Scott T. Avecilla

Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis

The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo−/−) or TPO receptor–deficient (Mpl−/−) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo−/− and Mpl−/− mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4+ megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.

Angiogenic Factors Reconstitute Hematopoiesis by Recruiting Stem Cells from Bone Marrow Microenvironment

Abstract: The mechanism by which angiogenic factors recruit bone marrow (BM)‐derived quiescent endothelial and hematopoietic stem cells (HSCs) is not known. Here, we report that functional vascular endothelial growth factor receptor‐1 (VEGFR1, Flt‐1) is expressed on a subpopulation of human CD34+ and mouse Lin−Sca‐1+c‐Kit+ BM‐repopulating stem cells, conveying signals for recruitment of HSCs and reconstitution of hematopoiesis. Inhibition of VEGFR1 signaling, but not VEGFR2 (Flk‐1, KDR), blocked HSC cell cycling, differentiation and hematopoietic recovery after BM suppression, resulting in the demise of the treated mice. Plasma elevation of placental growth factor (PlGF), which signals through VEGFR1, but not VEGFR2, restored hematopoiesis during the early and late phases following BM suppression. The mechanism whereby PlGF enhanced early phases of BM recovery was mediated directly through rapid chemotaxis of readily available VEGFR1+ BM‐repopulating and progenitor cells. The late phase of hematopoietic recovery was driven by PlGF‐induced upregulation of matrix metalloproteinase‐9 (MMP‐9) in the BM, mediating the release of soluble Kit‐ligand (sKitL). sKitL increased proliferation and motility of HSCs and progenitor cells, thereby augmenting hematopoietic recovery. PlGF promotes recruitment of VEGFR1+ HSCs from a quiescent to a proliferative microenvironment within the BM, favoring differentiation, mobilization, and reconstitution of hematopoiesis.

Thrombospondins deployed by thrombopoietic cells determine angiogenic switch and extent of revascularization

Thrombopoietic cells may differentially promote or inhibit tissue vascularization by releasing both pro- and antiangiogenic factors. However, the molecular determinants controlling the angiogenic phenotype of thrombopoietic cells remain unknown. Here, we show that expression and release of thrombospondins (TSPs) by megakaryocytes and platelets function as a major antiangiogenic switch. TSPs inhibited thrombopoiesis, diminished bone marrow microvascular reconstruction following myelosuppression, and limited the extent of revascularization in a model of hind limb ischemia. We demonstrate that thrombopoietic recovery following myelosuppression was significantly enhanced in mice deficient in both TSP1 and TSP2 (TSP-DKO mice) in comparison with WT mice. Megakaryocyte and platelet levels in TSP-DKO mice were rapidly restored, thereby accelerating revascularization of myelosuppressed bone marrow and ischemic hind limbs. In addition, thrombopoietic cells derived from TSP-DKO mice were more effective in supporting neoangiogenesis in Matrigel plugs. The proangiogenic activity of TSP-DKO thrombopoietic cells was mediated through activation of MMP-9 and enhanced release of stromal cell-derived factor 1. Thus, TSP-deficient thrombopoietic cells function as proangiogenic agents, accelerating hemangiogenesis within the marrow and revascularization of ischemic hind limbs. As such, interference with the release of cellular stores of TSPs may be clinically effective in augmenting neoangiogenesis.

Functional heterogeneity of the bone marrow vascular niche.

Sinusoidal endothelial cells (SECs) comprise the platform where trafficking into and out of the BM occurs and where hematopoietic stem and progenitor cells (HSPC) harbor and receive cues for self‐renewal, survival, and differentiation. Therefore, SECs are referred to as a bone marrow vascular niche (BMVN). Hematopoietic regeneration has been shown to occur only with concurrent angiogenic regeneration. However, there are still not sufficient means to identify and isolate SECs, therefore the “niche endothelial cell” remains incompletely characterized. VEGF‐receptor‐3 (VEGFR3) is expressed exclusively by the SECs, while Sca1 and Tie2 are only expressed on the VEGFR3− arteriolar endothelium. We previously demonstrated the importance of vascular recovery in hematopoietic regeneration from myelosuppression due to cytotoxic agents or whole‐body irradiation. Therefore to establish the functional importance of SECs, the mechanisms underlying BMVN regeneration were examined utilizing a 5‐fluorouracil (5‐FU) myelosuppression model of vascular damage. Injection of antibodies against murine VEGFR‐1 and ‐2 had no significant effect on hemangiogenic recovery. However, when soluble VEGFR‐1, a decoy receptor for VEGF‐A and PlGF, was injected after 5‐FU, both angiogenic remodeling and regeneration of megakaryopoiesis were delayed. In conclusion, we show that the bone marrow vasculature comprises heterogeneous compartments. SECs are distinguished from arterioles by unique immunophenotypes. Regeneration of damaged SECs is the rate‐limiting step in hematopoietic regeneration from myelosuppressive therapy. Novel, high‐efficiency VEGF‐binding drugs in combination with chemotherapeutic agents may lead to cases of prolonged cytopenia.

Tumor vasculature address book: Identification of stage-specific tumor vessel zip codes by phage display

Using in vivo phage display technology with murine tumorigenesis models, two reports have identified peptide motifs that selectively home to distinct molecular vascular targets in a tumor type- and stage-specific manner (Hoffman et al., 2003 and Joyce et al., 2003 [this issue of Cancer Cell]). By probing the surface-protein repertoire of these unique vascular beds, a pioneering draft of a molecular roadmap to decipher the heterogeneity of the vascular system in the context of carcinogenic progression has been plotted. Analysis of these phage peptides that differentially home to dysplastic or invasive tumor vasculature will lay the foundation for identifying unique functional tumor vascular-specific motifs that could potentially be applied to targeted therapeutic and imaging modalities.

35 – Molecular and Cellular Pathways Involved in the Recruitment of Proangiogenic Stem Cells from Bone Marrow Microenvironments

This chapter describes the mechanism by which angiogenic factors influence the localization of hematopoietic stem cells (HSCs) to various marrow niches, thereby facilitating their proliferation, differentiation, and recruitment to various tissues. The concept of an HSC niche was introduced by Schofield who hypothesized that HSCs are fixed within a specific microenvironment, providing conditions conducive to the survival and differentiation of HSCs. Self-renewal and differentiation of stem cells are regulated through interaction with cytokines and microenvironmental cues. Although much is known about the cytokines that modulate survival and lineage-specific differentiation of stem cells, the identities of the factors within the bone marrow microenvironments that modulate stem cell regeneration and differentiation remain unknown. Several lines of evidence suggest that most stem cells reside in the endosteal niche, where they are maintained in a quiescent state. Angiogenic factors, such as placental growth factor (PlGF) and vascular endothelial growth factor A (VEGF-A), through interaction with their tyrosine kinase receptor, VEGFR1, induce expression of active matrix metal-loproteinase-9 (MMP-9) in the marrow, mediating the release of stem cell active cytokines, such as soluble Kit-ligand. Using differential gene display technology, clusters of genes selectively expressed on organ-specific stem cells have been identified. Nonetheless, expression and function of these stem cell-specific genes may be influenced by the microenvironment (niche) in which they reside.