BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 175, No. 1, 1991
Pages 68-76
February 28, 1991
S P E C I F I C BINDING OF V A S C U L A R P E R M E A B I L I T Y FACTOR TO E N D O T H E L I A L CELLS Jitka V. Olander,
Daniel T. Connolly,
and J o s e p h E. DeLarco
H e a l t h Sciences Division, M o n s a n t o C o r p o r a t e Research, M o n s a n t o Company, St. Louis, M i s s o u r i 63167 Received December 21, 1990 SUMMARY: V a s c u l a r p e r m e a b i l i t y factor (VPF), also known as v a s c u l a r e n d o t h e l i a l cell growth factor, has r e c e n t l y been p u r i f i e d from guinea pig, human, and bovine sources. We show that v a r i o u s fetal or adult endothelial cell strains o r i g i n a t i n g from either c a p i l l a r y or large vessels possess specific high a f f i n i t y and s a t u r a b l e b i n d i n g sites for guinea pig t u m o r - d e r i v e d [IzsI]VPF. Two classes of sites with KDS of a p p r o x i m a t e l y i0 pM and 1 nM were d e t e c t e d for all endothelial cell types examined. Guinea pig [125I]VPF b i n d i n g to endothelial cells was inhibited by human VPF (IDs0 = 0.8 ng/ml) and by suramin (IDs0 = 75 ~g/ml) but not by heparin. C r o s s - l i n k i n g experiments revealed specific [125I]VPF-receptor complexes of two types. Most of the complexes m i g r a t e d v e r y slowing in SDS-PAGE, indicating that they were of very high m o l e c u l a r weight and p r o b a b l y h i g h l y cross-linked. A p o r t i o n of the m o l e c u l e s m i g r a t e d as 270 kDa complexes, indicating that the m o l e c u l a r w e i g h t of the endothelial cell VPF r e c e p t o r is about 230 kDa. ©1991AcademicPress,lnc.
Vascular permeability
factor
(VPF)
is a m u l t i - f u n c t i o n a l
40
kDa p r o t e i n known to be secreted by some tumors and by p i t u i t a r y f o l l i c u l a r cells
(1-8).
VPF acts as a potent and fast acting
m e d i a t o r of v a s c u l a r p e r m e a b i l i t y
(9,10), as an e n d o t h e l i a l cell
specific growth factor
and as an a n g i o g e n i c agent i__nn
vivo
(4,8,10).
(3,5,8,10)
Initially,
in the media of tumor cells
the p e r m e a b i l i t y (9,11)
factor was identified
and has since been p u r i f i e d
from c o n d i t i o n e d media of the human h i s t i o c y t i c line U937
lymphoma cell
(i), bovine p i t u i t a r y follicular stellate cells
and a m o u s e p i t u i t a r y cell line AtT-20
(8).
(3,5),
A role for VPF in
The a b b r e v i a t i o n s used are: aFGF, acid fibroblast growth factor; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; EGF, epidermal growth factor; ELISA, enzyme linked i m m u n o s o r b a n t assay; FBS, fetal bovine serum; gVPF, guinea pig v a s c u l a r p e r m e a b i l i t y factor; HUVEC, human u m b i l i c a l vein endothelial cells; hVPF, human v a s c u l a r p e r m e a b i l i t y factor; PAGE, p o l y a c r y l a m i d e gel electrophoresis; PDGF, p l a t e l e t derived growth factor; SDS, sodium dodecyl sulfate. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, blc. All rights of reproduction in any form reserved.
68
Vol. 175, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the ascites fluid a c c u m u l a t i o n caused by certain tumors has been suggested
(9) and it is likely that VPF also p a r t i c i p a t e s
in
non-tumor associated processes
involving increased vessel
p e r m e a b i l i t y and angiogenesis,
such as during i n f l a m m a t i o n or
w o u n d healing. A l t h o u g h the m e c h a n i s m of VPF's action is unknown, activities
e n d o t h e l i a l cells.
We recently reported p r e l i m i n a r y evidence for
the p r e s e n c e of high affinity receptors cells
its
suggest that it could interact d i r e c t l y with
(i0).
