Proteases and receptors in the recruitment of endothelial progenitor cells in neovascularization

ARTICLE

Auteur(s) : Robert E Verloop¹, Pieter Koolwijk¹, Anton Jan van Zonneveld², Victor WM van Hinsbergh¹

¹Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center Amsterdam, Amsterdam, The Netherlands
²Department of Nephrology and the Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands

accepté le 12 Juin 2009

Once the vascular tree has grown to maturity, the endothelium remains in a rather quiescent state. However, during tissue repair after injury, in response to tissue growth or in pathological conditions, neovascularization occurs, often as sprouting from the existing microvessels. In addition to this angiogenic process, it is generally believed that vasculogenesis can also contribute to neovascularization in the adult. This process is driven by the recruitment of circulating endothelial progenitor cells (EPC), and the subsequent participation of these cells in the growing vessels [1, 2]. Asahara et al. [1] described for the first time the isolation and characterization of EPC and their contribution to therapeutic neovascularization in animals. This work shifted the existing view on neovascularization, since at that time the overall consensus was that new vessel formation, in adult life, was solely the result of proliferation and rearrangement of mature cells of the existing vasculature. The interest in these cells was further stimulated by the observation that alterations in the number or function of EPC have been associated with different manifestations of cardiovascular disorders and atherosclerosis [3, 4]. However, subsequent studies pointed to a limited participation of these circulating cells in the endothelial lining of newly grown vessels [5] and challenged the existence of EPC [6, 7].

At present, two different phenotypes of EPC have been recognized, with predominant characteristics of either monocytic or endothelial cells. Here, we will briefly describe the characteristics of the different cell types that have been indicated as EPC. Subsequently, we will discuss current insights into the molecular mechanisms that are involved in mobilization and recruitment of progenitor cells to areas of neovascularization, with specific emphasis on the role of proteases and receptors.

Identity of endothelial progenitor cells

Cell culture and characterization of EPC

Assuming circulating EC would be similar to the embryonic angioblasts, Asahara et al. [1] plated cells enriched for CD34⁺ and VEGFR-2⁺ on fibronectin in an “endothelial medium”. In co-culture with CD34⁺-depleted mononuclear cells, clusters with round cells centrally and sprouts of spindle-shaped cells at the periphery appeared within three days. These attaching cells endocytosed acetylated-LDL, stained with Ulex europaeus lectin (UEA-1), and displayed surface expression of endothelial markers CD31, Tie-2, E-selectin and VEGFR-2. Later, the protocol was adopted by Ito et al. [11] and Hill et al. [12], who cultured unselected peripheral blood mononuclear cells (with additional re-plating selection) in endothelial medium. Similarly, round cells centrally, with spindle-shaped cells at the periphery appeared and were named colony-forming unit endothelial cells (CFU-EC, also known as CFU-Hill). This and comparable assays became very popular because of the correlation of CFU-EC with endothelial function and cumulative indexes of cardiovascular risk (Framingham risk factor score) [3, 12-14]. However, from subsequent studies it became clear that these colonies were in fact not endothelial cells, but consisted of a core of round, hematopoietic cells, including myeloid progenitor cells, monocytes and T lymphocytes, and spindle-shaped monocytes/macrophages that display some features of endothelial cells, such as CD31, CD105, CD144, CD146, vWF, VEGFR-2, and UEA-1 [15-19]. Furthermore, proteomic analysis showed that these colonies had been contaminated by platelet microparticles, explaining the presence of several endothelial markers [20].

Although the CFU-EC were not composed of endothelial progeny, it does not exclude CFU-EC from being involved in angiogenesis or serving as a biomarker for clinical outcome in cardiovascular diseases. Indeed, it has been shown that monocytes/macrophages are potent circulating regulators of angiogenesis and arteriogenesis, and play an important role in the initiation of angiogenesis during wound healing, tissue ischemia and tissue remodeling without actually being incorporated into the endothelial lining [21-27]. Thus, early-outgrowth cells can support angiogenesis in an indirect manner by producing essential growth factors and cytokines, similar to monocytes [21, 24, 27-31].

