Biology of Blood and Marrow Transplantation 13:43-46 (2007) 䊚 2007 American Society for Blood and Marrow Transplantation 1083-8791/07/1301-0001$32.00/0 doi:10.1016/j.bbmt.2006.10.007
Endothelial Biology and the Bone Marrow Andrew R. Cuddihy, Gay M. Crooks Division of Research Immunology/BMT, Childrens Hospital Los Angeles, Los Angeles, CA Correspondence and reprint requests: Gay M. Crooks, MB, BS, Children’s Hospital of Los Angeles, Los Angeles, CA (e-mail:
[email protected]).
KEY WORDS Bone marrow
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Endothelium
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Vascular niche
INTRODUCTION: THE NICHE CONCEPT Key aspects of adult stem cell biology include not just identifying multipotent cells, but also determining these cells’ physical location within the organism itself, the entity referred to as the stem cell “niche.” Schofield first proposed the concept of a hematopoietic stem cell (HSC) niche in 1978, suggesting that stem cells are fixed tissue cells located within a defined microenvironment and function to regulate proliferation and differentiation of the HSC [1]. Work by many groups since then has shown that the supporting “stromal” cells, in addition to providing a tether for stem cells, play a key role in helping maintain the stem cell population by providing paracrine signals that provide the checks and balances of self-renewal, differentiation, and mobilization.
OSTEOBLASTIC AND VASCULAR BONE MARROW NICHES It has been established that rather than being randomly distributed throughout the bone marrow (BM) cavity, HSCs are more specifically localized to the inner surface (endosteum) of the trabecular area of bone and around blood vessels. Within the endosteal niche, the specific cell type most closely associated with HSCs is the osteoblast. Osteoblasts are normally involved in osteogenesis, working in opposition to osteoclasts, which are involved in the resorption of bone. A transgenic mouse model in which osteoblasts are specifically ablated causes a depletion of murine HSCs in vivo. Conversely, increasing the number of osteoblasts increases the number of HSCs; in this setting, the numbers of mature myeloid and lymphoid cells do not seem to increase, suggesting that the pathways that control HSC differentiation prevent inappropriate hemato-
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Engraftment
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Transplantation
poiesis even in the face of increasing numbers of HSCs. Osteoblasts are an extremely rich source of cytokines, providing one mechanism by which they affect HSC survival. However, these cytokines are not sufficient to ensure HSC survival in vitro, demonstrating that the microenvironment provides other factors, including extracellular matrix (ECM) proteins and intercellular signalling pathways, necessary to support HSC survival in vivo. In recent years, another niche for HSCs within the BM cavity has been identified and studied: the BM vasculature. Unlike the osteoblastic niche, which is thought to be largely concerned with self-renewal, the vascular niche appears to regulate differentiation of stem and progenitor cells, as well as the ingress and egress of circulating hematopoietic cells [2]. The hematopoietic and vascular systems are closely related developmentally, being derived from a single common precursor, the hemangioblast. Vascular precursor cells are required for the development of blood islands in the yolk sac. At day 35 of embryonic life, human CD34⫹ cells can be found in the wall of the aorta. Understanding the vascular architecture of the BM provides further insight into the functional intimacy of endothelium and hematopoietic cells that extends into postnatal life. After entering the marrow, arteries divide into arterioles and capillaries, ultimately ending in sinusoids distributed around a central sinus. The sinusoids consist of a single layer of endothelium and lack the normal supporting stromal cells that surround blood vessels found in other organs. In fact, the supporting perivascular tissue comprises mainly of hematopoietic cells. The absence of a complex vascular wall explains the high level of permeability seen in BM endothelium. Similar sinusoidal endothelium is found in the spleen and liver, 2 other organs involved in hematopoiesis. 43
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The vascular niche is also the gateway into the peripheral blood circulation for HSC, progenitors, and fully differentiated blood cells. Owing to the lack of supporting stroma and pericyte coverage, some cells, such as megakaryocytes, appear to be able to transmigrate through the cytoplasm of a single endothelial cell (EC) to enter the circulation. Other cells may enter the circulation in this fashion or through fenestrations between individual ECs. Conversely, and perhaps more importantly within the auspices of BM transplantation (BMT), circulating donor HSCs and hematopoietic progenitor cells (HPCs) can enter the BM through the vasculature, engraft in the host marrow, and establish de novo hematopoiesis after transplantation. Several excellent reviews describe this process in more detail and provide specific references [2-4]. Many questions surround the role of the vascular niche in regulation of hematopoiesis and engraftment and, conversely, the role of BM-derived cells in endothelial differentiation and angiogenesis. If, under normal conditions, HSCs and HPCs, as well as fully mature myeloid and erythroid cells, are able to enter and exit the circulation relatively freely, why then is engraftment largely unsuccessful without some form of conditioning therapy? Do different conditioning regimens make the BM endothelium be more permissive to homing, extravasation, and engraftment of donor HSCs? What is the mechanism by which BM provides the pool of circulating endothelial progenitors that in turn contribute to the vasculature of other organs during steady state, tissue remodeling, and tumor angiogenesis? Studies into the endothelial biology of the BM provide essential information on the processes of engraftment and hematopoiesis and have wide implications for future developments in HSC therapy.
