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ObjectiveVascular smooth muscle cells (VSMCs) are the primary contractile component of blood vessels and can undergo phenotypic switching from a contractile to a synthetic phenotype in vascular diseases such as coronary artery disease (CAD). This process leads to decreased expression of SMC lineage genes and increased proliferative, migratory and secretory abilities that drive disease progression. Super-enhancers (SE) and occupied transcription factors are believed to drive expression of genes that maintain cell identify and homeostasis. The goal of this study is to identify novel regulator of VSMC homeostasis by screening for SE-regulated transcription factors in arterial tissues.Approach and ResultsWe characterized human artery SEs by analyzing the enhancer histone mark H3K27ac ChIP-seq data of multiple arterial tissues. We unexpectedly discovered the transcription factor PRDM16, a GWAS identified CAD risk gene with previously well-documented roles in brown adipocytes but with an unknown function in vascular disease progression, is enriched with artery-specific SEs. Further analysis of public bulk RNA-seq and scRNA-seq datasets, as well as qRT-PCR and Western blotting analysis, demonstrated that PRDM16 is preferentially expressed in arterial tissues and in contractile VSMCs but not in visceral SMCs, and down-regulated in phenotypically modulated VSMCs. To explore the function ofPrdm16in vivo, we generatedPrdm16SMC-specific knockout mice and performed histological and bulk RNA-Seq analysis of aortic tissues. SMC-deficiency ofPrdm16does not affect the aortic morphology but significantly alters expression of many CAD risk genes and genes involved in VSMC phenotypic modulation. Specifically,Prdm16negatively regulates the expression ofTgfb2that encodes for an upstream ligand of TGF-β signaling pathway, potentially through binding to the promoter region ofTgfb2. These transcriptomic changes likely disrupt VSMC homeostasis and predispose VSMCs to a disease state.ConclusionsOur results suggest that the CAD risk genePRDM16is preferentially expressed in VSMCs and is a novel regulator of VSMC homeostasis. Future studies are warranted to investigate its role in VSMCs under pathological conditions such as atherosclerosis.

This study aimed to investigate left atrium (LA) strain components in the assessment of cardiac function and its clinical correlates in pre-eclampsia (PE). With the use of speckle tracking echocardiography, phasic LA strain and (LASr)/(E/e'), the surrogate of LA compliance, were compared between healthy pregnant women (n = 70) and those with PE (n = 146) and among different diastolic dysfunction (DD) grades in PE. Receiver operating characteristic curves and logistic regression analysis were used to identify the role of strain components in distinguishing DD grades and predicting cardiac complications. LA reservoir strain, conduit strain and LA compliance reduced significantly in PE (p0.01). LASr/(E/e') gradually decreased with worsening DD and LASr/(E/e')3.40 was the independent risk factor for cardiac events in PE (p0.01). This study observed significantly decreased LA strain and compliance in PE. Notably, LA compliance decreased progressively with the severity of DD, and LASr/(E/e')3.40 is the independent risk factor for cardiac complications during PE pregnancy.

As a common air pollutant, formaldehyde is widely present in nature, industrial production and consumer products. Endogenous formaldehyde is mainly produced through the oxidative deamination of methylamine catalysed by semicarbazide‐sensitive amine oxidase (SSAO) and is ubiquitous in human body fluids, tissues and cells. Vascular endothelial cells and smooth muscle cells are rich in this formaldehyde‐producing enzyme and are easily damaged owing to consequent cytotoxicity. Consistent with this, increasing evidence suggests that the cardiovascular system and stages of heart development are also susceptible to the harmful effects of formaldehyde. Exposure to formaldehyde from different sources can induce heart disease such as arrhythmia, myocardial infarction (MI), heart failure (HF) and atherosclerosis (AS). In particular, long‐term exposure to high concentrations of formaldehyde in pregnant women is more likely to affect embryonic development and cause heart malformations than long‐term exposure to low concentrations of formaldehyde. Specifically, the ability of mouse embryos to effect formaldehyde clearance is far lower than that of the rat embryos, more readily allowing its accumulation. Formaldehyde may also exert toxic effects on heart development by inducing oxidative stress and cardiomyocyte apoptosis. This review focuses on the current progress in understanding the influence and underlying mechanisms of formaldehyde on cardiovascular disease and heart development.

Congenital left atrial appendage aneurysm is an extremely rare anomaly. It is seen at any time ranging from fetal to ninth decade of life. Patients with left atrial appendage aneurysm may be asymptomatic, or present with dyspnea, arrhythmias, thromboembolic phenomenon. We report a case of prenatal diagnosis at 26 weeks of gestation , postnatal management and outcome of left atrial appendage aneurysm.

