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Congenital coronary artery anomalies occur in up to 5% of the human population. Such anomalies can lead to myocardial ischemia, myocardial infarction and sudden death. Coronary vessels are composed of endothelial and smooth muscle cells. The primary function of vascular endothelial cells is to provide a barrier between the vessel lumen and surrounding tissue. Vascular repair processes often recapitulate developmental mechanisms. Elucidation of signaling mechanisms that govern formation of nascent coronary endothelial cells is required to develop therapeutic strategies aimed at regrowth of congenitally malformed or damaged coronary vessels. Embryonic mice with epicardial‐specific deletion of Tbx5 (Tbx5epi−/−) exhibit delayed epicardial cell attachment to the myocardium and impaired coronary vessel formation. This leads to punctate cardiac hemorrhaging that may stem from deficits in vascular integrity. Expression of Reln mRNA, that encodes the Reelin extracellular matrix glycoprotein, is also reduced in embryonic Tbx5epi−/− mouse hearts. Reelin localizes to some, but not all, coronary vascular endothelial cells during mammalian cardiogenesis. These findings lead us to speculate that expression of Reelin in coronary vascular endothelial cells may be critical for establishing vascular integrity.Study ObjectiveThe goal of this study was to elucidate contributions of Reelin to endothelial cell function for establishing vascular integrity during coronary vessel formation.MethodsWe utilized human microvascular endothelial cells (HMECs) to assess contributions of Reelin to endothelial cell function as these cells are appropriate in vitro models of vessel formation and have endogenous Reelin expression. We achieved RELN gene silencing through a small interfering RNA‐mediated approach that led to >90% reduction in both RELN mRNA and Reelin protein expression in comparison to negative control‐transfected HMECs. Next, control and RELN‐silenced endothelial cells were subjected to physiological assays to assess their ability to establish vascular integrity. First, we examined the ability of RELN‐silenced HMECs to adhere to the basement membrane through exposure of these cells to various matrices. Second, we assessed cell membrane permeability of RELN‐silenced HMECs that were plated as a monolayer on a collagen‐coated semi‐permeable barrier. This served as an indicator of in vitro vascular permeability. The cell monolayer was treated with a permeability factor and fluorescent‐labeled Dextran was added on top of the monolayer. Fluorescent Dextran passed through the cell monolayer at a rate proportional to membrane permeability. Permeability was measured by quantifying the fluorescence of the media on the basolateral side of the monolayer.ResultsOur results indicate that RELN silencing alters in vitro endothelial cell adhesion and cell membrane permeability.ConclusionsWe conclude that Reelin plays a critical role during coronary vessel development as it establishes vascular integrity through maintenance of cell adhesions and membrane permeabilitySupport or Funding InformationAHA 17AIREA3360773 and PCOM Center for Chronic Disorders of AgingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The coronary vasculature forms to supply oxygen and nutrients to the contractile myocardium when it can no longer be supported by simple diffusion of blood from the cardiac lumen. This is an important step in cardiovascular development that is preceded by formation of the epicardium that contributes epicardial‐derived cells (EPDCs). These EPDCs proceed to form some cells within the nascent coronary vessels. Mice with proepicardium (PE)‐specific deletion of Tbx5 (Tbx5epi−/−) have delayed attachment of the epithelial‐like epicardial cells to the myocardium. This delay precipitates a cascade of deleterious events that impair coronary vessel formation and lead to punctate cardiac hemorrhaging and myocardial hypoxia in Tbx5epi−/− mice. In addition, Tbx5epi−/− mice exhibit reduced cardiac Ajap1 mRNA expression. We previously demonstrated that Ajap1 is expressed within the PE, epicardium and coronary vessels of the developing mouse heart, but little is known about its contribution to cardiovascular morphogenesis other than it is speculated to function as a cell adhesion molecule.Study ObjectiveThe goal of this study was to elucidate contributions of Ajap1 to epithelial cell behaviors that are integral to formation of the epicardium and the subsequent development of the coronary vessels. Based upon our preliminary in vivo data, we hypothesized that the reduction in cardiac Ajap1 expression