Hemangioblast Activity Research Articles

Contribution of the Local RAS to Hematopoietic Function: A Novel Therapeutic Target

The renin-angiotensin system (RAS) has long been a known endocrine system that is involved in regulation of blood pressure and fluid balance. Over the last two decades, evidence has accrued that shows that there are local RAS that can affect cellular activity, tissue injury, and tissue regeneration. There are locally active ligand peptides, mediators, receptors, and signaling pathways of the RAS in the bone marrow (BM). This system is fundamentally involved and controls the essential steps of primitive and definitive blood-cell production. Hematopoiesis, erythropoiesis, myelopoiesis, thrombopoiesis, formation of monocytic and lymphocytic lineages, as well as stromal elements are regulated by the local BM RAS. The expression of a local BM RAS has been shown in very early, primitive embryonic hematopoiesis. Angiotensin-converting enzyme (ACE-1, CD143) is expressed on the surface of hemangioblasts and isolation of the CD143 positive cells allows for recovery of all hemangioblast activity, the first endothelial and hematopoietic cells, forming the marrow cavity in the embryo. CD143 expression also marks long-term blood-forming CD34+ BM cells. Expression of receptors of the RAS is modified in the BM with cellular maturation and by injury. Ligation of the receptors of the RAS has been shown to modify the status of the BM resulting in accelerated hematopoiesis after injury. The aim of the present review is to outline the known functions of the local BM RAS within the context of primitive and definitive hematopoiesis as well as modification of BM recovery by administration of exogenous ligands of the RAS. Targeting the actions of local RAS molecules could represent a valuable therapeutic option for the management of BM recovery after injury as well as neoplastic disorders.

Acute Myeloid Leukemia Cells Generate Leukemic Endothelial Cells with Leukemogenic Potential: Blood Vessels As Sanctuaries for Leukemia Relapse

Abstract 241Human hematopoietic stem cells (HSCs) possess hemangioblast activity, which is defined as the ability to generate both blood and endothelium. Whether malignant HSC counterparts such as acute myeloid leukemia (AML) also display this bipotentiality remains to be defined. To test the hemangioblast potential of AML cells we first cultured primary human AML bone marrow in conditions established by Yoder and colleagues that support the growth of functional endothelial cell (EC) progenitors. AML cultured in endothelial colony forming cell (ECFC) media generated endothelial progenitor cell colonies that showed uptake of acetylated LDL and expressed several EC surface proteins, including CD105, CD146, UEA-1 and CD144. Importantly, ECFCs derived from AML bone marrow no longer expressed CD45 or myeloid surface proteins such as CD14. When placed in Matrigel, these AML derived ECFC generated capillary-like, tubular structures. Moreover, these ECFCs contained cytogenetic mutations associated with their parental leukemias. Thus, under the appropriate conditions, AML bone marrow cells can generate cells with an endothelial-like phenotype and harboring leukemia specific mutations that will be referred to as ‘L-ECFC.’ To functionally define leukemia hemangioblast activity, a xenograft model of AML was employed. Sublethally irradiated NOD/scid/IL2Rγ−/− (NSG) mice were transplanted with primary human AML cells and then sacrificed at 8–36 weeks after transplant. Significant accumulations of human AML cells were found in perivascular regions of the liver. Both tight coupling and bona fide cell fusion between AML and ECs was observed. AML derived EC that were integrated into portal vein endothelium showed induction of CD105 expression Follow-up AML xenotransplant experiments with BrdU labeling revealed almost four-fold fewer (6%) of the AML cells incorporated within blood vessels were BrdU+, as compared to AML cells not integrated in blood vessels (22%) (P=0.01). These results suggest that AML cell incorporation within the endovascular lining induces cell quiescence. Thus, leukemia-integrated ECs may be less susceptible to cell cycle active agents like cytarabine. Results from these experiments also raised the possibility that AML cells adopting an endothelial-like phenotype may serve as a reservoir for leukemic relapse. To test this hypothesis, we injected CD105+CD45- L-ECFC derived from AML patients into NSG mice. These L-ECFC generated colonies of human CD105+CD45- within spleens and bone marrow of recipient mice. We also found a distinct population of human CD45+CD19- cells comprising 5–10% of bone marrow cells. Leukemia-derived cells were confirmed by detection of cytogenetically mutant cells consistent with the parent leukemia (e.g., MLL duplications). In conclusion, this study demonstrates that AML cells can functionally generate leukemic ECs that become quiescent after incorporation in blood vessel networks and can re-emerge with a leukemogenic phenotype. Together, our results raise the strong possibility that AML cells exhibit functional hemangioblast activity and that vascular endothelium may serve as a clinically important sanctuary for occult leukemia. Our data also support endothelial cell targeting strategies as a means to eradicate AML. Disclosures:No relevant conflicts of interest to declare.

