Date of Award

2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Steven W. Kubalak

Second Advisor

Scott Argraves

Third Advisor

Amy Bradshaw

Fourth Advisor

Dhan Kuppuswamy

Fifth Advisor

Andy Wessels

Abstract

The normal formation of the epicardium, the outer cell layer of the heart, is critical for subsequent development of the heart to proceed normally. Abnormalities in the epicardium lead to cardiac defects as shown in mice deficient in retinoid X receptor α (RXRα-/-), a model of congenital heart disease that exhibits many cardiac malformations including epicardial defects. The RXRα-/- epicardium is slower to form and once formed, it detaches from the myocardium. Previously an elevation of transforming growth factor β2 (TGFβ2) has been observed in RXRα-/- hearts at midgestation and an alteration in vascular cell adhesion molecule 1 (VCAM-1) was found at E11.5. Based on these findings it was hypothesized that proper expression of VCAM-1 is essential for normal cardiac morphogenesis and is regulated by TGFβ2 and/or retinoid signaling. VCAM-1 is a transmembrane protein known to be involved in epicardial cell adhesion and has been reported to inhibit epithelial to mesenchymal transformation (EMT) of epicardial cells. In this study the expression of VCAM-1 was analyzed from E9.5-E13.5 in the wild type (WT) and RXRα-/- mice using real time quantitative PCR, immunohistochemistry and western blotting. At E11.5, VCAM-1 protein expression levels were similar to WT in the RXRα-/- mouse, but later (E12.5 and E13.5) misexpression of VCAM-1 was found in the epicardium of RXRα-/- mice. Specifically, VCAM-1 mRNA and protein were increased in the myocardium of the RXRα-/- heart compared to the WT at E12.5 and E13.5. Elevation of VCAM-1 protein was also found in E13.5 epicardial explants from RXRα-/- embryos. To investigate possible involvement of TGFβ2 in VCAM-1 regulation, E11.5 epicardial explants were treated with TGFβ2 and the treatment was found to promote upregulation of VCAM-1 in the epicardial cells. Treatment of embryos in whole embryo culture with TGFβ2 resulted in elevation of VCAM-1 and also caused epicardial detachment after 18 hours of treatment. RXRα and Smad4 were shown to bind to the mouse VCAM-1 promoter using ChIP analysis and the VCAM-1 promoter can be activated by TGFβ2 treatment (shown through use of a luciferase expression plasmid containing the VCAM1 promoter). Together the findings show that VCAM-1 is elevated in the hearts of RXRα-/- mice and TGFβ2 can regulate VCAM-1 expression in the embryonic heart, particularly in the epicardium. Elevated TGFβ2 in the heart, such as that observed in the RXRα-/- mouse, can cause upregulation of VCAM-1 in the myocardium and epicardium. Upregulation of VCAM-1 could decrease epicardial EMT, which is also observed in the RXRα. From our study we show that proper expression (levels and location) of VCAM-1 is essential for normal heart development and that misexpression of VCAM-1 can negatively affect formation of the heart.

Rights

All rights reserved. Copyright is held by the author.

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