Receptors of Vascular Endothelial Growth Factor/Vascular ...

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of Texas. Health. Sciences. Center at San Antonio. San. Antonio,. TX. Correspondence ..... of the Medical. School of Marburg. University. Immunofluorescence and ... immunofluorescence and double-label immunohistochemistry tech- niques. .... San Diego, ... by auto- radiography with. Hyperfilm-3H. (Amersham-Buchler,.

Receptors of Vascular Endothelial Growth Factor/Vascular Permeability Factor (VEGF/VPF) in Fetal and Adult Human Kidney: Localization and [1251]VEGF Binding Sites MATTHIAS ELISABETH EBERHARD *I,istittjte

SIMON,* WOLFGANG ROCKL,t CARSTEN HORNIG,t F. GRONE,* HENNER THEIS,* HERBERT A. WEICH, FUCHS, AVNER YAYON, and HERMANN-JOSEF GRONE*

of Pathology,

Differentiation,

German

of Neurobiology, Weizmann

institute

Vascular

endothelial

important

function

in renal

expressed

growth

in podocytes

implications for mans, the cellular specific

receptors:

to localize

(1)

genesis;

(2)

kidney;

and

(3) by

and

aims

binding

into

only

of

binds

was

proteins

possesses a complex vascular and morphologic characteristics.

pressure vessel kines

(2).

The

system and

Vascular

and

their

receptors

endothelial

to

be

the have

and

integrity

regulated

by

known aim

placenta

of

lointerstitial

solely

to

fenestrated

Department

of Molecular

In developing

Cell

glomerubi,

network

(Kd)

Kd: gbomeruli

of

38.6

±

(fK, ii binding receptor cells.

(aK)

Biology,

VEGF

A

n

19.4

±

coexpression

shown,

with

receptor

binding

of

receptor

sites

2.6

(aK,

both

VEGF

togen

for

endothelial

cells

permeability

factor

(VPF)

kidneys

was:

± 7. 1 (fK, n

5),

=

binding

the

in the kidney.

1998)

(1K)

n

5);

=

1 1.6

± 7.0

growth factor-2 displaced VEGF by approximately 60%. VEGF found only in renal endothelial

a function for of angiogenesis.

1044,

developmental

fetal

5), 36.3

=

Flt- 1 demonstrating of

different

and

1 1 .2 (aK,

5) pmol. Placenta in all renal structures proteins thus were

=

in

adult

tubulointerstitium

microvaseubar

renal

blood

factors/cyto-

most

These

VEGF (J Am

is a potent

mi-

mice

an important

function

sites

could

abundant studies

be

VEGF

support

the

in adult kidney that is Soc Nephrol 9: 1032-

Four

and

[indicating

The

(9).

readily the

larger

associated.

This

homologous

chain

increase

brane

binding

studies gene

and

phenotypes

for VEGF

point

to

ontogenesis

a molecular

of VEGF

of amino

VEGF

NH, to

(PDGF)-A

with

and

also

exon

6

enriched

have

are

display

a typical

these because

of

two

to glycosaminoglycans

and sequence

are acid

growth on

that the

are

Although

leader

residues

i89,

detected

isoforms

of a 24-amino

basic

i65.

been

cells.

platelet-derived

with

of 34 to

secreted

of transfected

terminus,

is probably

weight

(VEGF121

acids])

isoforms

in medium forms

the

to increase

in vascular

isoforms

smaller

measurable

two

VEGF

protein

number

preceding

as vascular

ability

its

(3,7,8).

molecular

two

known

of

a functional

for

is a dimeric

46 kD.

It is also

because

(6). In situ

lacking

development

206

(4,5).

permeability

of transgenic kidney

Received June 20, 1997. Accepted November 26, 1997. Drs. Simon and Rock! contributed equally to this work. Dr. Simon’s current address: Division of Nephrology. Department of Medicine, University of Texas. Health Sciences Center at San Antonio. San Antonio, TX. Correspondence to Dr. Hermann-Josef GrOne. Department of Pathology, Klinikum Lahnberge. Universit#{228}tMarburg, Conradistrasse. D-35043 Marburg. Germany. 1046-6673/0906l032$03.00/0 Journal of the American Society of Nephrology Copyright 0 1998 by the American Society of Nephrology

and

Germany;

Departinent

capillary

Affinity

hypothesis independent

transcapillary

of this

(VEGF)

Braunschweig, and

vessels.

