In this report, we have demonstrated that the elevation of VEGF165 and/or Ang-1 plasma levels by Ad vectors results in robust mobilization of CEPs and hematopoietic progenitors and HSCs (CFU-S, marrow repopulating cells) to the peripheral circulation. Although VEGF165 alone could also induce splenomegaly and increase BM cellularity, the sustained elevation of both VEGF165 and Ang-1 was essential to induce significant remodeling of BM vascular architecture, with concomitant mobilization to the extramedullary organs, resulting in profound splenomegaly. Elevation of Ang-1 alone did not affect the size of the spleen or cellularity of the BM, suggesting that combined signaling through VEGFR2/VEGF and Ang1/Tie-2 signaling pathways plays a key role in the regulation of organ-specific hematopoietic activity.
The overexpression of VEGF165
induces a rapid (24–48 h) mobilization of CEPs and HSCs, suggesting that the effect of VEGF165
is mediated through a chemokinetic process. However, plasma elevation of Ang-1 requires at least 7 d to induce mobilization of CEPs and HSCs. The physiological effect of VEGF is most likely not mediated through BM endothelial cell leakiness since studies have shown that Ang-1 prevents VEGF-induced vascular permeability 1318
. In this regard, Ang-1 should have inhibited the VEGF-induced mobilization of the HSCs and CEPs. In contrast, Ang-1 promoted the mobilization of HSCs and CEPs and synergized with VEGF in augmenting its effect. Collectively, these data suggest that VEGF alone or in combination with Ang-1 induce mobilization by directly interacting with stem cells or promoting remodeling of the BM vasculature. It is conceivable that Ang-1 may alter the adhesion molecule repertoire of BM endothelial cells or HSCs, resulting in enhanced mobilization. Alternatively, Ang-1 may sustain or promote the survival of Tie-2+
HSCs and CEPs, inducing cell cycle shifts that may favor their mobilization to the peripheral circulation.
VEGF has been shown to exert its pleiotropic effects on endothelial cells through its receptors VEGFR1 and VEGFR2. VEGFR2 has been shown to be the principal mitogenic receptor for VEGF 2122
, whereas VEGFR1 conveys signals for vascular remodeling. However, VEGFR1 is expressed by various mature hematopoietic cells, including dendritic and monocytic cells 2324
, whereas VEGFR2 is expressed by HSCs and CEPs 4
. Therefore, VEGF165
-induced mobilization of mature cells such as monocytes may be mediated though VEGFR1, whereas mobilization of HSCs is most likely mediated through VEGFR2 6
. Neutralizing mAb to VEGFR2 inhibited VEGF165
-induced mobilization and splenomegaly, suggesting that interaction of VEGF165
with VEGFR2 is essential for mobilization and recruitment of HSCs. Delayed mobilization in response to elevation in circulating Ang-1 is most likely induced by interaction with either Tie-2+
on the stem cells 19
or because of remodeling of the BM microenvironment.
Most in vivo experiments assessing the chemoattractant properties of VEGF165
have been performed with injection of recombinant VEGF165
. However, given that the half-life of VEGF165
in the circulation is only several minutes, it has been difficult to evaluate the role of chronic VEGF165
release, as it may occur during tumor growth and metastasis or myelodysplastic syndromes. Likewise, recombinant Ang-1 is unstable and previous studies using Ang-1 protein have failed to demonstrate a functional endpoint 21
. It is suggested that in contrast to VEGF165
, whereby injection of recombinant protein can induce mobilization of stem cells, Ang-1 protein does not have enough biochemical stability to exert any biological effect. Furthermore, given the delayed mobilization seen with Ang-1 treatment, injection of AdAng-1 into SCID mice as opposed to immunocompetent mice was the only means of sustaining the level of Ang-1 long enough in vivo to observe its effects. Therefore, the use of Ad vectors as regional delivery vehicles for VEGF165
and Ang-1 provides ideal tools for studying the role of these angiogenic factors in mobilization and recruitment of HSCs and CEPs. In addition, given that VEGF189
is matrix bound, Ad vectors producing VEGF189
provide an ideal mechanism to deliver this matrix-bound isoform of VEGF regionally to the extramedullary hematopoietic microenvironments such as the liver.
We show that long-term overexpression of both VEGF165
and Ang-1 is essential to induce an initial increase in BM cellularity followed by remodeling of the BM architecture, with depletion of sinusoidal spaces of hematopoietic cells and a parallel increase in BM vascularization and splenomegaly. These results suggest that regional expression of VEGF165
and Ang-1 in extramedullary organs may act synergistically to modulate postnatal hematopoiesis by inducing extramedullary shifts in hematopoiesis or remodeling of vascular architecture of sinusoidal BM endothelial cells. Recently, it has been shown that plasma VEGF levels are significantly elevated in myelodysplastic syndromes 17
, where the BM microenvironment is damaged by fibrotic tissue. This raises the possibility that elevation of VEGF may serve as one of the primary adaptive responses to compensate for BM myelofibrosis, by promoting the relocalization and recruitment of HSCs to other hematopoietic microenvironments such as the spleen, resulting in splenomegaly.
Mobilization of CEPs to the peripheral circulation may play a critical role in regulating postnatal angiogenesis and vasculogenesis. Based on the data presented here, high levels of VEGF produced by tumors may result in the mobilization of BM-derived CEPs to the peripheral circulation and enhance their recruitment into the tumor vasculature. Given that CEPs may also contribute to vascular healing, overexpression of VEGF165 may promote the peripheral blood mobilization and recruitment of CEPs to the injured vascular bed, thereby accelerating wound healing.
As evidenced by the relative plasma levels of Ang-1 and VEGF, higher levels of Ang-1 are necessary to induce mobilization of HSCs and CEPs. Indeed, at doses >1.5 × 108 PFU of AdVEGF165, elevations of plasma VEGF were associated with significant toxicity and resulted in the death of the treated mice. Ang-1 does not exert any major physiological effect at the lower doses (5 × 108 PFU); however, when administered at higher doses (109 PFU) it had a potent synergistic effect with VEGF in mobilizing HSCs and CEPs. These differences in the dose response may be due to the variable receptor densities or differential physiological effects of the VEGF receptors and Tie-2. It is also conceivable that expression of VEGFR2 (Flk-1) on HSCs and CEPs is significantly higher than Tie-2.
These data set forth a novel paradigm for efficient mobilization of HSCs and CEPs to the peripheral circulation. This strategy facilitates recovery of large numbers of pluripotent HSCs and CEPs, which may be used for transplantation or treatment of a wide variety of genetic and malignant disorders. Whether transplantation of mobilized CEPs in conjunction with mobilized HSCs will exert a synergistic effect to enhance engraftment and reconstitution of hematopoiesis is the subject of future studies.