Gina C. Schatteman

Endothelial Cells of Hematopoietic Origin Make a Significant Contribution to Adult Blood Vessel Formation

Granulation tissue formation is an example of new tissue development in an adult. Its rich vascular network has been thought to derive via angiogenic sprouting and extension of preexisting vessels from the surrounding tissue. The possibility that circulating cells of hematopoietic origin can differentiate into vascular endothelial cells (ECs) in areas of vascular remodeling has recently gained credibility. However, no quantitative data have placed the magnitude of this contribution into a physiological perspective. We have used hematopoietic chimeras to determine that 0.2% to 1.4% of ECs in vessels in control tissues derived from hematopoietic progenitors during the 4 months after irradiation and hematopoietic recovery. By contrast, 8.3% to 11.2% of ECs in vessels that developed in sponge-induced granulation tissue during 1 month derived from circulating hematopoietic progenitors. This recruitment of circulating progenitors to newly forming vessels would be difficult to observe in standard histological studies, but it is large enough to be encouraging for attempts to manipulate this contribution for therapeutic gain.

Blood-derived angioblasts accelerate blood-flow restoration in diabetic mice

Endothelial cell progenitors, angioblasts, have been detected in the peripheral blood of adult humans, mice, and rabbits. These cells have been shown to incorporate into the endothelium of newly forming blood vessels in pathological and nonpathological conditions. Here we investigated the possibility that the CD34-expressing leukocytes (CD34(+) cells) that appear to be enriched for angioblasts could be used to accelerate the rate of blood-flow restoration in nondiabetic and diabetic mice undergoing neovascularization due to hindlimb ischemia. CD34(+) cells did not accelerate the restoration of flow in nondiabetic mice, but dramatically increased it in diabetic mice. Furthermore, CD34(+) cells derived from type 1 diabetics produced fewer differentiated endothelial cells in culture than did their type 2 diabetic- or nondiabetic-derived counterparts. In vitro experiments suggest that hyperglycemia per se does not alter the ability of angioblasts to differentiate or of angioblast-derived endothelial cells to proliferate. In contrast, hyperinsulinemia may enhance angioblast differentiation but impair angioblast-derived endothelial cell survival or proliferation. Our findings suggest that CD34(+) cells may be a useful tool for therapeutic angiogenesis in diabetics.

Hypoxia-induced paracrine regulation of vascular endothelial growth factor receptor expression.

Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF), an endothelial cell (EC)-specific mitogen, stimulates angiogenesis in vivo, particularly in ischemic regions. VEGF/VPF expression by cells of hypoxic tissues coincides with expression of its two receptors, KDR and flt-1, by ECs in the same tissues. We investigated whether hypoxia or hypoxia-dependent conditions operate in coordinating this phenomenon. Human umbilical vein and microvascular ECs were exposed to direct hypoxia or to medium conditioned (CM) by myoblasts maintained in hypoxia for 4 d. Control ECs were maintained in normoxia or normoxia-CM. Binding of 125I-VEGF to ECs was then evaluated. Hypoxic treatment of ECs had no effect on 125I-VEGF binding. However, treatment of ECs with hypoxia-CM produced a threefold increase in 125I-VEGF binding, with peak at 24 h (P < 0.001, ANOVA). Scatchard analysis disclosed that increased binding was due to a 13-fold increase in KDR receptors/cell, with no change in KDR affinity (Kd = 260 +/- 51 pM, normoxia-CM versus Kd = 281 +/- 94 pM, hypoxia-CM) and no change in EC number (35.6 +/- 5.9 x 10(3) ECs/cm2, normoxia-CM versus 33.5 +/- 5.5 x 10(3) ECs/cm2, hypoxia-CM). Similar results were obtained using CM from hypoxic smooth muscle cells. KDR upregulation was not prevented by addition to the hypoxia-CM of neutralizing antibodies against VEGF, tumor necrosis factor-alpha, transforming growth factor beta 1 or basic fibroblast growth factor. Similarly, addition of VEGF or lactic acid to the normoxia-CM had no effect on VEGF binding. We conclude that mechanism(s) initiated by hypoxia can induce KDR receptor upregulation in ECs. Hypoxic cells, normal or neoplastic, not only can produce VEGF/VPF, but can also modulate its effects via paracrine induction of VEGF/VPF receptors in ECs.

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: Role in vasculogenic mimicry

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which “mimics” embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.

CD34− Blood-Derived Human Endothelial Cell Progenitors

A subset of adult peripheral blood leukocytes functions as endothelial cell progenitors called angioblasts. They can incorporate into the vasculature in animal models of neovascularization and accelerate the restoration of blood flow to mouse ischemic limbs. Earlier reports suggested that CD34‐expressing (CD34+) but not CD34+ cell‐depleted (CD34−) leukocytes can differentiate into endothelial cells (EC) in vitro and in vivo. Recent findings suggest that CD14+ cells, which are typically CD34−, also have angioblast‐like properties in vitro. To determine the identity of angioblasts, the potential of CD34+, CD34−, CD34−CD14+, and CD34−CD14− cells to produce EC was compared. We show that a subset of monocyte (CD34−CD14+)‐enriched cells can take on an EC‐like phenotype in culture, but that the EC‐like cells also express dendritic cell antigens. These findings suggest that monocytes differentiate into macrophages, dendritic cells, or EC depending on environmental cues. The data also demonstrate that angioblasts are more abundant in the blood than previously thought. Finally, we demonstrate that CD34− and CD34−CD14+ cells incorporate into the endothelium of blood vessels in mouse ischemic limbs. However, incorporation of these cells requires co‐injection with CD34+ cells, indicating that leukocyte‐leukocyte interactions may play a critical role in governing angioblast behavior in vivo.

Angiogenesis: New insights and therapeutic potential

Angiogenesis, the formation of vessels from pre‐existing vessels, is of critical importance not only during normal growth, but also in pathological situations. In the latter, some diseases are enhanced by excessive vascular growth (e.g., tumors), whereas in others inadequate vascular growth contributes to morbidity and mortality (e.g., ischemic heart disease). Our current state of knowledge makes it clear that the cascade of angiogenic events depends on complex processes that include cell‐cell interactions, various intracellular signaling pathways, and the appropriate extracellular microenvironment. The literature regarding angiogenesis has increased exponentially during the last decade. Progress in this area is largely a consequence of advances in our understanding of angiogenic growth factor and cytokine function, in part due to the determination of their complete amino acid sequences and cloning of their genes. Other factors also play key roles in angiogenesis, including the extracellular matrix, adhesion molecules and their inhibitors, and metabolic and mechanical factors. The potential for developing therapeutic protocols has been enhanced by data from both in vitro and in vivo studies and has provided the rationale for clinic trials. Angiogenic therapy strategies include inhibition of aberrant angiogenesis, as seen in tumors or diabetes, as well as stimulation of angiogenesis in conditions of ischemia, such as ischemic heart or peripheral vascular disease. Anat Rec (New Anat) 261:126–135, 2000.

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.

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.

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 (Leprdb) mice. The viability, proliferation, and differentiation of EC progenitors were unaffected in Leprdb cell cultures under basal condition. However, Leprdb‐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, Leprdb‐derived progenitors inhibited it in nondiabetic mice. Additionally, although treatment with Leprdb‐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.

Leprdb Diabetic Mouse Bone Marrow Cells Inhibit Skin Wound Vascularization but Promote Wound Healing

Abstract —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. (Circ Res. 2003;92:1247–1253.)