The

influence

of

low-grade

glioma

on

resting

state

oscillatory

brain

activity

a

magnetoencephalography

study

Purpose

In

the

present

MEG-study

power

spectral

analysis

of

oscillatory

brain

activity

was

used

to

compare

resting

state

brain

activity

in

both

low-grade

glioma

-LRB-

LGG

-RRB-

patients

and

healthy

controls

We

hypothesized

that

LGG

patients

show

local

as

well

as

diffuse

slowing

of

resting

state

brain

activity

compared

to

healthy

controls

and

that

particularly

global

slowing

correlates

with

neurocognitive

dysfunction

Patient

and

methods

Resting

state

MEG

recordings

were

obtained

from

LGG

patients

and

age

sex

and

education-matched

healthy

controls

Relative

spectral

power

was

calculated

in

the

delta

theta

upper

and

lower

alpha

beta

and

gamma

frequency

band

A

battery

of

standardized

neurocognitive

tests

measuring

neurocognitive

domains

was

administered

Results

LGG

patients

showed

a

slowing

of

the

resting

state

brain

activity

when

compared

to

healthy

controls

Decrease

in

relative

power

was

mainly

found

in

the

gamma

frequency

band

in

the

bilateral

frontocentral

MEG

regions

whereas

an

increase

in

relative

power

was

found

in

the

theta

frequency

band

in

the

left

parietal

region

An

increase

of

the

relative

power

in

the

theta

and

lower

alpha

band

correlated

with

impaired

executive

functioning

information

processing

and

working

memory

Conclusion

LGG

patients

are

characterized

by

global

slowing

of

their

resting

state

brain

activity

and

this

slowing

phenomenon

correlates

with

the

observed

neurocognitive

deficits

Introduction

Low

grade

glioma

-LRB-

LGG

-RRB-

patients

constitute

-LSB-

-RSB-

of

the

glioma

patient

population

and

have

a

survival

of

years

-LSB-

-RSB-

The

optimal

treatment

for

this

patient

group

is

still

a

matter

of

debate

-LSB-

-RSB-

The

first

clinical

feature

of

this

disease

is

epilepsy

in

two-third

of

the

patients

-LSB-

-RSB-

The

majority

of

LGG

patients

also

suffer

from

cognitive

deficits

which

tend

to

have

a

global

character

and

can

not

only

be

explained

by

the

tumor

localization

alone

-LSB-

-RSB-

Higher

cognitive

functions

depend

on

the

integrated

activity

of

several

specialized

brain

areas

Functional

imaging

techniques

such

as

electroencephalography

-LRB-

EEG

-RRB-

and

magnetoencephalography

-LRB-

MEG

-RRB-

are

used

to

characterize

oscillatory

activity

in

patients

with

neurodegenerative

diseases

including

Alzheimer

's

-LSB-

AD

-RSB-

and

Parkinson

's

disease

-LSB-

PD

-RSB-

Oscillations

are

a

feature

of

neuronal

brain

activity

and

the

synchronization

of

the

oscillatory

activity

-LRB-

reflecting

the

interactions

of

neuronal

activity

-RRB-

is

a

likely

mechanism

for

neuronal

communication

For

an

overview

of

the

relation

between

oscillatory

brain

activity

and

neurocognitive

function

-LSB-

see

-RSB-

Magnetoencephalography

-LRB-

MEG

-RRB-

is

a

relatively

novel

way

to

capture

the

dynamics

of

the

electromagnetic

fields

within

the

brain

and

combines

excellent

temporal

and

spatial

resolution

Comparison

of

MEG

in

AD

patients

and

healthy

controls

showed

more

delta

and

theta

activity

in

the

patient

population

especially

in

the

temporo-parietal

region

-LSB-

-RSB-

Another

study

compared

spectral

power

in

four

different

groups

-LRB-

severe

AD

moderate

AD

Lewy

Body

dementia

and

healthy

controls

-RRB-

and

showed

that

the

healthy

control

population

had

the

largest

rhythmic

activity

in

the

alpha

band

The

moderate

AD

population

and

the

patients

with

Lewy

Body

dementia

had

the

rhythmic

predominance

in

the

pre-alpha

frequency

band

-LRB-

Hz

-RRB-

whereas

the

severe

AD

showed

a

shift

towards

the

slow-band

-LRB-

Hz

-RRB-

-LSB-

-RSB-

In

PD

patients

MEG

has

been

used

by

Kotini

-LSB-

-RSB-

who

showed

slowing

of

the

alpha

rhythm

in

the

patient

population

compared

to

healthy

controls

and

Bosboom

-LSB-

-RSB-

who

demonstrated

slowing

in

the

theta

beta

and

gamma

frequency

band

in

non-demented

PD

patients

with

a

further

slowing

-LRB-

and

involvement

of

delta

and

alpha

band

-RRB-

in

the

demented

PD

patient

population

It

is

suggested

that

the

increase

in

slow

wave

activity

corresponds

with

neurocognitive

deficits

in

AD

as

well

as

in

PD

-LSB-

-RSB-

MEG-studies

have

previously

been

performed

in

brain

tumor

patients

Oshino

-LSB-

-RSB-

found

an

increase

in

delta

and

theta

band

activity

especially

in

the

vicinity

of

the

tumor

and

in

the

surrounding

regions

of

edema

In

the

majority

of

these

patients

the

increase

correlated

with

the

clinical

symptoms

of

these

patients

assessed

with

a

routine

neurological

examination

but

neurocognitive

function

were

not

formally

investigated

Although

the

focal

lesion

in

brain

tumor

patients

contrasts

with

the

generalized

brain

degeneration

in

AD

and

PD

there

is

evidence

that

brain

tumors

can

cause

functional

disturbances

in

areas

remote

from

the

tumor

-LSB-

-RSB-

De

Jongh

-LSB-

-RSB-

found

focal

clusters

of

delta

activity

near

the

tumor

area

but

also

in

the

contralateral

hemisphere

An

evaluation

of

fast

MEG

waves

in

brain

tumor

patients

showed

dipoles

located

in

the

parietal

and

occipital

area

whereas

the

tumors

were

located

in

the

parietal

temporal

and

frontal

area

-LSB-

-RSB-

In

this

study

these

dipoles

were

found

remote

from

the

tumor

area

and

even

in

the

contralateral

hemisphere

Bartolomei

-LSB-

-RSB-

found

differences

in

resting

state

functional

connectivity

in

the

same

brain

tumor

population

within

several

frequency

bands

when

compared

to

healthy

controls

Interestingly

in

agreement

with

de

Jongh

's

results

these

differences

were

not

limited

to

the

tumor

area

and

were

more

obvious

in

those

with

a

tumor

in

the

left

hemisphere

Since

higher

cognitive

functions

are

presumed

to

depend

on

the

integrated

activity

of

several

specialized

brain

areas

it

is

suggested

that

neurocognitive

deficits

may

have

a

stronger

correlation

with

diffuse

alterations

in

resting

state

brain

oscillatory

activity

than

focal

abnormalities

in

the

brain

tumor

population

In

the

present

MEG-study

power

spectral

analysis

of

oscillatory

brain

activity

was

used

to

compare

resting

state

brain

oscillatory

activity

in

both

LGG

patients

and

healthy

controls

We

hypothesize

that

-LRB-

-RRB-

LGG

patients

show

in

addition

to

the

local

abnormalities

at

the

tumor

site

global

slowing

of

the

resting

state

brain

activity

compared

to

healthy

controls

which

will

vary

between

patients

with

a

tumor

in

the

left

or

right

hemisphere

-LRB-

-RRB-

changes

in

resting

state

brain

oscillatory

activity

reflect

an

intermediate

level

between

the

impact

of

tumor

and

tumor-related

treatment

on

the

one

hand

-LRB-

input

-RRB-

and

the

neurocognitive

deficits

-LRB-

output

-RRB-

on

the

other

hand

and

that

diffuse

slowing

is

correlated

with

higher

neurocognitive

dysfunction

Materials

and

methods

Patients

and

controls

Twenty-three

LGG

patients

were

asked

to

participate

in

this

study

Patients

were

eligible

if

-LRB-

a

-RRB-

they

had

a

suspected

or

histologically

confirmed

LGG

-LRB-

b

-RRB-

there

was

no

radiological

-LRB-

confirmed

by

MR

or

CT

scan

-RRB-

and/or

clinical

tumor

progression

in

the

previous

months

-LRB-

c

-RRB-

they

did

not

use

medication

possibly

interfering

with

neurocognitive

function

other

than

anti-epileptic

drugs

-LRB-

AEDs

-RRB-

Patients

were

recruited

from

the

VU

University

Medical

Center

-LRB-

VUmc

-RRB-

and

the

Academic

Medical

Center

-LRB-

AMC

-RRB-

both

tertiary

referral

centres

in

Amsterdam

for

brain

tumor

patients

after

the

institutional

ethical

review

boards

of

both

participating

hospitals

approved

the

study

protocol

Relatives

of

the

patients

were

asked

to

participate

as

healthy

controls

Healthy

controls

were

eligible

if

they

-LRB-

a

-RRB-

did

not

suffer

from

any

neurological

disease

-LRB-

b

-RRB-

did

not

use

any

medication

that

might

influence

cognitive

function

For

patients

who

could

not

provide

a

healthy

control

participant

VU

University

Medical

Center

staff

members

were

included

Magnetoencephalography

MEG

recordings

were

obtained

using

a

whole-head

MEG

system

-LRB-

CTF

systems

Port

Coquitlam

British

Columbia

Canada

-RRB-

while

participants

were

seated

inside

a

magnetically

shielded

room

-LRB-

Vacuumschmelze

GmbH

Hanau

Germany

-RRB-

Magnetic

fields

were

recorded

during

a

no-task

eyes-closed

resting

state

Metal

artefacts

were

avoided

as

much

as

possible

A

third-order

software

gradient

-LSB-

-RSB-

was

used

with

a

recording

passband

of

Hz

and

a

sample

frequency

of

Hz

At

the

beginning

middle

and

end

of

each

recording

the

head

position

relative

to

the

coordinate

system

of

the

helmet

was

recorded

by

leading

small

alternating

currents

through

three

head

position

coils

attached

to

the

left

and

right

preauricular

points

and

the

nasion

on

the

subject

's

head

Head

position

changes

up

to

approximately

cm

during

a

recording

condition

were

accepted

For

this

study

of

the

channels

could

be

used

MEG

recordings

were

converted

to

ASCII

files

From

these

ASCII

files

four

artefact

free

epochs

of

s

per

subject

-LRB-

samples

-RRB-

were

carefully

selected

by

visual

analysis

by

one

of

the

authors

-LRB-

IB

-RRB-

Magnetic

field

frequencies

ranging

from

to

Hz

were

recorded

The

MEG

data

were

digitally

filtered

off-line

in

the

following

frequency

bands

delta

-LRB-

Hz

-RRB-

theta

-LRB-

Hz

-RRB-

lower

alpha

-LRB-

Hz

-RRB-

upper

alpha

-LRB-

Hz

-RRB-

beta

-LRB-

Hz

-RRB-

gamma

-LRB-

Hz

-RRB-

The

MEG

channels

were

grouped

into

distinct

regions

respectively

left

and

right

frontal

region

left

and

right

temporal

region

left

and

right

parietal

region

left

and

right

occipital

region

and

left

and

right

central

region

as

shown

in

Fig.

Fast

Fourier

transformation

was

separately

applied

for

every

patient

and

control

on

the

four

epochs

in

the

above

mentioned

frequency

bands

The

results

of

the

four

epochs

were

averaged

for

each

participant

and

the

mean

relative

power

for

each

MEG

region

were

used

for

the

analysis

Fig.

of

MEG

regions

Neurocognitive

assessment

Patients

and

controls

participated

in

neurocognitive

assessment

-LRB-

see

Table

-RRB-

The

total

duration

of

the

assessment

varied

between

and

h.