C r o s s - l i n k i n g experiments
for VPF on e n d o t h e l i a l showed that the b i n d i n g of
[12SI]VPF to human umbilical vein endothelial
(HUVEC)
cells was
inhibited by u n l a b e l e d VPF and p r e v e n t e d by p o l y c l o n a l anti-VPF antiserum,
w h i l e v a r i o u s other growth and p e r m e a b i l i t y
were w i t h o u t effect. factor
These included acidic fibroblast g r o w t h
(aFGF), basic FGF
(bFGF), epidermal growth factor
p l a t e l e t - d e r i v e d growth factor histamine,
serotonin,
interleukin-2.
(PDGF), tumor necrosis
bradykinin,
platelet-activating
(EGF),
factor, factor and
In this report we c h a r a c t e r i z e the VPF receptor
on v a r i o u s types of endothelial in v i t r o
factors
cells and p r e s e n t evidence that
b i n d i n g can be inhibited by suramin but not by heparin.
MATERIALS
AND
METHODS
Cell Culture. Human umbilical vein endothelial cells (HUVEC) were isolated by p u b l i s h e d p r o c e d u r e s (12,13), e s t a b l i s h e d in M C D B 131 m e d i u m (14) and subcultured in MCDB 107 m e d i u m (15) s u p p l e m e n t e d with 10% fetal bovine serum (FBS), 90 ~g/ml Na heparin, 30 ~g/ml endothelial cell growth supplement, i0 ng/ml (EGF), and 1 ~g/ml h y d r o c o r t i s o n e on human f i b r o n e c t i n - c o a t e d tissue culture vessels. The ECGS, hydrocortisone, EGF, and h u m a n f i b r o n e c t i n were obtained from C o l l a b o r a t i v e Research. FBS was from H i C l o n e Laboratories Inc. and sodium h e p a r i n from Sigma Chemical Co. Fetal bovine aortic endothelial (FBAE) and b o v i n e calf adrenal capillary endothelial cells (BAC) were isolated and m a i n t a i n e d as d e s c r i b e d in O l a n d e r et. al. (16). Bovine calf p u l m o n a r y artery endothelial cells (CPAE) were o b t a i n e d from the ATCC and carried in DMEM s u p p l e m e n t e d w i t h 10% FBS w h i l e the adult human aortic endothelial cells (HAEC) were a kind gift of Dr. P. DiCorleto (Cleveland Clinic Foundation) and were m a i n t a i n e d in MCDB 107 with the same s u p p l e m e n t s as the HUVEC. of VPF. Highly p u r i f i e d guinea pig VPF (gVPF) (i0) was stored frozen in 0.1% t r i f l u o r o a c e t i c acid and approxim a t e l y 30% acetonitrile. A sample c o n t a i n i n g 2-4 ~g was dried in a m i c r o f u g e tube using a Savent SpeedVac and r e d i s s o l v e d in 50 ~i 0.1M p o t a s s i u m p h o s p h a t e pH 8.2, 1.0% T r i t o n X-100. One mCi [12SI]diiodo B o l t o n - H u n t e r reagent (DuPont-NEM) was used to radiolabel the VPF as d e s c r i b e d by Bolton and H u n t e r (17). R e a c t i o n p r o d u c t s were separated from the labeled p r o t e i n by p a s s a g e over a S e p h a d e x G-25 column. The t r i c h l o r o a c e t i c acid Radioiodination
69
Vol. 175, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(TCA) p r e c i p i t a b i l i t y of e a c h b a t c h of r a d i o l a b e l e d l i g a n d w a s d e t e r m i n e d a n d w a s a p p r o x i m a t e l y 80%. A n a l y s i s on 1 0 - 1 5 % g r a d i ent s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s (SDS-PAGE) P h a s t G e l (Pharmacia) a n d a u t o r a d i o g r a p h y confirmed t h a t t h e 40 k D a V P F b a n d w a s labeled. The permeability activity of t h e r a d i o i o d i n a t e d V P F w a s t e s t e d u s