Advanced

glycation

end

products

cause

increased

CCN

family

and

extracellular

matrix

gene

expression

in

the

diabetic

rodent

retina

Aims/hypothesis

Referred

to

as

CCN

the

family

of

growth

factors

consisting

of

cystein-rich

protein

-LRB-

also

known

as

-RRB-

connective

tissue

growth

factor

-LRB-

CTGF

also

known

as

-RRB-

nephroblastoma

overexpressed

gene

-LRB-

NOV

also

known

as

-RRB-

and

signalling

pathway

proteins

and

-LRB-

and

also

known

as

and

-RRB-

affects

cellular

growth

differentiation

adhesion

and

locomotion

in

wound

repair

fibrotic

disorders

inflammation

and

angiogenesis

AGEs

formed

in

the

diabetic

milieu

affect

the

same

processes

leading

to

diabetic

complications

including

diabetic

retinopathy

We

hypothesised

that

pathological

effects

of

AGEs

in

the

diabetic

retina

are

a

consequence

of

AGE-induced

alterations

in

CCN

family

expression

Introduction

Diabetic

retinopathy

is

a

major

complication

of

diabetes

and

a

leading

cause

of

blindness

-LSB-

-RSB-

Despite

recent

progress

in

understanding

the

pathogenesis

of

diabetic

retinopathy

further

research

is

warranted

as

the

disease

remains

neither

preventable

nor

curable

Diabetic

retinopathy

is

preceded

by

an

asymptomatic

preclinical

phase

in

which

a

microangiopathy

develops

which

is

characterised

by

diffusely

increased

vascular

permeability

and

capillary

basement

membrane

thickening

resulting

from

excess

accumulation

of

extracellular

matrix

components

-LSB-

-RSB-

In

later

stages

of

preclinical

diabetic

retinopathy

endothelial

cell

and

pericyte

loss

leads

to

vascular

cell

death

and

the

development

of

acellular

capillaries

Experimental

prevention

of

basement

membrane

thickening

has

been

shown

to

ameliorate

these

retinal

vascular

changes

-LSB-

-RSB-

Thus

in

galactose-fed

rats

a

model

of

diabetes

downregulation

of

fibronectin

synthesis

partly

prevented

retinal

basement

membrane

thickening

and

also

reduced

pericyte

and

endothelial

cell

loss

-LSB-

-RSB-

Combined

downregulation

of

mRNA

levels

of

the

extracellular

matrix

components

fibronectin

collagen

type

IV

alpha

-LRB-

-RRB-

and

laminin

beta

-LRB-

-RRB-

not

only

prevented

increases

in

their

protein

levels

but

also

reduced

vascular

leakage

in

the

retina

of

rats

with

streptozotocin-induced

diabetes

-LSB-

-RSB-

These

findings

suggest

that

basement

membrane

thickening

is

not

just

an

epiphenomenon

of

the

diabetic

state

but

may

be

instrumental

in

the

progression

of

sight-threatening

diabetic

retinopathy

Modulation

of

basement

membrane

thickening

may

therefore

have

a

preventive

effect

on

the

development

of

diabetic

retinopathy

However

the

mechanisms

leading

to

diabetes-induced

basement

membrane

thickening

remain

largely

unknown

One

of

the

postulated

mechanisms

is

the

formation

of

AGEs

in

the

diabetic

milieu

Inhibition

of

AGE

formation

by

aminoguanidine

has

been

shown

to

protect

against

retinal

capillary

basement

membrane

thickening

-LSB-

-RSB-

AGE

have

also

been

shown

to

induce

extracellular

matrix

synthesis

in

the

diabetic

rat

kidney

A

similar

induction

of

extracellular

matrix

synthesis

was

also

shown

to

be

mediated

by

connective

tissue

growth

factor

-LRB-

CTGF

also

known

as

-RRB-

-LSB-

-RSB-

a

member

of

the

family

of

proteins

referred

to

as

CCN

-LRB-

for

cystein-rich

protein

-LSB-

also

known

as

-RSB-

CTGF

and

nephroblastoma

overexpressed

gene

-LSB-

NOV

also

known

as

-RSB-

-RRB-

CTGF

leads

to

accumulation

of

extracellular

matrix

by

induction

of

collagen

fibronectin

and

laminin

synthesis

as

well

as

decreased

proteolysis

of

extracellular

matrix

components

as

a

result

of

increased

production

of

tissue

inhibitors

of

metalloproteases

-LRB-

TIMP

-RRB-

-LSB-

-RSB-

Recently

we

observed

that

Ctgf

mice

-LRB-

lacking

one

functional

allele

for

Ctgf

-RRB-

are

protected

from

diabetes-induced

basement

membrane

thickening

of

retinal

and

kidney

glomerular

capillaries

-LRB-

-LSB-

-RSB-

P.

Roestenberg

F

A.

Van

Nieuwenhoven

R.

