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[Cancer Research 62, 1809-1814, March 15, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Nuclear Factor {kappa}B Dependency of Platelet-activating Factor-induced Angiogenesis1

Hyun-Mi Ko, Kook Heon Seo, Su-Ji Han, Kyu Youn Ahn, Il-Hwan Choi, Gou Young Koh, Hern-Ku Lee, Myung Suk Ra and Suhn-Young Im2

Department of Biological Sciences, The Institute of Basic Sciences [H-M. K., K. H. S., S-J. H., S-Y. I], Research Institute of Medical Science [K. Y. A.], Chonnam National University, Kwangju 500-757; National Creative Research Initiatives Center for Endothelial Cells, Department of Life Science, Pohang University of Science and Technology, Pohang 790-784 [G. Y. K.]; Department of Immunology and Institute for Medical Sciences, Chonbuk National University Medical School, Chonju 561-756 [I-H. C., H-K. L.]; and Department of Food Environment and Health, Kwangju Women’s University, Kwangju 500-757 [M. S. R.], Republic of Korea


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study investigated the mechanisms of platelet-activating factor (PAF)-induced angiogenesis in a mouse model of Matrigel implantation. PAF induced a dose- and time-dependent angiogenic response. Inhibitors of nuclear factor (NF) {kappa}B expression or action, including antisense oligonucleotides to the p65 subunit of NF{kappa}B (p65 antisense) and antioxidants such as {alpha}-tocopherol and N-acetyl-L-cysteine, significantly reduced PAF-induced angiogenesis. In human umbilical vein endothelial cells, PAF-induced mRNA expression and protein synthesis of various NF{kappa}B-dependent angiogenic factors, such as tumor necrosis factor-{alpha}, interleukin-1{alpha}, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF). The PAF-induced expression of the above mentioned factors was inhibited by p65 antisense or antioxidants. A significant inhibition of the angiogenic effect of PAF was achieved by anti-VEGF antibodies or soluble VEGF receptors such as KDR and flt-1 but not by antibodies against tumor necrosis factor-{alpha}, interleukin-1{alpha}, or basic fibroblast growth factor. These data indicate that PAF enhances angiogenesis through inducing NF{kappa}B activation, which in turn promotes the production of angiogenic factors such as VEGF.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neovasculization or angiogenesis is required to sustain primary tumor enlargement as well as metastasis. The induction of tumor angiogenesis is mediated by the increased production of various angiogenic molecules released by both tumor and host cells (1 , 2) . PAF,3 which is produced by a variety of cells involved in inflammatory reactions, is a potent lipid first messenger active in general cell activation, fertilization, intracellular signaling, apoptosis, and diverse inflammatory reactions (3, 4, 5, 6, 7) . Recent studies have demonstrated that PAF has the capacity to enhance tumor metastasis (8) , to induce in vitro migration of human endothelial cells, and promote in vivo angiogenesis (9) . PAF-induced angiogenesis may occur via its ability to induce the expression of angiogenic factors such as TNF-{alpha} (10) and hepatocyte growth factor (11) . Furthermore, a role for PAF has been suggested in neoangiogenesis observed in tumors (12, 13, 14) and chronic inflammatory disease such as rheumatoid arthritis (15) . However, the mechanism of PAF-induced angiogenesis remains largely unknown.

The transcription factor NF{kappa}B is normally present in the cytosol in an inactive complex with a class of inhibitory proteins known as I{kappa}Bs. Phosphorylation of I{kappa}Bs triggers their degradation and dissociation from NF{kappa}B. NF{kappa}B subsequently translocates to the nucleus where it transactivates various genes for proinflammatory cytokines and immunoregulatory genes (16 , 17) . Recent studies have demonstrated that PAF is an inducer of NF{kappa}B (18 , 19) , and we have identified PAF as a proximal mediator in the inflammatory cascade via its ability to activate NF{kappa}B (20 , 21) . Furthermore, several investigators have reported a role for NF{kappa}B in angiogenesis (22, 23, 24) . Therefore, these findings suggest a linkage between PAF-induced NF{kappa}B activation and angiogenesis.

In this study, we investigated the role for PAF-mediated NF{kappa}B activity during the process of angiogenesis. We found that PAF induced angiogenesis through NF{kappa}B activation, which in turn promoted the expression of key effector angiogenic factors, including VEGF.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Specific pathogen-free female BALB/c mice were obtained from the Korean Institute of Chemistry Technology (Daejeon, Korea) and were kept in our animal facility for at least 2 weeks before use. All of the mice were used at 8–10 weeks of age.

