PIBF1 regulates trophoblast syncytialization and promotes cardiovascular development.

Proper placental development in early pregnancy ensures a positive outcome later on. The developmental relationship between the placenta and embryonic organs, such as the heart, is crucial for a normal pregnancy. However, the mechanism through which the placenta influences the development of embryonic organs remains unclear. Trophoblasts fuse to form multinucleated syncytiotrophoblasts (SynT), which primarily make up the placental materno-fetal interface. We discovered that endogenous progesterone immunomodulatory binding factor 1 (PIBF1) is vital for trophoblast differentiation and fusion into SynT in humans and mice. PIBF1 facilitates communication between SynT and adjacent vascular cells, promoting vascular network development in the primary placenta. This process affected the early development of the embryonic cardiovascular system in mice. Moreover, in vitro experiments showed that PIBF1 promotes the development of cardiovascular characteristics in heart organoids. Our findings show how SynTs organize the barrier and imply their possible roles in supporting embryogenesis, including cardiovascular development. SynT-derived factors and SynT within the placenta may play critical roles in ensuring proper organogenesis of other organs in the embryo.

Generation of heart and vascular system in rodents by blastocyst complementation.

Generating organs from stem cells through blastocyst complementation is a promising approach to meet the clinical need for transplants. In order to generate rejection-free organs, complementation of both parenchymal and vascular cells must be achieved, as endothelial cells play a key role in graft rejection. Here, we used a lineage-specific cell ablation system to produce mouse embryos unable to form both the cardiac and vascular systems. By mouse intraspecies blastocyst complementation, we rescued heart and vascular system development separately and in combination, obtaining complemented hearts with cardiomyocytes and endothelial cells of exogenous origin. Complemented chimeras were viable and reached adult stage, showing normal cardiac function and no signs of histopathological defects in the heart. Furthermore, we implemented the cell ablation system for rat-to-mouse blastocyst complementation, obtaining xenogeneic hearts whose cardiomyocytes were completely of rat origin. These results represent an advance in the experimentation towards the in vivo generation of transplantable organs.

Cardiovascular fetal-to-neonatal transition: an in silico model.

BACKGROUND: Previous models describing the fetal-to-neonatal transition often lack oxygen saturation levels, homeostatic control mechanisms, phasic hemodynamic signals, or describe the heart with a time-varying elastance model. METHODS: We incorporated these elements in the adapted CircAdapt model with the one-fiber model for myocardial contraction, to simulate the hemodynamics of the healthy term human fetal circulation and its transition during the first 24 h after birth. The fetal-to-neonatal model was controlled by a time- and event-based script of changes occurring at birth, such as lung aeration and umbilical cord clamping. Model parameters were based on and validated with human and animal data. RESULTS: The fetal circulation showed low pulmonary blood flow, right ventricular dominance, and inverted mitral and tricuspid flow velocity patterns, as well as high mean ductus venosus flow velocity. The neonatal circulation showed oxygen saturation levels to gradually increase to 98% in the first 15 min after birth as well as temporary left ventricular volume overload. CONCLUSIONS: Hemodynamics of the term fetus and 24-h-old neonate, as well as the events occurring directly after birth and the transition during the first 24 h after birth, were realistically represented, allowing the model to be used for educational purposes and future research. IMPACT: With the addition of oxygen saturation levels, homeostatic pressure-flow control mechanisms, and the one-fiber model for myocardial contraction, a new closed-loop cardiovascular model was constructed to give more insight into the healthy term human fetal circulation and its cardiovascular transition during the first 24 h after birth. Extensive validation confirmed that the hemodynamics of the term fetus and the fetal-to-neonatal transition were realistically represented with the model. This well-validated and versatile model can serve as an education as well as a research platform for in silico investigation of fetal-to-neonatal hemodynamic changes under a wide range of physiological and pathophysiological conditions.

Long-Term Hypoxia Maintains a State of Dedifferentiation and Enhanced Stemness in Fetal Cardiovascular Progenitor Cells.

