TWEAK Induces Angiogenesis and Proliferation of Endothelial Cells

TWEAK is a recently described member of theTumor Necrosis Factor (TNF) ligand family whose transcripts are present in a wide variety of human tissues (Chicheportiche, Y., Bourdon, P. R., Xu, H., Hsu Y. M., Scott, H., Hession, C., Garcia, I., and Browning, J. L. (1997)J. Biol. Chem. 272, 32401–32410). TWEAK is a weak inducer of apoptosis in transformed cells when administered with interferon-γ or cycloheximide (Chicheportiche, Y., Bourdon, P. R., Xu, H., Hsu Y. M., Scott, H., Hession, C., Garcia, I., and Browning, J. L. (1997) J. Biol. Chem. 272, 32401–32410; Masters, S. A., Sheridan, J. P., Pitti, R. M., Brush, A. G., and Ashkenazi, A. (1998) Curr. Biol.8, 525–528) and also promotes IL-8 secretion in cultured cells. We report here that picomolar concentrations of recombinant soluble TWEAK induce proliferation in a variety of normal human endothelial cells and in aortic smooth muscle cells and reduce culture requirements for serum and growth factors. Blocking antibodies to VascularEndothelial Growth Factor (VEGF) do not significantly inhibit TWEAK-induced proliferation, indicating that TWEAK does not function indirectly through up-regulation of VEGF. Pellets containing TWEAK induce a strong angiogenic response when implanted in rat corneas, suggesting a role for TWEAK in vasculature formation in vivo. TWEAK is a recently described member of theTumor Necrosis Factor (TNF) ligand family whose transcripts are present in a wide variety of human tissues (Chicheportiche, Y., Bourdon, P. R., Xu, H., Hsu Y. M., Scott, H., Hession, C., Garcia, I., and Browning, J. L. (1997)J. Biol. Chem. 272, 32401–32410). TWEAK is a weak inducer of apoptosis in transformed cells when administered with interferon-γ or cycloheximide (Chicheportiche, Y., Bourdon, P. R., Xu, H., Hsu Y. M., Scott, H., Hession, C., Garcia, I., and Browning, J. L. (1997) J. Biol. Chem. 272, 32401–32410; Masters, S. A., Sheridan, J. P., Pitti, R. M., Brush, A. G., and Ashkenazi, A. (1998) Curr. Biol.8, 525–528) and also promotes IL-8 secretion in cultured cells. We report here that picomolar concentrations of recombinant soluble TWEAK induce proliferation in a variety of normal human endothelial cells and in aortic smooth muscle cells and reduce culture requirements for serum and growth factors. Blocking antibodies to VascularEndothelial Growth Factor (VEGF) do not significantly inhibit TWEAK-induced proliferation, indicating that TWEAK does not function indirectly through up-regulation of VEGF. Pellets containing TWEAK induce a strong angiogenic response when implanted in rat corneas, suggesting a role for TWEAK in vasculature formation in vivo. The family of TNF 1The abbreviations used are:TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; HUVEC, human umbilical vein endothelial cells; HMVEC-d, normal human dermal microvasculature endothelial cells; AOSMC, aortic smooth muscle cells; NHDF-neo, neonatal normal human dermal fibroblasts; CSF, colony-stimulating factor.ligands, with the exception of lymphotoxin-α, are type II membrane spanning proteins whose extracellular C-terminal domains interact to form oligomeric complexes. These ligands, either presented on cell surfaces or shed to produce soluble molecules, initiate a variety of biological activities by cross-linking cognate members of the parallel family of TNF receptors (2Masters S.A. Sheridan J.P. Pitti R.M. Brush A.G. Ashkenazi A. Curr. Biol. 1998; 8: 525-528Abstract Full Text Full Text PDF PubMed Google Scholar, 3Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1838) Google Scholar). These activities include T-cell co-stimulation (4Goodwin R.G. Din W.S. Davis-Smith T. Anderson D.M. Gimpel S.D. Sato T.A. Maliszewski C.R. Brannan C.I. Copeland N.G. Jenkins N.A. Farrah T. Armitage R.J. Fanslow W.C. Smith C.W. Eur. J. Immunol. 1993; 23: 2631-2641Crossref PubMed Scopus (286) Google Scholar, 5Smith C.A. Gruss H.J. Davis T. Anderson D. Farrah T. Baker E. Sutherland G.R. Brannan C.I. Copeland N.G. Jenkins N.A. Grabstein K.H. Gliniak B. McAlister I.B. Fanslow W. Alderson M. Falk B. Gimpel S. Gillis S. Din W.S. Goodwin R.G. Armitage R.J. Cell. 1993; 73: 1349-1360Abstract Full Text PDF PubMed Scopus (513) Google Scholar, 6Goodwin R.G. Alderson M.R. Smith C.A. Armitage R.J. VandenBos T. Jerzy T.R. Tough T.W. Schoenborn M.A. Davis-Smith T. Hennen K. Falk B. Cosman D. Baker E. Sutherland G.R. Grabstein K.H. Farrah T. Giri J.G. Beckman M.P. Cell. 1993; 73: 447-456Abstract Full Text PDF PubMed Scopus (273) Google Scholar), apoptosis (7Daniel P.T. Krammer P.H. J. Immunol. 