Introduction

E-cigarettes are frequently perceived as safer than traditional cigarettes. However, there is a growing body of evidence that shows e-cigarette vapor (ECV) exposure has led to lung injury and a spate of sudden deaths in recent years. Recent studies have shown that the plasma of e-cigarette users had elevated biomarkers such as platelet-derived growth factor (PDGF) and tumor necrosis factor alpha compared to non-smokers. Currently, the cellular and molecular mechanism of ECV-mediated lung vascular damage is poorly defined. Here, we investigate the behavior of immune cells in the lung microcirculation following nicotine-free ECV exposure. The goal of this study is to define the role of PDGF in recruiting neutrophils and enhancing neutrophil-platelet aggregates. Neutrophils are the most abundant type of white blood cells, and when stimulated, release neutrophil extracellular traps (NETs). These webs of extruded DNA fibers provoke an inflammatory response and vasculature damage. We hypothesize that PDGF, released from activated platelets, binds to PDGF receptors (PDGFR) on the surface of neutrophils to initiate phosphorylation, reactive oxygen species generation through the NADPH oxidase pathway, neutrophil-platelet aggregation and subsequent NET release.  

Methods

Male and female BALB/c mice underwent 2.5 hrs/day of nicotine-free ECV exposure (1 puff/min at 30 watts, 50% vegetable glycerin (VG)/ 50% propylene glycol (PG)) for 3 days, 6 days, or 9 days. Control mice received room air. To activate PDGFR, positive control mice received 9 days of PDGF-BB treatment. Negative control mice received imatinib mesylate, a pharmacological inhibitor of PDGFR tyrosine kinase, for the same duration. The last experimental group received both ECV exposure and imatinib mesylate for 9 days. Analytes were collected from mice one day later. Lung intravital microscopy (IVM) of the pulmonary microvasculature (n=3 mice per group) was performed. Fluorescent labels were retro-orbitally injected to stain vasculature, neutrophils, and platelets. Neutrophil and platelet presence was assessed by measuring the average cellular area per image. Plasma and perfused lungs will be examined for RNA and protein expression by RT-PCR and western blot, respectively, to assess the presence of PDGF-BB, neutrophil elastase, NOX2, and myeloperoxidase.

Results

Lung IVM revealed that: (1) Male mice produced more severe neutrophil-platelet aggregations than female mice. (2) Duration of ECV exposure modulates neutrophil-platelet aggregates. A significant increase of aggregates in male mice were observed after 6 or 9 days with ECV exposure, whereas only 9 days ECV exposure were significant in female mice when compared to room air control groups. (3) Female mice injected with PDGF-BB for 9 days mimicked the aggregations seen in 9 days of ECV exposure. (4) Imatinib mesylate showed slightly decreased neutrophil-platelet aggregates versus room air exposed mice. RT-PCR and western blot results are in progress.

Conclusion

In conclusion, our data indicates that the immune response is intensified in the pulmonary microvasculature relative to the duration of ECV exposure, with PDGF-BB treatment alone replicating the aggregation observed from ECV exposure. ECV promotes a greater aggregation response in male mice versus female mice. This work aims to explore the biomolecular pathway in which PDGF contributes to neutrophil-platelet interactions after EC use to better educate the public about the dangers of e-cigarettes.

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