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First published online September 28, 2006
Stem Cells Vol. 25 No. 1 January 2007, pp. 252 -261
doi:10.1634/stemcells.2006-0020; www.StemCells.com
© 2007 AlphaMed Press

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TRANSLATIONAL AND CLINICAL RESEARCH

Placental Growth Factor-1 Potentiates Hematopoietic Progenitor Cell Mobilization Induced by Granulocyte Colony-Stimulating Factor in Mice and Nonhuman Primates

Carmelo Carlo-Stellaa,b, Massimo Di Nicolaa, Paolo Longonia, Loredana Clerisc, Cristiana Lavazzaa, Raffaella Milanid, Marco Milanesia, Michele Magnia, Virgilio Pacee, Francesco Colottae, Maria A. Avanzinif, Franca Formellic, Alessandro M. Giannia,b

a"Cristina Gandini" Medical Oncology Unit, Istituto Nazionale Tumori, Milano, Italy;
bChair of Medical Oncology, University of Milano, Milano, Italy;
cDepartment of Experimental Oncology, Istituto Nazionale Tumori, Milano, Italy;
dHematology and Bone Marrow Transplantation Unit, Istituto Nazionale Tumori, Milano, Italy;
eDompé S.p.A., Research and Development, L'Aquila, Italy;
fDepartment of Pediatric Hematology-Oncology, Laboratory of Transplant Immunology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy

Key Words. Stem cell mobilization • Stem cell transplantation • Placental growth factor • Granulocyte colony-stimulating factor

Correspondence: Carmelo Carlo-Stella, M.D., "C. Gandini" Bone Marrow Transplantation Unit, Istituto Nazionale Tumori, Via Venezian, 1, 20133 Milano, Italy. Telephone: +39 02 2390 2717; Fax: +39 02 2390 3461; e-mail: carmelo.carlostella{at}unimi.it

Received January 10, 2006; accepted for publication September 20, 2006.
First published online in STEM CELLS EXPRESS   September 28, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
The complex hematopoietic effects of placental growth factor (PlGF) prompted us to test in mice and nonhuman primates the mobilization of peripheral blood progenitor cells (PBPCs) elicited by recombinant mouse PlGF-2 (rmPlGF-2) and recombinant human PlGF-1 (rhPlGF-1). PBPC mobilization was evaluated by assaying colony-forming cells (CFCs), high-proliferative potential-CFCs (HPP-CFCs), and long-term culture-initiating cells (LTC-ICs). In mice, both rmPlGF-2 and rhPlGF-1 used as single agents failed to mobilize PBPCs, whereas the combination of rhPlGF-1 and granulocyte colony-stimulating factor (rhG-CSF) increased CFCs and LTC-ICs per milliliter of blood by four- and eightfold, respectively, as compared with rhG-CSF alone. rhPlGF-1 plus rhG-CSF significantly increased matrix metalloproteinase-9 plasma levels over rhG-CSF alone, suggesting a mechanistic explanation for rhPlGF-1/rhG-CSF synergism. In rhesus monkeys, rhPlGF-1 alone had no mobilization effect, whereas rhPlGF-1 (260 µg/kg per day) plus rhG-CSF (100 µg/kg per day) increased rhG-CSF-elicited mobilization of CFCs, HPP-CFCs, and LTC-ICs per milliliter of blood by 5-, 7-, and 15-fold, respectively. No specific toxicity was associated with the administration of rhPlGF-1 alone or in combination. In conclusion, our data demonstrate that rhPlGF-1 significantly increases rhG-CSF-elicited hematopoietic mobilization and provide a preclinical rationale for evaluating rhPlGF-1 in the clinical setting.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Mobilized peripheral blood progenitor cells (PBPCs) have an established role in the management of patients with a variety of cancers undergoing autologous [1–4] or allogeneic [5–7] stem cell transplantation (SCT). In the autologous setting, PBPCs are efficiently mobilized by the administration of short courses of recombinant human granulocyte colony-stimulating factor (rhG-CSF) alone or during recovery from cytotoxic chemotherapy [8, 9]. Due to prior chemo-radiotherapy, disease stage, or disease-intrinsic factors, a substantial proportion of cancer patients (10%–30%) either mobilize suboptimal amounts of CD34+ cells (i.e., ≤5 x 106 CD34+ cells per kilogram) or fail any CD34+ cell mobilization [10–12].

Although suboptimal mobilization represents a substantial limitation to the completion of cell therapy programs requiring high amounts of CD34+ cells (i.e., haploidentical bone marrow transplanation, tandem autografts, gene therapy on hematopoietic stem cells), the lack of autologous stem cells raises important issues for the clinical management of patients for whom autologous SCT has proven to be clinically beneficial. Therefore, any procedure applicable to cancer patients and capable of increasing the yield of circulating progenitors in the absence of added toxicity, is expected to have a profound impact on the feasibility, toxicity, and costs of hematopoietic transplants. Despite several attempts to improve PBPC mobilization by molecules capable of interfering with the mechanism(s) regulating hematopoietic stem cell trafficking [13–16], or by using combinations of cytokines [17–23], so far substitutes or adjuncts to rhG-CSF either failed to substantially improve the mobilization of blood progenitors obtained with rhG-CSF alone or resulted in limited improvement [24, 25]. Recent data suggest that new agents, such as the CXCR4 antagonist, AMD3100, or the recombinant human growth hormone (GH) significantly improve PBPC mobilization in normal donors [16, 26, 27] or poor mobilizers [28, 29]. Exploitation of the full clinical potential of these new agents is still under evaluation, and no general consensus exists for a therapy capable of improving stem cell mobilization.

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family, occurring in at least four splicing isoforms, PlGF-1 to PlGF-4, which differ in size and binding properties [30–33]. PlGF functions as an angiogenic amplifier by signaling through VEGF receptor-1 (VEGFR-1), which is predominantly expressed by vascular endothelial cells, monocytes, macrophages, but also by human CD34+ and mouse Sca-1+c-kit+ marrow repopulating stem cells [34–36]. Recently, administration of an adenoviral vector expressing human PlGF was shown to exert complex hematopoietic effects, including restoration of early and late bone marrow hematopoiesis in 5-fluorouracil-suppressed mice, and mobilization of hematopoietic stem/progenitor cells into the bloodstream [37].