VEGF

factor

Regulation

protein appeared as soon as endothelial cells were positive for von Willebrand factor. Specific ‘25I-VEGF binding could be localized to renal arteries and veins, glomerubi, and the tubu-

(3).

growth

of Gene

and post-

are

growth

glomerular

cortical

system with distinct The endothelial to a high

Biotechnology,

were:

immunohis-

glomeruli,

Departtnent

Germany;

stages.

autoradiography

localized

for

In huto two

latter and

capillaries

are exposed

development

seems

vessels,

peritubular

capillaries

two

VEGF

were

The kidney functional

of glomerular

its

By double-label

receptor

of preglomerular

Glomerular

ability

study

The

by

cells

(I).

an

to Flt- I . Quantification

performed

endothelial

cells

The

ofthis

Flt- 1 receptors.

densitometry. VEGF

kidney.

has is consti-

and to increase activator may

binding

sites

computerized

tochemistry,

and

The

the

and

which

binding

(VEGF)

proteins in human renal ontobinding in human fetal and adult

competitive

factor-2,

‘25IVEGF

receptor VEGF

KDR

factor

Germany;

Israel.

and renal disease. depend on binding

KDR.

to dissect

the

achieved

growth

and

VEGF to quantify

components: was

Flt-l

Gottingen,

Rehovot,

of the adult

renal development actions of VEGF

Center

Center,

ontogenesis

for monocytes plasminogen

Marburg,

Research

Primate

of Science,

vascular

of VEGF to be chemotactic activity of collagenase and

University,

National

German

Abstract.

tutively

Philipps

cellregion factor

apparently

plasma

mem-

(10).

VEGF

mRNA

and protein

have

been

demonstrated

in meso-

VEGF/VPF

and metanephros in glomerular leeting duct epithelia by in situ tochemistry ney

cortex

leads

activity

by

ability

increase

the also

and

to development beads

tor)

selectively

receptors;

and

fik-

to mediate tions

are

have

cell-to-cell KDR

and

and

Flt-

mesangial

cells,

exert

effects

VEGF

may

of endothelial

cell

for

that

VEGF

For been

the

other

receptor

receptors

are

human

localized,

and

data

for

kidney.

Cultured

sources

have

binding

characteristics

VEGF activity

aspects an

been

analysis

cannot

always

VEGF/VPF

adult

rat,

conditioned cells

(27).

With we

between

of

rodents,

and receptor

and

been

receptor

that

highly

are

for

developed

in

in

mammals

the

from ovary

vasoactive

important

receptor

proteins

pobyclonal

and

binding the

by

binding

kidney

quantitative into

study

two

were:

in amino

is an endothelial acid

sequence

to localize using

antibodies; during

components,

cell growth to VEGF

(2)

ontogeny

radiographic one

to quantify and

method;

to Fbt- 1 . The third aim was achieved VEGF and placenta growth factor VEGF,

(1)

immunohistochemistry,

monocbonal

in human

an in situ

ofour

due

and to KDR

in the (3) and

g;

pling

humans

column

was

VEGF adult

by

to dissect due

by

1). VEGF

glycosylated

to sKDR

analysis

eluted

at least

with

0.1

M glycin,

dodecyl

purified serum

with

other

VEGF

mined by SDS-lO% polyacrylamide ing conditions. followed by Western proteins

were

membranes

transferred

(Millipore, was

then

completed

anti-rabbit Mouse

IgG

recombinant Freund’s intervals,

of

Antibodies BALB!c

extracellular

mouse

injection.

myeloma

sKDR

hypoxanthine.

cells

Hybridization

was

10% 4

X

the

gel elecblot

reactivity proteins

of the was

deter-

difluoride the Western

(3 1), using

affinity-purified

The

were

bands

alkaline

were

visualized

phosphatase-labeled

Booster spleen was

M aminopterin,

Soluble

FIt-I

with

FIt- I (sFlt-

25

Prog

of

I ) emulsified

in

injections were given at 3-wk cells of immunized mice and

performed

induced

heat-inactivated lO

and

immunized

or soluble

glycol (MW 1500). in Dulbecco’s modified with

in

with I was

Western

the transfer,

to sKDR

mice

complete adjuvant. and fusion between

NS-l

pH 2.4,

antibody.

