QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (25) Citing Articles via Google Scholar Google Scholar Articles by Muñoz, F. J. Articles by Inestrosa, N. C. Search for Related Content PubMed PubMed Citation Articles by Muñoz, F. J. Articles by Inestrosa, N. C.

 Previous Article  |  Next Article 

The Journal of Neuroscience, April 15, 2002, 22(8):3081-3089

Vitamin E But Not 17-Estradiol Protects against Vascular Toxicity Induced by -Amyloid Wild Type and the Dutch Amyloid Variant

Francisco J. Muñoz¹, Carlos Opazo⁴, Gabriel Gil-Gómez², Gladys Tapia³, Virginia Fernández³, Miguel A. Valverde¹, and Nibaldo C. Inestrosa⁴

¹ Unitat de Senyalització Cel·lular, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona E-08003, Spain, ² Institut Municipal d'Investigació Mèdica (Universitat Pompeu Fabra), Barcelona E-08003, Spain, ³ Programa de Farmacología Molecular y Clínica, Instituto de Ciencias Biomédicas, Universidad de Chile, Santiago, Chile, and ⁴ Centro de Regulación Celular y Patología, Fondo de Investigación Avanzada en Áreas Prioritarias-Biomedicina and Millenium Institute for Fundamental and Applied Biology, Pontificia Universidad Católica de Chile, Santiago, Chile

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Amyloid -peptide (A) fibril deposition on cerebral vessels produces cerebral amyloid angiopathy that appears in the majority of Alzheimer's disease patients. An early onset of a cerebral amyloid angiopathy variant called hereditary cerebral hemorrhage with amyloidosis of the Dutch type is caused by a point mutation in A yielding A_Glu22Gln. The present study addresses the effect of amyloid fibrils from both wild-type and mutated A on vascular cells, as well as the putative protective role of antioxidants on amyloid angiopathy. For this purpose, we studied the cytotoxicity induced by A_{1-40 Glu22Gln} and A_{1-40 wild-type} fibrils on human venule endothelial cells and rat aorta smooth muscle cells. We observed that A_Glu22Gln fibrils are more toxic for vascular cells than the wild-type fibrils. We also evaluated the cytotoxicity of A fibrils bound with acetylcholinesterase (AChE), a common component of amyloid deposits. A_{1-40 wild-type}-AChE fibrillar complexes, similar to neuronal cells, resulted in an increased toxicity on vascular cells. Previous reports showing that antioxidants are able to reduce the toxicity of A fibrils on neuronal cells prompted us to test the effect of vitamin E, vitamin C, and 17-estradiol on vascular damage induced by A_wild-type and A_Glu22Gln. Our data indicate that vitamin E attenuated significantly the A-mediated cytotoxicity on vascular cells, although 17-estradiol and vitamin C failed to inhibit the cytotoxicity induced by A fibrils.

Key words: Alzheimer's disease; CAA; HCHWA-D; amyloid; vitamin E; 17-estradiol; vitamin C; oxidative stress; acetylcholinesterase; endothelial cells; vascular smooth muscle cells

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cerebral amyloid angiopathy (CAA) is linked to most cases of Alzheimer's disease (AD). CAA is characterized by the deposition of amyloid -peptide (A) mainly in the media and adventitia of both leptomeningeal and intracortical vessels (Vinters et al., 1988). An early onset of CAA occurs in the hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D) (van Duinen et al., 1987). HCHWA-D is caused by a point mutation in the A-encoding gene, which produces the substitution of GluGln at position 22 (Levy et al., 1990), which renders A more fibrillogenic (Wisniewski et al., 1991; Alvarez et al., 1997).

Vascular amyloid deposits of both CAA and HCHWA-D, similar to senile plaques from the brain parenchyma of AD patients, contain molecules other than A (Snow et al., 1988; van Duinen et al., 1995; Verbeek et al., 1998). The enzyme acetylcholinesterase (AChE) has been reported in CAA deposits (Mesulam et al., 1992), which could be relevant for CAA pathology because AChE is able to induce A aggregation into fibrils (Inestrosa et al., 1996; Alvarez et al., 1998). Moreover, those A-AChE fibrillar complexes are more toxic for neuronal cells than A fibrils alone (Alvarez et al., 1998; Muñoz and Inestrosa, 1999).

Amyloid-associated pathophysiology has been reported to involve oxidative stress (Behl, 1997; Miranda et al., 2000). The role of oxidative stress in AD is strengthened by in vitro findings showing that A increases H₂O₂ in cells (Behl et al., 1994), whereas catalase, an enzyme that converts H₂O₂ to O₂ and H₂O, blocks A toxicity (Behl et al., 1994), although there is controversy on the role of H₂O₂ in A-mediated cell damage (Zhang et al., 1996). Moreover, neuroblastoma cells (Neuro 2a), which are resistant to A, glutamate, and H₂O₂, contain high levels of the antioxidant glutathione (Calderón et al., 1999). In accordance with the oxidative pathophysiological hypothesis, vitamin E (vit E) and other antioxidants have demonstrated protective properties on neuronal cells against the A-mediated neurotoxicity (Behl et al., 1992; Pappolla et al., 1997). The hormone 17-Estradiol (E₂), with well known antioxidant properties, also protects neuronal cells against A-mediated neurotoxicity (Goodman et al., 1996; Behl et al., 1997a; Bonnefont et al., 1998). Moreover, vit E and E₂ have been associated with the retardation of the onset and progression of AD (Tang et al., 1996; Kawas et al., 1997; Sano et al., 1997; Morris et al., 1998).

