Acute Exposure to E‐Cigarette Vapor Promotes Neutrophil‐Platelet Aggregation in Murine Pulmonary Microvasculature

Abstract

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. Neutrophils and platelets are known to undergo activation and proinflammatory phenotypic changes when exposed to ENDS vapor, and their aggregation could promote vascular occlusion and further inflammation. We hypothesize 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. To investigate lung vascular pathology following ENDS use, we have applied an innovative spinning disk confocal microscopy approach to visualize real-time interactions of blood cells 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 were 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. Mice subjected to room air sham inhalation constituted the control group. 24 hours after completion of the inhalation course, mice were anesthetized to facilitate microsurgery enabling fluorescence microscopy of the living lung. Neutrophils and platelets were labeled with fluorescently tagged antibodies to compare overall presence and aggregation within the lung microvasculature (Fig. 1). 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 consequences of ENDS use.