CHAPTER 1
INTRODUCTION
Cardiovascular pathologies, such as myocardial infarction and ischemic diseases, are the number one cause of death globally. Vascular occlusions, resulting in tissue ischemia, are the main cause of cardiovascular events such as myocardial infarction. Hence, current standard treatments focus on the relief of ischemia, predominantly by restoring vascular perfusion, to prevent ongoing tissue damage. However, such treatments fail to induce tissue regeneration. Therefore, the search for new therapies that induce tissue regeneration is warranted, although these therapies do not abolish the underlying causes of cardiovascular disease. Immediately after ischemic damage, such as myocardial infarction, a local inflammatory response is mounted, which is important for the clearance of cell debris and to form the scar that preserves tissue integrity [1,2]. Tissue perfusion has an important role during this inflammatory reaction and scar formation [3,4]. Preservation of the vasculature, or even induction of vascularization in the ischemic region might decrease the lesion size, prevent loss of cells through apoptosis [5] and eventually might inhibit the development of organ failure [6]. In this regard the last decade has seen a huge interest in the field of regenerative biology, with particular emphasis on the use of isolated or purified stem and progenitor cells to try to restore structure and function to damaged organs by inducing neovascularization.
Adult neovascularization, or the de novo formation of blood vessels, is classically regarded to be a consequence of angiogenesis, rather than vasculogenesis. Angiogenesis is the process whereby new blood vessels are formed from preexisting vessels (reviewed in [7,8]). Sprouting angiogenesis encompasses an increase in vasopermeability, leading to extravasation of plasma proteins that function as a temporary scaffold for migrating endothelial cells (EC). Matrix metalloproteases, secreted by the endothelium, break down the vascular basement membrane and allow the migration of EC. Proliferation and subsequent migration of newly formed EC results in the formation of a
solid endothelial cord. Lumen formation and stabilization are the final processes of sprouting angiogenesis (Fig. 1a). Vasculogenesis, the generation of new blood vessels by stem or progenitor cells (Fig. 1b), was long-regarded to be confined to embryogenesis. However, the discovery of endothelial progenitor cells (EPC) in adult bone marrow and peripheral blood has challenged this theory [9].
FIG.1 Mechanisms of angiogenesis and vasculogenesis. (a) Sprouting angiogenesis originates from the pre-existing vasculature (i)
and encompasses the secretion of matrix metalloproteases that breakdown the vascular basement membrane (ii) and allow migration of endothelial cells (ECs, yellow) (iii). Proliferation and subsequent migration of newly-formed ECs results in the formation of a solid endothelial cord (iv). Lumen formation and stabilization are the final processes of sprouting angiogenesis (v). (b) Vasculogenesis begins with the formation of a primary vascular plexus by endothelial progenitor cells (EPCs) (i,ii). Matrix deposition (iii) and lumen formation (iv) by EPCs result in the formation of immature capillaries
1.1 ENDOTHELIAL PROGENITOR CELLS (EPC)
The first molecular evidence for circulating endothelial progenitor cells (EPC) was described by Asahara et al. in 1997 [9]. EPC are believed to be the postnatal equivalent of angioblasts, i.e. those
cells found in embryonic life which form the walls of “blood island that gradually develop into vasculature contributing to repair of vascular damage in vivo [10,11].
Bone marrow (BM) has been considered to be the major reservoir of EPC. An EPC is not a cell with an invariant phenotype but one with preserved full plasticity, which may transdifferentiate into other cell types under diverse microenvironments, [12,13] in which cell-cell interaction has been postulated to play a vital role. [14] Indeed, the difficulty in defining what an EPC is due to the multiple origins and whereabouts of EPC, implicating the existence of multiple identifiers for this cell type. In earlier studies relatively simple markers to identify EPC were used, amongst which Dil-labelled acetyl low-density lipoprotein (Dil-Ac-LDL) and lectin (either Ulex or BSI) double-positive, cobblestone shaped cells. However the method is not a very precise one to define EPC, as it also includes mature endothelial cells, monocytes and macrophages.
