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Expression of pleiotrophin an embryonic growth and differentiation factor in rheumatoid arthritis.

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Vol. 48, No. 3, March 2003, pp 660–667
DOI 10.1002/art.10839
© 2003, American College of Rheumatology
Expression of Pleiotrophin, an Embryonic Growth and
Differentiation Factor, in Rheumatoid Arthritis
Thomas Pufe, Michaela Bartscher, Wolf Petersen, Bernhard Tillmann, and Rolf Mentlein
Objective. Pleiotrophin (PTN), a 15.3-kd heparinbinding peptide, is expressed in mesodermal and neuroectodermal cells during development, but rarely in
adult tissues. Since developmentally regulated factors
often reappear during disease, we sought to determine
whether there was PTN expression in the synovial
membranes of patients with rheumatoid arthritis (RA).
Methods. PTN messenger RNA expression was
assayed by quantitative reverse transcriptase–
polymerase chain reaction. The protein was localized by
immunohistochemistry and quantified by enzymelinked immunosorbent assay (ELISA). Effects of PTN
on cell proliferation in vitro were determined by DNA
Results. PTN expression in normal adult synovial
membranes and cartilage was barely detectable. However, PTN was strongly up-regulated in synovial tissues
from patients with RA. In contrast, samples from patients with pyogenic arthritis had moderate PTN levels,
and those from patients with osteoarthritis had only a
slight increase in PTN, as measured by ELISA. In RA
patients, PTN was localized primarily in synoviocytes
but was also found in endothelial cells of blood vessels.
In cultured mouse fibroblasts used as a model, PTN
expression was up-regulated by tumor necrosis factor ␣
and was more weakly up-regulated by epidermal growth
factor. Recombinant PTN stimulated the proliferation
of cultured human synoviocytes and the monocyte cell
line THP-1, but not human dermal fibroblasts, in which
PTN increased the synthesis of vascular endothelial
growth factor.
Conclusion. In addition to certain types of cancer,
the embryonic growth and differentiation factor PTN is
expressed in adults with inflammatory diseases, in
particular, RA. Proinflammatory cytokines enhance the
expression of PTN. Thus, we propose that PTN is a
further paracrine angiogenesis and growth factor for
synovial cells in RA.
Pleiotrophin (PTN), which is also known as
heparin-binding growth-associated molecule, heparinbinding growth factor 8, heparin-binding neurotrophic
factor, and osteoblast-specific factor 1, is an 136–amino
acid (15.3-kd) secreted growth/differentiation cytokine
that is developmentally regulated (for review, see refs.
1–3). Mature PTN is a nonglycosylated lysine-rich peptide that migrates anomalously as an 18-kd band on
sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and is therefore called p18 (4). The
mature form of PTN is derived from a 168-residue
precursor with a 32–amino acid signal sequence. The
molecule is called “pleiotrophin” because of its function
as a differentiation factor or growth factor for various
cell types. PTN promotes angiogenesis, stimulates neurite outgrowth from cultured neurons, induces a bipolar
form of glial cell precursors, and induces cell migration
(5–9). Together with midkine, PTN forms a family of
heparin-binding proteins that are normally expressed
during embryogenesis but not (or only at low levels) in
healthy adult tissues (10). However, PTN is reexpressed
in some human tumors, e.g., meningiomas (11), gliomas
(12), some breast cancers (13), and pancreatic cancers
(14). There is also evidence that PTN is involved in
tumorigenesis by enhancing the angiogenesis or proliferation of the tumor cells themselves (13–16).
The hyperplastic synovial pannus in rheumatoid
arthritis (RA) resembles a solid tumor in certain ways,
Supported by grants from the Muskel- und Skelettsystem of
the Medical Faculty of the University Kiel and the Verein zur
Förderung und Bekämpfung Rheumatischer Erkrankungen, Bad
Bramstedt, Germany.
Thomas Pufe, PhD, Michaela Bartscher, Wolf Petersen, MD,
Bernhard Tillmann, MD, Rolf Mentlein, PhD: University of Kiel, Kiel,
Dr. Pufe and Ms Bartscher contributed equally to this work.
