* Department of Microbiology-Immunology and Robert H. Lurie Cancer Center, Northwestern University Medical School,
Chicago, Illinois 60611; Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St.
Louis, Missouri 63110; and § Department of Medicine, Division of Hematology-Oncology, Cornell University Medical College,
New York 10021
Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that is able to make
normal endothelial cells unresponsive to a wide variety
of inducers. Here we use both native TSP-1 and small
antiangiogenic peptides derived from it to show that
this inhibition is mediated by CD36, a transmembrane
glycoprotein found on microvascular endothelial cells.
Both IgG antibodies against CD36 and glutathione-S-transferase-CD36 fusion proteins that contain the TSP-1
binding site blocked the ability of intact TSP-1 and its
active peptides to inhibit the migration of cultured microvascular endothelial cells. In addition, antiangiogenic TSP-1 peptides inhibited the binding of native
TSP-1 to solid phase CD36 and its fusion proteins, as
well as to CD36-expressing cells. Additional molecules
known to bind CD36, including the IgM anti-CD36 antibody SM, oxidized (but not unoxidized) low density
lipoprotein, and human collagen 1, mimicked TSP-1 by
inhibiting the migration of human microvascular endothelial cells. Transfection of CD36-deficient human umbilical vein endothelial cells with a CD36 expression plasmid caused them to become sensitive to TSP-1 inhibition of their migration and tube formation. This work
demonstrates that endothelial CD36, previously
thought to be involved only in adhesion and scavenging
activities, may be essential for the inhibition of angiogenesis by thrombospondin-1.
THROMBOSPONDIN-1 (TSP-1)1 is one of a small number of molecules found naturally in vertebrates that
can inhibit angiogenesis, the sprouting of new blood
vessels from preexisting ones (Bouck et al., 1996 TSP-1 is an extremely potent inhibitor of angiogenesis.
It is effective at subnanomolar concentrations in vitro and
able to prevent neovascularization in vivo in the rodent
cornea and polyvinyl sponge assays (Good et al., 1990 Soluble TSP-1 inhibits angiogenesis by interacting directly with endothelial cells. At concentrations below 20 nM, which is about forty times that seen in normal plasma
(Saglio and Slayter, 1982 TSP-1 can render endothelial cells refractory to a wide
variety of inducers from prostaglandins to VEGF (Volpert
et al., 1995 CD36, also called glycoprotein IV, is an 88-kD transmembrane glycoprotein with a large extracellular domain
(Greenwalt et al., 1992 Reagents
FA6-152 monoclonal antibody against CD36 was purchased from Immunotech (Westbrook, ME), and SM Purified human platelet TSP-1 was kindly provided by Jack Lawler
(Harvard University, Cambridge, MA). TSP-1 peptides were synthesized,
purified, and dialyzed as previously described (Tolsma et al., 1993
Preparation of Glutathione-S-transferase-CD36
Fusion Proteins
CD36-glutathione-S-transferase (GST) recombinant fusion proteins
spanning ~98% of the human CD36 sequence were previously generated
in a bacterial expression system (Pearce et al., 1995 Binding Studies
Cell binding assays were performed with Bowes melanoma cells stably
transfected with human CD36 cDNA, or with a control plasmid, prepared
and maintained as previously described (Silverstein et al., 1992 Solid phase binding assays were used to measure TSP-1 interactions
with CD36 or CD36-GST fusion proteins in the presence of TSP-1 peptides. CD36 or CD36-GST fusion protein was immobilized on wells in a
detachable 96-microwell plate by overnight incubation at 4°C. CD36 was
adsorbed at 4 µg/ml in PBS, while fusion proteins were adsorbed at 10 µg/
ml in carbonate buffer (100 mM NaHCO3, 1 mM Mg Cl2, 0.02% NaN3, pH
9.8). Total protein coating the wells ranged from 200-280 ng. Wells were
washed three times with 20 mM Tris, 150 mM NaCl, pH 7.4, containing
0.05% Tween-20 (TBS-T) and then blocked with TBS-T containing 0.5%
BSA. Radiolabeled TSP-1 (20 µg/ml) along with two concentrations of
peptide (at the Kd and five times the Kd as predicted by the cell binding studies) were added in TBS-T, and the mixture was incubated for 2 h at
22°C. Wells were then washed thoroughly three to four times with TBS-T
and dried, and bound radioactivity was measured by gamma counting.
The addition of 5 mM EDTA was used to determine nonspecific binding.
Migration Assays
Bovine adrenal capillary endothelial cells (BCECs), kindly provided by
Judah Folkman, were grown in DME with 10% donor calf serum (Flow
Laboratories, McLean, VA) and 1% endothelial cell mitogen (Biomedical
Technologies, Inc., Stoughton, MA) and used at passage 15. Human dermal microvascular endothelial cells (HMVECs) were purchased from
Clonetics (San Diego, CA), grown in Clonetics' endothelial cell growth
media, and used between passages 6 and 9. Human umbilical vein endothelial cells (HUVECs), generously provided by Dr. L. Cornelius (Washington University, St. Louis, MO), were grown in 199 Earles medium with
20% FCS and 10 µg/ml endothelial cell mitogen and used between passages 6-11.
