Electronic Cigarette Extracts Alter Endothelial Cell Inflammatory Responses

Abstract

Electronic cigarettes (e‐cigarettes) have recently become a popular alternative to traditional tobacco cigarettes. The effects of traditional cigarettes on endothelial cells (ECs) have been well‐documented and illustrate that tobacco products predispose ECs towards cardiovascular pathologies. However, the effects of e‐cigarettes on ECs have not been studied even though the composition of e‐vapor has been shown to resemble the composition of tobacco smoke in a number of important parameters that have been related to cardiovascular disease progression. We hypothesized that exposure to e‐cigarette extracts would elicit a pro‐inflammatory response through the activation of the complement cascade, a down‐regulation of the negative regulators of complement and an increase in the production/release of pro‐inflammatory cytokines. We also hypothesized that the increase in pro‐inflammatory state would be dependent on the dose of the e‐vapor exposure (e.g. nicotine concentration in the e‐cigarette) and that exposure to e‐vapor would induce an overall reduction in EC culture conditions. In order to test this hypothesis, human umbilical vein endothelial cells (HUVECs) were incubated with extracts of tobacco smoke, e‐cigarette vapors, and pure nicotine for 2–3 days. A solid‐phase ELISA technique was used to measure the deposition of various complement proteins, including C1q, iC3b, C4d and C5b‐9, onto the cell surface, along with the expression of the complement C1q receptor (gC1qR and cC1qR) and the negative regulators of complement. All conditions – tobacco smoke, e‐cigarette vapor, and pure nicotine – led to an increase in C1q receptor expression. In contrast to studies performed on other cell lines and results of tobacco smoke exposure, the deposition of complement proteins decreased after exposure to e‐cigarette vapor extracts. Interestingly, we also observed a decrease in the expression/release of negative regulators of complement for most of the e‐cigarette vapor extracts that were investigated here. After exposure to e‐vapor, ECs up‐regulated the production/release of pro‐inflammatory cytokines. To quantify changes in cell culture conditions, EC metabolic activity, cell viability and cell density were measured using a standard MTT assay and a live/dead cytotoxicity assay. After exposure to e‐cigarette vapor extracts, we observed a decrease in metabolic activity, in some cases to a further extent than that caused by mainstream tobacco smoke exposure. A slight decrease in cell viability was also observed, combined with a marked decrease in cell density. Thus, it is possible that the observed reductions in complement deposition (or complement inhibitor expression), were due to a decrease in the cell number as compared with the control conditions. Overall, these results indicate that e‐cigarettes present a significant risk to vascular endothelial cells and may alter the progression of cardiovascular diseases.