Congenital valve defects promote Calcific Aortic Valve Disease (CAVD). Bone morphogenetic protein 2 (BMP2) is a key signal in valve development. In diseased adult valves, the pro-calcific BMP2 and downstream responses stimulate calcification. Thus, aberrant BMP2 patterns and levels influence CAVD indirectly by disrupting valve shape and directly by inducing ectopic bone formation. Our overall hypothesis is that controlling BMP2 levels would be an effective medical therapy for CAVD. Our strategy is to elucidate how BMP2 levels are controlled in the aortic valve during normal valve development and under conditions that promote CAVD.
Aim 1. To test DNA sequences that control BMP2 synthesis at the transcriptional level in the valves of mice that model CAVD and during valve development.
This aim will use an existing collection of reagents and mice that were created to survey bone regulatory sequences. One region includes a conserved bone enhancer that binds the master bone differentiation transcription factor RUNX2 and other potential regulators. We will test ECR1 and map other cis-elements that control Bmp2 expression in mice that develop CAVD when fed a high cholesterol diet and during the early stages of normal aortic valve development.
Aim 2. To test RNA sequences that control BMP2 synthesis at the post-transcriptional level in developing valves and in the valves of mice that model CAVD. Both positive and negative mechanisms influence BMP2 synthesis in the aortic valve. A sequence conserved between mammals and fishes strongly represses BMP2 reporter gene repression in the developing and adult aortic valve. We will test the hypothesis that this element and its flanking RNA prevent BMP2 synthesis and ectopic bone in aortic valves in the Notch-compromised CAVD model. We will also test this regulatory mechanism during normal aortic valve development.
Aim 3. To identify trans-regulatory molecules that control Bmp2 transcription through ECR1. The trans-regulatory factors that bind ECR1 may promote BMP2 synthesis in calcifying aortic valves. Molecular studies will be used to define factors interacting with the ECR1 sequence. The association of RUNX2 and Hes1 will be examined in MC3T3-E1 pre-osteoblasts that express BMP2 in response to FGF2. These findings will be confirmed in primary aortic valve interstitial cells and diseased valves from the CAVD mouse model.
Aim 4. To identify trans-regulatory molecules that control BMP2 synthesis at the post- transcriptional level. We will test the influence of microRNAs that target the BMP2 transcript MC3T3-E1 cells and primary valve interstitial cells. This study will provide fundamental knowledge regarding the spatial and temporal control of Bmp2 in a new model of CAVD and in the developing aortic valve. These studies will direct future tests of anti-BMP2 therapies in CAVD. This proposal explores these Specific Areas of Research Interest: Genetics of CAVD and bicuspid aortic valve, Infrastructure (improved animal models), Cell biology (signaling pathways, mechanisms, and regulation of calcification), and Diagnosis.

Public Health Relevance

Calcific Aortic Valve Disease (CAVD) is the most common valve disease and, because medical options are limited , the only therapeutic approach is costly and invasive surgery. Our goal is to determine the mechanisms that control the synthesis of bone morphogenetic protein 2 (BMP2), because BMP2 regulates valve development and stimulates the defining feature of late stage disease - ectopic bone formation in the valve. This project will generate increased understanding of aortic valve biology, potentially reveal biomarkers for early diagnosis, and explore novel therapeutic targets for medical therapy.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZHL1)
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Evans, Frank
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Vanderbilt University Medical Center
Schools of Medicine
United States
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Shah, Tapan A; Zhu, Youhua; Shaikh, Nadia N et al. (2017) Characterization of new bone morphogenetic protein (Bmp)-2 regulatory alleles. Genesis 55:
Fotinos, Anastasios; Fritz, David T; Lisica, Steven et al. (2016) Competing Repressive Factors Control Bone Morphogenetic Protein 2 (BMP2) in Mesenchymal Cells. J Cell Biochem 117:439-47
Rogers, Melissa B; Shah, Tapan A; Shaikh, Nadia N (2015) Turning Bone Morphogenetic Protein 2 (BMP2) on and off in Mesenchymal Cells. J Cell Biochem 116:2127-38
Pregizer, Steven K; Mortlock, Douglas P (2015) Dynamics and cellular localization of Bmp2, Bmp4, and Noggin transcription in the postnatal mouse skeleton. J Bone Miner Res 30:64-70
Yutzey, Katherine E; Demer, Linda L; Body, Simon C et al. (2014) Calcific aortic valve disease: a consensus summary from the Alliance of Investigators on Calcific Aortic Valve Disease. Arterioscler Thromb Vasc Biol 34:2387-93