Address correspondence and reprint requests to Professor
Rolf Mentlein, PhD, Department of Anatomy, University of Kiel,
Olshausenstrasse 40, Kiel D-24098, Germany. E-mail: rment@
Submitted for publication June 7, 2002; accepted in revised
form December 2, 2002.
especially its intrinsic cellular proliferation and invasive
properties and the association of angiogenesis with the
development of this highly vascularized tissue (17–19).
Some oncogenes and angiogenesis factors constitutively
produced by malignant cells have been detected in cells
of the synovium and synovial fluid in RA (20–22). We
therefore investigated whether PTN, a growth and angiogenesis factor of tumors, is produced in RA and
whether it also affects the proliferation of synoviocytes.
Peptides and antibodies. Goat anti-PTN (affinitypurified IgG fraction; catalog no. AF-252-PB) and biotinylated
anti-PTN (catalog no. BAF252) were purchased from R&D
Systems (Minneapolis, MN). Secondary antibodies were obtained from Sigma (Taufkirchen, Germany). Recombinant
human PTN, tumor necrosis factor ␣ (TNF␣), and epidermal
growth factor (EGF) were obtained from PeproTech (Rock
Hill, NJ).
Tissues and cell cultures. Synovial tissues and synoviocytes were obtained from patients with RA, pyogenic arthritis
(PA), and osteoarthritis (OA) at the time of therapeutic
synovectomy, which was performed at the Department of
Orthopaedic Surgery, Christian-Albrechts University (Kiel,
Germany). Samples from RA patients were obtained during
the early phase of disease (1–5 years from disease onset) from
joints with radiographic Larsen scores of 0–2. These patients
had been treated with sulfasalazine and a low dose of cortisone
(5 mg/day). Healthy control samples were obtained during
routine autopsies performed at the Department of Anatomy,
University of Kiel (Kiel, Germany). Synovial fluids were
obtained from RA, PA, and OA patients who were undergoing
arthroscopy at the Ambulantes Operationszentrum am Eichkoppelweg (Kiel, Germany).
Human THP-1 cells were obtained from the National
Institutes of Health (Bethesda, MD) and normal human
dermal fibroblasts from PromoCell (Heidelberg, Germany).
Cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal calf serum (FCS). Synoviocytes
were isolated without enzymatic digestion from synovial membranes obtained from cadavers. Synovium was cut into small
pieces, transferred to petri dishes, and kept in DMEM–50%
FCS for 24 hours. Then, the pieces were removed, and the cells
that migrated out of the tissue and adhered to the bottoms of
the petri dishes were cultured for 48 hours in DMEM–10%
FCS. Cells were subcultured in trypsin–EDTA and used for
proliferation assays after a further 48 hours of culture.
Reverse transcriptase–polymerase chain reaction (RTPCR). RNA was isolated by a standard guanidinium
thiocyanate–phenol–chloroform extraction method, and digested with DNase. For standard RT-PCR, complementary
DNA was generated and amplified for PTN with the primers
5⬘-CCT-TCT-TGG-CAT-TCA-TTT-TCA-TAC-3⬘ (sense) and
5⬘-GAG-GTT-TGG-GGT-TGG-TCA-GT-3⬘ (antisense) at an
annealing temperature of 60°C for 40 cycles, according to
previous protocols (23). The RT-PCR product was identified
by agarose gel electrophoresis and by direct sequencing with
the DyeDeoxy Terminator Cycle Sequencing method using an
ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster
City, CA) as described elsewhere (23). A separate RT-PCR for
GAPDH with an intron-spanning primer pair served as control
for the intactness of RNA and the absence of contaminating
DNA (23).
For real-time RT-PCR, 100 ng of total RNA was
reverse transcribed and amplified in the presence of SYBR
Green with a commercial 1-step system (QuantiTect SYBR
Green RT-PCR; Qiagen, Hilden, Germany) using the above
primers. Amplification was monitored with an iCycler (BioRad, Munich, Germany) according to standard procedures.
