Placenta
Volume 31, Issue 1 , Pages 1-10, January 2010

Endothelial Progenitor Cells: Their Potential in the Placental Vasculature and Related Complications

  • P.I. Sipos

      Affiliations

    • Maternal and Fetal Health Research Group, The University of Manchester, Research Floor, St Mary's Hospital, Oxford Road, Manchester, M13 9WL, UK
    • Corresponding Author InformationCorresponding author. Tel.: +44 447931254176.
    • ,
  • I.P. Crocker

      Affiliations

    • Maternal and Fetal Health Research Group, The University of Manchester, Research Floor, St Mary's Hospital, Oxford Road, Manchester, M13 9WL, UK
    • ,
  • C.A. Hubel

      Affiliations

    • Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, PA 15213, USA
    • ,
  • P.N. Baker

      Affiliations

    • Maternal and Fetal Health Research Group, The University of Manchester, Research Floor, St Mary's Hospital, Oxford Road, Manchester, M13 9WL, UK

Accepted 12 October 2009. published online 18 November 2009.

Article Outline

Abstract 

Endothelial progenitor cells (EPCs) have received significant attention in recent times. A role for EPCs has been suggested in a range of pathologies and some recent studies of EPCs in pregnancy have been published. This review provides a guide to the confusing field of EPCs. Attention is paid to their phenotyping, as although elementary this remains a highly debated topic. The current understanding of different subtypes and physiological role of EPCs in the placenta, fetus and adult are also considered. An overview is given as to role of EPC's in the pathophysiology of different disease states and the possible therapeutic and diagnostic applications expected from EPC-related research in obstetrics.

Keywords: Endothelial progenitor cells, Embryogenesis, IUGR, Pre-eclampsia, Placenta

 

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1. Introduction 

In 1997, Asahara et al. [1] proposed a new concept of neovasculogenesis and suggested that a cell type exists in the adult, which resides in the bone marrow and migrates to ischaemic sites to form new endothelium. They also proposed a culture technique for the isolation of such cells. These outgrowth cells were referred to as putative Endothelial Progenitor Cells (EPCs). Since then, more than 2000 EPC-related articles have been published and a relationship has been found between endothelial progenitors and dozens of disease states from pulmonary hypertension [2] to moya-moya disease [3]. EPCs are thought to be diagnostic of cardiovascular risk [4], [5], [6], [7], [8], [9], [10] and are therapeutic in animal ischaemia models [11], [12], [13], [14]. In hoping to find a cure for atherosclerosis, the main stream of EPC-related research has been conducted in the field of adult cardiovascular medicine, despite the fact that the presence of these cells has been known for much longer in the fetus and that vessel-related complications represent a significant clinical problem in pregnancy. This article provides a critical overview of the current literature and discusses avenues for further obstetric research. In doing so, we propose a potential role for EPCs in the pathophysiology of pre-eclampsia, intrauterine growth restriction (IUGR), gestational diabetes and increased life-long cardiovascular risk associated with these conditions. The potential for EPCs to be used as diagnostic tools or therapeutic agents in pregnancy-related complications is also briefly considered.

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2. Physiology of EPCs 

Vascularisation is a new described model of vessel formation - as opposed to the previously recognised vasculogenesis and angiogenesis. This model was created once the presence of EPCs in the adult circulation was suspected. Vasculogenesis refers to the de novo formation of blood vessels from progenitor cells. Angiogenesis, in contrast, means the sprouting of new vessels or elongation from existing ones through the remodelling of differentiated endothelial cells. Vascularisation in many ways resembles angiogenesis; however, in this case, EPCs contribute to neovessel formation by their incorporation into vessel walls, their secretion of paracrine hormones and subsequent angiogenic stimulation.

Endothelial and blood cell precursors originate from the same ancestor: the haemangioblast [15], [16]. EPCs were originally thought to be derivates of the haemangioblast and were defined as cells capable of proliferation, incorporation into forming blood vessels, and differentiation into mature endothelial cells. They were thought to reside in the bone marrow, leave after recruitment, and have the capacity to migrate and participate in vasculogenesis and vascularisation.

