Article Text
Abstract
Systemic sclerosis (SSc) is characterised by a progressive microangiopathy that contributes significantly to the morbidity of patients with SSc. Besides insufficient angiogenesis, defective vasculogenesis with altered numbers of endothelial precursor cells (EPCs) might also contribute to the vascular pathogenesis of SSc. However, different protocols for isolation, enrichment, culture and quantification of EPCs are currently used, which complicate comparison and interpretation of the results from different studies.
The aim of the European League Against Rheumatism Scleroderma Trials and Research (EUSTAR) group expert panel was to provide recommendations for standardisation of future research on EPCs. Consensus statements and recommendations were developed in a face to face meeting by an expert panel of the basic science working group of EUSTAR.
The findings were: cardiovascular risk factors and medications such as statins should be described in detail. A detailed description of methods considering isolation, culture, enrichment and detection of EPCs should be given. For in vitro culture of EPCs, no protocol has been shown to be superior to another, but coating with laminin and type IV collagen would resemble most closely the situation in vivo. The endothelial phenotype should be confirmed in all in vitro cultures at the end of the culture period. We recommend using CD133, vascular endothelial growth factor type 2 receptor (VEGFR2) and CD34 in combination with a viability marker for quantification of EPCs in the blood. Finally, exact standard operating procedures for fluorescence-activated cell sorting (FACS) analysis are given that should be strictly followed.
In summary, the EUSTAR recommendations will help to unify EPC research and allow better comparison between the results of different studies.
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The first histopathological hallmark of systemic sclerosis (SSc) is apoptosis of endothelial cells and subsequent vasculopathy. The vasculopathy in patients with SSc results in a decreased capillary blood flow, which can manifest clinically as fingertip ulcers.1 Despite the presence of several stimuli that induce the formation of new vessels such as tissue hypoxia and increased levels of vascular endothelial growth factor (VEGF), appropriate vessel formation does not occur in patients with SSc.2 ,3
New vessels can be formed by angiogenesis and vasculogenesis. Angiogenesis is defined as sprouting of fully differentiated endothelial cells from pre-existing vessels. By contrast, vasculogenesis describes the formation of new vessels de novo by circulating progenitor cells. Accumulating evidence suggests that vasculogenesis occurs in the adult and that endothelial precursor cells (EPCs) play an important role for the homeostasis of the vascular network. EPCs might not only be involved in the formation of new vessels in ischaemic tissues, but might also contribute to the repair of pre-existing vessels.4 ,5 Thus, EPCs might be interesting candidates for novel therapeutic approaches. This is indirectly supported by a recent study indicating that stem cell transplantation might improve microvascular disease in SSc.6 – 8 Furthermore, EPCs could also serve as biomarkers for the individual capacity for vascular repair, new vessel formation and cardiovascular prognosis.9 Recent studies suggest that defective vasculogenesis might also contribute to the vascular pathogenesis of SSc.1
However, different protocols for isolation, enrichment, culture and quantification of EPCs are currently used. This complicates the interpretation of the results and might even lead to contradictory results. We summarise herein the current pitfalls of EPC research and propose recommendations for standardisation of future research on EPCs. These recommendations are on the level of expert opinion and were generated during a face to face meeting of the basic science working group of EUSTAR (European League Against Rheumatism (EULAR) Scleroderma Trials and Research group). The aim of EUSTAR is to foster the awareness, understanding and research of scleroderma and its management throughout Europe.10 This includes clinical11 and basic science12 research in SSc. The following recommendations were also approved by the EULAR Standing Committee for International clinical Studies Including therapeutic Trials (ESCISIT). They might not only be of interest for EPC research in SSc, but also for other rheumatic diseases such as rheumatoid arthritis (RA) and systemic lupus erythaematosus (SLE), in which perturbed vasculogenesis has also been demonstrated.13 – 19
EPCS BECOME IMPORTANT: SUMMARY OF CURRENT KNOWLEDGE ABOUT EPCS IN SSC
Kuwana et al first investigated whether vasculogenesis is impaired in patients with systemic sclerosis.20 In this study, CD34-positive cells were enriched from peripheral blood mononuclear cells by an immunomagnetic technique with a monoclonal antibody to CD34 coupled to magnetic beads. EPCs were defined as circulating cells positive for CD34, CD133 and the type 2 receptor for VEGF (VEGFR2), and quantified by three-colour flow cytometry. Using this definition and method of detection, the absolute numbers of EPCs were found to be lower in patients with SSc than in patients with RA and controls. In patients with SSc, the EPC count did not correlate with the disease subset, disease duration or the modified Rodnan Skin Score (RSS). However, the numbers of EPCs were significantly lower in patients with SSc with pitting scars. Active fingertip ulcers were observed exclusively in patients with the lowest numbers of EPCs. More than 80% of the adherent, CD133 and VEGFR2 positive cells also expressed the endothelial cell markers CD31 and the angiopoietin receptor Tie-2 and took up acetylated low-density lipoprotein (LDL). By contrast, the expression of markers for mature endothelial cells such as vascular endothelium cadherin (VE-cadherin), CD146 and, von Willebrand factor was faint or lacking. In a follow-up study using the same techniques, the authors showed an increase of circulating EPCs by atorvastatin treatment in patients with SSc. However, the impaired functional capacity of EPCs in patients with SSc was not improved by atorvastatin.21