i n g t h e M i l e s a s s a y (18) a n d f o u n d to b e e s s e n t i a l l y t h e s a m e as s t a r t i n g m a t e r i a l . Additionally, its m i t o g e n i c a c t i v i t y or fetal b o v i n e a o r t i c e n d o t h e l i a l c e l l s w a s u n c h a n g e d f r o m t h a t of u n l a b e l e d g V P F (ECs0 ~ 18 pM). E i t h e r 1% b o v i n e s e r u m a l b u m i n (BSA) or 2.5 m g / m l g e l a t i n w a s a d d e d to p r o t e c t t h e V P F f r o m r a d i a t i o n d a m a g e a n d f r o m n o n - s p e c i f i c l o s s on s u r f a c e s . T h e V P F c o n c e n t r a t i o n of t h e p o o l w a s d e t e r m i n e d b y e n z y m e l i n k e d i m m u n o s o r b e n t a s s a y (ELISA). S p e c i f i c a c t i v i t y r a n g e d b e t w e e n i0 - 49 ~ C i / ~ g or b e t w e e n 0.i 0.4 m o l e s B o l t o n - H u n t e r label p e r m o l e VPF. Binding Experiments. M o n o l a y e r s of n e a r c o n f l u e n t e n d o t h e l i a l c e l l s w e r e w a s h e d 3 t i m e s w i t h w a r m s e r u m - f r e e m e d i u m or B i n d i n g B u f f e r (Hank's B a l a n c e d S a l t S o l u t i o n w i t h 25 m M H E P E S a n d 0.1% BSA) b e f o r e b r i n g i n g t h e c e l l s to t h e t e m p e r a t u r e of t h e assay. T h e n r a d i o l a b e l e d V P F w a s a d d e d in m i n i m u m v o l u m e a n d t h e b i n d ing a l l o w e d to p r o c e e d for t h e d e s i g n a t e d t i m e on a r o t a t i n g platform. Unlabeled VPF that had been partially purified by heparin-Sepharose chromatography and concentrated by ultraf i l t r a t i o n w a s a d d e d in 1 0 0 - f o l d e x c e s s to s o m e c u l t u r e w e l l s to e s t i m a t e t h e a m o u n t of n o n - s p e c i f i c b i n d i n g . Binding was terminated by removing unbound radiolabeled gVPF by washing each p l a t e 4 t i m e s w i t h c o l d T y r o d e ' s b u f f e r c o n t a i n i n g 25 m M H E P E S a n d 0.5% BSA. B o u n d c o u n t s w e r e s o l u b i l i z e d b y a d d i t i o n of L y s i n g b u f f e r (0.01 M T r i s p H 7.4, 0.001 M E D T A 0.5% SDS) a n d c o u n t e d a l o n g w i t h one w a s h of t h e p l a t e w i t h t h e L y s i n g b u f f e r in a P a c k a r d 7 - c o u n t e r . E a c h d e t e r m i n a t i o n w a s p e r f o r m e d in duplicate. T h e s p e c i f i c c o n d i t i o n s for e a c h e x p e r i m e n t a r e g i v e n in t h e f i g u r e legends.
RESULTS
A total ability
of s e v e n
to b i n d cells
regardless
of w h e t h e r
or w h e t h e r
they
from
or a d u l t
fetal
thelial cell
cells
strains
antigen,
cell
strains
the
ability
of h u m a n
tissue.
This
isolates.
cells
The HUE6 bound
strain, more
cell
is a c o m m o n
though
there
or endo-
endothelial Factor
VIII
and refractile
and could
be passaged
was
from these
property
70
expresses
I),
origin,
vessels,
other
0.2
(Table
of u m b i l i c a l
than
which
for t h e
between
or b o v i n e
25 p a s s a g e s )
it is c l e a r
examined.
bound
large
strain VPF
elongated
lines
(about
Even
tested,
more
tested
[125I]gVPF
from capillaries,
somewhat
to b i n d V P F
endothelial
were
in c u l t u r e
among
lines
the cells
the other
umbilical
of t h e
were
0.i n M
examined.
generations
lines
with
consistently
than
All
cell
incubated
originated
exhibited
morphology more
when
DISCUSSION
endothelial
[125I]gVPF.
