An acute negative bystander effect of γ-irradiated recipients on transplanted hematopoietic stem cells.

Ultimate success of hematopoietic stem cell transplantation (HSCT) depends not only on donor HSCs themselves but also on the host environment. Total body irradiation is a component in various host conditioning regimens for HSCT. It is known that ionizing radiation exerts "bystander effects" on nontargeted cells and that HSCs transplanted into irradiated recipients undergo proliferative exhaustion. However, whether irradiated recipients pose a proliferation-independent bystander effect on transplanted HSCs is unclear. In this study, we found that irradiated mouse recipients significantly impaired the long-term repopulating ability of transplanted mouse HSCs shortly (∼ 17 hours) after exposure to irradiated hosts and before the cells began to divide. There was an increase of acute cell death associated with accelerated proliferation of the bystander hematopoietic cells. This effect was marked by dramatic down-regulation of c-Kit, apparently because of elevated reactive oxygen species. Administration of an antioxidant chemical, N-acetylcysteine, or ectopically overexpressing a reactive oxygen species scavenging enzyme, catalase, improved the function of transplanted HSCs in irradiated hosts. Together, this study provides evidence for an acute negative, yet proliferation-independent, bystander effect of irradiated recipients on transplanted HSCs, thereby having implications for HSCT in both experimental and clinical scenarios in which total body irradiation is involved.

Hematopoietic stem cell regeneration enhanced by ectopic expression of ROS-detoxifying enzymes in transplant mice.

High levels of reactive oxygen species (ROS) can exhaust hematopoietic stem cells (HSCs). Thus, maintaining a low state of redox in HSCs by modulating ROS-detoxifying enzymes may augment the regeneration potential of HSCs. Our results show that basal expression of manganese superoxide dismutase (MnSOD) and catalase were at low levels in long-term and short-term repopulating HSCs, and administration of a MnSOD plasmid and lipofectin complex (MnSOD-PL) conferred radiation protection on irradiated recipient mice. To assess the intrinsic role of elevated MnSOD or catalase in HSCs and hematopoietic progenitor cells, the MnSOD or catalase gene was overexpressed in mouse hematopoietic cells via retroviral transduction. The impact of MnSOD and catalase on hematopoietic progenitor cells was mild, as measured by colony-forming units (CFUs). However, overexpressed catalase had a significant beneficial effect on long-term engraftment of transplanted HSCs, and this effect was further enhanced after an insult of low-dose γ-irradiation in the transplant mice. In contrast, overexpressed MnSOD exhibited an insignificant effect on long-term engraftment of transplanted HSCs, but had a significant beneficial effect after an insult of sublethal irradiation. Taken together, these results demonstrate that HSC function can be enhanced by ectopic expression of ROS-detoxifying enzymes, especially after radiation exposure in vivo.

Vascular endothelial growth inhibitor (VEGI; TNFSF15) inhibits bone marrow-derived endothelial progenitor cell incorporation into Lewis lung carcinoma tumors.

Bone marrow (BM)-derived endothelial progenitor cells (EPC) have a critical role in tumor neovascularization. Vascular endothelial growth inhibitor (VEGI) is a member of the TNF superfamily (TNFSF15). We have shown that recombinant VEGI suppresses tumor angiogenesis by specifically eliminating proliferating endothelial cells (EC). We report here that treatment of tumor bearing mice with recombinant VEGI leads to a significantly decreased population of BM-derived EPC in the tumors. We transplanted whole bone marrow from green fluorescent protein (GFP) transgenic mice into C57BL/6 recipient mice, which were then inoculated with Lewis lung carcinoma (LLC) cells. Intraperitoneal injection of recombinant VEGI led to significant inhibition of tumor growth and decrease of vasculature density compared to vehicle-treated mice. Tumor implantation yielded a decrease of BM-derived EPC in the peripheral blood, while VEGI-treatment resulted in an initial delay of such decrease. Analysis of the whole bone marrow showed a decrease of Lin(-)-c-Kit(+)-Sca-1(+) hematopoietic stem cell (HSC) population in tumor bearing mice; however, VEGI-treatment caused a significant increase of this cell population. In addition, the number of BM-derived EPC in VEGI-treated tumors was notably less than that in the vehicle-treated group, and most of the apoptotic cells in the VEGI-treated tumors were of bone marrow origin. These findings indicate that VEGI inhibits BM-derived EPC mobilization and prevents their incorporation into LLC tumors by inducing apoptosis specifically of BM-derived cells, resulting in the inhibition of EPC-supported tumor vasculogenesis and tumor growth.

