The CD40-Induced Signaling Pathway in Endothelial Cells Resulting in the Overexpression of Vascular Endothelial Growth Factor Involves Ras and Phosphatidylinositol 3-Kinase¹

Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a characteristic component of cell-mediated immune inflammatory reactions (1, 2, 3). In immune-mediated angiogenesis, commonly called leukocyte-induced angiogenesis, it has been shown that cytokines or cell surface molecules expressed by circulating leukocytes can mediate the angiogenic reaction (4, 5, 6, 7). Of the many angiogenesis factors produced in the course of an inflammatory response, vascular endothelial growth factor (VEGF) ⁴ has emerged as a dominant factor for immune-mediated angiogenesis (1, 2, 8, 9, 10).

VEGF is a 34- to 45-kDa glycoprotein expressed by leukocytes (including macrophages and activated T cells), endothelial cells (EC), as well as a variety of other cell types (2, 8, 11, 12, 13). It exists in at least five different isoforms of 206, 189, 165, 145, and 121 aa, which arise from the alternative splicing of a single gene (12). VEGF is expressed in association with many inflammatory diseases (9), including arthritis (14), atherosclerosis (15), and allograft rejection (16, 17), and VEGF antagonism has been shown to protect against tissue injury in several disease models (14, 15, 18, 19). VEGF also induces the expression of EC adhesion molecules and has chemoattractant properties suggesting that it may directly promote leukocyte recruitment (15, 20, 21, 22). Thus, independent of its effects on angiogenesis, VEGF is also a proinflammatory cytokine, and its expression may represent an intermediary between cell-mediated immune inflammation and angiogenesis.

CD40, a 50-kDa type I transmembrane glycoprotein member of the TNFR superfamily, is expressed on many cells in the immune system, including EC and monocytes (23, 24, 25, 26, 27). The expression of CD40 has been reported to be prominent in processes known to be associated with angiogenesis and inflammation. Interactions between CD40 and its cognate ligand, CD40 ligand (CD40L, also called CD154), have repeatedly been found to be of importance in the activation of EC for the expression of adhesion molecules and the production of several proinflammatory cytokines and chemokines in vitro and in vivo (24, 25, 28, 29). Moreover, ligation of CD40 results in the production of several angiogenesis factors, including metalloproteinases (30), fibroblast growth factor (10), and VEGF (31), and promotes a VEGF-dependent angiogenesis reaction (10). Together these findings are consistent with observations that blockade of CD40 or CD40L in vivo attenuates the development of several vascular diseases, including atherosclerosis and allograft rejection (15, 32, 33), known to be associated with both immune inflammation and angiogenesis (1).

Nevertheless, relatively little is reported on CD40 signaling pathways in EC. Bavendiek et al. (34) examined the ability of CD40-CD40L interactions to regulate the expression of tissue factor, and found that ligation of CD40 on EC results in the activation of the transcription factors AP-1, NF-κB, and Egr-1. Reyes et al. (35) determined that cell surface CD40L on B cells interacts with EC CD40, resulting in the dephosphorylation and rephosphorylation of focal adhesion kinase, paxcillin, and extracellular signal-regulated kinase-2. And, more recently, Deregibus et al. (36) reported that ligation of CD40 on EC results in phosphorylation of Akt, Akt-dependent EC proliferation, and tube formation. Using a pharmacological inhibitor (wortmanin), they also demonstrated that this Akt-dependent angiogenesis response involved the phosphatidylinositol 3-kinase (PI3K) pathway. However, these authors did not test the specificity of PI3K for angiogenesis factor production, nor did they define a mechanism whereby the PI3K pathway might regulate angiogenesis.

In this study, we define a CD40 signaling pathway in EC that results in the expression of the angiogenesis factor VEGF. Specifically, we find that ligation of CD40 on EC results in the activation of the Ras pathway, and that Ras associates with PI3K to result in the functional expression of VEGF. Thus, for the first time, these findings detail the mechanism by which CD40 may signal for the activation of VEGF in EC. Our findings also provide a framework for the understanding of how CD40-induced signals may result in the expression of additional proinflammatory genes in EC of importance in immunity.

Soluble CD40L (sCD40L) was a gift of Y.-M. Hsu of Biogen (Cambridge, MA), and was used for all assays at 3.0 μg/ml. Wortmanin was purchased from Calbiochem (La Jolla, CA).

