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Division of Pathophysiological and Experimental Pathology, Department of Pathology, and
Department of Orthopedic Surgery, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan;
DNAVEC Research, Tsukuba, Ibaraki, Japan; and
AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Angiogenesis, the formation of new blood vessels, may also critically affect the disease progression in RA (9, 10, 11, 12). Various angiogenic factors, including vascular endothelial growth factor (VEGF) and fibroblast growth factors (FGFs), have been noted in synovial tissue or the joint fluid of human RA, suggesting that these angiogenic growth factors may contribute to disease initiation/progression of RA (12, 13, 14, 15, 16, 17, 18, 19, 20, 21). FGF-2 is a member of the family of heparin-binding growth factors, which show specifically enhanced expression in RA synovial fluid, compared with that in osteoarthritis in human subjects (13). In addition, FGF-2 stimulates angiogenic events partly by up-regulating VEGF (22), and FGF-2 directly accelerates osteoclast maturation to promote bone resorption, as well as osteoclastogenesis in murine bone marrow cultures (23, 24, 25, 26), suggesting its disease-modulatory role in the progression of RA. However, information regarding direct evidence for the role of FGF-2 in RA in vivo is scanty.
To determine the exact role of overexpression of FGF-2 in the RA joint in vivo, we conducted in vivo gene transfer of FGF-2 using a recombinant Sendai virus vector (SeV), which has shown effective transfer potential to various organs (27, 28). We show in this study that the endogenous FGF-2 level is elevated in rat joints of adjuvant-induced arthritis (AIA), as well as in human RA fluid. Furthermore, the SeV-mediated overexpression of FGF-2 enhanced, and inversely, inhibition of endogenous FGF-2 via administration of neutralizing Ab attenuated the disease severity with regard to joint swelling and destruction in AIA. To our knowledge, this is the first direct in vivo evidence indicating the disease-modulatory role of FGF-2 in AIA, and we conclude that FGF-2 deserves further attention as a possible therapeutic target in care of human RA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synovial fluid was obtained from knee joints of 12 RA Japanese
patients with severe joint edema, who were under the care of Showa
Central Hospital (Kitakyushu, Japan). The diagnosis of RA was based on
the American Rheumatism Association (1987) criteria for RA
(29). All the patients were classified grade IV or V in
accordance with Larsen and Thoen classification in terms of the degree
of knee joint destruction seen on plain x-ray photographs
(30). Synovial fluids were aspirated under aseptic
conditions using 18-gauge needles, and were treated with or without
25% hyaluronidase for 1 h at 37°C before measurements.
Quantitation of human FGF-2 and human VEGF proteins was done by ELISA
using commercially available systems (R&D Systems, Minneapolis, MN).
The lower limit of sensitivity of the systems was consistently 
3
pg/ml. Written informed consent for the study was obtained from all the
patients.
Rat model of AIA
One hundred and twenty-four inbred male Lewis rats, which were Charles-River grade (8 wk of age; purchased from KBT Oriental, Tosu, Saga, Japan), were used. Eighty-two rats were immunized s.c. at the base of the tail with 1 mg Mycobacterium Butyricum desiccated (MBD; Difco, Detroit, MI) dissolved in 100 µl mineral oil (NACALAI TESQUE, Kyoto, Japan) on day 0 to prepare the AIA model (31). The onset of AIA was approximately 10 days after the adjuvant treatment. These experiments were reviewed by the Committee of the Ethics on Animal Experiment in Faculty of Medicine, Kyushu University, and conducted under the control of the Guideline for Animal Experiment in Faculty of Medicine, Kyushu University, and The Law (number 105) and Notification (number 6) of the Government.
