br /><small>Circ Res: 05 May 2023; epub ahead of print</small></div>

<div><h4>Mechanism of Tumor-Platelet Communications in Cancer.</h4><i>Dudiki T, Veleeparambil M, Zhevlakova I, Biswas S, ... Podrez EA, Byzova TV</i><br /><b>Background</b><br />Thrombosis is one of the main complications in cancer patients often leading to mortality. However, the mechanisms underlying platelet hyperactivation are poorly understood.<br /><b>Methods and results</b><br />Murine and human platelets were isolated and treated with small extracellular vesicles (sEVs) from various cancer cell lines. We demonstrate that platelets very effectively take up sEVs from aggressive prostate cancer cells. The process of uptake is fast, proceeds effectively in circulation in mice, and is mediated by the abundant sEV-membrane protein-CD63. The uptake of cancer-sEVs leads to the accumulation of cancer cell-specific RNA in platelets in vitro and in vivo. The human prostate cancer-sEV-specific RNA marker PCA3 is detected in platelets of ~70% of prostate cancer patients. This was markedly reduced after prostatectomy. In vitro studies showed that platelet uptake of cancer-sEVs induces strong platelet activation in a CD63-RPTPα (receptor-like protein tyrosine phosphatase alpha)-dependent manner. In contrast to physiological agonists ADP and thrombin, sEVs activate platelets via a noncanonical mechanism dependent upon active translation. Intravital studies demonstrated accelerated thrombosis both in murine tumor models and in mice that received intravenous injections of cancer-sEVs. The prothrombotic effects of sEVs were rescued by blocking CD63.<br /><b>Conclusions</b><br />Tumors communicate with platelets by means of sEVs, which deliver cancer markers and activate platelets in a CD63-dependent manner leading to thrombosis. This emphasizes the diagnostic and prognostic value of platelet-associated cancer markers and identifies new pathways for intervention.<br /><br /><br /><br /><small>Circ Res: 05 May 2023; epub ahead of print</small></div>

<div><h4>Oxidized LDL receptors: a recent update.</h4><i>Khan MA, Mohammad I, Banerjee S, Tomar A, ... Chandele A, Arockiasamy A</i><br /><b>Purpose of review</b><br />LDL in its oxidized form, or \'oxLDL\', is now generally acknowledged to be highly proatherogenic and to play a significant role in atherosclerotic plaque formation. Therefore, there has been increasing interest in understanding the significance of oxLDL and its receptors in different phases of atherosclerosis, leading to the accumulation of additional data at the cellular, structural, and physiological levels. This review focuses on the most recent discoveries about these receptors and how they influence lipid absorption, metabolism, and inflammation in various cell types.<br /><b>Recent findings</b><br />Two crystal structures of lectin-like oxLDL receptor-1 (LOX-1), one with a small molecule inhibitor and the other with a monoclonal antibody have been published. We recently demonstrated that the \'surface site\' of LOX1, adjacent to the positively charged \'basic spine region\' that facilitates oxLDL binding, is a targetable site for drug development. Further, recent human studies showed that soluble LOX-1 holds potential as a biomarker for cardiovascular disease diagnosis, prognosis, and assessing the efficacy of therapy.<br /><b>Summary</b><br />Receptor-mediated oxLDL uptake results in cellular dysfunction of various cell types involved in atherogenesis and plaque development. The current advancements clearly demonstrate that targeting oxLDL-LOX-1 axis may lead to development of future therapeutics for the treatment of atherosclerotic cardiovascular and cerebrovascular diseases.<br /><br />Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.<br /><br /><small>Curr Opin Lipidol: 05 May 2023; epub ahead of print</small></div>

<div><h4>BIN1, Myotubularin, and Dynamin-2 Coordinate T-Tubule Growth in Cardiomyocytes.</h4><i>Perdreau-Dahl H, Lipsett DB, Frisk M, Kermani F, ... Morth JP, Louch WE</i><br /><b>Background</b><br />Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca<sup>2+</sup> homeostasis. The membrane bending and scaffolding protein BIN1 (amphiphysin-2) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3\'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission.<br /><b>Methods</b><br />We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca<sup>2+</sup> release was recorded using Fluo-4.<br /><b>Results</b><br />We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca<sup>2+</sup> channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca<sup>2+</sup> release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms.<br /><b>Conclusions</b><br />These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.<br /><br /><br /><br /><small>Circ Res: 04 May 2023; epub ahead of print</small></div>

