Tài liệu Báo cáo Y học: Toxicity of substrate-bound amyloid peptides on vascular smooth muscle cells

Toxicity of substrate-bound amyloid peptides on vascular smooth

muscle cells is enhanced by homocysteine

Su San Mok1,2, Bradley J. Turner1,2, Konrad Beyreuther3

, Colin L. Masters1,2, Colin J. Barrow4

and David H. Small1,2

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
age, a process by which Ab is

induced to aggregate over several hours to days. Oxidative

stress via hydrogen peroxide (H2O2) 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

(reviewed in [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: [email protected]

Abbreviations: Ab, amyloid-b-protein; CAA, cerebral amyloid

angiopathy; APP, amyloid protein precursor; VSMC, vascular

smooth muscle cell; H2O2, 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) FEBS 2002 doi:10.1046/j.1432-1033.2002.02976.x