Benzene metabolites increase vascular permeability by activating heat shock proteins and Rho GTPases

Benzene is a hazardous air pollutant and environmental contaminant emitted into the atmosphere from motor vehicle exhaust, fuel evaporation at gas stations, and during man-made and natural disasters such as wildfires and military burn pit incinerations. Chronic exposure to benzene, even at lower levels, is associated with elevated risk for cardiovascular diseases, including atherosclerosis and cardiac dysfunction. However, the mechanisms of benzene-induced cardiovascular complications remain unknown. Our data suggest that intradermal injections of benzene metabolite trans,trans-muconaldehyde (MA) increased vascular permeability by 54% in C57BL6 mice, while intravenous administration of MA propagated endothelial injury measured by elevated circulating endothelial-specific microparticles. The exposure of primary cardiac microvascular endothelial cells to MA increased vascular permeability, which was detected by transendothelial monolayer electrical resistance and by fluorescently labeled dextran’s diffusion. To investigate the molecular mechanisms of MA-induced endothelial permeability, we exposed mouse cardiac microvascular endothelial cells (CMVEC) to 10 μM MA for 6 hours and human aortic endothelial cells (HAEC) for 2, 6, and 18 hours. Then, we analysed differentially regulated genes (DEG) using bulk RNA sequencing. The transcriptomic analysis indicated that MA increased the expression of stress-response and chaperone genes and perturbed GTPase regulation in endothelial cells. The functional importance of HSP and GTPase signaling pathway in MA-induced endothelial permeability was confirmed in vitro and in vivo. In conclusion, benzene metabolites increased vascular permeability through activation of HSP and GTPase signaling pathways.

Methods
Cell culture: Mouse cardiac microvascular endothelial cells (mCMVEC) and human aortic endothelial cells (HAEC) were purchased from CellBiologics, Chicago, IL. Cells were cultured in a complete endothelial growth medium without phenol red (CellBiologics) and used up to 8 passages for the experiments.

RNA Isolation and RNAseq analysis: HAEC and mCMVEC were incubated with MA (10 µM in 0.1% DMSO) or vehicle (0.1% DMSO) for 2, 6, and 24 hours (mCMVEC were incubated only for 6 hours), and total RNA was isolated using an RNeasy Mini Kit (Qiagen).  RNA quality was measured by Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA), and samples with high RNA integrity were used for subsequent RNAseq analysis. RNA samples were processed by Novogene using mRNA sequencing services (Novogene, Beijing, China). The resultant raw reads of the FASTQ files were aligned to the human genome (hg38) or mouse genome (mm39) using the HISAT2 R package. The mRNA differentially regulated genes (DEG) and pathway enrichment analysis were performed using DESeq2 and ReactomePA enrichPathway R packages.