Tài liệu Báo cáo Y học: Toxicity of substrate-bound amyloid peptides on vascular smooth muscle cells is enhanced by homocysteine docx

Toxicity of substrate-bound amyloid peptides on vascular smooth
muscle cells is enhanced by homocysteine
Su San Mok
1,2
, Bradley J. Turner
1,2
, Konrad Beyreuther
3
, Colin L. Masters
1,2
, Colin J. Barrow
4
and David H. Small
1,2
1
Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia;
2
The Mental Health Research Institute
of Victoria, Royal Park Hospital, Parkville, Victoria, Australia;
3
ZMBH, The University of Heidelberg, Heidelberg, Germany;
4
The School of Chemistry, The University of Melbourne, Parkville, Victoria, Australia
The main component of cerebral amyloid angiopathy
(CAA) in Alzheimer’s disease is the amyloid-b protein (Ab),
a 4-kDa polypeptide derived from the b-amyloid protein
precursor (APP). The accumulation of Ab in the basement
membrane has been implicated in the degeneration of adja-
cent vascular smooth muscle cells (VSMC). However, the
mechanism of Ab toxicity is still unclear. In this study, we
examined the effect of substrate-bound Ab on VSMC in

culture. The use of substrate-bound proteins in cell culture
mimics presentation of the proteins to cells as if bound to the
basement membrane. Substrate-bound Ab peptides were
found to be toxic to the cells and to increase the rate of cell
death. This toxicity was dependent on the length of time the
peptide was allowed to ÔageÕ, a process by which Ab is
induced to aggregate over several hours to days. Oxidative
stress via hydrogen peroxide (H
2
O
2
) release was not involved
in the toxic effect, as no decrease in toxicity was observed in
the presence of catalase. However, substrate-bound Ab sig-
nificantly reduced cell adhesion compared to cells grown on
plastic alone, indicating that cell–substrate adhesion may be
important in maintaining cell viability. Ab also caused an
increase in the number of apoptotic cells. This increase in
apoptosis was accompanied by activation of caspase-3.
Homocysteine, a known risk factor for cerebrovascular
disease, increased Ab-induced toxicity and caspase-3 acti-
vation in a dose-dependent manner. These studies suggest
that Ab may activate apoptotic pathways to cause loss of
VSMC in CAA by inhibiting cell–substrate interactions. Our
studies also suggest that homocysteine, a known risk factor
for other cardiovascular diseases, could also be a risk factor
for hemorrhagic stroke associated with CAA.
Keywords: amyloid-b; vascular smooth muscle cell; toxicity;
homocysteine; caspase-3.
Cerebral amyloid angiopathy (CAA) is one of the morpho-

logical hallmarks of Alzheimer’s disease. However, CAA is
also seen in normal ageing. There is increasing evidence that
CAA may underlie certain forms of vascular dementia and
intracranial hemorrhage associated with ageing [1]. The
major form of CAA consists of proteinaceous deposits of
amyloid-b protein (Ab) that occur adjacent to vascular
smooth muscle cells (VSMC). Ab consists of 39–43 amino
acids and is proteolytically derived from its larger precursor,
the amyloid protein precursor (APP) [2,3]. APP is cleaved
by a transmembrane aspartic protease named BACE (b-site
APP cleaving enzyme) at the N-terminus of Ab [4,5] and by
an as yet unidentified c-secretase at the C-terminus of Ab
(reviewedin[6]).Ab is the main component of vascular
amyloid in Alzheimer’s disease, Down’s Syndrome and
hereditary cerebral hemorrhage with amyloidosis-Dutch
(HCHWA-D).
The accumulation of Ab in the cerebral vasculature
increases the risk of stroke due to intracranial hemorrhage
[1,7]. For example, in patients with HCHWA-D, in which
there is a point mutation at amino acid 22 in the Ab region,
Ab deposits occur in small and medium-sized arteries and
arterioles of the cerebral cortex and leptomeninges [8].
Patients often die from severe intracranial hemorrhage.
Other mutations within the Ab sequence also result in severe
cerebrovascular pathology [9–11].
A major feature of CAA is the degeneration of vascular
smooth muscle cells at sites of Ab deposition. Ultrastruc-
tural and immunocytochemical studies on autopsy tissue
show Ab deposition in walls of cerebral blood vessels and
the degeneration and disappearance of cells suggests that

