Abstract Body: Introduction: Micro- and nanoplastics (MNPs) are linked to atherosclerotic disease, but underlying mechanisms are not understood. Autophagy is a complex, critical cellular process that maintains homeostasis by removing misfolded proteins, damaged organelles and foreign invaders. Induction of autophagy machinery takes many forms, but all involve conjugation of intracellular membranes with Atg8/LC3 complexes (Atg8ylation). We previously observed upregulation of autophagy-related genes in vascular smooth muscle cells (VSMCs) from human carotid plaque high in MNPs and in animal experiments of MNP-induced aortic atherosclerosis. Disturbances in autophagy are known to influence VSMC phenotype toward a pro-atherogenic state but the precise effects of MNPs on Atg8ylation in VSMCs specifically remain obscure.
Hypothesis: We hypothesize that MNPs modulate autophagy pathways and induce Golgi apparatus disruption in VSMCs.
Methods: Primary human aortic VSMCs were exposed to polystyrene fluorescent nanospheres (200 nm), with or without V-ATPase blocker bafilomycin A1 (BafA1). Intracellular membrane Atg8ylation was measured by LC3 immunofluorescence to quantify LC3 intensity and colocalization with particles. Results were compared with fluorescent-labeled ocean-derived natural MNPs (<20 µm) of mixed polymer content. Golgi fragmentation was quantified by immunostaining for the trans-Golgi marker TGN46 and cis-Golgi marker GM130.
Results: Control cells had mean 6.2 LC3+ puncta/cell compared to 19.4 (3-fold) and 52 (8-fold) LC3+ puncta/cell after exposure to 10 µg/mL MNPs and 100 µg/mL MNPs, respectively. With BafA1, LC3 significantly decreased, a known indicator of non-canonical autophagy. In cells treated with ocean MNPs, LC3 intensity again significantly increased with plastic exposure. However, addition of BafA1 produced variable effects depending on MNP concentration, suggesting that polymer type may influence the precise autophagic response mechanism. Both types of MNPs did not cause Golgi fragmentation, with no significant difference in TGN46 and GM130 puncta.
Conclusions: These data provide the first evaluation of the direct effects of nanoplastics on human VSMCs, linking MNP-induced autophagic dysfunction to Golgi-independent non-canonical autophagy, which we term Nanoplastic-Associated Membrane Atg8ylation (NAMA), and suggesting polymer-specific modulation of cellular responses. NAMA influence on VSMC phenotype and atherosclerosis is the subject of future work.