Modeling Cardiovascular Risks of E-Cigarettes With Human-Induced Pluripotent Stem Cell-Derived Endothelial Cells

Abstract

Background: Electronic cigarettes (e-cigarettes) have experienced a tremendous increase in use. Unlike cigarette smoking, the effects of e-cigarettes and their constituents on mediating vascular health remain understudied. However, given their increasing popularity, it is imperative to evaluate the health risks of e-cigarettes, including the effects of their ingredients, especially nicotine and flavorings.

Objectives: The purpose of this study was to investigate the effects of flavored e-cigarette liquids (e-liquids) and serum isolated from e-cigarette users on endothelial health and endothelial cell-dependent macrophage activation.

Methods: Human-induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and a high-throughput screening approach were used to assess endothelial integrity following exposure to 6 different e-liquids with varying nicotine concentrations and to serum from e-cigarette users.

Results: The cytotoxicity of the e-liquids varied considerably, with the cinnamon-flavored product being most potent and leading to significantly decreased cell viability, increased reactive oxygen species (ROS) levels, caspase 3/7 activity, and low-density lipoprotein uptake, activation of oxidative stress-related pathway, and impaired tube formation and migration, confirming endothelial dysfunction. Upon exposure of ECs to e-liquid, conditioned media induced macrophage polarization into a pro-inflammatory state, eliciting the production of interleukin-1β and -6, leading to increased ROS. After exposure of human iPSC-ECs to serum of e-cigarette users, increased ROS linked to endothelial dysfunction was observed, as indicated by impaired pro-angiogenic properties. There was also an observed increase in inflammatory cytokine expression in the serum of e-cigarette users.

Conclusions: Acute exposure to flavored e-liquids or e-cigarette use exacerbates endothelial dysfunction, which often precedes cardiovascular diseases.

Keywords: e-cigarette aerosol; e-liquid flavoring; endothelial dysfunction; iPSC-ECs.

Figure 1.. Assessment of e-liquid flavor-induced cytotoxicity in iPSC-ECs.

Effects of 6 e-liquid flavors with different nicotine concentrations on iPSC-EC viability after 48-hour treatment were determined using a luminescent CellTiter-Glo 2.0 assay. The data were obtained using iPSC-ECs from 3 biological donors and the assay was repeated twice. Data are represented as mean ± SD. ^ap < 0.05, compared to controls within 0 mg nicotine/ml group; ^bp < 0.05, compared to controls within 6 mg nicotine/ml group; ^cp < 0.05, compared to control within 18 mg nicotine/ml group. Statistically significant from controls (Bonferroni-adjusted P<0.05). VG: vegetable glycerin; PG: propylene glycol.

Figure 2.. Assessment of e-liquid flavor-induced ROS production in iPSC-ECs.

Effects of 6 e-liquid flavors with different nicotine concentrations on intracellular ROS production in iPSC-ECs after 48-hour treatment were determined using a ROS-Glo^TM H₂O₂ assay. The data were obtained using iPSC-ECs from 3 biological donors and the assay was repeated twice. Data are represented as mean ± SD. ^*p < 0.05 and ^**p < 0.001, compared to controls within each group. Statistically significant from controls (Bonferroni-adjusted P<0.05). VG: vegetable glycerin; PG: propylene glycol.

Figure 3.. Assessment of e-liquid flavor-induced caspase 3/7 activity in iPSC-ECs.

Effects of 6 e-liquid flavors with different nicotine concentration on caspase 3/7 activity for apoptosis in iPSC-ECs after 48-hour treatment were determined using a Caspase-Glo® 3/7. The data were obtained using iPSC-ECs from 3 biological donors and the assay was repeated twice. Data are represented as mean ± SD. *p < 0.05 and **p < 0.001, compared to controls within each group. Statistically significant from controls (Bonferroni-adjusted P<0.05). VG: vegetable glycerin; PG: propylene glycol.

Figure 4.. Effect of e-liquid treatment on endothelial function of iPSC-ECs.