Ab has a toxic effect on these cells in vivo [12,13]. The
accumulation of Ab occurs principally in the basement
membrane between smooth muscle cells resulting in damage
to the basement membrane and leading to the eventual
destruction of the cells [12]. The loss of VSMC may result in
weakening of the vessel wall, its subsequent rupture and
ultimately hemorrhage. Amyloid deposition and VSMC
degeneration has also been observed in transgenic mice that
overexpress APP [14–17].
Several mechanisms may contribute to CAA. Smooth
muscle cells themselves have been shown to synthesize APP
and produce Ab both in vivo [12,13,18] and in vitro [19–21].
Correspondence to D. H. Small, Department of Pathology,
The University of Melbourne, Parkville, Victoria 3010, Australia.
Fax: + 61 3 8344 4004, Tel.: + 61 3 8344 4205,
E-mail:
Abbreviations: Ab, amyloid-b-protein; CAA, cerebral amyloid
angiopathy; APP, amyloid protein precursor; VSMC, vascular
smooth muscle cell; H
2
O
2
, hydrogen peroxide; HCHWA-D,
hereditary cerebral hemorrhage with amyloidosis-Dutch;
DMEM, Dulbecco’s modified Eagle’s medium; MTS,
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2
(4-sulfophenyl)-2H-tetrazolium].
(Received 4 February 2002, revised 25 April 2002,
accepted 3 May 2002)
Eur. J. Biochem. 269, 3014–3022 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02976.x

However, recent studies from transgenic mouse models of
Alzheimer’s Disease suggest that most of the Ab in CAA
can be derived from central neurons [14–17].
Although the role of Ab in neuronal toxicity has been
extensively studied in recent years, the mechanism of this
toxicity is unclear. Ab peptides have been shown to be
neurotoxic both in vivo [22] and in vitro [23,24]. Several
studies have shown that Ab disrupts calcium homeostasis
and that increases in intracellular calcium cause cellular
damage [25–27]. Increases in oxidative stress due to Ab have
also been widely studied [28,29]. Ab hasalsobeenshownto
induce apoptosis in neurons and smooth muscle cells
[30–33]. In addition, Ab peptides with the Dutch E22Q
and Iowa D23N mutations have been shown to be toxic to
human leptomeningeal smooth muscle cells in culture
[31,34–36].
As binding of Ab to the basement membrane is an early
step in Ab-induced VSMC toxicity, we have examined the
effect of substrate-bound Ab on the growth of vascular
smooth muscle cells in culture. The use of proteins in
substrate-bound form mimics certain features of their
presentation as if bound to the extracellular matrix [37].
In this study, we demonstrate that substrate-bound Ab is
toxic to VSMC by the activation of apoptotic cell death
pathways and that a known risk factor for cerebrovascular
disease, homocysteine, makes VSMC more vulnerable to
Ab toxicity.
EXPERIMENTAL PROCEDURES
Materials
Dulbecco’s modified Eagle’s medium (DMEM) was pur-

chased from Gibco Life Technologies (Mulgrave, Vic,
Australia). Foetal bovine serum, trypsin-versene and
penicillin/streptomycin were obtained from Common-
wealth Serum Laboratories Biosciences Pty. Ltd.
(Parkville, Vic, Australia). Matrigel basement membrane
matrix was purchased from Becton Dickinson (Franklin
Lakes, NJ, USA).
D
,
L
-Homocysteine, pepstatin, leupeptin,
aprotinin, catalase and phenylmethansulfonyl fluoride were
purchased from Sigma–Aldrich (Castle Hill, NSW,
Australia). Glutaraldehyde was purchased from Ajax
Chemicals (Auburn, NSW, Australia). The lactate
dehehydrogenase detection kit was purchased from Roche
Molecular Biochemicals (Castle Hill, NSW, Australia). The
CellTiter 96 AQueous One Solution Cell Proliferation
Assay kit was from Promega Corporation (Madison, WI,
USA). The fluorescent Hoechst dye 33258 was purchased
from Molecular Probes (Eugene, OR, USA). Etoposide
and the colorimetric caspase-3 substrate I was from
Calbiochem (Croydon, Vic, Australia). Plastic 96-well
and 24-well tissue culture plates were obtained from Nunc
(Naperville, IL, USA).
Synthesis of Ab peptides
Human sequence Ab1–40 and Ab1–42 peptides were
synthesized using manual solid-phase Boc amino acid
synthesis, as previously described [38]. Peptides were
released from the resin using anhydrous hydrogen fluoride

