Joan Fernández Esmerats

Ph.D. Thesis Defense

Date: Monday, August 27, 2018

Time: 12:30PM

Location: Health Sciences Research Building (HSRB) room E-160 (Emory)

Thesis committee members:

Advisor: Dr. Hanjoong Jo, PhD (Georgia Institute of Technology and Emory University)

Dr. Ajit Yoganathan, PhD (Georgia Institute of Technology)

Dr. Robert Nerem, PhD (Georgia Institute of Technology)

Dr. Andres Garcia, PhD (Georgia Institute of Technology)

Dr. Robert Taylor, MD, PhD (Emory University)

Dr. Keith Wilkinson, PhD (Emory University)

Title: The role of flow-sensitive miRNAs and UBE2C-dependent HIF1α pathway in calcific aortic valve disease

Abstract: Calcific aortic valve disease (CAVD), characterized by AV sclerosis and calcification, is a major cause of death especially in the aging population. Except for valve replacement or repair, there are no effective medical therapies for CAVD; therefore, novel, non-invasive medical therapies are urgently needed. Limited understanding of the pathogenic mechanisms of the disease and lack of reliable animal models have been major challenge in developing new medical therapies for CAVD. The goal of my PhD thesis work is to address these problems.

Interestingly, AV calcification occurs preferentially on the fibrosa side, which is exposed to disturbed and oscillatory flow conditions (OS); whereas the ventricularis side exposed to stable and laminar flow conditions (LS) remains mostly protected. Therefore, we hypothesize that OS on the fibrosa plays key roles by regulating coding and non-coding genes including miRNAs in endothelial dysfunction leading to CAVD. Identification of these key miRNAs and their action mechanisms may lead to novel therapeutics.

In this dissertation I studied the role of flow-sensitive miRNAs in endothelial biology and CAVD. To identify flow-sensitive miRNAs, we conducted two microRNA arrays in human aortic valve endothelial cells (HAVECs) exposed to OS or LS, and endothelial-enriched RNAs from the fibrosa and ventricularis of porcine AVs. Here, we identified two novel flow-sensitive miRNAs in the valvular endothelium: miR-181b and miR-483. miR-181 was upregulated in OS conditions and it inhibits tissue inhibitor of metalloproteinases 3 (TIMP3) expression. Silencing of miR-181b by anti-miR-181b prevented flow-induced extracellular matrix degradation—an important step in pathophysiology of CAVD, suggesting that targeting miR-181b could be a novel therapy for AV sclerosis. We found that miR-483 is upregulated in LS conditions and downregulates UBE2C and the HIF1α pathway preventing flow-induced inflammation and endothelial-to-mesenchymal transition. Therapeutic studies using porcine AVs exposed to pro-osteogenic media showed that upregulation of miR-483 or inhibition of HIF1α can significantly decrease AV calcification.

In addition, we developed a novel mouse model of CAVD by combining two important risk factors of CAVD: bicuspid aortic valve (BAV) and hypercholesterolemia. By inducing hypercholesterolemia in GATA5 knockout mice, which present BAV phenotype, we anticipate to generate robust AV sclerosis and microcalcification within four months.

Overall, this dissertation work provides critical insights into the mechanisms of flow-induced AV calcification and development of an accelerated mouse model of CAVD, which will be useful in translating novel therapeutics from the lab to the clinic.