Journal: Circ Res

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Abstract

Cardiac Resident Macrophages Prevent Fibrosis and Stimulate Angiogenesis.

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




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

Myofilament Phosphorylation in Stem Cell Treated Diastolic Heart Failure.

Soetkamp D, Gallet R, Parker SJ, Holewinski R, ... Marban E, Van Eyk JE
Rationale: Phosphorylation of sarcomeric proteins has been implicated in heart failure with preserved ejection fraction (HFpEF); such changes may contribute to diastolic dysfunction by altering contractility, cardiac stiffness, Ca2+-sensitivity and mechanosensing. Treatment with cardiosphere-derived cells (CDCs) restores normal diastolic function, attenuates fibrosis and inflammation, and improves survival in a rat HFpEF model.Objective: Phosphorylation changes that underlie HFpEF and those reversed by CDC therapy, with a focus on the sarcomeric subproteome were analyzed.
Methods and results:
Dahl salt-sensitive rats fed a high-salt diet, with echocardiographically-verified diastolic dysfunction, were randomly assigned to either intracoronary CDCs or placebo. Dahl salt-sensitive rats receiving low salt diet served as controls. Protein, and phosphorylated Ser, Thr and Tyr residues from left ventricular tissue, were quantified by mass spectrometry. HFpEF hearts exhibited extensive hyperphosphorylation with 98% of the 529 significantly changed phospho-sites increased compared to control. Of those 39% were located within the sarcomeric subproteome, with a large group of proteins located or associated with the Z-disk. CDC treatment partially reverted the hyperphosphorylation, with 85% of the significantly altered 76 residues hypophosphorylated. Bioinformatic upstream analysis of the differentially phosphorylated protein residues revealed PKC as the dominant putative regulatory kinase. PKC isoform analysis indicated increases in PKC α, β and δ concentration, whereas CDC treatment led to a reversion of PKCβ. Use of PKC isoform specific inhibition and overexpression of various PKC isoforms strongly suggests PKCβ is the dominant kinase involved in hyperphosphorylation in HFpEF and is altered with CDC treatment. Conclusions: Increased protein phosphorylation at the Z-disk is associated with diastolic dysfunction, with PKC isoforms driving most quantified phosphorylation changes. Because CDCs reverse the key abnormalities in HFpEF and selectively reverse PKCβ upregulation, PKCβ merits being classified as a potential therapeutic target in HFpEF, a disease notoriously refractory to medical intervention.




Circ Res: 12 Oct 2021; epub ahead of print
Soetkamp D, Gallet R, Parker SJ, Holewinski R, ... Marban E, Van Eyk JE
Circ Res: 12 Oct 2021; epub ahead of print | PMID: 34641704
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Abstract

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

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




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

ANKRD36 Is Involved in Hypertension by Altering ENaC Genes Expression.

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




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

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

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




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

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

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




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

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

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




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

PCSK9 Activity Is Potentiated Through HDL Binding.

Burnap SA, Sattler K, Pechlaner R, Duregotti E, ... Levkau B, Mayr M
Rationale: Proprotein convertase subtilisin/kexin type 9 (PCSK9) circulates in a free and lipoprotein-bound form, yet the functional consequence of the association between PCSK9 and high-density lipoprotein (HDL) remains unexplored. Objective: This study sought to interrogate the novel relationship between PCSK9 and HDL in humans.
Methods and results:
Comparing lipoprotein and apolipoprotein profiles by nuclear magnetic resonance and targeted mass spectrometry measurements with PCSK9 levels in the community-based Bruneck (n=656) study revealed a positive association of plasma PCSK9 with small HDL, alongside a highly significant positive correlation between plasma levels of PCSK9 and apolipoprotein-C3, an inhibitor of lipoprotein lipase. The latter association was replicated in an independent cohort, the SAPHIR study (n=270). Thus, PCSK9-HDL association was determined during the postprandial response in two dietary studies (n=20 participants each, 8 times points). Peak triglyceride levels coincided with an attenuation of the PCSK9-HDL association, a loss of apolipoprotein-C3 from HDL and lower levels of small HDL as measured by nuclear magnetic resonance. Crosslinking mass spectrometry (XLMS) upon isolated HDL identified PCSK9 as a potential HDL-binding partner. PCSK9 association with HDL was confirmed through size-exclusion chromatography and immuno-isolation. Quantitative proteomics upon HDL isolated from patients with coronary artery disease (n=172) returned PCSK9 as a core member of the HDL proteome. Combined interrogation of the HDL proteome and lipidome revealed a distinct cluster of PCSK9, phospholipid transfer protein, clusterin and apolipoprotein-E within the HDL proteome, that was altered by sex and positively correlated with sphingomyelin content. Mechanistically, HDL facilitated PCSK9-mediated low-density lipoprotein receptor degradation and reduced low-density lipoprotein uptake through the modulation of PCSK9 internalisation and multimerisation. Conclusions: This study reports HDL as a binder of PCSK9 and regulator of its function. The combination of -omic technologies revealed postprandial lipaemia as a driver of PCSK9 and apolipoprotein-C3 release from HDL.