Objective: Myh11 encodes a myosin heavy chain protein that is specifically expressed in smooth muscle cells (SMCs) and is important for maintaining vascular wall stability. The goal of this study is to generate a Myh11 dual reporter mouse line for definitive visualization of MYH11 + SMCs in vivo. Approach and Results: We generated a Myh11 knock-in mouse model by inserting LoxP-nlacZ-4XpolyA-LoxP-H2B-GFP-polyA-FRT-Neo-FRT reporter cassette into the Myh11 gene locus. The nuclear (n) lacZ-4XpolyA cassette is flanked by 2 LoxP sites followed by H2B-GFP (histone 2B fused green fluorescent protein). Upon Cre-mediated recombination, nlacZ-stop cassette is removed thereby permitting nucleus localized H2B-GFP expression. Expression of the nuclear localized lacZ or H2B-GFP is under control of the endogenous Myh11 promoter. Nuclear lacZ was expressed specifically in SMCs at embryonic and adult stages. Following germline Cre-mediated deletion of nuclear lacZ, H2B-GFP was specifically expressed in the nuclei of SMCs. Comparison of nuclear lacZ expression with Wnt1 Cre and Mef2c Cre mediated-H2B-GFP expression revealed heterogenous origins of SMCs from neural crest and second heart field in the great arteries and coronary vessels adjacent to aortic root. Conclusions: The Myh11 knock-in dual reporter mouse model offers an exceptional genetic tool to visualize and trace the origins of SMCs in mice.

Yin Yang 1 (YY1) regulates gene transcription in a variety of biological processes. In this study, we aim to determine the role of YY1 in vascular smooth muscle cell (VSMC) phenotypic modulation both in vivo and in vitro. Here we show that vascular injury in rodent carotid arteries induces YY1 expression along with reduced expression of smooth muscle differentiation markers in the carotids. Consistent with this finding, YY1 expression is induced in differentiated VSMCs in response to serum stimulation. To determine the underlying molecular mechanisms, we found that YY1 suppresses the transcription of CArG box-dependent SMC-specific genes including SM22α, SMα-actin and SMMHC. Interestingly, YY1 suppresses the transcriptional activity of the SM22α promoter by hindering the binding of serum response factor (SRF) to the proximal CArG box. YY1 also suppresses the transcription and the transactivation of myocardin (MYOCD), a master regulator for SMC-specific gene transcription by binding to SRF to form the MYOCD/SRF/CArG box triad (known as the ternary complex). Mechanistically, YY1 directly interacts with MYOCD to competitively displace MYOCD from SRF. This is the first evidence showing that YY1 inhibits SMC differentiation by directly targeting MYOCD. These findings provide new mechanistic insights into the regulatory mechanisms that govern SMC phenotypic modulation in the pathogenesis of vascular diseases.

Left atrial (LA) adaptive changes associated with pre-eclampsia (PE) have not been adequately addressed. This study aimed to evaluate the usefulness of LA myocardial function indices in detecting early cardiac alterations in PE. Using speckle tracking echocardiography (STE), LA volumetric and myocardial parameters were acquired in PE women with preserved left ventricular (LV) systolic and diastolic function based on standard criteria and normotensive controls. LA compliance was assessed by the ratio of reservoir strain (LASr) to the estimated LA pressure (E/e’). Subtle systolic and diastolic abnormalities were identified as LV global longitudinal strain

Background & aimsVisceral smooth muscle cells (SMCs) are an integral component of the gastrointestinal (GI) tract and are critical for regulating motility. SMC contraction is regulated by changes in post-translational signaling and the state of differentiation. Impaired SMC contraction is associated with significant morbidity and mortality but the mechanisms regulating the expression levels of SMC-specific contractile proteins, including the role of long non-coding RNAs (lncRNAs), remains largely unexplored. Herein, we have uncovered an important role of Carmn (Cardiac mesoderm enhancer-associated noncoding RNA), a SMC-specific lncRNA, in regulating the phenotype of visceral SMCs of the GI tract.MethodsAnalysis of GTEx and publicly available single-cell RNA sequencing (scRNA-seq) datasets from embryonic, adult human and mouse GI tissues were used to identify SMC-specific lncRNAs. The functional role of Carmn was investigated using a novel GFP knock-in (KI) reporter/knockout (KO) mouse model. Bulk RNA sequencing (RNA-seq) and single nuclei RNA sequencing (snRNA-seq) of colonic muscularis were used to investigate underlying mechanisms.ResultsUnbiased in silico analyses and GFP expression patterns in Carmn GFP KI mice revealed that Carmn is specifically expressed in SMCs in human and mouse GI tract. Premature lethality was observed in global Carmn KO (gKO) and inducible SMC-specific KO (iKO) mice due to colonic pseudo-obstruction, severe distension of the GI tract with blockages in cecum and colon segments. Histology, whole-gut GI transit time and muscle myography analysis revealed severe dilation, significantly delayed GI transit and impaired GI contractility in Carmn KO mice versus control mice. Bulk RNA-seq of colonic muscularis revealed that Carmn deficiency promotes SMC de-differentiation as evidenced by up-regulation of extracellular matrix genes and down-regulation of SMC contractile genes including Mylk, a key regulator of SMC contraction. SnRNA-seq further revealed SMC Carmn deficiency not only compromised myogenic motility by reducing expression of contractile genes but also impaired neurogenic motility by disrupting cell-cell connectivity in the colonic muscularis. These findings may have translational significance as silencing CARMN in human colonic SMCs significantly attenuated contractile gene expression including MYLK and decreased SMC contractility. Luciferase reporter assays showed that CARMN enhances the transactivation activity of the master regulator of SMC contractile phenotype, myocardin, thereby maintaining the GI SMC myogenic program.ConclusionOur data suggest that Carmn is indispensable for maintaining GI SMC contractile function in mice, and that loss of function of CARMN may contribute to human visceral myopathy. To our knowledge this is the first study showing an essential role of lncRNA in the regulation of visceral SMC phenotype.

