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*
First Department of Internal Medicine, Showa University School of Medicine, Tokyo, Japan; and
Department of Bacteriology, Osaka City University Medical School, Osaka, 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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Neovascularization is a complex process, involving endothelial cell
division, selective degradation of vascular basement membranes and
surrounding extracellular matrix, and endothelial cell migration.
Several polypeptide growth factors have been identified based on their
ability to stimulate the proliferation of endothelial cells
(4), and a number of angiogenic factors may be important
in the neovascularization found in the RA joint. These include TNF-
,
acidic and basic fibroblast growth factor, and IL-8 (5).
Another important mediator of neovascularization is vascular
endothelial growth factor (VEGF), which is a secreted, heparin-binding,
homodimeric glycoprotein with several protein variants resulting from
alternative mRNA splicing (6, 7). VEGF is an endothelial
cell-specific mitogen in vitro and an angiogenic growth factor in vivo.
It is known to play an important role in the female reproductive system
as well as in such pathological conditions as diabetic retinopathy,
certain tumors, and RA (8, 9, 10, 11). However, the location and
time course of VEGF expression during with the development of CIA have
not been delineated, nor has it been determined whether VEGF is
directly involved in the induction of the synovitis seen in
CIA.
In the present study we show that VEGF is expressed during the development of CIA and that the synovitis can be attenuated by passive immunization with a neutralizing anti-VEGF Ab. Our data support the hypothesis that the expression of specific angiogenic factors during the development of CIA is a key pathologic-component mediating neovascularization in the pannus and the surrounding joint tissue, making VEGF a potentially important factor in the pathogenesis of CIA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male DBA/1J mice (8–10 wk) were purchased from Charles River Japan (Yokohama, Japan), chick type II collagen was obtained from Sigma (St. Louis, MO), recombinant murine VEGF (mVEGF) was purchased from PeproTech (London, U.K.), and goat anti-mVEGF and anti-human von Willebrand factor (vWF) polyclonal Abs (IgG fraction) were obtained from R&D Systems (Minneapolis, MN) and Enzyme Research Laboratories (South Bend, IN), respectively. Monoclonal anti-Mac-1 Ab detects murine macrophages (Cedarlane, Hornby, Ontario, Canada). Monoclonal anti-fibroblast Ab detects murine fibroblasts (Biogenesis, Poole, U.K.). All reagents were tested for endotoxin contamination using a Limulus amebocyte lysate test kit (Limulus HS-F test, Wako, Tokyo, Japan). The concentration of endotoxin was consistently below 0.03 EU/ml in all reagents.
Endothelial cell proliferation assays
Endothelial cell proliferation was assayed using HUVECs purchased from Clonetics (Walkersville, MD) and cultured according to the provided protocol. HUVECs were seeded in triplicate in 24-well plates at 5 x 104 cells/well. The following day, the culture medium was replaced with serum-free RPMI medium (Dai-Nippon, Tokyo, Japan), and then mVEGF was added. VEGF inhibition studies, in which a specific anti-mVEGF neutralizing Ab (10 µg/ml; R & D Systems) was also added to the cultures, were conducted in parallel. After 60 h of culture in the presence of VEGF, [3H]thymidine (1 mCi/ml) was added, and its incorporation into DNA was measured by liquid scintillation counting after an additional 12 h.
Preparation of polyclonal anti-mVEGF antiserum
Rabbit polyclonal anti-mVEGF antiserum was prepared according to the methods of Tilton et al. (12). Briefly, the synthesized N-terminal 24 aa of mVEGF were coupled to keyhole limpet hemocyanin (KLH) via the maleimide-activated carrier protein that cross-links cysteine to KLH. New Zealand White rabbits were then immunized by intradermal injection of 200 µg of the KLH-linked peptide. After Abs against the carrier protein (KLH) were deleted by passage over a KLH-conjugated column, the antiserum titer was tested for reactivity to mVEGF by ELISA, as described previously (13). Neutralizing activity of anti-mVEGF-peptide antisera was assessed by evaluating inhibition of mVEGF-stimulated HUVEC proliferation; at a dilution of 1/1000, the antiserum inhibited 20 ng of mVEGF in this assay (data not shown).