Individual

patient

's

test

scores

were

converted

to

z-scores

using

the

means

and

standard

deviations

of

the

age

sex

and

education-matched

healthy

controls

as

a

reference

Table

of

neuropsychological

test

batteryLetter-digit

substitution

test

-LSB-

-RSB-

This

test

provides

a

measure

of

psychomotor

performance

that

is

relatively

unaffected

by

intellectual

prowess

and

is

suitable

for

groups

with

an

age

range

exceeding

years

The

number

of

items

written

down

in

s

is

registered

as

in

the

decrease

in

performance

when

graphomotor

speed

is

involvedVisual

verbal

learning

test

-LSB-

-RSB-

This

version

of

the

Rey

Auditory

Verbal

Learning

Test

calls

for

various

aspects

of

verbal

learning

and

recall

Measures

used

for

analysis

are

memory

performance

on

trial

as

indicator

of

immediate

recall

total

recall

after

five

trials

delayed

recall

and

recognition

after

min

as

indicators

of

memory

consolidation

into

long-term

memory

and

a

delta

score

as

a

measure

of

learning

capacityStroop

color-word

test

-LSB-

-RSB-

This

test

is

a

selective

attention

task

aiming

at

measuring

interference

susceptibility

and

consists

of

three

subtasks

with

increasing

task

complexityCategoric

word

fluency

-LSB-

-RSB-

A

simple

task

requiring

the

generation

of

words

from

semantic

categories

-LRB-

animals

-RRB-

within

a

limited

timeConcept

shifting

test

-LSB-

-RSB-

This

test

which

has

two

conditions

of

complexity

predominately

measures

functions

associated

with

executive

function

especially

visual

scanning

and

conceptual

tracking

The

motor

component

of

this

task

is

measured

by

three

dummy

conditions

in

which

no

cognitive

capacity

except

for

graphomotor

speed

is

requiredMemory

comparison

test

-LRB-

MCT

-RRB-

Selective

attention

mental

concentration

memory

and

information

processing

To

reduce

data

individual

scores

on

these

tests

were

summarized

into

six

cognitive

domains

namely

information

processing

speed

psychomotor

function

attention

verbal

memory

working

memory

and

executive

functioning

Construction

of

these

domains

has

previously

been

reported

-LSB-

-RSB-

and

was

based

on

a

Principal

Component

Analysis

using

Varimax

rotation

with

Kaiser

normalization

performed

on

the

z-scores

of

a

large

group

of

healthy

controls

-LSB-

-RSB-

The

domains

found

are

commonly

used

in

neurocognitive

practice

and

research

Statistical

analysis

Differences

between

both

groups

in

the

distribution

of

age

sex

and

education

were

analyzed

by

means

of

chi-square

tests

Mann

Whitney

nonparametric

U-tests

were

used

to

investigate

whether

patients

standardised

z-scores

on

neurocognitive

tests

in

the

overall

measure

of

cognition

differed

significantly

from

healthy

control

z-scores

Because

of

the

non-normal

distribution

of

the

relative

power

Mann

Whitney

nonparametric

U-tests

were

used

to

determine

possible

differences

between

the

patient

population

and

healthy

controls

To

assess

the

association

between

relative

power

and

cognition

within

the

patient

group

Spearman

's

correlation

coefficient

-LRB-

ρ

-RRB-

was

calculated

between

the

relative

power

in

the

different

MEG

regions

and

z-scores

of

the

six

neurocognitive

domains

for

all

separate

frequency

bands

Results

Patient

characteristics

From

the

initial

patient

group

six

patients

were

excluded

four

patients

due

to

metal

artefacts

on

the

MEG

measurements

one

due

to

severe

epileptic

seizures

and

one

due

to

tumor

progression

at

the

time

of

registration

The

final

analyses

were

performed

on

a

sample

of

patients

and

matched

healthy

control

participants

Due

to

the

matching

procedure

there

were

no

significant

differences

between

patients

and

healthy

controls

in

age

-LRB-

M

SD

in

patients

M

SD

in

healthy

controls

p

-RRB-

and

educational

level

-LRB-

M

SD

in

patients

M

SD

in

healthy

controls

p

-RRB-

The

male

female

ratio

between

the

two

groups

did

not

differ

significantly

-LRB-

p

-RRB-

Fourteen

of

the

LGG

patients

had

a

histologically

confirmed

LGG

clinically

and

radiologically

stable

for

more

than

six

months

before

inclusion

whereas

the

other

patients

were

suspected

of

having

LGG

and

stable

for

more

than

six

months

The

patients

in

our

study

were

diagnosed

several

years

ago

-LRB-

mean

years

range

years

-RRB-

One

year

after

MEG

registration

two

of

the

three

patients

with

suspected

LGG

were

operated

on

because

of

increasing

epilepsy

frequency

Eleven

of

those

patients

underwent

debulking

whereas

patients

underwent

a

stereotactic

biopsy

and

another

two

patients

had

an

open

biopsy

Of

the

patients

with

a

histologically

confirmed

LGG

the

pathological

diagnosis

was

grade

II

astrocytoma

in

ten

patients

oligodendroglioma

grade

II

in

four

patients

and

oligoastrocytoma

grade

II

in

two

patients

Seven

of

the

seventeen

patients

underwent

radiotherapy

with

prior

chemotherapy

in

two

patients

-LRB-

patient

with

cycles

of

PCV

and

patient

with

cycles

of

PCV

and

cycles

of

temozolomide

-RRB-

Eleven

patients

had

left

hemisphere

tumors

and

patients

had

right-sided

tumors

The

specific

localisation

of

the

tumor

is

shown

in

Table

Table

lateralization

and

localizationLeft

hemisphereRight

hemisphereTumor

locationNo

of

patientsTumor

locationNo

of

patientsLeft

insular

parieto-occipital

In

the

patient

group

all

but

one

patient

used

AED

mono

or

poly-therapy

Six

of

the

patients

on

AED

were

free

of

seizures

while

the

other

patients

were

still

having

seizures

Differences

between

patient

and

healthy

control

group

Neurocognitive

functioning

As

expected

patients

performed

poorer

than

healthy

controls

on

the

neurocognitive

test

battery

-LRB-

controls

M

-RRB-

More

specifically

patients

showed

a

significantly

lower

psychomotor

function

-LRB-

M

SD

versus

SD

p

-RRB-

working

memory

capacity

-LRB-

M

SD

versus

SD

p

-RRB-

information

processing

speed

-LRB-

M

SD

versus

SD

p

-RRB-

and

attention

-LRB-

M

SD

versus

SD

p

-RRB-

Patients

performance

on

the

other

two

cognitive

domains

did

not

differ

significantly

from

controls

-LRB-

Fig.

-RRB-

Fig.

z-scores

on

the

six

neurocognitive

domains

and

on

total

neurocognitive

functioning

Note

p

p

A

attention

EF

executive

functioning

IPS

information

processing

speed

PF

psychomotor

function

VM

verbal

memory

WM

working

memory

Performance

is

relative

to

that

of

age

sex

and

education-matched

healthy

controls

-LRB-

represented

by

the

-RRB-

A

higher

score

-LRB-

i.e.

approaching

-RRB-

means

better

performance

Spectral

analysis

Global

spectral

analysis

The

mean

relative

power

of

the

LGG

patient

population

showed

a

significant

decrease

-LRB-

Mann

Whitney

U

p

-RRB-

in

the

gamma

band

-LRB-

Hz

-RRB-

compared

with

healthy

controls

Spectral

analysis

of

frequency

bands

within

each

MEG

region

An

increase

-LRB-

Mann

Whitney

U

p

-RRB-

in

relative

power

in

the

LGG

patients

compared

to

the

healthy

controls

was

found

in

the

left

parietal

region

within

the

theta

frequency

band

-LRB-

Hz

-RRB-

A

decrease

in

relative

power

was

seen

in

the

left

and

right

central

region

-LRB-

Mann

Whitney

U

both

p

-RRB-

and

left

and

right

frontal

region

-LRB-

Mann

Whitney

U

both

p

-RRB-

of

the

LGG

patient

population

compared

to

the

healthy

controls

within

the

gamma

band

-LRB-

Hz

-RRB-

as

shown

in

Table

and

Fig.

Table

differences

in

relative

power

between

patients

and

controls

per

frequency

band

and

accompanying

statisticsPatientsControlspMSDMSDThetaLeft

GammaLeft

Right

Left

Right

Significant

higher

relative

power

is

depicted

in

bold

and

in

italicsFig

significant

differences

in

relative

power

between

the

patient

group

and

the

healthy

controls

within

the

different

frequency

bands

Green

area

significant

higher

relative

power

in

the

patient

group

compared

to

the

healthy

controls

Red

area

significant

lower

relative

power

in

the

patient

group

compared

to

the

healthy

controls

Influence

of

tumor

lateralization

on

relative

power

Eleven

patients

had

left

hemisphere

tumors

and

patients

had

a

tumor

in

the

right

hemisphere

Patients

with

a

tumor

in

the

left

hemisphere

showed

an

increase

in

theta

activity

in

the

left

and

right

central

region

-LRB-

Mann

Whitney

U

respectively

p

and

p

-RRB-

left

and

right

parietal

region

-LRB-

respectively

p

and

p

-RRB-

and

left

and

right

temporal

region

-LRB-

respectively

p

and

p

-RRB-

when

compared

to

the

healthy

control

population

Patients

with

left-sided

tumors

also

showed

a

decrease

in

relative

power

in

the

gamma

band

in

the

left

and

right

central

region

-LRB-

respectively

p

and

p

-RRB-

and

left

and

right

frontal

region

-LRB-

respectively

p

and

p

-RRB-

Patients

with

a

tumor

in

the

right

hemisphere

showed

a

significant

increase

in

relative

power

of

delta

activity

in

the

right

parietal

region

-LRB-

Mann

Whitney

U

p

-RRB-

compared

to

the

healthy

control

population

These

patients

also

showed

a

decrease

in

relative

power

in

the

gamma

band

in

the

left

and

right

frontal

region

-LRB-

respectively

p

and

p

-RRB-

right

temporal

region

-LRB-

p

-RRB-

and

left

and

right

occipital

region

-LRB-

respectively

p

and

p

-RRB-

as

shown

in

Table

and

Fig.