Verheul

et

al.

unpublished

results

-RRB-

We

hypothesised

in

the

present

study

that

AGE-induced

basement

membrane

thickening

observed

in

the

retina

is

at

least

partly

mediated

by

CTGF

To

establish

the

role

of

CTGF

in

the

AGE-induced

production

of

vascular

basement

membrane

components

and

their

mediators

we

investigated

the

effects

of

aminoguanidine

on

the

levels

of

CTGF

the

other

CCN

family

members

-LRB-

NOV

and

signalling

pathway

protein

and

-LSB-

and

also

known

as

and

-RSB-

-RRB-

-LSB-

-RSB-

and

vascular

basement

membrane-related

molecules

in

the

retina

of

rats

with

streptozotocin-induced

diabetes

as

well

as

in

the

retina

of

mice

infused

with

AGE

Materials

and

methods

Animals

All

animal

studies

were

carried

out

in

accordance

with

the

Guide

for

the

Care

and

Use

of

Laboratory

Animals

of

the

National

Institutes

of

Health

All

experiments

involving

rats

were

reviewed

and

approved

by

the

ethics

committee

for

animal

care

and

use

of

the

Free

University

Medical

Centre

Amsterdam

the

Netherlands

All

experiments

involving

mice

were

carried

out

in

accordance

with

British

Home

Office

regulations

Streptozotocin-induced

diabetic

rat

model

Adult

Wistar

rats

-LRB-

Charles

River

Maastricht

the

Netherlands

-RRB-

weighing

approximately

g

were

randomly

divided

into

three

experimental

groups

a

control

group

-LRB-

n

-RRB-

a

diabetic

group

-LRB-

n

-RRB-

and

a

diabetic

group

treated

with

aminoguanidine

hydrogen

carbonate

-LRB-

n

Fluka

Buchs

Switzerland

-RRB-

Diabetes

was

induced

by

a

single

i.p.

injection

of

mg/kg

streptozotocin

-LRB-

Sigma

St

Louis

MO

USA

-RRB-

Immediately

prior

to

use

streptozotocin

was

dissolved

in

cold

mol/l

citrate

buffer

pH

Control

rats

received

a

single

i.p.

injection

of

mol/l

citrate

buffer

only

Aminoguanidine

was

administered

from

day

at

a

dose

of

g/l

in

drinking

water

Serum

glucose

levels

and

body

weight

were

monitored

at

the

start

and

end

of

the

experiment

Diabetes

was

verified

by

a

serum

glucose

level

mmol/l

At

weeks

half

of

the

rats

were

randomly

selected

from

the

three

experimental

groups

and

killed

with

a

lethal

dose

of

pentobarbital

sodium

-LRB-

i.p.

-RRB-

At

weeks

the

remaining

rats

were

killed

Eyes

from

each

rat

were

rapidly

enucleated

one

being

snap-frozen

in

liquid

nitrogen

and

stored

at

°C

while

the

contralateral

eye

was

fixed

in

paraformaldehyde

Additionally

blood

samples

were

collected

and

plasma

levels

of

Nɛ

-LRB-

carboxymethyl

-RRB-

lysine

-LRB-

CML

-RRB-

were

measured

by

stable-isotope

dilution

tandem

mass

spectrometry

-LSB-

-RSB-

In

vivo

administration

of

exogenous

AGE

Female

mice

-LRB-

weeks

old

-RRB-

were

randomly

assigned

to

two

groups

of

equal

size

and

injected

i.p.

with

either

native

mouse

serum

albumin

-LRB-

MSA

-RRB-

or

glycoaldehyde-modified

MSA

-LRB-

mg/kg

-RRB-

daily

for

seven

consecutive

days

At

h

after

the

final

injections

mice

were

killed

eyes

enucleated

and

retinas

dissected

freshly

before

being

snap-frozen

in

liquid

nitrogen

Preparation

of

AGE-modified

albumin

The

glycoaldehyde-modified

MSA

preparation

was

made

according

to

Nagai

et

al.