Migrations were performed as previously described (Polverini et al.,
1991 Transfections and Flow Cytometry
The plasmid pCDM8-CD36 was a kind gift of Dr. B. Seed (Massachusetts
General Hospital, Boston, MA). The HindIII-NotI fragment of CD36 was
ligated to the vector pcDNAneo (Invitrogen, San Diego, CA) predigested
with HindIII and NotI to produce a CD36 mammalian expression vector
containing an enhancer/promoter sequence of the immediate early gene
of the human cytomegalovirus and a SV-40 polyadenylation signal. HUVECs at passage 2 were transfected with this pcDNAneo-CD36 using lipofectin reagent (GIBCO BRL, Gaithersburg, MD). 30 µg of plasmid
DNA was preincubated in 100 µl lipofectin for 15 min at room temperature. Cells grown to 30-50% confluency were washed with serum-free media and refed with 4 ml of Opti-MEM medium (GIBCO BRL) plus the
plasmid/lipofectin mix. The cells were then incubated for 5 h in 5% CO2 at
37°C, subsequently rinsed twice with growth medium, allowed to recover
in normal growth medium for 2 d, and finally split into selection medium
with 100 µg/ml G418. Single colonies were picked with cloning rings
(Bellco Glass, Inc., Vineland, NJ), expanded, and screened.
CD36-positive clones were characterized and analyzed by flow cytometric
analysis. Cells were washed twice with cold PBS, incubated with anti-CD36
monoclonal antibody SM Tube Formation Assay
Matrigel was purchased from Biomedical Technologies, Inc. 150 µl of
Matrigel matrix was added to each well of a 48-well plate and incubated at
37°C for 30 min to allow for gel formation. HUVECs at 80-90% confluency
were trypsinized, washed twice with PBS, plated on the Matrigel at 3.5 × 104
cells/well, incubated in 5% CO2 at 37°C for 24 h, and then photographed.
CD36 Fusion Proteins Blocked the Inhibitory Activity
of TSP-1 and Its Antiangiogenic Peptides
To determine if an interaction between CD36 and TSP-1
could block the antiangiogenic activity of TSP-1, soluble
GST-CD36 fusion proteins were tested for their ability to
block the inhibition of bovine capillary endothelial cell migration by TSP-1. This assay measures the migration of
cultured endothelial cells toward a known angiogenic factor (bFGF) and consistently parallels angiogenic activity
seen in vivo (Folkman and Klagsbrun, 1987
To more stringently link the TSP-1-CD36 interaction
with inhibition of angiogenesis, blocking studies with the
GST-CD36 fusion proteins were repeated with small TSP-1
peptides known to inhibit endothelial cell migration and
neovascularization in vivo (Tolsma et al., 1993 Antiangiogenic TSP-1 Peptides Physically Associated
with CD36
The Mal III and Col overlap peptides physically interacted
with CD36 because both were able to competitively displace 125I -TSP-1 from CD36 expressed in its natural context on the surface of CD36 cDNA-transfected Bowes
melanoma cells. TSP-1 peptides Mal III and Col overlap,
as well as the Mal III variant that lacks the VTCG motif
but still inhibits endothelial cell migration (Tolsma, S., and
N. Bouck, personal communication), were all able to inhibit binding of TSP-1 to CD36 (Fig. 3 A). Inhibition constants (IC50's) were 8.60 ± 1.12 µM for Mal III, 30.08 ± 8.88 µM for Col overlap, and 15.78 ± 2.08 µM for Mal III
variant. A control peptide did not inhibit binding demonstrating specificity. Melanoma cells not transfected with
CD36 do not bind TSP-1 (Silverstein et al., 1992
To demonstrate that TSP-1 peptides interacted only
with CD36 fusion proteins containing the TSP-1 binding
domain (Pearce et al., 1995 CD36 Antibodies Specifically Blocked the Inhibitory
Activity of TSP-1 and Its Antiangiogenic Peptides
To demonstrate more directly that TSP-1 inhibits angiogenesis via an interaction with CD36, two anti-CD36 monoclonal antibodies, OKM-5 and FA6-152, known to map to
an immunodominant epitope on the extracellular region
of CD36 (Daviet et al., 1995
Engagement of CD36 by Other Ligands Also Inhibited
Endothelial Cell Migration
SM
CD36-transfected HUVECs Became Responsive to
TSP-1 Inhibition
CD36 is typically expressed on human microvascular endothelial cells such as HMVECs, but it is absent from large
vessel endothelial cells such as HUVECs (Swerlick et al.,
1992
When tested in a migration assay, clones 31 and 36 were
sensitive to TSP-1 inhibition, and this inhibition was abrogated by a CD36-blocking antibody (Fig. 7). Clone 35, which expressed only very low levels of CD36 (FACS®
analysis in Fig. 6 and Western blotting with anti-CD36
mAb Mo-91, not shown), was refractory to TSP-1 inhibition (Figs. 6 and 7). Thus, introduction of CD36 into HUVECs rendered them susceptible to inhibition of chemotaxis by TSP-1 and induced, either directly or indirectly,
changes in growth rate, morphology, and ability to organize into tubular structures in Matrigel.