These observations suggesting that PlGF might exert a relevant clinical impact on SCT by improving stem cell mobilization prompted us to test the capability of PlGF to mobilize PBPCs in BALB/c mice and in a nonhuman primate model allowing the simulation of PBPC mobilization as occurs in a clinical situation. Hematopoietic mobilization was analyzed by assaying committed colony-forming cells (CFCs), high-proliferative potential progenitors (HPP-CFCs), as well as long-term culture-initiating cells (LTC-ICs) in mice and nonhuman primates treated with PlGF alone or in combination with rhG-CSF.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Studies in Mice

Animals.   Six- to 8-week-old female BALB/c mice, with a body weight of 20–25 g, were purchased from Charles River Laboratories (Milan, Italy, http://www.criver.com). Experimental procedures performed on animals were approved by the Ethical Committee for Animal Experimentation of the Istituto Nazionale Tumori (Milan, Italy) and were carried out in accordance with the guidelines of the United Kingdom Coordinating Committee on Cancer Research [38].

Cytokines.   rhG-CSF (Neupogen; Amgen, Thousand Oaks, CA, http://www.amgen.com) was from Roche S.p.A. (Milan, Italy, http://www.roche.it), recombinant mouse PlGF-2 (rmPlGF-2) was purchased from R&D Systems Europe Ltd. (Abingdon, U.K., http://www.rndsystems.com) and recombinant human PlGF-1 (rhPlGF-1) was a kind gift of Dr. A. Mion (Geymonat S.p.A., Anagni, Italy, http://www.geymonat.com).

Mobilization Protocols.   The standard mobilization protocol known to elicit a maximal PBPC mobilization consisted of i.p. injection of 10 µg/day rhG-CSF (days 1–5) [13, 39, 40]. Single-agent treatments with PlGF consisted of i.p. injection of 5 µg/day rmPlGF-2 (days 1–5) or 10 µg/day rhPlGF-1 (days 1–5). Combination treatments consisted of 5-day treatments with rhG-CSF (10 µg/day) plus either rmPlGF-2 (2.5, 5, or 10 µg/day) or rhPlGF-1 (5, 10, or 15 µg/day). The dose ranges of PlGF used in combination studies had been identified in preliminary experiments as the doses inducing a maximal enhancement of rhG-CSF-elicited PBPC mobilization. Control mice were injected with low-endotoxin phosphate-buffered saline (PBS) containing 0.1% mouse serum albumin (MSA) as a carrier. Each experiment was performed on at least three separate occasions, and four mice per group and time point were used. Unless otherwise stated, all animal groups were sacrificed 2–3 hours after the last treatment.

Cell Harvesting and Separation.   Peripheral blood was harvested from the orbital plexus into heparin-containing tubes. After white blood cell (WBC) counting, blood was diluted (1:4, vol/vol) with PBS, and mononuclear cells (MNCs) were separated by centrifugation (280g, 30 minutes, room temperature) on a Ficoll discontinuous density gradient [41]. Cells were then washed twice in Iscove's modified Dulbecco's medium (Cambrex Bio Science Verviers, S.p.r.l., Verviers, Belgium, http://www.cambrex.com) supplemented with 10% fetal bovine serum (FBS) (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), 2 mM L-glutamine, and antibiotics.

Colony-Forming Cell Assay.   Total colony-forming cells (CFCs)—granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and multilineage CFU (CFU-GEMM)—were assessed in methylcellulose cultures [13]. Briefly, 1-ml aliquots of blood (5 x 104 to 2 x 105 MNCs) were plated in 35-mm Petri dishes in methylcellulose-based medium (HCC-3434; StemCell Technologies) supplemented with stem cell factor (rmSCF; 50 ng/ml), interleukin-3 (rmIL-3; 10 ng/ml), interleukin-6 (rhIL-6; 10 ng/ml), and erythropoietin (rhEpo, 3 U/ml). Four plates were scored for each data point per experiment, and colonies were scored after 12–14 days of incubation (37°C, 5% CO2) [41, 42].

LTC-IC Assay.   LTC-ICs were assessed in bulk cultures [41]. Briefly, test cells (4–8 x 106) were resuspended in complete medium (Myelocult 5100; StemCell Technologies) and seeded into cultures containing a feeder layer of irradiated (2,000 cGy) murine AFT024 cells (kindly provided by Dr. K. Moore, Princeton University, Princeton, NJ) [43]. Complete medium consisted of {alpha}-medium supplemented with FBS (12.5%), horse serum (12.5%), L-glutamine (2 mM), 2-mercaptoethanol (10–4 M), inositol (0.2 mM), folic acid (20 µM), and freshly dissolved hydrocortisone (10–6 M). Cultures were fed weekly by replacement of half of the growth medium with fresh complete medium. After 4 weeks in culture, nonadherent cells and adherent cells harvested by trypsinization were pooled, washed, and assayed together for clonogenic cells in methylcellulose cultures. The total number of clonogenic cells present in 4-week-old LTC provides a relative measure of the number of LTC-ICs originally present in the test suspension. Absolute LTC-IC values were calculated by dividing the total number of clonogenic cells by 4, which is the average output of clonogenic cells per LTC-IC [44].

Assay for Radioprotective Cells.   Radioprotective cells were studied by comparing the survival of lethally irradiated (850 cGy) BALB/c mice transplanted with MNCs obtained from syngeneic donors that had been treated for 5 days with PBS/MSA, rhG-CSF (10 µg/day), or rhPlGF-1 (10 µg/day) plus rhG-CSF (10 µg/day). Ten recipient mice were injected with control MNCs from PBS/MSA-treated animals, and 15 mice were injected with 2 x 105 or 5 x 105 rhG-CSF- or rhPlGF-1/rhG-CSF-mobilized cells. Control irradiated mice were included in all experiments. A dose of irradiation (850 cGy) that in preliminary experiments resulted in 100% mortality by day 15 was chosen. Recipient mice were placed in a polymethylmetaacetate (PMMA) box and given total body irradiation by a Radgil x-ray treatment unit (Gilardoni, Milan, Italy, http://www.gilardoni.it), supplied with a 200-kV x-ray unit, at a dose rate of 35 cGy/minute. Recipient mice were injected via the tail vein 2–3 hours after irradiation [45].