PCR was performed using Hot StarTaq DNA polymerase,
which is activated by an initial heating step, whereas Omniscript Reverse Transcriptase is deactivated. The temperature
profile included an initial denaturation for 15 minutes at 95°C,
followed by 37 cycles at 95°C for 15 seconds, annealing at 60°C
for 30 seconds, elongation at 72°C (elongation time depended
on the size of the fragment; the number of basepairs divided by
25 yielded the time in seconds), and fluorescence monitoring at
For analysis of the PCR data, iCycler Data Analysis
software (Bio-Rad) was used. The specificity of the amplification reaction was determined by performing a melting curve
analysis. Relative quantification of the signals was performed
by normalizing the signals of the different genes with a ␤-actin
signal, with the primers 5⬘-TGC-CAT-CCT-AAA-AGCCAC-3⬘ (sense) and 5⬘-TCA-ACT-GGT-CTC-AAG-TCAGTG-3⬘ (antisense).
Western blots and immunohistochemistry. Western
blotting was performed as previously described (12,23). Briefly,
samples were reduced and boiled in SDS sample buffer with
2-mercaptoethanol, separated on 15% SDS-PAGE gels, and
transferred by semidry method onto nitrocellulose membranes. Bands were detected by chemiluminescence.
For immunohistochemistry, tissues were fixed with 4%
formaldehyde in phosphate buffered saline (PBS; 30 minutes
at room temperature), dehydrated, and embedded in paraffin.
Sections (8 ␮m) were deparaffinized in xylol (3 times for 10
minutes each), organic solvent was removed with decreasing
concentrations of ethanol (100%, 90%, 80%, 70%, 60%, and
50%; 2 minutes each), then sections were immersed in doubledistilled water for 10 seconds. After washing 3 times with Tris
buffered saline (TBS; pH 7.4), sections were demasked with
trypsin (0.1% in TBS with 0.1% CaCl2), washed 3 times with
TBS, and incubated overnight at 4°C with anti-PTN (diluted
1:500 in PBS). Sections were then washed 3 times with TBS,
incubated for 30 minutes at room temperature with
peroxidase-labeled anti-goat IgG (1:250 in PBS), washed another 3 times with TBS, stained for 7 minutes at room
temperature with 3-amino-9-ethylcarbazole/H2O2 (Universal
Peroxidase Detection kit; Coulter Immunotech, Hamburg,
Germany), and then counterstained with Mayer’s hemalum.
Enzyme-linked immunosorbent assay (ELISA). For
ELISA, tissues were homogenized in 0.14M NaCl, 20 mM
HEPES (pH 7.4), centrifuged at 15,000g for 15 minutes at 4°C,
and the supernatants were analyzed. The ELISA was performed with Nunc-Immuno Maxisorp plates (Nunc, Roskilde,
Denmark) that had been coated overnight at room temperature with anti-PTN (50 ng/well), washed 3 times with 0.05%
Tween 20 in PBS (washing buffer), blocked for 1 hour at room
temperature with 1% BSA in PBS, and washed with washing
buffer. Wells were incubated for 2 hours at room temperature
with samples, standards, or blanks (300 ␮l, diluted with PBS),
washed 3 times with washing buffer, and incubated for another
2 hours at room temperature or overnight at 4°C with biotinylated anti-PTN (50 ng/well). After washing 3 times with
washing buffer, wells were incubated for 20 minutes at room
temperature with a 1:20,000 dilution of a 1.25-mg/ml solution
Figure 2. Pleiotrophin (PTN) protein concentrations in synovial
membranes from patients with rheumatoid arthritis (RA), pyogenic
arthritis (PA), and osteoarthritis (OA), as well as in normal cadavers.
PTN protein concentrations are highly elevated in synovial membranes
from RA patients, moderately elevated in those from PA patients, but
only slightly elevated in those from OA patients. Tissues were homogenized, and PTN concentrations were determined by enzyme-linked
immunosorbent assay (related to wet tissue weight). Values are the
mean ⫾ SD of triplicate determinations of samples from individual
subjects. The following between-group comparisons were statistically
significant: RA versus normal (P ⬍ 0.01), PA versus normal (P ⬍ 0.05),
and RA versus OA (P ⬍ 0.05).