Del Papa et al identified EPCs in whole blood by expression of CD34, VEGFR2 and CD133 with fluorescence-activated cell sorting (FACS).22 Despite using the same surface markers (with, however, antibodies from a different manufacturer), the results were different and EPCs numbers were found significantly increased. Further subgroup analysis showed a negative correlation between the number of EPCs and disease duration. Apart from disease duration, no significant correlations were observed between EPC count and clinical parameters, in particular with digital ulcers. In addition to circulating EPCs, Del Papa et al determined also the number of EPCs in the bone marrow. The numbers of cells positive for CD133 were significantly decreased in SSc. To evaluate the ability of bone marrow cells enriched for CD133 positive cells to differentiate into endothelial cells, cells were cultured in M199 medium supplemented with 10% fetal bovine serum (FBS), VEGF, basic fibroblast growth factor (bFGF) and insulin-like growth factor 1 (IGF-1) on fibronectin-coated flasks. In a second step, cells were purified using fluorescein isothiocyanate (FITC)-labelled agglutinin-1 and anti-FITC coated magnetic beads. After 21 days of cultures, colonies obtained from SSc were rare and smaller than colonies obtained from healthy individuals and showed signs of senescence and stress.
Allanore et al investigated EPC counts in whole blood and potential correlations with clinical parameters in patients with SSc, osteoarthritis (OA) and active RA.23 The authors measured the numbers of cells positive for CD34 and CD133 cells, but did not analyse the expression of VEGFR2. The numbers of CD34/CD133 double positive cells were significantly higher in SSc than in patients with OA, but lower than in patients with RA. The numbers of CD34/CD133 double positive cells increased in parallel with the European Disease Activity Score (DAS). In agreement with the results of Del Papa et al, numbers of CD34/CD133 double cells tended to be higher in early stages of SSc. In another study, this group used a method to enrich immature mononuclear cells: Negative lineage (Lin–) mononuclear cells were obtained by enrichment using a human progenitor cell enrichment cocktail. After incubation with an Fc receptor (FcR) blocking reagent, these cells were then subjected to triple labelling with anti-CD133, anti-CD34 and anti-VEGFR-2. Furthermore, 7-aminoactinomycin D (7AAD) was used for viability staining. EPCs, defined as Lin–/7AAD–/CD34+/CD133+/VEGFR-2+ cells, were quantified in by cell sorting/flow cytometry and by counting late-outgrowth colony-forming units (CFU). Patients with SSc displayed higher circulating EPC counts than controls. Lower EPC counts were associated with higher Medsger severity scores and with digital ulcers. The number of colonies correlated with levels of EPCs, validating the combination of FACS surface markers.24
DIFFERENT SUBPOPULATIONS OF EPCS
Two main populations of EPCs with different origins and function and morphological characterisations have been identified, namely a CD14-positive and a CD14-negative subset. Short-term cultures contain mainly CD14-positive cells. This subset of EPCs consists of transdifferentiated monocytes, which are capable of developing into an endothelial phenotype in certain culture conditions.25 By contrast, long-term cultures consist of CD14-negative EPCs, which form late outgrowth cultures. The CD14-negative subset of EPCs is often referred to as “angioblast-like EPCs” and possesses a high proliferation capacity.26 CD14-positive and CD14-negative EPCs can both form capillary-like structures in vitro. Furthermore, both subsets can mediate re-endothelialisation after vessel injury, improve neovascularisation, and can be incorporated into vessels after short-term culture under conditions promoting their differentiation into EPCs.4
IN VITRO CULTURE OF EPCS
Current methods of detection and quantification of EPCs by flow cytometry rely on the surface expression of CD34, CD133 and VEGFR2. When assessed by this method, the frequency of EPCs in the blood appears quite low (0.01%–0.0001% of peripheral blood mononuclear cells (PBMCs)). The low yield obtained by the method is a major limitation for functional studies. To increase the yield of putative EPCs, in vitro outgrowth techniques are widely used. However, the protocols for culture vary remarkably (fig 1). The vast majority of studies used one of the following three culture media: Medium 199 (Gibco, Carlsbad, California, USA) has been used for the CFU assay by Hill et al without supplements other than fetal bovine serum.9 By contrast, Shintani et al added an undefined “endothelial cell growth supplement”.27 Several other studies used the so-called endothelial growth medium (EGM; Clonetics, San Diego, California, USA) supplemented with bovine brain extract and human epidermal growth factor. Most recently, EGM-2 (Clonetics, San Diego, California, USA) has been introduced as an advanced endothelial culture medium system. In contrast to EGM, EGM-2 contains well defined concentrations of VEGF-A, human fibroblast growth factor 2, human epidermal growth factor, insulin-like growth factor 1, ascorbic acid, heparin and hydrocortisone.
