Embryonic coronary vasculogenesis and angiogenesis are regulated by interactions between multiple FGFs and VEGF and are influenced by mesenchymal stem cells.

In embryonic hearts explanted on collagen gels, epicardial cells delaminate and form vascular tubes, thus providing a model for coronary tubulogenesis. Using this model, we show that fibroblast growth factors (FGFs) 1, 2, 4, 8, 9, and 18 contribute to tubulogenesis and that the availability of multiple FGFs provides the optimal tubulogenic response. Moreover, the FGF effects are vascular endothelial growth factor (VEGF) -dependent, while VEGF-induced tubulogenesis requires FGF signaling. The number of endothelial cells (ECs) is increased by all of the FGFs, while EC migration is significantly enhanced only by FGF-2 and FGF-18. Finally, addition of embryonic mesenchymal stem cells (EMSC) to the explants markedly enhances EC numbers and a 23-fold increase in stromal derived factor-1α (SDF-1α), which is FGF dependent. Both explants and EMSCs produce SDF-1α. In conclusion, coronary tubulogenesis of embryonic epicardium: (1) is responsive to many FGF family members, (2) requires both FGF and VEGFA signaling, and (3) is responsive to EMSCs.

Lin- cells mediate tissue repair by regulating MCP-1/CCL-2.

Exogenous bone marrow-derived cells (BMDCs) are promising therapeutic agents for the treatment of tissue ischemia and traumatic injury. However, until we identify the molecular mechanisms that underlie their actions, there can be no rational basis for the design of therapeutic strategies using BMDCs. The pro-healing effects of BMDCs are apparent very shortly after treatment, which suggests that they may exert their effects by the modulation of acute inflammation. We investigated this hypothesis by taking advantage of the fact that BMDCs from healthy, young, but not obese, diabetic mice stimulate vascular growth. By comparing both in vitro secretion and in vivo local induction of acute phase inflammatory cytokines by these cells, we identified monocyte chemoattractant factor 1 and tumor necrosis factor α as potential mediators of BMDC-induced tissue repair. In vivo analysis of BMDC-treated ischemic limbs and cutaneous wounds revealed that the production of monocyte chemoattractant factor 1 by exogenous and endogenous BMDCs is essential for BMDC-mediated vascular growth and tissue healing, while the inability of BMDCs to produce tumor necrosis factor α appears to play a lesser but still meaningful role. Thus, measurements of the secretion of cytokines by BMDCs may allow us to identify a priori individuals who would or would not be good candidates for BMDC-based therapies.

Diabetes and aging alter bone marrow contributions to tissue maintenance.

Bone marrow-derived cells contribute to repair of injured tissue and to the maintenance of tissue homeostasis, but the extent to which perturbations of systemic homeostasis modulate this contribution is unknown. Accordingly, hematopoietic chimeras were used to determine contributions of bone marrow-derived cells to hepatocytes, skeletal muscle myocytes, and cardiomyocytes in healthy young, healthy old, and young obese diabetic mice. Mice with multiple genomic copies of a non-expressed ß-globin/pBR322 sequence served as bone marrow donors. Because detection of the integrated sequence does not involve gene expression and many copies of the sequence are present, the sensitivity of detection is high and is not influenced by the state of cell differentiation. Our data indicate that bone marrow contributes a significant fraction of hepatocytes in old and diabetic mice, but half as many in young mice. They also show that bone marrow is a significant source of new cardiomyocytes at all ages and that this contribution is unaffected by diabetes. Additionally we found that bone marrow makes a substantial contribution to skeletal myocyte replacement that decreases with age. In summary, bone marrow-derived cells contribute significantly to normal non-hematopoietic cell replacement, a contribution that is altered by overall homeostatic state in a tissue specific manner. These data are significant if we are to understand if, and if so how, bone marrow-derived cell dysfunction contributes to tissue damage and senescence.

Biology of bone marrow-derived endothelial cell precursors.

Over the past decade, the old idea that the bone marrow contains endothelial cell precursors has become an area of renewed interest. While some still believe that there are no endothelial precursors in the blood, even among those who do, there is no consensus as to what they are or what they do. In this review, we describe the problems in identifying endothelial cells and conclude that expression of endothelial nitric oxide synthase may be the most reliable antigenic indicator of the phenotype. The evidence for two different classes of endothelial precursors is also presented. We suggest that, though there is no single endothelial cell precursor, we may be able to use these phenotypic variations to our advantage in better understanding their biology. We also discuss how a variety of genetic, epigenetic, and methodological differences can account for the seemingly contradictory findings on the physiological relevance of bone marrow-derived precursors in normal vascular maintenance and in response to injury. Data on the impact of tumor type and location on the contribution of bone marrow-derived cells to the tumor vasculature are also presented. These data provide hope that we may ultimately be able to predict those tumors in which bone marrow-derived cells will have a significant contribution and design therapies accordingly. Finally, factors that regulate bone marrow cell recruitment to and function in the endothelium are beginning to be identified, and several of these, including stromal derived factor 1, monocyte chemoattractant factor-1, and vascular endothelial growth factor are discussed.