VASCULAR ENDOTHELIAL GROWTH FACTOR AND ANGIOGENESIS Vascular endothelial growth factor (VEGF)-A, a critical mediator of vascular growth and differentiation, is one of a family of vascular growth factors that also includes VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). The members of the VEGF family are key regulators of EC migration, survival, and cellular proliferation. VEGF acts as a mitogen for ECs that express its receptor (VEGF-R2) and causes formation of new capillaries and sprouting or branching in blood vessels. VEGF is required for angiogenesis during normal organ development in the embryo. Deletion of a single VEGF allele is embryonically lethal, and deletion of VEGF-R2 leads to defects in organ angiogenesis [5]. Dr. Donald McDonald’s laboratory at the University of Califor-
A. R. Cuddihy and G. M. Crooks
nia, San Francisco has been investigating the VEGF’s role in ECs and in angiogenesis in both normal and tumor cells. His group was among the first to characterize the age-related dependency of ECs on VEGF. VEGF-responsive vessels typically demonstrate dense branching of capillary networks, and their endothelium is fenestrated and more permeable to macromolecules. The loss of VEGF dependency is associated with increased pericyte coverage and decreased branching, fenestrations, and permeability. Baffert et al. [6] found that VEGF dependency does not persist throughout postnatal life. During development, as blood vessels become less responsive to VEGF, another growth factor, angiopoietin-1 (Ang-1), takes over vascular remodeling, increasing blood vessel diameter and decreasing EC fenestrations [7]. The receptor for Ang-1 is Tie-2, a receptor tyrosine kinase expressed on both ECs and HSCs [8]. VEGF inhibition causes an increase in EC apoptosis in mouse tracheal mucosa, regression of blood vessels, and a decrease in VEGF receptor expression [9,10]. The McDonald group was able to demonstrate that VEGF inhibitors cause similar effects in the vasculature of tumors, including decreased fenestrations and overall decreased extravasation of macromolecules [11,12].
INTERACTIONS OF BONE MARROW ENDOTHELIAL CELLS AND HEMATOPOIESIS Recent work by Rafii and others has provided some unique insight into the role of the BM vasculature in the regulation of hematopoiesis and control of the movement of hematopoietic cells in and out of the marrow. The isolation and characterization of BM ECs (BMECs) has made it possible to elucidate in vitro how BMECs and HSCs regulate and respond to each other. BMECs can support proliferation and differentiation of multipotent CD34⫹ cells in vitro, and direct contact enhances survival of HPCs [13]. These studies have also revealed that BM endothelium regulates HSC differentiation and mobilization, and also that mobilized HSCs themselves are able to act directly on BMECs. More recently, Kiel et al. [14] were able to use the signaling lymphocytic activation molecule family of cell surface markers to determine that HSCs themselves are directly associated with the sinusoidal endothelium in BM and spleen in vivo. This evidence supports a role for the endothelium in regulating various aspects of HSC maintenance and differentiation [14]. Monolayers of BMECs have also been used to build models for the migration of circulating HSCs into the BM, demonstrating that homing of cord blood CD34⫹ cells and their subsequent transendothelial migration through a BMEC monolayer de-
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Endothelial Biology and the Bone Marrow
pends on a chemokine gradient from stromal cell– derived factor and on the expression of E-selectin on BMECs [15]. Interestingly, Rafii’s group and others have shown that HSCs themselves, as well as more mature progenitor cells, such as mast cells, express VEGF and its receptor VEGF-R2, thus allowing an autocrine loop to support cell survival [16,17]. In the case of more mature progenitor cells, VEGF expression is itself driven by cytokines such as kit ligand and interleukin (IL)-3. VEGF in turn acts on the BMECs to induce changes in the EC layer, including an increase in fenestrations. This in turn facilitates transendothelial migration of progenitor cells into the sinusoidal lumen [18], from where cells then migrate to sites of angiogenesis and/or wound healing. These studies beg the question of how endothelium is affected by the various conditioning regimens used in BMT. The tissue damage caused by irradiation or cytotoxic agents may mobilize HSCs from the osteoblastic niche to the vascular niche mediated by cytokines released in response to cellular stress. A recent article has described the role of radiation in enhancing mobilization of hematopoietic and endothelial progenitors from the BM through an matrix metalloproteinase-9 – dependent mechanism that also involves VEGF and kit ligand [19]. It is perhaps possible that the increased fenestrations induced by mobilized endogenous HSCs may also make the BM more permissive for engraftment.
