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The epicardium, the mesothelial envelope of the vertebrate heart, is the source of multiple cardiac cell lineages during embryonic development and provides signals that are essential to myocardial growth and repair. Here we generate self-organizing human pluripotent stem cell-derived epicardioids that display retinoic acid-dependent morphological, molecular and functional patterning of the epicardium and myocardium typical of the left ventricular wall. By combining lineage tracing, single-cell transcriptomics and chromatin accessibility profiling, we describe the specification and differentiation process of different cell lineages in epicardioids and draw comparisons to human fetal development at the transcriptional and morphological levels. We then use epicardioids to investigate the functional cross-talk between cardiac cell types, gaining new insights into the role of IGF2/IGF1R and NRP2 signaling in human cardiogenesis. Finally, we show that epicardioids mimic the multicellular pathogenesis of congenital or stress-induced hypertrophy and fibrotic remodeling. As such, epicardioids offer a unique testing ground of epicardial activity in heart development, disease and regeneration. The lineage of human epicardium is studied using time course single-cell analysis of epicardioids. [ABSTRACT FROM AUTHOR]

Cardiogenic growth factors play important roles in heart development. Placental growth factor (PLGF) has previously been reported to have angiogenic effects; however, its potential role in cardiogenesis has not yet been determined. We analyze single-cell RNA-sequencing data derived from human and primate embryonic hearts and find PLGF shows a biphasic expression pattern, as it is expressed specifically on ISL1+ second heart field progenitors at an earlier stage and on vascular smooth muscle cells (SMCs) and endothelial cells (ECs) at later stages. Using chemically modified mRNAs (modRNAs), we generate a panel of cardiogenic growth factors and test their effects on enhancing cardiomyocyte (CM) and EC induction during different stages of human embryonic stem cell (hESC) differentiations. We discover that only the application of PLGF modRNA at early time points of hESC-CM differentiation can increase both CM and EC production. Conversely, genetic deletion of PLGF reduces generation of CMs, SMCs and ECs in vitro. We also confirm in vivo beneficial effects of PLGF modRNA for development of human heart progenitor-derived cardiac muscle grafts on murine kidney capsules. Further, we identify the previously unrecognized PLGF-related transcriptional networks driven by EOMES and SOX17. These results shed light on the dual cardiomyogenic and vasculogenic effects of PLGF during heart development. Growth factors play key roles during heart development. Here they show that PLGF has both autocrine and paracrine roles during cardiomyogenesis and vasculogenesis, suggesting it may have therapeutic potential for heart disease. [ABSTRACT FROM AUTHOR]

Aims Retinoic acid (RA) signalling is essential for heart development, and dysregulation of the RA signalling can cause several types of cardiac outflow tract (OFT) defects, the most frequent congenital heart disease (CHD) in humans. Matthew-Wood syndrome is caused by inactivating mutations of a transmembrane protein gene STRA6 that transports vitamin A (retinol) from extracellular into intracellular spaces. This syndrome shows a broad spectrum of malformations including CHD, although murine Stra6 -null neonates did not exhibit overt heart defects. Thus, the detailed mechanisms by which STRA6 mutations could lead to cardiac malformations in humans remain unclear. Here, we investigated the role of STRA6 in the context of human cardiogenesis and CHD. Methods and results To gain molecular signatures in species-specific cardiac development, we first compared single-cell RNA sequencing (RNA-seq) datasets, uniquely obtained from human and murine embryonic hearts. We found that while STRA6 mRNA was much less frequently expressed in murine embryonic heart cells derived from the Mesp1+ lineage tracing mice (Mesp1Cre/+ ; Rosa26tdTomato), it was expressed predominantly in the OFT region-specific heart progenitors in human developing hearts. Next, we revealed that STRA6 -knockout human embryonic stem cells (hESCs) could differentiate into cardiomyocytes similarly to wild-type hESCs, but could not differentiate properly into mesodermal nor neural crest cell-derived smooth muscle cells (SMCs) in vitro. This is supported by the population RNA-seq data showing down-regulation of the SMC-related genes in the STRA6 -knockout hESC-derived cells. Further, through machinery assays, we identified the previously unrecognized interaction between RA nuclear receptors RARα/RXRα and TBX1, an OFT-specific cardiogenic transcription factor, which would likely act downstream to STRA6-mediated RA signalling in human cardiogenesis. Conclusion Our study highlights the critical role of human-specific STRA6 progenitors for proper induction of vascular SMCs that is essential for normal OFT formation. Thus, these results shed light on novel and human-specific CHD mechanisms, driven by STRA6 mutations. [ABSTRACT FROM AUTHOR]

