IntroductionThe angiopoietin/Tie receptor system, which consists of 4 angiopoietin (Ang) ligands and the Tie-1 and Tie-2 receptors, plays a nonredundant role in embryonic vascularization and tumor angiogenesis. 1,2 An autocrine Ang-1/Tie-2 axis modulates blood vessel plasticity and contributes to vascular maintenance. Accordingly, Ang-1 enhances survival, migration, and network formation of endothelial cells (ECs) in vitro 3 and induces neovascularization in vivo. 4,5 Drm, a member the Dan family of bone morphogenic protein (BMP) antagonists, 6,7 interacts with signaling EC receptors in a BMP-independent manner. 8 The productive interaction stimulates EC sprouting and migration in vitro and induces angiogenesis in vivo. 8 Drm is overexpressed in human cancers by tumor and stromal cells, thus suggesting that Drm may contribute to tumor neovascularization. [8][9][10] Here, we investigated the role of the Ang/Tie-2 system in mediating the angiogenic activity of Drm. The results demonstrate that Drm up-regulates Ang-1 expression in EC via activation of the transcription factor NF-B. This leads to an autocrine loop of stimulation that mediates Drm-induced EC sprouting in vitro and angiogenesis in vivo.
Methods
Cell culturesApproval for these studies was obtained from the Ethical Committee of the School of Medicine of the University of Brescia. Subcutaneous murine microvascular EC (SIE cells) and vascular endothelial growth factor (VEGF) receptor-2-transduced murine aortic EC (VEGFR2-MAE cells) were cultured in DMEM supplemented with 10% calf serum (Invitrogen, Carlsbad, CA). Human umbilical vein ECs (HUVECs) were grown as described. 11 ECs were transfected (70%-80% efficiency) with siRNAs using the SiRNA Transfection Reagent (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer's instructions.
Western blottingWestern blotting was performed according to standard procedures on the conditioned media (0.5 mL), total cell lysates (20 g), nuclear extracts (5 g), and cytoplasmic fractions (10 g) from serum-starved EC cultures stimulated with 50 ng/mL of recombinant Drm (rDrm, R&D Systems, Minneapolis, MN).
Electrophoretic mobility shift assayNuclear extracts (2 g) were incubated with biotin-labeled NF-B doublestranded DNA oligonucleotide probe (5Ј-AGTTGAGGGGACTTTCCCAGGC) according to LightShift Chemiluminescent EMSA Kit instructions (Pierce, Rockford, IL) and analyzed on a native 6% polyacrylamide gel.
EC sproutingFibrin gel and type I collagen gel invasion assays were performed on VEGFR2-MAE and HUVEC cell aggregates, respectively. 12,13 In addition, HUVEC cell aggregates were immunostained with anti-Ang-1 antibodies (Santa Cruz Biotechnology) and analyzed under a confocal laser microscope.
Artery ring assayOne-millimeter human umbilical artery rings were embedded in fibrin gel 14 and cultured in human EC medium SFM (Invitrogen) with different stimuli. After 3 days, EC sprouts were counted under an inverted microscope at ϫ10 magnification.
Gene expression analysiscDNA was generated from total RNA using t...