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From Paper, www.bloodjournal.org by guest on June 14, 2017. personal only. Blood First Edition prepublished online October 2, For 2003; DOI use 10.1182/blood-2003-04-1284 -1- Erythropoietin Regulates Endothelial Progenitor Cells Ferdinand H. Bahlmann1*, Kirsten de Groot1*, Jens-Michael Spandau1, Aimee L. Landry1, Barbara Hertel1, Thorsten Duckert1, Sascha M. Boehm1, Jan Menne2, Hermann Haller1, Danilo Fliser1 1 Division of Nephrology, Department of Internal Medicine, Hannover Medical School, Hannover, Germany 2 Phenos GmbH, Hannover, Germany *both authors contributed equally to the study Correspondence address: Ferdinand H. Bahlmann, MD Dept. Int. Med., Hannover Medical School Carl-Neuberg-Strasse 1, 30625 Hannover, Germany Phone: (49)-511-532-6319 / Fax: (49)-511-552366 E-mail: [email protected] Hemostasis, Thrombosis, and Vascular Biology Short title: Erythropoietin and EPC Word count: [3.072] Sources of support The study was supported by a Hanover Medical School Young Investigator Grant (Kirsten de Groot). We also thank Hoffman-La Roche AG for financial support. Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells Copyright (c) 2003 American Society of Hematology From www.bloodjournal.org by guest on June 14, 2017. For personal use only. -2Abstract [197] Background: Circulating bone marrow-derived endothelial progenitor cells (EPCs) promote vascular reparative processes and neo-angiogenesis, and their number in peripheral blood correlates with endothelial function and cardiovascular risk. We tested the hypothesis that the cytokine erythropoietin (EPO) stimulates EPC in humans. Methods and Results: We studied 11 patients with renal anemia and 4 healthy subjects who received standard doses of recombinant human EPO (rhEPO). Treatment with rhEPO caused a significant mobilization of CD34+/45+ circulating progenitor cells in peripheral blood (flow-cytometry), and increased the number of functionally active EPCs (in-vitro assay) in patients (week 2: 312 ± 31%; week 8: 308 ± 40%;both p<0.01 vs. baseline) as well as in healthy subjects (week 8: 194 ± 15%, p<0.05 vs. baseline). The effect on EPCs was already observed with a rhEPO dose of about 30 IU/kg/week. Administration of rhEPO increased the number of functionally active EPCs by differentiation in vitro in a dose dependent manner, assessed in cell culture and by tube formation assay. Furthermore, rhEPO activates the Akt protein kinase pathway in EPCs. Conclusions: Erythropoietin increases the number of functionally active EPCs in humans. Administration of rhEPO or EPO analogues may open new therapeutic strategies in regenerative cardiovascular medicine. [email protected] Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. -3Key words Angiogenesis Vasculogenesis Endothelial Progenitor Cells Endothelium Erythropoietin Vasculature Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. -4Introduction Stem cell therapy emerges as a promising approach in cardiovascular medicine. Current research focuses on bone marrow-derived endothelial progenitor cells (EPCs), which promote vascular reparative processes 1-3. EPCs are considered to origin from CD34-positive (CD34+) stem cells 3. These cells differentiate via separate pathways into erythrocytes, thrombocytes, various lineages of leukocytes, and also into endothelial cells. EPCs are mainly found in the bone marrow, but may also circulate in the vasculature where they home and incorporate into sites of active neovascularization 1,4-7 . In experimental studies increased neovascularization by these cells improves cardiac function after myocardial ischemia 8-10 . In patients with myocardial infarction the clinical outcome is strongly correlated to the number of mobilized EPCs from the bone marrow 11 . Thus, the search for substances which modulate the number and/or function of EPCs is a matter of considerable interest. For example, vascular endothelial growth factor (VEGF) has been shown to regulate EPC proliferation and differentiation 12. Erythropoietin (EPO) is a cytokine stimulating erythrocyte differentiation. It is mainly produced in the renal interstitium in response to hypoxic stimuli. Currently the main indication for use of recombinant human EPO (rhEPO) is