# Maintenance chemotherapy in children with ALL exerts metronomic-like thrombospondin-1 associated anti-endothelial effect

Nicolas Andre, Sylvie Cointe, Vincent Barlogis, Laurent Arnaud, Romaric Lacroix, Eddy G Pasquier, Francoise Dignat-George, Gérard Michel, Florence Sabatier

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Nicolas Andre, Sylvie Cointe, Vincent Barlogis, Laurent Arnaud, Romaric Lacroix, et al. Maintenance chemotherapy in children with ALL exerts metronomic-like thrombospondin-1 associated anti-endothelial effect. Oncotarget, 2015, 6 (26), 10.18632/oncotarget.3984. [https://doi.org/10.18632/oncotarget.3984](https://doi.org/10.18632/oncotarget.3984). [hal-01787272](https://HAL.inistre.fr/hal-01787272)

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# Maintenance chemotherapy in children with ALL exerts metronomic-like thrombospondin-1 associated anti-endothelial effect

Nicolas Andre<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Sylvie Cointe<fnref n="4" />,<fnref n="5" />, Vincent Barlogis<fnref n="1" />, Laurent Arnaud<fnref n="5" />, Romaric Lacroix<fnref n="4" />,<fnref n="5" />, Eddy Pasquier<fnref n="2" />,<fnref n="3" />, Françoise Dignat-George<fnref n="4" />,<fnref n="5" />, Gérard Michel<fnref n="1" /> and Florence Sabatier<fnref n="4" />,<fnref n="5" />

**Correspondence to:** Nicolas Andre, email: nicolas.andre@ap-hm.fr

**Keywords:** leukemia, maintenance therapy, metronomic chemotherapy, immune system, angiogenesis

Received: February 10, 2015

Accepted: April 15, 2015

Published: May 04, 2015

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

## ABSTRACT

Maintenance chemotherapy is an important part of the treatment of ALL in children. It relies on the long-term oral administration of daily low-dose mercaptopurin and weekly low-dose methotrexate. Although it has been used in the clinic for decades, its mechanisms of action remain unclear. Here, we investigated different angiogenic and immune biomarkers to gain insights into the mechanisms of action of maintenance therapy in children with ALL. We thus monitored circulating endothelial cells (CEC), endothelial progenitor cells (EPC) and endothelial microparticles (EMP), pro-angiogenic factors (VEGF, VEGFR-1 and Ang-2), anti-angiogenic factor thrombospondin-1 (THBS1) and regulatory T lymphocytes (Treg) in 47 children with ALL during the maintenance phase of their treatment (at treatment initiation and after 6, 12 and 18 months). We observed a statistically significant decrease in EPC and EMP counts throughout the maintenance phase associated with a significant increase in THBS1 levels. No significant change was detected in other angiogenic markers or in Treg numbers.

The results presented here indicate that maintenance therapy in children with ALL exerts its antitumor activity at least in part through anti-angiogenic effects, similar to those induced by metronomic chemotherapy. Larger studies are now warranted to validate these findings and determine their clinical implications.

## INTRODUCTION

Maintenance chemotherapy is an important part of the treatment of acute lymphoid leukemia (ALL) in children to prevent relapse by eliminating minimal residual disease [1, 2]. The cornerstone of ALL maintenance therapy relies on the oral administration of daily low-dose mercaptopurin and weekly low-dose methotrexate for a period of time ranging from 2 to 3 years [1]. Interestingly, metronomic chemotherapy (MC), which relies on the frequent administration of chemotherapy at low doses,

significantly below the single maximal tolerated dose and with no prolonged drug-free breaks [3, 4], has recently emerged as an alternative strategy to conventional high-dose chemotherapy. Despite accumulating clinical evidence in a variety of human cancers [4], the efficacy of MC is yet to be validated in large randomized phase III trials. However, as originally theorized by Barton Kamen, maintenance therapy in leukemia can be considered, in retrospect, as the early prototype of successful MC [5].