Background: Cell identify is established by strict control of cell type-specific gene expression programs through regulatory enhancers binding of transcriptional factors. Vascular smooth muscle cells (VSMCs) are critical in maintaining the vascular homeostasis by expressing an array of contractile genes that are primarily governed by SRF/MYOCD complex via binding to the DNA motif called CArG element. However, most of these previous studies regarding regulatory mechanism underlying SM-specific gene expression were performed in vitro, outside of their in situ genomic contexts. Methods and Results: To define the regulatory atlas of VSMC, we analyzed epigenetic datasets of multiple human tissues and cell types. We found CARMN , that we previously reported as a SMC-specific lncRNA and a critical regulator of SMC contractile phenotype by binding to MYOCD, is enriched with SMCs-specific enhancers. Co-expression analysis using RNA-seq datasets of different human tissues identified CARMN expression is positively correlated with both MYOCD and SRF . We further demonstrated overexpressing MYOCD activates, while silencing MYOCD or SRF inhibits CARMN expression in human coronary VSMCs. Subsequent analysis of SRF ChIP-seq data revealed 4 SRF binding peaks with CARMN locus, including 2 located within promoter (namely CArG 1 and 2) and the other 2 (namely CArG 3 and 4) located within distal enhancers. Further luciferase reporter assays revealed mutation of CArG 1 or 3 dramatically abolish MYOCD-induced reporter activity. To confirm these findings in vivo, we generated single CArG element mutant mice for each of the 4 CArG elements and CArG 3/4 double mutant mice by CRISPR-Cas9 genome editing approach. Analysis of Carmn expression in SMC-enriched tissues from these mutant mice revealed a ~50% decrease of Carmn expression in tissues of CArG 1 mutant mice while mutation of other CArG elements appears no effect on Carmn expression. Conclusions: Our results suggest the SM-specific expression of Carmn is dependent, at least partially, on SRF/MYOCD complex and CArG 1 is critical for this regulation. Together with our previous findings that CARMN potentiates MYOCD function, we propose CARMN and SRF/MYOCD complex form a feedforward loop promoting SMC contractile phenotype.

Background: Aortic aneurysm is a life-threatening vascular disease characterized by vascular smooth muscle cell (VSMC) depletion, ECM degradation, and infiltration of immune cells. Previous studies have shown that Carmn , a SMC-enriched long non-coding RNA, is critical for maintaining VSMC contractile phenotype. However, its functional role in aortic aneurysm remains completely unknown. Methods and Results: We re-analyzed the publicly available aortic aneurysm bulk RNA-seq and scRNA-seq datasets in mouse and human. These unbiased analyses revealed that Carmn is downregulated in the SMCs of aortic aneurysm samples. To examine the potential role of Carmn in aortic aneurysm, we generated Carmn global knockout (gKO) mice or inducible SMC-specific KO (iKO) mice driven by Myh11 -Cre ER T2, respectively. Unexpectedly, both Carmn gKO and iKO mice exhibited premature lethality, due to a severe colonic pseudo-obstruction resulting from deletion of Carmn in visceral SMCs. Despite the dominant gastrointestinal phenotype observed in Carmn KO mice, morphological analysis of the Carmn KO thoracic aorta revealed thinner vascular wall compared to that of controls. Results from TEM showed that the VSMC in the Carmn KO thoracic aorta are disorganized and degradative with aneurysmal dissection. Data from bulk RNA-seq of Carmn -deficient aorta revealed that the down-regulated genes are associated with degradation of ECM while the up-regulated genes are associated with cell death. Moreover, scRNA-seq of Carmn -deficient aorta revealed that the percentage of the SMCs is decreased while the percentage of infiltrated immune cells is increased. These data indicated that Carmn deficiency may play a critical role in promoting the development of aortic aneurysm. To circumvent the lethal visceral SMC phenotype, we are in the progress of crossing Carmn flox mice with the novel vascular SMC-specific inducible Itga8 -Cre ER T2 mouse to generate VSMC-specific Carmn KO mice. Conclusions: Our data suggest that Carmn is indispensable for maintaining gastrointestinal contractile function, and implicate that Carmn deficiency plays a potential role in contributing the development of aortic aneurysm.