In the present work, we studied the vascular toxicity induced by different types of A fibrils involved in amyloid angiopathy. First, we investigated the toxicity induced by A_1-40 wild type (A_wt) and A_Glu22Gln on endothelial and vascular smooth muscle cells (VSMC). Second, the effect of the presence of AChE in the A fibrillar complexes on these target cells was assessed. Finally, we evaluated the protective effect of vit E, vitamin C (vit C), and E₂ on vascular cells challenged by A fibrils.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. Synthetic A peptides corresponding to the human A_wt sequence and the A_1-40 Dutch variant that contains a glutamic acid to glutamine substitution (A_Glu22Gln) were used in the present work. All of the peptides were obtained from Chiron (Emeryville, CA), Sigma (St. Louis, MO), and Calbiochem (Postfach, Germany). Chemicals, culture media, and sera were obtained from Sigma, Roche (Alameda, CA), Merck (Darmstadt, Germany), Invitrogen (Paisley, UK), and Molecular Probes (Leiden, The Netherlands).

Cell lines. Rat aorta smooth muscle cells (A7r5) were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Human venule endothelial cells (HUVEC) were grown in M-199 medium supplemented with 10% FBS, 3.2 mM glutamine, and antibiotics.

AChE purification. The tetrameric G₄ AChE form (sedimentation coefficient of 10.7 S) was purified from bovine caudate nucleus, using acridine-affinity chromatography as described previously (Inestrosa et al., 1987). The specific activity (6000 U/mg protein) was determined by the method of Ellman et al. (1961). The staining intensity after SDS-PAGE (a single band of 66 kDa) was used to verify its purity.

Aggregation assay (turbidity). The aggregation assay was performed as described previously (Muñoz and Inestrosa, 1999). Briefly, peptide stock solutions were prepared by dissolving freeze-dried aliquots of A_Glu22Gln and A_wt in dimethylsulfoxide. Peptide stock aliquots were diluted in 0.1 M Tris-HCl, pH 7.4, to a final concentration of 96.6 µM A. For the aggregation assays with AChE, peptide stock aliquots were added to 0.1 M Tris-HCl containing AChE (100 nM). The solutions were stirred continuously (210 rpm) at room temperature for 48 hr. Aggregation was measured by turbidity at 405 nm versus buffer blank.

Amyloid fibril isolation. Preformed fibrils were washed four times with PBS by centrifugation at 14,000 rpm for 30 min to remove the soluble A and AChE. Pellets were homogenized in PBS. Aliquots of the homogenate were transferred to a denaturing buffer and subjected to Tris-tricine SDS-PAGE (Schagger et al., 1988) to quantify the concentrations of A peptide contained in the fibrils. This was achieved by densitometric scanning using A and AChE with known concentrations as standards. Data were processed by the GS365W program from Hoefer Scientific Instruments (San Francisco, CA).

Congo red staining. The A fibrils and A-AChE complexes were mounted on slides coated with 0.5% gelatin. Samples were dried overnight at room temperature and stained by using the alkaline Congo red (CR) method as described previously (Puchtler et al., 1961). Briefly, a saturated CR solution was prepared with 80% ethanol and 0.005% NaOH and NaCl saturated and was then filtered. Samples were incubated with CR solution for 20 min, dehydrated with increasing alcohol grades, cleared with xylene, and mounted in Canadian balsam. Slides were observed with a Nikon (Tokyo, Japan) Optiphot microscope configured for polarized light.

Electron microscopy. The amyloid fibrils were placed on Formvar carbon-coated 300 mesh nickel grids and negatively stained with 3% phosphotungstic acid solution for 1 min (Inestrosa et al., 1996). Grids were examined under a Philips EM-300 electron microscope at 60 kV.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and lactic dehydrogenase release assays. Cells were seeded in 96-well plates in serum- and phenol red-free medium with 2 µM insulin at a density of 4 × 10³ cells/100 µl per well. PBS (10 µl) (control), A fibrils, and AChE-A complexes were added at different concentrations to wells. Experiments to study the effect of AChE alone on vascular cells were performed by adding the enzyme to cell cultures at a range of concentrations (0.1-25 nM). In the experiments with antioxidants, 1 mM vit E, 100 µM vit C, or E₂ (0, 1, or 10 µM) was added 2 hr before the amyloid fibrils. The hydrophobic vit E and E₂ were dissolved in ethanol to a final concentration of 5 nM ethanol per well. Cells were incubated for 48 hr at 37°C, after which cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction method (Mosmann, 1983) and lactic dehydrogenase (LDH) release as described previously (Muñoz and Inestrosa, 1999). Briefly, after the addition of 11 µl of MTT stock solution (5 mg/ml), the reaction was terminated 4 hr later with 110 µl of stop solution (50% dimethylformamide and 20% SDS at pH 4.7). MTT reduction was determined in a Labsystems (Espoo, Finland) Uniskam I spectrophotometer at 540 and 650 nm. LDH release was measured in the medium of cytotoxicity assays. Fifty microliters of culture supernatants were collected from each well, and LDH activities were determined with a colorimetric LDH assay kit (Promega, Madison, WI). Total cellular LDH activity was determined by lysing the cells with the kit lysis buffer.

Phosphatydylserine translocation and O₂· production measurements. HUVEC and A7r5 cells were seeded in 24-well plates in serum- and phenol red-free medium with 2 µM insulin at a density of 50 × 10³ cells/300 µl per well. Cells were preincubated with 1 mM vit E or 10 µM E₂, after which PBS (control), A (10 µM final concentration), or H₂O₂ (50 mM) were added. Cells were incubated for 4 or 48 hr at 37°C, and translocation of plasma membrane phosphatydylserine (a marker of apoptosis) was detected by annexin-V-Fluos binding according to the protocol of the manufacturer. O₂· production was measured by the intracellular oxidation of dihydroethydium (100 nM). Annexin-V binding and ethydium production were quantified on >4000 cells per experimental condition with a Becton-Dickinson (Franklin Lakes, NJ) FACScan cytometer. Debris was excluded on the basis of forward and side light-scattering properties. Experiments run in parallel also showed that cells analyzed presented intact membranes, demonstrated by the exclusion of propidium iodide. The putative effect of lot-to-lot variation of A in aggregation and toxicity was discarded by using different lots purchased from different companies. In all cases, the Dutch variant was more toxic than the wild-type A, because it has been demonstrated previously in PC12 cells, a well known cell target model for A-mediated cytotoxicity (our unpublished observations).