Angiogenesis plays a role in the pathogenesis and progression of hematological malignancies [6] including

<footnote n="1">¹ Service d'Hématologie et Oncologie Pédiatrique, Centre Hospitalo-Universitaire Timone Enfants, AP-HM, Marseille, France</footnote>

<footnote n="2">² Aix-Marseille Université, INSERM, CRO2 UMRS-911, Marseille, France</footnote>

<footnote n="3">³ Metronomics Global Health Initiative, Marseille, France</footnote>

<footnote n="4">⁴ Aix-Marseille Université INSERM, Vascular Research Center of Marseille UMRS-1076, Marseille, France</footnote>

<footnote n="5">⁵ Laboratoire d'Hématologie, Centre Hospitalo-Universitaire Conception, AP-HM, Marseille, France</footnote>
leukemia and MC has initially been demonstrated to be
an antiangiogenic therapy [3]. Tumor angiogenesis can
be easily and non-invasively monitored by analysis of
circulating endothelial cells (CEC), endothelial progenitor
cells (EPC) and endothelial microparticles (EMP), which
are established biomarkers of endothelial integrity in
peripheral blood. However, new mechanisms of action
of MC have been unveiled, such as i) stimulation of
anticancer immune response by depletion of regulatory
T lymphocytes (Treg) or activation of dendritic cells, ii)
re-induction of tumor dormancy, and iii) potential direct
effects against cancer stem cells [4]. Thus, maintenance
therapy in leukemia, whose mechanisms of action remain
unclear [1, 2], could exert metronomic-like effects and
represent a form of multi-targeted therapy.

The aim of this study was to monitor the impact
of a maintenance treatment on endothelial biomarkers,
angiogenic cytokines, and Treg in 47 children with ALL.

RESULTS

In the 47 children enrolled in this study, endothelial
biomarkers analysis indicated no statistically significant
change in CEC and platelet counts during maintenance
therapy (Figure 1). In contrast, EMP counts decreased at
E12 and E18 with a significant change after 18 months of
maintenance therapy compared to baseline counts (11.3
+/- 12.84 EMP/µL vs 5.47 +/- 1.39 ; *p* < 0.05) (Figure
2A). In line with EMP levels, EPC counts decreased
throughout the maintenance phase with values dropping
from 507 +/- 272 EPC/mL at E0 to 112.9 +/- 126.6 (*p* <
0.001), 91.06 +/- 83.97 (*p* < 0.05) and 92.32 +/- 181.1
(*p* < 0.01) at E6, E12 and E18, respectively (Figure
2B). Moreover, all of EMP values measured during
maintenance treatment correlated with those of EPC (*p* <
0.05) (Figure 1C). Interestingly, THBS-1, which is a well-
established mediator of the antiangiogenic activity of MC

<figure><img src="image_2.png" /><figcaption>**Figure 1:** Evolution of CEC and platelet levels during maintenance therapy. Evolution of CEC and platelets was monitored at treatment initiation and after 6, 12 and 18 months of maintenance therapy and is shown in panels A. and C. Correlation between EMP and EPC during the maintenance phase of treatment is shown in panel B. Columns, mean value from 47 patients; Bars, SEM. Abbreviations used: CEC: circulating endothelial cells, EMP: endothelial microparticles, EPC: endothelial progenitor cells</figcaption></figure>
[7], increased at E12 and E18 with a significant change at E18 compared to baseline (6341 +/- 4121 ng/mL vs 9319 +/- 4095; *p* < 0.01) (Figure 2C). In sharp contrast, analysis of other angiogenic cytokines indicated no statistically significant change in VEGF, VEGF-R1 and ANG-2 levels throughout the maintenance phase (Figure 3). There were no significant correlations between biomarkers.

Only one patient (n°14-Table 1) relapsed during the maintenance phase. Noteworthily, this patient displayed high levels of EPC (3092/ml) shortly before relapsing.

The role of the immune system is gaining a considerable interest in solid tumors [8] and is a well-established actor of leukemia treatment. More specifically, Tregs are being increasingly documented as key players in the antitumor activity of MC [4]. However, in this study, Treg counts did not significantly vary during maintenance therapy (Figure 3D).

DISCUSSION

How maintenance therapy operates in children with ALL remains highly speculative [1, 2]. Several mechanisms of action have been proposed. They include direct long-term cytotoxicity on leukemia cells and inhibition of leukemia stem cell maintenance. The results of the present study provide interesting new insights into how maintenance therapy might affect residual leukemia disease.