Single donor HUVEC were purchased from Clonetics (Walkersville, MD) and were cultured in complete endothelial medium (EGM BulletKit; Clonetics), as supplied, according to the recommended instructions. The cells were subcultured and used at passage 3–5. All EC used in these studies were assessed by FACS analysis for constitutive cell surface expression of CD40 such that they are responsive to stimulation by sCD40L.

A VEGF promoter-luciferase construct (in pGL2 basic vector; Promega, Madison, WI) containing the 2.6-kb full-length VEGF promoter sequence (bp −2361 to +298 relative to the transcription start site) was used in transfection assays, as described (31, 37, 38). The plasmids expressing the dominant-inhibitory mutant of Ras (Ras17N), and the Ras effector loop mutants were obtained as generous gifts from R. Khosravi-Far (Beth Israel Deaconess Medical Center, Boston, MA; Ref. 39). The pDCR-ras (12V,35S), pDCR-ras (12V,37G), and pDCR-ras (12V,40S) mammalian constructs encode effector domain mutants of Ha-Ras (12V) (39). The dominant-inhibitory mutant of PI3K (GST-Δp85) was obtained as a generous gift from A. Toker (Beth Israel Deaconess Medical Center; Ref. 40).

HUVEC were cultured at 2.5 × 10⁵ cells/well in six-well plates. The cells were transfected with the VEGF promoter-luciferase construct and expression plasmids using the Effectene transfection reagent (Qiagen, Valencia, CA), according to the manufacturer’s protocol. A 1:25 ratio of DNA to Effectene was used for all experiments. The total amount of transfected plasmid DNA was normalized using a control empty expression vector. Cells were harvested 24 h after transfection, and luciferase activity was measured using a standard assay kit (Promega, Madison, WI). Transfection efficiency was determined by cotransfection of the β-galactosidase gene under control of CMV immediate early promoter and by measurement of β-galactosidase activity. For all the transfection assays, we analyzed the average results of three independent experiments performed in triplicate.

HUVEC were washed twice with cold PBS, lysed with ice-cold lysis buffer (50 mM Tris, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM Na₃VO₄, 2 mM EGTA, 1 mM PMSF, 10 μg/ml leupeptine, 0.5% aprotinin, and 2 mM pepstatin A), incubated for 10 min on ice, and centrifuged for 10 min at 4°C. Immunoprecipitations were performed with 0.3 mg of total protein at Ab excess using either an anti-Raf (Genex, Hayward, CA), anti-Rho (Upstate Biotechnology, Lake Placid, NY), or anti-PI3K (Biosource International, Camarillo, CA) Ab. Immunocomplexes were captured with protein A-agarose beads (Amersham Pharmacia Biotech, Piscataway, NJ). After three washes with cell lysis buffer, bead-bound proteins were subjected to Western blot analysis.

Protein samples were mixed with 2× sample buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 10% 2-ME, 4% SDS, and 0.0025% bromphenol blue), boiled, and run on 10% polyacrylamide gel with Tris-glycine-SDS running buffer (Bio-Rad, Hercules, CA). Agarose beads with bound proteins were prepared in the same manner and directly loaded on the gel. Size-separated proteins were transferred to a polyvinylidene difluoride membrane (NEN, Boston, MA) at 60 V for 1 h. The membranes were blocked with 5% milk in PBS-Tween 20 (PBST) and coated with anti-Ras (BD Biosciences, San Diego, CA), anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Akt1 (Santa Cruz Biotechnology), or anti-phospho Akt (Cell Signaling Technology, Beverly, MA) Ab. As an internal control, the membranes were coated with anti-β-tubulin Ab (Oncogene Research Products, San Diego, CA). After three washes, the membranes were finally incubated with peroxidase-linked secondary Ab, and the reactive bands were detected by chemiluminescence (Pierce, Rockford, IL). Expression was quantified by densitometry using an Alpha Imager 2000 system (Alpha Innotech, San Leandro, CA). The signals were standardized to the expression of the internal control (β-tubulin).