-LSB-

-RSB-

Following

dialysis

against

PBS

endotoxin

was

removed

using

an

endotoxin-removing

column

-LRB-

Pierce

Rockford

IL

USA

-RRB-

Glycoaldehyde-MSA

and

native

MSA

were

passed

three

times

through

separate

columns

to

ensure

that

all

contaminating

endotoxin

was

removed

Analysis

of

the

CML

content

of

glycoaldehyde-MSA

and

native

MSA

was

performed

using

gas

chromatography

mass

spectrometry

The

lysine

content

of

the

samples

was

analysed

by

cation

exchange

chromatography

and

the

values

for

CML

were

corrected

for

lysine

loss

and

expressed

as

mmol

CML/mol

lysine

as

previously

reported

-LSB-

-RSB-

RNA

isolation

and

mRNA

quantification

Snap-frozen

rat

and

mouse

eyes

were

allowed

to

thaw

in

ice-cold

RNAlater

-LRB-

Ambion

Austin

TX

USA

-RRB-

The

anterior

chambers

of

the

eyes

were

removed

and

the

retinas

were

carefully

dissected

Total

RNA

was

extracted

with

TRIzol

reagent

-LRB-

Invitrogen

Carlsbad

CA

USA

-RRB-

The

amount

of

total

retinal

RNA

isolated

was

approximately

μg

per

retina

-LRB-

spectrophotometric

measurements

at

nm

-RRB-

with

no

significant

differences

between

the

experimental

groups

The

integrity

of

the

RNA

samples

was

verified

using

an

automated

electrophoresis

system

-LRB-

Experion

Bio-Rad

Hercules

CA

USA

-RRB-

All

samples

had

sharp

ribosomal

RNA

bands

with

no

sign

of

degradation.A

aliquot

of

total

RNA

was

treated

with

DNAse

I

-LRB-

amplification

grade

Invitrogen

-RRB-

and

reverse-transcribed

into

first

strand

cDNA

with

Superscript

II

and

oligo

-LRB-

dT

-RRB-

-LRB-

Invitrogen

-RRB-

Details

of

the

primers

are

given

in

the

Electronic

supplementary

material

-LRB-

ESM

-RRB-

-LRB-

ESM

Table

-RRB-

Specificity

of

the

primers

was

confirmed

by

a

nucleotide

nucleotide

BLAST

-LRB-

-RRB-

search

The

presence

of

a

single

PCR

product

was

verified

by

both

the

presence

of

a

single

melting

temperature

peak

and

detection

of

a

single

band

of

the

expected

size

on

a

agarose

gel.Real-time

quantitative

PCR

-LRB-

qPCR

-RRB-

was

performed

in

a

sequence

detection

system

-LRB-

ABI

Prism

Applied

Biosystems

Foster

City

CA

USA

-RRB-

For

each

primer

set

a

mastermix

was

prepared

consisting

of

SYBR

Green

PCR

buffer

-LRB-

Eurogentec

Seraing

Belgium

-RRB-

mmol/l

μmol/l

each

of

dATP

dGTP

and

dCTP

μmol/l

dUTP

U

AmpliTaq

Gold

-LRB-

Eurogentec

-RRB-

and

pmol

primers

All

cDNA

samples

were

diluted

and

amplified

using

the

following

PCR

protocol

min

at

°C

followed

by

cycles

of

s

at

°C

and

s

at

°C

and

a

melting

program

-LRB-

°C

-RRB-

Relative

gene

expression

was

calculated

using

the

equation

R

E

Ct

where

E

is

the

mean

efficiency

of

all

samples

for

the

gene

being

evaluated

and

Ct

is

the

cycle

threshold

for

the

gene

as

determined

during

real-time

PCR

All

qPCR

experiments

were

performed

at

least

twice.Real-time

qPCR

data

from

the

mouse

experiments

were

normalised

using

rRNA

which

was

determined

to

be

stably

expressed

in

all

experimental

groups

For

the

rat

experiments

no

suitable

housekeeping

genes

that

were

not

regulated

by

the

diabetic

background

could

be

found

Therefore

the

rat

data

were

normalised

using

the

relative

starting

amounts

of

cDNA

which

was

determined

using

a

novel

technique

recently

developed

in

our

laboratory

-LRB-

J.

M.

Hughes

I.

Klaassen

W.

Kamphuis

C.

J.

F.

Van

Noorden

and

R.

O.

Schlingemann

unpublished

results

-RRB-

In

brief

reverse

transcription

reactions

were

carried

out

in

duplicate

with

one

set

of

reactions

containing

the

normal

dNTP

mix

and

the

parallel

set

of

reactions

containing

a

dNTP

mix

with

dCTP

From

each

sample

μl

of

the

dCTP-incorporated

cDNA

were

pipetted

on

to

separate

nitrocellulose

filters

which

were

allowed

to

air-dry

After

washing

with

mol/l

phosphate

buffer

radioactivity

of

the

filters

was

measured

using

a

scintillation

counter

-LRB-

Beckman

Coulter

Fullerton

CA

USA

-RRB-

Western

blotting

Protein

was

isolated

from

paraformaldehyde-fixed

retinal

tissue

as

described

by

Shi

et

al.

-LSB-

-RSB-

In

brief

retinas

were

dissected

from

the

paraformaldehyde-fixed

rat

eyes

and

pooled

in

ml

Eppendorf

vials

in

antigen-retrieval

buffer

-LRB-

mmol/l

Tris

SDS

pH

-RRB-

The

pooled

samples

were

then

dissociated

using

a

pestle

and

incubated

at

°C

for

min

followed

by

h

at

°C

Supernatant

fractions

were

collected

after

centrifugation

at

°C

for

min

at

g.