Reagents.
Water soluble PAF (1–0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine), NAC and Vit. E were purchased from Sigma Chemical Co. (St. Louis, MO). PAF antagonist CV 6209 was purchased from WAKO Chemical (Kyoto, Japan). Matrigel, an extract of murine basement membrane proteins, consisting predominantly of laminin, collagen IV, heparin sulfate proteoglycans, and nidogen/entactin, was purchased from Collaborative Research Inc. (Bedford, MA). Human bFGF and mouse recombinant cytokines, such as VEGF, TNF-{alpha}, IL-1{alpha} were purchased from R & D Systems (Minneapolis, MN). PECAM-1 (CD31) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antirabbit IgG-FITC secondary antibody was purchased from Vector Laboratories (Burlingame, CA). Neutralizing antibodies against VEGF, bFGF, and goat IgG were purchased from R & D Systems, and neutralizing antibodies against TNF-{alpha} and IL-1{alpha} were from Endogen (Minneapolis, MN). Rabbit IgG was purchased from Sigma Chemical Co. Recombinant human flt-1/Fc chimera and KDR/Fc chimera were purchased from R & D Systems. ELISA kits for detecting VEGF and bFGF were purchased from R & D Systems. ELISA kits for detecting TNF-{alpha} and IL-1{alpha} were purchased from Endogen.

Antisense Oligonucleotides.
The following phosphorothioate oligonucleotides were synthesized for use in antisense inhibition of gene expression (Peptron, Korea): p65 antisense of the 5' end of the NF{kappa}B gene (5'-GAAACAGATCGTCCATGGT-3') and p65 nonsense (scrambled control) oligonucleotide (5'-GTACTACTCTGAGCAAGGA-3'). The NF{kappa}B antisense oligonucleotide includes the ATG initiation codon.

Cell Culture.
HUVECs were isolated from human umbilical cord veins and were cultured as described previously (25) .

Electrophoretic Mobility Shift Assay.
The nuclear extracts were prepared from the cells as described previously (20 , 21) . To inhibit endogenous protease activity, 1 mM phenylmethylsulfonyl fluoride was added. As a probe for the gel retardation assay, an oligonucleotide containing the immunoglobulin{kappa}-chain binding site ({kappa}B, 5'-CCGGTTAACAGA GGGGGCTTTCCGAG-3') and containing AP-1 binding site (AP-1, 5'-AAGGCG CTTGATGACTCAGCCGGAA-3') were synthesized. The two complementary strands were annealed and labeled with [{alpha}-32P]dCTP. Labeled oligonucleotides (10,000 cpm), 10 µg of nuclear extracts, and binding buffer [10 mM Tris-HCl (pH 7.6), 500 mM KCl, 10 mM EDTA, 50% glycerol, 100 ng of poly(dI·dC), and 1 mM DTT] were incubated for 30 min at room temperature in a final volume of 20 µl. The reaction mixture was analyzed by electrophoresis on a 4% polyacrylamide gel in 0.5 x tris-borate buffer. Specific binding was controlled by competition with a 50-fold excess of cold {kappa}B or CRE oligonucleotide (20 , 21) .

RT-PCR.
RNA was prepared as described previously (20 , 26) . Reverse transcription was performed using 1 µl of total RNA in a 10 µl reaction mixture (Promega, Madison, WI) containing oligo (dT)15 and avian myeloblastosis virus reverse transcriptase. cDNA (1 µl) was amplified by PCR in a thermal cycler Perkin-Elmer System 2400 (Norwalk, CT; denaturation for 30 s at 95°C, annealing for 30 s at 62°C, and elongation for 30 s at 72°C). The primers used in these analysis are as follows: VEGF; 5'-GCAGAATCATCACGAAGTGG-3' and 5'-GCAACGCGAGTC TGTGTTTTTG-3'; TNF-{alpha}; 5'-CCTGTAGCCCACGTCGTAGC-3' and 5'-TTGACC TCAGCGCTGAGTTG-3'; IL-1{alpha}, 5'-GTCTCTGAATCAGAAATCCTTCTATC-3' and 5'-CATGTCAAATTTCACTGCTTCATCC-3'; bFGF, 5'-CAAGCGGCTGTACTG CAAAAAC-3' and 5'-CAGCTCTTAGCAGACATTGG-3'; and ß-actin, 5'-GGGTCA GAACTCCTATG-3' and 5'-GTAACAATGCCATGTTCAAT-3'. RT-PCR products were quantified by staining the gel with ethidium bromide, and the density of each band was determined using the densitometry Fluor-STM Imager (Bio-Rad, Muncher Germany). The level of expression was quantified by calculating the ratio of densitometric reading of the bands for cytokines and ß-actin from the same cDNA.

