(E-168) Effects of Extracellular Vesicles Derived from Non-Irradiated or Irradiated Skeletal Muscle Stem Cells on Myogenesis and Angiogenesis

Introduction:: Introduction: The hazard of ionizing radiation exposure, intentional or accidental, is a threat to public health and safety and a growing concern for the Federal Emergency Management Agency and the United States Military [1]. Moreover, cancer patients receiving radiation therapy suffer less severe but long-term side effects of irradiation, such as chronic inflammation and tissue fibrosis [2]. Ionizing radiation causes cellular dysfunction by inducing DNA damage, pro-inflammatory responses, and altered intercellular signaling, especially in rapidly proliferating cells [3]. Satellite cells (SCs) or skeletal muscle stem cells are sensitive to radiation and can lose their ability to self-renew and differentiate into muscle fibers upon irradiation [4]. SCs give rise to new muscle fibers (myogenesis) in response to routine wear and tear or injury of the muscle tissue and produce paracrine factors that stimulate the growth of new blood vessels (angiogenesis). Currently, there is no effective therapeutic strategy to rescue satellite cells from radiation damage. Consequently, radiation survivors often suffer from muscle atrophy in the years following the irradiation event. Therefore, there is a critical need to develop a therapeutic approach to reduce the adverse effects of ionizing radiation on satellite cells and support their native function. Intercellular communication is essential for myogenesis, angiogenesis, and maintenance of skeletal muscle function. Extracellular vesicles (EVs) are a heterogeneous group of nano-sized vesicles that play a central role in cell-to-cell communication. The therapeutic potential of satellite cell-derived extracellular vesicles for the treatment of radiation injury in skeletal muscle was examined in these studies.

Materials and Methods:: Materials and Methods: Skeletal muscle satellite cells were isolated from four to six months old healthy Sprague Dawley rats. Precision CellRad (Precision X-Ray Inc, Madison, CT) benchtop X-ray irradiator was used to irradiate SCs using an automated dose control setting at 130 kV and 5 mA. Rad-Sure irradiation indicator labels were used to verify SC irradiation. SCs were exposed to 10 Gy irradiation (Rad-SCs) or no irradiation (NoRad-SCs) before EVs were collected from serum-free media. Cell viability (acridine orange/propidium iodide - live/dead staining, AOPI), cell proliferation (BrdU incorporation assay), and myogenic differentiation (immunocytochemistry and quantitative PCR) were analyzed after SC-EV treatment of NoRad-SCs and Rad-SCs. Angiogenic potentials of NoRad-EVs and Rad-EVs were evaluated by an adipose tissue-derived microvascular fragment (MVF) fibrin-based hydrogel model (3D). Sequential doses of 25 mg EV protein, NoRad-EVs, or Rad-EVs were added on days 1, 2, and 4. On day 8, the MVF gels were fixed and stained with Rhodamine-labeled GS Lectin I to visualize vessel formation. MVF growth and density were determined using confocal microscopy. To quantify the extent of angiogenesis and measure the vessel density, the percentage of the well coverage was determined using the Leica three-dimensional (3D) analysis toolkit using Otsu thresholding.

Results, Conclusions, and Discussions:: Results: Satellite cell viability at 48 hours after irradiation (a single X-ray dose of 10 Gy) was decreased by 48% in the SC cultures (Fig. 1A, p < 0.0001). NoRad-EVs treatment significantly increased the viability of irradiated SCs from 48% to 71% (% of non-irradiated control, p < 0.05). Rad-EVs or vehicle control treatment had no significant effect on irradiated SC viability (p > 0.05). Similarly, compared to non-irradiated SCs, there was an 85% decrease in cell proliferation levels in irradiated cells at 72 hours (Fig. 1B, p < 0.0001). NoRad-EVs treatment significantly improved the proliferation of irradiated SCs at 72 hours from 15 % to 31 % (% of non-irradiated control, p < 0.05), while Rad-EVs or vehicle control treatment had no significant effect on irradiated SC proliferation (p > 0.05). The effect of EV treatment on microvascular fragments growth (in 3D) shows a denser MVF network among NoRad-EVs treated MVFs than after Rad-EVs treatment (Fig. 1C). Qualitative analysis was supported by quantitative analysis; NoRad-EVs treatment increased the microvascular density by about three-fold compared to Rad-EVs treatment (Fig. 1D, p < 0.05).

Conclusions: Our in vitro data suggests that treatment with NoRad-EVs mediated increased viability and proliferation of irradiated SCs. This may improve myogenesis due to a larger population of SCs being available to grow and differentiate into functional muscle fibers. The pro-angiogenic properties of NoRad-EVs may further improve myogenesis in the irradiated skeletal muscle tissue, as muscle regeneration is highly dependent on a robust vasculature to ensure an adequate supply of oxygenated blood, nutrients, and growth factors to the growing muscle. Therefore, with their myogenic and angiogenic potentials, NoRad-EVs may have therapeutic value in mitigating the negative effects of ionizing radiation exposure on skeletal muscle growth and regeneration.


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