Endothelial Inflammatory Transcriptional Responses to an Altered Serum Exposome Following Inhalation of Diesel Emissions

Air pollution, especially emissions derived from traffic sources, is associated with adverse cardiovascular outcomes. However, it remains unclear how inhaled factors drive an extrapulmonary pathology, as the lung is an effective barrier for solid particulates and many gases. Previously, using plasma from healthy human subjects exposed to diesel exhaust under controlled conditions, we found that canonical inflammatory response transcripts of interleukin-8 (IL-8), intracellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) were elevated in endothelial cells treated wih plasma obtained after exposure compared with pre-exposure samples or filtered air (sham) exposures. While the findings confirmed the presence of bioactive factor(s) in the plasma after diesel inhalation, we wanted to better examine the complete genomic response to investigate 1) major responsive transcripts and 2) collected response pathways and ontogeny that may help to refine this method. Thus, we assayed previousy collected RNA with microarray chips, examining the responses of cultured endothelial cells to plasma obtained from 6 healthy human subjects exposed to 100 μg/m3 diesel exhaust or filtered air for 2 h on separate occasions. In addition to pre-exposure baseline samples, we investigated samples obtained immediately post and 24h post exposure. Primary human coronary artery endothelial cells were grown to confluence and treated with 10% plasma for 24 h, followed by isolation of RNA for microarrays. Microarray analysis of the coronary artery endothelial cells challenged with plasma identified 855 probes that change over time following diesel exhaust exposure. Over-representation analysis identified inflammatory cytokine pathways were upregulated both at the 2 and 24 h condition. These outcomes are consistent with our recent findings that plasma contains bioactive and inflammatory factors following pollutant inhalation and provide a novel pathway to explain the well-reported extrapulmonary toxicity of ambient air pollutants. There are 48 total samples representing three time points from two different conditions (air or diesel emissions) from eight study subjects.

空气污染，尤其是交通源排放物，与不良心血管结局密切相关。然而，目前尚不清楚吸入性因素如何驱动肺外病理改变，因为肺部是固体颗粒物与多数气体的有效物理屏障。既往研究中，我们在受控实验条件下让健康人体受试者暴露于柴油机尾气，采集其血浆样本；结果显示，与暴露前样本或过滤空气（假暴露）样本相比，经暴露后血浆处理的内皮细胞（endothelial cells）中，白细胞介素-8（IL-8）、细胞间黏附分子-1（ICAM-1）以及血管细胞黏附分子-1（VCAM-1）的经典炎症反应转录本水平均显著升高。尽管上述发现证实柴油机尾气吸入后血浆中存在生物活性因子，但我们希望更全面地分析全基因组应答，以探究两个研究方向：1）主要应答转录本；2）可用于优化该实验方法的已鉴定应答通路与发生发育轨迹（ontogeny）。因此，我们利用此前采集的RNA样本通过微阵列芯片（microarray chips）进行检测，分析培养的内皮细胞对血浆的应答情况。这些血浆样本来自6名健康人体受试者，他们分别在不同时段暴露于100 μg/m³的柴油机尾气或过滤空气，暴露时长为2小时。除暴露前基线样本外，我们还分析了暴露后即刻以及暴露后24小时采集的样本。我们将原代人冠状动脉内皮细胞（primary human coronary artery endothelial cells）培养至汇合状态，用10%体积分数的血浆处理24小时，随后分离RNA用于微阵列检测。对经血浆处理的冠状动脉内皮细胞的微阵列分析显示，共有855个探针在柴油机尾气暴露后随时间发生表达变化。富集分析（over-representation analysis）发现，炎症细胞因子通路在暴露后2小时与24小时两个时间点均出现上调。上述结果与我们近期的研究发现一致，即污染物吸入后血浆中存在生物活性与炎症因子，并为解释已被广泛报道的环境空气污染物肺外毒性提供了全新的分子通路。本数据集共包含48份样本，来自8名研究受试者，涵盖两种暴露条件（空气暴露或柴油机尾气排放暴露）下的三个时间点。