Management

of

Hepatic

Angiomyolipoma

Preoperative

diagnosis

of

hepatic

angiomyolipoma

is

difficult

and

the

treatment

for

it

remains

controversial

The

aim

of

this

study

is

to

review

our

experience

in

the

treatment

of

hepatic

angiomyolipoma

and

to

propose

a

treatment

strategy

for

this

disease

We

retrospectively

collected

the

clinical

imaging

and

pathological

features

of

patients

with

hepatic

angiomyolipoma

Immunohistochemical

studies

with

antibodies

for

actin

cytokeratin

vimentin

and

c-kit

were

performed

Treatment

experience

and

long-term

follow-up

results

are

summarized

During

a

period

of

years

patients

with

hepatic

angiomyolipoma

were

treated

at

our

hospital

There

was

marked

female

predominance

-LRB-

nine

patients

-RRB-

Nine

patients

received

surgical

resection

without

complications

One

patient

received

nonoperative

management

with

biopsy

and

follow-up

One

patient

died

months

after

surgery

because

of

recurrent

disease

We

propose

all

symptomatic

patients

should

receive

surgical

resection

for

hepatic

angiomyolipoma

Conservative

management

with

close

follow-up

is

suggested

in

patients

with

asymptomatic

tumors

and

meet

the

following

criteria

-LRB-

-RRB-

tumor

size

smaller

than

cm

-LRB-

-RRB-

angiomyolipoma

proved

through

fine

needle

aspiration

biopsy

-LRB-

-RRB-

patients

with

good

compliance

and

-LRB-

-RRB-

not

a

hepatitis

virus

carrier

Introduction

Hepatic

angiomyolipoma

-LRB-

AML

-RRB-

is

a

rare

mesenchymal

tumor

of

the

liver

composed

of

smooth

muscle

cells

adipose

tissue

and

proliferating

blood

vessels

Since

its

first

description

by

Ishak

in

approximately

cases

have

been

reported

in

the

English

literature

This

type

of

tumor

is

usually

seen

in

kidneys

associated

with

tuberous

sclerosis

Definite

pathologic

diagnosis

is

made

by

identification

of

the

three

different

components

and

positive

staining

In

the

past

this

tumor

has

been

considered

an

entirely

benign

and

slow-growing

lesion

without

the

possibility

of

malignant

transformation

Therefore

several

authors

have

suggested

that

this

disease

can

be

managed

with

conservative

treatment

However

since

several

reports

have

revealed

that

this

kind

of

tumor

can

be

malignant

with

evidence

of

recurrence

Although

the

combination

of

ultrasonography

computed

tomography

-LRB-

CT

-RRB-

magnetic

resonance

-LRB-

MR

-RRB-

imaging

and

angiography

increases

the

accuracy

in

diagnosis

of

hepatic

AML

the

correct

preoperative

diagnostic

rate

of

imaging

studies

has

been

reported

to

be

less

than

Even

the

postoperative

pathologic

diagnosis

has

been

easily

mistaken

as

hepatocellular

carcinoma

-LRB-

HCC

-RRB-

Many

patients

have

been

treated

with

surgical

resection

of

the

tumor

Therefore

the

proper

treatment

of

hepatic

AML

has

remained

controversial

The

purpose

of

this

study

is

to

retrospectively

review

the

clinical

imaging

and

pathological

features

of

patients

with

hepatic

AML

treated

at

our

hospital

and

to

summarize

our

experience

in

the

diagnosis

and

treatment

of

this

disease

We

also

review

the

literature

to

highlight

the

important

questions

concerning

hepatic

AML

-LRB-

-RRB-

Is

hepatic

AML

a

pure

benign

tumor

-LRB-

-RRB-

What

is

the

natural

course

of

this

tumor

Does

the

tumor

size

enlarge

frequently

during

observation

-LRB-

-RRB-

What

difficulties

exist

in

preoperative

diagnosis

with

imaging

studies

and

fine

needle

aspiration

biopsy

-LRB-

FNAB

-RRB-

-LRB-

-RRB-

Is

it

proper

for

a

hepatitis-carrier

patient

with

hepatic

AML

to

be

treated

with

conservative

management

-LRB-

-RRB-

What

are

the

criteria

for

patients

with

hepatic

AML

to

be

treated

with

surgical

resection

or

conservative

management

Materials

and

Methods

The

clinical

imaging

and

pathological

features

of

patients

with

hepatic

AML

treated

at

the

authors

institute

were

retrospectively

reviewed

The

follow-up

information

was

obtained

in

each

case

All

tumor

tissue

was

paraffin-embedded

for

routine

hematoxylin

and

eosin

-LRB-

H&E

-RRB-

staining

Immunohistochemical

assays

were

performed

using

a

three-step

indirect

peroxidase

complex

technique

with

the

following

antibodies

-LRB-

DAKO

dilution

-RRB-

actin

-LRB-

DAKO

dilution

-RRB-

-LRB-

DAKO

dilution

-RRB-

cytokeratin

-LRB-

Biogenix

dilution

-RRB-

vimentin

-LRB-

DAKO

dilution

-RRB-

and

c-kit

-LRB-

MBL

dilution

-RRB-

Results

Patients

and

Clinical

Data

Ten

patients

with

hepatic

angiomyolipoma

were

diagnosed

at

National

Taiwan

University

Hospital

from

July

to

June

There

was

marked

female

predominance

-LRB-

-RRB-

The

median

age

was

years

old

with

a

range

from

to

years

Most

patients

-LRB-

-RRB-

presented

no

symptoms

and

were

detected

incidentally

by

health

check-ups

or

during

medical

exams

for

other

diseases

Four

of

patients

had

symptoms

caused

by

the

space-occupying

effect

of

the

tumors

such

as

abdominal

pain

abdominal

fullness

and

palpable

mass

or

other

nonspecific

symptoms

such

as

fever

general

malaise

or

body

weight

loss

-LRB-

Tables

and

-RRB-

of

them

had

a

history

of

renal

AML

or

tuberous

sclerosis

Two

patients

were

hepatitis

B-virus

-LRB-

HBV

-RRB-

carriers

The

plasma

levels

of

α-FP

and

CEA

were

within

normal

limits

in

all

patients

Table

Presentation

of

Hepatic

AngiomyolipomaClinical

FeatureNo

of

years

-LRB-

median

years

-RRB-

Gender

-LRB-

female

male

-RRB-

Symptoms

No

Abdominal

Abdominal

Palpable

Body

weight

location

Right

Left

size

-LRB-

cm

-RRB-

-LRB-

-RRB-

Based

on

radiological

Based

on

tumor

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Associated

liver

diseaseHBV

Profile

of

Patients

with

Hepatic

AngiomyolipomaCaseSex/AgeTumor

Size

-LRB-

cm

-RRB-

lobeSymtoms/SignsIncidental

FindingTreatmentOutcome/F/U

CAtypical

mass

abdominal

fullness

BW

loss

feverExtended

left

mon

recurrent

liver

and

lung

CFNAB

and

F/ULost

fullness

malaise

BW

loss

feverLeft

lateral

of

CCaudate

LNilF/U

echo

due

to

colon

cancer

s/pLeft

lateral

CLeft

health

check-up

BW

body

weight

F/U

follow-up

FNAB

fine

needle

aspiration

biopsy

mon

month

mets

metastasis

s/p

postoperation

Imaging

Studies

Based

on

the

combined

imaging

studies

of

abdominal

ultrasonography

CT

MR

imaging

and

angiography

the

diagnostic

accuracy

of

hepatic

AML

in

this

series

was

only

-LRB-

Table

-RRB-

Other

preoperative

imaging

impressions

included

hepatocellular

carcinoma

angiosarcoma

hemangioma

and

metastatic

lesions

Two

other

cases

were

diagnosed

by

fine

needle

aspiration

biopsy

-LRB-

FNAB

-RRB-

The

accurate

preoperative

diagnostic

rate

was

-LRB-

-RRB-

after

imaging

studies

and

FNAB

-LRB-

Table

-RRB-

Pathologic

Study

All

patients

had

a

single

tumor

Five

tumors

were

in

the

right

lobe

of

the

liver

and

four

were

in

the

left

lobe

One

tumor

was

located

in

the

caudate

lobe

Most

tumor

sizes

were

larger

than

cm

-LRB-

-RRB-

The

median

tumor

size

was

cm

ranging

from

cm

to

cm

-LRB-

Tables

and

-RRB-

Gross

pathology

identified

all

tumors

as

a

well-circumscribed

nonencapsulated

tumor

masses

consisting

of

soft

to

elastic

tissue

The

cut

surface

in

tumors

varied

from

yellow

to

dark

brown

Histopathologic

studies

of

these

tumors

showed

a

picture

of

hepatic

angiomyolipoma

composed

of

myoid

and

vascular

components

with

a

variant

content

of

fatty

tissue

Hematopoiesis

was

noted

in

two

cases

Immunohistochemical

studies

were

performed

in

all

patients

except

one

-LRB-

case

-RRB-

Most

tumors

were

found

positive

for

-LRB-

-RRB-

SMA

-LRB-

-RRB-

-LRB-

-RRB-

Vimentin

-LRB-

-RRB-

but

negative

for

cytokeratin

-LRB-

-RRB-

Only

three

tumors

were

found

positive

for

c-kit

-LRB-

Table

-RRB-

Table

strongly

staining

positivity

weakly

staining

positivity

no

staining

or

positivity

Treatment

and

Follow-up

One

patient

-LRB-

case

-RRB-

was

confirmed

with

AML

through

fine

needle

aspiration

biopsy

Nonoperative

management

with

close

follow-up

was

performed

However

this

patient

was

lost

after

months

of

follow-up

The

other

nine

patients

underwent

hepatectomy

with

tumor

resection

These

nine

patients

except

for

one

patient

-LRB-

case

-RRB-

had

no

postoperative

complications

or

disease

recurrence

and

were

regularly

followed

up

at

our

outpatient

department

follow-up

ranging

from

to

months

-LRB-

Table

-RRB-

The

very

unusual

patient

-LRB-

case

-RRB-

was

a

woman

with

a

cm

large

tumor

at

the

left

lobe

of

the

liver

receiving

extended

left

lobectomy

-LRB-

Fig.