Stepwise Development of Hematopoietic Stem Cells from Embryonic Stem Cells

The cellular ontogeny of hematopoietic stem cells (HSCs) remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC) differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs) as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit+CD41+CD45− phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. We propose an HSC developmental model in which pre-HSCs and embryonic HSCs sequentially give rise to adult types of HSCs in a stepwise manner.

Activation of Bone Marrow-Derived Microglia Promotes Photoreceptor Survival in Inherited Retinal Degeneration

The role of microglia in neurodegeneration is controversial, although microglial activation in the retina has been shown to provide an early response against infection, injury, ischemia, and degeneration. Here we show that endogenous bone marrow (BM)-derived microglia play a protective role in vascular and neural degeneration in the retinitis pigmentosa model of inherited retinal degeneration. BM-derived cells were recruited to the degenerating retina where they differentiated into microglia and subsequently localized to the degenerating vessels and neurons. Inhibition of stromal-derived factor-1 in the retina reduced the number of BM-derived microglia and accelerated the rate of neurovascular degeneration. Systemic depletion of myeloid progenitors also accelerated the degenerative process. Conversely, activation of BM-derived myeloid progenitors by systemic administration of both granulocyte colony-stimulating factor and erythropoietin resulted in the deceleration of retinal degeneration and the promotion of cone cell survival. These data indicate that BM-derived microglia may play a protective role in retinitis pigmentosa. Functional activation of BM-derived myeloid progenitors by cytokine therapy may provide a novel strategy for the treatment of inherited retinal degeneration and other neurodegenerative diseases, regardless of the underlying genetic defect.

The Hematopoietic Stem Cell Is a Source of Endothelial Cells in Cancer Vasculogenesis and Depends on SDF-1.