Immunoassays of Cytokines.   Plasma levels of human PlGF and mouse promatrix metalloproteinase-9 (pro-MMP-9) were evaluated using commercial enzyme-linked immunosorbent assay (R&D Systems Europe Ltd.) in accordance with the manufacturer's instructions.

Studies in Nonhuman Primates

Animals.   Male rhesus monkeys (Macaca mulatta) ranging in age from 4 to 6 years and with a mean weight of 5 ± 0.6 kg were housed in individual stainless steel cages in conventional holding rooms at the Biomedical Primate Research Centre (BPRC) (Rijswijk, The Netherlands, http://www.bprc.nl). Animals had no experimental history of previous antibody or cytokine administration. Monkeys were housed in accordance with guidelines of the Animal Care and Use Committee of the BPRC and were provided with commercial monkey chow (Hope Farms, Woerden, The Netherlands, http://www.hopefarms.nl) supplemented with fresh fruit, vegetables, and bread. Tap water was available ad libitum via an automatic watering system. The study was approved by the Animal Care and Use Committee of the BPRC.

Mobilization Protocols.   A cohort of rhesus monkeys (n = 4) received four consecutive mobilization cycles separated from one another by a 6-week washout period. At cycle 1, animals received a standard mobilization protocol consisting of subcutaneous injection of rhG-CSF (100 µg/kg per day for 5 days). This regimen is known to elicit a maximal PBPC mobilization [14, 46, 47]. Subsequently, rhG-CSF (100 µg/kg per day) plus i.v. rhPlGF-1 at either 130 (cycle 2) or 260 µg/kg per day (cycle 3) were concomitantly administered for 5 days. Cycle 4 consisted of a 5-day treatment with rhPlGF-1 alone (260 µg/kg per day). According to the study design, the kinetics of mobilization after cycle 1 served as intramonkey control to assess the mobilization after cycles 2–4. Doses of rhPlGF-1 used in monkeys (i.e., 130 and 260 µg/kg per day) were calculated using an appropriate conversion factor to obtain the monkey dose equivalent, from mice studies in which a maximal enhancement of rhG-CSF-elicited PBPC mobilization by injecting 10 µg/day rhPlGF-1 was demonstrated [48]. Blood samples to be analyzed in Milan were kept at 4°C and processed within 24 hours from withdrawal. Previous studies had shown that the procedure had no effect on cell counts or in vitro colony assays [46].

Evaluation of Rhesus Monkey Blood.   Mobilization was evaluated by complete blood counts (CBCs), differential counts, and frequency and absolute numbers of CFCs, HPP-CFCs, and LTC-ICs. Mobilization parameters were analyzed daily during treatment (days 1–5) and 3 and 5 days after cessation of therapy. Peripheral blood samples were obtained from the femoral vein of anesthetized primates (ketamin, 10 mg/kg, intramuscularly) using aseptic techniques.

CBCs and Differential Counts.   CBCs were performed using EDTA-anticoagulated blood and an automated counter (Sysmex SF-3000; Goffin Meyvis, Ettenleur, The Netherlands, http://www.goffinmeyvis.com). Differential counts were performed on Wright-Giemsa-stained blood smears.

CFC and HPP-CFC Assays.   Total CFCs (i.e., CFU-GM, BFU-E, plus CFU-GEMM) and HPP-CFCs were assayed according to a previously described technique [46, 47]. Briefly, MNCs obtained by centrifugation on a Ficoll discontinuous gradient (density, 1.077 g/ml) were plated (1 x 104 to 2 x 105 per milliliter) in quadruplicate in 35-mm Petri dishes in methylcellulose-based medium (HCC-4100; Stem Cell Technologies) supplemented with rhSCF (50 ng/ml,), rhIL-3 (20 ng/ml), rhIL-6 (20 ng/ml), rhG-CSF (20 ng/ml), rhGM-CSF (20 ng/ml), and rhEpo (3 U/ml). All cytokines were purchased from StemCell Technologies. CFCs were scored after 12–14 days of incubation (37°C, 5% CO2) according to standard criteria. HPP-CFCs, defined as macroscopically visible colonies of more than 2 mm in diameter of compact colony growth, were scored after 28 days of incubation from methylcellulose cultures supplemented with rhSCF (50 ng/ml), rhIL-3 (20 ng/ml), rhIL-6 (20 ng/ml), rhG-CSF (20 ng/ml), rhGM-CSF (20 ng/ml), and rhEpo (3 U/ml) [14]. The absolute number of circulating CFCs or HPP-CFCs in blood is a function of the frequency of CFCs or HPP-CFCs multiplied by the total number of MNCs per milliliter of blood.

LTC-IC Assays.   The frequency of LTC-ICs was assessed under limiting dilution conditions [46, 49]. Briefly, serial dilutions of test cells (2 x 105 to 3 x 103) were resuspended in 150 µl of complete medium (Myelocult 5100) and plated in 96-well flat-bottom plates. For each test cell dose, 16–22 replicates were plated. Test cells were seeded into plates containing a feeder layer of irradiated (8,000 cGy) murine M2-10B4 cells (3 x 104/cm2; kindly provided by Dr. C. Eaves, Terry Fox Laboratory, Vancouver, BC, Canada) engineered by retroviral gene transfer to produce human IL-3 and G-CSF [50]. Cultures were fed weekly by replacement of half of the growth medium with fresh complete medium. After 5 weeks in culture, nonadherent and adherent cells from individual wells were harvested by trypsinization, washed, and assayed together for the growth of CFCs. After 12–14 days of incubation, cultures were scored as positive (≥1 colony) or negative (no colony), and the LTC-IC frequencies were calculated by using L-Calc software (StemCell Technologies). The absolute numbers of circulating LTC-ICs were assessed in bulk cultures [46]. Briefly, test cells (5–8 x 106) were resuspended in complete medium and seeded into cultures containing a feeder layer of irradiated murine M2-10B4 cells (3 x 104/cm2). After 5 weeks in culture, nonadherent cells and adherent cells harvested by trypsinization were pooled, washed, and assayed together for clonogenic cells. The total number of clonogenic cells (i.e., CFU-GEMM plus BFU-E plus CFU-GM) present in 5-week-old LTC provides a relative measure of the number of LTC-ICs originally present in the test suspension. Absolute LTC-IC values were calculated by dividing the total number of clonogenic cells by 4, which is the average output of clonogenic cells per LTC-IC [44].