The expression “putative EPC” is rather historical, and the EPC population consists of two distinct sub-populations with very different phenotypes and functional capabilites, both closely involved in vessel formation. The first group is a haemopoietic sub population, the so called Circulating Angiogenetic Cells (CACs), which have strong paracrine and hormonal activities and stimulate cell migration and proliferation [17], [18], [19], [20]. The second, Endothelial Colony Forming Cells (ECFC), have more endothelial-like characteristics and are profoundly influenced by CACs. They are highly proliferative and migrate to sites of vessel formation, before differentiating into mature endothelial cells, thus forming de novo endothelium [21], [22], [23].

In isolating EPCs and defining these subtypes, cell culture techniques have been widely used. In some protocols, fibronectin coated dishes are employed and adherent cells are typically harvested within one week after seeding. These cells are frequently referred to as “early outgrowth cells”. The most common approaches include: the Ashara technique [1], Hill-Colony Forming technique [4] and Vasa technique [24]. A different approach, using collagen-coated dishes and adherent cells harvested several weeks later, generates the so called “late outgrowth cells”. This technique was orginally described by Lin et al. 2000 [21] (See Fig. 1).

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  • Fig. 1 

    Bright-field images of outgrowth cells from human fetal peripheral blood mononuclear cells (PBMNCs). A: CFU-Hill technique. Round cells in the centrum of the colony, spindle shaped cells in the periphery. The colonies appear to be smaller than in adult and spindle shaped cells migrate away from the centre rapidly. (Scale bar: 50μm) B: Vasa technique: monolayer of round adherent cells. (Scale bar: 100μm) C: Lin-technique: Colony formed by a monolayer of cells with cobble stone appearance. The colonies are larger and appear earlier than those in the adult. (Scale bar: 100μm) Unpublished images of the authors.

As the phenotypes and functions of early and late outgrowth cells are now clarified, cells obtained with early outgrowth techniques are practically considered CACs, while those produced with late outgrowth protocols are deemed ECFCs. In accord with current opinion, these terms will be used throughout the rest of this article: using the term EPC, where the actual sub-population is not determined, or where the comment applies equally to both CAC and ECFC phenotypes.

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3. EPC-phenotypes 

Although ECFCs and CACs can be isolated by the above culture techniques and by flow cytometry, no standardised and accepted means of phenotyping has yet been defined, and there are several different, partly contradictory, approaches found in the literature. Consequently, confirming cell-phenotype is a core agenda and remains a major issue. The identity of outgrowth cells or those acquired by flow cytometry is determined by the expression of tissue-specific surface markers. In addition, functional assays can contribute to the verification of endothelial or progenitor progeny.

3.1. Flow cytometry techniques 

To date no EPC-specific antigen has been defined. Instead, various combinations of haematopoietic and endothelial stem-markers are proposed. It has been postulated that at the angioblast stage of development, i.e. before the loss of haematopoietic markers, cells start expressing endothelial antigens. The three antigenes most commonly considered are CD34, CD133 and KDR/VEGFR-2.

•CD34 is a single pass transmembrane sialomucin. As a cell-cell adhesion factor, it may mediate the attachment of stem cells to bone marrow extracellular matrix or directly to stromal cells. It is expressed on numerous cell types of mesodermal progeny, including blood cells, endothelial and fibroblast cells, epithelial and cancer stem cell populations. It is expressed by all haematopoietic stem cells, but it is subsequently lost upon differentiation [25], [26]. It is also expressed by many cells of endothelial progeny, including differentiated endothelial cells in the adult [27].

•CD133 or prominin-1 is a 5-transmembrane domain cell-surface glycoprotein. It is localised on membrane protrusions of epithelial, haematopoietic and cancer stem cells. It is strongly expressed on haematopoietic stem cells, but also disappears during haematopoietic differentiation [28].

•KDR or VEGFR-2 (Human) is a thyrosine-kinase transmembrane receptor of vascular endothelial growth factor (VEGF). It is expressed by both early haemopoietic stem cells and endothelial cell-lines, but its expression also ceases with haematopoietic differentiation [29], [30].

It was Asahara et al. that showed that CD34 enriched KDR+ cells localise to sites of vasculogenesis [1], and Peichev et al. that suggested the addition of CD133 to differentiate EPCs from mature endothelial cells. They concluded that circulating CD34+/KDR+/CD133+ cells represent a distinct population with a role in neo-angiogenesis and CD34+/KDR+/CD133- represent a more mature population of progenitors [31]. In 2006, Friedrich et al. identified a new CD34–/CD133+/KDR+ EPC sub-population which differentiated into CD34+/133+/KDR+-defined EPCs in culture [32]. Currently, most available research is based on these phenotypes; however direct clonal evidence is not usually provided and these techniques notably fail to distinguish CACs and ECFCs.