Cirrhotic cardiomyopathy (CCM) is a common complication of liver cirrhosis. Many patients with cirrhotic livers do not die from liver failure but from abnormal hemodynamics secondary to liver cirrhosis. Liver transplantation is one of the most effective treatments for liver diseases. Recent studies have found that liver transplantation can reverse CCM and improve cardiac function; however, its role and remedial mechanism remain unclear. Circular RNAs (circRNAs) have become an important marker for diagnosing diseases. The differential expression of circRNAs is associated with heart diseases. In this study, we used gene sequencing to detect the circRNA expression profile of patients with CCM before and after liver transplantation and predicted the differential circRNA target genes. The results showed that a total of 1495 circRNAs were dysregulated after liver transplantation, 1319 genes were downregulated, and 176 were upregulated (P 0.05, log2 (fold change) 2.0). The qRT-PCR results showed that circ-ASAP1, circ-N4BP2L2, circ-EXOC6B were significantly downregulated (P 0.05), which were consistent with the RNA sequencing data, and circ-ASAP1 had the most significant difference. Bioinformatics analysis suggested that mTOR and MAPK signaling pathways might be involved in the pathogenesis of CCM. By constructing a circRNA-miRNA-mRNA interaction network, hsa-miR-197-3p, hsa-miR-483-3p, and hsa-miR-885-3p, particularly key miRNA (hsa-miR-483-3p), were found to be the major potential genes involved in CCM regulation. In summary, this study suggested that circRNAs play a crucial regulatory role in the occurrence of CCM before and after liver transplantation, and their potential biological function might be the key to diagnosis and treatment.

Aortic dissection (AD) is a lethal cardiac disorder and one of the most concerning cardiovascular diseases (CVDs). Increasing evidence indicates that human aortic vascular smooth muscle cells (VSMCs) play a crucial role in the pathogenesis of AD, especially related to phenotypic transformation. And notablely, the development of AD is also accompanied by inflammation.By using quantitative real-time PCR and fluorescence in situ hybridization (FISH), we detected the expression levels of miR-564 in vitro and in vivo. The effects of miR-564 proliferation and migration were investigated in VSMCs. The downstream targets of miR-564 were found by bioinformatics analyse, and verified in the regulation on VSMCs. An AD murine model was constructed and clinical evaluation was performed to explore the critical roles of miR-564 in vivo. At the same time, the level of inflammation was detected using quantitative real-time PCR and immunofluorescence.Overexpression of miR-564 inhibited cell proliferation and migration, as well as phenotype switch, with or without platelet-derived growth factor BB (PDGF-BB) treatment, whereas downregulation of miR-564 led to opposite results. Mechanistically, miR-564 directly interacted with the target genes proto-oncogene (SKI) and neurogranin (NRGN) to regulate the biological functions of VSMCs. In particular, animal experiments demonstrated that miR-564 can alleviate the progression of AD mainly through mediating phenotypic swithing and inflammation which was consistent with clinical evaluation.Our study identified miR-564 as a significant molecule that attenuates AD progression by inhibiting inflammation and VSMCs proliferation, migration and phenotypic transformation, suggesting that it may be a potential therapeutic target for AD.

Background: Vascular homeostasis is maintained by the differentiated phenotype of vascular smooth muscle cells (VSMCs). The landscape of protein coding genes comprising the transcriptome of differentiated VSMCs has been intensively investigated but many gaps remain including the emerging roles of noncoding genes. Methods: We reanalyzed large-scale, publicly available bulk and single-cell RNA sequencing datasets from multiple tissues and cell types to identify VSMC-enriched long noncoding RNAs. The in vivo expression pattern of a novel smooth muscle cell (SMC)–expressed long noncoding RNA, Carmn (cardiac mesoderm enhancer-associated noncoding RNA), was investigated using a novel Carmn green fluorescent protein knock-in reporter mouse model. Bioinformatics and quantitative real-time polymerase chain reaction analysis were used to assess CARMN expression changes during VSMC phenotypic modulation in human and murine vascular disease models. In vitro, functional assays were performed by knocking down CARMN with antisense oligonucleotides and overexpressing Carmn by adenovirus in human coronary artery SMCs. Carotid artery injury was performed in SMC-specific Carmn knockout mice to assess neointima formation and the therapeutic potential of reversing CARMN loss was tested in a rat carotid artery balloon injury model. The molecular mechanisms underlying CARMN function were investigated using RNA pull-down, RNA immunoprecipitation, and luciferase reporter assays. Results: We identified CARMN , which was initially annotated as the host gene of the MIR143/145 cluster and recently reported to play a role in cardiac differentiation, as a highly abundant and conserved, SMC-specific long noncoding RNA. Analysis of the Carmn GFP knock-in mouse model confirmed that Carmn is transiently expressed in embryonic cardiomyocytes and thereafter becomes restricted to SMCs. We also found that Carmn is transcribed independently of Mir143/145 . CARMN expression is dramatically decreased by vascular disease in humans and murine models and regulates the contractile phenotype of VSMCs in vitro. In vivo, SMC-specific deletion of Carmn significantly exacerbated, whereas overexpression of Carmn markedly attenuated, injury-induced neointima formation in mouse and rat, respectively. Mechanistically, we found that Carmn physically binds to the key transcriptional cofactor myocardin, facilitating its activity and thereby maintaining the contractile phenotype of VSMCs. Conclusions: CARMN is an evolutionarily conserved SMC-specific long noncoding RNA with a previously unappreciated role in maintaining the contractile phenotype of VSMCs and is the first noncoding RNA discovered to interact with myocardin.