b

-RRB-

Pathology

revealed

a

picture

of

hepatic

AML

-LRB-

Fig.

b

-RRB-

Figure

woman

-LRB-

case

-RRB-

presented

with

fever

and

palpable

abdominal

mass

-LRB-

a

-RRB-

The

axial

view

of

contrast-enhanced

CT

scans

on

portal

venous

phase

shows

a

huge

hepatic

tumor

at

the

left

hepatic

lobe

with

heterogeneous

enhancement

Notice

the

engorged

vessels

within

the

tumor

are

vividly

identified

-LRB-

arrow

-RRB-

-LRB-

b

-RRB-

The

MR

coronal

Tru

FISP

fast

imaging

with

steady-state

precession

-LRB-

TR/TE/FA

-RRB-

shows

engorged

vessels

in

the

tumor

The

right

portal

vein

-LRB-

arrow

-RRB-

is

displaced

by

the

tumor

-LRB-

c

-RRB-

After

months

of

extended

left

lobectomy

the

abdominal

ultrasonography

reveals

a

huge

recurrent

tumor

-LRB-

arrows

-RRB-

in

the

previous

location

of

left

hepatic

lobe

and

numerous

smaller

tumors

in

the

right

lobe

-LRB-

d

-RRB-

Celiac

angiography

also

demonstrates

the

recurrent

huge

tumor

and

other

multiple

smaller

ones

in

the

right

lobe

of

liver

Note

the

early

drainage

vein

-LRB-

arrow

-RRB-

Figure

appearance

of

the

hepatic

angiomyolipoma

in

case

-LRB-

a

-RRB-

The

primary

tumor

is

composed

of

polygonal

to

spindle

cells

arranged

in

solid

sheets

or

trabecular

pattern

with

endothelial

lining

Some

of

the

tumor

cells

have

eosinophilic

cytoplasm

and

some

have

large

fat

vacuoles

Some

of

the

nuclei

are

bizarre

and

some

have

large

eosinophilic

nucleoli

-LRB-

H&E

stain

original

magnification

-RRB-

-LRB-

b

-RRB-

The

tumor

cells

are

strongly

immunoreactive

for

-LRB-

original

magnification

-RRB-

Recurrent

tumor

was

noted

months

later

and

the

patient

received

fine

needle

aspiration

biopsy

-LRB-

c

-RRB-

Microscopically

it

shows

tumor

cells

with

clear

to

ample

eosinophilic

cytoplasm

arranged

in

trabecular

pattern

-LRB-

H&E

stain

original

magnification

-RRB-

-LRB-

d

-RRB-

Immunohistochemical

staining

shows

the

tumor

cells

are

also

positive

for

-LRB-

original

magnification

-RRB-

Unfortunately

months

later

ultrasonography

showed

recurrent

hepatic

lesions

at

the

right

lobe

of

the

liver

-LRB-

Fig.

-RRB-

MRI

also

confirmed

a

large

tumor

in

the

caudate

lobe

and

numerous

smaller

nodules

in

the

right

lobe

of

the

liver

Angiography

also

revealed

multiple

tumor

stains

-LRB-

Fig.

-RRB-

Fine

needle

aspiration

biopsy

was

performed

The

biopsy

specimen

was

immunoreactive

to

antibody

-LRB-

Fig.

