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(The FASEB Journal. 2004;18:300-310.)
© 2004 FASEB

Cyclooxygenase 2 is a key enzyme for inflammatory cytokine-induced angiogenesis

TAKASHI KUWANO*, SHINTARO NAKAO*, HIDETAKA YAMAMOTO{dagger}, MASAZUMI TSUNEYOSHI{dagger}, TOMOYA YAMAMOTO{ddagger}, MICHIHIKO KUWANO§ and MAYUMI ONO*,1

* Departments of Medical Biochemistry,
{dagger} Anatomic Pathology, Pathological Sciences, and
{ddagger} Otorhinolaryngology, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; and
§ Research Center for Innovative Cancer Therapy, Kurume University, Kurume, Fukuoka 830-0011, Japan

1 Correspondence: Department of *Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: mayumi{at}biochem1.med.kyushu-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclooxygenase1 (COX1) and COX2 mediate the rate-limiting step in arachidonic acid metabolism. Expression of COX2 mRNA and protein is often enhanced in various human cell types by inflammatory cytokines such as interleukin-1ß (IL-1ß) and tumor necrosis factor {alpha} (TNF{alpha}). IL-1ß enhanced expression of various prostanoids and this expression was blocked by COX2 selective inhibitors. IL-1ß markedly induced angiogenesis in vitro and in vivo, which was significantly inhibited by COX2 selective inhibitors but not by a vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor. In contrast, COX2 selective inhibitors only partially blocked VEGF-induced angiogenesis. EP2, EP4 (prostaglandin E2 receptors) agonists and thromboxane A2 (TXA2) receptor agonists induced angiogenesis in vitro and in vivo; IL-1ß-induced angiogenesis was blocked by an EP4 antagonist and a TXA2 receptor antagonist. IL-1ß induced much less angiogenesis in cornea of COX2 knockout mice than that of wild-type mice. This is the first report that COX2 and some prostanoids play a key role in IL-1ß-induced angiogenesis.—Kuwano, T., Nakao, S., Yamamoto, H., Tsuneyoshi, M., Yamamoto, T., Kuwano, M., Ono, M. Cyclooxygenase 2 is a key enzyme for inflammatory cytokine-induced angiogenesis.


Key Words: COX2 • vascular endothelial growth factor • prostanoid • inflammatory disease


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BOTH ISOFORMS of cyclooxygenase (COX), constitutive COX1 and inducible COX2, catalyze the production of prostanoids from arachidonic acid (1) . COX2-induced production of prostanoids is often implicated in inflammatory diseases, characterized by edema and tissue injury due to the release of many inflammatory cytokines and chemotactic factors, prostanoids, leukotrienes, and phospholipase (2 , 3) . Enhanced COX2-induced synthesis of prostaglandins stimulates cancer cell proliferation (4) , promotes angiogenesis (5 , 6) , inhibits apoptosis (7) , and increases metastatic potential (8) . COX2 is also closely involved in the carcinogenesis process (9) and is overexpressed in adenocarcinoma in comparison with noncancerous mucosal regions in colon cancers (10) and gastric cancers (11) . Elevated levels of mRNA and protein of COX2 are known to be associated with esophageal, head and neck, breast, lung, prostate, and other cancers, indicating a close involvement of COX2 in tumor progression and other pathological phenotypes in various malignant tumors (6 , 9) . COX2 is also known to be associated with lymph node metastasis in gastric cancer (12) and to affect the prognosis in primary lung adenocarcinoma (13) .

On the other hand, ovulation is closely controlled by prostaglandins (14) . Mice deficient in COX2 fail to ovulate, and this ovulatory failure could be restored by prostaglandin E2 (PGE2) or IL-1ß and gonadotropins (15) , suggesting that IL-1ß/PGE2 plays a key role in ovulation. Numerous cytokines, hormones, growth factors, and chemical stimuli up-regulate expression of COX2 in various cell types including malignant cells, stromal cells, epithelial cells, and nonepithelial cells (9) . Of these many stimuli, IL-1ß has been well known to stimulate COX2 expression and/or PGE2 production in various cell types including monocytes/macrophages (16) , vascular endothelial cells (17) , colon fibroblasts (18) , neuroblastoma cells (19) , and osteoblasts (20) . IL-1ß-induced activation of the COX2 gene is modulated by various transcription factors such as NF-{kappa}B, IL-6, CRE (17 , 21 , 22) . We have reported that potent angiogenic factors such as VEGF, IL-8, basic fibroblast growth factor (bFGF), metalloproteinases, and plasminogen activators are up-regulated in response to a representative inflammatory cytokine, TNF{alpha} in endothelial cells (23) . In human cancer cells, IL-1{alpha}/ß resulted in enhanced production of angiogenic factors VEGF and IL-8 (24) . These findings led us to theorize that angiogenesis induced by TNF{alpha} and/or IL-1{alpha}/ß is partially attributable to the production of such angiogenic factors (25) . It remains unclear, however, whether representative inflammation-related substances such as prostanoids play any role in inflammatory angiogenesis.

A recent highlight is the development of COX2 inhibitors, known as nonsteroidal anti-inflammatory drugs (NSAIDs). Clinical trials of these NSAIDs have been performed for some inflammatory diseases such as rheumatoid arthritis and osteoarthritis (26 , 27) . NSAIDs have been shown to inhibit the growth of human colon tumor cells expressing higher levels of COX2 in vitro as well as in vivo (28) . Treatment with NSAIDs decreased polyp number and size in familial adenomatous polyposis patients, indicating that NSAIDs may be chemopreventive against human polyposis (29) . COX2 inhibitors markedly reduced polyposis in adenomatous polyposis coli (Apc) mice (30 , 31) , suggesting that selective COX2 inhibitors have potential as chemopreventive agents against human intestinal and colon cancer. One possible mechanism by which NSAIDs could modulate carcinogenesis, tumor growth, and other malignancy-related phenotypes in various tumors is their effects on tumor angiogenesis (9) . Using animal models with Apc mutations during polyp formation, selective COX2 inhibitors decreased the expression of VEGF, a potent angiogenic factor (31) . Overexpression of COX2 in colon cells is accompanied by up-regulation of VEGF, bFGF, nitric oxide synthases, and angiogenesis (6) . COX2 inhibitors suppress both angiogenesis and tumor growth of xenografts of cancer cells in vivo (32) . It was reported that a COX2 inhibitor suppressed angiogenesis induced by bFGF in rat corneas (33) . Their studies suggest that NSAIDs may modulate tumor growth and carcinogenesis through antiangiogenesis. However, the underlying mechanism by which NSAIDs inhibit angiogenesis remains unclear. In our present study, we examine whether COX2 is directly associated with angiogenesis using various angiogenesis models and present a plausible model in which a COX2 inhibitor specifically induces antiangiogenic activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Agents used
One COX2 inhibitor (DFU) was obtained from Banyu Pharmaceutical Co., Tokyo, and another (JTE-522) from Japan Tobacco Inc., Tokyo. The characteristics of these COX2 inhibitors were previously reported (34 , 35) . The chemical structures of DFU and JTE522 are shown in Fig. 1 . ONO-AE1-259 (a PGE2 receptor EP2 agonist), ONO-AE1-329 (a PGE2 receptor EP4 agonist), ONO-AE3-208 (an EP4 antagonist), ONO-NT-126 (TXA2 receptor antagonists, used for in vitro), and ONO-8809 (orally active type of ONO-NT-126, used for in vivo) were obtained from ONO Pharmaceutical Co, Tokyo. U46619 (a TXA2 receptor agonist) was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). IL-1ß and VEGF were purchased from R&D Inc. (Minneapolis, MN, USA).



