Since endothelial damage and dysfunction play a critical role in atherosclerotic disease, research interest has aimed at evaluating the role of endothelial progenitor cells (EPCs) in vascular endothelial layer maintenance. In contrast to local resident endothelial cells, which have a poor proliferative rate, the regenerative capacity of EPCs and their ability to integrate into blood vessels have been interpreted as playing a protective role in vascular homeostasis. Indeed, the number and function of EPCs correlates with the progression of atherosclerosis, and the accumulation of cardiovascular risk factors or an increased overall risk is inversely associated with the number and function of EPCs. This article focuses on the potential value of blood-circulating EPCs as biomarkers for cardiovascular disease and progression.
Phenotypic and Functional Characterisation of Endothelial Progenitor Cells
The first descriptions of EPCs were by Asahara et al.1 and Shi et al.,2 who demonstrated that, even in adults, bone-marrow-derived haematopoietic progenitor cells (PCs) can give rise to endothelial cells (ECs) that contribute to active neovascularisation in ischaemic tissues. These cells were termed EPCs, and since their discovery intense effort has been focused on defining the role of EPCs in the regeneration of injured endothelium, neovascularisation of ischaemic tissue and cancer angiogenesis. The best characterised source of EPCs are the bone marrow hematopoietic stem cells (HSCs), which are located in the stem cell niche and released into the peripheral blood on mobilisation by chemokines such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor 2 (bFGF-2), stromal-cell-derived factor-1 (SDF-1) or granulocyte macrophage-colony-stimulating factor (GM-CSF), which are synthesised by ischaemic tissues.3–5 Later studies demonstrated that several tissues may be sources of circulating EPCs (reviewed in reference 6).
Until now, the best source of EPCs has been the cord blood, which contains 10-fold more PCs than adult peripheral blood,7 where EPCs remain extremely rare (0.0001%). The paucity of these circulating cells has contributed to the difficulty of identifying these cells and in understanding their biology in vitro and in vivo. Isolation and characterisation of circulating EPCs has been hampered by the absence of a specific gold standard marker. The methods for isolating these cells include adherence culture of total mononuclear cells obtained from fresh blood by density gradient centrifugation, and positive pre-selection of mononuclear cells by antibodies against surface marker (CD133, CD34, CD31 or VEGF receptor-2).1,8–10
EPCs express several markers, including VEGF receptors-2 (VEGFR2, KDR, Flk-1), Ve-cadherin, CD34, platelet endothelial cell adhesion molecule (PECAM; CD31) and von Willebrand factor (VWF), and are also able to incorporate acetylated low-density lipoprotein (AcLDL) and to bind lectins such as BS-1 and ulex europaeus agglutinin-1 (UEA-1), which are usually considered endothelial-specific.11
Identification of a surface marker, AC133, capable of distinguishing between EPCs and vessel-wall-derived ECs has facilitated the ability to isolate and study EPCs in vitro. In fact, AC133 is expressed on subsets of EPCs and HSCs, but in vitro differentiation of these cells results in the loss of AC133 expression, suggesting that EPCs with angioblast potential may be marked selectively with AC133.9,12 After isolation from peripheral blood, the most common method for culturing EPCs is to plate the cells on fibronectin-coated plates using commercially available tissue-culture media with specific growth factors.1,8–10,13 During the culture, cells develop a spindle-shaped, EC-like morphology, and by the end of culture form a cobblestone-like monolayer.
A distinctive feature of freshly isolated CD34+ VEGFR-2+ cells is their lack of ability to adhere to extracellular matrix at the time of isolation; this means they are able to respond to chemotactic factors such as SDF-1 and VEGF. To add confusion, two different types of EPC from peripheral blood mononuclear cells – called ‘early’ and ‘late’ EPCs according to their time-dependent appearance – can be obtained through different culture media. Early EPCs with spindle shapes showed peak growth at two to three weeks and died at four weeks, whereas late EPCs with cobblestone shapes appeared late at two to three weeks, showed exponential growth at four to eight weeks and lived for up to 12 weeks. They shared some endothelial phenotypes, but had different morphologies, proliferation rates and survival features. They also had different gene expression profiles. The two types of EPC show functional differences in the production of nitric oxide (NO) in response to VEGF, tube formation on matrigel and incorporation to the human umbilical vein endothelial cell (HUVEC) monolayer in vitro. Despite these functional differences in vitro, both types of EPC similarly contributed to vasculogenesis in vivo.14,15
Therefore, many studies have reported EPCs in different ways in terms of cell surface markers or culture methods. This suggests that there are heterogeneous cells that have been called EPCs without a clear definition, and as yet there is no standard method of obtaining EPCs from peripheral blood. The heterogeneity of outgoing cell populations, including monocytic cells with no proliferative potential (so-called ‘circulating angiogenic cells’), as well as sub-populations of true endothelial precursors, generating late outgrowth, make the scenario even more complicated.16,17
It is noteworthy that the endothelial characteristics of double-positive cells are different, but these are not always mentioned. For example, a recent work18 demonstrated that the vast majority of peripheral-blood-derived acLDL+ UEA-I+ cells, obtained from short-term cultured mononuclear cells, despite sharing certain characteristics with ECs such as the expression of CD31, express only a very low percentage of the endothelium-specific marker VE-cadherin. This is not the only study showing very low expression of a typical endothelial marker. In another study,19 although the spindle-shaped cells often aligned to form long linear threads after 20 days of culture of CD34+ cells, the expression of VE-cadherin, endothelial NO synthase (eNOS) and vWF was not detectable until 39 days of culture.