Determination of superoxide dismutase activity. Superoxide dismutase (SOD) activity was evaluated by measuring the inhibition of cytochrome c reduction by SOD from homogenized vascular cells. Cytochrome c reduction was induced by O₂· (superoxide radical) generated by the xanthine-xanthine oxidase system (Fridovich, 1985). The reaction was initiated by adding xanthine oxidase (10 µl of a 0.7 U/ml solution) and 5-150 µl of SOD standard (44.8 U/ml) or 20-200 µl of homogenized vascular cell samples to 1 ml of reaction buffer (50 mM potassium phosphate at pH 7.8, 0.1 mM EDTA, 50 µM xanthine, and 10 µM cytochrome c). Reaction was followed at 550 nm for 2-6 min at 25°C with a Lambda 2 spectrophotometer (PerkinElmer Life Sciences, Ueberlingen, Germany). Results are expressed as units of SOD per milligram of protein. Proteins were quantified in homogenized vascular cells as described previously (Lowry et al., 1951).

Determination of nitric oxide synthase activity. Nitric oxide synthase (NOS) was evaluated by measuring the oxidation of oxyhemoglobin to methemoglobin by NO (Knowles et al., 1990). Homogenized vascular cell samples (100 µl) were preincubated for 5 min with the reaction buffer (40 mM potassium phosphate at pH 7.2, 1.6 µM oxyhemoglobin, and 1 mM MgCl₂) at 37°C. The reaction was initiated by the addition of 1 mM L-arginine and 1 mM NADPH. Oxidation of oxyhemoglobin was measured for 2 min at 37°C by the change in absorbance at 401 versus 411 nm registered with a Lambda 2 spectrophotometer. The results are expressed as nanomoles of NO per milligram of protein per minute. Proteins were quantified in homogenized vascular cells as described previously (Lowry et al., 1951).

Statistical analysis. Data were expressed as the mean ± SEM of the values from the number of experiments performed in triplicate as indicated in the corresponding figures. Data were evaluated statistically by using the Student's t test. For multiple comparisons, a one-way ANOVA test by the addition of the Kruskal-Wallis test was used. p < 0.05 was the minimum significance level.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Characterization of A fibrils and A-AChE complexes

In the present work, we used A_1-40 instead of A_1-42 because both CAA and HCHWA-D vascular deposits are mostly composed of the A_1-40 type (Castaño et al., 1996; Hamano et al., 1997). To avoid anomalous results is essential to characterize the amyloid nature of the A_Glu22Gln fibrils and A_Glu22Gln-AChE complexes, because it was performed when AChE was described as a fibrillogenic agent for A_wt1-40 (Inestrosa et al., 1996; Alvarez et al., 1998) and A_wt1-42 (Muñoz and Inestrosa, 1999). The following set of criteria were applied: turbidometry, electron microscopy, and Congo red staining. Turbidometric assays showed an increase of the maximal amount of aggregates formed in the presence of AChE on the A_Glu22Gln fibrillation (Fig. 1). The morphology of A_Glu22Gln fibrils was examined by electron microscopy, showing typical unbranched fibrils with no morphological differences regardless of the presence or absence of AChE (Fig. 2), as has been reported previously for A_wt and A_wt-AChE complexes (Inestrosa et al., 1996). The amyloid quality of the fibrils was further assessed by staining with Congo red, which produced birefringent Maltese crosses in both types of fibrils observed under polarized light (Fig. 2, insets). The presence of AChE in the fibrillar complexes was demonstrated by the hydrolysis of the specific substrate acetylthiocholine (Fig. 3A) and by SDS-PAGE in which AChE bands appeared in both types of complexes with A_wt and A_Glu22Gln (Fig. 3B). The amount of AChE bound to the fibrils differed depending on the type of peptides used, with A_wt binding more AChE than A_Glu22Gln (Fig. 3A, inset).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Aggregation assay of AGlu22Gln and AGlu22Gln with AChE followed by turbidometry at 405 nm. Data are from one representative experiment from the initial time at 0 up to 360 min.

View larger version (106K): [in this window] [in a new window]   Figure 2.   Electronic micrographs of negatively stained AGlu22Gln fibrils (A) and AGlu22Gln-AChE complexes (B). Scale bar, 0.15 µm. Insets are pictures of Maltese crosses obtained for both AGlu22Gln fibrils (A) and AGlu22Gln-AChE complexes (B) by staining with Congo red. Pictures were taken with a Nikon Optiphot microscope configured for polarized light. Scale bar, 15 µm.

View larger version (36K): [in this window] [in a new window]   Figure 3.   Presence of AChE in AGlu22Gln-AChE complexes. It was demonstrated by the hydrolysis of the substrate acetylthiocholine (A) following the method of Ellman et al. (1961). Briefly, aliquots of 3 µl of AGlu22Gln fibrils and AGlu22Gln-AChE complexes were incubated in triplicate with acetylthiocoline, and the reaction was stopped with tacrine. Absorbances were determined at 412 nm. In the inset in A, the amount of AChE bound to 10 µg of both amyloid fibril types is shown. The AChE bound to the complexes was calculated by densitometric scanning of the SDS-PAGE bands of AChE compared with known concentrations of AChE. Data are mean ± SEM (error bars) values of five to nine separate experiments. *p < 0.05 by nonpaired Student's t tests versus the respective values of Awt-AChE complexes. B shows an SDS-PAGE with samples from both types of fibrils and complexes used in the present work. The different lanes were occupied as follows: lane 1, purified Awt fibrils; lane 2, Awt-AChE complexes; lane 3, AGlu22Gln fibrils; and lane 4, AGlu22Gln-AChE complexes.

