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. coli, thus LPS contamination could PAR-2-/- segments was lower (Fig. 2C). Thus, the effect of asTF potentially influence sprout formation. However, inclusion of the on angiogenesis is not dependent on VIIa-mediated activation of LPS inhibitor polymyxin B did not affect aortic sprouting induced PAR-2. Importantly, the effect of asTF on sprouting was much by asTF (Fig. S2A), and similar sprouting was observed in a more profound than that observed using sTF, either in wild-type serum-free system (Fig. S2B), a condition that does not allow for aortas or PAR-2-/- aortas (Fig. 2C), suggesting that asTF is the main TF isoform that mediates angiogenesis. efficient LPS signaling (22). asTF Binds Endothelial Integrins. Since flTF was previously shown to asTF-Dependent Angiogenesis Is Not Dependent on PAR-2. Next, we bind integrins, the effect of asTF on angiogenesis may be dependent examined the mechanism behind asTF-induced angiogenesis. Un- on integrin ligation. To study this, tissue culture plates were coated der basal conditions, endothelial cells express low levels of PAR-2, with asTF and blocked with BSA, after which ECRF cells were but these levels go up during angiogenesis (10). flTF in complex seeded. ECRF cells bound time-dependently to asTF-coated plates, with VIIa was previously shown to promote PAR-2-dependent whereas cells bound very poorly to BSA-only treated plates (Fig. angiogenesis, presumably through activation of, among others, the 3A). Cells bound to asTF displayed enhanced phosphorylation of MAP kinase signal transduction pathway. To study the influence of Focal Adhesion Kinase (FAK), p42/p44 MAP kinase, p38 MAP asTF on PAR-2 activation in endothelial cells, we used an endo- kinase, and Akt—events commonly elicited by integrin ligation thelial cell line (ECRF) adenovirally transduced to express PAR-2, (Fig. 3B). Inclusion of polymyxin B did not influence cell adhesion after which these cells were stimulated with asTF, VIIa, or the to asTF (Fig. S4A). Depletion of asTF from our preparation using combination of asTF and VIIa. A truncated soluble form of flTF nickel-NTA abolished cell adhesion, whereas incubation of asTF (sTF) in complex with VIIa and a PAR-2 agonist, SLIGRL, were preparations with non-nickel bound control beads did not prevent used as positive controls. VIIa, asTF, or the combination of asTF cell adhesion (Fig. S4B). Moreover, cell adhesion to asTF was fully
19498 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905325106 van den Berg et al. Downloaded by guest on September 26, 2021 The identity of integrins involved in asTF-enhanced migration was determined using integrin-blocking antibodies. 1, 2, and 4 blocking antibodies, as well as a control antibody, were unsuccessful in blocking migration, but 3 blockade fully inhibited migration (Fig. 4D). An anti-␣v3 antibody (LM609) also blocked asTF- dependent migration, demonstrating that ␣v3 is required for asTF-dependent migration (Fig. 4E). Similarly, ␣v3 blockade inhibited migration of primary HUVECs (Fig. S5A). It is important to note that ␣v3 integrin blockade by LM609 induces apoptosis in endothelial cells (23), which may be responsible for the inhibiting effect on asTF-induced migration, but apoptosis was not observed (Fig. S5B). Polymyxin B did not affect migration, confirming that possible traces of LPS did not contribute to this effect (Fig. 4E). To investigate the signal transduction pathways involved in asTF-dependent migration, we preincubated cells with specific kinase inhibitors. The p38 MAP kinase inhibitor SB203580 and the PI3 kinase inhibitor LY294002, but not the p42/p44 MAP kinase inhibitor, completely abolished migration (Fig. 4F). An Akt inhib- itor only partially inhibited asTF-dependent migration (Fig. 4G), suggesting that PI3-kinase acts through Akt-dependent and inde- pendent pathways.
