E-cigarette exposure augments murine abdominal aortic aneurysm development: role of Chil1

Abstract

Aims: Abdominal aortic aneurysm (AAA) is a common cardiovascular disease with a strong correlation to smoking, although underlying mechanisms have been minimally explored. Electronic cigarettes (e-cigs) have gained recent broad popularity and can deliver nicotine at comparable levels to tobacco cigarettes, but effects on AAA development are unknown.

Methods and results: We evaluated the impact of daily e-cig vaping with nicotine on AAA using two complementary murine models and found that exposure enhanced aneurysm development in both models and genders. E-cigs induced changes in key mediators of AAA development including cytokine chitinase-3-like protein 1 (CHI3L1/Chil1) and its targeting microRNA-24 (miR-24). We show that nicotine triggers inflammatory signalling and reactive oxygen species while modulating miR-24 and CHI3L1/Chil1 in vitro and that Chil1 is crucial to e-cig-augmented aneurysm formation using a knockout model.

Conclusions: In conclusion our work shows increased aneurysm formation along with augmented vascular inflammation in response to e-cig exposure with nicotine. Further, we identify Chil1 as a key mediator in this context. Our data raise concerns regarding the potentially harmful long-term effects of e-cig nicotine vaping.

Keywords: Abdominal aortic aneurysm; Aortic aneurysm; E-cigarette; Nicotine; Vascular inflammation.

Graphical Abstract

Figure 1

AAA progression and gene expression, miR-24, and Chil1 response to E-cig exposure during AAA development. (A) Schematic timeline of E-cig vaping experiments. Aortic ultrasound scans were performed at baseline, at day 3 and day 7 after AAA induction, and then weekly. (B) Daily exposure to E-cig in male C57BL/6J mice containing nicotine (24 mg/mL, 9 s puffs per minute, 60 min/d) increased AAA diameter by ultrasound vs. Room air-exposed control animals after PPE and vs. sham surgery with or without E-cig (n = 5-13 per group). Sham surgery did not lead to aortic dilation in E-cig or room air-exposed animals. (C) Gene expression of key inflammatory markers (Il6, Ccl2) and ECM-related genes (Mmp9, Eln) (n = 5–6 per group) in males at day 7 (top) and day 28 (bottom) in aortic tissue derived from untreated animals, or after sham or PPE surgery with or without E-cig exposure. (D and E) Gene expression of miR-24 and its target Chil1 in the same tissues as in C quantified by qRT–PCR at day 7 (D left) and 28 (E right) (n = 5–6 per group). (F) Aortic aneurysm sections from male animals were harvested after 28 days and stained with hematoxylin and eosin, and IHC was used for anti-F4/80 and anti-Chil1. Positive areas were quantified. Scale bar is 100 µm. Arrows indicate positively stained areas. (G) IHC of F4/80- (top) and Chil1- (bottom) positive areas were quantified in high power fields (HPF) (4 HPF per slide, 3 slides per animal, 3 AAA per group). (H) Male ApoE^−/− mice were exposed to E-cig according to the same protocol as used in A. After 2 weeks of pre-exposure to E-cig or Room-air, osmotic minipumps filled with Ang-II were implanted and suprarenal aortic growth was measured via ultrasound (n = 8 per group). (I) miR-24 and Chil1 gene expression levels in AAA tissues from E-cig-exposed (vs. Room-air-exposed, Ang-II-treated-) ApoE^−/− mice using qRT–PCR at Day 28 after pump implantation (n = 5–6 per group). Graphs (B and H) show aortic aneurysm diameter (AAD) % increase vs. baseline. *P < 0.05; **P < 0.01 AAA E-cig vs. AAA room air. ##P < 0.01 both PPE groups vs. both sham surgery groups. All by two-way ANOVA with multiple comparison (B and H). Gene expression data are presented as fold change (FC) vs. unoperated, room air-exposed control. *P < 0.05 or **P < 0.01 vs. Sham; #P < 0.05 or ##P < 0.01 vs. unoperated, e-cig-exposed control; §P < 0.05 or §§P < 0.01 vs. sham-operated, room air-exposed control; †P < 0.05 or ††P < 0.01 vs. sham-operated, e-cig-exposed control; ßP < 0.05 or ßßP < 0.01 vs. PPE-operated, room air-exposed. Two tailed student’s t-test (C, D, E, and I). *P < 0.05; **P < 0.01 Two tailed student’s t-test (G) Data are mean ± SEM. Down-regulated genes are shown as −1/FC.

