Defibrotide Protects Endothelium from Radiation Induced Injury: A Potential New Strategy in the Armamentarium against Radiation Toxicity

ABSTRACT

Radiation induced toxicity (RIT) has long been a notorious limit of radiotherapy. RIT can only be circumvented by patient selection or dose de-escalation. No effective treatment of RIT is available. Endothelium injury (EnI) has been recognized as the key pathogenesis in radiotoxicity. EnI initiates the vicious cascade and propagates to various RIT. Defibrotide (DF), a mixture of oligonucleotides, is a potent endothelium protector and is the only medication approved for the treatment of post-HSCT severe hepatic sinusoid obstructive syndrome (SOS), with survival benefits and minimal toxicity. The protective effect of DF has been exhibited in chemo-toxicity, hypoxia, physical injury and infection, but not yet in radiation (RT). We hypothesize that DF alleviates RIT through a similar mechanism. Here, we present the first report and primitive results of an in vitro study of DF in RT setting. Primary human hepatic sinusoidal endothelial cells (HHSEC) and human umbilical vein endothelial cells (HUVEC) were cultured and RT for a single 4Gy or 8Gy with or without DF at the final concentration of 300ug/ml. Assays for cell viability, survival and proliferation were evaluated. Molecule expression pattern was analyzed by rt-PCR. All the assays were replicated 3 times. After RT, HHSECs became sparse and deformed under the microscope. In contrast, HHSECs cultured with DF before RT generally appeared normal. In accordance, a cell counting kit assay showed that viability of HHSECs was preserved after RT at the presence of DF, while it was dramatically reduced by RT alone (p<0.001). Flow cytometry analysis of programed cell death 48h post-RT confirmed that DF significantly reduced both early and late apoptosis (8.9% vs 14.6%, p<0.05). In the proliferation assay, RT alone almost quartered the number of HUVEC clones, while DF co-treatment partially prevented the extermination. DF alone had no deleterious effect compared with control groups, in concordance with published studies. RT-PCR revealed elevated expression of pro-inflammatory and pro-coagulant molecules. In detail, raised mRNA levels of von Willebrand factor (vWF), ICAM-1, VCAM-1, IL-1a, IL-1b, IL-6, TNF-a and eNOS were detected after RT, which were down regulated by DF. DNA break assay by γ-H2AX formation was performed, which showed no drastic difference of γ-H2AX staining after RT with or without DF, implicating other mechanisms. Besides, DF prophylaxis before RT rather than salvage after RT revealed better endothelium protection. We then built a rat model of radiation induced endothelium injury (RIEI) by whole liver exposure. However, due to the COVID-19 outbreak and temporary un-availability of DF, further study is in waiting. Effective therapy of RIT is a huge unmet medical need. DF, an approved, well-tolerated orphan drug for post-HSCT SOS, exhibited definitive protective effect in RIEI as well. Further studies, not only pre-clinically but also in clinical scenarios are warranted and in eager expectation. [ FROM AUTHOR] Copyright of International Journal of Radiation Oncology, Biology, Physics is the property of Pergamon Press - An Imprint of Elsevier Science and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)