Protein

concentrations

were

determined

with

a

bicinchoninic

acid

protein

assay

kit

-LRB-

Perbio

Etten-Leur

the

Netherlands

-RRB-

and

adjusted

to

μg/μl

For

SDS-PAGE

and

western

blots

proteins

were

separated

using

mini

gels

under

reducing

conditions

Following

gel

electrophoresis

proteins

were

transferred

to

a

nitrocellulose

filter

-LRB-

Whatman

Schleicher

Schuell

Brentford

Middlesex

UK

-RRB-

using

a

semi-dry

transfer

cell

-LRB-

Bio-Rad

-RRB-

At

the

end

of

the

transfer

the

filter

was

blocked

in

blocking

buffer

-LRB-

non-fat

skimmed

milk

powder

BSA

mmol/l

in

Tris-buffered

saline

and

Tween

-RRB-

overnight

at

°C

while

being

gently

rocked

The

filter

was

incubated

in

blocking

solution

for

h

at

room

temperature

with

the

relevant

antibodies

as

defined

below

Following

three

washes

in

the

blots

were

incubated

in

blocking

solution

with

horseradish

peroxidase-conjugated

goat

anti-rabbit

or

goat

anti-mouse

antibodies

for

h

at

room

temp

After

extensive

washing

blots

were

developed

using

a

chemiluminescent

kit

-LRB-

SuperSignal

West

Pico

Perbio

-RRB-

Filters

were

exposed

to

X-ray

film

-LRB-

Kodak-Biomax

Herts

UK

-RRB-

Primary

antibodies

-LRB-

ESM

Table

-RRB-

were

diluted

with

skimmed

milk

powder

in

TBS/Tween

and

horseradish

peroxidase-conjugated

goat

anti-rabbit

or

goat

anti-mouse

-LRB-

Perbio

-RRB-

was

diluted

Intensity

of

bands

was

quantified

by

densitometry

using

AlphaEase

software

-LRB-

AlphaInnotech

San

Leandro

CA

USA

-RRB-

Immunohistochemistry

Cryostat

sections

-LRB-

thick

-RRB-

were

stained

using

an

indirect

immunoperoxidase

procedure

as

previously

described

-LSB-

-RSB-

Primary

antibodies

are

listed

in

ESM

Table

Primary

antibody

was

omitted

for

negative

controls

Indirect

immunoperoxidase

staining

was

performed

using

histostaining

reagents

-LRB-

Powervision

ImmunoVision

Daly

City

CA

USA

-RRB-

for

all

sections

except

those

incubated

with

the

antibody

The

sections

were

indirectly

stained

using

horseradish

peroxidase-labelled

rabbit

anti-goat

antibody

-LRB-

Dako

Glostrup

Denmark

-RRB-

Statistics

CML

data

were

transformed

to

obtain

a

normal

distribution

Significant

differences

-LRB-

p

-RRB-

in

glucose

and

CML

plasma

levels

and

in

gene

expression

levels

among

groups

were

calculated

with

single

ANOVA

The

Bonferroni

post

hoc

test

was

used

to

perform

pairwise

comparisons

of

groups

Results

Glucose

and

CML

levels

in

control

and

diabetic

rats

Induction

of

diabetes

and

the

degree

of

hyperglycaemia

in

streptozotocin-treated

rats

was

established

by

serum

glucose

levels

-LRB-

Fig.

-RRB-

Streptozotocin

treatment

resulted

in

a

three

to

fourfold

increase

in

serum

glucose

concentration

after

and

weeks

irrespective

of

aminoguanidine

treatment

Fig.

glucose

-LRB-

a

-RRB-

and

CML

-LRB-

b

-RRB-

levels

in

control

and

diabetic

rats

at

and

weeks

of

streptozotocin-induced

diabetes

White

bars

control

rats

black

bars

diabetic

rats

cross-hatched

bars

diabetic

rats

treated

with

aminoguanidine

p

and

p

for

difference

between

experimental

and

control

group

The

aminoguanidine

group

was

only

significantly

different

from

the

groups

with

diabetes

at

weeks

-LRB-

p

-RRB-

The

error

bars

show

the

standard

deviation

for

each

groupPlasma

levels

of

CML

were

measured

to

determine

the

efficacy

of

aminoguanidine

treatment

CML

plasma

levels

were

elevated

by

twofold

at

and

weeks

after

streptozotocin-treatment

-LRB-

Fig.

-RRB-

Aminoguanidine

treatment

had

no

effect

on

CML

levels

at

weeks

but

at

weeks

the

CML

levels

were

decreased

by

approximately

CCN

family

gene

expression

in

control

and

diabetic

rats

After

weeks

of

diabetes

mRNA

levels

in

the

diabetic

retina

were

increased

by

threefold

against

control

retina

Treatment

with

aminoguanidine

reduced

expression

to

levels

that

were

not

significantly

different

from

control

levels

-LRB-

Fig.