In cardiovascular pathologies, the significance of endothelial biomarkers has been clearly established [9]. Indeed, elevated counts of CEC and EMP in the peripheral blood can be considered as markers of endothelial injury and vascular dysfunction, while decrease in EPC, which contribute to endothelium repair processes, plays a critical role in progression of cardiovascular diseases [10]. In clinical oncology, interpretation of these biomarkers levels and kinetics is more complex [11]. CEC and EPC can be considered as surrogate biomarkers of

<figure><img src="image_3.png" /><figcaption>**Figure 2: Evolution of EMP, EPC, and THBS-1 levels during maintenance therapy.** Evolution of EMP, EPC and THBS-1 was monitored at treatment initiation and after 6, 12 and 18 months of maintenance therapy and is shown in panels A, B, and C, respectively. Columns, mean value from 47 patients; Bars, SEM. Statistical analysis was performed using Wilcoxon matched paired test; \*p* < 0.05; \*\*p* < 0.01; \*p* < 0.001. Abbreviations used: EMP: endothelial microparticles, EPC: endothelial progenitor cells, THBS-1: Thrombospodin-1</figcaption></figure>
angiogenesis and used for instance to monitor the activity
of antiangiogenic agents [12]. The results presented here
indicate a significant impact of maintenance therapy on
endothelium integrity. The significant decrease in EMP
counts and circulating EPC levels underlined a reduction
in endothelium activation. Taken together, decreases in
these cellular biomarkers and increase in THBS-1 levels,
suggest that maintenance therapy reduces endothelium
turn-over and restores its resting state. The mechanisms
by which maintenance therapy exerts this anti-endothelial
effect remains uncertain but several can be proposed.
A direct effect on endothelial cells has been previously
demonstrated for the 3 anticancer drugs involved here
[3, 13,15]. In addition, this effect could also be mediated
by VEGF inhibition and/or THBS-1 up-regulation [3,
7]. In the patients included in this study, VEGF levels
remained low and unchanged throughout maintenance
therapy; it is thus unlikely that VEGF plays a key role in
this setting. In contrast, we observed a significant increase
in the level of THBS-1, an antiangiogenic factor and
established mediator of MC efficacy [3, 4, 7]. Platelets
contain significant amount of bioactive factors such as
THBS-1 that can be released during platelet activation
and influence neovascularization [14]. It is important
to note however that the increase in THBS-1 levels

was not associated with significant changes in platelet
counts, suggesting that platelets may not be involved in
this context. However our study failed to demonstrate a
significant inverse correlation between EPCs or EMPs and
THBS-1 levels suggesting that THBS-1 may act in concert
with other potential mechanisms.

Tregs have recently gained considerable interest
from oncologists, as they have been associated with
decreased efficacy of treatment in pre-clinical and clinical
studies investigating MC [4, 15]. However, no change in
Treg numbers was observed as a result of maintenance
therapy in our study, suggesting that Tregs are not
implicated in the long-term control of ALL. Consistently,
Brtnický and al. [16] reported no significant change
in Treg numbers in patients with ovarian carcinoma
receiving maintenance MC with cyclophosphamide or
etoposide. Other immune cell types, such as dendritic cells
or myeloid-derived suppressors cells may be affected by
maintenance therapy [4, 15]. For instance, Hasnis and
al. [17] reported that metronomic gemcitabine improved
pancreatic adenocarcinoma outcome by inhibiting the
pro-tumorigenic effects induced by myeloid-derived
suppressor cells. Future studies are required to characterize
the evolution of these cell types during ALL maintenance
therapy.