Cancer growth and metastasis depend upon a rich supply of oxygen and nutrients from blood vessels. Previously we found that the hematopoietic stem cell (HSC) is capable of contributing to vasculogenesis in settings of physiologic repair, and given the relative hypoxia within tumor microenvironments, we subsequently hypothesized that the HSC also acts as a pathologic hemangioblast. First, we injected cancers (lung, pancreatic, melanoma, lymphoma) into cohorts of C57BL/6 mice which were previously transplanted green fluorescent protein (GFP)-tagged whole bone marrow. All cancer specimens demonstrated blood vessels with marrow-derived endothelial cells. Approximately 25% of tumor vessels contained marrow-derived endothelial cells as demonstrated by GFP, CD31 and vWF expression. We further questioned whether the tumor neovasculogenesis is from a clonal, self-renewing HSC. Single HSC transplanted mice were used as donors for secondary transplant mice in order to select for the true HSC. Lung cancers grown in recipients of single cell and serially transplanted hematopoietic stem cells (n=9) demonstrate clonal, donor-derived endothelial cells in 5% of tumor vasculature, matching hematopoietic engraftment. Thus, our results indicate that the self-renewing, clonal adult HSC exhibits pathologic hemangioblast activity. We further hypothesized that factors that affect leukocyte trafficking likely affect the pathologic hemangioblast activity of HSC. To test this hypothesis, we made slight modifications to our transplant and tumor inoculation model by administering GCSF and SCF to mobilize marrow derived EPC. Over the ensuing 14 days, the tumors in the cytokine treated group grew at a much faster rate and to a much larger size than tumors in the control mice. After 14 days of cytokine treatment and tumor growth, microvessel density was not different between cytokine treated mice (n=4) and control mice (n=4); however, marrow-derived tumor vasculogenesis was markedly elevated in the cytokine treated compared to controls (63% vs. 26%). Given that the SDF1/CXCR4 axis is pivotal for marrow cell homing and migration, we hypothesized that blocking this axis would block marrow-derived blood vessels in cancer. To test this hypothesis, we transplanted green fluorescent protein (GFP) marrow into wild-type C57BL/6 mice and then inoculated these mice with lung cancer. An experimental cohort of mice (n=4) received intra-tumoral anti-SDF1. A control cohort included mice receiving intra-tumoral PBS (n=4). Over the ensuing 14 days, tumors in the anti-SDF1 treated group grew at a much slower rate and to a much smaller size, if at all. After 14 days of injections and tumor growth, microvessel density was markedly decreased in the anti-SDF1 cohort compared to the control cohort. Moreover, marrow-derived tumor vasculogenesis was decreased in the anti-SDF1 treated tumors compared to controls (18% of vessels with marrow-derived endothelial cells vs. 26%, respectively). Lung cancer cells grew normally in vitro in the presence of anti-SDF-1. In conclusion, the results of our studies indicate that the HSC contributes to blood vessels within a variety of cancers and that strategies targeting HSC and EPC mobilization and homing potentially represent excellent ant-neoplastic opportunities. Indeed, perturbing the SDF1/CXCR4 axis inhibits marrow-derived tumor vasculogenesis. These studies serve as the preclinical basis for anti-SDF-1 antibody therapy as an adjunct to anti-cancer therapy.

Tumor Vasculogenesis Can Be Derived from the Hematopoietic Stem Cell.

Blood vessel development is essential to cancer growth and metastasis. Based on our recent findings that the hematopoietic stem cell (HSC) provides functional hemangioblast activity in repairing the ischemic retina, we questioned if the HSC also acts as a pathologic hemangioblast - contributing to tumor vasculogenesis. Using a transplant model of green fluorescent protein (GFP) marrow into wild-type C57BL/6 mice, we injected lung cancer (LLC) and allowed the tumor to grow for 14 days. All tumor specimens demonstrated tumor vessels with marrow-derived endothelial cells. Approximately 25% of tumor vessels contained marrow-derived endothelial cells as demonstrated by GFP, CD31 and vWF expression. Confocal microscopy was used to identify true marrow-derived endothelial cells lining the vascular lumen versus marrow-derived pericytes juxtaposed to endothelial cells. We further questioned whether the tumor neovasculogenesis is from a clonal, self-renewing HSC. Lung cancers grown in recipients of single cell and serially transplanted hematopoietic stem cells (n=9) demonstrate clonal, donor-derived endothelial cells in 5% of tumor vasculature, matching hematopoietic engraftment. Our results indicate that the self-renewing, clonal adult hematopoietic stem cell exhibits pathologic hemangioblast activity, capable of producing both blood and blood vessels within tumors. Given that the HSC and its EPC progeny can contribute to tumor vasculogenesis, we further hypothesized that factors that affect leukocyte trafficking likely affect the pathologic hemangioblast activity of HSC. Thus, we made slight modifications to our transplant and tumor inoculation model by administering GCSF and SCF to mobilize marrow derived EPC. Over the ensuing 14 days, the tumors in the cytokine treated group grew at a much faster rate and to a much larger size than tumors in the control mice. After 14 days of cytokine treatment and tumor growth, microvessel density was not different between cytokine treated mice (n=4) and control mice (n=4); however, marrow-derived tumor vasculogenesis was markedly elevated in the cytokine treated compared to controls (63% vs. 26%). In conclusion, the HSC contributes to blood vessels within lung cancer and strategies targeting HSC/EPC mobilization (such as during the recovery phase of chemotherapy) potentially represent excellent ant-neoplastic opportunities.