Cytotoxicity of A fibrils and A-AChE complexes on vascular cells

We used endothelial cells and VSMC because they degenerate in both CAA and HCHWA-D (Wisniewski et al., 1992; Wisniewski and Wegiel, 1994; Kalaria, 1997; Zhang et al., 1998). Cytotoxicity on vascular cells was evaluated at 6, 48, and 120 hr in the presence of 10 µM A_Glu22Gln fibrils (Fig. 4A). A series of experiments was also performed using different concentrations of A (Fig. 4B). The incubation time and fibril concentration for subsequent experiments were set at 48 hr and 10 µM A fibrils, because there were no significant differences between 48 and 120 hr of incubation and between 10 and 25 µM A fibrils. HUVEC cells (Fig. 4C) showed a higher sensitivity to the toxic action of the A_wt and A_Glu22Gln fibrils than A7r5 cells (Fig. 4E), and the highest cytotoxicity was produced by the Dutch A variant on both types of cells. The effect of A-AChE complexes depended on the type of A; thus, with A_wt, the complexes showed higher toxicity than with A_wt alone on both types of vascular cells, measured by MTT reduction (Fig. 4C,E). No significant differences were obtained in A7r5 cells with A_Glu22Gln fibrils and A_Glu22Gln-AChE complexes (Fig. 4E), whereas on HUVEC cells, A_Glu22Gln-AChE complexes resulted less toxic than fibrils alone (Fig. 4C). Controls run with AChE alone at a range of concentrations, including those bound to the fibrils, did not induce a loss of vitality in either type of cell (Fig. 4D).

View larger version (25K): [in this window] [in a new window]   Figure 4.   Cytotoxicity of A fibrils and A-AChE complexes on HUVEC (C) and A7r5 (E) cells are expressed as MTT reduction percentages obtained from the incubation with 10 µM fibrils alone (black bars) and complexes with AChE (white bars) for 48 hr. Data are mean ± SEM (error bars) values of five experiments performed in triplicate. *p < 0.05 by nonpaired Student's t test versus the respective experiments performed with fibrils without AChE. A, MTT reduction percentages obtained with 10 µM AGlu22Gln fibrils at different incubation times. Data are mean ± SEM (error bars) values of three experiments performed in triplicate. B, MTT reduction percentages obtained after 48 hr of treatment with increasing concentrations of AGlu22Gln fibrils. Data are mean ± SEM (error bars) values of five to eight experiments performed in triplicate. D, MTT reduction percentages obtained by incubating A7r5 and HUVEC cells for 48 hr with increasing concentrations of AChE alone in the range that is bound to the fibrils. Data are mean ± SEM (error bars) values of three experiments performed in triplicate.

A and oxidative stress

Oxidative stress has been proposed as one of the possible mechanisms of A-mediated toxicity in both neuronal (Calderón et al., 1999) and endothelial (Thomas et al., 1996) cells. The different sensitivity of A7r5 and HUVEC cells to the amyloid peptides might indicate differences in the antioxidant cellular protection, a hypothesis also proposed for several neuronal cell lines (Calderón et al., 1999). To investigate whether A-mediated toxicity involves the production of free radicals and subsequent apoptosis in vascular cells, we performed experiments to determine the O₂· production and the presence of phosphatydylserine in the outer leaflet of the plasma membrane, a specific marker for apoptosis, on HUVEC and A7r5 cells challenged with A. The annexin-V (apoptosis marker) versus dihidroethydium (O₂· production) dot plots of Figure 5 show that A incubation induced a marked increase in O₂·-positive HUVEC cells (Fig. 5A) but not in A7r5 cells (Fig. 5B). However, both cell types showed a significant increase in the number of cells positive to annexin-V, demonstrating that the A-induced cytotoxicity on vascular cells is mediated by apoptosis. The effect of H₂O₂, a well known prooxidant, was also evaluated on HUVEC cells (Fig. 5C). The dot plots obtained with H₂O₂ were similar to those obtained with A (Fig. 5A), reinforcing the hypothesis that oxidative stress is directly involved in A-mediated vascular cytotoxicity.

View larger version (35K): [in this window] [in a new window]   Figure 5.   Oxidative stress and A-mediated vascular cytotoxicity. Phosphatydylserine translocation to the outer membrane and O2· production on HUVEC cells (A) and A7r5 cells (B) exposed to 10 µM Awt fibrils and HUVEC cells (C) with 50 µM H2O2 at 48 hr. Percentages indicate cells positive for annexin-V binding (top quadrants) and O2· production (right quadrants) in regard to the controls (bottom left quadrant). Activity of SOD (D) and NOS (E) in A7r5 and HUVEC cells. Results are expressed in relation to milligrams of protein. Data are mean ± SEM (error bars) values of seven to eight cell samples analyzed in triplicate. *p < 0.05 by nonpaired Student's t test versus the respective results on A7r5 cells.

The different O₂· production between HUVEC and A7r5 cells in response to A might be related to a differential balance of the prooxidant and antioxidant systems on each cell type. Therefore, we studied the SOD activity, the enzyme responsible to eliminate O₂· on vascular cells, and NOS activity. The interest in studying NOS activity lies on the observation that superoxide radicals can interact with NO to produce the highly prooxidant peroxynitrite radical (Beckman, 1996). HUVEC cells showed a lower SOD activity (twofold lower than that of A7r5 cells) (Fig. 5D). On the other hand, NO generation was fourfold higher in HUVEC cells than in A7r5 cells (Fig. 5E), as expected for endothelial cells (Moncada et al., 1988). Both results are consistent with a lower antioxidant protection of HUVEC cells compared with A7r5 cells.