Circ Res: 03 Oct 2021; epub ahead of print
Burnap SA, Sattler K, Pechlaner R, Duregotti E, ... Levkau B, Mayr M
Circ Res: 03 Oct 2021; epub ahead of print | PMID: 34601896
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Abstract

Growth Arrest Specific-6 and Axl Coordinate Inflammation and Hypertension.

Van Beusecum JP, Ruggeri Barbaro N, Smart CD, Patrick DM, ... Shibao CA, Harrison DG
Rationale: There is an intimate relationship between the endothelium and monocytes, and activated endothelial cells promote monocyte transformation to macrophages and dendritic cells (DCs). Recently, a subset of human DCs expressing the receptor tyrosine kinase, Axl and the lectin Siglec-6 has been described and termed AS DCs. Objective: We sought to determine if circulating AS DCs are increased in human hypertension and to examine how Axl signaling contributes to this disease.
Methods and results:
We demonstrated that circulating AS DCs are increased in hypertensive humans compared to normotensive controls. Pulse pressure in humans also correlated with plasma levels of the Axl agonist growth arrest specific 6 (GAS6). Exposure of human endothelial cells to 10% cyclical stretch increased release of the GAS6, promoted Axl signaling and caused AS DC formation; events that were inhibited by blockade of Axl with R428 or by siRNA knockdown of either endothelial GAS6 or Axl. GAS6/Axl signaling in human monocytes potentiated interleukin 1-beta production through NLRP3/caspase-1 and caused accumulation of immunogenic isolevuglandin (isoLG)-protein adducts. In mice, the Axl inhibitor R428 or global deletion of Axl attenuated hypertension and renal inflammation caused by angiotensin II (Ang II) infusion. Bone marrow transplant studies demonstrated a role of both stromal and immunological Axl in Ang II-induced hypertension. Lastly, in freshly harvested human endothelial cells, a striking correlation was observed between the degree of endothelial cell activation as reflected by intracellular adhesion molecule 1 (ICAM-1), isoLG-adduct accumulation and intracellular GAS6 levels. Conclusions: We define a previously unrecognized interaction of human endothelial cells and monocytes that promote formation of AS DCs in hypertension and show a critical role of GAS6 and Axl signaling in both immune cells and endothelial cells. This pathway is potentially a novel therapeutic target to reduce inflammation and end organ damage in hypertension.




Circ Res: 26 Sep 2021; epub ahead of print
Van Beusecum JP, Ruggeri Barbaro N, Smart CD, Patrick DM, ... Shibao CA, Harrison DG
Circ Res: 26 Sep 2021; epub ahead of print | PMID: 34565181
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Abstract

A Novel Mechanism Underlying Inflammatory Smooth Muscle Phenotype in Abdominal Aortic Aneurysm.

