Fig 1.
eNOS activation is impaired in endothelial cells from individuals who use pod-based e-cigarettes to a similar degree as combustible cigarette use.
Ser-1177 phosphorylation was compared between the unstimulated and acetylcholine stimulated endothelial cells and evaluated as a percentage change. Data are plotted as individual measurements for each participant, mean and standard error of the mean compared by ANOVA with Tukey post-hoc testing for between group comparisons. Cigarette = combustible cigarette.
Table 1.
Clinical characteristics.
Fig 2.
The JUUL e-liquid and its individual components induce cellular death.
The percentage of cells staining positive for TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) were assessed with dilutions from 1 x 10−5 to 1% (n = 3). DNase was used as a positive control. The percentage of apoptosis for all dilutions of each e-liquid were compared to control (* indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.005). Additionally, a dose-dependent nonparametric trend test was performed for each e-liquid type (indicated by horizontal line p-value). Data are plotted as mean and SEM.
Fig 3.
JUUL e-liquids and nicotine salt impair nitric oxide production.
4,5-diaminofluorescein diacetate fluorescence was assessed as a percentage change with A23187 stimulation. HAECs were exposed to one of the tested e-liquids at 1 x 10−5 and 1 x 10−4% dilutions (indicated as 10−5 and 10−4, respectively; n = 5), and comparisons were made between control and the tested dilutions of each e-liquid (* indicates p<0.05 and ** indicates p<0.01, *** indicates p<0.001). Data are expressed as relative change in NO production upon A23187 stimulation normalized to controls. RQ = relative quantification.
Fig 4.
Heated particle fractions of JUUL e-liquids impair nitric oxide production.
HAECs were exposed to 0.001% dilution of heated particle fractions collected at stage 6 of the 10-stage mini-MOUDI cascade impactor. 4,5-diaminoflourescein diacetate fluorescence was assessed as a percentage change with A23187 stimulation. Comparisons were made between control to each heated particle fraction (n = 4). Standard Cig = 3R4F (standard nicotine cigarette); Low Yield Cig = 1R5F (low yield nicotine cigarette). Data are plotted as mean and SEM. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.005.
Fig 5.
Inflammatory gene expression and pod e-liquid exposure.
Interleukin-6 (IL-6) expression in endothelial cells was quantified using reverse transcription-quantitative PCR, respectively. All 2-ΔΔCT values were computed relative to untreated cells, and comparisons were made between the control and tested dilutions of each e-liquid (n = 5). Data are expressed as gene expression levels normalized to matched controls. RQ = relative quantification. ** indicates p<0.01.
Fig 6.
Menthol-containing JUUL e-liquids induce oxidative stress.
MitoSox fluorescence was quantified in HAECs exposed to 1 x 10−5% dilutions of JUUL e-liquids, and the constituents PG:VG and nicotine salt (n = 4). Each e-liquid was compared to control. Data are expressed as fluorescence intensity normalized to control, dots represent individual experiments, columns and error bars are mean and SEM. * indicates p<0.05.