Figure 2

Nicotine induces and augments pro-inflammatory cytokines and ROS in human AoSMC and murine RAW264.7 cells. (A) Nicotine increases expression levels of inflammatory genes (Ccl2, Il6 and Tnf) in murine RAW264.7 cells. (B) Nicotine dose-dependently augments inflammatory gene (Ccl2, Tnf) responses to recombinant IL-6 treatment (20 ng/mL) in RAW264.7 cells. (C) Nicotine dose-dependently increases total ROS in RAW264.7 cells (DCFDA Assay, Ex/Em = 485/535 nm), a process reversed by TEMPOL. (D) Nicotine dose-dependently increases expression levels of inflammatory genes (CCL2, IL6 and IL8) in human AoSMCs. (E) Nicotine dose-dependently augments inflammatory gene (CCL2, IL6 and IL8) responses to recombinant IL-6 treatment (20 ng/mL) in human AoSMCs. (F) Nicotine dose-dependently increases ROS response to IL6 (20 ng/mL) in human AoSMCs (Abcam/Rhodamine, Ex/Em = 550/620 nm). (G) Corresponding pictures to (F) of human AoSMCs stained for ROS under green channel using ROS Detection Assay (Abcam/Rhodamine). Gene expression data are qRT–PCR presented as fold change vs. control. §P < 0.05 vs. control; vs. §§ control and IL6; † vs. control, IL6, and IL6 + nicotine 10 nM (A, B, D, and E). *P < 0.05 vs. control; #P < 0.05 vs. other group (C). *P < 0.05 or **P < 0.01 vs. IL6, #P < 0.05 vs. other group (F). All two-tailed Student’s t-test. Data are mean ± SEM (n = 4–6/treatment group).

Figure 3

Nicotine down-regulates miR-24-1. (A) Nicotine (10 nM) down-regulates miR-24 expression in human AoSMCs via the NF-kB pathway. Down-regulation is neutralized with siRNA to RELA. (B) Nicotine (10 nM) down-regulates pri-miR-24-1 but not pri-miR-24-2 in RAW264.7 cells. (C) Nicotine reduces pri-miR-24-1 expression in RAW264.7 cells via ROS. Addition of ROS-scavenger TEMPOL leads to an increase in pri-miR-24-1 expression despite nicotine treatment. (D and E) Recombinant IL-6 treatment lowers pri-miR-24-1 in RAW264.7 cells and human AoSMCs, a process which is augmented dose-dependently by nicotine. Gene expression data are presented as fold change vs. control. *P < 0.05 vs. untreated control (A, B, and C). §P < 0.05 vs. control; §§ vs. control and IL6; † vs. control, IL6, and IL6 + Nic10 nM (D and E). All Student’s t-test. Data are mean ± SEM (n = 3–8/treatment group). Down-regulated genes are shown as −1/FC.

Figure 4

Nicotine-induced CHI3L1/Chil1 is crucial to the augmentation of both the inflammatory response and AAA development. (A and B) Nicotine augments IL6-induced increase of CHI3L1/Chil1 gene expression in human AoSMCs and RAW264.7 cells by qRT–PCR. (C) Overexpression of miR-24 using transfection with pre-miR-24 (pre-24) reduces nicotine-augmented cytokine (IL6, CCL2 and IL8) gene expression in human AoSMCs. Scr-miR = scrambled miR control. (D–F) Nicotine augments the effects of IL6 in increasing inflammatory cytokines CCL2 (D), IL6 (E), and IL8 (F) gene expression in human AoSMCs. These effects are reduced or reversed with miR-24 overexpression (pre-miR-24 transfection). Conversely, further down-regulation of miR-24 with anti-miR-24 transfection augmented the effects on inflammatory gene expression. However, simultaneous silencing of CHI3L1 (siRNA transfection) reduces or reverses the effects of the combination of IL6, nicotine and anti-miR-24. (G) Smoking augments the increase in aortic CHI3L1 gene expression, and the decrease in miR-24 in human AAA tissue (n = 6–9 per cohort). (H) Globally induced Chil1^−/− suppresses AAA growth after PPE surgery in male (left) and female mice (right) exposed to E-cig vapour for a total of 6 weeks (n = 5–13 for males; 5–10 for females). Gene expression qRT–PCR data are presented as fold change vs. control. §P < 0.05 vs. control; vs. §§ control and IL6; † vs. control, IL6, and IL6 + Nic10 nM (A–F), two-tailed student’s t-test, data are mean ± SEM (n = 3–6/treatment group). *P < 0.05 two-tailed Student’s t-test (G). Down-regulated genes are shown as −1/FC. Graphs show aortic aneurysm diameter (AAD) % increase vs. baseline. *P < 0.05 or **P < 0.01 in two-way ANOVA with multiple comparison (H).