From a functional point of view, EPCs also demonstrate some properties typical of both haematopoietic cells and ECs. For example, one feature of EPCs is their ability to establish colony-forming units (CFUs), a property typical of cells of haematopoietic origin in general.20 Moreover, after a culture period of several weeks, EPCs formed monolayers with a typical ‘cobblestone’ endothelial appearance.21,22
Methods of Counting Endothelial Progenitor Cells
When the number of circulating EPCs is determined, most studies refer to freshly isolated EPCs, usually identified as CD34+KDR+ cells. Counting is performed by fluorescent-activated cell sorter (FACS) analysis of blood samples or peripheral blood mononuclear cells stained with monoclonal antibodies against KDR and CD34. After gating for CD34+ cells, double-positive cells are numbered. However, there are many studies where a different cell population is referred to as EPCs: CD133+KDR+ cells or CD34+KDR+CD133+ cells (immature EPCs), and CD34+ or CD34+CD133+ cells (PCs and immature PCs, with the potential to differentiate into both haematopoietic and endothelial cells). The way of expressing the results also differs among studies, with EPC numbers expressed as cells/ml, % of mononuclear cells, % of cells x white blood cell count/100, cells/103 cytometric events, logarithmic transformation in base 10 of EPC numbers and so on.
When EPCs are counted after culture, their number is determined by DiI-Ac-LDL uptake/FITC-UEA-I lectin binding or by colony formation.23,24 For colony quantification, EPC colonies – defined as flat cells surrounding a cluster of round cells – are counted by two blinded investigators using an inverted microscope.
Major Cardiovascular Risk Factors and Endothelial Progenitor Cells
The number and/or functional activity of circulating EPCs have been associated with classic risk factors for cardiovascular disease and have been shown to be modulated by different pathologies. The impairment in EPC numbers and/or function might contribute to a vicious circle resulting in endothelial dysfunction. Recent studies suggest that EPCs seem to be lowered in number or impaired in function by various risk factors, leading to reduced regenerative capacity and increasing the modified integrity of the endothelium. The interference by risk factors with EPC-mediated vascular protection, although occurring via various possible mechanisms, may thereby modulate the endothelial regeneration process. Inadequate repair may have an effect on the atherosclerotic process, involving initiation, progression and lesion destabilisation. EPC dysfunction could have consequences on the pathologies of patients whose intrinsic capacity for arterial repair has been already eroded by risk factors. In these patients, autologous cell therapy would not be effective; instead, they require an additional treatment through drug therapy and/or PC processing.
In any case, assessing circulating EPC numbers only would not be sufficient. As yet, there is no consensus on the possibility of evaluating circulating or cultured EPCs and on their phenotype, so it is not possible to compare all of the studies on this subject.
In hypertensive patients, the degree of hypertension-induced organ damage has been found to be negatively correlated with telomerase activity and positively correlated with EPC senescence.26 The mechanisms by which hypertension could accelerate the senescence of EPCs may be due to an effect of angiotensin II, which was recently demonstrated in vitro to induce EPC senescence through augmentation of oxidative stress25 or to a more general vascular oxidative stress. One limitation of the study is that the hypertensive patients had a number of additional risk factors that could influence EPC senescence, such as hyperlipidaemia, diabetes and smoking.
Diabetes is another major cardiovascular risk factor that causes impairment of neovascularisation. In 20 patients with type 1 diabetes, the number of cultured EPCs was reduced compared with 20 age- and sexmatched control subjects, and was inversely related with levels of glycated haemoglobin (HbA1c).27 The function of EPCs was also impaired (in vitro angiogenesis assay).