CD36 is necessary for the inhibition of endothelial cell migration and tube formation by TSP-1. Soluble GST-CD36
proteins blocked the inhibition of migration by either intact TSP-1 or small TSP-1 peptides only when they contained the TSP-1 binding site. Blocking antibodies against
CD36 prevented TSP-1 from inhibiting migration, while
an IgM monoclonal antibody against CD36 mimicked TSP-1 and directly inhibited migration. Large vessel endothelial cells that lacked CD36 became sensitive to TSP-1
inhibition of both migration and tube formation after
CD36 transfection. The inhibition mediated by CD36 in
vitro likely reflects the in vivo situation because inhibition
of migration is a consistent predictor of inhibition of neovascularization in vivo (Folkman and Klagsbrun, 1987 These results indicate that the antiangiogenic activity of
TSP-1 is receptor mediated. They are not consistent with
TSP-1 inhibiting angiogenesis by sequestering inducers, as
has been suggested by the finding that TSP-1 can bind directly to inducers of angiogenesis, such as scatter factor
(Lamszus et al., 1996 While CD36 has often been implicated in adhesion and
scavenging (Greenwalt et al., 1992 Although the CSVTCG motif on TSP-1 has previously
been thought to mediate its interactions with CD36 (Asch
et al., 1992 The identification of CD36 as a mediator of the inhibitory effects of TSP-1 helps to explain the unusual biphasic
dose response curve generated when TSP-1 effects on endothelial cell migration are measured. TSP-1 inhibits endothelial cell migration at low concentrations less than 20 nM, yet stimulates migration at higher concentrations
(Taraboletti et al., 1990 The binding activity of CD36 expressed on platelets has
been shown to be regulated by the extracellular phosphorylation state of its ectodomain, with increasing phosphorylation decreasing its binding to TSP-1 and increasing its
affinity for collagen and vice versa (Asch et al., 1993 A variety of antiangiogenic compounds are now entering clinical trials as anticancer agents (Folkman, 1995a). It is secreted at high levels by a variety of normal cells (Frazier,
1991
; Lahav, 1993
; Bornstein, 1995
) and can decrease the
density of the vasculature that forms during embryonic development (Stellmach et al., 1997
) and wound healing in
granulation tissue (Polverini, P.J., L.A. DiPietro, V.M.
Dixit, R.O. Hynes, and J. Lawler. 1995. FASEB [Fed. Am.
Soc. Exp. Biol.] J. 9:272a). In addition, TSP-1 can be an
antiangiogenic barrier to the development of malignant
tumors (Dameron et al., 1994
; Weinstat-Saslow et al.,
1994
; Hsu et al., 1996
; Volpert et al., 1997
), which must induce a vigorous angiogenic response to grow and progress
(Folkman, 1995b
).
;
Tolsma et al., 1993
). The 50-kD central stalk region of the
180-kD subunit of homotrimeric TSP-1 is sufficient for its
full antiangiogenic activity. Molecules that retain either
the procollagen homology region or the properdin type 1 repeat region from this stalk also retain antiangiogenic activity (Tolsma et al., 1993
; Volpert et al., 1995
), and small peptides derived from each of these domains are independently able to block endothelial cell migration in vitro and
neovascularization in vivo (Tolsma et al., 1993
).
; Dawes et al., 1983
; Harker et al.,
1983
; Browne et al., 1996
), it inhibits endothelial cells in
vitro, blocking their proliferation (Bagavandoss and
Wilkes, 1990
; Good et al., 1990
; Taraboletti et al., 1992
;
Vogel et al., 1993
; Raychaudhury et al., 1994
) and migration (Rastinejad et al., 1989
; Good et al., 1990
; Taraboletti
et al., 1990
, 1992
; Tolsma et al., 1993
, 1997
; Volpert et al.,
1995
) and their ability to form lumens (Tolsma et al., 1997
)
and tubes (Iruela-Arispe et al., 1991
; DiPietro et al., 1994
;
Canfield and Schor, 1995
), although at higher concentrations it can be stimulatory for endothelial cell migration
(Taraboletti et al., 1990
; Tolsma et al., 1993
; Gao et al.,
1996
).
), all of which stimulate cells through different
receptors. To address the mechanism by which TSP-1 induces this unresponsiveness in the endothelial cell, we
have sought a receptor through which it might act. The intact TSP-1 molecule binds to at least 12 different receptors
(Bornstein, 1995
), many of which are present on the surface of the endothelial cell. Some, including integrin
v
3 (Lawler, 1993
), integrin-associated protein (also referred
to as CD47 [Gao et al., 1996
]), and possibly low-density lipoprotein related receptor protein (Godyna et al., 1995
;
Mikhailenko et al., 1995
), interact with TSP-1 motifs that
lie outside the 50-kD stalk and thus seem unlikely to be involved in mediating the antiangiogenic activity of TSP-1.