More recently the use of Fluorescence Activated Cell Sorting (FACS) and PCR has shown that ECFC from peripheral or umbilical blood are inevitably CD34 positive and CD45 negative, and that they also express KDR but not CD133 [33]. This population represents approximately 2% of all CD34+ mononuclear cells. Timmermans et al. also showed that CACs belong to the CD14+ monocytic sub-population of CD34+/CD45+ cells and although they co-express CD133 they never express KDR [33]. This CD34+/KDR+/CD133+ triple-positive population has since been shown to be neither CAC, nor ECFC in origin, but nevertheless they are primitive haematopoietic progenitors [34], and some suggest they fit with a more broader definition of CACs [35], [36].

In summary, ECFCs can be characterised by the surface marker-combination of CD31+/CD34+/CD45-/KDR+/CD133-, but it must be noted that this combination does not differentiate ECFCs from circulating endothelial cells. Alternatively, CACs can be characterised by the surface marker-combination of CD31+/CD34+/CD45+/CD14+/CD133+/KDR-. However, some suggest that CD34+/KDR+/CD133+ may also be considered CACs [35], [36]. It should also be noted that the specificity of these flow cytometry techniques, as based on these antigen-combinations, can be significantly improved by excluding false-positive events, i.e. those of non-vital cells and CD3 positive T-lymphocytes. Fig. 2, Fig. 3 show typical gating strategies in flow cytometry, and they demonstrate the current understanding of surface antigen expression patterns of the two EPC sub populations, ECFCs and CACs.

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  • Fig. 2 

    Flow cytometry technique for surface marker distribution of ECFCs in peripheral blood. A: Typical Foreward Scatter-Side Scatter histogram of lysed blood; Gate 1 set over mononuclear leucocytes. B: 7AAD-monohistogram. Gate 1 applied. Gate 2 set over 7AAD-negative (viable) cells. Non-viable cells have the tendency to non-specifically bind antibodies. Exclusion of non-vital cells helps reduce the number of false-positive events. C: CD45 monohistogram. Gates 1 and 2 applied. Gate 3 set over the CD45 negative population. D: CD31 monohistogram. Gates 1, 2 and 3 applied. Gate 4 set over the CD31 bright population. E: X-axis represents CD133; Y-axis represents CD34. Gates 1, 2, 3 and 4 are applied. Gate 5 set over CD34+ and CD133- populations, while Gate 6 segregates CD34+ and CD133+ dual positive cells. Gate 5 highlights CD45-/CD31Bright/CD133-/CD34+ events, consistence with an ECFC/CEC identity. Non-vital cells are excluded. Unpublished images of the authors. Flow cytometry technique modified from Duda et al. 2007 [111].

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  • Fig. 3 

    Flow cytometry technique for surface marker distribution of CACs in peripheral blood. A: Typical Foreward Scatter-Side Scatter histogram of lysed blood; Gate 1 set over mononuclear leucocytes. B: 7AAD-monohistogram. Gate 1 applied. Gate 2 set over 7AAD-negative (vital) cells. Non-vital cells may non-specifically bind antibodies. Exclusion of non-vital cells helps reduce false-positive events. C: CD45 monohistogram. Gates 1 and 2 applied. Gate 3 set over CD45 dim population. D: CD31 monohistogram. Gates 1, 2 and 3 applied. Gate 4 set over CD31-positive population. E: X-axis represents CD133; Y-axis represents CD34. Gates 1, 2, 3 and 4 applied. Gate 5 set over CD133+ and CD34Bright population. This population is CD45Dim/CD31+/CD133+/CD34Bright. Non-vital cells are excluded. These features are characteristic to CACs. Unpublished images of the authors. Flow cytometry technique modified from Duda et al. 2007 [111].

3.2. Cell culture techniques 

The confirmation of the phenotype of outgrowth cells in culture is another essential validation process. Differentiating CACs from ECFCs requires distinct methods and many authors fail to achieve this sufficiently or even differentiate EPCs from epithelial or mononuclear cells. Originally it was believed that a combination Dil-AcLDL-uptake and positive Ulex europaeus-1 Agglutinin binding test [37], [38] (two probes traditionally used to identify vascular tissues) was sufficient to confirm EPC-identity. However, the specificity of this dual-positivity test has now been discredited [39], [40], [41], [42], [43].

