AJAP1 and Epithelial Cell Adhesion for Epicardial and Coronary Vessel Formation

Abstract

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.