Statistical Analysis.   Four plates were scored for each data point per experiment, and the results were expressed as the mean ± 1 SEM. The Student's t test for unpaired or paired data (two-tail) was used as appropriate to test the probability of significant differences among different mobilization cycles. Differences were considered significant if p ≤ .05. Statistical analysis was performed with the statistical package Prism 4 (GraphPad Software, Inc., San Diego, http://www.graphpad.com) run on a Macintosh G4 personal computer (Apple Computer, Inc., Cupertino, CA, http://www.apple.com). The LTC-IC frequencies were calculated from the proportion of wells that were negative using L-Calc software, which uses Poisson statistics and the method of maximum likelihood.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
Effect of Murine PlGF-2 on rhG-CSF-Elicited PBPC Mobilization in BALB/c Mice
In contrast to previous studies demonstrating that administration of an adenoviral vector expressing PlGF induced a significant PBPC mobilization [37], a 5-day i.p. treatment of BALB/c mice with rmPlGF-2 alone had no effect on hematopoietic mobilization (Table 1). However, the complex hematopoietic and microenvironmental effects of PlGF strongly suggesting that this cytokine may affect stem cell mobilization, prompted us to investigate whether rmPlGF-2 could eventually enhance rhG-CSF-elicited PBPC mobilization.


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  		Table 1. Hematopoietic mobilization in BALB/c mice upon treatment with rhG-CSF and rmPlGF-2

 
A 5-day combination therapy with rmPlGF-2 (2.5 and 5 µg/day) and rhG-CSF (10 µg/day) had no effect on WBC counts but enhanced in a dose-dependent manner rhG-CSF-elicited PBPC mobilization (Table 1). As compared with rhG-CSF alone, a 5-day course of 5 µg/day rmPlGF-2 plus 10 µg/day rhG-CSF increased CFC frequency and absolute numbers and LTC-IC absolute numbers by twofold (p ≤ .0002), twofold (p ≤ .01), and sevenfold (p ≤ .0002), respectively. Using this combination regimen (i.e., 10 µg/day of rhG-CSF plus 5 µg/day of rmPlGF-2), the median absolute numbers of CFCs and LTC-ICs were approximately 6,000 and 1,200 per milliliter, respectively. Increasing the dose of rmPlGF-2–10 µg/day failed to further improve rhG-CSF-elicited PBPC mobilization (data not shown).

Effect of Human PlGF-1 on rhG-CSF-Elicited PBPC Mobilization in BALB/c Mice
Similarly to what was observed for rmPlGF-2, treatment of BALB/c mice with rhPlGF-1 alone had no mobilizing activity (Table 2). However, rhPlGF-1 (5 or 10 µg/day) strongly synergized with rhG-CSF in increasing progenitor cell mobilization. Again, such a hematopoietic activity was dose-dependent, with the combined injection of 10 µg/day rhPlGF-1 plus rhG-CSF resulting in average increases of threefold for CFC frequency (p ≤ .0001), fourfold for CFC absolute numbers (p ≤ .001), and eightfold for LTC-IC absolute numbers (p ≤ .001), as compared with rhG-CSF alone. Upon combination, the highest absolute CFC and LTC-IC counts were 12,000 and 1,700 per milliliter, respectively.


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  		Table 2. Hematopoietic mobilization in BALB/c mice upon treatment with rhG-CSF and rhPlGF-1

 
Although increasing the dose of rhPlGF-1 up to 15 µg/mouse per day failed to further improve rhG-CSF-elicited PBPC mobilization (data not shown), prolonging the combined rhPlGF-1/rhG-CSF administration up to 12 days resulted in a persistent and statistically significant enhancement of rhG-CSF-elicited PBPC mobilization (Table 3), with absolute counts of CFCs as high as 32,000 per milliliter.


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  		Table 3. Effects of a 12-day mobilization with rhG-CSF alone or in combination with rhPlGF-1 in BALB/c mice

 
To determine whether cells mobilized by rhPlGF-1 plus rhG-CSF could rescue irradiated animals, BALB/c mice were lethally irradiated (850 cGy) and injected with blood MNCs obtained from syngeneic donors that had been treated for 5 days with PBS/MSA, rhG-CSF (10 µg/day), or rhPlGF-1 (10 µg/day) plus rhG-CSF (10 µg/day). There was no survival in the irradiation control group (n = 10) or in the group (n = 10) injected with 5 x 105 PBS/MSA MNCs (Fig. 1). Injection of 2 x 105 cells mobilized by rhG-CSF or rhPlGF-1/rhG-CSF rescued 13% and 37% of recipient mice (n = 15 per group, p = .2), respectively. Injection of 5 x 105 cells mobilized by rhG-CSF or rhPlGF-1/rhG-CSF rescued 41% and 87% of recipient mice (n = 15 per group, p = .08), respectively. Although survivals of mice rescued by rhG-CSF- or rhPlGF-1/rhG-CSF-mobilized cells were not statistically different, a trend in favor of higher survival of mice rescued by rhPlGF-1/rhG-CSF-mobilized cells was evident at both cell dose levels.


Figure 1
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  		Figure 1. Radioprotective activity of recombinant human placental growth factor-1 (rhPlGF-1)/recombinant human granulocyte colony-stimulating factor (rhG-CSF)-mobilized cells. Survival of lethally irradiated (850 cGy) BALB/c mice at 60 days after transplantation with blood mononuclear cells (MNCs) from PBS/MSA-, rhG-CSF-, or rhPlGF-1/rhG-CSF-treated mice. Donor mice were treated once daily for 5 days with phosphate-buffered saline/mouse serum albumin (PBS/MSA), rhG-CSF (10 µg/mouse), or rhPlGF-1 (10 µg/mouse) plus rhG-CSF (10 µg/mouse). Control mice received irradiation only. Donor MNCs (2 x 105 or 5 x 105 cells per mouse) were injected into recipient mice via the tail vein 2–3 hours after irradiation. {triangledown}, irradiation only; {triangleup}, MNCs (5 x 105 cells per mouse) from PBS/MSA-treated mice; {circ} MNCs (2 x 105 cells per mouse) from rhG-CSF-mobilized mice; bullet, MNCs (2 x 105 cells per mouse) from rhPlGF-1/rhG-CSF-mobilized mice; {square}, MNCs (5 x 105 cells per mouse) from rhG-CSF-mobilized mice; {blacksquare}, MNCs (5 x 105 cells per mouse) from rhPlGF-1/rhG-CSF-mobilized mice.