Further confirmation is therefore essential, and this is typically achieved by surface marker recognition. ECFCs grown by the Lin-technique express endothelial surface markers, such as CD31, CD141, CD105, CD146, CD144, vWF, and KDR, but are invariably negative for haemopoietic surface markers such as CD45 and CD14. Morphologically they resemble endothelial cells with a cobble stone appearance. They form capillary-like tubes in three-dimensional culture, a feature unique to endothelial cells, and also incorporate into newly forming blood vessels both in vitro [22] and in animal models of xenograft implantation [44].

CACs obtained from culture are monocytic cells, positive for CD45 and CD14 and the classic EPC markers CD34 and CD133; they are also negative for KDR. They readily incorporate Dil-AcLDL and stain with UAE-1 and unlike ECFCs, have a monocytic morphology in culture. (See Fig. 4.)

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  • Fig. 4 

    A comparison of CAC and ECFC characteristics. ECFCs form tubes on matrix, while CACs fail to do so. Both CACs and ECFCs ingest AC-LDL, and express CD31and CD34. CACs are CD45 and CD133 positive; whilst ECFCs fail to express these antigenes. (Scale bar: 100μm (A-H, K-M); Scale bar: 10μm (I-J)) Unpublished images of the authors.

3.2.1. Functional capabilities of endothelial progenitors 

EPCs are thought to behave similarly to previously known progenitor cells, i.e. (i) they interact with each other and other cell types, (ii) they are recruited and mobilised mainly from the bone marrow, (although residing ECFCs have been isolated from human large vessel walls [45] and cells with CAC-characteristics are also isolated from the spleen [46], liver [47] and adipose tissue [48], and equally, it cannot be excluded that EPCs are produced in the placenta itself, as it is a site of fetal haematopiesis [49], [50], [51]), and (iii) they migrate to sites of vessel formation, invade locally, proliferate and differentiate into mature endothelial cells. Each of these basic functions is elucidated below:

3.2.1.1. EPC cell-cell interactions and signals 

EPCs have dominant hormonal activity and a main consideration is the co-work that occurs between ECFC and CAC sub populations. As mentioned, CACs are thought to play an essential role in stimulating ECFCs. However, feedback mechanisms also exist. Vascular Endothelial Growth Factor (VEGF), Stromal Cell Derived Factor-1 (SDF-1) and Interleukin-8 (IL-8) [52], [53] are secreted by CACs and these act on ECFCs as stimulants and chemoattractants [54]. Furthermore, IL-8 is also released by ECFCs, which is chemoattractant to CACs [55], and mitogenic to ECFCs in an autocrine/paracrine fashion [56]. The activation of the thrombin receptor, Protease Activated Receptor 1 (PAR-1), increases ECFC-proliferation and their organisation into tubal structures [57], [58]. Moreover, stimulation of PAR-1 significantly increases the amount of IL-8 excreted by ECFCs [53]. These findings suggest a complex stimulatory positive feedback mechanism which helps maintain and enhance the proliferative capability of ECFCs in situ.

The mobilisation of progenitor cells from the bone marrow requires the degradation of extracellular matrix. In this context, Matrix metalloproteinase-9 (MMP-9) is secreted by CACs and Matrix metalloproteinase-2 (MMP-2) is produced by ECFCs, and assists in the breakdown of collagen-IV [52]. In response to VEGF and IL-8, ECFCs increase MMP-2 production and CACs increase MMP-9 secretion [52]. This may partly explain the increased invasiveness and vasculogenic activity of both cell types in co-culture, as the interaction between the MMP subtypes may also be significant [59].

In general terms, fat-soluble substances are less volatile in the circulation and their concentrations remain higher in the local micro-environment, therefore their effect on neighbouring cells may be more pertinent. Consequently, fat-soluble substances like endocannabinoids, produced by EPCs, may represent important paracrine signals. EPCs produce and release two subtypes of endocannabinoids: anandamide (arachidonyl-ethanol-amide; AEA) and 2-arachidonoyl-glycerol (2-AG). CACs secrete higher levels of 2-AG, while ECFCs secrete higher levels of anandamide. Adult blood derived ECFCs secrete more anandamide than cord blood ECFCs [60].