d

-RRB-

The

clinical

and

histologic

picture

demonstrated

recurrent

malignant

hepatic

angiomyolipoma

At

the

postoperative

month

chest

CT

scans

revealed

multiple

metastatic

nodules

Three

months

later

the

woman

died

due

to

hepatic

failure

and

renal

failure

Discussion

In

the

past

hepatic

AML

has

been

considered

as

a

benign

mesenchymal

tumor

However

in

Dalle

reported

the

first

case

of

malignant

hepatic

AML

with

vascular

invasion

and

recurrence

with

multiple

liver

metastases

and

suspected

portal

vein

thrombosis

months

after

primary

tumor

resection

Another

two

cases

have

been

reported

with

hepatic

recurrence

after

operation

One

case

was

a

girl

with

hepatic

AML

receiving

left

lobectomy

with

late

recurrence

noted

years

after

operation

The

other

one

was

in

the

Flemming

's

report

Recurrent

hepatic

tumors

were

noted

years

after

operation

Flemming

also

suggested

that

a

proliferation

index

exceeding

and

multicentric

growth

indicate

a

propensity

for

recurrence

In

this

study

we

reported

a

woman

with

left

hepatic

AML

A

recurrent

hepatic

mass

was

noted

months

after

tumor

resection

and

multiple

lung

metastases

were

noted

later

The

patient

died

months

after

diagnosis

To

our

knowledge

this

case

is

the

fourth

reported

case

of

recurrence

in

the

literature

and

the

tumor

in

this

case

behaved

as

the

most

malignant

one

Therefore

hepatic

AML

should

not

be

considered

as

an

entirely

benign

tumor

at

least

it

has

malignant

transformation

potential

Accordingly

conservative

treatment

should

be

performed

carefully

especially

for

patients

with

poor

compliance

who

are

unable

to

undergo

a

strict

follow-up

regimen

There

were

few

reports

concerning

the

growth

velocity

of

hepatic

AML

in

long-term

follow-up

In

one

retrospective

study

of

patients

there

were

six

patients

who

were

followed

up

for

more

than

year

and

finally

decided

to

receive

operation

because

of

the

enlargement

of

the

lesions

In

that

study

the

tumor

size

of

one

patient

increased

from

to

cm

during

the

follow-up

Another

patient

had

a

tumor

increasing

from

to

cm

in

years

In

another

case

report

of

a

patient

the

tumor

size

enlarged

from

to

cm

over

a

follow-up

period

Irie

also

reported

that

a

woman

had

hepatic

AML

with

tumor

size

increasing

in

size

from

cm

to

cm

during

a

follow-up

period

Although

hepatic

AML

seems

slow-growing

the

probability

of

tumor

enlargement

and

hence

an

induced

mass-compression

effect

is

not

uncommon

in

the

long-term

follow-up

period

In

the

present

series

the

median

age

of

patients

was

years

old

and

of

patients

were

below

years

If

all

of

these

patients

had

received

nonoperative

management

the

mass

effect

of

tumor

enlargement

might

have

been

presented

during

a

long-term

follow-up

period

especially

in

younger

patient

groups

with

longer

remaining

years

of

life

Moreover

the

difficulties

and

complications

of

operation

at

later

years

would

increase

when

the

tumor

enlarges

especially

for

those

patients

with

an

original

larger

tumor

-LRB-

cm

-RRB-

Similar

to

the

patients

presented

in

this

series

most

patients

with

hepatic

AML

are

not

symptomatic

Usually

these

patients

are

diagnosed

during

health

check-ups

Most

symptoms

are

mass-compression

effects

including

upper

abdominal

pain

abdominal

fullness

and

palpable

mass

There

are

also

some

vague

symptoms

such

as

body

weight

loss

general

malaise

and

fever

In

one

review

article

with

a

collection

of

patients

the

incidence

of

symptoms

or

signs

dramatically

increased

when

tumor

size

was

larger

than

cm

Twenty-one

percent

-LRB-

-RRB-

of

patients

with

a

tumor

smaller

than

cm

present

symptoms/signs

however

the

incidence

increases

to

-LRB-

-RRB-

when

tumor

size

is

between

and

cm

The

incidence

increases

to

when

tumor

size

is

larger

than

cm

In

our

series

-LRB-

-RRB-

of

patients

were

symptomatic

and

all

four

of

these

patients

had

a

tumor

larger

than

cm

Accordingly

we

suggested

that

patients

with

tumor

larger

than

cm

should

receive

tumor

resection

because

most

patients

in

this

group

were

predisposed

toward

being

symptomatic

The

typical

findings

in

imaging

studies

of

hepatic

AML

are

as

follows

-LRB-

-RRB-

heterogeneously

hyperechoic

mass

in

US

-LRB-

-RRB-

heterogeneously

low

density

with

low

attenuation

value

-LRB-

less

than

HU

-RRB-

in

plain

CT

-LRB-

-RRB-

high

intensity

on

and

weighted

MRI

and

-LRB-

-RRB-

hypervascularity

and

tumor

stain

on

angiography

Although

a

combination

of

US

CT

MRI

and

angiography

is

able

to

increase

the

accuracy

in

preoperative

diagnosis

hepatic

AML

usually

shows

various

patterns

in

imaging

studies

The

differences

in

imaging

studies

occur

because

the

relative

proportions

of

vessels

muscles

and

fatty

tissue

vary

widely

from

one

tumor

to

another

Consequently

hepatic

AML

is

sometimes

difficult

to

diagnose

based

on

imaging

studies

Therefore

fine

needle

aspiration

biopsy

has

been

reported

to

be

useful

in

the

preoperative

diagnosis

of

this

tumor

However

more

attention

should

be

paid

to

the

tumor

's

various

morphologic

appearances

when

minute

samples

are

interpreted

With

the

combined

tools

of

imaging

studies

and

FNAB

the

preoperative

diagnostic

accuracy

has

been

smaller

than

-LRB-

ranging

from

to

-RRB-

in

larger

series

In

a

collaborative

study

reported

by

Tsui

including

cases

from

nine

international

hepatology

centers

were

primarily

misdiagnosed

as

carcinoma

or

sarcoma

either

by

imaging

studies

or

by

needle

biopsy

In

Flemming

's

series

only

one

preoperative

case

was

diagnosed

correctly

In

the

present

series

only

four

preoperative

cases

-LRB-

-RRB-

were

correctly

diagnosed

by

combined

imaging

studies

and

FNAB

Definite

pathologic

diagnosis

of

this

tumor

is

usually

made

by

identification

of

the

three

different

components

of

smooth

muscle

cells

adipose

tissue

and

blood

vessels

positive

staining

of

myoid

cells

has

been

used

as

a

pathologic

characteristic

of

hepatic

AML

Because

of

the

rarity

and

pleomorphism

of

the

histological

features

of

hepatic

AML

histologic

diagnosis

may

be

difficult

especially

with

needle

biopsy

Many

features

in

AML

can

mislead

the

unwary

pathologist

to

a

diagnosis

of

hepatocellular

carcinoma

polygonal

cells

in

trabecular

arrangement

peliosis

nuclear

pleomorphism

prominent

eosinophilic

nucleoli

deficient

reticulin

framework

presence

of

glycogen

eosinophilic

globules

and

tumor

necrosis

In

Zhong

's

series

of

of

the

cases

were

correctly

diagnosed

before

operation

Furthermore

five

cases

were

misdiagnosed

as

hepatocellular

carcinoma

or

sarcoma

by

pathologists

even

after

operation

Therefore

we

should

be

cautious

when

using

FNAB

as

a

diagnostic

tool

In

an

endemic

area

of

hepatocellular

carcinoma

such

as

Taiwan

conservative

management

is

risky

because

cases

of

fat-rich

minute

hepatocellular

carcinoma

will

make

the

differential

diagnosis

more

difficult

Furthermore

Chang

reported

one

case

with

hepatic

AML

and

concomitant

hepatocellular

carcinoma

In

this

series

two

patients

were

carriers

of

hepatitis

B

virus

with

a

high

risk

for

hepatoma

formation

Not

only

would

these

hepatitis-carrier

patients

bear

more

risk

but

physicians

would

also

bear

more

risk

and

psychological

pressure

during

a

long-term

follow-up

period

if

conservative

management

were

adopted

Because

of

the

small

patient

number

we

could

not

get

definitely

conclusive

management

suggestions

solely

from

the

results

of

this

retrospective

study

But

a

combination

of

our

experience

and

a

review

of

the

literature

we

suggest

all

symptomatic

patients

should

receive

surgical

resection

for

hepatic

angiomyolipoma

Conservative

management

with

close

follow-up

is

suggested

in

patients

with

asymptomatic

tumors

and

meet

the

following

criteria

-LRB-

-RRB-

tumor

size

smaller

than

cm

-LRB-

-RRB-

angiomyolipoma

proved

through

fine

needle

aspiration

biopsy

-LRB-

-RRB-

patients

with

good

compliance

and

-LRB-

-RRB-

not

a

hepatitis-virus

carrier

O O O B 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 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 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 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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


Modulation

of

muscle

contraction

by

a

cell-permeable

peptide

In

contrast

to

immortal

cell

lines

primary

cells

are

hardly

susceptible

to

intracellular

delivery

methods

such

as

transfection

In

this

study

we

evaluated

the

direct

delivery

of

several

cell-permeable

peptides

under

noninvasive

conditions

into

living

primary

adult

rat

cardiomyocytes

We

specifically

monitored

the

functional

effects

of

a

cell-permeable

peptide

containing

the

amino

acid

N-terminal

peptide

from

human

ventricular

light

-LRB-

-RRB-

on

contraction

and

intracellular

signals

after

electrical

stimulation

in

primary

adult

cardiomyocytes

The

transducible

variant

was

taken

up

by

cardiomyocytes

within

min

with

more

than

efficiency

and

localized

to

sarcomeric

structures

Analysis

of

the

functional

effects

of

the

cell-permeable

revealed

an

enhancement

of

the

intrinsic

contractility

of

cardiomyocytes

without

affecting

the

intracellular

Therefore

peptide

transduction

mediated

by

cell-penetrating

peptides

represents

not

only

a

unique

strategy

to

enhance

heart

muscle

function

with

no

secondary

effect

on

intracellular

but

also

an

invaluable

tool

for

the

modulation

and

manipulation

of

protein

interactions

in

general

and

in

primary

cells

Introduction

Therapeutical

delivery

of

protein

or

peptides

into

living

primary

cells

is

hampered

by

the

lack

of

a

suitable

and

efficient

method

for

the

introduction

of

macromolecules

Most

commonly

used

transfections

methods

work

in

immortalized

cells

but

mostly

fail

in

primary

cells

or

require

specialized

and

time-consuming

protocols

i.e.

