EPCs and pathological angiogenesis: when good cells go bad

Abstract

Bone-marrow-derived endothelial progenitor cells (EPCs) contribute to angiogenesis-mediated pathological neovascularization, and recent studies have begun to recognize the biological significance of this contribution. This review will discuss the ability of EPCs to contribute to neovascularization in both physiological and pathological conditions. Circulating EPCs were originally identified in 1997 by Asahara as CD34(+) VEGFR2(+) mononuclear cells. These cells differentiated into an endothelial phenotype, expressed endothelial markers, and incorporated into neovessels at sites of ischemia (Asahara et al., 1997). EPCs provide both instructive (release of pro-angiogenic cytokines) and structural (vessel incorporation and stabilization) functions that contribute to the initiation of neo-angiogenesis. EPC populations can be characterized based on surface markers of freshly isolated cells, or they can be described by their in vitro characteristics once placed in culture. However, a major stumbling block to progress in the field has been the lack of consensus among investigators as to the optimal characterization of EPCs. This review intends to address the role of both EPC classes and evaluate how they interact in the setting of pathological angiogenesis. Since the EPCs may be responsible for turning on the "angiogenic switch," strategies have been employed to keep this switch in the "off" position for diseases like cancer, retinopathy, and wet AMD. The expectation is that EPCs will evolve into clinically useful prognostic and predictive tools in cancer and in ocular diseases associated with pathological neovascularization and that targeting this cell type is a key to successful management of patients suffering from diseases associated with pathological neovascularization.

Figure 1. Adult stem cells of the bone marrow

The bone marrow hosts at least two known types of adult stem cells, the mesenchymal stem cells (MSCs) and the hematopoietic stem cells (HSCs); the most prominent adult stem cell in the bone marrow, together with the EPC, is the focus of this review. The HSC can give rise to the hematopoietic progenitor cells (HPCs) which in turn give rise to the lymphoid progenitor cell, the myeloid progenitor cells, and likely the EPC. The precise origin of the EPC is under debate as this cell may directly arise from the HSC or from the HPC. The bone marrow microenvironment is composed of bone marrow stromal cells (which are the source of SDF-1), adipocytes, and cells of the bone matrix, osteoblasts and osteoclasts. The vessels within the bone marrow, composed of pericytes and endothelium, function to provide a barrier between the hematopoietic compartment and the circulatory system discussed in greater detail in Figure 2. Figure adapted from Domen, et al. (Domen et al., 2006).

Figure 2. Bone marrow and hematopoietic stem cell niches

The maintenance of HSC self-renewal and differentiation is dependent on the specific micro-environment in which they reside. These specialized microenvironments or “niches” include the endosteal or osteoblast niche and the vascular niche. In the endosteal niche, stem cells reside in close proximity to endosteal linings of the bone marrow cavities of the trabecular regions of long bones. HSCs dock to spindle-shaped N-cadherin⁺ CD45^- osteoblasts (SNO) (Upper inset) and to endothelial cells (Lower inset) through a variety of ligand/receptor binding (Opn/α₁-integrin; m-SCF/c-Kit; Ang-1/Tie-2; N-cad/n-cad; SDF-1/CXCR-4. Upper inset); (Vla-4/VCAM-1; Tie-2/Ang-1; CXCR-4/SDF-1. Lower inset). The vascular niche is where stem and progenitor cells have been identified to reside close to sinusoidal endothelium. The endosteal and vascular niches seem to cooperate and both niches are indispensible in controlling HSC quiescence and self renewal activities. Opn: Osteopontin; m-SCF: membrane-bound stem cell factor; Ang-1: angiopoietin-1; N-cad: neural cadherin; SDF-1: stromal cell-derived factor-1; CXCR-4: chemokine (C-X-C motif) receptor 4; CaR: calcium receptor; Vla-4: very late antigen 4; VCAM-1: vascular cell adhesion molecule 1. Figure adapted from Wilson and Trumpp (Wilson and Trumpp, 2006).