Induction and evaluation of CIA
Type II CIA was elicited in mice by an immunization with chick type II collagen as previously described (14, 15, 16). Paws were individually scored on a scale of 0–3 based on the amount of erythema, swelling, and joint rigidity (arthritis index); the maximum score was thus 12/mouse (16). In addition, joint swelling was quantitated by measuring footpad thickness with an engineer’s micrometer (Mitsutoyo, Tokyo, Japan). The percent increases in swelling were calculated by measuring the difference in thickness before and after the onset of arthritis. The joints were also histologically evaluated for the presence of arthritic lesions. Histopathologic assessment was coded for blind observation, and the histopathologic score assigned to each joint was based upon the extent of inflammation, pannus formation, cartilage damage, and bone erosion, each using a scale of 1 (minimal) to 5 (severe) (17).
Preparation of aqueous joint extracts
Joint tissues were prepared by first removing the skin and separating the limb below the ankle joint. They were then homogenized on ice in 3 ml of lysis buffer (PBS containing 2 mM PMSF and 1 mg/ml (final concentration) each of aprotinin, antipain, leupeptin, and pepstatin A) using a Polytron (Brinkmann Instruments, Westbury, NY). The homogenized tissues were centrifuged at 2000 x g for 10 min. Supernatants were sterilized by passage through a 0.45-µm filter (Millipore, Bedford, MA) and were stored at -80°C until analyzed. The extracts usually contained 0.5–2.5 mg protein/ml, as measured by protein assay kit (Pierce, Rockford, IL).
Isolation of fibroblast-like cells from joints
Fibroblast-like cells were isolated from affected joints by collagenase digestion, as described previously (18). The isolated cells were cultured in DMEM supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies, Grand Island, NY), and 10% FBS (Life Technologies). The cultured cells were used from passages 3 to 5, during which time they morphologically resembled fibroblast-like synoviocytes, were positive for anti-murine fibroblast Ab, and were negative for anti-Mac-1 Ab, indicating that they were not macrophage-like synoviocytes (data not shown).
Measurements of VEGF and vWF proteins
VEGF and vWF proteins were measured using commercially available
ELISAs for mVEGF (Quantikine M mouse VEGF immunoassay kit, R & D
Systems) and human vWF (Asserachrom vWF kit, Roche, Tokyo, Japan),
according to the manufacturer’s recommendations. The limit of
sensitivity of the VEGF ELISA was consistently 
3 pg/ml, whereas vWF
levels were expressed as percentages in which the detected vWF was
compared with that present in a human plasma calibration standard,
arbitrarily assigned a value of 100%.
Immunohistochemistry
VEGF and vWF Ags were visualized immunohistochemically as previously described (16). Joint specimens were initially decalcified for 2 wk in EDTA-containing buffer (19) and then were embedded in OCT compound (Miles, Elkhart, IN). Before staining, 5-µm frozen sections were fixed for 30 min in ice-cold acetone. Endogenous peroxidase activity was quenched by incubating the slides for an additional 30 min in absolute methanol and 3% hydrogen peroxide. The slides were then incubated with polyclonal goat anti-mVEGF Ab (1/500 dilution), goat anti-vWF Ab, or preimmune goat IgG. Biotinylated rabbit anti-goat IgG (BioGenex, San Ramon, CA) and peroxidase-conjugated streptavidin were used as second and third reagents, respectively, while the red color was developed using 3-amino-9-ethylcarbazole in N,N-dimethylformamide. After rinsing with distilled water, the slides were counterstained with Mayer’s hematoxylin. Tissue sections were also examined the immunostaining with anti-Mac-1 Ab and anti-fibroblast Ab (data not shown).
