Mir-210 Encapsulated Extracellular Vesicles Contributes To Ischemic Injury Of The Skin
Ayan Biswas, Subhadip Ghatak, Mohamed El Masry, Savita Khanna, Sashwati Roy, Chandan K. Sen.
Indiana University, Indianapolis, IN, USA.
Background: Chronic wounds are commonly associated with peripheral vasculopathies causing wound ischemia. Limitations in the ability of the vasculature to deliver O2-rich blood to the wound tissue leads to hypoxia. It has been reported that hypoxia induces release of extracellular vesicles (EV). The significance of such release in wound repair remain unclear. Methods: To determine the significance of keratinocyte-specific miR-210 during ischemic injury, animal model with keratinocyte specific knockout of miR-210 (K14cremiR-210Δ/Δ) was developed. Keratinocytes were captured using laser capture microdissection (LCM) from the skin. Mitochondrial respiration was measured through Seahorse XF96. Results: A significant knockdown (~50%) of miR-210 was noted in the skin epithelium of the K14cremiR-210Δ/Δ mice compared to their wildtype littermates. Using a mono-pedicle ischemic flap model, in C57BL/6 mice, it was noted that at day 3 post-surgery, miR-210 was induced commensurate with graded levels of hypoxia (n=5, p<0.05). The extent of hypoxia was categorically characterized by dividing the flap into three parts (proximal, intermediate and distal). The level of hypoxia gradually increased from the proximal to the distal part. Consistent findings were observed for miR-210 expression from different region of flap tissue (n=5, p<0.05). Interestingly, under similar condition, K14cremiR-210Δ/Δ mice showed increased perfusion resulting in better flap survival 3 days post-surgery (n=6, p<0.05). Furthermore, it was observed that the EV production from ischemic tissue was proportionate to the extent of hypoxia. Such influence of hypoxia on EV production was also verified in vitro in keratinocytes (n=4, p<0.05). qRT-PCR analysis of such EV showed high abundance of miR-210. Treatment with such miR-210-rich EV showed decreased mitochondrial respiration in normoxic keratinocytes. Conclusions: miR-210-rich EV may be viewed as paracrine mediator that minimizes oxygen cost by economizing metabolism of the cell. Inhibition of keratinocyte specific miR-210 may be an effective strategy to improve EV mediated ischemic chronic wound outcomes.
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