Cai D, Sun C, Zhang G, Que X, ... Weintraub NL, Chen SY
Rationale: Abdominal aortic aneurysm (AAA) is a permanent and localized dilatation of abdominal aorta with potentially fatal consequence of aortic rupture. No effective pharmacological approach has been identified to limit AAA progression and rupture. AAA is characterized by extensive aortic wall matrix degradation that contributes to arterial wall remodeling and eventual rupture, in which smooth muscle cell (SMC) phenotypic transition and matrix metalloproteinases (MMP), especially MMP2 and MMP9, play critical roles. Objective: Our previous study showed that adenosine deaminases acting on RNA 1 (ADAR1) regulates SMC phenotype, which prompted us to study if ADAR1 is involved in AAA development.
Methods and results:
We used angiotensin II (Ang II) infusion ApoE-/- mouse model combined with ADAR1 global and SMC-specific knockout to study the role of ADAR1 in AAA formation/dissection. Aortic transplantation was conducted to determine the importance of vascular cell ADAR1 in AAA development/dissection. Primary cultured SMC were used to study how ADAR1 regulates the inflammatory SMC phenotype and MMP production/activity. Patient specimens were obtained to investigate the relevance of ADAR1 expression to human AAA disease. ADAR1 was induced in abdominal aortic SMC in both mouse and human AAA tissues. Heterozygous knockout of ADAR1 diminished the Ang II-induced AAA/dissection in ApoE-/- mice. Mouse aortic transplantation showed that ADAR1 in vascular cells was essential for AAA formation. SMC-specific ADAR1 knockout reduced experimental AAA formation/dissection. Mechanistically, ADAR1 interacted with HuR to increase the stability of MMP2 and MMP9 mRNA, leading to increased MMP levels and activities. Conclusions: ADAR1 is novel regulator of AAA development/dissection, and thus may represent a potentially new therapeutic target to hinder AAA growth and rupture.




Circ Res: 22 Sep 2021; epub ahead of print
Cai D, Sun C, Zhang G, Que X, ... Weintraub NL, Chen SY
Circ Res: 22 Sep 2021; epub ahead of print | PMID: 34551587
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Abstract

Chaperone-Mediated Autophagy of eNOS in Myocardial Ischemia-Reperfusion Injury.

Subramani J, Kundumani-Sridharan V, Das KC
Rationale: Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) protects against myocardial ischemia-reperfusion injury (I/R). However, reperfusion of myocardium results in superoxide (O2•-) generation, which promotes eNOS glutathionylation that produces O2•- instead of NO. It is unclear whether glutathionylated eNOS (SG-eNOS) continues to produce O2•- indefinitely or undergoes a time-dependent degradation. Objective: To determine whether SG-eNOS continues to produce O2•- in I/R for a prolonged period causing accentuated I/R injury or it undergoes a time-dependent degradation.
Methods and results:
Since SG-eNOS produces significant O2•- instead of NO, we sought to determine the time-course of SG-eNOS levels in the HCAEC in hypoxia/reoxygenation (H/R) by western analysis and immunoprecipitation. SG-eNOS was degraded by chaperone-mediated autophagy (CMA), as inhibitors of CMA rescued eNOS expression. We further confirmed CMA by high resolution confocal and electron microscopy. We showed that SG-eNOS is targeted by HSC70 chaperone via its interaction with glutathionylated-cysteine 691 and 910. Glutathionylation of cysteine 691 residue in H/R exposes 735QRYRL739 motif for interaction with HSC70, and consequent transportation to LAMP2A vesicle, where it is degraded by lysosomal proteases. Mutagenesis of these residues in eNOS inhibited its CMA. Using contrast echocardiography and electron paramagnetic resonance spectrometry (EPR), we found that Trx-Tg mice show improved myocardial perfusion and decreased myocardial apoptosis in I/R due to deglutathionylation of SG-eNOS and restoration of NO generation. Further, WT mice treated with recombinant human Trx (rhTrx) were protected against eNOS CMA, and restored NO production with improved myocardial perfusion and decreased I/R injury. Conclusions: SG-eNOS undergoes degradation via CMA, following prolonged retention in the cytosol. CMA of SG-eNOS terminates O2•- generation preventing further tissue damage but causes irreversible loss of eNOS and NO availability. Prompt deglutathionylation of SG-eNOS prevents CMA, promotes NO production, and improved myocardial perfusion, resulting in amelioration of reperfusion injury.




Circ Res: 21 Sep 2021; epub ahead of print
Subramani J, Kundumani-Sridharan V, Das KC
Circ Res: 21 Sep 2021; epub ahead of print | PMID: 34547902
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Abstract

Fli1 Promotes Vascular Morphogenesis by Regulating Endothelial Potential of Multipotent Myogenic Progenitors.