1.2 ng/108
AND
than
for
other
some variability data
of all of t h e
that
the
-
Vol. 175, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I Binding
of VPF to V a r i o u s
Endothelial
CELL S T R A I N
Cell Strains
ng VPF/IO 6 cells
HUE6 (p.10) BAECI7 (p.12) BACIOA (p.12)
1.21 0.21 0.31
HUE6 (p.18) HAEC (p.8) CPAE (p.17)
1.38 0.94 0.31
HUE2A HUE2A
0.40 0.36
(p.7) (p.9)
HUE173 (p.4) HUE6 (p.10)
0.41 1.61
0.i nM [12SI]gVPF in 2.5 ml Binding Buffer was incubated with subconfluent monolayers of various human and bovine endothelial cells in 60mm tissue culture plates for 18 hours at 4°C. Nonspecific binding, which was approximately 20% in this experiment, was determined in the presence of 100-fold excess unlabeled VPF, subtracted and the ng/106 cells calculated. HUE6, HUE2A, and HUE173 are strains of human umbilical vein endothelial cells. HAEC are human adult aortic endothelial cells. BAECI7 are fetal bovine aortic endothelial cells; BACIOA are bovine calf adrenal capillary endothelial cells; and CPAE are calf pulmonary artery endothelial cells. Passage number is designated as (p.#).
The kinetics examined
of
[12SI]gVPF b i n d i n g
at different
temperatures.
equilibrium
after
temperature
o r 37°C.
equilibrium
(approximately
at all t h r e e Figure was high binding
3-4 h o u r s
IA s h o w s
affinity,
The total
that
of 9 p M
(25,000
Similar
experiments
sites/cell) were
at r o o m at
was
the
higher
numbers
Bovine
brain
types,
and
same
an endothelial pituitary (8). with
Two
cells
out
classes
affinity
cell
classes
dissociation
and
growth
of v a s c u l o t r o p i n constants 7!
KDS
lines
similar
significantly
recently
factor which
binding
of 4 p M
with
HUVEC
low affinity
"vasculotropin"
IB)
sites/cell).
of s i t e s w e r e cells had
(Fig.
sites,
for two other
cells were
specific
termed
(180,000
cells
of n o n - s p e c i f i c
analysis
of b i n d i n g
1.5 n M
endothelial
amount
Scatchard
but the HUE6
of both high
capillary
[12SI]VPF t o H U V 6
and that the
carried
cell
detected
reached
[125I]VPF b o u n d
of
of t w o c l a s s e s
amongst
to gVPF
binding 1-2 h o u r s
0.i n g / 1 0 B cells)
9% a n d 23%.
T h e KDS f o r b o t h
to bind
of
the binding
(Table II).
AtT-20
amount
0.7 + / -
saturable
between
the presence
the
Specific
at 4°C a n d a f t e r
cells was
temperatures.
varied
indicated
to HUVEC
receptors. reported
from mouse is h o m o l o g o u s sites were
(900 s i t e s / c e l l )
Vol. 175, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.869 B
w !1~
"'
20.
j
t,
A
total
0.
I I
//
u. 0.5
i'-
on spec
-~
0.4350 0.00
e-"
0"~
I L
I
I
I
I
0.50
1.00
1.50
2.00
2.50
n m
0
I 24
0
I 48
73
I-gVPF Bound (pM)
125
Fig. 1. The s a t u r a t i o n b i n d i n g curve and S c a t c b a r d plot of VPF b i n d i n g to HUE6 cells. A. Binding was carried out on subconfluent HUE6 cells in 60 mm plates for 18 hours at 4°C in the p r e s e n c e of increasing concentrations of [zzSI]gVPF in 2.5 ml
Binding
Buffer.
Non-specific
binding
(9-23% o f t h e
total
binding) was d e t e r m i n e d at each c o n c e n t r a t i o n in the p r e s e n c e of 100-fold excess unlabeled VPF. B. Scatchard plot of data in A using the "LIGAND" program developed by M u n s o n and Rodbard (19) to fit the data to a two-site model.
and
41 p M
(4600
sites/cell)
constant
was
essentially
affinity
constant
significantly detected Similar VPF
on the
were
of
as t h e
number
receptors
experiments
two
same
orders
brain
we
high
as r e p o r t e d
receptors
higher.
e t al. cells same
will
be
required
Parameters
of
[~SI]gVPF Binding to V a r i o u s
lower Also,
classes cells
(24)
were
.
with
except
order
on HUVECs.
to determine
the
of both
endothelial
are the
found
affinity
here,
of magnitude
endothelial
report
of receptors
the
by Vaisman
aortic
they
While
capillary
obtained
to bovine
number
the
vasculotropin
bovine
results
binding
was
fewer
(20).