Differential healing activities of CD34+ and CD14+ endothelial cell progenitors.

OBJECTIVE: Peripheral blood contains primitive (stem cell-like) and monocytic-like endothelial cell progenitors. Diabetes apparently converts these primitive progenitors, from a pro-angiogenic to anti-angiogenic phenotype. Monocytic progenitors seem to be less affected by diabetes, but potential pro-angiogenic activities of freshly isolated monocytic progenitors remain unexplored. We compared the ability of primitive and monocytic endothelial cell progenitors to stimulate vascular growth and healing in diabetes and investigated potential molecular mechanisms through which the cells mediate their in vivo effects. METHODS AND RESULTS: Human CD34+ primitive progenitors and CD14+ monocytic progenitors were injected locally into the ischemic limbs of diabetic mice. CD14+ cell therapy improved healing and vessel growth, although not as rapidly or effectively as CD34+ cell treatment. Western blot analysis revealed that cell therapy modulated expression of molecules in the VEGF, MCP-1, and angiopoietin pathways. CONCLUSIONS: Injection of freshly isolated circulating CD14+ cells improves healing and vascular growth indicating their potential for use in acute clinical settings. Importantly, CD14+ cells could provide a therapeutic option for people with diabetes, the function of whose CD34+ cells may be compromised. At least some progenitor-induced healing probably is mediated through increased sensitivity to VEGF and increases in MCP-1, and possibly modulation of angiopoietins.

Old bone marrow cells inhibit skin wound vascularization.

Local injection of hematopoietic stem cell-enriched cells, including mouse lin- cells, accelerates vascularization in animal injury models, apparently by release of angiogenic factors. Locally injected lin- cells from nondiabetic mice dramatically improve, but those from obese diabetic mice inhibit vascular growth in obese diabetic mouse skin wounds. Because of similarities between diabetes and aging and because autologous bone marrow-derived cells are currently being tested in clinical trials involving older patients, we investigated the effects of old lin- cells on skin wound vascularization in nondiabetic and obese diabetic mice. Treatment with old lin- bone marrow cells resulted in decreased vessel size and numerical density, leading to profoundly reduced vascular volume density in wounds of non-diabetic and diabetic mice. Our data suggest that bone marrow-derived cells may be poor candidates for therapeutic use in older patients and could actually harm them.

Obese diabetic mouse environment differentially affects primitive and monocytic endothelial cell progenitors.

Two classes of adult bone marrow-derived endothelial cell (EC) progenitors have been described, primitive hematopoietic stem cell-related cells and monocytic cells. Both differentiate into ECs and promote vascular growth in vivo but have distinct characteristics. Despite the association of obesity and type 2 diabetes with cardiovascular disease, their effects on primitive EC progenitors (prECPs) have not been examined, and the limited data on monocytic EC progenitors are conflicting. We investigated functional parameters of primitive and monocytic EC progenitors from obese diabetic (Lepr(db)) mice. The viability, proliferation, and differentiation of EC progenitors were unaffected in Lepr(db) cell cultures under basal condition. However, Lepr(db)-derived prECPs, but not monocytic EC progenitors, were less able to cope with hypoxia and oxidative stress, conditions likely present when EC progenitors are most needed. Intrinsic prECP dysfunction was also apparent in vivo. Whereas injection of nondiabetic prECPs promoted vascularization of skin wounds, Lepr(db)-derived progenitors inhibited it in nondiabetic mice. Additionally, although treatment with Lepr(db)-derived prECPs did not significantly reduce blood flow restoration to ischemic limbs, it resulted in increased tissue necrosis and autoamputation. Thus, type 2 diabetes coupled with obesity seems to induce intrinsic EC progenitor dysfunction that is exacerbated by stress. prECPs are more affected than monocytic progenitors, exhibiting a reduced ability to survive or proliferate. The proangiogenic phenotype of prECPs also seems to convert to an antiangiogenic phenotype in obese diabetic mice. These data suggest that therapies involving prECPs or stem-like cells in diabetic patients may be inadvisable at this time.

Non-classical mechanisms of heart repair.

