Alternatively Spliced Tissue Factor Induces Angiogenesis Through Integrin Ligation
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Alternatively spliced tissue factor induces angiogenesis through integrin ligation Y. W. van den Berga, L. G. van den Hengela, H. R. Myersa, O. Ayachia, E. Jordanovab, W. Rufc, C. A. Spekd, P. H. Reitsmaa, V. Y. Bogdanove, and H. H. Versteega,1 aThe Einthoven Laboratory for Experimental Vascular Medicine and bDepartment of Pathology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands; cDepartment of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; dCenter for Experimental and Molecular Medicine, Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands; and eDivision of Hematology/Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 Edited by Charles T. Esmon, Oklahoma Medical Research Foundation, Oklahoma City, OK, and approved September 25, 2009 (received for review May 15, 2009) The initiator of coagulation, full-length tissue factor (flTF), in complex pancreatic cancer cells transfected to express asTF produce more with factor VIIa, influences angiogenesis through PAR-2. Recently, an blood vessels (20), but it remained mechanistically unclear if and alternatively spliced variant of TF (asTF) was discovered, in which part how angiogenesis is regulated by asTF. One possibility is that asTF of the TF extracellular domain, the transmembrane, and cytoplasmic stimulates cancer cells to produce angiogenic factors, but it is also domains are replaced by a unique C terminus. Subcutaneous tumors plausible that asTF enhances angiogenesis via paracrine stimulation produced by asTF-secreting cells revealed increased angiogenesis, but of endothelial cells. Moreover, the role of VIIa, PAR-2 activation it remained unclear if and how angiogenesis is regulated by asTF. and downstream coagulation activation remains obscure. Here, we show that asTF enhances angiogenesis in matrigel plugs in In this study, we report that asTF enhances angiogenesis in vivo, mice, whereas a soluble form of flTF only modestly enhances angio- ex vivo, and in vitro independent of downstream coagulation factors genesis. asTF dose-dependently upregulates angiogenesis ex vivo or activation of PAR-2, but dependent on ␣v3 and ␣61 integrin independent of either PAR-2 or VIIa. Rather, asTF was found to function. ligate integrins, resulting in downstream signaling. asTF-␣V3 inte- grin interaction induces endothelial cell migration, whereas asTF- Results dependent formation of capillaries in vitro is dependent on ␣61 asTF Induces Angiogenesis In Vivo and Ex Vivo. To study a potential integrin. Finally, asTF-dependent aortic sprouting is sensitive to 1 effect of asTF on angiogenesis, we evaluated the properties of and 3 integrin blockade and a TF-antibody that disrupts asTF- recombinant asTF. Purified asTF migrated as a single band of integrin interaction. We conclude that asTF, unlike flTF, does not approximately 26 kDa, corresponding to its predicted molecular affect angiogenesis via PAR-dependent pathways but relies on inte- weight, on SDS/PAGE (Fig. S1A). The preparation reacted with a grin ligation. These findings indicate that asTF may serve as a target monoclonal antibody against the N-terminal part (9C3), but not to prevent pathological angiogenesis. with antibodies against the C-terminal part of full-length TF (flTF) (10H10, 5G9) (Fig. S1B), confirming that the expressed protein was cancer ͉ coagulation ͉ endothelial cells ͉ integrins indeed asTF. Analogous immunoreactivity of these antibodies was observed with asTF expressed in eukaryotic cells (Fig. S1C). he development of blood vessels out of existing vessels, Whether asTF induced angiogenesis was tested in a matrigel plug Ttermed angiogenesis, occurs in embryonic development, assay. Matrigel supplemented with asTF, VEGF, or buffer control wound healing, and cancer (1). Angiogenesis depends on initial was injected into C57BL/6 mice, and angiogenesis was analyzed 7 tip cell migration from the existing vessel, followed by migration days later. Both asTF and VEGF enhanced angiogenesis compared of stalk cells, which divide and form a capillary (2), and the to the buffer control (Fig. 1A), showing that asTF can influence recruitment of pericytes aligning the capillary. Angiogenesis is angiogenesis in the absence of tumor cells. Similar results were regulated by such proteins as vascular endothelial growth factor obtained in an ex vivo aortic sprouting model (21) in which (VEGF), metalloproteinases, and interleukin-8 (1, 3, 4), but also segmented aortas isolated from C57BL/6 mice were implanted in depends on integrins. In particular, 1- and 3-type integrins matrigel containing asTF, VEGF, or control buffer (Fig. 1B). play a role in endothelial and pericyte migration as well as Similar ex vivo experiments revealed that asTF concentration- capillary formation (5, 6). dependently enhanced the formation of sprouts at concentrations Upon vessel damage, tissue