Table

differences

in

relative

power

between

patients

with

a

tumor

in

the

left

or

right

hemisphere

and

controls

per

frequency

band

and

accompanying

statisticsPatientsControlspMSDMSDLeft

hemisphereThetaLeft

Right

Left

Right

Left

Right

GammaLeft

Right

Left

Right

Right

hemisphereDeltaRight

GammaLeft

Right

Right

Left

Right

Significant

higher

relative

power

is

depicted

in

bold

and

in

italicsFig

significant

differences

in

relative

power

between

the

patients

with

a

tumor

in

the

left

or

right

hemisphere

compared

to

the

healthy

controls

within

the

different

frequency

bands

Green

area

significant

higher

relative

power

in

the

patient

group

compared

to

the

healthy

controls

Red

area

significant

lower

relative

power

in

the

patient

group

compared

to

the

healthy

controls

Since

these

results

could

be

influenced

by

differences

in

tumor

volume

we

evaluated

the

tumor

size

of

participating

patients

We

used

MRI

and

defined

tumor

size

as

the

product

of

the

two

largest

perpendicular

diameters

of

tumor

hypointensity

-LSB-

-RSB-

We

found

no

significant

differences

in

the

tumor

size

between

left

and

right-sided

tumors

Associations

between

patient

's

neurocognitive

functioning

and

relative

power

in

distinct

MEG

regions

In

the

patient

population

negative

associations

between

neurocognitive

functioning

and

relative

power

were

found

in

the

theta

and

lower

alpha

band

An

increase

in

the

relative

power

in

the

theta

band

within

the

left

frontal

-LRB-

p

-RRB-

and

the

right

frontal

region

-LRB-

p

-RRB-

was

correlated

with

a

poorer

executive

functioning

In

the

same

frequency

band

-LRB-

Hz

-RRB-

an

increase

in

theta

band

power

was

correlated

with

poorer

information

processing

within

the

left

central

and

left

frontal

region

-LRB-

respectively

p

and

p

-RRB-

Within

the

lower

alpha

band

in

the

right

temporal

region

an

increase

in

relative

power

was

correlated

with

a

disturbed

working

memory

-LRB-

p

-RRB-

Discussion

The

primary

goal

of

the

present

study

was

to

evaluate

whether

LGG

patients

show

-LRB-

in

addition

to

the

well

known

MEG

slowing

around

the

tumor

-RRB-

diffuse

slowing

in

resting

state

brain

activity

The

secondary

goal

of

the

study

was

to

investigate

whether

this

slowing

is

correlated

with

neurocognitive

dysfunction

By

means

of

MEG-registrations

we

have

demonstrated

that

LGG

patients

have

slowing

of

the

resting

state

brain

activity

when

compared

to

healthy

controls

The

decrease

in

relative

power

was

mainly

found

in

the

gamma

frequency

band

in

the

bilateral

frontocentral

MEG

regions

Regarding

the

low

frequency

bands

an

increase

in

relative

power

was

found

in

the

theta

frequency

band

in

the

left

parietal

region

Correlations

of

neurocognitive

functioning

with

the

relative

power

in

the

patient

population

showed

clear

associations

in

the

lower

alpha

and

theta

band

increased

slowing

correlating

with

poorer

performance

We

chose

to

use

the

relative

power

instead

of

the

absolute

power

because

relative

power

is

less

influenced

by

the

distance

between

the

MEG

sensor

and

the

underlying

neural

populations

The

distance

is

variable

due

to

head

position

in

the

helmet

and

the

thickness

of

the

skull

Lower

variance

of

power

values

in

subject

groups

are

expected

by

using

the

relative

power

instead

of

the

absolute

power

A

second

reason

for

us

to

use

this

method

is

the

fact

that

by

using

this

method

we

are

able

to

compare

our

results

with

those

of

other

patient

cohorts

at

our

MEG

center

-LSB-

-RSB-

This

includes

comparisons

with

other

brain

tumor

data

sets

but

also

the

comparison

of

our

results

with

neurodegenerative

diseases

-LRB-

including

AD

and

Parkinson

's

disease

-LRB-

PD

-RRB-

-RRB-

This

will

give

us

an

opportunity

to

learn

more

about

differences

in

resting

state

oscillatory

brain

activity

in

the

different

diseases

and

at

different

disease

stages

A

limitation

of

this

method

of

analysis

is

that

an

increase

in

one

frequency

band

can

cause

a

decrease

in

another

band

although

this

might

not

be

the

observed

pattern

in

absolute

measures

In

previous

studies

MEG

has

been

used

for

power

analysis

in

patients

with

neurodegenerative

diseases

such

as

AD

and

PD

They

showed

similar

results

namely

a

rhythmic

predominance

in

the

lower

frequency

bands

as

was

found

in

our

study

with

LGG

patients

-LSB-

-RSB-

There

are

only

few

studies

performed

in

brain

tumor

patients

however

De

Jongh

-LSB-

-RSB-

demonstrated

that

both

a

higher

tumor

malignancy

and

larger

tumor

volume

were

associated

with

higher

signal

powers

in

the

delta

band

In

our

study

we

did

not

find

increased

oscillatory

brain

activity

in

the

delta

band

It

might

be

that

the

conflicting

results

are

due

to

differences

in

methodology

of

both

studies

In

contrast

with

De

Jongh

's

study

-LSB-

-RSB-

we

analysed

the

relative

power

which

means

that

the

power

is

influenced

by

the

power

in

the

other

frequency

bands

and

therefore

group

differences

in

a

specific

frequency

band

could

results

from

changes

in

that

specific

band

or

in

the

absolute

power

of

the

other

frequency

bands

Furthermore

we

evaluated

diffuse

delta

activity

instead

of

clusters

of

dipoles

in

the

vicinity

of

the

tumor

Apart

from

these

technical

differences

De

Jongh

's

patient

population

consisted

of

a

more

heterogeneous

group

of

brain

tumor

patients

They

found

a

higher

delta

power

in

the

high-grade

gliomas

compared

to

those

patients

with

a

low-grade

glioma

and

a

delta

signal

power

decrease

after

surgery

Our

patient

population

consisted

exclusively

of

patients

with

a

low-grade

glioma

and

all

but

three

MEG

registrations

were

done

after

surgery

which

might

explain

the

lack

of

changes

in

the

delta

band

in

the

present

study

It

also

suggests

that

changes

in

the

gamma

and

theta

band

may

be

more

sensitive

indicators

of

brain

dysfunction

in

LGG

In

another

study

of

De

Jongh

-LSB-

-RSB-

the

authors

found

focal

clusters

of

fast

MEG

waves

outside

the

tumor

area

and

even

in

the

contralateral

hemisphere

They

concluded

that

the

asymmetry

possibly

reflects

features

of

normal

background

activity

since

asymmetry

has

already

been

demonstrated

in

healthy

subjects

for

the

alpha

activity

which

is

usually

dominant

in

the

right

hemisphere

Since

De

Jongh

used

a

single

dipole

analysis

instead

of

our

analysis

of

diffuse

power

changes

and

again

analysed

a

group

of

patients

with

diverse

brain

tumors

it

is

difficult

to

compare

the

results

of

both

studies

Oshino

and

colleagues

-LSB-

-RSB-

used

synthetic

aperture

magnetometry

-LRB-

SAM

-RRB-

to

analyze

oscillatory

activity

recorded

by

the

MEG

SAM

is

an

alternative

method

investigating

the

distribution

of

reconstructed

sources

over

different

cortical

regions

In

contrast

to

other

source

localization

methods

beamforming

does

not

rely

on

averaging

and

therefore

allows

analysis

of

evoked

and

induced

brain

activity

In

a

group

of

patients

with

various

types

of

primary

or

secondary

brain

tumors

they

found

an

increase

in

delta

and

theta

band

activity

especially

in

the

vicinity

of

the

tumor

and

in

the

surrounding

regions

of

edema

In

the

majority

of

these

patients

the

increase

in

the

delta

band

correlated

well

with

the

clinical

symptoms

assessed

with

a

routine

neurological

examination

The

results

were

not

correlated

with

neurocognitive

functioning

because

they

stated

that

there

is

much

individual

variation

in

brain

activity

related

to

neurocognitive

function

and

they

expected

it

difficult

and

sometimes

impossible

for

these

patients

to

undergo

the

neurocognitive

battery

In

contrast

to

their

results

we

only

found

an

increase

in

theta

activity

and

decrease

in

the

gamma

frequency

band

As

mentioned

before

we

analyzed

the

relative

power

and

also

our

patient

population

with

only

LGG

patients

is

not

comparable

to

the

patients

with

various

brain

tumors

in

the

study

of

Oshino

Although

different

methods

to

reconstruct

sources

can

show

significant

discrepancies

we

also

found

spectral

changes

in

the

contralateral

hemisphere

By

evaluating

separately

those

patients

with

a

tumor

localisation

in

the

left

or

the

right

hemisphere

we

found

more

significant

differences

in

patients

with

a

tumor

in

the

left

hemisphere

compared

to

healthy

controls

In

agreement

with

our

previous

study

-LSB-

-RSB-

in

which

we

evaluated

the

functional

connectivity

-LRB-

functional

connectivity

is

a

statistical

correlation

between

time

series

of

brain

activity

recorded

over

distinct

regions

which

are

assumed

to

reflect

interactions

between

the

brain

regions

-RRB-

in

patients

with

diverse

primary

brain

tumors

we

also

found

that

patients

with

a

tumor

in

the

left

hemisphere

showed

more

differences

in

this

functional

connectivity

compared

to

those

with

right-sided

tumors

Why

those

patients

with

a

tumor

in

the

left

hemisphere

do

show

more

differences

in

oscillatory

activity

is

not

known

It

is

possible

that

the

left

hemisphere

-LRB-

usual

the

dominant

hemisphere

-RRB-

acts

differently

to

the

tumor

compared

to

those

located

in

the

right

hemisphere

It

is

also

possible

that

patients

with

a

tumor

in

the

left

hemisphere

are

in

a

different

stage

of

their

disease

compared

to

those

with

a

tumor

in

the

right

hemisphere

Since

our

sample

size

is

rather

small

this

could

be

reflected

on

the

observed

results

On

the

other

hand

is

it

possible

that

our

results

reflect

hemispheric

asymmetry

which

has

been

found

in

the

healthy

population

previously

Gootjes

et

al.