1994; 152: 5624-5632PubMed Google Scholar, 8Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.P. Nicholl J.K. Sutherland G.R. Smith T.D. Rauch C. Smith C.A. Goodwin R.G. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2656) Google Scholar), B-cell proliferation and isotype switching (9Allen R.C. Armitage R.J. Conley M.E. Rosenblatt H. Jenkins N.A. Copeland N.G Bedell M.A. Edelhoff S. Disteche C.M. Simoneaux D.K. Fanslow W.C. Belmont J. Spriggs M.K. Science. 1993; 259: 990-993Crossref PubMed Scopus (766) Google Scholar, 10Alderson M.R. Tough T.W. Davis-Smith T. Braddy S. Falk B. Schooley K.A. Goodwin R.G. Smith C.A. Ramsdell F. Lynch D.H. J. Exp. Med. 1995; 181: 71-77Crossref PubMed Scopus (868) Google Scholar), and development of peripheral lymph nodes (11De Togni P. Goellner J. Ruddle N.H. Streeter P.R. Fick A. Mariathasan S. Smith S.C. Carlson R. Shornick L.P. Strauss-Schoenberger J. Russel J.H. Karr R. Chaplin D.D. Science. 1994; 264: 703-707Crossref PubMed Scopus (876) Google Scholar). TNF itself was originally identified as an activity that degrades blood vessels within solid tumors, thereby causing necrotic death by hypoxia, a phenomenon called hemorrhagic necrosis (12Carswell E.A. Old L.J. Kassel R.L. Green S. Fiore N. Williamson B. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3666-3670Crossref PubMed Scopus (3760) Google Scholar). Blood vessels are lined by endothelial cells that play a role in modulation of blood pressure, immune function, and inflammation. Proliferation of endothelial cells is one step in the multi-step process of angiogenesis, the mechanism by which blood vessels are formed. Not surprisingly, major inducers of angiogenesis in vivo, such as Vascular EndothelialGrowth Factor (VEGF) and basicFibroblast Growth Factor (bFGF), also promote proliferation of cultured endothelial cells (13Rusnati M. Presta M. Int. J. Clin. Lab. Res. 1996; 26: 15-23Crossref PubMed Scopus (115) Google Scholar, 14Bussolino F. Mantovani A. Persico G. Trends Biochem. Sci. 1997; 22: 251-256Abstract Full Text PDF PubMed Scopus (417) Google Scholar). Angiogenesis is required for normal biological processes such as development, wound healing, and regrowth of the uterine epithelium after menstruation but also contributes to several conditions such as diabetic retinopathy (15Aiello L.P. Avery R.L. Arrigg P.G. Keyt B.A. Jampel H.D. Shah S.T. Pasquale L.R. Thieme H. Iwamoto M.A. Park J.E. N. Engl. J. Med. 1994; 331: 1480-1487Crossref PubMed Scopus (3407) Google Scholar, 16Paques M. Massin P. Gaudric A. Diabetes Metab. 1997; 23: 125-130PubMed Google Scholar), psoriasis (17Detmar M. Brown L.F. Claffey K.P. Yeo K.T. Kocher O. Jackman R.W. Berse B. Dvorak H.F. J. Exp. Med. 1994; 180: 1141-1146Crossref PubMed Scopus (647) Google Scholar), contact dermatitis (18Brown L.F. Olbricht S.M. Berse B. Jackman R.W. Matsueda G. Tognazzi K.A. Manseau E.J. Dvorak H.F. Van de Water L. J. Immunology. 1995; 154: 2801-2807PubMed Google Scholar), restenosis (19Pels K. Labinaz M. O'Brien E.R. Jpn. Circ. J. 1997; 61: 893-904Crossref PubMed Scopus (23) Google Scholar), and tumor growth (20Bikfalvi A. Eur. J. Cancer. 1995; 31A: 1101-1104Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 21Battegay E.J. J. Mol. Med. 1995; 73: 333-346Crossref PubMed Scopus (497) Google Scholar). Although the effect of TNF-α on cultured endothelial cells is inhibitory or apoptotic (22Schweigerer L. Malerstein B. Gospodarowicz D. Biochem. Biophys. Res. Commun. 1987; 143: 997-1004Crossref PubMed Scopus (74) Google Scholar, 23Robaye B. Mosselmans R. Fiers W. Dumont J.E. Galand P. Am. J. Pathol. 1991; 138: 447-453PubMed Google Scholar), TNF also indirectly stimulates angiogenesis by inducing production of angiogenic molecules such as heparin binding epidermal growth factor-like growth factor (24Yoshizumi M. Kourembanas S. Temizer D.H. Cambria R.P. Quertermous T. Lee M.E. J. Biol. Chem. 1992; 267: 9467-9469Abstract Full Text PDF PubMed Google Scholar), B.61 (25Pandey A. Shao H. Marks R.M. Polverini P.J. Dixit V.M. Science. 