Isolation of tissue RNA, RT-PCR, and Southern blotting
Total RNA was extracted from joint tissues using the guanidinium isothiocyanate method, and RT-PCR was performed as previously described (16). Briefly, 5 mg of total RNA was reverse transcribed using AMV reverse transcriptase (Life Technologies). PCR was conducted for 35 cycles, after which the amplified DNA fragments were subjected to 2% agarose gel electrophoresis and were transferred to nylon filters. The filters were hybridized with synthetic 32P 5' end-labeled internal oligoprobes that recognized sequences between the two primers. The primers and internal probe sequences were as follows: VEGF sense, 5'-CGA GAC CCT GGT GGA CAT CT; antisense, 5'-CAC CGC CTC GGC TTG TCA C; VEGF internal probe, 5'-CAC CAA AGC CAG CAC ATA GA (20); murine Flt-1 sense, 5'-CCT GAT TTC CTA CAG TTT CCA; antisense, 5'-TCC GGG GTT CTC ATC CGC ATG; Flt-1 internal probe, 5'-AGG ACC ACA CAT CGC TCT TGG (21); Flk-1 sense, 5'-CTT AGG TGC CTC CCC ATA CCC TGG G; antisense, 5'-TGG CCG GCT CTT TCG CTT ACT GTT C; and Flk-1 internal probe, 5'-CCA TGC TCA GTG TCT CTG ACA TTG GAA GAA CAA TAT AGT CTT TGC C (22).
Statistical analysis
Data were expressed as the mean ± SEM. Differences between
VEGF and vWF levels were compared by one-way ANOVA. The incidence and
severity of arthritis were evaluated using 
2
contingency analysis and Mann-Whitney U test, respectively.
Differences were considered significant if p <
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We initially examined whether arthritic joints contained elements
capable of stimulating endothelial cell proliferation. Aqueous joint
extracts obtained from mice with severe arthritis (4 days after onset)
or from controls without arthritis were tested for their ability to
stimulate HUVEC proliferation. In preliminary experiments HUVEC
proliferation was elicited by mVEGF (Table I
), most likely because cDNAs of human
and murine VEGF are highly homologous (20). In addition,
the viability of HUVEC at 72 h, as estimated by trypan blue dye
exclusion, ranged from 58 to 66% for cultures without serum-free
medium. Aqueous extracts from arthritic joints similarly stimulated the
proliferation of HUVEC compared with extracts from control joints
(Table I). Moreover, addition of neutralizing anti-mVEGF Ab
inhibited the activity by 59.3%, confirming that biologically active
VEGF was expressed within the arthritic joints.
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). Mice with or without type II collagen
immunization were sacrificed every 4 days, and joint tissue was
prepared for VEGF analysis. The amount of VEGF Ag present in the joints
of arthritic mice reached a maximum on day 4 after the onset of
arthritis, which correlated with footpad swelling, and then gradually
declined until stabilizing on day 24. Thus, VEGF was expressed
primarily during the acute stage of arthritis development, suggesting
that VEGF may play a role in the induction and maintenance of CIA. No
increase in VEGF was observed in control mice, immunized without type
II collagen, at any time (data not shown).
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To examine the extent to which VEGF expression correlates with
neovascularization in arthritic lesions, the vWF content of affected
joints was assessed using an appropriate ELISA. Because vWF is
considered to be a marker for endothelial cells (23), we
considered vWF levels to be an index of neovascularization. As shown in
Fig. 2, vWF levels reached a maximum on
day 4 after the onset of arthritis and then gradually declined;
neovascularization thus correlated temporally with both VEGF expression
and footpad swelling.
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VEGF transcription in joint tissue was assessed using RT-PCR and
Southern blotting. Several alternative splice variants of VEGF have
been described (6), and in fact, the primers used in the
present experiments were capable of yielding five spliced forms, 279,
304, 350, 411, and 483 bp in length (24). We found that
VEGF transcripts, especially the 279-bp form, were constitutively
expressed in joint tissues (Fig. 3A). After the onset of
arthritis, however, transcription of the 279- and 304-bp forms was
significantly increased until day 24 after onset, which correlates well
with the time course of expression of the protein (Fig. 1
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Transcription of Flt-1 and Flk-1, the cell surface receptors for mVEGF,
was also significantly elevated during the period between days 4 and 12
after the onset of arthritis, after which it gradually declined (Fig. 3, B and C).
