Wild-type amyloid beta 1-40 peptide induces vascular smooth muscle cell death independently from matrix metalloprotease activity

Wild-type amyloid beta 1-40 peptide induces vascular smoothmuscle cell death independently from matrixmetalloprotease activity

Regis Blaise,1 Veronique Mateo,1* Clotilde Rouxel,1

Francois Zaccarini,1 Martine Glorian,1 Gilbert Bereziat,1

Vladislav S. Golubkov2 and Isabelle Limon1

1University Paris 6, UR4, Vieillissement, Stress et Inflammation 7 quai

Saint-Bernard, 75252 Paris, France2Cancer Research Center, Sanford-Burnham Medical Research Institute,

La Jolla, CA 92037, USA

Summary

Cerebral amyloid angiopathy (CAA) is an important cause of

intracerebral hemorrhages in the elderly, characterized by amy-

loid-b (Ab) peptide accumulating in central nervous system

blood vessels. Within the vessel walls, Ab-peptide deposits

[composed mainly of wild-type (WT) Ab1-40 peptide in sporadic

forms] induce impaired adhesion of vascular smooth muscle

cells (VSMCs) to the extracellular matrix (ECM) associated with

their degeneration. This process often results in a loss of blood

vessel wall integrity and ultimately translates into cerebral

ischemia and microhemorrhages, both clinical features of CAA.

In this study, we decipher the molecular mechanism of matrix

metalloprotease (MMP)-2 activation in WT-Ab1-40-treated VSMC

and provide evidence that MMP activity, although playing a

critical role in cell detachment disrupting ECM components, is

not involved in the WT-Ab1-40-induced degeneration of VSMCs.

Indeed, whereas this peptide clearly induced VSMC apoptosis,

neither preventing MMP-2 activity nor hampering the expres-

sion of membrane type1-MMP, or preventing tissue inhibitors

of MMPs-2 (TIMP-2) recruitment (two proteins evidenced here

as involved in MMP-2 activation), reduced the number of dead

cells. Even the use of broad-range MMP inhibitors (GM6001

and Batimastat) did not affect WT-Ab1-40-induced cell apoptosis.

Our results, in contrast to those obtained using the Ab1-40

Dutch variant suggesting a link between MMP-2 activity, VSMC

mortality and degradation of specific matrix components, indi-

cate that the ontogenesis of the Dutch familial and sporadic

forms of CAAs is different. ECM degradation and VSMC degen-

eration would be tightly connected in the Dutch familial form

while being two independent processes in sporadic forms of

CAA.

Key words: amyloid beta peptide; cell death; matrix metal-

loproteases; vascular smooth muscle cell.

Introduction

Cerebral amyloid angiopathy (CAA) refers to sporadic and hereditary

cerebrovascular disorders frequently associated with cognitive impair-

ment in the elderly, including Alzheimer’s disease. From a histopathologi-

cal point of view, it is characterized by the amyloid accumulation in the

media and adventitia of small and large arteries irrigating the central ner-

vous system and ⁄ or amyloid deposition of Ab peptides around the capil-

laries perfusing the cerebellum, cerebral cortex and leptomeninges

(Smith & Greenberg, 2009). The Ab-peptide accumulation within arteries

induces vascular smooth muscle cell (VSMC) death (referred to, in the lit-

erature, as VSMC degeneration) and results in a loss of blood vessel wall

integrity. This possibly translates into cerebral ischemia and microhemor-

rhages, both clinical features of CAA (Knudsen et al., 2001; Maia et al.,

2007). The Ab-induced VSMC degeneration is associated with impaired

VSMC adhesion to the extracellular matrix (ECM) because of elevated

pericellular proteolysis of the ECM components (Maruyama et al., 1990;

Kawai et al., 1993; Mok et al., 2006).

