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: davidhs@unimelb.edu.au 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. REFERENCES 1. Vinters, H.V., Reave, S., Costello, P., Girvin, J.P. & Moore, S.A. (1987) Isolation and culture of cells derived from human cerebral microvessels. Cell Tissue Res. 249, 657–667. 2. Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.H., Multhaup, G., Beyreuther, K. & Muller- Hill, B. (1987) The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature 325, 733–736. 3. Selkoe, D.J. (1994) Normal and abnormal biology of the b-amy- loid precursor protein. Annu.Rev.Neurosci.17, 489–517. 4. Sinha, S., Anderson, J.P., Barbour, R., Basi, G.S., Caccavello, R., Davis,D.,Doan,M.,Dovey,H.F.,Frigon,N.,Hong,J.et al. (1999) Purification and cloning of amyloid precursor protein b-secretase from human brain. Nature 402, 537–540. 5. Vassar, R., Bennett, B.D., Babu-Khan, S., Kahn, S., Mendiaz, E.A.,Denis,P.,Teplow,D.B.,Ross,S.,Amarante,P.,Loeloff,R. et al. (1999) b-Secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286, 735–741. 6. Nunan, J. & Small, D.H. (2000) Regulation of APP cleavage by a-, b-andc-secretases. FEBS Lett. 483, 6–10. 7. Greenberg, S.M. (1998) Cerebral amyloid angiopathy: prospects for clinical diagnosis and treatment. Neurology 51, 690–694. 8. Yamaguchi, H., Yamazaki, T., Lemere, C.A., Frosch, M.P. & Selkoe, D.J. (1992) b-amyloid is focally deposited within the outer basement membrane in the amyloid angiopathy of Alzheimer’s disease. An immunoelectron microscopic study. Am. J. Pathol. 141, 249–259. 9. Grabowski, T.J., Cho, H.S., Vonsattel, J.P., Rebeck, G.W. & Greenberg, S.M. (2001) Novel amyloid precursor protein muta- tion in an Iowa family with dementia and severe cerebral amyloid angiopathy. Ann. Neurol. 49, 697–705. 10. Nilsberth, C., Westlind-Danielsson, A., Eckman, C.B., Condron, M.M., Axelman, K., Forsell, C., Stenh, C., Luthman, J., Teplow, D.B., Younkin, S.G., Naslund, J. & Lannfelt, L. (2001) The ÔArcticÕ APP mutation (E693G) causes Alzheimer’s disease by enhanced Ab protofibril formation. Nat. Neurosci. 4, 887–893. 11. Hendriks, L., van Duijn, C.M., Cras, P., Cruts, M., Van Hul, W., van Harskamp, F., Warren, A., McInnis, M.G., Antonarakis, S.E., Martin, J.J., et al. (1992) Presenile dementia and cerebral haemorrhage linked to a mutation at codon 692 of the beta- amyloid precursor protein gene. Nat. Genet. 1, 218–221. 12. Wisniewski, H.M. & Wegiel, J. (1994) b-Amyloid formation by myocytes of leptomeningeal vessels. Acta Neuropathol. 87,233– 241. 13. Wisniewski, H.M., Frackowiak, J., Zoltowska, A. & Kim, K.S. (1994) Vascular b-amyloid in Alzheimer’s disease angiopathy is produced by proliferating and degenerating smooth muscle cells. Amyloid 1, 8–10. 14. Calhoun, M.E., Burgermeister, P., Phinney, A.L., Stalder, M., Tolnay, M., Wiederhold, K.H., Abramowski, D., Sturchler- Pierrat,C.,Sommer,B.,Staufenbiel,M.&Jucker,M.(1999) Neuronal overexpression of mutant amyloid precursor protein results in prominent deposition of cerebrovascular amyloid. Proc. Natl Acad. Sci. USA 96, 14088–14093. 15. Van Dorpe, J., Smeijers, L., Dewachter, I., Nuyens, D., Spittaels, K., Van Den Haute, C., Mercken, M., Moechars, D., Laenen, I., Kuiperi, C. et al. (2000) Prominent cerebral amyloid angiopathy in transgenic mice overexpressing the London mutant of human APP in neurons. Am. J. Pathol. 157, 1283–1298. 16. Christie, R., Yamada, M., Moskowitz, M. & Hyman, B. (2001) Structural and functional disruption of vascular smooth muscle cells in a transgenic mouse model of amyloid angiopathy. Am. J. Pathol. 