Monoclonal Groups

p.g!ml. for

The

Bound

at once purification

cross

After

as described

substrate

NaCI,

polyvinylidene

MA).

of 0.55

buffer.

and

receptor

was

pH 8.0.

gel electrophoresis under reducblotting. Fractionated receptor

to Immobilon-P Bedford,

a chromogenic

The

a cou-

(r2l2)

same

staining

to KDR.

protein

with

NaCI.

150 mM

silver

in an

KDR

(SDS)-polyacrylamide

by

r212

mM

were neutralized of the antibody

sulfate

followed

bled.

to DNBr-activat-

antiserum

10 vol of the

after

was

dilution

Germany)

500

A total

the rabbit;

the animal

4 ml of rabbit

eluted proteins The efficiency

sodium

of the

Freiburg,

isolated

(r212). into

was coupled

Tris,

mo-

Germany). 5) was

Receptor

0.2 mg of protein,

Then,

puri-

an apparent

shown by N-terminal chemistry on an auto-

injected

domain)

overex-

were

1 through

was

of KDR

Weiterstadt,

(domains

was Gerinsect

and

with

Biosystems,

with

presence

supernatants

protein

protein

with

analysis

plemented

(30,3

washed were

checked

cell

domains Heidelberg. into Sf158

the

sKDR was degradation

in 100 mM

aliquots. The M Tris-HCI, pH 8.0.

factor

identity

Insect

Biotech,

column

S00-p.l

teins.

specific

one

boosts

of 50%.

to this

using

VEGF

for virus

protein

(Pharmacia

efficiency

to polyethylene were grown

a 53%

sepa-

KDR

extracellular (Clontech, transfection amplification

(Applied

sKDR

Soluble

for

1 10 kD extracellular

by competitive binding of (PLGF)-2. PLGF-2, like with

KDR

1 and

revealed a half-maximal titer at a I :7500 immunosorbent assay for sKDR. Purification of r212 Antiserum. Purified

applied

goat

the aims

These

Flt-

by screening (sKDR).

FIt-I

r21 2 at a concentration

(28,29).

Therefore,

two

The serum enzyme-linked Affinity

protocol

differences

and

(32).

to quantify

used

purified as described (34). Rabbit Polyclonal Antibody

affinity-purified

hamster

binding

there

affinity

Receptors. KDR was cloned cDNA library. A 2.3-kb BgIII IgG-like

1 10 kD with using Edman

soluble

trophoresis

studies

VEGFI#{212}Sobtained Chinese

in

situation.

performed

and

extracellular

it possible

the

of the purified

sequencer

antibodies

and

features

binding

to the in vito have

human

cells

functional

identity

ed-Sepharose

combined As

gas

were

KDR

mass of analysis,

human

(900

not

tissues

the

lecular sequence

The

VEGF placenta

obtained

of soluble

(33).

against

vector pBacPAK9 expression. After

clones

an additional

is part of

(26).

fled

were

These

muscle

characterized.

define

receptors

high

make

receptors

for the first seven

of 0.5 mg of purified

cells.

in

clones

pression

and

suggest

function

coding

matic

In situ

receptors

to the

Methods

of Soluble from a human

of pan-

have

different

generally

to

shown

been

from

transfected

regard

have

not

recombinant

medium

peptides,

kidney

VEGF

these

studies

human

in human

VEGF

be transferred

binding

using

(23-25).

VEGF

ofAntibodies Receptors

transferred into the many) for baculovirus

The

to modulation

integrity

proteins

culture can change their morphologic compared with the in vivo situation, vitro

smooth

receptor

of

to of

cells

on endothelial

cells

used

be expressed

beta

uterine

has

kiKDR/

interac-

found

of renal

endothelial

by

may

in addition

and

exclusively

their

1 also

with

1033

(VEGF-Rl)

binds

of PLGF

of the

and

message.