Early-stage mammalian embryos survive within a low oxygen tension environment and develop into fully functional, healthy organisms despite this hypoxic stress. This suggests that hypoxia plays a regulative role in fetal development that influences cell mobilization, differentiation, proliferation, and survival. The long-term hypoxic environment is sustained throughout gestation. Elucidation of the mechanisms by which cardiovascular stem cells survive and thrive under hypoxic conditions would benefit cell-based therapies where stem cell survival is limited in the hypoxic environment of the infarcted heart. The current study addressed the impact of long-term hypoxia on fetal Islet-1+ cardiovascular progenitor cell clones, which were isolated from sheep housed at high altitude. The cells were then cultured in vitro in 1% oxygen and compared with control Islet-1+ cardiovascular progenitor cells maintained at 21% oxygen. RT-PCR, western blotting, flow cytometry, and migration assays evaluated adaptation to long term hypoxia in terms of survival, proliferation, and signaling. Non-canonical Wnt, Notch, AKT, HIF-2α and Yap1 transcripts were induced by hypoxia. The hypoxic niche environment regulates these signaling pathways to sustain the dedifferentiation and survival of fetal cardiovascular progenitor cells.

Dynamic Expression of Membrane Type 1-Matrix Metalloproteinase (Mt1-mmp/Mmp14) in the Mouse Embryo.

MT1-MMP/MMP14 belongs to a subgroup of the matrix metalloproteinases family that presents a transmembrane domain, with a cytosolic tail and the catalytic site exposed to the extracellular space. Deficient mice for this enzyme result in early postnatal death and display severe defects in skeletal, muscle and lung development. By using a transgenic line expressing the LacZ reporter under the control of the endogenous Mt1-mmp promoter, we reported a dynamic spatiotemporal expression pattern for Mt1-mmp from early embryonic to perinatal stages during cardiovascular development and brain formation. Thus, Mt1-mmp shows expression in the endocardium of the heart and the truncus arteriosus by E8.5, and is also strongly detected during vascular system development as well as in endothelial cells. In the brain, LacZ reporter expression was detected in the olfactory bulb, the rostral cerebral cortex and the caudal mesencephalic tectum. LacZ-positive cells were observed in neural progenitors of the spinal cord, neural crest cells and the intersomitic region. In the limb, Mt1-mmp expression was restricted to blood vessels, cartilage primordium and muscles. Detection of the enzyme was confirmed by Western blot and immunohistochemical analysis. We suggest novel functions for this metalloproteinase in angiogenesis, endocardial formation and vascularization during organogenesis. Moreover, Mt1-mmp expression revealed that the enzyme may contribute to heart, muscle and brain throughout development.

In Vivo Characterization of Endogenous Cardiovascular Extracellular Vesicles in Larval and Adult Zebrafish.

Objective: Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterization of endogenously produced EVs is still in its infancy. Because of their genetic tractability and live imaging amenability, zebrafish represent an ideal but under-used model to investigate endogenous EVs. We aimed to establish a transgenic zebrafish model to allow the in vivo identification, tracking, and extraction of endogenous EVs produced by different cell types. Approach and Results: Using a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labeled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Additionally, cryo electron microscopy reveals the full morphological diversity of larval and adult EVs. Importantly, we demonstrate the utility of this model by showing that different cell types exchange EVs in the adult heart and that ischemic injury models dynamically alter EV production. Conclusions: We describe a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labeling approach allows cell-type specific tracing of EV origin without bias toward the expression of individual protein markers and will allow detailed future examination of their function.

When form meets function: the cells and signals that shape the lymphatic vasculature during development.

The lymphatic vasculature is an integral component of the cardiovascular system. It is essential to maintain tissue fluid homeostasis, direct immune cell trafficking and absorb dietary lipids from the digestive tract. Major advances in our understanding of the genetic and cellular events important for constructing the lymphatic vasculature during development have recently been made. These include the identification of novel sources of lymphatic endothelial progenitor cells, the recognition of lymphatic endothelial cell specialisation and heterogeneity, and discovery of novel genes and signalling pathways underpinning developmental lymphangiogenesis. Here, we review these advances and discuss how they inform our understanding of lymphatic network formation, function and dysfunction.

Maternal iron deficiency perturbs embryonic cardiovascular development in mice.

