DOI: 10.5061/dryad.12jm63z8q
title: Benzene metabolites increase vascular permeability by activating heat shock proteins and Rho GTPases

description: 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.
description: <strong>Cell culture:</strong> 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.
 <strong>RNA Isolation and RNAseq analysis:</strong>
 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.
description: # Benzene metabolites increase vascular permeability by activating heat
 shock proteins and Rho GTPases Dataset DOI:
 [10.5061/dryad.12jm63z8q](10.5061/dryad.12jm63z8q) ## Description of the
 data and file structure We analyzed gene expression profiles in mouse
 cardiac microvascular endothelial cells (mCMVEC) and human aortic
 endothelial cells (HAEC) exposed to 10 μM of trans,trans-muconaldehyde
 (MA) for the indicated time using bulk RNA sequencing. Specific analysis
 details are available in the associated manuscript. Description of the
 data and file structure Raw fastq files Name  Cell type         
 Treatment       Time Z1_1.fq.gz       mCMVEC       Control             6h
 Z1_2.fq.gz       mCMVEC       Control             6h Z2_1.fq.gz      
 mCMVEC       Control             6h Z2_2.fq.gz       mCMVEC       Control
             6h Z3_1.fq.gz       mCMVEC       Control             6h
 Z3_2.fq.gz       mCMVEC       Control             6h Z4_1.fq.gz      
 mCMVEC       Control             6h Z4_2.fq.gz       mCMVEC       Control
             6h Z5_1.fq.gz       mCMVEC       MA                  6h
 Z5_2.fq.gz       mCMVEC       MA                  6h Z6_1.fq.gz      
 mCMVEC       MA                  6h Z6_2.fq.gz       mCMVEC       MA     
             6h Z7_1.fq.gz       mCMVEC       MA                  6h
 Z7_2.fq.gz       mCMVEC       MA                  6h Z8_1.fq.gz      
 mCMVEC       MA                  6h Z8_2.fq.gz       mCMVEC       MA     
             6h   C1_1.fq.gz      HAEC  Control             2h
 C1_2.fq.gz      HAEC  Control             2h C2_1.fq.gz      HAEC  Control
             2h C2_2.fq.gz      HAEC  Control             2h
 C3_1.fq.gz      HAEC  Control             2h C3_2.fq.gz      HAEC  Control
             2h C4_1.fq.gz      HAEC  Control             6h
 C4_2.fq.gz      HAEC  Control             6h C5_1.fq.gz      HAEC  Control
             6h C5_2.fq.gz      HAEC  Control             6h
 C6_1.fq.gz      HAEC  Control             6h C6_2.fq.gz      HAEC  Control
             6h C7_1.fq.gz      HAEC  Control             24h
 C7_2.fq.gz      HAEC  Control             24h C8_1.fq.gz      HAEC 
 Control             24h C8_2.fq.gz      HAEC  Control             24h
 C9_1.fq.gz      HAEC  Control             24h C9_2.fq.gz      HAEC 
 Control             24h   T1_1.fq.gz       HAEC  MA      2h
 T1_2.fq.gz       HAEC  MA                  2h T2_1.fq.gz       HAEC 
 MA                  2h T2_2.fq.gz       HAEC  MA                  2h
 T3_1.fq.gz       HAEC  MA                  2h T3_2.fq.gz       HAEC 
 MA                  2h T4_1.fq.gz       HAEC  MA                  6h
 T4_2.fq.gz       HAEC  MA                  6h T5_1.fq.gz       HAEC 
 MA                  6h T5_2.fq.gz       HAEC  MA                  6h
 T6_1.fq.gz       HAEC  MA                  6h T6_2.fq.gz       HAEC 
 MA                  6h T7_1.fq.gz       HAEC  MA                  24h
 T7_2.fq.gz       HAEC  MA                  24h T8_1.fq.gz       HAEC 
 MA                  24h T8_2.fq.gz       HAEC  MA                  24h
 T9_1.fq.gz       HAEC  MA                  24h T9_2.fq.gz       HAEC 
 MA                  24h Counts data were analyzed using the DESeq2
 R-package: * File "DEG.MA_mCMVEC_6h.csv" contains differential
 expression data generated from DESeq2 comparing normalized counts for
 mCMVEC following 6 hours of exposure to 10 uM MA vs vehicle (0.1% DMSO). *
 File "DEG.MA_HAEC_2h.csv" contains differential expression data
 generated from DESeq2 comparing normalized counts for HAEC following 2
 hours of exposure to 10 uM MA vs vehicle (0.1% DMSO). * File
 "DEG.MA_HAEC_6h.csv" contains differential expression data
 generated from DESeq2 comparing normalized counts for HAEC following 6
 hours of exposure to 10 uM MA vs vehicle (0.1% DMSO). * File
 "DEG.MA_HAEC_24h.csv" contains differential expression data
 generated from DESeq2 comparing normalized counts for HAEC following 24
 hours of exposure to 10 uM MA vs vehicle (0.1% DMSO). In each of the
 files, column "baseMean" represents the mean of normalized
 counts for all samples, log2FoldChange represents the Log base 2 of the
 fold change, lfcSE represents the standard error of the log base 2 of the
 fold change, column “stat” represents the difference in deviance between
 the reduced model and the full model, which is compared to a chi-squared
 distribution to generate a p-value, pvalue represents the Wald test
 p-value: condition treated vs untreated, and padj represents the BH
 adjusted p-values. The files above were used to identify significant
 enrichment of upstream regulators and signaling pathways among the
 differentially expressed genes in the datasets. The file
 **"Reactome_mCMVEC.csv"** contains the results of the Reactome
 analysis of DEGs in the mCMVEC_6h (MA vs Control) dataset. Description of
 column names: ID: The unique identifier for the Reactomr term Description:
 The descriptive name of the Reactome term GeneRatio: The ratio of genes in
 this list that are annotated with this term, formatted as (M/N) (where (M)
 is the number of your genes in the set and (N) is the total number of your
 genes in the set). BgRatio: The ratio of the number of genes in the
 background universe that are annotated with this term, formatted as (M/N)
 (where (M) is the number of background genes in the set and (N) is the
 total number of background genes). pvalue: over-representation assessed
 using hypogeometric distribution, which is equivalent to a one-sided
 Fisher's exact test. p.adjust: The adjusted p-value, which corrects
 for multiple testing to control the false discovery rate (FDR). qvalue: An
 estimate of the FDR calculated from the p-values. geneID: The gene IDs
 that overlap with the functional term. Count: Total number of genes from
 the input gene list that match the functional term. The file
 **"enrichGO_HAEC.csv"** contains the results of the analysis
 using the enrichGO function in the clusterProfiler R package. The analysis
 was performed on all three time points for HAEC exposed to MA vs Control.
 Description of column names: ID: The unique identifier for the GO term
 Description: The descriptive name of the GO term GeneRatio: The ratio of
 genes in this list that are annotated with this term, formatted as (M/N)
 (where (M) is the number of your genes in the set and (N) is the total
 number of your genes in the set). BgRatio: The ratio of the number of
 genes in the background universe that are annotated with this term,
 formatted as (M/N) (where (M) is the number of background genes in the set
 and (N) is the total number of background genes). pvalue:
 over-representation assessed using hypogeometric distribution, which is
 equivalent to a one-sided Fisher's exact test. p.adjust: The adjusted
 p-value, which corrects for multiple testing to control the false
 discovery rate (FDR). qvalue: An estimate of the FDR calculated from the
 p-values. geneID: The gene IDs that overlap with the functional term.
 Count: Total number of genes from the input gene list that match the
 functional term.