TWEAK Induces Angiogenesis and Proliferation of Endothelial Cells*

Carolyn N. LynchDagger , Yi Chun WangDagger , Jennifer K. LundDagger , Yung-Wu ChenDagger , Juan A. LealDagger , and Steven R. Wiley§

From the Dagger  Abbott Laboratories, Abbott Park, Illinois 60064

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

TWEAK is a recently described member of the Tumor 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-gamma 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 Vascular Endothelial 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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The family of TNF1 ligands, with the exception of lymphotoxin-alpha , 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 (3). These activities include T-cell co-stimulation (4-6), apoptosis (7, 8), B-cell proliferation and isotype switching (9, 10), and development of peripheral lymph nodes (11). 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 (12).

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 Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF), also promote proliferation of cultured endothelial cells (13, 14). 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 (15, 16), psoriasis (17), contact dermatitis (18), restenosis (19), and tumor growth (20, 21).

Although the effect of TNF-alpha on cultured endothelial cells is inhibitory or apoptotic (22, 23), TNF also indirectly stimulates angiogenesis by inducing production of angiogenic molecules such as heparin binding epidermal growth factor-like growth factor (24), B.61 (25), platelet-activating factor (26), and nitric oxide (27). 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 (28). 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cell Culture-- Normal human aortic endothelial cells, normal Human Umbilical Vein Endothelial Cells (HUVEC), normal Human Dermal Microvasculature Endothelial Cells (HMVEC-d), Aortic Smooth Muscle Cells (AOSMC), and neonatal Normal Human Dermal Fibroblasts (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.

Proliferation Assays-- 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.

TWEAK Purification-- 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 (29) 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 MgCl2 to 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 on A280 nm measurement.

RNase Protection Assays-- 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-alpha (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 [alpha -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).

Rat Cornea Angiogenesis Assay-- 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.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Titration of Purified TWEAK on HMVEC-d Cells-- 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, B and 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 (30).


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Fig. 1.   Effect of TWEAK titration on endothelial cells. A, HMVEC-d were seeded with the following concentrations of TWEAK: 1.56, 3.12, 6.25, 12.5, 25, 50, 100, 200, 400, and 800 ng/ml. After 5 days, living cells were quantitated by metabolism of calcein AM dye. Control cultures with no TWEAK had a calcein fluorescence of 12.0. Calcein AM-stained cells either treated with 50 ng/ml of TWEAK (B) or untreated (C) were photographed at ×100 magnification.


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Fig. 2.   TWEAK 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 labeled A, T, and U indicate samples treated with TNF-alpha , samples treated with TWEAK, or untreated samples, respectively. Undigested probes are labeled along the left.

Proliferative Activity on Primary Human Cells-- 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 (31, 32), bFGF (33), platelet-derived endothelial cell growth factor (34, 35), and scatter factor/hepatocyte growth factor (36). 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 (37).

                              
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Table I
Proliferative effects of TWEAK on various primary cell types
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.

TWEAK Is Not Acting Indirectly by Inducing VEGF-- VEGF is a selective mitogen for endothelial cells. Several other angiogenic factors including platelet-derived growth factor-BB (38), keratinocyte growth factor (fibroblast growth factor 7), epidermal growth factor, TNF-alpha (39), transforming growth factor-beta 1 (38-40), IL-1beta (41), and scatter factor/hepatocyte growth factor (36) 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-alpha 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-alpha treatment. Cytokines GM-CSF, M-CSF, IL-6, and SCF were up-regulated in TNF-alpha treated cells. Thrombin receptor transcript levels decreased after TNF-alpha treatment as described previously (42). 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.

                              
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Table II
Table represents the angiogenesis (Fig. 2A) and the cytokine panels (Fig. 2B)
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.


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Fig. 3.   TWEAK-induced proliferation does not require VEGF. HUVEC were seeded into media supplemented as indicated with 50 ng/ml VEGF, 50 ng/ml TWEAK, and/or 2.0 mg/ml VEGF neutralizing antibody. Living cells were quantified after 5 days by metabolism of calcein AM dye. Each data point represents the average value over six wells with standard deviation between the wells used to create error bars.

TWEAK Induces Angiogenesis in Rat Corneas-- Given the proliferative effect of TWEAK on cultured endothelial cells, TWEAK was also tested for its ability to induce angiogenesis in vivo by placing TWEAK-containing pellets in rat corneas and measuring neovascularization after 7 days. Fig. 4A 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. 4B 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. 4C) 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.


