Bone marrow stem and progenitor cell contribution to neovasculogenesis is dependent on model system with SDF-1 as a permissive trigger.

Adult bone marrow (BM) contributes to neovascularization in some but not all settings, and reasons for these discordant results have remained unexplored. We conducted novel comparative studies in which multiple neovascularization models were established in single mice to reduce variations in experimental methodology. In different combinations, BM contribution was detected in ischemic retinas and, to a lesser extent, Lewis lung carcinoma cells, whereas B16 melanomas showed little to no BM contribution. Using this spectrum of BM contribution, we demonstrate the necessity for site-specific expression of stromal-derived factor-1alpha (SDF-1alpha) and its mobilizing effects on BM. Blocking SDF-1alpha activity with neutralizing antibodies abrogated BM-derived neovascularization in lung cancer and retinopathy. Furthermore, secondary transplantation of single hematopoietic stem cells (HSCs) showed that HSCs are a long-term source of neovasculogenesis and that CD133(+)CXCR4(+) myeloid progenitor cells directly participate in new blood vessel formation in response to SDF-1alpha. The varied BM contribution seen in different model systems is suggestive of redundant mechanisms governing postnatal neovasculogenesis and provides an explanation for contradictory results observed in the field.

The role of the donor in the repair of the marrow vascular niche following hematopoietic stem cell transplant.

Bone marrow sinusoids maintain homeostasis between developing hematopoietic cells and the circulation, and they provide niches for hematopoietic progenitors. Sinusoids are damaged by chemotherapy and radiation. Hematopoietic stem cells (HSCs) have been shown to produce endothelial progenitor cells that contribute to the repair of damaged blood vessels. Because HSCs home to the marrow during bone marrow transplant, these cells may play a role in repair of marrow sinusoids. Here, we explore the role of donor HSCs in the repair of damaged sinusoids following hematopoietic stem cell transplant. We used three methods to test this role: (a) expression of platelet endothelial cell adhesion molecule to identify endothelial progenitors and the presence of the Y chromosome to identify male donor cells in female recipients; (b) presence of the Y chromosome to identify male donor cells in female recipients, and expression of the panendothelial marker mouse endothelial cell antigen-32 to identify sinusoidal endothelium; and (c) use of Tie-2/green fluorescent protein mice as donors or recipients and presence of Dil-Ac-LDL to identify sinusoids. We found that sinusoids were predominantly host-derived posttransplant. Donor cells spread along the marrow vasculature early post-transplant in a pattern that matched stromal-derived factor-1 expression. Furthermore, these engrafting progenitors were positioned to provide physical support, as well as growth and survival signals in the form of vascular-endothelial growth factor-A. Occasionally, donor cells provide cellular "patches" in the damaged sinusoids, although this occurred at a low level compared with hematopoietic engraftment. Donor support for the repair of the marrow vascular niche may be a critical first step of hematopoietic engraftment.

Bone marrow contributes to epithelial cancers in mice and humans as developmental mimicry.

Bone marrow cells have the capacity to contribute to distant organs. We show that marrow also contributes to epithelial neoplasias of the small bowel, colon, and lung, but not the skin. In particular, epithelial neoplasias found in patients after hematopoietic cell transplantations demonstrate that human marrow incorporates into neoplasias by adopting the phenotype of the surrounding neoplastic environment. To more rigorously evaluate marrow contribution to epithelial cancer, we employed mouse models of intestinal and lung neoplasias, which revealed specifically that the hematopoietic stem cell and its progeny incorporate within cancer. Furthermore, this marrow involvement in epithelial cancer does not appear to occur by induction of stable fusion. Whereas previous claims have been made that marrow can serve as a direct source of epithelial neoplasia, our results indicate a more cautionary note, that marrow contributes to cancer as a means of developmental mimicry. Disclosure of Potential Conflicts of Interest is found at the end of this article.

Nitric oxide synthases modulate progenitor and resident endothelial cell behavior in galactosemia.