production

of

viral

vectors

Moreover

the

limited

life

span

of

primary

cells

often

does

not

provide

sufficient

time

for

expression

of

gene

products

and

subsequent

analysis

of

their

effects

Physical

methods

such

as

microinjection

and

bead

loading

are

highly

invasive

procedures

and

are

not

tolerated

by

several

cell

types

It

has

been

known

for

quite

some

time

that

basic

proteins

like

histones

or

poly-ornithine

as

well

as

stretches

of

basic

amino

acids

within

proteins

aid

the

uptake

of

proteins

into

mammalian

cells

-LSB-

-RSB-

In

the

transducible

peptides

like

from

the

homeodomain

of

Antennapedia

-LSB-

-RSB-

and

amino

acids

from

the

transactivator

of

transcription

of

human

immunodeficiency

virus

-LRB-

HIV

-RRB-

-LSB-

-RSB-

were

identified

and

exploited

to

introduce

drugs

or

biological

macromolecules

into

mammalian

cells

Together

with

other

native

and

synthetic

transducing

peptides

they

are

referred

to

as

cell-penetrating

peptides

-LRB-

CPPs

-RRB-

-LSB-

-RSB-

However

it

became

evident

that

the

invasive

methods

used

to

analyze

the

transduction

mediated

by

CPPs

or

effects

of

interconnected

cargoes

had

led

to

overestimated

transduction

results

and

overrated

effects

of

cargoes

fused

to

CPPs

-LSB-

-RSB-

Nevertheless

CPPs

mediate

the

introduction

of

fused

cargoes

into

living

cells

with

cargo-dependent

mechanistical

differences

One

uptake

mechanism

can

be

described

as

a

slow

adsorptive

endocytosis

and

is

preferentially

used

by

globular

proteins

whereas

small

compounds

like

peptides

linked

to

CPPs

favor

a

rapid

membrane

potential-dependent

uptake

with

overall

intracellular

availability

of

the

transduced

species

-LSB-

-RSB-

CPP-mediated

peptide

transduction

has

recently

been

used

to

deliver

cardioprotective

peptides

derived

from

protein

kinase

C

-LSB-

-RSB-

the

antiapoptotic

peptide

derived

from

the

apoptosis

regulator

protein

Bcl-xL

-LSB-

-RSB-

and

a

peptide

-LSB-

-RSB-

into

isolated

cardiomyocytes

or

whole

hearts

We

have

tested

the

feasibility

of

this

approach

to

study

muscle

function

in

living

adult

primary

cardiomyocytes

Therefore

we

targeted

the

interaction

between

actin

and

the

essential

myosin

light

chain

-LRB-

-RRB-

of

cardiac

type

II

myosin

by

a

peptide

competition

approach

Transgenic

overexpression

of

the

N

terminus

of

the

human

ventricular

-LRB-

residues

hereafter

termed

-RRB-

which

binds

to

actin

and

targets

actin/MLC

interaction

significantly

increased

the

magnitude

and

kinetics

of

the

contraction

of

isolated

perfused

hearts

-LSB-

-RSB-

We

fused

the

same

human

peptide

to

the

TAT

CPP

-LRB-

-RRB-

and

investigated

its

uptake

intracellular

distribution

and

functional

consequences

in

primary

living

adult

rat

cardiomyocytes

Materials

and

methods

Isolation

of

adult

rat

cardiomyocytes

Primary

cultures

of

cardiomyocytes

from

male

WKY

rats

aged

months

were

performed

as

described

previously

-LSB-

-RSB-

Peptides

The

peptides

consisted

of

either

l-amino

acids

-LRB-

capital

letters

-RRB-

or

of

d-amino

acids

-LRB-

lower

case

letters

-RRB-

In

case

of

d-amino

acid

peptides

functional

motifs

were

synthesized

as

retro-all

d-variants

-LRB-

underlined

-RRB-

to

maintain

their

functionality

Peptides

were

either

labeled

with

the

fluorophore

-LRB-

TAMRA

-RRB-

or

fluorescein

isothiocyanate

-LRB-

FITC

-RRB-

TAT

-LRB-

TAMRA

or

FITC-rrrqrrkkrg

-RRB-

-LSB-

-RSB-

-LRB-

TAMRA-araqraaaray

-RRB-

and

-LRB-

TAMRA-rrrqrrkkrgaaAGRKRRQTSMTDFYHSKRRLIFSa-amide

-RRB-

-LSB-

-RSB-

were

labeled

directly

at

their

N

termini

with

the

indicated

fluorophores

-LRB-

Peptide

Speciality

Laboratories

Heidelberg

-RRB-

In

the

case

of

-LRB-

MAPKKPEPKKSSAKA-C-TAMRA-ME-amide

-RRB-

and

-LRB-

MAPKKPEPKDDAKAPAGRKKRRQRRR-C-TAMRA-ME-amide

Biosyntan

Berlin

-RRB-

the

TAMRA

fluorophore

was

coupled

via

an

additional

cysteine

at

the

C

terminus

leaving

the

N

terminus

free

for

the

interaction

with

actin

Uptake

of

transducible

peptides

and

controls

To

analyze

the

transduction

ability

of

different

compounds

into

living

adult

ventricular

cardiomyocytes

the

primary

cells

were

plated

into

a

laminin-coated

μ-slide

eight-well

ibiTreat

-LRB-

ibidi

Martinsried

-RRB-

or

four-well

labtek

-LRB-

Nunc

Wiesbaden

-RRB-

microscopy

observation

chamber

The

respective

peptides

and

controls

were

added

directly

to

Hank

's

balanced

salts

solution

buffered

with

mM

-LRB-

-RRB-

acid

at

pH

-LRB-

HBSS

-RRB-

or

medium

and

gently

shaken

to

yield

a

final

concentration

of

μM

in

case

of

the

peptides

and

Trypan

blue

-LRB-

Sigma-Aldrich

-RRB-

was

added

to

a

final

concentration

of

-LRB-

v/v

-RRB-

to

the

HBSS

buffer

For

the

experiments

summarized

in

Fig.

confocal

images

were

acquired

h

after

addition

of

the

respective

compounds

to

living

cardiomyocytes

To

study

the

intracellular

localization

of

transducible

peptides

in

Fig.

after

min

of

peptide

incubation

the

medium

was

removed

and

the

cells

were

washed

twice

with

the

HBSS

buffer

For

subsequent

confocal

image

collection

the

cells

were

kept

in

medium

or

buffer

To

study

the

effect

of

the

peptide

on

muscle

contraction

and

intracellular

-LRB-

Fig.

-RRB-

cardiomyocytes

were

electrically

stimulated

-LRB-

see

below

-RRB-

to

permit

the

access

of

the

peptides

to

all

actin

molecules

with

potential

binding

sites

During

confocal

image

collection

the

electric

stimulation

was

temporarily

switched

off

Fig.

uptake

and

intracellular

distribution

in

living

adult

cardiomyocytes

To

study

the

uptake

capacity

of

different

compounds

into

primary

rat

cardiomyocytes

different

fluorescent

compounds

were

directly

added

for

h

to

the

medium

or

buffer

of

freshly

isolated

cardiomyocytes

After

the

incubation

period

confocal

images

of

the

cells

were

taken

without

exchange

of

the

medium

and

after

removal

of

the

fluorescent

solution

and

exchange

against

the

fresh

medium

Internalization

into

healthy

adult

cardiomyocytes

occurred

after

application

of

μM

of

the

transducible

peptides

FITC-TAT

shown

at

low

-LRB-

a

-RRB-

and

high

magnification

-LRB-

b

-RRB-

and

shown

at

low

-LRB-

c

-RRB-

and

high

magnification

-LRB-

d

-RRB-

but

not

with

application

of

μM

of

a

basic

control

peptide

-LRB-

e

-RRB-

Moreover

of

the

vital

dye

trypan

blue

-LRB-

f

-RRB-

or

μM

of

the

TAMRA-fluorophore

-LRB-

g

-RRB-

itself

were

not

able

to

enter

healthy

cardiomyocytes

-LRB-

rod-shaped

cells

in

culture

-RRB-

Scale

bar

μm

Stars

indicate

the

nuclei

and

arrowheads

the

nucleoli

-LRB-

N

-RRB-

in

the

higher

magnification

images

PC

Phase

contrast

NP

nucleoplasm

Microscopy

image

acquisition

and

analysis

Confocal

images

were

acquired

with

a

Zeiss

laser

scanning

microscope

Meta

mounted

on

an

Axiovert

inverted

microscope

using

a

phase-contrast

oil

immersion

plan-apochromat

objective

or

a

phase-contrast

oil

immersion

plan-neofluar

objective

For

all

settings

the

main

beam

splitter

was

HFT

and

the

specific

parameters

for

the

single

fluorophores

were

FITC

excited

at

nm

light

detected

with

a

bandpass

filter

TAMRA

or

rhodamine

excited

at

nm

detected

with

bandpass

filter

and

trypan

blue

excited

with

nm

detected

with

longpass

filter

Phase-contrast

images

were

recorded

with

excitation

at

nm

and

detection

in

the

transmission

channel

Laser

power

for

observation

was

typically

-LRB-

nm

mW

-RRB-

and

-LRB-

nm

mW

-RRB-

unless

otherwise

indicated

Settings

were

adjusted

in

a

way

that

image

pixels

were

not

over

or

underexposed

with

the

range

indicator

function

in

the

Zeiss

LSM

image

acquisition

and

examiner

software

version

Measurement

of

cardiomyocyte

shortening

and

transients

Attached

cardiomyocytes

were

washed

with

HBSS

Cells

were

loaded

with

for

min

at

room

temperature

in

the

dark

The

dye

solution

was

removed

and

cells

were

left

on

HBSS

for

another

min

Only

cardiomyocytes

of

optically

intact

rod-shaped

morphology

with

clear

cross-striation

were

analyzed

Cardiomyocytes

were

electrically

stimulated

until

a

stable

steady-state

contraction

and

signal

could

be

monitored

Cell

shortening

and

signals

were

simultaneously

measured

at

°C

on

an

Ionoptix

Contractility

and

Fluorescence

System

-LRB-

Ionoptix

-RRB-

Cardiomyocytes

were

electrically

stimulated

with

bipolar

pulses

of

ms

duration

at

Hz

Cell

shortening

expressed

as

percentage

of

resting

cell

length

was

measured

using

the

video-edge

technique

at

a

sampling

rate

of

per

second

transients

were

monitored

as

ratio

of

fluorescence

emission

at

nm

was

obtained

by

alternate

excitation

at

and

nm

-LRB-

ratio

-RRB-

Data

files

from

consecutive

beats

recorded

at

intervals

were

averaged

for

analysis

Subsequently

the

cardiomyocytes

were

incubated

with

the

peptides

for

min

without

electrical

stimulation

The

peptide

was

then

removed

by

replacing

the

peptide-containing

HBSS

buffer

with

normal

HBSS

buffer

without

the

peptide

The

cardiomyocytes

were

subsequently

electrically

stimulated

and

both

the

shortening

and

signals

were

simultaneously

recorded

The

effect

of

the

peptide

on

shortening

amplitude

and

the

signal

was

expressed

in

percent

change

compared

to

the

steady-state

signals

obtained

in

the

preincubation

period

Results

and

discussion

We

recently

demonstrated

by

live-cell

confocal

microscopy

that

the

TAT

CPP

was

able

to

shuttle

fused

peptides

into

primary

blood

cells

and

subsequently

modulated

their

functional

properties

-LSB-

-RSB-

In

this

study

we

tested

whether

TAT

CPP

could

penetrate

and

if

a

peptide

derived

from

the

N

terminus

of

fused

to

TAT

is

able

to

modulate

the

contractile

function

of

primary

adult

cardiomyocytes

Different

basic

peptides

were

incubated

with

freshly

prepared

cultures

of

cardiomyocytes

and

peptide

internalization

was

analyzed

live

by

laser-scanning

confocal

microscopy

The

fluorescently

labeled

minimal

transduction

domain

TAT

CPP

of

the

transactivator

of

transcription

from

was

readily

taken

up

into

cardiomyocytes

and

displayed

a

strong

affinity

to

the

nuclear

compartment

-LRB-

Fig.

-RRB-

It

could

also

be

visualized

in

a

punctate

pattern

throughout

the

entire

cardiomyocyte

-LRB-

Fig.

