Proangiogenic Function of CD40 Ligand-CD40
Interactions
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J Immunol 2003; 171:1534-1541; ;
doi: 10.4049/jimmunol.171.3.1534
http://www.jimmunol.org/content/171/3/1534
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References
Marlies E. J. Reinders, Masayuki Sho, Stuart W. Robertson,
Christopher S. Geehan and David M. Briscoe
The Journal of Immunology
Proangiogenic Function of CD40 Ligand-CD40 Interactions1,2
Marlies E. J. Reinders, Masayuki Sho, Stuart W. Robertson, Christopher S. Geehan, and
David M. Briscoe
A
ngiogenesis, the generation of new blood vessels from
pre-existing ones, is a complex process involving endothelial cell (EC)3 division, degradation of the vascular
basement membrane and surrounding extracellular matrix, and EC
migration (1–3). It is a characteristic component of many normal
physiologic processes and is pathologic in various diseases, including cancer, arthritis, atherosclerosis, and some forms of blindness (3, 4). Angiogenesis is regulated by a balance between the
relative expression and function of pro- vs antiangiogenic mediators. To this end, it is proposed that pathologic angiogenesis results
either from an overproduction of proangiogenic factors or an underproduction or lack of function of antiangiogenic factors.
Multiple factors have been identified to possess angiogenic effects, but vascular endothelial growth factor (VEGF) is recognized
as the most potent angiogenesis factor described to date. VEGF is
a 45-kDa protein produced by EC, monocyte/macrophages, and a
variety of other cell types, including activated T cells (2, 5–10). As
its name suggests, VEGF stimulates EC proliferation in vitro and
angiogenesis in vivo, and it has important roles in both normal
physiological as well as pathological vasculogenesis and angiogenesis (11). It exists in at least five different isoforms of 206, 189,
Division of Nephrology, Department of Medicine, Children’s Hospital, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115
Received for publication November 19, 2002. Accepted for publication May
23, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI46756 and
AI50157 (to D.M.B.). M.R. was supported in part by a grant from Stichting Dr.
Catharine van Tussenbroek.
2
Address correspondence and reprint requests to Dr. David M. Briscoe, Division of
Nephrology, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115. E-mail
address: [email protected]
3
Abbreviations used in this paper: EC, endothelial cell; CD40L, CD40 ligand; FGF,
fibroblast growth factor; PCNA, proliferating cell nuclear Ag; ; VEGF, vascular endothelial growth factor; vWf, von Willebrand factor.
Copyright © 2003 by The American Association of Immunologists, Inc.
164, 145, and 121 aa, which arise from the alternative splicing of
a single gene (12). Despite significant physical differences, the
several VEGF isoforms bind VEGF receptors 1 and 2 (called Flt-1
and KDR, respectively) and function as growth and survival factors for EC (2, 12, 13). However, only the VEGF164 isoform binds
VEGF receptor 3 (also called neuopilin) (14), and recent studies
suggests that there may be isoform-specific differences in function,
especially with regard to developmental angiogenesis (15–17).
VEGF may act synergistically with other factors, such as fibroblast
growth factor (FGF), in vitro to induce endothelial growth and
capillary formation (18 –20). VEGF and FGF also act as survival
factors for endothelium, and their withdrawal causes apoptosis in
vitro and regression of newly formed vessels in vivo (21).
It has been established for over 30 years that lymphocytes and
monocytes produce angiogenesis factors and stimulate angiogenesis in the course of inflammatory responses (7, 22–24). For example, activated T cells are a source of angiogenesis factors, including VEGF, and can stimulate angiogenesis in association with
early lymphocyte recruitment (25). Products of activated macrophages, including VEGF, TNF-␣, TGF-␤, NO, and FGF-2, are
most potent to induce angiogenesis (26 –29). Thus, it is not surprising that angiogenesis is characteristic of delayed-type hypersensitivity (1) and that excessive production of angiogenesis factors occurs in many chronic inflammatory disorders (11).