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  		Figure 1. Chemical structures of COX2 inhibitors, DFU (a) and JTE522 (b). DFU and JTE522 are selective inhibitors of COX2 without significant activity on COX1. The chemical name of DFU (C19H17FO4S; mol wt 360. 4) is 3-(3-fluorophenyl)-4(4-[methylsulfonyl] phenyl)-5, 5-dimethyl-5H-furan-2-one, and that of JTE522 (C16H19FN2O3S; mol wt 338. 4) is 4-(4-cyclohexyl-2-methyl-1, 3-oxazol-5-yl)-2-fluorobenzenesulfonamide.

Cell culture
HUVECs (Clonetics Inc., San Francisco, CA, USA) were cultured according to the manufacturer’s instructions (36 , 37) .

Western blot analysis
Confluent KB3-1 cells were cultured in medium containing 2% NBS and HUVECs in medium containing 0.5% FBS for 24 h. The cells were then preincubated with COX2 inhibitors for 4 h before 1 ng/mL IL-1ß or 20 ng/mL VEGF and incubated for 24 h at 37°C. Cells were then rinsed with ice-cold PBS and lysed in Triton X-100 buffer (50 µM HEPES, 150 µM NaCl, 1% Triton X-100, and 10% glycerol containing 1 µM PMSF, 1 mg/mL aprotinin, 1 mg/mL leupeptin, and 2 µM sodium vanadate). Cell lysates were subjected to SDS-PAGE and transferred to Immobilon membranes (Millipore, Bedford, MA, USA). After transfer, blots were incubated with the blocking solution and probed with anti-COX1 antibody, or anti-COX2 antibody, followed by washing. The protein content was visualized using HRP-conjugated secondary antibodies, followed by enhanced chemiluminescence (ECL, Amersham).

Migration assay of HUVECs
This assay was performed using a multiwell chamber. Polycarbonate filters (8 µm pores) were coated with 1.33 µg/mL fibronectin for 1 h at 37°C and used as the inner chamber (36 , 37) . HUVECs (3x105 cells) were suspended in EBM containing 0.5% FBS and seeded into the inner chamber. In the outer chamber, we added an EP4 agonist (ONO-AE1-329, 1 or 10 µM), a TXA2 receptor agonist (U46619, 1 or 10 µM), PGF2 (1 or 10 µM). VEGF (20 ng/mL), or IL-1ß (1 ng/mL), with or without serial dilutions of DFU (10 or 100 µM) or JTE522 (10 or 100 µM). IL-1ß, with or without EP4 antagonist (ONO-AE3-208, 1 or 10 µM) or TXA2 receptor antagonist (ONO-NT-126, 1 or 10 µM) in the same medium, was added. After incubation for 5 h at 37°C, nonmigrated cells on the upper surface of the filter were removed and cells that had migrated under the filter were counted. Cells were counted using average numbers from assays of three chambers.

Corneal micropocket assay in mice and quantification of corneal neovascularization
The corneal micropocket assay in mice has been described (36 , 37) . Briefly, 0.3 µL of hydron pellets (IFN Sciences, New Brunswick, NJ, USA) containing IL-1ß (30 ng/pellet), the EP2 agonist ONO-AE1-259 (1 or 10 µg/pellet), the EP4 agonist ONO-AE1-329 (1 or 10 µg/pellet), the TXA2 receptor agonist U46619 (50 or 100 µg/pellet), PGF2 (50 or 100 µg/pellet), or VEGF (200 ng/pellet) was prepared and implanted in the corneas of male BALB/c mice. DFU (50 mg · kg-1 · day-1), JTE522 (50 or 100 mg · kg-1 · day-1), the EP4 antagonist ONO-AE3-208 (1 mg · kg-1 · day-1), and the TXA2 receptor antagonist ONO-8809 (1 mg · kg-1 · day-1) were administered orally on days 1–6, and the VEGF receptor tyrosine kinase inhibitor SU5416 was administered intraperitoneally on days 1–6. On day 6, the mice were killed and their corneal vessels were photographed. Images of the corneas were recorded using Nikon Coolscan software. Areas of corneal neovascularization were analyzed using the software package NIH Image 1.61 (36 , 37) and expressed in mm2. The corneal micropocket assay was also performed with COX2 knockout mice. COX2 knockout mice (C57BL/6, 129P2-Ptgs2 tm1smi) (38) were purchased from Taconic Farms Inc. (Germantown, NY, USA). In this assay, 0.3 µL of hydron pellets containing IL-1ß (30 ng/pellet) or VEGF (200 ng/pellet) were implanted in the corneas of male COX2 knockout or wild-type mice. As wild-type counterpart, C57 Black mouse was used.

ELISA assays of PGE2 and TXA2/TXB2
Concentrations of PGE2 and TXA2/TXB2 in the condition medium of HUVECs were measured using commercially available ELISA kits. Cells were plated in 24-well dishes in medium containing 2% FBS. When cells were subconfluent, the medium was replaced with 0.5% serum medium for 24 h. The cells were then preincubated with various concentrations of DFU or JTE522 for 4 h, followed by 1 ng/mL IL-1ß at 37°C. Assays were performed after 24 h of incubation with 0.5% serum medium. Results were normalized for the number of cells and reported as picograms of growth factor/104 cells/24 h.

