Atherogenic mechanisms of SVEP1, a Novel Human Coronary Artery Disease Locus

项目摘要/摘要 心血管疾病是世界上导致死亡的主要原因。发展非脂类食品至关重要 治疗心血管疾病的方法，因为成功降脂后仍有显著风险。通过 利用人类疾病的发现作为实验研究的起点，我们可以将资源集中在 最适用于人类疾病的机制、途径和治疗策略。 斯蒂齐尔实验室在细胞外基质基因SVEP1中发现了一种变体，它与 患有冠状动脉疾病。为了测试SVEP1是否是风险基因中的因果基因，实验室首先生成了 Svep1单倍体缺乏的易患动脉粥样硬化的小鼠。研究发现，这些小鼠的动脉粥样硬化程度较低 斑块负担明显高于对照组。同样，在成熟的血管平滑肌细胞中有条件地缺失Svep1 小鼠(VSMCs)的斑块负担显著减少。越来越多的证据表明，VSMCs 在动脉粥样硬化中发挥核心作用，包括现在与这些细胞相关的几个疾病位点。除了……之外 产生SVEP1的VSMCs含有Notch和整合素受体，我们假设这些受体与SVEP1结合。我 发现在重组SVEP1上生长的VSMC增加了Notch和整合素信号，以及 参与细胞增殖和分化的基因转录增加。SVEP1诱导健壮 原代VSMC的增殖依赖于Notch和整合素α9β1信号。这些 初步研究结果证实SVEP1可能通过影响VSMC在动脉粥样硬化中起作用 以细胞自主的方式进行增殖和分化。尽管有这些有希望的线索，但机制 SVEP1及其变异体导致疾病的原因尚不完全清楚。 这个项目将通过以下方式回答关于分子和细胞机制的关键、突出的问题 哪个SVEP1促进动脉粥样硬化的形成。我将使用互补的分子技术，细胞培养模型和 动物模型来解决这些问题。我首先的目标是确定SVEP1是否直接与Notch和整合素结合 受体，以及，如果是的话，蛋白质的哪些区域有助于结合亲和力。这项实验也将澄清 每条信号通路在SVEP1对VSMC的整体作用中的作用。领先的风险变量 将被包括在这些研究中，因为变异残基位于假定的整合素结合域 SVEP1。然后，我将使用一种小鼠疾病来询问SVEP1在动脉粥样硬化形成中的细胞机制 模特。这将包括执行谱系追踪和单细胞RNA测序 新生内膜VSMCs内源性SVEP1的产生这种活体方法是对建议的 分子技术，侧重于其病理生理背景下的机制。成功完成 这些目的将揭示SVEP1的常见和危险等位基因促进动脉粥样硬化的机制 同时提供了对世界上最致命疾病的发病机制的洞察，并有可能揭示出新的 治疗候选人。

PROJECT SUMMARY/ABSTRACT Cardiovascular disease is the leading cause of mortality in the world. It is critical to develop non-lipid therapies to address cardiovascular disease since significant risk remains after successful lipid reduction. By using human disease findings as a starting point for experimental investigation, we can focus our resources on the mechanisms, pathways and therapeutic strategies that are the most applicable to human disease. The Stitziel Lab discovered a variant in the extracellular matrix gene, SVEP1, that positively associates with coronary artery disease. To test if SVEP1 is the causal gene in the risk locus, the lab first generated athero-prone mice that were haploinsufficient for Svep1. These mice were found to exhibit less atherosclerotic plaque burden than controls. Similarly, conditional deletion of Svep1 in mature vascular smooth muscle cells (VSMCs) of mice resulted in dramatically less plaque burden. There is a growing body of evidence that VSMCs play a central role in atherosclerosis, including several disease loci now linked to these cells. In addition to producing SVEP1, VSMCs contain Notch and integrin receptors that we hypothesize bind to SVEP1. I discovered that VSMCs grown on recombinant SVEP1 have increased Notch and integrin signaling, as well as increased transcription of genes involved in cell proliferation and differentiation. SVEP1 induces robust proliferation of primary VSMCs, which is dependent on both Notch and integrin α9β1 signaling. These preliminary findings confirm the contribution of SVEP1 to atherosclerosis, potentially by influencing VSMC proliferation and differentiation in a cell-autonomous manner. Despite these promising leads, the mechanisms by which SVEP1 and its variants contribute to disease have yet to be fully characterized. This project will answer critical, outstanding questions about the molecular and cellular mechanisms by which SVEP1 promotes atherogenesis. I will use complementary molecular techniques, cell culture models and animal models to address these questions. I first aim to determine if SVEP1 binds directly to Notch and integrin receptors and, if so, which regions of the protein contribute to binding affinity. This experiment will also clarify the contribution of each signaling pathway to the overall effects of SVEP1 on VSMCs. The leading risk variant will be included in these studies, since the variant residue is within the putative integrin binding domain of SVEP1. I will then interrogate the cellular mechanisms of SVEP1 in atherogenesis using a murine disease model. This will include performing lineage tracing and single cell RNA sequencing with and without the endogenous production of SVEP1 by neointimal VSMCs. This in vivo approach complements the proposed molecular techniques by focusing on mechanisms in their pathophysiologic context. Successful completion of these aims will reveal the mechanisms by which the common and risk allele of SVEP1 promote atherosclerosis while providing insight into the pathogenesis of the world’s deadliest disease with potential to reveal new therapeutic candidates.