E-Cigarette Aerosol Condensate Leads to Impaired Coronary Endothelial Cell Health and Restricted Angiogenesis

Figure 1. Cell viability in HCAEC exposed to (A) Direct effects of EAC. MTT Assay was performed on HCAEC after exposure to various concentration of EAC generated from: (i) a PG/VG solution (non-flavoured), (ii) 0 mg nicotine (tobacco flavoured), and (iii) 18 mg nicotine (tobacco flavoured) for 24 h. (B) Indirect effects of EAC. A549 epithelial lung cells were exposed to EAC under the same conditions. Media from the treated A549 cells were then used to treat HCAEC on a separate plate for 24 h before cell viability was assessed via MTT assay. Results are expressed as mean ± SEM (n = 4 biological replicates). One-way ANOVA with Bonferroni post-tests was used for statistical analysis; * p < 0.05, ** p < 0.01, *** p < 0.001 versus Ctrl.

Figure 2. Reactive oxygen species levels in HCAEC after (A) Direct EAC exposure. ROS levels were measured in HCAEC after exposure to various concentration of EAC generated from: (i) a PG/VG standard (non-flavoured), (ii) 0 mg nicotine (tobacco flavoured), and (iii) 18 mg nicotine (tobacco flavoured) at for 24 h Data shown is expressed as a mean ± SEM (n = 3 biological replicates). (B) Indirect effects of EAC. A549 epithelial lung cells were exposed to EAC under the same conditions. Media from the treated A549 cells were then used to treat HCAEC on a separate plate for 24 h before a DCF assay was performed. Data shown is expressed as a mean ± SEM (n = 5 biological replicates). One-way ANOVA with Bonferroni post-tests was used for statistical analysis, ** p < 0.01; **** p < 0.0001 versus Ctrl.

Figure 3. Expression of cellular adhesion molecules after exposure to EAC treatment. HCAEC were exposed to various concentrations of EAC generated from: (i) 0 mg nicotine (tobacco flavoured) and (ii) 18 mg nicotine (tobacco flavoured) for 24 h. (A) VCAM-1 protein expression. (B) ICAM-1 protein expression. (C) Indirect effects of EAC on ICAM-1 protein exposure. A549 epithelial lung cells were exposed to EAC under the same conditions. Media from the treated A549 cells were then used to treat HCAEC on a separate plate for 24 h before measuring ICAM-1 protein levels. Results are expressed as mean ± SEM (n = 3 biological replicates). One-way ANOVA with Bonferroni post-tests was used for statistical analysis, ** p < 0.01 versus Ctrl.

Figure 4. (A) Changes in membrane conduction of tethered bilayer lipid membranes (tBLM) in response to EAC (1% and 10%) in 100 mM NaCl 10 mM tris pH 7 buffer (n = 3). EAC solutions containing nicotine increase membrane conduction (membrane permeability). The effect of the nicotine-containing EAC rapidly falls away following a buffer wash. (B) In contrast, only minor changes of the membrane capacitances are observed in the same tBLMs, suggesting permeability changes aren’t related to large membrane structural changes.

Figure 5. Cardiac VCAM1, ICAM1, and CD31 mRNA expression following treatment of mice with e-cigarettes with or without nicotine. RT-qPCR was performed on the left ventricle of mice exposed to ambient air (SHAM) or e-Cig aerosol (0 mg, 18 mg nicotine). (A) FKBPL. (B) CD31. (C) VCAM-1. (D) ICAM-1. All data expressed as mean fold change ± SEM (n = 5–9). One-way ANOVA with Bonferroni post-test was used for statistical analysis, * p < 0.05 versus Sham.

Figure 6. (A) Immunohistochemical on seven-week-old Balb/c female mice left ventricle sections (Scale bar = 20 μm). Mice were treated in 3 groups: SHAM (ambient air), 0 mg (no nicotine), and 18 mg (nicotine) treatment groups. Sections were stained for FKBPL (green), CD31 (red), and DAPI (blue) and images were taken at 20×. (B) FKBPL staining intensity was quantified as the mean greyscale value in three images per sample, ** p < 0.005 (SHAM vs. 18 mg). (C) CD31 staining intensity was quantified as the mean greyscale value in three images per sample, * p < 0.05 (SHAM vs. 18 mg). Results are expressed as mean ± SEM (n = 5–9) compared to SHAM. One-way ANOVA with Kruksal-Wallis post-tests was used for statistical analysis.

Figure 7. Experimental setup for e-cigarette aerosol condensate collection.