Interactions between CD40 ligand (CD40L; also called CD154)
and its counter-receptor CD40 are well established to promote T
cell activation, T cell-B cell interactions, T cell-dependent macrophage activation, as well as endothelial activation (30 –32). We
recently demonstrated that interactions between CD40L and CD40
were the most potent for the expression of VEGF in EC and monocytes in vitro (33). Moreover, we found that injection of soluble
CD40L into skin resulted in enhanced VEGF expression as well as
angiogenesis in the athymic nude mouse. CD40 signals also regulate VEGF expression in fibroblasts (34) and other cells (35) and
have been demonstrated to induce the expression of matrix metalloproteinases of functional importance in EC tube formation
0022-1767/03/$02.00
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Angiogenesis is a characteristic component of cell-mediated immune inflammation. However, little is known of the immunologic
mediators of angiogenesis factor production. Interactions between CD40 ligand (CD40L) and CD40 have been shown to have
pluripotent functions in inflammation, including the production of cytokines, chemokines, as well as the angiogenesis factor,
vascular endothelial growth factor (VEGF), by endothelial cells. In this study we found that treatment of cultured human endothelial cells with an anti-CD40 Ab (to ligate CD40) resulted in the expression of several other angiogenesis factors, including
fibroblast growth factor-2 and the receptors Flt-1 and Flt-4. To determine the proangiogenic effect of CD40L in vivo, human skin
was allowed to engraft on SCID mice for 6 wk. These healed human skins express CD40 on resident endothelial cells and
monocyte/macrophages, but not on CD20-expressing B cells. Skins were injected with saline, untransfected murine fibroblasts, or
murine fibroblasts stably transfected with human CD40L. We found that the injection of CD40L-expressing cells, but not control
cells, resulted in the in vivo expression of several angiogenesis factors (including VEGF and fibroblast growth factor) and a marked
angiogenesis reaction. Mice treated with anti-VEGF failed to elicit an angiogenesis reaction in response to injection of CD40Lexpressing cells, suggesting that the proangiogenic effect of CD40L in vivo is VEGF dependent. These observations imply that
ligation of CD40 at a peripheral inflammatory site is of pathophysiological importance as a mediator of both angiogenesis and
inflammation. The Journal of Immunology, 2003, 171: 1534 –1541.
The Journal of Immunology
1535
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FIGURE 1. CD40 ligation induces FGF-2, Flt-4, and Flt-1 in
EC in a time- and dose-dependent
manner. Confluent cultures of human EC were treated for 6 h with
different concentrations of stimulating anti-CD40 (A, C, and D) or
as a time course using 1 ␮g/ml antiCD40 (B) as indicated. Total RNA
was harvested, and the expression
of angiogenesis factors was analyzed by RNase protection assay
and PCR. Anti-CD40 induced the
expression of FGF-2, Flt-4, and
Flt-1 expression in EC (A and B),
whereas several other angiogenesis
factors and receptors had a constitutive level of expression and no
detectable change after treatment
with anti-CD40 (C and D). mRNA
expression was quantified by densitometry as the relative expression
of the angiogenesis factor compared with the expression of the
housekeeping GAPDH signal. Bar
graphs on the right of A and B illustrate the relative expression of
each gene examined (mean ⫾ 1
SD of three blots, including the illustrated blots). The expression of
GAPDH or ␤-actin served as internal housekeeping gene controls.
All autoradiographs are representative of three experiments with
similar results.
(36). In addition, some of the intracellular signaling pathways for
CD40-dependent angiogenesis have recently been defined (35, 37).
Together, these in vitro studies point to a major function for
CD40L-CD40 interactions in angiogenesis.
