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The Journal of Immunology, 2003, 171: 1534-1541.
Copyright © 2003 by The American Association of Immunologists

Proangiogenic Function of CD40 Ligand-CD40 Interactions 1,2

Marlies E. J. Reinders, Masayuki Sho, Stuart W. Robertson, Christopher S. Geehan and David M. Briscoe

Division of Nephrology, Department of Medicine, Children’s Hospital, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 CD40L-expressing 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Angiogenesis, 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, 2, 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, 6, 7, 8, 9, 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, 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, 16, 17). VEGF may act synergistically with other factors, such as fibroblast growth factor (FGF), in vitro to induce endothelial growth and capillary formation (18, 19, 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, 23, 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-{alpha}, TGF-{beta}, NO, and FGF-2, are most potent to induce angiogenesis (26, 27, 28, 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, 31, 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 (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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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, 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).

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.

In vivo assessment of angiogenesis

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. Full-thickness human neonatal foreskin grafts were transplanted onto CB.17 (SCID) mice as previously described (25, 39). Following engraftment for 4–6 wk, mice were divided into different experimental groups. In each group the human skin was injected intracutaneously with 35 µl of Matrigel (growth factor-reduced Matrigel; BD Biosciences, Bedford, MA) and either 35 µl of saline (as a control) or 2.5 x 106 murine fibroblasts stably transfected with human CD40L (CD40L cells) or mock transfectants (mock cells) each in 35 µl 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 µl 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 x400 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'. {beta}-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 µg) 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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.



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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 µg/ml anti-CD40 (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 {beta}-actin served as internal housekeeping gene controls. All autoradiographs are representative of three experiments with similar results.

 
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.



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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 x400 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, x400; F and G, ~x800.

 
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 CD20-expressing B cells in any of the untreated, mock-injected, or CD40L transfectant-injected skins. CD40L was minimal or absent 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.



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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: x200 (upper left panel) and x400.

 
CD40L induces the expression of multiple angiogenesis factors in vivo

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).



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  		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. *, p < 0.05.

 
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.



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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, x400. 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 {beta}-actin is illustrated as an internal control. Data are representative of two experiments with similar results.

 
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, 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 anti-VEGF 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.



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  		FIGURE 6. VEGF is a critical angiogenesis factor in CD40L-induced angiogenesis. SCID mice bearing human skin grafts were treated with a neutralizing anti-VEGF Ab (n = 9) or control IgG (n = 4) by i.p. injection (5 mg/kg on days -2, 0, 2, 4, and 6). Skins were treated with either saline as a control (n = 4) or CD40L-transfected cells (n = 9). The mean vessel density was evaluated in skin grafts by standard calibrated grid counting of vWf-positive vessels at x400 magnification. Bars illustrate the mean vessel counts ± 1 SEM. The CD40L cells failed to elicit an angiogenesis reaction in animals treated with anti-VEGF Ab.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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, 31, 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, 43, 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 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, 34, 35, 36, 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 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, 53, 54, 55, 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 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 CD40L-transfected fibroblasts.


   Footnotes
 
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. Back

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: david.briscoe{at}tch.harvard.edu Back