-LSB-

-RSB-

found

hemispheric

differences

in

functional

connectivity

in

healthy

young

adults

during

resting

state

The

neurocognitive

domains

of

psychomotor

function

working

memory

information

processing

speed

and

attentional

tasks

were

impaired

in

LGG

patients

These

results

corroborate

previous

studies

indicating

a

decline

in

neurocognitive

functioning

in

LGG

patients

-LSB-

-RSB-

Several

research

groups

have

suggested

that

the

increase

in

slow

wave

activity

corresponds

with

neurocognitive

deficits

in

AD

and

PD

Fernandez

-LSB-

-RSB-

found

right

parietal

delta

activity

to

be

significantly

associated

with

variability

in

the

Cambridge

Examination

for

Mental

Disorders

in

the

elderly

-LRB-

CAMCOG

-RRB-

score

whereas

the

left

temporal

theta

activity

predicted

the

variability

of

Mini

Mental

State

Examination

-LRB-

MMSE

-RRB-

results

Sinanovic

-LSB-

-RSB-

found

a

positive

correlation

between

diffuse

changes

in

spectral

power

and

MMSE

scores

in

the

PD

patients

with

dementia

Bosboom

-LSB-

-RSB-

found

a

negative

correlation

in

the

non-demented

PD

patients

between

theta

power

and

CAMCOG

scores

in

both

occipital

and

right

temporal

region

but

expected

these

associations

given

the

large

number

of

relations

that

were

analyzed

likely

to

be

caused

by

coincidence

In

the

demented

PD

group

they

found

no

significant

correlations

Stoffers

-LSB-

-RSB-

showed

a

negative

association

between

lower

alpha

power

and

the

performance

on

perseveration-related

tasks

in

the

non-demented

PD

patients

Evidently

this

study

has

its

limitations

First

of

all

the

patient

group

is

rather

small

as

mentioned

before

Secondly

although

our

patient

population

consisted

of

only

LGG

patients

they

did

not

all

receive

the

same

treatment

In

our

study

we

hypothesized

changes

in

oscillatory

brain

activity

to

be

the

intermediate

between

the

impact

of

the

tumor

and

its

treatment

on

the

one

hand

and

the

neurocognitive

deficits

as

the

output

on

the

other

hand

This

study

showed

that

irrespective

of

the

different

treatment

options

changes

in

oscillatory

brain

activity

can

be

found

in

brain

tumor

patients

which

is

associated

with

neurocognitive

function

To

get

informed

on

the

influence

of

the

different

tumor

treatments

on

brain

activity

and

its

relation

with

neurocognitive

function

is

very

interesting

A

longitudinal

study

of

these

effects

-LRB-

e.g.

