Tumor Necrosis Factor-a Upregulates Angiopoietin-2 in Human Umbilical Vein Endothelial Cells Injune Kim, Ji-Hye Kim, Young Shin Ryu, Mingzhu Liu, and Gou Young Koh 1 National Creative Research Initiatives Center for Cardiac Regeneration and Institute of Cardiovascular Research, Chonbuk University School of Medicine, Chonju, Korea Received January 11, 2000 The angiopoietin-Tie2 system is an important regulator of vasculogenesis and vascular integrity. Angiopoietin-2 (Ang2) disrupts blood vessel formation in the developing embryo by antagonizing the effects of angiopoietin-1 (Ang1) on the Tie2 receptor. In this study, we examined the effect of a well-known proin- flammatory cytokine, tumor necrosis factor-a (TNF-a), on Ang2 expression in human umbilical vein endothe- lial cells. Reverse transcriptase-polymerase chain re- action and Northern blot analyses indicated that TNF-a induced Ang2 mRNA expression in a time- and dose-dependent manner. Western blot analyses re- vealed that TNF-a treatment increased cellular Ang2 protein. TNF-a induced less Ang2 mRNA expression in the presence of nuclear factor-kB (NF-kB) inhibitor. These results suggest that TNF-a-induced inflamma- tory angiogenesis might be facilitated by the induc- tion of Ang2. © 2000 Academic Press The recent discovery of Ang1 and Ang2 has provided insight into the molecular and cellular mechanisms of blood vessel formation (1, 2). Ang1 and Ang2 share about 60% amino acid identity and bind with similar affinity to the endothelial cell tyrosine kinase receptor, Tie2 (1, 2). In vivo analysis by targeted gene inactiva- tion reveals that Ang1 recruits and sustains periendo- thelial support cells (3). Overexpression of Ang1 re- sults in increased vascularization (4), whereas over- expression of Ang2 disrupts blood vessel formation in the developing embryo by antagonizing the effects of Ang1 on Tie2 (2). Thus, Ang2 is a naturally occurring antagonist of Ang1 that competes for binding to Tie2 and blocks Ang1-induced Tie2 autophosphorylation (2). While Ang1 mRNA is mainly expressed in periendo- thelial cells, including vascular smooth muscle (1, 3, 5), Ang2 mRNA is selectively expressed in endothelial cells (2). To date, the regulation of Ang1 mRNA expres- sion is an unknown, but we do have some information about the regulation of Ang2. Hypoxia, vascular endo- thelial growth factor (VEGF) and basic fibroblast growth factor upregulate Ang2 expression in endothe- lial cells (6, 7). High levels of Ang2 mRNA are observed in highly vascularized glioblastoma and hepatocellular carcinoma (8, 9). Furthermore, histological analyses indicate that Ang2 expression plays a key role in tumor angiogenesis, both at early and later stages of vascular activation (10). Angiogenesis is also an essential pro- cess during inflammation (11). However, it is unknown whether Ang2 is involved during inflammation. In this study, we examined the effect of a well-known proinflammatory cytokine, TNF-a, on Ang2 expression in human umbilical vein endothelial cells (HUVECs). The present results indicate that TNF-a induces Ang2 expression, at least in part, through intracellular NF-kB activation. Our results suggest that TNF-a- induced Ang2 expression might be involved in inflam- matory angiogenesis. MATERIALS AND METHODS Materials and cell culture. Recombinant human TNF-a was pur- chased from R&D systems (Minneapolis, MN). NF-kB SN50 and NF-kB SN50M were purchased from Calbiochem (La Jolla, CA). Media and sera were obtained from Life Technology, Inc (Gaithers- burg, MD). Most other biochemical reagents were purchased from Sigma (St. Louis, MO), unless otherwise specified. HUVECs were prepared from human umbilical cords by collagenase digestion as described previously (12). The endothelial origin of the cultures was confirmed by immunofluorescent staining with an anti-von Wille- brand factor antibody; acceptable cultures had .95% fluorescent cells. These endothelial cells were maintained in M-199 medium supplemented with 20% (vol/vol) heat-inactivated fetal bovine serum at 37°C in 5% CO 2 . The primary cultured cells used in this study were between passage 2 and 4. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis. Total RNA (100 ng) from HUVECs was suspended in 100 ml of RT buffer (10 mM Tris-HCl, pH 8.3; 50 mM 1 To whom correspondence should be addressed at National Cre- ative Research Initiatives Center for Cardiac Regeneration, Chon- buk University School of Medicine, San 2-20, Keum-Am-Dong, Chonju, 560-180, Republic of Korea. Fax: 82-652-270-4071. E-mail: gykoh@moak.chonbuk.ac.kr. Biochemical and Biophysical Research Communications 269, 361–365 (2000) doi:10.1006/bbrc.2000.2296, available online at http://www.idealibrary.com on 361 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.