<figure><img src="image_4.png" /><figcaption>**Figure 3: Evolution of angiogenic cytokines and regulatory T lymphocytes during maintenance therapy.** Evolution of Ang-2, VEGFR-1, VEGF and Treg levels was monitored at treatment initiation and after 6, 12 and 18 months of maintenance therapy and is shown in panels A., B., C. and D., respectively. Columns, mean value from 47 patients; Bars, SEM. Ang-2: Angiopoietin-2, VEGFR-1: Vascular Endothelial Growth Factor Receptor-1, VEGF: Vascular Endothelial Growth Factor, Treg: Regulatory T Lymphocyte</figcaption></figure>
<table><caption>Table 1: Patients characteristics.</caption><thead><tr><th>patient number</th><th>age</th><th>phenotype (months)</th><th>CNS Involvement</th><th>hyperleucytosis</th><th>Karyotype</th><th>FISH</th><th>Corticosensitivity</th><th>Chemosensitivity</th></tr></thead><tbody><tr><td>1</td><td>154</td><td>LAL T</td><td>no</td><td>yes</td><td>abnormal</td><td>tel-aml1</td><td>yes</td><td>yes</td></tr><tr><td>2</td><td>82</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>3</td><td>114</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>4</td><td>52</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>no</td><td>yes</td></tr><tr><td>5</td><td>35</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>6</td><td>53</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>not evaluable</td><td>yes</td></tr><tr><td>7</td><td>107</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>8</td><td>45</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>negative</td><td>no</td><td>yes</td></tr><tr><td>9</td><td>190</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>tel-aml1</td><td>yes</td><td>yes</td></tr><tr><td>10</td><td>65</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>negative</td><td>not</td><td>yes</td></tr><tr><td>11</td><td>91</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>12</td><td>196</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>tel-aml1</td><td>yes</td><td>yes</td></tr><tr><td>13</td><td>35</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>not done</td><td>no</td><td>yes</td></tr><tr><td>14</td><td>180</td><td>LAL T</td><td>no</td><td>no</td><td>Failure</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>15</td><td>76</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>16</td><td>61</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>17</td><td>183</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>18</td><td>91</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>19</td><td>38</td><td>LAL B</td><td>no</td><td>yes</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>20</td><td>50</td><td>LAL B</td><td>no</td><td>yes</td><td>normal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>21</td><td>195</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>tel-aml1</td><td>yes</td><td>yes</td></tr><tr><td>22</td><td>78</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>not evaluable</td><td>yes</td></tr><tr><td>23</td><td>23</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>tel-aml1</td><td>not evaluable</td><td>yes</td></tr><tr><td>24</td><td>72</td><td>LAL B</td><td>yes</td><td>no</td><td>normal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>25</td><td>39</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>negative</td><td>not evaluable</td><td>yes</td></tr><tr><td>26</td><td>35</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>tel-aml1</td><td>no</td><td>yes</td></tr><tr><td>27</td><td>57</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>28</td><td>55</td><td>LAL B</td><td>no</td><td>yes</td><td>normal</td><td>negative</td><td>not evaluable</td><td>yes</td></tr><tr><td>29</td><td>149</td><td>LAL T</td><td>no</td><td>no</td><td>normal</td><td>tel-aml1</td><td>yes</td><td>yes</td></tr><tr><td>30</td><td>28</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>31</td><td>51</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>32</td><td>23</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>33</td><td>97</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>tel-aml1</td><td>not evaluable</td><td>yes</td></tr><tr><td>34</td><td>75</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>35</td><td>30</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>mll+</td><td>no</td><td>yes</td></tr><tr><td>36</td><td>184</td><td>LAL T</td><td>no</td><td>yes</td><td>normal</td><td>negative</td><td>not evaluable</td><td>yes</td></tr><tr><td>37</td><td>107</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>38</td><td>52</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>tel-aml1</td><td>no</td><td>yes</td></tr><tr><td>39</td><td>43</td><td>LAL B</td><td>no</td><td>yes</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>40</td><td>158</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>41</td><td>47</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>42</td><td>58</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>43</td><td>147</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>44</td><td>157</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>negative</td><td>yes</td><td>yes</td></tr><tr><td>45</td><td>145</td><td>LAL B</td><td>no</td><td>no</td><td>normal</td><td>tel-aml1</td><td>not evaluable</td><td>yes</td></tr><tr><td>46</td><td>57</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>not evaluable</td><td>yes</td></tr><tr><td>47</td><td>62</td><td>LAL B</td><td>no</td><td>no</td><td>abnormal</td><td>negative</td><td>not evaluable</td><td>yes</td></tr></tbody></table>

The results presented here indicate that maintenance treatment in children with ALL not only pharmacologically but also mechanistically represents a form of metronomic treatment, acting at least in part by decreasing endothelium activity. Larger studies are mandatory to confirm these effects and evaluate their clinical implications. These studies may lead to the identification of novel biomarkers and pave the way to strengthen the antiangiogenic effect of maintenance therapy.

## PATIENTS AND METHODS

### Patients

Details of the population and the underlying leukemia are given in table 1. Children were all included in the FRALLE protocol and treated accordingly. Maintenance therapy consisted in daily oral purinethol (75 mg/m²/day), weekly oral methotrexate (25 mg/m²/1x/week) and vincristine (1,5 mg/m²) once a month for 12 months and dexamethasone (6 mg/m²/day) for 5 days/month for 12 month. All family and/or patients gave informed consent. The study was approved by ethical and
institutional committee. Blood samples were taken before
starting maintenance therapy (E0), and respectively after
6 (E6), 12 (E12), and 18 (E18) months of maintenance.