The human matrix metalloprotease (MMP) family encoded by 24 genes

is composed of Zn2+-dependent proteases known to degrade a large vari-

ety of ECM components and a number of bioactive molecules at the prox-

imity of the cell surface. Among all the MMPs, six members are anchored

to the cell membrane (membrane-type MMPs, MT-MMPs), whereas the

other members are soluble and secreted into the extracellular space. Of

note, there is increasing evidence that soluble MMPs (such as MMP-2) are

recruited to the local cell environment by interacting with cell surface pro-

teins (including MT-MMPs) and via the pericellular matrix (Murphy &

Nagase, 2011). MMP activity is regulated at transcriptional and post-

translational levels. All MMPs are synthesized as inactive zymogens. The

cysteine switch containing N-terminal propeptide of the latent pro-

enzyme interacts with the Zn2+ at the active site, blocking proteolytic

activity (Van Wart & Birkedal-Hansen, 1990). The first post-translational

modification cleaves the propeptide allowing the latent proenzyme to

become activated, enabling proteolysis of its substrate molecules. The

second one involves the broad-spectrum proteinase inhibitor b2-macro-

globulin and four specific inhibitors named tissue inhibitors of MMPs

(TIMPs-1 to 4, Visse & Nagase, 2003). A 1:1 stoichiometric interaction of

MMPs with TIMPs inhibits the enzyme activity, whereas unbalancing this

ratio in favor of the proteases results in increasing MMP activity.

Ab-peptides are produced by proteolytic processing of the amyloid pro-

tein precursor (APP) by b- and c-secretases. Amyloid deposits within the

vessel walls are mainly composed of wild-type (WT) in sporadic CAAs or

mutated forms in familial CAAs of the 40 amino acids Ab-peptide species

(Ab1-40) (Alonzo et al., 1998). Previous studies have demonstrated that a

mutated form, the Dutch mutant E22Q Ab1-40 (Ab1-40D) involved in a rare

but severe hereditary CAA (Levy et al., 1990), triggers the expression and

activation of MMP-2 in human smooth muscle cells (HSMC, Davis & Van

Nostrand, 1996; Jung et al., 2003); they also suggest that it may contrib-

ute to the Ab1-40D-induced HSMC death. However, the pathogenicity of

WT Ab1-40 (WT-Ab1-40) and the possible role of MMPs in sporadic CAAs

(which represent more than 90% of CAAs) have not been defined. In this

study, we demonstrate that WT-Ab1-40 induces both VSMC apoptosis

Correspondence

Isabelle Limon, University Paris 6, UR4, Vieillissement, Stress et Inflammation 7

quai Saint-Bernard, Bat A 5eme etage, 75252 Paris, France.

Tel.: +33 144 273 716; fax: +33 144 274 140; e-mail: isabelle.limon@snv.

jussieu.fr

*Present address: Paris 6 Pierre et Marie Curie University, CNRS UMR 7211 and

CNRS U959, 75651 Paris, France.

Accepted for publication 23 December 2011

384 ª 2012 The AuthorsAging Cell ª 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland

Aging Cell (2012) 11, pp384–393 Doi: 10.1111/j.1474-9726.2012.00797.xAg

ing

Cell

and the MT1-MMP-dependent MMP-2 activation in vitro and decipher

the molecular mechanism of this activation. We also prove that there is

no cause-to-effect relationship between WT-Ab1-40-induced cell death

and MMP activity suggesting that MMP proteolytic activity is not the pri-

mary cause of VSMC apoptosis observed in sporadic CAA.

Results

WT-Ab1-40 decreases VSM cells viability

Comparing cell morphology of WT-Ab1-40 with that of inverted Ab1-40-

treated VSMC revealed modifications of their adhesion to the ECM evi-

dent by cell shape alteration (Fig. 1A). Hoechst nuclei labeling followed

by immunofluorescence microscopy evidenced pycnotic nuclei, character-

istic of cells undergoing apoptosis. Consistently, the percentage of hypo-

diploid nuclei [measured by propidium iodide flow cytometric assay

(Mateo et al., 2007)] was more than 70% after a 72 h-treatment with

WT-Ab1-40 peptide; inverted Ab40-1 peptide has no effect on spontaneous

cell death (Fig. 1B). Apoptosis induced by WT-Ab1-40 was also measured

by evaluating phosphatidylserine (PS) exposure in the cytoplasmic outer

leaflet membrane using APC-conjugated Annexin-V in combination with

7-amino-actinomycin D exclusion dye (data not shown).