We used knockout animals of either inducible nitric oxide synthase (iNOS(/)) or endothelial NOS (eNOS(/)) to characterize the role of NOS in galactosemia, a model of diabetic retinopathy. NADH oxidase and nitrotyrosine were used as biomarkers of oxidative stress and vascular dysfunction. These animals were engrafted with hematopoietic stem cells (HSC) expressing green fluorescence protein (gfp(+)) to characterize the contribution of HSC and endothelial progenitor cells to neovascularization. Increased NADH oxidase activity and superoxide generation occurred in all galactose-fed mice. eNOS(/) mice demonstrated increased iNOS immunoreactivity in their retinal vasculature. Nitrotyrosine levels were low at baseline in the wild-type (WT) mice, eNOS(/) and iNOS(/) mice, and the galactose-fed iNOS mice and increased following galactose feeding in eNOS(/) and WT. Galactose-fed WT.gfp and iNOS(/).gfp chimeric animals had areas of perfused new vessels composed of gfp(+) cells. In contrast, galactose-fed eNOS(/).gfp mice produced copious, unbranched, nonperfused tubes. Thus, nitric oxide modulates HSC behavior and vascular phenotype in the retina. Although there is increased NADH oxidase and superoxide in galactosemic mice of all isoforms, iNOS is the source of nitric oxide responsible for peroxynitrite and nitrotyrosine formation that leads to the pathology observed in galactosemic mice.

SDF-1 is both necessary and sufficient to promote proliferative retinopathy.

Diabetic retinopathy is the leading cause of blindness in working-age adults. It is caused by oxygen starvation in the retina inducing aberrant formation of blood vessels that destroy retinal architecture. In humans, vitreal stromal cell-derived factor-1 (SDF-1) concentration increases as proliferative diabetic retinopathy progresses. Treatment of patients with triamcinolone decreases SDF-1 levels in the vitreous, with marked disease improvement. SDF-1 induces human retinal endothelial cells to increase expression of VCAM-1, a receptor for very late antigen-4 found on many hematopoietic progenitors, and reduce tight cellular junctions by reducing occludin expression. Both changes would serve to recruit hematopoietic and endothelial progenitor cells along an SDF-1 gradient. We have shown, using a murine model of proliferative adult retinopathy, that the majority of new vessels formed in response to oxygen starvation originate from hematopoietic stem cell-derived endothelial progenitor cells. We now show that the levels of SDF-1 found in patients with proliferative retinopathy induce retinopathy in our murine model. Intravitreal injection of blocking antibodies to SDF-1 prevented retinal neovascularization in our murine model, even in the presence of exogenous VEGF. Together, these data demonstrate that SDF-1 plays a major role in proliferative retinopathy and may be an ideal target for the prevention of proliferative retinopathy.

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

We 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.

Analysis of the vascular potential of hematopoietic stem cells.

The hematopoietic stem cells residing in the bone marrow have tremendous proliferative and self-renewing capacity, and until recently these cells were thought to produce only progeny of the blood lineages. We have recently demonstrated that these cells are capable of producing endothelial cells of blood vessels. This chapter will outline the methodology for producing chimeric mice through labeled bone marrow transplantation and induction of these donor cells in order to track their plasticity, or their ability to produce non-hematopoietic tissues, specifically blood vessels.

Adult human hematopoietic cells provide functional hemangioblast activity.

The 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.

PU.1 supports proliferation of immature erythroid progenitors.

Despite normal levels of erythropoiesis in PU.1(-/-) embryos, PU.1(-/-) fetal hematopoietic progenitors are unable to establish sustained erythropoiesis in the adult bone marrow. This study demonstrates that PU.1(-/-) fetal erythroid progenitors are synergistically expanded by TPO plus SCF, but not combinations of EPO plus SCF, IL-3 or GM-CSF. The EPO defect is not corrected by a constitutively active variant of EPOR. Microarray analysis identified several candidate PU.1 target genes known to affect cytokine signaling and gene regulation in the erythroid lineage. These data suggest that PU.1 plays an important role in regulating the proliferation of immature erythroid progenitors.

An overview of stem cell research and regulatory issues.

Stem cells are noted for their ability to self-renew and differentiate into a variety of cell types. Some stem cells, described as totipotent cells, have tremendous capacity to self-renew and differentiate. Embryonic stem cells have pluripotent capacity, able to form tissues of all 3 germ layers but unable to form an entire live being. Research with embryonic stem cells has enabled investigators to make substantial gains in developmental biology, therapeutic tissue engineering, and reproductive cloning. However, with these remarkable opportunities many ethical challenges arise, which are largely based on concerns for safety, efficacy, resource allocation, and methods of harvesting stem cells. Discussing the moral and legal status of the human embryo is critical to the debate on stem cell ethics. Religious perspectives and political events leading to regulation of stem cell research are presented and discussed, with special attention directed toward the use of embryonic stem cells for therapeutic and reproductive cloning. Adult stem cells were previously thought to have a restricted capacity to differentiate; however, several reports have described their plasticity potential. Furthermore, there have been close ties between the behavior of stem cells and cancer cells. True eradication of cancer will require a deeper understanding of stem cell biology. This article was written to inform medical scientists and practicing clinicians across the spectrum of medical education about the research and regulatory issues affecting the future of stem cell therapy.

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.