Cyclic Hypoxia Conditioning Alters the Content of Myoblast-Derived Extracellular Vesicles and Enhances Their Cell-Protective Functions

Abstract

Remote ischemic conditioning (RIC) is a procedure that can attenuate ischemic-reperfusion injury by conducting brief cycles of ischemia and reperfusion in the arm or leg. Extracellular vesicles (EVs) circulating in the bloodstream can release their content into recipient cells to confer protective function on ischemia-reperfusion injured (IRI) organs. Skeletal muscle cells are potential candidates to release EVs as a protective signal during RIC. In this study, we used C2C12 cells as a model system and performed cyclic hypoxia-reoxygenation (HR) to mimic RIC. EVs were collected and subjected to small RNA profiling and proteomics. HR induced a distinct shift in the miRNA profile and protein content in EVs. HR EV treatment restored cell viability, dampened inflammation, and enhanced tube formation in in vitro assays. In vivo, HR EVs showed increased accumulation in the ischemic brain compared to EVs secreted from normoxic culture (N EVs) in a mouse undergoing transient middle cerebral artery occlusion (tMCAO). We conclude that HR conditioning changes the miRNA and protein profile in EVs released by C2C12 cells and enhances the protective signal in the EVs to recipient cells in vitro.

Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification

“…From one 175 mL flask, 3.0 ± 0.2 × 10 12 EVs/mL with a total of 0.3 mL were collected after SEC purification, with an average size of 128 ± 0.7 nm according to nanoparticle tracking analysis (NTA), which is in agreement with previous observations (see the Materials and Methods section). In accordance with International Society for Extracellular Vesicles (ISEV) guidelines, Western blotting of EV markers was carried out, confirming that CD81 and TSG101 were enriched in the EV fraction, while Calnexin and RPL22 were enriched in the cellular fraction (see Supporting Figure 2). In addition, the EVs and liposomes were visualized using negative-stain transmission electron microscopy (nsTEM) (Supporting Figures 3–5).…”

“…For EV Western blotting, the proteinase inhibitor was added after EV purification. 37 Protein concentration of cell lysate and EVs was determined using a Qubit Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA), and 30 μg of protein was used. For blotting CD81 and Calnexin, we used a nonreducing protocol, and for blotting TSG101 and RPL22, we used a reducing protocol.…”

“…For blotting CD81 and Calnexin, we used a nonreducing protocol, and for blotting TSG101 and RPL22, we used a reducing protocol. 37 For the nonreducing protocol, the proteins were prepared in 4× NuPAGE LDS sample buffer, and for the reducing protocol, proteins were prepared in 4× NuPAGE LDS sample buffer with NuPAGE sample reducing agent (all purchased from Invitrogen, Waltham). The samples were loaded on a 4−12% NuPAGE Novex Bis-Tris gel (Invitrogen, Waltham) with 6 μL of protein ladder (cat.…”

“…Protein concentration of cell lysate and EVs was determined using a Qubit Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA), and 30 μg of protein was used. For blotting CD81 and Calnexin, we used a nonreducing protocol, and for blotting TSG101 and RPL22, we used a reducing protocol . For the nonreducing protocol, the proteins were prepared in 4× NuPAGE LDS sample buffer, and for the reducing protocol, proteins were prepared in 4× NuPAGE LDS sample buffer with NuPAGE sample reducing agent (all purchased from Invitrogen, Waltham).…”

“…Extracellular vesicles (EVs) were isolated from a C2C12 cell culture. To harvest EVs, 9 T175 flasks of C2C12 cells with 90% confluency , were washed twice in 2 × 10 mL phosphate-buffered saline (PBS), followed by replacement of 20 mL EV collection media (DMEM, supplemented with 10% EV-depleted FBS) and cultured for 24 h before collection.…”