Title

Reelin Signaling in Coronary Vasculogenesis

Date of Award

6-2017

Degree Type

Thesis

First Advisor

Cathy J. Hatcher, PhD

Second Advisor

Ruth C. Borghaei, PhD

Third Advisor

Heather L. Montie, PhD

Abstract

Coronary vascular anomalies occur in almost 1% of the population. These anomalies can lead to myocardial ischemia, myocardial infarction and heart failure. Coronary blood vessels supply oxygen and nutrients to the contractile myocardium of the heart to maintain homeostasis. Identification of the cellular origins of the coronary vascular smooth muscle and endothelial cells and the molecular signals associated with coronary vascular morphogenesis are fundamental for understanding the etiology of cardiovascular disease. The proepicardium (PE) is a transitory structure that lies dorsal to the primitive heart tube and it is the progenitor to the epicardium and epicardial-derived cells (EPDCs). Most coronary vascular smooth muscle cells are derived from EPDCs, but the coronary vascular endothelial cells arise from multiple progenitors. Nearly 20% of embryonic coronary arterial and capillary endothelial cells are derived from EPDCs. The EPDCs differentiate into vascular cells via events regulated by transcription factors including Tbx5. PE-specific deletion of Tbx5 impairs epicardium and coronary vessel formation in the embryonic mouse heart and decreases epicardial Reln mRNA expression. Thus, Tbx5 contributes to epicardium and coronary vessel formation.

Study Objective: Reelin, which is encoded by the Reln gene, is a secreted, extracellular matrix glycoprotein that is involved in neural and lymphatic system development where it regulates cell migration and positioning. Its role in cardiovascular development is unknown. The goal was to identify the functional contributions of Reelin to structural formation of the developing heart and to embryonic cardiac cell behavior. iv

Methods: First, the ability of TBX5 to regulate RELN transcription was examined. A luciferase reporter plasmid containing a human RELN promoter with three putative T-box DNA binding elements was transfected into human lung epithelial cells that stably express human TBX5 and we measured the resultant luciferase activity. Second, Reelin expression in embryonic and adult mouse hearts was examined through immunofluorescent staining along with co-staining for markers of specific cell lineages. Third, the physiological role of Reelin in endothelial cells was explored by assessing in vitro cell behavior in a primary human microvascular endothelial cell (HMEC) line that models vasculogenesis and has endogenous Reelin. Functional assays on HMECs transfected with RELN specific small interfering RNAs were performed to inhibit Reelin translation.

Results: These data show that TBX5 regulates RELN transcription. Reelin is expressed in the PE, epicardium and endocardium of the mouse heart by embryonic day (E) 9.5. Co-staining for cell-specific markers enabled us to determine that Reelin localizes to epicardial cells and appears in nascent coronary vessels by E13.5. Reelin is also expressed in mature coronary vessels at E18.5. Functional analysis of RELN-silenced cells provides insight into how Reelin contributes to cardiovascular development. Conclusions: TBX5 regulates RELN transcription. The in vitro and in vivo results shed light on Reelin expression during development and its contribution to cellular mechanisms required for coronary vessel formation.

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