HUVEC were serum starved and treated with sCD40L, washed twice in cold PBS, and lysed with cold lysis buffer containing 137 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1 mM CaCl₂, 1 mM MgCl₂, 0.1 mM sodium orthovanadate, 1% Nonidet P-40, and 1 mM PMSF. Protein content of the lysates was quantified, and equal amount of proteins was used for immunoprecipitation assays. Immunoprecipitations were performed, as described above, using the anti-PI3K Ab (Biosource International); and the immunoprecipitated enzymes were subjected to a kinase assay by competitive ELISA, according to the manufacturer’s instructions (Echelon Biosciences, Salt lake City, UT). Briefly, the bead-bound enzymes were incubated with 100 pmol of phosphatidylinositol (4, 5) bisphosphate (PI(4, 5)P₂) substrate in kinase reaction buffer (4 mM MgCl₂, 20 mM Tris, pH 7.4, 10 mM NaCl, and 25 μM ATP) for 2 h at room temperature. After the kinase reaction, the mixtures were incubated with phosphatidylinositol (3, 4, 5) trisphosphate (PI(3, 4, 5)P₃) detector for 1 h at room temperature in the dark. The reaction mixtures were subsequently added to PI(3, 4, 5)P₃-coated microplate wells, and incubated for 30 min at room temperature in the dark. After thorough washings, the manufacturer’s peroxidase-linked secondary detection reagent was added, and PI(3, 4, 5)P₃ detector protein binding to the plate was assessed by colorimetric analyses. The data for the kinase activity are expressed as fold induction in treated cells compared with the activity in untreated cells.

Nuclear extracts were prepared from HUVEC, as described (41). Cells were washed in cold PBS, and suspended in a buffer containing 10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl₂, 0.1 mM EDTA, 10 μg/ml aprotinin, 3 mM DTT, and 0.1 mM PMSF for 15 min on ice. Cells were then lysed with 0.5% Nonidet P-40, and the pellets were resuspended for 25 min in a buffer, containing 50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol, 3 mM DTT, and 0.1 mM PMSF. The samples were then centrifuged at 14,000 rpm for 10 min, and clear supernatants containing the nuclear proteins were collected and stored at −70°C until use.

EMSA was performed, as described previously (42). Briefly, an EMSA-binding reaction mixture (25 μl) was prepared with 20 mM HEPES (pH 8.4), 100 mM KCl, 20% glycerol, 0.1 mM EDTA, 0.05% Nonidet P-40, and 1 μg of BSA. Extract protein and 200 ng of poly(dA-dT).poly(dA-dT) were added to the mixture for 10 min at room temperature before addition of ∼0.1 ng of radiolabeled oligonucleotide probe. After 20 min of incubation at 4°C, samples were run on 7% acrylamide gel in 1× TAE (40 mM Tris acetate, 1 mM EDTA) buffer. For quantification by densitometry, signals were standardized to the intensity of the free probe.

Data were compared by nonparametric analysis using the Mann-Whitney U test for data analysis. Differences with p < 0.05 were considered statistically significant.

For these studies, human EC were transfected with a 2.6-kb full-length VEGF promoter-luciferase construct (or controls), as we have described (31), and the effect of CD40 agonists on VEGF transcription was assessed by measurement of luciferase activity in cell lysates. Using this model system, we found that ligation of CD40 with sCD40L was potent for the transcriptional activation of VEGF (31). To determine a role for Ras in CD40-induced VEGF overexpression, EC were cotransfected with the 2.6-kb full-length VEGF promoter-luciferase construct and a dominant-inhibitory mutant of Ras (Ras17N). We found that the Ras dominant-inhibitory mutant alone lowered basal levels of VEGF transcriptional activation and significantly inhibited the ability of sCD40L to induce VEGF transcription (Fig. 1,A). Cotransfection of EC with a plasmid containing the activated form of Ras, Ha-Ras (12V), resulted in a similar induction of luciferase activity as that found following treatment with sCD40L (Fig. 1 A). Thus, CD40 signals in EC result in the induced expression of VEGF in part via the activation of Ras.

To further examine whether Ras is an intermediary in CD40-induced transcriptional activation of VEGF, we used the CD40-inducible region of the VEGF promoter (bp −50 to +157), relative to transcription start site (43) in EMSAs. We found that nuclear protein extracts from sCD40L-treated EC, but not the untreated controls, formed a strong complex with the VEGF probe (Fig. 1,B, lanes 3 and 5). In contrast, nuclear protein extracts from EC transfected with the dominant-inhibitory mutant of Ras, and then stimulated with sCD40L, only formed a weak complex with the VEGF promoter sequence (Fig. 1 B, lanes 4 and 6). Again, these findings are suggestive that Ras is a mediator of CD40-induced transcription of VEGF in EC.

