TWEAK Induces Angiogenesis and Proliferation of Endothelial
Cells*
Carolyn N.
Lynch
,
Yi Chun
Wang
,
Jennifer K.
Lund
,
Yung-Wu
Chen
,
Juan A.
Leal
, and
Steven R.
Wiley§
From the
Abbott Laboratories,
Abbott Park, Illinois 60064
 |
ABSTRACT |
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-
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 |
The family of TNF1
ligands, with the exception of lymphotoxin-
, 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-
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 |
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-
(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
[
-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 |
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- , 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-
(39), transforming growth factor-
1 (38-40), IL-1
(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-
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-
treatment.
Cytokines GM-CSF, M-CSF, IL-6, and SCF were up-regulated in TNF-
treated cells. Thrombin receptor transcript levels decreased after
TNF-
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.
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|
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-
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.
 |
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