Immunohistochemistry of mouse cornea
After stimulation by IL-1ß for 6 days, cornea of Balb/cN mouse was formalin-fixed, paraffin-embedded, and sliced into sections (4 µm thick) as described (39) . Tissue sections were immunohistochemically stained using polyclonal primary antibody and the Streptavidin-biotin-peroxidase method (Histofine SABPO Kit; Nichirei, Tokyo, Japan). COX2 polyclonal antibody (Dilution 1:200; Cayman) was used as a primary antibody.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Enhanced expression of COX2 by IL-1ß and effect of COX2 inhibitors
A representative inflammatory cytokine, IL-1ß, induces up-regulation of COX2 in various cell types. Western blot analysis was performed with specific antibodies against COX1 and COX2 to examine expression of both isoforms in human head and neck cancer KB3-1 cells and HUVECs. In KB3-1 cells and HUVECs, IL-1ß did not enhance COX1 protein expression, indicating a constitutive expression of COX1 gene (Fig. 2 a). Two COX2 inhibitors, DFU and JTE522, also had no effect on expression of COX1 protein. In contrast, there marked increases appeared in COX2 levels in KB3-1 cells as well as HUVECs by IL-1ß. However, two COX2 inhibitors, DFU and JTE522, up to 50 µM did not block IL-1ß-induced up-regulation of COX2 protein (Fig. 2b ). Other inflammatory cytokines such as TNF{alpha} and IL-1{alpha} also enhanced production of COX2 protein but not that of COX1 (data not shown). IL-1ß potently up-regulated expression of COX2 in various cell types used in this study. In contrast, VEGF, a representative angiogenic factor, did not enhance COX2 protein expression (Fig. 2c ).



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  		Figure 2. Effect of COX2 inhibitors on expression of COX1 and COX2 protein. Effects of COX2 inhibitors on protein expression of COX1 and COX2 protein in KB3-1 and HUVECs were compared by Western blot analysis. KB3-1 cells and HUVECs were incubated in the absence or presence of 1 ng/mL IL-1ß with or without indicated doses of DFU or JTE522. The same amount of cellular protein was separated by SDS-PAGE and Western blot analysis was performed with specific antibody against COX1 (a) and COX2 (b). c) Protein levels of COX2 in KB3-1 cells and HUVECs were also compared when exposed to 20 ng/mL VEGF or 1 ng/mL IL-1ß.

Production of prostanoids by IL-1ß and effect of COX2 inhibitors
We first investigated whether IL-1ß could enhance the production of prostanoids through the up-regulation of COX2 gene. HUVECs produce little if any prostanoids without the exogenous addition of cytokines (Table 1 ). IL-1ß, however, induced marked production of prostanoids, including PGE2 and TXB2/TXA2. As TXA2 is very unstable, we therefore measured TXB2 (a relatively stable prostanoids derived from TXA2). Cellular production of both PGE2 and TXB2 was increased ~10-fold over the control by IL-1ß alone. This IL-1ß-induced production of these prostanoids was markedly inhibited by the addition of DFU or JTE-522 (Table 1) . These two COX2 inhibitors were found to inhibit the production of prostanoids at similar dosages as used in this study when human cancer cells or monocytic cells were treated with IL-1ß (data not shown).


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  		Table 1. IL-1ß-induced production of prostanoids by vascular endothelial cells and inhibition of COX2 inhibitorsa

Cell migration by vascular endothelial cells in response to IL-1ß or prostanoids and effect of COX2 inhibitors
Cell migration of vascular endothelial cells is a key step in the process of neovascularization. We first examined whether IL-1ß or prostanoids could stimulate cell migration and whether COX2 inhibitors could affect the IL-1ß-induced migration of vascular endothelial cells. IL-1ß at 1 ng/mL stimulated cell migration ~2.5-fold higher than the control, whereas VEGF at 20 ng/mL stimulated cell migration ~3.5-fold higher (Fig. 3 a). DFU or JTE522 at a concentration of 100 µM resulted in marked inhibition of IL-1ß-induced migration by vascular endothelial cells (Fig. 3a ). In contrast, there was no evidence of inhibition by DFU or JTE52 at 100 µM on cell migration stimulated by VEGF (Fig. 3a ). Exogenous addition of a PGE2 receptor EP2 agonist (ONO-AE1-259), EP4 agonist (ONO-AE1-329), or a TXA2 receptor agonist (U46619) at a concentration of 10 µM stimulated cell migration ~twofold over the control (Fig. 3b ). There was, however, no apparent stimulation of cell migration by PGF2 (data not shown). The stimulatory effects of prostanoids were comparable to those of VEGF or IL-1ß, and their stimulatory effects on cell migration were reproducibly observed. We then investigated which prostanoid was responsible for IL-1ß-induced angiogenesis in vitro. IL-1ß-induced cell migration was significantly inhibited by an EP4 antagonist (ONO-AE3-208, 10 µM) and a TXA2 receptor antagonist (ONO-NT-126, 10 µM) (Fig. 3c ). These findings suggest that COX2 activity is closely associated with IL-1ß-induced angiogenesis in vitro. Moreover, TXA2 and PGE2 appeared to play critical roles in IL-1ß-induced angiogenic activity in vitro.



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  		Figure 3. Effects of COX2 inhibitors and prostanoids on cell migration. a) Effects of COX2 inhibitors on endothelial cell migration by VEGF and IL-1ß were assayed using HUVECs in vitro. The migrated cell number was the mean of triplicate dishes. Relative activity (%) was recorded as 100% when the cell number (133±9.7) in the absence of any factor was subtracted from that in the presence of VEGF (20 ng/mL) or IL-1ß (1 ng/mL) alone. b) Effects of a TXA2 receptor agonist (U46619, 1 or 10 µM), a PGE2 receptor EP2 agonist (ONO-AE1-259, 1 or 10 µM) and EP4 agonist (ONO-AE1-329, 1 or 10 µM) on cell migration were determined by using HUVECs. c) Effects of a TXA2 receptor antagonist (ONO-NT-126) and an EP4 antagonist (ONO-AE3-208) on IL-1ß-induced vascular endothelial cell migration. Both agents were found to significantly inhibit IL-1ß-induced cell migration in vitro. Each column gives the average value ±SD when 3 independent assays were performed. *Statistically significant difference (P<0.01) to value for IL-1ß alone.

