Endothelial Proteotoxic Stress Drives Inhaled Benzene-Induced Vascular Inflammation in Mice

Abstract Benzene exposure is associated with increased cardiovascular disease (CVD) risk, yet the mechanisms linking benzene to vascular injury remain incompletely understood. Here, we investigated the effects of benzene and its reactive metabolite trans,trans-muconaldehyde (MA) on endothelial activation and leukocyte recruitment, early events in atherogenesis. Wild-type and endothelial-specific heat shock protein A1B overexpressing (EC-HSPA1B-TG) mice were exposed to inhaled benzene (1 ppm), and leukocyte dynamics were assessed by intravital microscopy. Complementary studies in vitro examined MA-induced endothelial activation via leukocyte adhesion assays, RNA sequencing, pharmacological interventions, and siRNA-mediated gene silencing. Benzene inhalation increased leukocyte rolling (16-21-fold) and adhesion (11-44-fold) in vivo with female mice exhibiting greater responses than males. In vitro, MA enhanced monocyte adhesion (1.7-fold) and transmigration (1.4-fold) and induced a conserved transcriptional program characterized by activation of oxidative stress, unfolded protein response (UPR), MAPK signaling, and heat shock pathways. MA stimulated phosphorylation of p38, JNK, and eIF2α, increased XBP1 splicing, elevated reactive oxygen species generation, and depleted glutathione. Alleviation of ER stress with 4-phenylbutyric acid attenuated MA-induced expression of HSPA1B, ATF3, and ICAM1. Conversely, HSPA1B silencing exacerbated endothelial activation and stress signaling, whereas endothelial-specific overexpression of HSPA1B significantly reduced benzene-induced leukocyte recruitment in vivo. Collectively, these findings identify proteotoxic stress as a central mechanism of benzene-induced vascular toxicity. Further, we demonstrate that activation of the heat shock pathway (HSF1-HSPA1B) serves as a protective response that limits endothelial inflammation. These results provide new mechanistic insight into how environmental benzene exposure may promote vascular injury and CVD in humans.