The role of vitamin E, vitamin C, and E₂ on A-mediated vascular cytotoxicity

The protective role of different antioxidants on HUVEC cells challenged with A_wt fibrils was studied by measuring apoptosis by the annexin-V method (Fig. 6A). The percentage of annexin-V-positive cells doubled in the presence of A. Preincubation of HUVEC cells with vit E reduced significantly the percentage of annexin-V-positive cells, whereas preincubation with E₂ showed no statistically significant reduction of annexin-V staining. Similarly, vit E, but not E₂, attenuated significantly the A-mediated cytotoxicity for both wild type and Dutch variant on HUVEC cells measured by MTT reduction (Figs. 6B, 7A). The lack of protection by E₂ against A-mediated cytotoxicity was also confirmed by measuring the LDH release on both types of cells (Fig. 7B). LDH release in response to A was similar in the presence or absence of E₂. The other classic vitamin with antioxidant activity, vit C, at 100 µM did not show any protective effect on vascular cells in response to the insult with A fibrils (Fig. 6B). Higher concentrations of vit C were discarded because preliminary experiments at 1 mM produced a significant decrease in the viability of the vascular cells (data not shown). The protective effect of the three antioxidants against A was also tested on A7r5 cells (Figs. 6B, 7). As described previously, these cells were more resistant to A toxicity than HUVEC cells. Consequently, no major differences in the A toxicity were identified in the presence of the antioxidants.

View larger version (27K): [in this window] [in a new window]   Figure 6.   A-mediated vascular cytotoxicity and protection by vit E. A, Annexin-V-positive HUVEC cells obtained after preincubating 1 mM vit E and 10 µM E2 for 2 hr before adding 10 µM Awt. Cells were incubated with the fibrils for 48 hr. Cell controls (18 ± 4% annexin-V-positive cells) were assumed as 100%. Data are mean ± SEM (error bars) values of four experiments performed in duplicate. *p < 0.05 by a one-way ANOVA test by the addition of the Kruskal-Wallis test to compare the respective treatments versus fibrils alone. B, MTT reduction percentages obtained by preincubating HUVEC and A7r5 cells with 1 mM vit E and 100 µM vit C for 2 hr before adding 10 µM Awt or AGlu22Gln fibrils. Cells were incubated with the fibrils for 48 hr. Data are mean ± SEM (error bars) values of four to six experiments performed in triplicate. *p < 0.05 by a one-way ANOVA test by the addition of the Kruskal-Wallis test to compare the respective treatments versus fibrils alone. C, H2O2 MTT reduction percentages obtained by preincubating vascular cells for 2 hr with 1 mM vit E, 100 µM vit C, and 10 µM E2. Cells were challenged for 48 hr with 100 µM (A7r5 cells) and 50 µM (HUVEC cells) H2O2. Data are mean ± SEM (error bars) values of three to five experiments performed in triplicate. *p < 0.05 by a one-way ANOVA test by the addition of the Kruskal-Wallis test to compare the respective treatments versus H2O2 alone.

View larger version (36K): [in this window] [in a new window]   Figure 7.   Effect of E2 on the cytotoxicity induced by Awt and AGlu22Gln fibrils on vascular cells. A, MTT reduction percentages obtained by preincubating HUVEC and A7r5 cells with 0.1 and 10 µM E2 for 2 hr before adding 10 µM Awt or AGlu22Gln fibrils. Cells were incubated with fibrils for 48 hr. Data are mean ± SEM (error bars) values of 7-14 experiments performed in triplicate. B, LDH release obtained by preincubating HUVEC and A7r5 cells with 0.1 and 10 µM E2 for 2 hr before adding 10 µM Awt or AGlu22Gln fibrils. Cells were incubated with fibrils for 48 hr. Results are expressed as percentages with respect to the total LDH assumed as 100%. Data are mean ± SEM (error bars) values of three to five experiments performed in triplicate.

A similar pattern of protection with the three antioxidants was obtained when both cell types were challenged with H₂O₂ instead of A (Fig. 6C). First, HUVEC cells were more sensitive to the oxidative stress than A7r5 cells (Fig. 6C), even when the concentration of H₂O₂ used for the experiments performed on HUVEC cells (50 µM) was lower than that used for the experiments performed on A7r5 cells (100 µM). Second, as shown with the A challenge, only vit E was able to attenuate the H₂O₂-mediated oxidative insult by increasing the cell viability on both types of cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CAA is a pathology frequently linked to Alzheimer's disease (Vinters et al., 1988). The amyloid vascular deposits from CAA are similar to the brain senile plaques, including the presence of several molecules such as AChE (Mesulam et al., 1992), which has been reported to increase the neurotoxicity of A_wt fibrils (Muñoz and Inestrosa, 1999). In addition, CAA is the key pathological feature of patients suffering from HCHWA-D, although in this case, the angiopathy is more extensive and develops at early ages, without the presence of mature senile plaques in the cerebral parenchyma (van Duinen et al., 1987; Maat-Schieman et al., 1992). The pathophysiology underlying the A-mediated vascular damage is not fully understood, but oxidative stress could play a key role as in the case of the A-mediated neuronal degeneration (Behl, 1997; Miranda et al., 2000).

In the present study, we determined the cytotoxic effect of A_wt and A_Glu22Gln fibrils on vascular cells and the modulation of the cytotoxicity by AChE. Furthermore, we evaluated whether well known antioxidants such as vit E, vit C, and E₂ might reverse the vascular toxicity conferred by A fibrils.