Although endocannabinoid secretion-patterns of EPC sub-populations show marked differences, the significance of this pattern and the exact role of different endocannabinoids in various cell types remain unclear. What is known is that endocannabinoids suppress the induction of the pro-inflammatory Vascular Cell Adhesion Molecule 1 (VCAM-1), which is a facilitator of leukocyte-endothelium adhesion. In this respect, these EPC derivatives may act as anti-inflammatory agents. For cord blood ECFCs, pro-inflammatory Tumor Necrosis Factor-α (TNF-α) increases anandamide-excretion but decreases endocannabinoid-release from CACs [60]. In as yet undefined ways, this differential affect may hold significance in the pathogeneses of certain pregnancy-related conditions.

3.2.1.2. EPC mobilisation and recruitment 

Progenitor cells reside in stem cell niches of the bone marrow from which they leave and differentiate in response to micro-environmental changes. As suggested, MMP-9 has a crucial role in mobilising progenitor cells from this stem cell niche. Kit Ligand (also called Stem Cell Factor: KitL or SCF) is an adhesive molecule which keeps EPCs bound to the osteoblastic niche of the bone marrow. MMP-9 catalyses the proteolysis of KitL from the cell membrane and a soluble form is released. The proteolysis of KitL improves the mobility of progenitors and their transfer to the vascular-enriched niche, which is a favourable environment for further differentiation and egress into the peripheral circulation [61]. The expression of proMMP-9 and active MMP-9 is nitric oxide (NO)-dependent [62], and VEGF also activates the enzyme [61]. Therefore, the mobilisation of EPCs is considered NO-dependent and stimulated by VEGF. This pathway is similarly exploited by estradiol which likewise augments the mobilisation and proliferation of bone marrow–derived EPCs and their incorporation into a recovering endothelium [63], [64].

Other stimuli for the recruitment of EPCs are Stromal Cell Derived Factor-1 (SDF-1), a significant chemoattractant released by platelets in large quantities [65], and reactive oxygen species (ROS), end products of NOX-2 activity - the enzymatic core of NADPH-oxidase. In reply to endothelial activation, ROS stimulate diverse signalling pathways leading to angiogenic-related responses. Numerous stimuli including angiogenic growth factors, cytokines, shear stress, hypoxia, and G protein–coupled receptor agonists are implicated [66]. Specifically, VEGF has an important role in signal-induced migration, proliferation and reparative angiogenesis [67] whilst, NOX-2 derived ROS are involved in the homing, chemotaxis, invasion, and actin-polarisation of bone marrow derived EPCs [68].

3.2.1.3. EPC homing, migration, invasion, proliferation and differentiation 

Once in the vicinity of ongoing vasculogenesis, progenitor cells interact with the pre-existing endothelial monolayer. The adhesion molecules P-selectin and E-selectin mediate the initial steps of this interaction, with the activation of Eph-B4 receptor by Ephrin B2 increasing EPC-affinity [69]. Integrins on the surface of EPCs further mediate adhesion and facilitate transmigration [69]. In this context, high-mobility group box 1 (HMGB1) activates β1 and β2 integrins, encouraging homing to hypoxic areas [70], whilst α-4 integrin promotes recruitment to sites of tissue remodelling [71].

To achieve transmigration, extracellular matrix degradation by EPCs must occur. In this context, ECFC derived matrix metalloproteinase-2 (MMP-2), secreted in response to increased VEGF, plays a role in the degradation of collagen-IV [59] and Cathepsin L is an essential protease in CAC invasion [72].

For resident EPCs, VEGF, SDF-1 and mechanical forces may initiate differentiation, with Homebox transcription factor A9 (HoxA9) acting as a master switch in the expression of endothelial-committed genes, i.e. endothelial nitric oxide synthase (eNOS), VEGFR-2, and VE-cadherin [73], [74]. Peroxisoma-proliferator activated receptor-δ (PPAR-δ) is also considered to induce proliferation of EPCs and has reported anti-apoptotic effects [75].

Overall, EPCs not only have complicated cell-cell interactions, but have well defined functional roles. Impairment in any of these functional steps could result in defected vasculogenesis. These steps can be influenced by agents targeting the messengers or ligands known to be involved in these processes. Testing of these functionalities is the basis of currently used EPC assessments, i.e. combinations of migration, invasion, tube forming assays and xenograft models of implantation.

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4. Endothelial progenitors and the developing fetus 

Haemangioblasts reside in the blood islands of the early embryo [15] and during embryogenesis undergo differentiation, with those in the periphery giving rise to endothelial progenitors, and those in the centrum of the blood islands generating haematopoietic precursors - having potentially undergone a prior haemotogenic endothelial stage [76].