treatment of anemia due to EPO deficiency in patients with chronic renal failure. EPO appears to have also direct biological effects on endothelial cells 13,14 . Furthermore, both VEGF and EPO share important activities with respect to (neo-)angiogenesis 12,15 . The main target of both cytokines seems to be the vasculature 16. Thus EPO could affect EPC proliferation and differentiation as well. We tested the hypothesis that EPO modulates the number of functionally active EPCs in humans. For this purpose we assessed circulating CD34+ cells in whole blood using flow cytometry, and the number of functionally active EPCs in an in-vitro assay during 8 weeks of treatment with standard rhEPO doses in 11 patients with renal anemia and in 4 healthy subjects. Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. -5Methods Study participants and protocol The study protocols were approved by the Hannover Medical School Ethics Committee, and informed consent was obtained from all participants. We studied 11 patients with advanced renal failure (6 males, 5 females, mean age 57 ± 6 years), who were non-smoking Caucasians and had stable renal function for at least 2 months before enrollment. At study entry their serum creatinine concentration was 470 ± 166 µmol/l and EPO blood level 11.3 ± 1.4 U/l (normal range 5 - 25 U/l). Patients with malignant diseases, bleeding conditions, recent cardiovascular events (e.g. myocardial infarction) or active inflammation were excluded from the study. All patients studied received a standard rhEPO therapy for treatment of renal anemia (Erythropoietin beta, NeoRecormon, Hofmann-La Roche AG). None of the patients had received blood transfusions for at least 3 months before study entry and in all of them iron stores were replenished before rhEPO treatment. The starting weekly dose was chosen according to the severity of anemia present in the individual patient, and the mean starting rhEPO dose was 5000 ± 674 IU per week. This dose was adjusted only to a minor extent within the 8 weeks of treatment. The dose of concomitant medications was kept constant during the treatment period. We took blood samples for study purposes during regular outpatient visits before and after 2, 4, 6 and 8 weeks of rhEPO treatment. VEGF blood levels (normal range: 62 – 707 pg/ml) were measured before and after rhEPO therapy using ELISA (R&D Systems). In addition, we analyzed circulating hematopoietic progenitor cells and EPCs in 11 healthy age and gender matched subjects (mean age 57 ± 5 years). Furthermore, 4 healthy subjects (mean age 28 ± 2 years) received 30 IU rhEPO/kg/week (n = 2) and 90 IU rhEPO/kg/week (n = 2) for 8 weeks. In these subjects we took blood samples for study purposes before and after 2, 4, 6 and 8 weeks of rhEPO administration. Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. -6Flow cytometry of circulating stem cells We first analyzed the effects of rhEPO on the total number of circulating hematopoietic progenitor cells (cHPC) before and at indicated time points during rhEPO therapy. These cells are a small population bearing the CD34 and the CD45 surface antigen a gating strategy for flow cytometry on the basis of the ISHAGE guidelines 17 . We adopted 17 , and used the CD34 and CD45 expression patterns as well as their morphological qualities for detection (Figure 1). For this purpose we stained whole EDTA blood within 6 hours after drawing the blood. Thereafter we incubated a volume of 100µl with an appropriate amount of FITClabeled monoclonal mouse anti-human-CD45 antibody (Coulter Beckman) for 20 minutes. For detection of cHPC we added PE-labeled monoclonal mouse anti-human-CD34 antibody (Coulter Beckman) to the sample after titration of the optimal antibody concentration. In addition, we added a PE-labeled mouse IgG1-antibody (Coulter Beckman) to a second antiCD45 stained blood sample as the isotype control. Subsequent lysis was done with ammonium chloride. We acquired at least 200.000 CD45+ cells using an Epics XL cytometer (Coulter Beckman). The absolute number of cHPC was expressed per 100.000 mono- and lymphocytes. Two blinded investigators independently assessed the number of cHPC. Isolation and cultivation of EPCs We isolated peripheral blood mononuclear cells from 14 ml of patients blood