Amyloid fibrils and cytotoxicity

Our results show that A_wt and A_Glu22Gln fibrils induce toxicity on vascular cells with characteristics similar to that reported on neuronal cells (Bonnefont et al., 1998). A_Glu22Gln fibrils were more toxic than those of A_wt on vascular cells. These findings suggest a key role for fibril stability on the cytotoxicity, because previous studies have shown that the Gln mutation stabilizes the fibrils and hence increases the fibrillogenic nature of this A type (Wisniewski et al., 1991; Fraser et al., 1992), despite the absence of any major folding difference between the wild-type and the mutated peptide (George and Howlett, 1999). Therefore, a plausible explanation could be related to an increased attachment of A_Glu22Gln to the extracellular matrix of vascular cells, which has demonstrated the ability to bind and assemble amyloid, hence enhancing its toxicity (Watson et al., 1997; van Nostrand et al., 1998).

A-AChE complexes

We found that the AChE-A complexes formed with A_wt are significantly more toxic than A_wt fibrils alone for both target cells, consistent with previous reports on neuronal cells (Alvarez et al., 1998; Muñoz and Inestrosa, 1999). These results might be related to the enhancement of the A_wt fibril stability by AChE (Inestrosa et al., 1996). Those findings suggest that the AChE bound to the amyloid vascular deposits could act as an enhancer of the vascular degeneration observed in CAA, because AChE has been reported to be present in amyloid vascular deposits (Mesulam et al., 1992).

On the other hand, A_Glu22Gln-AChE complexes showed no differences on VSMC compared with the A_Glu22Gln fibrils alone, and endothelial cells showed a significant increase in cell survival. The lack of morphological differences observed for either kind of fibril by electron microscopy analysis suggests that the differences could be in the fibril stability, A_Glu22Gln-AChE complexes being less stable than A_Glu22Gln fibrils. This might be the result of an anomalous pattern in the association between A_Glu22Gln and AChE compared with other amyloid peptides (Inestrosa et al., 1996; Alvarez et al., 1997; Muñoz et al., 1999). Our data also showed a lower binding of AChE to A_Glu22Gln, an observation that it might be related to the fast fibrillation of A_Glu22Gln, resulting in fewer molecules of AChE being bound or to a decrease in the binding affinity attributable to the changes in the A sequence. The pathophysiological relevance of A_Glu22Gln-AChE complexes is unknown at present because there is no evidence for the presence of AChE in HCHWA-D vascular deposits.

Free radicals and A-mediated cytotoxicity

Several studies have reported a release of O₂· in response to A in vascular cells (Thomas et al., 1996; Suo et al., 1997), linking the toxicity of A to oxidative stress, as was observed on HUVEC cells. We found that endothelial cells were more sensitive to H₂O₂ and A fibrils than VSMC, an observation that correlates with the higher rate of O₂· production on HUVEC cells. Furthermore, endothelial cells showed low levels of SOD activity, which could render these cells more vulnerable to oxidative insults and, therefore, to the action of A. This observation is in agreement with previous studies reporting that exogenous addition of SOD reverses the effect of A in endothelial cells (Crawford et al., 1997; Thomas et al., 1997; Price et al., 1997). NO production was also higher in endothelial cells than in VSMC. NO can react with the prooxidant O₂· to form peroxynitrite (Beckman, 1996), which is considered a powerful oxidant in AD-associated brain damage (Smith et al., 1997). All of these results point to a correlation between oxidative stress and the cytotoxicity ratio. We hypothesize that endothelium is more sensitive to A-mediated apoptosis attributable to the lower intracellular antioxidant activity and the high production of O₂· and NO.

Protection with antioxidants

Both vit E and vit C have antioxidant properties. In the case of the former, it seems to be associated with the prevention of lipid peroxidation (Halliwell and Gutteridge, 1984) by trapping the peroxyl radicals (Naiki et al., 1998). In the case of vit C, its involvement as a protective antioxidant agent is controversial. On one hand, its physiological role appears to be mainly directed to restore the antioxidant properties of vit E (Tappel, 1968), whereas at high concentrations it acts as a prooxidant (Halliwell, 1999).

As discussed above, the oxidative stress hypothesis is well suited to explain the toxic effect of -amyloid in both neuronal cells (Miranda et al., 2000) and vascular cells (this study). Additional support for the oxidative stress hypothesis was provided by several studies showing the neuroprotective effects of different antioxidants, such as vit E (Behl et al., 1992), vit C (Yallampalli et al., 1998), E₂ (Goodman et al., 1996), melatonin (Pappolla et al., 1997), and lazaroids (Behl et al., 1997b), against -amyloid-induced toxicity. Biological effects found for many of these antioxidants in laboratory experiments have also been matched by epidemiological studies reporting the beneficial effects of antioxidants (Tang et al., 1996; Sano et al., 1997).

Our data show that vit E protects endothelial cells against A challenge, whereas vit C failed to inhibit the A- and H₂O₂-mediated cytotoxicity. In the case of vit C, reports showing a lack of neuroprotection have also appeared (Lockhart et al., 1994).

The protective effect of E₂ against A has been shown previously on neuronal cells (Goodman et al., 1996; Behl et al., 1997a; Bonnefont et al., 1998). However, E₂ was unable to reverse the toxicity of A or H₂O₂ in vascular cells (this study), despite its antioxidant properties (Sugioka et al., 1987). These findings do not rule out that E₂, via one or more of its different mechanisms of action (Nadal et al., 2001), plays a protective role in AD. E₂ exerts a wide range of positive effects on both the CNS (Honjo et al., 1992; Bonnefont et al., 1998; Inestrosa et al., 1998; Toran-Allerand, 2000) and the vascular system (Ruehlmann et al., 1998; Mendelsohn and Karas 1999; Valverde et al., 1999). All or part of these effects of E₂ may be related to the protective role on neuronal cells challenged with A (Goodman et al., 1996; Behl et al., 1997a; Bonnefont et al., 1998). However, they appear to be ineffective at the vascular level for amyloid-associated pathology.