Apart from different culture media, different extracellular matrix proteins have been used for the coating of cell culture dishes, which might also influence the outcome. Some studies have been performed on cells expanded on culture dishes coated with collagen, whereas others used fibronectin or gelatine coated dishes.
The different culture protocols might be enriching for different cell populations, leading to analysis of different cells. In the first protocol summarised in fig 1, all cell populations of the PBMC fraction are cultured together. This implies the risk of contamination with mature circulating endothelial and monocytic cells. To minimise contamination, some authors included a pre-plating step. The rationale behind this pre-plating step is that mature endothelial cells should adhere to the culture surface, whereas EPCs should remain in suspension. In this protocol, non-adherent cells are removed after 24 to 48 h of culture and replated on coated culture dishes. The replated cells are kept in culture for various time periods before analysis.
Besides contamination, another limitation of outgrowth techniques has recently been demonstrated. George et al showed that the frequency of EPCs quantified by this culture method does not correlate with the number of CD34/CD133/VEGFR2 positive cells determined by flow cytometry.28
ENRICHMENT TECHNIQUES BEFORE FACS
Immunomagnetic enrichment of cells positive for CD34 and CD133
CD34 or CD133-positive cells may be enriched from peripheral blood mononuclear cells by immunomagnetic techniques with monoclonal antibodies to CD34 or CD133 coupled to magnetic beads. These magnetically labelled cells are then retained on a column placed in a separator, while unlabeled cells pass through. The retained cells are then eluted after removal from the magnet.
Enrichment of lineage negative cells
Lineage negative cells can be enriched from peripheral blood after the depletion of mature haematopoietic cells (positive lineage cells). For depletion, lineage positive mononuclear cells may be magnetically labelled by using a cocktail of biotin-conjugated antibodies against a panel of so-called “lineage” antigens (CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123 and CD235a). Lineage negative cells may also be obtained by enrichment from whole blood using a human progenitor cell enrichment cocktail. This antibody cocktail crosslinks unwanted lineage positive cells to red blood cells to form immunorosettes. This increases the density of the unwanted (rosetted) cells, such that they pellet along with the free red blood cells when centrifuged over the buoyant density medium.24
FACS MARKERS FOR EPCS
Attempts to enumerate EPCs centred on the use of antibodies to VEGFR2 (also known as KDR) in combination with CD34 to identify EPCs. Because neither is specific for EPCs alone or together, it is unclear whether the data accurately reflect the number of true EPCs in the circulation. The advent of CD133 for use in flow cytometry provided a means for detecting primitive stem cells in the circulation. Gehling et al demonstrated that purified CD133 positive stem cells have the capacity to differentiate into endothelial cells.29 More recent studies have demonstrated that cells positive for CD133 and VEGFR2 in the circulation have functional properties of EPCs.30 ,31 Expression of CD45, which is a marker of differentiated haematopoietic cells, on these cells has been reported by various groups to be positive or negative.31 ,32 Dim expression of CD45 by these cells might be the cause of this confusion; hence, the use of CD45 as a gating reagent for EPCs is not supported.