asTF Enhances Endothelial Capillary Formation. Next, we determined asTF’s potential to support capillary formation by seeding ECRF Fig. 3. asTF binds integrins on endothelial cells. (A) Cells were seeded on cells on matrigel supplemented with asTF or control buffer. asTF BSA- or asTF-coated culture wells and adhered cells were counted at the enhanced the formation of capillaries 2.5-fold (Fig. 5 A and B). indicated times. (B) Cells were left to adhere to BSA- or asTF-coated wells for When asTF was added to the medium, capillary formation was the indicated times, lysed, and the lysates were checked for FAK, p42/22 inhibited, most likely by binding and blocking the integrins required MAPK, p38 MAPK, and c-Akt phosphorylation on Western blot. Total levels of for capillary formation on matrigel. Surprizingly, 1 but not 3 these proteins were assessed to verify equal loading. (C) Culture wells were blockade inhibited capillary formation to subbasal levels in ECRF coated with 50 g/mL asTF or sTF and cell adhesion was assayed in the and primary HUVECs (Fig. 5B and Fig. S6). Apparently, endo- absence/presence of 100 g/mL 6B4. (D) Endothelial cells were preincubated thelial cell migration and capillary formation induced by asTF were with integrin-blocking antibodies and seeded onto BSA- or asTF-coated wells. dependent on different integrin heterodimers. Blockade of ␣3 and Adhered cells were counted. ␣6 integrin subunits that were previously shown to interact with flTF (10) was also tested. Integrin ␣6 blockade inhibited asTF- inhibited in the presence of the anti-TF antibody 6B4, which induced capillary formation to subbasal levels (Fig. 5C). In contrast, ␣ interferes with TF-integrin binding (14) (Fig. 3C). Thus, enhance- 3 blockade modestly inhibited capillary formation, suggesting that ␣ ment of cell adhesion was entirely dependent on the presence of 3 integrins do not play a major role in this process. ␣ asTF. The use of sTF yielded similar results in this assay. Matrigel primarily consists of collagen and laminin, thus 3 and ␣6 may also participate in the attachment of cells to matrigel. To To identify the responsible integrins, ECRF cells were preincu- ␣ ␣ bated with specific blocking antibodies. 2or4 blockade did not verify whether the inhibition of capillary formation by 3 and 6  blockade was due to diminished adhesion to asTF, we tested ␣3 and inhibit cell adhesion to asTF, 3 blockade modestly inhibited cell ␣ ␣ ␣ adhesion, and 1 blockade resulted in a 50% reduction of cell 6 blockade on cell adhesion to asTF. 6, but not 3, blockade partially inhibited cell binding to asTF (Fig. 5D). Cell adhesion was adhesion (Fig. 3D). The combination of 1 and 3 blockade reduced to basal levels upon inclusion of a 3 blocking antibody, reduced cell adhesion to basal levels. Similar results were obtained supporting the conclusion that endothelial cells employ different using a murine endothelial cell line (Fig. S4C), validating the use of integrin heterodimers, namely ␣61 and ␣v3, but only require human asTF in murine models such as the matrigel plug assay and ␣    6 1 integrin for asTF-induced capillary formation. Preincubation the aortic ring assays. In conclusion, both 1 and 3 integrins bind of ECRF cells with SB203580, LY294002 and PD98059 showed that to asTF to mediate cell adhesion. asTF-induced capillary formation was dependent on p42/p44 MAP kinase and PI3 kinase action, but not the promigratory p38 MAP asTF Induces Endothelial Cell Migration. During angiogenesis, en- kinase cascade (Fig. 5E), demonstrating that distinct integrin- MEDICAL SCIENCES dothelial tip cells initially migrate out of the intima and subse- dependent pathways are involved in asTF-dependent migration and quently, endothelial cell proliferation and differentiation result capillary formation. In support of that, cells allowed to form in capillary formation. Using a transwell assay, we assessed capillaries on matrigel supplemented with asTF showed higher whether asTF induces cell migration. asTF induced a 4-fold levels of p42/p44 and Akt, but not p38 phosphorylation. upregulation in ECRF migration when present in the lower Similar to what was observed in aortic sprouting experiments, compartment, and cells appeared rounded (Fig. 4A). When the VIIai, hirudin, and polymyxin B did not influence capillary forma- lower sides of transwells were coated with asTF, a 10-fold tion (Fig. 5 C and E), demonstrating that asTF-dependent capillary upregulation of cell migration was observed while cells displayed formation was not dependent on coagulation activation and/or LPS a flattened morphology. contamination. Pericytes also contribute to angiogenesis. We found that asTF did not facilitate pericyte migration, whereas a mix of extracellular asTF-Enhanced Aortic Sprouting Is Dependent on Integrins. We next matrix proteins optimized for pericyte binding supported potent assessed the relative contributions of 1 and 3 integrins to migration (Fig. 4B). Thus, asTF does not induce pericyte migration, asTF-dependent sprout formation in matrigel, using integrin block- but selectively leads to migration of endothelial cells. Interestingly, ing antibodies. Blockade of 1 inhibited basal sprouting and endothelial migration was reduced on sTF when compared to asTF, asTF-induced sprouting to similar levels (Fig. 6A), suggesting that which may explain the lower angiogenic potential of sTF (Fig. 4C). both matrigel and asTF ligation of integrins was inhibited. Blockade