Human Myeloid Leukemia Xenotransplant Model Demonstrates Leukemia Blood Vessels from Both Human and Mouse Origin.

Leukemia stem cell activity has been extensively studied and has been approached mainly in terms of self-renewal within the hematopoietic compartment. However, given the recent findings that the adult hematopoietic stem cell can provide hemangioblast activity - giving rise to both blood and blood vessels - we questioned whether leukemia also exhibits hemangioblast activity. To test this hypothesis we established a xenotransplant model whereby K562 human myeloid leukemia cells suspended in Matrigel are injected into sublethally irradiated NOD/scid mice. Leukemia loaded Matrigel plugs reliably grew in size in all (n=6) immunocompromised animals. After four weeks of leukemia growth the Matrigel plugs were excised and blood vessels evaluated. Out of six animals, two animals demonstrated human endothelial cells within the Matrigel plug, confirmed by LAMP-1 staining. Interestingly, six out of six animals demonstrated mouse endothelial cells within the Matrigel. This in vivo model demonstrates that human myeloid leukemia can provide hemangioblast activity. Moreover, it appears that within this Matrigel xenotransplant model, host blood vessels are also recruited. The significance of these results highlights the importance in recognizing the hemangioblast potential of leukemia stem cells. Ultimately, complete eradication of leukemia may depend on targeting leukemia-derived blood vessels in addition to cytotoxic therapy directed at the hematopoietic compartment.

Marrow Contributes to Kidney Vasculogenesis in Humans.

The hematopoietic stem cell (HSC) is capable of contributing to vasculogenesis in response to ischemic injury. We have previously demonstrated that adult hemangioblast activity also exists within the capabilities of human hematopoietic stem/progenitor cells (HSC/HPC). However, we question the clinical relevance of adult hemangioblast activity. Thus, to address this question, we sought to analyze the marrow contribution to blood vessels within damaged vascular beds. Given the nephrotoxic side effects of chemotherapy and hematopoietic cell transplantation regimens, we identified kidney tissues from women (n=4) who received hematopoietic cell transplantations from male donors. Kidney tissues were stained for CD31, CD45, and XY FISH. One hundred forty vascular fields were analyzed. Fields were divided into three zones based on physiologic functions: glomerular, arteriolar (afferent & efferent), and medullary. In ever zone we identified male/marrow-derived endothelial cells contributing to kidney vasculature. However, each zone differed as to the degree of male/marrow-derived vascular contribution. In the glomerular regions, 36% of glomeruli contained one or more marrow-derived endothelial cells, as determined by orientation at the lumen edge, CD31 surface expression, and Y chromosome staining. In contrast, only 4% of arterioles contained male/marrow-derived endothelial cells. In the medulla, 9% of blood vessels contained male/marrow-derived blood vessels. The median number of marrow-derived endothelial cells in all regions was 1 male cell per blood vessel in field. In conclusion, human marrow can contribute modestly to kidney vasculature, especially in glomeruli. These results expose a potential strategy of kidney vessel repair by illuminating marrow-derived vasculogenesis within the kidney.

Blocking the SDF1/CXCR4 Axis Inhibits Marrow Derived Vasculogenesis in Cancer.