In this study we extended upon these observations in an in vivo
model and tested the mechanism of function of CD40L for angiogenesis in vivo. Our findings clearly demonstrate that the provision
of CD40L to tissues in vivo is a physiologic stimulus for the production of several angiogenesis factors, but that the CD40L-induced angiogenesis reaction is VEGF dependent.
Santa Cruz, CA). Humanized anti-human VEGF for in vivo studies was a
gift from Genentech (South San Francisco, CA). Human IgG was purchased from Sigma-Aldrich (St. Louis, MO). The proliferating cell nuclear
Ag (PCNA) kit was purchased from Novocastra (Burlingame, CA). Murine
fibroblasts stably transfected with human CD40L (CD40L cells) or untransfected cells (mock cells) were a gift from C. van Kooten (38).
Materials and Methods
In vivo assessment of angiogenesis
Reagents
The following Abs were used in these studies: anti-human CD31, anti-von
Willebrand factor (anti-vWf), anti-CD20, anti-CD68, and anti-mouse
CD31 (DAKO, Carpenteria, CA); anti-human CD40 (G28.5) and CD40L
(106; gifts from D. Hollenbaugh, Bristol Myers Squibb, Princeton, NJ);
and anti-human CD40L, VEGF, and FGF-2 (Santa Cruz Biotechnology,
Cell culture
Single-donor human umbilical vein EC were purchased from Clonetics
(Walkersville, MD) and were cultured in complete endothelial medium
(EGM Bulletkit; Clonetics) as supplied and according to the recommended
instructions. EC were subcultured and used at passages four to six.
CB.17 SCID mice were purchased from Taconic Farms (Germantown,
NY) and were used at 6 – 8 wk of age. The SCID mice used in these studies
were occasionally monitored for “leakiness” by assessment of circulating T
cells, and in all cases were found to be immunodeficient and stable. Fullthickness human neonatal foreskin grafts were transplanted onto CB.17
(SCID) mice as previously described (25, 39). Following engraftment for
1536
CD40-DEPENDENT ANGIOGENESIS
4 – 6 wk, mice were divided into different experimental groups. In each
group the human skin was injected intracutaneously with 35 ␮l of Matrigel
(growth factor-reduced Matrigel; BD Biosciences, Bedford, MA) and either 35 ␮l of saline (as a control) or 2.5 ⫻ 106 murine fibroblasts stably
transfected with human CD40L (CD40L cells) or mock transfectants
(mock cells) each in 35 ␮l of saline. Before injection, mock and CD40L
cells were irradiated at 7500 rad.
SCID mice were also treated with a neutralizing anti-human VEGF Ab
(Genentech) or human IgG at a dose of 5 mg/kg in 100 ␮l of saline i.p. Ab
therapy was begun 2 days before and every other day after the injection of
CD40L cells into the skin. Animals were sacrificed, and skin grafts were
harvested after 7 days. Animal care and anesthesia were performed in
compliance with guidelines established by the animal care and use committee at Children’s Hospital (Boston, MA).
Immunohistochemistry
Frozen specimens were fixed in acetone, and formalin-fixed specimens
were deparaffined. All specimens were then incubated in primary and secondary Abs as previously described (25, 33) using the Vectastain Kit (Vector Laboratories, Burlingame, CA). Finally, specimens were counterstained
in Gill’s hematoxylin and mounted in glycerol gelatin. The PCNA kit was
used according to the manufacturer’s instructions.
Quantification of angiogenesis
Vessels were identified by staining for human vWf, and angiogenesis was
quantified by a standard grid-counting method at ⫻400 magnification as
previously described (40). Four to six adjacent nonoverlapping fields of
each specimen were analyzed in a blinded manner, and the mean vessel
counts for each specimen were calculated.