-RRB-

protein

was

mainly

localised

in

the

ganglion

cell

layer

-LRB-

Fig.

-RRB-

No

differences

in

staining

patterns

were

found

between

experimental

groups

Fig.

expression

of

CCN

family

members

Fold

change

compared

with

control

values

in

retinal

mRNA

levels

of

CCN

family

members

in

streptozotocin-induced

diabetic

rats

at

-LRB-

white

bars

-RRB-

and

weeks

-LRB-

cross-hatched

bars

-RRB-

after

streptozotocin-induction

and

in

aminoguanidine-treated

streptozotocin-induced

diabetic

rats

at

-LRB-

black

bars

-RRB-

and

weeks

-LRB-

grey

bars

-RRB-

of

diabetes

p

for

difference

between

experimental

group

and

control

group

p

for

difference

between

aminoguanidine-treated

diabetic

group

and

diabetes-only

groupFig

staining

patterns

of

a

b

CTGF

and

c

in

control

rat

retinas

Intense

staining

of

and

CTGF

was

present

in

large

cell

bodies

of

the

ganglion

cell

layer

-LRB-

GCL

-RRB-

and

weak

staining

in

the

inner

plexiform

layer

-LRB-

IPL

-RRB-

Intense

uniform

immunostaining

of

was

found

in

the

GCL

and

weak

staining

in

the

IPL

INL

inner

nuclear

layer

OPL

outer

plexiform

layer

ONL

outer

nuclear

layer

RCL

rod

and

cones

layer

RPE

retinal

pigment

epithelium

Magnification

mRNA

levels

were

elevated

by

twofold

at

weeks

of

streptozotocin-induced

diabetes

Aminoguanidine

treatment

almost

completely

prevented

this

increase

-LRB-

Fig.

-RRB-

Western

blotting

showed

a

increase

in

CTGF

protein

levels

in

the

retina

of

diabetic

rats

at

weeks

whereas

aminoguanidine

treatment

also

prevented

this

effect

-LRB-

Fig.

-RRB-

CTGF

immunostaining

was

mainly

found

in

the

ganglion

cell

layer

and

was

more

diffuse

throughout

the

outer

plexiform

layer

inner

nuclear

layer

and

inner

plexifrom

layer

-LRB-

Fig.

-RRB-

Differences

in

staining

between

experimental

groups

were

not

observed

Fig.

protein

levels

in

retina

of

control

and

diabetic

rats

a

Western

blots

of

CTGF

and

GAPDH

as

loading

control

Samples

were

pooled

for

each

group

A

prominent

band

of

CTGF

protein

is

present

in

the

diabetic

group

-LRB-

n

-RRB-

whereas

protein

bands

were

similar

in

all

other

groups

-LRB-

control

rats

at

-LSB-

n

-RSB-

and

-LSB-

n

-RSB-

weeks

diabetic

rats

at

-LSB-

n

-RSB-

weeks

and

diabetic

rats

treated

with

aminoguanidine

at

-LSB-

n

-RSB-

and

-LSB-

n

-RSB-

weeks

-RRB-

b

The

blots

were

quantified

by

densitometry

and

expressed

as

a

ratio

of

CTGF

and

mRNA

levels

were

low

in

retinas

of

all

groups

of

rats

The

levels

never

differed

more

than

between

experimental

and

control

groups

Due

to

small

standard

deviations

significant

differences

were

found

-LRB-

Fig.

-RRB-

but

it

is

doubtful

whether

these

small

differences

are

biologically

meaningful

Nov

and

mRNA

expression

levels

were

too

low

to

be

detected

in

all

groups

of

rats

Expression

of

transforming

growth

factor

beta

and

in

control

and

diabetic

rats

To

determine

whether

-LRB-

-RRB-

transforming

growth

factor

beta

-LRB-

TGFB

-RRB-

an

upstream

regulator

of

CTGF

production

is

induced

by

diabetes

and

-LRB-

-RRB-

this

induction

is

prevented

by

aminoguanidine

in

rat

retina

we

examined

the

mRNA

levels

of

its

two

most

common

isoforms

and

mRNA

expression

was

decreased

by

approximately

in

retina

of

rats

with

diabetes

for

weeks

-LRB-

Fig.

-RRB-

Whether

this

difference

is

biologically

relevant

remains

to

be

determined

In

all

other

experimental

groups

and

mRNA

levels

were

similar

to

control

levels

-LRB-

Fig.

-RRB-

Immunohistochemical

analysis

of

revealed

a

vascular

pattern

of

staining

in

all

experimental

groups

-LRB-

Fig.