Circular RNAs (circRNAs) are a novel type of long non-coding RNAs that can regulate gene expression in heart development and heart disease. However, the expression pattern of circRNAs in congenital heart disease (CHD) induced by formaldehyde exposure is still unknown. We detected circRNAs expression profiles in heart tissue taken from six neonatal rat pups with formaldehyde exposure group and normal group using RNA-sequencing. Results revealed that a total of 54 circRNAs were dysregulated in the formaldehyde exposure group compared to the normal group. Among them, 31 were upregulated and 23 were downregulated (fold change = 2.0, p 0.0 5). The qRT-qPCR results showed that expressions of 12:628708|632694, 18:77477060|77520779, 5:167486001|167526275 were significantly upregulated, while that of 7:41167312|4116775 and 20:50659751|5068786 were notably downregulated; the expression pattern was consistent with the RNA sequencing data. Bioinformatics analysis shows that the pathogenesis of formaldehyde exposure-induced CHD may involve Hippo-YAP pathway、Notch signaling pathway and other pathways. A key miRNA (rno-miR-665) was identified by constructing a circRNA-miRNA-mRNA co-expression network. In summary, the study illustrated that circRNAs differentially expressed in fetal heart tissues during formaldehyde exposure has potential biological functions and may be a biomarker or therapeutic target for CHD.

Knowledge regarding the relationship between the molecular mechanisms underlying atherosclerosis (AS) and transfer RNA‐derived small RNAs (tsRNAs) is limited. This study illustrated the expression profile of tsRNAs, thus exploring its roles in AS pathogenesis. Small RNA sequencing was performed with four atherosclerotic arterial and four healthy subject samples. Using bioinformatics, the protein‐protein interaction network and cellular experiments were constructed to predict the enriched signalling pathways and regulatory roles of tsRNAs in AS. Of the total 315 tsRNAs identified to be dysregulated in the AS group, 131 and 184 were up‐regulated and down‐regulated, respectively. Interestingly, the pathway of the differentiated expression of tsRNAs in cell adhesion molecules (CAMs) was implicated to be closely associated with AS. Particularly, tRF‐Gly‐GCC might participate in AS pathogenesis via regulating cell adhesion, proliferation, migration and phenotypic transformation in HUVECs and VSMCs. In conclusion, tsRNAs might help understand the molecular mechanisms of AS better. tRF‐Gly‐GCC may be a promising target for suppressing abnormal vessels functions, suggesting a novel strategy for preventing the progression of atherosclerosis.

The Hippo signaling effector, TEAD1 plays an essential role in cardiovascular development. However, a role for TEAD1 in postmitotic cardiomyocytes (CMs) remains incompletely understood. Herein we reported that TEAD1 is required for postmitotic CM survival. We found that adult mice with ubiquitous or CM-specific loss of Tead1 present with a rapid lethality due to an acute-onset dilated cardiomyopathy. Surprisingly, deletion of Tead1 activated the necroptotic pathway and induced massive cardiomyocyte necroptosis, but not apoptosis. In contrast to apoptosis, necroptosis is a pro-inflammatory form of cell death and consistent with this, dramatically higher levels of markers of activated macrophages and pro-inflammatory cytokines were observed in the hearts of Tead1 knockout mice. Blocking necroptosis by administration of necrostatin-1 rescued Tead1 deletion-induced heart failure. Mechanistically, genome-wide transcriptome and ChIP-seq analysis revealed that in adult hearts, Tead1 directly activates a large set of nuclear DNA-encoded mitochondrial genes required for assembly of the electron transfer complex and the production of ATP. Loss of Tead1 expression in adult CMs increased mitochondrial reactive oxygen species, disrupted the structure of mitochondria, reduced complex I-IV driven oxygen consumption and ATP levels, resulting in the activation of necroptosis. This study identifies an unexpected paradigm in which TEAD1 is essential for postmitotic CM survival by maintaining the expression of nuclear DNA-encoded mitochondrial genes required for ATP synthesis.

Constitutive explorations have demonstrated that circular RNAs (circRNAs) are closely related to cardiovascular diseases. However, few studies have identified regulatory roles of circRNAs in endothelial repair. In particular, circTMEM165 has first been reported to be highly expressed in hypoxic human umbilical vein endothelial cells (HUVECs), indicating it to be closely linked with endothelial functions and atherosclerosis. Here, we explored the main biological functions of circTMEM165 and its regulatory mechanism in atherosclerosis. We identified that circTMEM165 was downregulated in patients with atherosclerosis and negatively associated with atherosclerosis progression. Functionally, circTMEM165 was found to be abundant in endothelial cells, inhibiting inflammation and adhesion in HUVECs. Particularly, we first observed that lipopolysaccharide (LPS) could induce HUVEC apoptosis and mitochondrial fission, and circTMEM165 was later found to alleviate these effects. Mechanistically, circTMEM165, as a miR-192-3p sponge, upregulated the downstream expression of SCP2, which serves as critical regulator of biological functions of HUVECs. Moreover, the classic rat carotid artery balloon injury model demonstrated that circTMEM165 reduced apoptosis and intimal hyperplasia in vivo. These findings demonstrated the specific role of the circTMEM165/miR-192-3p/SCP2 pathway in regulating endothelial function and indicated that these molecules served as promising targets for the prediction and diagnosis of vascular diseases. Funding Information: This work was supported by The National Natural Science Foundation of China (grant no. 81870331), and The Qingdao municipal science and technology bureau project (grant no. 21-1-4-rkjk-12-nsh). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: This study was approved by the Ethical Committee of Qingdao University and conducted in accordance with the Declaration of Helsinki. All human subjects provided signed informed consent. And the animal experiments approved by Qingdao University Laboratory Animal Welfare Ethics Committee (No. 201809SD18202012014).