158, 1065–1071. 17. Winkler, D.T., Bondolfi, L., Herzig, M.C., Jann, L., Calhoun, M.E., Wiederhold, K.H., Tolnay, M., Staufenbiel, M. &Jucker, M. 3020 S. S. Mok et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (2001) Spontaneous hemorrhagic stroke in a mouse model of cerebral amyloid angiopathy. J. Neurosci. 21, 1619–1627. 18. Kawai, M., Kalaria, R.N., Cras, P., Siedlak, S.L., Velasco, M.E., Shelton, E.R., Chan, H.W., Greenberg, B.D. & Perry, G. (1993) Degeneration of vascular muscle cells in cerebral amyloid angio- pathy of Alzheimer disease. Brain Res. 623, 142–146. 19. Davis-Salinas, J., Saporito-Irwin, S.M., Cotman, C.W. & Van Nostrand, W.E. (1995) Amyloid b-protein induces its own pro- duction in cultured degenerating cerebrovascular smooth muscle cells. J. Neurochem. 65, 931–934. 20. Davis-Salinas, J. & Van Nostrand, W.E. (1995) Amyloid b-pro- tein aggregation nullifies its pathologic properties in cultured cerebrovascular smooth muscle cells. J. Biol. Chem. 270, 20887– 20890. 21. Van Nostrand, W.E., Rozemuller, A.J.M., Chung, R., Cotman, C.W. & Saporito-Irwin, S.M. (1994) Amyloid b-protein precursor in cultured leptomeningeal smooth muscle cells. Amyloid. 1,1–7. 22. Geula, C., Wu, C.K., Saroff, D., Lorenzo, A., Yuan, M. & Yankner, B.A. (1998) Aging renders the brain vulnerable to amyloid b-protein neurotoxicity. Nat. Med. 4, 827–831. 23. Howlett, D.R., Jennings, K.H., Lee, D.C., Clark, M.S., Brown, F., Wetzel, R., Wood, S.J., Camilleri, P. & Roberts, G.W. (1995) Aggregation state and neurotoxic properties of Alzheimer b-amyloid peptide. Neurodegeneration 4, 23–32. 24. Yankner, B.A., Dawes, L.R., Fisher, S., Villa-Komaroff, L., Oster-Granite, M.L. & Neve, R.L. (1989) Neurotoxicity of a fragment of the amyloid precursor associated with Alzheimer’s disease. Science 245, 417–420. 25. Mattson, M.P., Cheng, B., Davis, D., Bryant, K., Lieberburg, I. & Rydel, R.E. (1992) b-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J. Neurosci. 12, 376–389. 26. Mattson, M.P., Barger, S.W., Cheng, B., Lieberburg, I., Smith- Swintosky, V.L. & Rydel, R.E. (1993) b-Amyloid precursor protein metabolites and loss of neuronal Ca 2+ homeostasis in Alzheimer’s disease. Trends Neurosci. 16, 409–414. 27. Mattson, M.P., Tomaselli, K.J. & Rydel, R.E. (1993) Calcium- destabilizing and neurodegenerative effects of aggregated b-amy- loid peptide are attenuated by basic FGF. Brain Res. 621, 35–49. 28. Behl, C., Davis, J.B., Lesley, R. & Schubert, D. (1994) Hydrogen peroxide mediates amyloid b protein toxicity. Cell 77, 817–827. 29. Goodman, Y. & Mattson, M.P. (1994) Secreted forms of b-amyloid precursor protein protect hippocampal neurons against amyloid b-peptide-induced oxidative injury. Exp. Neurol. 128, 1–12. 30. Kruman, I., Bruce-Keller, A.J., Bredesen, D., Waeg, G. & Mattson, M.P. (1997) Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis. J. Neurosci. 17, 5089–5100. 31. Davis, J., Cribbs, D.H., Cotman, C.W. & Van Nostrand, W.E. (1999) Pathogenic amyloid b-protein induces apoptosis in cultured human cerebrovascular smooth muscle cells. Amyloid 6, 157–164. 32. Mattson, M.P., Keller, J.N. & Begley, J.G. (1998) Evidence for synaptic apoptosis. Exp. Neurol. 153, 35–48. 33. Loo, D.T., Copani, A., Pike, C.J., Whittemore, E.R., Wale- ncewicz, A.J. & Cotman, C.W. (1993) Apoptosis is induced by b-amyloid in cultured central nervous system neurons. Proc. Natl Acad.Sci.USA90, 7951–7955. 