Cardiac organoids are in vitro self-organizing and three-dimensional structures composed of multiple cardiac cells (i.e., cardiomyocytes, endothelial cells, cardiac fibroblasts, etc.) with or without biological scaffolds. Since cardiac organoids recapitulate structural and functional characteristics of the native heart to a higher degree compared to the conventional two-dimensional culture systems, their applications, in combination with pluripotent stem cell technologies, are being widely expanded for the investigation of cardiogenesis, cardiac disease modeling, drug screening and development, and regenerative medicine. In this mini-review, recent advances in cardiac organoid technologies are summarized in chronological order, with a focus on the methodological points for each organoid formation. Further, the current limitations and the future perspectives in these promising systems are also discussed. [ABSTRACT FROM AUTHOR]

The human heart has the least regenerative capabilities among tissues and organs, and heart disease continues to be a leading cause of mortality in the industrialized world with insufficient therapeutic options and poor prognosis. Therefore, developing new therapeutic strategies for heart regeneration is a major goal in modern cardiac biology and medicine. Recent advances in stem cell biology and biotechnologies such as human pluripotent stem cells (hPSCs) and cardiac tissue engineering hold great promise for opening novel paths to heart regeneration and repair for heart disease, although these areas are still in their infancy. In this review, we summarize and discuss the recent progress in cardiac tissue engineering strategies, highlighting stem cell engineering and cardiomyocyte maturation, development of novel functional biomaterials and biofabrication tools, and their therapeutic applications involving drug discovery, disease modeling, and regenerative medicine for heart disease. [ABSTRACT FROM AUTHOR]

Major cardiovascular events including myocardial infarction (MI) continue to dominate morbidity rates in the developed world. Although multiple device therapies and various pharmacological agents have been shown to improve patient care and reduce mortality rates, clinicians and researchers alike still lack a true panacea to regenerate damaged cardiac tissue. Over the previous two to three decades, cardiovascular stem cell therapies have held great promise. Several stem cell-based approaches have now been shown to improve ventricular function and are documented in preclinical animal models as well as phase I and phase II clinical trials. More recently, the cardiac progenitor cell has begun to gain momentum as an ideal candidate for stem cell therapy in heart disease. Here, we will highlight the most recent advances in cardiac stem/progenitor cell biology in regard to both the basics and applied settings. [ABSTRACT FROM AUTHOR]

The latest discoveries and advanced knowledge in the fields of stem cell biology and developmental cardiology hold great promise for cardiac regenerative medicine, enabling researchers to design novel therapeutic tools and approaches to regenerate cardiac muscle for diseased hearts. However, progress in this arena has been hampered by a lack of reproducible and convincing evidence, which at best has yielded modest outcomes and is still far from clinical practice. To address current controversies and move cardiac regenerative therapeutics forward, it is crucial to gain a deeper understanding of the key cellular and molecular programs involved in human cardiogenesis and cardiac regeneration. In this review, we consider the fundamental principles that govern the 'programming' and 'reprogramming' of a human heart cell and discuss updated therapeutic strategies to regenerate a damaged heart. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC+CD31+CD34+CD14−KDRhigh endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR+ mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs. [ABSTRACT FROM AUTHOR]