Recombinant SeVs
Recombinant SeVs were constructed, as described (32, 33). In brief, 18 bp of spacer sequence
5'-(G)-CGGCCGCAGATCTTCACG-3' with a NotI restriction site
were inserted between the 5' nontranslated region and the initiation
codon of the nucleoprotein (N) gene. This cloned SeV genome also
contains a self-cleaving ribosome site from the antigenomic strand of
the hepatitis 
virus. The entire cDNA-coding Escherichia coli
lacZ with a nuclear localizing signal (for
SeV-NLS-lacZ), luciferase (for SeV-luciferase), and murine
FGF-2 (for SeV-FGF-2) were amplified by PCR, using primers with a
NotI site and new sets of SeV E and S signal sequence tags
for an exogenous gene, then inserted into the NotI site of
the cloned genome. The entire length of the template SeV genomes,
including exogenous genes, was arranged in multiples of six nucleotides
(so-called "rule of six") (34). Template SeV genomes
with an exogenous gene and plasmids encoding N, P, and L proteins
(plasmids pGEM-N, pGEM-P, and pGEM-L) were complexed with commercially
available cationic lipids, then cotransfected with vaccinia virus vT7-3
into CV-1 or LLMCK cells. Forty hours later, the cells were disrupted
by three cycles of freezing and thawing and injected into the
chorioallantoic cavity of 10-day-old embryonated chicken eggs.
Subsequently, the virus was recovered and the vaccinia virus was
eliminated by a second propagation in eggs. Virus titer was determined
using chicken RBCs in hemagglutination assay (35), and
viruses were kept frozen at -80°C until use.
SeV-mediated reporter gene transfer to rat joints
Characterization of SeV-mediated gene transfer was determined in
AIA ankle and knee joints, using SeVs encoding reporter genes
(n = 8 joints for lacZ, and
n = 36 joints for luciferase). The ankle joints were
held at 20° of plantar flexion, and a 30-gauge needle on a plastic
syringe was inserted anterolaterally at the ankle joint line 1 mm
distal to the lateral malleolus. The knee joints were held at 90° of
flexion, and the needle was inserted at the femorotibial joint line
1 mm medial to the patellar tendon. Four rats for AIA were treated
with 1 mg MBD on day 0, and injected intraarticularly with
SeV-NLS-lacZ (5 x 107 PFU) into
the ankle and the knee joints at 7 days after the adjuvant treatment.
Two days later, each joint was dissected out en bloc, incised
transversally to expose the joint space. These en bloc were incubated
with the 
-galactosidase substrate
5-bromo-4-chloro-3-indolyl--D-galactoside
(X-gal) for 3 h at room temperature (36, 37, 38). All
samples were photographed using a stereoscopic dissecting microscope
(Carl Zeiss, Oberkochen, Germany).
Sixteen rats were intraarticularly injected with 5 x 107 PFU of SeV-luciferase into bilateral ankle joints, and eight, four, and four rats were killed on 2, 7, and 14 days after the virus injection, respectively. Two naive rats, i.e., four ankle joints were prepared for negative control. Each ankle joint was dissected out and incised transversally to expose the joint space, and incubated with 500 µl 1x cell culture lysis buffer (Promega, Madison, WI) for 5 min. Twenty microliters of the supernatant were exposed to 100 µl luciferase assay buffer (Promega), and the luciferase activity was measured using a luminometer (model LB9507; EG&G Berthold, Bad Wildbad, Germany). Data were expressed as relative light units/mg protein. Protein concentrations were determined by Bradford’s method with a commercially available protein assay system (Bio-Rad, Hertfordshire, U.K.).
Measurement of FGF-2 and VEGF in rat joint extracts
Twenty-four rats were immunized with 1 mg MBD on day 0 for AIA
rats. Among them, 12 rats were intraarticularly injected with 5 x
107 PFU of SeV-FGF-2 into bilateral ankle joints
for AIA + FGF-2 group; the others were injected with 50 µl PBS into
bilateral ankle joints on day 7 for AIA. Each ankle joint was dissected
out and incised transversally to expose the joint space, then incubated
with 500 µl 1x cell culture lysis buffer (Promega) for 5 min on day
9. Protein levels of murine FGF-2 and VEGF were measured using
commercially available ELISA systems (R&D Systems). Data were expressed
as FGF-2 or VEGF protein ng/mg protein. The lower limit of sensitivity
of the FGF-2 and VEGF ELISA was consistently 3 pg/ml.
Treatment protocol
Experimental groups and experimental protocols for FGF-2
overexpression are summarized in Fig. 1
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Rats were divided into two groups: control rats (C rats:
n = 24) and AIA rats (n = 36). C rats
were injected with 100 µl mineral oil at the base of the tail, and
AIA rats were immunized with MBD on day 0. The C rats were further
divided into two groups: C + FGF-2 group injected SeV-FGF-2
(n = 12) and C + luciferase group injected
SeV-luciferase (n = 12). AIA rats were further
separated into three groups: AIA group given PBS injection, AIA + FGF-2
group given SeV-FGF-2, and AIA + luciferase group given SeV-luciferase
(eight joints for each group). Twelve rats of AIA group were killed on
days 14, 21, and 28 (four rats, i.e., eight joints for each day).
