ORIGINAL ARTICLE

Vascular endothelial growth factor corrected for platelet countand hematocrit is associated with the clinical course of aplasticanemia in children

Yuichi Kodama • Yasuhiro Okamoto • Teruto Hashiguchi •

Yuichi Shinkoda • Takuro Nishikawa • Takayuki Tanabe •

Yoshifumi Kawano

Received: 21 October 2011 / Revised: 4 April 2012 / Accepted: 5 April 2012 / Published online: 22 April 2012

� The Japanese Society of Hematology 2012

Abstract The wide variety of clinical courses that lead to

the development of severe aplastic anemia (AA) makes it

difficult to speculate whether treatment for AA is required in

the early phase. The objective of this study was to identify a

method for predicting the clinical course of AA at the onset of

the disease. First, in healthy adults, vascular endothelial

growth factor (VEGF) released per platelet was measured by

the activation of platelet-rich plasma (PRP) and platelet-poor

plasma (PPP). Serum concentration of VEGF, serum con-

centration of VEGF corrected for platelet count, and serum

concentration of VEGF corrected for both platelet count and

hematocrit (corrected VEGF) were then compared to VEGF

released per platelet. Corrected VEGF showed the best cor-

relation with VEGF released per platelet by the activation of

PRP in healthy subjects (R2 = in a single 0.806, p = 0.001).

Next, corrected VEGF was assayed in 11 pediatric patients

with AA at the time of diagnosis. Corrected VEGF in AA

patients was significantly greater than that in age-matched

control subjects [1.32 9 10-6 pg (range 0.36–1.85) vs.

0.18 9 10-6 pg (range 0.12–0.94)] (p = 0.002). Moreover,

corrected VEGF in AA patients who did not require treatment

for more than 2 years was significantly greater than that in

AA patients who required earlier treatment [1.67 9 10-6 pg

(range 1.32–1.85) vs. 0.87 9 10-6 pg (0.36–1.34)]

(p = 0.011). These data indicate that a compensatory

mechanism for increasing VEGF and preventing disease

progression might play a role in AA. Corrected VEGF may be

useful for predicting the clinical course of AA.

Keywords Aplastic anemia � VEGF � Clinical course �Biomarker

Introduction

Most patients with aplastic anemia (AA) deteriorate to

severe AA and require transfusions. However, some cases

of AA show stable disease or resolve spontaneously [1–3].

Treatment is often begun after the patient has become

transfusion dependent or their disease has met the criteria

of severe AA because it can be difficult to decide whether

to initiate immunosuppressive therapy (IST) or hemato-

poietic stem cell transplantation (HSCT), which are asso-

ciated with many adverse effects and a risk of therapy-

related mortality [4]. For physicians, the wide variety of

clinical courses that lead to the development of severe AA

makes it difficult to judge if treatment for AA is required in

the early phase [2]. Therefore, a procedure should be

established for predicting the clinical course at the onset of

AA, especially mild and moderate AA.

Based on an immunohistochemical analysis, it has been

reported that AA is associated with the reduced expression

of vascular endothelial growth factor (VEGF) in mega-

karyocyte and immature myeloid progenitor cells and with

a reduced serum concentration of VEGF levels compared

to those in normal controls [5]. The report also noted that

VEGF expression recovered after the treatment of AA.

Since VEGF in serum is known to be exclusively released

from platelets [6], it is reasonable that the serum

Y. Kodama � Y. Okamoto (&) � Y. Shinkoda � T. Nishikawa �T. Tanabe � Y. Kawano

Department of Pediatrics, Kagoshima University Graduate

School of Medical and Dental Sciences,

8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan

e-mail: okamoto@m2.kufm.kagoshima-u.ac.jp

T. Hashiguchi

Laboratory and Vascular Medicine, Kagoshima University

Graduate School of Medical and Dental Sciences,

Kagoshima, Japan

123

Int J Hematol (2012) 95:494–499

DOI 10.1007/s12185-012-1074-1

concentration of VEGF is low in AA patients. To under-

stand the significance of low VEGF in AA, VEGF released

per platelet, rather than simply VEGF in the serum, should

be measured to exclude the effect of a low platelet count in

AA patients. The most accurate and physiological method

for assessing VEGF released per platelet is to measure

VEGF by the activation of platelet-rich plasma (PRP) and

platelet-poor plasma (PPP) [7]. However, the activation of

PRP and PPP is time-consuming and requires a large

amount of blood, which is not feasible in children with AA.