The

Introduction: Restenosis after percutaneous intervention is predominantly attributed to proliferation and migration of vascular smooth muscle cells (VSMCs). However, the key regulators responsible for VSMC proliferation and migration remain to be identified. Hypothesis: We previously reported that the novel high mobility group (HMG) nuclear protein HMGXB4 (HMG-Box containing 4) plays a critical role in the de-differentiation of vascular smooth muscle cells in vitro and in acute inflammatory response to septic shock. We hypothesize that HMGXB4 is critical for neointimal hyperplasia in response to inflammatory stimuli. Methods and Results: We found that the expression of HMGXB4 is dramatically induced in ligation or wire injury-induced neointimal hyperplasia and correlated with the activation of inflammatory signaling in mice. Using an inducible smooth muscle-specific Hmgxb4 KO (knockout) mice model, we found specific KO of Hmgxb4 in VSMCs ameliorates ligation- or wire- injury induced neointimal formation. Among an array of growth factors and inflammation cytokines, we found that TNFα and INFγ effectively induces the expression of HMGXB4 in VSMCs and correlates with the VSMC proliferation in vitro. Furthermore, we found deletion of HMGXB4 attenuates while over-expression of HMGXB4 promotes inflammation cytokines-induced VSMC proliferation in vitro. These results suggest injury-induced inflammatory signal triggers HMGXB4 induction, which, in turn, promotes the VSMC proliferation and neointimal formation. Conclusions: Our study not only demonstrates a critical role of HMGXB4 in promoting neointimal hyperplasia in response the arterial injury, but also suggests HMGXB4 is a potential novel target for the management of restenosis in human.

We have previously demonstrated that the transcription co-factor yes-associated protein 1 (YAP1) promotes vascular smooth muscle cell (VSMC) de-differentiation. Yet, the role and underlying mechanisms of YAP1 in neointima formation in vivo remain unclear. The goal of this study was to investigate the role of VSMC-expressed YAP1 in vascular injury-induced VSMC proliferation and delineate the mechanisms underlying its action. Experiments employing gain- or loss-of-function of YAP1 demonstrated that YAP1 promotes human VSMC proliferation. Mechanistically, we identified platelet-derived growth factor receptor beta (PDGFRB) as a novel YAP1 target gene that confers the YAP1-dependent hyper-proliferative effects in VSMCs. Furthermore, we identified TEA domain transcription factor 1 (TEAD1) as a key transcription factor that mediates YAP1-dependent PDGFRβ expression. ChIP assays demonstrated that TEAD1 is enriched at a PDGFRB gene enhancer. Luciferase reporter assays further demonstrated that YAP1 and TEAD1 co-operatively activate the PDGFRB enhancer. Consistent with these observations, we found that YAP1 expression is upregulated after arterial injury and correlates with PDGFRβ expression and VSMC proliferation in vivo. Using a novel inducible SM-specific Yap1 knockout mouse model, we found that the specific deletion of Yap1 in adult VSMCs is sufficient to attenuate arterial injury-induced neointima formation, largely due to inhibited PDGFRβ expression and VSMC proliferation. Our study unravels a novel mechanism by which YAP1/TEAD1 promote VSMC proliferation via transcriptional induction of PDGFRβ, thereby enhancing PDGF-BB downstream signaling and promoting neointima formation.

Background Heart failure is a leading cause of human morbidity and mortality. Circular RNAs (circRNAs) are a newly discovered class of RNA that have been found to have important physiological and pathological roles. In the current study, we de novo analyzed existing whole transcriptome data from 5 normal and 5 dilated cardiomyopathy (DCM) human heart samples and compared the results with circRNAs that have been previously reported in human, mouse and rat hearts. Results Our analysis identifies a list of cardiac circRNAs that are reliably detected in multiple studies. We have also defined the top 30 most abundant circRNAs in healthy human hearts which include some with previously unrecognized cardiac roles such as circHIPK3_11 and circTULP4_1. We further found that many circRNAs are dysregulated in DCM, particularly transcripts originating from DCM-related gene loci, such as TTN and RYR2. In addition, we predict the potential of cardiac circRNAs to sponge miRNAs that have reported roles in heart disease. We found that circALMS1_6 has the highest potential to bind miR-133, a microRNA that can regulate cardiac remodeling. Interestingly, we detected a novel class of circRNAs, referred to as read-though (rt)-circRNAs which are produced from exons of two different neighboring genes. Specifically, rt-circRNAs from SCAF8 and TIAM2 were observed to be dysregulated in DCM and these rt-circRNAs have the potential to sponge multiple heart disease-related miRNAs. Conclusions In summary, this study provides a valuable resource for exploring the function of circRNAs in human heart disease and establishes a functional paradigm for identifying novel circRNAs in other tissues.

