Polystyrene microplastic particles induce endothelial activation

Abstract

Due to its increasing production, durability and multiple applications, plastic is a material we encounter every day. Small plastic particles from the μm to the mm range are classified as microplastics and produced for cosmetic and medical products, but are also a result of natural erosion and decomposition of macroplastics. Although being omnipresent in our environment and already detected in various organisms, less is known about the effects of microplastics on humans in general, or on vascular biology in particular. Here we investigated the effects of carboxylated polystyrene microplastic particles (PS, 1 μm) on murine endothelial and immune cells, which are both crucially involved in vascular inflammation, using in vitro and in vivo approaches. In vitro, PS induced adhesion molecule expression in endothelial cells with subsequent adhesion of leukocytes both under static and flow conditions. In monocytic cells, PS enhanced pro-inflammatory cytokine expression and release. Accordingly, administering mice with PS led to enhanced aortic expression of cytokines and adhesion molecules. Furthermore, we identified neutrophils as the PS-clearing blood leukocyte population. The findings from this study for the first time indicate polystyrene microplastic as a new environmental risk factor for endothelial inflammation.

Fig 1. Effects of PS particles on cell viability and inflammatory gene expression.

(A) Cell viability (%) of J774A.1 and MyEND cells after 16 hours of PS particles stimulation (10³−10⁷ particles/mL) was determined using alamarBlue cell viability assay, n = 3–5. (B) Il1-β, Tnf-α expression and TNF-α levels after 3 hours of PS particles stimulation (10⁷ particles/mL) in J774A.1 cells (above) and Vcam-1, Icam-1 expression and sVCAM-1 levels after 6 hours of PS stimulation (10⁷ particles/mL) in MyEND cells (below) were determined by real-time PCR and by ELISA, respectively, n = 7–10. *P<0.05, **P<0.01, ***P<0.001 vs. control (con). Data were analyzed by Student t-test and are depicted as mean±SD.

Fig 2. Effects of PS particles on monocytic cell adhesion to endothelial cells.

Fluorescence microscopy images depicting Calcein-AM-labelled J774A.1 cells on a MyEND monolayer after stimulation with PS particles for 16 hours (10⁷ particles/mL), one hour adhesion under static (A) and two hours under flow (B) conditions and subsequent washing. Scale bars = 100 μm. Adherent cells per high power field (HPF) were quantified. N = 4–6. **P<0.01 vs. control (con). Data were analyzed by Student t-test and are depicted as mean±SD.

Fig 3. Effects of PS particles in vivo: Hepatic accumulation and mRNA expression.

(A) Confocal microscopy composite of liver sections from mice 3 hours after injection with 2.5 mg TRITC-conjugated PS particles. blue: DAPI, red: Alexa Fluor 488-WGA, green: TRITC-PS (appear intracellularly in yellow). Scale bars: overview = 50 μm, insertion = 10 μm, z-stack in right panel = 5 μm. (B) Hepatic Saa1, Saa2 and Saa3 expression of mice administered with either 2.5 mg TRITC-conjugated PS particles or PBS as control were determined by real-time PCR, n = 8–9 mice/group. **P<0.01, ***P<0.001 vs. control (con). Data were analyzed by Student t-test and are depicted as mean±SD.

Fig 4. Effects of PS particles in vivo: Peripheral blood and aortic tissue.

(A) Overlay of brightfield and epiflourescence (left) and rendered confocal image stack (right) of peripheral blood cells 3 hours after administration of 2.5 mg TRITC-conjugated PS particles. Whole blood was collected and red blood cells were lysed prior to staining. Scale bar = 10 μm. Blue: DAPI, red: FITC-Ly6G, green: TRITC-PS. (B) Il-1β, Vcam-1 and Icam-1 expression in aortic tissue of mice administered with either 2.5 mg TRITC-conjugated PS particles or PBS as control were determined by real-time PCR, n = 7 mice/group. *P<0.05, **P<0.01 vs. control (con). Data were analyzed by Student t-test and are depicted as mean±SD.