with p-cresol and p-thiocresol as scavengers. After elimin-
ating hydrogen fluoride, the peptides were solubilized in
trifluoroacetic acid and precipitated with ether. Peptides
were purified using a reverse-phase preparative Zorbax high
performance liquid chromatography (HPLC) column hea-
tedto60°C based on an acetonitrile/water (0.01%
trifluoroacetic acid) gradient [38]. Analytical HPLC, elec-
trospray mass spectrometry and amino-acid analysis were
performed to validate peptide purity. Ab1–40 and Ab1–42
peptides were solubilized in distilled water by trituration and
sonication at 42 kHz for 5 min. In some experiments,
peptides were incubated for 5 days at 37 °C in distilled
water to induce aggregation into fibrils (a process known as
ÔageingÕ) before being used.
Preparation of tissue culture plates
Plastic 96-well tissue culture plates were coated with 10 lL
of freshly solubilized Ab peptides (1 mgÆmL
)1
) unless
otherwise indicated. Sterile distilled water (10 lL per well)
was used in control wells. The peptides were dried onto the
well surface by storing the plates for 4 h in a sterile laminar
flow hood. To coat plates with Matrigel, 50 lLofMatrigel
basement membrane matrix [3.4 mgÆmL
)1
protein in Dul-
becco’s modified Eagle’s medium (DMEM)] was aliquoted
into 96-well microtitre plates and allowed to polymerize for
30 min at 37 °C. DMEM (50 lL) was aliquoted into
control wells.

Vascular smooth muscle cell culture
Aortae were dissected from Wistar-Kyoto or Sprague-
Dawley rats and VSMC isolated by incubation in
collagenase and elastase according to the method of
Hadrava et al. [39]. VSMC were plated at a density of
4 · 10
3
cells per well in 100 lL of DMEM containing
10% (v/v) fetal bovine serum, 3.7 mgÆmL
)1
sodium
bicarbonate and 1% (v/v) penicillin/streptomycin. Cells
were cultured on Ab or Matrigel substrates for 24 h at
37 °C unless otherwise stated. Where indicated, homocy-
steine, catalase or etoposide was added to cells 1–2 h after
plating.
Cytotoxicity assay
Release of the cytoplasmic enzyme lactate dehydrogenase
into the culture medium was used as a measure of
cytotoxicity. Lactate dehydrogenase was determined using
an lactate dehydrogenase detection kit (Roche Molecular
Biochemicals). Medium was removed from cells, samples
centrifuged at 10 000 g in a Hermle Z160M microfuge for
5 min and supernatant fractions assayed for lactate dehy-
drogenase activity. Diaphorase/NAD
+
(catalyst) was dilu-
ted in iodotetrazolium chloride/lactate (dye) and 100 lLof
this reagent was added to 100 lL of culture medium. The
plate was gently shaken for 20–30 min in the dark at room

temperature. The absorbance of samples was then read at a
wavelength of 490 nm. Total lactate dehydrogenase was
determined by lysing cells in 0.2% Triton X-100 in DMEM/
10% fetal bovine serum and measuring the total amount of
lactate dehydrogenase in the cell lysate and medium.
Absorbance values were expressed as a percentage of total
cellular lactate dehydrogenase after correction for the
amount of endogenous lactate dehydrogenase activity
present in the medium.
Ó FEBS 2002 Ab is toxic to vascular smooth muscle cells (Eur. J. Biochem. 269) 3015
Cell adhesion assay
Cell–substrate adhesion was tested by plating VSMC at
4 · 10
3
cells per well in a 96-well tissue culture plate. After a
30-min incubation at 37 °C, the medium was aspirated and
wells rinsed three times with 200 lLofNaCl/P
i
to remove
poorly adherent cells. The remaining attached cells were
fixed in 2.5% (v/v) glutaraldehyde, permeabilized with 0.1%
(v/v) Triton X-100 and stained with haematoxylin-eosin.
The total number of cells in three fields in each of three
treatment groups was counted, then averaged and expressed
as a percentage of total seeding density per well.
MTS assay of cell viability
Cellular viability was measured using the CellTiter 96
AQueous One Solution Cell Proliferation Assay kit. After a
24-h treatment period, 10 lL of AQueous One solution
containing the compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium]
(MTS) was added to 100 lL of sample in the wells and
allowedtoincubatefor2hat37°C. The absorbance of the
samples was then read at a wavelength of 560 nm.
Absorbance values were expressed as a percentage of the
untreated controls.
Apoptosis assay
The percentage of cells undergoing apoptosis was assessed
by staining with the fluorescent DNA-binding dye Hoechst
33258. The culture medium was removed, cells washed twice
with 100 lLofNaCl/P
i
and fixed in 100 lLof4%(w/v)
paraformaldehyde in NaCl/P
i
for 20 min. The fixative was
then aspirated and after two washes with NaCl/P
i
, cells were
permeabilized with 100 lL of 100% methanol ()20 °C) for
20 min at room temperature. Cells were then rinsed three
times with NaCl/P
i
and stained with 100 lLof
0.12 lgÆmL
)1
Hoechst 33258 in NaCl/P
i
for 15 min in the
dark. This was followed by five washes with NaCl/P