Angiogenesis Assay.
Angiogenesis was assayed as growth of blood vessels from s.c. tissue into a solid gel of basement membrane containing the test sample. Matrigel (10 mg/ml), in liquid form at 4°C, was mixed with 64 units/ml heparin plus the experimental substances or vehicle alone and injected (0.2 ml) into the dorsal s.c. tissue of mice (9) . Matrigel rapidly forms a solid gel at body temperature, thus allowing any incorporated substances to be released slowly and continuously for prolonged periods of time. After 6 days, mice were killed, and the gels were recovered and processed for the measurement of angiogenesis. Briefly, the assay works by measuring the amount of hemoglobin in the vessels that have invaded the Matrigel (27 , 28) using the Drabkin reagent kit 525 (Sigma Chemical Co.). Matrigels were reliquefied by being placed at 4°C on ice with red cell lysing reagent (Sigma Chemical Co.) for 24 h. After brief centrifugation, 20 µl of supernatant was added to 100 µl of Drabkin’s solution. The mixture was allowed to stand 30 min at room temperature, and absorbance was measured at 540 nm. The results were expressed as mg hemoglobin/g Matrigel pellet.

Histology.
The Matrigels were removed and immersed in 4% paraformaldehyde overnight at 4°C. The Matrigels were washed in PBS, dehydrated in a graded series of ethanol washes, and embedded in paraffin. Tissue sections were cut at 6 µm and mounted on gelatin-coated glass slides and stained with H&E. Vessel area and the total Matrigel area were planimetrically assessed from stained sections (10 , 29) . Only vessels presenting a patent lumen containing RBCs were included in the vessel area measurement.

Immunohistochemistry.
Serial sections of paraffin-embedded Matrigel cut at 6 µm were deparaffinized in xylene, rehydrated in a graded series of ethanol, rinsed twice in PBS, and then blocked in PBS containing 5% normal goat serum for 1 h. The sections were incubated for 12–14 h with the antibodies for CD31 (Santa Cruz Biotechnology) diluted 1:200 in PBS with 0.3% BSA. For a negative control, the sections were incubated in PBS containing only 5% normal goat serum. The sections were then rinsed three times in PBS and incubated sequentially for 1 h with the antirabbit IgG-FITC secondary antibody (Vector Laboratories). Finally, the tissue sections were examined and photographed on a fluorescence microscope.

Quantitation of Cytokines by ELISA.
The quantitative determination of cytokines in culture supernatants and cell lysates from HUVEC was performed by ELISA according to the manufacturer’s instructions. Briefly, after pretreatment of antisense oligonucleotides or antioxidants, medium containing 0.5 µg/ml of PAF was added, and culture supernatants and cell lysates were prepared after 3 h. Cell lysates were prepared using 200 µl of radioimmunoprecipitation assay buffer (0.1% SDS, 1% igepal, 0.5% sodium deoxycholate, and 1 mM phenylmethylsulphonyl fluoride).

Statistical Analysis.
The data are represented as the mean ± SE. Statistical significance was determined by the Student t test when two data sets were analyzed or, alternatively, by ANOVA followed by the appropriate post-hoc test for multiple data sets with the statistical software StatView (version 4.5). All of the experiments were conducted two or more times. Reproducible results were obtained, and representative data are therefore shown in the figures.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PAF-induced NF{kappa}B-dependent Angiogenesis.
In the first set of experiments, we determined the in vivo effect of PAF on angiogenesis in a murine model in which Matrigel was injected s.c. Injection of Matrigel containing PAF caused neovascularization in dose- and time-dependent manners (Fig. 1, A and B)Citation . Angiogenesis progressively increased from day 2 and reached its maximal at day 6, and the minimal effective dose of PAF was 1 µg/0.2 ml Matrigel. PAF-induced angiogenesis was blocked by the PAF antagonist CV 6209 (Fig. 1C)Citation .