In this study, we first attempted to identify a more fea-

sible, alternative method for assessing VEGF released per

platelet in healthy controls. Next, we examined the asso-

ciation between VEGF measured at the onset of AA by this

new method and the clinical course.

Methods

Blood samples

Platelet-rich plasma, PPP and serum samples were taken

from healthy adult donors. In contrast, only serum samples

were taken from AA patients and control subjects. Plasma

samples were collected in sterile tubes containing sodium

citrate as an anticoagulant. PRP was obtained by centri-

fugation (120 g for 5 min). PPP and serum samples were

also obtained by centrifugation (2,000 g for 20 min).

Plasma samples and serum samples were stored at -40 �C

(Sanyo, Tokyo, Japan) until the time of assay.

Measurement of VEGF

The VEGF concentration in serum or plasma was measured

in duplicate with a commercially available human VEGF

quantitative enzyme-linked immunosorbent assay (ELISA:

Catalogue No. DVE00, R&D Systems, Minneapolis, MN).

This system can measure as little as 9 pg/ml and does not

cross-react with platelet-derived growth factor or other

homologous cytokines. The optical density at 450 nm was

measured on an ImmunoMini NJ-2300 (Nippon Inter Med,

Tokyo, Japan) and the VEGF concentration was determined

by linear regression from a standard curve that was con-

structed using the VEGF supplied with the kit as a standard.

VEGF released per platelet by the activation of PRP

and PPP (VEGF per platelet) and serum concentration

of VEGF corrected for both the platelet count

and hematocrit (corrected VEGF)

Platelet-rich plasma and PPP were activated by the addition

of human thrombin (Sigma Chemical Co., St. Louis,

MO)/calcium chloride solution to a final concentration of

5 U/ml/10 mM to ensure maximal clotting (PRP VEGF and

PPP VEGF, respectively). After being allowed to stand at

room temperature for 30 min, samples were centrifuged at

750 g at 4 �C for 20 min, and the supernatants were trans-

ferred for measurement of the VEGF concentration. VEGF

per platelet was calculated as {PRP VEGF (pg/ml) - PPP

VEGF (pg/ml)}/platelet count in PRP (/ll) 9 1,000, as

described previously [7]. Since the measurement of VEGF

per platelet is time-consuming and requires a large amount

of blood as described above, the serum concentration of

VEGF was also measured. The serum concentration of

VEGF corrected for platelet count was calculated as the

serum concentration of VEGF (pg/ml)/platelet count (/ll) 9

1,000 to exclude the effect of the platelet count. Corrected

VEGF was calculated as the serum concentration of VEGF

(pg/ml) 9 {1-hematocrit}/platelet count (/ll) 9 1,000 to

exclude the effects of both the platelet count and hematocrit.

Subjects for the examination of VEGF per platelet

Eight healthy adult subjects were examined to evaluate the

correlation between VEGF per platelet, the serum con-

centration of VEGF, the serum concentration of VEGF

corrected for platelet count and corrected VEGF.

Patients and control subjects

Eleven AA patients (5 male and 6 female) diagnosed at

Kagoshima University Hospital between 2001 and 2007

whose serum at the onset was available were evaluated.

The median age of the patients was 10 years (range

8–14 years). All patients were diagnosed with acquired AA

and none had a family history or any physical malforma-

tions. None were post-hepatitis. Myelodysplastic syndrome

and paroxysmal nocturnal hemoglobinuria were excluded

by cytology and chromosome analysis, and/or by flow

cytometry using anti-CD55/59 [8]. None of the patients

showed an abnormal physical examination that would

indicate Fanconi anemia or chromosome fragility. Two

patients were classified with mild, 6 with moderate and 3

with severe AA, according to currently used criteria [2].

In our institution, treatments including HSCT or IST were

initiated if the patient became transfusion dependent or was

classified as severe AA. HSCT was performed if the patient

had a human leukocyte antigen (HLA)-matched/HLA one-

locus-mismatched sibling or an HLA-matched unrelated

donor. IST was performed for the remaining patients. If the

patient did not respond to IST, an HLA one-locus-mis-

matched unrelated donor was used as an alternative donor.

In this study, acute AA was defined as when the patient

required treatment within 2 years after diagnosis. Chronic

AA was defined as when the patient did not require treat-

ment more than 2 years after diagnosis.

Corrected VEGF is associated with the clinical course of AA 495

123

The control subjects consisted of 7 age-matched children

who did not have AA. They were not obese and their median

body mass index was 17.1 (range 16.5–21.7). Basically, they

were afebrile and the median C-reactive protein was 0.02

(range 0.02–0.05). The clinical protocols for measuring

serum VEGF were approved by the Institutional Review

Board at Kagoshima University Hospital. Written informed

consent was obtained from the respective parents.