34. Wang, Z., Natte, R., Berliner, J.A., van Duinen, S.G., Vinters, H.V. & Rosenblum, W.I. (2000) Toxicity of Dutch (E22Q) and Flemish (A21G) mutant amyloid b proteins to human cerebral microvessel and aortic smooth muscle cells. Stroke 31, 534–538. 35. Melchor, J.P. & Van Nostrand, W.E. (2000) Fibrillar amyloid b-protein mediates the pathologic accumulation of its secreted precursor in human cerebrovascular smooth muscle cells. J. Biol. Chem. 275, 9782–9791. 36. Van Nostrand, W.E., Melchor, J.P., Cho, H.S., Greenberg, S.M. & Rebeck, G.W. (2001) Pathogenic effects of D23N Iowa mutant amyloid b-protein. J. Biol. Chem. 276, 32860–32866. 37. Letourneau, P.C. (1975) Cell-to-substratum adhesion and guid- ance of axonal elongation. Dev. Biol. 44, 92–101. 38. He, W. & Barrow, C.J. (1999) The Ab 3-pyroglutamyl and 11-pyroglutamyl peptides found in senile plaque have greater b-sheet forming and aggregation propensities in vitro than full-length Ab. Biochemistry 38, 10871–10877. 39. Hadrava, V., Tremblay, J. & Hamet, P. (1989) Abnormalities in growth characteristics of aortic smooth muscle cells in sponta- neously hypertensive rats. Hypertension 13, 589–597. 40. Yankner, B.A. (1996) Mechanisms of neuronal degeneration in Alzheimer’s disease. Neuron 16, 921–932. 41. Pike, C.J., Burdick, D., Walencewicz, A.J., Glabe, C.G. & Cotman, C.W. (1993) Neurodegeneration induced by b-amyloid peptides in vitro: the role of peptide assembly state. J. Neurosci. 13, 1676–1687. 42. Pike, C.J., Walencewicz, A.J., Glabe, C.G. & Cotman, C.W. (1991) Aggregation-related toxicity of synthetic b-amyloid protein in hippocampal cultures. Eur. J. Pharmacol. 207, 367–368. 43. Pike, C.J., Walencewicz, A.J., Glabe, C.G. & Cotman, C.W. (1991) In vitro aging of b-amyloid protein causes peptide aggre- gation and neurotoxicity. Brain Res. 563, 311–314. 44. Mattson, M.P. & Goodman, Y. (1995) Different amyloidogenic peptides share a similar mechanism of neurotoxicity involving reactive oxygen species and calcium. Brain Res. 676, 219–224. 45. Postuma, R.B., He, W., Nunan, J., Beyreuther, K., Masters, C.L., Barrow, C.J. & Small, D.H. (2000) Substrate-bound b-amyloid peptides inhibit cell adhesion and neurite outgrowth in primary neuronal cultures. J. Neurochem. 74, 1122–1130. 46. Fernandes-Alnemri, T., Litwack, G. & Alnemri, E.S. (1994) CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 b-converting enzyme. J. Biol. Chem. 269, 30761– 30764. 47. Perlmutter, L.S., Barron, E., Saperia, D. & Chui, H.C. (1991) Association between vascular basement membrane components and the lesions of Alzheimer’s disease. J. Neurosci. Res. 30, 673–681. 48. Kusumoto, Y., Lomakin, A., Teplow, D.B. & Benedek, G.B. (1998) Temperature dependence of amyloid b-protein fibrilliza- tion. Proc. Natl Acad. Sci. USA 95, 12277–12282. 49. Estus, S., Tucker, H.M., van Rooyen, C., Wright, S., Brigham, E.F., Wogulis, M. & Rydel, R.E. (1997) Aggregated amyloid-b protein induces cortical neuronal apoptosis and concomitant ÔapoptoticÕ pattern of gene induction. J. Neurosci. 17, 7736–7745. 50. Kruman, I., Culmsee, C., Chan, S.L., Kruman, Y., Guo, Z., Penix, L. & Mattson, M.P. (2000) Homocysteine elicits a DNA damage response in neurons that promotes apoptosis and hypersensitivity to excitotoxicity. J. Neurosci. 20, 6920–6926. 51. Harada, J. & Sugimoto, M. (1999) Activation of caspase-3 in b-amyloid-induced apoptosis of cultured rat cortical neurons. Brain Res. 842, 311–323. 52. Troy, C.M., Rabacchi, S.A., Friedman, W.J., Frappier, T.F., Brown, K. & Shelanski, M.L. (2000) Caspase-2 mediates neuronal cell death induced by b-amyloid. J. Neurosci. 20, 1386–1392. 