Although there are uncharacterized receptors present on
endothelial cells that can bind to TSP-1 (Chen et al., 1996
)
and TSP-1-interacting molecules identified on other cell types that could also be present on endothelial cells
(Yabkowitz and Dixit, 1991
; Tuzinsky et al., 1993
), CD36
currently appears to be the most likely candidate to mediate the angioinhibitory activity of TSP-1.
). Originally found on platelets and
monocytes, it is also expressed on the microvascular cells
that give rise to neovascularization in vivo (Swerlick et al.,
1992
; Peltzbauer et al., 1993
). CD36 is well known as an
adhesion receptor for TSP-1 (Asch et al., 1987
, 1993
; Li et
al., 1993
; Pearce et al., 1995
) and Plasmodium falciparum
parasitized erythrocytes (Oquendo et al., 1989
; Baruch et
al., 1996
), facilitates the binding of platelets to collagen, monocytes, and the subendothelium, and contributes to
the activation of monocytes and platelets (Greenwalt et
al., 1992
). CD36 also serves as a scavenger receptor. It mediates the uptake of anionic phospholipids (Rigotti et al.,
1995
) and oxidized low-density lipoprotein (OxLDL) (Endemann et al., 1993
; Nicholson et al., 1995
; Nozaki et al.,
1995
), an activity that may contribute to the formation of
foam cells involved in the genesis of artherosclerotic plaques, and plays a key role in the phagocytosis of apoptotic cells (Savill et al., 1992
; Ren et al., 1995
; Stern et al.,
1996
) and rod outer segments in the retina (Ryeom et al.,
1996a
,b). Although not usually considered to be a signaling receptor, it coprecipitates from platelets and endothelial
cells with several src-related kinases (Huang et al., 1991
;
Bull et al., 1994
). Here we demonstrate that CD36 is an essential mediator of the antiangiogenic action of TSP-1 on
endothelial cells in vitro. This is the first indication that engagement of CD36 can produce a biological response in
endothelial cells and the first identification of a receptor able to mediate the effects of a broad spectrum inhibitor
of angiogenesis like thrombospondin-1.
Materials and Methods
monoclonal antibody against CD36
and IgG1 and IgM control monoclonal antibodies, MOPC-21 and MOPC-104E, were from Sigma Chemical Co. (St. Louis, MO). OKM-5 monoclonal antibody against CD36 was kindly provided by Dr. Mary Makowski
(Ortho Diagnostic Systems, Raritan, NJ). Before use, all monoclonal antibodies were extensively dialyzed against DME using a 30-kD Centricon®
concentrator (Amicon Corp., Beverly, MA).
) and included Col overlap, NGVQYRN representing TSP-1 amino acid residues
303-309; Mal III, SPWDIASVTAGGGVQKRSK representing TSP-1
amino acid residues 481-499 with the cysteines present in the native molecule replaced with alanines; and Mal III variant, SPWDIASTSAGGGVQRSK, containing Mal III residues with an altered VTCG sequence.
The position of these peptides in the intact TSP-1 molecule is shown in
Fig. 1. Angiostatin was kindly provided by Michael O'Reilly and Judah
Folkman (Harvard University). Recombinant human basic fibroblast
growth factor (bFGF) was purchased from R & D Systems Inc. (Minneapolis, MN), human type I collagen was from Collaborative Biomedical
Products (Bedford, MA), and human LDL was from Sigma Chemical Co.
LDL was oxidized by dialyzing 200 µg/ml LDL against 5 µM CuSO4 in
PBS for 24 h at 37°C (Nicholson et al., 1995
).
Fig. 1.
The relationship of small TSP-1 peptides to the 180-kD
monomer of TSP-1 and of GST-CD36 fusion proteins to the
whole CD36 receptor protein. Numbers indicate amino acid residues present in the peptide or fusion protein. Shading on the
whole CD36 molecule defines the minimal region of CD36 required for binding to TSP-1 (Pearce et al., 1995). Actual peptide
sequences and detailed description of the fusion proteins are included in Materials and Methods.
[View Larger Versions of these Images (14 + 9K GIF file)]
). In this study, we used
fusion proteins containing the TSP-1 binding region and spanning amino
acids 67-157, 93-120, and 93-298. As a control, we used a fusion protein
unable to bind TSP-1 spanning CD36 amino acids 298-439. For the relative positions of these peptides on CD36, see Fig. 1. As described previously (Pearce et al., 1995
), all plasmid constructs were mapped and insertion sites sequenced to confirm that the fusion protein sequences were correct and in frame. Generated fusion proteins were examined by SDS-PAGE, Western blot, gel filtration chromatography, and ELISA to confirm size and document CD36 immunoreactivity. All molecular weights as
determined by gel filtration chromatography under nondenaturing conditions varied no more than 5% from calculated values (Pearce et al., 1995
).
These assays also showed that the fusion proteins did not form dimers or
large multimers.