It has been shown that the activation of Ras triggers several associated signaling molecules, including Raf, Rho, and PI3K (44, 45). To evaluate whether these molecules associate with Ras in EC, we treated cells with sCD40L and immunoprecipitated whole cell lysates with either an anti-Raf, anti-Rho, or anti-PI3K Ab. Subsequently, the lysates were run in SDS-PAGE and subjected to immunoblotting with an anti-Ras Ab (Fig. 2). Our findings were that the activation of EC with sCD40L increased the association among Ras and all three of its effector molecules, beginning at 20 min and increasing up to 1 h after treatment (Fig. 2). These observations indicate that Ras may interact with Raf, Rho, and/or PI3K to mediate CD40 signals in EC.

We next wished to determine which effector molecule is functional for CD40-induced and Ras-mediated expression of VEGF. EC were cotransfected with the 2.6-kb VEGF promoter-luciferase construct and one of three effector loop mutants of Ras. Ras (12V,35S) retains full-length Raf-1-binding activity, Ras (12V,37G) retains Rho-binding activity, and Ras (12V,40S) retains PI3K-binding activity (39, 46). We found that cotransfection with the Ras (12V,40S) resulted in a significant increase in VEGF promoter-reporter activity, while the other two mutants failed to promote VEGF transcriptional activation (Fig. 3 A). Ras (12V), which was used as a positive control, also significantly increased luciferase activity. Because Ras (12V,40S) fails to bind Raf or Rho, these data suggest that PI3K may be the critical effector molecule channeling Ras-mediated signals for VEGF transcriptional activation in EC.

To establish whether PI3K is capable of mediating CD40-induced VEGF overexpression in EC, we next transfected EC with the VEGF promoter construct and treated them with sCD40L in the absence or presence of wortmanin (a pharmacological inhibitor of PI3K). Although wortmanin resulted in some inhibition, the results were inconsistent in the promoter-luciferase assay possibly due to some toxicity to the cells (data not shown). To further address this question, we obtained a specific dominant-inhibitory mutant of PI3K (Δp85) for cotransfection assays in EC with the 2.6-kb VEGF promoter-reporter construct. We found that the dominant-inhibitory mutant of PI3K inhibited basal levels of VEGF transcription, and consistently reduced CD40-induced VEGF transcriptional activation in a dose-dependent manner (Fig. 3,B). Also, we measured CD40-induced PI3K activity in EC by ELISA and found that sCD40L treatment of EC resulted in induced PI3K activity as compared with untreated controls (Fig. 3,C). Furthermore, the phosphorylation of the PI3K substrate Akt was found to be markedly increased in EC following CD40 ligation (Fig. 3 D). Together, these findings indicate that PI3K is a key molecule in CD40-dependent activation of VEGF in EC.

CD40-CD40L interaction promotes a dose-dependent increase in VEGF mRNA expression (31). By Western blot analysis, we also found a significant increase in VEGF protein expression following 24-h stimulation of EC with sCD40L as compared with untreated controls (Fig. 4,A). We first evaluated the effect of Ras on protein expression by transfecting cells with the Ras dominant-inhibitory mutant or the empty expression vector and stimulating cells with sCD40L, as described above. This inhibitory mutant significantly reduced CD40-induced VEGF protein expression (Fig. 4,B). To evaluate the effect of the PI3K pathway in CD40-induced VEGF expression, we treated EC with wortmanin before stimulation with sCD40L. Our findings were that wortmanin reduced the ability of sCD40L to stimulate VEGF protein expression (Fig. 4 B). In addition, we found that the specific dominant-inhibitory mutant of PI3K also reduced CD40-induced VEGF protein overexpression. Thus, it appears that Ras and PI3K are functional for CD40-induced VEGF transcription and protein expression in EC. We suggest that these findings are consistent with the interpretation that the immune response (represented by CD40L) can induce VEGF expression in EC; and thus VEGF-dependent angiogenesis through the Ras-PI3K signaling pathway.

