Lymphatic Endothelial Cell Response to XRay Irradiation
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INTRODUCTIONThe lymphatics play a key role in the redistribution of interstitial fluids, cytokines, and immune cells via lymph formation. Severe impairment in lymphatic function causes swelling and inflammation, diagnosed as lymphedema. Secondary lymphedema is a known side effect cancer patients experience after radiation therapy; however, the pathogenesis is unknown. Furthermore astronauts develop edema while in space, being exposed to chronic, lowdose, high LET background radiation. The lymphatics response radiation has not been extensively investigated. This is one of the first investigations characterizing lymphatic endothelium responses to radiation via viability and cell morphological measures.METHODSPrimary, rat mesenteric lymphatic endothelial cells were exposed to a broad range of Xray (250 kVp, ~0.75 Gy/min) doses: 0, 0.5, 1, 1.5, 2, 4, 16 Gy, and travel sham. Cell viability was analyzed by XTT viability kit between 6268 hours postirradiation at multiple time points. 24 and 72hours postirradiation, immunofluorescence was performed for catenin, Factin, VEcadherin, DRAQ7, and measured by confocal microscopy (20 + 63 average zstack projections, 0.5 m slices). No measurable differences were seen between travel sham and 0 Gy; therefore, statistical comparisons of treatment groups were made with 0 Gy group by 1way ANOVA and nonparametric Wilcoxon test (p<0.05).RESULTSCell viability dropped in > 1 Gy groups at day 3, but became comparable to 0 Gy values after day 7. Cell morphometry showed radiation sensitivity at all doses. Notably there was a loss of celltocell adhesion with irradiated cells increasing in surface area coverage, marked reduction in nuclei per equivalent field of view, and increased cellular roundness. This was coupled with decreased catenin & VEcadherin intensity and altered Factin anisotropy leading to a loss of intercellular contact.CONCLUSIONSLymphatics show radiation sensitivity in the context of these cell culture experiments. Our results may have functional implications of lymphatics in tissue, with potential endothelial barrier dysfunction leading to leakage and edema. These preliminary experiments will build the framework for future investigations towards clinical and spaceflight relevant lymphatic radiation exposure response.Support or Funding InformationAN is supported by the NASANational Space Biomedical Research Institute Predoctoral Fellowship through NCC 958.