The late-outgrowth ECFC have the classical endothelial cobblestone phenotype and display a wide range of vascular endothelial markers, but do not express CD45, CD14, or CD115, nor do they ingest bacteria (a monocyte/macrophage characteristic). In comparison with the early-outgrowth CFU-EC, late-outgrowth ECFC show exponential growth and a high proliferative capacity [8, 15, 30, 32, 33]. Furthermore, in contrast to CFU-EC, the late-outgrowth ECFC spontaneously form blood vessels that associate with the nearby vessels and become a part of the systemic circulation in mice [15, 34, 35]. On the basis of these and other experiments, it has been suggested that the late-outgrowth ECFC are derived from circulating cells that best approximate the true “endothelial progenitor cell” definition of EPC [9, 15, 36].

Further characterization of different types of EPC

In contrast to CD34⁺CD45⁺ cells, culturing CD34⁺CD45⁻ cells resulted in colonies of highly proliferative endothelial cells [37, 38]. These endothelial colonies expressed VEGFR-2, but not CD133. They manifested as blood-outgrowth endothelial cells (BOEC), and likely represent the same cell population as the late outgrowth ECFC mentioned above.

Additional evidence for the existence of two different EPC populations came from studies in patients with chronic myeloproliferative disorders (CMPD) [44]. ECFC derived from CML patients, or Ph-negative CMPD were not clonally related to the cells that gave rise to the hematopoietic disorder. However, the disease marker was present in all CFU-EC derived from the blood of these patients, thus confirming that CFU-EC are derived from myeloid cells.

From the foregoing, it is clear that a distinction between the two populations is needed for understanding the contribution of EPC in angiogenesis. However, as literature data on the recruitment of EPC from the bone marrow and their contribution to angiogenesis often fail to make this discrimination, we shall refer in the forthcoming discussion to CFU-EC for early-outgrowth myeloid cells that have acquired endothelial markers, and to ECFC for the few cases where late-outgrowth progenitor cells have been used, which have a true endothelial nature. Furthermore, we shall use EPC to indicate the overall populations of circulating or non-defined EPC, which de facto largely consist of CFU-EC progenitors.

Mobilization of endothelial progenitor cells from the bone marrow

Proteolytic modification of receptors and cytokines in the mobilization of progenitor cells

Following the seminal observations of Heissig et al. [51], it became clear that MMP-9 plays a central role in the mobilization of EPC. It was demonstrated that, after fluorouracil-induced bone marrow ablation in mice, myelosuppression was accompanied by elevated plasma levels of stromal cell-derived factor-1 (SDF-1) and vascular endothelial cell growth factor (VEGF), which upregulated the expression of MMP-9 in bone marrow cells (both HSC and stromal cells). MMP-9 liberated sKitL (also known as stem cell factor [SCF]) from its membrane precursor KitL on bone marrow stromal cells. Signaling of sKitL, through c-Kit (SCF receptor) recruited c-Kit⁺ stem/progenitor cells to the circulation, including VEGFR-2⁺ EPC (figure 1). These observations were underscored by experiments in MMP-9^−/− mice, which demonstrated impaired release of sKitL and HSC motility. This deficiency resulted in impairment of hematopoietic recovery and increased mortality after bone marrow ablation, while exogenous sKitL restored hematopoiesis and survival. Of note, HSC, cardiac, epithelial and EPC all express the receptor for sKitL, c-Kit [52, 53].

Subsequent studies in endothelial nitric oxide synthase (eNOS)-knockout mice identified nitric oxide (NO) as a crucial cofactor in VEGF-induced MMP-9 activation [54]. Mice deficient in eNOS showed reduced VEGF-induced mobilization of EPC (CFU-EC) and increased mortality after myelosuppression. Mechanistically, MMP-9 activity was reduced in the bone marrow of eNOS ^-/- mice, because of reduced S-nitrosylation of MMP-9 [55]. The importance of NO in progenitor cell function was further highlighted by the work from Sasaki et al. [56], who treated dysfunctional bone marrow cells from ischemic cardiomyopathy (ICMP) patients with the novel eNOS transcription enhancer AVE9488. AVE9488 increased eNOS mRNA levels and eNOS activity, which at least partially reversed the impaired functional activity of BMC, improving the neovascularization capacity of infused BMC in an in vivo, ischemic hindlimb model.

Apart from VEGF and SDF-1, which are considered to be among the most effective mobilizers of EPC, a vast number of growth factors and cytokines have been described to modulate mobilization of EPC (CFU-EC) [57]. Granulocyte colony-stimulating factor (G-CSF), which has a strong stem/progenitor cell mobilizing potential, induces the release of elastase and cathepsin G from neutrophils, leading to the cleavage of adhesive bonds on bone marrow stromal cells [58, 59]. Moreover, these proteases can cleave and inactivate SDF-1, which is released by stromal cells and signals through its receptor CXCR4 on stem/progenitor cells [59, 60]. Similarly, G-CSF induced upregulation of the exopeptidase CD26 on CXCR4⁺ stem cells. CD26 is able to splice the N-terminal dipeptide from SDF-1, which causes inhibition of CXCR4 activation and reduced cell retention, resulting in an additional mechanism of mobilization [61, 62].