1 is essential

cell-to-matrix

and

mRNA

kidney,

of these

basic

sites

Preparation and Flt-J

cells,

kinase

that

Flt-

as monocytes.

proliferation

VEGF

Knowledge the

such

=

recep-

tyrosine

suggest

whereas

ereatic

studies

features

binding

fragment

characterized with

mice

vasculogenesis,

Because

domain

domains

cells

cells,

plasma

III tyrosine

are

knockout

on nonendothelial cells,

type

to Flt-l

PLGF

of Fit- I , but not to KDR,

Generation from cDNA

( I 9,20).

intracellular on

endothelial

islet

to two

insert

domains repeats

for

(2 1 ,22).

development

affinity

(kinase

receptors

Results

1 is important

high

or KDR

immunoglobulin-like

receptors

kidney

(15-18).

with

extracellular

activity.

activator

called Flt- 1 (fms-like tyrosine kinase1 I ) and fik- I (fetal liver kinase = VEGF

These

their

normal

disease

affinity

high

in contrast,

Kidney

rately.

and to

plasminogen

with

in Human

cap-

for monocytes

and

for

of renal

receptors receptor [RI

Both

lacking

bind

(VEGF-R2);

receptor-binding the

of VEGF

gbomeruli

of collagenase

implications

in humans.

nase

kid-

in organotypie

inhibition

in vito

to abnormal

[R] 2) in mouse

seven

embryonic

of capillaries

to be chemotactic

activity

binds

receptor

KDR

Materials

of VEGF

have

VEGF VEGF

to rabbit

(1 3); in contrast,

the pathogenesis

membrane

given

(14).

The may

protein

antibody

tufts

homodimers domain

VEGF

conditions

iblary

and in cobimmunohis-

(11,12).

Recombinant culture

visceral epithelia hybridization and

Receptors

3 d after

by exposure

After

fusion,

Eagle’s horse and

the third of the

mixed

medium serum

cells

cell cultures (DMEM)

containing

1.6 X l0

booster

mixed

suplO

M thymidine

M

1034

Journal

(HAT

of the American

medium).

Cultures

Society

were

refed

with

every 2 to 3 d. After 10 to 14 d, culture specific antibodies by enzyme-linked munoblotting,

using

were

KDR-l

(IgG-l

determined

using

DMEM-HAT

supernatants immunosorbent

were

) and

clone

the Sigma

190.1

1 for anti

Immunotype

TM

sFlt-4,

for im-

domain

Positive wells were isolated, The mouse immunoglobulin

sFlt-l , sFlt-4, or cross-react with

by incubation

with

SO p.1 of unspecific

for 30 mm supernatant

at RT and centrifuged was then incubated

(diluted

1 :20)

1 h at RT.

The pH

(extracellular

pellet

8.0),

buffered buffer

the

was

0. 1%

domain,

washed Triton,

gels.

rabbit

was

jugated

After used,

goat

twice

with

once

Human

control,

TSA

with

blotting,

an

followed

IgG

adult

the

with

removal frozen

(n

due to renal in isopentane,

The

fetal

formed tissue. for this be well

serum

2.5 tg

and

once

dissolved gel

affinity-purified

diluted

Tris,

to use

5) were

=

was

applied

(diluted

for S serum

body

for

the

of purified

10 mM

with

NaC1,

phosphate-

After

tions

for

monoclonal

dilutions

from

peroxidase-con-

surgical

autopsies

per-

was

structures could listed in Table I. obtained

of Marburg

and

Immunohistochemistry

from

the

University.

Immunohistochemistry

was

in acetone

immunofluorescence

at

and

carried -

out

10#{176}C for

double-label

on

S-p.m

10 mm,

frozen

using

by

Hamburg, goat Germa-

(FCS;

factor

10%)

antibody

at 37#{176}C followed

I :20;

Sigma,

by

Munich,

medium.

8.4,

body were

tissue

double-label

immunohistochemistry

tech-

or

antibody

mouse

of 20

antibody

was

in PBS,

then

pH

(Z 259,

7.6.

For

DAKO)

mM),

naphthol-AS-BI-phosphate and levamisole 146 mM

sections

new

(0.5

(S mM)

NaC1

at 22#{176}C for

staining,

(28

for

seemouse

incubated

nitrite

p.g

to the tissue

1 h; alkaline-phosphatase

of sodium

containing

anti-

the

fuchsin

mM),

in SO mM

dim-

TnsIHC1

15 mm.

were then washed in H2O and PBS, followed by an with FCS (10%). Anti-von Willebrand factor rabbit anti-

(DAKO), washed

diluted 1 :500, was applied for 18 h at 4#{176}C. Sections three times with PBS at 22#{176}C.A mouse anti-rabbit

two

diluted antibody

antibodies

R (Camon,

incubated

rabbit

IgG,

to produce

for the with

respectively,

without

antibody.