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. Back

Received for publication November 19, 2002. Accepted for publication May 23, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cotran, R. S.. 1994. Inflammation and repair. R. S. Cotran, and V. Kumar, and S. L. Robbins, eds. Pathologic Basis of Disease 51.-92. Saunders, Philidelphia. Chapt. 3..
  2. Brown, L. F., M. Detmar, K. Claffey, J. A. Nagy, D. Feng, A. M. Dvorak, H. F. Dvorak. 1997. Vascular permeability factor/vascular endothelial growth factor: a multifunctional angiogenic cytokine. EXS 79:233.[Medline]
  3. Folkman, J.. 1995. Seminars in Medicine of the Beth Israel Hospital, Boston: clinical applications of research on angiogenesis. N. Engl. J. Med. 333:1757.[Free Full Text]
  4. Carmeliet, P., R. K. Jain. 2000. Angiogenesis in cancer and other diseases. Nature 407:249.[Medline]
  5. Leung, D. W., G. Cachianes, W. J. Kuang, D. V. Goeddel, N. Ferrara. 1989. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246:1306.[Abstract/Free Full Text]
  6. Tischer, E., R. Mitchell, T. Hartman, M. Silva, D. Gospodarowicz, J. C. Fiddes, J. A. Abraham. 1991. The human gene for vascular endothelial growth factor: multiple protein forms are encoded through alternative exon splicing. J. Biol. Chem. 266:11947.[Abstract/Free Full Text]
  7. Polverini, P. J.. 1997. Role of the macrophage in angiogenesis-dependent diseases. EXS 79:11.[Medline]
  8. Bottomley, M. J., N. J. Webb, C. J. Watson, P. J. Holt, A. J. Freemont, P. E. Brenchley. 1999. Peripheral blood mononuclear cells from patients with rheumatoid arthritis spontaneously secrete vascular endothelial growth factor (VEGF): specific up-regulation by tumour necrosis factor-alpha (TNF-{alpha}) in synovial fluid. Clin. Exp. Immunol. 117:171.[Medline]
  9. Freeman, M. R., F. X. Schneck, M. L. Gagnon, C. Corless, S. Soker, K. Niknejad, G. E. Peoples, M. Klagsbrun. 1995. Peripheral blood T lymphocytes and lymphocytes infiltrating human cancers express vascular endothelial growth factor: a potential role for T cells in angiogenesis. Cancer Res. 55:4140.[Abstract/Free Full Text]
  10. Peoples, G. E., S. Blotnick, K. Takahashi, M. R. Freeman, M. Klagsbrun, T. J. Eberlein. 1995. T lymphocytes that infiltrate tumors and atherosclerotic plaques produce heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor: a potential pathologic role. Proc. Natl. Acad. Sci. USA 92:6547.[Abstract/Free Full Text]
  11. Ferrara, N.. 2000. VEGF: an update on biological and therapeutic aspects. Curr. Opin. Biotechnol. 11:617.[Medline]
  12. Ferrara, N., K. A. Houck, L. B. Jakeman, J. Winer, D. W. Leung. 1991. The vascular endothelial growth factor family of polypeptides. J. Cell Biochem. 47:211.[Medline]
  13. Zeng, H., H. F. Dvorak, D. Mukhopadhyay. 2001. Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) receptor-1 down-modulates VPF/VEGF receptor-2-mediated endothelial cell proliferation, but not migration, through phosphatidylinositol 3-kinase-dependent pathways. J. Biol. Chem. 276:26969.[Abstract/Free Full Text]
  14. Soker, S., S. Takashima, H. Q. Miao, G. Neufeld, M. Klagsbrun. 1998. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 92:735.[Medline]
  15. Carmeliet, P., Y. S. Ng, D. Nuyens, G. Theilmeier, K. Brusselmans, I. Cornelissen, E. Ehler, V. V. Kakkar, I. Stalmans, V. Mattot, et al 1999. Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Nat. Med. 5:495.[Medline]
  16. Stalmans, I., Y. S. Ng, R. Rohan, M. Fruttiger, A. Bouche, A. Yuce, H. Fujisawa, B. Hermans, M. Shani, S. Jansen, et al 2002. Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms. J. Clin. Invest. 109:327.[Medline]
  17. Stalmans, I., D. Lambrechts, F. De Smet, S. Jansen, J. Wang, S. Maity, P. Kneer, M. Von Der Ohe, A. Swillen, C. Maes, et al 2003. VEGF: a modifier of the del22q11 (DiGeorge) syndrome?. Nat. Med. 9:173.[Medline]
  18. Pepper, M. S., N. Ferrara, L. Orci, R. Montesano. 1992. Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Biochim. Biophys. Acta 189:824.