INTEGRINS, ENDOTHELIAL CELL PROLIFERATION, AND PROGENITOR CELL HOMING Another important interaction between endothelium and HSC is the regulation of homing of circulating HSCs and HPCs to target tissues and organs. Clues to better understanding the process of homing to BM endothelium may come from studies on tumorinduced angiogenesis. Integrins are a family of heterodimeric cell surface– expressed proteins consisting of ␣ and  subunits in various combinations. These subunits play a key role in facilitating the ability of migrating cells to attach to ligands found on the ECM. The binding of integrins to their ligands triggers signaling pathways that inhibit apoptotic pathways and thus promote cell survival and proliferation [20]. Varner’s group at the University of California, San Diego has studied the role of integrins in neovascularization during angiogenesis in tumors. Zhong et al. [21] first characterized the role of the ECM protein Del-1 in neovascularization. Del-1 was known to promote extracellular migration and angiogenesis in cooperation with the integrin ␣v3, but the biological mechanisms were unclear. Using ischemic animal
models in which purified Del-1 or a plasmid encoding Del-1 had been injected into the ischemic limb, these authors observed that Del-1 initially bound to another integrin, ␣v5, causing the transcriptional activation of several genes, including cyclin D1, Hox3, and ␣v3, which promotes extracellular proliferation and subsequent new vessel growth [21]. Dr. Varner’s laboratory has extensively studied the role of ␣41 (previously known as VLA-4) in neovascularization. Her group has shown that ␣41 is expressed in proliferating ECs rather than in quiescent ECs. During this proliferation period, ␣41 mediates a critical interaction with its ligand, vascular cell adhesion molecule (VCAM)-1, expressed on proliferating pericytes. In this way, formation of the new vessel itself and the supporting pericytes go hand in hand. Inhibiting either ␣41 or VCAM-1 through the use of specific antibodies leads to apoptosis of both ECs and pericytes and to angiogenic failure [22,23]. The ability of myeloid cells and macrophages to target tumors appears to depend on their expression of ␣41, which also promotes the ability of macrophages to invade the tumor space. Once in the tumor space, macrophages can express growth factors such as VEGF, which promote angiogenesis [23]. CD34⫹ BM-derived progenitor cells also express ␣41, which facilitates homing of HPCs to VCAM-1– expressing cells in actively remodeling neovasculature. These progenitor cells can then differentiate into the ECs that make up the vasculature [24]. Interestingly, some recent studies have demonstrated that integrins play a key role in homing of HSCs and HPCs to the BM from the circulation [25] and that VEGF inhibition down-regulates integrin expression in ECs [26]. The complex processes by which progenitor cell mobilization, growth factor signaling, transendothelial migration, homing, and maturation occur during tumor angiogenesis provides possible insight into how engraftment occurs after BMT and also how it may be enhanced. For example, localized expression of VEGF in the BM endothelium may increase fenestrations that allow HSC extravasation from the circulation into the BM space. Engineering the expression of particular integrins and/or their ECM ligands may improve homing of HSCs to the BM endothelium. Therefore, it may be possible to take cues from the study of tumor angiogenesis and neovascularization and apply them to BMT and HSC biology in general.
SUMMARY Until recently, the vascular niche of the BM has received relatively little interest in the stem cell biology community. Although it is probably less impor-
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tant for stem cell renewal than the osteoblastic niche, the vascular niche interacts with HSCs in other critical ways; it provides a tightly regulated gateway for both ingress from and egress to the circulation, and also may control stages of stem cell and progenitor differentiation. The fields of vascular and tumor biology have revealed mechanisms for angiogenesis and metastasis that can be used as a platform from which to study mobilization and engraftment of HSCs. The interactions of HSCs and endothelium should be considered in the design of more specific and targeted conditioning regimens for BMT.