For competition

I h.

a blue

color.

1:40) Blue

Sections

medium.

Control experiments entailed immunohistology antigens,

followed by a 1:40. Each of

monoclonal antibody (diluted 1 h and developed with Vector

Germany)

in aqueous

applied, diluted

at 22#{176}C for

mouse at 22#{176}C for

Wiesbaden,

mounted

1 :50; DAKO) was (Z 259; DAKO)

was

Alkaline-phosphatase was then incubated were

monoclonal

IgG!ml

at a concentration

primary

I :40)

done

(2 1 mM),

mouse

p.g

anti-mouse

(diluted

(64 mM),

pH

the 15

at 22#{176}C for

were

fuchsin)

buffer,

of the

to a solution

these

Immunofluorescence fixed

antibody

exposed

(basic

with

190. 1 1 to flt-l

was applied

Sections incubation

specimens

serum

Willebrand

(diluted

of

2 h at 22#{176}C, a rabbit 1:40

during

School

(con-

Eching,

calf

in aqueous

incubated

application

obtained

of these

mounted

were

(n

Medical

were

antibody

IgG!ml.

1 h. All

abortion and were handled as described for adult of fetuses of I 7-wk gestation or older were taken

of the

fetal

for 45 mm

IgG

primary

at 37#{176}C,followed

37#{176}C; Biozol,

incubated

the dilution

(M 737, DAKO, by rhodamine-labeled

PBS,

at a concentration

monoclonal

mune

sections

was

anti-rabbit

antibody (M 737, rabbit anti-mouse

5) were

mm

at 22#{176}C.Von

sheep

parts were excised, and stored at -70#{176}C.

=

mm

with

DAKO)

KDR-l

ethylformamide

IgG

after

20

sections

anti-KDR

immediately

45

used

at a 1:10

Immunohistochemistry

Tissue

were

horseradish

washes

Sections

Double-Label

I :2500.

received

Germany).

for

1 :20, 45 mm,

three

for

1 :25;

incubated

(diluted

after

Tumor-free nitrogen,

sections

committee

Then,

in 30 j.d of sample electrophoresis with

study; in these, renal glomerular and tubular visualized. Gender and age of patients are

Approval

IgG

ny).

(r212)

monoclonal antibody mm at 37#{176}C, followed

cell carcinoma. cooled in liquid

kidneys

shortly after Only kidneys

IgG!ml)

mouse for 45

diluted

(10 mM

TSA,

by anti-rabbit

(Promega)

kidneys

j.g

with serum

Kidneys

The

35

experiments

antibody

over

I 10 kD).

saline (PBS). The pellet was and used for SDS-polyacrylamide

7.5%

ethics

For

serum

incubated bovine

in a microcentrifuge with rabbit anti-KDR

(50 pA) was preincubated

protein

rabbit

(35). The lysate was then slurry (saturated with

albumin) mm. The

KDR

taming

anti-KDR

FITC-labeled

1 h at room temperature (RT) SO pA of protein A-Sepharose

immunoprecipitation

immunofluorescence

polyclonal

anti-mouse

The

or PDGF-R.

over

rabbit

(IgG-1)

sFlt-l

Immunofluorescence

Double-label

anti-rabbit Germany)

Isolation of KDR from Porcine Endothelial Cells Overexpressing the KDR Receptor. Cell lysate from 1 X lO cells (800 pA) was precleared

Double-Label

and cells isotypes

Kit (150-1).

KDR-l monoclonal antibody did not cross-react with PDGF-R3. The Flt-l monoclonal antibody did not sKDR,

medium screened assay and

KDR extracellular

baculovirus-expressed

or sFIt-l protein, respectively. were cloned by limiting dilution. of the clone

of Nephrology

immunhistochemical nonimmune mouse and

primary

with

antibody,

sKDR

added

to the section,

together

higher

concentration

than

the primary

used

for

as competing

alkaline

the respective

with

that

or sFlt-1

or without

experiments,

demonstrations IgG or nonimphosphatase competitor

antibody,

was

at a SO-fold

the antibody.

niques.

VEGF/VPF

in Vitro

Binding Table

I.