Ferdous A, Singh S, Luo Y, Abedin MJ, ... Trojanowska M, Hill JA
Rationale: Fetal growth and survival depend critically on proper development and integrity of the vascular system. Fli1 (Friend leukemia integration 1), a member of the Ets family of transcription factors, plays critical roles in vascular morphogenesis and homeostasis at mid-gestation, the developmental stage at which expression of its upstream regulator, Etv2, ceases. However, molecular mechanisms of Fli1 action in vascular morphogenesis remain incompletely understood. Objective: To dissect molecular mechanisms of vascular morphogenesis governed by Fli1.
Methods and results:
Utilizing Fli1 promoter-driven lineage-specific LacZ expression, Fli1 loss-of-function strategies, and a series of molecular techniques, we demonstrate that Fli1 expression in multipotent myogenic progenitor cells (MPCs) occurs independent of Etv2, and loss of Fli1 expression results in a significant increase in LacZ+ cells in mesoderm within somites and limb buds, leading to reciprocal regulation of the expression of several key endothelial and myogenic genes and vascular abnormalities. Conversely, embryos with conditional Fli1 gain-of-function in MPCs manifested aberrant vasculogenesis with lack of myogenesis. Mechanistically, elevated Fli1 activity in myoblasts and in adult MPCs (also called satellite cells) of X-linked muscular dystrophic mdx mice markedly induced endothelial, but attenuated myogenic, gene expression and differentiation. Importantly, ectopic expression of Myf5 or MyoD, two key myogenic regulators, in Fli1-expressing myoblasts restored their differentiation potential, indicating that levels of Fli1 and myogenic regulators in MPCs inversely regulate their endothelial versus myogenic potential. Conclusions:Fli1 governs vascular morphogenesis by regulating endothelial potential by inversely regulating endothelial versus myogenic programs in MPCs. Our data uncover an important and previously unrecognized mechanism of vascular morphogenesis governed by Fli1 and highlight the physiological significance of the fine tuning of Fli1 activity in multipotent progenitors for proper vascular and muscle morphogenesis during development and disease.




Circ Res: 20 Sep 2021; epub ahead of print
Ferdous A, Singh S, Luo Y, Abedin MJ, ... Trojanowska M, Hill JA
Circ Res: 20 Sep 2021; epub ahead of print | PMID: 34544261
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Abstract

Germline and Somatic Variants Convergently Promote Endothelial-to-Mesenchymal Transition in Simplex Brain Arteriovenous Malformation.

Li H, Nam Y, Huo R, Fu W, ... Jiguang W, Cao Y
Rationale: Brain arteriovenous malformations (bAVMs) are abnormal entanglement of blood vessels in brain, with direct connections from arteries to veins, lacking functional capillary bed. Although several somatic mutations were reported, the molecular mechanism and genetic disposition of bAVM remain poorly understood. Objective: We aim to identify transcriptional anomalies and critical functional pathways in bAVM lesions, and explore their association with key de novo germline and somatic variants in bAVM patients.
Methods and results:
We established a comprehensive bAVM dataset from 269 patients, by performing single-cell sequencing of 17 bAVM lesions, whole-exome sequencing of germline DNA from 60 case-unaffected-parental trios, and genomic/transcriptomic sequencing of 231 bAVM lesions. We found abnormal expression of endothelial and mesenchymal markers in bAVM at both bulk and single-cell level, which was validated by flow-cytometric analysis and immunofluorescence staining, suggesting an involvement of Endothelial-to-Mesenchymal transition (EndMT) process in AVM. Using data from the 60 trios we identified non-synonymous de novo germline mutations (DNMs) affecting 46 genes, including EXPH5 (detected in two independent cases), and vessel-related genes such as EPAS1 and ENG. Interestingly, knockdown of epas1 in zebrafish embryo showed AVM-like phenotype exclusively in brain. Subsequent computational and experimental analyses demonstrated that expression of genes affected by DNMs was enriched in vascular cell types and was involved in EndMT-relevant behaviours including cell migration, angiogenesis and cell-marker transition. Moreover, we detected somatic KRAS mutations in 129 of 179 (72%) cases, and showed that KRAS mutations were associated with bleeding as the first symptom (p=0.0072). Following experimental studies demonstrated that KRAS mutations independently regulated EndMT features, consolidating the involvement of EndMT in this disease. Lastly, we showed that Lovastatin reversed EndMT features in vitro and ex vivo. Conclusions: Our results suggest the convergent role of DNMs and somatic mutations in regulating EndMT in bAVM and provided a potential therapeutic option.




Circ Res: 16 Sep 2021; epub ahead of print
Li H, Nam Y, Huo R, Fu W, ... Jiguang W, Cao Y
Circ Res: 16 Sep 2021; epub ahead of print | PMID: 34530633
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Abstract

Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG.