Vasculogenesis was long considered to be a mechanism of blood vessel formation limited to the embryo, and not used in adult vascular repair. Similarly, myocardial proliferation was generally thought to cease in the adult. Yet there is an extensive older literature suggesting that blood might be a source of adult angioblasts and somewhat more recent data indicate that cardiomyocytes may proliferate. Only recently has the significance of this literature been recognized and the findings more closely investigated. It is now clear that bone marrow-cells move into the blood as angioblasts and contribute to vascular repair and neovascularization in the adult. Bone marrow cells can also move into the damaged myocardium, proliferate, and repair the damaged heart. This review will discuss data relevant to these non-classical mechanisms of heart repair, principally vascular repair, and some of the scientific history that led to modern thinking. Though the literature in this field is replete with examples of conclusions not warranted by the data, despite oft-exaggerated claims, the data do show that these non-classical mechanisms can contribute to cardiovascular maintenance and repair. What remains to be deciphered is the physiological importance of the mechanisms.

Adult bone marrow-derived hemangioblasts, endothelial cell progenitors, and EPCs.

Long before their existence was proven, work with blood islands pointed to the existence of hemangioblasts in the embryo, and it was widely accepted that such cells existed. In contrast, though evidence for adult hemangioblasts appeared at least as early as 1932, until quite recently, it was commonly assumed that there were no adult hemangioblasts. Over the past decade, these views have changed, and it is now generally accepted that a subset of bone marrow cells or their progeny can and do function as adult hemangioblasts. This chapter will examine the basic biology of bone marrow-derived hemangioblasts and endothelial cell progenitors (angioblasts) and the relationship of these adult cells to their embryonic counterparts. Efforts to define the endothelial cell progenitor phenotype will also be discussed, though to date, there is no consensus on the definitive adult phenotype, probably because there are multiple phenotypes and because the cells are plastic. Also examined are the putative roles of bone marrow-derived cells in vascular homeostasis and repair, including both their ability to differentiate and contribute directly to vascular repair, as well as to promote vascular growth by secreting pro-angiogenic factors. Finally, the use of bone marrow cells as therapeutic tools will be addressed.

Epidermal cells accelerate the restoration of the blood flow in diabetic ischemic limbs.

Epidermal progenitor cells (EpPCs) were long thought to be unipotent, giving rise only to other keratinocytes but recent studies question this assumption. Here, we investigated whether mouse EpPCs can adopt other antigenic and functional phenotypes. To test this, we injected freshly isolated and cultured EpPCs and transient amplifying cells into diabetic and non-diabetic mouse ischemic hindlimb and followed the cells' fate and the recovery of the ischemic limb blood flow over time. Both freshly isolated and cultured EpPCs and transient amplifying cells were incorporated into the vasculature of the ischemic limb 2 and 5 weeks post-injection, and some expressed endothelial cell but not keratinocyte antigens. Additionally, in the non-diabetic animals, first transient amplifying cells and then EpPCs accelerated the restoration of the blood flow. By contrast, in diabetic animals, only injected EpPCs or unsorted epidermal cells accelerated the restoration of the blood flow. These data indicate that epidermal cells can adopt non-skin phenotypes and functions, and that this apparent pluripotency is not lost by differentiation of EpPCs into transient amplifying cells. They also suggest that epidermal cell therapy might be of therapeutic value in the treatment of diabetic ischemia. Finally, because epidermal cells are readily accessible and expandable, they appear to be ideally suited for use as a non-viral gene delivery therapy.

Mechanical, cellular, and molecular factors interact to modulate circulating endothelial cell progenitors.

It appears that there are two classes of human circulating endothelial cell (EC) progenitors, CD34+ and CD34-CD14+ cells. Attention has focused on CD34+ cells, yet CD34-CD14+ monocytic cells are far more abundant and may represent the most common class of circulating EC progenitor. Little is known about molecular or physiological factors that regulate putative CD34-CD14+ EC progenitor function, although factors secreted by other blood and cardiovascular cells to which they are exposed probably affect their behavior. Hypoxia and stretch are two important physiological stimuli known to trigger growth factors in cardiovascular cells and accordingly may modulate EC progenitors. To investigate the impact of these environmental parameters on EC progenitors, EC production in CD34-CD14+ cultures was evaluated. Our data indicate that neither stretch nor hypoxia alters EC production by EC progenitors directly but do so indirectly through their effects on cardiovascular cells. Conditioned media (CM) from coronary artery smooth muscle cells inhibit EC production in culture, and this inhibition is stronger if the coronary smooth muscle cells have been subjected to cyclic stretch. In contrast, cardiomyocyte CM increases EC cell number, an effect that is potentiated if the myocytes have been subjected to hypoxia. Significantly, EC progenitor responses to CM are altered by the presence of CD34-CD14- peripheral blood mononuclear cells (PBMCs). Moreover, CD34-CD14- PBMCs attenuate EC progenitor responsiveness to the angiogenic factors basic fibroblast growth factor (FGF-2), vascular endothelial cell growth factor-A165, and erythropoietin while inducing EC progenitor death in the presence of transforming growth factor-beta1 in vitro

Hemangioblasts, angioblasts, and adult endothelial cell progenitors.