Inhibition of endothelial progenitor cell differentiation by VEGI.

Endothelial progenitor cells (EPCs) play a critical role in postnatal and tumor vasculogenesis. Vascular endothelial growth inhibitor (VEGI; TNFSF15) has been shown to inhibit endothelial cell proliferation by inducing apoptosis. We report here that VEGI inhibits the differentiation of EPCs from mouse bone marrow-derived Sca1(+) mononuclear cells. Analysis of EPC markers indicates a significant decline of the expression of endothelial cell markers, but not stem cell markers, on VEGI-treated cells. Consistently, the VEGI-treated cells exhibit a decreased capability to adhere, migrate, and form capillary-like structures on Matrigel. In addition, VEGI induces apoptosis of differentiated EPCs but not early-stage EPCs. When treated with VEGI, an increase of phospho-Erk and a decrease of phospho-Akt are detected in early-stage EPCs, whereas activation of nuclear factor-kappaB, jun N-terminal kinase, and caspase-3 is seen in differentiated EPCs. Furthermore, VEGI-induced apoptosis of differentiated EPC is, at least partly, mediated by death receptor-3 (DR3), which is detected on differentiated EPC only. VEGI-induced apoptosis signals can be inhibited by neutralizing antibodies against DR3 or recombinant extracellular domain of DR3. These findings indicate that VEGI may participate in the modulation of postnatal vasculogenesis by inhibiting EPC differentiation.

Mouse hematopoietic stem cell transplantation.

Hematopoietic stem cells (HSCs) are capable of self-renewal and multi-lineage reconstitution of hematopoiesis in irradiated transplant recipient mice. As such, bone marrow transplantation (BMT) is a major assay commonly used to examine murine HSC activity. BMT traditionally involves injection of HSCs into lethally irradiated recipients via the tail vein, then subsequently analyzing donor engraftment. Here, we describe the methods for assaying HSC reconstitution in direct, competitive, and serial BMT.

Bid is a positive regulator for donor-derived lymphoid cell regeneration in γ-irradiated recipients.

OBJECTIVE: Hematopoietic regeneration is regulated by cell survival proteins, such as the Bcl-2 family. Bid, a BH3-only protein of the Bcl-2 family, has multiple cellular functions and is involved in a variety of physiological or pathological conditions. We attempted to define its role in hematopoietic cell repopulation under the stress condition of bone marrow transplantation. MATERIALS AND METHODS: We performed conventional or competitive bone marrow transplantation with donor hematopoietic cells from Bid(-/-) or Bid(+/+) mice. Flow cytometry was used for quantification of hematopoietic stem cells, hematopoietic progenitor cells, and differentiated cells in different lineages (T, B, and myeloid cells). Single cell culture and homing assays were performed to further evaluate hematopoietic stem cell functions. Hematopoietic progenitor cells were also measured by the colony-forming cell culture. RESULTS: Contrary to the widely recognized role of Bid as a pro-apoptotic protein, the absence of Bid significantly reduced the reconstitution of donor hematopoietic cells in γ-irradiated recipients. Interestingly, however, numbers of hematopoietic stem cells and hematopoietic progenitor cells and their functions were not overtly altered. Instead, the regeneration of donor T and B cells was significantly impaired in the absence of Bid. Further analysis indicated an accumulation of the triple-negative T-cell population in the thymus, and pro-B cells in the bone marrow. CONCLUSIONS: Our current study demonstrates a positive impact of Bid on hematopoietic regeneration mainly due to its unique effects on donor lymphopoiesis in the transplant recipients.