RT-PCR and RNase protection assays
RNA was isolated from cultured EC or skin samples using the Ultraspec
RNA isolation system (Biotecx, Houston, TX) and was reverse transcribed,
and PCR was performed using standard techniques (33). Sequence-specific
primers for PCR of human KDR (also called VEGF receptor-2) were:
sense, 5⬘-CGAAGCATCAGCATAAGAAAC-3⬘; and antisense, 5⬘ACATACACAACCAGAG-AGACC-3⬘. ␤-Actin (Stratagene, La Jolla,
CA) was used as an internal control. The PCR conditions were 94°C for 5
min, followed by 35 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C
for 1 min. The last cycle was extended to 7 min at 72°C. The amplified
products were resolved by electrophoresis in an ethidium bromide-stained
1.5% agarose gel.
RNase protection assays were performed using the RiboQuant multiprobe template system (BD PharMingen, San Diego, CA), according to the
manufacturer’s instructions and as previously described (33). Briefly, equal
amounts of RNA (10 ␮g) were hybridized with [32P]UTP-labeled riboprobes that were synthesized from the template. After hybridization, samples were digested with RNase A, RNase T1, and proteinase K, and protected RNA was resolved on a denaturing polyacrylamide gel. Relative
signals were detected by autoradiography with Kodak MR film (Eastman
Kodak, Rochester, NY), and expression was quantified by densitometry by
means of an AlphaImager 2000 system (Alpha Innotech, San Leandro,
CA). For quantification, signals were standardized to the internal housekeeping gene GAPDH.
Statistical analyses
Data were compared by nonparametric analysis using the Mann-Whitney
test for data analysis. Differences with p ⬍ 0.05 were considered statistically significant.
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FIGURE 2. Ligation of CD40 induces angiogenesis in vivo. We used murine fibroblasts stably transfected with human CD40L to test the function of
CD40L-CD40 interactions for the development of angiogenesis in vivo. A and B, Flow cytometry illustrating the expression of human CD40L on
CD40L-transfected cells, but not on control-transfected mock cells. C–E, SCID mice bearing human skin transplants received intracutaneous injections of
saline, mock cells, or CD40L cells as described in Materials and Methods. Skin specimens were harvested after 7 days and were evaluated for angiogenesis
by immunohistochemical staining for vWf. Illustrated is representative staining of vWf (rose-red color) in a skin injected with mock cells (C) or CD40L
cells (D). E, The mean vessel density was evaluated in skin grafts by standard calibrated grid counting of vWf-positive vessels at ⫻400 magnification. Bars
illustrate the vessel counts from skin specimens injected with saline (n ⫽ 4), mock cells (n ⫽ 8), or CD40L cells (n ⫽ 11) ⫾ 1 SD. F and G, Skin specimens
were stained for PCNA to identify cells undergoing active proliferation. Shown is enhanced PCNA staining of EC in a skin treated with CD40L cells (G)
compared with skin treated with mock cells (F). Magnification: C and D, ⫻400; F and G, ⬃⫻800.
The Journal of Immunology
1537
Results
Ligation of CD40 induces FGF-2, Flt-4, and Flt-1 mRNA
expression in human EC in vitro
We first assessed the effect of CD40L-CD40 interactions on the
expression of a panel of angiogenesis factors and receptors in cultured EC in vitro using RNase protection assays. Human EC were
starved overnight in 0.5% FCS and were subsequently treated with
stimulating anti-CD40. Anti-CD40 induced the expression of
FGF-2, Flt-1 and Flt-4 in a dose- and a time-dependent manner
(Fig. 1, A and B). FGF-2, Flt-1, and Flt-4 increased in expression
as early as 3 h following activation with anti-CD40, with peak
expression occurring after 6 h (Fig. 1B). Similar results were obtained following treatment with soluble CD40L, and under all conditions resulted in the expression of VEGF (data not shown) (33).
In contrast, the mRNA expression of angiopoietin, endoglin,
CD31, TIE, TIE2, thrombin receptor, and KDR was unaltered after
treatment with anti-CD40 (Fig. 1, C and D). These findings suggest
that CD40L-CD40 interactions have the potential to be potent for
angiogenesis in vivo.