A role for the plasminogen activator system in the recruitment and homing of CD34⁺ progenitor cells has also been suggested. Selleri et al. [63] documented the co-mobilization of myeloid and monocytic cells with an increased uPAR expression, as well as the increase in serum levels of soluble uPAR and cleaved soluble uPAR after G-CSF-induced CD34+ HSC mobilization. Both fragments were able to attract CD34+ progenitor cells via activation of the high-affinity fMet-Leu-Phe (fMLP) receptor (FPR) and desensitization of the SDF-1 receptor CXCR4 (which facilitates bone marrow retention of HSC) both in vitro and in vivo [64]. Recently, compelling data from Tjwa et al. [65] demonstrated that membrane-anchored uPAR is present on a subpopulation of HSC, regulating stem cell-cycle status, adhesion to the bone marrow microenvironment, and homing and engraftment in vivo. During G-CSF treatment, plasmin is most likely to be responsible for cleavage of uPAR and increased plasma levels of soluble uPAR. The concomitant loss of the interaction of uPAR with α₄β₁ integrin thereby promoted stem cell mobilization. Similarly, impaired stem cell mobilization as well as homing and engraftment of transplanted stem and progenitor cells occurred in uPAR-deficient mice [65].

Mobilization of ECFC progenitors from the bone marrow

Homing of EPC to areas of neovascularization

Homing and incorporation of EPC in the endothelial lining or sub-endothelial space is a multi-step process, which involves chemo-attraction, rolling and tethering of progenitor cells, subsequent firm adhesion and, finally, extravasation (figure 2). Various degrees of blood vessel and tissue injury may result in the production and release of cytokines and chemokines creating a gradient within the vessel wall. A large number of cytokines and growth factors, including VEGF [24, 68, 69], insulin-like growth factor 2 (IGF2) [70], monocyte chemotactic protein 1 (MCP-1/CCL2) [71], interleukin-8, CXCL1 and CXCL7 [72, 73], bradykinin [74], macrophage migration inhibitory factor (MIF) [75], and SDF-1/CXCL12 have been described as participating in EPC (CFU-EC) recruitment.

The latter one, SDF-1, and its specific receptor CXCR4 play a major role in stem cell recruitment and retention to ischemic areas [76-78]. In physiological conditions, SDF-1 is constitutively expressed, but a range of stimuli such as inflammation [79], tissue damage and hypoxia rapidly increase SDF-1 levels. SDF-1 is expressed or is surface-bound at injured smooth muscle and endothelial cells, and is released by activated platelets [80-83]. Activated platelets secrete high levels of SDF-1. Platelets are probably the first responders to vascular trauma and, after their adherence to subendothelial matrix structures and subsequent activation, release the necessary factors for progenitor cell mobilization and homing to the damaged area [81]. Reduced tissue and plasma levels of SDF-1 have been correlated with unstable coronary artery disease [84] and impaired wound healing in diabetes [85]. The gene expression of SDF-1 is regulated by the transcription factor hypoxia-inducible factor-1α (HIF-1α) and is also induced by VEGF overexpression, thus mediating SDF-1-dependent recruitment of CXCR4⁺ progenitor cells to injured arteries and hypoxic regions [24, 86, 87].

As with the impaired SDF-1/CXCR4 signaling in diabetic patients, Kränkel et al. [74], reported that circulating progenitor cells from cardiovascular disease patients showed low levels of the kinin B2 receptor (B2R) and decreased migratory capacity toward bradykinin (BK), suggesting that impaired homing and migration might contribute to impaired neovascularization after ischemic complications.