 
PlGF and MMP-9 Plasma Levels
Under our experimental conditions, failure of rhPlGF-1 alone to mobilize PBPCs was associated with PlGF plasma levels' peaking 2 hours after cytokine injection and declining to undetectable levels within 24 hours (Fig. 2A). PlGF plasma levels achieved after i.p. administration might explain the lack of any mobilizing activity using rhPlGF-1 as a single agent. However, rhPlGF-1 strongly synergized with rhG-CSF in mobilizing PBPCs, thus allowing the hypothesis that the combined mobilization therapy may eventually enhance protease release/activation. Because MMP-9 has a critical role in PBPC mobilization by either releasing soluble Kit ligand (sKitL) [37, 51] or degrading stromal cell-derived factor-1 (SDF-1) [52–54], we quantified MMP-9 plasma levels in BALB/c mice receiving different mobilization regimens (Fig. 2B). Whereas rhPlGF-1 alone had a modest effect on MMP-9 plasma levels (p = .2), treatment with rhG-CSF or rhPlGF-1/rhG-CSF was associated with a twofold (p ≤ .008) and threefold (p ≤ .0001) increase of MMP-9 over baseline, respectively. As compared with rhG-CSF alone, the combined rhPlGF-1/rhG-CSF therapy induced significantly higher (p ≤ .003) levels of MMP-9, thus suggesting that the effect at the protease level might represent the biological basis for the synergistic mobilizing activity of rhPlGF-1/rhG-CSF therapy.


Figure 2
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  		Figure 2. Plasma levels of human PlGF and mouse MMP-9 in BALB/c mice mobilized by recombinant human (rh)PlGF-1, (rh)G-CSF, or both. Cohorts of three BALB/c mice per group per experiment were injected i.p. for 4 days with control vehicle (PBS/MSA), rhPlGF-1 (10 µg/day), rhG-CSF (10 µg/day), or a combination of rhG-CSF (10 µg/day) and rhPlGF-1 (10 µg/day). Data are expressed as mean ± SEM. Statistical differences were evaluated using the Student's t test for unpaired data (two-tail). Combined data from three separate experiments are shown. (A): Plasma levels of human PlGF determined by enzyme-linked immunosorbent assay (ELISA). Collection of blood samples started 2 hours after the last injection of rhPlGF-1. (B): Plasma concentration of mouse MMP-9 determined by ELISA. Blood samples were collected 2 hours after the last injection of cytokine. *, p < .008 compared with control. °, p = .2 compared with control. °°, p < .003 compared with rhG-CSF. Abbreviations: Ctrl, control; G-CSF, human granulocyte colony-stimulating factor; MMP-9, matrix metalloproteinase; PBS/MSA, phosphate-buffered saline/mouse serum albumin; PlGF-1, human placental growth factor-1.

 
Effects of rhPlGF-1 and rhG-CSF on Circulating WBCs, MNCs, and Platelets in Rhesus Monkeys
Due to the significant activity of rhPlGF-1/rhG-CSF in mice, we further explored the capability of rhPlGF-1 to mobilize PBPCs in nonhuman primates. Rhesus monkeys received four mobilization cycles at 6-week intervals. After a standard mobilization with rhG-CSF alone (100 µg/kg per day for 5 days) (cycle 1) [14, 46, 47], monkeys received rhG-CSF in combination with rhPlGF-1 at either 130 (cycle 2) or 260 µg/kg per day (cycle 3). A fourth cycle consisting of rhPlGF-1 alone (260 µg/kg per day) was finally administered (cycle 4). Because the 5-day treatment with rhPlGF-1 alone had no mobilization effect (data not shown), only the results observed in monkeys receiving rhG-CSF alone or in combination with rhPlGF-1 are reported here.

When administered alone, rhG-CSF induced an average increment of fivefold for WBCs (8,708 vs. 43,523, p ≤ .0008) and threefold for MNCs (5,543 vs. 14,738, p ≤ .04), whereas platelets (PLTs) were slightly reduced (Fig. 3A–3C). As compared with rhG-CSF alone, the combined rhPlGF-1/rhG-CSF therapy further increased day-5 WBCs at both dose levels (i.e., 130 [60,040 vs. 43,523, p ≤ .001] or 260 µg/kg per day [49,048 vs. 43,523, p ≤ .001] [Fig. 3A]), whereas MNC and PLT values were not significantly changed (Fig. 3B, 3C). Five days after cessation of mobilization therapy, WBC, MNC, and PLT counts had returned to pretreatment levels.


Figure 3
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  		Figure 3. Effects of rhPlGF-1 and/or rhG-CSF on WBC, MNC, and PLT counts. Rhesus monkeys (n = 4) received 5-day mobilization cycles at 6-week intervals. Mobilization was elicited at cycle 1 ({circ}) by rhG-CSF alone (100 µg/kg per day, s.c.), at cycle 2 (bullet) by a combination of rhPlGF-1 (130 µg/kg, i.v.) plus rhG-CSF (100 µg/kg per day, s.c.), at cycle 3 ({square}) by a combination of rhPlGF-1 (260 µg/kg, i.v.) plus rhG-CSF (100 µg/kg per day, s.c.), and at cycle 4 (data not shown) by rhPlGF-1 alone (260 µg/kg, i.v.). Data are expressed as mean ± SEM. Statistical differences were evaluated using the Student's t test for paired data (two-tail). (A): WBC counts. The average pretreatment WBC counts per microliter of blood at cycles 1, 2, and 3 were 8,708 ± 1,299, 13,498 ± 2,757, and 8,370 ± 793, respectively. (B): MNC counts. The average pretreatment MNC counts per microliter of blood at cycles 1, 2, and 3 were 5,543 ± 973, 3,325 ± 519, and 4,975 ± 554, respectively. (C): PLT counts. The average pretreatment PLT counts per microliter of blood at cycles 1, 2, and 3 were 349,500 ± 11,637, 365,000 ± 31,507, and 355,500 ± 40,556, respectively. *, p < .001 compared with rhG-CSF. Abbreviations: G-CSF, granulocyte colony-stimulating factor; MNC, mononuclear cell; PlGF, placental growth factor; PLT, platelet; rhG-CSF, recombinant human granulocyte colony-stimulating factor; rhPlGF-1, recombinant human placental growth factor-1; WBC, white blood cell.