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Immunolocalization showed VEGF to be mainly associated with
infiltrating macrophage- and fibroblast-like cells, identified by
morphology and by the reactivity with anti-Mac-1 Ab and
anti-fibroblast Ab, respectively, and chondrocytes in chronically
inflamed joint tissues (Fig. 4B, arrow); tissue sections
stained with preimmune, control IgG showed little or no nonspecific
staining (Fig. 4A). Of particular interest to us, VEGF also
appeared to be expressed by vascular endothelial cells, but to only a
small degree (Fig. 4D, arrow). Identified by their
expression of vWF (Fig. 4C, arrowhead), this was in contrast
to earlier findings suggesting that vascular endothelial cells are the
primary source of VEGF (6, 7).
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, the
fibroblast-like cells expressed significant amounts of VEGF (Fig. 5, arrowhead), which suggests that the
cellular sources of VEGF are probably the macrophage- and
fibroblast-like cells that infiltrate the joint during CIA development
as well as in the synovium of human RA (10).
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The results described above show that VEGF is expressed within
arthritic joints during the development of CIA and may have a crucial
role in the evolution of the ailment. To obtain more direct evidence of
VEGF-induced neovascularization, we injected anti-mVEGF antiserum
(0.75 ml/time/mouse) i.p. every other day between days 30 and 38 along
with type II collagen immunization. The mice were then observed daily,
and the incidence and severity of arthritis were assessed. As shown in
Fig. 6, we found that administration of
anti-mVEGF antiserum attenuated both the incidence and the severity
of joint inflammation (arthritis index) in CIA mice. Moreover,
histopathologic examination of joints from mice treated with
anti-mVEGF antiserum showed less cartilage and bone destruction,
less pannus formation, and less fibroblast proliferation than observed
in mice treated with control serum (Figs. 7, A and B), and
neovascularization (vWF levels) was also significantly reduced (Fig. 7C).
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). This suggests that VEGF acts
primarily during the initiation of CIA by regulating neovascularization
during that period.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We detected the presence of elements that stimulated endothelial cell
proliferation in the extracts of inflamed mouse joints (Table I).
Proliferation of vascular endothelial cells is induced not only VEGF,
but also by cytokines such as IL-1, TNF-, IL-8, and fibroblast
growth factor (25, 26). Although we did not assess the
specific contributions made by other factors, the fact that inflamed
synovitis was markedly (59.3%) inhibited by anti-VEGF Ab strongly
suggests that it has a central role in the process.
An unexpected finding was that vascularization reached a maximum during
the acute stage of the arthritis and then gradually declined (Fig. 2).
Because both vWF and VEGF levels were normalized to total joint protein
(percent increased or milligrams per milligrams of protein), one
possible explanation is that total joint protein increased relative to
vWF and VEGF at later stages (early stage, 0.79 ± 0.09 mg/ml;
late stage, 1.92 ± 0.22 mg/ml).
VEGF was originally thought to be an endothelial cell-derived,
angiogenic, autocrine factor. However, recent reports have shown that
fibroblasts (24, 27, 28), macrophages (10, 29, 30), alveolar epithelial cells (31), vascular
smooth muscle cells (32), neutrophils (33),
and even chondrocytes (34) also express significant
amounts of VEGF. Indeed, most of the cells expressing VEGF in arthritic
joints were macrophage- and fibroblast-like cells and chondrocytes, but
not endothelial cells (Fig. 4). Furthermore, that TNF- evoked
significant VEGF expression in cultured fibroblast-like cells from
arthritic joints (Fig. 5) suggests that inflammatory cells infiltrating
inflamed sites are stimulated to express VEGF by other cytokines
present there.
