Abstract 16844: Relative Endothelial Toxicity of Tobacco Smoke and E-Cigarette Aerosol: A Functional and Mechanistic Assessment

Abstract

Background: Smoking cigarettes decreases expression of eNOS in the endothelium, resulting in lower nitric oxide (NO) secretion and decreased flow-mediated dilation in the conducting arteries. In contrast, the effects of e-cigarettes (e-cigs) on endothelial function are just beginning to be studied and the mechanisms of action are unclear. Aim: To test the hypothesis that circulating factors from e-cigarette users decrease endothelial eNOS protein levels and NO secretion in primary endothelial cell cultures, relative to nonsmokers and cigarette smokers. Methods: 36 healthy individuals were recruited and grouped as nonsmokers (n=12), cigarette smokers (n=7), and e-cig users (n=17). Human umbilical vein endothelial cells (HUVECs) were cultured at 20,000 cells per well in 24-well culture plate. Serum samples from individual subjects and endothelial growth media were added at a 1:1 ratio to confluent cells and incubated for 12 hours (basal condition), followed by fresh medium containing VEGF at 50 ng/ml for 30 min (stimulated condition). The amount of NO liberated from cells was measured in culture supernatants by the chemilumin- escence method using a NO analyzer. Endothelial NO synthase (eNOS) protein levels in HUVECs was measured in cell lysates by ELISA. NO and eNOS results were normalized to cell number. Results: The cells treated with serum from e-cig users vs. nonsmokers produced less NO upon stimulation (P<.03) and contained less eNOS protein (P<.03). eNOS protein level was lower in the e-cig user serum group than in the cigarette smoker serum group (P<.05), although stimulated NO production was decreased less by e-cig user serum than by smoker serum. Lower eNOS levels in the cigarette group vs. non-smoker group was not significant, but stimulated NO was significantly lower in the cigarette group vs. nonsmoker (P<.006; see figure). Conclusion: Exposure of cultured endothelial cells to circulating factors from e-cig users, relative to non-users, leads to lower eNOS protein levels and decreased NO production.