Ma S, Jiang W, Liu X, Lu WJ, ... Wang Y, Lan F
Rationale: Genetic editing has shown great potential for the treatment of human hereditary disorders via the elimination of mutations in embryos. However, the efficiency and safety of germline gene editing are not well understood. Objective: We aimed to examine the preclinical efficacy/safety of embryonic base editing in a mouse model of hypertrophic cardiomyopathy (HCM) using a novel adenine base editor (ABE) platform.
Methods and results:
Here, we described the use of an ABEmax-NG to directly correct the pathogenic R404Q/+ mutation (Myh6 c.1211C>T) in embryos for a mouse model of HCM, increasing the number of wild-type embryos for in vitro fertilization. Delivery of the ABEmax-NG mRNA to embryos from R404Q/+ HCM mice resulted in 62.5-70.8% correction of the Myh6 c.1211C>T, reducing the level of mutant RNA and eliminating HCM in the post-natal mice as well as their offspring. In addition, the same sgRNA was also used to target an intronic locus (TGG PAM) with an overall editing rate of 86.7%, thus confirming that ABEmax-NG can efficiently edit target loci with different PAMs (NG) and genomic distribution in vivo. Compared with CRISPR/ssODN-mediated correction, ABEmax-NG displayed a much higher correction rate without introducing indels. DNA and RNA off-target analysis did not detect off-target editing in treated embryos and founder mice. In utero injection of adeno-associated virus 9 (AAV9) encoding the ABEmax-NG also resulted in around 25.3% correction of the pathogenic mutation and reduced of mutant RNA, thereby indicating ABEmax-NG has the potential to correct the HCM mutation in vivo. Conclusions: We developed an ABEmax-NG system, which efficiently corrected a pathogenic Myh6 HCM mutation in mouse embryos without off target lesions, thus safely eliminating HCM in derived mice and their progeny.




Circ Res: 15 Sep 2021; epub ahead of print
Ma S, Jiang W, Liu X, Lu WJ, ... Wang Y, Lan F
Circ Res: 15 Sep 2021; epub ahead of print | PMID: 34525843
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Abstract

PDE1 Inhibition Modulates Ca1.2 Channel to Stimulate Cardiomyocyte Contraction.

Muller GK, Song J, Jani V, Wu Y, ... Anderson ME, Kass DA
Rationale: Cyclic adenosine monophosphate (cAMP) activation of protein kinase A (PKA) stimulates excitation-contraction coupling, increasing cardiac contractility. This is clinically achieved by beta-adrenergic receptor stimulation (b-ARs) or inhibition of phosphodiesterase type-3 (PDE3i), though both approaches are limited by arrhythmia and chronic myocardial toxicity. Phosphodiesterase type-1 inhibition (PDE1i) also augments cAMP and enhances contractility in intact dogs and rabbits. Unlike b-ARs or PDE3i, PDE1i-stimulated inotropy is unaltered by b-AR blockade and induces little whole-cell Ca2+ ([Ca2+]i) increase. Positive inotropy from PDE1i was recently reported in human heart failure. However, mechanisms for this effect remain unknown. Objective: Define the mechanism(s) whereby PDE1i increases myocyte contractility.
Methods and results:
We studied primary guinea pig myocytes that express the PDE1C isoform found in larger mammals and humans. In quiescent cells, the potent, selective PDE1i (ITI-214) did not alter cell shortening or [Ca2+]i whereas b-ARs or PDE3i increased both. When combined with low-dose adenylate cyclase stimulation, PDE1i enhanced shortening in a PKA-dependent manner but unlike PDE3i, induced little [Ca2+]i rise nor augmented b-ARs. b-ARs or PDE3i reduced myofilament Ca2+ sensitivity, and increased SR Ca2+ content and phosphorylation of PKA-targeted serines on troponin-I, myosin binding protein C, and phospholamban. PDE1i did not significantly alter any of these. However, PDE1i increased Cav1.2 channel conductance similarly as PDE3i (both PKA-dependent), without altering NCX current density. Cell shortening and [Ca2+]i augmented by PDE1i were more sensitive to Cav1.2 blockade and premature or irregular cell contractions and [Ca2+]i transients less frequent than with PDE3i. Conclusions: PDE1i enhances contractility by a PKA-dependent increase in Cav1.2 conductance with less total [Ca2+]i increase, and no significant changes in SR [Ca2+], myofilament Ca2+-sensitivity, or phosphorylation of critical EC-coupling proteins as observed with b-ARs and/or PDE3i. PDE1i could provide a novel positive inotropic therapy for heart failure without the toxicities of b-ARs and PDE3i.