van den Berg et al. PNAS ͉ November 17, 2009 ͉ vol. 106 ͉ no. 46 ͉ 19499 Downloaded by guest on September 26, 2021 Fig. 4. asTF induces ␣v3-dependent endothelial cell migration. (A) Transwell inserts were coated with BSA (negative control), 50 g/mL asTF, or 1% gelatin (positive control). Endothelial cells were left to migrate for 5 h. Cells were also seeded in uncoated inserts and migration was induced by placing 100 nM asTF into the lower well. Note the rounded-up morphology of cells migrating toward a gradient of asTF versus the flattened morphology of cells migrating through asTF-coated inserts. The graph on the right shows a quantification of these results (n ϭ 9). (B) Pericytes were seeded into asTF-coated inserts or inserts coated with a pericyte extracellular matrix protein mix (Cell Systems) and left to migrate for 5 h. (C) Effect of asTF versus sTF coating on migration of ECRF cells. (D) Endothelial cells were preincubated with  integrin-blocking antibodies and seeded into asTF-coated transwell inserts. Migration was assessed as described above. (E) Endothelial cells were preincubated with 3or␣v3-blocking antibodies. asTF-induced cell migration was assessed as described. Inclusion of PM in the assay did not inhibit migration. (F) Effects of MAPK pathway inhibitor PD98059 (20 M), p38 MAPK inhibitor SB203580 (10 M), or the PI-3 kinase inhibitor LY294002 (10 M) on asTF-induced migration. (G) Effect of c-Akt inhibitor (10 M) on asTF-induced migration.
of 3 did not affect basal sprouting, but reduced asTF-dependent Discussion sprouting to basal levels. This is expected because matrigel contains Here, we show that asTF induces angiogenesis in a matrigel plug relatively little 3-ligating matrix proteins. 6B4, which inhibits assay and aortic sprouting in matrigel and fibrin. asTF induced asTF-integrin interaction, similarly reduced asTF-dependent aortic angiogenesis in a VIIa/PAR-2-independent manner but acted sprouting (Fig. 6B). through ligation of 1 and 3 integrins, at concentrations as low as asTF is found in thrombi and could therefore potentiate angio- 1 nM. asTF-induced endothelial cell migration and capillary for- genesis in a fibrin matrix. asTF dose-dependently upregulated mation was dependent on ␣v3 and ␣61, respectively. Our sprouting in a fibrin milieu, similarly to that observed in matrigel conclusion that asTF binds integrins is based on the following (Fig. 6C); sTF also enhanced sprouting, but at much higher observations: (i) endothelial cell adhesion to asTF as well as concentrations. Inclusion of 6B4 and blockade of 1 and 3 asTF-induced capillary formation and aortic sprouting were po- integrins led to inhibition of asTF-dependent sprouting (Fig. 6D). tently inhibited by integrin blocking antibodies, (ii) inclusion of In contrast to what was observed in matrigel, 3 but not 1 asTF in the medium blocked formation of capillaries, most likely by blockade inhibited sprouting to subbasal levels, reflecting the blocking integrins, and (iii) an antibody that inhibits TF-integrin requirement of 3 integrin activation by fibrin. The asTF/integrin- interaction similarly inhibited sprouting and in vivo angiogenesis. Only the combination of 1 and 3 integrin blocking antibodies blocking antibody 6B4 reduced vessel formation in vivo, and  although sTF modestly induced vessel formation, 6B4 did not have completely abrogated asTF-dependent cell adhesion, thus both 1 and 3 integrin appear to physically bind to asTF. This distinguishes an effect (Fig. 6E). Thus, integrins are instrumental in asTF- asTF from matrigel and fibrin which predominantly activate 1 and dependent sprouting in matrigel and fibrin and in vivo angiogenesis. 3 integrins, respectively. Although the importance of other inte- grin ligands, for example fibronectin and vitronectin, cannot for- Intratumoral asTF Concentrations. To test whether asTF concentra- mally be excluded, they are not the constituents of the tumor tions that induce angiogenesis are pathologically relevant, we extracellular matrix (matrigel) or the provisional angiogenic fibrin assessed intratumoral asTF concentrations in a set of cervical matrix, and therefore play no role in our model. Endothelial cell tumors. We chose cervical cancer since high levels of asTF mRNA migration was dependent on ␣v3 integrin, p38 MAP kinase, and were observed in tumors from seven patients (Fig. 7A). Ten tumor PI3-kinase, whereas capillary formation was dependent on ␣61, specimens were homogenized and analyzed for asTF expression by p42/p44 MAP kinase, and PI3-kinase. It is noteworthy that tip cell Western blot using a specific anti-asTF antibody. Bands were migration and capillary formation represent different stages of quantified using an asTF concentration curve. All but one specimen angiogenesis and involves different integrins (24). Concordantly, revealed asTF concentrations that fell within the range that induces ␣v3 ligation results in directional migration, whereas 1 ligation angiogenesis (Fig. 7B), indicating that the observed asTF-induced induces non-directional migration that is likely to be more impor- angiogenesis is pathobiologically relevant. tant for capillary formation (25). In wound healing, ␣v3 is focally