i
. Cells
were visualized under ultraviolet light using a Leica DMIRB
microscope. Three fields in each well were photographed
with an Olympus DP10 digital camera and apoptotic nuclei
quantified. Cells with condensed or fragmented nuclear
chromatin were considered apoptotic. The number of
apoptotic cells was expressed as a percentage of the total
number of cells counted in each field.
Caspase-3 assay
Caspase-3 activity was measured by a colorimetric assay
using the substrate DEVD-pNA [32]. Culture medium was
removed from wells and cells washed briefly with warm
NaCl/P
i
. The cells were then extracted with 20 m
M
Tris/HCl
pH 7.4 containing 0.25
M
sucrose, 1 m
M
EDTA, 1% (v/v)
Triton X-100, 1 m
M
dithiotreitol, 0.5 m
M
phenyl-
methansulfonyl fluoride, 1 lgÆmL
)1

pepstatin, 1 lgÆmL
)1
aprotinin and 1 lgÆmL
)1
leupeptin for 15 min at 4 °C.
Samples were then centrifuged at 10 000 g for 5 min at
4 °C, the supernatant fractions collected and cell pellets
discarded. DEVD-pNA (100 lL of a 200 l
M
solution) was
thenaddedto100lL aliquots of cell extracts and samples
incubated at 37 °C for 24 h. The absorbance of samples was
then read at a wavelength of 415 nm.
RESULTS
Effect of substrate-bound Ab on VSMC
To determine whether culture of VSMC on a substrate of
Ab peptides induces a cytotoxic response, VSMC were
grown on Ab-coated 96-well microtitre plates. Lactate
dehydrogenase activity in the medium was measured 24 h
after plating. VSMC cultured on Ab1–40 and Ab1–42
released significantly more lactate dehydrogenase into the
medium than cells cultured on plastic alone (Fig. 1,
P <0.05 and P <0.005 for Ab1–40 and Ab1–42,
respectively), indicating that Ab1–40 and Ab1–42 were both
toxic in substrate-bound form. Cells grown on Matrigel, a
commercial basement membrane preparation, did not
show a significant increase in lactate dehydrogenase activity
in the medium compared with uncoated plates (Fig. 1).
Effect of Ab ÔageingÕ on toxicity
Incubation of Ab in solution for several days (a process

known as ÔageingÕ) causes the peptide to aggregate into
fibrils and increases its neurotoxic potential [24,40–43]. To
test the effect of ageing Ab on VSMC toxicity, peptides were
incubated at 37 °C, for various periods of time, prior to
being coated onto 96-well culture plates. Twenty-four hours
after plating VSMC, lactate dehydrogenase release was
measured as an index of cell death (Fig. 2). Lactate
dehydrogenase release from VSMC treated with Ab1–40
aged for 24 h was not increased compared to untreated cells.
However, ageing the Ab1–40 peptide for 72 h (P ¼ 0.011)
or 120 h induced an increase in lactate dehydrogenase
Fig. 1. Effect of substrate-bound Ab on VSMC. Ab peptides or Mat-
rigelwereallowedtodryorgelontothesurfaceofwells.VSMCwere
plated on to substrates and cultured for 24 h. Culture medium was
analysed for lactate dehydrogenase activity. The relative amounts of
lactate dehydrogenase in the medium was calculated by expressing the
absorbance as a percentage of total lactate dehydrogenase in the cul-
tures. Bars represent the mean of triplicate values ± SEM (n ¼ 5).
*Significantly different from cells grown on plastic (P <0.05 and
P <0.005forAb1–40 and Ab1–42, respectively) by a Student’s t-test.
LDH, lactate dehydrogenase.
3016 S. S. Mok et al. (Eur. J. Biochem. 269) Ó FEBS 2002
release. Similar results were obtained with Ab1–42. The
toxic effect was again increased by ageing the Ab1–42
peptide for 72 (P ¼ 0.004) or 120 h (P ¼ 0.003) relative to
untreated VSMC (Fig. 2).
Role of oxidative stress
A number of studies have reported that Ab fibrils can
generate H
2