Background The purpose of this paper was to evaluate the difference in postoperative outcomes following multidetector computed tomography (MDCT) and transesophageal echocardiography (TEE)‐based annulus sizing for transcatheter aortic valve replacement (TAVR). Methods Electronic search of PubMed, Biomed Central, Scopus, and Google Scholar databases was conducted until August 15, 2019. We included all types of studies comparing MDCT‐based annulus sizing with TEE‐based annulus sizing and assessing paravalvular regurgitation (PVR). Data were summarized using the Mantel‐Haenszel odds ratio (OR) with 95% confidence intervals (CI). Results A total of six studies were included. Pooled analysis of 431 participants in the MDCT group and 509 participants in the TEE group demonstrated that MDCT‐based annulus sizing is associated with a significantly lower incidence of more than moderate PVR as compared to 2DTEE‐based sizing (OR: 0.31, 95% CI: 0.18‐0.54, P

Differentiated vascular smooth muscle cells (VSMCs) are critical in maintaining vascular homeostasis by expressing a unique repertoire of contractile genes. Despite the well-defined coding transcriptome in differentiated VSMCs, little is known about the non-coding gene expression signature. Herein, by de novo analyzing publicly available RNA-seq and single cell RNA-seq datasets generated from different tissues and cell types, we unambiguously identifiedCARMN(CARdiac Mesoderm Enhancer-associated Non-coding RNA) as an evolutionarily conserved, SMC-specific lncRNA.CARMNwas initially annotated as the host gene ofMIR143/145cluster and recently reported to play roles in cardiac differentiation. Here, we generated aCarmnGFP knock-in reporter mouse model and confirmed its specific expression in SMCsin vivo. In addition, we foundCarmnis transcribed independently fromMir143/145and only expressed transiently in embryonic cardiomyocytes and thereafter becomes restricted to adult SMCs in both human and mouse. Furthermore, we demonstrated thatCARMNexpression is not only dramatically decreased in human vascular diseases but functionally critical in maintaining VSMC contractile phenotypein vitro. In conclusion, we provided the first evidence showing thatCARMNis an evolutionarily conserved SMC-specific lncRNA, down-regulated in different human vascular diseases, and a key lncRNA for maintaining SMC contractile phenotype.

Background: Pre-eclampsia (PE) is a severe obstetric complication associated with many cardiovascular disorders. Left atrial (LA) function and its relevance to the cardiovascular adaptation changes associated with PE have not been adequately addressed. The aim of this study was to quantify LA myocardial deformation characteristics in women with PE using speckle tracking echocardiography (STE). Methods: Using STE, LA global longitudinal strain (LAGLS), phasic LA strain values and LASr/(E/e'), which was the value of LA reservoir strain (LASr) divided by the quotient of early mitral flow velocity (E) and diastolic mitral ring velocity (e'), were compared among nonpregnant controls (n=39), normotensive pregnant women (n=40), and PE (n=43) patients. Results: LAGLS, LASr and LA conduit strain reduced in PE group in spite of no difference of LA contraction strain among the three groups. LASr/(E/e'), the surrogate of LA compliance, was decreased during PE pregnancy. The multivariate regression analyses showed LAGLS was independently correlated with LASr/(E/e'), left ventricular global longitudinal strain and LA maximum volume. Conclusion: PE cases undergo impaired LA myocardial function and decreased atrial compliance. STE-derived LA strain may be a powerful diagnostic parameter to evaluate diastolic function with the progression of PE pregnancy.

Additional file 12. Figure S7. Correlation of expression between 24 the most abundant circRNAs and their host genes. (A) CircRNAs with expression significantly correlated with the expression of host genes (Pearson correlation, p < 0.05). (B) CircRNAs with expression independent of their host genes (Pearson correlation, p > 0.05).

Additional file 9. Figure S5. Comparison of circRNAs with 2 unique back-spliced reads observed only once in this study with human circRNAs reported by 3 previous studies and 4 circRNA databases. (A) Four-set venn diagram showing the number of over-lapping circRNAs between the 12,099 circRNAs with 2 unique back-spliced reads observed only in one sample in this study, and human cardiac circRNAs reported by 3 previous studies. (B) Five-set venn diagram showing the number of over-lapping circRNAs between the 12,099 circRNAs with 2 unique back-spliced reads observed only in one sample in this study, and human circRNAs reported by 4 circRNA databases. (C) Three-set venn diagram showing the number of over-lapping circRNAs between the 12,099 circRNAs with 2 unique back-spliced reads observed only in one sample in this study, and 24,256 and 263,738 non-redundant circRNAs obtained by merging circRNAs reported by study of Werfel et al. (n = 16,427), Tan et al. (n = 15,303), and Van et al. (n = 8878), as well as 4 circRNA datasets including circBase (n = 91,986), circBank (n = 140,331), circRNADB (n = 32,883), and CIRCpedia (n = 183,943).

Additional file 4. Figure S2. Validation of the public RNA-seq dataset. (A) Principal Component Analysis (PCA) of the 10 samples (5 normal and 5 DCM hearts) using expression of genes with raw count > 10 in at least 5 samples. (B) Expression of NPPA in normal and DCM hearts from the RNA-seq dataset. PFKM: Fragments Per Kilobase of exon per Million fragments mapped. *p < 0.05 (edgeR).

Additional file 8. Figure S4. Relationship between numbers of circRNAs and exons of their host genes. (A) Schematic diagram of all the circRNAs originated from TTN and (B) RYR2 gene. Arrows point to the transcriptional directions of TTN and RYR2 genes. (C) The numbers of circRNAs increased proportionally with the numbers of exons per gene (binned to 10). (D) Distribution of exon numbers to produce circRNAs.