  19. Couper, L. L., S. R. Bryant, J. Eldrup-Jorgensen, C. E. Bredenberg, V. Lindner. 1997. Vascular endothelial growth factor increases the mitogenic response to fibroblast growth factor-2 in vascular smooth muscle cells in vivo via expression of fms-like tyrosine kinase-1. Circ. Res. 81:932.[Abstract/Free Full Text]
  20. Seghezzi, G., S. Patel, C. J. Ren, A. Gualandris, G. Pintucci, E. S. Robbins, R. L. Shapiro, A. C. Galloway, D. B. Rifkin, P. Mignatti. 1998. Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: an autocrine mechanism contributing to angiogenesis. J. Cell Biol. 141:1659.[Abstract/Free Full Text]
  21. Alon, T., I. Hemo, A. Itin, J. Pe’er, J. Stone, E. Keshet. 1995. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat. Med. 1:1024.[Medline]
  22. Sidky, Y. A., R. Auerbach. 1975. Lymphocyte-induced angiogenesis: a quantitative and sensitive assay of the graft-vs.-host reaction. J. Exp. Med. 141:1084.[Abstract/Free Full Text]
  23. Polverini, P. J., P. S. Cotran, M. A. Gimbrone, E. R. Unanue. 1977. Activated macrophages induce vascular proliferation. Nature 269:804.[Medline]
  24. Auerbach, R., Y. A. Sidky. 1979. Nature of the stimulus leading to lymphocyte-induced angiogenesis. J. Immunol. 123:751.[Abstract/Free Full Text]
  25. Moulton, K. S., R. J. Melder, V. R. Dharnidharka, J. Hardin-Young, R. K. Jain, D. M. Briscoe. 1999. Angiogenesis in the huPBL-SCID model of human transplant rejection. Transplantation 67:1626.[Medline]
  26. Leibovich, S. J., P. J. Polverini, H. M. Shepard, D. M. Wiseman, V. Shively, N. Nuseir. 1987. Macrophage-induced angiogenesis is mediated by tumour necrosis factor-{alpha}. Nature 329:630.[Medline]
  27. Koch, A. E., P. J. Polverini, S. J. Leibovich. 1986. Induction of neovascularization by activated human monocytes. J. Leukocyte Biol. 39:233.[Abstract]
  28. Wiseman, D. M., P. J. Polverini, D. W. Kamp, S. J. Leibovich. 1988. Transforming growth factor-{beta} (TGF{beta}) is chemotactic for human monocytes and induces their expression of angiogenic activity. Biochim. Biophys. Acta 157:793.
  29. Leibovich, S. J., P. J. Polverini, T. W. Fong, L. A. Harlow, A. E. Koch. 1994. Production of angiogenic activity by human monocytes requires an L-arginine/nitric oxide-synthase-dependent effector mechanism. Proc. Natl. Acad. Sci. USA 91:4190.[Abstract/Free Full Text]
  30. Yellin, M. J., J. Brett, D. Baum, A. Matsushima, M. Szabolcs, D. Steern, L. Chess. 1995. Functional interactions of T cells with endothelial cells: the role of CD40L-CD40-mediated signals. J. Exp. Med. 182:1857.[Abstract/Free Full Text]
  31. Alderson, M. R., R. J. Armitage, T. W. Tough, L. Strockbine, W. C. Fanslow, M. K. Spriggs. 1993. CD40 expression by human monocytes: regulation by cytokines and activation of monocytes by the ligand for CD40. J. Exp. Med. 178:669.[Abstract/Free Full Text]
  32. Grewal, I. S., R. A. Flavell. 1996. A central role of CD40 ligand in the regulation of CD4+ T-cell responses. Immunol. Today 17:410.[Medline]
  33. Melter, M., M. E. Reinders, M. Sho, S. Pal, C. Geehan, M. D. Denton, D. Mukhopadhyay, D. M. Briscoe. 2000. Ligation of CD40 induces the expression of vascular endothelial growth factor by endothelial cells and monocytes and promotes angiogenesis in vivo. Blood 96:3801.[Abstract/Free Full Text]
  34. Cho, C. S., M. L. Cho, S. Y. Min, W. U. Kim, D. J. Min, S. S. Lee, S. H. Park, J. Choe, H. Y. Kim. 2000. CD40 engagement on synovial fibroblast up-regulates production of vascular endothelial growth factor. J. Immunol. 164:5055.[Abstract/Free Full Text]
  35. Tai, Y. T., K. Podar, D. Gupta, B. Lin, G. Young, M. Akiyama, K. C. and Anderson. 2002. CD40 activation induces p53-dependent vascular endothelial growth factor secretion in human multiple myeloma cells. Blood 99:1419.
  36. Mach, F., U. Schonbeck, R. P. Fabunmi, C. Murphy, E. Atkinson, J. Y. Bonnefoy, P. Graber, P. Libby. 1999. T lymphocytes induce endothelial cell matrix metalloproteinase expression by a CD40L-dependent mechanism: implications for tubule formation. Am. J. Pathol. 154:229.[Abstract/Free Full Text]
  37. Deregibus, M. C., S. Buttiglieri, S. Russo, B. Bussolati, G. Camussi. 2002. CD40-dependent activation of phosphatidylinositol 3-kinase/Akt pathway mediates endothelial cell survival and in vitro angiogenesis. J. Biol. Chem. 277:25195.[Abstract/Free Full Text]