Angiogenesis in vivo in response to IL-1ß and the effect of selective COX2 inhibitors
We investigated whether IL-1ß could induce angiogenesis in vivo in mouse corneas. Implantation of IL-1ß at doses of 10–30 ng into mouse corneas was found to induce neovascularization in a nonvascular area of the cornea at rates comparable to 200 ng of VEGF. Apparent angiogenesis in the cornea in mice when either IL-1ß at 30 ng or VEGF at 200 ng was implanted (Fig. 4 b, f). Oral administration of DFU (50 mg · kg-1· day-1) or JTE522 (100 mg · kg-1 · day-1) dramatically inhibited IL-1ß-induced angiogenesis (Fig. 4c, d ) but not VEGF-induced angiogenesis (Fig. 4 h). In contrast, intraperitoneal administration of SU5416, a specific inhibitor of both VEGF receptors, KDR/Flk-1 and Flt-1 (40) , almost completely inhibited VEGF-induced angiogenesis (Fig. 4g ) but had no effect on IL-1ß-induced angiogenesis in vivo (Fig. 4e ). Quantitative analysis using three or four mice for each assay showed almost complete inhibition of IL-1ß-induced neovascularization when DFU or JTE-522 was orally administered (Fig. 4i ). Almost no inhibition by DFU (Fig. 4h, i ) or JTE-522 (data not shown) of VEGF-induced neovascularization was observed, whereas SU5416 significantly inhibited VEGF-induced neovascularization. IL-1ß-induced angiogenesis was blocked by selective COX2 inhibitors in vitro and in vivo.



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  		Figure 4. Effects of COX2 inhibitors on angiogenesis in vivo. Photographs of angiogenesis in mouse corneas. Mice were treated with DFU (50 mg ·kg-1 · day-1, orally), JTE522 (50 or 100 mg · kg-1 · day-1, orally), or SU5416 (25 mg · kg-1 · day-1, intraperitoneally) on day 1 to 6. Six days later, vessels in the region of the pellet implant were photographed. Representative photographs of mouse corneas; a) buffer alone, b) IL-1ß (30 ng), c) IL-1ß with DFU (50 mg · kg-1 · day-1), d) IL-1ß with JTE522 (100 mg ·kg-1 · day-1), e) IL-1ß with SU5416 (25 mg · kg-1 · day-1), f) VEGF (200 ng), g) VEGF with SU5416 (25 mg · kg-1· day-1), h) VEGF with DFU (50 mg · kg-1 · day-1). i) Quantification of corneal neovascularization in mice after administration of DFU, JTE522, or SU5416. Neovascular areas developed in mouse corneas (a–h) were quantified as described in Materials and Methods. Columns are mean (±SD) of 3 or 4 independent experiments. *Statistically significant difference (P<0.01) to value for IL-1ß alone.

Angiogenesis in vivo by prostanoid receptor agonists
We next investigated whether prostanoids could also induce neovascularization in mouse corneas in vivo. Corneal implantation of a TXA2 receptor agonist (U46619, 50 or 100 µg), an EP2 receptor agonist (ONO-AE1-259, 1 or 10 µg) or an EP4 receptor agonist (ONO-AE1-329, 1 or 10 µg) as a pellet was found to induce angiogenesis, although its neovascularization activity was less than that of IL-1ß (Fig. 5 c–e). In contrast, PGF2 did not induce angiogenesis (data not shown). Quantitative analysis using three or four mice for each assay showed angiogenic activity of TXA2 and PGE2 when their respective agonists were implanted (Fig. 5 f).



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  		Figure 5. Angiogenesis in vivo by prostanoid receptor agonists. Photographs of angiogenesis in mouse corneas. Hydron pellets containing of a) buffer alone, b) IL-1ß (30 ng/pellet), c) TXA2 receptor agonist (U46619, 100 µg/pellet), d) EP2 agonist (ONO-AE1-259, 10 µg/pellet), e) EP4 agonist (ONO-AE1-329, 10 µg/pellet) were implanted into the corneas of Balb/c mice. Six days later vessels in the region of the pellet implanted were photographed. Quantitative analysis was performed on data in panel f with 3 or 4 mice corneas.

Effect of prostanoid receptor antagonists on angiogenesis in vivo by IL-1ß
We further investigated which prostanoid was most closely involved in IL-1ß-induced angiogenesis. Oral administration of the TXA2 receptor antagonist ONO-8809 (1 mg · kg-1 · day-1) or the EP4 receptor antagonist ONO-AE3-208 (1 mg · kg-1 · day-1) was found to reduce IL-1ß-induced angiogenesis in mouse corneas (Fig. 6 a–c). Quantitative analysis showed that ONO-8809 and ONO-AE3-208 both significantly inhibited IL-1ß-induced angiogenesis by ~50% (Fig. 6 d).



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  		Figure 6. Inhibition of IL-1ß-induced angiogenesis by prostanoid receptor antagonists. Photographs of angiogenesis in mouse corneas. Hydron pellets containing IL-1ß (30 ng/pellet) were implanted into the corneas of Balb/c mice. TXA2 receptor antagonist (ONO-8809, 1 mg · kg-1 · day-1) or the PGE2 receptor EP4 antagonist (ONO-AE3-208, 1 mg · kg-1 · day-1) was administered orally on day 1 to 6. Representative photographs of mouse corneas a) IL-1ß (30 ng/pellet), b) IL-1ß with ONO-8809 (1 mg · kg-1 · day-1), and c) IL-1ß with ONO-AE3-208 (1 mg ·kg-1 · day-1). d) Quantitative analysis was performed on data in panels a–c with 3 or 4 mice corneas. *Statistically significant difference (P<0.01) to value for IL-1ß alone.

Localization of COX2 in infiltrating cells within IL-1ß-treated corneas
We examined whether COX2-positive cells were infiltrated in IL-1ß-treated corneas. The cornea developed new vessels by IL-1ß, immunohistochemical analysis of the corneas was performed with anti-COX2 antibody. Many infiltrating cells stained with COX2 appeared in the stroma and anterior chamber when treated with IL-1ß (Fig. 7 ). By contrast, there appeared to be no infiltrating cells within the untreated corneas (data not shown). Cells infiltrated near new vessels in the cornea consisted of inflammatory cells, including monocyte/macrophage. Immunohistochemical analysis with murine macrophage-recognizing monoclonal antibody (F4/80) showed many F4/80-positive cells in IL-1ß-treated cornea (data not shown).



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  		Figure 7. Identification of COX2-positive cells infiltrating in corneas treated with IL-1ß. Immunohistochemical staining was preformed with the sections of IL-1ß-treated cornea in mice with anti-COX2 antibody (a, b). The black arrows represent new vessels and the red arrows represent COX2 positive cells. Compared with untreated group (data not shown), section of IL-1ß-treated corneas shows numerous immunopositive cells in stroma and anterior chamber. Magnification, 40x (a), 100x (b).