Cancer growth and metastasis depend upon a rich supply of oxygen and nutrients from blood vessels. Our previous work found that the hematopoietic stem cell (HSC) is capable of contributing to vasculogenesis in settings of physiologic repair (i.e., retinal ischemia) as well as tumor vasculogenesis. Given that the HSC and its EPC progeny can contribute to tumor vasculogenesis, we hypothesized that factors that affect leukocyte trafficking likely affect the pathologic hemangioblast activity of HSC. The SDF1/CXCR4 axis is pivotal for marrow cell homing and migration, thus was targeted in these set of experiments. To test this hypothesis, we transplanted green fluorescent protein (GFP) marrow into wild-type C57BL/6 mice and then inoculated these mice with lung cancer (LLC). An experimental cohort of mice (n=4) received intra-tumoral anti-SDF1. A control cohort included mice receiving intra-tumoral PBS (n=4). Over the ensuing 14 days, tumors in the anti-SDF1 treated group grew at a much slower rate and to a much smaller size, if at all. After 14 days of injections and tumor growth, microvessel density was markedly decreased in the anti-SDF1 cohort compared to the control cohort. Moreover, marrow-derived tumor vasculogenesis was decreased in the anti-SDF1 treated tumors compared to controls (18% of vessels with marrow-derived endothelial cells vs. 26%). As a complementary approach, we used the same transplant and tumor model, but administered anti-CXCR4 in the experimental group (n=4), compared to controls receiving PBS injections (n=4). The tumors in the anti-CXCR4 group had the same growth kinetics as the control; however, had lower tumor microvessel density and markedly decreased marrow-derived tumor vasculogenesis compared to controls (12% of vessels with marrow-derived endothelial cells vs. 26%). In conclusion, perturbing the SDF1/CXCR4 axis inhibits marrow-derived tumor vasculogenesis and may represent an excellent target for future anti-neoplastic therapy.

Rhesus EB cultures exhibit functional hemangioblast activity during hematopoietic differentiation

Journal of the American College of Surgeons 203(3):p S41-S42, September 2006. | DOI: 10.1016/j.jamcollsurg.2006.05.105

A Regenerative Role for Bone Marrow Following Experimental Colitis: Contribution to Neovasculogenesis and Myofibroblasts

Bone marrow (BM) cells form differentiated adult lineages within nonhematopoietic tissues, with a heightened propensity with increasing regenerative pressure dictated by disease. We have previously shown that BM cells engraft into the gut and contribute substantially to the subepithelial intestinal myofibroblast population in the lamina propria. To investigate the reparative capacity of BM in inflammatory bowel disease (IBD), a well-established model of experimental colitis was used. Lethally irradiated female mice were rescued by a BM transplant from male donors. Colitis was induced 6 weeks posttransplantation by injection of trinitrobenzene sulfonic acid (TNBS), and tissues were analyzed 1-14 days later. Donor-derived cells were detected by in situ hybridization using a Y chromosome-specific probe, and their phenotype was determined by immunohistochemistry. TNBS-induced colitis was manifest as patchy lesions that increased in severity between days 1 and 8, and the mucosa gradually regenerated between days 8 and 14. The contribution of BM to intestinal myofibroblasts was significantly increased in regions of colitis compared with noninflamed regions. Furthermore, BM-derived endothelial cells, pericytes, and vascular smooth muscle cells were frequently interspersed throughout blood vessels, suggesting that these cells facilitate angiogenesis in tissue repair, substantiated by a significant increase in the incidence of BM-derived vascular smooth muscle cells in colitic compared with noninflamed regions. Blood vessels formed entirely from BM-derived cells were also seen, suggesting a role for BM in neovasculogenesis. Our data show that BM contributes to multiple intestinal cell lineages in colitis, with an important function in tissue regeneration and vasculogenesis after injury.

The nitric oxide pathway modulates hemangioblast activity of adult hematopoietic stem cells

AbstractWe have previously established a model inducing hematopoietic stem cell (HSC) production of circulating endothelial progenitor cells (EPCs) to revascularize ischemic injury in adult mouse retina. The unique vascular environment of the retina results in new blood vessel formation primarily from HSC-derived EPCs. Using mice deficient (–/–) in inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS), we show that vessel phenotype resulting from hemangioblast activity can be altered by modulation of the NO/NOS pathway. iNOS–/– or eNOS–/– animals were engrafted with wild-type (WT) HSCs expressing green fluorescence protein (gfp+) and subjected to our adult retinal ischemia model. WT hemangioblast activity in adult iNOS–/– recipients resulted in the formation of highly branched blood vessels of donor origin, which were readily perfused indicating functionality. In contrast, eNOS–/– recipients produced relatively unbranched blood vessels with significant donor contribution that were difficult to perfuse, indicating poor functionality. Furthermore, eNOS–/– chimeras had extensive gfp+ HSC contribution throughout their vasculature without additional injury. This neovascularization, via EPCs derived from the transplanted HSCs, reveals that the NO pathway can modulate EPC activity and plays a critical role in both blood vessel formation in response to injury and normal endothelial cell maintenance.

