Oral Polystyrene Consumption Potentiates Atherosclerotic Lesion Formation in ApoE^−/− Mice

Derived from the physicochemical degradation of disposed polymers or synthesized for commercial purposes, plastic particles of the micro- or nano-scales have become wide-spread 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 microplastics (MP) exposures. Using a murine model of exposure, the consumption of drinking water containing polystyrene (PS) 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 PS 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 (CVD), we now asked whether oral PS bead consumption promoted overt CVD, focusing on atherosclerosis.

To do this, atherosclerosis-prone, apolipoprotein E-deficient (ApoE^−/−) male mice at 8wk of age were maintained on a chow diet and randomly selected to receive normal water (n=14) or that containing 0.5μm PS beads at a dose of 1μg/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 (20wk), we observed significantly increased lipid accumulation in the heart valves of mice consuming the PS-containing water compared to mice consuming normal water (Figure 1A). These PS-exposed mice demonstrated increased levels of fasting plasma glucose (p=7.0x10⁻⁴), but lower levels of plasma insulin (p=0.038) (Figure 1B). However, there were no differences in HOMA-IR scores between the groups (Figure 1C), nor were there significant changes in plasma lipids or cytokines (Figure 1B).

Figure 1. Polystyrene consumption potentiates atherosclerotic lesion development.

ApoE^−/− mice were supplied with a chow diet and either normal water (N) or that containing polystyrene beads (PS: 0.5μm, 1μg/ml) for 20wks and then euthanized. A) Upper: Illustrated are representative heart valve sections stained with oil red O. Lower: 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 a Student’s t-Test. B) Listed are plasma and metabolic parameters measured in the two groups of mice, their values, and statistical significance. C) Illustrated are the HOMA-IR scores for the two treatment groups. The groups were not significantly different as determined using a Mann-Whitney-Wilcoxon test. At euthanasia, aortae were collected from the exposed mice and used for a transcriptomics analysis. Illustrated (D) is a gene ontology (GO) analysis of top 30 impacted functions, where the ordinate is the GO term and the abscissa is 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 up-regulated and down-regulated genes in the PS-exposed mice versus mice consuming normal water.

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 rate using the Benjamini-Hochberg method. DEGs were defined as those with a |log2 fold change|≥ 1 and a padj ≤ 0.05. In this analysis, we found that there were 1622 DEGs between the PS and normal water exposure groups. These consisted of 414 up-regulated genes and 1208 down-regulated 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 1D. These pathways consist largely of those impacting immune cell function (T-cell/leukocyte activation, regulation of T-cell/lymphocyte activation, leukocyte proliferation, regulation of cell adhesion). Changes in these pathways are consistent with a pro-inflammatory, atherosclerotic phenotype. The top 10 most up-regulated and down-regulated genes are listed in Figure 1E. The most down-regulated gene was vasoactive intestinal peptide (Vip), 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 Vip reduced the number and size of atherosclerotic plaques in a preclinical disease model.⁵ The altered expression of other genes in our exposure model are 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 accumulation; 2) the downregulation of Dynlrb2, which upregulates cholesterol efflux; 3) the upregulation of group 1B phospholipase A2 (Pla2g1b) which disrupts glucose and lipid homeostasis and promotes atherosclerotic lesion formation; and 4) the upregulation of certain matrix-degrading enzymes (Cela3b, Ctrb1) which accelerate vascular extracellular matrix remodeling. The contribution of PS consumption to atherogenesis thorough accentuated inflammatory responses, intestinal barrier disruption, or altered cholesterol and lipid handling awaits clarification in future studies. Additionally, whether PS 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 PS, may potentiate the development of atherosclerotic lesions and that such exposures are a heretofore unrecognized contributor to CVD. Further defining the pathological outcomes of MP exposure and the underlying mechanisms thereof are priority research areas given 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 Availability.

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