O
2
and that oxidative stress may be the cause
of Ab toxicity [44]. To determine if the generation of H
2
O
2
by Ab causes VSMC toxicity, cells were incubated with Ab
in the absence or presence of the antioxidant catalase
(1000 UÆmL
)1
)for24hat37°C. The MTS assay of
mitochondrial function was used to measure changes in cell
redox potential. While Ab peptides decreased cell viability
compared to untreated controls (P < 0.05), no significant
protection in toxicity was observed in the presence of
catalase (Table 1). In contrast, cells treated with 5 l
M
H
2
O
2
showed a decrease in viability that could be reversed
by the presence of catalase (P < 0.005). The failure of
catalase to reverse cellular redox potential suggested that
although Ab increases cellular oxidation, the direct
generation of extracellular H
2
O
2

does not play a major
role in this effect.
Effect of substrate-bound Ab on VSMC adhesion
and toxicity
Inhibition of cellular adhesion to substrate-bound Ab has
been shown to affect neurite outgrowth in vitro [45]. To
determine whether the effects of Ab on cell viability were
due to the disruption of cell–substrate adhesion, cell
adherence was tested by plating VSMC for 30 min.
Weakly attached cells were removed by washing the plates
and then cells that remained attached were counted
(Fig. 3A). Substrate-bound Ab1–40 and Ab1–42 both
significantly reduced cell adhesion compared with cells
grownonplastic(P ¼ 0.009 and P ¼ 0.005, respectively).
In contrast, the cells adhered strongly to Matrigel-coated
wells, with less adherence observed when Ab was present
with the Matrigel (Fig. 3A). A correlation was observed
between cell adhesion and cytotoxicity (Fig. 3B). Lactate
dehydrogenase release into the medium was increased
when cells were cultured on Ab substrates (P <0.001)
compared with cells cultured on uncoated plastic. Simi-
larly, cells cultured on Ab peptides and Matrigel released
more lactate dehydrogenase into the medium than Matrigel
alone (P ¼ 0) (Fig. 3B).
Effect of homocysteine and Ab on VSMC
Increased plasma homocysteine has been shown to be a
risk factor for cardiovascular disease and Alzheimer’s
disease. Therefore, the effect of homocysteine on
Ab-induced VSMC toxicity was examined. VSMC were
incubated with various concentrations of homocysteine for

24 h and the amount of lactate dehydrogenase in the
medium measured (Fig. 4A). Homocysteine elicited a dose-
dependent increase in lactate dehydrogenase release. At
250 l
M
homocysteine, a significant increase in lactate
dehydrogenase was observed compared with cells grown
on plastic alone (Fig. 4B, P < 0.001). When homocysteine
was added to VSMC grown on 10 lg of substrate-bound
Ab1–40 or Ab1–42, this toxicity was enhanced. In the
presence of homocysteine, Ab1–40 caused a 40% increase
in toxicity over that with homocysteine alone, while
Ab1–42 caused a further 50% increase in toxicity (Fig. 4B,
P <0.05).
Measurement of apoptosis
To determine whether Ab-induced cell death was in part due
to apoptosis, cells were treated with Ab1–40 or Ab1–42 for
24 h at 37 °C. Cellular nuclei were then stained with the
fluorescent DNA-binding dye Hoechst 33258. Apoptotic
cells were identified by condensation of their nuclear
chromatin or fragmentation of their nuclei. In the presence
of Ab1–40 or Ab1–42, there was a significant increase
(P < 0.001) in the number of cells undergoing apoptosis
(Fig. 5). This increase in apoptosis was also seen when
homocysteine was added to the VSMC (P ¼ 0.015).
However, no further increase in apoptosis over that of Ab
Table 1. Catalase does not protect vascular smooth muscle cells from
toxicity induced by Ab. VSMC were treated with Ab at 37 °Cfor24h
in the presence or absence of catalase (1000 UÆmL
)1

