Derived from the physicochemical degradation of disposed polymers or synthesized for commercial purposes, plastic particles of the microscales or nanoscales have become widespread environmental contaminants, finding their way into water supplies and the food chain. Human exposure to these particles through inhalation, ingestion, or absorption is inevitable, and this is evidenced by their detection in multiple tissues.¹ However, relatively little is known about the health consequences of microplastic exposures. Using a murine model of exposure, the consumption of drinking water containing polystyrene beads, we have previously found that such exposures led to a dose- and size-dependent potentiation of weight gain, increased body fat, alterations in glucose homeostasis, and changes in the gut microbiome that were consistent with increased adiposity and impaired metabolism.² We also observed that polystyrene consumption impacted the gut-liver-adipose axis, perhaps through alterations of nuclear receptor signaling.³ Studies from other groups have identified similar responses.⁴ As these outcomes are risk factors for cardiovascular disease, we now asked whether oral polystyrene bead consumption promoted overt cardiovascular disease, focusing on atherosclerosis.

To do this, atherosclerosis-prone, ApoE^−/− (apolipoprotein E-deficient) male mice aged 8 weeks were maintained on a chow diet and randomly selected to receive normal water (n=14) or that containing 0.5-µm polystyrene beads at a dose of 1 μg/mL (n=15). These exposure conditions promoted maximal weight gain and hyperglycemia in our previous study and, when considering daily murine water consumption, approximated estimates of human particle exposure. At the end of treatment duration (20 weeks), we observed significantly increased lipid accumulation in the heart valves of mice consuming the polystyrene-containing water compared with mice consuming normal water (Figure [A]). These polystyrene-exposed mice demonstrated increased levels of fasting plasma glucose (P=7.0×10⁻⁴) but lower levels of plasma insulin (P=0.038; Figure [B]). However, there were no differences in homeostatic model assessment for insulin resistance (HOMA-IR) scores between the groups (Figure [C]), nor were there significant changes in plasma lipids or cytokines (Figure [B]).

Figure. Polystyrene consumption potentiates atherosclerotic lesion development. ApoE^−/− (apolipoprotein E-deficient) mice were supplied with a chow diet and either normal water (N) or that containing polystyrene (PS) beads (0.5 µm, 1 μg/mL) for 20 weeks and then euthanized. A, Top, Illustrated are representative heart valve sections stained with oil red O. Bottom, Illustrated are group data (n=14–15). A Shapiro-Wilk test was used to determine normality, and statistical significance (*P=0.016) was determined using the Student t test. B, Listed are plasma and metabolic parameters measured in the 2 groups of mice, their values, and statistical significance. C, Illustrated are the homeostatic model assessment for insulin resistance (HOMA-IR) scores for the 2 treatment groups. The groups were not significantly different as determined using a Mann-Whitney U test-Wilcoxon test. At euthanasia, aortas were collected from the exposed mice and used for a transcriptomics analysis. Illustrated (D) is a gene ontology (GO) analysis of the top 30 impacted functions, where the ordinate is the GO term and the abscissa is the ratio of the differential gene number to the total number of differential genes on the GO term; count, number of genes annotated to a given GO term. E, Listed are the top 10 most upregulated and downregulated genes in the PS-exposed mice vs mice consuming normal water. ALT indicates alanine transaminase; AST, aspartate transaminase; CCR, C-C motif receptor; G-CSF, granulocyte colony-stimulating factor; HDL, high-density lipoprotein; IFNɣ, interferon gamma; IL, interleukin; IP-10, interferon gamma-induced protein 10; KC, keratinocyte-derived chemokine; LDL, low-density lipoprotein; LIX, LPS-induced C-X-C motif chemokine 5; M-CSF, macrophage colony-stimulating factor; MCP-1, monocyte chemoattractant protein-1; MIG, monokine induced by gamma; MIP, macrophage inflammatory protein; RANTES, chemokine (C-C motif) ligand 5; TNFɑ, tumor necrosis factor alpha; VEGF, vascular endothelial growth factor; and VLDL, very low-density lipoprotein.

