Aerosolized vitamin E acetate causes oxidative injury in mice and in alveolar macrophages

Abstract

Although vitamin E acetate (VEA) is suspected to play a causal role in the development of electronic-cigarette, or vaping, product use-associated lung injury (EVALI), the underlying biological mechanisms of pulmonary injury are yet to be determined. In addition, no study has replicated the systemic inflammation observed in humans in a murine EVALI model, nor investigated potential additive toxicity of viral infection in the setting of exposure to vaping products. To identify the mechanisms driving VEA-related lung injury and test the hypothesis that viral infection causes additive lung injury in the presence of aerosolized VEA, we exposed mice to aerosolized VEA for extended times, followed by influenza infection in some experiments. We used mass spectrometry to evaluate the composition of aerosolized VEA condensate and the VEA deposition in murine or human alveolar macrophages. Extended vaping for 28 days versus 15 days did not worsen lung injury but caused systemic inflammation in the murine EVALI model. Vaping plus influenza increased lung water compared with virus alone. Murine alveolar macrophages exposed to vaped VEA hydrolyzed the VEA to vitamin E with evidence of oxidative stress in the alveolar space and systemic circulation. Aerosolized VEA also induced cell death and chemokine release and reduced efferocytotic function in human alveolar macrophages in vitro. These findings provide new insights into the biological mechanisms of VEA toxicity.

Keywords: E-cigarette or vaping product use-associated lung injury; acute respiratory distress syndrome; alveolar macrophage; oxidative stress; vitamin E acetate.

Figure 1.

Dose-dependent pulmonary toxicity by aerosolized vitamin E acetate (VEA) reaches plateau during prolonged exposure and does not lead to more severe lung injury. Nine-to-eleven-week-old C57BL/6 mice were exposed to aerosolized vitamin E acetate (VEA) for 1 h twice daily for up to 28 days. Following 6, 15, or 28 days of exposure, mice were euthanized and assessed for endpoints. A: excess extravascular lung water (ELW) was dose-dependently increased up to 15 days and did not increase further at 28 days (n = 5/time point, P < 0.0001 ANOVA). In contrast, bronchoalveolar lavage (BAL) protein concentrations increased significantly by 6 days (B), plateaued at 15 days, then decreased at 28 days (P < 0.0001 ANOVA). BAL neutrophils numbers were increased significantly at 15 days (P = 0.014 ANOVA) (C), BAL monocyte/macrophage had an increasing trend relative to control but significantly decreased at 28 days (P = 0.054 ANOVA) (D). E–H: BAL monocyte chemokine monocyte chemotactic protein-3 (MCP3), neutrophil chemokine keratinocyte chemoattractant (KC), and proinflammatory cytokine interleukin (IL) 6 were uniformly elevated at 28 days relative to control (E–G). Although not statistically significant, IL10 had trend to be elevated at 28 days, suggesting increased anti-inflammatory compensation mechanism by day 28 (H). I: representative cytology photomicrograph (×600 magnification) showed vacuolated macrophages after 28 days of exposure with (J) significant increase in diameter for all time points relative to the control, P < 0.0001 by ANOVA. K: representative histology photomicrographs revealed similar degrees of mixed acute and chronic inflammation in the bronchovascular bundle in a bronchiolocentric pattern (top, ×40 magnification) and thickened septa, foamy macrophages, and proteinaceous debris filling alveolar spaces (bottom, ×600 magnification) at 15 and 28 days. n = 10/treatment groups, 5 for ELW measurement and 5 for BAL and histology. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Tukey’s multiple-comparison test (MCT) following ANOVA.

Figure 2.

Prolonged exposure to vitamin E acetate (VEA) aerosol leads to perivascular cuffing by mononuclear cells and increased markers of endothelial injury and systemic inflammation. A: representative photomicrographs (control, left column; VEA 15 days, middle; VEA 28 days, right column) demonstrated mononuclear cell aggregates around blood vessels (arrowheads, bottom) following 28 days of exposure (top, ×40 magnification; bottom, ×400 magnification). B–D: plasma angiopoietin (Ang) 2, plasma intercellular adhesion molecule (ICAM) 1, and matrix metalloproteinase (MMP) 8 were all significantly increased following 28 days of exposure to VEA. n = 5/treatment groups. P = 0.0001, = 0.0003, and = 0.0059, respectively, by ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001 by Tukey’s MCT. MCT, multiple-comparison test.

Figure 3.

