Proteomic Analysis Unveiled Exercise-induced Changes In Extracellular Vesicles From Balf And Blood In Cigarette Smoke Exposed Mice

INTRODUCTION: Cigarette smoke exposure increases expression of extracellular vesicles (EV) in the pulmonary system and circulation, which are implicated in development of chronic obstructive pulmonary disease (COPD). Exercise, a core component of pulmonary rehabilitation, also induces circulating EVs and promotes anti-inflammatory signaling. PURPOSE: To evaluate the effect of acute exercise on smoke induced EVs in mice. METHODS: C57BL/6 mice were exposed to air (n = 20) or cigarette smoke (CS) (n = 20; 10 cigarettes/day) from 2 to 6 months of age. Ten mice from each group ran for 1 hour while remaining 10 rested, producing 4 groups: Air+Rest; Air+Exercise; CS + Rest; CS + Exercise. Serum and bronchoalveolar lavage fluid (BALF) were collected and, EVs isolated by immunoprecipitation. EVs were quantified by nanoparticle tracking analysis and EV protein content was profiled by liquid chromatography tandem mass spectrometry (LC-MS/MS). Significant differential enrichment was assessed by π-value >1.3 (where π considers both fold-change and p-value). RESULTS: In BALF, EV concentration was lower in CS than Air (ANOVA; F = 8.92; p = 0.007) but there was no effect of Exercise or interaction. In serum, Exercise increased EV concentration in Air, but reduced it in CS (F = 3.87; p = 0.085). CS exposure (vs Air) resulted in 269 differentially enriched proteins in BALF and 6 in serum (none in common). Exercise (vs Rest) resulted in 10 differentially enriched proteins in BALF and 3 in serum (none in common). In BALF EVs, proteins such as Vim (3.5-fold), Psap (2.8-fold), Lmna (2.6-fold), Lcp1 (2.5-fold), Asah1 (2.3-fold) and Gpnmb (5.4-fold) were positively enriched by CS, but unaffected by Exercise. However, enrichment of mitochondrial proteins Pdha1 and Etfa was ~0.6-fold less in CS and 1.4- to 1.6-fold greater following Exercise. In Air, Exercise resulted in 0.4-fold enrichment of Man2c1, but not in CS. In serum EVs, RBM10 was 0.1-fold enriched in CS, but was 40-fold enriched by Exercise. Also, SAA1 was 0.1-fold less in CS, but was not affected by Exercise. CONCLUSION: Acute exercise modified protein enrichment of CS-induced EVs in both serum and BALF. Immune system and mitochondrial related proteins were among the most significantly enriched pathways. TRDRP (28FT-0017), NIH (1R43 HL167289-01), Johnny Carson Foundation