After decades of speculation, proof of embryonic hemangioblasts finally emerged a few years ago. Surprisingly, at about the same time, evidence for adult hemangioblasts began to appear, and recent single-cell bone marrow transplants have confirmed their existence. Embryonic and adult hemangioblasts appear to share antigenic determinants, including CD34, ACC133, and VEGFR2, although their phenotype may be plastic. They also respond to similar factors, prominent among them vascular endothelial growth factor (VEGF). In the adult, hemangioblasts reside principally in the bone marrow, although they may subsequently leave that niche to reside in nonhematopoietic tissues. A number of studies indicate that these cells or their progeny may be a significant source of endothelial cells in adult pathologic and nonpathologic vascularization, and may participate in vascular repair. In addition to hemangioblasts, a more differentiated source of endothelial cell progenitors may be present in the blood, namely, monocytes or monocytic-like cells. The relative importance of the two cell types in vivo is not clear, though endothelial cells derived from the two sources may not be identical, and hemangioblasts seem to provide a stimulus for differentiation of the monocytes. Treatment with exogenous bone marrow-derived cells can promote neovascularization, accelerate restoration of blood flow to ischemic tissues, and improve cardiac function after infarct. Hence, there is great hope that either alone, in combination with angiogenic factors, or as gene therapy vectors, we can harness these cells to treat ischemic and vascular diseases in the relatively near future.

Leprdb diabetic mouse bone marrow cells inhibit skin wound vascularization but promote wound healing.

Bone marrow stem cells participate in tissue repair processes and may have roles in skin wound repair. Diabetes is characterized by delayed and poor wound healing, and type 1 diabetes seems to lead to stem cell dysfunction. Hence, stem cell dysfunction could contribute to poor healing, and stem cell-based therapies may be efficacious in diabetic wounds. We investigated the potential of exogenous stem cells to promote skin healing and possible effects of type 2 diabetes on stem cell function. Mouse bone marrow cells from nondiabetic and diabetic mice were enriched for putative stem cells and injected under skin wounds of nondiabetic or type 2 diabetic Leprdb mice. Using histology and morphometry, vascularization and healing in treated and untreated mice were analyzed. We anticipated a correlation between improved wound healing and vascularization, because therapies that increase tissue vascularization tend to enhance wound healing. Our data indicate that exogenous nondiabetic bone marrow-derived cells increase vascularization and improve wound healing in Leprdb mice but have little effect on nondiabetic controls. In contrast, Leprdb-derived marrow cells inhibit vascularization but promote wound healing in Leprdb mice. Thus, adult stem cell function may be impaired by type 2 diabetes; the ability to promote vascularization and wound healing are distinct functions of bone marrow cells; and neovascularization and wound healing may not be tightly coupled. Additionally, we observed little incorporation of injected cells into wound structures, suggesting that improved healing is mediated through mechanisms other than direct differentiation and incorporation of the cells.

Expression of multiple molecular phenotypes by aggressive melanoma tumor cells: role in vasculogenic mimicry.

Cutaneous melanoma has been increasing at an alarming rate over the past two decades, however, there are no acceptable histopathological markers that classify various stages of melanoma progression. Recently, the molecular analysis of cancer has contributed significantly to our understanding of the cellular and molecular underpinnings of tumor progression. The data summarized in this review describe the molecular signature of aggressive cutaneous melanoma cells as that of multiple phenotypes which may be similar to a pluripotent, embryonic-like phenotype. An example of the plasticity of this phenotype is demonstrated by the ability of aggressive melanoma cells to engage in vasculogenic mimicry and neovascularization. A review of the current data demonstrating important cellular and molecular determinants of human melanoma vasculogenic mimicry is presented. These findings should stimulate additional studies to address the biological relevance of the multiple molecular phenotypes expressed by aggressive melanoma cells which may lead to the development of new diagnostic markers and therapeutic targets for clinical intervention.

Transendothelial function of human metastatic melanoma cells: role of the microenvironment in cell-fate determination.

On the basis of the ability of aggressive melanoma cells to participate in vasculogenic mimicry, particularly their expression of endothelial-associated genes, we examined the plasticity of human metastatic cutaneous melanoma cells with respect to vascular function. Fluorescently labeled metastatic melanoma cells were challenged to an ischemic microenvironment surgically induced in the hind limbs of nude mice. The data reveal the capability of these melanoma cells to express cell-fate determination molecules, normally expressed during embryonic vasculogenesis, and to participate in the neovascularization of circulation-deficient muscle. These results demonstrate the powerful influence of the microenvironment on the transendothelial differentiation of aggressive melanoma cells, and may provide new perspectives on tumor cell plasticity that could be exploited for novel therapeutic strategies.