Project 4: Impact of cardiovascular disease on proliferation and genetic diversity of hematopoietic stem cells

在衰老过程中，单个造血干细胞(HSC)克隆的后代可以开始主导显著 外周血的一部分，这种现象称为克隆性造血(CH)。CH最近一直在 显示出与心血管疾病风险增加相关。这种联系背后的机制才刚刚开始 有待探索。现有的研究集中在检查司机基因是如何频繁突变的 CH影响驱动CVD的外周免疫细胞的炎症表型。在此，我们建议 调查反向因果关系的可能性。我们将研究CVD如何影响HSC的进化和CH 涌现，使用数学建模和新颖的实验方法。在目标1中，我们将构建一个 体细胞稳态演化和心血管疾病的数学模型。已发现心肌梗死 使HSCs的增殖率翻倍。此外，我们未公布的初步数据，收集于 与其他PPG项目的合作表明，动脉粥样硬化同样可以增加血管的增殖 高达2.5倍的HSCs。我们假设，HSC间隔区的慢性增殖增加 由于中性漂移和/或由于具有选择性优势的克隆的加速出现而加速了CH。 为了检验这些假设，我们将构建一个遗传多样性的随机分枝过程模型。 人类一生中的HSC数量。我们将从单独研究中性漂移开始，然后仔细研究 扩展我们的模型，将有益突变的发生包括在内。这一目标的结果将阐明 HSC增殖在慢性肝炎发生中的作用，为未来研究提供了坚实的定量基础 CH.的作用机理在目标2中，我们将通过实验研究CVD是否会导致克隆性造血。 老鼠。我们将建立CH的小鼠模型，并验证我们的假设，即HSC增殖率增加 由CVD引起的增强CH。我们将使用聚鸟氨酸基因分型，这是一种依赖于非编码的遗传分析， 快速积累插入/缺失突变的超可变DNA重复序列，以测量高 是否在衰老小鼠的血液中发生克隆性扩张的敏感性。然后我们将确定是否 在两种CVD模型(动脉粥样硬化和心肌梗死)中，CH的发生率显著增加。在……里面 目的2B，我们将在移植环境中研究CVD和CH。我们将分离和转导高度纯化的 携带慢病毒的LT-HSC群体携带一个遗传条形码文库，以及七个 可用于跟踪体内克隆动态的荧光蛋白。然后我们将移植标记的造血干细胞 进入去髓细胞的宿主，滴定它们的数量，使CH在相对较短的时间内出现。 最后，我们将诱发心肌梗死和动脉粥样硬化，并对CH的发生率进行比较 年龄匹配的对照组。结合我们在目标1中的电子计算机方法，我们预计这些 实验将提供关于心血管疾病如何影响HSC种群动态的基本见解。

During aging, descendants of individual hematopoietic stem cell (HSC) clones can begin dominating significant portions of the peripheral blood, a phenomenon called clonal hematopoiesis (CH). CH has recently been shown to correlate with an increased risk for CVD. The mechanisms behind this association are only beginning to be explored. Existing studies have focused on examining how driver genes that are frequently mutated in CH affect the inflammatory phenotype of peripheral immune cells that drive CVD. Here, we propose to investigate the possibility of reverse causality. We will study how CVD influences HSC evolution and CH emergence, using mathematical modeling and novel experimental methods. In aim 1, we will construct a mathematical model of somatic HSC evolution in steady state and CVD. Myocardial infarction has been shown to double the proliferation rate of HSCs. Furthermore, our unpublished preliminary data, collected in collaboration with other PPG projects, show that atherosclerosis can similarly increase the proliferation of HSCs by up to 2.5-fold. We hypothesize that chronically elevated proliferation in the HSC compartment accelerates CH due to neutral drift and/or due to accelerated emergence of clones with a selective advantage. To test these hypotheses, we will construct a stochastic branching process model of genetic diversity in the HSC population over a human lifetime. We will begin by studying neutral drift alone and will then carefully expand our model to include the occurrence of beneficial mutations. Results from this aim will elucidate the role of HSC proliferation in CH emergence and provide a firm quantitative basis for future research into the mechanisms of CH. In aim 2, we will investigate experimentally whether CVD causes clonal hematopoiesis in mice. We will establish murine models of CH and test our hypothesis that increased HSC proliferation rates caused by CVD enhance CH. We will use polyguanine genotyping, a genetic analysis that relies on non-coding, hypermutable DNA repeats which rapidly accumulate insertion/deletion mutations, to measure with high sensitivity whether clonal expansions occur in the blood of aging mice. We will then determine whether the incidence of CH is significantly increased in two CVD models (atherosclerosis and myocardial infarction). In Aim 2B, we will study CVD and CH in a transplantation setting. We will isolate and transduce highly purified LT-HSC populations with a lentivirus carrying a library of genetic barcodes, along with a collection of seven fluorescent proteins that can be used to track clonal dynamics in vivo. We will then transplant tagged HSCs into myeloablated hosts, titrating their numbers such that CH emerges within a relatively short time frame. Finally, we will induce myocardial infarction and atherosclerosis and score the incidence of CH in comparison to age-matched controls. In conjunction with our in silico approach in aim 1, we anticipate that these experiments will provide fundamental insights into how HSC population dynamics are influenced by CVD.