Congenital heart disease (CHD) is the most common class of human birth defects, with a prevalence of 0.9% of births. However, two-thirds of cases have an unknown cause, and many of these are thought to be caused by in utero exposure to environmental teratogens. Here we identify a potential teratogen causing CHD in mice: maternal iron deficiency (ID). We show that maternal ID in mice causes severe cardiovascular defects in the offspring. These defects likely arise from increased retinoic acid signalling in ID embryos. The defects can be prevented by iron administration in early pregnancy. It has also been proposed that teratogen exposure may potentiate the effects of genetic predisposition to CHD through gene-environment interaction. Here we show that maternal ID increases the severity of heart and craniofacial defects in a mouse model of Down syndrome. It will be important to understand if the effects of maternal ID seen here in mice may have clinical implications for women.

Relevance of N6-methyladenosine regulators for transcriptome: Implications for development and the cardiovascular system.

N6-methyladenosine (m6A) is the most abundant and well-studied internal modification of messenger RNAs among the various RNA modifications in eukaryotic cells. Moreover, it is increasingly recognized to regulate non-coding RNAs. The dynamic and reversible nature of m6A is ensured by the precise and coordinated activity of specific proteins able to insert ("write"), bind ("read") or remove ("erase") the m6A modification from coding and non-coding RNA molecules. Mounting evidence suggests a pivotal role for m6A in prenatal and postnatal development and cardiovascular pathophysiology. In the present review we summarise and discuss the major functions played by m6A RNA methylation and its components particularly referring to the cardiovascular system. We present the methods used to study m6A and the most abundantly methylated RNA molecules. Finally, we highlight the possible involvement of the m6A mark in cardiovascular disease as well as the need for further studies to better describe the mechanisms of action and the potential therapeutic role of this RNA modification.

Acute exposure to N-Ethylpentylone induces developmental toxicity and dopaminergic receptor-regulated aberrances in zebrafish larvae.

N-Ethylpentylone (NEP) is one of the most recent novel stimulants, and there is limited understanding of its toxicity. Here we employed zebrafish model for analyzing the effects of NEP on early embryos and cardiovascular and nervous systems at late developmental stages. We first observed multi-malformations in early embryos and larvae after NEP administration, together with significant deregulations of brain and heart development-associated genes (neurog1, her6, elavl3, nkx2.5, nppa, nppb, tnnt2a) at transcriptional level. Low-dosed NEP treatment induced an anxiety-like phenotype in zebrafish larvae, while higher doses of NEP exerted an inhibitory effect on locomotion and heart rate. Besides, the expression of th (tyrosine hydroxylase) and th2 (tyrosine hydroxylase 2), identifying dopamine (DA) release, were significantly increased during one-hour free swimming after effective low-dosed NEP administration, along with the upregulation of gene fosab and fosb related to stress and anxiety response. D1R antagonist SCH23390 and D2R antagonist sulpiride partially alleviated the aberrances of locomotion and heart rate, indicating dopaminergic receptors were involved in the bidirectional dosage-dependent pattern of NEP-induced performance. Meanwhile, sulpiride offset the upregulated expression of th, th2 and fosab in the group of 1.5 µM NEP, which highlighted the significant role of D2R in NEP-induced locomotive effects. This study systematically described the developmental, neuronal and cardiac toxicity of NEP in zebrafish, and identified the dopaminergic receptors as one of the downstream effectors of NEP administration.

A zebrafish toolbox for biomechanical signaling in cardiovascular development and disease.

PURPOSE OF REVIEW: The zebrafish embryo has emerged as a powerful model organism to investigate the mechanisms by which biophysical forces regulate vascular and cardiac cell biology during development and disease. A versatile arsenal of methods and tools is available to manipulate and analyze biomechanical signaling. This review aims to provide an overview of the experimental strategies and tools that have been utilized to study biomechanical signaling in cardiovascular developmental processes and different vascular disease models in the zebrafish embryo. Within the scope of this review, we focus on work published during the last two years. RECENT FINDINGS: Genetic and pharmacological tools for the manipulation of cardiac function allow alterations of hemodynamic flow patterns in the zebrafish embryo and various types of transgenic lines are available to report endothelial cell responses to biophysical forces. These tools have not only revealed the impact of biophysical forces on cardiovascular development but also helped to establish more accurate models for cardiovascular diseases including cerebral cavernous malformations, hereditary hemorrhagic telangiectasias, arteriovenous malformations, and lymphangiopathies. SUMMARY: The zebrafish embryo is a valuable vertebrate model in which in-vivo manipulations of biophysical forces due to cardiac contractility and blood flow can be performed. These analyses give important insights into biomechanical signaling pathways that control endothelial and endocardial cell behaviors. The technical advances using this vertebrate model will advance our understanding of the impact of biophysical forces in cardiovascular pathologies.