murine
that
the
of magnitude
Further
if t h e s e
differences
Table II
Dissociation Constants HUVEC S T R A I N
KDI(pM)
HUE2A (p.8) HUE6 (p.10) HUE6 (p.8) HUE173 (p.4)
8 19 8 2
± ± ± ±
3 5 2 3
Receptors/Cell
KD2(nM)
3.4 1.7 0.9 0.i
± ± ± ±
H U V E C Strains
Bm
2.0 2.0 0.2 0.i
5,040 24,000 25,000 1,610
± ± ± ±
B~
1,430 5,040 3,800 1,000
These were d e t e r m i n e d as described for Figure 2. 72
13,800 112,500 250,000 13,700
± 3,500 ± 56,300 ± 24,000 ± 2,900
VoI. 175, No. 1, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
120" c 0
100"
0
/
80. o
60"
5
e.-
o
40"
II1 If. ~. >
20-
~
Non-reduced
v'i'~,T
T',
\
200 - 116 -97-66-45--
O.L
I
-20
10
®
I
-10
10
-9
I 10
I
-8
I -6 10
10 -7
I -5 10
Reduced
I -4 10
I -3 10
(~
I n h i b i t o r (g/ml)
1 2
3
4
Fig. 2 . Effect of gVPF, hVPF, Suramin, and H e p a r i n on the B i n d i n g of [nSI]gVPF to HUE6 cells. Binding experiments were p e r f o r m e d on subconfluent HUE6 cells in 24-well plates. Competitors were always added to the monolayers first in 0.5 ml Binding Buffer and equilibrated for 1 hour at room temperature. Then 0.i nM [125I]gVPF was added and the binding c o n t i n u e d for two hours longer. Since the off rate is very slow at room t e m p e r a t u r e (>6 hours; data not shown), the binding in these competitive reactions does not reach equilibrium but favors the c o m p e t i t o r binding. Data represent competition by u n l a b e l e d gVPF O --- O, u n l a b e l e d hVPF • --- *, and suramin • --- e, Na Heparin •
------
•.
Fig. 3. A u t o r a d i o g r a p h of SDS-PAGE analysis of affinity labeled HUE6 cells w i t h [~SI]gVPF. HUE6 cells grown to n e a r - c o n f l u e n c y in 60-mm plastic tissue culture dishes were incubated with 0. inM [125I]gVPF at 4°C for 4 hours (lanes 1 and 3) or in the presence of [IzSI]gVPF plus i0 nM u n l a b e l e d gVPF (lanes 2 and 4). At the end of the binding period, the cell layers were w a s h e d 3 times and then crosslinked with 0.25 mM disuccinimidyl suberate for 15 minutes. T h e reaction was quenched with 200 ~i 0.2M glycine and the cells scraped into 1 ml PBS plus protease inhibitors (imN EDTA, 1 mM p h e n y l m e t h a n e s u l f o n y l fluoride, 1 ~g/ml leupeptin, 1 ~g/ml p e p s t a t i n A and 20 KIU/ml aprotinin). Cell m e m b r a n e s were spun down and solubilized into 75 ~i PBS with inhibitors plus 1% Triton X-100, separated under non-reducing (lanes 1 and 2) and reducing (lanes 3 and 4) conditions on 4-12% gradient SDS-PAGE and a u t o r a d i o g r a p h e d for 5 days at -70°C. The top of the gel coincides w i t h the very dark bands.
are
a reflection
differences,
of
A competition potential VPF
inhibitors
competed
receptor. inhibit to their
binding
receptors
centration
differences,
assay on
endothelial
was
used
to test
[12SI]gVPF b i n d i n g
as e f f e c t i v e l y
Suramin,
the
species
cell
or both.
as g u i n e a
an anionic of growth (21,22)
of approximately
the
pig VPF
anti-trypanosomal factors
blocked
2).
of Human
for binding drug
as b F G F ,
[12SI]gVPF b i n d i n g
75 ~ g / m l 73
such
effects
(Figure
(IDs0 = 50 ~ M ) .
to the
reported
PDGF
and
at a conHeparin,
to
EGF
Vol. 175, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a n o t h e r anion with high charge density, was tested in the r a d i o r e c e p t o r assay and did not compete for
[IzSI]gVPF binding.
Our p r e v i o u s results indicated that w h e n c h e m i c a l l y c r o s s l i n k e d to HUVEC, barely entered a 7.5% gel.