factor (TF) together with factor as low as 1 nM (Fig. 1C). Active site-blocked VIIa (VIIai) or VII(a), generates factor Xa, thrombin, and fibrin, yielding a hemo- hirudin, inhibitors of VIIa and thrombin, respectively, did not inhibit asTF-dependent sprouting, indicating that generation of static plug (7). TF is indispensable for life since TF-deficient MEDICAL SCIENCES embryos die in utero (8). Moreover, TF affects tumor angiogenesis coagulation proteases was not required (Fig. 1C). Moreover, adding in colorectal cancer and breast cancer models, through TF:VIIa- asTF in combination with VIIa did not enhance aortic sprouting dependent protease-activated receptor-2 (PAR-2) activation (9– compared to asTF alone (Fig. 1D), supporting our finding that asTF 11), resulting in expression of VEGF, IL-8, MMP-7, and CXCL-1 induced angiogenesis independent of coagulation activation. Pres- (10–13). In addition, TF binds ␣31 and ␣61 integrins, and ence of asTF in the matrigel, but not in the media placed on top of disruption of this complex downregulates pro-angiogenic signaling the matrigel, enhanced angiogenesis, suggesting that asTF’s pres- and suppresses tumor growth in vivo (11, 14). Recently, a TF isoform, asTF, which results from alternative Author contributions: Y.W.v.d.B., P.H.R., and H.H.V. designed research; Y.W.v.d.B., splicing, was discovered, in which the transmembrane and cyto- L.G.v.d.H., H.R.M., O.A., and E.J. performed research; W.R., C.A.S., and V.Y.B. contributed plasmic domains are replaced by a unique 40 amino acid, C- new reagents/analytic tools; Y.W.v.d.B., L.G.v.d.H., H.R.M., O.A., E.J., and H.H.V. analyzed terminal domain, rendering asTF soluble (15). asTF incorporates in data; and Y.W.v.d.B., P.H.R., V.Y.B., and H.H.V. wrote the paper. thrombi and asTF secreted by endothelium exhibits pro-coagulant The authors declare no conflict of interest. activity (16). While some groups failed to detect pro-coagulant This article is a PNAS Direct Submission. activity of asTF in transfected cells (15–17), native asTF can be 1To whom correspondence should be addressed. E-mail: [email protected]. pro-coagulant (16) and is expressed in malignancies such as pan- This article contains supporting information online at www.pnas.org/cgi/content/full/ creatic and lung cancer (18, 19). Hobbs et al. demonstrated that 0905325106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905325106 PNAS ͉ November 17, 2009 ͉ vol. 106 ͉ no. 46 ͉ 19497–19502 Downloaded by guest on September 26, 2021 Fig. 2. asTF does not activate PAR-2. (A) PAR-2-expressing ECRF cells were stimulated with 100 nM asTF Ϯ 100 nM VIIa, VIIa alone, 100 nM truncated flTF (sTF) ϩ VIIa, or SLIGRL (100 M). MAPK phosphorylation was determined by Western blotting. (B) Cells were serum starved and stimulated with asTF Ϯ VIIa, VIIa alone, or 15% FCS. Cell proliferation was assessed using MTT assays. (C) Aortic segments from wild-type or PAR-2-/- C57BL/6 mice were implanted Fig. 1. asTF induces angiogenesis in vivo and ex vivo. (A) Matrigel containing in matrigel supplemented with solvent control, asTF, or sTF, and the number 100 nM asTF, 50 ng/mL VEGF, or buffer control was injected s.c. into mice. of sprouts was determined as described. FITC-dextran was tail vein-injected before sacrifice. Extracted plugs were examined using MZ 16FA stereo microscope and DFC 420C camera (Leica), and the number of invading vessels was counted. The graph shows quantification of this experiment (n ϭ 10 Ϯ SEM) (B) Aortic segments from C57BL/6 mice were and VIIa were not able to induce phosphorylation of MAP kinase, implanted into matrigel supplemented with solvent control, asTF, or VEGF. whereas VIIa in complex with sTF or SLIGRL elicited robust Outgrowing sprouts were visualized and counted on day 5. The graph shows phosphorylation within 10 min (Fig. 2A). Similar results were quantification of this experiment (n ϭ 8 Ϯ SEM). (C) Aortic segments in obtained using primary endothelial cells (HUVECs, Fig. S3). matrigel supplemented with solvent control or various concentrations of asTF. Since angiogenesis is dependent on endothelial cell prolifer- The coagulation inhibitors VIIai (100 nM) or hirudin (500 nM) were included in ation, we also determined whether asTF induced proliferation in some of the conditions. (D) Aortic segments in matrigel supplemented with PAR-2 transduced cells. FCS induced significant cell prolifera- 100 nM VIIa, 100 nM asTF, or the combination of asTF and VIIa. (E) Sprouting tion, but incubation with asTF, VIIa or the combination of asTF occurs in asTF-containing matrigel, but not when media overlying the matri- B gel contains asTF. and VIIa were without effect (Fig. 2 and Fig. S3). In agree- ment, we found that aortic segments isolated from PAR-2-/- mice displayed the same increase in asTF-dependent sprout ence in the extracellular matrix is required (Fig. 1E). Recombinant formation as wild-type segments, although basal sprouting in asTF was produced in E.