 
CFC Mobilizing Effects of rhPlGF-1 and rhG-CSF in Rhesus Monkeys
As compared with baseline, the mean frequency of blood CFCs at peak was increased by 16-fold (p ≤ .0009), 33-fold (p ≤ .0002), and 41-fold (p ≤ .0001) under rhG-CSF alone, rhG-CSF plus rhPlGF-1 at 130 µg/kg per day, and rhG-CSF plus rhPlGF-1 at 260 µg/kg per day, respectively (Table 4). The combined rhPlGF-1/rhG-CSF treatment induced a significant increase of CFC frequencies over those detected under rhG-CSF alone, with increments of twofold (p ≤ .004) and 2.5-fold (p ≤ .004) when rhPlGF-1 was injected at 130 and 260 µg/kg per day, respectively (Table 4). Five days after cessation of treatment, CFCs had almost returned to baseline values.


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  		Table 4. Hematopoietic mobilization in rhesus monkeys upon treatment with rhG-CSF and rhPlGF-1

 
Absolute numbers of circulating CFCs in blood were calculated as a function of the frequency of CFCs multiplied by the total number of MNCs per milliliter of blood. Whereas under steady-state conditions 141 ± 26 CFCs per milliliter of blood could be detected, this value was increased by 72-fold (p ≤ .01), 322-fold (p ≤ .001), and 388-fold (p ≤ .004) under rhG-CSF alone, rhG-CSF plus rhPlGF-1 at 130 µg/kg per day, and rhG-CSF plus rhPlGF-1 at 260 µg/kg per day, respectively (Fig. 4A). Thus, rhG-CSF-elicited mobilization of CFCs per milliliter of blood was increased by fourfold (p ≤ .006) and fivefold (p ≤ .006) under rhG-CSF plus rhPlGF-1 at 130 and 260 µg/kg per day, respectively.


Figure 4
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  		Figure 4. Absolute values of circulating CFCs and HPP-CFCs in monkeys mobilized with recombinant human placental growth factor-1 (rhPlGF-1) and/or recombinant human granulocyte colony-stimulating factor (rhG-CSF). Rhesus monkeys (n = 4) received 5-day mobilization cycles at 6-week intervals. Mobilization was elicited at cycle 1 ({circ}) by rhG-CSF alone (100 µg/kg per day, s.c.), at cycle 2 (bullet) by a combination of rhPlGF-1 (130 µg/kg, i.v.) plus rhG-CSF (100 µg/kg per day, s.c.), at cycle 3 ({square}) by a combination of rhPlGF-1 (260 µg/kg, i.v.) plus rhG-CSF (100 µg/kg per day, s.c.), and at cycle 4 (data not shown) by rhPlGF-1 alone (260 µg/kg, i.v.). Data are expressed as mean ± SEM derived from quadruplicate cultures on samples from each animal at each time point. Statistical differences were evaluated using the Student's t test for paired data (two-tail). (A): Absolute values per milliliter of blood of circulating CFCs. CFCs include granulocyte-macrophage CFC (CFU-GM), erythroid burst-forming unit (BFU-E), and multipotent CFC (CFU-Mix). The absolute number of circulating CFCs in blood is a function of the frequency of CFCs multiplied by the total number of mononuclear cells (MNCs) per milliliter of blood. The average CFC count in control monkeys was 141 ± 26 per milliliter of blood. (B): HPP-CFCs were defined as macroscopically visible colonies of more than 2 mm in diameter of compact colony growth and were scored after 28 days of incubation from methylcellulose cultures. Absolute values per milliliter of blood of circulating HPP-CFCs. The absolute number of circulating HPP-CFCs in blood is a function of the frequency of HPP-CFCs multiplied by the total number of MNCs per milliliter of blood. The average HPP-CFC count in control monkeys was 32 ± 7 per milliliter of blood. °, p < .01 compared with phosphate-buffered saline/mouse serum albumin (PBS/MSA). °°, p < .006 compared with rhG-CSF. *, p < .002 compared with PBS/MSA. **, p < .001 compared with rhG-CSF. Abbreviations: CFC, colony-forming cells; HPP-CFC, high-proliferative potential colony-forming cells.

 
HPP-CFC Mobilizing Effects of rhPlGF-1 and rhG-CSF in Rhesus Monkeys
In addition to CFCs, macroscopically visible colonies of more than 2 mm in diameter of compact growth (HPP-CFCs) were assessed in methylcellulose culture scored after 28 days of incubation [46]. On day 5 of mobilization, HPP-CFC frequency was increased by 6-fold (p ≤ .03), 12-fold (p ≤ .002), and 16-fold (p ≤ .01) under rhG-CSF alone, rhG-CSF plus rhPlGF-1 at 130 µg/kg per day, and rhG-CSF plus rhPlGF-1 at 260 µg/kg per day, respectively (Table 4). The combined rhPlGF-1/rhG-CSF treatment induced a significant increase of HPP-CFC frequencies over those detected under rhG-CSF alone, with increments of twofold (p ≤ .001) and 2.5-fold (p ≤ .001) when rhPlGF-1 was injected at 130 and 260 µg/kg per day, respectively (Table 4).