1995; 268: 567-569Crossref PubMed Scopus (345) Google Scholar), platelet-activating factor (26Bussolino F. Albini A. Camussi G. Presta M. Viglietto G. Ziche M. Persico G. Eur. J. Cancer. 1996; 32A: 2401-2412Abstract Full Text PDF PubMed Scopus (87) Google Scholar), and nitric oxide (27Montrucchio G. Lupia E. de Martino A. Battaglia E. Arese M. Tizzani A. Bussolino F. Camussi G. Am. J. Pathol. 1997; 151: 557-563PubMed Google Scholar). Recently, agonistic antibodies to FAS, a member of the family of TNF receptors, have been shown to induce capillary formation in vivo, although this effect was heparin dependent, implicating a requirement for heparin-binding growth factors; no direct effects on cultured endothelial cells were reported (28Biancone L. Martino A.D. Orlandi V. Conaldi P.G. Toniolo A. Camussi G. J. Exp. Med. 1997; 186: 147-152Crossref PubMed Scopus (108) Google Scholar). In contrast, data presented here support the hypothesis that a recently discovered TNF ligand family member, TWEAK, is a direct inducer of angiogenesis by the dual criteria that 1) picomolar concentrations of TWEAK promote proliferation of normal endothelial cells in tissue culture and that 2) TWEAK induces angiogenesis in an in vivo rat cornea model with potency similar to bFGF and VEGF. Normal human aortic endothelial cells, normalHuman Umbilical VeinEndothelial Cells (HUVEC), normalHuman Dermal MicrovasculatureEndothelial Cells (HMVEC-d), AorticSmooth Muscle Cells (AOSMC), and neonatal Normal Human DermalFibroblasts (NHDF-neo) were obtained from Clonetics Corp. (San Diego, CA). Normal human brain microvasculature endothelial cells were obtained from Applied Cell Biology Research Institute (Kirkland, WA). Basal media and growth factor supplements were purchased from Clonetics Corp. and used as recommended by the manufacturer. All endothelial cells, except HMVEC-d, were grown in endothelial base medium containing the following growth factors and supplements: bovine brain extract, hydrocortisone, and 2% fetal bovine serum. HMVEC-d were cultured in endothelial base-2 medium containing the following growth factors and supplements: hydrocortisone, bFGF, VEGF, R3-IGF-1, ascorbic acid, heparin, and EGF with 10% fetal bovine serum. AOSMC were grown in smooth muscle base medium containing bFGF, EGF, dexamethasone, and 5% fetal bovine serum, and NHDF-neo were grown in fibroblast base medium containing insulin, FGF, and 2% serum. Cells were trypsinized and seeded onto 96-well plates at a density of 1500 cells per well into medium with reduced serum and growth factors. Media for endothelial cells, smooth muscle cells, and fibroblasts were, respectively, endothelial base medium with bovine brain extract and 1% serum, smooth muscle base medium 3 with 0.5% FGF and 0.5% serum, and fibroblast base medium with FGF and 1% serum. Indicated factors were added at the time of cell seeding. Rabbit anti-human VEGF neutralizing antibodies were purchased from Research Diagnostics (Flanders, NJ) and incubated with the indicated factors 30 min prior to cell seeding. After incubation at 37 °C with 5% CO2 for 5 days in a humidified chamber, cell density was determined by replacing the medium with 100 μl of 0.4 μm calcein AM (Molecular Probes, Eugene OR) in medium lacking serum. Diesterase activity was measured by fluorescence in a cytofluor 2300 system (Millipore, Bedford MA) using an excitation wavelength of 485 nm and emission wavelength of 530 nm. Unless otherwise indicated, each data point represents the average value over four wells with standard deviation between the wells used to create error bars. Photographs were taken at ×100 magnification on a microscope with a mercury light source filtered through an excitation filter of 450–490 nm with 520 nm emission. Soluble TWEAK protein was engineered as follows. The leader sequence from the UL4 protein of cytomegalovirus (amino acids 1–27) followed by a synthetic octapeptide FLAG epitope (29Hopp T.P. Prickett K.S. Price V.L. Liggy R.T. March C.J. Cerretti D.P. Urdal D.L,. Conlon P.J. Bio/Technology. 