surgery

radiotherapy

and

chemotherapy

-RRB-

on

brain

activity

is

currently

under

way

The

goal

of

that

study

is

to

explore

correlations

between

changes

in

neurocognitive

function

and

changes

in

functional

brain

dynamics

during

the

disease

course

Our

study

is

the

first

to

correlate

the

relative

power

with

neurocognitive

functioning

in

brain

tumor

patients

The

observed

correlations

were

quite

strong

and

showed

that

an

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


Modulation of calcification of vascular smooth muscle cells in culture by calcium antagonists , statins , and their combination Background Vascular calcification is an organized process in which vascular smooth muscle cells -LRB- VSMCs -RRB- are implicated primarily . The purpose of the present study was to assess the effects of calcium antagonists and statins on VSMC calcification in vitro . Methods VSMC calcification was stimulated by incubation in growth medium supplemented with 10 mmol/l β-glycerophosphate , 8 mmol/l CaCl2 , 10 mmol/l sodium pyruvate , 1 μmol/l insulin , 50 μg/ml ascorbic acid , and 100 nmol/l dexamethasone -LRB- calcification medium -RRB- . Calcification , proliferation , and apoptosis of VSMCs were quantified . Results Calcium deposition was stimulated dose-dependently by β-glycerophosphate , CaCl2 , and ascorbic acid -LRB- all P < 0.01 -RRB- . Addition of amlodipine -LRB- 0.01 -- 1 μmol/l -RRB- to the calcification medium did not affect VSMC calcification . However , atorvastatin -LRB- 2 -- 50 μmol/l -RRB- stimulated calcium deposition dose-dependently . Combining treatments stimulated calcification to a degree similar to that observed with atorvastatin alone . Both atorvastatin and amlodipine inhibited VSMC proliferation at the highest concentration used . Only atorvastatin -LRB- 50 μmol/l -RRB- induced considerable apoptosis of VSMCs . Conclusion In vitro calcification of VSMCs is not affected by amlodipine , but is stimulated by atorvastatin at concentrations ≥ 10 μmol/l , which could contribute to the plaque-stabilizing effect reported for statins . Introduction Atherosclerosis is the principal cause of coronary artery disease , stroke , and peripheral artery disease , and is the major cause of mortality in the Western hemisphere . Vascular calcification is a prominent feature of atherosclerosis , and it is associated with an increased risk of myocardial infarction -LSB- 1 -RSB- . Vascular calcification refers to the deposition of calcium phosphate mineral , most often in the form of hydroxyapatite , in the vessel wall . Calcification of the vessel wall and heart valves is associated with ageing , diabetes , and uremia -LSB- 2 -- 4 -RSB- . Vascular calcification is now considered to be an organized , regulated process comparable to bone mineralization . The presence of various components associated with bone mineralization , such as bone morphogenetic protein , osteocalcin , osteopontin , osteoblast-like cells , and matrix vesicles in atherosclerotic lesions supports this concept -LSB- 5 -- 7 -RSB- . Vascular cells such as vascular smooth muscle cells -LRB- VSMCs -RRB- and pericyte-like cells play an important role in vascular calcification -LSB- 8 -RSB- . The lipophilic calcium antagonist -LRB- CA -RRB- amlodipine has been shown to restore cholesterol-induced membrane bilayer abnormalities in VSMCs derived from the atherosclerotic rabbit aorta -LSB- 9 , 10 -RSB- , thereby restoring normal calcium homeostasis . Other mechanisms , through which CAs may affect atherosclerosis development include inhibition of proliferation and migration of VSMCs -LSB- 11 , 12 -RSB- , and inhibition of lipoprotein oxidation -LSB- 13 -RSB- . In addition , CAs modify binding of monocytes to the endothelium , and activate synthesis of matrix components -LSB- 14 -RSB- . The effects of CAs on atherosclerotic calcification have not been widely studied , however . Lipids may contribute to atherosclerotic calcification -LSB- 15 , 16 -RSB- . Recently statins have been shown to decrease the progression of coronary artery calcification and aortic valve calcification -LSB- 17 , 18 -RSB- . Therapy with a combination of a CA and a statin might be more atheroprotective than either treatment alone -LSB- 19 , 20 -RSB- . Combination therapy with CA and statin was shown to improve endothelial function and arterial compliance , to diminish LDL atherogenicity , and to slow the progression of atherosclerosis . So far , the effects of the combination therapy with CA and statin on vascular calcification have not been studied extensively . We used an in vitro model of vascular calcification by neonatal rat VSMCs , isolated by outgrowth from aortic explants . The purpose of this investigation was to identify the factors , and their concentrations , that are mandatory to induce calcification of aortic VSMCs in vitro . Since calcium is such a major component of advanced atherosclerotic lesions , we furthermore studied whether the CA amlodipine and the statin atorvastatin , alone and in combination , affected calcification of aortic VSMCs in vitro . Materials and methods Cell culture Vascular smooth muscle cells -LRB- VSMCs -RRB- were obtained from segments of aortas explanted from 2-day old Wistar rats . Aortic segments were obtained aseptically and cut open longitudinally . The endothelium was removed by gently rubbing the luminal side of the aortas over the surface of a tissue culture dish -LRB- Falcon -RRB- . Subsequently , the aortas were placed , lumen side down , on the bottom of a tissue culture flask -LRB- Greiner -RRB- , and allowed to adhere for approximately 3 h . Then tissues were immersed in growth medium consisting of Dulbecco 's modified Eagle 's medium -LRB- DMEM -RRB- -LRB- Life Technologies -RRB- supplemented with 10 % heat-inactivated fetal bovine serum -LRB- FBS -RRB- -LRB- Life Technologies -RRB- , penicillin -LRB- 100 U/ml -RRB- , and streptomycin -LRB- 100 μg/ml -RRB- -LRB- both supplied by BioWhittaker Europe -RRB- . Seven days later the aortic tissues were removed , and the VSMCs that had grown out were detached by trypsinization . The detached cells were resuspended in growth medium and seeded in tissue culture flasks -LRB- Greiner -RRB- , 6 or 12-well plates or on glass cover slips . Immunocytochemistry Subconfluent cultures at early passage were examined for the presence of α-smooth muscle actin , SM myosin heavy chain and calponin using specific antibodies -LRB- anti-SM actin clone 1A4 -LRB- 1:400 -RRB- , Sigma ; anti-SM myosin heavy chain -LRB- 1:100 -RRB- , SanverTech ; anti-calponin -LRB- 1:10,000 -RRB- , Sigma -RRB- . After fixation in 1 % formalin in PBS on ice for 30 min , cells were permeabilized with 0.1 % Triton X-100 for 30 min , and subsequently incubated with the primary antibody for several hours at 4 °C . Secondary antibodies were conjugated with fluorescein isothiocyanate -LRB- FITC -RRB- or Cy3 -LRB- Sigma -RRB- . To identify cell nuclei , the cells were also stained with Hoechst 33342 -LRB- 10 μg/ml ; Molecular Probes -RRB- . Immunofluorescent images were obtained using a fluorescence microscope -LRB- Nikon Eclipse -RRB- equipped with 20 × , 40 × , and 100 × objectives and a digital camera -LRB- Nikon DXM1200 -RRB- . In vitro calcification of VSMCs Calcification of VSMC cultures was induced by the method of Shioi et al. -LSB- 21 -RSB- with minor modifications . When confluent , the incubation medium of VSMCs was switched from growth medium to calcification medium . Calcification medium consisted of DMEM -LRB- high glucose , 4.5 g/l -RRB- supplemented with 15 % FBS , penicillin -LRB- 100 U/ml -RRB- , streptomycin -LRB- 100 μg/ml -RRB- , 8 mmol/l CaCl2 , 10 mmol/l sodium pyruvate , 1 μmol/l insulin , 50 μg/ml ascorbic acid , 10 mmol/l β-glycerophosphate , and 100 nmol/l dexamethasone . The medium was replaced with fresh medium every 2 -- 3 days . In several experiments , the concentrations of CaCl2 , ascorbic acid , β-glycerophosphate , and dexamethasone were varied . In another series of experiments , calcification medium was supplemented with amlodipine -LRB- 0.01 -- 1 μmol/l -RRB- , atorvastatin -LRB- 2 -- 50 μmol/l -RRB- , or a combination of these drugs . The CA amlodipine was dissolved in absolute ethanol at a concentration of 1 mmol/l . This stock solution was to be diluted at least 1:1,000 in culture medium . Atorvastatin , an inhibitor of HMG-CoA reductase , was dissolved in absolute ethanol at a concentration of 10 mmol/l . This stock solution was to be diluted at least 1:200 in culture medium . Cells treated with calcification medium without added drugs were used as controls . Appropriate amounts of solvent -LRB- ethanol -RRB- were added to these controls . Addition of ethanol at these levels did not affect cell growth . After 3 weeks of incubation , calcification was quantified . Assessment of calcium deposition VSMCs were decalcified with 0.6 N HCl for 24 h . The calcium contents of the supernatants were determined by spectrophotometer using the o-cresolphthalein method -LRB- Roche Diagnostics -RRB- . After decalcification , the cells were washed with PBS and scraped from the culture plate . The protein content was measured using the BCA protein assay -LRB- Pierce -RRB- . The calcium content of the cell layer was normalized to protein content . Proliferation assay VSMCs were seeded at a density of 5 × 103 -- 2 × 104 cells/well in a 96-well plate and allowed to attach overnight . Subsequently , the standard culture medium -LRB- DMEM , 10 % FBS , antibiotics -RRB- was replaced by calcification medium supplemented with amlodipine , atorvastatin , or a combination of both . Cell proliferation was assessed at day 4 and day 9 using the Cell Proliferation Kit II -LRB- XTT , Roche -RRB- , which is a colorimetric assay for the non-radioactive quantification of cell proliferation and viability . Detection of apoptosis To identify apoptotic cells by assessment of condensation of nuclear chromatin , VSMC cultures were stained with Hoechst 33342 -LRB- 10 μg/ml ; Molecular Probes -RRB- for 10 min in the dark . Immunofluorescent images were obtained using a fluorescence microscope -LRB- Nikon Eclipse -RRB- equipped with 20x , 40x , and 100x objectives and a digital camera -LRB- Nikon DXM1200 -RRB- . In 6 frames per treatment , an average of 250 nuclei per frame were assessed for the presence of apoptosis . Statistical analysis Results are expressed as mean ± SEM . For statistical analysis , SPSS 10.0 for Windows was used . Since the data were not normally distributed , non-parametric tests were used for comparisons between groups . Overall comparisons between groups were performed with the Kruskall -- Wallis test . If only two groups were compared , Mann -- Whitney rank sum tests were used . P values less than 0.05 were regarded as significant . Results VSMCs that have been cultured from rat aortic explants for approximately 7 days stained positive for α-smooth muscle-actin , smooth muscle myosin heavy chain , and calponin , confirming the smooth muscle cell nature of these cells -LRB- Fig. 1 , online supplement -RRB- . VSMCs of passage 3 -- 6 were subsequently used in calcification experiments . Fig. 1 -LRB- A -RRB- Rat aortic VSMC isolated by outgrowth , phase contrast . Immunofluorescent staining of neonatal rat VSMC incubated with -LRB- B -RRB- -LRB- and insert -RRB- anti-smooth muscle actin Ab , -LRB- C -RRB- anti-smooth muscle myosin Ab , and -LRB- D -RRB- anti-calponin Ab . Original magnification B and C × 100 , D and insert × 200 . VSMC = vascular smooth muscle cell Calcium deposition and dependence of extracellular Ca2 + concentration Cells were incubated with calcification medium supplemented with various Ca2 + concentrations for 21 days . At Ca2 + concentrations ≤ 3 mmol/l , hardly any calcium deposition was observed , but at Ca2 + concentrations > 3 mmol/l , a dose-dependent increase in calcium deposition was observed -LRB- Fig. 2A -RRB- . The amount of calcium deposition was positively correlated to calcium concentration in the culture medium -LRB- P < 0.01 -RRB- . We chose to continue with 8 mmol/l of Ca2 + - ions added to the calcification medium . Fig. 2 -LRB- A -RRB- Dose-dependent effects of CaCl2 on calcification of neonatal rat VSMCs . -LRB- B -RRB- Dose-dependent effects of β-glycerophosphate on calcification of neonatal rat VSMCs . VSMCs were treated for 21 days with calcification medium containing varying concentrations of Ca2 + ions or β-glycerophosphate . Control cultures -LRB- = con -RRB- were incubated with DMEM , 10 % FBS , and antibiotics . Calcium deposition was quantified by o-cresolphthalein method . The data are presented as mean ± SEM -LRB- n = 3 -- 9 -RRB- . * P < 0.05 vs. control cultures . ◆ P < 0.05 vs. 2 and 3 mM calcium in culture medium . # P < 0.05 vs. all other treatments Calcium deposition and dependence of extracellular phosphate concentration Shioi et al. have demonstrated that β-glycerophosphate accelerates in vitro calcification of VSMCs and induces extensive calcium deposition in a manner analogous to in vitro mineralization by osteoblasts -LSB- 21 -RSB- . In our model of vascular calcification , the amount of calcium deposition was positively correlated to the β-glycerophosphate concentration in the calcification medium -LRB- P < 0.01 ; Fig. 2B -RRB- . Calcium deposition and dependence of extracellular dexamethasone concentration To determine the contribution of added dexamethasone , we incubated the VSMCs with various concentrations of dexamethasone -LRB- 10 -- 1,000 nmol/l -RRB- . No significant differences in calcium deposition were observed between the cells incubated with 0 , 10 , 100 , and 1,000 nmol/l dexamethasone -LRB- Fig. 3A -RRB- . Fig. 3 -LRB- A -RRB- Dose-dependent effects of dexamethasone on calcification of neonatal rat VSMCs . -LRB- B -RRB- Dose-dependent effects of ascorbic acid on calcification of neonatal rat VSMCs . VSMCs were treated for 21 days with calcification medium containing varying amounts of dexamethasone or ascorbic acid . Calcium deposition was quantified by o-cresolphthalein method . The data are presented as mean ± SEM -LRB- n = 6 -- 12 -RRB- . # P < 0.05 vs. all other treatments . ◆ P < 0.05 vs. 0 and 5 μg/ml ascorbic acid Calcium deposition and dependence of extracellular ascorbic acid concentration To determine the contribution of ascorbic acid to calcium deposition , we incubated VSMCs with various concentrations of ascorbic acid -LRB- 0 , 5 , 50 , and 500 μg/ml -RRB- . At the highest concentration -LRB- 500 μg/ml -RRB- ascorbic acid was associated with significantly more calcium deposition than at all other concentrations -LRB- Fig. 3B -RRB- . However , 500 μg/ml ascorbic acid caused a considerable decrease in pH of the culture medium . Since calcium deposition was positively correlated with ascorbic acid