). Cells
were allowed to attach to 12-well plates. Radiolabeled TSP-1 was prepared as previously described (Pearce et al., 1995
) with specific activity
ranging from 0.5-1 mCi/mg. Inhibition studies were carried out by combining 125I-TSP-1 (20 µg/ml) with increasing concentrations of peptide
(0.1-1000 µM). After incubation for 2 h at 37°C, the cells were washed
five times with cold PBS and lysed with 0.1 N NaOH. The amount of
bound radioactivity was determined by gamma counting. The peptide
LYPQHKT, obtained from a domain of CD36 that does not bind TSP,
was used as a control. IC50's were calculated using the curve fitting software program Enzfitter (Elsevier Biosoft, Cambridge, UK).
). Briefly, confluent flasks of endothelial cells were starved overnight
in control medium (DME containing 0.1% BSA). Cells were harvested,
resuspended in control medium, and plated at 3 × 104 cells/well on the
bottom side of a gelatinized 5 µM (for BCECs) or 8 µM (for HMVECs
and HUVECs) porous membrane (Nucleopore Corp., Pleasanton, CA) in
an inverted modified Boyden chamber. Cells were allowed to attach to the
membrane for 2 h at 37°C, after which the chamber was reinverted, test
samples were added to the top wells, and cells were allowed to migrate towards the top well for 4 h at 37°C. Membranes were removed, fixed, and
stained, and the number of cells migrating to the top side of the membrane per 10 high-power fields was counted. Test samples containing GST
fusion proteins were first preincubated for 2 h at 4°C and then warmed to
room temperature before addition to the chamber. With the exception of
SM
, monoclonal antibodies, when used, were added to both the upper
and lower wells. Because OKM-5 is unable to recognize bovine CD36
(Ockenhouse et al., 1989
), human HMVECs were used instead of bovine
BCECs for migrations with anti-CD36 monoclonal antibodies. Control
medium alone was used as a negative control to measure background resulting from random cell movement. Samples in each experiment were
tested in quadruplicate, and experiments were repeated at least twice.
Data presented in figures have been normalized to maximum migration,
where 100% was calculated as the migration towards bFGF minus the
background migration towards control medium alone. Background was
consistently less than 60% of migration towards bFGF. Negative percentages appear when test samples suppressed random cell movement. Where
indicated, statistical significance between sample means was determined
using a two-tailed t test on initial raw data within a single experiment, before background subtraction and normalization.
or with an IgM control at 5 µg/ml for 45 min
on ice, washed, incubated with FITC-conjugated goat anti-mouse IgM (5 µg/ml) for an additional 45 min on ice, washed again, and finally analyzed
on a Becton-Dickinson FACScan® flow cytometer (Mountain View, CA).
Results
; Klagsbrun and D'Amore, 1991
; Bouck et al., 1996
). TSP-1 was previously shown to interact in a specific, saturable, and reversible manner with a minimal region of CD36 encompassing
amino acids 93-120 (Pearce et al., 1995
). As shown in Fig.
2 A, GST-CD36 fusion proteins FP93-120 and FP93-298,
both of which contained the TSP-1 binding domain, were
able to block TSP-1 inhibition of bFGF-induced migration in a dose-dependent fashion, whereas GST alone (data not
shown) and fusion protein FP298-439, which lacked the
TSP-1 binding domain (Fig. 2 A), were ineffective.
Fig. 2.
Interference with the activity of TSP-1 and its antiangiogenic peptides by soluble GST-CD36 fusion proteins. Increasing concentrations of CD36 fusion proteins that contain a TSP-1 binding site, FP93-120 (circles) or FP93-298 (triangles), or a CD36 fusion protein that lacks a TSP-1 binding site, FP298-439 (squares), were preincubated for 2 h at 4°C with (A) 2 nM TSP-1, (B) 10 µM Mal III peptide, (C) 30 µM Col overlap peptide, or (D) control media. Each mixture was then tested for the ability to block bovine capillary
endothelial cell migration towards bFGF (solid symbols) or influence background migration in the absence of bFGF (D, open symbols).
Data, accumulated from nine experiments, are reported as a percentage of maximum migration, where 100% represents the number of
cells migrating towards the inducer bFGF alone, and 0% corresponds to the number of cells migrating randomly in the absence of inducer (Bkgd). 100% varied between experiments from 32 to 81 cells migrated/10 high-power fields. Bars indicate standard errors.
[View Larger Versions of these Images (16 + 15 + 14 + 19K GIF file)]
). Concentrations of inhibiting TSP-1 peptides were deliberately
chosen to be less than 100% effective so that both positive
and negative effects on migration could be observed. CD36 fusion proteins containing TSP-1 binding sites interfered with the antiangiogenic activity of Mal III, a 19-mer
containing a homologue of the CSVTCG sequence previously reported to interact with CD36 (Asch et al., 1992
;
Leung et al., 1992
; Li et al., 1993
), and of Col overlap, a 7-mer
lacking the CSVTCG sequence (Figs. 2, B and C). Loss of
inhibition could not be attributed to induction of migration by the fusion proteins themselves as each was neutral when tested alone in this assay (Fig. 2 D). The inhibitory
activities of the peptides were quenched only by CD36 fusion proteins that also bound TSP-1 (Figs. 2, B and C).