Gender were

and

age

obtained

of patients for

from

‘25I-VEGF

binding

Adult Gender

kidneys studiesa

(yr)

Gender

bovine medium Age

(wk)

x

cells

were

washed

19

buffer

20

VEGF165,

Female

22

mately

46

Female

57

Female

65

Female

22

(31,36). PLGF-2

a

VEGF,

vascular

endothelial

growth

factor.

(bFGF)

lO

serum

cells

(36).

with

(Sigma)

Diego,

similar

pM

were

(Life

Technologies, plates.

of

VEGF amounts

of

PDGF-BB, were

used

human and

for

seeded Paisley,

competition

Scot-

3 to 4 d, the

Hepes,

and

1 mg/mI

recombinant activity

fibroblast experiments

human of

Bensheim,

recombinant basic

for

in EGM

for 3 h at 4#{176}C in binding

a specific

(Immundiagnostik,

pas-

described

After

25 mM

‘25I-labeled with

between

to those

cells

with

microvascular

CA)

in 12-well

DMEM

25I-VEGF,

j.tCi/ng

(3 1 ,33),

FCS

human

pH 7.4, and incubated 23

named Different

5%

using

San out

Assay on

Briefly,

per well

albumin,

containing 2000

Clonetics, carried

cells

Binding

performed

were

(Clonetics) at 2

Female

Male

8 and

land)

Male

Male

were

(MVEC;

endothelial

bovine

42

cells

6 and

I7

Female

63

endothelial sages

Fetal Age

Male

whom

Receptor

experiments

approxiGermany)

VEGF165 growth (see

(33), factor Figure

VEGF/VPF

5). Radioactivity

present

in the lysates

was

quantified

using

a gamma

the

counter.

human

with

KDR

purified

in Situ

Frozen

sections

thaw-mounted uum

Receptor

(10

p.m;

or

Control

periodic

pathologic sites

In short.

consecutive

DMEM

supplemented

4 .tg!ml

leupeptin,

then with

and

the

labeled

binding

was

of a 700-fold

excess

were

twice

washed

to remove

For

unspecific

the specificity

‘251-VEGF VEGF and

binding

incubated

The

standards

sections

Imaging,

tissue

using

The

sites generated

and Pad and

dry

(Bniax) with

After

San

Diego, more

incubation were

Rochester,

NY).

consecutive

(Figure that

von

covered

fixed

sections

with

were

densito-

of

Kodak

stored

Prism,

‘25I-VEGF

we used

the coverslip

for

tech-

‘251-VEGF.

the

(Kodak,

but

and

In

human

and

4c)

positive

more

label

for

4a) colocalized

the

latter

antigen

(Figure

ex-

2, e and also

Willebrand

were

in In

capillaries obtained

d,

had

factor.

a

Compa-

with

the

or Fit- 1 protein

KDR

as

appeared

factor.

VEGF-R

von

glomeruli for

depicted

be demonstrated

(Figure

Peritubular

was

as soon

factor

not

with

than

specific

which 1 ),

(FIt-

Willebrand

and KDR

4a).

fetal

(Figures

(Figure

3b

3c)

were

monoclonal also

in peritubular

cells

and

kidney,

mono-

(Figures

3a

cells

VEGF-R. endothelial (Figures

smooth

glomerubar

endotheliab

for only

capillaries

vascular

VEGF

adult and

positive

antibodies,

muscle

cells

capilof

As

shown

cells

were

3d

and

4b).

did

not

exhibit

arteries by

the

stained, Mesangial staining

receptors.

Characteristics

counterstained

with

VEGF To

binds

receptors,

the

MVEC.

The

specifically

specific

to

radioactive

binding PDGF

could

was

be

was

achieved bFGF

Fifty with

did

whether

cell

surface

incubated

with

human

in

percent 58 pM

not

iodinated

VEGF/VPF

competed

or PLGF-2. and

Receptors analyze

the

ligand

binding

VEGFI6S

PLGF-2.

eosin.

of

MVEC in Culture.

VEGF165

unlabeled

10 to 14 d. developed

and

could

factor, and

1

convolutes,

3a),

for

r2l2,

Willebrand

von

strongly

antibodies

Binding

binding

2 emulsion

at 4#{176}C for

Readymatic,

the

Scatchard

(GraphPad

NTB

antibody

von

for

of VEGF-R

results

laries

for

experiments.

