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Rapid and large increase of the frequency of circulating endothelial colony-forming cells (ECFCs) generating late outgrowth endothelial cells in patients with acute myocardial infarction

2008; Elsevier BV; Volume: 37; Issue: 1 Linguagem: Inglês

10.1016/j.exphem.2008.09.007

1873-2399

Margherita Massa, Rita Campanelli, Elisa Bonetti, Maurizio Ferrario, Barbara Marinoni, Vittorio Rosti,

Immune cells in cancer

A spontaneous mobilization of endothelial progenitor cells (EPCs) was described in the early phase of acute myocardial infarction (AMI) [1Shintani S. Murohara T. Ikeda H. et al.Mobilization of endothelial progenitor cells in patients with acute myocardial infarction.Circulation. 2001; 103: 2776-2779Crossref PubMed Scopus (1045) Google Scholar, 2Massa M. Rosti V. Ferrario M. et al.Increased circulating hematopoietic and endothelial progenitor cells in the early phase of acute myocardial infarction.Blood. 2005; 105: 199-206Crossref PubMed Scopus (426) Google Scholar, 3Wojakowski W. Tendera M. Michalowska A. et al.Mobilization of CD34/CXCR4+, CD34/CD117+, c-met+ stem cells, and mononuclear cells expressing early cardiac, muscle, and endothelial markers into peripheral blood in patients with acute myocardial infarction.Circulation. 2004; 110: 3213-3220Crossref PubMed Scopus (406) Google Scholar]. In these studies, EPCs were investigated either by cytofluorimetric analysis and/or by in vitro culture (so-called colony-forming unit endothelial [CFU-End]), according to the original methods by Asahara et al. [4Asahara T. Murohara T. Sullivan A. et al.Isolation of putative progenitor endothelial cells for angiogenesis.Science. 1997; 275: 964-967Crossref PubMed Scopus (7665) Google Scholar].While immunophenotyping of circulating EPCs is still a matter of debate [5Case J. Mead L.E. Bessler W.K. et al.Human CD34+AC133+VEGFR-2+ cells are not endothelial progenitor cells but distinct, primitive hematopoietic progenitors.Exp Hematol. 2007; 35: 1109-1118Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar], in vitro culture of EPCs has been recently clarified by Ingram et al. [6Ingram D.A. Mead L.E. Tanaka H. et al.Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.Blood. 2004; 104: 2752-2760Crossref PubMed Scopus (1306) Google Scholar], who clearly showed that CFU-End are derived from the hematopoietic system retaining some myeloid progenitor activity with no ability to form secondary endothelial colonies in vitro, although they can facilitate vasculogenesis in vivo. On the contrary, true EPCs can be grown in vitro from adherent mononuclear circulating cells, very likely from their CD45–CD34+ fraction [6Ingram D.A. Mead L.E. Tanaka H. et al.Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.Blood. 2004; 104: 2752-2760Crossref PubMed Scopus (1306) Google Scholar]. The frequency of these progenitors (called endothelial colony-forming cell [ECFC]) is very low compared to that of CFU-End in the peripheral blood (PB) of adult individuals, whereas it is increased in umbilical cord blood (CB) [6Ingram D.A. Mead L.E. Tanaka H. et al.Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.Blood. 2004; 104: 2752-2760Crossref PubMed Scopus (1306) Google Scholar]. Therefore, true EPCs have never been matter of in vitro culture in patients with AMI. We used this approach to investigate whether in AMI patients a mobilization of such a type of progenitor cell occurs and whether the frequency of ECFCs correlates with the frequency of EPCs assessed on the base of their phenotype. According with local institutional review board approval, 13 patients (all male; median age 67 years; range, 42–81 years) with AMI, diagnosed according to standard criteria [2Massa M. Rosti V. Ferrario M. et al.Increased circulating hematopoietic and endothelial progenitor cells in the early phase of acute myocardial infarction.Blood. 2005; 105: 199-206Crossref PubMed Scopus (426) Google Scholar], were studied within a median of 3 hours from the onset of symptoms. Six age- and sex-matched healthy controls (CTRLs), and 12 CB samples were also evaluated. By cytofluorimetric analysis EPCs were evaluated on 50 μL PB or CB collected in ethylene diamine tetraacetic acid-containing tube, incubated with 5 μL mouse anti-human allophycocyanin-CD45, fluorescein isothiocyanate-CD34 (Becton Dickinson Pharmingen, San Jose, CA, USA), phycoerythrin-CD133 (Miltenyi Biotech, Bergisch Gladbach, Germany), or biotin-conjugated anti-vascular endothelial growth factor receptor–2 (VEGFR-2) (Sigma Chemical, St Louis, MO, USA) revealed by 5 μL peridinin-chlorophyll-protein-streptavidin for 30 minutes at 4°C. The appropriate isotype controls were used. After red cells lysis, the cells were centrifuged and the pellets resuspended in 300 μL phosphate buffer with 0.5% fetal calf serum. Cells (2 × 105) were acquired by flow cytometer (FACSCanto, Becton Dickinson), and analyzed by FACSDiva software (BD Bioscience). For ECFC cultures, mononuclear cells (MNCs) obtained from 15 mL heparinized PB from patients with AMI or CB, and at least 50 mL from CTRLs were plated on collagen-coated