-RRB-

Fig.

change

of

and

expression

in

diabetic

rats

at

-LRB-

white

bars

-RRB-

and

weeks

-LRB-

cross-hatched

bars

-RRB-

after

streptozotocin-induction

and

in

aminoguanidine-treated

diabetic

rats

at

-LRB-

black

bars

-RRB-

and

weeks

-LRB-

grey

bars

-RRB-

after

streptozotocin-induction

was

significantly

decreased

in

the

diabetic

rats

after

weeks

-LRB-

p

vs

control

-RRB-

This

difference

was

not

observed

in

the

aminoguanidine-treated

group

at

weeks

expression

was

not

significantly

altered

at

either

time

pointFig

staining

patterns

of

-LRB-

a

d

-RRB-

laminin

-LRB-

b

e

-RRB-

and

fibronectin

-LRB-

c

f

-RRB-

in

retina

of

control

rats

-LRB-

a

c

-RRB-

and

rats

weeks

after

streptozotocin-induced

diabetes

-LRB-

STZ

-RRB-

-LRB-

d

f

-RRB-

Immunostaining

of

laminin

and

fibronectin

was

confined

to

the

retinal

microvasculature

and

did

not

notably

differ

between

control

and

streptozotocin

sections

Magnification

Expression

of

extracellular

matrix

molecules

in

control

and

diabetic

rats

CTGF

and

are

known

modifiers

of

the

extracellular

matrix

Therefore

we

investigated

expression

patterns

of

various

extracellular

matrix

components

mRNA

levels

in

the

rat

retina

were

elevated

by

threefold

after

weeks

of

diabetes

Aminoguanidine

treatment

inhibited

this

induction

of

mRNA

levels

by

-LRB-

Fig.

-RRB-

mRNA

levels

showed

a

increase

in

diabetic

rats

which

was

virtually

unaffected

by

aminoguanidine

treatment

Fibronectin

mRNA

levels

were

not

affected

by

streptozotocin-induced

diabetes

mRNA

levels

were

not

affected

either

but

mRNA

levels

were

elevated

by

in

retina

of

diabetic

rats

this

increase

being

completely

prevented

by

aminoguanidine

treatment

-LRB-

Fig.

-RRB-

Laminin

and

fibronectin

were

localised

immunohistochemically

in

microvessels

in

rat

retina

-LRB-

Fig.

-RRB-

This

staining

pattern

was

similar

in

all

groups

immunostaining

was

restricted

to

the

ganglion

cell

layer

in

all

groups

of

rats

-LRB-

Fig.

-RRB-

Fig.

expression

of

extracellular

matrix

components

Fold

change

of

extracellular

matrix

gene

expression

as

indicated

in

diabetic

rats

at

-LRB-

white

bars

-RRB-

and

weeks

-LRB-

cross-hatched

bars

-RRB-

after

streptozotocin-induction

and

in

aminoguanidine-treated

diabetic

rats

at

-LRB-

black

bars

-RRB-

and

weeks

-LRB-

grey

bars

-RRB-

after

streptozotocin-induction

p

for

difference

between

experimental

and

control

group

p

for

difference

between

aminoguanidine-treated

diabetic

group

and

diabetes-only

group

CCN

family

mRNA

expression

in

control

and

AGE-treated

mice

AGE-treated

MSA

infusion

of

mice

induced

an

increased

retinal

expression

of

and

Ctgf

mRNA

by

and

twofold

respectively

compared

with

control

mice

-LRB-

Fig.

-RRB-

and

mRNA

expression

was

not

affected

by

AGE-treated

MSA

infusion

-LRB-

data

not

shown

-RRB-

Fig.