To gain mechanistic insight, we performed a transcriptomic analysis of aortic lesions. We identified differentially expressed genes (DEGs) using the edgeR software, controlling for false discovery rates using the Benjamini-Hochberg method. DEGs were defined as those with |log2-fold change|≥ 1 and P_adj≤0.05. In this analysis, we found that there were 1622 DEGs between the polystyrene and normal water exposure groups. These consisted of 414 upregulated genes and 1208 downregulated genes. We used the clusterProfiler software to perform a functional analysis of these DEGs. A gene ontology enrichment analysis of the top biological processes impacted by these DEGs is depicted in Figure D. These pathways consist largely of those impacting immune cell function (T-cell/leukocyte activation, regulation of T-cell/lymphocyte activation, leukocyte proliferation, and regulation of cell adhesion). Changes in these pathways are consistent with a proinflammatory, atherosclerotic phenotype. The top 10 most upregulated and downregulated genes are listed in Figure E. The most downregulated gene was vasoactive intestinal peptide, which has vasodilatory properties, protects from intestinal barrier disruption, and suppresses leukocyte activation and migration. Presumably owing to this regulation of leukocyte function and inflammatory responses, exogenously delivery of vasoactive intestinal peptide reduced the number and size of atherosclerotic plaques in a preclinical disease model.⁵ The altered expression of other genes in our exposure model is also consistent with an atherogenic phenotype. Among these include: (1) the upregulation of carboxyl ester lipase (Cel), which limits reverse cholesterol transport but promotes lipoprotein metabolism and LDL (low-density lipoprotein) accumulation; (2) the downregulation of Dynlrb2 (dynein light chain roadblock-type 2), which upregulates cholesterol efflux; (3) the upregulation of Pla2g1b (group 1B phospholipase A2), which disrupts glucose and lipid homeostasis and promotes atherosclerotic lesion formation; and (4) the upregulation of certain matrix-degrading enzymes (Cela3b [chymotrypsin-like elastase family, member 3b] and Ctrb1 [chymotrypsinogen B1]), which accelerates vascular extracellular matrix remodeling. The contribution of polystyrene consumption to atherogenesis through accentuated inflammatory responses, intestinal barrier disruption, or altered cholesterol and lipid handling awaits clarification in future studies. Additionally, whether polystyrene consumption contributes to lesion instability and facilitates rupture, an initiating event of adverse cardiovascular events in humans also awaits further study.

Limitations of the study are a lack of multiple testing and the use of a single animal sex. Nevertheless, despite an uncertain mechanistic definition, these results suggest that exposure to polystyrene may potentiate the development of atherosclerotic lesions and such exposures are a heretofore unrecognized contributor to cardiovascular disease. Further defining the pathological outcomes of microplastic exposure and the underlying mechanisms thereof is a priority research area, given the unabated use of plastics in the modern world, their continued deposition in aquatic and terrestrial ecosystems, their accelerated breakdown owing to increases in global temperatures and UV exposures, and inevitable exposure in humans.

Data for this article are available at doi: 10.5061/dryad.j3tx95xnw.

This study was supported by funding from the National Institutes of Health (grants R01ES019217 and P30ES030283) and had approval from the University of Louisville Institutional Animal Care and Use Committee (No. 23265).

Nonstandard Abbreviations and Acronyms

ApoE−/−	apolipoprotein E-deficient
DEG	differentially expressed gene
LDL	low-density lipoprotein
Pla2g1b	group 1B phospholipase A2

apolipoprotein E-deficient

differentially expressed gene

low-density lipoprotein

group 1B phospholipase A2

Disclosures None.

For Sources of Funding and Disclosures, see page 1230.