More specific EPC markers are summarized in Table 1. Importantly, BM-EPC has been shown to share origin and markers with haematopoietic stem cells. [15] Kiel et al. recently reported a method to distinguish HSC from progenitors, [16] namely by staining the ‘SLAM’ membrane receptors (CD150,CD244,CD48) that are not present on EPC. [17,18] Furthermore, it has become clear that EPC undergo at least three stages during their specific journey of maturation into the endothelial lineage, namely BM-EPC, early and late circulating EPC (Fig. 2). Late EPC, characterised by CD34+/CD45-, are different from early EPC in secreted growth factors and possess outgrowth capabilities. [19,20] In contrast, early circulating EPC is a myeloid-derived endothelial-like cell with a limited vascular tube formation capacity on Matrigel. [21]
FIG 2 EPC mobilisation and interactions between bone marrow niches and ischaemic niches in the heart. In peripheral organs ischaemic injury provokes increases of angiogenic factors, such as HIF-1〈, SDF-1 and VEGF, which in turn causes activation of matrix metalloproteinase-9 (MMP-9) and increases the cleavage of soluble kit ligand (sKitL, such as stem cell factor), and thus initiates recruitment and mobilisation of bone marrow endothelial progenitor cells (BM-EPCs) from bone marrow niches into the peripheral circulation. The initiation is followed by shedding of c-Kit+/VEGFR2+/CXCR-4+/Sca-1+ progenitors from endosteal or bone marrow stromal niches to the vascular sinusoidal of the bone marrow and their subsequent transmembrane movement compels BM-EPCs to differentiate into circulating EPCs. In bone marrow, after receiving peripheral signals, stem cells divide into a mother cell (self-renewal: semi-circular arrow) and a daughter cell (in this figure a haemangioblast) by asymmetric division. BM-EPCs are of haematopoietic origin and possibly derived from these haemangioblasts, which are characterised by c-Kit+/Sca-1+Lin-/ CD133+/CD34+/VEGFR-2+/CXCR-4+, and capable of giving rise to late endothelial outgrowth. The early circulating EPC is characterised by CD34+/CD45+/CD14+ whereas the late circulating EPC is negative for CD45 and CD14.
More recently, studies from Ingram’s group redefined the concept of EPC, [22] using angiogenic potency as a determinator. They distinguished endothelial cell colony-forming units (CFU-ECs), descendents of myeloid cells, from endothelial colony-forming cells (ECFCs), which derive from mononuclear cells. These latter are believed to be genuine endothelial progenitor cells, (less than 1% of circulating EPC) able to form perfused vessels in vivo. [22] This finding underscores that use of different definitions and consequently different subsets of cells may have contributed to the ambiguous outcomes of BM-EPC therapy in the ongoing clinical trials. [23,24] Moreover, this complicates the search for suitable pharmacological targets and should be cautiously considered in future cell therapy studies.
Transplantation of these progenitor cells successfully promotes therapeutic neovascularization in both ischemic hind limbs as well as acute myocardial infarction models. [5,10,25-27]
Mechanistically, these cells can either induce angiogenesis by incorporation into vascular structures depicting phenotypes of endothelial cells or may induce neovascularization by production of pro-angiogenic factors acting in a paracrine manner. [28]
Physiological EPC-induced neovascularization is initiated by hypoxia. In short, EPC-driven neovascularization starts with the mobilization of EPC from the bone marrow to the circulation in response to stress- and/or damage-related signals (e.g. vascular endothelial growth factor [VEGF], stromal cell-derived factor 1 [SDF-1] and monocyte chemotactic protein 1 [MCP-1] [29,30], followed by migration of EPC through the bloodstream and extravasation of EPC through the endothelium. The EPC migrate towards the site of neovascularization, where they contribute to the formation of the neo-vessels. So these progenitor cells represent appropriate candidates for cell therapies for the treatment of various ischemic diseases. Here, EPC are isolated from the peripheral blood and implanted into the ischemic tissue, where they are involved in restoration of vascular perfusion (reviewed in [30]). However, controversy exists regarding the mechanism by which human EPC induce neovascularization in such a therapeutic setting.
