Extension of Endocardium-Derived Vessels Generate Coronary Arteries in Neonates.

BACKGROUND: Unraveling how new coronary arteries develop may provide critical information for establishing novel therapeutic approaches to treating ischemic cardiac diseases. There are 2 distinct coronary vascular populations derived from different origins in the developing heart. Understanding the formation of coronary arteries may provide insights into new ways of promoting coronary artery formation after myocardial infarction. METHODS: To understand how intramyocardial coronary arteries are generated to connect these 2 coronary vascular populations, we combined genetic lineage tracing, light sheet microscopy, fluorescence micro-optical sectioning tomography, and tissue-specific gene knockout approaches to understand their cellular and molecular mechanisms. RESULTS: We show that a subset of intramyocardial coronary arteries form by angiogenic extension of endocardium-derived vascular tunnels in the neonatal heart. Three-dimensional whole-mount fluorescence imaging showed that these endocardium-derived vascular tunnels or tubes adopt an arterial fate in neonates. Mechanistically, we implicate Mettl3 (methyltransferase-like protein 3) and Notch signaling in regulating endocardium-derived intramyocardial coronary artery formation. Functionally, these intramyocardial arteries persist into adulthood and play a protective role after myocardial infarction. CONCLUSIONS: A subset of intramyocardial coronary arteries form by extension of endocardium-derived vascular tunnels in the neonatal heart.

Coronary blood vessels from distinct origins converge to equivalent states during mouse and human development.

Most cell fate trajectories during development follow a diverging, tree-like branching pattern, but the opposite can occur when distinct progenitors contribute to the same cell type. During this convergent differentiation, it is unknown if cells 'remember' their origins transcriptionally or whether this influences cell behavior. Most coronary blood vessels of the heart develop from two different progenitor sources-the endocardium (Endo) and sinus venosus (SV)-but whether transcriptional or functional differences related to origin are retained is unknown. We addressed this by combining lineage tracing with single-cell RNA sequencing (scRNAseq) in embryonic and adult mouse hearts. Shortly after coronary development begins, capillary endothelial cells (ECs) transcriptionally segregated into two states that retained progenitor-specific gene expression. Later in development, when the coronary vasculature is well established but still remodeling, capillary ECs again segregated into two populations, but transcriptional differences were primarily related to tissue localization rather than lineage. Specifically, ECs in the heart septum expressed genes indicative of increased local hypoxia and decreased blood flow. Adult capillary ECs were more homogeneous with respect to both lineage and location. In agreement, SV- and Endo-derived ECs in adult hearts displayed similar responses to injury. Finally, scRNAseq of developing human coronary vessels indicated that the human heart followed similar principles. Thus, over the course of development, transcriptional heterogeneity in coronary ECs is first influenced by lineage, then by location, until heterogeneity declines in the homeostatic adult heart. These results highlight the plasticity of ECs during development, and the validity of the mouse as a model for human coronary development.

Dach1 Extends Artery Networks and Protects Against Cardiac Injury.

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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.

Fsp1 cardiac embryonic expression delineates atrioventricular endocardial cushion, coronary venous and lymphatic valve development.

Fsp1 (a.k.a S100A4 or Metastatin) is an intracellular and secreted protein widely regarded as a fibroblast marker. Recent studies have nonetheless shown that Fsp1 is also expressed by other cell types, including small subsets of endothelial cells. Since no detailed and systematic description of Fsp1 spatio-temporal expression pattern in cardiac vascular cells is available in the literature, we have used a transgenic murine line (Fsp1-GFP) to study Fsp1 expression in the developing and postnatal cardiac vasculature and endocardium. Our work shows that Fsp1 is expressed in the endocardium and mesenchyme of atrioventricular valve primordia, as well as in some coronary venous and lymphatic endothelial cells. Fsp1 expression in cardiac venous and lymphatic endothelium is progressively restricted to the leaflets of cardiac venous and lymphatic valves. Our results suggest that Fsp1 could play a role in the development of atrioventricular valves and participate in the patterning and morphogenesis of cardiac venous and lymphatic vessel valves.

Dynamic changes in endoglin expression in the developing mouse heart.

