(160d) Acute Exposure to E-Cigarette Vapor Promotes Neutrophil/Platelet Aggregation in Pulmonary Microvasculature

Background & Rationale: More than 1 in 4 high school students use electronic nicotine delivery systems (ENDS) in the United States, with usage rising over 130% since 2017. It is well known that smoking can cause cardiovascular disease and thrombosis; however, few studies have investigated the adverse effects of ENDS use on the cardiovascular system, especially in the lungs. The public perception of ENDS as a “safe” alternative to smoking and high rates of use particularly amongst youth demand that ENDS safety be rigorously studied in order to inform appropriate regulations. ENDS use has been linked to hypercoagulation and a persistent pro-inflammatory state, particularly in the lungs. Neutrophils and platelets are known to undergo activation and proinflammatory phenotypic changes when exposed to ENDS vapor, and their aggregation promotes vascular occlusion and further inflammation. We have pioneered a revolutionary live lung imaging technique that can visualize real-time interactions between blood cells within the pulmonary microvasculature of live mice. Herein, we will utilize lung intravital imaging to test our hypothesis that platelet and neutrophil recruitment will be increased in mice exposed to ENDS vapor, and that higher rates of aggregate formation will be observed in the pulmonary vasculature of these subjects.

Methods: To investigate lung vascular pathology following ENDS use, we have applied an innovative spinning disk confocal microscopy approach to visualize real-time interactions of neutrophils and platelets in the lungs of live mice. Identifying the presence of platelet-neutrophil aggregates in the lung due to ENDS vapor exposure would suggest that ENDS users may be at higher risk of atherosclerotic and thrombotic complications that with chronic use could eventually result in respiratory disease. Mice will be subjected to 3 consecutive days of ENDS vapor inhalation using a Juul device with Virginia Classic Tobacco e-liquid (5% nicotine) for 3 hours each day for each topographical profile. 3 profiles will be investigated, varying puff frequency and time of imaging post-exposure. Mice subjected to room air sham inhalation will constitute the control group. Platelet aggregometry on whole blood collected post-imaging will identify potential clotting pathways active in ENDS-induced thrombogenesis.

For the preliminary data illustrated here, mice were anesthetized to facilitate microsurgery enabling fluorescence microscopy of the living lung 24 hours after completion of the inhalation course- in this case, mice were exposed to ENDS vapor for three continuous hours at a rate of 1 puff per 2 minutes for three consecutive days. Neutrophils and platelets were labeled with fluorescently tagged antibodies to compare overall presence and aggregation within the lung microvasculature (Fig. 1). 15 fields of view (FOVs) were collected for each mouse and quantified using Nikon General Analysis. FOVs from saline injected mice which underwent intravital imaging constituted the sham group.

Results & Conclusions: Preliminary data suggest significant (p<0.01) increases (Fig. 2) in platelet (2.59-fold), neutrophil (2.06-fold), and platelet-neutrophil aggregate (2.02-fold) presence in mice exposed to ENDS vapor compared to untreated mice. Collectively, this illustrates that ENDS vapor induces a pro-inflammatory state in the pulmonary vasculature in as little as one day after limited exposure. These findings will prove useful in future studies seeking to model the consequences of long-term ENDS use, which will ultimately enhance both clinician monitoring of patients at risk of such complications and public awareness of the potential dangers of ENDS use.