In summary, A_Glu22Gln fibrils are more toxic for vascular cells than A_wt wild-type fibrils, suggesting that the early onset of HCWA-D is related to the high toxicity rate induced by A_Glu22Gln fibrils. Additional work is needed to verify that A_Glu22Gln toxicity is related to the stability of the fibrils. The presence of AChE in the A_wt fibrils could also be a risk factor for CAA attributable to the enhancer effect of the amyloid toxicity by the enzyme. Finally, we postulate that one of the mechanisms of action of A fibrils is the generation of oxidative stress on vascular cells and that the high sensitivity of endothelial cells to A fibrils is related to their lower protection against oxidative stress (low levels of SOD and high levels of NOS activity), a hypothesis that receives additional support from the observation that the antioxidant vit E reverses the A-mediated cytotoxicity.

	FOOTNOTES

Received Oct. 22, 2001; revised Feb. 4, 2002; accepted Feb. 5, 2002.

This work was supported by grants from Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) (Ministerio de Educación, Chile Grant 3980024) and Fondo de Investigación Sanitaria (Ministerio de Sanidad, Spain Grant 01-1029) to F.J.M.; Dirección General de Investigación Científica y Técnica (Ministerio de Ciencia y Tecnología, Spain Grant BIO99-0508) to G.G.-G.; Human Frontier Science Program and Distinció de la Generalitat de Cataluña (Spain) to M.A.V.; FONDECYT Grant 2990087 to C.O.; and Fondo de Investigación Avanzada en Áreas Prioritarias-Biomedicina Grant 1389001, Millenium Institute for Fundamental and Applied Biology Grant P-99-007-F, and the Presidential Chair in Science from the Chilean Government to N.C.I. We acknowledge Aoife Currid for proofreading this manuscript.

Correspondence should be addressed to Dr. Francisco J. Muñoz, Unitat de Senyalització Cel·lular, Universitat Pompeu Fabra, Calle Dr. Aiguader, 80, Barcelona 08003, Spain. E-mail: paco.munoz{at}cexs.upf.es.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