Endoglin (ENG) is essential for cardiovascular development and is expressed in the heart from its earliest developmental stages. ENG expression has been reported in the cardiac crescent, endocardium, valve mesenchyme and coronary vascular endothelial cells. However, its expression in these cell types is non-uniform and the dynamic changes in ENG expression during heart development have not been systematically studied. Using immunofluorescent staining we tracked ENG protein expression in mouse embryonic hearts aged from 11.5 to 17.5 days, and in postnatal and adult hearts. ENG is expressed in the endocardium and in venous endothelial cells throughout these developmental stages. ENG protein is down-regulated by approximately two-fold as a subset of early coronary veins reprogram to form arteries within the developing myocardium from E13.5. This two-fold higher ratio of ENG protein in veins versus arteries is maintained throughout cardiac development and in the adult heart. ENG is also down-regulated two-fold following mesenchymal transition of endocardial cells to form cardiac valve mesenchyme, whilst expression of the pan-endothelial marker CD31 is completely lost. A subset of epicardial cells (which do not express ENG protein) delaminate and undergo a similar mesenchymal transition to form epicardially derived cells (EPDCs). This transient intra-myocardial mesenchymal cell population expresses low levels of ENG protein, similar to valve mesenchyme. In conclusion, ENG shows dynamic changes of expression in vascular endothelial cells, endocardial cells and mesenchymal cells in the developing heart that vary according to cardiovascular cell type.

Three-dimensional alignment of microvasculature and cardiomyocytes in the developing ventricle.

While major coronary artery development and pathologies affecting them have been extensively studied, understanding the development and organization of the coronary microvasculature beyond the earliest developmental stages requires new tools. Without techniques to image the coronary microvasculature over the whole heart, it is likely we are underestimating the microvasculature's impact on normal development and diseases. We present a new imaging and analysis toolset to visualize the coronary microvasculature in intact embryonic hearts and quantify vessel organization. The fluorescent dyes DiI and DAPI were used to stain the coronary vasculature and cardiomyocyte nuclei in quail embryo hearts during rapid growth and morphogenesis of the left ventricular wall. Vessel and cardiomyocytes orientation were automatically extracted and quantified, and vessel density was calculated. The coronary microvasculature was found to follow the known helical organization of cardiomyocytes in the ventricular wall. Vessel density in the left ventricle did not change during and after compaction. This quantitative and automated approach will enable future cohort studies to understand the microvasculature's role in diseases such as hypertrophic cardiomyopathy where misalignment of cardiomyocytes has been observed in utero.

Sox17 Controls Emergence and Remodeling of Nestin-Expressing Coronary Vessels.

RATIONALE: The molecular mechanisms underlying the formation of coronary arteries during development and during cardiac neovascularization after injury are poorly understood. However, a detailed description of the relevant signaling pathways and functional TFs (transcription factors) regulating these processes is still incomplete. OBJECTIVE: The goal of this study is to identify novel cardiac transcriptional mechanisms of coronary angiogenesis and vessel remodeling by defining the molecular signatures of coronary vascular endothelial cells during these complex processes. METHODS AND RESULTS: We demonstrate that Nes-gfp and Nes-CreERT2 transgenic mouse lines are novel tools for studying the emergence of coronary endothelium and targeting sprouting coronary vessels (but not ventricular endocardium) during development. Furthermore, we identify Sox17 as a critical TF upregulated during the sprouting and remodeling of coronary vessels, visualized by a specific neural enhancer from the Nestin gene that is strongly induced in developing arterioles. Functionally, genetic-inducible endothelial deletion of Sox17 causes deficient cardiac remodeling of coronary vessels, resulting in improper coronary artery formation. CONCLUSIONS: We demonstrated that Sox17 TF regulates the transcriptional activation of Nestin's enhancer in developing coronary vessels while its genetic deletion leads to inadequate coronary artery formation. These findings identify Sox17 as a critical regulator for the remodeling of coronary vessels in the developing heart.

Hypoxia-induced downregulation of Sema3a and CXCL12/CXCR4 regulate the formation of the coronary artery stem at the proper site.