using density gradient centrifugation with Bicoll (Biochrome)3, and seeded 107 cells on 6-well plates coated with human fibronectin (Sigma) in endothelial basal medium (EBM-2, Clonetics). The medium was supplemented with EGM-2 Single Quots containing fetal bovine serum, human VEGF-A, human fibroblast growth factor-B, human epidermal growth factor, insulin-like growth factor-1 and ascorbic acid in appropriate amounts. After 4 days in culture, we removed non-adherent cells by washing the plates with PBS. We trypsinated the remaining adherent cells and reseeded 106 cells on fibronectin coated 6-well plates. New media was applied and the cell culture was maintained through day 7. Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. -7Characterization of EPCs We performed fluorescent chemical detection in order to determine the cell type of the attached human peripheral blood mononuclear cells after 7 days in culture. To detect the uptake of 1,1´-dioctadecyl-3,3,3´,3´-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein (acLDL-DiI, Molecular Probes), we incubated the cells with acLDL-DiI (6 µg/ml) at 37°C for 2 hours. Cells were then fixed with 1% paraformaldehyde for 10 minutes and incubated with FITC-labeled Ulex europaeus agglutinin-1 (UEA-1, Sigma) for 1 hour. After the staining, we viewed the samples with an inverted fluorescent microscope (Leica). We counted double stained cells for both UEA-1 and acLDL-DiI as EPCs. Two blinded investigators counted at least four randomly selected high-power fields. Dose dependency of rhEPO action on EPCs Isolation and cultivation of EPCs was performed as mentioned above in 8 experiments, i.e. we studied cells from two healthy volunteers on 4 separate days. After reseeding the cells on day 4 we added different doses of rhEPO to the media. The doses applied were chosen in correspondence to standard therapeutical weekly rhEPO doses for treatment of renal anemia (e.g. 0 IU, 0.2 IU/ml ≈ 1000 IU/week, 0.6 IU/ml ≈ 3000 IU/week, and 1.2 IU/ml ≈ 6000 IU/week). Characterization of EPCs on day 7 was performed with fluorescent chemical detection as described above. Effect of rhEPO on tube formation We used a tube formation test as described previously 18 . Briefly, DiI-labeled EPCs (2x104) were co-plated with HUVECs (4x104) on a 4 well glass slide precoated with 250 µL of ECMatrixTM (Chemicon International) in 500 µl EBM-2 with addition of 0.2, 0.6, 1.2 or 2.4 U/ml of rhEPO or without rhEPO. After 6 hours of incubation in 5% CO2 humidified atmosphere at 37°C, the three-dimensional organization of the cells was examined under an inverted phase-contrast photomicroscope using following grades: 0: individual cells, well separated; 1: cells begin to migrate and align themselves; 2: capillary tubes visible, no sprouting; 3: sprouting of new capillary tubes visible; 4: closed polygons begin to form; 5: Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. -8complex mesh like structures develop. The proportion of EPCs in tubes was determined. Two blinded investigators examined 10 randomly selected high-power fields; the inter-assay variability was below 6% (10 repeated experiments under identical conditions). We performed 4 experiments on separate days with cells obtained from 2 healthy subjects. Proliferation assay in cultured EPCs In order to clarify whether the increase in EPC number results from EPC proliferation or cell mobilization and differentiation, we performed a CFDA SE assay for tracing asynchronous cell divisions in cultured EPCs. For this purpose we stained peripheral blood mononuclear cells from healthy subjects with 0.1 µM CFDA SE (Vybrant CFDA SE Cell Tracer Kit, Molecular Probes) 19 . From these cells EPCs were isolated as described above and cultured in the presence of EGM-2 in three separate batches. We added the proliferation inhibitor mitomycin C (10µg/ml) to the second batch and 1.2 IU rhEPO/ml to the third batch. Proliferation of cells was detected by flow cytometry. Activation of Akt protein kinase We assessed the influence of rhEPO on the intracellular activation of Akt protein kinase in vitro using cultured day 7 EPCs from healthy volunteers. Polyclonal antibodies against Phospho-Akt (Ser473) and Total-Akt (both Cell Signaling) were used to assess Akt activation by Western Immunoblotting protocol as described in detail elsewere 20. Statistical analysis We analyzed data on whole blood cHPC and the number of EPCs in culture during 8 weeks of rhEPO treatment using one way ANOVA for non-parametrical comparison of repeated observations, i.e. a Kruskal-Walis-Test (InStat software, GraphPad Software Inc.). If not stated otherwise, data from all other experiments were analyzed using the same test. The statistical significance was set at a p level of <0.05. Data are given as mean ± SEM. Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. -9Results Effect of rhEPO on circulating peripheral blood hematopoietic progenitor cells The absolute number of cHPC before the start of rhEPO treatment in renal patients ranged from 46 to 139 cells per 100.000 analyzed CD45+ mononuclear cells. These values were set as baseline, namely 100%. Accordingly, we observed a significant 1.5-fold increase of the total number of circulating hematopoietic progenitor cell after 2 weeks (152 ± 11%; p<0.01 vs. baseline) and 4 weeks (140 ± 14%; p<0.05 vs. baseline) of rhEPO treatment. Thereafter, the total number of these cells decreased towards baseline value (week 6: 120 ± 8%; week 8: 105 ± 10%, n.s. vs. baseline). An increase in the total number of cHPC was seen in every patient. Effect of rhEPO on EPCs To evaluate the effect of rhEPO on EPC number and function, we isolated mononuclear cells from each patient’s and healthy subject´s blood before and at 2, 4, 6 and 8 weeks after starting rhEPO administration. The culture conditions were in favor of selective EPC plate adherence. After culturing the cells for 7 days, we identified adherent EPCs by acLDL-DiI uptake and concomitant UEA-1 binding 21,22. The absolute number of functionally active EPCs before the start of rhEPO treatment in the group of patients studied ranged from 31 to 90 double positive cells per high-power field. The absolute number of functionally active EPCs in the 11 age and gender matched healthy subjects was significantly (p<0.01) higher; it ranged from 135 to 452 double positive EPCs per high-power field (Figure 2). The absolute number of functionally active EPCs before the start of rhEPO treatment in the group of patients were set as baseline, namely 100%. Already at 2 weeks of rhEPO treatment the total number of functionally active EPCs increased about 3-fold, namely to 312 ± 31%. This highly significant increase was maintained throughout the study (week 4: 276 ± 40%; week 6: 318 ± 56%; week 8: 308 ± 40%) (Figure 3). We observed no difference in the EPC response to rhEPO therapy in those patients who Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 10 had received a weekly rhEPO dose above 5.000 IU (n = 6; 6667 ± 422 IU/week) and in those patients who received a rhEPO dose below 5.000 IU per week (n = 5; 3200 ± 735 IU/week). In both groups of patients EPC numbers increased after 8 weeks of rhEPO therapy to a similar extent, i.e. to 294 ± 62% in the former group and to 311 ± 58% in the latter group (Figure 3). The absolute number of EPCs in rhEPO treated patients after 8 weeks of therapy was comparable to the absolute EPC number in matched untreated control subjects (238 ± 28 vs. 217 ± 27 EPCs per high power field; Figure 2). We found a marked increase in EPC number under rhEPO therapy in every patient studied, and representative high power fields from one patients´ EPC cultures before and after 8 weeks on rhEPO treatment are shown in Figure 4. For comparison, we present the EPC culture of an age- and sex-matched healthy subject. In 4 healthy subjects treated with rhEPO we observed a 2-fold increase in the absolute number of EPCs already after 2 weeks of treatment, namely to 196 ± 22%. This increase was maintained throughout the treatment period (week 4: 229 ± 5%; week 6: 217 ± 23%; week 8: 194 ± 15%). The absolute number of EPCs after 8 weeks of rhEPO application was significantly higher as compared with baseline (p<0.01; paired t-test). Again, we observed a similar increase in the number of EPCs with the lower and higher dose of rhEPO used. In vitro experiments with rhEPO Figure 5a shows the in vitro effect of rhEPO on EPCs. The number of double positive cells increased steadily with escalating rhEPO doses in the cell culture medium from baseline (100%) to a maximum of 192% with 1.2 IU/ml rhEPO (p<0.05 vs. baseline). In the CFDA SE assay we observed a two-fold increase in the number of attached EPCs in the presence of 1.2 IU rhEPO/ml compared to EGM-2 alone. This increase was not the result of EPC proliferation, because we did not detect cell divisions by analyzing CFDA SE fluorescence using flow cytometry. Mitomycin C treated cells served as a negative control. Supplementation of 1.2 IU/ml rhEPO to the cell culture medium caused a marked and time dependent phosphorylation of the Akt protein kinase in EPCs (Figure 5b). Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 11 Figure 6 presents data from the tube formation assay. Supplementation of rhEPO significantly stimulated the formation of tube like structures in a dose-dependent manner (1.2 IU/ml vs. baseline; p<0.05). EPCs made a substantial contribution to the cellular network. Blood count and VEGF blood levels in study patients Table 1 summarizes data on blood counts during 8 weeks of rhEPO therapy in renal patients. As expected, the mean hemoglobin concentrations increased steadily and reached the anticipated target level after 8 weeks of therapy. In accordance with the European Renal Association guidelines on the treatment of renal anemia with rhEPO 23 , we achieved a target hematocrit level of 33.3% after 8 weeks of follow-up. The mean total number of leukocytes did not change, but we observed a small but non-significant increase in the number of thrombocytes. There was no significant change in VEGF blood levels with rhEPO therapy in renal patients (before: 430 ± 72 pg/ml; at the end: 356 ± 115 pg/ml). Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 12 - Discussion Our main finding was that rhEPO significantly regulates EPC number in humans. Importantly, the effect of rhEPO on EPCs in renal patients and in healthy subjects was achieved with a standard therapeutic dose, contrasting most previous observations from in-vitro studies on cardiovascular effects of EPO, in which supratherapeutic doses were used 13,14,24. Although in vitro the effect of rhEPO on EPCs was dose dependent, our in vivo observations in renal patients as well as in healthy subjects permit the conclusion that even low rhEPO doses with respect to treatment of renal anemia have significant effects on EPCs in humans. The effect of rhEPO on EPCs in renal patients was by far more marked than the increase in total erythrocyte number respectively hematocrit. These observations together with the results of the tube formation assay confirm that EPO has a potent effect on the number of functionally active EPCs in humans. Moreover, our observations in the CFDA SE cell proliferation assay are in line with findings from a recent study in laboratory animals showing a marked effect of rhEPO on mobilization of EPCs from the bone marrow25. In accordance with data from experiments using statins we have shown that rhEPO activates the intracellular Akt protein kinase pathway in human EPCs 26. We hypothesized that in adults EPO remains a key molecule in the process of vascular repair and (neo-) angiogenesis by stimulating EPCs. Indeed, our findings are in line with results of experiments using VEGF as a stimulus, i.e. a major regulatory cytokine in the process of (neo-) angiogenesis 12 . VEGF plasma levels in our patients did not change with rhEPO treatment, however, pointing to direct effect of rhEPO on EPCs. Thus the vasculature seems to be the main target for both VEGF and EPO 15,16. Emerging data on the beneficial role of EPCs in patients at high risk for cardiovascular events make our results all the more relevant. Patients with renal failure are an important case in point 27 . Most die of complications related to atherosclerosis, namely myocardial infarction and stroke. Possibly, impaired vascular repair mechanisms may contribute to the problem. Treatment with rhEPO reduces left ventricular mass, ameliorates exercise related cardiac ischemia, and improves outcome in patients with advanced renal Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 13 failure 28,29 . These salutary actions are thought to be the consequence of improved tissue oxygenation. A reduced number and/or impaired function of EPCs due to EPO deficiency