Figure 1. Detection of pleiotrophin (PTN) mRNA in synovial tissues
by A, reverse transcriptase–polymerase chain reaction (RT-PCR) in
inflamed synovial tissue and B, real-time RT-PCR, showing higher
expression in rheumatoid arthritis (RA) synovium. RNA was extracted
from the synovial membranes of normal cadavers and patients with
osteoarthritis (OA), pyogenic arthritis (PA), or RA, and then reverse
transcribed. A, For RT-PCR, cDNA was amplified with primers
specific for PTN or GAPDH (control for intactness of RNA and
absence of contaminating DNA). Products were separated by agarose
gel electrophoresis and stained with ethidium bromide. C ⫽ control
(without RNA). The PTN PCR products correspond to the predicted
size of 402 bp, as estimated from the 100-bp markers (M); their
identity was further verified by sequencing. B, Real-time RT-PCR was
performed in the presence of SYBR Green as described in Materials
and Methods. Relative quantification of the PTN signals was performed by normalizing them to a ␤-actin signal. Values are the mean
and SD of 3 experiments.
of streptavidin–peroxidase (catalog no. 43-4323; Zymed, Burlingame, CA), washed 3 times with washing buffer, and then
incubated for 30 minutes at room temperature with 100 ␮l of
tetramethylbenzidine–H2O2 (TMB Substrate kit, catalog no.
34021; Pierce, Rockford, IL). The reaction was stopped by the
addition of 50 ␮l of 0.5M H2SO4, and the yellow dye was
measured at an absorbance of 450 nm. The assay was linear for
0.05–1.5 ng of PTN (detection limit ⬃0.02 ng).
Stimulation of cells and assays for proliferation and
vascular endothelial growth factor (VEGF). Cells (106) were
seeded into fresh dishes and cultured for 24 hours in DMEM
plus 10% FCS. The medium was replaced with DMEM plus
0.5% FCS, and the cells were exposed to the stimulators for 24
hours. Conditioned medium was removed, and aliquots were
assayed for PTN or VEGF content. The cells were then
washed with PBS, lysed, and the DNA content was measured
fluorometrically with the CyQuant reagent (Molecular Probes,
Eugene, OR) and related to a measurement of microscopically
counted number of trypsinized cells. VEGF content was
Figure 3. Strong immunostaining of pleiotrophin (PTN) in synovial membranes of rheumatoid arthritis
(RA) patients, with much weaker staining in osteoarthritis (OA) and normal synovium. In normal
synovium, only a few single cells, mostly endothelial cells, are PTN positive (red). The synovial surface
and the lining cells are immunonegative. In OA synovium, the lining cells are PTN immunoreactive.
Staining in RA synovium is strongly immunopositive, with immunoreactivity in lining cells as well as
endothelial cells. RA synovium is immunonegative after absorption of the antibody with recombinant
PTN (antibody control [Co]). Blue areas indicate nuclear counterstaining with Mayer’s hemalum. Bars ⫽
10 ␮m.
determined with a sandwich ELISA as described previously
Proliferation assays were performed in DMEM plus
0.5% FCS. 3H-thymidine incorporation (72 hours of stimulation; 6 hours of incorporation in the presence of stimulators)
was measured in adherent subconfluent cells, as described
elsewhere (12). Nonadherent THP-1 cells were subjected to 48
hours of stimulation, and DNA was quantified with the CyQuant method (24).
Statistical analysis. Statistical significance was evaluated by Dunnett’s multiple comparisons test or by t-test.
Detection of pleiotrophin in RA synovial tissue
by RT-PCR and quantification by real-time RT-PCR
and ELISA. Using sequence-specific primers, we detected PTN messenger RNA (mRNA) in synovial tissues
from RA patients (Figure 1A), as well as from patients
with PA and OA. Quantitative measurements by realtime RT-PCR showed considerably higher PTN mRNA
expression in RA synovium than in normal synovium,
whereas in OA synovium, only a moderate increase was
detected (Figure 1B). Quantification of PTN protein by
ELISA yielded similar results (Figure 2). Synovial tissues from controls (cadavers without signs of RA or joint
diseases) showed a low, but measurable, PTN content.
In samples from RA patients, the PTN content was
elevated 3–5-fold over that in the normal controls. In
samples from PA patients, a lower (up to 2.5-fold)
increase in PTN concentration was detected, whereas
the PTN content was increased only in some of the
samples from patients with OA.
