Human primary endothelial cells are impaired in nucleotide excision repair and sensitive to benzo[a]pyrene compared with smooth muscle cells and pericytes

Joana M Kress,Bernd Kaina,Alessandra Pulliero,Lorella Di Dio,Alberto Izzotti,Larissa Heck,Gerhard Fritz,Kathrin Laarmann

doi:10.1038/s41598-019-49953-w

Abstract

The endothelium represents the inner cell layer of blood vessels and is supported by smooth muscle cells and pericytes, which form the vessel structure. The endothelium is involved in the pathogenesis of many diseases, including the development of atherosclerosis. Due to direct blood contact, the blood vessel endothelium is inevitably exposed to genotoxic substances that are systemically taken up by the body, including benzo[a]pyrene, which is a major genotoxic component in cigarette smoke and a common environmental mutagen and human carcinogen. Here, we evaluated the impact of benzo[a]pyrene diol epoxide (BPDE), which is the reactive metabolite of benzo[a]pyrene, on the three innermost vessel cell types. Primary human endothelial cells (HUVEC), primary human smooth muscle cells (HUASMC) and primary human pericytes (HPC) were treated with BPDE, and analyses of cytotoxicity, cellular senescence and genotoxic effects were then performed. The results showed that HUVEC were more sensitive to the cytotoxic activity of BPDE than HUASMC and HPC. We further show that HUVEC display a detraction in the repair of BPDE-induced adducts, as determined through the comet assay and the quantification of BPDE adducts in post-labelling experiments. A screening for DNA repair factors revealed that the nucleotide excision repair (NER) proteins ERCC1, XPF and ligase I were expressed at lower levels in HUVEC compared with HUASMC and HPC, which corresponds with the impaired NER-mediated removal of BPDE adducts from DNA. Taken together, the data revealed that HUVEC exhibit an unexpected DNA repair-impaired phenotype, which has implications on the response of the endothelium to genotoxicants that induce bulky DNA lesions, including the development of vascular diseases resulting from smoking and environmental pollution.

Highlights

The inner layer of blood vessels is lined with more than 10 billion endothelial cells, which regulate, among other features, coagulation and blood pressure and play an important role in wound healing[1]
HUVEC are more sensitive to BPDE than human primary smooth muscle cells (HUASMC) and human primary pericytes (HPC)
To compare the effect of BPDE on vascular cells, we first determined the cytotoxicity of BPDE in primary HUVEC, HUASMC and HPC through the MTT viability assay and apoptosis and necrosis measurement through annexin V/PI flow cytometry

Summary

Introduction

The inner layer of blood vessels is lined with more than 10 billion endothelial cells, which regulate, among other features, coagulation and blood pressure and play an important role in wound healing[1]. The wide environmental distribution of B[a]P and the existence of defence systems, including DNA repair, encouraged us to question the extent to which the various blood vessel cell types are protected against BPDE and the differences in their sensitivity to DNA insults resulting from BPDE exposure. To address these questions, we compared the responses of human primary endothelial cells (HUVEC), human primary smooth muscle cells (HUASMC) and human primary pericytes (HPC) to BPDE. Our findings revealed that HUVEC are more sensitive to BPDE than HPC and HUASMC and display an unexpected DNA repair-impaired phenotype

Methods

Results

Conclusion