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• W2012060832 abstract "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 subjected to RNase protection analysis using the human angiogenesis multiprobe set (catalog number 45606P), which contains templates for the following RNA transcripts: FLT1, FLT4, TIE, thrombin receptor, TIE2, CD31, endoglin, angiopoietin, VEGF, and VEGF-C; and the housekeeping genes L32 and glyceraldehyde-3-phosphate dehydrogenase. RNA was also subjected to analysis using the hCK4 template set (catalog number 45034P) which contains templates for the chemokines IL-3, IL-7, GM-CSF, M-CSF, IL-6, SCF, LIF, OSM, and the housekeeping genes L32 and glyceraldehyde-3-phosphate dehydrogenase. The probes were labeled with [α -32P]UTP using T7 RNA polymerase. 3 × 106 cpm of labeled probe was hybridized to 5 μg of total RNA for 16 h at 56 °C. mRNA probe hybrids were treated with RNase mixture and phenol-chloroform extracted. Protected hybrids were resolved on a 6% denaturing polyacrylamide sequencing gel and read on a STORM 860 phosphoimager, and the bands were quantified using ImageQuant software (Molecular Dynamics, Sunnyvale CA). TWEAK, bFGF, or VEGF was mixed with equal volumes of 12% hydron (Sigma). Ten μl of the mixture were pipetted into the tip of a sterile Teflon rod. After drying for 1–2 h, the pellets were stored at 4 °C. A small (approximately 2 mm) incision at 1 mm from the center of the cornea was performed on anesthetized Sprague-Dawley rats. Using a curved iris spatula, an intrastromal pocket was made to a distance of 1 mm from the limbus, the circular blood vessels that surround the cornea. A single pellet was implanted, containing the indicated factors. Antibiotic ointment (Neosporin) was applied post-surgery to the operated eye to prevent infection and decrease inflammation. Seven days later, neovascularization was measured through slitlamp biomicroscopy (Nikon NS-1) connected to an image analysis system (Leica QWin). Angiogenesis was calculated by measuring the area of new blood vessels. This experimental protocol was approved by the institutional animal care and use committee. To determine the concentration range in which TWEAK is biologically active, various concentrations of TWEAK were used to treat HMVEC-d cultures. Cells were grown in serum and growth factor-rich medium and then split into medium with reduced serum and growth factors (Fig.1) with or without TWEAK. Under these conditions, cells that were not treated with TWEAK adhered to the plate but did not proliferate and did begin to die at day 2 to 3. Cells that were treated with TWEAK proliferated and continued to grow over the 5-day period of the assay. Similar results were obtained using direct cell counting as a means of quantifying the effect of TWEAK treatment on cell number. Photographs of cells treated with or without 50 ng/ml TWEAK are shown in Fig. 2, Band C, respectively. Although TWEAK was originally identified as an apoptosis-inducing factor, under these conditions TWEAK reduces the serum and growth factor requirements for HMVEC-d proliferation at a concentration which is consistent with that of other angiogenic factors such as VEGF and bFGF. This is not without precedent because other TNF family members such as FAS ligand can either induce apoptosis or proliferation depending upon experimental conditions (30Alderson M.R. J. Exp. Med. 1993; 178: 2231-2253Crossref PubMed Scopus (527) Google Scholar).Figure 2TWEAK does not increase RNA transcript levels of genes involved in angiogenesis. Five μg of total RNA were subjected to RNase protection analysis using radiolabeled probes for several genes involved in angiogenesis (A) and cytokines (B). Protected fragments were resolved on a 6% polyacrylamide gel. In each panel, lanes labeledA, T, and U indicate samples treated with TNF-α, samples treated with TWEAK, or untreated samples, respectively. Undigested probes are labeled along theleft.View Large Image Figure ViewerDownload (PPT) To further investigate the activity of TWEAK, several other types of primary cells were tested. HUVEC and three types of microvasculature cells (normal human aortic endothelial cells, human brain microvasculature endothelial cells, and HMVEC-d) were tested along with AOSMC and NHDF-neo. As shown in Table I, TWEAK appears to be active on the endothelial cells and smooth muscle cells tested, but no effect was observed on the dermal fibroblasts. Endothelial and smooth muscle cells are both components of vascular tissue and have similar basal growth requirements. They are highly responsive to serum, and respond to some of the same growth factors, such as EGF (31Carpenter G. Wahl M. Sporn I.M.B. Roberts A.B. Peptide Growth Factors and Their Receptors. Springer-Verlag New York Inc., New York1990: 89Google Scholar, 32Carpenter G. Curr. Opin. Cell Biol. 1993; 5: 261-264Crossref PubMed Scopus (28) Google Scholar), bFGF (33Goldfarb M. Cell Growth Differ. 