1988; 6: 1204-1210Crossref Scopus (753) Google Scholar) and the extracellular domain of human TWEAK (amino acids 98–249) were placed in the pcDNA3 expression plasmid multiple cloning site (Invitrogen, Carlsbad, CA). This construct was used to create a stably expressing clone in Chinese hamster ovary cells by selection with G418 from Life Technologies, Inc. (Grand Island, NY). 500 ml of conditioned medium from this clone was incubated with 500 μl of M2 anti-FLAG-agarose beads (Kodak, Rochester, NY) overnight at 4 °C on a rotator wheel. Beads were harvested by centrifugation and washed several times with phosphate-buffered saline containing 2 mm MgCl2. TWEAK was eluted in 1 ml of 1 mm FLAG peptide (Kodak, Rochester, NY) and dialyzed against phosphate-buffered saline containing 2 mm MgCl2to remove FLAG peptide. The apparent molecular mass of the resulting protein as calculated by SDS-PAGE analysis was approximately 24 kDa, which is somewhat larger than the predicted molecular mass of 18 kDa after cleavage of the signal peptide. The concentration of the purified protein was estimated to be 500 μg/ml based onA 280 nm measurement. RNase protection assays were performed by PharMingen (San Diego, CA) using their RiboQuant Multi-Probe RNase protection assay system. Total RNA was isolated from HUVEC treated with 50 ng/ml TNF-α (Collaborative Biomedical, Bedford MA), 50 ng/ml soluble TWEAK, or left untreated for 9 h with RNAeasy (Qiagen, Chatsworth, CA). RNA samples were to RNase protection analysis using the human angiogenesis which for the following RNA VEGF, and and the and RNA was also to analysis using the which for the and the and The were with using RNA 3 of was to 5 of RNA for h at were treated with RNase and were on a and on a and the were using (Molecular CA). TWEAK, bFGF, or VEGF was with of μl of the were into the of a After for the were at 4 2 at 1 mm from the of the cornea was performed on a an was to a of 1 mm from the the blood vessels that the was containing the indicated factors. was to the to and inflammation. days was measured through to an analysis system Angiogenesis was calculated by the of blood This was by the and the concentration in which TWEAK is concentrations of TWEAK were used to HMVEC-d Cells were grown in serum and growth medium and into medium with reduced serum and growth factors with or cells that were not treated with TWEAK to the but not and to at 2 to Cells that were treated with TWEAK and to over the of the were obtained using direct cell as a of the effect of TWEAK on cell Photographs of cells treated with or 50 ng/ml TWEAK are shown in Although TWEAK was originally identified as an conditions TWEAK the serum and growth factor requirements for HMVEC-d proliferation at a concentration which is with that of angiogenic factors such as VEGF and This is not TNF family members such as ligand either induce apoptosis or proliferation conditions M.R. J. Exp. Med. 1993; PubMed Scopus Google does not RNA of in of RNA were to RNase protection analysis using for several in angiogenesis and were on a In each and samples treated with samples treated with TWEAK, or untreated are the activity of TWEAK, several of cells were HUVEC and of microvasculature cells human aortic endothelial cells, human brain microvasculature endothelial cells, and were with AOSMC and shown in TWEAK to be on the endothelial cells and smooth muscle cells but no effect was on the dermal and smooth muscle cells are of vascular tissue and have similar growth are to serum, and to of the growth such as EGF G. M. Growth and Scholar, G. Curr. Cell Biol. 1993; PubMed Scopus Google Scholar), bFGF M. Cell Growth Google Scholar), endothelial cell growth factor F. K. U. H. C. K. K. F. W. PubMed Scopus Google Scholar, K. K. J. Cell. Biochem. 1991; PubMed Scopus Google Scholar), and growth factor E. B. D. M. L. R. 1998; PubMed Scopus Google Scholar). not that smooth muscle cells also to NHDF-neo cells not TWEAK-induced This VEGF, TWEAK as a vascular growth factor K.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google effects of TWEAK on cell ng/ml human brain endothelial cells HMVEC-d, HUVEC, AOSMC, or NHDF-neo cells were seeded with or 50 ng/ml cells were 5 days after by of calcein in a human brain endothelial cells HMVEC-d, HUVEC, AOSMC, or NHDF-neo cells were seeded with or 50 ng/ml cells were 5 days after by of calcein VEGF is a for endothelial cells. angiogenic factors growth E. T. A. 1994; PubMed Scopus Google Scholar), growth factor growth factor epidermal growth TNF-α S. G. G. S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), growth E. T. A. 1994; PubMed Scopus Google Scholar, S. G. G. S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, L. A. T. A. N. O. K. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), J. M.A. M. C. F. Lee J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), and growth factor E. B. D. M. L. R. 1998; PubMed Scopus Google Scholar) have been shown to induce expression of VEGF in a variety of cultured cells, which in for their role in effect is through of in angiogenesis, RNA from HUVEC treated for 9 h with 50 ng/ml of TWEAK, 50 ng/ml of TNF-α or untreated to RNase protection analysis with for in 2 and II the of was by and to RNA of several of the were by TNF-α and were in TNF-α treated cells. after TNF-α as described Mol. Cell. Biol. 1988; 8: PubMed Scopus Google Scholar). TWEAK, not to significantly of several in angiogenesis such as VEGF, either of the VEGF receptors or or or TWEAK not of the with the of and represents the angiogenesis and the from 2 were using a (Molecular CA). The were against and are presented as of to in a from 2 were using a (Molecular CA). The were against and are presented as of to the of the RNase protection used VEGF neutralizing antibodies to the effect of TWEAK is by VEGF. were into reduced serum and growth factor medium with growth factors and neutralizing antibodies as Cell was by calcein fluorescence after 5 The data in that TWEAK and VEGF are to proliferation of HMVEC-d, VEGF neutralizing VEGF proliferation, but does not significantly reduce TWEAK-induced The in cell in treated with and TWEAK as with TWEAK the of VEGF in the The of neutralizing antibodies against VEGF to significantly TWEAK-induced proliferation of cells that TWEAK-induced proliferation does not VEGF. the effect of TWEAK on cultured endothelial cells, TWEAK was also for to induce angiogenesis in in rat and after the effect of with the indicated of TWEAK, bFGF, or implanted with These that TWEAK induces with that by similar concentrations of 4 the of the TWEAK to VEGF. the of the proteins to induce is approximately of rat days after of growth and are shown in and The vessels between the and the vein the cornea 4 are of the and be from the vessels of the cornea by their These data that TWEAK promote angiogenesis in vivo. TNF are to induce production from their cells B. A. Immunol. PubMed Scopus Google Scholar). TWEAK been shown to induce IL-8 secretion in cell and Y. P.R. H. Hsu H. C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). TWEAK induce secretion of and in Although with ng/ml TNF-α for h production of TWEAK in IL-8 and and was no in not In members of the TNF ligand family C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1838) Google Scholar, R.G. Din W.S. Davis-Smith T. Anderson D.M. Gimpel S.D. Sato T.A. Maliszewski C.R. Brannan C.I. Copeland N.G. Jenkins N.A. Farrah T. Armitage R.J. Fanslow W.C. Smith C.W. Eur. J. Immunol. 1993; 23: 2631-2641Crossref PubMed Scopus (286) Google Scholar, 5Smith C.A. Gruss H.J. Davis T. Anderson D. Farrah T. Baker E. Sutherland G.R. Brannan C.I. Copeland N.G. Jenkins N.A. Grabstein K.H. Gliniak B. McAlister I.B. Fanslow W. Alderson M. Falk B. Gimpel S. Gillis S. Din W.S. Goodwin R.G. Armitage R.J. Cell. 1993; 73: 1349-1360Abstract Full Text PDF PubMed Scopus (513) Google Scholar, 6Goodwin R.G. Alderson M.R. Smith C.A. Armitage R.J. VandenBos T. Jerzy T.R. Tough T.W. Schoenborn M.A. Davis-Smith T. Hennen K. Falk B. Cosman D. Baker E. Sutherland G.R. Grabstein K.H. Farrah T. Giri J.G. Beckman M.P. Cell. 1993; 73: 447-456Abstract Full Text PDF PubMed Scopus (273) Google Scholar), TWEAK was also for this Although a strong in was with treated with no was in cells with TWEAK not In data that TWEAK a effect on a variety of endothelial cells and AOSMC in In in rat that TWEAK is a strong inducer of the of the TNF family in immune and the of vascular to immune processes such as is not that TNF vascular data presented here that TWEAK a direct effect on angiogenesis and endothelial cell proliferation than been to member of the TNF The of this response is similar to angiogenic factors such as VEGF and We and Smith for the and of Clonetics for