Figure 3. Mobilization and homing are two processes closely related

Mobilization involves the exodus of HSC/HPC from the bone marrow into the circulation while homing is the “opposite” of this event. HSC mobilize from the endosteal niche, move to the vascular niche, and ultimately into the circulation. This normally occurs when stress induces changes of SDF-1 levels in the bone marrow. The mechanism of stress-induced mobilization as occurs following irradiation or G-CSF-induced mobilization is not fully known, but is, in part, accomplished by the upregulation of proteases such as MMP-2, MMP-9, cathepsin-G and elastase. These proteases cleave niche retention signals like membrane-bound stem cell factor (mSCF), SDF-1, VCAM-1 and osteopontin (Opn). Gradients of fibroblast growth factor 4 (FGF-4) also regulate mobilization. For homing events, key steps are needed. Upon reaching the bone marrow vasculature, SDF-1-stimulated circulating HSC/HPC express integrins such as very late antigen 4 (VLA-4) and hyaluronan binding–cellular adhesion molecule (CD44). These integrins, in turn, interact with vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-and P-selectins expressed on bone marrow endothelial cells which slows down the circulating HSC/HSP in a process known as “rolling.” Following rolling, firm adhesion and subsequent endothelia trans- migration into the hematopoietic compartment is mainly accomplished by VLA-4 interactions. Once extravasated, the cells migrate along extravascular hematopoietic cords toward specific niches through as SDF-1 gradient or receding oxygen gradient originating from the supporting osteoblastic or endothelia niches. BMEC: Bone marrow microvascular endothelial cell. Figure adapted from Wilson and Trumpp (Wilson and Trumpp, 2006) and from Yin, et al. (Yin and Li, 2006).

Figure 4. Tunneling and vascular mimicry by MC/Mph in Matrigel plugs in vivo

A. Mph-lined, capillary-like structures (arrows) detected at 1 week in a Matrigel plug in a Tie2-βgal mouse (no Tie2-βgal⁺ cells present). Note the branching (arrowheads), and that the cells in the lumen are F4/80⁺. Insert: Enlargement of the F4/80⁺ cell marked with dashed lines, showing a lumen-like vacuole and a low-density tunnel in Matrigel (arrowhead). B. Negative control for the F4/80 immunostaining (omission of the primary antibody). Cell columns line a collagen bundle cut longitudinally, visible in the background of this phase contrast micrograph (arrow). In this field, a Tie2-ßgal⁺ progenitor cell (light blue) is wrapped by another cell (arrowhead). Counterstaining is with hematoxylin. Original magnifications: A-B, ×120. (From Anghelina et al., AJP, 2006). C. Peripheral blood CD14⁺ cells (green, visualized by reaction to anti-human nuclear antigen) isolated from a diabetic patient injected within the vitreous of a mouse that had STZ-induced diabetes for 11 months show vascular association with presumably damaged capillaries. Original magnification: ×20.

Figure 5. Schematic of the hypotheses for the pathogenesis of PDR and the involvement of EPCs

In physiological conditions, CD34⁺ EPCs contribute to routine blood vessel maintenance through eNOS activation. In diabetes, initially, cytokines like stem cell factor (SCF), monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α released by dysfunctional CD34⁺ EPCs) and iNOS activation initiate CD14⁺ EPC-mediated aberrant vascular repair and CD34⁺ EPC dysfunction which ultimately results in retinal ischemia. This phase is referred to as non proliferative diabetic retinopathy (NPDR). The vasodegenerative phase of diabetic retinopathy associated with reduced reparative function of EPCs evolves in the proliferative diabetic retinopathy (PDR). PDR is associated with marked iNOS activation in the CD14⁺ EPC population. Increased levels of NO generated by a change of NOS isoform expression (from eNOS in health to iNOS in late diabetes) is associated with a phenotypic switch in these cells to a more inflammatory cell with secretion of proangiogenic growth factors and cytokines. This phase is characterized by pathological neovascularization seen in the diabetic retina.

Figure 6. EPCs in tumor angiogenesis

Bone marrow-derived hematopoietic stem cells supply endothelial cells that directly contribute to neovessel formation and perivascular cells. Putative VEGFR2⁺ EPCs mobilize into the peripheral circulation in response to tumor cytokines, including VEGF, and move to the tumor bed where they incorporate into sprouting neovessels. Several specific populations of bone marrow derived hematopoietic cells have been reported to contribute to tumor angiogenesis and invasion including GR1⁺CD11b⁺ myeloid progenitors, F4/80⁺ CD11b⁺ tumor-associated macrophages (TAMs), Tie2-expressing monocytes (TEMs), CXCR4⁺VEGFR1⁺ hemangiocytes, VE-cadherin⁺ CD45⁺ vascular leukocytes, and infiltrating mast cells and neutrophils. However, exactly how each of these cells contributes remains a topic of great debate. Figure adapted from Murdoch, et al. (Murdoch et al., 2008).

Figure 7. Laser injury promotes recruitment of bone-marrow derived GFP⁺HSCs into the adult retinal vasculature

Confocal image of a retinal flatmount from a chimeric mouse that underwent laser retinal vessel occlusion injury, double stained with rhodamine-agglutinin (A) to depict retinal blood vessels and anti-GFP (B) to reveal GFP⁺ c-Kit⁺, Sca-1⁺ HSCs. C: Composite image of red (A) and green (B) channels. Scale bar: 10μm.