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  		Fig. 1. The angiogenic effect of PAF. A, the Matrigel plugs mixed with indicated concentrations of PAF were injected s.c. On day 6, the Matrigel plugs were excised and used for quantification of angiogenesis by measuring the hemoglobin content in the Matrigel matrix. Hemoglobin was measured by using the Drabkin reagent kit 525 as described in "Materials and Methods." Matrigel containing 64 units/ml heparin and the vehicle alone was used as a control. B, Matrigel was mixed with PAF (1 µg) and injected s.c. The angiogenesis assay was performed at the indicated days. C, the Matrigel plugs mixed with PAF with or without various concentrations of CV 6209 were injected s.c., and angiogenesis assay was performed on day 6. The results were expressed as mg of hemoglobin/g of Matrigel pellet. *, P < 0.0001 compared with control group; **, P < 0.001, and ***, P < 0.0001 compared with PAF-treated group. Values are expressed as means; bars, ±SE.

 
PAF is a potent inducer of NF{kappa}B in vivo and in vitro (18, 19, 20, 21) , and NF{kappa}B activity has been associated with the process of angiogenesis (22, 23, 24) . Therefore, it was our hypothesis that PAF may induce angiogenesis via the activation of NF{kappa}B. To assess this possibility, the effects of an antisense oligonucleotide to the p65 subunit of NF{kappa}B (p65 antisense) and antioxidant treatment (Vit. E and NAC) on PAF-induced angiogenesis were examined. NF{kappa}B antisense oligonucleotides have been used as a tool to block the specific activity of NF{kappa}B (30 , 31) . It is well known that ROI are strong inducers of NF{kappa}B (32 , 33) , and we have demonstrated recently that PAF-induced NF{kappa}B activation is an exclusively ROI-dependent process (26) . We first examined the efficacy of p65 antisense and antioxidants as NF{kappa}B inhibitors. As shown in Fig. 2ACitation , p65 antisense inhibited the PAF-induced NF{kappa}B activation in a dose-dependent manner in HUVEC cultures, whereas p65 nonsense (scrambled control) oligonucleotide had no significant effect. Moreover, p65 antisense treatment did not alter PAF-induced AP-1 activation (Fig. 2B)Citation , thereby confirming the specificity of the antisense treatment protocol. Antioxidants also significantly inhibited PAF-induced NF{kappa}B activation in a dose-dependent manner (Fig. 2C)Citation . Both p65 antisense, but not scramble, and antioxidants significantly inhibited PAF-induced angiogenesis in dose-dependent manners (Fig. 2, D and ECitation , respectively), suggesting a critical role for NF{kappa}B activity in PAF-induced angiogenesis.



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  		Fig. 2. Involvement of NF{kappa}B activation in PAF-induced angiogenesis. A and B, HUVEC were plated at 1 x 105 cells/dish and were pretreated with p65 antisense or scrambled control oligonucleotide at indicated concentrations 5 days before PAF (0.5 µg/ml) treatment. C, HUVEC (1 x 106 cells/dish) were pretreated with Vit. E, or NAC 30 min before PAF treatment. Nuclear extracts were prepared 1 h after PAF treatment and were incubated with {alpha}-32P-labeled {kappa}B, AP-1, or CRE oligonucleotide and electrophoresed on a 4% polyacrylamide gel. D, Matrigel containing 64 units/ml heparin was mixed with PAF (1 µg) in the presence or absence of indicated concentrations of p65 antisense or scrambled control oligonucleotide. E, Matrigel containing 64 units/ml heparin was mixed with PAF (1 µg) with or without the addition of Vit. E (5 µg) or NAC (10 µg). The Matrigel plugs were excised and processed on day 6 for quantification of angiogenesis by measuring the hemoglobin content. The results were expressed as mg of hemoglobin/g of Matrigel pellet. *, P < 0.001 compared with control group; **, P < 0.001 compared with PAF-treated group. Values are expressed as means; bars, ±SE.