Under steady-state conditions, 32 ± 7 HPP-CFCs were detected per milliliter of blood. This value was increased by 50-fold (p ≤ .002), 280-fold (p ≤ .0007), and 337-fold (p ≤ .0007) under rhG-CSF alone, rhG-CSF plus rhPlGF-1 at 130 µg/kg per day, and rhG-CSF plus rhPlGF-1 at 260 µg/kg per day, respectively (Fig. 4B). The peak levels of HPP-CFCs per milliliter of blood induced by rhG-CSF were increased by sixfold (p ≤ .001) and sevenfold (p ≤ .001) upon administration of rhG-CSF plus rhPlGF-1 at 130 and 260 µg/kg per day, respectively (Fig. 4B).

LTC-IC Mobilizing Effects of rhPlGF-1 and rhG-CSF in Rhesus Monkeys
To further evaluate the mobilizing activity of the combined rhPlGF-1/rhG-CSF treatment, we investigated the frequency and the absolute numbers per milliliter of blood of the primitive hematopoietic progenitors capable of forming colonies in long-term culture (LTC-IC). As compared with rhG-CSF alone, the combined administration of rhG-CSF and rhPlGF-1 at 130 µg/kg per day increased the mean frequency of LTC-ICs by 14-fold (i.e., from 1 LTC-IC in 83,237 up to 1 in 5,829 [p ≤ .04] MNCs) (Fig. 5A). Increasing the dose of rhPlGF-1 at 260 µg/kg per day did not result in a further increase of LTC-IC frequency (data not shown). As compared with rhG-CSF alone, the absolute numbers of circulating LTC-ICs detected under rhG-CSF plus rhPlGF-1 at 130 and 260 µg/kg per day were increased by 12-fold (p ≤ .006) and 15-fold (p ≤ .007), respectively (Fig. 5B).


Figure 5
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  		Figure 5. Frequency and absolute values of circulating LTC-ICs in monkeys mobilized with recombinant human (rh)PlGF-1 and/or rhG-CSF. Rhesus monkeys (n = 4) received 5-day mobilization cycles at 6-week intervals. Mobilization was elicited at cycle 1 ({circ}) by rhG-CSF alone (100 µg/kg per day, s.c.), at cycle 2 (bullet) by a combination of rhPlGF-1 (130 µg/kg, i.v.) plus rhG-CSF (100 µg/kg per day, s.c.), at cycle 3 ({square}) by a combination of rhPlGF-1 (260 µg/kg, i.v.) plus rhG-CSF (100 µg/kg per day, s.c.), and at cycle 4 (data not shown) by rhPlGF-1 alone (260 µg/kg, i.v.). Data are expressed as mean ± SEM. Statistical differences were evaluated using the Student's t test for paired data (two-tail). (A): Frequency of circulating LTC-ICs (per 105 mononuclear cells [MNCs]). The frequency of LTC-ICs was assayed under limiting dilution conditions using the murine M2-10B4 cell line (kindly provided by Dr. C. Eaves, Terry Fox Laboratory, Vancouver, BC, Canada) as stromal layer. Blood samples were collected on day 5 of mobilization therapy. Serial dilutions of test cells (2 x 105 to 3 x 10) were cultured for 5 weeks, and 16–22 replicates were plated for each test cell dose. After 5 weeks, nonadherent and adherent cells from individual wells were assayed for clonogenic cells, and the LTC-IC frequencies were calculated using Poisson statistics and the method of maximum likelihood. (B): Absolute values per milliliter of blood of circulating LTC-ICs. The absolute number of circulating LTC-ICs in blood is a function of the frequency of LTC-ICs multiplied by the total number of MNCs per milliliter of blood. The average LTC-IC count in control monkeys was 21 ± 6 per milliliter of blood. *, p < .02 compared with PBS/MSA. °, p < .006 compared with rhG-CSF. °°, p < .007 compared with rhG-CSF. Abbreviations: G-CSF, granulocyte colony-stimulating factor; LTC-IC, long-term culture-initiating cells; PBS/MSA, phosphate-buffered saline/mouse serum albumin; PlGF-1, placental growth factor-1.

 
Toxicity
No specific toxicity was associated with the administration of rhPlGF-1 alone or in combination with rhG-CSF. Mobilization therapy did not affect body weight, body temperature, or blood pressure. In monkeys receiving rhPlGF-1 alone or rhPlGF-1/rhG-CSF, no significant changes in serum values of electrolytes, blood urea nitrogen, creatinine, hepatic aminotransferases, bilirubin, alkaline phosphatase, coagulation profiles, serum chemistry, or urine analysis could be detected.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
Attempts to enhance the quality, quantity, and kinetics of cytokine-induced mobilization of PBPCs have been pursued by using a variety of cytokine combination protocols, including rhGM-CSF plus rhG-CSF [19], rhIL-3 plus rhG-CSF or rhGM-CSF [20], and PIXY-321 [17]. Enhancement of PBPC mobilization has also been pursued by incorporating in the standard mobilization regimens early-acting cytokines, such as rhSCF of the flt-3 ligand, capable of expanding marrow progenitors susceptible to subsequent mobilization by rhG-CSF [13–16]. So far, these strategies have resulted in a limited clinical application, due to either the failure to improve hematopoietic mobilization achieved with rhG-CSF alone or increased toxicity. The need to develop new agents for effective and safe mobilization of PBPCs is critically important for the clinical practice of hematopoietic SCT. Recently, AMD3100 and recombinant human GH were shown to significantly improve rhG-CSF-elicited PBPC mobilization in normal donors and poor mobilizers, respectively [16, 28, 29]. In this scenario, rhPlGF-1 might represent a new cytokine to be exploited due to its potent enhancing effect on PBPC mobilization when used in combination with rhG-CSF.