中文翻译：

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口服聚苯乙烯消耗增强 ApoE−/− 小鼠动脉粥样硬化病变的形成

微米级或纳米级的塑料颗粒源自废弃聚合物的物理化学降解或出于商业目的而合成，已成为广泛的环境污染物，进入供水系统和食物链。人类通过吸入、摄入或吸收而接触这些颗粒是不可避免的，在多个组织中检测到这些颗粒就证明了这一点。¹然而，人们对微塑料暴露对健康的影响知之甚少。使用小鼠暴露模型，即饮用含有聚苯乙烯珠的饮用水，我们之前发现，这种暴露会导致剂量和大小依赖性的体重增加、体脂增加、葡萄糖稳态改变以及血糖水平的变化。肠道微生物群与肥胖增加和代谢受损一致。²我们还观察到，聚苯乙烯的消耗可能通过改变核受体信号传导来影响肠-肝-脂肪轴。³其他团体的研究也发现了类似的反应。⁴由于这些结果是心血管疾病的危险因素，我们现在询问口服聚苯乙烯珠是否会促进明显的心血管疾病，重点关注动脉粥样硬化。

为此，8 周龄的易患动脉粥样硬化的 ApoE ^−/−（载脂蛋白 E 缺陷）雄性小鼠维持普通饮食，并随机选择接受正常水 (n=14) 或含有 0.5 µm 聚苯乙烯珠的水，温度为剂量为 1 μg/mL (n=15)。在我们之前的研究中，这些暴露条件促进了最大体重增加和高血糖，并且在考虑小鼠每日饮水量时，对人类颗粒暴露进行了近似估计。在治疗结束时（20周），我们观察到与饮用普通水的小鼠相比，饮用含聚苯乙烯水的小鼠心脏瓣膜中的脂质积累显着增加（图[A]）。这些暴露于聚苯乙烯的小鼠表现出空腹血糖水平升高（P =7.0×10 ^-4），但血浆胰岛素水平降低（P =0.038；图[B]）。然而，各组之间的胰岛素抵抗稳态模型评估（HOMA-IR）评分没有差异（图[C]），血浆脂质或细胞因子也没有显着变化（图[B]）。

数字。 聚苯乙烯的消耗会加剧动脉粥样硬化病变的发展。 ApoE ^−/−（载脂蛋白 E 缺陷）小鼠被喂食饲料和普通水 (N) 或含有聚苯乙烯 (PS) 珠（0.5 µm，1 µg/mL）的水 20 周，然后安乐死。A，上图所示是用油红 O 染色的代表性心脏瓣膜切片。下图所示是组数据（n=14-15）。使用 Shapiro-Wilk 检验来确定正态性，并使用 Student t检验确定统计显着性 (* P =0.016) 。B，列出了两组小鼠中测量的血浆和代谢参数、它们的值和统计显着性。C，图示为 2 个治疗组的胰岛素抵抗稳态模型评估 (HOMA-IR) 评分。使用 Mann-Whitney U检验-Wilcoxon 检验确定各组没有显着差异。安乐死时，从暴露的小鼠身上收集主动脉并用于转录组学分析。图( D )为受影响前30个功能的基因本体(GO)分析，纵坐标为GO项，横坐标为GO项上差异基因数与差异基因总数的比值； count，给定 GO term 注释的基因数量。E，列出了暴露于 PS 的小鼠与消耗正常水的小鼠中上调和下调最多的 10 个基因。 ALT表示丙氨酸转氨酶； AST，天冬氨酸转氨酶； CCR，CC基序受体； G-CSF，粒细胞集落刺激因子； HDL，高密度脂蛋白； IFNɣ，干扰素γ； IL，白细胞介素； IP-10，干扰素γ诱导蛋白10； KC，角质形成细胞来源的趋化因子； LDL，低密度脂蛋白； LIX，LPS 诱导的 CXC 基序趋化因子 5； M-CSF，巨噬细胞集落刺激因子； MCP-1，单核细胞趋化蛋白-1； MIG，由伽马诱导的单核因子； MIP，巨噬细胞炎症蛋白； RANTES，趋化因子（CC 基序）配体 5； TNFɑ，肿瘤坏死因子α； VEGF，血管内皮生长因子；和 VLDL（极低密度脂蛋白）。

