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-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
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-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
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-3Key words
Angiogenesis
Vasculogenesis
Endothelial Progenitor Cells
Endothelium
Erythropoietin
Vasculature
Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells
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-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.
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-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.
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-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.
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-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
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-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.
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-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
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- 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).
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- 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).
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- 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
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- 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.
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- 14 -
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28. Silverberg DS, Wexler D, Blum M, Keren G, Sheps D, Leibovitch E, Brosh
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Charlesworth A, Hampton JR, van Veldhuisen DJ. Renal function, neurohormonal
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D, Yachnin T, Steinbruch S, Shapira I, Laniado S, Iaina A. The effect of correction of
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Finkel T. Circulating endothelial progenitor cells, vascular function, and
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- 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
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- 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
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- 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 -
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Bahlmann et al. Erythropoietin Regulates Endothelial Progenitor Cells
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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
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