Twenty-four rats of AIA + FGF-2 and AIA + luciferase groups were
intraarticularly injected with SeV-FGF-2 or SeV-luciferase into the
right or left ankle joint, respectively, on day 7, and killed on days
14, 21, and 28 (eight rats for each day, i.e., eight joints for each
group per each day).
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The extent of swelling in the hind paw was measured using a volume meter (MK-550; Muromachi Kikai, Tokyo, Japan) at each time point by two individual examiners (A. Yamashita and T. Irisa) in a blinded fashion. After macroscopic examinations, the hind paws were amputated at the middle of the leg and imaged on industrial x-ray film (Fuji, Tokyo, Japan) to evaluate bone and joint destruction radiologically (31), using Softex imaging equipment (CMB-2; Softex, Tokyo, Japan). The severity of bone and joint destruction was scored for each ankle joint, according to the Barbier et al. (31, 39) method, but with some modifications. Briefly, based on soft tissue swelling, joint space narrowing, extent of bone atrophy (osteoporosis), erosion, and bone destruction due to osteolysis of each ankle joint was scored, using a scale of 0–4 termed the radiological index.
Histopathological assessment of arthritis
Ankle joints were resected en bloc and fixed in 4% paraformaldehyde for 4 days. After decalcification with 10% EDTA for 14 days, the ankle joints were sectioned on the sagittal plane through the center of the joint line. Samples were embedded in paraffin, and 3-µm sections were prepared for H&E staining. Whole sections were histopathologically analyzed for the degree of synovitis, proliferation of synovial granulation tissue known as a pannus, and associated destruction of bone and cartilage. For an objective evaluation of synovitis, infiltrating granulocytes were counted at the adjacent synovium to talo-tibial joint in a high power field (x400) by two individual pathologists (A. Yamashita and Y. Yonemitsu) in a blinded fashion.
Immunohistochemical study
To detect vascular endothelial cells, an immunohistochemical examination for rat von Willebrand factor (vWF) was done using dextran polymer conjugate two-step visualization, so-called the EnVision system (40). In brief, deparaffinized sections were digested by 0.1% trypsin for 30 min at 37°C and incubated with 10% normal goat serum for 10 min to minimize nonspecific binding of the primary Ab, then were incubated with the primary rabbit anti-vWF (DAKO, A/S, Glostrup, Denmark) Ab overnight at 4°C in a moisture chamber. To inhibit endogenous peroxidase activity, the sections were incubated with 1% H2O2 in absolute methanol for 30 min. Thereafter, the sections were incubated with EnVision (EnVision+, rabbit/HRP; Dako) for 30 min. The EnVision was a polymeric conjugate consisting of a large number of secondary Abs (goat anti-rabbit) bound directly to a dextran backbone containing HRP. Visualization of a positive reaction was developed with a peroxidase substrate solution containing 0.02% (w/v) H2O2 and 0.1% (w/v) 3,3'-diaminobenzidine tetrahydrochloride (Merck, Darmstadt, Germany) in PBS to give the reaction product a brown color, then the sections were lightly counterstained with hematoxylin. Small vessels consisting of vWF-positive endothelial cells were counted at three optional lesions for every ankle joint by two individual pathologists (A. Yamashita and Y. Yonemitsu) in a blinded fashion, and numbers of vessels in each ankle joint were expressed as the average value.
Tartrate-resistant acid phosphatase stain for osteoclasts
The tissue sections were further subjected to tartrate-resistant acid phosphatase (TRAP) staining. TRAP staining was done using a leukocyte acid phosphatase kit, a cell staining kit for the detection of TRAP (Sigma, St. Louis, MO). Multinucleated macrophages and mononuclear cells showing a positive reaction in TRAP staining were defined as osteoclasts or precursor cells (41). TRAP-positive osteoclasts and their precursor cells were counted at three optional pannus sites for every ankle joint by two individual pathologists (A. Yamashita and Y. Yonemitsu) in a blinded fashion, and the number of osteoclasts and precursor cells in each ankle joint were expressed as the average value.