In this study, we have investigated the intracellular signals that regulate CD40-CD40L-mediated induction of VEGF in EC. We show that ligation of CD40 results in activation of the Ras signal transduction pathway and its effector molecules Raf, Rho, and PI3K. Moreover, we find that PI3K is a functional intermediary for CD40-induced VEGF expression in EC. We suggest that these findings for the first time provide a mechanism by which the immune system (represented by CD40-CD40L interactions) can regulate angiogenesis.

Many studies examining the CD40 signaling pathways have used B cells (47, 48, 49, 50), and to date, little has been reported on CD40-induced signaling in EC. Moreover, only one previous report has determined that CD40-dependent signaling in EC may regulate the process of angiogenesis (36). Members of the TNFR family, including CD40, display homology in their extracellular ligand-binding domains, which are composed of tandemly repeated cysteine-rich modules (51, 52). The TNF ligand family trimerizes, thereby allowing their cognate receptors to aggregate upon binding. This receptor aggregation, in turn, activates signal transduction cascades that facilitate gene expression (53, 54). The cytoplasmic domain of CD40 lacks intrinsic catalytic activity, but associates with the TNFR-associated factor (TRAF) adaptor proteins (55, 56). Nevertheless, it is not yet known whether CD40 may regulate angiogenesis in part via interactions with the TRAF family of molecules. Also, several other kinases have been implicated in the CD40 signaling pathway in B cells (50, 57, 58, 59). In initial studies, we have found that ligation of CD40 on EC is associated with TRAF activation (data not shown). However, we do not yet know whether EC use these effector molecules in activation responses or whether they are of importance in immune-mediated angiogenesis.

Our findings in this study lead to the possibility that Ras is the key intermediary in CD40-dependent activation of VEGF in EC. Indeed, Ras has been shown to play a critical role in regulating VEGF expression in solid tumors (40, 60). There are at least three separate, ubiquitously expressed Ras genes: H-Ras, N-Ras, and K-Ras (61), all of which are proposed to act as molecular switches and essential components of cell growth and differentiation (61, 62). Each Ras protein cycles between its active and inactive GTP-bound forms that relate to its function via interactions with effector molecules that mediate the critical signal. The Ras effector molecule PI3K is composed of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit, which is directly activated by binding to GTP-bound Ras (63, 64). Other effectors are the serine-threonine kinase c-Raf1 and the closely related A-Raf and B-Raf kinases (61, 62, 63), and some studies suggest that Ras may also act through members of the Rho family of Ras-related proteins (62, 65). In this study, we demonstrate that the PI3K-specific mutant (Ras (12V,40S)) significantly increases VEGF transcriptional activation in EC, while Ras effector loop mutants specific for Raf or Rho fail to increase VEGF transcription. We also show that PI3K is functional in CD40-induced VEGF protein expression using both wortmanin and a specific kinase-inactive dominant-negative mutant. Thus, our experimental data illustrate a major role for PI3K as an effector of Ras for CD40-induced signaling and VEGF expression in EC.

As CD40 signaling in B cells has been shown to be highly variable depending on, among other things, cellular differentiation and activation (66), it is likely that CD40 signaling also differs among different cell types. Therefore, one cannot presume that EC and B cells share identical CD40 signaling pathways. Also, human EC, but not mouse EC, expresses CD40, suggesting that there are interspecies differences in the expression of CD40. Thus, in contrast to B cells (in which CD40 is expressed in the human and the mouse), the analysis of CD40 signals in human EC is most likely of great importance to the understanding of human vascular diseases. Indeed, CD40-CD40L interactions have been implicated in a wide range of chronic inflammatory conditions, including arthritis, atherosclerotic disease, and allograft rejection (9, 14, 15, 16). Furthermore, these processes are all characterized by interactions between inflammatory cells of the immune system (expressing CD40L) and the vascular endothelium (expressing CD40). Ligation of CD40 on EC is potent for the transcriptional activation of many proinflammatory genes in addition to VEGF (29, 32). We suggest that the CD40-induced Ras-PI3K pathway warrants consideration as a future target for therapies to inhibit immune-mediated activation of EC and angiogenesis, both of which are critical components of several chronic inflammatory disease processes.

We thank Yen-Ming Hsu for the gift of sCD40L; Roya Khosravi-Far for the gift of the Ras dominant-inhibitory and Ras effector domain mutant constructs; Alex Toker for the PI3K dominant-inhibitory mutant construct; and Debabrata Mukhopadhyay for helpful suggestions.