Although no direct evidence is available to confirm their origin, the angioblasts observed in the fetus are identical with ECFCs, and their phenotype and capacity to proliferate and differentiate are undoubtedly analogous. These angioblasts express Vascular Growth Factor Receptor (VEGFR), Vascular Endothelial-cadherin (endothelial junction molecule, CD144), but not CD45 (pan-haemopoietic) surface markers. While conversely, haemopoietic progenitors (haematoblasts) express CD45 and c-Kit (a cytokine receptor on the surface of haematopoietic stem cells, also known as CD117). With the diverging point in development of haematoblast and angioblast at the VEGFR+/VE-cadherin-/CD4- stage [16].

During the maturation of blood vessels, the expression of surface markers follows a specific sequence: TAL1 (T-cell acute Lymphocytic Leukaemia-1) and VEGFR are expressed first, followed by the Platelet Endothelial Cell Adhesion Molecule (PECAM; CD31), CD34, VE-cadherin, and later Tie-2. Subsequently, TAL1 is down-regulated in the endothelial cells of mature vessels [77]. Loose aggregations of cells expressing endothelial markers have been observed throughout the entire embryonic mesoderm, except on the notochord and precordal plate, and these aggregations have been shown to form definitive blood vessels [78]. Some endothelial lineages may also develop directly from the mesothelium without passing through the haemangioblast stage. However, this mode of vessel formation is primarily present in the somites [79].

Although several signals are known to control vasculogenesis in the fetus, none have yet been defined for the induction of angioblasts or haemangioblasts. Whilst, endodermal signals (e.g. those which influence the Indian Hedgehog and Wnt signalling pathways) have been recognised in the formation of tubal structures [80], Fibroblast Growth Factor (FGF) and Bone Morphogenetic Proteins 2 and 4 (BMP 2 and 4) are proposed modulators of haemato-endothelial differentiation [81] and early vasculogenesis [82]. The most proximal signalling molecule in endothelial development is Vascular Endothelial Growth Factor (VEGF) [83], which forms a complex with neurophilin-1(NP1), a semaphorin receptor, that enhances binding to VEGF receptor-2 (VEGFR-2) resulting in anti-apoptotic signals, and the promotion of endothelial progenitor survival [84].

Additional intracellular and extracellular signals for vessel formation include, Hypoxia Inducible factor-1 (HIF-1), Endothelial Growth Factor-like-domain, multiple 17 (Egfl-17), neurophilins and plexins and Ephrin-B2. Of these, HIF-1 induces blood vessel growth at sites of hypoxia [85], whilst Egfl-17 is essential in the linear arrangement of angioblasts. Neurophilins and plexins, two classes of semaphorine receptors, direct microvessel growth and branching to their targets [86], whilst Ephrin-B2, a membrane ligand exclusive to arterial cells, matches the arterial and venous ends of growing capillaries through reciprocal signalling with its opposing receptor, Eph-B4, which is uniquely expressed on the venous endothelia [87].

Finally, platelet derived growth factor (PDGF) and tumor necrosis factor (TNF) act as chemoattractants, recruiting pericytes and smooth muscle cells which ultimately form into complex vascular structures [88].

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5. EPCs in pregnancy-related clinical conditions 

Aberrant vasculature and abnormal endothelial function on both the maternal and fetal side are thought to play a role in the pathogenesis of certain pregnancy-related complications and in the pathogenesis of pre-eclampsia and intrauterine growth restriction in particular. The pathogenesis of aberrant vessel formation may be the impaired bioavailability or function of the EPCs forming them. Therefore, altered EPC function may contribute to both these pregnancy complications.

It is clear from the literature that many authors have worked with different hypotheses regarding the role of EPCs, and that the EPC-phenotypes investigated have been often wrongly identified and insufficiently verified. Therefore, many past studies should be interpreted and compared with caution.