  38. van Kooten, C., X. van der Linde, A. M. Woltman, L. A. van Es, M. R. Daha. 1999. Synergistic effect of interleukin-1 and CD40L on the activation of human renal tubular epithelial cells. Kidney Int. 56:41.[Medline]
  39. Briscoe, D. M., V. R. Dharnidharka, C. Isaacs, G. Downing, S. Prosky, P. Shaw, N. L. Parenteau, J. Hardin-Young. 1999. The allogeneic response to cultured human skin equivalent in the hu-PBL-SCID mouse model of skin rejecton. Transplantation 67:1590.[Medline]
  40. Weidner, N., J. P. Semple, W. R. Welch, J. Folkman. 1991. Tumor angiogenesis and metastasis: correlation in invasive breast carcinoma. N. Engl. J. Med. 324:1.[Abstract]
  41. Ng, Y. S., P. A. D’Amore. 2001. Therapeutic angiogenesis for cardiovascular disease. Curr. Control Trials Cardiovasc. Med. 2:278.
  42. Folkman, J.. 1995. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med. 1:27.[Medline]
  43. Celletti, F. L., J. M. Waugh, P. G. Amabile, A. Brendolan, P. R. Hilfiker, M. D. Dake. 2001. Vascular endothelial growth factor enhances atherosclerotic plaque progression. Nat. Med. 7:425.[Medline]
  44. Lu, J., T. Kasama, K. Kobayashi, Y. Yoda, F. Shiozawa, M. Hanyuda, M. Negishi, H. Ide, M. Adachi. 2000. Vascular endothelial growth factor expression and regulation of murine collagen-induced arthritis. J. Immunol. 164:5922.[Abstract/Free Full Text]
  45. Denton, M. D., R. M. Reul, V. R. Dharnidharka, J. C. Fang, P. Ganz, D. M. Briscoe. 1998. Central role for CD40/CD40 ligand (CD154) interactions in transplant rejection. Pediatr. Transplant. 2:6.[Medline]
  46. Van Kooten, C., J. Banchereau. 1996. CD40-CD40-ligand: a multifunctional receptor-ligand pair. Adv. Immunol. 61:1.[Medline]
  47. Mach, F., U. Schonbeck, J. Y. Bonnefoy, J. S. Pober, P. Libby. 1997. Activation of monocyte/macrophage functions related to acute atheroma complication by ligation of CD40: induction of collagenase, stromelysin, and tissue factor. Circulation 96:396.[Abstract/Free Full Text]
  48. Urbich, C., E. Dernbach, A. Aicher, A. M. Zeiher, S. Dimmeler. 2002. CD40 ligand inhibits endothelial cell migration by increasing production of endothelial reactive oxygen species. Circulation 106:9816.
  49. Mach, F., U. Schonbeck, P. Libby. 1998. CD40 signaling in vascular cells: a key role in atherosclerosis?. Atherosclerosis 137:(Suppl.):S89.
  50. Reul, R. M., J. C. Fang, M. D. Denton, C. Geehan, C. Long, R. N. Mitchell, P. Ganz, D. M. Briscoe. 1997. CD40 and CD40 ligand (CD154) are coexpressed on microvessels in vivo in human cardiac allograft rejection. Transplantation 64:1765.[Medline]
  51. Hansson, G. K., P. Libby, U. Schonbeck, Z. Q. Yan. 2002. Innate and adaptive immunity in the pathogenesis of atherosclerosis. Circ. Res. 91:281.[Abstract/Free Full Text]
  52. Larsen, C. P., D. Z. Alexander, D. Hollenbaugh, E. T. Elwood, S. C. Ritchie, A. Aruffo, R. Hendrix, T. C. Pearson. 1996. CD40-gp39 interactions play a critical role during allograft rejection. Transplantation 61:4.[Medline]
  53. Larsen, C. P., E. T. Elwood, D. Z. Alexander, S. C. Ritchie, R. Hendrix, C. Tucker-Burden, H. R. Cho, A. Aruffo, D. Hollenbaugh, P. S. Linsley, et al 1996. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381:434.[Medline]
  54. Hancock, W. W., M. H. Sayegh, X. G. Zheng, R. Peach, P. S. Linsley, L. A. Turka. 1996. Costimulatory function and expresson of CD40 ligand, CD80, CD86 in vascularized murine cardiac allograft rejection. Proc. Natl. Acad. Sci. USA 93:13967.[Abstract/Free Full Text]
  55. Kirk, A. D., D. M. Harlan, N. N. Armstrong, T. A. Davis, Y. Dong, G. S. Gray, X. Hong, D. Thomas, J. H. Fechner, Jr, S. J. Knechtle. 1997. CTLA4-Ig and anti CD40 ligand prevent renal allograft rejection in primates. Proc. Natl. Acad. Sci. USA 94:8789.[Abstract/Free Full Text]
  56. Schonbeck, U., G. K. Sukhova, K. Shimizu, F. Mach, P. Libby. 2000. Inhibition of CD40 signaling limits evolution of established atherosclerosis in mice. Proc. Natl. Acad. Sci. USA 97:7458.[Abstract/Free Full Text]



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