Angiogenesis by IL-1ß in COX2 knockout mice
We finally asked whether COX2 was directly involved in IL-1ß-induced angiogenesis in vivo. We compared angiogenesis by IL-1ß between COX2 knockout mice and wild-type mice. Implantation of IL-1ß at a dose of 30 ng into cornea of wild-type mouse induced neovascularization in a nonvascular area. By contrast, a marked reduction in the angiogenesis in cornea of COX2 knockout mice was demonstrated (Fig. 8 b). We observed angiogenesis at similar levels between wild-type and COX2 knockout mice by VEGF at a dose of 200 ng (Fig. 8c, d ). Quantitative analysis using three to four mice for each assay showed very low (~20%) angiogenesis by IL-1ß in COX2 knockout mice in comparison with wild-type mice (Fig. 8e ). A similar level of angiogenic activity in vivo by VEGF between COX2 knockout and wild-type mice was observed (Fig. 8e ).



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  		Figure 8. Angiogenesis in cornea of COX2 knockout mice Photographs of angiogenesis in mouse corneas. IL-1ß or VEGF was implanted in corneas of COX2 knockout mice and wild-type mice on day 1. Six days later, vessels in the region of the pellet implanted were photographed. Representative photographs of angiogenesis in corneas of wild-type and COX2 knockout mice a) wild-type mouse: IL-1ß (30 ng); b) COX2 knockout mouse: IL-1ß (30 ng); wild-type mouse: VEGF (200 ng); and d) COX2 knockout mouse: VEGF (200 ng). e) Quantitative analysis was performed on data in panels a–d with 3 or 4 mice corneas. Black bars represent wild-type mice; blue bars represent COX2 knockout mice. *Statistically significant difference (P<0.01) to the value for IL-1ß in wild-type mouse.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Voronov et al. recently reported that IL-1 is required for both angiogenesis and tumor invasiveness using IL-1ß or IL-1{alpha} knockout mice (41) . Other independent studies have reported that IL-1ß promotes tumor growth, invasion, and angiogenesis in animal models with concomitant enhanced production of VEGF, MMP-2, IL-8, and adhesion molecules (42 , 43) . IL-1{alpha} also promotes angiogenesis in vivo through VEGF receptor pathway possibly by inducing VEGF synthesis (44) . However, it remains unclear how VEGF or other angiogenesis-related factors could be involved in IL-1-induced angiogenesis and tumor invasion. In our present study, IL-1ß was found to markedly enhance production of prostanoids such as PGE2 and TXA2/TXB2. IL-1ß-induced production of prostanoids was almost completely blocked by both COX2-selective inhibitors DFU and JTE522. Both cell migration by vascular endothelial cells in vitro and corneal neovascularization in vivo were markedly induced in response to IL-1ß at rates similar to VEGF. Administration of COX2 inhibitors resulted in a dramatic reduction of IL-1ß-induced angiogenesis in vivo as well as cell migration in vitro. A TXA2 receptor agonist (U46619), an EP2 agonist (ONO-AE1-259), and an EP4 agonist (ONO-AE1-329) stimulated cell migration in vitro and induce corneal neovascularization in mice. TXA2 receptor antagonists (ONO-8809) and EP4 antagonist (ONO-AE3-208) also inhibited IL-1ß-induced angiogenesis in vivo. We therefore present a model that some prostanoids such as TXA2 and PGE2 directly induce angiogenesis through interaction with their cognate receptors on vascular endothelial cells (Fig. 9 ).



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  		Figure 9. A model for IL-1ß-induced angiogenesis and effect of COX2 inhibitor. We previously reported that IL-1ß and TNF-{alpha} enhance production of angiogenesis-related factors such as VEGF, IL-8, bFGF, plasminogen activator, and metalloproteinases from vascular endothelial cells and other cell types, resulting in angiogenesis through autocrine and/or paracrine control. In the present study, we demonstrated that inflammatory cytokines such as IL-1ß and probably TNF-{alpha} induce angiogenesis through the direct interaction of prostanoids with vascular endothelial cells. COX2 induced by IL-1ß catalyzes the process of arachidonic acid cascade in vascular endothelial cells. PGE2 and TXA2 are prostanoids, final products of the arachidonic acid cascade thought to be critical factors for angiogenesis through PGE2 receptors (EP2, EP4) and the TXA2 receptor.

We also observed apparent reduction in IL-1ß-induced angiogenesis in corneas of the COX2 knockout mice in comparison with wild-type mice. This experiment with knockout mice strongly suggests a direct involvement of COX2 and relevant prostanoids in IL-1ß-induced angiogenesis. However, some neovascularized area was observed in the COX2 knockout mice by IL-1ß, suggesting an involvement of different factors or pathways in IL-1ß-induced angiogenesis.

COX2 overexpresssion up-regulates expression of several angiogenic factors, VEGF and bFGF, and COX2 inhibitors significantly inhibited production of VEGF and bFGF as well as angiogenesis in vivo (32 , 45) . Administration of COX2 inhibitors blocked expression of VEGF and bFGF in vitro as well as angiogenesis and tumor growth in vivo (32) . It has been reported that prostaglandins stimulate production of VEGF and bFGF (46) . We have reported that IL-1{alpha} and TNF{alpha} significantly enhance production of VEGF, IL-8, bFGF (23 , 24) , and COX2 protein in endothelial cells and cancer cells (Fig. 9) . Such potent angiogenic factors are expected to be involved in angiogenesis through the IL-1{alpha}/ß-COX2 pathway (17) . It seems likely there are at least two pathways in the IL-1ß-induced production of VEGF and other factors through either COX2-dependent or COX2-independent pathways. IL-1ß-induced production of VEGF and IL-8 was, however, inhibited by only ~50% when treated with COX2 inhibitors at concentrations of 50 to 100 µM (T. Kuwano and M. Ono, unpublished data). In contrast, COX2 inhibitors at low concentrations markedly inhibited IL-1ß-induced production of PGE2 and TXB2/TXA2 (Table 1) . Our in vivo study further demonstrated that IL-1ß-induced corneal angiogenesis in vivo was blocked, but not completely, by DFU and JTE522. This IL-1ß-induced angiogenesis was blocked only slightly if at all by SU5416. This in vivo study suggests specific involvement of prostanoids in IL-1ß-induced angiogenesis rather than in angiogenesis by the VEGF/VEGF receptor pathway (Fig. 9) .