-RRB-

To

determine

whether

the

TAT

CPP

is

able

to

shuttle

an

attached

peptide

into

cardiomyocytes

we

used

the

TAMRA-labeled

CIP

-LRB-

-RRB-

fusion

peptide

-LSB-

-RSB-

which

in

addition

to

the

transducing

moiety

contains

amino

acids

derived

from

the

C

terminus

of

human

protein

The

uptake

of

this

cargo

peptide

-LRB-

assessed

by

appearance

in

nucleoli

-RRB-

occurred

on

a

timescale

of

to

min

after

peptide

application

Its

pattern

of

localization

inside

the

cardiomyocytes

was

similar

than

that

seen

for

TAT

alone

-LRB-

Fig.

-RRB-

Basic

amino

acid

residues

are

a

prerequisite

for

the

transduction

ability

of

CPPs

-LSB-

-RSB-

and

peptides

containing

a

minimum

of

six

basic

amino

acids

have

been

shown

to

traverse

biological

membranes

-LSB-

-RSB-

As

a

nontransducing

control

peptide

we

chose

the

TAMRA-labeled

peptide

which

contains

three

arginines

in

total

-LSB-

-RSB-

More

than

h

after

application

of

this

peptide

to

the

cardiomyocyte

culture

of

the

healthy

intact

cardiomyocytes

took

up

the

fluorescent

peptide

-LRB-

Fig.

-RRB-

Similar

results

were

obtained

for

the

vital

dye

trypan

blue

-LRB-

Fig.

-RRB-

and

the

TAMRA

fluorophore

-LRB-

Fig.

-RRB-

Approximately

of

cardiomyocytes

in

the

cultures

were

dead

or

harmed

-LRB-

visible

in

the

phase-contrast

images

as

not

rod

shaped

and

with

no

striations

-RRB-

These

cells

were

flooded

with

both

the

CPPs

as

well

as

any

other

control

dyes

-LRB-

Fig.

e

g

-RRB-

The

cell-permeable

TAT-derived

peptides

displayed

a

similar

enrichment

in

the

nuclear

compartment

with

strong

accumulation

inside

nucleoli

-LRB-

arrowheads

-RRB-

as

can

be

seen

in

the

higher

magnification

images

-LRB-

Fig.

d

-RRB-

This

intranuclear

distribution

is

basically

determined

by

the

TAT

moiety

which

includes

a

nuclear

localization

sequence

-LSB-

-RSB-

Having

shown

that

CPP

TAT

was

able

to

shuttle

fused

peptides

into

living

cardiomyocytes

we

designed

a

transducible

peptide

comprising

the

actin-binding

element

of

the

ventricular

isoform

of

human

-LRB-

-RRB-

i.e.

the

N-terminal

residues

-LRB-

peptide

-RRB-

connected

to

TAT

termed

-LRB-

Fig.

-RRB-

The

hearts

of

transgenic

animals

harboring

minigenes

encoding

for

the

peptide

revealed

enhanced

myosin

motor

activity

and

positive

inotropic

effects

-LSB-

-RSB-

A

possible

molecular

mechanism

for

the

increase

in

myosin

motor

activity

and

contractility

is

illustrated

in

the

scheme

in

Fig.

isoforms

of

vertebrate

striated

muscle

play

a

regulatory

role

in

myosin

motor

function

The

N-terminal

part

of

contains

a

sticky

element

-LRB-

SE

-RRB-

which

consists

of

several

positively

charged

amino

acids

that

bind

to

the

C

terminus

of

actin

-LSB-

-RSB-

The

N-terminal

amino

acids

of

have

previously

been

modeled

on

the

basis

of

contemporary

crystallographic

data

-LSB-

-RSB-

Interactions

between

the

sticky

element

and

actin

serve

as

a

negative

modulator

and

decrease

the

activity

of

the

myosin

motor

domain

Intracellular

delivery

of

a

synthetic

sticky

element

by

TAT-mediated

transduction

therefore

would

antagonize

actin

interaction

We

predict

this

would

increase

myosin

motor

activity

and

enhance

the

shortening

amplitude

without

change

of

the

systolic

free

activation

level

of

intact

cardiomyocytes

Fig.

hypothesis

for

the

enhancement

of

cardiomyocyte

contractility

by

the

cell-permeable

peptide

a

Primary

sequence

of

peptide

color-coded

orange

for

the

and

yellow

for

the

TAT-moiety

b

Schematic

sliding

filament

model

displaying

actin

in

violet

and

the

MHC

in

red

The

higher

magnification

shows

the

catalytic

domain

-LRB-

CD

-RRB-

and

the

neck

domain

-LRB-

ND

-RRB-

of

one

molecule

of

the

MHC

MLC

proteins

are

colored

green

with

the

N-terminal

SE

inside

the

essential

protein

highlighted

in

red

Upon

intramolecular

conformational

changes

in

the

CD

of

MHC

the

ND

swings

out

exposing

the

SE

of

to

an

actin

molecule

in

direction

to

the

Z-line

Treatment

of

cardiomyocytes

with

the

transducible

TAT

peptide

-LRB-

yellow

ball

-RRB-

fused

to

the

SE-peptide

-LRB-

orange

-RRB-

blocks

potential

binding

sites

of

the

native

N

terminus

After

the

MHC

rearrangement

the

protein

can

no

longer

interact

with

actin

and

downregulate

the

myosin

motor

activity

The

scheme

is

based

on

the

pre

and

postpower

stroke

model

-LSB-

-RSB-

developed

with

respect

to

crystallographic

data

on

the

Dicytostelium

myosin

motor

domain

-LSB-

-RSB-

To

first

test

whether

the

peptide

itself

might

be

taken

up

by

isolated

cardiomyocytes

because

of

its

basic

charge

and

were

applied

for

min

to

freshly

isolated

cardiomyocytes

under

resting

conditions

or

in

another

set

of

experiments

constant

electric

stimulation

-LRB-

Hz

V

-RRB-

After

the

fluorescent

peptides

were

washed

away

the

cells

were

analyzed

live

by

confocal

laser-scanning

microscopy

-LRB-

Fig.

-RRB-

alone

was

not

internalized

into

healthy

cardiomyocytes

and

stained

only

compromised

cells

-LRB-

upper

panel

Fig.

-RRB-

To

ensure

that

low

concentrations

of

intracellular

were

not

missed

the

laser

intensity

was

increased

to

its

maximum

and

still

no

fluorescent

signals

were

measured

-LRB-

lower

panel

Fig.

-RRB-

In

contrast

reached

the

intracellular

compartments

of

more

than

of

adult

cardiomyocytes

-LRB-

Fig.

-RRB-

Fig.

and

intracellular

localization

of

in

isolated

adult

cardiomyocytes

a

Scheme

for

the

application

of

the

peptides

and

confocal

imaging

Peptides

were

applied

at

a

concentration

of

μM

incubated

for

min

to

freshly

isolated

cardiomyocytes

After

removal

of

the

peptide

solution

the

cells

were

washed

twice

and

kept

in

buffer

or

medium

for

direct

live-cell

confocal

imaging

microscopy

Subsequently

the

transduced

cardiomyocytes

were

electrically

stimulated

with

Hz

for

maximally

h.

b

The

negative

control

peptide

TAMRA-labeled

stained

only

dead

cardiomyocytes

-LRB-

low

magnification

upper

panel

-RRB-

but

failed

to

get

internalized

into

healthy

cardiomyocytes

even

when

laser

power

was

used

to

detect

potentially

weak

signals

-LRB-

lower

panel

-RRB-

c

Application

of

to

living

cardiomyocytes

led

to

rapid

internalization

of

the

peptide

-LRB-

upper

panel

-RRB-

The

lower

panel

shows

transduced

at

higher

magnification

in

an

overlay

of

the

phase

contrast

and

the

TAMRA-fluorescence

The

arrow

indicates

the

direction

of

the

intensity

profile

of

gray

and

red

intensities

in

the

line

scan

for

the

localization

analysis

of

which

was

present

at

sarcomeric

structures

with

enrichment

at

the

actin-containing

I-bands

-LRB-

light

bands

in

phase-contrast

image

-RRB-

Unlike

the

localization

of

the

TAT

and

peptides

-LRB-

Fig.

-RRB-

the

was

not

detected

in

the

cell

nucleus

Detailed

analysis

of

the

intracellular

distribution

of

transduced

and

correlation

of

the

fluorescent

signals

with

the

phase

contrast

images

-LRB-

merge

image

lower

panel

Fig.

-RRB-

demonstrated

that

the

peptide

associates

with

sarcomeric

structures

It

showed

strong

enrichment

at

the

I-bands

representing

the

nonoverlapping

zones

of

the

thin

actin

filaments

with

the

thick

myosin

filaments

-LRB-

linescan

lower

panel

Fig.

-RRB-

After

transduction

the

peptide

remained

inside

the

sarcomeric

compartments

of

the

cardiomyocytes

for

at

least

h

after

transferring

the

loaded

cardiomyocytes

into

the

peptide-free

buffer

solution

Using

the

same

incubation

protocol

we

studied

the

effects

of

the

cell-permeable

peptide

on

muscle

function

by

simultaneously

monitoring

the

shortening

amplitude

and

the

systolic

and

diastolic

cytoplasmic

fluctuations

-LRB-

measured

by

fluorescence

-RRB-

of

paced

adult

rat

cardiomyocytes

-LRB-

Fig.

