Title

AJAP1 and Epithelial Cell Adhesion in Epicardial and Coronary Vessel Formation

Date of Award

2019

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Cathy Hatcher, PhD

Second Advisor

Ruth Borghaei, PhD

Third Advisor

Heather Montie, PhD

Abstract

The coronary vasculature forms to supply oxygen and nutrients to the contractile myocardium when it can no longer be supported by simple diffusion from the cardiac lumen. This is an important step in cardiovascular development that is preceded by formation of the epicardium, which eventually contributes epicardial-derived cells (EPDCs) to the nascent coronary vessels. Mice with proepicardium (PE)-specific deletion of Tbx5 (Tbx5epi-/-) have delayed attachment of epithelial-like epicardial cells to the myocardium. This delay precipitates a cascade of events that impair coronary vessel formation in Tbx5epi-/- mice. This leads to punctate cardiac hemorrhaging and myocardial hypoxia. In addition, Tbx5epi-/- mice exhibit reduced cardiac Ajap1 mRNA expression. We demonstrated previously that Ajap1 is expressed within the PE, epicardium, and coronary vessels of the developing mouse heart. However, little is known about its role in cardiovascular morphogenesis other than it is speculated to function as a cell adhesion molecule. The goal of this study was to elucidate contributions of Ajapl to epithelial cell behaviors that are integral to the formation of the epicardium and subsequent development of the coronary vessels. Based upon our preliminary in vivo data in Tbx5epi-/- mice, we hypothesized that the reduction in cardiac epithelial Ajap1 expression led to impaired formation of the epicardium and,subsequently, the coronary vessels.

Methods: To test this hypothesis, we first analyzed the contributions of AJAP1 to cell behavior in an in vitro system designed to model mammalian epicardial cells. We utilized the MCF-7 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 MCF-7s through a small interfering RNA (siRNA)-mediated approach that led to a significant reduction in AJAP1 mRNA expression and qualitatively less AJAP1 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 the number of adherent cells was quantified. Finally, we analyzed Ajap1 protein expression in embryonic and adult mouse hearts to gauge changes in its expression levels throughout cardiovascular development and maturation.

Results: Our data indicated that cardiac Ajap1 mRNA expression changed substantially throughout cardiovascular development and maturation. Also, data from our gene-silenced epithelial cells revealed a significant difference in the cell adhesive properties of control versus AJAP1 knockdown siRNA- transfected cells on certain matrices. IV

Conclusion: We concluded that AJAP1 contributes to the cell adhesion properties of epithelial cells, proven in a context that models mammalian epicardial cell behavior during cardiovascular development. These findings may prove to be instrumental for us to develop a more complete understanding of the mechanisms of coronary vessel formation.

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