 
The angiogenic activity of PAF was also evaluated by H&E and immunofluorescence staining for CD31. A significant number of erythrocyte-containing canalized vessels and CD31-positive endothelial cells were observed within the Matrigel in PAF-treated mice but not in Matrigel only control group. Furthermore, both p65 antisense, but not scramble, and Vit. E significantly inhibited the number of PAF-induced increased CD31-positive endothelial cells and canalized vessels (Fig. 3, A and BCitation , respectively). These data indicate that the extent of vessel number and intensity of FITC paralleled the angiogenic pattern measured by hemoglobin contents.



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  		Fig. 3. Histological and immunohistochemistry analysis of Matrigel plugs. A, the Matrigel plugs were mixed with 1 µg of PAF plus oligonucleotide (10 µM) or Vit. E (5 µg) and injected s.c. On day 6, the Matrigel plugs were excised for H&E (a, c, e, g, and i) and immunofluorescence staining for CD31 (b, d, f, h, and j). a and b, control Matrigel; c and d, PAF-treated group; e and f, PAF + p65 scrambled control oligonucleotide-treated group; g and h, PAF + p65 antisense oligonucleotide-treated group; i and j, PAF + Vit. E-treated group. Immunoreactivity was detected using an immunofluorescence procedure as described in "Materials and Methods." Arrows (c–f) indicate the capillary endothelial cells. All magnifications are x80. B, quantitation of neovascularization was performed on five fields (x200) of nonseriated H&E stained sections from three animals, and results were expressed as percentage of the vessel area to the total Matrigel area; bars, ±SE. *, P < 0.02 compared with control group; **, P < 0.02 compared with PAF-treated group.

 
Effects of NF{kappa}B-dependent Angiogenic Factors on PAF-induced Angiogenesis.
The critical role for NF{kappa}B activity in PAF-induced angiogenesis derived from the Matrigel implies that specific angiogenic molecules under the transcriptional regulation by NF{kappa}B may be involved in the process. Thus, we examined whether various known NF{kappa}B-dependent angiogenic factors such as TNF-{alpha} (34) , IL-1{alpha} (35) , bFGF (36) , and VEGF (37, 38, 39, 40) were regulated by PAF. We first questioned whether PAF is able to induce mRNA expression and protein synthesis of the angiogenic factors, and, if so, p65 antisense treatment can block the PAF-dependent regulation of angiogenic factor expression. Treatment of HUVEC cultures with PAF resulted in the mRNA expression of all of the cytokines examined (Fig. 4A)Citation . Both p65 antisense oligonucleotide, but not scrambled control oligonucleotide, and antioxidants blocked the PAF-induced mRNA expression of the cytokines (Fig. 4, B and CCitation , respectively). Likewise, treatment of HUVEC with PAF induced the synthesis of the cytokine proteins (Fig. 5)Citation . The TNF-{alpha}, IL-1{alpha}, and bFGF proteins were detected in the culture supernatants, but VEGF protein was detected in only cell lysate. Both p65 antisense and antioxidants inhibited cytokine synthesis (Fig. 5, A and BCitation , respectively), indicating that PAF-induced mRNA expression and protein synthesis of these cytokines occurs via a NF{kappa}B-dependent process.



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  		Fig. 4. PAF-induced expression of TNF-{alpha}, IL-1{alpha}, bFGF, and VEGF mRNA is inhibited by p65 antisense oligonucleotides and antioxidants. A, HUVEC (1 x 106 cells/dish) were treated with 0.5 µg/ml of PAF for the times indicated. B, HUVEC (1 x 105 cells/dish) were pretreated with p65 antisense or scrambled control oligonucleotide 5 days before PAF treatment. C, HUVEC (1 x 106 cells/dish) were pretreated with Vit. E or NAC 30 min before PAF treatment. RNA was prepared 3 h after PAF treatment. cDNA was reverse transcribed from total RNA of the cells (0.01 µg) and amplified as described in "Materials and Methods." Signal intensity of these amplified cDNA was analyzed quantitatively using Fluor-STM Imager (Bio-Rad). These results of RT-PCR are shown (top panels) and quantitated by calculating the ratio of densitometric reading of the bands for cytokines and ß-actin (bottom panels).