The Way to Man's Heart Is through the Stomach.

Organ systems do not exist in a vacuum. However, in an era of increasingly specialized medicine, the focus is often on the organ system alone. Many symptoms are associated with differential diagnoses from upper gastrointestinal (GI) and cardiovascular medical and surgical specialties. Furthermore, a large number of rare but deadly conditions cross paths between the upper GI tract and cardiovascular system; a significant proportion of these are iatrogenic injuries from a parallel specialty. These include unusual fistulae, herniae, and embolisms that transcend specialties. This review highlights these conditions and the shared anatomy and embryology of the two organ systems.

Exposure to the natural alkaloid Berberine affects cardiovascular system morphogenesis and functionality during zebrafish development.

The plant-derived natural alkaloid berberine displays therapeutic potential to treat several pathological conditions, including dyslipidemias, diabetes and cardiovascular disorders. However, data on berberine effects during embryonic development are scarce and in part controversial. In this study, using zebrafish embryos as vertebrate experimental model, we address the effects of berberine treatment on cardiovascular system development and functionality. Starting from the observation that berberine induces developmental toxicity and pericardial edema in a time- and concentration-dependent manner, we found that treated embryos display cardiac looping defects and, at later stages, present an abnormal heart characterized by a stretched morphology and atrial endocardial/myocardial detachment. Furthermore, berberine affected cardiac functionality of the embryos, promoting bradycardia and reducing the cardiac output, the atrial shortening fraction percentage and the atrial stroke volume. We also found that, during development, berberine interferes with the angiogenic process, without altering vascular permeability. These alterations are associated with increased levels of vascular endothelial growth factor aa (vegfaa) mRNA, suggesting an important role for Vegfaa as mediator of berberine-induced cardiovascular defects. Altogether, these data indicate that berberine treatment during vertebrate development leads to an impairment of cardiovascular system morphogenesis and functionality, suggesting a note of caution in its use during pregnancy and lactation.

Fluid flow as a driver of embryonic morphogenesis.

Fluid flow is a powerful morphogenic force during embryonic development. The physical forces created by flowing fluids can either create morphogen gradients or be translated by mechanosensitive cells into biological changes in gene expression. In this Primer, we describe how fluid flow is created in different systems and highlight the important mechanosensitive signalling pathways involved for sensing and transducing flow during embryogenesis. Specifically, we describe how fluid flow helps establish left-right asymmetry in the early embryo and discuss the role of flow of blood, lymph and cerebrospinal fluid in sculpting the embryonic cardiovascular and nervous system.

Early Life Oxidative Stress and Long-Lasting Cardiovascular Effects on Offspring Conceived by Assisted Reproductive Technologies: A Review.

Assisted reproductive technology (ART) has rapidly developed and is now widely practised worldwide. Both the characteristics of ART (handling gametes/embryos in vitro) and the infertility backgrounds of ART parents (such as infertility diseases and unfavourable lifestyles or diets) could cause increased oxidative stress (OS) that may exert adverse influences on gametogenesis, fertilisation, and foetation, even causing a long-lasting influence on the offspring. For these reasons, the safety of ART needs to be closely examined. In this review, from an ART safety standpoint, the origins of OS are reviewed, and the long-lasting cardiovascular effects and potential mechanisms of OS on the offspring are discussed.

Endothelial cells and angiogenesis in the horse in health and disease-A review.