Infiltration of tissues with CD40L-expressing cells induces
angiogenesis in vivo
We next assessed whether cell surface CD40L on infiltrating cells
might function to mediate immune-mediated angiogenesis in vivo.
It is well established that activated platelets and lymphocytes expressing CD40L are present in tissues in cell-mediated immune
inflammation. We used an established model of angiogenesis in
which human skin is transplanted onto SCID mice (25). Skin is
allowed to heal for 4 – 6 wk to enable stabilization of the healing
angiogenesis reaction in the skin graft (39). Untransfected murine
fibroblasts (mock cells) or murine fibroblasts stably transfected
with human CD40 ligand (CD40L cells; Fig. 2, A and B) were
injected intracutaneously into the healed human skins on the SCID
mice. Injection of saline served as a negative control. The skins
were harvested after 7 days and were evaluated for the development of angiogenesis. By quantitative grid counting, we found that
the number of vessels in skins injected with saline or mock cells
was comparable to that in untreated skins, as determined by standard immunostaining with vWF. In contrast, the mean vessel density was significantly increased in the skins injected with CD40L
cells vs controls (2.3-fold vs saline and 2.1-fold vs mock cells,
both p ⬍ 0.01; Fig. 2, C–E). Similar to untreated human skins (39),
the skins injected with mock-transfectant cells had an approximately equal number of human and mouse EC, as determined by
immunohistochemical analysis of human vs mouse CD31. In contrast, we found a greater percentage of cells expressing human
CD31 in skins treated with CD40L cells (not shown). Furthermore,
in the CD40L-injected skins there were numerous EC expressing
PCNA in their nucleus, a marker that identifies cells that have
entered the cell cycle (Fig. 2, F and G). This suggests active angiogenesis in skins that received CD40L cells.
We also wished to establish which intragraft cells may respond
to the injected CD40L cells. By immunohistochemistry, CD40 was
expressed by multiple cells in untreated skins, including EC, resident macrophages, and keratinocytes with a similar pattern of expression in all groups of skins (Fig. 3). CD68-expressing monocyte/macrophages were present in both untreated and treated skins
in variable numbers, but overall there appeared to be slightly more
cells in the mock- and CD40L transfectant-injected skins than in
uninjected skins (Fig. 3). In contrast, we did not find any CD20expressing B cells in any of the untreated, mock-injected, or
CD40L transfectant-injected skins. CD40L was minimal or absent
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FIGURE 3. Expression pattern of CD40-expressing cells within skin grafts: representative immunohistochemical staining of CD40, CD40L, CD20, and
CD68 in untreated skins (Untreated) or skins treated
with CD40L-transfected cells (Treated). CD40 (upper
panels) was expressed on EC and resident macrophages in all skins examined. There was no difference
in the pattern or intensity of expression of CD40 in
untreated or treated skins. In contrast, CD40L was
present only in the skins treated with the CD40L
transfectants, where its expression was most apparent
on groups of cells in clusters (middle panel, right).
There was no staining of CD20 in any of the skin
samples (middle panel, left), whereas CD68-expressing monocyte/macrophages (lower panels) were
present in all skins examined. The expression of
CD68 was similar in mock- and CD40L-treated skins.
Data are representative of three skins examined in
each group. Magnification of micrographs: ⫻200 (upper left panel) and ⫻400.
1538
in untreated skins and in skins injected with mock transfectant
cells. In contrast, staining for CD40L was found on discrete clusters of cells in the skins injected with CD40L transfectants (Fig. 3).
By double immunohistochemical staining (not shown), there was
no distinct pattern of contact between the CD40L-expressing clusters and EC or macrophages. We interpret this to suggest that the
injected CD40L-expressing cells may interact with several resident
CD40-expressing skin cells spatially associated with the CD40L
cell clusters. Alternatively, it is possible that CD40L shed from
these cells may bind the several CD40-expressing cells within the
skin to mediate the angiogenesis reaction.