Recent clinical studies suggest that restoration of blood flow in peripheral artery disease and recovery of left ventricular function can be enhanced after autologous transplantation of bone marrow–derived cells (BMCs) or cultured EPC in patients with coronary artery disease. [31,32] However, the numbers and function of these EPC might be affected as a result of the disease. In this regard many studies have described functional impairment of EPC in various diseases, including inflammatory diseases [33], renal diseases [34], and cardiovascular diseases [35], and Krankel and coworkers [37], recently described decreased migratory activity of EPC from patients with cardiovascular disease. However, in their experiments, the cells that did migrate showed no reduction in their angiogenic capacity. These data suggest that although there is a numerical dysfunction of EPC in patients with cardiovascular diseases, the remaining EPC have proper angiogenic function. Hence, the therapeutic success is determined by functional properties of transplanted cells, [38,39], providing the basis for improvement of functional activities, eg, by
pharmacological stimulation of surface receptors, in order to enhance homing of these progenitors cells and to overcome the current limitations observed in EPC-mediated therapy.
1.2 EPC AND ANGIOGENESIS
Although EPC are no longer thought to be genuine endothelial progenitors (and therefore several authors have recommended that the term EPC should not be used as a descriptor for this cell population) there is no doubt that they enhance angiogenesis and vascular repair in a variety of experimental models. Rehman et al. [40] were amongst the first to show that EPC secreted angiogenic factors including VEGF. Zhang et al. [41] found that both early outgrowth EPC and late outgrowth ECFC also secrete pro-inflammatory cytokines, though with different profiles.
In vivo experimental studies such as those of Anghelina et al. [42] have emphasised the primary role of monocytic cells in localising and driving neoangiogenesis, acting via secretion of angiogenic factors and enzymes that can degrade matrix. A recent review by Krenning et al [43] summarises evidence that EPC can act similarly. They conclude that new adult blood vessel formation is, as originally conceived, primarily due to sprouting and replication of endothelial cells from existing local blood vessels, but is stimulated and facilitated by the prior arrival of blood-derived monocytic EPC. However, they also cite evidence suggesting that a fraction of the new endothelial cells may be bone marrow-derived, presumably from the rarer CD34-positive CD14-negative circulating cells. This remains controversial: at least four studies in animals with reconstituted genetically marked bone marrow (one examining collateral vessel formation in response to ischemia, two VEGF-driven angiogenesis, and one tumor-induced angiogenesis) have concluded that bone marrow-derived cells are present only as perivascular cells, and not as luminal lining cells, in new blood vessels. [44-47]
1.3 EPC AND VASCULAR REPAIR
Krenning et al., [43] additionally cite work indicating that the CD14-positive EPC have antithrombotic properties and may form a temporary blind-ended capillary-like structure as they initiate neoangiogenesis by crossing the existing vessel wall. Support for the potential ability of blood-borne monocytic cells to form the lining cells for vascular conduits comes particularly from studies of vascular grafts. Stump [48] et al. first described the luminal coverage by non-thrombogenic cells of large dacron grafts inserted in pig aortas, and Shi et al. [49] by using bone marrow transplanted dogs demonstrated that the cells lining interposed non-porous synthetic arterial grafts were of donor origin. More recently Xu et al. [50] showed in tie-2/lacZ bone marrow reconstituted mice that when a homologous vein segment is placed in the carotid artery, host endothelial cells are rapidly lost and replaced with a monolayer of lacZ-positive cells.
There is evidence that EPC can enhance vascular repair and reduce neo-intima formation and atherogenesis. Early results consistent with this view were presented by Werner et al, [51] who found that after wire injury to murine carotid arteries bone marrow-derived cells could be detected adhering to the vessel area denuded of endothelium. Statin treatment increased circulating EPC and adherent bone marrow-derived cells, and concomitantly reduced neointima formation and increased reendothelialisation. In high fat diet-induced atherosclerosis in ApoE knockout mice, repeated infusion of bone marrow-derived monocytes significantly reduced the extent of disease. [52] Griese et al. [53] showed that delivery of purified circulating CD34-positive cells to the site of balloon-induced injury in rabbit carotid arteries enhanced re-endothelialisation and reduced neointima formation. Similar results were obtained in this model by Ma et al. [54] using culture-derived EPC labelled with superparamagnetic iron oxide, enabling them to monitor the presence of cells at the injury site non-invasively by magnetic resonance (MR) imaging, and finding reduced stenosis over up to 15 weeks, but a detectable MR signal for only 2 weeks after cell delivery.