1990; 1: 439-445PubMed Google Scholar), platelet-derived endothelial cell growth factor (34Ishikawa F. Miyazono K. Hellman U. Drexler H. Wernstedt C. Hagiwara K. Usuki K. Takaku F. Risau W. Heldin C.H. Nature. 1989; 338: 557-562Crossref PubMed Scopus (693) Google Scholar, 35Heldin C.H. Usuki K. Miyazono K. J. Cell. Biochem. 1991; 47: 208-210Crossref PubMed Scopus (20) Google Scholar), and scatter factor/hepatocyte growth factor (36Van Belle E. Witzenbichler B. Chen D. Silver M. Chang L. Schwall R. Isner J.M. Circulation. 1998; 97: 381-390Crossref PubMed Scopus (410) Google Scholar). It is, therefore, not surprising that smooth muscle cells might also respond to TWEAK. However, NHDF-neo cells did not show TWEAK-induced proliferation. This may imply that, like VEGF, TWEAK has some specificity as a vascular growth factor (37Thomas K.A. J. Biol. Chem. 1996; 271: 603-606Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar).Table IProliferative effects of TWEAK on various primary cell typesUntreated50 ng/ml TWEAKHAEC6.7 ± 1.715.1 ± 1.5HBE6.8 ± 3.612.0 ± 1.9HMVEC-d5.5 ± 0.623.3 ± 2.6HUVEC−0.4 ± 0.620.9 ± 5.6AOSMC4.2 ± 0.411.8 ± 2.6NHDF-neo20.6 ± 2.022.0 ± 3.5HAEC, human brain microvascular endothelial cells (HBE), HMVEC-d, HUVEC, AOSMC, or NHDF-neo cells were seeded with or without 50 ng/ml TWEAK. Living cells were quantitated 5 days after seeding by metabolism of calcein AM. Open table in a new tab HAEC, human brain microvascular endothelial cells (HBE), HMVEC-d, HUVEC, AOSMC, or NHDF-neo cells were seeded with or without 50 ng/ml TWEAK. Living cells were quantitated 5 days after seeding by metabolism of calcein AM. VEGF is a selective mitogen for endothelial cells. Several other angiogenic factors including platelet-derived growth factor-BB (38Brogi E. Wu T. Namiki A. Isner J.M. Circulation. 1994; 90: 649-652Crossref PubMed Scopus (536) Google Scholar), keratinocyte growth factor (fibroblast growth factor 7), epidermal growth factor, TNF-α (39Frank S. Hubner G. Breier G. Longaker M.T. Greenhalgh D.G. Werner S. J. Biol. Chem. 1995; 270: 12607-12613Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar), transforming growth factor-β1 (38Brogi E. Wu T. Namiki A. Isner J.M. Circulation. 1994; 90: 649-652Crossref PubMed Scopus (536) Google Scholar, 39Frank S. Hubner G. Breier G. Longaker M.T. Greenhalgh D.G. Werner S. J. Biol. Chem. 1995; 270: 12607-12613Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar, 40Pertovaara L. Kaipainen A. Mustonen T. Orpana A. Ferrara N. Saksela O. Alitalo K. J. Biol. Chem. 1994; 269: 6271-6274Abstract Full Text PDF PubMed Google Scholar), IL-1β (41Li J. Perrella M.A. Tsai J.-C. Yet S.-F. Hsieh C.-M. Yoshizumi M. Patterson C. Endege W.O. Zhou F. Lee M.-E. J. Biol. Chem. 1995; 270: 308-312Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), and scatter factor/hepatocyte growth factor (36Van Belle E. Witzenbichler B. Chen D. Silver M. Chang L. Schwall R. Isner J.M. Circulation. 1998; 97: 381-390Crossref PubMed Scopus (410) Google Scholar) have been shown to induce expression of VEGF in a variety of cultured cells, which may account in part for their role in angiogenesis. To test whether TWEAK's effect is mediated through induction of other genes involved in angiogenesis, we subjected 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 probes for various genes involved in angiogenesis. Fig. 2 and Table II show the results of these experiments. Each band was quantified by densitometry and normalized to GAPDH levels (Table II). RNA levels of several of the genes tested were modulated by TNF-α treatment. Cytokines GM-CSF, M-CSF, IL-6, and SCF were up-regulated in TNF-α treated cells. Thrombin receptor transcript levels decreased after TNF-α treatment as described previously (42Conway E.M. Rosenberg R.D. Mol. Cell. Biol. 1988; 8: 5588-5592Crossref PubMed Scopus (334) Google Scholar). TWEAK, however, did not appear to significantly alter transcript levels of several genes involved in angiogenesis such as VEGF, either of the VEGF receptors (flt1/VEGFR or Flt4/VEGFR3), angiopoietin, or it's receptor TIE, TIE2, thrombomodulin, endoglin, or CD31. Furthermore, TWEAK did not affect transcript levels of the cytokines tested with the possible exceptions of IL-6 and OSM.Table IITable represents the angiogenesis (Fig. 2A) and the cytokine panels (Fig. 2B)flt1flt4TIEThrombin receptorTIE2CD31EndoglinAngiopVEGFVEGF-CL32GAPDHAngiogenesisTNF-α0.500.640.560.680.840.640.60ND0.77ND0.851.0TWEAK0.921.091.020.970.901.071.05ND1.06ND0.751.0IL3IL7GM-CSFM-CSFIL6SCFLIFOSML32GAPDHCytokineTNF-αNDND20.6010.124.041.77ND1.781.01.0TWEAKNDND2.471.201.660.97ND1.780.901.0Radiolabeled fragments from Fig. 2 were quantified using a STORM860 PhosphorImager (Molecular Dynamics, Sunnyvale CA). The values were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels and are presented as ratio of experimental sample to untreated. Open table in a new tab Radiolabeled fragments from Fig. 2 were quantified using a STORM860 PhosphorImager (Molecular Dynamics, Sunnyvale CA). The values were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels and are presented as ratio of experimental sample to untreated. To confirm the results of the RNase protection assay, we used VEGF neutralizing antibodies to test whether the proliferative effect of TWEAK is mediated by VEGF. HMVEC cultures were split into reduced serum and growth factor medium supplemented with growth factors and anti-VEGF neutralizing antibodies as indicated. Cell number was quantitated by calcein fluorescence after 5 days. The data in Fig.3 show that while TWEAK and VEGF are both able to stimulate proliferation of HMVEC-d, VEGF neutralizing antibody eliminates VEGF induced proliferation, but does not significantly reduce TWEAK-induced proliferation. The slight decrease in cell number in cultures treated with anti-VEGF antibody and TWEAK as compared with TWEAK alone may reflect the action of endogenous VEGF in the HMVEC sample. The inability of neutralizing antibodies against VEGF to significantly block TWEAK-induced proliferation of HMVEC cells demonstrates that TWEAK-induced proliferation does not require VEGF. Given the proliferative effect of TWEAK on cultured endothelial cells, TWEAK was also tested for its ability to induce angiogenesis in vivoby placing TWEAK-containing pellets in rat corneas and measuring neovascularization after 7 days. Fig.4 A summarizes the effect of pellets coated with the indicated amounts of TWEAK, bFGF, or implanted with vehicle only. These results demonstrate that TWEAK induces neovascularization comparable with that induced by similar concentrations of bFGF. Fig. 4 B shows the results of the analogous experiment comparing TWEAK to VEGF. Again, the ability of the two proteins to induce neovascularization is approximately equivalent. Representative slitlamp micrographs of rat corneas 7 days after implantation of growth factor-free, VEGF-containing, bFGF-containing, and TWEAK-containing pellets are shown in Fig. 4, C,E, D, and F, respectively. The minor winding vessels between the control pellet and the large circular limbus vein surrounding the cornea (Fig. 4 C) are part of the iris and can be distinguished from the straighter vessels of the cornea (Fig. 4, D–F) by their morphology. These data provide clear evidence that TWEAK can promote angiogenesis in vivo. Many TNF ligands are able to induce cytokine production from their target cells (43Beutler B. Cerami A. Annu. Rev. Immunol. 1989; 7: 625-655Crossref PubMed Scopus (1498) Google Scholar). TWEAK has been shown to induce IL-8 secretion in three cell lines, HT29 (colon), A375 (melanoma), and WI-38 (fibroblast) (1Chicheportiche Y. Bourdon P.R. Xu H. Hsu Y.M. Scott H. Hession C. Garcia I. Browning J.L. J. Biol. Chem. 1997; 272: 32401-32410Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar). Therefore, we evaluated whether TWEAK can induce secretion of IL-8, IL-6, and GM-CSF in HUVEC. Although stimulation with 10 ng/ml TNF-α for 28 h increased production of all three cytokines, TWEAK induced only small increases in IL-8 and GM-CSF, and there was no increase in IL-6 (data not shown). In addition, because many members of the TNF ligand family co-stimulate T-cells (3Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1838) Google Scholar, 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), TWEAK was also tested for this activity. Although a strong increase in tritiated thymidine uptake was seen with anti-CD3 treated T-cells co-stimulated with anti-CD28, no increase was seen in cells co-stimulated with TWEAK (data not shown). In conclusion, these data show that TWEAK has a proliferative effect on a variety of endothelial cells and AOSMC in culture. In vivostudies in rat corneas demonstrate that TWEAK is a strong inducer of angiogenesis. Given the importance of the TNF family in many immune responses, and the contribution of vascular endothelium to immune processes such as inflammation, it is not surprising that TNF ligands can affect vascular tissue. However, data presented here show that TWEAK has a more direct effect on angiogenesis and endothelial cell proliferation than has previously been ascribed to any other member of the TNF family. The magnitude of this response is similar to more thoroughly characterized angiogenic factors such as VEGF and bFGF. We thank Connie Faltynek, Ray Goodwin, and Craig Smith for critically reading the manuscript and Alexa Dillberger of Clonetics for excellent technical advice." @default.
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• W2012060832 title "TWEAK Induces Angiogenesis and Proliferation of Endothelial Cells" @default.
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