<div><h4>Super Enhancer-Associated Circular RNA-CircKrt4 Regulates Hypoxic Pulmonary Artery Endothelial Cell Dysfunction in Mice.</h4><i>Ma C, Wang X, Zhang L, Zhu X, ... Qu L, Zhu D</i><br /><b>Background</b><br />Circular RNAs (circRNAs) have been implicated in pulmonary hypertension progression through largely unknown mechanisms. Pulmonary artery endothelial cell (PAEC) dysfunction is a hallmark in the pathogenesis of pulmonary hypertension. However, the specific role of circular RNAs in PAEC injury caused by hypoxia remains unclear.<br /><b>Methods</b><br />In this study, using the Western blotting, RNA pull down, Dual-luciferase reporter assay, immunohistochemistry, and immunofluorescence, we identified a novel circular RNA derived from alternative splicing of the keratin 4 gene (circKrt4).<br /><b>Results</b><br />CircKrt4 was upregulated in lung tissues and plasma and specifically in PAECs under hypoxic conditions. In the nucleus, circKrt4 induces endothelial-to-mesenchymal transition by interacting with the transcriptional activator protein Pura (Pur-alpha) to promote N-cadherin gene activation. In the cytoplasm, increased circKrt4 leads to mitochondrial dysfunction by inhibiting cytoplasmic-mitochondrial shuttling of mitochondrial-bound Glpk (glycerol kinase). Intriguingly, circKrt4 was identified as a super enhancer-associated circular RNA that is transcriptionally activated by a transcription factor, CEBPA (CCAAT enhancer binding protein alpha). Furthermore, RBM25 (RNA-binding-motif protein 25) was found to regulate circKrt4 cyclization by increase the back-splicing of <i>Krt4</i> gene.<br /><b>Conclusions</b><br />These findings demonstrate that a super enhancer-associated circular RNA-circKrt4 modulates PAEC injury to promote pulmonary hypertension by targeting Pura and Glpk.<br /><br /><br /><br /><small>Arterioscler Thromb Vasc Biol: 04 May 2023; epub ahead of print</small></div>

<div><h4>An interferon gamma response signature links myocardial aging and immunosenescence.</h4><i>Ashour D, Rebs S, Arampatzi P, Saliba AE, ... Streckfuß-Bömeke K, Ramos GC</i><br /><b>Aims</b><br />Aging entails profound immunological transformations that can impact myocardial homeostasis and predispose to heart failure. However, preclinical research in the immune-cardiology field is mostly conducted in young healthy animals, which potentially weakens its translational relevance. Herein, we sought to investigate how the aging T-cell compartment associates with changes in myocardial cell biology in aged mice.<br /><b>Methods and results</b><br />We phenotyped the antigen-experienced effector/memory T cells purified from heart-draining lymph nodes of 2-, 6-, 12-, and 18-month-old C57BL/6J mice using single-cell RNA/T cell receptor (TCR) sequencing (sc-seq). Simultaneously, we profiled all non-cardiomyocyte cell subsets purified from 2- and 18-month-old hearts and integrated our data with publicly available cardiomyocyte sc-seq datasets. Some of these findings were confirmed at the protein level by flow cytometry. With aging, the heart-draining lymph node and myocardial T cells underwent clonal expansion and exhibited an up-regulated pro-inflammatory transcription signature, marked by an increased interferon-γ (IFN-γ) production. In parallel, all major myocardial cell populations showed increased IFN-γ responsive signature with aging. In the aged cardiomyocytes, a stronger IFN-γ response signature was paralleled by the dampening of expression levels of transcripts related to most metabolic pathways, especially oxidative phosphorylation. Likewise, induced pluripotent stem cells-derived cardiomyocytes (iPSC-CM) exposed to chronic, low grade IFN-γ treatment showed a similar inhibition of metabolic activity.<br /><b>Conclusions</b><br />By investigating the paired age-related alterations in the T cells found in the heart and its draining lymph nodes, we provide evidence for increased myocardial IFN-γ signaling with age, which is associated with inflammatory and metabolic shifts typically seen in heart failure.<br /><br />© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.<br /><br /><small>Cardiovasc Res: 04 May 2023; epub ahead of print</small></div>