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Fig. 4.   TWEAK-induced angiogenesis in rat corneas. A, pellets containing the indicated amounts of TWEAK or bFGF were implanted into corneas of Sprague-Dawley rats. Growth factor-free pellets were implanted as controls. Neovascularization as measured through a slitlamp microscope using an image analysis system is expressed in µm2. Each data point represents the average value over six corneas with standard deviation between the corneas used to create error bars. B, same as in panel A but VEGF was mixed in the pellets in place of bFGF. Representative cornea images used to measure neovascularization are shown for a growth factor free pellet (C), a pellet containing 200 ng of VEGF (D), a pellet containing 200 ng of bFGF (E), and a pellet containing 200 ng of TWEAK (F).

TWEAK Does Not Strongly Induce Cytokines or Co-stimulate T Cells-- Many TNF ligands are able to induce cytokine production from their target cells (43). TWEAK has been shown to induce IL-8 secretion in three cell lines, HT29 (colon), A375 (melanoma), and WI-38 (fibroblast) (1). 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-alpha 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 (3-6), 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 vivo studies 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.

    ACKNOWLEDGEMENTS

We thank Connie Faltynek, Ray Goodwin, and Craig Smith for critically reading the manuscript and Alexa Dillberger of Clonetics for excellent technical advice.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Immunex Corp., 51 University St., Seattle, WA. 98101. To whom correspondence should be addressed. Tel.: 206-587-0430, ext. 4670; Fax: 206-233-9733.

