(E-200) Mesenchymal Stromal Cells as a Potential Cell-Therapeutic Option for Endothelial Dysfunction Caused by Exposure to Ionizing Radiation

Introduction:: One area of growing concern for the war fighter in the field is the threat of exposure to ionizing radiation due to the spread of nuclear weapons and material and the ever-present danger posed by terrorist organizations and rogue states. Ionizing radiation, in particular gamma rays and x-rays, is extremely harmful to biological tissues due to the deep penetrating power of the high energy particles emitted. These high energy particles can cause widespread cellular damage causing a range of life-threatening symptoms. The intestine is especially radio-sensitive, particularly the microvasculature of the small intestine that acts as a barrier between the gut lumen and the systemic circulation. The tight and adherens junctions that constitute the endothelial barrier between adjacent cells become compromised, causing abnormal translocation of biomolecules and cells. Mesenchymal stromal cells (MSCs) have shown promise for some patients because of activities such as immune modulation, angiogenesis, and wound healing. This study tests whether these cells and/or their secreted products could reverse the loss of endothelial integrity after exposure to ionizing radiation. Electric Cell-Substrate Impedance Sensing (ECIS) was employed to quantitate monolayer integrity and immunofluorescence was used to visualize tight and adherens junction protein expression and distribution.

Materials and Methods:: Conditioned media (CM) was collected from human bone marrow or adipose MSCs (Rooster Bio) for 72 hours using serum free rat endothelial media (Cell Biologics) and then frozen at -20oC. For the ECIS assay, rat small intestine microvascular endothelial cells (Cell Biologics) in rat endothelial cell media (Cell Biologics) were seeded at 15,000/well onto gelatin coated 96-well ECIS plates (Applied Biophysics) and loaded onto the ECIS instrument in a 37oC incubator with 5% CO2. Cells were allowed to proliferate and form tight and adherens junctions for 72 hours. After 72 hours, wells were exposed to either 0, 5, 10 or 15Gy doses of x-ray radiation. TNF-α (50ng/mL) was used as a positive control and unirradiated wells served as negative controls. Human bone marrow and adipose MSC CM was added at 25% (50µL) of total well volume. Resistance was measured for an additional 24-48 hours to ascertain how x-ray exposure affected monolayer integrity, with and without the addition of MSC CM. For the immunofluorescent staining assay, rat small intestine microvascular endothelial cells (Cell Biologics) were seeded at 15,000/well onto gelatin coated 8-well chamber slides (Thermo Fisher) and cultured for 72 hours. Cells were exposed to 0, 5 or 15Gy doses of x-ray radiation. Cells exposed to TNF-α (50ng/mL) were used as positive controls. Cells were cultured an additional 24 hours with or without 25% MSC CM. Cells were fixed, permeabilized, and stained with antibodies for zona occludin-1 (ZO-1), claudin-5, VE-cadherin, and β-catenin. All visualizations were captured using a Keyence BZ-X810 microscope.

Results, Conclusions, and Discussions:: According to the ECIS assay, there was an initial improvement in monolayer resistance for irradiated cells after treatment with bone marrow MSC CM compared to untreated controls (10% better), but the advantage declined over time (5-10% worse) falling below control cells after approximately 30 hours post radiation exposure. Adipose MSC CM appeared to perform slightly below controls (5-10% worse) for the entire time course post radiation exposure. When the cells were treated with the pro-inflammatory mediator TNF-α, neither bone marrow nor adipose MSC CM offered any recovery. The immunofluorescence staining results suggest that bone marrow MSC-derived CM was able to partially restore cell morphology and junctional protein distribution to resembling that of unirradiated controls for ZO-1, Claudin-5, and β-Catenin upon visual inspection. The results were inconclusive for VE-Cadherin. MSC conditioned media had little to no effect on TNF-α exposed wells.

ECIS and immunofluorescence assays showed recovery of monolayer integrity and restoration of adherens and tight junctions after exposure to ionizing radiation with the addition of bone marrow conditioned media from human MSCs within the the first 30 hours after radiation exposure. This was not observed for adipose MSC conditioned media, however. This lends evidence that MSCs can help repair the loss of monolayer integrity suffered after exposure to ionizing radiation during the initial acute phase but might be MSC tissue of origin dependent. Further investigation is warranted to better understand the underlying mechanisms at play following exposure to radiation and how cellular therapies can be utilized to help repair damage to the intestinal endothelial barrier function.

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