We demonstrate here that human and murine HSCs with long-term BM-repopulating capacity express VEGFR1, conveying signals regulating cell cycling and motility. Inhibition of VEGFR1, but not VEGFR2, blocked recruitment of HSCs from their resting microenvironment, thereby retarding hematopoietic reconstitution after myelosuppression. PlGF augmented early phases of hematopoietic recovery by promoting recruitment, chemotaxis and mobilization of VEGFR1+ HSCs and progenitors, while during late phases of BM recovery PlGF-mediated upregulation of MMP-9 facilitated the release of sKitL thereby accelerating hematopoietic reconstitution.
Our data suggest that PlGF activates non-angiogenic pathways and thus serves functions other than regulating angiogenesis. Based on our data, the accumulation of angioblasts within the yolk sac in VEGFR1−/−
mice may not only be due to enhanced commitment to angioblasts17
, but may be due to impaired cell migration. This appears to be the case in Drosophila
, where VEGF-A activation is directly linked to hemocyte motility23
How does VEGFR1 activation increase HSC motogenic potential? Chemotaxis of VEGFR1+
HSCs and progenitors accounts for the WBC increase in the early phase of PlGF mobilization. PlGF mobilized CFU-Cs and CFU-Ss in MMP-9+/+
, but to a lesser extent in MMP-9−/−
mice. In later phases, PlGF-induced cell mobilization was impaired in MMP-9−/−
mice, suggesting that PlGF recruits HSCs and progenitors by a different mechanism. PlGF induces the release of sKitL through MMP-9 activation. sKitL increases motility by promoting cell cycle transition through interaction with its receptor c-Kit29,30
. Activation of this pathway accounts for the hematopoietic reconstitution during later phases of BM recovery. Indeed, neutralizing anti-VEGFR1 profoundly diminished plasma elevation of sKitL in myelosuppressed mice during BM recovery, while introduction of sKitL into myelosuppressed mice treated with anti-VEGFR1 completely restored hematopoiesis.
The increase in cell cycling and proliferation of VEGFR1+
cells after BM suppression is most likely not a direct effect of PlGF, because activation of VEGFR1 did not change HSC survival or colony formation in vitro
. These data support other reports in which VEGF-A or PlGF had no major effect on the proliferation or survival of HSCs or progenitors31–33
. However, these studies do not rule out the possibility that VEGF/VEGFR internal autocrine signaling pathways may synergize with other cytokines to convey survival signals for repopulating cells.
Although VEGFR2 is expressed on human NOD/SCID-repopulating cells14
, the functional role of VEGFR2 in the regulation of post-natal hematopoiesis remains unclear. Transplantation of murine VEGFR2+
BM cells failed to reconstitute hematopoiesis in lethally irradiated recipients15
. These data suggest that murine BM-derived VEGFR2+
cells may mark endothelial progenitors1,34
rather than repopulating HSCs. Supporting these observations, we showed only a transient delay in the recovery of lymphoid and erythroid precursors in mice treated with neutralizing anti-VEGFR2. This delay in hematopoietic recovery was not sufficient to induce life-threatening complications, since all mice treated with 5FU and anti-VEGFR2 survived. Anti-VEGFR2 may delay the recovery of certain lineages through interference with the production of lineage-specific cytokines, such as GM-CSF and IL-6 (ref. 35
In contrast, mice treated with 5FU and neutralizing anti-VEGFR1 showed a striking impaired tri-lineage cell recovery with prolonged pancytopenia, which resulted in the demise of 70% of treated mice kept under germ-free conditions. The lack of a more profound defect in myelosuppressed mice treated with anti-VEGFR2 seems to be in disagreement with the dramatic defect in blood-island formation observed in VEGFR2-null mice. It is difficult to compare the role of VEGFR2 signaling in the regulation of hematopoiesis in the embryo and adult mice, as there are fundamental differences between the established post-natal BM microenvironment and fragile embryonic blood islands. Adult BM stromal cells provide a fortified cellular scaffold that may be resistant to effects of anti-VEGFR2. Since stromal cells express VEGFR136
, it is conceivable that anti-VEGFR1 block MMP-9 expression leading to failure of the recruitment of HSCs. This may explain why anti-VEGFR1 compromises hematopoietic recovery, while inhibition of VEGFR2 has only a marginal effect on stress hematopoiesis.
We have shown that plasma elevation of VEGF165
with or without angiopoietin-1 promoted mobilization of HSCs and CFU-Cs (ref. 22
). Here, we show that, under steady-state conditions, chronic inhibition of VEGFR1 or VEGFR2 had no major effect on hematopoiesis. This is in sharp contrast to the failure of hematopoietic reconstitution and demise of the treated mice by inhibiting VEGFR1 during BM suppression. These data suggest that during steady-state hematopoiesis, VEGFR1 inhibition does not result in a profound impairment in hematopoiesis, because the demand for HSC recruitment is low. In contrast, during stress hematopoiesis activation of VEGFR1 is necessary for hematopoietic reconstitution. This phenomenon is similar to other physiological processes requiring rapid tissue vascularization, where stress angiogenesis driven by the mobilization of VEGFR2+
BM-derived cells is necessary to accelerate neo-angiogenesis1
. These data suggest that in stress hematopoiesis the molecular switch requires activation of VEGFR1, whereas during stress angiogenesis the molecular switch is driven by VEGFR2 activation.
Based on our data, the clinical use of VEGFR1 inhibitors, but to a lesser degree VEGFR2, delivered in combination with myelosuppressive agents, may result in a life-threatening prolongation of BM suppression. In this respect, suppression of hematopoiesis through VEGFR1 inhibition has two ramifications. On one hand, blocking VEGFR1 may inhibit tumor angiogenesis and growth, but on the other hand, it may introduce unwanted BM toxicity. Nonetheless, since VEGFR1 toxicity is dose-dependent, VEGFR1 inhibitors can be delivered with a manageable toxicity profile. Simultaneous administration of sKitL may reduce BM toxicity caused by blocking VEGFR1 and therefore rescue patients from infection or hemorrhage.
Stress promotes mobilization of BM-derived stem cells that ultimately incorporate, although at very low levels, into specific organs. This low efficiency of incorporation may be reflected in the paucity of readily motile stem cells capable of being recruited from the BM to circulation. PlGF, endowed with a low toxicity profile, allows for mobilization of stem cells to the circulation, facilitating recovery of many stem cells that may ultimately be used for organ restoration.
Our findings introduce the novel paradigm that determination of VEGFR1 and MMP-9 expression should be considered as surrogate markers for evaluating the efficacy and success of stem cell mobilization and engraftment in transplantation settings. It is conceivable that dysregulated MMP-9, VEGFR1 or PlGF expression may be responsible for defects in the stem-cell microenvironment leading to BM failure. In this regard, therapeutic strategies to upregulate VEGFR1, PlGF and MMP-9 may provide an effective means to restore motogenic potential of BM-repopulating cells and help to replenish the stem cell pool after BM transplantation.