19500 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905325106 van den Berg et al. Downloaded by guest on September 26, 2021 Fig. 5. asTF enhances ␣61-dependent endothelial capillary formation. (A) Endothelial cells were seeded on matrigel supplemented with solvent control or 100 nM asTF. Photographs were taken after O/N incubation. (B) Cells were Fig. 6. asTF induces integrin-dependent angiogenesis in vivo and ex vivo. (A) seeded on matrigel containing solvent control or asTF. Alternatively, asTF was Wild-type aortic segments were implanted in solvent control-containing ma- added tot the medium containing the cells and the mix was added onto a trigel or asTF-containing matrigel. Murine 1or3-blocking antibodies were matrigel layer without asTF. Cells were also preincubated with  integrin added to the matrigel. The number of sprouts was determined as described. blocking antibodies and seeded onto asTF-containing matrigel. Total capillary (B) Wild-type aortic segments were implanted in solvent control-containing length was quantified. (C) Cells were preincubated with ␣3or␣6-blocking matrigel or matrigel containing 6B4-preincubated (100 g/mL) asTF. (C) Wild- antibodies and seeded onto asTF-containing matrigel. Non-treated cells were type aortic segments in fibrin supplemented with various concentrations of also seeded onto asTF-cotaining matrigel in the presence of VIIai or hirudin. asTF or sTF. (D) Wild-type aortic segments in fibrin Ϯ 100 nM asTF, in the (D) Cells were preincubated with ␣3or␣6-blocking antibodies in the presence presence/absence of 50 g/mL integrin-blocking antibodies or 100 g/mL 6B4. or absence of 3-blocking antibody before seeding of cells onto immobilized (E) In vivo matrigel plug assay; matrigel was supplemented with control buffer asTF. Adhered cells were counted after 4 h. (E) Effect of PD98059, SB203580 or VEGF, asTF or sTF. asTF and sTF were also preincubated with 100 g/mL 6B4 LY294002 on asTF-induced capillary formation. (F) asTF-dependent phosphor- before addition to the matrigel. Vessels were counted as described above. ylation of MAPK, p38, and Akt in capillary-forming endothelial cells. Cells were left to form capillaries for the times indicated on matrigel in the presence/absence of 100 nM asTF. Another important finding is that asTF is a much more potent inducer of angiogenesis that a truncated form of flTF (sTF), probably because asTF is more pro-migratory than sTF, rendering expressed at the tips of invading capillary sprouts whereas 1 extracellular asTF the principal candidate to engage in integrin- integrins are broadly expressed on the entire capillary (26). It appears that asTF-driven migration through ␣v3 integrin mimics dependent angiogenesis, especially because sTF is not known to endothelial tip cell migration, whereas 1-dependent capillary occur naturally in vivo. The underlying basis may be the differential MEDICAL SCIENCES formation through ␣61 recapitulates the formation of vessels from non-tip cell endothelial cells through migration and differentiation. In aortic sprouting assays, which combine these features, asTF- induced angiogenesis was indeed sensitive to both 1 and 3 blockade. flTF on tumor cells has also been shown to induce angiogenesis, but in a mechanistically different manner. Like asTF, flTF interacts with integrins, but rather than serving as receptors to TF, integrin ␣31, and ␣61 control flTF-VIIa signaling via PAR-2 (10). The fact that flTF is membrane bound and binds integrins expressed on the same cell surface, whereas asTF is a soluble protein capable of being deposited into the extracellular matrix; that is, at sites not Fig. 7. Cervical tumors express high levels of asTF. (A) mRNA obtained from accessible to flTF, suggests flTF and asTF may bind integrins in seven cervival tumors was subjected to RT-PCR using asTF- and -actin-specific distinct orientations, thus determining specificity of integrins for primers. (B) Ten cervical tumor specimens were homogenized and lysed in these two TF forms. Our results, showing a lack of asTF-␣31 sample buffer. Lysates were analyzed for asTF expression on Western blot, and integrin interaction, support this concept. asTF bands were quantified using an asTF concentration curve.
