Title

Role of AJAPl in Epithelial Cell Proliferation and Apoptosis for Cardiovascular Development

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

Jocelyn Lippman-Bell, PhD

Abstract

The hea1i is formed during embryogenesis through a series of molecular processes that modify specific cells. Congenital heart defects (CHDs) occur when these processes are disrupted and this compromises normal cardiovascular function. Coronary vascular anomalies are a subcategory of Cl-IDs accounting for 15-34% of sudden cardiac death in adolescents. Coronary vessels are formed during embryogenesis from a cluster of epithelial-like mesothelial cells within the proepicardium (PE). PE cells undergo proliferation or apoptosis, and migrate onto the myocardium to form the epicardium. Under the influence of transcription factors, epicardial cells differentiate to form coronary vascular cells. The Tbx5 transcription factor is expressed in the PE and epicardium. Mice deficient in proepicardial Tbx5 exhibit delayed epicardial formation, cardiac hemorrhaging, myocardial hypoxia, and reduced mRNA expression of adherens junctionassociated protein l, or AJAP 1. Immunofluorescent studies showed that Ajapl is expressed in the mouse PE, epicardium and primitive coronary vessels. Ajap 1 overexpression inhibits hepatocellular carcinoma in mouse models while its downregulation is associated with increased metastasis. However, the role of AJAP 1 in coronary vessel formation remains unknown. This study was designed to elucidate the contributions of A!AP 1 to epithelial cell proliferation and apoptosis that are essential to epicardium and coronary vascular formation.

Methods: Human mammary epithelial cells (HMEpiCs) were utilized to assess the contribution of endogenous AJAP 1 expression to in vitro cell behavior. AJAP 1 gene silencing through small interfering RN As ( siRNAs) caused a significant reduction in AJAP 1 mRNA expression in these cells. Control and AJAP I-silenced epithelial cells were analyzed for differences in cell morphology and density by visual comparisons. These cells were examined for altered cell proliferation and apoptosis markers by qPCR.

Results: AJAP I-silenced HMEpiCs exhibited a >75% decrease inAJAP 1 mRNA expression compared to control HMEpiCs. Reduced AJAP 1 expression produced distinct cellular changes, including decreased density and the presence of membrane blebbing indicating increased apoptosis. AJAP I-silenced HMEpiCs exhibited increased expression of certain cell cycle and apoptotic markers.

Conclusion: AJAP 1 contributes to the growth and viability of epithelial cells in vitro. AJAPl-silenced HMEpiCs may provide a solid model system to improve our understanding of mammalian epicardium formation that is critical for coronary vessel development.

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