P208 Extracellular vesicles from COPD small airway fibroblasts spread senescence to healthy fibroblasts

Background

COPD is associated with cellular senescence and fibrosis. Extracellular vesicles (EVs) are membrane-derived vesicles involved in intercellular communication. EVs contain miRNAs, mRNA and proteins and have been implicated in COPD to induce senescence and the transition of fibroblast to myofibroblasts. This study examined whether EVs derived from COPD fibroblasts drive senescence in healthy recipient fibroblasts. Changes in expression of p21^CIP1 and alpha-smooth muscle actin (αSMA) were chosen as markers of senescence and transition of fibroblasts to myofibroblasts respectively.

Methods

Large EVs, and small EVs were isolated from media from non-smoker (NS) and COPD fibroblasts cultured with or without H₂O_2. EVs were labelled with phk67 and uptake measured by flow cytometry. Healthy recipient fibroblasts were cultured with EVs or EV-free media for 24h and 48h and protein expression of p21^CIP1 and αSMA measured using western blots and CXCL8 release by ELISA.

Results

There was a time-dependent uptake of EVs into recipient cells with no difference between EVs from control or COPD fibroblasts with 91.8 ± 3.8% of recipient cells phk67 positive by 48h (n=4). Incubation of recipient fibroblasts (n=2–5) with large EVs from either non-smokers or COPD subjects did not alter the expression of p21^CIP1 or αSMA at 24h. Similarly large EVs from fibroblasts exposed to H₂O₂ had no effect on these markers in recipient cells. By contrast, at 48h (figure 1), small EVs from COPD cells showed a trend to increased expression of p21^CIP1 and EVs from both non-smokers and COPD subjects increased expression of αSMA. Incubation of recipient cells with large EVs from non-smoker fibroblasts that had been cultured with or without H₂O₂ increased release of CXCL8 (0.36±0.15ng/ml to 5.43±3.92ng/ml and 5.44±5.23ng/ml respectively) and small EVs from COPD fibroblasts induced CXCL8 release at 48h (0.36±0.15ng/ml to 3.75±3.16ng/ml).

Conclusions

Large and small EVs tend to increase the expression of p21^CIP1 and αSMA in recipient fibroblasts. These results are confirmed by the uptake analysis showing that maximum uptake of EVs from both NS and COPD fibroblasts is reached after 48h. Altogether, these data suggest that EVs participate in COPD pathophysiology by spreading senescence in recipient fibroblasts.