Administration of anti-FGF-2 Ab
Eighteen rats were prepared to evaluate the suppressive effect
of the FGF-2 neutralization in AIA, right ankle joints were used for
AIA + anti-FGF-2 Ab group, and ipsilateral left ankle joints were
used for AIA + nonimmunized IgG group, as the control. These 18 rats
were treated on day 0 with MBD, as described above. Right ankle joints
were intraarticularly injected with 50 µg anti-FGF-2 Ab
(anti-FGF basic Ab; R&D Systems), while left ankle joints were also
intraarticularly injected with 50 µg nonimmunized rabbit IgG on days
7, 10, 14, and 17, respectively. Thus, the total amount of
administrated anti-FGF-2 Ab and nonimmunized rabbit IgG was 200
µg/rat (Fig. 8A). Anti-FGF-2 Ab was produced in rabbits
immunized with bovine FGF-2, and neutralized the biological activity of
bovine and recombinant human FGF-2. Measurements of hind paw volume and
macroscopic examination were made on days 0, 7, 14, 21, and 28. On day
28, these rats were killed, and bilateral ankle joints were rejected.
Under methods described above, radiological examination and
histological evaluations were made.
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Data were expressed as the mean ± SEM, and for statistical analysis we used the one-way ANOVA or the nonparametric Mann-Whitney U test, as appropriate, according to data form. Differences were considered significant at a level of p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As this is apparently the first attempt to apply SeV to joint gene
transfer, we first assessed the in vivo gene transfer efficiency using
reporter genes. Two days after 5 x 107 PFU
of SeV-NLS-lacZ injection, knee and ankle joints were
subjected to in situ X-gal staining. As shown in Fig. 2, all rats macroscopically had
representative diffuse and widespread blue staining, in the synovial
surface of knee (data not shown) and ankle joint (Fig. 2, A
and B). Surface of the cartilage did not show blue stain,
indicating that this cartilage was resistant to SeV-mediated gene
transfer (Fig. 2, A and B). These findings were
also noted in joints of naive rats (data not shown). Histological
examination revealed gene expression of NLS-lacZ in the
nuclei of synoviocytes and sublining cells (Fig. 2C).
Control rat joints received SeV-luciferase for X-gal staining showing
negative results (data not shown).
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D). A time course study determined a
significant transgene expression that persisted for 14 days after
injection of the virus. Expression of angiogenic growth factors in the synovial fluids
To assess the relevance of angiogenic gene transfer, we first
measured levels of VEGF and FGF-2 using ELISAs. As shown in Fig. 3A, the expression level of
VEGF in human synovial fluids was 1.2 ± 0.4 and 1.1 ± 0.5
ng/ml in original samples and hyaluronidase-digesting samples,
respectively. Marked elevation of FGF-2 (2.3 ± 0.5 ng/ml) was
also noted in human RA synovial fluid digested with hyaluronidase,
while no significant FGF-2 protein was detected in undigested fluid,
suggesting that FGF-2 may be stabilized by interaction with colloids
present in the fluid.
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B).
VEGF expression was also shown to be enhanced by FGF-2 to achieve
synergistic angiogenic properties (22), and we examined
VEGF protein concentrations, using the same synovial extract samples.
As shown in Fig. 3B, endogenous VEGF in AIA joints was
detected (0.3 ± 0.1 ng/mg protein, n = 12), while
negative results were seen in naive rat joints. In contrast, AIA +
FGF-2 rats showed a marked elevation of endogenous VEGF, approximately
16 times greater than that of AIA-related expression (4.9 ± 0.6
ng/mg protein, n = 12, p < 0.0001),
thus indicating a rough parallel increase with FGF-2. These
observations suggest that FGF-2 enhances VEGF expression in AIA
joints.
Progressive effect of FGF-2 overexpression on AIA
Hind paw volume.
In another set of time course experiments, we examined macroscopic paw
swelling and hind paw volume (Fig. 1). Although neither macroscopic
findings of arthritis such as paw swelling, redness, nor ulceration of
the skin were found in C + FGF-2 and C + luciferase groups throughout
the examination (Fig. 4A),
these untoward events were recognized in AIA (Fig. 4B), AIA
+ FGF-2 (Fig. 4C), and AIA + luciferase (Fig. 4C)
groups, and were accentuated from 10 days after the adjuvant treatment.