). The calculated IC50 of each peptide closely approximated its previously reported ED50 for inhibition of endothelial cell migration (Tolsma et al., 1993
).
Fig. 3.
Inhibition of TSP-1 binding to CD36 and its fusion proteins by antiangiogenic TSP-1 peptides. (A) Binding of 125I-TSP-1 (20 µg/
ml) to a confluent monolayer of Bowes melanoma cells expressing CD36 was determined in the presence of increasing concentrations of
antiangiogenic TSP-1 peptides Mal III (circles, lowest curve), Mal III variant (triangles, second lowest curve), and Col overlap (squares,
third lowest curve) or of control peptide LYPQHKT (diamonds, top curve). Data were normalized as a percentage of TSP-1 bound under control conditions. (B) The effects of TSP-1 peptides Mal III and Col overlap on the binding of 125I-TSP-1 (20 µg/ml) to solid phase
CD36 and CD36 fusion proteins FP67-157 and FP93-120, which contain a TSP-1 binding site, and to FP298-439, which does not, are
shown. Nonspecific binding was determined in the presence of 5 mM EDTA. Bars indicate standard error (n = 3 for A and n = 5 for B).
[View Larger Versions of these Images (24 + 38K GIF file)]
), their ability to block 125I-TSP-1
binding to immobilized CD36 and CD36-GST fusion proteins was tested (Fig. 3 B). Mal III at 10 µM reduced the
specific binding of TSP-1 to CD36 and to two CD36-GST
fusion proteins by 53-65%. Col overlap was similarly effective at inhibiting the binding of TSP-1 to intact CD36
and to the longer fusion protein FP67-157. The CD36 fusion protein FP298-439, which lacks the TSP binding site,
showed only background binding equal to that seen in the presence of 5 mM EDTA. The clear dose response relationship seen for peptide binding to CD36 expressed on
cells in Fig. 3 A was much less evident in the cell-free binding experiments (Fig. 3 B), likely caused in part by immobilization of CD36 and its fusion proteins on plastic and in
part by the use of high peptide concentrations where a significant dose response effect might not be expected. There
is an interesting discrepancy in that the Col overlap peptide failed to block TSP-1 binding to the CD36-GST fusion protein containing the minimal TSP-1 binding domain
(FP93-120; Fig. 3 B), although this same fusion protein
blocked the inhibitory effects of Col overlap on endothelial cell migration (Fig. 2 C). This discrepancy suggests that
Col overlap has a much lower affinity for solid phase
FP93-120 than soluble FP93-120, perhaps because of constraints imposed on this short fusion protein when bound
to plastic.
) as well as to physically displace TSP-1 from CD36 (Asch et al., 1987
; Kieffer et al.,
1989
), were tested. Both antibodies blocked the inhibition
of bFGF-induced migration of HMVECs by TSP-1 (Fig. 4
A) and by TSP-1 peptides (Fig. 4 B). This block was specific to TSP-1 as neither antibody affected the inhibition of
endothelial cell migration by angiostatin (Fig. 4 A), another well-established inhibitor of endothelial cell migration in vitro (Gately et al., 1996
) and of angiogenesis in
vivo (O'Reilly et al., 1994
). An isotype-matched control
monoclonal antibody was without effect in the assay (Fig.
4 A). The two anti-CD36 antibodies were also able to
block TSP-1 inhibition of migration induced by angiogenic molecules other than bFGF. For example, antibody FA6-152 blocked TSP-1 inhibition of endothelial cell migration
induced by either scatter factor (Lamszus et al., 1996
) or
vascular endothelial growth factor (Koch et al., 1994
)
(data not shown).
Fig. 4.
Interference with the activity of TSP-1 and its antiangiogenic peptides by monoclonal IgG antibodies against CD36. Monoclonal IgG antibodies against CD36, FA6-152, and OKM-5 or an isotype-matched control were tested at 10 µg/ml for ability to block the
inhibition of human microvascular endothelial cell migration towards bFGF by (A) TSP-1 (2 nM) or (B) TSP-1 peptides Mal III (30 µM) or Col overlap (50 µM). Angiostatin (2 µg/ml) served as a control inhibitor. Data from three separate experiments were normalized and reported as in Fig. 1. 100% varied between experiments from 32 to 53 cells migrated/10 high-power fields. *Samples significantly different from parallel condition using control media, P < 0.02.
[View Larger Versions of these Images (32 + 36K GIF file)]
, an anti-CD36 monoclonal antibody of the IgM
class, mimicked the activity of TSP-1 and inhibited bFGF-induced HMVEC migration, while an isotype-control
monoclonal antibody had no significant effect (Fig. 5 A).