Circ Res: 14 Sep 2021; epub ahead of print
Muller GK, Song J, Jani V, Wu Y, ... Anderson ME, Kass DA
Circ Res: 14 Sep 2021; epub ahead of print | PMID: 34521216
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Abstract

NLRP3 Inflammasome Mediates Immune-Stromal Interactions in Vasculitis.

Porritt RA, Zemmour D, Abe M, Lee Y, ... Arditi M, Noval Rivas M
Rationale: NLRP3 activation and IL-1β production are implicated in Kawasaki Disease (KD) pathogenesis, however a detailed and complete characterization of the molecular networks and cellular subsets involved in the development of cardiovascular lesions is still lacking. Objective: Here, in a murine model of KD vasculitis, we used single-cell RNA sequencing and spatial transcriptomics to determine the cellular landscape of inflamed vascular tissues.
Methods and results:
We observe infiltrations of innate and adaptive immune cells in murine KD cardiovascular lesions, associated with increased expression of Nlrp3 and Il1b. Monocytes, macrophages and dendritic cells were the main sources of IL-1β, whereas fibroblasts and vascular smooth muscle cells (VSMCs) expressed high levels of IL-1 receptor. VSMCs type 1 surrounding the inflamed coronary artery undergo a phenotype switch to become VSMCs type 2, which are characterized by gene expression changes associated with decreased contraction, and enhanced migration and proliferation. Genetic inhibition of IL-1β signaling on VSMCs efficiently attenuated the VSMCs type 2 phenotypic switch and the development of cardiovascular lesions during murine KD vasculitis. In addition, pharmacological inhibition of NLRP3 prevented the development of cardiovascular inflammation. Conclusions: Our studies unravel the cellular diversity involved in IL-1β production and signaling in murine KD cardiovascular lesions and provide the rationale for therapeutic strategies targeting NLRP3 to inhibit cardiovascular lesions associated with KD.




Circ Res: 13 Sep 2021; epub ahead of print
Porritt RA, Zemmour D, Abe M, Lee Y, ... Arditi M, Noval Rivas M
Circ Res: 13 Sep 2021; epub ahead of print | PMID: 34517723
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Abstract

Macrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4.

Liu M, Yan M, He J, Lv H, ... Zhu Y, Ai D
Rationale: Timely inhibition of inflammation and initiation of resolution are important to repair injured tissues. Mammalian STE20-like protein kinase 1/2 (MST1/2) acts as a regulator of macrophage-associated immune responses to bacterial infections. However, the role of MST1/2 in regulating macrophage phenotype and function in myocardial infarction (MI) remains unclear. Objective: To determine the function and underlying mechanism of macrophage MST1/2 in cardiac repair post-MI.
Methods and results:
Using LysMCre-mediated Mst1/2-deficient mice, we found that MST1 deficiency exacerbated cardiac dysfunction after MI. Single-cell RNA sequencing assay indicated that the effect was attributed to a shift of macrophage subtypes from those expressing Cxcl2 and Cd163 toward Ccl2 and Ccl4 expression. Mass spectrometry identified leukotriene B4 (LTB4) as the lipid mediator that was upregulated in the absence of MST1. We found that MST1 phosphorylated 5-lipoxygenase (5-LOX) at its T218 residue, disrupting the interaction between 5-LOX and 5-LOX-activating protein, resulting in a reduction of LTB4 production. In contrast, a 5-LOXT218A variant showed no response to MST1. Moreover, treatment of peritoneal macrophages with LTB4 or medium conditioned by Mst1-deficient macrophages resulted in high Ccl2 and Ccl4 expression and low Cxcl2 and Cd163 expression, except when the cells were co-treated with the LTB4 receptor 1 (BLT1) antagonist CP105696. Furthermore, CP105696 ameliorated cardiac dysfunction in LysMCre-mediated Mst1/2-deficient mice and enhanced cardiac repair in wild-type mice treated with XMU-MP-1 after MI. Conclusions: Taken together, our results demonstrate that inhibition of MST1/2 impaired post-MI repair through activating macrophage 5-LOX-LTB4-BLT1 axis.




Circ Res: 12 Sep 2021; epub ahead of print
Liu M, Yan M, He J, Lv H, ... Zhu Y, Ai D
Circ Res: 12 Sep 2021; epub ahead of print | PMID: 34515499
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This program is still in alpha version.