could be another causal factor for the high cardiovascular morbidity and mortality in renal patients, however. Replacement therapy with rhEPO may ameliorate this problem via the endothelial cell pathway. Impaired renal function of any degree emerged as an important independent cardiovascular risk factor 30,31 , however, and many of these patients have renal failure due to cardiovascular pathology such as heart failure, atherosclerosis or hypertension. Two studies in patients with impaired renal function due to heart failure suggested that rhEPO treatment may significantly improve outcome in these patients 28,32. Thus administration of rhEPO could be beneficial in other cardiovascular high-risk populations as well. The results of a recent experimental study are of considerable interest in this respect: application of EPO reduced the extent of stroke in laboratory animals 33. In conclusion, our results document that EPO markedly mobilizes functionally active EPCs in humans. Since even in subjects without manifest cardiovascular complications the number of EPCs significantly correlates with endothelial function and cardiovascular risk 34 , stimulation of EPC number and/or function with rhEPO or EPO analogues may open new therapeutic strategies in vascular medicine 16,24,35,36. Acknowledgements We thank Drs Dumann, Haubitz, Hiss, Lonnemann and Paetow for referring patients to the study. We also thank Dr. Koksch (Beckman Coulter) for fruitful discussions. Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 14 - References 1. Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M, Kearne M, Magner M, Isner JM. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res. 1999;85:221-228 2. Peichev M, Naiyer AJ, Pereira D, Zhu Z, Lane WJ, Williams M, Oz MC, Hicklin DJ, Witte L, Moore MA, Rafii S. Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. Blood. 2000;95:952-958 3. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275:964-967 4. Crosby JR, Kaminski WE, Schatteman G, Martin PJ, Raines EW, Seifert RA, Bowen-Pope DF. Endothelial cells of hematopoietic origin make a significant contribution to adult blood vessel formation. Circ Res. 2000;87:728-730 5. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, Li T, Isner JM, Asahara T. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A. 2000;97:34223427 6. Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, Magner M, Isner JM, Asahara T. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med. 1999;5:434-438 7. Schatteman GC, Hanlon HD, Jiao C, Dodds SG, Christy BA. Blood-derived angioblasts accelerate blood-flow restoration in diabetic mice. J Clin Invest. 2000;106:571-578 8. Edelberg JM, Tang L, Hattori K, Lyden D, Rafii S. Young adult bone marrow-derived endothelial precursor cells restore aging-impaired cardiac angiogenic function. Circ Res. 2002;90:E89-93 9. Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida S, Masuda H, Silver M, Ma H, Kearney M, Isner JM, Asahara T. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation. 2001;103:634-637 10. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, Itescu S. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med. 2001;7:430-436 11. Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dimmeler S. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001;89:E1-7 12. Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, Inai Y, Silver M, Isner JM. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. Embo J. 1999;18:3964-3972 13. Carlini RG, Dusso AS, Obialo CI, Alvarez UM, Rothstein M. Recombinant human erythropoietin (rHuEPO) increases endothelin-1 release by endothelial cells. Kidney Int. 1993;43:1010-1014 14. Haller H, Christel C, Dannenberg L, Thiele P, Lindschau C, Luft FC. Signal transduction of erythropoietin in endothelial cells. Kidney Int. 1996;50:481-488 Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 15 15. Jaquet K, Krause K, Tawakol-Khodai M, Geidel S, Kuck K. Erythropoietin and VEGF exhibit equal angiogenic potential. Microvasc Res. 2002;64:326 16. Goldman SA, Nedergaard M. Erythropoietin strikes a new cord. Nat Med. 2002;8:785-787 17. Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering. J Hematother. 1996;5:213-226 18. Tepper OM, Galiano RD, Capla JM, Kalka C, Gagne PJ, Jacobowitz GR, Levine JP, Gurtner GC. Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation. 2002;106:2781-2786 19. Bronner-Fraser M. Alterations in neural crest migration by a monoclonal antibody that affects cell adhesion. J Cell Biol. 1985;101:610-617 20. Bommakanti RK, Vinayak S, Simonds WF. Dual regulation of Akt/protein kinase B by heterotrimeric G protein subunits. J Biol Chem. 2000;275:38870-38876 21. Voyta JC, Via DP, Butterfield CE, Zetter BR. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol. 1984;99:2034-2040. 22. Jackson CJ, Garbett PK, Nissen B, Schrieber L. Binding of human endothelium to Ulex europaeus I-coated Dynabeads: application to the isolation of microvascular endothelium. J Cell Sci. 1990;96:257-262. 23. Cameron JS. European best practice guidelines for the management of anaemia in patients with chronic renal failure. Nephrol Dial Transplant. 1999;14 Suppl 2:61-65 24. Siren AL, Fratelli M, Brines M, Goemans C, Casagrande S, Lewczuk P, Keenan S, Gleiter C, Pasquali C, Capobianco A, Mennini T, Heumann R, Cerami A, Ehrenreich H, Ghezzi P. Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proc Natl Acad Sci U S A. 2001;98:4044-4049 25. Heeschen C, Aicher A, Lehmann R, Fichtlscherer S, Vasa M, Urbich C, Mildner-Rihm C, Martin H, Zeiher AM, Dimmeler S. Erythropoietin is a potent physiological stimulus for endothelial progenitor cell mobilization. Blood. 2003 26. Llevadot J, Murasawa S, Kureishi Y, Uchida S, Masuda H, Kawamoto A, Walsh K, Isner JM, Asahara T. HMG-CoA reductase inhibitor mobilizes bone marrow-derived endothelial progenitor cells. J Clin Invest. 2001;108:399-405 27. Drueke TB. Aspects of cardiovascular burden in pre-dialysis patients. Nephron. 2000;85 Suppl 1:9-14 28. Silverberg DS, Wexler D, Blum M, Keren G, Sheps D, Leibovitch E, Brosh D, Laniado S, Schwartz D, Yachnin T, Shapira I, Gavish D, Baruch R, Koifman B, Kaplan C, Steinbruch S, Iaina A. The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe, resistant congestive heart failure improves cardiac and renal function and functional cardiac class, and markedly reduces hospitalizations. J Am Coll Cardiol. 2000;35:1737-1744 29. Metivier F, Marchais SJ, Guerin AP, Pannier B, London GM. Pathophysiology of anaemia: focus on the heart and blood vessels. Nephrol Dial Transplant. 2000;15 Suppl 3:14-18 30. Mann JF, Gerstein HC, Pogue J, Bosch J, Yusuf S. Renal insufficiency as a predictor of cardiovascular outcomes and the impact of ramipril: the HOPE randomized trial. Ann Intern Med. 2001;134:629-636 31. Hillege HL, Girbes AR, de Kam PJ, Boomsma F, de Zeeuw D, Charlesworth A, Hampton JR, van Veldhuisen DJ. Renal function, neurohormonal Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 16 activation, and survival in patients with chronic heart failure. Circulation. 2000;102:203-210 32. Silverberg DS, Wexler D, Sheps D, Blum M, Keren G, Baruch R, Schwartz D, Yachnin T, Steinbruch S, Shapira I, Laniado S, Iaina A. The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study. J Am Coll Cardiol. 2001;37:1775-1780 33. Cerami A. Beyond erythropoiesis: novel applications for recombinant human erythropoietin. Semin Hematol. 2001;38:33-39 34. Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003;348:593-600 35. Celik M, Gokmen N, Erbayraktar S, Akhisaroglu M, Konakc S, Ulukus C, Genc S, Genc K, Sagiroglu E, Cerami A, Brines M. Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury. Proc Natl Acad Sci U S A. 2002;99:2258-2263 36. Calvillo L, Latini R, Kajstura J, Leri A, Anversa P, Ghezzi P, Salio M, Cerami A, Brines M. Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling. Proc Natl Acad Sci U S A. 2003 Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 17 - Figure legends Figure 1 Gating strategy for detection of circulating hematopoietic progenitor cells (cHPC) on the basis of the ISHAGE guidelines17. We used CD34 and CD45 expression as well as the morphological qualities of cHPC for their detection. The upper panel (A, B, C and D) represents a patient sample stained with anti-CD45-FITC and anti-CD34-PE. The lower panel (E, F, G and H) shows the same sample using an isotype control for anti-CD34. We first counted 200.000 CD45 positive cells (A and E). From this primary gate, cHPC were identified using the additional expression of