Protocol for the isolation and single-nuclei multiomic analyses of the human fetal epicardium.

The characterization of cell populations that reside in the outer layer of the heart has been hindered by difficulties in their isolation. Here, we present a protocol for isolation and single-nuclei multiomic analyses of the human fetal epicardium. We describe steps for microdissection, isolation, and enrichment of epicardial cells by mechanical dissociations and direct lysis. We then detail procedures for integrating transcriptome and chromatin accessibility datasets. This approach allows the analysis of diverse cell populations, marked by unique cis-regulatory elements. For complete details on the use and execution of this protocol, please refer to Travisano et al.1.

Single-nuclei multiomic analyses identify human cardiac lymphatic endothelial cells associated with coronary arteries in the epicardium.

Cardiac lymphatic vessels play important roles in fluid homeostasis, inflammation, disease, and regeneration of the heart. The developing cardiac lymphatics in human fetal hearts are closely associated with coronary arteries, similar to those in zebrafish hearts. We identify a population of cardiac lymphatic endothelial cells (LECs) that reside in the epicardium. Single-nuclei multiomic analysis of the human fetal heart reveals the plasticity and heterogeneity of the cardiac endothelium. Furthermore, we find that VEGFC is highly expressed in arterial endothelial cells and epicardium-derived cells, providing a molecular basis for the arterial association of cardiac lymphatic development. Using a cell-type-specific integrative analysis, we identify a population of cardiac lymphatic endothelial cells marked by the PROX1 and the lymphangiocrine RELN and enriched in binding motifs of erythroblast transformation specific (ETS) variant (ETV) transcription factors. We report the in vivo molecular characterization of human cardiac lymphatics and provide a valuable resource to understand fetal heart development.

Molecular mechanisms of coronary artery disease risk at the PDGFD locus.

Platelet derived growth factor (PDGF) signaling has been extensively studied in the context of vascular disease, but the genetics of this pathway remain to be established. Genome wide association studies (GWAS) for coronary artery disease (CAD) have identified a risk locus at 11q22.3, and we have verified with fine mapping approaches that the regulatory variant rs2019090 and PDGFD represent the functional variant and putative functional gene. Further, FOXC1/C2 transcription factor (TF) binding at rs2019090 was found to promote PDGFD transcription through the CAD promoting allele. Employing a constitutive Pdgfd knockout allele along with SMC lineage tracing in a male atherosclerosis mouse model we mapped single cell transcriptomic, cell state, and lesion anatomical changes associated with gene loss. These studies revealed that Pdgfd promotes expansion, migration, and transition of SMC lineage cells to the chondromyocyte phenotype and vascular calcification. This is in contrast to protective CAD genes TCF21, ZEB2, and SMAD3 which we have shown to promote the fibroblast-like cell transition or perturb the pattern or extent of transition to the chondromyocyte phenotype. Further, Pdgfd expressing fibroblasts and pericytes exhibited greater expression of chemokines and leukocyte adhesion molecules, consistent with observed increased macrophage recruitment to the plaque. Despite these changes there was no effect of Pdgfd deletion on SMC contribution to the fibrous cap or overall lesion burden. These findings suggest that PDGFD mediates CAD risk through promoting SMC expansion and migration, in conjunction with deleterious phenotypic changes, and through promoting an inflammatory response that is primarily focused in the adventitia where it contributes to leukocyte trafficking to the diseased vessel wall.

Molecular mechanisms of coronary artery disease risk at the PDGFD locus.

Genome wide association studies for coronary artery disease (CAD) have identified a risk locus at 11q22.3. Here, we verify with mechanistic studies that rs2019090 and PDGFD represent the functional variant and gene at this locus. Further, FOXC1/C2 transcription factor binding at rs2019090 is shown to promote PDGFD transcription through the CAD promoting allele. With single cell transcriptomic and histology studies with Pdgfd knockdown in an SMC lineage tracing male atherosclerosis mouse model we find that Pdgfd promotes expansion, migration, and transition of SMC lineage cells to the chondromyocyte phenotype. Pdgfd also increases adventitial fibroblast and pericyte expression of chemokines and leukocyte adhesion molecules, which is linked to plaque macrophage recruitment. Despite these changes there is no effect of Pdgfd deletion on overall plaque burden. These findings suggest that PDGFD mediates CAD risk by promoting deleterious phenotypic changes in SMC, along with an inflammatory response that is primarily focused in the adventitia.

The Lymphatic System in Zebrafish Heart Development, Regeneration and Disease Modeling.

Heart disease remains the single largest cause of death in developed countries, and novel therapeutic interventions are desperately needed to alleviate this growing burden. The cardiac lymphatic system is the long-overlooked counterpart of the coronary blood vasculature, but its important roles in homeostasis and disease are becoming increasingly apparent. Recently, the cardiac lymphatic vasculature in zebrafish has been described and its role in supporting the potent regenerative response of zebrafish heart tissue investigated. In this review, we discuss these findings in the wider context of lymphatic development, evolution and the promise of this system to open new therapeutic avenues to treat myocardial infarction and other cardiopathologies.