 


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  		Fig. 5. p65 antisense oligonucleotide or antioxidant treatment inhibits the PAF-induced increase in expression of proinflammatory cytokine. A, HUVEC (1 x 105 cells/dish) were pretreated with p65 antisense or scrambled control oligonucleotide at the indicated concentrations 5 days before PAF (0.5 µg/ml) treatment. B, HUVEC (1 x 106 cells/dish) were pretreated with Vit. E or NAC 30 min before PAF treatment. TNF-{alpha}, IL-1{alpha}, and bFGF proteins were detected in the culture supernatants, and VEGF protein was detected in cell lysate. The supernatants and cell lysates were prepared 3 h after PAF treatment, and the contents of VEGF, TNF-{alpha}, IL-1{alpha}, and bFGF were measured by ELISA. *, 0.0001<P < 0.05 compared with control group; **, 0.0001<P < 0.05 compared with PAF-treated group. Values are expressed as means; bars, ±SE.

 
The extent of the involvement of these cytokines in PAF-induced angiogenesis was subsequently analyzed through the use of neutralizing antibodies. We performed preliminary experiments and used a concentration of antibody that resulted in the neutralization of 50% maximal angiogenic effect of each cytokine in Matrigel (maximum angiogenic effect of TNF-{alpha} was 50 ng; IL-1{alpha}, 50 ng; bFGF, 1 ng; and VEGF, 10 ng). Fig. 6Citation showed that using antibodies against TNF-{alpha}, IL-1{alpha}, or bFGF showed very little inhibitory effect on PAF-induced angiogenesis. In contrast, anti-VEGF antibody exhibited the greatest angiogenic effect. To additionally clarify the effect of VEGF, the effect of soluble VEGF receptors, sKDR and sflt-1 on PAF-induced angiogenesis was examined. sKDR or sflt-1 chimera almost completely inhibited the angiogenic effect of PAF at a concentration as low as 1 ng/0.2 ml Matrigel, indicating that VEGF is the most potent effector molecule in PAF-induced angiogenesis.



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  		Fig. 6. Effects of neutralizing antibodies against NF{kappa}B-dependent angiogenic factors on the PAF-induced angiogenesis. Matrigels containing 64 units/ml heparin were mixed with PAF (1 µg) in the presence or absence of sKDR (1 or 10 ng) and sFlt-1 (1 or 10 ng) and neutralizing antibodies for TNF-{alpha} (90 ng), IL-1{alpha} (100 ng), bFGF (80 ng), and VEGF (10 ng). Isotype-matched rabbit IgG (100 ng) or goat IgG (100 ng) were used as negative controls. The results are expressed as mg of hemoglobin/g of Matrigel pellet. *, P < 0.0001 compared with control group; **, P < 0.0001 compared with PAF-treated group. Values are expressed as means; bars, ±SE.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PAF has been shown recently to induce in vitro migration of human endothelial cells and promote in vivo angiogenesis (12) . In these studies, PAF was implicated as a mediator of angiogenesis by its capacity to induce expression of angiogenic factors such as TNF-{alpha} (10) and hepatocyte growth factor (11) . Furthermore, a role for PAF has been suggested in neoangiogenesis observed in tumors (12, 13, 14) and chronic inflammatory disease such as rheumatoid arthritis (15) . Consistent with these findings, we observed an angiogenic effect of PAF in this study in which Matrigel was used as a vehicle. Importantly, we demonstrated that the activation of NF{kappa}B appears to be essential to the angiogenic activity of PAF. This was substantiated by the findings that NF{kappa}B inhibitors such as antisense oligonucleotide to the p65 subunit of NF{kappa}B and antioxidants (Vit. E and NAC) significantly inhibited the angiogenic effect of PAF (Fig. 2)Citation . In recent studies, NF{kappa}B antisense oligonucleotides have been used as a tool to block the specific activity of NF{kappa}B (30 , 31) . Additionally, we demonstrated recently that PAF induced NF{kappa}B activation via generation of ROI (26) . These two tools (p65 antisense and antioxidants) were used in the current study to inhibit NF{kappa}B activation. Our data are the first to show that PAF induces angiogenesis through activating NF{kappa}B in a ROI-dependent manner. Limited data exist concerning the possible contribution of NF{kappa}B to angiogenesis. An arachidonic acid metabolite, 12(R)-hydroxy-5,5,14-eicosatrienoic acid (22) , oxidative stress (23) , and hypoxia (24) have been reported to be associated angiogenesis through the activation of NF{kappa}B. It is not clear whether there is a basic mechanism common to these mechanisms. However, given that ROI are potent inducers of NF{kappa}B (32 , 33) and arachidonic acid metabolites, oxidative stress, and hypoxia cited above have ability to generate ROI (22 , 40 , 41) , any conditions and molecules including PAF that are capable of generating ROI appear to induce angiogenesis through the activation of NF-{kappa}B.