• Alvarez A, Opazo C, Alarcón R, Garrido J, Inestrosa NC (1997) Acetylcholinesterase promotes the aggregation of amyloid-beta-peptide fragments by forming a complex with the growing fibrils. J Mol Biol 272:348-361[ISI][Medline].
• Alvarez A, Alarcón R, Opazo C, Campos EO, Muñoz FJ, Calderón FH, Dajas F, Gentry MK, Doctor BP, De Mello FG, Inestrosa NC (1998) Stable complexes involving acetylcholinesterase and amyloid-beta peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer's fibrils. J Neurosci 18:3213-3223[Abstract/Free Full Text].
• Beckman JS (1996) Oxidative damage and tyrosine nitration from peroxynitrite. Chem Res Toxicol 9:836-844[ISI][Medline].
• Behl C (1997) Amyloid beta-protein toxicity and oxidative stress in Alzheimer's disease. Cell Tissue Res 290:471-480[ISI][Medline].
• Behl C, Davis JB, Cole GM, Schubert D (1992) Vitamin E protects nerve cells from amyloid  protein toxicity. Biochem Biophys Res Commun 186:944-950[ISI][Medline].
• Behl C, Davis JB, Lesley R, Schubert D (1994) Hydrogen peroxide mediates amyloid  protein toxicity. Cell 77:817-827[ISI][Medline].
• Behl C, Skutella T, Lezoualc'h F, Post A, Widmann M, Newton CJ, Holsboer F (1997a) Neuroprotection against oxidative stress by estrogens: structure-activity relationship. Mol Pharmacol 51:535-541[Abstract/Free Full Text].
• Behl C, Trapp T, Skutella T, Holsboer F (1997b) Protection against oxidative stress-induced neuronal cell death-a novel role for RU486. Eur J Neurosci 9:912-920[ISI][Medline].
• Bonnefont AB, Muñoz FJ, Inestrosa NC (1998) Estrogen protects neuronal cells from the cytotoxicity induced by acetylcholinesterase-amyloid complexes. FEBS Lett 441:220-224[ISI][Medline].
• Calderón FH, Bonnefont A, Muñoz FJ, Fernández V, Videla LA, Inestrosa NC (1999) PC12 and Neuro 2a cells have different susceptibilities to acetylcholinesterase-amyloid complexes amyloid_25-35 fragment glutamate hydrogen peroxide. J Neurosci Res 56:620-631[ISI][Medline].
• Castaño EM, Prelli F, Soto C, Beavis R, Matsubara E, Shoji M, Frangione B (1996) The length of amyloid-beta in hereditary cerebral hemorrhage with amyloidosis Dutch type. Implications for the role of amyloid-beta 1-42 in Alzheimer's disease. J Biol Chem 271:32185-32191[Abstract/Free Full Text].
• Crawford F, Suo Z, Fang C, Sawar A, Su G, Arendash G, Mullan M (1997) The vasoactivity of A beta peptides. Ann NY Acad Sci 826:35-46[Abstract/Free Full Text].
• Ellman GL, Courtney KD, Andres V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88-95[ISI][Medline].
• Fraser PE, Nguyen JT, Inouye H, Surewicz WK, Selkoe DJ, Podlisny MB, Kirschner DA (1992) Fibril formation by primate rodent Dutch-haemorrhagic analogues of Alzheimer amyloid beta-protein. Biochemistry 31:10716-10723[Medline].
• Fridovich I (1985) In: CRC Handbook of methods for oxygen radical research (Greenwald RA, ed), pp 213-220. Boca Raton, FL: CRC.
• George AR, Howlett DR (1999) Computationally derived structural models of the beta-amyloid found in Alzheimer's disease plaques and the interaction with possible aggregation inhibitors. Biopolymers 50:733-741[ISI][Medline].
• Goodman Y, Bruce AJ, Cheng B, Mattson MP (1996) Estrogens attenuate and corticosterone exacerbates excitotoxicity oxidative injury amyloid -peptide toxicity in hippocampal neurons. J Neurochem 66:1836-1844[ISI][Medline].
• Halliwell B (1999) Vitamin C: poison, prophylactic or panacea? Trends Biochem Sci 24:255-259[ISI][Medline].
• Halliwell B, Gutteridge JM (1984) Lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy. Lancet 1:1396-1397[ISI][Medline].
• Hamano T, Yoshimura M, Yamazaki T, Shinkai Y, Yanagisawa K, Kuriyama M, Ihara Y (1997) Amyloid -protein (A) accumulation in the leptomeninges during aging and in Alzheimer disease. J Neuropathol Exp Neurol 56:922-932[Medline].
• Honjo H, Tamura T, Matsumoto Y, Kawata M, Ogino Y, Tanaka K, Yamamoto T, Ueda S, Okada H (1992) Estrogen as a growth factor to central nervous cells. Estrogen treatment promotes development of acetylcholinesterase-positive basal forebrain neurons transplanted in the anterior eye chamber. J Steroid Biochem Mol Biol 41:633-635[ISI][Medline].
• Inestrosa NC, Roberts WL, Marshall TL, Rosenberry TL (1987) Acetylcholinesterase from bovine caudate nucleus is attached to membranes by a novel subunit distinct from those of acetylcholinesterases in other tissues. J Biol Chem 262:4441-4444[Abstract/Free Full Text].
• Inestrosa NC, Alvarez A, Perez CA, Moreno RD, Vicente M, Linker C, Casanueva O, Soto C, Garrido J (1996) Acetylcholinesterase accelerates assembly of amyloid-beta-peptides into Alzheimer's fibrils: possible role of the peripheral site of the enzyme. Neuron 16:881-891[ISI][Medline].
• Inestrosa NC, Marzolo MP, Bonnefont AB (1998) Cellular and molecular basis of estrogen's neuroprotection. Potential relevance for Alzheimer's disease. Mol Neurobiol 17:73-86[ISI][Medline].
• Kalaria RN (1997) Cerebrovascular degeneration is related to amyloid-beta protein deposition in Alzheimer's disease. Ann NY Acad Sci 826:263-271[Abstract/Free Full Text].
• Kawas C, Resnick S, Morrison A, Brookmeyer R, Corrada M, Zonderman A, Bacal C, Lingle DD, Metter E (1997) A prospective study of estrogen replacement therapy and the risk of developing Alzheimer's disease: the Baltimore Longitudinal Study of Aging. Neurology 48:517-521.
• Knowles RG, Merrett M, Salter M, Moncada S (1990) Differential induction of brain lung and liver nitric oxide synthase by endotoxin in the rat. Biochem J 270:833-836[ISI][Medline].
• Levy E, Carman MD, Fernandez-Madrid IJ, Power MD, Lieberburg I, van Duinen SG, Bots GT, Luyendijk W, Frangione B (1990) Mutation of the Alzheimer's disease amyloid gene in hereditary cerebral hemorrhage Dutch type. Science 248:1124-1126[Abstract/Free Full Text].
• Lockhart BP, Benicourt C, Junien JL, Privat A (1994) Inhibitors of free radical formation fail to attenuate direct beta-amyloid25-35 peptide-mediated neurotoxicity in rat hippocampal cultures. J Neurosci Res 39:494-505[Medline].
• Lowry OH, Rosebrough NJ, Farr L, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:267-275.
• Maat-Schieman ML, van Duinen SG, Haan J, Roos RA (1992) Morphology of cerebral plaque-like lesions in hereditary cerebral hemorrhage with amyloidosis (Dutch). Acta Neuropathol (Berl) 84:674-679[Medline].
• Mendelsohn ME, Karas RH (1999) The protective effects of estrogen on the cardiovascular system. N Engl J Med 340:1801-811[Free Full Text].
• Mesulam M-M, Carson K, Price B, Geula C (1992) Cholinesterases in the amyloid angiopathy of Alzheimer's disease. Ann Neurol 31:565-569[Medline].
• Miranda S, Opazo C, Larrondo LF, Muñoz FJ, Ruiz F, Leighton F, Inestrosa NC (2000) The role of oxidative stress in the toxicity induced by amyloid beta-peptide in Alzheimer's disease. Prog Neurobiol 62:633-648[ISI][Medline].
• Moncada S, Radomski MW, Palmer RM (1988) Endothelium-derived relaxing factor. Identification as nitric oxide and role in the control of vascular tone and platelet function. Biochem Pharmacol 37:2495-2501[ISI][Medline].
• Morris MC, Beckett LA, Scherr PA, Hebert LE, Bennett DA, Field TS, Evans DA (1998) Vitamin E and vitamin C supplement use and risk of incident Alzheimer disease. Alzheimer Dis Assoc Disord 12:121-126[ISI][Medline].
• Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55-63[ISI][Medline].
• Muñoz FJ, Inestrosa NC (1999) Neurotoxicity of acetylcholinesterase-amyloid--peptide aggregates is dependent on the type of A-peptide and the AChE concentration present in the complexes. FEBS Lett 450:205-209[ISI][Medline].
• Nadal A, Díaz M, Valverde MA (2001) The estrogen trinity: membrane, cytosolic and nuclear effects. News Physiol Sci 16:251-255[Abstract/Free Full Text].
• Naiki H, Hasegawa K, Yamaguchi I, Nakamura H, Gejyo F, Nakakuki K (1998) Apolipoprotein E and antioxidants have different mechanism of inhibiting Alzheimer's -amyloid fibril formation in vitro. Biochemistry 37:17882-17889[Medline].
• Pappolla MA, Sos M, Omar RA, Bick RJ, Hickson-Bick DL, Reiter RJ, Efthimiopoulos S, Robakis NK (1997) Melatonin prevents death of neuroblastoma cells exposed to the Alzheimer amyloid peptide. J Neurosci 17:1683-1690[Abstract/Free Full Text].
• Price