of our Tbx5epi−/− mice acts as a catalyst to impair epicardium and, subsequently, coronary vessel formation.MethodsTo test this hypothesis, we first analyzed the contribution of AJAP1 to cell behavior in an in vitro system that models mammalian epicardial cells. We utilized the stably transformed MCF7 human breast adenocarcinoma cell line to assess contributions of AJAP1 to epithelial cell function as these cells retain epithelial characteristics in vitro and have endogenous AJAP1 expression. We achieved AJAP1 gene silencing in MCF7s through a small interfering RNAmediated approach that led to a significant reduction in both AJAP1 mRNA and protein expression in these cells. Next, control and AJAP1‐silenced epithelial cells were subjected to cell adhesion assays to assess the ability of these cells to adhere to various basement membrane matrices. Cells were allowed to adhere to these matrices for 1 – 24 hours and adherent cells were quantified. Finally, we analyzed Ajap1 protein expression in embryonic and adult mouse hearts to gauge how its levels change throughout important stages of cardiovascular development.ResultsData from our gene‐silenced epithelial cells revealed a significant difference in cell adhesion abilities between the control and AJAP1‐transfected cells. Cardiac Ajap1 expression changed notably throughout mouse development and after gestation.ConclusionsWe concluded that AJAP1 contributes to the in vitro cell adhesion properties in an epithelial model system of the mammalian epicardium. These data shed light on a mechanism by which AJAP1 contributes to cell adhesion in a mammalian model of epicardial cell behavior. This may be instrumental to understanding coronary vessel formation.Support or Funding InformationSupport: PCOM Center for Chronic Disorders of AgingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Introduction The epicardium and coronary vessels of the embryonic mammalian heart are derived from epithelial‐like progenitor cells of the proepicardium (PE). These epithelial cells migrate from the PE during development to envelop and adhere to the myocardium to form the epicardium. Epithelial cells maintain their cellular integrity through adherens and tight junctions before undergoing epithelial‐mesenchymal transition (EMT) to become epicardial‐derived cells that invade the myocardium and transform into the vascular smooth muscle cells and vascular endothelial cells of the coronary vessels. Adherens junction‐associated protein 1 (AJAP1) is a cell adhesion molecule that is expressed in the epicardium and coronary vessels, and it regulates epithelial cell behaviors. Study Objective Our goal was to identify and assess the molecular target(s) of AJAP1‐modifiable epithelial cell behaviors that are important for formation of the epicardium and coronary vessels during cardiovascular development. We speculate that AJAP1 regulates epithelial cell behaviors by altering the expression of transcripts of adherens junctions, tight junctions and EMT markers. Methods Primary human mammary epithelial cells (HMEpiCs) retain characteristics of epithelial cells in vivo. We examined the in vitro contributions of AJAP1 to epithelial cell behaviors necessary for epicardium formation using HMEpiCs. We silenced AJAP1 mRNA expression in HMEpiCS with small interfering RNAs (siRNAs) and this produced changes in HMEpiC behavior. We obtained qPCR array plates to assay AJAP1‐gene‐silenced versus control HMEpiCs for differential expression of transcripts classified as markers of adherens junctions, tight junctions and EMT. This array expression data was subjected to gene ontology (GO) analysis to query the targeted genes and classify them by distinct biologic functions. Results A reduction in AJAP1 mRNA expression produced discernible changes in epithelial cell morphology and functions. A comparison of adherens junction, tight junction and EMT qPCR arrays revealed transcripts with altered expression in AJAP1‐silenced versus control HMEpiCs. GO analysis showed these transcripts are involved in several biological processes including blood vessel development, cell migration, cell‐cell adhesion, cytoskeleton organization and cell junction organization. This analysis correlates with our previous cell behavioral data. Conclusion Our results indicate that AJAP1 plays an important role in epithelial cells to enable expression of genes involved in an array of biological processes such as cell migration, cell‐cell adhesion, cytoskeleton organization and cell junction organization. These biological processes are important for formation of the epicardium and coronary vessels during cardiovascular development.