By contrast, others have shown that bone marrow-derived EPC can exacerbate injury or disease progression. In a very similar study to that of Rauscher et al., [52] George et al. [55] found that infusion of EPC led to incorporation of bone marrow-derived cells into the atherosclerotic lesions and increased lesion formation. Langwieser et al. [56] studied restenosis following wire injury in strains of mice that differed in their mobilisation of monocytic cells from bone marrow in response to the injury: the degree of restenosis and the presence of leukocytes in the vessel wall were greater in the strain with higher cell mobilisation, and in bone marrow transfer experiments the extent of neointima formation related to the phenotype of the donor rather than the recipient. At least part of the difficulty in reconciling these disparate results is likely to be due to differences in composition of the cell populations used, even though they are all regarded as EPC. It is reasonable to presume that increased neointimal hyperplasia will be found if the test cells include significant numbers of either proinflammatory monocyte precursors, or mesenchymal precursors that can home and differentiate to smooth muscle cells. Blockade of leukocyte recruitment in mouse models of arterial injury (using Mac-1 knockout animals) or vein graft into artery (ICAM1 knockout animals) reduces neointimal growth [57-59] reported that smooth muscle progenitors are recruited from the bloodstream at the site of mouse arterial wire injury and contribute to neointimal hyperplasia in a manner dependent on SDF1α expression in the vessel and its receptor CXCR4 on the progenitors. In this context the author noted in his previous review that the enthusiasm for a new generation of stents designed to capture CD34-positive circulating cells, and thus accelerate re-endothelialisation, should be tempered by the understanding that only a small fraction of these cells will be EPC and of potential benefit, whereas others may promote inflammation or hyperplasia. [60] This argument has been independently put forward by Wendel et al. [61] in response to the first report of thrombosis on an anti-CD34 coated stent when dual antiplatelet therapy was withdrawn after 6 months. Since more rapid endothelial cell regrowth reduces intimal hyperplasia, the desired cell therapy should ideally deliver genuine endothelial progenitors to the site of injury, or deliver other cell types that can selectively encourage local regrowth and repair of the denuded endothelium. Despite the ability
of blood-derived cells to maintain an antithrombotic lining in large vascular grafts, it is much less certain that EPC contribute directly to the re-endothelialisation of injured vessels. In agreement with classic studies by Reidy and colleagues [62], who demonstrated that in minor wire injury, restricted to removal of relatively small numbers of endothelial cells, re-endothelialisation takes place by migration and replication of endothelial cells at the edges of the wound, Tsuzuki [63], has used genetically tagged bone marrow transplants in mice to show there is no contribution of bone marrow-derived cells to re-endothelialisation in this model. In an attempt to restore endothelial cell function in eNOS knockout mice, Perry et al. [64], reconstituted the bone marrow with wild-type cells transgenically expressing EGFP. However, they found no restoration of endothelial function in the mice over 4 months, and no detectable EGFP- or eNOS-positive endothelial cells in isolated blood vessels. Earlier studies in normal mice had found a detectable, but low (around 1%), contribution of bone marrow-derived cells to vascular endothelium over a similar time period [65], Even with 4 mm length loss of endothelium in a nude mouse carotid artery injury model, where re-endothelialisation was significantly promoted by infusion of EPC from healthy humans (but significantly less by EPC from diabetics or EPC in which eNOS gene transcription had been knocked down), it is clear that accelerated re-endothelialisation was due to regrowth of host endothelium from the upstream and downstream uninjured vessel [66], Last, in the context of rejection of an aortic transplant in mismatched rats, where the donor endothelium is lost and completely replaced with host endothelium, Hillebrands et al. [67], were unable to find evidence that bone marrow derived cells contributed to the new endothelial layer. Taken together, these results strongly suggest that in most models studied to date, beneficial effects of EPC have been due to paracrine mechanisms that stimulate local endothelial cell mobility and growth, and not due to transformation of circulating EPC into endothelial cells.