(A and C) The cells were incubated with each e-liquid flavor for 16 hours to allow the formation of capillary-like structures on Matrigel and then imaged by phase-contrast microscopy (x20). (B and D) Quantitative data from the tube-formation assay. (E) LDL uptake was measured in iPSC-ECs after treatment of the e-liquid for 2 days. (F) Representative images and (G) quantitative data from the migration assay at 0, 4, and 10 hours in the presence of the e-liquid flavors Marcado and RY4. The migration data were normalized to time point 0. Data are represented as mean ± SEM. *p < 0.05 and **p < 0.001, compared to controls; and ^#p<0.05, compared to groups treated with 0 mg nicotine/ml.

Figure 5.. Effect of conditioned media from iPSC-ECs treated with e-liquid on macrophage polarization.

(A) Conditioned media was collected from iPSC-ECs after 48 hours of incubation with each e-liquid flavor, and then exposed to macrophage-like cells for 48 hours. Phenotype of macrophages was analyzed by flow cytometry for M1 (CD40) and M2 (CD163) markers. (B) After 48 hours incubation with conditioned media, the media was replaced with the fresh macrophage culture media for 16 hours to determine cytokine expression and ROS levels. Expression of cytokines produced by macrophages was determined by quantitative real-time RT-PCR. (C) Intracellular ROS levels produced by macrophages was determined using a ROS-Glo H₂O₂ assay. Data are represented as mean ± SEM. Statistically significant from controls (Bonferroni-adjusted P<0.05). MAR0: Marcado e-liquid without nicotine; MAR18: Marcado e-liquid with 18 mg/ml of nicotine; RY0: RY4 e-liquid without nicotine; RY18: RY4 e-liquid with 18 mg/ml of nicotine.

Figure 6.. Effects of sera from active e-cigarette users and cigarette smokers on ROS generation and tube formation of iPSC-ECs in vitro.

iPSC-ECs were incubated with 10% serum from nonsmokers (n=5), e-cigarette users (n=4), and cigarette smokers (n=5) for 48 hours. (A) Intracellular ROS production in iPSC-ECs treated with sera collected from nonsmokers, e-cigarette users, or cigarette smokers at baseline. (B) Serial changes of ROS production in iPSC- ECs treated with serum collected from smokers before (−1 hours; shown in detail in A) and after (0, 1, and 3 hours) smoking. (C) iPSC-ECs (1 × 10⁴) treated with serum samples from nonsmokers or smokers were seeded onto μ-Slide Angiogenesis containing Matrigel® Basement Membrane Matrix for 16 hours, and the formation of capillary-like structures were then imaged by phase-contrast microscopy (x 20) and quantified. Data are represented as mean ± SD. *p < 0.05 and **p < 0.001, compared to nonsmokers or baseline; and ^#p < 0.05, compared to pre-smoking within each group.

Figure 7.. Serum cytokine levels in active e-cigarette users and cigarette smokers obtained before and after smoking.

Concentration of IL-6, ICAM-1, MCSF, and MCP-1 in serum from e-cigarette users (A) or cigarette smokers (B) were compared before (Pre) and 3 hours after smoking (Post). Data are represented as mean ± SEM. *p < 0.05, compared to pre-smoking. IL-6 = interleukin-6; ICAM-1 = intracellular adhesion molecule-1; MCSF = macrophage colony- stimulating factor; MCP-1 = monocyte chemoattractant protein-1.

Central Illustration.. Human Induced Pluripotent Stem Cell-Derived Endothelial Cells for evaluating e-cigarette risk.

Mechanistic overview by which e-cigarette use might case acute endothelial dysfunction. Exposure of endothelial cells to e-cigarette flavorings or serum of e-cigarette users leads to endothelial dysfunction associated with increased apoptosis, ROS, and inflammation. IL-6 = interleukin-6; ICAM-1 = intracellular adhesion molecule-1; MCSF = macrophage colony-stimulating factor; MCP-1 = monocyte chemoattractant protein-1.