Epigenetic-mediated Notch pathway activation promotes elastin aortopathy

项目摘要 瓣上主动脉瓣狭窄（SVAS）是一种发育性心血管疾病，单独发生或 威廉姆斯博伦综合征（WBS），并导致过度的动脉平滑肌细胞（SMC）增殖 和管腔阻塞。我们的长期目标是阐明如何减轻这种病理。 弹性蛋白基因ELN的杂合性功能丧失突变或缺失导致SVAS。Eln（-/-）胚胎和 Eln（+/-）新生小鼠发生具有与人SVAS 4 -6相似特征的动脉疾病。弹性蛋白形成了 动脉中弹性膜的组成部分。有缺陷的椎板与过度发育有关， SVAS中的SMC增殖。如果不治疗，SVAS会导致心力衰竭和猝死风险增加， 而风险很大的大手术是唯一的治疗方法。缺乏药物治疗，因为 连接有缺陷的弹性蛋白和SMC过度肌肉化的机制还不完全清楚。我们的目标是 阐明弹性蛋白纤维变性的分子和细胞机制。 Notch通路在调节SMC生物学中至关重要，我们最近报道了Notch在调节SMC生物学中的作用。 SVAS发病机制（JCI，2022）。SMC中通过Notch配体Jagged 1（JAG 1）和NOTCH 3受体的信号传导 激活增殖。我们的研究表明，JAG 1-NOTCH 3通路组分在 弹性蛋白耗竭重要的是，我们确定在Eln（-/-）胚胎中抑制NOTCH 3途径会减弱 在Eln（+/-）幼仔中，主动脉肌化过度和狭窄，并逆转已建立的肌化过度。 表观遗传修饰通过改变染色质可及性影响基因表达，但在我们的研究之前， JCI的论文中，尚未探讨弹性蛋白缺乏症。我们的初步数据表明，缺乏弹性蛋白的腰椎 SMC显示DNA甲基化降低，组蛋白乙酰化升高，DNA表达降低， 甲基转移酶1（DNMT 1）和组蛋白脱乙酰酶1（HDAC 1）。我们假设弹性蛋白缺乏 减弱DNMT 1和HDAC 1介导的Notch途径基因抑制，以促进主动脉 肌肉过度化和狭窄。拟议的研究使用细胞培养、小鼠模型、去识别的人类 样本和先进的基因组和表观遗传技术，以揭示SVAS的机制， 治疗靶向。我们将从两个方面来检验我们的假设。目的1将确定如何弹性蛋白缺乏症 改变人和鼠SMC中的表观遗传介质和表观遗传景观，包括1a）鉴定 弹性蛋白调节的表观遗传酶，1b）确定弹性蛋白缺乏调节的机制 表观遗传调节因子，1c）整合的全基因组表观遗传和转录组学分析，以鉴定新的 调节机制和1d）人类样品中表观遗传酶和标记的表征。目的2 将阐明弹性蛋白、染色质重塑和Notch途径之间的关系，包括2a） 鉴定因果表观遗传机制，2b）动力学和2c）测试HAT抑制作为小鼠中的治疗 SVAS模型。这些研究有望为这种致命的遗传疾病提供新的治疗方法。

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.