In 20 patients with type 2 diabetes, the proliferation of EPCs was decreased compared with 20 age-matched control subjects, and was inversely correlated with patient levels of HbA1c.28 EPCs in subjects with diabetes had reduced adherence to activated HUVECs and decreased tubule incorporation in a Matrigel assay. In another study on 40 patients with type 2 diabetes (24 with peripheral vascular disease [PVD] and 16 without PVD), reduced levels of circulating PCs (CPCs; CD34+) and EPCs (CD34+KDR+) were observed compared with 17 healthy control subjects, and PVD was associated with an extremely low number of CPCs and EPCs in both diabetics and 11 non-diabetics with PVD.29 In our study on pre-diabetes and type 2 diabetes in 219 middle-aged individuals with no pre-diagnosed alterations in carbohydrate metabolism, the number of CPCs and EPCs was significantly reduced in individuals who were found to have diabetes, and was negatively correlated with both fasting and post-challenge glucose in the whole population. While CPCs – but not EPCs – were significantly reduced in pre-diabetic individuals, post-challenge glucose was an independent determinant of the levels of both kinds of cell.30
Peripheral Vascular Disease
A report regarding EPCs in PVD used characterisation by reverse transcriptase polymerase chain reaction (RT-PCR) in only four patients with peripheral obstructive arterial disease who qualified for bone marrow transplantation.31 Expression of EPC/EC-specific molecules (VEGF-R1, CD133, VE-cadherin, KDR, CD31, vWF) in bone marrow mononuclear cells (BM-MNCs) decreased compared with five healthy controls. The same result was obtained for KDR, CD31 and VWF expression in peripheral-blood-derived mononuclear cells (PB-MNCs). Circulating CD34+CD133+ PCs and both circulating and marrow CD34+CD133+KDR+ EPCs were also reduced. In our experience, patients with critical limb ischaemia (CLI) have lower levels of EPC compared with healthy controls. A four-week treatment with iloprost, a prostacyclin analogue, increased EPC numbers in 23 patients with stage III and IV CLI, and also improved clinical and instrumental parameters.32
In patients with stable coronary artery disease (CAD) undergoing coronary artery bypass grafting (CABG), pre-operative values of circulating CD34+CD133+ PCs decreased with increasing age, similar to plasma VEGF levels. These age-associated decreases could not be explained by differences in atherosclerotic risk factors, the cumulative number of risk factors or cardiac function.20 Another study comparing 20 old and 20 young (average age 61 and 25 years, respectively) healthy individuals without major cardiovascular risk factors found no differences in the numbers of circulating CD34/KDR or CD133/KDR EPCs.33 In contrast, lower survival, migration and proliferation of cultured EPCs were observed in the older subjects. The flow-mediated dilation (FMD) correlated univariately with EPC migration and EPC proliferation. Multivariate analysis showed that both functional features were independent predictors of endothelial function.
In hypercholesterolaemia, the number of cultured EPCs is significantly reduced and inversely correlated with total cholesterol and low-density lipoprotein (LDL) cholesterol.34 EPC proliferative, migratory, adhesive and in vitro vasculogenesis capacity is also impaired. The hypothesis is that the reduction in the number of EPCs and their decreased functional activity may represent a novel pathophysiological mechanism of hyper-cholesterolaemia, influencing the endothelial repair process and ultimately contributing to endothelial dysfunction. However, this may not be the only explanation, since bone marrow depletion, increased senescence or apoptosis could be induced by oxidised LDL.35
The number of circulating CD45lowCD34+CD133+ (PCs) and CD45lowCD34+CD133+VEGFR2+ (EPCs) was reduced in 15 chronic smokers (10 light smokers [<20 cigarettes per day] and five heavy smokers [20 cigarettes per day]) compared with 14 non-smokers.36 Smoking cessation led to a rapid restoration of PC/EPC levels. The recovery of EPC levels was greater in light smokers than in heavy smokers. In a culture assay, the number of attaching EPCs was greater in non-smokers than in light smokers, and EPCs from heavy smokers could not be cultured at all. An in vitro study on the influence of nicotine on EPC number and activity showed complex effects: nicotine may induce the augmentation of EPCs with enhanced functional activity at relatively low concentrations.37
Nicotine dose dependently increased the number of EPCs and their proliferative, migratory, adhesive and in vitro vasculogenesis capacity at nicotine concentrations of 10-12–10-8mol/l, so the question of the effect of nicotine on EPCs remains open.