dishes and cultured in EGM-2 MV medium (Cambrex, East Rutherford, NJ, USA) at 37° C in 5% CO2 for up to 30 days. Nonadherent cells were discarded after 2 days and the medium changed twice a week. The number of ECFCs, expressed as frequency of ECFCs/107 MNCs plated, is shown as median, range. In vitro capillary-formation assay was performed by plating 15 × 103 ECFC-derived endothelial cells on 40 μL Cultrex (Trevigen, Gaithersburg, MD, USA) into a 96-well plate and incubated as for ECFC growth. Plates were scored every 2 hours. Frequency of ECFCs/107 MNCs in patients with AMI (median = 1.9; range, 0–5.3) was higher than in CTRLs (median = 0; range, 0–0.3) and CB (median = 0.17; range, 0–2.2) (p < 0.006 and p < 0.03, respectively). In patients with AMI, the percentage of CD45– on gated CD34+ cells (median = 26.8%; range, 5.6–50) was higher (p = 0.001) than in CTRLs (median = 2.3%; range, 0–2.8) and increased with respect to CB (median = 20.0%; range, 7.1–30.9). There was a significant correlation between the frequency of ECFCs and both the percentage of CD45–CD34+ (R = 0.62; p = 0.024) and the percentage of CD34+ VEGFR 2+ EPCs (R = 0.59; p = 0.030) in patients with AMI, while no correlation was found with the CD34+CD133+ hemopoietic cell subset. ECFCs grown in culture from AMI patients, CB, or healthy CTRLs did not show any difference neither in their ability to form capillary like structures in vitro nor in their antigenic profile (not shown). This is the first report showing that severe tissue damage, such as AMI, can largely increase the ECFC frequency in PB to values at least 10 times higher than those found in both CB and PB from healthy CTRLs; the ECFC frequency in patients is also higher than that observed in the PB of a swine model of AMI recently described, where the number of ECFCs/100 mL only tripled within 45 minutes after the onset of AMI with respect to the baseline condition [7Huang L. Hou D. Thompson M.A. et al.Acute myocardial infarction in swine rapidly and selectively releases highly proliferative endothelial colony forming cells (ECFCs) into circulation.Cell Transplant. 2007; 16: 887-897Crossref PubMed Scopus (40) Google Scholar]. These data suggest that AMI induces either an acute release of the ECFCs from the damaged area or the release of mobilizing molecules (i.e., cytokines) that recruits ECFC progenitors from host vessels or extravascular marrow sites into the circulation. This may represent a temporally related event aimed at fast repair/recovery of tissue damage through angiogenic processes related to an increased number of circulating EPCs more than to a greater efficiency in forming new vessels. Alternatively, ECFC frequency may be physiologically increased as a consequence of severe coronary artery disease not yet associated to ischemia [8Guven H. Sheperd R.M. Bach R.G. Capoccia B.J. Link D.C. The number of endothelial progenitor cell colonies in the blood is increased in patients with angiographically significant coronary artery disease.J Am Coll Cardiol. 2006; 48: 1579-1587Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar], suggesting that these cells may play a role in maintaining homeostasis of the endothelium and possibly reflecting the vascular condition of a subject. The correlation of ECFC frequency/107 MNCs with the spontaneously increased percentage of circulating CD34+CD45–cells suggests that true EPCs belong to this cell subset, and is in keeping with recent studies showing that CD34+CD45– cells sorted from CB or PB of granulocyte colony-stimulating factor–mobilized humans originate ECFCs [6Ingram D.A. Mead L.E. Tanaka H. et al.Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.Blood. 2004; 104: 2752-2760Crossref PubMed Scopus (1306) Google Scholar]. In addition, in patients with AMI, receiving an EPC mobilizer (Darbepoetin), circulating CD34+CD45– cells were found significantly increased 72 hours after the drug administration [9Lipsic E. Voors A.A. van Veldhuisen D.J. Serum erythropoietin levels and infarct size.J Am Coll Cardiol. 2006; 47: 464-471Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar]. The fact that 34+ VEGFR 2+ cells, which differently from CD34+CD45– cells may include hematopoietic progenitors and mature endothelial cells [10Ziegler B.L. Valtieri M. Porada G.A. et al.KDR receptor: a key marker defining hematopoietic stem cells.Science. 1999; 285: 1553-1558Crossref PubMed Scopus (410) Google Scholar], also correlates with ECFC frequency appears to be consistent with the emerging concept that several blood cell populations with angiogenic activity exist. In conclusion, EPC mobilization characterizing the early phase of AMI involves an increase of late outgrowing "true" EPCs represented by ECFCs; further studies are needed to understand the signals stimulating angiogenic cell proliferation or release, the role of these cells in the progression/stabilization of severe coronary artery disease, and their possible impact on clinical outcome. This work has been partly supported by grants from Fondazione IRCCS (Istituto di Ricovero e Cura a Carattere Scientifico) Policlinico S. Matteo 08013001/08.