The cardiovascular system is the first functional organ in the embryo, and its blood vessels form a widespread conductive network within the organism. Blood vessels develop de novo, by the differentiation of endothelial progenitor cells (vasculogenesis) or by angiogenesis, which is the formation of new blood vessels from existing ones. This review presents an overview of the current knowledge on physiological and pathological angiogenesis in the horse including studies on equine endothelial cells. Principal study fields in equine angiogenesis research were identified: equine endothelial progenitor cells; equine endothelial cells and angiogenesis (heterogeneity, markers and assessment); endothelial regulatory molecules in equine angiogenesis; angiogenesis research in equine reproduction (ovary, uterus, placenta and conceptus, testis); angiogenesis research in pathological conditions (tumours, ocular pathologies, equine wound healing, musculoskeletal system and laminitis). The review also includes a table that summarizes in vitro studies on equine endothelial cells, either describing the isolation procedure or using previously isolated endothelial cells. A particular challenge of the review was that results published are fragmentary and sometimes even contradictory, raising more questions than they answer. In conclusion, angiogenesis is a major factor in several diseases frequently occurring in horses, but relatively few studies focus on angiogenesis in the horse. The challenge for the future is therefore to continue exploring new therapeutic angiogenesis strategies for horses to fill in the missing pieces of the puzzle.

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia (HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization - WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 °C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 °C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Growth and cardiovascular development are repressed by florfenicol exposure in early chicken embryos.

Florfenicol (FLO) is one of the most popular antibacterial drugs used in veterinary clinics and aquaculture. The drug was found to decrease the hatchability of eggs laid by treated hens in veterinary clinics and research work. However, the pathological changes in developing embryos and their cardiovascular system and the mechanism underlying FLO-induced embryonic death remain unclear. In the present study, fertilized eggs laid by hens treated with a therapeutic dose of FLO were collected and incubated. Results showed that FLO exposure repressed embryonic development and induced early embryonic death. As a result, FLO decreased the hatchability and increased the proportion of weak chicks. Moreover, FLO exposure led to embryonic lethality and inhibited the development of chick embryos as characterized by decreased weights, lagging distribution of Hamburger-Hamilton stages, and dysplastic eyes. Pathological examination indicated that FLO exposure affected the normal development of the heart in 4.5-day-old chick embryos, as characterized by shorter transverse cardiac diameter, disordered arrangement of trabecular muscles in ventricles, and reduced thickness of ventricular walls. Furthermore, FLO decreased blood vascular densities and downregulated the expression levels of key angiogenesis-related genes, including the vascular endothelial growth factor and fibroblast growth factor, in the yolk sac membrane. These findings indicated that FLO exposure restricted vascular development during early embryonic development. In summary, our data suggest that the restricted growth and abnormal cardiovascular development may be responsible for FLO-induced early embryonic death. Thus, these findings can be useful for guiding the proper use of FLO and in laying a foundation for further studies on the mechanism of FLO-induced embryonic toxicity.

Significance of sphingosine-1-phosphate in cardiovascular physiology and pathology.

Sphingosine-1-phosphate (S1P) is a signaling lipid, synthetized by sphingosine kinases (SPHK1 and SPHK2), that affects cardiovascular function in various ways. S1P signaling is complex, particularly since its molecular action is reliant on the differential expression of its receptors (S1PR1, S1PR2, S1PR3, S1PR4, S1PR5) within various tissues. Significance of this sphingolipid is manifested early in vertebrate development as certain defects in S1P signaling result in embryonic lethality due to defective vasculo- or cardiogenesis. Similar in the mature organism, S1P orchestrates both physiological and pathological processes occurring in the heart and vasculature of higher eukaryotes. S1P regulates cell fate, vascular tone, endothelial function and integrity as well as lymphocyte trafficking, thus disbalance in its production and signaling has been linked with development of such pathologies as arterial hypertension, atherosclerosis, endothelial dysfunction and aberrant angiogenesis. Number of signaling mechanisms are critical - from endothelial nitric oxide synthase through STAT3, MAPK and Akt pathways to HDL particles involved in redox and inflammatory balance. Moreover, S1P controls both acute cardiac responses (cardiac inotropy and chronotropy), as well as chronic processes (such as apoptosis and hypertrophy), hence numerous studies demonstrate significance of S1P in the pathogenesis of hypertrophic/fibrotic heart disease, myocardial infarction and heart failure. This review presents current knowledge concerning the role of S1P in the cardiovascular system, as well as potential therapeutic approaches to target S1P signaling in cardiovascular diseases.