Enumeration of circulating endothelial cells
(CEC)

As previously described [18], CECs were isolated
by immuno-magnetic separation using CD146-coated
Dynabeads (Life Technologies, Carlsbad, California,
USA) from 1mL of whole blood samples collected into
EDTA. Isolated cells were then incubated with fluorescein
isothiocyanate (FITC) - coupled *Ulex europeaus* lectin-1
(Sigma-Aldrich, St Louis, MO, USA), and quantified with
fluorescence microscopy at 533nm. CECs were identified
according to the following consensus morphologic and
immunologic criteria: rosetted cells with sizes over 15µm
in diameter, bearing more than 5 beads and binding *Ulex
europeaus* lectin-1. The number of CECs was expressed as
cells per mL of blood.

Enumeration of endothelial microparticles
(EMPs)

CD31+ CD41- EMPs were quantified in platelet-poor citrated plasma (PPP). Briefly, PPP was prepared using serial centrifugations (15 min at 1500 × g, 2 min at 13 000 × g) and stored at -80 °C until use. Thirty µL of thawed PPP were labeled 30min at room temperature with 10µl of CD41-FITC (Biocytex, Marseille, France), and 10µl of CD31-PE (Beckman Coulter, Miami, FL, USA). 20µl of concentration-matched PE-conjugated murine IgG1 antibody was used as CD31 fluorescence minus one (FMO) control. After incubation, samples were diluted in 500µl of PBS (Life Technologies) and 30µl of flowcount beads (Beckman Coulter) were added for calculation of microparticles absolute values. CD31+ CD41- EMPs were quantified with a FC500 Cytometer (Beckman Coulter) with a previously described standardized flow cytometry protocol for microparticles counting [19]. Results were expressed as EMP per µl of PPP.

Enumeration of endothelial progenitor cells
(EPCs)

Enumeration of CD34+KDR+ EPCs was
performed with three-color flow cytometry after direct
immunolabeling of peripheral blood mononuclear cells
(PBMCs) As previously described [10], PBMCs were
isolated from 5 mL of heparinized peripheral blood
by density gradient centrifugation with lymphocyte
separation medium (PAA Laboratories, Pasching, Austria),
and labeled with 10 µL of 7-AAD Viability Dye, 10 µL
of FITC-CD34 antibody (Beckman Coulter), and 10 µL

of PE-KDR antibody (R&D Systems, Abingdon, UK). 10µl of concentration-matched PE-conjugated murine IgG1 antibody was used as fluorescence minus one (FMO) control. After incubation for 30 min at room temperature, cells were washed, resuspended in 500 µL of phosphate-buffered saline (PBS) (Life Technologies), and analyzed with a FC 500 Cytometer. After selection of 7-AAD-negative cells, KDR+ cells were identified within CD34+ cells displaying FSC/SSC characteristics corresponding to the lymphocyte cluster. At least 5 × 10⁵ viable cells were acquired per run. The percentage of KDR+ cells among CD34+ cells was determined, and absolute values were calculated by multiplying the percentage of KDR+ cells by the absolute values of CD34+ cells. Absolute values of circulating CD34+ cells were determined in whole blood using the single plateform sequential gating strategy according to the standardized ISHAGE protocol [20].

Enumeration of regulatory T lymphocytes (Tregs)

CD4+ CD25+ Tregs were quantified with a flow
cytometry method from whole blood samples collected
into EDTA [21]. One hundred µl of samples were
labeled 20min at room temperature with 10 µl PE-CD25,
Phycoerythrin-Texas Red (ECD)-CD4, and Phycoerythrin-
Cyanin5 (PC5)-CD3 antibodies (Beckman Coulter). After
lysis of Erythrocytes, cells were washed, resuspended in
500µl of PBS, and analyzed with a FC500 Cytometer.
The proportion of CD4+ CD25+ Tregs among CD3+
lymphocytes was determined. Absolute values of Tregs
in element mm⁻³ were calculated using the proportion
of CD3+ cells obtained by flow cytometry and the
lymphocytes numeration obtained from Hemocytometer
Instrument.

Analysis of angiogenic cytokines

Circulating levels of VEGF, VEGF-R1,
Thrombospondin-1 (THBS-1), and angiopoietin-2 (ANG-
2) were measured in serum using commercially available
ELISA kits from R&D System Inc. (Minneapolis,
MN, USA). Assays were performed according to
manufacturer's instructions.

Statistical analyses

Statistical analyses were performed using GraphPad
Prism Software v.4.0. (GraphPad Software, San Diego,
CA, USA). Wilcoxon matched paired test was used
to compare different times of maintenance therapy.
Differences were considered significant when *p* was <
0.05. To determine the correlation between biomarkers,
Spearman correlation test was used with a confidence
interval of 95%.
## CONFLICTS OF INTEREST

None declared.

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