-RRB-

As

shown

in

Figs.

and

c

μM

but

not

μM

of

significantly

increased

the

shortening

amplitude

of

adult

cardiomyocyte

while

the

cytosolic

signal

remained

unchanged

The

same

concentrations

of

control

TAT

peptide

alone

did

not

change

the

contractile

state

or

the

signals

if

compared

to

the

basal

state

during

the

preincubation

period

Higher

concentrations

of

both

control

and

test

peptides

induced

deleterious

effects

on

the

cardiomyocyte

contraction

It

is

interesting

to

note

that

not

only

the

maximal

amplitude

of

shortening

rose

but

also

the

maximal

rate

of

shortening

and

the

maximal

relaxation

velocity

increased

significantly

Because

the

signal

remained

unchanged

the

selective

effect

of

the

peptide

on

the

contraction

parameters

suggests

a

selective

and

direct

effect

on

the

function

of

the

myosin

cross-bridges

The

sensitizing

effect

of

the

peptide

could

be

predicted

from

the

mechanism

of

action

as

the

inhibition

of

the

actin

interaction

may

rise

selectively

myosin

motor

activity

and

therefore

muscle

contraction

at

a

given

activating

free

concentration

i.e.

an

inotropic

effect

without

additional

recruitment

of

It

should

be

noted

that

the

peptide

left

the

intracellular

handling

system

unchanged

as

seen

by

the

fact

that

the

kinetic

parameters

of

the

fluorescence

signals

remained

normal

-LRB-

Fig.

-RRB-

Fig.

effects

of

cell

permeable

in

living

adult

cardiomyocytes

a

Original

registration

of

cell

length

-LRB-

top

-RRB-

and

cytosolic

signals

-LRB-

signal

expressed

as

ratio

R

between

and

excitation

bottom

-RRB-

of

electrically

paced

adult

rat

cardiomyocytes

before

-LRB-

basal

-RRB-

and

after

incubation

with

μM

or

μM

TAT

as

control

b

Statistical

evaluation

of

the

effects

of

different

concentrations

of

transducible

peptides

-LRB-

μM

-RRB-

on

fractional

shortening

-LRB-

FS

given

in

percent

of

basal

contraction

obtained

before

incubation

with

the

peptide

-RRB-

maximal

rate

of

FS

-LRB-

dFS/dtmax

-RRB-

and

maximal

relaxation

rate

-LRB-

dFS/dtmax

both

given

in

percent

of

basal

values

obtained

before

peptide

incubation

-RRB-

of

control

peptide

-LRB-

TAT

-RRB-

and

c

Statistical

evaluation

of

the

effects

of

different

concentrations

of

transducible

peptides

-LRB-

μM

-RRB-

on

peak

fluorescence

signal

-LRB-

expressed

as

ratio

R

between

and

excitation

given

in

percent

of

basal

obtained

before

incubation

with

the

peptide

-RRB-

maximal

rate

of

-LRB-

-RRB-

and

maximal

rate

of

fluorescence

decline

-LRB-

both

given

in

percent

of

basal

values

obtained

before

peptide

incubation

-RRB-

of

control

peptide

-LRB-

TAT

-RRB-

and

Values

are

means

SEM

n

different

cardiomyocytes

at

each

peptide

concentration

used

Double

asterisk

p

triple

asterisk

p

-LRB-

t

test

for

paired

values

-RRB-

In

summary

we

demonstrate

by

live-cell

microscopy

that

CPPs

and

CPPs

fused

to

peptides

were

taken

up

by

adult

cardiomyocytes

with

high

efficiency

and

localize

to

their

targets

inside

the

cytoplasm

and/or

nucleus

The

cell-permeable

peptide

accumulated

in

the

actin-containing

I-band

of

the

sarcomeres

and

was

able

to

enhance

the

contractility

of

isolated

adult

cardiomyocytes

without

changing

the

myoplasmic

levels

Importantly

the

fact

that

did

not

affect

intracellular

concentration

and

that

it

only

has

targets

in

striated

muscle

cells

makes

this

peptide

uniquely

suited

as

a

novel

potential

therapeutic

tool

to

modulate

heart

function

is

a

powerful

new

drug

candidate

to

improve

the

contractile

state

of

the

failing

heart

O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B 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 B O O O O O O O O B 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 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 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 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 B 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 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 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 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 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 B 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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 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 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 B 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 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 B 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 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 B 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 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 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 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 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 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 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 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 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