The high valency of this pentameric IgM presumably allowed for oligomerization of receptors that did not occur
when divalent anti-CD36 antibodies of the IgG class were
used (Fig. 4). Similar differential antibody effects of IgGs
and IgMs are also seen with FAS and TNF receptors (Nagata, 1997
). Two other known ligands of CD36, human
collagen I and OxLDL, also inhibited bFGF-induced HMVEC migration (Fig. 5 B) and showed maximal inhibitory activity at concentrations similar to those at which they
saturably bind CD36 (Tandon et al., 1989
; Endemann et
al., 1993
). These effects were mediated via CD36, for they
were blocked by anti-CD36 monoclonal antibodies OKM-5
or FA6-152 (Fig. 5 B). Inhibition was concentration dependent, as both collagen I and OxLDL were ineffective
at inhibiting migration at concentrations below 0.1 nM
(data not shown). Unoxidized LDL, which is unable to
bind CD36 (Endemann et al., 1993
), failed to influence
HMVEC migration (Fig. 5 B).
Fig. 5.
Inhibition of endothelial cell migration by additional CD36 ligands. (A) A murine anti-CD36 IgM monoclonal antibody SM
and an isotype-matched control monoclonal antibody were tested at 1 µg/ml for ability to inhibit human microvascular endothelial cell
migration towards bFGF. Data were normalized and reported as in Fig. 1. *Significant inhibition compared with bFGF tested alone, P < 0.001. (B) Human collagen I, LDL, and OxLDL were tested at 2 µg/ml in the presence or absence of 10 µg/ml of monoclonal antibodies
against CD36 for ability to inhibit migration of human microvascular cells. Data from two experiments were normalized and reported as
in Fig 1. When tested alone, collagen I, OxLDL, and LDL had no significant effect on migration. 100% varied between experiments
from 29 to 42 cells migrated/10 high-power fields. *Samples significantly different from bFGF tested with control media, P < 0.001.
[View Larger Versions of these Images (24 + 37K GIF file)]
; Peltzbauer et al., 1993
), thus providing a useful way
to assess CD36 function. HUVECs negative for CD36 as
determined by FACS® (Fig. 6), by immunoprecipitation
followed by Western blot, and by Northern blot (data not
shown) were transfected with a CD36 expression vector,
and a series of clones expressing increasing levels of CD36
as judged by FACS® and by Western blots were selected
(Fig. 6). It was noted that clones expressing any appreciable level of CD36 had slower growth rates than parental
HUVECs. For example, the relative increases in cell number after 8 d of culture for each clone were HUVEC, 6.2;
clone 35, 5.5; clone 31, 2.1; and clone 36, 1.4. The transfectants also exhibited a progressively aberrant, very highly
spread morphology as seen in the middle panels of Fig. 6
(photographed at confluency). When plated in Matrigel,
the degree of inhibition of tube formation was correlated
with level of CD36 expression (Fig. 6). In total, 30 clones
expressing various levels of CD36 were characterized and
found to have growth impairment and morphological
changes proportional to their CD36 levels. An additional
five clones were analyzed for tube formation, and they fit
the pattern seen in Fig. 6. All of the CD36-positive clones
maintained high levels of TSP-1 expression as determined
by Western blotting of serum-free culture media collected
over 24 h. Relative amounts of TSP-1 as determined by
densitometry were HUVEC, 6.8; clone 35, 8.1; clone 31, 6.3; and clone 36, 6.0.
Fig. 6.
Sensitivity to TSP-1 inhibition of tube formation induced by CD36 expression in CD36-deficient HUVECs. The first column
shows FACS® analysis for CD36 expression performed on untransfected HUVECs, a transfectant expressing very low levels of CD36
(clone 35), and two transfectants expressing higher levels of CD36 (clones 31 and 36). Fluorescence is plotted vs. cell number. The dotted line in the clones reproduces the HUVEC CD36 null pattern. The morphology of each of the transfectants grown in two-dimensional cell culture is shown in the second column. In the third column, tube formation in a three-dimensional culture of Matrigel is pictured for each line.
[View Larger Version of this Image (107K GIF file)]
Fig. 7.
Sensitivity to TSP-1
inhibition of migration after
transfection of HUVECs
with CD36. Clones described in Fig. 5 were tested for sensitivity to inhibition
of migration by 2 nM TSP-1
in the presence and absence
of 10 µg/ml anti-CD36 monoclonal antibody OKM-5.
Data from four separate experiments were normalized
and reported as in Fig 1.
100% varied between experiments from 37 to 82 cells migrated/10 high-power fields.
*Samples that differ significantly from bFGF tested
alone, P < 0.02.
[View Larger Version of this Image (23K GIF file)]
Discussion
; Klagsbrun and D'Amore, 1991
; Bouck et al., 1996
). Furthermore, experiments to be reported elsewhere show that
inhibition of corneal neovascularization by TSP-1 but not
by angiostatin is defective in CD36 null animals (Febbraio,
M., O.V. Volpert, N.P. Bouck, and R.L. Silverstein, unpublished data).
). In fact, in this study TSP-1 inhibition of scatter factor-induced migration could be completely blocked by an antibody to CD36. Our results are
also not consistent with models suggesting that TSP-1 inhibits bFGF-induced angiogenesis by competing with it for
binding proteoglycans on endothelial cells, as suggested by experiments with heparin-binding TSP-1 peptides (Vogel
et al., 1993
), because migration induced by bFGF was effectively blocked in the absence of TSP-1 by agents that
directly activated CD36, and inhibition by TSP-1 was antagonized by antibodies that blocked CD36 engagement.
