Epigenetic-mediated Notch pathway activation promotes elastin aortopathy

项目总结 主动脉瓣上狭窄(SVAS)是一种发育性心血管疾病，单独或合并 Williams Beuren综合征(WBS)，并导致动脉平滑肌细胞(SMC)过度增殖 还有管腔梗阻。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杂合性功能丧失突变或缺失导致SVAS。ELN(-/-)胚胎和 ELN(/-)新生小鼠出现动脉疾病，其特征类似于人类SVAS4-6。弹性蛋白是主要的 动脉中弹力板的组成部分。板层缺陷与过度发育有关 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， 而大手术是唯一的治疗方法，它有很大的风险。缺乏药物治疗是因为 弹性蛋白缺陷和SMC过度肌化之间的联系机制尚未完全确定。我们的目标是 阐明弹性蛋白大动脉病变的分子和细胞机制。 Notch通路在调节SMC生物学中起关键作用，我们最近报道了Notch在SMC生物学中的作用。 SVAS发病机制(JCI，2022)。Notch配体Jagged1(JAG1)和NOTCH3受体在SMC中的信号转导 激活了扩散。我们的研究表明，JAG1-NOTCH3途径组件在 弹性蛋白耗尽。重要的是，我们确定抑制ELN(-/-)胚胎中的NOTCH3途径会减弱 ELN(/-)幼鼠的主动脉过度肌化和狭窄，并逆转已建立的过度肌化。 表观遗传修饰通过改变染色质可及性来影响基因表达，但在我们的 JCI论文中，还没有对弹性蛋白缺乏症进行过探讨。我们的初步数据显示弹性蛋白缺乏的大动脉 SMC表现为DNA甲基化减少，组蛋白乙酰化增加，DNA表达减少 甲基转移酶1(DNMT1)和组蛋白脱乙酰基酶1(HDAC1)。我们假设弹性蛋白缺乏症 减弱DNMT1和HDAC1介导的Notch途径基因抑制促进主动脉 肌肉化和狭窄。这项拟议的研究使用了细胞培养、小鼠模型、去鉴定人类 样本和先进的基因组和表观遗传学技术，以揭示SVAS的机制， 在治疗上有针对性。我们将在两个目标上检验我们的假设。目标1将确定弹性蛋白缺乏症 改变人类和小鼠SMC的表观遗传介体和表观遗传格局，包括1)识别 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 表观遗传调节剂，1c)整合的全基因组表观遗传和转录分析，以识别新的 人类样本中表观遗传酶和标记的调节机制和一维特征。目标2 将阐明弹性蛋白、染色质重塑和Notch途径的关系，包括2a) 确定因果表观遗传机制，2b)动力学和2c)测试HAT抑制作为治疗方法 SVAS的模型。这些研究有望为这种致命的遗传病找到新的治疗方法。

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.