Statistical analysis

Data are presented as medians (ranges). Paired data were

compared using the Mann–Whitney U test. A simple linear

regression analysis was used to evaluate the correlation

between two parameters. All statistical analyses were

performed using SPSS statistical software (SPSS Co.,

Tokyo, Japan), and p \ 0.05 was considered to be statis-

tically significant.

Results

VEGF per platelet in healthy adults

First, in healthy adults, VEGF released per platelet was

measured by the activation of PRP and PPP, which is

considered to be the most accurate and physiological

method as described in ‘‘Methods’’. The correlations

between VEGF per platelet, the serum concentration of

VEGF, the serum concentration of VEGF corrected for

platelet count and corrected VEGF were then examined.

Corrected VEGF showed the best correlation (R2 = 0.806,

p = 0.001) with VEGF per platelet among these parameters

[serum concentration of VEGF (R2 = 0.624, p = 0.020)

and the serum concentration of VEGF corrected for platelet

(R2 = 0.663, p = 0.014)] (Fig. 1). Therefore, in the fol-

lowing experiments in AA patients, we measured corrected

VEGF as a substitute for VEGF per platelet, since the

measurement of VEGF per platelet is time-consuming and

requires a large amount of blood.

AA patients

Three patients with severe AA and 3 with moderate AA

were classified as being of the acute type. On the other

hand, 3 patients with moderate AA and 2 with mild AA

were classified as being of the chronic type (Table 1).

Subjects with acute and chronic AA were compared

with regard to sex, age and absolute neutrophil, platelet and

absolute reticulocyte counts, as summarized in Table 2.

The median platelet count in acute AA (10.5 9 109/l) was

significantly less than that in chronic AA (37.0 9 109/l)

(p = 0.011).

Fig. 1 VEGF released from platelets by PRP activation in healthy

adults. The correlations between VEGF per platelet and the serum

concentration of VEGF (a), the serum concentration of VEGF

corrected by the platelet count (b) and corrected VEGF (c) are shown

496 Y. Kodama et al.

123

Corrected VEGF in AA patients and control subjects

The median corrected VEGF in AA patients [1.32 9

10-6 pg (range 0.36–1.85)] was significantly greater

(p = 0.002) than that in control subjects [0.18 9 10-6 pg

(range 0.12–0.94)] (Fig. 2a). The median corrected VEGF

levels in chronic AA patients [1.67 9 10-6 pg (range

1.32–1.85)] were significantly greater (p = 0.011) than

those in acute AA patients [0.87 9 10-6 pg (0.36–1.34)]

(Fig. 2b).

Corrected VEGF in moderate AA patients

To exclude the effect of the severity of the disease, we

compared corrected VEGF only in moderate AA patients.

The corrected VEGF values in 3 moderate/acute and 3

moderate/chronic AA patients were 0.36, 0.98 and

0.86 9 10-6 pg and 1.76, 1.32 and 1.54 9 10-6 pg,

respectively (Fig. 2b). The corrected VEGF in moderate/

chronic AA patients was higher than that in moderate/acute

AA patients.

Discussion

Vascular endothelial growth factor in platelets is derived

from the megakaryocytes in bone marrow [9]. Therefore,

we believe that VEGF could reflect bone marrow function.

Another group reported that AA was associated with the

reduced expression of VEGF in megakaryocyte and

immature myeloid progenitor cells, and with a low serum

concentration of VEGF compared with normal controls [5].

They also reported that VEGF expression recovered after

the treatment of AA. We presume that a low serum con-

centration of VEGF level may not merely reflect bone

marrow function in AA. Therefore, in this study we tried to

appreciate the significance of a low VEGF in AA by

measuring VEGF released per platelet. VEGF assayed by

the activation of PRP and PPP should be quite accurate,

since the maximal VEGF released from platelets by the

activation of PRP and PPP is actually measured [7].

