Publication Date

2014-10-20

Availability

Open access

Embargo Period

2014-10-20

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2014-04-21

First Committee Member

Joy Lincoln

Second Committee Member

Danuta Szczesna-Cordary

Third Committee Member

Michael Kim

Fourth Committee Member

Jae Lee

Fifth Committee Member

Vladen Slepak

Abstract

Mitral valve prolapse (MVP) affects more than 2% of the population in the United States, and is the most common cause of chronic mitral valve regurgitation (MVR) in developed countries. It is estimated that MVP affects more than 150 million people worldwide. When left untreated, MVP can lead to severe MVR resulting in a myriad of cardiac complications. The mortality rate of MVP patients with severe MVR is over 6% per year. Despite clinical significance, there remains a lack of medical therapies for MVP, with surgical intervention being the most effective treatment to date. Therefore, understanding normal heart valve development, and how it is altered during disease, may provide novel insights into new therapeutic targets. MVP associated with myxomatous mitral valve degeneration (MMVD) is the most common cause of MVR requiring surgery. Healthy mature heart valves are highly organized and composed of stratified layers of elastins, collagens and proteoglycans that collectively provide all of the necessary biomechanical properties for structure-function relationships throughout life. In contrast, diseased valves that suffer from MMVD are pathologically thickened and characterized by an abnormal abundance of extracellular matrix (ECM) proteoglycans, which prevents the valves from closing properly and leads to regurgitation and functional prolapse. Although the ECM composition of mature tri-laminar valves has been well described, little is known about the molecular mechanisms that establish and maintain these highly organized structures. However, the etiology of MVP has historically been associated with genetic connective tissue disorders including Marfan syndrome (MFS). A deeper understanding of ECM regulation in normal valve development will help elucidate conserve pathways in disease as a potential means of therapy. In the current studies, we show that the basic helix-loop-helix (bHLH) transcription factor Scleraxis (Scx) regulates ECM deposition, with a loss of Scx being largely attributed to a significant decrease in the expression and contribution of chondroitin sulfate proteoglycans (CSPGs) to the mature valve leaflets. In addition, we determine that Scx is sufficient to promote CSPG expression in both embryonic and mature valve cells. We further delineate that canonical Tgf-beta-Smad signaling positively regulates Scx and CSPG expression, while activated MAPK attenuates this pathway in a Tgf-beta-independent manner. We show that MAPK activation is sufficient to stabilize the bHLH activator/repressor Twist1, however conclude that Twist1 does not bind to or transcriptionally regulate Scx. We also show that Scx is increased in a MFS mouse model of MMVD, and overexpression can promote myxomatous phenotypes in otherwise normal human mitral valve interstitial cells. Using this model, we explore the idea that reduced Scx function may potentially rescue myxomatous valve phenotypes in vivo. Furthermore, we have identified previously unappreciated protein-coding and non-protein-coding mRNAs that are differentially expressed in the absence of Scx during valve remodeling stages. We report an enrichment of mRNAs associated with processes related to gene regulation and cellular development. Furthermore, bioinformatics analysis predicted known (Tgf-beta-2) and novel (Onecut1) upstream regulators of Scx during valve remodeling. In addition, we show that the loss of Scx leads to differential changes in mRNA transcripts and alternative splicing of several genes. Together, these findings provide insights into molecular signaling pathways that regulate Scx, and identify novel genes and hierarchical networks that are regulated by Scx during valve development, which may be altered in MMVD.

Keywords

Heart Valve; MAPK; Scleraxis; Proteoglycan; TGF-beta; Myxomatous

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