Calcific aortic valve disease (CAVD) affects 25% of those aged >65 years, and its incidence is growing inevitably as our population ages. Increased transforming growth factor beta1 (TGF1) protein is found in human CAVD and pericardial fibrosis and calcification associated with pericarditis, and TGF1 stimulates calcification in vito in cells with osteoblastic potential. TGF1 can induce calcification in vitro in porcine and ovine valve interstitial cells (VIC). TGF1 is anti-inflammatory and pro-fibrotic growth factor cytokine which regulates transcription through its downstream Smad2/3 mediators. TGF1 is implicated in both inflammatory and non-inflammatory cardiovascular diseases, including cardiac fibrosis and atherosclerosis. Despite the strong association between increased TGF1 and aortic valve calcification, the role of TGF1 and its downstream signaling mechanisms in the development and progression of CAVD in vivo remain unknown. Our data shows that conditional overexpression of TGF1 in VICs in adult mice results in calcification of peri-aortic and aortic valve hinge-region, as seen in the Klotho-deficient mice. It is known that TGF1 signaling is dependent upon secreted Klotho protein, an anti-aging hormone. TGF1 overexpression also leads to increased expression of osteochondrogenic transcription factor Sox4. Moreover, SOX4 is misexpressed in the human CAVD, and Sox4 knockdown (via siRNA) in vitro in mouse valve progenitor cells decreases the expression of calcification markers such as Runx2. These findings suggest the novel hypothesis that the CAVD occur as a result of augmented TGF1 signaling via canonical Smad and activation of Sox4 expression in the VICs. Rigorous analyses of TGF1 transgenic mice will be done over time as a model of aging. In addition, we will use genetic (Smad3 heterozygous deletion) and pharmacologic (anti-TGF1 drugs) approaches to rescue the onset and progression of CAVD in TGF1 transgenic mice (Aim 1). Next, we will test the hypothesis that Klotho critically mediates TGF1-dependent inputs within the VICs to regulate the onset and progression of CAVD (Aim 2). R26RlacZ and TGF1-responsive SBE-luc reporter mice will be integrated into these models to permit cell fate mapping and measurement of the cell autonomous or cell-cell actions of TGF1. Finally, we will use conditional transgenic and in vitro valve progenitor cell micromass culture to test the hypothesis that Sox4 critically mediates TGF1-dependent transcriptional inputs to regulate the pathogenesis of CAVD (Aim 3). Our experiment will identify the signaling network for TGF1 within the valve interstitial cells, which will provide a novel perspective to designing safer treatment strategies for the CAVD patients. For example, instead of targeting TGF1 signaling in general for treatment of CAVD (e.g., Losartan), which runs the risk of inducing increased inflammatory activity, targeting TGF1/Smad3 might reduce the unintentional overall TGF1/Smad hyperactivity while at the same time removing the pro-inflammatory side-effects. Thus, this project will provide information useful for practical applications that could enhance human health and well-being.

Public Health Relevance

Calcific aortic valve disease (CAVD) is common with age and that there is no effective treatment for CAVD. Despite the strong association between increased TGF1 and CAVD in humans, the role of TGF1 in the development and progression of CAVD is not known. By using TGF1 overexpressing mouse model we will determine if increased TGF1 causes CAVD, identify the mechanism of TGF1 function in CAVD, and develop novel anti-TGF1 strategies to block the progression of CAVD.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
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Evans, Frank
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University of South Carolina at Columbia
Anatomy/Cell Biology
Schools of Medicine
United States
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Chopra, Sunita; Al-Sammarraie, Nadia; Lai, Yimu et al. (2017) Increased canonical WNT/?-catenin signalling and myxomatous valve disease. Cardiovasc Res 113:6-9
Azhar, Mohamad; Ware, Stephanie M (2016) Genetic and Developmental Basis of Cardiovascular Malformations. Clin Perinatol 43:39-53