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

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.

Werner syndrome (WRN) DNA helicase and base excision repair (BER) factors maintain endothelial homeostasis.

The accelerated ageing disease Werner Syndrome (WRN) is characterized by pronounced atherosclerosis. Here, we investigated the influence of WRN downregulation on the functionality of non-replicating human endothelial cells. RNAi-mediated downregulation of WRN reduces cell motility and enhances the expression of factors regulating adhesion, inflammation, hemostasis and vasomotor tone. Moreover, WRN influences endothelial barrier function and Ca2+-release, while cell adhesion, Dil-acLDL-uptake and the mRNA expression of NO-synthases (eNOS, iNOS) remained unaffected. Regarding motility, we propose that WRN affects Rac1/FAK/ß1-integrin-related mechanisms regulating cell polarity and directed motility. Since oxidative DNA base damage contributes to aging and atherosclerosis and WRN affects DNA repair, we investigated whether downregulation of base excision repair (BER) factors mimics the effects of WRN knock-down. Indeed, downregulation of particular WRN-interacting base excision repair (BER) proteins (APE1, NEIL1, PARP1) imitates the inhibitory effect of WRN on motility. Knock-down of OGG1, which does not interact with WRN, does not influence motility but increases the mRNA expression of E-selectin, ICAM, VCAM, CCL2 and VEGFR and stimulates adhesion. Thus, individual BER factors themselves differently impact endothelial cell functionality and homeostasis. Impairment of endothelial activities caused by genotoxic stressor (tBHQ) remained largely unaffected by WRN. Summarizing, both WRN, WRN-associated BER proteins and OGG1 promote the maintenance of endothelial cell homeostasis, thereby counteracting the development of ageing-related endothelial malfunction in non-proliferating endothelial cells.

The protein kinase IKKepsilon contributes to tumour growth and tumour pain in a melanoma model.

Inhibitor-kappaB kinase epsilon (IKKε) constitutes a non-canonical I-κB kinase, which amongst others modulates NF-κB activity. IKKε and NF-κB have both been described for their role in cell proliferation and their dysregulation has been associated with tumourigenesis and metastasis in multiple cancer types. Accordingly, overexpression and constitutive activation of NF-κB have also been shown in melanoma, however, the role of IKKε in this cancer type has not been investigated so far. Thus, we determined IKKε expression in malignant melanoma cells and we were able to show a significant overexpression of IKKε in tumour cells in comparison to melanocytes. Inhibition of IKKε either by shRNA or the pharmacological inhibitor amlexanox resulted in reduced cell proliferation associated with a cell cycle block in the G1-phase. Functional analysis indicated that NF-κB, Akt1 and MAPK pathways might be involved in the IKKε-mediated effects. In vivo, we applied a mouse melanoma skin cancer model to assess tumour growth and melanoma-associated pain in IKKε knockout mice as well as C57BL/6 mice after inoculation with IKKε-negative cells. In IKKε knockout mice, tumour growth was not altered as compared to IKKε wild type mice. However, melanoma associated pain was strongly suppressed accompanied by a reduced mRNA expression of a number of pain-relevant genes. In contrast, after inoculation of IKKε-depleted tumour cells, the development of melanoma was almost completely prevented. In conclusion, our data suggest that IKKε in the tumour plays an essential role in tumour initiation and progression while IKKε expression in tumour surrounding tissues contributes to melanoma-associated pain.