5.1. Clinical significance of EPCs in pregnancy 

There have been limited reports of EPC number and function in normal pregnancy and in pregnancies complicated with pre-eclampsia, and the results obtained have been somewhat contradictory. Sugawara et al 2005, found that the number of outgrowing EPCs increased gradually with gestation in normal pregnancy and correlated with the levels of serum estradiol [89], whilst Buemi et al 2007 showed similar results by flow cytometry [90]. In the latter, the antibodies to detect EPCs were inconsequently selected and results expressed as a percentage of the total white cell counts (instead of absolute numbers), something inappropriate given the physiological variations between pregnant and non-pregnant states. In opposition to these findings, Savvidou et al. found that EPC numbers are decreased in normal and multiple pregnancies compared to non-pregnant individuals. They also found a gradual decrease across gestation. EPC numbers in this study were determined as a ratio of ULEX/Ac-LDL double positive cells compared to all fibronectin-adherent peripheral blood mononuclear cells (PBMNCs), but no further verification was attempted [91].

One of the first studies monitoring maternal EPCs hypothesised a role in the pathogenesis of pre-eclampsia. Sugawara et al. found that the number of circulating EPCs was decreased in women with pre-eclampsia as defined by the CFU-HILL count. The rate of cellular senescence was also significantly increased in these pre-eclamptic patients [92]. Nevertheless, a more recent study failed to distinguish a difference between EPC numbers in peripheral blood in normal and pre-eclamptic pregnancies, but the proliferation of pre-eclampsia-derived EPCs in culture was significantly exaggerated [93].

Overall, it could be said that the assessment of EPCs in normal pregnancy and pre-eclampsia requires significant input, as even the basic issues regarding their number and function remain unanswered.

5.2. Clinical significance of EPCs in the developing fetus 

It would seem biologically plausible that progenitor cells are more readily available in fetal life and more active than those in the adult. Ingram et al. performed a head to head comparison of umbilical and adult ECFCs. They found that EPC concentrations were 15 times higher in cord blood than in the adult peripheral circulation. In culture, fetal colonies emerged a week earlier than adult colonies and they were consistently larger, while the cells forming them were smaller in size. Cord blood ECFC-populations doubled at least 100 times without any signs of senescence, while adult ECFC did not exceed more than 20–30 population doublings. The population doubling time was 2.5 fold shorter in cases of fetal ECFCs. Moreover, they responded with more active DNA-synthesis to endothelial mitogenic stimulants [23].

These results not only confirm that fetal ECFCs are more abundant and more active, but also suggest that fetal and adult ECFCs may not be clonally identical entities. This was further elucidated in a single cell culture model, where three different cell populations with different phenotypes have been established. The first cell population forms large colonies, which after re-plating form secondary colonies and tertiary colonies in 9% of cases. One single cell gives rise to as many as 107-1012 descendent cells and their telomerase-activity is high, but they are not immortalised. The second cell population forms colonies larger than 50 cells, but they cannot be successfully re-plated. The third population forms colonies of less than 50 cells. The terms High Proliferative Potential Endothelial Colony Forming Cell (HPP-ECFC), Low Proliferative Potential Endothelial Colony Forming Cell (LPP-ECFC) and Endothelial Cell Cluster (ECC) were suggested to describe these identities. Notably, HPP-ECFC differentiate into LPP-ECFC and ECC, while LPP-ECFC differentiate into ECCs. Strikingly, the most active form - HPP-ECFC - is not present in the adult, and is unique to umbilical blood.

Fetal ECFC numbers appear to change during pregnancy, gradually increasing with advancing gestation. Javed et al. found that ECFC numbers - determined as ECFC colonies per umbilical blood volume - are constant between 24 and 31 weeks of gestation, increase to double this between 32 and 36 weeks, and triple this between 37 and 40 weeks [94]. Whether this reflects an increase in production or reduced consumption in the advanced stages of pregnancy is still unclear.

In addition, Case et al. [95] evaluated the effect of oxidative stress on the proliferative potential of different classes of ECFCs in a single cell culture assay. Oxidative stress reduced the proliferative potential of each sub-population, but the proliferative potential of HPP-ECFCs was affected more significantly than those of LPP-ECFC and EC clusters. Oxidative stress also reduced the vasculogenetic capacity of ECFCs in tubulogenesis assays in vitro and in murine xenograft implantation models. Surprisingly, adult ECFCs were more sensitive to oxidative stress in this respect.

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6. Perspectives and possibilities 

Considering the available evidence, it could be envisaged that EPCs have a yet unrecognised role in the patho-mechanistic origins of placental-derived pre-eclampsia and IUGR, and perhaps also the associated increase in long-term cardiovascular risk. In fact, EPCs could pose a plausible explanation linking these pregnancy complications and Barker's hypothesis [96], i.e. the associated connection between sub-optimal intrauterine growth and adult chronic disease.