There is a strongly negative relationship between smoking – either active or passive – and the endothelium. Smoking promotes several alterations consisting of functional and structural disorders such as impaired NO release, inflammatory response, platelet activation and atherosclerotic plaque and thrombus formation. Cigarette smoking induces impairment in the repair of endothelial damage, as EPCs, irrespective of their number, are dysfunctional and possibly incapable of restoring endothelial function. However, the effects of tobacco on EPCs are still incompletely known, since there are more than 4,000 smoke constituents potentially affecting EPC biology, either directly or indirectly.38
Multiple Risk Factors
Recent studies have found an association not only between EPCs and single risk factors (age, hypertension, diabetes, hypercholesterolaemia, history of CAD), but also with the number of risk factors and a cumulative risk score, although this is not always calculated in the same way.
Compared with 15 healthy volunteers, in 45 patients with CAD there were reduced levels of both cultured EPCs and circulating CD34+KDR+ cells and impaired migratory response of EPCs, which correlated with risk factors for CAD.39 The number of risk factors significantly correlated with a reduction of EPC levels and number of CD34+KDR+ cells. Analysis of the individual risk factors demonstrated that smokers had significantly reduced levels of EPCs and CD34+KDR+ cells. Moreover, a positive family history of CAD was associated with a reduced number of CD34+KDR+ cells. The impaired migratory response of EPCs was inversely correlated with the number of risk factors. By multivariate analysis, hypertension was identified as a major independent predictor of impaired EPC migration.
Endothelial Progenitor Cells as Prognostic Markers
EPCs have also been suggested to be a marker for cardiovascular risk.40 In 45 healthy men with various degrees of cardiovascular risk but no history of CAD, the level of endothelial PCs was shown to be a potential surrogate biologic marker for vascular function and cumulative cardiovascular risk.
A strong correlation between the number of CFUs of EPCs and the combined Framingham risk factor score of the subjects was found. Endothelial function and the number of PCs were significantly related, with the level of circulating EPCs being a better predictor of vascular reactivity than the presence or absence of conventional risk factors. Moreover, EPCs from subjects at high risk of cardiovascular events had higher rates of in vitro senescence than cells from subjects at low risk.
A more recent study suggested EPCs as biomarkers with prognostic value, with their number being an independent predictor of clinical outcome.41 In a large setting, the level of circulating CD34+KDR+ EPCs and CD133+ immature PCs and the number of CFU-ECs in 519 patients with CAD predicted the occurrence of cardiovascular events, death from cardiovascular causes, revascularisation and hospitalisation, with the potential of helping to identify patients at increased cardiovascular risk and improving risk stratification. However, the univariate analyses showed different correlation between patient variables and CD34+KDR+ cells, CD133+ cells and CFU-ECs. A great variability in CD34+KDR+ EPC levels (range: 12–1,039 CD34+KDR+ cells; mean: 86.3±71.9) was observed. EPC counts were categorised into three groups – low, medium and high EPC levels – and, in univariate analyses, smoking, diuretic and statin therapy were associated with high levels of EPCs whereas low levels were associated with a high left ventricular ejection fraction and treatment with angiotensin-receptor blockers. The association between high EPC levels and smoking is noteworthy, but is in contrast with previous studies showing that the number of circulating PCs and EPCs was reduced in chronic smokers36 and in patients with CAD,39 with smoking having a significant role as an individual risk factor. Therapy with statins and angiotensin-converting enzyme (ACE) inhibitors was associated with high baseline levels of CD133+ cells, whereas low levels were associated with increased LDL cholesterol levels, advanced age and high systolic blood pressure. In a subgroup of 203 patients, the number of CFU-ECs was measured. Therapy with statins and ACE inhibitors was associated with increased numbers of CFU-ECs, whereas reduced numbers were associated with increased LDL cholesterol levels, advanced age, diabetes, smoking and a family history of premature coronary artery disease.
In another recent study, 120 individuals (33 patients with acute coronary syndromes, 44 with stable CAD and 43 healthy controls) were followed up for a median period of 10 months.42 Reduced levels of circulating CD34+KDR+ EPCs independently predicted atherosclerotic disease progression, thus supporting an important role for endogenous vascular repair in modulating the clinical course of coronary artery disease. By univariate analysis for the entire cohort, the classic risk factors (age, hypertension, smoking, family history of CAD) were inversely correlated with EPC numbers.
Because a wide array of methodologies has been used, there is a great confusion regarding data interpretation, which perhaps explains the inconsistent results obtained in different studies. Thus, a reasonable question is: different ingredients, same results? Or, in other words: how we can compare cells obtained using so many different culture conditions and give them the same name? Studies on the effect of risk factors are therefore sometimes a little contradictory, even if they all point in the same direction. Another point of debate is the number of subjects included in the studies, which may compromise the possibility of comparison between the results. For this reason it is very important to compare different methodologies for EPC assessment and the extent of the population of the studies in order to evaluate the experimental results.