ventricular light chain-1 (vlc-1)
cell penetrating peptide (cpp)
drug delivery


Impact of headache in Europe : a review for the Eurolight project A recent health economic survey in Europe has suggested that migraine is the costliest among the neurological disorders . According to many studies , migraine and other disorders lead to widespread suffering , reduction of quality of life , and marked impairment of participation , both in work and social activities . The present literature survey was made in order to summarize what is known on the subject , as a preparation for a EU-supported study to assess the impact in several EU countries with similar methodology and the same research instrument . Previous studies have yielded relatively reliable data only for migraine , whereas the impact of tension-type headache is virtually unknown or only very incompletely known for most dimensions of headache impact . Some data do suggest , however , that this headache may be as important from a health economic and a public health perspective as migraine . In future studies it is important to get population-based data from various countries relevant for estimation of indirect -LRB- mostly absenteeism from work and reduced working efficiency when having headache -RRB- and direct costs -LRB- related to medication , consultations , investigations and hospitalisations -RRB- . Also , the impact on ability to get education and participate in the workforce is very relevant , as is the impact on love life and family planning . The quality of life of headache patients should be measured by validated instruments . To get a complete picture , one should also ask about the effect on the life of partners and children , and on the possible impact even when headache-free -LRB- e.g. fear of the next attack -RRB- . Introduction For a just and rational distribution of means to health-care services and health-related research , reliable data on the individual and societal impact of different disorders are crucial . In recent years several initiatives have been launched to raise the awareness that headache is not only a nuisance for some individuals , but also that it entails widespread suffering and loss of opportunities for patients and their families , and large cost for the society . The recently published report on the prevalence and burden of headache -LSB- 1 -RSB- is a premise for the campaign `` Lifting the burden : The Global Campaign to reduce the burden of headache '' -LSB- 2 -RSB- . In Europe , much data on both the economic costs of migraine have been collected and presented in connection with the `` Cost of Brain Disorders in Europe '' project -LSB- 3 -RSB- , in which migraine is treated along with many of the other -LRB- neurological and psychiatric -RRB- `` brain disorders '' . The Eurolight project -LRB- http://www.eurolight-online.eu -RRB- is an initiative supported by the EC Public Health Excecutive Agency launched in May 2007 . Its objectives are to bring together the relevant medical , scientific and lay organizations , and to gather updated reliable comparable information regarding migraine , tension-type and chronic headache . It will be the first data collection on headaches at EU level focusing on a holistic , patient-driven and scientifically validated approach , aiming to fill in the main holes in our knowledge by performing comparable studies on headache prevalence and its impact in selected European countries -LRB- Austria , France , Germany , Italy , Lithuania , the Netherlands , Spain , UK , Ireland , Luxembourg -RRB- . A pilot study has already been performed in Luxembourg . The present review of the existing literature on both economic and non-economic impact was performed as a part of the Eurolight project , to assess the current state of knowledge , and to build up a questionnaire to measure all the most relevant aspects of headache impact . This study presents the results of this review and a suggestion of the main dimensions that ought to be covered in the headache impact tool to be used in the Eurolight study . Economic impact of headache Headache may have considerable economic consequences , both for the patient and for the society as a whole . There are more studies about the societal costs than about the individual economic losses of the patients . Relation to socioeconomic status , education and employment In a large Norwegian study -LRB- the HUNT study -RRB- , both migraine and headache in general was associated with low socioeconomic status -LSB- 4 -RSB- , which has also been found in North America -LSB- 5 , 6 -RSB- but not in some smaller European studies -LSB- 7 -- 11 -RSB- . The question whether this is a consequence or a cause of headache is not satisfactorily answered , but in one Swedish study , half of the patients reported a negative influence of migraine on their ability to pursue studies and one third a negative influence on their finances -LSB- 9 -RSB- . In a US study it was found that headache patients have somewhat reduced labour force participation -LSB- 12 -RSB- , but employment status has not been found to be related to headache in some European studies -LSB- 7 , 13 -RSB- . Absenteeism from work In two relatively old studies , one from Finland in 1979 -LSB- 14 -RSB- and one from San Marino in 1986 -LSB- 15 -RSB- , 7 % of working individuals had been absent from work in the previous year due headache . In a Danish study of 1992 -LSB- 16 -RSB- , it was found that 43 % of migraineurs -LRB- 5 % of the population -RRB- and 12 % of TTH patients -LRB- 9 % of the population -RRB- had been absent from work during the previous year due to headache , i.e. a total of 14 % of the population . In a Swedish study of 2004 -LSB- 9 -RSB- , it was found that 65 % of migraineurs reported some degree of absence from either school or work during the previous year . These data are , however , of relatively limited interest from an economical viewpoint as they do not indicate the number of days that the headache sufferers are away from work . The number of days with work absence due to headache is relatively consistent across studies from different countries . In some previous studies it has varied between 2 and 6 days per year among headache patients in general -LSB- 17 -RSB- , and between 1.5 and 4.2 days per year in migraineurs -LSB- 9 -RSB- . A study among migraineurs in Sweden revealed that 35 % were never absent from work due to migraine , and 54 % were absent 1 -- 2 days per year -LSB- 9 -RSB- . Compared to headache-free individuals , migraine patients in the HUNT study from Norway lost on average 4.4 workdays per year , and persons with non-migrainous headache lost 2.5 workdays per year -LSB- 18 -RSB- . In the Danish study from Copenhagen -LSB- 16 -RSB- , the TTH patients who had been absent seem to have been as much or more absent from work than the migraine patients , and the number of workdays lost due to migraine was 270 and to TTH 820 per 1,000 persons per year , i.e. a total of 1,090 days . In a study from England in 2003 -LSB- 13 -RSB- , 15 % had been absent from work or had reduced ability to work due to headaches in the previous 3 months . Per year , headache accounted for 1,327 missed and 5,213 reduced ability days per 1,000 workers per year , representing 0.5 and 2.0 % of all working days in the adult population , irrespective of headache status . This study did not relate absenteeism to different headache diagnoses . In an English study of 2003 , an estimated 5.7 workdays per year was missed by migraineurs working or attending school -LSB- 8 -RSB- . This seems to be higher than in France where a diary-based registration of absenteeism published in 1999 showed that migraineurs were away from work 2.18 days per year due to headache -LSB- 19 -RSB- . Effectiveness when working with headache Working with migraine results in a 35 % productivity loss on average according to some European studies -LSB- 20 -RSB- . This figure is , however , largely based on migraineurs ' self-report , which may give a too high estimate according to a recent US study from a workplace -LSB- 21 -RSB- . In this study , it was found that the working ability assessed by self-report was much lower than the objectively measured working efficiency -LRB- 20 vs. 8 % -RRB- . The relatively small decline in working ability led the authors to conclude that workers with even relatively severe headache find creative ways to cope with the pain and maintain standards . Health economic studies For the headache part of the `` Cost of Brain Disorders in Europe '' project , a literature search for studies containing cost data for migraine and other headaches identified eight European studies evaluating the direct or indirect costs of migraine from a societal perspective -LSB- 20 -RSB- , from France -LSB- 22 , 23 -RSB- , Germany -LSB- 24 -RSB- , The Netherlands -LSB- 25 -RSB- , Spain -LSB- 26 -RSB- , Sweden -LSB- 27 -RSB- and the UK -LSB- 28 , 29 -RSB- . No studies analysing the cost of TTH or other non-migraineous headaches were found . There were large variations in costs across the six European countries where data were available , ranging from around $ 100 per patient per year in Sweden to nearly $ 900 in Germany . These variations are probably mostly due to different methodologies and differences in the year when the studies were conducted . An important finding was that the vast majority of total costs , between 72 and 98 % , was indirect costs , due to lost productivity , either in the form of work absence or reduced efficiency levels when working with migraine . Women tended to lose more workdays than men , but indirect costs were similar due to lower salaries and labour force participation amongst women . The direct costs , related to consultation , diagnostic investigations , treatments , and hospital admissions accounted for less than 30 % of total costs in most studies . The cost estimate for migraine in the European report was based on an average of the most representative cost estimates , from the UK , Germany and France . An average annual cost of $ 585 per migraine patient was estimated for these Western European countries . The 1-year prevalence of migraine was 14 % among adults in Europe according to the review of epidemiological studies -LSB- 1 -RSB- , i.e. 41 million adult Europeans with active migraine . Per patient migraine was the least costly disorder among the brain disorders . However , due to the high prevalence , the total cost of migraine was estimated to be $ 27 billion for whole Europe in 2004 , which was the highest cost among the purely neurological disorders . Many of the psychiatric afflictions were even more costly according to this review . It is , however , likely that the available cost data in Europe would tend to underestimate the actual costs of headache , mainly because no cost data existed on the most common headache type -LRB- TTH -RRB- , but also because children and adolescents were not considered , and because cost connected with more expensive medication -LRB- triptans -RRB- were not included , since most cost studies were performed before this class of drugs was introduced . In a separate paper summarizing the prevalence and cost data for headache in Europe -LSB- 30 -RSB- a more speculative estimate for the cost of headache , rather than migraine alone , was derived by using the results of the Danish -LSB- 31 -RSB- and British -LSB- 32 -RSB- population-based studies which demonstrated that around 1,100 -- 1,300 days per 1,000 workers were missed due to headache each year . The British study also suggested that the number of days with reduced efficacy was around four times higher than the number of days missed . Assuming a reduced efficiency of 35 % when working with headache , and that the direct costs of headache constitute the same proportion of the total costs as for migraine , the average total cost per headache patient was estimated to be roughly $ 420 per year -LRB- of which $ 390 would be due to indirect costs and $ 30 due to direct medical costs -RRB- . Since headache in general was found to affect nearly 50 % of Europeans , this estimate , if true , would make headache a much more costly disorder than migraine alone . It is of interest to compare the European cost study -LSB- 3 -RSB- with more recent cost studies in some individual European countries . In one study from Spain -LSB- 33 -RSB- the annual costs of migraine was only about 50 % of the sum given in the European Cost study for the same country . The difference may partly be explained by somewhat lower prevalence figures -LRB- 12 vs. 14 % -RRB- for migraine used in the Spanish study , but the main difference may be that this study did not employ a bottom-up design , but used published statistics and data to estimate resource use and productivity losses , which may have led to an underestimation of some costs . A recent study from France -LSB- 34 -RSB- , restricted to the direct costs in 1999 , found that these costs were at least twice as high -LRB- $ 128 -RRB- as in the European migraine cost study -LRB- < $ 60 -RRB- . This study included both `` strict '' migraine -LRB- IHS 1.1 and 1.2 -RRB- and `` migraineous disorder '' -LRB- IHS 1.7 , corresponding to 1.6 in ICHD-2 -RRB- , which together affected 17 % of the population . For the whole country the direct costs amounted to more than 1 billion $ , which was 0.068 % of the gross national product . Non-migraineous episodic headache , affecting 9.2 % of the population , entailed a considerably lower cost of $ 28 . It may also be of interest to compare the European studies with one US study using a quite different methodology to assess direct costs . In this study , all types of medical care costs -LRB- not only those related to headache -RRB- were derived from the claims records of a large health plan , whereas diagnostic status -LRB- migraine or not -RRB- and comorbid and demographic status was ascertained using a telephone interview among members of the health plan -LSB- 35 -RSB- . Migraineurs incurred on average $ 700 more per year in total medical care costs than the controls . Interestingly , this statistically significant difference disappeared when psychiatric comorbidity variables -LRB- anxiety and depression -RRB- were entered into the model . The much higher costs per patient reflected in this study than in the French study -LSB- 34 -RSB- and Spanish study -LSB- 33 -RSB- may therefore at least partly be due to the differences in cost assessment methodology , indicating that the direct costs specifically related to migraine and not to comorbid disorders are most reliably assessed by a direct method , questioning patients about use of health-care resources . Medication for headache constitutes an important part of the direct costs . In France in year 2000 , the most frequently used acute