In this study, we demonstrated for the first time that

VEGF per platelet showed the best correlation with cor-

rected VEGF, which is VEGF corrected for platelet count

Table 1 Clinical features at diagnosis of 11 children with aplastic anemia

Case no. Course Age/sex Severity ANC (9109/l) Platelets (9109/l) ARC (9109/l) Treatment

Case 1 Acute 10/F Severe 1.132 6 10.20 HSCT at 1 month

Case 2 Acute 13/M Severe 0.000 8 5.60 HSCT at 1 month

Case 3 Acute 8/M Severe 0.450 10 36.30 IST at 0.5 month

Case 4 Acute 9/M Moderate 0.588 11 42.66 HSCT at 2 months

Case 5 Acute 12/M Moderate 0.398 30 31.95 HSCT at 3 months

Case 6 Acute 11/F Moderate 1.320 18 35.40 IST at 1 month, HSCT at 6 months

Case 7 Chronic 12/M Moderate 1.240 37 37.53 No therapy for 30 months

Case 8 Chronic 10/F Moderate 0.481 22 77.66 No therapy for 33 months

Case 9 Chronic 8/M Moderate 0.820 33 20.86 No therapy for 33 months

Case 10 Chronic 7/F Mild 1.835 63 52.48 No therapy for 26 months

Case 11 Chronic 14/F Mild 1.220 51 57.00 No therapy for 28 months

ANC absolute neutrophil count, ARC absolute reticulocyte count, M male, F female, HSCT hematopoietic stem cell transplantation, ISTimmunosuppressive therapy

Table 2 Comparison of acute and chronic aplastic anemia with regard to sex, age, ANC, platelet count and ARC

Acute Chronic p value

Sex (male:female) 3:3 2:3

Median age at diagnosis 10.5 (8–13) 10.0 (7–14) 0.783

Median ANC for each patient (9109/l) 0.59 (0.40–1.38) 1.22 (0.48–1.84) 0.251

Median ARC for each patient (9109/l) 33.7 (5.6–42.6) 52.5 (20.9–77.7) 0.068

Median platelet count for each patient (9109/l) 10.5 (6–30) 37.0 (22–63) 0.011

Median time to require treatment (months) 1 (0.5–3) 30 (26–33) 0.006

The results are presented as median values (ranges)

ANC absolute neutrophil count, ARC absolute reticulocyte count

Corrected VEGF is associated with the clinical course of AA 497

123

and hematocrit. Since most AA patients are anemic to

different degrees, it is reasonable to correct VEGF by

hematocrit. Previous reports did not directly assess VEGF

per platelet or account for anemia [6, 10, 11]. Since the

measurement of VEGF per platelet is time-consuming and

requires a large amount of blood, it is more convenient to

measure VEGF in serum than in plasma, especially in

children. In this regard, our approach which uses corrected

VEGF is more accurate than the measurement of the serum

concentration of VEGF and more feasible than the mea-

surement of VEGF in plasma.

Second, we examined the association between corrected

VEGF at the onset of AA and the clinical course. Howard

and co-workers reported that 67 % of moderate AA

patients progressed to severe disease. They stated that the

timing of progression was unpredictable; some patients

progressed to severe AA within a few months of diagnosis,

while others progressed after years of persistent moderate

AA [2]. Furthermore, it has also been reported that none of

the potential risk factors, including age at diagnosis, sex,

absolute neutrophil count, platelet count, absolute reticu-

locyte count, hemoglobin content, mean corpuscular vol-

ume and initial diagnosis of idiopathic thrombocytopenia,

were significantly associated with disease progression [3].

A new finding in the present study was that corrected

VEGF in patients with chronic AA was much greater than

that in patients with acute AA. This finding makes it much

easier to anticipate the clinical course of AA. To confirm

this result, a prospective analysis in a larger population of

patients is currently planned to determine if corrected

VEGF predicts the clinical course of moderate AA.

We believe that a breakdown of the compensatory

mechanisms of VEGF production in megakaryocytes is a

factor that triggers the disease rather than a consequence of

the disease, because we have demonstrated that corrected

VEGF in chronic AA is much greater than that in control

subjects. Some compensatory mechanisms that result in

increased VEGF production might occur at the beginning

of the clinical course in chronic AA. Once these compen-

satory mechanisms cease to be effective, patients show an

accelerated clinical course.

There are several possible compensatory mechanisms

that could be related to the pathogenesis and clinical course

of AA, based on the reported evidence. For example,

hypoxia-inducible factor-1a (HIF-1a) is a primary tran-

scription factor for VEGF [12]. Both HIF-1a and VEGF

have been shown to be induced in anemic rats [13].

Therefore, anemia in AA patients could induce HIF-1aexpression, which results in increased VEGF production.

AA patients have been reported to have up-regulated

thrombopoietin [14], which has been shown to stabilize

HIF-1a protein [15]. This would also result in increased

VEGF production. Further studies are necessary to more

fully investigate the regulation of HIF-1a in AA patients.

Acknowledgments The authors gratefully acknowledge the excel-

lent laboratory assistance of the research technologist Mami

Sakaemura.

Conflict of interest The authors declare that they have no com-

peting interests

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