PROJECT SUMMARY Supravalvular aortic stenosis (SVAS) is a developmental cardiovascular disease, occurring alone or in Williams Beuren Syndrome (WBS), and results in excessive arterial smooth muscle cell (SMC) proliferation and lumen obstruction. Our long-term objective is to elucidate how this pathology can be attenuated. Heterozygous loss-of-function mutations or deletions of the elastin gene ELN cause SVAS. Eln(-/-) embryos and Eln(+/-) neonatal mice develop arterial disease with features similar to human SVAS4-6. Elastin forms the major component of the elastic lamellae in arteries. Defective lamellae are associated with excessive developmental SMC proliferation in SVAS. If untreated, SVAS results in heart failure and an increased risk of sudden death, and major surgery, which carries substantial risk, is the only treatment. Medical therapies are lacking because mechanisms linking defective elastin and SMC hypermuscularization are incompletely defined. We aim to elucidate molecular and cellular mechanisms underlying elastin aortopathy. The Notch pathway is critical in regulating SMC biology, and we recently reported a role for Notch in SVAS pathogenesis (JCI, 2022). Signaling via Notch ligand Jagged1 (JAG1) and NOTCH3 receptor in SMCs activates proliferation. Our studies reveal that JAG1-NOTCH3 pathway components are upregulated after elastin depletion. Importantly, we determined that inhibiting the NOTCH3 pathway in Eln(-/-) embryos attenuates aortic hypermuscularization and stenosis and reverses established hypermuscularization in Eln(+/-) pups. Epigenetic modifications influence gene expression by altering chromatin accessibility but prior to our JCI paper, have not been explored in elastin deficiency. Our initial data indicate that elastin deficient aortas and SMCs display reduced DNA methylation, elevated histone acetylation and reduced expression of DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1). We hypothesize that elastin deficiency attenuates DNMT1- and HDAC1-mediated repression of Notch pathway genes to promote aortic hypermuscularization and stenosis. The proposed studies use cell culture, mouse models, de-identified human samples and advanced genomic and epigenetic techniques to uncover mechanisms of SVAS that can be therapeutically targeted. We will test our hypothesis in two aims. Aim 1 will determine how elastin deficiency alters epigenetic mediators and the epigenetic landscape in human and murine SMCs, including 1a) identifying elastin-regulated epigenetic enzymes, 1b) determining mechanisms by which elastin deficiency modulates epigenetic regulators, 1c) an integrated genome-wide epigenetic and transcriptomic analysis to identify new regulatory mechanisms and 1d) characterization of epigenetic enzymes and marks in human samples. Aim 2 will elucidate the relationship between elastin, chromatin remodeling and the Notch pathway, including 2a) identifying causal epigenetic mechanisms, 2b) kinetics and 2c) testing HAT inhibition as a therapy in mouse models of SVAS. These studies promise to yield new treatments for this lethal genetic disease.

PROJECT NARRATIVE Supravalvular aortic stenosis (SVAS) is a devastating developmental genetic disease characterized by accumulation of excess smooth muscle cells which narrows the aorta, reducing blood flow and putting a heightened load on the heart. We recently discovered that a specific set of molecules, known as the Notch pathway, plays a key role in this disease and is modulated by a gene regulatory system known as epigenetics. We propose to use mouse models as well as human cells and tissue samples and cutting-edge epigenetic approaches to study underlying mechanisms and thus delineate concrete steps towards developing novel therapeutic approaches for SVAS and other vascular diseases.