为了获得机制见解，我们对主动脉病变进行了转录组分析。我们使用edgeR软件识别差异表达基因（DEG），并使用Benjamini-Hochberg方法控制错误发现率。 DEG 定义为 |log2 倍变化 |≥ 1 且P _adj ≤ 0.05。在此分析中，我们发现聚苯乙烯组和正常水暴露组之间存在 1622 DEG。其中包括 414 个上调基因和 1208 个下调基因。我们使用 clusterProfiler 软件对这些 DEG 进行功能分析。图 D 描述了受这些 DEG 影响的主要生物过程的基因本体富集分析。这些途径主要由影响免疫细胞功能的途径组成（T 细胞/白细胞激活、T 细胞/淋巴细胞激活调节、白细胞增殖、和细胞粘附的调节）。这些途径的变化与促炎症、动脉粥样硬化表型一致。图E列出了前10个最上调和下调的基因。最下调的基因是血管活性肠肽，它具有血管舒张特性，防止肠道屏障破坏，并抑制白细胞活化和迁移。据推测，由于白细胞功能和炎症反应的这种调节，外源性递送血管活性肠肽减少了临床前疾病模型中动脉粥样硬化斑块的数量和大小。⁵我们的暴露模型中其他基因表达的改变也与致动脉粥样硬化表型一致。其中包括：（1）羧基酯脂肪酶（Cel）的上调，限制胆固醇反向转运，但促进脂蛋白代谢和LDL（低密度脂蛋白）积累； (2) Dynlrb2（动力蛋白轻链路障 2 型）的下调，从而上调胆固醇流出； (3) Pla2g1b（1B族磷脂酶A2）上调，破坏葡萄糖和脂质稳态，促进动脉粥样硬化病变形成； (4) 某些基质降解酶（Cela3b [胰凝乳蛋白酶样弹性蛋白酶家族，成员 3b] 和 Ctrb1 [胰凝乳蛋白酶原 B1]）的上调，加速血管细胞外基质重塑。聚苯乙烯消耗通过加剧炎症反应、破坏肠道屏障或改变胆固醇和脂质处理而导致动脉粥样硬化形成，有待未来研究的澄清。此外，聚苯乙烯的消耗是否会导致病变不稳定并促进破裂（人类不良心血管事件的始发事件）也有待进一步研究。

该研究的局限性是缺乏多重测试和使用单一动物性别。然而，尽管机械定义不确定，但这些结果表明，接触聚苯乙烯可能会加剧动脉粥样硬化病变的发展，并且这种接触是迄今为止未被认识到的心血管疾病的促成因素。鉴于现代世界塑料的使用有增无减、塑料在水生和陆地生态系统中的持续沉积、以及由于全球气温和紫外线的增加而加速分解，进一步确定微塑料暴露的病理后果及其潜在机制是一个优先研究领域暴露，以及人类不可避免的暴露。

本文的数据可从 doi 获取：10.5061/dryad.j3tx95xnw。

这项研究得到了美国国立卫生研究院的资助（拨款 R01ES019217 和 P30ES030283），并得到了路易斯维尔大学机构动物护理和使用委员会的批准（编号 23265）。

非标准缩写词和首字母缩略词

ApoE −/−	载脂蛋白E缺乏
二甘醇	差异表达基因
低密度脂蛋白	低密度脂蛋白
Pla2g1b	1B 族磷脂酶 A2

载脂蛋白E缺乏

差异表达基因

低密度脂蛋白

1B 族磷脂酶 A2

披露无。

有关资金来源和披露信息，请参阅第 1230 页。