Aims Angiotensin-converting enzyme 2 (ACE2) is known as a negative regulator of the renin–angiotensin system. We aimed to determine the roles of ACE2 on the development of vascular diseases. Methods and results Using two diversely different models of vascular diseases, hyperlipidaemia-induced atherosclerosis in apolipoprotein E knockout (KO) mice and mechanical injury-induced arterial neointimal hyperplasia in C57Bl6 mice, we examined whether ACE2 deficiency could affect formation of the vascular lesions. ACE2 deficiency resulted in significantly larger vascular lesions in both aortic atherosclerotic plaques and arterial neointima formation, compared with ACE2+ control. These ACE2-deficient vascular lesions exhibited enhanced accumulation of macrophages into the lesions and proliferation of vascular smooth muscle cells (VSMCs), accompanied with increased angiotensin-II (Ang-II) levels and enhanced expression of vascular inflammation-related genes, including vascular cell adhesion molecule (VCAM)-1, monocyte chemoattractant protein (MCP)-1, and matrix metalloproteinase (MMP)9 in aorta/artery tissues. Primary bone marrow macrophages and aortic VSMCs isolated from ACE2 KO mice also displayed enhanced pro-inflammatory responsiveness such as up-regulated gene/protein expression of VCAM-1, MCP-1, and MMP9 to stimulation with tumour necrosis factor-α and Ang-II. The similar phenotype was shown in human macrophages and aortic VSMCs that were transfected with ACE2-specific siRNA. In ACE2-deficient VSMCs, inhibition of c-Jun N-terminal kinase (JNK) by pharmacological blockade with SP600125 or genetic knockdown with JNK-specific siRNA significantly attenuated their pro-inflammatory phenotype. Conclusion ACE2 deficiency promotes the development of vascular diseases associated with Ang-II-mediated vascular inflammation and activation of the JNK signalling, leading to the notion that ACE2 potentially confers protection against vascular diseases. [ABSTRACT FROM AUTHOR]

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart. [ABSTRACT FROM AUTHOR]

Most of the mammalian heart is formed from mesodermal progenitors in the first and second heart fields (FHF and SHF), whereby the FHF gives rise to the left ventricle and parts of the atria and the SHF to the right ventricle, outflow tract and parts of the atria. Whereas SHF progenitors have been characterized in detail, using specific molecular markers, comprehensive studies on the FHF have been hampered by the lack of exclusive markers. Here, we present Hcn4 (hyperpolarization-activated cyclic nucleotide-gated channel 4) as an FHF marker. Lineage-traced Hcn4+/FHF cells delineate FHF-derived structures in the heart and primarily contribute to cardiomyogenic cell lineages, thereby identifying an early cardiomyogenic progenitor pool. As a surface marker, HCN4 also allowed the isolation of cardiomyogenic Hcn4+/FHF progenitors from human embryonic stem cells. We conclude that a primary purpose of the FHF is to generate cardiac muscle and support the contractile activity of the primitive heart tube, whereas SHF-derived progenitors contribute to heart cell lineage diversification. [ABSTRACT FROM AUTHOR]

Background: An antianginal KATP channel opener nicorandil has various beneficial effects on cardiovascular systems; however, its effects on pulmonary vasculature under pulmonary arterial hypertension (PAH) have not yet been elucidated. Therefore, we attempted to determine whether nicorandil can attenuate monocrotaline (MCT)-induced PAH in rats. Materials and Methods: Sprague-Dawley rats injected intraperitoneally with 60 mg/kg MCT were randomized to receive either vehicle; nicorandil (5.0 mg·kg-1·day-1) alone; or nicorandil as well as either a KATP channel blocker glibenclamide or a nitric oxide synthase (NOS) inhibitor N®-nitro-L-arginine methyl ester (L-NAME), from immediately or 21 days after MCT injection. Four or five weeks later, right ventricular systolic pressure (RVSP) was measured, and lung tissue was harvested. Also, we evaluated the nicorandil-induced anti-apoptotic effects and activation status of several molecules in cell survival signaling pathway in vitro using human umbilical vein endothelial cells (HUVECs). Results: Four weeks after MCT injection, RVSP was significantly increased in the vehicle-treated group (51.0±4.7 mm Hg), whereas it was attenuated by nicorandil treatment (33.2±3.9 mm Hg; P<0.01). Nicorandil protected pulmonary endothelium from the MCT-induced thromboemboli formation and induction of apoptosis, accompanied with both upregulation of endothelial NOS (eNOS) expression and downregulation of cleaved caspase-3 expression. Late treatment with nicorandil for the established PAH was also effective in suppressing the additional progression of PAH. These beneficial effects of nicorandil were blocked similarly by glibenclamide and L-NAME. Next, HUVECs were incubated in serum-free medium and then exhibited apoptotic morphology, while these changes were significantly attenuated by nicorandil administration. Nicorandil activated the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in HUVECs, accompanied with the upregulation of both eNOS and Bcl-2 expression. Conclusions: Nicorandil attenuated MCT-induced vascular endothelial damage and PAH through production of eNOS and anti-apoptotic factors, suggesting that nicorandil might have a promising therapeutic potential for PAH. [ABSTRACT FROM AUTHOR]