Adult human hematopoietic cells provide functional hemangioblast activity

AbstractThe murine adult hematopoietic stem cell is able to function as a hemangioblast, contributing both to blood reconstitution and to blood vessel repair in response to ischemic injury. We developed a novel mouse xenotransplantation model of retinal neovascularization to test human hematopoietic cell plasticity. Immunocompromised nonobese diabetic (NOD)/scid mice underwent myeloablative conditioning and transplantation with human CD34+ umbilical cord blood. After multilineage reconstitution was established, retinal ischemia was induced to promote neovascularization. Our results demonstrate human retinal neovascularization, thus revealing the functional hemangioblast activity of human hematopoietic cells.

The role of adult bone marrow-derived stem cells in choroidal neovascularization.

Age-related macular degeneration (ARMD) is the primary cause of blindness in people aged of 50 years or more. The wet form leads to severe loss of central vision. Recent evidence supports that adult hematopoietic stem cells (HSCs) contribute to preretinal neovascularization. In the current study, it was determined whether HSCs, by producing both blood and blood vessels, provide functional hemangioblast activity during choroidal neovascularization (CNV) in mice. Gfp chimeric mice were developed by bone marrow ablation of C57BL/6J mice and reconstitution with donor tissue from gfp(+/+) transgenic mice. Gfp chimeric mice underwent laser rupture of Bruch's membrane and were killed and eyes enucleated at 1, 2, 3, and 4 weeks after laser injury. CNV was examined by confocal microscopy of retinal flatmounts. Because endothelial progenitor cells (EPCs) derive from HSCs, immunocytochemistry was used to quantify relative the EPC contribution to CNV. Laser injury alone was sufficient to induce stem cell recruitment and subsequent CNV. Gfp+ cells formed part of the functional vasculature in the choroid as early as 1 week after injury and were present for the duration of the study. The relative EPC contribution to CNV remained fairly constant throughout the study and constituted almost 50% of the total vasculature. Adult stem cells are recruited to the choroid in a model of CNV, where they contribute to forming aberrant new vessels. This observation suggests that targeting stem cell recruitment to the eye may offer a novel therapeutic strategy for ARMD.

Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Grant MB, May WS, Caballero S, Brown GAJ, Guthrie SM, Mames RN, Byrne BJ, Vaught T, Spoerri PE, Peck AB, Scott EW. Nat Med 2002;6:607-612.

Bone marrow cells or hematopoetic stem cells were obtained from green fluorescent protein transgenic mice and introduced via intravenous injection into irradiated C57BI/6 mice. This chimeric C57BL/6 mice then underwent argon green laser application to selected venous sites next to the optic nerve to produce venous occlusions. The mice were sacrificed 3 weeks later and retinal flat-mounts were examined by confocal microscopy and immunocytochemistry. Results showed clonal differentiation of the hematopoietic stem cells to all hematopoietic cell lineages as well as endothelial cells that revascularized the adult murine retina. There was also recruitment of endothelial precursors to sites of ischemic injury.—Hans E. Grossniklaus

Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization.

Adults maintain a reservoir of hematopoietic stem cells that can enter the circulation to reach organs in need of regeneration. We developed a novel model of retinal neovascularization in adult mice to examine the role of hematopoietic stem cells in revascularizing ischemic retinas. Adult mice were durably engrafted with hematopoietic stem cells isolated from transgenic mice expressing green fluorescent protein. We performed serial long-term transplants, to ensure activity arose from self-renewing stem cells, and single hematopoietic stem-cell transplants to show clonality. After durable hematopoietic engraftment was established, retinal ischemia was induced to promote neovascularization. Our results indicate that self-renewing adult hematopoietic stem cells have functional hemangioblast activity, that is, they can clonally differentiate into all hematopoietic cell lineages as well as endothelial cells that revascularize adult retina. We also show that recruitment of endothelial precursors to sites of ischemic injury has a significant role in neovascularization.