[CANCER RESEARCH 59, 4574 – 4577, September 15, 1999] Advances in Brief Thromboxane A 2 Is a Mediator of Cyclooxygenase-2-dependent Endothelial Migration and Angiogenesis 1 Thomas O. Daniel, 2 Hua Liu, Jason D. Morrow, Brenda C. Crews, and Lawrence J. Marnett Departments of Medicine, Cell Biology, Biochemistry, and Pharmacology, Divisions of Nephrology and Hypertension and Clinical Pharmacology, The Vanderbilt Center for Vascular Biology, Vanderbilt Cancer Center, Vanderbilt University, Nashville, Tennessee 37232 Abstract Cyclooxygenase-2 (COX-2) inhibitors reduce angiogenic responses to a variety of stimuli, suggesting that products of COX-2 may mediate critical steps. Here, we show that thromboxane A 2 (TXA 2 ) is one of several eicosanoid products generated by activated human microvascular endo- thelial cells. Selective COX-2 antagonists inhibit TXA 2 production, endo- thelial migration, and fibroblast growth factor-induced corneal angiogen- esis. Endothelial migration and corneal angiogenesis are similarly inhibited by a TXA 2 receptor antagonist, SQ29548. A TXA 2 agonist, U46619, reconstitutes both migration and angiogenesis responses under COX-2-inhibited conditions. These findings identify TXA 2 as a COX-2 product that functions as a critical intermediary of angiogenesis. Introduction Recent evidence suggests that COX-2 3 metabolic products contrib- ute to neovascularization and may support vasculature-dependent solid tumor growth and metastasis. Selective COX-2 inhibitors are antiangiogenic (1), and COX-2-null mice are substantially protected in a genetic model of human familial adenomatous polyposis (2). Forced COX-2 overexpression enhances the metastatic potential of CaCo-2 colon carcinoma cells through processes that are sensitive to COX-2 inhibitors (3). Coculture of endothelial cells with tumor cells promotes COX-2-dependent endothelial motility and assembly into capillary-like structures (4), an effect that is attributed to tumor cell release of angiogenic peptides and nitric oxide. Alternatively, eico- sanoids synthesized by endothelial COX-2 may contribute to this effect. COX-2 expression or function is induced in cultured endothelial cells in response to phorbol esters (5, 6), basic FGF (7), hypoxia (8), cyclic strain (9), thrombin, interleukin 1 (10), or interleukin 1 (11). Hypoxia (12) or lipopolysaccharide administration (13) induce micro- vascular endothelial COX-2 expression in situ. Moreover, COX-2 inhibitors have been shown to decrease urinary excretion of prosta- cyclin, a major product of vascular endothelium in human subjects (14). These findings motivated our efforts to identify a COX-2 prod- uct or products that are capable of functioning as intermediaries of angiogenesis. Materials and Methods Eicosanoids and Quantitation. All of the eicosanoids and eicosanoid agonists were purchased from Cayman Chemical, (Ann Arbor, MI). Eico- sanoids were quantified by gas chromatographic negative ion chemical ioni- zation mass spectrometric assays using the precise and accurate stable isotope dilution technique (15). Endothelial Migration. Confluent human renal microvascular endothelial cells were grown to confluency and serum-depleted in medium containing 1% (w/v) bovine albumin for 18 h prior to assay (16). Triplicate circular “wounds” (600 –900 m in diameter) were generated in confluent endothelial monolayers within a single well, using a rotating silicon-tipped drill bit mounted on a drill press, to avoid scoring subjacent surfaces. Medium was supplemented at the time of wounding with test agents at concentrations indicated in the figures. Residual fractional wound areas were measured using a Bioquant (Nashville, TN) software package calibrated to a Nikon Diaphot microscope. Mean frac- tional residual areas of three wounds, calculated at each of two or three time points (see Fig. 1b), were used to derive linear regressions, reflecting migration rates (expressed as percentage closure per h  95% confidence intervals). Mouse Corneal Angiogenesis Assay. Hydron pellets incorporating sucral- fate with vehicle alone, basic FGF (a kind gift from Scios, Inc.), or bFGF in combination with other agents indicated in the figure legends were surgically implanted into corneal stromal micropockets, created 1 mm medial to the lateral corneal limbus of C57BL mice (7–10 weeks old), as described previ- ously (17). On day 5, corneas were photographed at an incipient angle of 35–50° from the polar axis in the meridian containing the pellet, using a Zeiss slit lamp. Images were digitalized and processed by subtractive color filters (Adobe Photoshop Version 4.0), as displayed (see Figs. 3 and 4). Images were analyzed using Bioquant image analysis software to determine the fraction of the two-dimensional total corneal image that was vascularized and the fraction of pixels within that area (regional density) or within the corneal perimeter (total density) that exceeded a threshold matching visible capillaries. The dose of VU08 used (5 mg/kg) was selected based on anti-inflammatory responses in a carrageenan foot pad assay and on effects of this dose to suppress TXA 2 production by endogenous COX activity in prostate tumor tissue issue (85% inhibition at 3 days daily i.p. injection). Concentrations of SQ29548 were selected based on a dose-response experiment optimizing for inhibition of FGF-induced angiogenesis (data not shown). Results and Discussion Fig. 1a provides the profile of eicosanoids produced by cultured microvascular endothelial cells under COX-2-induced conditions, fol- lowing stimulation by PMA. Prostaglandin E 2 , TXA 2 (measured indirectly as its thromboxane B 2 metabolite), and prostaglandin F-2 were the dominant PMA-induced products, and induced endothelial production of each was blocked by coincident exposure to a COX-2- selective inhibitor. Notably, basal endothelial capacity to produce these metabolites was maintained in the presence of COX-2 inhibi- tion, consistent with production by constitutive endothelial COX-1. To assess functional consequences of endothelial COX-2 inhibition, we evaluated endothelial motility. The rates at which endothelial cell migration closed replicate circular wounds in confluent monolayers were determined by quantitating residual wound areas in digital images that were captured at multiple points during a 12-h time Received 5/21/99; accepted 8/2/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by USPHS Awards RO1 DK38517 and P50 DK39261(to T. O. D.), CA47479 (to L. J. M.), and DK48831, GM42056, DK26657, and GM15431 (to J. D. M.) as well as a by a center grant from the National Cancer Institute (Grant CA68485) . J. D. M. is the recipient of a Burroughs-Wellcome Fund Clinical Scientist Award in Translational Research. The T. J. Martell Foundation provided support critical to this work. 2 To whom requests for reprints should be addressed, at Division of Nephrology, MCN S3223, Vanderbilt University Medical Center, Nashville, TN 37232-2372. Phone: (615) 343-8496; Fax: (615) 343-7156; E-mail: tom.daniel@mcmail.vanderbilt.edu. 3 The abbreviations used are: COX-2, cyclooxygenase-2; FGF, fibroblast growth factor; bFGF, basic FGF; TXA 2 , thromboxane A 2 ; PMA, phorbol myristate acetate. 4574 Research. on December 8, 2015. © 1999 American Association for Cancer cancerres.aacrjournals.org Downloaded from