By using two different animal models (i.e., BALB/c mice and rhesus monkeys) that allow simulation of PBPC mobilization as occurs in a clinical situation, we demonstrate herein that rhPlGF-1 synergizes with rhG-CSF in enhancing the frequencies and absolute numbers of a broad spectrum of circulating hematopoietic progenitors, including committed CFCs, HPP-CFCs, primitive LTC-ICs, and radioprotective cells. In BALB/c mice, rhPlGF-1 enhanced rhG-CSF-elicited mobilization of CFCs and LTC-ICs per milliliter of blood by fourfold and eightfold, respectively. In rhesus monkeys, rhPlGF-1 enhanced rhG-CSF-elicited mobilization of CFCs, HPP-CFCs, and LTC-ICs per milliliter of blood by 5-fold, 7-fold, and 15-fold, respectively. Our data significantly extend previous observations in myelosuppressed mice showing that injection of an adenovector expressing PlGF enhances early phases of marrow recovery directly by recruitment of VEGFR-1+ marrow repopulating cells, whereas in later stages PlGF indirectly supports hematopoiesis through release of sKitL mediated by MMP-9 [37]. The preferential mobilization of primitive LTC-IC progenitors observed in our animal models might be explained by the PlGF-induced upregulation of MMP-9 on bone marrow stromal cells which releases sKitL, thus contributing to the deadhesion and mobilization into the circulation of the more primitive stroma-adherent progenitor cells [51, 55].

The lack of WBC and progenitor cell release into the bloodstream under rhPlGF-1 alone which we observed in our model systems is in contrast with previous data [37] and is likely to be due to differences in PlGF plasma levels. Injection of an adenovector expressing PlGF allowed long-lasting and elevated PlGF plasma levels; in contrast, injection of the recombinant protein elicited only transient effects (Fig. 2A). Alternatively, in vivo injection of an adenoviral vector might induce the release of inflammatory cytokines cooperating with PlGF in inducing PBPC mobilization [56]. Under our experimental conditions, rhPlGF-1 administration was associated with a modest, if any, increase of plasma MMP-9, further corroborating the lack of any mobilizing activity of rhPlGF-1 alone. Interestingly, the combined rhPlGF-1/rhG-CSF administration had an additive effect on MMP-9 plasma levels, suggesting that this protease might be critically involved in the molecular mechanism(s) underlying the synergism of rhPlGF-1 and rhG-CSF. Because MMP-9 plays a key role in PBPC mobilization by either releasing sKitL [37, 51] or degrading SDF-1 [52–54], it is possible to hypothesize that the release of proteases by either stromal cells or neutrophils accumulating in the bone marrow during rhG-CSF administration is significantly enhanced by the concomitant rhPlGF-1/rhG-CSF injection, thus resulting in a synergistic effect on hematopoietic mobilization [57–59].

The lower dose of rhPlGF-1 tested in monkeys (i.e., 130 µg/kg per day) was calculated from mice studies using an appropriate conversion factor. The monkey dose was biologically equivalent to the dose of rhPlGF-1 (10 µg/day) which induced a maximal enhancement of rhG-CSF-elicited PBPC mobilization in BALB/c mice [48]. Although two different rhPlGF-1 doses were then investigated in monkeys, we were not aiming to perform a dose-finding study, and the formal identification of the maximal tolerated dose or the optimal therapeutical dose will result from already planned phase I/II studies in humans. In rhesus monkeys, addition of rhPlGF-1 to rhG-CSF did not significantly modify the levels of circulating WBCs or PLTs at either of the two rhPlGF-1 doses used here (130 and 260 µg/kg), whereas the combined regimen resulted in higher values of blood progenitors mobilized throughout the duration of treatment without changes in the mobilization kinetics, and in the absence of clinical and blood chemistry side effects. Therefore, the combined rhPlGF-1/rhG-CSF mobilization regimen might significantly increase the total amount of PBPCs collected during a standard mobilization procedure with rhG-CSF alone in cancer patients or healthy donors without inducing potential side effects related to massive WBC increases. Because rhG-CSF therapy mobilizes similar amounts of WBCs, CFCs, and LTC-ICs in human and nonhuman primates [15, 47, 53], translating the results of the combined rhPlGF-1/rhG-CSF therapy from monkeys to humans would allow us to predict that at least a 1-log increase of committed and primitive progenitors should be achievable both in cancer patients and normal donors in the absence of any additional toxicity.

According to our study design in monkeys, the second and the third mobilization cycles (i.e., rhPlGF-1 plus rhG-CSF) were compared with the first mobilization cycle (i.e., rhG-CSF alone), with each mobilization treatment being separated by a 6-week washout period. For each animal, the kinetic of mobilization after cycle 1 served as intramonkey control to assess the mobilization after cycles 2 and 3. This study design was aimed at preventing the effects the interanimal variability and was based on previous data showing that repeated mobilization cycles have no enhancing or detrimental effect on hematopoietic mobilization provided that an adequate washout period was included between each mobilization procedure [46, 60]. Based on this hypothesis, we did not include a control group receiving rhG-CSF alone at cycles 2 and 3.

The PlGF receptor, VEGFR-1, is expressed by endothelial cells but is also present on inflammatory cells such as monocytes and macrophages [34–36]. Although the precise function of VEGFR-1 remains as unclear as that of its ligands PlGF and VEGF-B, recent studies implicated PlGF binding to VEGFR-1 as an important mediator of stem cell recruitment and mobilization, angiogenesis, and inflammation and also suggested that PlGF treatment may prove more effective, and produce fewer side effects, than application of VEGF-B itself [61, 62]. The application of VEGFR-1 ligands for hematopoietic stem cell recruitment and mobilization could be useful not only in patients undergoing SCT but also in patients with suppressed bone marrow function after irradiation and chemotherapy. However, problems could arise with systemic VEGFR-1 therapies in that VEGFR-1 stimulation of neovascularization could also accelerate retinopathy, plaque formation, or possibly tumor growth. All these issues should be carefully addressed in future clinical studies aimed at exploring the hematopoietic modulating effects of rhPlGF-1.

Our data demonstrate that rhPlGF-1 significantly enhances rhG-CSF-elicited hematopoietic mobilization and provide a preclinical rationale for evaluating the potential clinical benefit of rhPlGF-1 in conjunction with rhG-CSF to mobilize PBPCs for application in peripheral blood SCT, gene therapy, and/or immunotherapy. A dose-finding study aimed at identifying the dose of rhPlGF-1 capable of improving rhG-CSF-elicited mobilization is planned in patients with cancer.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
This work was supported in part by grants from Ministero dell'Istruzione, dell'Università e della Ricerca (Rome), Ministero della Salute (Rome), and Michelangelo Foundation for Advances in Cancer Research and Treatment (Milan, Italy). We thank Dr. A. Mion (Geymonat S.p.A.) for the kind gift of rhPlGF-1.


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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