). The MTS re-
duction assay was then used to measure cell viability. * Significantly
different from untreated controls (P < 0.05). Catalase did not protect
cells from toxicity of these treatments. # Significantly different from
incubations with H
2
O
2
+ catalase (P < 0.005, Student’s t-test).
Cell viability (% of untreated
control) ± SEM (n ¼ 3)
– Catalase + Catalase
Control 100 ± 2.1 100.7 ± 0.7
Ab 1–40 87.8 ± 2.3 * 84.1 ± 4.8
Ab 1–42 86.4 ± 1.8 * 88.5 ± 1.5
H
2
O
2
(5 l
M
) 88.8 ± 1.7 * # 102.2 ± 2.2
Fig. 2. Effect of aged Ab on VSMC toxicity. Ab1–40 or Ab1–42
peptides solubilized in DMEM (0.1 mgÆmL
)1
) were aged by incuba-
tion at 37 °C for 24, 72 or 120 h and aliquots allowed to dry on to
wells. VSMC were plated and cultured for 24 h at 37 °C, and super-
natant fractions analysed for lactate dehydrogenase activity. The rel-
ative amount of lactate dehydrogenase in the medium is shown as a

percentage of total lactate dehydrogenase in the cultures. Bars repre-
sent the mean of triplicate values ± SEM (n ¼ 3). * Significantly dif-
ferent from plastic by Student’s t-test, P < 0.05. LDH, lactate
dehydrogenase.
Ó FEBS 2002 Ab is toxic to vascular smooth muscle cells (Eur. J. Biochem. 269) 3017
alone or homocysteine alone was observed when Ab1–40
(P ¼ 0.014) or Ab1–42 (P ¼ 0.012) was added together
with homocysteine. In the presence of the topoisomerase II
inhibitor, etoposide, a potent inducer of apoptosis in many
cells, approximately 40% of VSMC were observed to
undergo apoptosis (P <0.001).
Effect of Ab1–40 or Ab1–42 on caspase-3 activity
As caspase-3 is normally activated during apoptosis in all
cellular systems [46], the protease can be used as an indicator
of apoptosis. VSMC were exposed to various concentrations
of homocysteine in the absence and presence of Ab1–40 or
Ab1–42. Caspase-3 activity was then measured using the
synthetic caspase-3 substrate DEVD-pNA. Levels of
caspase-3 activity increased with increasing concentrations
of homocysteine (Fig. 6). In the presence of substrate-bound
Fig.4. EffectofAb and homocysteine on VSMC toxicity. VSMC were
plated and allowed to attach on to Ab coated wells before homocy-
steine was added to the cultures. After 24 h, culture medium was
removed and assayed for lactate dehydrogenase activity. The amount
of lactate dehydrogenase in the medium is shown as a percentage of the
total lactate dehydrogenase in the cultures. (A) Plot shows that
increasing concentrations of homocysteine are toxic to VSMC. Values
are means ± SEM (n ¼ 3). (B) Cultures exposed to 250 l
M
homo-

cysteine in the presence of Ab peptides show an increase in lactate
dehydrogenase activity. Values are means ± SEM (n ¼ 7).
* P < 0.001 compared to plastic, ** P < 0.05 compared to homo-
cysteine alone (Student’s t-test). LDH, lactate dehydrogenase.
Fig.3.EffectofAb peptides and Matrigel on VSMC adhesion and
toxicity. (A) VSMC were plated on to Ab1–40, Ab1–42 or Matrigel
coated wells and allowed to attach for 30 min at 37 °C. Adherent cells
were fixed, stained and counted. The proportion of adherent cells is
shown as a percentage of total cells plated per well. Values are means
±SEM (n ¼ 3). * Statistically significant decrease compared with
untreated plastic (P <0.01andP < 0.005 for Ab1–40 and Ab1–42).
# Statistically significant decrease compared with Matrigel alone
(P < 0.05, determined by Student’s t-test). (B) VSMC were plated on
to substrates and cultured for 24 h. Lactate dehydrogenase activity
was measured in the culture medium. The amount of lactate dehy-
drogenase in the medium is shown as a percentage of the total lactate
dehydrogenase in the cultures. Values represent means ± SEM
(n ¼ 3). *Significantly different from plastic (P < 0.001). # Sig-
nificantly different from Matrigel alone (P <0.05,Student’st-test).
LDH, lactate dehydrogenase.
3018 S. S. Mok et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Ab, the level of caspase-3 activity also increased significantly
with increasing concentrations of homocysteine (P < 0.001
and P <0.01forAb1–40 and Ab1–42, respectively). These
data suggest that caspase activation occurs in the presence of
Ab, and caspase-3 levels are further increased in the presence
of homocysteine.
DISCUSSION
InCAA,Ab deposition occurs principally in association
with the vascular basement membrane [8,12,47]. Binding to