Arachidonic acid metabolites have been known to modulate endothelial cell proliferation or migration and capillary formation in vivo (47) . We previously reported that arachidonic acid metabolism inhibitors block angiogenesis in vivo as well as in vitro, suggesting a close association between prostanoids and angiogenesis. Of the various prostanoids produced by COX2 in response to IL-1ß, PGE2 appears to play a key role in inflammatory angiogenesis (5) . PGI2 (48) and TXA2 (49) were reported to demonstrate some angiogenic activity in vivo. Our study indicated that PGE2 and TXA2 stimulate cell migration by vascular endothelial cells. Cell migration in response to IL-1ß, but not to VEGF, was blocked by DFU or JTE522. Angiogenesis assay in vivo also demonstrated induction of angiogenesis in corneas by a TXA2 receptor agonist (U46619) and an EP4 agonist (ONO-AE-329). Taken together, these results consistently support the notion that inflammatory cytokine-elicited angiogenesis is induced mainly by TXA2, PGE2, and other undetermined prostanoids through COX2 activation. Both COX2 inhibitors at low concentrations significantly inhibited the production of PGE2 and TXA2/TXB2 whereas at high concentrations inhibited the IL-1ß-induced migration of vascular endothelial cells (Table 1 and Fig. 3 ). Concerning this discrepancy, experimental conditions for the two assay systems are different. 1) In the migration assay, IL-1ß and COX2 inhibitors were added simultaneously while COX2 inhibitors were added 4 h before exposure to IL-1ß in the prostanoid production assay. 2) Migration assay was performed for only 5 h whereas prostanoid production assay was performed for 24 h. We still favor the idea that PGE2 and TXA2/TXB2 play key roles in IL-1ß-induced cell migration. However, further study is required to determine the involvement of prostanoids other than PGE2 and TXA2/TXB2 and/or angiogenic factors in the IL-1ß-induced cell migration.

The tumor microenvironment consists mainly of various inflammatory cells and closely affects proliferation, survival and migration in the neoplastic process (50) . Of these inflammatory cell types, macrophage is a significant component of inflammatory infiltrates, affecting angiogenesis as well as malignant characteristics of cancer. Infiltration of macrophages is closely associated with microvascular density and malignant status in various human tumor types (24) . Activated macrophages are thought to play a key role in angiogenesis of inflammatory diseases and in malignant tumors. Activated macrophages infiltrating tumor stroma and inflammatory regions produce various angiogenesis factors, including prostanoids (25) . Related studies have reported that activation of macrophage is accompanied by induction of COX2 and IL-1ß (16) and that high expression of COX2 is often observed in macrophages infiltrating in tumor stroma (18) . We have reported that macrophage infiltration is maximized in mouse cornea within 4 to 5 days after inflammatory stimuli by chemical cauterization and that the kinetics of macrophage infiltration is similar to that of neovascularization (39) . Consistent with this study, inflammatory cytokine IL-1ß could induce infiltration of COX2-positive macrophages and/or other inflammatory cells in cornea. If such macrophages/monocytes actively produce prostanoids, these prostanoids could also play a key role in angiogenesis under certain inflammatory conditions.

NSAIDs block tumor development in some animal carcinogenesis models (30) . In a recent study, COX2 overexpression in the skin of transgenic mice resulted in suppression of tumor development, again suggesting a key role for COX2 and elevated prostaglandin levels in the development of skin tumor (51) . On the other hand, angiogenesis, a risk factor for metastasis and recurrence (52) , is closely involved in the tumor development process. Angiogenesis is a prerequisite for the early switch-on of tumor development in several animal carcinogenesis models. Anticarcinogenic and antineoplastic effects of NSAIDs are known to be mediated through COX2-dependent and -independent pathways (53) . Anticarcinogenesis and antineoplastic effects through inhibition of COX2 dependent pathways could be attributable at least in part to inhibition of angiogenesis by NSAIDs. The clinical application of COX2 inhibitors will provide new information as to whether COX2 is a useful molecular target.


   ACKNOWLEDGMENTS
 
We would like to thank Banyu Pharmaceutical Co. and Japan Tobacco Inc. for COX2 inhibitors and ONO Pharmaceutical Co. for prostanoid receptor agonists and antagonists. This work was supported by grants for cancer research from the Ministry of Education, Science, Sport and Culture, Japan (M.O., M.K.), and the Ministry of Human Health Labor and Welfare, Japan (M.K.).