REFERENCES 1. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells. 1978;4:7-25. 2. Kopp HG, Avecilla ST, Hooper AT, et al. The bone marrow vascular niche: home of HSC differentiation and mobilization. Physiology (Bethesda). 2005;20:349-356. 3. Wilson A, Trumpp A. Bone marrow haematopoietic stem cell niches. Nat Rev Immunol. 2006;6:93-106. 4. Yin T, Li L. The stem cell niches in bone. J Clin Invest. 2006;116:1195-1201. 5. Olsson AK, Dimberg A, Kreuger J, et al. VEGF receptor signalling: in control of vascular function. Nat Rev Mol Cell Biol. 2006;7:359-371. 6. Baffert F, Thurston G, Rochon-Duck M, et al. Age-related changes in vascular endothelial growth factor dependency and angiopoietin-1–induced plasticity of adult blood vessels. Circ Res. 2004;94:984-992. 7. Li LY, Barlow KD, Metheny-Barlow LJ. Angiopoietins and Tie2 in health and disease. Pediatr Endocrinol Rev. 2005;2:399408. 8. Sato A, Iwama A, Takakura N, et al. Characterization of TEK receptor tyrosine kinase and its ligands, angiopoietins, in human hematopoietic progenitor cells. Int Immunol. 1998;10: 1217-1227. 9. Baffert F, Le T, Sennino B, et al. Cellular changes in normal blood capillaries undergoing regression after inhibition of VEGF signaling. Am J Physiol Heart Circ Physiol. 2006;290: H547-H559. 10. Kamba T, Tam BY, Hashizume H, et al. VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature. Am J Physiol Heart Circ Physiol. 2006;290:H560H576. 11. Inai T, Mancuso M, Hashizume H, et al. Inhibition of vascular endothelial growth factor (VEGF) signaling in cancer causes loss of endothelial fenestrations, regression of tumor vessels, and appearance of basement membrane ghosts. Am J Pathol. 2004;165:35-52.
A. R. Cuddihy and G. M. Crooks
12. Nakahara T, Norberg SM, Shalinsky DR, et al. Effect of inhibition of vascular endothelial growth factor signaling on distribution of extravasated antibodies in tumors. Cancer Res. 2006; 66:1434-1445. 13. Rafii S, Shapiro F, Rimarachin J, et al. Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. Blood. 1994;84:10-19. 14. Kiel MJ, Yılmaz OH, Iwashita T, et al. 2005. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell. 2005;121: 1109-1121. 15. Naiyer AJ, Jo DY, Ahn J, et al. Stromal derived factor-1– induced chemokinesis of cord blood CD34(⫹) cells (long-term culture-initiating cells) through endothelial cells is mediated by E-selectin. Blood. 1999;94:4011-4019. 16. Bautz F, Rafii S, Kanz L, et al. Expression and secretion of vascular endothelial growth factor-A by cytokine-stimulated hematopoietic progenitor cells: possible role in the hematopoietic microenvironment. Exp Hematol. 2000;28:700-706. 17. Gerber HP, Malik AK, Solar GP, et al. VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism. Nature. 2002;417:954-958. 18. Mohle R, Rafii S, Moore MA. The role of endothelium in the regulation of hematopoietic stem cell migration. Stem Cells 1998;16(Suppl 1):159-165. 19. Heissig B, Rafii S, Akiyama H, et al. Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9 –mediated progenitor cell mobilization. J Exp Med. 2005;202:739-750. 20. Jin H, Varner J. Integrins: roles in cancer development and as treatment targets. Br J Cancer. 2004;90:561-565. 21. Zhong J, Eliceiri B, Stupack D, et al. Neovascularization of ischemic tissues by gene delivery of the extracellular matrix protein Del-1. J Clin Invest. 2003;112:30-41. 22. Garmy-Susini B, Jin H, Zhu Y, et al. Integrin ␣41–VCAM1–mediated adhesion between endothelial and mural cells is required for blood vessel maturation. J Clin Invest. 2005;115: 1542-1551. 23. Jin H, Su J, Garmy-Susini B, et al. Integrin ␣41 promotes monocyte trafficking and angiogenesis in tumors. Cancer Res. 2006;66:2146-2152. 24. Jin H, Aiyer A, Su J, et al. A homing mechanism for bone marrow– derived progenitor cell recruitment to the neovasculature. J Clin Invest. 2006;116:652-662. 25. Qian H, Tryggvason K, Jacobsen SE, et al. Contribution of ␣& integrins to hematopoietic stem and progenitor cell homing to bone marrow and collaboration with ␣4 integrins. Blood. 2006;107:3503-3510. 26. Yao VJ, Ozawa MG, Varner AS, et al. Antiangiogenic therapy decreases integrin expression in normalized tumor blood vessels. Cancer Res. 2006;66:2639-2649.