van den Berg et al. PNAS ͉ November 17, 2009 ͉ vol. 106 ͉ no. 46 ͉ 19501 Downloaded by guest on September 26, 2021 integrin activation by secreted asTF and membrane bound flTF. Cell Migration Assays. Cell migration was assessed using a transwell assay. Indeed, asTF differs from flTF such that asTF displays a decreased Polycarbonate membrane transwell inserts (8.0 m) (Corning Costar, Corning Life affinity for VII. Sciences, Corning) were coated with 1% gelatin or asTF (50 g/mL). Cells (25,000) were seeded per insert after a 20-min pretreatment with integrin blocking mAb’s, In our experiments, asTF induced angiogenesis at concentrations when appropriate. Cells were allowed to migrate for5hat37°C;migrated cells as low as 1 nM. Interestingly, in a panel of 10 cervical tumors, we were fixed, stained with crystal violet for 10 min, and counted per field of 40ϫ found that nine out of 10 tumors contained asTF at levels in the magnification. range within which asTF is pro-angiogenic, rendering asTF a potential key player in tumor angiogenesis. In agreement, asTF is In Vitro Capillary Formation Assay. Ninety-six-well plates were coated with 50 L expressed in pancreatic cell lines (18), squamous cell carcinoma of matrigel, supplemented with asTF according to the experimental conditions. the lung (27), and advanced stages of non-small cell lung cancer Endothelial cells (20,000) were seeded after a 20-min pretreatment with blocking antibodies when appropriate, allowed to form capillaries for 18 h (ECRF) or 6 h (19); asTF appears to drive tumor growth in xenograft models (20). (HUVECs), and the lengths of tubular networks was measured. It remains controversial whether asTF always displays coagulant activity and can be effectively secreted (16). Regardless of whether Mouse Aortic Ring Assay. Mouse thoracic aortas were isolated and cleaned of the asTF is procoagulant in vivo, we did not observe extensive thrombus surrounding tissue in serum-free RPMI (Invitrogen) containing 50 g/mL penicillin formation in our in vivo experiments using either asTF or sTF, and 50 g/mL streptomycin. Dissected aortas were flushed, cut into equal seg- ruling out that these proteins induce thrombus formation in newly ments, embedded in matrigel, and covered with EBM containing 2% serum and formed vessels. Szotowski et al. (17) showed that asTF can be penicillin/streptomycin. Sprouts were counted on day 5. Aortas were also em- bedded in fibrin that was prepared by mixing 2 mg/mL fibrinogen with 0.1 U/mL secreted after appropriate stimulation and asTF is found in normal thrombin. Aortas were then overlayed with medium containing 5 U/mL hirudin. human plasma, making up approximately 30% of the total blood- borne TF pool (15). Although the cue to asTF secretion in tumors Matrigel Plug Assay. Eight-week-old C57Bl6 mice (n ϭ 10 per group) were remains to be identified, hypoxia and/or tumor-expressed cytokines anesthetized with isoflurane and injected s.c. into the flank with 0.5 mL ice-cold may contribute to this phenomenon. matrigel. Matrigel was either supplemented with 100 nM asTF or sTF in the presence/absence of 100 g/mL 6B4 and 50 ng/mL mouse recombinant VEGF or Materials and Methods PBS. After 7 days, 150 L FITC-Dextran (30 mg/mL) was injected into the tail vein. After 15 min the animals were killed, implants were extracted, fixed in 10% Materials, Cell Lines. Protein Expression, and Survival Assay. Materials, cell lines, formalin, and analyzed on a Leica MZ16 FA stereomicroscope. protein expression and survival assays are described in detail in the SI Text.
Statistics. Data are mean ϩ/- SD unless otherwise stated. Statistical analysis Endothelial Cell Adhesion Assays. Ninety-six-well plates were coated with 50 was performed using Student’s t-test. *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. g/mL asTF or 10% BSA as negative control. Uncoated areas were blocked with 10% BSA. Cells (20,000) were seeded per well and plates were incubated at 37 °C ACKNOWLEDGMENTS. We acknowledge Lars C. Petersen (Novo Nordisk) for the for 4 h. In some experiments, cells were preincubated with 50 g/mL integrin gift of VIIai. H.H.V. is supported by The Netherlands Scientific Organisation (grant blocking antibodies. Photographs were taken and cells adopting a flattened no. 916.76.012). W.R. is supported by the National Institutes of Health (NIH) (grant morphology were counted. Data are shown as mean Ϯ SD, n ϭ 4. nos. HL060742 and HL016411). V.Y.B. is supported by NIH (grant no. HL094891).
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