53. Ivins, K.J., Bui, E.T. & Cotman, C.W. (1998) b-Amyloid induces local neurite degeneration in cultured hippocampal neurons: evidence for neuritic apoptosis. Neurobiol. Dis. 5, 365–378. 54. Allen, J.W., Eldadah, B.A., Huang, X., Knoblach, S.M. & Faden, A.I. (2001) Multiple caspases are involved in b-amyloid-induced neuronal apoptosis. J. Neurosci. Res. 65, 45–53. 55. Xu, J., Chen, S., Ku, G., Ahmed, S.H., Chen, H. & Hsu, C.Y. (2001) Amyloid b peptide-induced cerebral endothelial cell death involves mitochondrial dysfunction and caspase activation. J. Cereb. Blood Flow Metab. 21, 702–710. Ó FEBS 2002 Ab is toxic to vascular smooth muscle cells (Eur. J. Biochem. 269) 3021 56. Nakagawa,T.,Zhu,H.,Morishima,N.,Li,E.,Xu,J.,Yankner, B.A. & Yuan, J. (2000) Caspase-12 mediates endoplasmic- reticulum-specific apoptosis and cytotoxicity by amyloid-b. Nature 403, 98–103. 57. Gervais, F.G., Xu, D., Robertson, G.S., Vaillancourt, J.P., Zhu, Y.,Huang,J.,LeBlanc,A.,Smith,D.,Rigby,M.,Shearman,M.S. et al. (1999) Involvement of caspases in proteolytic cleavage of Alzheimer’s amyloid-b precursor protein and amyloidogenic Ab peptide formation. Cell 97, 395–406. 58. Uetsuki, T., Takemoto, K., Nishimura, I., Okamoto, M., Niinobe, M., Momoi, T., Miura, M. & Yoshikawa, K. (1999) Activation of neuronal caspase-3 by intracellular accumulation of wild- type Alzheimer amyloid precursor protein. J. Neurosci. 19, 6955–6964. 59. Verbeek, M.M., de Waal, R.M., Schipper, J.J. & Van Nostrand, W.E. (1997) Rapid degeneration of cultured human brain peri- cytes by amyloid b protein. J. Neurochem. 68, 1135–1141. 60. Roher, A.E., Lowenson, J.D., Clarke, S., Woods, A.S., Cotter, R.J., Gowing, E. & Ball, M.J. (1993) b-Amyloid-(1–42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of Alzheimer disease. Proc. Natl Acad. Sci. USA 90, 10836–10840. 61. Castillo, G.M., Ngo, C., Cummings, J., Wight, T.N. & Snow, A.D. (1997) Perlecan binds to the b-amyloid proteins (Ab)of Alzheimer’s disease, accelerates Ab fibril formation, and maintains Ab fibril stability. J. Neurochem. 69, 2452–2465. 62. Fraser, P.E., Levesque, L. & McLachlan, D.R. (1994) Alzheimer Ab amyloid forms an inhibitory neuronal substrate. J. Neurochem. 62, 1227–1230. 63. Matter, M.L., Zhang, Z., Nordstedt, C. & Ruoslahti, E. (1998) The a5b1 integrin mediates elimination of amyloid-b peptide and protects against apoptosis. J. Cell Biol. 141, 1019–1030. 64. Joosten, E., Lesaffre, E., Riezler, R., Ghekiere, V., Dereymaeker, L., Pelemans, W. & Dejaeger, E. (1997) Is metabolic evidence for vitamin B-12 and folate deficiency more frequent in elderly patients with Alzheimer’s disease? J.Gerontol.aBiol.Sci.Med. Sci. 52, M76–M79. 65. Ikeda, U., Ikeda, M., Minota, S. & Shimada, K. (1999) Homo- cysteine increases nitric oxide synthesis in cytokine-stimulated vascular smooth muscle cells. Circulation 99, 1230–1235. 66. White,A.R.,Huang,X.,Jobling,M.F.,Barrow,C.J.,Beyreuther, K., Masters, C.L., Bush, A.I. & Cappai, R. (2001) Homocysteine potentiates copper- and amyloid b peptide-mediated toxicity in primary neuronal cultures: possible risk factors in the Alzheimer’s- type neurodegenerative pathways. J. Neurochem. 76, 1509–1520. 67. Ho, P.I., Collins, S.C., Dhitavat, S., Ortiz, D., Ashline, D., Rogers, E. & Shea, T.B. (2001) Homocysteine potentiates b-amyloid neurotoxicity: role of oxidative stress. J. Neurochem. 78, 249–253. 3022 S. S. Mok et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . 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. 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