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

项目概要/摘要 心血管疾病是世界上导致死亡的主要原因。开发非脂质至关重要 治疗心血管疾病的疗法，因为成功降脂后仍然存在重大风险。由 使用人类疾病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。 Stitziel 实验室发现了细胞外基质基因 SVEP1 中的一个变体，该变体与 患有冠状动脉疾病。为了测试 SVEP1 是否是风险基因座中的致病基因，实验室首先生成了 Svep1 单倍体不足的易患动脉粥样硬化的小鼠。这些小鼠被发现表现出较少的动脉粥样硬化 斑块负担高于对照组。同样，成熟血管平滑肌细胞中 Svep1 的条件性缺失 小鼠的血管平滑肌细胞（VSMC）显着减少了斑块负担。越来越多的证据表明 VSMC 在动脉粥样硬化中发挥核心作用，包括目前与这些细胞相关的几个疾病位点。除了 产生 SVEP1 的 VSMC 含有 Notch 和整合素受体，我们假设它们与 SVEP1 结合。我 发现在重组 SVEP1 上生长的 VSMC 增加了 Notch 和整合素信号传导，并且 参与细胞增殖和分化的基因转录增加。 SVEP1 诱导鲁棒 原代 VSMC 的增殖，依赖于 Notch 和整合素 α9β1 信号传导。这些 初步研究结果证实 SVEP1 可能通过影响 VSMC 来促进动脉粥样硬化 以细胞自主的方式增殖和分化。尽管有这些有希望的线索，但机制 SVEP1 及其变体导致疾病的机制尚未完全确定。 该项目将通过以下方式回答有关分子和细胞机制的关键、悬而未决的问题 其中 SVEP1 促进动脉粥样硬化形成。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。我的第一个目标是确定 SVEP1 是否直接与 Notch 和整合素结合 受体，如果是的话，蛋白质的哪些区域有助于结合亲和力。这个实验也将澄清 每个信号通路对 SVEP1 对 VSMC 的总体影响的贡献。主要风险变体 将被纳入这些研究中，因为变体残基位于推定的整联蛋白结合域内 SVEP1。然后我将使用小鼠疾病来探究 SVEP1 在动脉粥样硬化形成中的细胞机制 模型。这将包括在有或没有的情况下进行谱系追踪和单细胞 RNA 测序。 新生内膜 VSMC 内源性产生 SVEP1。这种体内方法补充了所提出的 分子技术，重点关注其病理生理学背景中的机制。顺利完成 这些目标将揭示 SVEP1 的常见和风险等位基因促进动脉粥样硬化的机制 同时深入了解世界上最致命疾病的发病机制，有可能揭示新的 治疗候选者。

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.