), data presented here
show that CD36 expressed on endothelial cells is also a
signaling receptor able to trigger a biological response.
The response in this case is one that generates an inhibitory signal that blocks a positive response to inducers of
angiogenesis. It is reminiscent of receptor-mediated negative signals to which immune cells are particularly sensitive (Scharenberg and Kinet, 1996
). Such signals often require a coreceptor and are mediated by src family kinases.
It is not known if CD36 acts alone or in concert with an
unidentified coreceptor or other surface molecule, but it
does coprecipitate from endothelial cells in association
with the src family kinase p59fyn, and possibly other src
family kinases (Bull et al., 1994
). It is also not yet clear
how a signal emanating from CD36 might block the variety of stimulatory signaling cascades initiated by many different inducers of angiogenesis. However, the cytoplasmic
domain of CD36 has been seen to associate with focal adhesion kinase (Sheibani, N., R. Zhong, and W.A. Frazier,
manuscript in preparation), a kinase that can be essential
for the stimulation of cell movement (Cary et al., 1996
;
Gilmore and Romer, 1996
). If activation of CD36 were to
disable focal adhesion kinase and thereby prevent the adhesion-associated tyrosine phosphorylation that may be
essential for endothelial cell motility (Williams et al.,
1996
), the effectiveness of a wide variety of inducers of angiogenesis would be severely compromised.
; Catimel et al., 1992
; Li et al., 1993
), data presented here show that an additional TSP-1 motif can interact with CD36. The Col overlap peptide lacking CSVTCG
was antiangiogenic, was able to displace TSP-1 from
CD36-transfected melanoma cells, and was rendered inactive by CD36 fusion proteins. A Mal III variant also lacking CSVTCG was antiangiogenic and able to displace
TSP-1 from CD36-expressing cells. These peptides share a
central GVQXR sequence that could represent a second
CD36 binding motif. Two-step binding of TSP-1 to CD36
has been postulated by others (Leung et al., 1992
). Perhaps CSVTCG facilitates an initial interaction between
TSP-1 and CD36 and GVQXR mediates a signaling response. Such a role would fit well with our observation
that the Mal III peptide, which contains both motifs, had a
higher affinity for CD36 and is a more potent inhibitor of
migration (Tolsma et al., 1993
) than Col overlap, which
lacks CSVTCG.
; Tolsma et al., 1993
; Gao et al.,
1996
), a fact that has led to much confusion about its ultimate activity. Recent work has shown that a blocking antibody against the integrin-associated protein (IAP) prevents higher concentrations of TSP-1 from inducing
endothelial cell migration (Gao et al., 1996
). Thus, the biphasic dose response curve for TSP-1 can be explained as
resulting from the sum of activities of two distinct receptors, inhibitory CD36 and stimulatory IAP. CD36 mediates the inhibitory effects of TSP-1 at lower concentrations. The ability of CD36 to mediate phagocytosis (Ryeom
et al., 1996a
,b) and the rapid degradation of lipids (Nozaki
et al., 1995
) suggest that it may be rapidly cleared from the
cell surface upon engagement with TSP-1. Thus, at high
concentrations of TSP-1, CD36 may be sufficiently depleted
to allow IAP to act unopposed, resulting in the stimulation
of endothelial cell migration.
). The
in vitro sensitivity of CD36 to both ligands suggests that
cultured endothelial cells have both phosphorylated and
unphosphorylated CD36 molecules on their surface. In
vivo it is possible that changes in CD36 phosphorylation by extracellular phosphatases and kinases could regulate
the effect of both TSP-1 and other CD36 ligands on endothelial cells. This may be of particular importance at sites
of platelet secretion and inflammation.
;
Gradishar, 1997
). The identification of CD36 as an inhibitory signaling receptor for TSP-1 could be useful in the design and discovery of additional pharmacologic agents that
can inhibit pathologic neovascularization.
Received for publication 14 March 1997 and in revised form 14 May 1997.
Address all correspondence to Noël P. Bouck, Cancer Center, Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL 60611. Tel.: (312) 503-5934. Fax: (312) 908-1372. E-mail: n-bouck{at}nwu.eduThis work was supported in part by grants from the National Institutes of Health/National Cancer Institute (NIH/NCI) CA52750 and CA64239 (to N.P. Bouck) and CA65872 (to W.A. Frazier); NIH/NCI Institutional Training Grant 5T32CA09569 and Chicago Baseball Charities (to D.W. Dawson); NIH HL46403 and EY10967 and the Charles Fogarty Trust (to R.L. Silverstein); and a grant-in-aid from the American Heart Association, New York City Affiliate, and the Dorothy Rodbell Cohen Foundation (to S.F.A. Pearce).
BCEC, bovine capillary endothelial cell; bFGF, basic fibroblast growth factor; FP, fusion protein; GST, glutathione-S-transferase; HMVEC, human microvascular endothelial cell; HUVEC, human umbilical vein endothelial cell; IAP, integrin-associated protein; (Ox)LDL, (oxidized) low-density lipoprotein; TSP-1, thrombospondin-1.
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