BACKGROUND: During the formation of the coronary artery stem, endothelial strands from the endothelial progenitor pool surrounding the conotruncus penetrate into the aortic wall. Vascular endothelial growth factors (VEGFs) as well as CXCL12/CXCR4 signaling are thought to play a role in the formation of the coronary stem. However, the mechanisms regulating how endothelial strands exclusively invade into the aorta remain unknown. METHODS AND RESULTS: Immunohistochemistry showed that before the formation of endothelial strands, Sema3a was highly expressed in endothelial progenitors surrounding the great arteries. At the onset of/during invasion of endothelial strands into the aorta, Sema3a was downregulated and CXCR4 was upregulated in the endothelial strands. In situ hybridization showed that Cxcl12 was highly expressed in the aortic wall compared with in the pulmonary artery. Using avian embryonic hearts, we established two types of endothelial penetration assay, in which coronary endothelial strands preferentially invaded into the aorta in culture. Sema3a blocking peptide induced an excess number of endothelial strands penetrating into the pulmonary artery, whereas recombinant Sema3a inhibited the formation of endothelial strands. In cultured coronary endothelial progenitors, recombinant VEGF protein induced CXCR4-positive endothelial strands, which were capable of being attracted by CXCL12-impregnated beads. Monoazo rhodamine detected that hypoxia was predominant in aortic/subaortic region in ovo and hypoxic condition downregulated the expression of Sema3a in culture. CONCLUSION: Results suggested that hypoxia in the aortic region downregulates the expression of Sema3a, thereby enhancing VEGF activity to induce the formation of CXCR4-positive endothelial strands, which are subsequently attracted into the Cxcl12-positive aortic wall to connect the aortic lumen.

In Vitro Model of Coronary Angiogenesis.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

Cardiogenesis with a focus on vasculogenesis and angiogenesis.

The initial intraembryonic vasculogenesis occurs in the cardiogenic mesoderm. Here, a cell population of proendocardial cells detaches from the mesoderm that subsequently generates the single endocardial tube by forming vascular plexuses. In the course of embryogenesis, the endocardium retains vasculogenic, angiogenic and haematopoietic potential. The coronary blood vessels that sustain the rapidly expanding myocardium develop in the course of the formation of the cardiac loop by vasculogenesis and angiogenesis from progenitor cells of the proepicardial serosa at the venous pole of the heart as well as from the endocardium and endothelial cells of the sinus venosus. Prospective coronary endothelial cells and progenitor cells of the coronary blood vessel walls (smooth muscle cells, perivascular cells) originate from different cell populations that are in close spatial as well as regulatory connection with each other. Vasculo- and angiogenesis of the coronary blood vessels are for a large part regulated by the epicardium and epicardium-derived cells. Vasculogenic and angiogenic signalling pathways include the vascular endothelial growth factors, the angiopoietins and the fibroblast growth factors and their receptors.

Epicardial cell lineages and the origin of the coronary endothelium.

The embryonic epicardium generates a population of epicardial-derived mesenchymal cells (EPDC) whose contribution to the coronary endothelium is minor or, according to some reports, negligible. We have compared four murine cell-tracing models related to the EPDC in order to elucidate this contribution. Cre recombinase was expressed under control of the promoters of the Wilms' tumor suppressor (Wt1), the cardiac troponin (cTnT), and the GATA5 genes, activating expression of the R26REYFP reporter. We have also used the G2 enhancer of the GATA4 gene as a driver due to its activation in the proepicardium. Recombination was found in most of the epicardium/EPDC in all cases. The contribution of these lineages to the cardiac endothelium was analyzed using confocal microscopy and flow cytometry. G2-GATA4 lineage cells are the most frequent in the endothelium, probably due to the recruitment of circulating endothelial progenitors. The contribution of the WT1 cell lineage increases along gestation due to further endothelial expression of WT1. GATA5 and cTnT lineages represent 4% of the cardiac endothelial cells throughout the gestation, probably standing for the actual EPDC contribution to the coronary endothelium. These results suggest caution when using a sole cell-tracing model to study the fate of the EPDC.

Defective coronary vessel organization and reduction of VEGF-A in mouse embryonic hearts with gestational mild hypoxia.