CD40L induces the expression of multiple angiogenesis factors
in vivo
Function of VEGF in CD40L-induced angiogenesis
It is well established that angiogenesis factors mediate angiogenesis synergistically with VEGF (41). Thus, it is possible that
VEGF is a critical factor in CD40L-induced angiogenesis. To test
this possibility, human skins on SCID mice were injected with
either saline or CD40L cells, as described above. Simultaneously,
FIGURE 4. Ligation of CD40 induces mRNA expression of multiple angiogenic factors in vivo. A and
B, Analysis of angiogenesis factor expression by
RNase protection assay from skins treated with saline,
mock cells, or CD40L cells. C, mRNA expression was
quantified by densitometry as the relative expression
of the angiogenesis factor compared with the expression of the housekeeping GAPDH signal. Bar graphs
represent the mean expression (⫾1 SD) of three skin
specimens injected with saline (control), mock cells,
or CD40L cells. ⴱ, p ⬍ 0.05.
the mice were treated with either anti-human VEGF or IgG as a
control by i.p. injection. There was no change in baseline vessel
density in anti-VEGF or IgG-treated mice when skins were treated
with mock cells (not shown). In contrast, skins treated with CD40L
cells in IgG-treated mice had significantly increased vessel numbers over baseline saline-treated skins (Fig. 6; p ⬍ 0.01). However,
injection of CD40L cells into skins on mice treated with antiVEGF failed to result in an angiogenesis reaction (Fig. 6; p ⬍
0.03). Thus, the effect of CD40L for the stimulation of an angiogenesis reaction involves the critical expression of VEGF.
Discussion
In this study we show that local infiltration of skin with cells expressing human CD40L on their cell surface mediates the induction of VEGF and FGF in vivo and results in a marked angiogenesis reaction. These findings have important implications for the
function of CD40L-CD40 interactions in immunity and chronic
inflammation. First, it is well established that CD40L-CD40 interactions are of central importance in the development of an effective
immune response involving T cell activation, T cell-B cell interactions, and Ab switching as well as endothelial activation responses (30 –32). Our findings here extend the functions of
CD40L-CD40 to include regulation of VEGF and FGF as well as
the angiogenesis receptors Flt-1 and Flt-4 in vitro and in vivo.
Second, inflammatory cells, including lymphocytes and monocytes, can elicit an angiogenesis reaction, a process called leukocyte-induced angiogenesis (24, 25). This process is well characterized in the literature and is reported to be of pathological
significance in several chronic inflammatory diseases, including
arthritis, atherosclerosis, and diabetes (1, 3, 42– 44). Our finding
here that CD40L-expressing cells within a tissue can functionally
induce VEGF-dependent angiogenesis is consistent with these reports and further suggests that CD40L-CD40 interactions may represent an intermediary between cell-mediated immune inflammation and angiogenesis.
Activated T cells and platelets express CD40L, and CD40 is
expressed on multiple cell types, including EC, fibroblasts, monocytes, and epithelial cells (45, 46). Our findings demonstrate that
CD40L promotes the functional expression of several angiogenesis
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We next examined the mRNA expression of a panel of angiogenesis factors and receptors (including VEGF, FGF, and angiopoeitin-1) in skins harvested 7 days after intracutaneous injection of
saline, mock cells, or CD40L cells. There was a low and variable
expression of all factors in skins injected with saline or mock cells
(Fig. 4, A–C). In contrast, the angiogenesis factors VEGF, FGF,
and angiopoeitin-1 were significantly up-regulated after treatment
with CD40L cells. Also, the angiogenesis receptors TIE-2, Flt-4,
and Flt-1 as well as the EC markers endoglin and CD31 were
significantly increased in skins injected with CD40L cells (Fig. 4,
A–C).