[125I]VPF was
a g g r e g a t e s were formed w h i c h
We have r e p e a t e d these e x p e r i m e n t s
using the HUE6 strain of endothelial
cells w h i c h have an ex-
c e p t i o n a l l y high number of receptors per cell.
While the
m a j o r i t y of the r a d i o l a b e l e d material was still p r e s e n t a g g r e g a t e s which remained near the top of the gel, band of a p p r o x i m a t e l y
270 kDa was seen under both n o n r e d u c i n g
and r e d u c i n g conditions specific b i n d i n g of
in large
an additional
(Fig. 3).
This was the result of
[12SI]VPF since no r a d i o l a b e l e d band was
present w h e n the b i n d i n g took place in the p r e s e n c e of excess u n l a b e l e d gVPF.
Since the m o l e c u l a r w e i g h t of VPF is 40 kDa,
the r e c e p t o r is e s t i m a t e d to be a p p r o x i m a t e l y
230 kDa.
the receptors aggregate upon b i n d i n g VPF, thus m a k i n g for d i s u c c i n i m i d y l
Possibly, it p o s s i b l e
suberate to c r o s s l i n k m u l t i p l e r e c e p t o r - l i g a n d
complexes t o g e t h e r into large aggregates. m o l e c u l a r w e i g h t complex,
A l t e r n a t i v e l y the high
also s p e c i f i c a l l y tagged by
represents the second class of b i n d i n g sites.
[12SI]VPF,
A s i m i l a r l y large,
p r o t e o g l y c a n r e c e p t o r has been reported for tumor g r o w t h factor-~ (23).
C r o s s - l i n k i n g experiments p e r f o r m e d by V a i s m a n et al.
(24)
indicate a m o l e c u l a r w e i g h t of 180 kDa for the VPF r e c e p t o r on bovine aortic endothelial
cells.
The p r e s e n t data show that endothelial cells of d i f f e r e n t origins can bind VPF.
Analysis of the b i n d i n g data revealed two
d i s t i n c t a p p a r e n t classes of b i n d i n g sites. of these data is that the endothelial heterogeneous.
Alternatively,
One i n t e r p r e t a t i o n
cell VPF receptors are
multiple binding affinities
could be the result of h e t e r o g e n e i t y
in VPF itself.
As m a n y
as 12 isoforms of gVPF have been d e t e c t e d using 2 - d i m e n s i o n a l electrophoresis
(i0), thus the
[12SI]VPF used in the p r e s e n t
e x p e r i m e n t s c o n t a i n e d m u l t i p l e isoforms.
Even t h o u g h permea-
b i l i t y e n h a n c i n g and growth p r o m o t i n g activities are a s s o c i a t e d with all of the isoforms a c t i v i t i e s are identical. homogeneous
(i0), it is not known if the specific This issue will not be r e s o l v e d until
isoforms of VPF are available.
u n d e r l y i n g mechanism,
R e g a r d l e s s of the
the h e t e r o g e n e i t y of b i n d i n g o b s e r v e d here
raises the interesting p o s s i b i l i t y that q u a n t i t a t i v e d i f f e r e n c e s in b i n d i n g of VPF to e n d o t h e l i u m are p h y s i o l o g i c a l l y relevant. For example,
h a l f - m a x i m a l growth s t i m u l a t i o n of endothelial 74
cells
Vol. 175, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by VPF occurs in vitro in the 1.6-50 pM range permeability
enhancement does not occur until the VPF concen-
trations in solutions used for intradermal 1 nM (i,i0).
(3,5,10) whereas
injections are above
Even though the assay systems that measure these
two responses are quite different,
it is reasonable to hypo-
thesize that these events are quantitatively receptor interactions
regulated by VPF-
involving specific isoforms of VPF and its
receptor(s).
ACKNOWLEDGMENTS
We thank Rickey Nelson and Roger Monsell for assistance in growing large-scale cultures of cells, Deborah Heuvelman for the purification of VPF and Judith Sligar for culture of HUVEC cells. The support and helpful discussions of Joseph Feder in the initial stages of this work is gratefully acknowledged. We also wish to thank Arthur wittwer for his review of this manuscript and Patrick Lennon for drawing our attention to Suramin's effect on growth factor binding.
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