BACKGROUND: Vasculature is formed by responding to homeostatic tissue demands including in developing hearts. Hypoxia generally stimulates vascular formation in which vascular endothelial growth factor A (VEGF-A) plays a critical role. Gestational hypoxia increases the risk of low intrauterine growth and low birth weight, both of which are known to increase the risk of the fetus developing cardiovascular defects. In fact, continuous gestational mild hypoxia (14% O2 ) from the mid-embryonic stage causes cardiac anomalies accompanied by a thinning compact layer in mice in vivo. Because coronary vasculature formation is necessary for compact layers to thicken, we hypothesized that defective coronary vessel organization is related to the thinning compact layer under gestational hypoxia conditions. RESULTS: Continuous gestational mild hypoxia (14% O2 ) applied from embryonic day 10.5 (E10.5) reduced the expression of VEGF-A mRNA and proteins by over 60% in E12.5 hearts relative to control normoxic hearts. Formation of CD31-positive vascular plexus, blood islands, and microvessels in embryonic ventricles were stunted by gestational hypoxia compared to control E12.5 hearts. CONCLUSIONS: Our results suggest that mild hypoxia (14% O2 ) does not induce coronary vessel organization or VEGF-A expression in developing mouse hearts, opposing the general effects of hypoxia-triggering vascular organization and VEGF-A expression.

The Formation of Coronary Vessels in Cardiac Development and Disease.

Understanding how coronary blood vessels form and regenerate during development and progression of cardiac diseases will shed light on the development of new treatment options targeting coronary artery diseases. Recent studies with the state-of-the-art technologies have identified novel origins of, as well as new, cellular and molecular mechanisms underlying the formation of coronary vessels in the postnatal heart, including collateral artery formation, endocardial-to-endothelial differentiation and mesenchymal-to-endothelial transition. These new mechanisms of coronary vessel formation and regeneration open up new possibilities targeting neovascularization for promoting cardiac repair and regeneration. Here, we highlight some recent studies on cellular mechanisms of coronary vessel formation, and discuss the potential impact and significance of the findings on basic research and clinical application for treating ischemic heart disease.

Anatomy of the coronary arteries in fetal pigs: comparison with human anatomy.

In this study, 94 fetal pigs were used to comprehensively investigate the origins, number, location, and distribution of the coronary arteries to enrich knowledge on the coronary circulation in fetal pigs, and allow comparison with adult pigs and humans. In fetal pigs, the posterior interventricular sulcus branch always arose from the right coronary artery and the circumflex artery was rarely extended to the posterior interventricular sulcus, while it is variable in humans. In fetal pigs, there was sometimes anastomosis (8.5%) between the left and right conus branches as nutrient arteries of the pulmonary cone. Other branches were not significantly different between fetal pigs and humans, including the acute marginal branch, obtuse marginal branch, and sinoatrial nodal artery. Coronary dominance was also similar. In conclusion, compared with adult pigs, dissection of the coronary arteries in fetal pigs provided a more faithful overview of the porcine coronary circulation. The coronary arteries in fetal pigs were also more suitable for comparison with humans when pigs are used as experimental animals for studying the coronary vessels, which could be an important reference for investigation of clinical treatment of the coronary arteries. In summary, our data provide reliable information about the distribution and ramifications of the coronary arteries, and could be useful for clinicians and surgeons who wish to comprehensively understand coronary anatomy.

Inositol 1,4,5-trisphosphate receptor 2 as a novel marker of vasculature to delineate processes of cardiopulmonary development.

Congenital heart diseases (CHDs) involving the outflow tract (OFT), such as persistent truncus arteriosus (PTA), lead to mortality and morbidity with implications not only in the heart, but also in the pulmonary vasculature. The mechanisms of pulmonary artery (PA) development and the etiologies underlying PA disorders associated with CHD remain poorly understood partly because of a specific marker for PA development is nonexistent. The three subtypes of inositol 1,4,5-trisphosphate receptors (IP3R1, 2, and 3) are intracellular Ca2+ channels that are essential for many tissues and organs. We discovered that IP3R2 was expressed in the vasculature and heart during development using transgenic mice, in which a LacZ marker gene was knocked into the IP3R2 locus. Whole-mount and section LacZ staining showed that IP3R2-LacZ-positive cells were detectable exclusively in the smooth muscle cells, or tunica media, of PA, merging into αSMA-positive cells during development. Furthermore, our analyses suggested that IP3R2-LacZ positive PA smooth muscle layers gradually elongate from the central PA to the peripheral PAs from E13.5 to E18.5, supporting the distal angiogenesis theory for the development of PA, whereas IP3R2-LacZ was rarely expressed in smooth muscle cells in the pulmonary trunk. Crossing IP3R-LacZ mice with mice hypomorphic for Tbx1 alleles revealed that PTA of Tbx1 mutants may result from agenesis or hypoplasia of the pulmonary trunk; thus, the left and right central to peripheral PAs connect directly to the dorsal side of the truncus arteriosus in these mutants. Additionally, we found hypercellular interstitial mesenchyme and delayed maturation of the lung endoderm in the Tbx1 mutant lungs. Our study identifies IP3R2 as a novel marker for clear visualization of PA during development and can be utilized for studying cardiopulmonary development and disease.

