In this study, we found that HSCs or CD11blow
cells played critical roles in promoting stable blood vessel formation by their differentiation into MCs and by the production of Ang-1 in both physiological and pathological conditions. Although it has been reported that ECs are developed from HSC populations through the monocyte lineage (27
), we found that MCs are also differentiated from the hematopoietic lineage through the same pathway as ECs by using CD45 and CD11b, well-known specific markers expressed on HCs and the monocyte/macrophage lineage, respectively. In particular, we showed differences between CD11blow
cells and CD11bhigh
cells in blood vessel formation. One such difference was that CD11low
cells have a long-term regeneration capacity for neovascularization by differentiating into ECs and MCs, but CD11bhigh
cells do not. The other is that CD11bhigh
cells induced the formation of leaky blood vessels, but CD11blow
cells did not. Thus, CD11blow
cells might promote the stability of newly developed blood vessels.
Progenitor cells for ECs or MCs have been suggested to exist in adult BM, and several reports have suggested that these cells develop into endothelial progenitor cells (EPCs) in the neovascularization of tumors and ischemic regions (34
). Moreover, the HSC population in the adult BM was reported to incorporate into blood vessels as MCs in atherosclerotic plaques and in transplanted hearts. Sata et al. (20
) suggested that vascular SMC progenitors also originated from the Lin−
HSC population. At the moment, it is not clear whether single Lin−
cells can give rise to the three different cell types, namely, HCs, ECs, and MCs. However, in this study, we showed that the latter two populations develop from the HC lineage by using CD45 as a marker expressed on HCs but not on ECs nor MCs.
Although CD11b+ cells could give rise to MCs as well as ECs, there were differences between CD11bhigh and CD11blow cells. CD11bhigh cells rapidly differentiated into CD31+ EC-like cells in vitro and were found to incorporate into newly developed blood vessels as ECs in an in vivo ischemia model. However, they showed low frequency for differentiation into MCs and ECs, and MCs from CD11bhigh cells could not survive for the long term, both in vitro and in vivo. In contrast, although CD11blow cells took more time for their differentiation into ECs, they differentiated into MCs with high frequency. Both MCs and ECs from CD11blow cells could form vascular cells for the long term.
In the course of angiogenesis, vessel regression is part of the maturation process. Indeed, the number of newly developed blood vessels induced by CD11b+ cells gradually decreased between 2 wk and 6 mo. In the case of CD11bhigh cell injection, capillary density after 6 mo was ~70% of that present after 2 wk. On the other hand, in the case of CD11low cells, the capillary density was ~80% at 6 mo compared with that present at 2 wk. It is not clear whether this higher loss of capillary density associated with CD11bhigh cells reflects higher apoptosis in vivo of ECs generated by CD11bhigh cells or not; however, this is possible because ECs generated by CD11high cells declined because of apoptosis after 2 wk in vitro (Fig. S2).
Recently, two groups independently reported that there were two populations of cells with the capacity to differentiate into ECs from mononuclear cells in the PB (36
). They reported that CD14+
cells or spindle-shape cells in the PB differentiate into ECs rapidly, and such ECs could not survive for the long term. They termed these ECs as early EPCs. On the other hand, ECs derived from CD14−
cells or cells showing a cobblestone appearance grow exponentially, survive for a longer time, and are called late outgrowth ECs or late EPCs. Although they did not observe the differentiation of MCs from EPCs, we assume that the former corresponds to CD11bhigh
cells and the latter to CD11blow
cells. Based on a previous report showing that cycling HSCs express CD11b weakly (30
cells might belong to the HSC population. However, the results of limiting dilution analysis showing low frequency of differentiation of the HSC population into ECs or MCs suggest that only a part of the immature HSC population expressing CD11b, c-Kit, and Sca-1 can develop into vascular cells.
Additionally, we found that CD11bhigh
cells induced hyperpermeability in this ischemia model, but CD11blow
cells did not. In terms of leakiness of blood vessels, Ang-1 produced from MCs has been suggested to induce structurally stable blood vessels by promoting adhesion between MCs and ECs (9
). Relative to this, it has been reported that VEGF-mediated hyperpermeability can be suppressed by Ang-1 (33
). As far as we observed, CD11blow
cells, but not CD11bhigh
cells, express Ang-1 abundantly, but both CD11blow
cells express VEGF. We previously reported that the HSC population produces Ang-1 strongly and promotes the remodeling of blood vessels during embryogenesis (19
). Moreover, we recently reported that the CD45+
HSC population regulates caliber change and remodeling of blood vessels located in the fibrous cap of a murine tumor model (38
). Collectively, the data strongly suggest that the CD11blow
population contains higher amounts of HSCs and promotes remodeling and stability of newly developed blood vessels along with their differentiated MCs as well as the maintenance of Ang-1 production. Recently, there has been interest in the regeneration of blood vessels in ischemic patients using the transplantation of BM cells (39
). Our analysis shows that selective injection of BM HSC populations may be effective in achieving blood vessel regeneration.
Although several studies have shown the plasticity of HSCs, the problem of autonomous cell fusion between HSCs and tissue-specific cells has been considered (40
). When the HSC population differentiates into another lineage except hematopoietic lineage during embryogenesis or other physiological situations, we consider those events as the natural course of the differentiation pathway in the HSC population. Here we showed that the HSC population in the brain during embryogenesis easily differentiates into MCs as well as ECs. Moreover, coculturing of HSCs from the fetal liver with cells from the brains of embryos is necessary for the differentiation of the HSC population from the fetal liver into vascular cells. This is the first report showing that the HSC population can change their pathway of differentiation to something other than the hematopoietic lineage by receiving adequate molecular cues depending on the tissue demands, such as development and ischemia. Therefore, we propose that differentiation of vascular cells from the HSC population runs a physiologically natural course rather than being attributed to plasticity of the cells. At this moment, the molecular mechanism whereby the HSC population differentiates into MCs as well as ECs is not fully understood. Among various cytokines, we found that TGF-β has a weak capacity for changing the fate of the HSC population into MCs. In the present therapeutic approach to angiogenesis, larger and more structurally stable blood vessel formation, the so-called arteriogenesis, is thought to be important as a new strategy. To address this issue, MC management is essential. In this study, we could generate MC progenitors from the BM HSC population (Figs. S5 and S6, available at http://www.jem.org/cgi/content/full/jem.20050373/DC1
), although efficiency was low. Therefore, the use of molecules that are effective in promoting MC development from HSCs may enable the realization of the arteriogenesis goal.