CD34 (B and F). The CD45 antigen expression (C and G) and the characteristic light scatter properties (D and H) are shown. Figure 2 Absolute numbers (and box plots) of EPCs per high power field before and after 8 weeks of rhEPO treatment in 11 patients with renal anemia. For comparison we show absolute EPC numbers of 11 age and gender matched healthy subjects. The absolute number of functionally active EPCs in renal patients before rhEPO therapy was significantly lower than in healthy subjects (p<0.01), but increased to comparable levels during treatment with rhEPO. Figure 3 Quantitative assessment of cultured endothelial progenitor cells (EPCs) from 11 patients with renal anemia during rhEPO treatment. Administration of rhEPO clearly resulted in a marked increase in total EPC number within 8 weeks of therapy. * = p<0.01 – comparison EPCs number at week 2, 4, 6 and 8 vs. baseline. We observed no difference in the EPC response to rhEPO therapy in those patients who had received a weekly rhEPO dose above 5.000 IU (n = 6) and in those patients who received an rhEPO dose below 5.000 IU per week (n = 5). Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 18 Figure 4 Representative images of cultured endothelial progenitor cells in one patient before (A) and after 8 weeks of rhEPO treatment (B). An age- and sex-matched healthy subject is shown for comparison (C). Figure 5 (A) Quantitative assessment of cultured endothelial progenitor cells (EPCs) of 2 healthy subjects with supplementation of rhEPO to the cell culture medium. With supplementation of 1.2 IU rhEPO/ml to the medium we observed a significant, approximately two-fold increase in the total number of EPCs (* = p<0.05 vs. baseline). (B) Representative Western immunoblots of Akt phosphorylation are shown as time dependent changes in Akt phosphorylation at serine 473 after exposure of EPCs to rhEPO (1.2 IU/ml). Figure 6 Tube formation index of co-cultered EPCs (upper pannel). Supplementation of rhEPO significantly stimulated the formation of tube like structures in a dose-dependent manner (* = p<0.05 vs. baseline). Representative photomicrographs of tube formation with 1.2 IU/ml rhEPO (lower pannel). Fluorescent labeled EPCs (red) were co-plated with HUVECs (transparent) to form tubular structures. Both cell types were stained with endothelial cell specific UEA-1 (green fluorescence). Superimposed light and fluorescent images of identical fields reveal that EPCs made a substantial contribution to the cellular network. Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 19 Table 1: Blood counts from 11 renal patients before and during 8 weeks of rhEPO therapy. Erythrocytes Blood hemoglobin Hematocrit Thrombocytes Leukocytes [x10³ /µl] [g/l] [%] [x106 /µl] [x10³ /µl] Baseline 3.51 ± 0.13 9.6 ± 0.3 29.8 ± 1.0 247 ± 25 7.7 ± 1.1 2 weeks 3.48 ± 0.08 10.3 ± 0.3 32.0 ± 1.0 273 ± 45 6.9 ± 0.9 4 weeks 3.56 ± 0.12 10.7 ± 0.3 32.5 ± 0.9 296 ± 28 6.8 ± 0.7 6 weeks 3.62 ± 0.08 10.9 ± 0.3 34.0 ± 1.0 307 ± 34 6.6 ± 0.4 8 weeks 3.79 ± 0.11 11.1 ± 0.3a 34.6 ± 1.3a 311 ± 30 7.7 ± 0.6 a p < 0.05 – comparison of week 8 Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells vs. baseline - 20 - From www.bloodjournal.org by guest on June 14, 2017. For personal use only. Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 21 - Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 22 - Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 23 - Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 24 - Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. - 25 - Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells From www.bloodjournal.org by guest on June 14, 2017. For personal use only. Prepublished online October 2, 2003; doi:10.1182/blood-2003-04-1284 Erythropoietin regulates endothelial progenitor cells Ferdinand H Bahlmann, Kirsten de Groot, Jens-Michael Spandau, Aimee L Landry, Barbara Hertel, Thorsten Duckert, Sascha M Boehm, Jan Menne, Hermann Haller and Danilo Fliser Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Advance online articles have been peer reviewed and accepted for publication but have not yet appeared in the paper journal (edited, typeset versions may be posted when available prior to final publication). 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