Macroscopic joint swelling in AIA + FGF-2 group was the most severe
compared with findings in AIA and AIA + luciferase groups (Fig. 4A–C).
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D).
Radiological assessment of joint destruction.
Next, radiological examination was made when we killed the rats to
assess effects of FGF-2 gene transfer on joint and bone destruction. No
apparent radiological abnormality was seen in ankle joints both of C +
FGF-2 and C + luciferase groups in throughout the experimental course
(data not shown). On day 14, radiological findings of soft tissue
swelling and joint space narrowing were similar in ankle joints in all
of AIA, AIA + FGF-2, and AIA + luciferase groups (Fig. 5, A, D, and
G). On day 21, radiological findings mainly of joint space
narrowing and bone atrophy were found in ankle joints of AIA and AIA +
luciferase groups, while more destructive findings such as bone erosion
and osteolysis were found in ankle joints of AIA + FGF-2 group (Fig. 5, B, E, and H). On day 28, disappearance
of joint spaces, total bone atrophy, bone erosion, and focal and mild
osteolysis was similar in AIA and AIA + luciferase group (Fig. 5, C and F). More severe destructive findings,
including marked osteolysis, periosteal reaction, and disappearance of
joint structure, were seen in the AIA + FGF-2 group (Fig. 5I). We then divided ankle joints of AIA, AIA + FGF-2, and
AIA + luciferase groups into five subclasses radiologically on the
basis of degree of joint destruction (31, 39). As shown in
Fig. 5J, the radiological index of AIA + FGF-2 group was
higher (p < 0.05) than that of any other group
on days 21 and 28.
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, A–F). On the other hand, in the AIA + FGF-2 group,
histological joint structures such as demarcation of the bone cortex
and joint line composed of articular cartilages were unclear in low
power field (Fig. 6, G and H). In the high power
field, articular cartilages were totally destroyed by marked erosion;
moreover, inflamed granulation tissue associated with aggregates of
osteoclast-like multinucleated giant cells had invaded subchondral bone
tissue (Fig. 6I). On day 28, proliferative synovitis was
dominant, and tibial distal end, talus, and other tarsal bones were
largely invaded by inflamed granulation tissue so-called pannus
formation; these findings were most severe in the AIA + FGF-2 group
(data not shown).
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A), but not on day 21,
probably due to decline in cell number during repair process.
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B), which
suggests that SeV-mediated overexpression of FGF-2 in ankle joints
promotes neovascularization.
Furthermore, to determine whether FGF-2 gene transfer would affect
osteoclastogenesis in vivo, we determined the number of infiltrating
TRAP-positive osteoclasts. The number in the AIA + FGF-2 group on days
21 and 28 was larger than in the other two groups, with a statistical
significance (p < 0.01) (Fig. 7, C
and D), suggesting that overexpression of FGF-2 in ankle
joint also promotes osteoclastogenesis, especially in late phase.
Effects of neutralization by anti-FGF-2 IgG on AIA development
To obtain further in vivo evidence that FGF-2 is a key mediator in
AIA, we assessed the suppressive effect of neutralizing anti-FGF-2
Ab (Fig. 8A). As shown in Fig. 8B, administration of anti-FGF-2 Ab significantly
ameliorated both joint swelling and hind paw volume
(p < 0.05) as compared with rat joints with
the nonimmunized rabbit IgG in AIA rats at days 14 and 21 of the
experimental course. In radiological assessment of ankle joints
injected with anti-FGF-2 Ab showed less bone and joint destruction,
and a lower radiological index than was observed in ankle joints
treated with control IgG (Fig. 8C). Moreover, in
histopathological examinations, administration of anti-FGF-2 Ab
also significantly reduced vWF-positive vessel density (Fig. 9A) and the number of
osteoclasts (Fig. 9B) on day 28.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, VEGF and FGF-2 have received much attention from the viewpoint of the therapeutic control of RA. VEGF is considered to be a vascular endothelial cell-specific mitogen, as well as a vascular permeability factor. Recent studies showed that the level of VEGF protein in joint fluids of RA patients was significantly higher than that of non-RA fluids with osteoarthritis patients (14, 15), and its receptors, Flt-1 and KDR, were also induced in the active phase of RA (15). Administration of soluble VEGF receptor (17) or VEGF antisera (18, 19) after the onset of collagen-induced mice arthritis ameliorated not only joint inflammation, but also bone destruction, suggesting that the control of VEGF function can be an effective strategy to treat RA.