Retention of CD34⁺ progenitor cells by developing endothelial tubes in vitro

When the CD34⁺ cells were fixed with paraformaldehyde before adding them to the tube formation system, a complete arrest of the administered CD34⁺ cells on the stimulated endothelial monolayer was observed, indicating that the movement was indeed an active process of the cells. In contrast, fixation of the endothelial monolayer and tubular structures interfered neither with the random movement of the CD34⁺ cells, nor with the accumulation of CD34⁺ cells at the tubular structures. The response to fixed tube structures and the absence of a chemotactic response suggest that adhesion receptors rather than cytokines were involved. Preincubation of the endothelial monolayer with a cocktail of blocking antibodies against E- and P-selectin, ICAM-1 and VCAM-1 to study this process was not sufficient to block the accumulation of CD34⁺ cells at the tubular structures, suggesting that other adhesion factors may also be involved.
Table 1 Migration and tubular homing of peripheral and cord blood CD34⁺ cells

Receptors involved in EPC (CFU-EC) homing to areas of angiogenesis in vivo

Integrins expressed on the surface of EPC mediate the firm adhesion and transmigration of EPC to the damaged endothelium. Again, the murine model of hindlimb ischemia was utilized to demonstrate that progenitor cells from β₂-integrin-deficient mice are less capable of homing to sites of ischemia and of improving neovascularization. Preactivation of the β₂-integrins expressed on EPC by activating antibodies augments the EPC-induced neovascularization in vivo [93]. This work was supported by the fact that not only VLA-4 (α₄β₁ integrin) and LFA-1 (α_Lβ₂ integrin), but also their counterparts ICAM-1 and VCAM-1, were upregulated in ischemic tissue [94-96]. Neutralization of one of these factors reduced adhesion and migration of EPC in vitro and reduced recovery of hindlimb blood flow, capillary density and incorporation of EPC into ischemic tissues in vivo [96-98]. Mechanistically, cell adhesion molecule (CAM)-integrin-mediated firm adhesion was reported as being regulated by hypoxia as well as necrosis. High-mobility group box 1 (HMGB1) is a nuclear protein that is released extracellularly upon cell necrosis and tissue damage. Binding of HMGB1 to its receptor RAGE (receptor for advanced glycation end products) on EPC resulted in rapidly increased integrin affinity and induced integrin polarization, enhancing the in vitro adhesion and migration and in vivo homing and incorporation of EPC in the tumor vasculature [99].

Exposure of endothelial cells to hypoxia increased the endogenous amount and kinase activity of the protein integrin-linked kinase (ILK-1) in a NFκB- and HIF-1α-dependent manner. Overexpression of ILK-1 resulted in ICAM-1 upregulation, whereas blocking ILK-1 abrogated the expression of ICAM-1 under hypoxia, with co-committed reduction in EPC homing and poor neovascularization in vivo [100, 101]. In addition, the adipokine leptin, a published modulator of vascular remodeling and neointima formation, has recently been described as increasing the expression of α_Vβ₅- and α₄-integrins in EPC [102].

Proteases involved in homing of EPC (CFU-EC)

Urbich et al. [104] demonstrated the crucial role of cathepsin L in EPC-mediated neovascularization. Cathepsin L was highly expressed in EPC and was essential for matrix degradation and invasion by EPC in vitro. Cathepsin L-deficient mice showed impaired functional recovery following hindlimb ischemia. Infused cathepsin L-deficient progenitor cells neither homed to sites of ischemia nor augmented neovascularization. This could be reversed by forced expression of cathepsin L in mature endothelial cells. Recently, the same group revealed a decreased cathepsin L expression and activity in EPC from patients with type 2 diabetes, suggesting a novel mechanism for diabetes-related impairment of neovascularization [107].

Membrane-type-(MT-)1-MMP (MMP-14) is another powerful protease involved in migration and invasion of cells including CD34⁺ progenitor cells [106]. Recently, it was shown that the balance between the expression of MT1-MMP and its membrane-anchored inhibitor RECK [108], is involved in the regulation of homing, retention, egression, and mobilization of immature human CD34⁺ progenitor cells [109]. Steady-state egression of human CD34⁺ cells and, to a greater extent, their G-CSF-induced mobilization, is accompanied by an increase in MT1-MMP and a simultaneous reduction in RECK expression, which facilitate MT1-MMP-mediated CD44 cleavage and progenitor cell motility and mobilization. Furthermore, MMP-2 was found to affect the invasive properties of EPC. MMP-2^−/− mice responded poorly to hindlimb ischemia because of reduced neovascularization. Transplantation of MMP-2^+/+ bone marrow cells dramatically improved the recovery of these mice [110]. Stimulation with TNFα, IL-8 or SDF-1 resulted in increased MMP levels, facilitating the migration of EPC into Matrigel plugs or transwell systems [36, 111-113].