By immunohistochemistry, little FGF-2 and VEGF was found in
saline- and mock cell-treated skins (Fig. 5, A–D). In contrast, a
marked induction of both FGF-2 and VEGF expression was found
in skins treated with CD40L cells (Fig. 5, E and F). FGF-2 and
VEGF were expressed mainly by vascular EC. Thus, it is likely
that CD40L-induced angiogenesis in vivo involves the expression
and function of multiple angiogenesis factors.
CD40-DEPENDENT ANGIOGENESIS
The Journal of Immunology
1539
factors in vivo. These findings are consistent with in vitro observations by several groups that CD40L-CD40 interactions may result in angiogenesis via enhanced VEGF, tissue factor, or matrix
metalloproteinase expression and via signaling through the phosphoinositol 3-kinase/Akt pathway (33–37, 47). However, despite
these observations, a recent report by Urbich et al. (48) suggested
that CD40L may have antiangiogenesis effects. In the report the
authors cited that a possible reason for their different findings was
the cell culture conditions used in their analysis. Nevertheless, our
studies here demonstrate that cell surface CD40L is sufficient to
facilitate angiogenesis factor expression and angiogenesis in vivo.
Thus, we suggest that it is most likely that the physiological effect
of CD40L-CD40 interactions is proangiogenic. However, in light
of the report by Urbich (48), it will be important to determine
whether there is a factor or physiologic condition that can limit or
inhibit this proangiogenic effect of CD40L.
The expression of CD40 has been reported to be prominent in
processes known to be associated with angiogenesis and inflammation (32, 45, 46). These include atherosclerosis, arthritis, as well
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FIGURE 5. Ligation of CD40
induces the expression of FGF-2
and VEGF in vivo. A, Immunohistochemical staining of FGF-2
and VEGF in vivo in representative human skin grafts on SCID
mice following intracutaneous injection of saline (A and B), mock
cells (C and D), or CD40L cells
(E and F). There is enhanced expression of FGF-2 and VEGF
protein in skins treated with
CD40L cells compared with the
control skins. Data are representative of 10 similar experiments.
Magnification of A–F, ⫻400. B,
A representative skin from each
group was evaluated for VEGF
mRNA expression by RT-PCR.
The expression of VEGF164 and
VEGF121 (upper and lower
bands) is increased in skins
treated with CD40L cells. The
expression of ␤-actin is illustrated as an internal control. Data
are representative of two experiments with similar results.
as allografts undergoing rejection (49, 50). Interestingly, these
same disease processes have been found to be associated with
intense VEGF expression (43, 44). Moreover, FGF has been
shown to act directly on smooth muscle cells, which might have
important implications for inflammatory disorders, such as atherosclerosis and graft arteriosclerosis (51). Furthermore, blockade of
CD40L-CD40 interactions has been found to prevent the development of acute and chronic inflammation, including allograft rejection and atherosclerosis (52–56). It is thus possible that these effects may in part be related to the CD40L-dependent angiogenesis
response. Therefore, our findings here that local infiltration of
CD40L-expressing cells at a peripheral inflammatory site mediate
both angiogenesis factor production and angiogenesis are probably
of pathophysiological importance.
In conclusion, in this report we define a role for CD40L-CD40
interactions in angiogenesis in vitro and in vivo, and we suggest
that a major component of immune-mediated angiogenesis is
VEGF dependent. Our findings imply that the infiltration of cells
1540
CD40-DEPENDENT ANGIOGENESIS
14.
15.
16.
17.
18.
19.
20.
21.
22.
expressing CD40L at a peripheral site of inflammation is mechanistic for an interplay between the immune response and
angiogenesis.
Acknowledgments
We thank William Wong for technical assistance, Ingrid Vos, Ph.D., for
help with animal studies, and Cees van Kooten for the gift of CD40Ltransfected fibroblasts.
23.
24.
25.
26.
27.
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FIGURE 6. VEGF is a critical angiogenesis factor in CD40L-induced
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