FGF-2 is an alternative angiogenic growth factor acting on not only vascular endothelial cells, but also various mesenchymal cells. FGF-2 was specifically up-regulated in joint fluid from RA human subjects (13, 21), as well as in synovial extract from animal models (42), thus suggesting that FGF-2 has a crucial role in joint destruction of RA, affecting joint inflammation and bone destruction.
In this study, we compared VEGF and FGF-2 protein content levels from
human RA joint fluid treated with or without hyaluronidase. Joint fluid
from RA patients is hyaluronic acid rich, resulting in increased
viscosity and in gel formation. As shown in Fig. 3A, no
FGF-2 was detected in the solution phase of RA fluid without
hyaluronidase treatment, while a high level of FGF-2 was recovered in
the same fluid sample treated with hyaluronidase. These findings
suggest that FGF-2 may be bound to and stabilized by substances with
hyaluronic acid in human RA fluid. The value of FGF-2 detected in this
study was 2 to 3 logs higher than in a previous report
(13), and we concluded that this discrepancy was due to
hyaluronidase treatment.
Although little knowledge is available regarding the importance of these angiogenic factors, our current findings that FGF-2 overexpression resulted in VEGF up-regulation in the synovial tissue suggest hierarchy in the regulatory process of angiogenesis. Recent reports indicated that FGF-2 stimulates VEGF expression in some mesenchymal cells, and these two act synergistically for an efficient angiogenic response (22), which supports our current findings. Furthermore, we recently found that neutralization of endogenous VEGF activity completely abolished FGF-2-mediated angiogenesis (43), thereby suggesting that in vivo angiogenic properties of FGF-2 completely depend on VEGF. On the other hand, recent studies suggested that the nonangiogenic action of FGF-2 might even worsen RA. FGF-2 accelerated osteoclastogenesis and activated bone resorption by osteoclasts through activation of FGFR1 and p42/p44 mitogen-activated protein kinase and stimulated bone resorption at physiological or pathological concentrations in vitro (26).
Since the pathophysiology of RA has been thought to involve synovial proliferation and angiogenesis as well as bone destruction and absorption, blockade of VEGF, dominantly resulting in suppression of angiogenesis and fluid collection, might not be sufficient to prevent RA disease from the viewpoint of clinical treatment. Our current results suggest that FGF-2 seems to be a better molecular target of RA, because it might indirectly control VEGF expression via regulating FGF-2. Clinical prognosis of RA, for example necessity of surgical treatment and deterioration of quality of life, almost always depends on the degree of bone and joint destruction brought by pannus formation and bone absorption due to osteoclastogenesis. Thus, functional control or neutralization of FGF-2 would be a meaningful treatment approach to suppress RA progression and deteriorate RA severity. However, the limitation of the current study is that little is known regarding the initial events of RA and the relationship between FGF-2 and other proinflammatory cytokines specifically expressed in RA, including IL-17 (43, 44, 45, 46, 47, 48). Further studies on the understanding of RA pathophysiology are called for to clarify this complexed process of the disease to establish more effective therapeutic approaches.
In conclusion, FGF-2 appears to have an important role in the progression of AIA, namely, exacerbation of joint swelling and bone destruction. The control of FGF-2 may prove to be an effective therapeutic approach to modify RA in humans.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: RA, rheumatoid arthritis; AIA, adjuvant-induced arthritis; FGF-2, fibroblast growth factor-2; MBD, Mycobacterium Butyricum desiccated; NLS, nuclear localizing signal; SeV, Sendai virus; TRAP, tartrate-resistant acid phosphatase; VEGF, vascular endothelial cell growth factor; vWF, von Willebrand factor; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactoside.
Received for publication August 27, 2001. Accepted for publication October 25, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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antibody in rheumatoid arthritis. Mol. Pathol. 50:225.This article has been cited by other articles:
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