Platelet Mitochondrial Epigenetics: New CVD Markers of Particle and Metal Effects

描述(申请人提供)：生物标记物是细胞、生化或分子的改变，可以在人体组织中轻松和非侵入性地测量，并直接或间接地参与疾病的途径。心血管疾病(CVD)是美国的主要死亡原因，每年有80万人死亡。在流行病学研究中，短期和长期暴露于空气污染/有毒金属一直与心血管疾病的发病率和死亡有关。血小板参与动脉粥样硬化和动脉血栓形成的发展，而动脉血栓形成是导致心血管疾病的主要因素。血小板计数的增加被认为是调节颗粒物(PM)效应的手段。血小板是无核的，但线粒体DNA(MtDNA)可以很容易地从血浆中分离出来。线粒体对PM相关的氧化高度敏感，最重要的是，血小板严重依赖于线粒体中的能量产生。我们假设，空气污染物和金属暴露可能导致暴露诱导的全身氧化/炎症反应中血小板线粒体DNA甲基化(MtDNA)的改变。血小板线粒体DNA甲基化的改变可能导致或至少反映线粒体功能障碍，直接或间接地参与将毒性暴露与心血管疾病风险联系起来的途径。我们建议使用标准老龄化研究(NAS)的数据和生物样本来检验这些假设，NAS是一个大型的预期老年个体队列。我们已经修改和调整了从血浆血小板中分离mtDNA的方案，并成功地测试了我们的高度时间和成本效益的方法。700名参与者的血浆血小板将在相隔4年的两个时间点进行mtDNA氧化损伤(8-oxo-DG)、标准DNA甲基化(5-MC)和新兴羟甲基化(5-OH-MC)标记的线粒体全球含量以及mtDNA综合序列中的特定区域甲基化的测量。我们将确定空气污染和金属对mtDNA生物标记物的影响(目标1)；评估mtDNA生物标记物与心血管疾病风险的进一步关联(目标2)；以及进行一项探索性目标(目标3)，通过中介分析确定血小板mtDNA的变化是否直接或间接地在将毒性暴露与氧化/炎症标记物联系起来，并最终与心血管疾病风险联系起来。这项拟议的研究将是第一次表征与心血管疾病风险相关的血小板线粒体DNA甲基化的机制。该项目的重点是血小板，这是一个在心血管疾病病因学中具有关键作用的单一纯细胞群体，将提供对表观遗传学结果的解释，从而克服目前在大量人类群体中进行表观遗传学研究的主要局限性，这些群体因使用混合细胞群体，如白细胞或颊细胞而受到阻碍。由于血小板可以廉价和非侵入性地从血浆中分离出来，我们的研究具有很高的潜力，可以产生未来CVD的新的风险和诊断生物标记物。如果我们观察到假设的相关性，我们将寻求资金来扩大这一研究路线，在具有高心血管疾病风险的不同人群中进行大规模研究。

DESCRIPTION (provided by applicant): Biomarkers are cellular, biochemical, or molecular alterations that can be easily and non-invasively measured in human tissues and are directly or indirectly involved in the pathway of disease. Cardiovascular disease (CVD) is the leading cause of death in the United States accounting for 800,000 deaths every year. Both short- and long-term exposure to air pollution/toxic metals has been consistently associated with CVD morbidity and death in epidemiologic studies. Platelets contribute to the development of both atherosclerosis and arterial thrombosis, which are primary contributing events leading to CVD. Increased platelet counts have been suggested to mediate particulate matter (PM) effects. Platelets are anucleate, but mitochondrial DNA (mtDNA) can be easily isolated from plasma platelets. Mitochondria are highly sensitive to PM-related oxidation and, most importantly, platelets are critically dependent on energy production in mitochondria. We hypothesize that air pollutants and metals exposure may cause alteration in platelet mitochondrial DNA methylation (mtDNA) in response to exposure-induced systemic oxidation/inflammation. Platelet mtDNA methylation changes may lead to, or at a minimum reflect mitochondrial dysfunction participating directly or indirectly to the paths linking toxic exposures to CVD risk. We propose t test these hypotheses using data and biospecimens from the Normative Aging Study (NAS), a large prospective cohort of aging individuals. We have modified and adapted protocols to isolate mtDNA from plasma platelets, and successfully tested our highly time- and cost-effective approach. Plasma platelets from 700 participants will be measured at two time points separated by 4 years for mtDNA oxidative damage (8-oxo-dG), mitochondrial global content of both standard DNA methylation (5-mC) and the emerging hydroxymethylation (5-OH-mC) mark, and locus-specific methylation in a comprehensive panel of mtDNA sequences. We will determine the effects of air pollution and metals on mtDNA biomarkers (Aim 1); evaluate further association of mtDNA biomarkers with CVD risk (Aim 2); and conduct an exploratory aim (Aim 3) to determine via mediation analysis whether platelet mtDNA changes are directly or indirectly on the paths linking toxic exposures to oxidation/inflammation markers, and ultimately to CVD risk. The proposed study will be the first to characterize mechanisms in platelet mtDNA methylation related to CVD risk. The project's focus on platelets, a single pure cell population with critical roles in CVD etiology will provide ease of interpretation of epigenetic findings and thus overcome the major limitation of current epigenetic studies in large human population, which have been hampered by the use of mixed cell populations such as white blood cells or buccal cells. Because platelets can be inexpensively and non- invasively isolated from plasma, our study has high potential to generate novel risk and diagnostic biomarkers of future CVD. If we observed the hypothesized associations, we will seek funding to expand this line of research in large-scale studies in diverse populations with high risk of CVD.