The physiological role of Angiopoietin-like proteins (Angptls) in hematopoietic system is unknown. Here we showed that Angptl3 is expressed by both hematopoietic stem cells (HSCs) and bone marrow stromal cells. In particular, the expression of Angptl3 in the bone marrow stromal cells is significantly increased upon transplantation, suggesting that this protein may play an important role in the bone marrow under stress. We asked whether Angptl3 expression had a functional role in HSCs by utilizing mice ablated for Angptl3. Using Hoechst/pyronin Y staining and Brdu incorporation analysis, we found that HSCs in Angptl3-null mice exhibited significantly decreased quiescence compared to those in wild-type mice. To test the role of Angptl3 in the stress response of hematopoietic cells, we treated mice with 5-fluorouracil (5-FU), which is toxic to cycling cells and accelerates the entry of HSCs into the cell cycle. The survival of Angptl3-null mice was significantly lower than that of wild-type mice. To further identify the role of Angptl3 in stress response of HSCs, we examined whether Angptl3 affected DNA damage in HSCs upon transplantation. To this end, we transplanted WT bone marrow cells into lethally irradiated Angptl3 null recipients or WT mice. We found that HSCs in the Angptl3 null recipient mice had significantly increased gamma-H2AX foci compared to WT recipients, suggesting that Angptl3 protects HSCs from DNA damage. We further used the competitive reconstitution analysis to determine the roles of Angptl3 on HSC activity. Importantly, both Angptl3-null HSCs transplanted to wild-type recipients and wild-type HSCs transplanted to Angptl3-null recipients showed impaired repopulation. These results conclude that Angptl3 has both cell-autonomous and environmental effects that support the in vivo activity of HSCs.
To identify the intracellular target of Angptl3 in HSCs, we performed DNA microarray and real-time RT-PCR analyses to compare the gene expression in HSCs isolated from WT and Angptl3 null mice. We found that Angptl3-null HSCs had increased expression of transcription factor Ikaros. Consistently, extrinsic treatment of HSCs by Angptl3 also suppressed the expression of Ikaros. Ikaros is a zinc finger transcription factor important for differentiation of lymphoid, myeloid, and erythroid cells, and its expression is low in multi-potent HSCs, but high in progenitors with lymphoid-myeloid potential. Since Angptl3 downregulates the expression of Ikaros in HSCs, we examined the effect of forced expression of Ikaros on HSC activities. Indeed, overexpression of Ikaros enhanced HSC cycling and DNA damage, and diminished their repopulation activity, indicating the downregulation of Ikaros by extrinsic Angptl3 maintains the stemness of HSCs.
We studied the spatial relationship of Angptl3-expressing cells and the bone-marrow HSCs using immunohistochemical tools. We showed that 58.6% of Angptl3-producing cells were in contact with sinusoidal endothelial cells in bone marrow and that 60.8% of HSCs are adjacent to Angptl3-producing cells in the bone marrow. To directly test whether Angptl3-producing bone marrow stromal cells support HSC expansion, we co-cultured HSCs and CD45-SSEA4+ cells and used competitive reconstitution analysis to measure HSC activity. HSCs co-cultured with WT CD45-SSEA4+ cells had significantly increased repopulation relative to those co-cultured with Angptl3 null CD45-SSEA4+ cells (36% vs. 17%). This result demonstrated that bone marrow CD45-SSEA4+ cells support expansion of HSCs, and provided the functional evidence that Angptl3-producing stromal cells are a part of HSC niche in the bone marrow.
Thus, Angptl3-producing cells are an important component of the HSC niche. Our experiments demonstrate a unique example of the extrinsic control of stemness by cell-autonomous effects from stem cells per se and by environmental effects from the niche cells.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.