the extracellular matrix may therefore be an important step
for Ab accumulation and toxicity. However, the relation-
ship between the increase in Ab deposition in the basement
membrane and smooth muscle cell degeneration is unclear.
In this study, we used substrate-bound Ab to examine the
effect of Ab on VSMC. The use of substrate-bound proteins
in cell culture has been used extensively to mimic the
presentation of proteins as if they were bound to the
extracellular matrix [37]. This study shows that substrate-
bound Ab can increase apoptotic cell death in vascular
smooth muscle cell cultures in vitro and that the cardiovas-
cular risk factor homocysteine increases Ab-induced cell
death. The study also shows that the effects of Ab are likely
to be due to altered cell adherence to substrate that is
accompanied by a cytotoxic effect and an increase in
caspase-3 activity. When VSMC were cultured on a
substrate of Ab peptides, there was a decrease in cellular
adhesion properties and changes characteristic of apoptosis.
The extent of Ab aggregationwasshowntocorrelatewith
the toxic response in the VSMC. The duration of the Ab
ageing by incubation at 37 °C was related to the amount of
lactate dehydrogenase activity measured in the medium.
The effect of aggregation was observed with both Ab1–40
and Ab1–42. Longer ageing periods have been shown to
promote the formation of amyloid fibrils in solution [48].
The aggregation of Ab is thought to be significant in
Alzheimer pathogenesis since it correlates with neuronal
toxicity in vitro [41–43]. This may apply to myotoxicity as
Wisniewski & Wegiel [12] observed that leptomeningeal
myocyte destruction was also preceded by Ab fibrillogen-

esis. However, Davis-Salinas & Van Nostrand [20] showed
that preaggregation of Ab1–42 abolished its cytotoxic effect
on cultured human leptomeningeal smooth muscle cells.
The Ab had to be in a soluble form to aggregate at the cell
surface and exert its toxicity [20,35]. Our model demon-
strates that pre-aggregated Ab, which is first bound to its
substrate, is also toxic to VSMC. The form in which the
peptide is presented to cells thus plays a crucial role in
eliciting toxicity.
Ab-Induced apoptotic cell death is well documented.
There is increasing evidence that neurons die via apoptotic
mechanisms in a range of neurodegenerative conditions
including Alzheimer’s disease and stroke. Pro-apoptotic
genes have been shown to be induced in cultured cortical
neurons treated with Ab [33,49]. Kruman et al. [50] have
reported that homocysteine can induce neuronal apoptosis.
Our studies show that levels of caspase-3 activity are
increased in VSMC treated with Ab peptides. Ab has been
shown to induce activation of different caspases in different
cell types in vitro [51–56]. Caspase-3 cleaves APP at caspase
consensus sites and has been shown to increase Ab
production [57]. In addition, intracellular accumulation of
APP can lead to neuronal caspase-3 activation that in turn
leads to increased Ab production and cell death [58]. Thus
cytotoxic effects can arise from caspase cleavage of APP.
Oxidative stress has been widely implicated in Ab toxicity
[28,29,44]. Induction of oxidative stress can occur by the
generation of reactive oxygen species such as superoxide
(O
2

–
), hydrogen peroxide (H
2
O
2
), peroxynitrite (ONOO
–
)
and hydroxy radical (OH
•
). In our system, toxicity was not
mediated by H
2
O
2
generation.
Ab was also shown to interfere with the substrate-
adhesive properties of VSMC. Ab interfered with VSMC-
substrate adhesion and the inhibitory effect was more
prominent with Ab1–42 than Ab1–40. The inhibition of
adhesion and subsequent toxicity to the cells by Ab may be
important in cerebrovascular pathogenesis of amyloid
angiopathy. The greater toxicity of the longer Ab1–42
species is consistent with previous work [19,59] which shows
Fig. 5. Ab induces apoptosis in VSMC. VSMC were plated and
allowed to attach on to Ab coated wells before homocysteine
(0.25 m
M
) or etoposide (2.5 l
M