mRNA

levels

of

and

Ctgf

in

retinas

of

control

-LRB-

white

bars

-RRB-

and

AGE-treated

-LRB-

black

bars

-RRB-

mice

depicted

as

fold

change

in

comparison

with

control

mice

p

for

effect

of

AGE

treatment

on

and

Ctgf

mRNA

levels

Summary

of

results

In

the

retina

of

rats

with

streptozotocin-induced

diabetes

mRNA

levels

of

the

CCN

family

members

and

Ctgf

were

increased

threefold

at

weeks

and

twofold

at

weeks

respectively

whereas

expression

of

all

other

CCN

family

members

was

not

notably

affected

CTGF

protein

levels

in

retina

were

also

elevated

twofold

at

weeks

of

diabetes

In

the

aminoguanidine-treated

diabetic

rats

these

increases

were

partly

counteracted

by

treatment

with

this

AGE

inhibitor

In

line

with

these

findings

treatment

of

mice

with

exogenous

AGE

induced

elevated

retina

mRNA

levels

of

and

Ctgf

but

not

of

the

other

CCN

family

members

In

parallel

mRNA

levels

of

some

extracellular

matrix

components

were

also

increased

in

the

retina

of

diabetic

rats

an

effect

also

prevented

by

aminoguanidine

treatment

Discussion

We

present

here

a

comprehensive

expression

analysis

of

the

CCN

family

of

fibrosis-inducing

cytokines

in

the

retina

of

rats

with

streptozotocin-induced

diabetes

Messenger

RNA

and

protein

levels

of

and

CTGF

both

known

to

be

capable

of

modulating

the

extracellular

matrix

were

increased

in

diabetic

rats

whilst

the

AGE

inhibitor

aminoguanidine

attenuated

these

effects

of

diabetes

We

also

found

that

exogenously

administered

AGEs

are

capable

of

inducing

and

Ctgf

expression

in

the

adult

mouse

retina

in

vivo

Taken

together

these

data

present

evidence

that

AGEs

are

both

necessary

and

sufficient

to

cause

increased

levels

of

and

CTGF

in

the

diabetic

retina

Expression

of

CTGF

at

the

mRNA

or

protein

level

has

previously

been

demonstrated

in

vivo

in

normal

and

diabetic

rat

-LSB-

-RSB-

and

human

retina

-LSB-

-RSB-

as

well

as

in

cultured

retinal

microvascular

cells

-LSB-

-RSB-

and

astrocytes

-LSB-

-RSB-

Our

data

on

CTGF

are

in

agreement

with

a

previously

reported

twofold

increase

in

Ctgf

expression

in

the

diabetic

rat

retina

-LSB-

-RSB-

In

our

study

immunostaining

with

a

polyclonal

anti-CTGF

antibody

showed

staining

in

the

ganglion

cell

layer

-LSB-

-RSB-

and

diffusely

throughout

the

larger

part

of

the

inner

rat

retina

In

contrast

in

a

previous

study

of

the

human

diabetic

retina

employing

a

monoclonal

anti-CTGF

antibody

CTGF

was

detected

in

microglia

and

pericytes

in

the

microvasculature

of

the

inner

retina

-LSB-

-RSB-

These

varying

patterns

of

CTGF

protein

distribution

may

be

species-related

or

due

to

differences

in

specificity

of

the

antibodies

used

Gene

expression

analysis

was

also

performed

on

the

other

five

known

members

of

the

CCN

family

We

found

expression

in

the

normal

adult

rat

retina

and

upregulation

in

the

diabetic

retina

In

addition

to

CTGF

this

suggests

a

possible

role

for

in

the

development

of

diabetes-related

retinal

sequelae

The

lack

of

detectable

Nov

and

expression

argues

against

a

role

for

these

two

proteins

in

normal

or

diabetic

retina

This

is

in

agreement

with

a

previous

study

which

demonstrated

that

mRNA

was

not

present

in

tissues

of

the

adult

rat

-LSB-

-RSB-

Expression

of

which

is

much

less

studied

but

known

to

suppress

cancer

cell

growth

in

vivo

-LSB-

-RSB-

was

slightly

decreased

in

diabetic

retina

whereas

a

cell

growth

suppressor

and

inhibitor

of

angiogenesis

-LSB-

-RSB-

was

increased

The

significance

of

these

findings

remains

unclear

as

the

functions

of

the

CCN

family

members

are

complex

Still

such

opposing

actions

may

be

reflective

of

a

regulatory

balance

or

may

indicate

that

some

CCN

family

members

are

redundant

although

this

is

currently

not

considered

to

be

the

case

in

other

tissues

-LSB-

-RSB-

The

role

of

CTGF

in

diabetic

renal

pathology

has

been

clearly

established

CTGF

is

responsible

for

mesangial

expansion

-LSB-

-RSB-

and

increased

extracellular

matrix

deposition

-LSB-

-RSB-

as

observed

in

early

stages

of

diabetic

nephropathy

Therefore

CTGF

and/or

may

have

a

similar

role

in

diabetic

retina

and

be

responsible

for

the

thickening

of

microvascular

basement

membranes

observed

in

early

stages

of

diabetic

retinopathy

We

also

therefore

examined

expression

patterns

of

the

genes

encoding

several

extracellular

matrix-related

molecules

Expression

of

the

basement

membrane

component

was

found

to

be

increased

concomitantly

with

expression

after

weeks

of

diabetes

an

increase

significantly

reduced

in

aminoguanidine-treated

diabetic

rats

At

weeks

of

diabetes

and

expression

demonstrated

a

concomitant

increase