Immunostaining of PTN in the synovium of RA
patients. Consistent with the ELISA measurements,
synovial tissues from RA patients showed intense immunostaining for PTN (Figure 3). Immunostaining of OA
synovium was much weaker. In normal adult synovium
(obtained from cadavers), only single cells, mostly endothelial cells, were PTN immunopositive. Reactivity in
Figure 4. Staining of 18-kd and 36-kd bands in Western blots of
rheumatoid arthritis (RA) synovial proteins by pleiotrophin (PTN)
antibody. Synovial tissue from RA patients was boiled and reduced in
sodium dodecyl sulfate sample buffer. Equal amounts of protein from
2 different samples (lanes 1 and 2) were separated on 15% sodium
dodecyl sulfate–polyacrylamide gel electrophoresis gels, blotted onto
nitrocellulose paper, stained with anti-PTN followed by a peroxidaselabeled secondary antibody, and detected by chemiluminescence. A
control with PTN-inactivated antibody remained negative (lane C).
The positions of molecular mass markers (from the top, chicken
ovalbumin, bovine erythrocyte carbonic anhydrase, soybean trypsin
inhibitor, bovine milk ␣-lactalbumin, and bovine lung aprotinin) are
shown on the left. The estimated apparent molecular masses for PTN
and its putative dimer are shown on the right.
OA and RA synovium was confined to the lining cells,
immunostaining both type A (macrophage-like) and
type B (fibroblast-like) cells. These cells were negative in
normal synovium. Endothelial cells in RA and OA
synovium, as well as normal synovium, were also immunopositive for PTN (Figure 3). There was no immunoreactivity in control samples subjected to preadsorption
of the antibody with recombinant PTN (Figure 3), those
in which the primary antibody had been omitted (results
not shown), and those incubated with anti-goat IgG
control antibody (results not shown).
The specificity of the antibody was further evaluated by Western blotting experiments with RA synovium (Figure 4). The antibody stained an 18-kd protein
(an apparent molecular mass corresponding to the value
previously reported for PTN [4]) as well as a 36-kd
protein (probably the dimer [for comparison, see ref. 3]).
Staining of both bands could be suppressed by preadsorption of the antibody with recombinant PTN. No
other strong bands of staining were identified.
Induction of PTN in fibroblasts by TNF␣. Several cytokines are involved in the inflammatory process
of RA, in particular, TNF␣. Using human dermal fibroblasts and the human monocyte cell line THP-1 as a
model for fibroblast-like and macrophage-like synovial
cells, respectively, we determined whether TNF␣ could
induce the expression of PTN. Indeed, stimulation of
dermal fibroblasts with this cytokine yielded an ⬃9-fold
induction of PTN secretion into the culture medium
(Figure 5). Treatment with EGF had a considerably
smaller induction effect. Thus, expression of PTN in
fibroblasts (and probably cells derived from them) can
be induced by proinflammatory cytokines and growth
Stimulation of proliferation and gene expression
in synoviocytes by PTN. To identify target cells for PTN,
we investigated the effects of PTN on the proliferation
of synoviocytes as well as on the gene expression of
human dermal fibroblasts and a monocyte cell line that
served as a model for fibroblast-type and monocyte/
macrophage-type synovial cells. PTN increased the proliferation of synoviocytes ⬃2.5-fold at concentrations of
10–100 ng/ml (Figure 6). Proliferation of the human
monocyte cell line THP-1 was also stimulated by PTN,
but proliferation of human dermal fibroblasts was not
As an example of the effects of PTN on gene
induction, we investigated its possible effects on the
expression of the angiogenesis factor VEGF. Synthesis
of VEGF, as measured by its release into the culture
medium after 24 hours of stimulation, was increased by
PTN in dermal fibroblasts, but not in THP-1 cells
Figure 5. Induction of pleiotrophin (PTN) secretion in human dermal
fibroblasts by tumor necrosis factor ␣ (TNF␣) and epidermal growth
factor (EGF), and negligible PTN production in the human monocyte
cell line THP-1, which cannot be stimulated. Cells were stimulated
without (control [C]) and with 10 ng/ml of the cytokines for 24 hours
in medium containing 0.5% fetal calf serum, and PTN was measured in
the supernatants by enzyme-linked immunosorbent assay. Values are
the mean and SD of 4 samples. P ⬍ 0.01 for cytokines versus control
in the fibroblast studies.