Finally, a role for the plasminogen system in EPC homing and recruitment was anticipated. Xiang et al. [114] investigated the potential of the plasminogen system in vivo. Blocking the protease inhibitor plasminogen-activator inhibitor-1 (PAI-1) with a sequence-specific DNA enzyme at the time of myocardial infarction, resulted in increased engraftment of exogenously delivered CD34⁺ progenitors in the infarct zone. Abrogation of the natural inhibition of PAI-1 on u-PA was most likely responsible for the observed effect [114].

Homing of ECFC progenitors

While no information is presently available on homing receptors, it is likely that these cells use similar proteases as other endothelial cells, such as MT1-MMP, MMP-2, MMP-9 and the urokinase/plasmin system [116]. These proteases are indeed expressed in ECFC. Cultured ECFC displayed relatively high u-PA and MMP-2 levels compared to normal endothelial cells [105, 117]. Inhibition of ECFC-associated u-PA by monoclonal antibodies that block u-PA activity or binding to its receptor significantly reduced proliferation, migration and capillary-like tube formation in vitro [105, 117].

Participation of EPC in the newly formed vessels

Intussusception of circulating EPC in healing human blood vessels

Contribution of late-outgrowth ECFC, but not early EPC to endothelial tubular structures

In contrast, the ECFC incorporate into existing differentiated endothelial tubule structures in vitro, and are also able to form tubules readily, by themselves [15, 34, 117, 131]. This process proceeds similarly as the tube formation by microvascular endothelial cells, and depends on proper integrin-matrix interactions and the pericellular recruitment of proteases, including u-PA, the metalloproteinases MT1-MMP and MMP-2, and cathepsin L [104, 105, 117].

More convincingly as regards clinical applications, several reports have also shown that peripheral blood- and cord blood-derived ECFC can form functional blood vessels when implanted in vivo [15, 34, 132]. This opens avenues for the engineering of new vasculature in tissues with limited blood supply. However, Au et al. [133] reported that umbilical cord blood-derived ECFC, when co-transplanted with mesenchymal cells in mice, formed stable vascular structures, while similar cells obtained from peripheral blood formed only a limited number of unstable vascular structures. Thus, ECFC are able to participate in blood vessels as true endothelial cells, but much has still to be learned about their prerequisites and functional properties.

Conclusion

The first population involves the early-outgrowth EPC (CFU-EC), which have a myeloid origin. They represent monocytes with endothelial properties and a strong ability to provide factors to stimulate angiogenesis, but little capacity to incorporate persistently into the endothelial lining. The decrease in circulating EPC observed in diabetes and cardiovascular disease may reflect a contribution to vascular maintenance and repair in patients, but alternatively may merely reflect increased oxidative stress [134]. Future studies will clarify whether these cells reflect the course of the disease, making them suitable as a prognostic marker, or whether their reduction also contributes to the etiology of the vascular complications (causal), thus providing a perspective on new therapies.

The second population (ECFC or late-outgrowth EPC) grows out as colonies of real endothelial cells. Better identification of these cells in the blood by specific markers would help to improve the isolation, separation and amplification of these cells, and the possible use of these cells for autologous and possibly heterologous transplantation. Much has still to be learned; it may well be that these cells perform best in collaboration with other mesenchymal cells. Furthermore, the differences between ECFC obtained from cord blood and those from peripheral blood require further attention.

The mobilization and recruitment of the progenitor cells that develop into the myeloid CFU-EC and endothelial ECFC is determined in the bone marrow and formation of similar new interactions at the sites of homing and neovascularization. Proteases, such as MMP-9, play an important role in mobilization from the bone marrow by disintegration of existing cell adhesions and chemokine-receptor interactions. Once liberated, the circulating progenitor cells home via receptors that act as cell adhesion molecules and recognize cytokine gradients, such as SDF-1 in hypoxic areas. The subsequent invasion process requires proteolytic activity to squeeze the cells through the collagen-rich extracellular matrix and/or the fibrin matrix of a fibrinous exudate. In this latter process, MT1-MMP, MMP-2 and u-PA acting on a cellular uPA receptor are involved. Indeed, cultured ECFC have a high expression of u-PA, which appears to be involved in migration and tubule formation. Similarly roles for MT1-MMP, MMP-2 and cathepsin L are likely. Furthermore, these and other proteases also activate and modify cytokines, angiogenic growth factors and their receptors, thus fine-tuning the regulation of the neovascularization process.

Acknowledgments

Disclosure. The authors have no conflicts of interest to declare.

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