The Bartonella species have been recently recognized as important causative agents of culture-negative bacterial endocarditis. Antineutrophil cytoplasmic antibodies (ANCAs) have been associated with the spectrum of idiopathic small vessel vasculitis. However, a variety of infections can result in a false-positive ANCA test, and especially subacute bacterial endocarditis (SBE) with the presence of ANCAs occasionally mimics the clinical manifestations of an ANCA-associated vasculitis such as skin purpura and glomerulonephritis. In contrast, noninfectious endocardial involvement is known to be part of the spectrum of the manifestations of the ANCA-associated vasculitis. Therefore, it is crucial to distinguish an ANCA-positive SBE from an ANCA-associated vasculitis with endocardial compromise, because the misdiagnosis of an SBE as an ANCA-associated vasculitis can lead to an inappropriate immunosuppressive therapy with catastrophic consequences. The differential diagnosis is sometimes difficult, especially in the case of culture-negative infective endocarditis with a positive ANCA test. We describe here a case of a culture-negative SBE caused by Bartonellaquintana, accompanied with a positive cytoplasmic ANCA test and clinical findings masquerading as ANCA-associated vasculitis. Both a serological test for Bartonella and polymerase chain reaction restriction fragment length polymorphism analysis were helpful for a correct diagnosis and appropriate treatment. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Aims Although various predictors of in-stent restenosis (ISR) have been reported, the subject of parameters relating to ISR on intravascular ultrasound (IVUS) still leaves room for discussion. The aim of this study was to clarify the strongest predictors of ISR using IVUS. Methods and results Ninety-two native coronary lesions undergoing single bare-metallic stent implantation were investigated retrospectively. We classified them into the ISR (n=46) and non-ISR (n=46) groups using quantitative coronary angiography. On serial IVUS studies, plaque morphology, and areas and volumes of each component in vessel were evaluated. Among all parameters, diabetes mellitus and soft plaque appearing hypoechoic on IVUS were associated with ISR. By multivariate analysis, soft plaque was the only independent predictor of ISR (p=0.0057). Compared with non-soft plaque, soft plaque had a larger plaque reduction rate (–7.1% vs. –1.6%, p=0.0613) and smaller percent plaque volume (53.0% vs. 55.5%, p=0.0273) after stenting. Conversely, soft plaque had a larger neointimal area (4.39 vs. 3.33 mm2, p=0.0437) and percent plaque area (80.5% vs. 75.1%, p=0.0503) at follow-up. Conclusion Soft plaque detected on IVUS was the strongest predictor of ISR. Soft plaque was compressed more easily by stenting, however, causing more proliferation of neointima subsequently and resulted in a worse prognosis. [ABSTRACT FROM PUBLISHER]

The advent of pluripotent stem cell biology and facile genetic manipulation via CRISPR technology has ushered in a new era of human disease models for drug discovery and development. While these precision "super models" hold great promise for tailoring personalized therapy, their full potential and in vivo validation have remained elusive. [ABSTRACT FROM AUTHOR]

Corrections to the article "Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells" that was published in the 2015 issue are presented.

A 77-year-old woman, who had suffered from osteoporosis with compression fractures of vertebrae, was admitted to our hospital because of increasing bone pain. She had anemia and monocytosis, and was diagnosed as chronic myelomonocytic leukemia (CMML). Although chemotherapy was initiated, she died from pneumonia. Autopsy revealed multiple tumors composed of megakaryoblastic cells in her bone marrow and extramedullary organs and, therefore, the pathological diagnosis was made as megakaryoblastic transformation of CMML. Severe bone pain was considered to be a symptom of megakaryoblastic transformation of CMML. [ABSTRACT FROM AUTHOR]