Expression of VEGF is known to be regulated by other mediators, including platelet-derived growth factor (35), TGF-ß (36), IL-1 (28), oxygen radicals and nitric oxide (30, 37, 38, 39), and hypoxia (32, 38, 40). The synovium is chronically hypoxic in RA and probably also in CIA: biochemical evidence of anaerobic metabolism suggests that blood flow is insufficient to meet the high metabolic demands of inflamed synovial tissue (41). In that regard, the cytokines mentioned above are known to be involved in the development and maintenance of CIA (42, 43, 44). Thus, hypoxia, various cytokines, oxygen radicals, and nitric oxide may act in concert to stimulate angiogenesis in CIA synovitis through up-regulation of VEGF.
The inhibitory effect of administering neutralizing antiserum clearly
showed that VEGF-induced neovascularization is a crucial step in the
initiation and early development CIA (Figs. 1, 2, and 6). On the other
hand, for reasons that are less clear, arthritis severity was
unaffected by administering anti-VEGF antiserum after the onset of
the disease (late stage; Fig. 8). One possibility is that newly
organized vascular networks may be virtually complete by the chronic
stage, making VEGF less important than other immune/inflammatory
mediators.
We found that two specific VEGF receptors, Flt-1 and Flk-1, were
expressed at the site of synovitis and that the time course of their
expression paralleled that for VEGF itself (Fig. 3). This is in
agreement with an earlier report showing that Flt-1 and Flk-1 are
expressed at low levels in endothelial cells under normal conditions,
but are up-regulated at sites where there is a concomitant
up-regulation of VEGF (45). Activation of Flt-1 by VEGF
does not efficiently induce migration of endothelial cells without
simultaneous Flk/KDR activation (46). Nevertheless, a
recent report showed that activated monocytes and even dendritic cells,
which are APC present at inflamed sites, expressed only Flt-1 and not
Flk-1/KDR (47, 48, 49). VEGF may thus induce monocyte
activation and migration via Flt-1 (47, 50). This idea is
supported by the finding that during the development of CIA,
infiltration of the synovial tissue by monocytes and neutrophils occurs
predominantly during the acute stage (51). Our present
findings therefore suggest that infiltrating, activated monocytes
contribute to the neovascularization seen during CIA development by
acting directly on endothelial cells through secretion of various
cytokines, including VEGF, as well as by acting indirectly through
recruitment of additional monocytes through the VEGF-Flt-1 pathway.
In conclusion, VEGF appears to be an important endogenous mediator during the development of both RA and CIA synovitis. A more complete understanding the functions of VEGF and its receptors, Flt-1 and Flk-1, should contribute to our understanding of the pathophysiology of CIA and human RA. More broadly, it should contribute to our understanding of the role of the vascular system in chronic inflammatory responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tsuyoshi Kasama, First Department of Internal Medicine Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan.
3 Abbreviations used in this paper: CIA, collagen-induced arthritis; RA, rheumatoid arthritis; VEGF, vascular endothelial growth factor; mVEGF, murine VEGF; vWF, von Willebrand factor; KLH, keyhole limpet hemocyanin.
Received for publication November 18, 1999. Accepted for publication March 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Arthritis Rheum. 42:100.[Medline]
-dependent angiogenesis. Mol. Cell. Biol. 17:4015.[Abstract]
and transforming growth factor ß during induction of collagen type II arthritis in mice. Proc. Natl. Acad. Sci. USA 89:7375.This article has been cited by other articles:
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  C. Clapp, S. Thebault, M. C. Jeziorski, and G. Martinez De La Escalera Peptide Hormone Regulation of Angiogenesis Physiol Rev, October 1, 2009; 89(4): 1177 - 1215. [Abstract] [Full Text] [PDF]
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 B. P. Cheppudira, B. M. Girard, S. E. Malley, K. C. Schutz, V. May, and M. A. Vizzard Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis Am J Physiol Renal Physiol, September 1, 2008; 295(3): F826 - F836. [Abstract] [Full Text] [PDF]
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