E-cigarette exposure causes early pro-atherogenic changes in an inducible murine model of atherosclerosis

Abstract

Introduction: Evidence suggests that e-cigarette use (vaping) increases cardiovascular disease risk, but decades are needed before people who vape would develop pathology. Thus, murine models of atherosclerosis can be utilized as tools to understand disease susceptibility, risk and pathogenesis. Moreover, there is a poor understanding of how risk factors for atherosclerosis (i.e., hyperlipidemia, high-fat diet) intersect with vaping to promote disease risk. Herein, we evaluated whether there was early evidence of atherosclerosis in an inducible hyperlipidemic mouse exposed to aerosol from commercial pod-style devices and e-liquid. Methods: Mice were injected with adeno-associated virus containing the human protein convertase subtilisin/kexin type 9 (PCSK9) variant to promote hyperlipidemia. These mice were fed a high-fat diet and exposed to room air or aerosol derived from JUUL pods containing polyethylene glycol/vegetable glycerin (PG/VG) or 5% nicotine with mango flavoring for 4 weeks; this timepoint was utilized to assess markers of atherosclerosis that may occur prior to the development of atherosclerotic plaques. Results: These data show that various parameters including weight, circulating lipoprotein/glucose levels, and splenic immune cells were significantly affected by exposure to PG/VG and/or nicotine-containing aerosols. Discussion: Not only can this mouse model be utilized for chronic vaping studies to assess the vascular pathology but these data support that vaping is not risk-free and may increase CVD outcomes later in life.

Keywords: atherosclerosis; e-cigarette; hyperlipidaemia; mouse model; vaping.

FIGURE 1

Experimental design and mouse model of hyperlipidemia. (A) C57BL/6J male mice were injected with AAV-PCSK9^DY virus and started on high fat diet on the same day. 1 week following injection mice were exposed to either room air, PG/VG only, or JUUL aerosol with mango flavour and 59 mg/mL nicotine. Mice were exposed to 4 puffs per min for 20 min 3 times a day for 4 weeks. Created with BioRender.com (B) Serum cholesterol level where a red line indicates 200 mg/dL total cholesterol level, below which mice were excluded. n = 10 (C) Serum cotinine level was measured by ELISA after 24 h of last exposure in hyperlipidemic mice. There was a significant increase in cotinine level in the JUUL mango group compared to Air and PG/VG groups (**** = p < 0.0001; n = 5). (D) CYP2A5 western blot comparing uninfected, unexposed C57BL/6J mice and AAV-PCSK9^DY mice exposed to air, PG/VG, or JUUL mango shows no difference among exposure groups. Red stars indicate those mice infected with AAV-PCSK9^DY, but without hyperlipidemia (not included in densitometry). Densitometric data includes only those mice with cholesterol >200 mg/dL. Results are expressed as the mean ± SD. n = 3 (E) Body weight was measured before and after the exposure period. ** = p = 0.005 n = 3–5.

FIGURE 2

HDL-Chol (A) and glucose (B) levels are decreased in JUUL Mango-exposed AAV-PCSK9^DY mice compared to air control (* = p < 0.05). LDL-Chol (C) and triglycerides (D) were not altered. Red lines indicate average male C57BL/6 levels. HDL ratios are also shown in (E–G). Results are expressed as the mean ± SD. n = 6–9.

FIGURE 3

AAV-PCSK9^DY increases circulating soluble endothelial cell activation markers, which is not altered by JUUL aerosol exposure. Adhesion molecules (A) sICAM-1, (B) sPECAM-1, (C) sE-selectin, and (D) sP-selectin were assessed in the serum of control (open squares) and hyperlipidemic mice via multi-plex ELISA. AAV-PCSK9^DY mice were exposed to air (black circles), PG/VG (black squares), or Mango-flavored JUUL (black triangles). Soluble adhesion molecule levels for uninfected, unexposed C57BL/6J mice are shown for comparison. Results are expressed as the mean ± SD. n = 3–4.

FIGURE 4

JUUL aerosol exposure has no effect on VCAM-1 expression on the endothelial cells in either the BCA (A) or carotid (B) as assessed by IHC staining and quantified by measuring Mean DAB OD within the endothelial cell layer. Results are expressed as the mean ± SD. n = 2–4.

FIGURE 5

No significant changes were observed in plaque size within the aortic sinus. (A) Plaque size (plaque per sinus area) and (B) lipid content (staining per plaque area) were quantified in the aortic sinus after oil red O staining. Representative images from each group are shown in (C). Black arrows point toward small fatty streaks/plaques. Results are expressed as the mean ± SD. n = 6–9.

FIGURE 6

Effect of JUUL aerosol exposure on immune cell distribution of myeloid cells and B cells in the spleen. Using immunophenotyping, the following cell populations were identified: (A) neutrophils, (B) myeloid DCs, (C) monocytes, (D–F) immature, M1, and M2 macrophages, and (G) B cells. Results are expressed as the mean ± SD; individual data points represent individual mice. n = 6–9.

FIGURE 7

Effect of JUUL aerosol exposure on T cell distribution in the spleen. Using immunophenotyping, splenic T cell populations were analyzed as follows: (A) CD3⁺ total T-cells, (B) CD4⁺ T cells, (C) CD4 helper T cells, (D) CD25⁺ FoxP3+Tregs, and (E) CD8⁺ T cells. Results are expressed as the mean ± SD; individual data points represent individual mice. (* = p ˂ 0.05, ** = p ˂ 0.005) n = 6–9.