    ABBREVIATIONS

The 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.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
  1. 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[Abstract/Free Full Text]
  2. Masters, S. A., Sheridan, J. P., Pitti, R. M., Brush, A. G., and Ashkenazi, A. (1998) Curr. Biol. 8, 525-528[Medline] [Order article via Infotrieve]
  3. Smith, C. A., Farrah, T., and Goodwin, R. G. (1994) Cell 76, 959-962[Medline] [Order article via Infotrieve]
  4. Goodwin, 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., and Smith, C. W. (1993) Eur. J. Immunol. 23, 2631-2641[Medline] [Order article via Infotrieve]
  5. Smith, 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., and Armitage, R. J. (1993) Cell 73, 1349-1360[Medline] [Order article via Infotrieve]
  6. Goodwin, 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., and Beckman, M. P. (1993) Cell 73, 447-456[Medline] [Order article via Infotrieve]
  7. Daniel, P. T., and Krammer, P. H. (1994) J. Immunol. 152, 5624-5632[Abstract/Free Full Text]
  8. Wiley, 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., and Goodwin, R. G. (1995) Immunity 3, 673-682[Medline] [Order article via Infotrieve]
  9. Allen, 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., and Spriggs, M. K. (1993) Science 259, 990-993[Medline] [Order article via Infotrieve]
  10. Alderson, M. R., Tough, T. W., Davis-Smith, T., Braddy, S., Falk, B., Schooley, K. A., Goodwin, R. G., Smith, C. A., Ramsdell, F., and Lynch, D. H. (1995) J. Exp. Med. 181, 71-77[Abstract]
  11. De 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., and Chaplin, D. D. (1994) Science 264, 703-707[Medline] [Order article via Infotrieve]
  12. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., and Williamson, B. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 3666-3670[Abstract]
  13. Rusnati, M., and Presta, M. (1996) Int. J. Clin. Lab. Res. 26, 15-23[Medline] [Order article via Infotrieve]
  14. Bussolino, F., Mantovani, A., and Persico, G. (1997) Trends Biochem. Sci. 22, 251-256[CrossRef][Medline] [Order article via Infotrieve]
  15. Aiello, 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., and Park, J. E. (1994) N. Engl. J. Med. 331, 1480-1487[Abstract/Free Full Text]
  16. Paques, M., Massin, P., and Gaudric, A. (1997) Diabetes Metab. 23, 125-130[Medline] [Order article via Infotrieve]
  17. Detmar, M., Brown, L. F., Claffey, K. P., Yeo, K. T., Kocher, O., Jackman, R. W., Berse, B., and Dvorak, H. F. (1994) J. Exp. Med. 180, 1141-1146[Abstract]
  18. Brown, L. F., Olbricht, S. M., Berse, B., Jackman, R. W., Matsueda, G., Tognazzi, K. A., Manseau, E. J., Dvorak, H. F., and Van de Water, L. (1995) J. Immunology 154, 2801-2807[Abstract/Free Full Text]
  19. Pels, K., Labinaz, M., and O'Brien, E. R. (1997) Jpn. Circ. J. 61, 893-904[CrossRef][Medline] [Order article via Infotrieve]
  20. Bikfalvi, A. (1995) Eur. J. Cancer 31A, 1101-1104[CrossRef]
  21. Battegay, E. J. (1995) J. Mol. Med. 73, 333-346[Medline] [Order article via Infotrieve]
  22. Schweigerer, L., Malerstein, B., and Gospodarowicz, D. (1987) Biochem. Biophys. Res. Commun. 143, 997-1004[Medline] [Order article via Infotrieve]
  23. Robaye, B., Mosselmans, R., Fiers, W., Dumont, J. E., and Galand, P. (1991) Am. J. Pathol. 138, 447-453[Abstract]
  24. Yoshizumi, M., Kourembanas, S., Temizer, D. H., Cambria, R. P., Quertermous, T., and Lee, M. E. (1992) J. Biol. Chem. 267, 9467-9469[Abstract/Free Full Text]
  25. Pandey, A., Shao, H., Marks, R. M., Polverini, P. J., and Dixit, V. M. (1995) Science 268, 567-569[Medline] [Order article via Infotrieve]
  26. Bussolino, F., Albini, A., Camussi, G., Presta, M., Viglietto, G., Ziche, M., and Persico, G. (1996) Eur. J. Cancer. 32A, 2401-2412[CrossRef]
  27. Montrucchio, G., Lupia, E., de Martino, A., Battaglia, E., Arese, M., Tizzani, A., Bussolino, F., and Camussi, G. (1997) Am. J. Pathol. 151, 557-563[Abstract]
  28. Biancone, L., Martino, A. D., Orlandi, V., Conaldi, P. G., Toniolo, A., and Camussi, G. (1997) J. Exp. Med. 186, 147-152[Abstract/Free Full Text]
  29. Hopp, T. P., Prickett, K. S., Price, V. L., Liggy, R. T., March, C. J., Cerretti, D. P., Urdal, D. L,., and Conlon, P. J. (1988) Bio/Technology 6, 1204-1210
  30. Alderson, M. R. (1993) J. Exp. Med. 178, 2231-2253[Abstract]
  31. Carpenter, G., and Wahl, M. (1990) in Peptide Growth Factors and Their Receptors (Sporn, I. M. B., and Roberts, A. B., eds), p. 89, Springer-Verlag New York Inc., New York
  32. Carpenter, G. (1993) Curr. Opin. Cell Biol. 5, 261-264[Medline] [Order article via Infotrieve]
  33. Goldfarb, M. (1990) Cell Growth Differ. 1, 439-445[Medline] [Order article via Infotrieve]
  34. Ishikawa, F., Miyazono, K., Hellman, U., Drexler, H., Wernstedt, C., Hagiwara, K., Usuki, K., Takaku, F., Risau, W., and Heldin, C. H. (1989) Nature 338, 557-562[CrossRef][Medline] [Order article via Infotrieve]
  35. Heldin, C. H., Usuki, K., and Miyazono, K. (1991) J. Cell. Biochem. 47, 208-210[Medline] [Order article via Infotrieve]
  36. Van Belle, E., Witzenbichler, B., Chen, D., Silver, M., Chang, L., Schwall, R., and Isner, J. M. (1998) Circulation 97, 381-390[Abstract/Free Full Text]
  37. Thomas, K. A. (1996) J. Biol. Chem. 271, 603-606[Free Full Text]
  38. Brogi, E., Wu, T., Namiki, A., and Isner, J. M. (1994) Circulation 90, 649-652[Abstract]
  39. Frank, S., Hubner, G., Breier, G., Longaker, M. T., Greenhalgh, D. G., and Werner, S. (1995) J. Biol. Chem. 270, 12607-12613[Abstract/Free Full Text]
  40. Pertovaara, L., Kaipainen, A., Mustonen, T., Orpana, A., Ferrara, N., Saksela, O., and Alitalo, K. (1994) J. Biol. Chem. 269, 6271-6274[Abstract/Free Full Text]
  41. Li, J., Perrella, M. A., Tsai, J.-C., Yet, S.-F., Hsieh, C.-M., Yoshizumi, M., Patterson, C., Endege, W. O., Zhou, F., and Lee, M.-E. (1995) J. Biol. Chem. 270, 308-312[Abstract/Free Full Text]
  42. Conway, E. M., and Rosenberg, R. D. (1988) Mol. Cell. Biol. 8, 5588-5592[Medline] [Order article via Infotrieve]
  43. Beutler, B., and Cerami, A. (1989) Annu. Rev. Immunol 7, 625-655[CrossRef][Medline] [Order article via Infotrieve]


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