Received for publication May 13, 2004. Accepted for publication October 8, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Williams, C. S., DuBois, R. N. (1996) Prostaglandin endoperoxide synthase: why two isoforms?. Am. J. Physiol. 270,G393-G400
  2. Arslan, A., Zingg, H. H. (1996) Regulation of COX-2 gene expression in rat uterus in vivo and in vitro. Prostaglandins 52,463-481[CrossRef][Medline]
  3. Tanaka, Y., Takahashi, M., Kawaguchi, M., Amano, F. (1997) Delayed release of prostaglandins from arachidonic acid and kinetic changes in prostaglandin H synthase activity on the induction of prostaglandin H synthase-2 after lipopolysaccharide-treatment of RAW264.7 macrophage-like cells. Biol. Pharm. Bull. 20,322-326[Medline]
  4. Sheng, H., Shao, J., Washington, M. K., DuBois, R. N. (2001) Prostaglandin E2 increases growth and motility of colorectal carcinoma cells. J. Biol. Chem. 276,18075-18081[Abstract/Free Full Text]
  5. Ben-Av, P., Crofford, L. J., Wilder, R. L., Hla, T. (1995) Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: a potential mechanism for inflammatory angiogenesis. FEBS Lett. 372,83-87[CrossRef][Medline]
  6. Tsuji, S., Kawano, S., Tsujii, M., Michida, T., Masuda, E., Gunawan, E. S., Hori, M. (1998) Mucosal microcirculation and angiogenesis in gastrointestinal tract. Nippon Rinsho. 56,2247-2252[Medline]
  7. Sheng, H., Shao, J., Morrow, J. D., Beauchamp, R. D., DuBois, R. N. (1998) Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res. 58,362-366[Abstract/Free Full Text]
  8. Kakiuchi, Y., Tsuji, S., Tsujii, M., Murata, H., Kawai, N., Yasumaru, M., Kimura, A., Komori, M., Irie, T., Miyoshi, E., et al (2002) Cyclooxygenase-2 activity altered the cell-surface carbohydrate antigens on colon cancer cells and enhanced liver metastasis. Cancer Res. 62,1567-1572[Abstract/Free Full Text]
  9. Tsuji, S., Tsujii, M., Kawano, S., Hori, M. (2001) Cyclooxygenase-2 upregulation as a perigenetic change in carcinogenesis. J. Exp. Clin. Cancer Res. 20,117-129[Medline]
  10. Sano, H., Kawahito, Y., Wilder, R. L., Hashiramoto, A., Mukai, S., Asai, K., Kimura, S., Kato, H., Kondo, M., Hla, T. (1995) Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res. 55,3785-3789[Abstract/Free Full Text]
  11. Murata, H., Kawano, S., Tsuji, S., Tsuji, M., Sawaoka, H., Kimura, Y., Shiozaki, H., Hori, M. (1999) Cyclooxygenase-2 overexpression enhances lymphatic invasion and metastasis in human gastric carcinoma. Am. J. Gastroenterol. 94,451-455[CrossRef][Medline]
  12. Xue, Y. W., Zhang, Q. F., Zhu, Z. B., Wang, Q., Fu, S. B. (2003) Expression of cyclooxygenase-2 and clinicopathologic features in human gastric adenocarcinoma. World J. Gastroenterol. 9,250-253[Medline]
  13. Achiwa, H., Yatabe, Y., Hida, T., Kuroishi, T., Kozaki, K., Nakamura, S., Ogawa, M., Sugiura, T., Mitsudomi, T., Takahashi, T. (1999) Prognostic significance of elevated cyclooxygenase 2 expression in primary, resected lung adenocarcinomas. Clin. Cancer Res. 5,1001-1005[Abstract/Free Full Text]
  14. Richards, R. G., Almond, G. W. (1994) Lipopolysaccharide-induced increases in porcine serum cortisol and progesterone concentrations are not mediated solely by prostaglandin F2 alpha. Inflammation 18,203-214[CrossRef][Medline]
  15. Davis, B. J., Lennard, D. E., Lee, C. A., Tiano, H. F., Morham, S. G., Wetsel, W. C., Langenbach, R. (1999) Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E2 and interleukin-1beta. Endocrinology 140,2685-2695[Abstract/Free Full Text]
  16. Caivano, M., Cohen, P. (2000) Role of mitogen-activated protein kinase cascades in mediating lipopolysaccharide-stimulated induction of cyclooxygenase-2 and IL-1 beta in RAW264 macrophages. J. Immunol. 164,3018-3025[Abstract/Free Full Text]
  17. Kirtikara, K., Raghow, R., Laulederkind, S. J., Goorha, S., Kanekura, T., Ballou, L. R. (2000) Transcriptional regulation of cyclooxygenase-2 in the human microvascular endothelial cell line, HMEC-1: control by the combinatorial actions of AP2, NF-IL-6 and CRE elements. Mol. Cell. Biochem. 203,41-51[CrossRef][Medline]
  18. Bamba, H., Ota, S., Kato, A., Adachi, A., Itoyama, S., Matsuzaki, F. (1999) High expression of cyclooxygenase-2 in macrophages of human colonic adenoma. Int. J. Cancer 83,470-475[CrossRef][Medline]
  19. Fiebich, B. L., Mueksch, B., Boehringer, M., Hull, M. (2000) Interleukin-1beta induces cyclooxygenase-2 and prostaglandin E(2) synthesis in human neuroblastoma cells: involvement of p38 mitogen-activated protein kinase and nuclear factor-kappaB. J. Neurochem. 75,2020-2028[CrossRef][Medline]
  20. Wadleigh, D. J., Herschman, H. R. (1999) Transcriptional regulation of the cyclooxygenase-2 gene by diverse ligands in murine osteoblasts. Biochem. Biophys. Res. Commun. 264,865-870[CrossRef][Medline]
  21. Crofford, L. J., Tan, B., McCarthy, C. J., Hla, T. (1997) Involvement of nuclear factor kappa B in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes. Arthritis Rheum. 40,226-236[Medline]
  22. Inoue, H., Yokoyama, C., Hara, S., Tone, Y., Tanabe, T. (1995) Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin-6 expression site and cAMP response element. J. Biol. Chem. 270,24965-24971[Abstract/Free Full Text]
  23. Yoshida, S., Ono, M., Shono, T., Izumi, H., Ishibashi, T., Suzuki, H., Kuwano, M. (1997) Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis. Mol. Cell. Biol. 17,4015-4023[Abstract]
  24. Torisu, H., Ono, M., Kiryu, H., Furue, M., Ohmoto, Y., Nakayama, J., Nishioka, Y., Sone, S., Kuwano, M. (2000) Macrophage infiltration correlates with tumor stage and angiogenesis in human malignant melanoma: possible involvement of TNFalpha and IL-1alpha. Int. J. Cancer 85,182-188[Medline]
  25. Ono, M., Torisu, H., Fukushi, J., Nishie, A., Kuwano, M. (1999) Biological implications of macrophage infiltration in human tumor angiogenesis. Cancer Chemother. Pharmacol. 43,S69-S71
  26. Kawai, S. (1998) Drug delivery system of anti-inflammatory and anti-rheumatic drugs. Nippon Rinsho. 56,782-787[Medline]
  27. Dequeker, J., Hawkey, C., Kahan, A., Steinbruck, K., Alegre, C., Baumelou, E., Begaud, B., Isomaki, H., Littlejohn, G., Mau, J. (1998) Improvement in gastrointestinal tolerability of the selective cyclooxygenase (COX)-2 inhibitor, meloxicam, compared with piroxicam: results of the Safety and Efficacy Large-scale Evaluation of COX-inhibiting Therapies (SELECT) trial in osteoarthritis. Br. J. Rheumatol. 37,946-951[Abstract/Free Full Text]