medications for migraine were paracetamol , salicylates and NSAIDs . Triptans were used by 8 % of migraineurs , and prophylactic treatment was used by 6 % -LSB- 36 -RSB- . In Denmark 26 % of migraineurs had used triptans in 2001 , but less than 5 % of those with pure migraine had used prophylactic medication -LSB- 37 -RSB- . Non-economic impact From a purely humanitarian perspective , but also from a public health perspective , the pain , suffering and disability caused by headaches are as important as the economic consequences . In a study performed in young women in nine Western European countries , 86 % of migraineurs stated that their life would have been better if they did not suffer form migraine -LSB- 38 -RSB- . A German study showed that , on average , patients with migraine or TTH had around 1 month every year affected by headaches -LSB- 10 -RSB- . The main burden of headache is carried by a minority of sufferers , and a Swedish study has shown that 27 % of migraine patients had 68 % of all attacks -LSB- 9 -RSB- . Three to 4 % of the European population have headache half of the days or more per month -LSB- 1 -RSB- . Disability It has been calculated that in the US , 300,000 persons stay in bed each day -LRB- 24 h -RRB- due to headaches -LSB- 39 -RSB- . A Swedish study has shown that the disability is not only related to the attacks since many migraine patients report an impairment also between attacks -LSB- 40 -RSB- . Nine % of patients report that they have some residual disability since they do not recover completely between attacks , and in addition , many patients live in a constant worry about the next attack -LSB- 9 , 38 -RSB- . In some studies , the level of disability due to migraine has been evaluated with the Migraine Disability Assessment Scale -LRB- MIDAS -RRB- . With this instrument , days with work absence -LRB- job or household chores -RRB- , days with ≥ 50 % reduction in productivity , and days with inability to participate in social activities are counted during a 3-month period . In France , among those with active migraine , 22 % -LRB- 1.5 % of the whole population -RRB- had grades III or IV disability -LRB- moderate or severe disability , indicating 11 days or more during the last 3 months ' period when headache affected work/household chores 50 % or more , or leisure activities -RRB- -LSB- 36 -RSB- . MIDAS III or IV were about twice as common among migraineurs in one US study -LRB- 54 % -RRB- -LSB- 41 -RSB- , as it was in the multinational Latin American study -LRB- 50 % -RRB- -LSB- 42 -RSB- . Among patients with headache in general -LRB- both migraine and non-migraineous headache , comprising 70 % of the study population -RRB- , 10.3 % -LRB- 7.2 % of the population -RRB- had MIDAS grade III or IV disability -LSB- 13 -RSB- . Comparing the percentage of the general population in France with MIDAS disability grade III -- IV due to migraine -LRB- 1.5 % -RRB- -LSB- 36 -RSB- , with the percentage of population in England with same disability due to headache in general -LRB- 7.2 % -RRB- -LSB- 13 -RSB- , it seems that non-migraineous headache causes more disability on a population than does migraine . The headache-attributed lost time -LRB- HALT -RRB- index is a close derivative of MIDAS -LRB- http://www.liftingtheburden.org/ → Resources → Burden measure -RRB- to be used for headache burden studies , which will be conducted by the Lifting The Burden Campaign -LSB- 43 -RSB- . According to the World Health Organisation -LRB- WHO -RRB- , the preferred measure of disease burden is `` Disability Adjusted Life Years '' -LRB- DALYs -RRB- , which is a sum of the years of life lost -LRB- YLL -RRB- and the years lived with disability -LRB- YLDs -RRB- . The YLDs are determined by the incidence and duration of the disorder , and by a disability weight ranging between 0 and 1 -LSB- 44 -RSB- . Although migraine entails no increased mortality -LRB- i.e. YLL = 0 -RRB- , it ranked the 19th among the leading causes of DALYs among women aged between 15 -- 44 years , and with regard to YLDs , it was 19th for both sexes , and the 12th for women , irrespective of age . Using the WHO data for a calculation of the burden of `` brain disorders '' -LRB- i.e. the psychiatric and neurological disorders -RRB- in Europe , the weight of migraine was lower than that of the major psychiatric disorders , dementias , stroke and injuries , but higher than that of epilepsy , multiple sclerosis and Parkinson 's disease -LSB- 45 -RSB- . In a recent report on the global prevalence and burden of headache disorders , the burden of migraine and TTH were measured in a similar way as the DALYs by combining data on prevalence , mean number and duration of headache attacks , and headache intensity , from studies containing such information . For the world as a whole , it was demonstrated that TTH resulted in a higher population burden -LRB- approximately 55 % of total burden -RRB- than migraine -LRB- 45 % -RRB- . The data for Europe indicated an even higher burden due to TTH compared to migraine -LSB- 1 -RSB- . If one uses the European data from this study it can be calculated that the hours with migraine headache would add up to between 34 and 100 h per year , if distributed on each adult individual in the population . The data on TTH are too scarce to use for similar calculations . Studies using validated QoL-instruments The SF-36 is a validated instrument to measure quality of life -LRB- QoL -RRB- , containing eight dimensions . One US study -LSB- 46 -RSB- , recruiting migraine patients from a medication trial , demonstrated that migraineurs had lower QoL than the general US population , most marked for bodily pain , physical role limitations and social functioning . A Dutch population-based study found that migraine had a negative influence on all a dimensions compared to controls . The negative influence on QoL was larger than that of e.g. asthma , and it increased with increasing headache frequency -LSB- 47 -RSB- . Two population-based studies from Spain among chronic daily headache sufferers showed a marked negative influence , most marked for those with medication overuse , but similar for those with a headache of a migraine or a tension type -LSB- 48 -RSB- . One of these showed that the headache frequency may have a greater impact than headache intensity on QoL -LSB- 48 -RSB- , and the other that chronic headache with medication overuse was associated with a decrease in all QoL aspects studied with SF-36 , most marked for physical role and bodily pain -LSB- 49 -RSB- . A study from UK showed that migraineurs with high or moderate disability had a marked reduction on all dimensions on the SF-36 -LSB- 50 -RSB- . One Swedish study compared SF-36 results in the two sexes and in participants with different pain conditions . There was a gender difference for headache , which in men influenced physical function , physical role and bodily pain most , and in women vitality , social functioning , emotional functioning and mental health -LSB- 51 -RSB- . In a French study , migraineurs had significantly lower scores than headache-free controls on all SF-36 dimensions , and lower scores on the pain dimension than those with other headaches or with TTH -LSB- 52 -RSB- . One study comparing migraineurs in the US and the UK used a shorter QoL instrument , the SF-12 , which contains a physical and a mental component -LSB- 53 -RSB- . In both countries , migraineurs had lower scores than controls on both components also after adjusting for socioeconomic status and for depression . However , in those with both migraine and depression , the QoL was significantly reduced in comparison to those who were not depressed . In another French study , using a disease-specific QoL instrument called QVM , the QoL was found to be lowest among those with chronic headache , intermediate among migraineurs and highest among subjects with other forms of episodic headache -LSB- 54 -RSB- . The total burden of headache patients may not only be related to the headache per se , but also to comorbid conditions . European population-based studies have demonstrated that depression and/or anxiety occur two to three times more often among migraineurs than in the general population -LSB- 55 , 56 -RSB- . Depression adds to the reduction in QoL in migraine -LSB- 53 -RSB- . This comorbidity may be as important for non-migrainous headache -LSB- 57 -RSB- , but it is not known how this comorbidity influences the QoL in other headaches . In addition , it has been found that headache is also comorbid with other bodily pain , both in Finnish children -LSB- 58 -RSB- and Norwegian adults -LSB- 59 -RSB- . Family impact of migraine Migraine also affects the patients ' spouses and children . In a population-based Swedish study -LSB- 9 -RSB- , the percentage of migraine sufferers who reported a negative impact of migraine was 76 % for attendance to work , 67 % for family situation , 59 % for leisure time , 48 % for pursuing studies , 46 % for sexual life , 37 % for their social position , 31 % for love , 30 % for their financial situation , 27 % for making a career , and 11 % for making friends . One study has measured the impact on the family in two population samples of similar size in US and UK -LSB- 60 -RSB- . The impact was very similar in both countries . More than 60 % of patients reported a marked impact on the ability to do household chores because of their migraine during the past 3 months , and it was markedly reduced also in 20 % of the patients ' partners . Almost 46 % of patients , and 24 % of partners had missed days of family or social activities due to the proband 's migraine , and 16 % of patients and 12 % of partners had avoided making plans for family or social activities due to the proband 's migraine . As to the impact on the children of patients , more than 60 % stated that it had a moderate to marked influence on the relation with their children , 40 % stated that they would have been a better guardian or parent without migraine , more than 10 % stated that their children had missed school , and 10 % that their children had been late to school because of their headache . Forty-six percent of patients stated that they would have been better partners without headaches , and 5 % stated that they had had fewer children because of headache , 0.4 % that they had avoided having children , and 15 % that they had avoided oral contraception . Compared with a control group , the partners of migraine patients were significantly more dissatisfied with the demands , responsibility and duties placed upon them , and with their ability to perform . Conclusions Health economic studies have documented that the costs of headache disorders are huge ; the costs only for migraine amounting to $ 27 billion in the EU countries , and the cost for other headaches are probably as large . However , better population-based cost studies are needed to assess the cost involved with TTH . Headache sufferers tend to have lower income and education , and more of them may be unemployed , but it is still uncertain whether this is true for most European countries , and also whether it may be a cause of or an effect of headache . It is also amply documented that migraine confers a high degree of disability with more forced absence from work and leisure activities , and migraineurs also have a measurably reduced quality of life . In addition , there is a marked impact on family life , and headaches also put considerable strains on partners and children . A minority of headache sufferers chose to have fewer children than they would have had if they had not had headaches . Based on the present review we have identified some main domains , summarized in the Table 1 , that should be covered in order to capture as much as possible of the headache burden . The investigations should be performed in population-based samples , and to assess the whole burden , it is particularly important that not only migraine is included but also TTH and the chronic headaches . Different studies show marked variations between countries , both with regard to prevalence and burden of headache . At present it is not possible to determine with certainty whether these differences are real or due to variations in methodology . This highlights the need to study several countries with the same methodology and instrument , which will be done in the ongoing studies of the Eurolight . If further research shows that there are real and important differences between various countries and regions , investigations to determine the causes of such differences may elucidate ways to lower the burden of headache in a population . In any case , we believe that the Eurolight studies will provide the evidence needed to let headache disorders get the resources for treatment and research that they deserve according to the burden they place on people in Europe . Table 1The main domains that should be covered in headache burden studiesEconomic burden Direct costMedication , consultation , investigations , hospitalisations Indirect costsWorkdays lostDecreased effectiveness when working with headacheLost career and education opportunitiesNon-economic burden DisabilityMIDAS or HALTTime with disability : Headache frequency x duration x intensity/disability Impact outside attacksResidual disability and fear of next attack Quality of LifeSF 36 , SF 12 , WHOQual etc Family impactImpact on the life of partner and/or childrenImpact on marriage and love lifeImpact on family planning and/or contraception Psychiatric complaintsVarious scales to measure anxiety and/or depression as consequent or comorbid disorders -LRB- e.g. HADS -RRB-

B O B O O O O O O O B 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 B O O O B O O B O O O O O O O O O O O O B B 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 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 B 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 B 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 B 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 B O O O O O O O O O O O O B O O O O O O O B O B 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 B 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 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 B 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 B 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 B B 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 O O O O O O B O O O B O B 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 B 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 B 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 B 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 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 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 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