The phenotyping of EPCs requires a multicolour approach. The use of antibodies to CD133 and VEGFR2 is highly recommended to increase the specificity of the analysis. CD34 should also be added to the former combination although in some instances its lack of specificity might be confusing (table 1).
ISSUES OF QUALITY CONTROLS FOR FACS
Various recommendations for the detection of EPCs by flow cytometry can be made. The recommendations include:
The rigorous cleaning of the flow cytometer to avoid contamination of the sample by debris or residual cells from a previous analysis.
The setting and monitoring of fluorescence detectors sensitivity.
The mandatory collection of a large number of events (at least 500 000) to identify an adequate number of this scarce population.
The use of a real-time viability stain such as 7AAD or propidium iodide. Identification and exclusion of dead cells, which are a major source of non-specific staining, improve the resolution of the assay (fig 2).
The use of a blocking serum to decrease non-specific binding via Fc receptors, another source of non-specific staining.
The establishment of a dump channel to exclude cells not of interest from analysis, which is extremely useful.
EUSTAR RECOMMENDATIONS FOR STUDIES OF EPCS IN SSC
Accumulating evidence suggests that vasculogenesis occurs in the adult and might play a role for vascular homeostasis. However, in SSc, several issues remain unclear and should be in the focus of further research: (a) EPCs have only been demonstrated in vascular lesions in animal models of ischaemia, but not in SSc; (b) the mechanisms by which EPCs contribute to vascular repair and neovascularisation have not fully been elucidated. It remains to be determined whether EPCs mediate their effects in humans independently from mature endothelial cells or whether EPCs function more as bystanders of angiogenesis; (c) results addressing the numbers of EPCs in the blood of SSc are controversial. The initial study suggested a profound decrease of circulating EPCs, whereas subsequent studies found increased numbers of EPCs in patients with SSc. These results might be explained by different disease durations in the patient populations and by different techniques for enrichment of cells before FACS analysis. Other important confounding factor might be differences in the prevalence of coexisting cardiovascular risk factors. Several medications, in particular statins, also affect the numbers of EPCs. Thus, cardiovascular risk factors and medications should be given in detail in future studies on EPCs in patients with SSc. Considering the heterogeneity of the disease and the large number of confounding factors influencing the number of EPCs, studies with small number of patients are of limited help and should be avoided in future studies.
Methodical differences likely also account for different results in EPC studies. Different surface markers, enrichment techniques and operating procedures for FACS analysis, and specific culture conditions result in the characterisation of different subpopulations of EPCs. While we are aware that these different subpopulations of EPCs represent different aspects of EPC biology and that there is no clear cut “wrong” or “correct” way of identifying “true” EPCs, this expert panel believes that some basic methodical guidelines should be followed in future EPC studies.
Manuscripts should include a detailed description of methods considering the critical points mentioned above. For in vitro culture of EPCs, no protocol has been shown to be superior to another. However, as the contents of EGM-2 are best defined, we suggest using this medium for future experiments. Systematic studies on the different extracellular matrix components for coating of the culture dishes are not available, but we feel that coating with laminin and type IV collagen would be least artificial as they resemble most closely the vascular basal membrane. For all in vitro cultures, the endothelial phenotype should be confirmed at the end of the culture period. Regarding surface markers, we recommend evaluation of CD133, VEGFR2 and CD34 in combination with a viability marker in a multiparameter flow cytometer. Finally, standard operating procedures for FACS analysis as outlined above should be strictly followed. Our key recommendations are summarised in table 2.
These recommendations will help to unify future EPC research in the field of SSc and also might be of interest for other rheumatic diseases with abnormal vasculogenesis such as RA and SLE.
REFERENCES
Footnotes
Competing interests: RG, SG, FM, AA, UAW, AG, UM-L, AT and MM-C do not declare any specific conflicts of interest.
Funding: JHWD was supported by ELAN grant 53410022 of the University Erlangen-Nuremberg, grant A20 of the Interdisciplinary Center of Clinical Research (IZKF) in Erlangen and Career Support Award of Medicine of the Ernst Jung Foundation. JHWD received research grants and/or honoraria from Novartis, Cell Signal, Actelion, Encysive. OD has received research grants and/or served as a consultant and/or received honoraria from Encysive, Actelion, Ergonex, Array Biopharma and NicOx. YA and JA were supported by Association des Sclérodermiques de France, Société Française de Rhumatologie, INSERM, Agence Nationale pour la Recherche (grant number R07094KS). YA received honoraria (less than US$10 000) for participation in meetings organised by Actelion and by Encysive.