SO pM

to

were

and S-structures

glomerular

Adult Kidney.

per mg

between with

belong

as PDGF-R

and 4a).

of the

quantification

3a

coexpression

system

values

of

more

Figures

Human

precisely,

in Kodak

were

using

second

proteins

1B).

VEGF-R

VEGF-R

Willebrand often

rable

d).

FIt- 1 (Figure

clonal

of ‘25I-VEGF

programs,

the

immunofluorescence

for

negative

stages

VEGF-R, pressed

[i25I]

bound

analysis

level,

Proteins

with

positive

were

developed with

kidney)

with

saturation

but

(Figure

in comma-shaped

2, a through

cells

CA).

of renal

Slides

D19,

air.

in fmol for

from

relationships

structures

sections

derived

regression

the anatomical

renal

in dis-

amount

method

to a

lymphatic

with

such

with the anti-KDR

cells

has been established (37). (Kd) and the maximal num-

curve-fitting

nonparametric

Software,

in Kodak

were

the

expressed

of this

were

nique.

was

sections constant

To define sites

to determine

which

proteins

endothelial

fetal

Grey

in

receptor

receptor

kinases

Double-label

in the pre-

analysis

Canada).

led 1 A).

at a moderate

Other

VEGF-Receptor

cross-reactive

M) of unlabeled

image

Ontario,

validity

analysis Graph

a computerized

of

protein

of cold

films

antibody

(Figure

reactivity

VEGF

antibody

Kidney.

VEGF-R

performed by autoBraunschweig,

The

the tyrosine

the

slightly

once

for 2 d, together

binding sites in tissue equilibrium dissociation

ber of binding Data

exposed

used

sections,

equivalent.

peptide The

with

for

to l0

i2

binding sites was (Amersham-Buchler,

were

were

to tissue

(lO

cross

noticed.

by

Fetal VEGF-R

the

and

recognized

same

incubation,

in detail,

a weak to

the

(I 10 kD)

is expressed

I) was

related

1035

of

KDR

detected, that

Also,

III receptor

Localization

in I .5 M NaCl

(adult

not

class

also

(FLT-

used.

were

a stream

binding 40 pM

doses

St. Catharines,

[1251I-microscales bound

under

Amersham-Buchler).

analyzed

(MCID

After

are

not

up to

experiments sections

that

of

immunobands

a receptor

cells.

receptor

and

(microscales,

metrically

dried

with

Quantification of ‘251-VEGF radiography with Hyperfilm-3H Germany).

in the

to proteoglycans,

then

increasing

at RT pM

endothelial

Preincubation

specific

antibody

protein,

Kidney

MgCI,.

and

once

in

Sections

experiments

5 to 250

in PBS,

of ‘251-VEGF

were

and with PLGF-2.

0.5 mM

polyclonal

same

‘25I-VEGF.

fluoride.

VEGF.

10 mm

of

taken.

(35).

in Human

domain

of the

FLT-4

VEGF

free

were

Hepes,

on alternate

of unlabeled for

at RT for 30 s, and

sections

tissues with

saturation from

determined

slides

To test

25 mM

ranging

vac-

at RT for 30 mm

experiments

ligand.

sence

kidney

only

by incubation

FCS.

under

the

by hematoxylin-

microscopy,

preincubated

competition

Nonspecific

water

labeled 10%

that

light

up to I 2 h for association

concentrations

15 mm

ensure

This

on a cryostat,

placed

S nM phenylmethylsulfonyl

‘25I-VEGF,

tilled

stained

were

with

saturation

buffer

were

and

incubated

3 h for

and

were

by

Kidney

cut

slides,

to

sections

were

glass

determined

for VEGF

in Human

fetal)

sections

acid-Schiff

changes,

Binding

were

and

on gelatin-coated

at 4#{176}C overnight.

eosin

Binding

adult

protein

extracellular

disappearance

VEGF/VPF

Receptors

the

excess

inhibition VEGFI#{212}S and

compete

for

of of the

VEGF

8 nM

binding

(Figure 5). Statistical Data

Human

Analyses are

determine

given

as means

statistical

regarded

± SD.

significance

as a significant

The

with

difference

unpaired

t test

a two-tailed

between

groups

was

used

to

adult

P value

of

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