concentration -LRB- P < 0.01 -RRB- , we decided to use 50 μg/ml ascorbic acid in future calcification studies , the highest concentration of ascorbic acid that did not cause acidification of the medium . Effects of amlodipine on in vitro VSMC calcification To study the effect of the CA amlodipine on VSMC calcification , VSMCs were incubated for 2 -- 3 weeks with calcification medium supplemented with various concentrations of amlodipine -LRB- 0.01 -- 1 μmol/l -RRB- . Incubation of neonatal rat VSMCs with amlodipine had no effect on VSMC calcification , at none of the concentrations tested -LRB- Fig. 4A -RRB- . Fig. 4 -LRB- A -RRB- Dose-dependent effects of amlodipine on calcification of neonatal rat VSMCs . -LRB- B -RRB- Dose-dependent effects of atorvastatin on calcification of neonatal rat VSMCs . -LRB- C -RRB- Effects of amlodipine , atorvastatin and a combination of both treatments on neonatal rat VSMC calcification . VSMCs were treated for 21 days with calcification medium containing varying concentrations of atorvastatin or amlodipine , a combination of both , or none of them -LRB- control -RRB- . Calcium deposition was quantified by o-cresolphthalein method . The data are presented as mean ± SEM -LRB- n = 15 -RRB- . * P < 0.05 when compared to untreated control cultures . # P < 0.05 when compared to all other treatments Effects of atorvastatin on in vitro VSMC calcification To study the effect of the statin atorvastatin on VSMC calcification , VSMCs were incubated for 2 -- 3 weeks with calcification medium supplemented with various concentrations of atorvastatin -LRB- 2 -- 50 μmol/l -RRB- . Atorvastatin increased VSMC calcification dose-dependently -LRB- Fig. 4B -RRB- . At a concentration of 2 μmol/l atorvastatin , calcium deposition was increased by 30 % -LRB- P = 0.04 -RRB- when compared to VSMCs incubated with atorvastatin-free calcification medium . At concentrations of 10 and 50 μmol/l atorvastatin , calcium deposition was increased 2.3-fold and 6.1-fold , respectively -LRB- P < 0.001 -RRB- when compared to VSMCs incubated with atorvastatin-free calcification medium . Effect of a combination of CA and statin on in vitro VSMC calcification To study the effect of combining amlodipine and atorvastatin treatment on VSMC calcification , VSMCs were incubated for 2 -- 3 weeks with calcification medium supplemented with 0.1 μmol/l amlodipine , 10 μmol/l atorvastatin , or a combination of these drugs in the same concentrations . Incubation of VSMCs with 0.1 μmol/l amlodipine had no effect on VSMC calcification . Incubation of VSMCs with 10 μmol/l atorvastatin resulted in a 2.2-fold increased calcium deposition when compared to control cultures treated with calcification medium only -LRB- Fig. 4C -RRB- -LRB- P < 0.001 -RRB- . Treatment with a combination of amlodipine and atorvastatin also resulted in a 2.2-fold increased calcium deposition when compared to control cultures treated with calcification medium only -LRB- P = 0.026 -RRB- . The combination therapy resulted in significantly more calcium deposition than treatment with amlodipine alone -LRB- P = 0.003 -RRB- , and as much calcium deposition compared to treatment with atorvastatin alone -LRB- n.s. -RRB- . Effects of amlodipine , atorvastatin , and their combination on VSMC proliferation VSMC proliferation was assessed at day 4 and day 9 . After 4 days of incubation in calcification medium supplemented with amlodipine -LRB- 1 μmol/l -RRB- , VSMC proliferation was decreased by 21 % -LRB- P < 0.001 -RRB- as compared to proliferation of VSMCs cultured in amlodipine-free calcification medium -LRB- Fig. 6A , online supplement -RRB- . After 9 days of incubation , VSMC proliferation had increased by 14 % -LRB- P < 0.05 -RRB- when treated with 0.01 μmol/l amlodipine , but at an amlodipine concentration of 1 μmol/l , VSMC proliferation had decreased by 26 % -LRB- P < 0.01 -RRB- as compared to proliferation of VSMCs cultured in amlodipine-free calcification medium -LRB- Fig. 5A -RRB- . Atorvastatin , at a concentration of 50 μmol/l , decreased VSMC proliferation by 50 % -LRB- P < 0.001 ; Fig. 5B -RRB- . Treatment of VSMCs with the combination of 0.1 μmol/l amlodipine and 10 μmol/l atorvastatin did not result in significant changes in cell proliferation compared to VSMCs incubated in the absence of these drugs . Fig. 5Effect of amlodipine and atorvastatin on VSMC proliferation . VSMC proliferation was assessed at day 4 and day 9 using the Cell Proliferation Kit II -LRB- XTT -RRB- -LRB- Roche -RRB- . VSMC proliferation is depicted as percentage relative to untreated cells -LRB- = 100 % -RRB- -LRB- A -RRB- Effect of amlodipine . -LRB- B -RRB- Effect of atorvastatin . # P < 0.05 vs. control cultures . * P < 0.05 vs. control cultures Effects of amlodipine , atorvastatin , and their combination on VSMC apoptosis VSMCs incubated in calcification medium only and VSMCs incubated with amlodipine -LRB- 0.01 -- 1 μmol/l -RRB- or atorvastatin -LRB- 2 and 10 μmol/l -RRB- in calcification medium for 72 h hardly contained any apoptotic nuclei . Amlodipine at the highest concentration tested -LRB- 1 μmol/l -RRB- , caused apoptosis of on average 0.05 % of the cells -LRB- n.s. compared to control -RRB- . At the highest concentration of atorvastatin -LRB- 50 μmol/l -RRB- , however , 5.6 % of the nuclei were apoptotic -LRB- P < 0.05 vs. control , 2 and 10 μmol/l atorvastatin ; Fig. 6 -RRB- . Fig. 6VSMCs were treated with 1 μmol/l amlodipine -LRB- A , B -RRB- or 50 μmol/l atorvastatin -LRB- C , D -RRB- for 72 h. Nuclei were visualized with Hoechst 33342 . Apoptotic nuclei are circled in panel C and indicated with arrows in panel D. Microscopy : magnification A and C × 100 , B and D × 400 VSMCs treated with the combination of 0.1 μmol/l amlodipine and 10 μmol/l atorvastatin for 72 h had a number of apoptotic nuclei that did not significantly differ from those observed in 10 μmol/l atorvastatin only , 0.1 μmol/l amlodipine only , and VSMCs incubated in calcification medium in the absence of these drugs . Discussion In an in vitro model of vascular calcification of rat aortic VSMCs , calcium deposition was dependent upon the extracellular concentration of organic phosphate -LRB- P < 0.01 -RRB- , calcium ions -LRB- P < 0.01 -RRB- , and ascorbic acid -LRB- P < 0.01 -RRB- . While studying the effect of the CA amlodipine and the statin atorvastatin , alone and in combination , on calcification , we found that , at none of the concentrations tested -LRB- 0.01 -- 1 μmol/l -RRB- did amlodipine have any effect on VSMC calcification in this model , nor on development of apoptosis . In contrast , atorvastatin stimulated VSMC calcification at a concentration of ≥ 10 μmol/l -LRB- P < 0.05 -RRB- , and 50 μmol/l inhibited VSMC proliferation and induced apoptosis -LRB- P < 0.05 -RRB- . VSMCs retain remarkable plasticity , even in adult animals . VSMCs can undergo rapid and reversible changes in its phenotype in response to changes in local environmental conditions -LSB- 22 -RSB- . Due to production of a variety of cytokines by subendothelial macrophages , proliferation , and migration of VSMCs to the intimal layer can occur , thereby contributing to the process of intimal thickening and atherosclerosis . Neonatal VSMCs resemble VSMCs in atherosclerotic plaque , since they retain the proliferative phenotype . Neonatal aortic explants exhibit rapid outgrowth of VSMCs , and we chose to use these cells to develop a model of vascular