  28. Reddy, B. S., Rao, C. V. (2000) Colon cancer: a role for cyclo-oxygenase-2-specific nonsteroidal anti-inflammatory drugs. Drugs Aging 16,329-334[CrossRef][Medline]
  29. Giardiello, F. M., Hamilton, S. R., Krush, A. J., Piantadosi, S., Hylind, L. M., Celano, P., Booker, S. V., Robinson, C. R., Offerhaus, G. J. (1993) Treatment of colonic and rectal adenomas with sulindac in familial adenomatous polyposis. N. Engl. J. Med. 328,1313-1316[Abstract/Free Full Text]
  30. Oshima, M., Dinchuk, J. E., Kargman, S. L., Oshima, H., Hancock, B., Kwong, E., Trzaskos, J. M., Evans, J. F., Taketo, M. M. (1996) Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell 87,803-809[CrossRef][Medline]
  31. Oshima, M., Murai, N., Kargman, S., Arguello, M., Luk, P., Kwong, E., Taketo, M. M., Evans, J. F. (2001) Chemoprevention of intestinal polyposis in the Apcdelta716 mouse by rofecoxib, a specific cyclooxygenase-2 inhibitor. Cancer Res. 61,1733-1740[Abstract/Free Full Text]
  32. Sawaoka, H., Tsuji, S., Tsujii, M., Gunawan, E. S., Sasaki, Y., Kawano, S., Hori, M. (1999) Cyclooxygenase inhibitors suppress angiogenesis and reduce tumor growth in vivo. Lab. Invest. 79,1469-1477[Medline]
  33. Masferrer, J. L., Koki, A., Seibert, K. (1999) COX-2 inhibitors. A new class of antiangiogenic agents. Ann. N.Y. Acad. Sci. 889,84-86[Abstract/Free Full Text]
  34. Riendeau, D., Percival, M. D., Boyce, S., Brideau, C., Charleson, S., Cromlish, W., Ethier, D., Evans, J., Falgueyret, J. P., Ford-Hutchinson, A. W., et al (1997) Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor. Br. J. Pharmacol. 121,105-117[CrossRef][Medline]
  35. Wakitani, K., Nanayama, T., Masaki, M., Matsushita, M. (1998) Profile of JTE-522 as a human cyclooxygenase-2 inhibitor. Jpn. J. Pharmacol. 78,365-371[CrossRef][Medline]
  36. Hirata, A., Ogawa, S., Kometani, T., Kuwano, T., Naito, S., Kuwano, M., Ono, M. (2002) ZD1839 (Iressa) induces antiangiogenic effects through inhibition of epidermal growth factor receptor tyrosine kinase. Cancer Res. 62,2554-2560[Abstract/Free Full Text]
  37. Nakao, S., Kuwano, T., Ishibashi, T., Kuwano, M., Ono, M. (2003) Synergistic effect of TNF-{alpha} in soluble VCAM-1-induced angiogenesis through {alpha}4 integrins. J. Immunol. 170,5704-5711[Abstract/Free Full Text]
  38. Morham, S. G., Langenbach, R., Loftin, C. D., Tiano, H. F., Vouloumanos, N., Jennette, J. C., Mahler, J. F., Kluckman, K. D., Ledford, A., Lee, C. A., et al (1995) Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell 83,473-482[CrossRef][Medline]
  39. Ogawa, S., Yoshida, S., Ono, M., Onoue, H., Ito, Y., Ishibashi, T., Inomata, H., Kuwano, M. (1999) Induction of macrophage inflammatory protein-1{alpha} and vascular endothelial growth factor during inflammatory neovascularization in the mouse cornea. Angiogenesis 3,327-334[CrossRef][Medline]
  40. Itokawa, T., Nokihara, H., Nishioka, Y., Sone, S., Iwamoto, Y., Yamada, Y., McMahon, G., Shibuya, M., Kuwano, M., Ono, M., et al (2002) Antiangiogenic effect by SU5416 is partly attributable to inhibition of FIt-1 receptor signaling. Mol. Cancer Ther. 1,295-302[Abstract/Free Full Text]
  41. Voronov, E., Shouval, D. S., Krelin, Y., Cagnano, E., Benharroch, D., Iwakura, Y., Dinarello, C. A., Apte, R. N. (2003) IL-1 is required for tumor invasiveness and angiogenesis. Proc. Natl. Acad. Sci. USA 100,2645-2650[Abstract/Free Full Text]
  42. Yano, S., Nokihara, H., Goto, H., Ogawa, H., Kanematsu, T., Miki, T., Uehara, H., Saijo, Y., Nukiwa, T., Sone, S., et al (2003) Multifunctional interleukin-1ß promotes metastasis of human lung cancer cells in SCID mice via enhanced expression of adhesion-, invasion- and angiogenesis-related molecules. Cancer Sci. 94,244-252[CrossRef][Medline]
  43. Saijo, Y., Tanaka, M., Miki, M., Suzuki, T., Maemondo, M., Hong, X., Tazawa, R., Kikuchi, T., Matsushima, K., Nukiwa, T., et al (2002) Proinflammatory cytokine IL-1ß promotes tumor growth of Lewis lung carcinoma by induction of angiogenic factors: in vivo analysis of tumor-stromal interaction. J. Immunol. 169,469-475[Abstract/Free Full Text]
  44. Salven, P., Hattori, K., Heissig, B., Rafii, S. (2002) Interleukin-1{alpha} promotes angiogenesis in vivo via VEGFR-2 pathway by inducing inflammatory cell VEGF synthesis and secretion. FASEB J. 16,1471-1473[Abstract/Free Full Text]
  45. Majima, M., Hayashi, I., Muramatsu, M., Katada, J., Yamashina, S., Katori, M. (2000) Cyclo-oxygenase-2 enhances basic fibroblast growth factor-induced angiogenesis through induction of vascular endothelial growth factor in rat sponge implants. Br. J. Pharmacol. 130,641-649[CrossRef][Medline]
  46. Cheng, T., Cao, W., Wen, R., Steinberg, R. H., LaVail, M. M. (1998) Prostaglandin E2 induces vascular endothelial growth factor and basic fibroblast growth factor mRNA expression in cultured rat Muller cells. Invest. Ophthalmol. Vis. Sci. 39,581-591[Abstract/Free Full Text]
  47. Graeber, J. E., Glaser, B. M., Setty, B. N., Jerdan, J. A., Walenga, R. W., Stuart, M. J. (1990) 15-Hydroxyeicosatetraenoic acid stimulates migration of human retinal microvessel endothelium in vitro and neovascularization in vivo. Prostaglandins 39,665-673[CrossRef][Medline]
  48. He, H., Venema, V. J., Gu, X., Venema, R. C., Marrero, M. B., Caldwell, R. B. (1999) Vascular endothelial growth factor signals endothelial cell production of nitric oxide and prostacyclin through flk-1/KDR activation of c-Src. J. Biol. Chem. 274,25130-25135[Abstract/Free Full Text]
  49. Daniel, T. O., Liu, H., Morrow, J. D., Crews, B. C., Marnett, L. J. (1999) Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endothelial migration and angiogenesis. Cancer Res. 59,4574-4577[Abstract/Free Full Text]
  50. Coussens, L. M., Werb, Z. (2002) Inflammation and cancer (review). Nature (London) 420,860-867[CrossRef][Medline]
  51. Bol, D. K., Rowley, R. B., Ho, C. P., Pilz, B., Dell, J., Swerdel, M., Kiguchi, K., Muga, S., Klein, R., Fischer, S. M. (2002) Cyclooxygenase-2 overexpression in the skin of transgenic mice results in suppression of tumor development. Cancer Res. 62,2516-2521[Abstract/Free Full Text]
  52. Weidner, N., Semple, J. P., Welch, W. R., Folkman, J. (1991) Tumor angiogenesis and metastasis–correlation in invasive breast carcinoma. N. Engl. J. Med. 324,1-8[Abstract]
  53. Zhang, X., Morham, S. G., Langenbach, R., Young, D. A. (1999) Malignant transformation and antineoplastic actions of nonsteroidal antiinflammatory drugs (NSAIDs) on cyclooxygenase-null embryo fibroblasts. J. Exp. Med. 190,451-459[Abstract/Free Full Text]



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