In the first instance, it could be envisaged that anomalies in either CAC numbers of recruitment to the early placenta, or the secondary attraction and function of ECFCs, may impair formation of normal placental vessels and influence the embryonic endothelium. In this respect, the significant reduction in proliferative potential of HPP-ECFCs in response to oxidative stress may play an important role. Similarly, functional irregularities could impact on the maternal side, affecting uterine vessels, restricting or attenuating intrauterine blood flow and thus perpetuating placental disease. In this regard, the potential mechanisms are still unclear, but may involve hypoxia or oxidative stress, two proposed utero-placental features of pre-eclampsia and IUGR. In both cases, EPCs (most likely CACs) would be first attracted via HIF-mediated signals, followed by the secondary attraction of ECFCs. In pathological conditions, i.e. under excessive hypoxia, EPC numbers and function may be impaired; whilst in conditions of excessive ROS, peroxynitrite formation could reduce the bioavailability of NO, and thereby influence both EPC mobilisation and recruitment. Given these possibilities, further research is undoubtedly warranted.

In a study by Ingram et al. [44], in vitro hyperglycaemia and the diabetic intrauterine environment resulted in impaired ECFC-colony formation, reduced proliferative capacity and functional irregularities both in angiogenic and xenograft transplant assays. The sensitivity of ECFCs to the hyperglycaemic state may be one explanation for the increased risk of pre-eclampsia in diabetic pregnancies and again may link the diabetic environment to increased life-long cardiovascular risk.

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7. Summary 

The precise phenotype of endothelial progenitor cells remains to be determined, and different approaches make it difficult to compare previous studies. There is indirect evidence that both EPC subtypes play a role in the vasculogenesis of the human placenta. However, there is limited reliable literature about fetal EPCs and their role in the pathogenesis of pregnancy-related complications. Nevertheless, the existing evidence would suggest a potential role and both diagnostic and therapeutic clinical tools may be developed through a better understanding between EPCs and placental vascular complications.

It could be envisaged that a significant amount of clinical benefit would be expected from this field: (i) methods designed to assess EPC numbers and function in pregnancy may be developed into diagnostic tools, (ii) EPCs could be direct targets of medical interventions - medications known to improve EPC function are listed in Table 1, (iii) EPCs could be developed as therapeutic tools themselves, for either autologous or donor cell therapies in the pregnant women or fetus, (iv) autologous ECFCs may be utilised to form artificial blood vessels, which can bypass occlusions in the adult or replace larger vessels in cases of congenital vascular abnormalities in the fetus or new-born, and (v) in the case of pathologies associated with excessive vessel formation, such as malignancies, EPCs may be targets of therapy that impair their function or carry anti-tumor agents directly to sites of tumorigenesis. Time will tell whether these potential benefits will be realised.

Table 1. Pharmaceutical agents known to influence EPC number or function. The pharmaceutical agents included are accompanied by effects on EPCs and modes of action, where known.
Pharmaceutical agentEFFECT on EPCsMode of action
PPAR-δ agonists [97]Stimulates CAC-proliferation and mobilisation. Anti-apoptotic.PI3K/Akt pathway.
Nifedipine [98]Increases CAC number, function, resistence to oxidative stress.
Angiotensin-II [99]CAC senescenceAngiotensin type 1 receptor (AT1R)
Angiotensin-II inhibitors losartan [100], candesartan [101], olmesartan and ilbesartan [102]Increased EPC-numbers migration.C-kit-expression through anti-oxidative mechanisms.
Erythropoietin [103], [104]Increased CAC number, mobilisation, anti-apoptotic. NO-synthesisNO-dependent
StatinsElevated EPC number in bone marrow and circulation [105] Integrin-upregulation. [106]PI3K/Akt pathway.
Sildenafil [107]Increased EPC-numbers.
EstradiolMobilisation and proliferation. [63]NO and MMP-9-dependent [64]
Physical exercise [108], [109]Increased EPC numer, capacity and inhibited apoptosis.NO-dependent increase of VEGF level
Prostaglandin E1 [110]EPC mobilisation and improved function.NO-dependent, increased CXCR4 expression.

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PII: S0143-4004(09)00329-4

doi:10.1016/j.placenta.2009.10.006

Placenta
Volume 31, Issue 1 , Pages 1-10, January 2010

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