Bone Marrow Sinusoidal Endothelium Undergoes DNA Damage and Repair Following Lethal Irradiation.

In the light of the possibility that adult bone marrow cells possess hemangioblast ability, work from our laboratory demonstrates that the bone marrow sinusoids remain predominantly host-derived following bone marrow transplant when ionizing irradiation is used as the conditioning regimen. To determine the effect of lethal irradiation to the host sinusoidal endothelial cells, we performed four apoptosis related assays and two cell proliferation assays on bone marrow sections at various time points during the first two weeks post-irradiation. We found:

1. Phosphorylated H2AX was present in both hematopoietic and sinusoidal endothelial cells. However, only hematopoietic cells showed caspase-3 dependent apoptosis.

2. Three days after radiation, some sinusoidal endothelial cells became TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay) positive, but were activated caspase-3 and ISOL (in situ oligo ligation assay) negative, suggesting non-apoptotic DNA fragmentation.

3. TUNEL positive endothelial cells were present in non-transplanted irradiated bone marrow 7–13 days post-irradiation while after 7 days, there were almost no TUNEL positive endothelial cells in transplanted animal, demonstrating that donor cells support sinusoidal endothelial survival.

4. In some endothelial cells, TUNEL signal was concentrated in discrete areas of the nucleus, suggesting a repair process that involves the localization and removal of damaged DNA fragments.

5. Very few sinusoidal endothelial cells were Ki67 positive and even fewer were BrdU positive, demonstrating that endothelial cell division is not a major mechanism for the survival of bone marrow sinusoidal system after irradiation on the short term.

These results demonstrate that sinusoidal endothelial cells undergo DNA damage and repair after lethal irradiation for bone marrow transplant. These results may explain, in part, why patients with impaired DNA damage/repair mechanisms have engraftment defects.