Exploring Hypoxic and Normoxic Extracellular Vesicles in Murine Volumetric Muscle Loss Model: Implications for Immunomodulation and Regeneration

Introduction: Severe inflammation and fibrosis pose challenges to muscle regeneration post volumetric muscle loss (VML), with no definitive therapy available. Mesenchymal stem cells (MSCs) can promote muscle recovery by either directly differentiating into muscle tissue or by promoting angiogenesis, suppressing inflammation, and preventing fibrosis. However, their poor survival and tissue-specific differentiation rate post-transplantation limits their therapeutic use. MSCs exert their effects partly through paracrine signaling, suggesting that their secreted products, packaged in extracellular vesicles (EVs), could overcome these limitations. EVs are acellular membrane-bound nano-vesicles (30-150 nm) that act as intercellular shuttles for transporting cargo such as proteins, lipids, mRNAs, and microRNAs (miRNAs) between cells. Like parent MSCs, EVs can suppress cell apoptosis, tissue inflammation and fibrosis and promote angiogenesis and tissue regeneration. Low yield and poor biodistribution and bioavailability in vivo are major limitations we need to overcome to use MSC EVs as therapy. Exposing MSCs to a controlled low oxygen environment (hypoxia) can increase their proliferation rate and thereby secretion of EVs by them. Biomaterials such as hydrogels are being used for targeted and controlled release of drugs due to their biocompatibility and degradability. Fibrin hydrogels can act as a delivery vehicle for sustained and controlled release of EVs at the injury site. We hypothesize that EVs derived from hypoxic and normoxic MSC culture, when delivered via a fibrin gel at the injury site, will have different biological effects pertaining to immunomodulation and regeneration.

Materials and

Methods: EVs were isolated from cell-culture supernatants of murine bone-marrow derived MSCs that were cultured under normoxic (21% O2) or hypoxic (3% O2) conditions for 48 hours at 37°C. EVs were characterized using Nanoparticle Tracking Analysis (NTA) and surface marker expression. In vitro, macrophages were stimulated with lipopolysaccharide (LPS) for 24 hours in a 48 well plate at a density of 300,000 cells/well (n=3 per group). After 24 hours, LPS stimulated macrophages were washed and exposed to fresh media (control group), or media containing 25µg/mL of either normoxia or hypoxia EVs. After 72 hours of culture under standard conditions, cell-culture supernatants were harvested and used for ELISA. For the fabrication of PBS and EV encapsulated crosslinked fibrin gels, hypoxic or normoxic EVs (concentration- 4.48 X 1010 particles/ml) were mixed in a solution containing fibrinogen (35.5 mg/ml), 0.9% NaCl and 3.5 ul of protease inhibitor. Thrombin (20 U/ml) and calcium chloride (20mM) were added to this solution and incubated at 37°C for 20 minutes to form a crosslinked fibrin gel. A 3 mm biopsy punch was used to create a full thickness, ~20% VML defect in both gastrocnemius soleus (GAS) muscles of 129S1/SumJ mice. PBS or Hypoxic or normoxic EV-encapsulated fibrin gel (6 mm diameter) was placed atop the surface of the injured muscle directly after the injury, and the defect was closed with interrupted sutures (n=6-7/group). The mice were allowed to recover for 14 days and muscles were harvested for histological and biochemical analysis. Results were analyzed using one-way ANOVA.

Results, Conclusions, and Discussions: EVs showed characteristic surface marker expression (CD81, TSG101) and were in the characteristic size range of 30-150 nm (Fig 1). Macrophages exposed to normoxia and hypoxia MSC derived EVs showed differences in secretion of trophic factors (Fig 2). TNF-α and IL-6 are pro-inflammatory cytokines associated with M1 phenotype. Hypoxic and normoxic EVs suppressed the TNF-α, however, only hypoxic EVs inhibited the release of IL-6. Both normoxic and hypoxic EVs elevated the release of VEGF (M2 phenotype associated cytokine promoting angiogenesis and regeneration), but hypoxic EVs induced less VEGF release from macrophages compared to normoxic EVs. In vivo results showed that treatment with gel encapsulated normoxic-EVs resulted in higher muscle mass (p=0.0066) relative to both gel alone and gel encapsulated hypoxic-EVs group (Fig 3 a). Western blotting revealed no significant differences in the expression of myogenic markers, MyoD and Myogenin, but Desmin expression was higher in gel alone group compared to gel encapsulated normoxic (p=0.0217) or hypoxic EV (p=0.0339) group (Fig 3 b, c and d). No significant differences were observed in the expression of pro-inflammatory marker, iNOS or the anti-inflammatory marker arginase (Fig. 4).
Hypoxic preconditioning (HP) of MSCs increases the production of EVs by MSCs. In vitro results suggest that normoxic EVs are associated with both M1 and M2 phenotype. In vivo, gel encapsulated normoxic EVs promoted higher muscle mass recovery 14 day-post injury, suggesting that concerted actions of M1 and M2 macrophages are needed to promote VML repair in vivo. Interestingly, no differences in myogenic or immunogenic markers were observed between normoxic and hypoxic EV treated groups.
Post VML, a sustained inflammatory response coupled with excessive fibrotic tissue deposition can drive muscle into a myogenesis-inhibitive feedback loop, resulting in impaired regeneration and function. Strategies that can limit inflammation at the VML injury site are critically needed. Our results show that normoxic EVs promote muscle mass recovery. Future studies are needed to elucidate the mechanisms underlying these effects to optimize the dosage and delivery of EV based therapies for muscle regeneration. Future studies will also compare the effects of EVs on fibrotic tissue deposition and angiogenesis post VML injury.

Acknowledgements (Optional): This work is being supported by National Institute of General Medical Sciences (NIGMS) grant 2R15GM129731-02 awarded to Dr. Koyal Garg