The PAF-induced NF{kappa}B-dependent angiogenic factors used in this study included TNF-{alpha}, IL-{alpha}, bFGF, and VEGF. In response to PAF, VEGF protein was detected only in cell lysate. This implies that VEGF induced by PAF is the cell-associated VEGF 189 isoform (42) . Among the angiogenic factors, VEGF was found to be the most potent. Anti-VEGF antibodies or soluble VEGF receptors inhibited nearly all of the PAF-induced angiogenesis. VEGF is a potent peptide growth factor, specific for vascular endothelial cells, which promotes neovascularization and increases vascular permeability in vivo (43 , 44) . The critical role of VEGF in in vivo tumor angiogenesis was evidenced by experiments showing inhibition of tumor growth after treatment with anti-VEGF neutralizing antibodies (45) or by blocking signals provided by the VEGF receptors (46) . Several mediators including hypoxia (47) , ROI (48) , and proinflammatory cytokines such as TNF-{alpha}, and IL-1{alpha} (49) have been shown to induce VEGF gene expression. Although the role of PAF in the regulation of VEGF expression has not been documented, the fact that PAF is released from the hypoxic cells (50) , is an inducer of ROI generation (32 , 51) , and is an inducer of proinflammatory cytokine expression (18 , 20 , 21 , 52) supports the idea that PAF may be the initial inducer of VEGF.

It is well established that the transcription factor NF{kappa}B is essential for TNF-{alpha}, IL-1{alpha}, and bFGF expression, but it is not known whether NF{kappa}B regulates VEGF expression. Although VEGF promoter does not contain the NF{kappa}B binding site (53) , many investigators have reported that VEGF production and gene expression are inhibited by NF{kappa}B inhibition (34 , 37, 38, 39) . For example, VEGF promoter activity is significantly decreased in cancer cells transfected with mutated I{kappa}B{alpha}, which blocks NF{kappa}B activation (39) . In this study, we also observed that blocking of NF{kappa}B activity resulted in the inhibition of VEGF production and gene expression. More importantly, we have observed a significantly increased (4-fold) luciferase activity in the human endothelial cell line ECV304 when the cells were transfected with VEGF luciferase promoter-reporter and plasmids expressing p65 or p50 subunit of NF{kappa}B (data not shown). These observations suggest that VEGF promoter may contain NF{kappa}B-like binding site. Studies are required to define the NF{kappa}B binding site(s) in regulatory regions of VEGF gene.

Our present data demonstrated that: (a) PAF induced mRNA expression and protein synthesis of various angiogenic factors such as TNF-{alpha}, IL-1{alpha}, bFGF, and VEGF, which is inhibited by both p65 antisense and antioxidants; and (b) PAF-induced angiogenesis was significantly inhibited by blocking VEGF, which strongly suggests that PAF exerts its angiogenic effect through expressing NF{kappa}B-dependent angiogenic factors. In conclusion, this study demonstrated that PAF enhances angiogenesis via the activation of NF{kappa}B, which in turn promotes the expression of angiogenic factor(s) such as VEGF. Because PAF is a key inducer of NF{kappa}B in a wide range of cell types and/or organ systems, it is possible this is a major pathway leading to angiogenesis in inflammatory and tumorigenic processes.


   FOOTNOTES
 
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 Supported by the Korea Health 21 R & D project Grant 01-PJ1-PG3-21200-00003, Ministry of Health and Welfare, Republic of Korea. Back

2 To whom requests for reprints should be addressed, at Department of Biological Sciences, The Institute of Basic Sciences, Chonnam National University Kwangju 500-757, Republic of Korea. Back

3 The abbreviations used are: PAF, platelet-activating factor; NF{kappa}B, nuclear factor {kappa}B; I{kappa}B, inhibitor of nuclear factor {kappa}B; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; TNF-{alpha}, tumor necrosis factor-{alpha}; IL, interleukin; RT-PCR, reverse transcription-PCR; Vit. E, (+)-{alpha}-tocopherol acid succinate; NAC, N-acetyl-L-cysteine; CRE, cyclic AMP response element; ROI, reactive oxygen intermediates; HUVEC, human umbilical vein endothelial cell. Back

Received 2/ 6/01. Accepted 1/16/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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