<div><h4>Adjustment of acquisition arc in cardiac malposition during myocardial perfusion SPECT imaging: computer simulation based on deterministic modeling.</h4><i>Qutbi M</i><br /><b>Objectives</b><br />To simulate cardiac malpositions, leftward and rightward shift and dextrocardia, and also to compare distribution of activity of septal and lateral walls of left ventricle acquired in standard acquisition arc and after relevant adjustment.<br /><b>Methods</b><br />In this study, digital phantoms with cardiac malpositions are designed and procedure of acquisition of scan in standard arc (from right anterior oblique to left posterior oblique) and adjusted acquisition arc is simulated. The three situations of malposition including leftward and rightward shift and dextrocardia are considered. For all types, acquisition is conducted in standard and then adjusted arcs (from anterior to posterior and also from right to left for leftward and rightward shifts, respectively, and for dextrocardia, from left anterior oblique to right posterior oblique). All obtained projections are reconstructed using the algorithm of filtered back projection. During forward projection to obtain sinograms, radiation attenuation is also modeled by incorporation of a simplified transmission map to emission map. The resulting tomographic slices of the LV (septum, apex, and lateral wall) are presented visually and are compared by plotting intensity profiles of the walls. Finally, normalized error images are also computed. All the computations are performed in MATLAB software package.<br /><b>Results</b><br />In transverse slice, septum and lateral wall are attenuated progressively from apex, which is closer to the camera, to the base in similar fashion. In tomographic slices of standard acquisition arc, the septum shows remarkably higher activity compared to lateral wall. However, after adjustment, both seems equally intense and progressively being attenuated from apex to base, similar to that found in phantom with normally positioned heart. Likewise, for the phantom with rightward shift, when the scanning was done in standard arc, the septum is more intense than the lateral wall. And similarly, adjustment of the arc renders both walls equally intense. In dextrocardia, level of attenuation of basal parts of septum and lateral wall is higher in 360° arc compared to adjusted 180° arc.<br /><b>Conclusion</b><br />Adjustment of acquisition arc exerts perceptible changes in distribution of activity over LV walls which are more compatible with normally positioned heart.<br /><br />© 2023. The Author(s) under exclusive licence to American Society of Nuclear Cardiology.<br /><br /><small>J Nucl Cardiol: 04 May 2023; epub ahead of print</small></div>

<div><h4>Cardiac protein kinase D1 ablation alters the myocytes β-adrenergic response.</h4><i>Hernandez JM, Ko CY, Mandel AR, Shen EY, ... Bossuyt J, Bers DM</i><br /><AbstractText>β-adrenergic (β-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca<sup>2+</sup>/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca<sup>2+</sup> sparks, contraction and L-type Ca<sup>2+</sup> current in paced cardiomyocytes under acute β-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca<sup>2+</sup> load was assessed by rapid caffeine (10 mM) induced Ca<sup>2+</sup> release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca<sup>2+</sup> ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca<sup>2+</sup> spark frequency, SR Ca<sup>2+</sup> load, L-type Ca<sup>2+</sup> current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca<sup>2+</sup>]<sub>i</sub> decline, lower Ca<sup>2+</sup> spark rate and lower RyR phosphorylation, but with similar SR Ca<sup>2+</sup> load, L-type Ca<sup>2+</sup> current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte β-adrenergic responsiveness by allowing optimal enhancement in SR Ca<sup>2+</sup> uptake and RyR sensitivity, but not altering L-type Ca<sup>2+</sup> current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal β-adrenergic responses in Ca<sup>2+</sup> handling.</AbstractText><br /><br />Copyright © 2023. Published by Elsevier Ltd.<br /><br /><small>J Mol Cell Cardiol: 04 May 2023; epub ahead of print</small></div>