with

Ctgf

which

was

significantly

attenuated

in

the

aminoguanidine-treated

group

-LRB-

not

statistically

significant

for

-RRB-

These

findings

suggest

a

causal

role

of

AGEs

in

the

diabetes-induced

production

of

CTGF

and

in

the

retina

Whether

increased

expression

is

also

mediated

by

AGEs

remains

to

be

determined

However

this

possibility

is

supported

by

a

previous

study

showing

that

protein

and

mRNA

levels

of

the

ribosomal

protein

SA

-LRB-

previously

known

as

laminin

receptor

-LSB-

kD

ribosomal

protein

SA

-RSB-

-RRB-

are

upregulated

by

AGEs

in

cultured

retinal

microvascular

endothelial

cells

-LSB-

-RSB-

AGE

CML

plasma

levels

used

in

our

study

as

a

marker

of

AGE

formation

in

the

rat

diabetes

model

were

not

altered

at

weeks

in

diabetic

rats

making

an

effect

of

AGEs

at

this

time

point

questionable

However

it

should

be

noted

that

CML

is

merely

one

of

many

types

of

AGE

known

to

be

generated

under

hyperglycaemic

conditions

-LSB-

-RSB-

and

that

aminoguanidine

has

been

shown

to

decrease

serum

AGE

levels

in

diabetic

rats

as

early

as

weeks

after

diabetes

induction

-LSB-

-RSB-

In

previous

studies

in

the

diabetic

rat

kidney

Ctgf

and

fibronectin

gene

expression

both

increased

after

weeks

of

streptozotocin-induced

diabetes

-LSB-

-RSB-

These

changes

were

prevented

by

aminoguanidine

treatment

As

AGE

accumulation

in

the

diabetic

kidney

was

prevented

by

aminoguanidine

those

authors

surmised

that

the

anti-fibrotic

effects

of

aminoguanidine

could

be

at

least

partially

mediated

by

a

decrease

in

CTGF

expression

-LSB-

-RSB-

Our

study

may

have

been

too

short

to

observe

an

increase

of

fibronectin

in

the

diabetic

retina

but

otherwise

our

results

are

in

line

with

these

findings

in

kidney

Additional

studies

will

be

necessary

to

further

elucidate

the

ability

of

and

CTGF

to

directly

modulate

these

extracellular

matrix

molecules

in

retinal

vascular

cells

TGFB

considered

to

be

the

most

important

fibrotic

factor

has

been

shown

to

upregulate

CTGF

in

many

cell

types

in

vitro

-LSB-

-RSB-

and

in

vivo

-LSB-

-RSB-

Although

our

findings

indicate

that

TGFB

production

is

not

increased

in

the

diabetic

retina

a

role

for

TGFB

in

the

observed

upregulation

of

CTGF

can

not

be

ruled

out

as

the

regulation

of

TGFB

bioavailability

is

complicated

and

not

solely

dependent

on

the

level

of

TGFB

production

-LSB-

-RSB-

We

have

recently

demonstrated

that

vascular

endothelial

growth

factor

-LRB-

VEGF

-RRB-

increases

expression

of

Ctgf

and

in

the

rat

retina

in

vivo

as

well

as

in

retinal

vascular

endothelial

cells

in

vitro

-LRB-

E.

J.

Kuiper

J.

M.

Hughes

I.

M.

C.

Vogels

et

al.

unpublished

results

-RRB-

As

AGEs

are

known

inducers

of

VEGF

in

retinal

cells

-LSB-

-RSB-

it

is

possible

that

the

increases

in

CTGF

and

observed

in

our

animal

models

are

the

result

of

AGE-induced

VEGF

A

major

finding

of

our

study

was

the

ability

of

aminoguanidine

to

attenuate

the

increase

in

Ctgf

expression

observed

in

the

diabetic

rat

retina

Tikellis

et

al.

-LSB-

-RSB-

have

reported

that

perindopril

an

ACE

inhibitor

prevents

increased

Ctgf

expression

in

diabetic

rat

retina

-LSB-

-RSB-

This

suggests

that

both

interventions

may

affect

a

common

molecular

mechanism

leading

to

the

upregulation

of

CTGF

As

ACE

inhibition

has

been

shown

to

prevent

AGE

accumulation

in

diabetic

tissues

-LSB-

-RSB-

it

is

feasible

that

inhibition

of

AGE

formation

could

be

a

common

molecular

mechanism

allowing

both

ACE

inhibitors

and

aminoguanidine

to

inhibit

the

increase

of

retinal

CTGF

In

summary

this

study

provides

the

first

evidence

that

in

addition

to

CTGF

the

CCN

family

molecules

and

play

possible

roles

in

the

development

of

early

stages

of

experimental

diabetic

retinopathy

At

the

very

least

these

results

warrant

further

study

into

the

functional

aspects

of

these

molecules

in

the

eye

and

of

how

these

aspects

pertain

to

the

development

of

diabetic

retinopathy

Additionally

we

demonstrate

for

the

first

time

that

AGEs

directly

upregulate

both

CTGF

and

levels

in

the

retina

in

vivo

and

that

aminoguanidine

inhibits

these

diabetes-induced

increases

This

provides

the

first

evidence

that

CTGF

and

are

downstream

effectors

of

AGEs

in

the

diabetic

retina

and

implicates

them

as

possible

targets

for

future

intervention

strategies

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

B I I I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

advanced glycation end products
aminoguanidine
basement membrane
connective tissue growth factor
diabetic retinopathy
experimental
extracellular matrix

cystein-rich protein 61
diabetes mellitus
gene expression regulation