calcification . VSMCs were shown to migrate from the aortic explant within several days , and were identified as VSMCs by immunohistochemistry -LSB- 8 -RSB- . Dialysis patients have accelerated atherosclerosis , with extensive calcification of both the intima and the media -LSB- 23 -RSB- . Hyperphosphatemia has been implicated in this process -LSB- 24 -RSB- . Inorganic phosphate -LRB- Pi -RRB- levels have previously been shown to regulate human aortic smooth muscle cell -LRB- HSMC -RRB- culture mineralization in vitro -LSB- 25 , 26 -RSB- . In the present study , the amount of calcium deposition in neonatal rat VSMCs correlated positively to the β-glycerophosphate concentration in the calcification medium . In the present study , the amount of calcium deposition in rat aortic VSMCs correlated to the calcium concentration in the calcification medium . This observation was not a result of passive calcium deposition , since incubation of VSMCs with normal growth medium supplemented with 8 mmol/l CaCl2 -LRB- final concentration -RRB- did not result in calcium deposition by these cells . Shioi et al. -LSB- 21 -RSB- have previously demonstrated that culturing bovine VSMCs in the presence of β-glycerophosphate , ascorbic acid , and insulin can induce diffuse calcification in a manner analogous to in vitro mineralization of osteoblasts . The present study also showed that β-glycerophosphate is crucial for the induction of calcification in rat aortic VSMCs . Ascorbic acid augments this process . Dexamethasone was demonstrated to be less vital in this model of aortic VSMC calcification . Statins possess potent lipid-lowering effects . Besides , statins exert pleiotropic effects on vascular wall cells , which include improvement of endothelial function , stabilization of the atherosclerotic plaque , and suppression of inflammation -LSB- 27 , 28 -RSB- . Although several studies have failed to demonstrate an effect of statins on the progression of calcific aortic stenosis -LSB- 29 , 30 -RSB- , statins have also been shown to decrease the progression of coronary artery calcification and aortic valve calcification -LSB- 17 , 18 , 31 , 32 -RSB- . Accordingly , Kizu et al. have demonstrated that statins inhibit calcification in an in vitro model of inflammatory vascular calcification -LSB- 33 -RSB- . Using interferon-γ , 1α , 25-dihydroxyvitamin D3 , tumor necrosis factor-α , and oncostatin M to induce calcification in human VSMCs , it was demonstrated that cerivastatin and atorvastatin inhibited calcification dose-dependently . However , one should realize that this model differs from the model we have used in the present study . Whereas Kizu et al. used inflammatory mediators to induce calcification of the VSMCs , we used increased levels of CaCl2 and organic phosphate in the culture medium to promote calcification . Statins are known to have anti-inflammatory effects , and therefore , their inhibitory effect on calcification induced by inflammatory mediators is to be expected . In addition , low doses of statins have also been shown to protect human aortic VSMCs from inorganic phosphate-induced calcification -LSB- 34 -RSB- . In contrast to the aforementioned studies , the present study showed a dose-dependent stimulatory effect of atorvastatin -LRB- 10 and 50 μmol/l -RRB- on calcification of VSMCs incubated in calcification medium . However , the dose of atorvastatin used in the present study was much higher than in the study described by Son et al. -LRB- 500x higher at the highest dose -RRB- -LSB- 34 -RSB- , and most likely explains the discrepancy between the results obtained by Son et al. and the results of the present study . Reynolds et al. -LSB- 35 -RSB- have demonstrated that human VSMCs undergo vesicle-mediated calcification in response to changes in calcium and phosphate concentrations in the culture medium . Elevated calcium and phosphate concentrations resulted in increased release of vesicles and stimulation of apoptosis . Calcification was initiated by release of membrane-bound matrix vesicles from living cells and also by apoptotic bodies from dying cells . Vesicles released by VSMCs after prolonged exposure to calcium and phosphate contained preformed basic calcium phosphate , and calcified extensively . The present study confirms that statins stimulate apoptosis of VSMCs . Apoptotic bodies can calcify extensively -LSB- 36 -RSB- , so a likely mechanism of induction of calcification by atorvastatin is through induction of apoptosis . Indeed , atorvastatin stimulated both apoptosis and calcification in our model . Myocardial infarction and unstable angina usually arise from the disruption of mildly stenosed atherosclerotic lesions . Such vulnerable plaques typically contain a large amount of lipid and have a preponderance of inflammatory cells at the shoulders of the plaque and a thin fibrous cap . Calcium is found infrequently in the culprit lesion of ruptured plaque . Thus , plaque vulnerability is not associated with size , but with composition . Beckman et al. -LSB- 37 -RSB- presented the concept `` that calcium is not a critical substrate for plaque disruption and is , in fact , associated with more stable plaques . '' Therefore , statin-induced alterations in the plaque 's composition from lipid to fibrous tissue without or with calcification would decrease the plaque 's vulnerability to rupture , thereby decreasing the risk of acute coronary syndromes . Prolonged intensive lipid-lowering has been demonstrated to increase calcium content of plaques as assessed by MRI in vivo , and lowered plaque lipid content at about the same extent , without a pronounced effect on plaque size -LSB- 38 -RSB- . Since atherosclerotic plaque composition plays an important role in plaque stability , with lipids destabilizing the plaque and calcification stabilizing the plaque , the effect of statins on plaque composition may be favorable . In addition to lipid-lowering and calcification-inducing effects , we observed that statins have an anti-proliferative effect on VSMCs . Less proliferation of VSMCs may destabilize the plaque . However , this only occurred at high doses of atorvastatin , whereas calcification induction occurred at lower dose of atorvastatin . Although several in vitro and in vivo studies have demonstrated that combining CA and statin therapy might be more atheroprotective than either treatment alone -LSB- 19 , 20 , 39 -RSB- , the present study found no difference between the combination of amlodipine and atorvastatin therapy , and atorvastatin therapy alone on VSMC calcification . In conclusion , extracellular β-glycerophosphate and CaCl2 concentrations are important determinants of in vitro calcification of rat aortic VSMCs . Ascorbic acid stimulates this process . Dexamethasone was demonstrated to be less vital in this model of VSMC calcification . In vitro calcification of rat aortic VSMCs is not affected by amlodipine treatment , but is stimulated by atorvastatin treatment . As high concentrations of atorvastatin have a pro-apoptotic effect , the pro-calcification effect of atorvastatin may be explained by the production of apoptotic bodies that act as foci for calcium deposition . The latter finding may explain the plaque-stabilizing effect reported for statins .

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

calcification
atherosclerosis
vascular smooth muscle cells
in vitro model
calcium antagonist
statin