Exposure to vitamin E acetate (VEA) aerosol augment lung injury caused by influenza infection. A: both mice exposed to VEA for 6 days and unexposed mice were inoculated with 400 foci-forming units (FFUs) of influenza Puerto Rico/8/34 (PR8). On 7 days postinfection (dpi), mice were euthanized and assessed for endpoints. B–E: body weight loss from inoculation (0 dpi; B) and extravascular lung water (ELW; C) were significantly higher in VEA-exposed mice, whereas bronchoalveolar lavage (BAL) protein (D) and total cell (E) counts were not different between groups. n = 10/treatment groups; 5 for ELW measurement and 5 for BAL. **P < 0.01 by unpaired t test.

Figure 4.

Inflammation caused by vitamin E acetate (VEA) attenuates over time with morphological changes in alveolar macrophage. A: following 6 days of exposure to VEA aerosol, some mice were euthanized and analyzed 12 h after the last exposure for endpoints while the others were allowed to recover for 6 days to study the resolution kinetics. B: extravascular lung water (ELW) remained elevated after the 6-day recovery period (P < 0.0001, ANOVA), whereas bronchoalveolar lavage (BAL) protein (P < 0.0001, ANOVA) (C) and BAL total cells (P = 0.0185, ANOVA) (D) tended to decrease. By marked contrast, BAL neutrophils were decreased after 6 days (E), (*P = 0.0091 by Kruskal–Wallis test). Despite no changes in BAL macrophage/monocyte counts (F), representative photomicrographs (×600 magnification) (G) show that their diameters were significantly decreased (P < 0.0001 by Kruskal–Wallis test) (H) suggesting that phagocytosed VEA was hydrolyzed. I–K: BAL keratinocyte-derived chemokine (KC) (P < 0.0001, ANOVA), monocyte-chemotactic protein (MCP)-3 (P < 0.0001 by Kruskal–Wallis test), and interleukin (IL)-6 (P < 0.0001 by Kruskal–Wallis test) were uniformly decreased following recovery period. n = 10/treatment groups, 5 for ELW measurement and 3–5 for BAL. *P < 0.05, **P < 0.01, ****P < 0.0001 by Tukey’s MCT for normally distributed data or Dunn’s MCT for skewed data. MCT, multiple-comparison test.

Figure 5.

Vitamin E acetate (VEA) was hydrolyzed within airspace cells with evidence of systemic oxidative stress. A: mass spectrometry (time of flight) spectra from 70 to 1,700 mass units of aerosolized VEA condensate demonstrated a similar mass/charge (m/z) and mass transition pairs as seen in the authentic VEA standard (m/z, 473.4→207.1). Following 3 days exposure to VEA aerosol, mice were lavaged and cell pellets from bronchoalveolar lavage (BAL) fluid were analyzed for VEA (α -tocopheryl acetate) (B), α-T (α-tocopherol, Vit. E) (C), and α-TQ (α-tocopheryl quinone VEQ) (D). Shown are the ratios of the indicated compound to cholesterol (mmol/mol) to account for variable cells counts per BAL sample; column heights equal means ± SD with individual samples indicated (****P < 0.0001 by unpaired t test of log-transformed data). E: plasma malondialdehyde (MDA), a lipid peroxidization marker, was time-dependently increased following 6 or 15 days of exposure (means ± SD, ANOVA: P < 0.0001, ***P < 0.001, ****P < 0.0001 by Tukey’s MCT). n = 5/treatment groups. MCT, multiple-comparison test.

Figure 6.

Vitamin E acetate (VEA) exposure to human primary alveolar macrophages induced cell death, impaired function, and increased chemokine release. Primary human alveolar macrophages cultured in media were exposed to aerosolized VEA for an hour daily for three consecutive days, which increased lactate dehydrogenase (LDH) concentrations in culture supernatant (A) and impaired efferocytotic function (B), (P = 0.0065 unpaired t test). C: VEA and Vit. E were significantly increased in human alveolar macrophages exposed to aerosolized VEA. D: representative photomicrographs (×600 magnification) revealed vacuolated, multinucleated, and Oil Red O (ORO) positive macrophages after VEA exposure. E: after 3 days of VEA exposure, the culture supernatant contained significantly increased concentrations of monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α (but not MIP-1β), stromal cell-derived factor (SDF)-1α, growth-regulated oncogene (GRO)-α, regulated on activation, normal T cell expressed and secreted (RANTES), and eotaxin. n = 3–6/treatment groups. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t test.