Structural coronary artery remodelling in the rabbit fetus as a result of intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is a fetal condition that affects up to 10% of all pregnancies and is associated with cardiovascular structural and functional remodelling that persists postnatally. Some studies have reported an increase in myocardial coronary blood flow in severe IUGR fetuses which has been directly associated to the dilatation of the coronary arteries. However, a direct measurement of the coronaries' lumen diameter in IUGR has not been reported before. The aim of this paper is to perform, for the first time, a quantitative analysis of the effects of IUGR in cardiac geometry and coronary vessel size in a well-known rabbit model of IUGR using synchrotron-based X-ray Phase Contrast Tomography Imaging (X-PCI). Eight rabbit fetal hearts were imaged non-destructively with X-PCI. 3D reconstructions of the coronary arterial tree were obtained after semi-automatic image segmentation. Different morphometric features including vessel lumen diameter of the three main coronaries were automatically quantified. IUGR fetuses had more globular hearts and dilated coronary arteries as compared to controls. We have quantitatively shown that IUGR leads to structural coronary vascular tree remodelling and enlargement as an adaptation mechanism in response to an adverse environment of restricted oxygen and nutrients and increased perfusion pressure.

HIF-1α is required for development of the sympathetic nervous system.

The molecular mechanisms regulating sympathetic innervation of the heart during embryogenesis and its importance for cardiac development and function remain to be fully elucidated. We generated mice in which conditional knockout (CKO) of the Hif1a gene encoding the transcription factor hypoxia-inducible factor 1α (HIF-1α) is mediated by an Islet1-Cre transgene expressed in the cardiac outflow tract, right ventricle and atrium, pharyngeal mesoderm, peripheral neurons, and hindlimbs. These Hif1aCKO mice demonstrate significantly decreased perinatal survival and impaired left ventricular function. The absence of HIF-1α impaired the survival and proliferation of preganglionic and postganglionic neurons of the sympathetic system, respectively. These defects resulted in hypoplasia of the sympathetic ganglion chain and decreased sympathetic innervation of the Hif1aCKO heart, which was associated with decreased cardiac contractility. The number of chromaffin cells in the adrenal medulla was also decreased, indicating a broad dependence on HIF-1α for development of the sympathetic nervous system.

Formación de vasos coronarios a partir del proepicardio / Proepicardial origin of developing coronary vessels

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Sulodexide inhibits angiogenesis via decreasing Dll4 and Notch1 expression in mouse proepicardial explant cultures.

Sulodexide (SDX) is a mixed drug containing low-molecular-weight heparin sulfate and dermatan sulfate. It exerts mild anticoagulant action but can also affect leukocytes, macrophages, and cell-cell adhesion and may interact with growth factors although its direct influence on endothelial cells is not well described. Clinically, SDX is used for the treatment of cardiovascular diseases, where it exerts anti-inflammatory and endothelial protective effects. The aim of this study was to determine the influence of SDX on tubule formation and angiogenesis-related proteins' mRNA expression in endothelial cell line C166 and mouse proepicardial explants. C166 cells and explants were stimulated with a proangiogenic cocktail containing bFGF/VEGF-A120 /VEGF-A164 enriched with SDX. After stimulation, the number and morphology of tubules stained with anti-CD31 antibody were examined under confocal microscope and expression of mRNA for VEGF-A, VEGF-B, VEGF-C, bFGF, IGF-1, Dll4, and Notch1 was measured with real-time PCR. In C166 cell line, there was no difference in tubule formation and mRNA expression, but in proepicardial explants, we observed reduction in tubule number and in mRNA level for DLL4 and Notch1 after SDX administration. In conclusion, SDX indirectly inhibits angiogenesis in mouse proepicardial explant cultures but has no direct effect on the C166 endothelial cell line.