Additional file 7. Figure S3. Analysis of artifactual circRNAs from MYH6 and MYH7 gene. (A) Schematic diagram showing a number of rt-circRNAs originate from MYH6 and MYH7 genes in human heart identified in this study, study of Tan et al. [27] and Werfel et al. [29], and in (B) mouse heart identified in this study and study of Werfel et al. [29], as well as in (C) rat heart revealed in the study of Werfel et al. [29]. Arrows point to the transcriptional directions of MYH6 and MYH7 genes. (D) Heatmap illustrates the rank of the average expression across all studied samples for circRNAs produced from MYH6 and MYH7 genes in this study, and in human, mouse and rat hearts as identified by Tan et al. [27] and Werfel et al. [29]. The numbers in the colored boxes indicate the rank of the circRNAs within the indicated species. (E) Representative example of circMYH6_e35:MYH7_e37 that is likely artifact. The upper schematic diagram illustrating the formation of circMYH6_e35:MYH7_e37 due to read misalignment. The exon 35 of MYH6 (red, the donor exon) and exon 36 of MYH7 (yellow, upstream of the acceptor exon) are homologous. The exon 36 of MYH6 (green, downstream of the donor exon) and exon 37 of MYH7 (blue, the acceptor exon) are highly homologous. Considering a chimeric sequencing read that is supposedly to be aligned back to exon 35 (red line without arrow) and 36 (green line with arrow) of MYH6, due to the high sequence similarity, the down-stream part of the read (green line with arrow) may be misaligned to exon 37 of MYH7 (blue line with arrow), leading to the artifactual identification of back-spliced junction, and versa vice. Dashed lines indicate splicing junctions. The bottom panel shows the sequence alignment of linear MYH6_e35:MYH6_e36, MYH7_e36:MYH7_e37, and the predicted sequence of the circular MYH6_e35:MYH7_e37.

Heart failure is a leading cause of morbidity and mortality. Dilated cardiomyopathy (DCM) is a primary cause of heart failure and sudden cardiac death worldwide. Therefore, it is important to identify factors that function in both normal and diseased hearts for a better understanding of etiology of DCM. CircRNA is a newly discovered class of RNA molecule and has been found to play a critical role in both physiological and pathological conditions. In this study, we systematically analyzed whole transcriptome data of five normal and five DCM human heart samples to identify putative key circRNAs and predict their potential underlying mechanisms. Our results revealed that many circRNAs produced from known cardiac diseases-related gene loci are dysregulated in DCM, such as circRNAs originated from TTN and RYR2 . Furthermore, our analysis revealed 40 conserved circRNAs that are putatively important in normal or DCM human heart, including 14 circRNAs with potential to act as sponges for DCM-related miRNAs and 4 circRNAs with potential coding ability. Importantly, our results identified that a novel class of circRNA, fusion-circRNAs are originated from MYH6 and MYH7 genes and exhibit most abundant expression in human heart with high conservation between different species. Furthermore, these fusion-circRNAs were dysregulated in DCM and have strong potential to sponge DCM-related miRNAs. In summary, our findings serve as a valuable resource for exploration of the function of circRNAs in human heart diseases.

TEAD1 (TEA domain transcription factor 1), a transcription factor known for the functional output of Hippo signaling, is important for tumorigenesis. However, the role of TEAD1 in the development of vascular smooth muscle cell (VSMC) is unknown. To investigate cell-specific role of Tead1, we generated cardiomyocyte (CMC) and VSMC-specific Tead1 knockout mice. We found CMC/VSMC-specific deletion of Tead1 led to embryonic lethality by E14.5 in mice due to hypoplastic cardiac and vascular walls, as a result of impaired CMC and VSMC proliferation. Whole transcriptome analysis revealed that deletion of Tead1 in CMCs/VSMCs downregulated expression of muscle contractile genes and key transcription factors including Pitx2c and myocardin. In vitro studies demonstrated that PITX2c and myocardin rescued TEAD1-dependent defects in VSMC differentiation. We further identified Pitx2c as a novel transcriptional target of TEAD1, and PITX2c exhibited functional synergy with myocardin by directly interacting with myocardin, leading to augment the differentiation of VSMC. In summary, our study reveals a critical role of Tead1 in cardiovascular development in mice, but also identifies a novel regulatory mechanism, whereby Tead1 functions upstream of the genetic regulatory hierarchy for establishing smooth muscle contractile phenotype.

Background Acute ischemic stroke has been recognized as one key cause of vascular cognitive impairment (VCI). The purpose of this study was to evaluate the correlation between carotid artery stenosis and post VCI in acute ischemic stroke patients. Methods In this study, B-mode ultrasound was applied to measure the degree of carotid artery stenosis. After 1 year, the stroke patients’ cognitive function was assessed by the mini-mental state examination (MMSE) score. The relationship between the VCI and degree of carotid artery stenosis was evaluated by multivariate regression analysis. Results VCI was observed in 136 (37.2%) of the 365 participants. High degree of carotid artery stenosis was significantly correlated with VCI (p