Environment-Sensing Aryl Hydrocarbon Receptor Inhibits the Chondrogenic Fate of Modulated Smooth Muscle Cells in Atherosclerotic Lesions.

BACKGROUND: Smooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. METHODS: We combined RNA-sequencing, chromatin immunoprecipitation followed by sequencing, assay for transposase-accessible chromatin using sequencing, and in vitro assays in human coronary artery SMCs, with single-cell RNA-sequencing, histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease. RESULTS: Genomic studies coupled with functional assays in cultured human coronary artery SMCs revealed that AHR modulates the human coronary artery SMC phenotype and suppresses ossification in these cells. Lineage-tracing and activity-tracing studies in the mouse aortic sinus showed that the Ahr pathway is active in modulated SMCs in the atherosclerotic lesion cap. Furthermore, single-cell RNA-sequencing studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMCs expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term "chondromyocytes," were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMCs in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout in comparison with wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses. CONCLUSIONS: Overall, we conclude that AHR promotes the maintenance of lesion cap integrity and diminishes the disease-related SMC-to-chondromyocyte transition in atherosclerotic tissues.

Coronary arterial development is regulated by a Dll4-Jag1-EphrinB2 signaling cascade.

Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase Mfng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade.

Sequential Ligand-Dependent Notch Signaling Activation Regulates Valve Primordium Formation and Morphogenesis.

RATIONALE: The Notch signaling pathway is crucial for primitive cardiac valve formation by epithelial-mesenchymal transition, and NOTCH1 mutations cause bicuspid aortic valve; however, the temporal requirement for the various Notch ligands and receptors during valve ontogeny is poorly understood. OBJECTIVE: The aim of this study is to determine the functional specificity of Notch in valve development. METHODS AND RESULTS: Using cardiac-specific conditional targeted mutant mice, we find that endothelial/endocardial deletion of Mib1-Dll4-Notch1 signaling, possibly favored by Manic-Fringe, is specifically required for cardiac epithelial-mesenchymal transition. Mice lacking endocardial Jag1, Notch1, or RBPJ displayed enlarged valve cusps, bicuspid aortic valve, and septal defects, indicating that endocardial Jag1 to Notch1 signaling is required for post-epithelial-mesenchymal transition valvulogenesis. Valve dysmorphology was associated with increased mesenchyme proliferation, indicating that Jag1-Notch1 signaling restricts mesenchyme cell proliferation non-cell autonomously. Gene profiling revealed upregulated Bmp signaling in Jag1-mutant valves, providing a molecular basis for the hyperproliferative phenotype. Significantly, the negative regulator of mesenchyme proliferation, Hbegf, was markedly reduced in Jag1-mutant valves. Hbegf expression in embryonic endocardial cells could be readily activated through a RBPJ-binding site, identifying Hbegf as an endocardial Notch target. Accordingly, addition of soluble heparin-binding EGF-like growth factor to Jag1-mutant outflow tract explant cultures rescued the hyperproliferative phenotype. CONCLUSIONS: During cardiac valve formation, Dll4-Notch1 signaling leads to epithelial-mesenchymal transition and cushion formation. Jag1-Notch1 signaling subsequently restrains Bmp-mediated valve mesenchyme proliferation by sustaining Hbegf-EGF receptor signaling. Our studies identify a mechanism of signaling cross talk during valve morphogenesis involved in the origin of congenital heart defects associated with reduced NOTCH function.

Epithelial-to-mesenchymal transition in epicardium is independent of Snail1.

The epicardium is the outer epithelial covering the heart. This tissue undergoes an epithelial-to-mesenchymal transition (EMT) to generate mesenchymal epicardial-derived cells (EPDCs) that populate the extracellular matrix of the subepicardium and contribute to the development of the coronary vessels and cardiac interstitial cells. Although epicardial EMT plays a crucial role in heart development, the molecular regulation of this process is incompletely understood. Here we examined the possible role of the EMT regulator Snail1 in this process. Snail1 is expressed in the epicardium and EPDCs during mouse cardiac development. To determine the function of Snail1 in epicardial EMT, we deleted Snail1 in the epicardium using Wt1- and Tbx18-Cre drivers. Unexpectedly, epicardial-specific Snail1 mutants are viable and fertile and do not display any obvious morphological or functional cardiac abnormalities. Molecular analysis of these mice reveals that epicardial EMT occurs normally, and epicardial derivatives are established in these mutants. We conclude that Snail1 is not required for the initiation and progression of embryonic epicardial EMT.