Figure 6. Induction of the proliferation of synoviocytes and monocytes, but not fibroblasts, by pleiotrophin
(PTN). Human synoviocytes, the monocyte cell line THP-1, and human dermal fibroblasts were stimulated
without (control [C]) and with different concentrations of PTN for 24 hours in medium containing 0.5%
fetal calf serum, and cell numbers were determined by 3H-thymidine incorporation (synoviocytes,
fibroblasts) or DNA quantification (THP-1). Values are the mean and SD of 6 individual cultures each. P ⬍
0.01 for PTN versus control in the synoviocytes and in the THP-1 cells at 100 ng/ml of PTN.
(Figure 7). The results of these experiments show not
only that PTN is produced in synoviocytes, but also that
synoviocytes are the targets of PTN.
Figure 7. Induction of vascular endothelial growth factor (VEGF)
synthesis in fibroblasts by pleiotrophin (PTN). Human dermal fibroblasts or human THP-1 monocytes used as a model for fibroblast-type
or macrophage-type cells were stimulated without (control [C]) and
with 10 ng/ml of PTN for 24 hours in medium containing 0.5% fetal
calf serum, and VEGF was measured in the supernatants by enzymelinked immunosorbent assay. Values are the mean and SD of 4
samples. P ⬍ 0.05 for PTN versus control in the fibroblasts. TNF␣ ⫽
tumor necrosis factor ␣; EGF ⫽ endothelial growth factor.
Chronic inflammation is characterized by the
production of various cytokines and angiogenic factors,
e.g., TNF␣, interleukins 1 and 6, VEGF, and basic
fibroblast growth factor (20,21,25). We have shown that
PTN, a developmentally expressed growth and differentiation factor, is reexpressed and up-regulated in RA
synovial membranes, whereas it is not expressed or is
only weakly expressed in tissues from normal controls or
from patients with other joint diseases (PA and OA).
Induction experiments with fibroblasts showed that
proinflammatory cytokines such as TNF␣ up-regulate
the expression of PTN and that growth factors such as
EGF or platelet-derived growth factor (see ref. 26) have
smaller effects.
The stimulatory effects of PTN on the proliferation of synoviocytes demonstrated that this peptide acts
as a paracrine growth factor. Experiments with a monocyte cell line as a model suggested that PTN targets the
macrophage-type cells in the synovial membrane in RA,
whereas recombinant PTN was not mitogenic for dermal
fibroblasts in our experiments. Moreover, gene expression (e.g., expression of mRNA for VEGF) was upregulated by PTN. Since PTN has repeatedly been
reported to induce the proliferation of endothelial cells
(5,6,9,26), we think that besides other factors such as
VEGF (21), PTN could be involved in the extensive
vascularization of the synovium that is observed in RA.
Thus, PTN is a further paracrine mitogenic and angiogenic factor that contributes to the inflammatory process
in RA.
Receptors for PTN and their signal transduction
mechanisms have not yet been fully characterized. So
far, interaction of PTN with the receptor-type protein
tyrosine phosphatase ␨/␤, anaplastic lymphoma kinase,
and syndecan 3 have been shown (2,28,29). By disruption of the normal balance between tyrosine kinase and
phosphatase activities, phosphorylation of intracellular
proteins has been found to be increased, particularly the
phosphorylation of ␤-catenin (28), which is involved in
transcription and transformation of the cytoskeletal architecture and the phosphorylation of downstream effector molecules, such as insulin receptor substrate 1,
Shc, phospholipase C␥, and phosphatidylinositol 3⬘kinase (29). However, the mechanisms and biologic
effects differ significantly between the various target cell
The expression and effects of PTN have been
primarily studied during embryogenesis. So far, clinical
interest in PTN has focused on its expression in certain
types of cancer, especially breast cancer, pancreatic
cancer, and melanoma (11–16). Chronic inflammatory
diseases such as RA should be added to the list of
pathologic conditions in which there is PTN expression.
Whether PTN functions as a repair factor or an amplifier of inflammation may depend on the tissue.
We thank Martina Burmester, Dagmar Freier, Miriam
Lemmer, Frank Lichte, Karin Stengel, and Regine Worm for
their expert technical assistance and Clemens Franke for
drawing the illustrations.
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expressions, factors, growth, differentiation, embryonic, arthritis, pleiotrophin, rheumatoid
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