) was added to the cultures. After 24 h,
cells were fixed and stained with the dye Hoechst 33258. The number of
apoptotic cells is shown as a percentage of the total number of cells in
each field. Values are means ± SEM (n ¼ 3). *P < 0.001 compared
to plastic, **P < 0.05 compared to plastic (Student’s t-test). LDH,
lactate dehydrogenase.
Fig. 6. Ab and homocysteine increase the levels of caspase-3 activity in
VSMC. VSMC were allowed to attach on to Ab coated wells before
addition of homocysteine. After 24 h, cell pellets were extracted and
assayed for caspase-3 activity. Figure shows caspase-3 activity in cul-
tures treated with homocysteine or homocysteine and Ab.Valuesare
means ± SEM (n ¼ 3). *P < 0.001 and **P < 0.01 (paired Stu-
dent’s t-test). LDH, lactate dehydrogenase.
Ó FEBS 2002 Ab is toxic to vascular smooth muscle cells (Eur. J. Biochem. 269) 3019
that Ab1–42 was highly toxic to smooth muscle cells and
pericytes. As Ab1–42 is deposited early in the cerebrovas-
culature [60] and binds basement membrane with greater
affinity than Ab1–40 [61] suggests it represents the more
fibrillogenic and pathogenic species. The observation that
Ab decreases cell adhesion events is supported by Fraser
et al. [62] and Postuma et al. [45] who found that substrate-
bound Ab inhibited neurite outgrowth and cell adhesion.
Interestingly, Chinese hamster ovary cells transfected with
a5b1 integrin demonstrated reduced susceptibility to Ab-
induced apoptosis [63], implying the significant role of cell
adhesion in pathogenesis. This pathogenic phenomenon
may be relevant to smooth muscle cells. The observation
that basal lamina destruction precedes leptomeningeal
smooth muscle degeneration in amyloid angiopathy [12]
may implicate the loss of adhesive extracellular elements.

Davis et al. [31] observed that human cerebrovascular
smooth muscle cells undergo shrinkage and regression of
processes upon exposure to Ab, agreeing with our findings
that the antiadhesive properties of Ab may contribute to
cellular degeneration. Thus the disruption of cell adherence
properties may play a role in downstream signal transduc-
tion cascades and influence cell toxicity.
Increased plasma homocysteine has been shown to be a
major cardiovascular risk factor. High homocysteine levels
have also been shown to be associated with Alzheimer’s
disease patients [64] and other disorders of the nervous
system such as schizophrenia and Parkinson’s disease.
Homocysteine has also been shown to be toxic to neurons in
culture by increasing the vulnerability of these cells to
excitotoxic and oxidative injury [50]. In smooth muscle cells,
homocysteine can increase production of nitric oxide [65].
However, the exact mechanism by which homocysteine
exerts its effects is still not known. Patients with hyperhom-
ocysteinemia have homocysteine levels in the 0.1–0.25 m
M
range. In these studies, homocysteine was found to elicit a
dose-dependent increase in toxicity in VSMC. In the
presence of Ab peptides, this toxicity is exacerbated. This
effect of homocysteine and Ab hasalsobeenshownin
primary cortical neurons [66] and in neuroblastoma cells
[67]. This indicates that homocysteine may induce a cell
death pathway that contributes to cellular degeneration.
In summary, the use of substrate-bound amyloid peptides
to study the effect of CAA on VSMC function provides a
new approach to investigate the mechanisms of smooth

muscle cell loss in vascular amyloidosis. Our studies show
that homocysteine, a risk factor for certain cardiovascular
diseases, can increase susceptibility of VSMC to Ab toxicity.
Therefore, we hypothesize that homocysteine may increase
the risk of stroke due to CAA. In addition, our studies
provide a method by which potential therapeutic agents can
be tested for their abilities to inhibit Ab-induced VSMC
death.
ACKNOWLEDGEMENTS
This work is supported by grants from the National Health and
Medical Research Council (NH & MRC) of Australia. KB is supported
by the Deutsche Forschungsgemeinschaft and the Bundesministerium
fu
¨
r Forschung und Technologie. The authors thank Drs Greg Dusting
and Justin Bilszta (Howard Florey Institute of Experimental
Physiology and Medicine, Parkville, Australia) for rat aorta smooth
muscle cells.
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