WT-Ab1-40 induces MT1-MMP-dependent MMP-2 activation

MT1-MMP and MMP-2 activity are known to be essential for pericellular

proteolysis (Itoh & Seiki, 2006), whereas MMP-9 is rather involved in

inflammatory diseases, including peripheral arterial diseases (Busti et al.,

2010). MMP-2 and MMP-9 are the predominant soluble MMPs in VSMCs

(Newby, 2006). As both processes, pericellular proteolysis and inflamma-

tion, are the major pathogenic features in CAA, we next studied the

expression and activity of these MMPs in WT-Ab1-40-treated VSMCs. As

shown in Fig. 2A, left panel, MMP-2 mRNA level (estimated by quantita-

tive RT–PCR) was more than 6-fold increased (P < 0.001) in cells treated

with WT-Ab1-40, when compared to cells incubated with vehicle or the

inverted Ab40-1 control peptide. MMP-9 mRNA was undetectable either

in control, inverted Ab40-1 or WT-Ab1-40-treated cells (Fig. 2A, right

panel), while IL-1b induced the expression of MMP-9 transcripts in agree-

ment with earlier reports (Delbosc et al., 2008). The active form of the

MMP-2 protein (referred to as Act. MMP-2, Fig. 2B) could only be

detected in WT-Ab1-40-treated VSMCs. In vehicle or inverted Ab40-1-trea-

ted cells, the observed bands corresponded to nonmature forms of the

enzyme (Fig. 2B). Of note, the lower unidentified band (*) was always

present independently of the MMP-2 antibody used. Accordingly, MMP-

2 activity (revealed by a 68-kDa zymographic band referred to as Act.

MMP-2 in Fig. 2C) was only detectable in media conditioned by cells trea-

ted with WT-Ab1-40. The identity of the zymographic bands was con-

trolled by immunoprecipitation using the anti-MMP-2 antibody and naıve

Ig on conditioned media derived from rat lung incubated in serum-free

medium. The active form of MMP-2 was identified as the lowest band by

(aminomethyl) phosphinic acid (AMPA)-induced activation of a control

sample prior to zymography (data not shown). According to the level of

MMP-9 transcripts, WT-Ab1-40 did not trigger MMP-9 activity, although

IL-1b treatment significantly increased it.

We also demonstrated an increase of MT1-MMP expression in WT-Ab1-

40-treated cells at both mRNA and protein levels by quantitative RT–PCR

(Fig. 3A, left panel) and Western blot (Fig. 3A, right panel). Because

MMP-2 activation is dependent on MT1-MMP activity (Strongin et al.,

1995), we next examined whether WT-Ab1-40-induced MT1-MMP

expression is responsible for MMP-2 activation. To do so, VSMCs were

transfected with a plasmid encoding the catalytically inert E240A mutant

of MT1-MMP (Fig. 3B). MMP-2 status was analyzed by gelatin zymogra-

phy. The overexpression of MT1-E240A protein was confirmed by Wes-

tern blot (Fig. 3B, right panel). As expected, the forced expression of

MT1-MMP E240A completely blocked the WT-Ab1-40-induced MMP-2

activity (Fig. 3B, left panel). MMP-2 mRNA expression was not affected

by the expression of MT1-E240A constructs whether with or without WT-

Ab1-40 treatment (data not shown). Altogether, these data highlighted a

cause-to-effect relationship between the WT-Ab1-40 induction of MT1-

MMP expression and MMP-2 activity.

(A)

(B)

Fig. 1 Wild-type (WT)-Ab1-40 peptide induces vascular smooth muscle cells

apoptosis. Serum-starved cells were treated for 48 h with WT-Ab1-40 (50 lM),

inverted Ab40-1 (50 lM) or vehicle (control). (A) Cell morphology was evaluated by

phase contrast microscopy, and nuclei were stained with Hoechst (blue). Images are

representative of three independent experiments. (B) Apoptosis was determined as

described in Materials and methods section by propidium iodide staining. Data

represent means ± SD of four independent experiments performed in triplicate;

n.s, not significant and ***P < 0.001 compared with control (Ctl).

MMP activity and WT amyloid beta 1-40-induced cell death, R. Blaise et al.

ª 2012 The AuthorsAging Cell ª 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland

385

WT-Ab1-40-induced MMP-2 activation is regulated by

TIMP-2 accumulation at the cell surface

It has been shown that the MT1-MMP–dependent MMP-2 activation

requires strictly regulated amounts of TIMP-2 molecules (Strongin et al.,

1995). In this activation mechanism, TIMP-2 bound to the catalytic

domain of MT1-MMP acts as a ‘receptor’ for pro-MMP-2 at the cell sur-

face. Within this protein complex, the hemopexin domain of pro-MMP-2

binds to the C-terminal domain of TIMP-2 allowing proteolytic activation

of pro-MMP-2 by an adjacent molecule of MT1-MMP that is free of TIMP-

2 (see Fig. S1). In this scenario, the TIMP-2 molecule, part of the trimolec-

ular complex (composed of MT1-MMP ⁄ TIMP-2 ⁄ pro-MMP-2), acts as an

MMP-2 activator. Here, TIMP-2 implication in WT-Ab1-40-induced MMP-2

activation was evidenced by the drastic reduction of MMP-2 active forms

(Fig. 4A, left panel) visualized in cells transfected with TIMP-2 siRNA com-

pared with control siRNA-transfected cells. TIMP-2 siRNA efficiency was

attested by RT–PCR, Fig. 4A, left panel. To study the mechanism of MMP-

2 activation, we analyzed TIMP-2 protein levels in control and WT-Ab1-40-

treated whole cell extracts (as opposed to cell media) assuming that it

would reflect TIMP-2 recruitment to the cell surface. We also defined

TIMP-2 subcellular localization. TIMP-2 protein was not present in con-

trols (whether in vehicle- or inverted peptide-treated VSMCs), but highly

accumulated in the WT-Ab1-40 treated VSMCs (Fig. 4B left panel). Data

obtained from immunocytochemistry experiments evidenced that TIMP-2

staining defines the plasma membrane in WT-Ab1-40-treated cells

(Fig. 4C). Altogether, these results strongly suggest the WT-Ab1-40-

dependent accumulation of TIMP-2 at the cell surface. Because WT-Ab1-

40 activates MMP-2 through MT1-MMP, we next examined whether

TIMP-2 could be recruited at the VSMC surface by silencing MT1-MMP

expression. As shown in Fig. 4D, the intensity of the TIMP-2 band was

strongly reduced in MT1-MMP-silenced cells compared with control siR-

NA-treated cells. As neither the amount of TIMP-2 mRNA nor that of

secreted protein (Fig. 4B, right panel) was modified by the WT-Ab1-40

treatment, we concluded that the WT-Ab1-40-dependent accumulation

of TIMP-2 takes place at the cell surface, because of its interaction with

the upregulated membrane type 1-MMP (MT1-MMP).

MMPs activity is not involved in WT-Ab1-40 induced

cell death

Anoıkis is a form of programmed cell death induced by cell detach-

ment from the ECM. As MMP activity contributes to ECM degradation,

we questioned whether WT-Ab1-40-induced VSMC death is linked to

MT1-MMP ⁄ MMP-2 activation. We analyzed the effect of MMP-2 or

MT1-MMP silencing with siRNA on the WT-Ab1-40-induced VSMC

apoptosis. The efficiency of MMP-2 and MT1-MMP silencing was esti-

mated by RT–PCR and gelatin zymography (Fig. 5A,C). To our surprise,

MMP-2 silencing did not change the rate of apoptosis in cells treated

with the WT-Ab1-40 (Fig. 5B), neither did MT1-MMP siRNA (Fig. 5D), as

the percentage of apoptotic cells was very similar whether MT1-MMP

expression was silenced or not (siRNA MT1-MMP:79.2 ± 7.4; siRNA

control: 69.3 ± 5.5). Similar results were obtained in the MCF-7 breast

carcinoma cell subline deficient in MT1-MMP and MMP-2 expression

(Rozanov et al., 2001) (Fig. S2). Altogether, these experiments demon-

strated that MMP-2 and MT1-MMP are not involved in the WT-Ab1-40-

induced cell death.

(A)

(B) (C)

Fig. 2 Wild-type (WT)-Ab1-40 peptide increases expression and activation of matrix metalloprotease (MMP)-2, but not MMP-9 in vascular smooth muscle cells. Serum-starved

cells were treated for 48 h with IL-1b (10 ng mL)1), WT-Ab1-40 (50 lM), inverted Ab40-1 (50 lM), or vehicle (control). (A) Transcripts encoding MMP-2 (left panel) and MMP-9

(right panel) were assayed by RT–PCR. Data represent means ± SD of four independent experiments performed in triplicate; n.s, not significant, n.d, not detectable, and

***P < 0.001 compared with control (Ctl). (B) MMP-2 immunoblot on total proteins (20 lg). The Western blot is representative of two independent experiments. Int. MMP-

2, intermediate form of MMP-2; Act. MMP-2, Active form of MMP-2; *Nonspecific band. (C) Culture medium was recovered after treatment; 45 lL were used for

determination of secreted MMP-2 and MMP-9 activities on gelatin zymography as described in Materials and methods. The lysis bands corresponding to the pro- or active

form (Act.) of MMP-2 and MMP-9 were visualized after Coomassie Blue staining and decoloration. The gelatin zymography image is representative of four independent

experiments.



386

As MMPs without gelatinase activity could be expressed in VSMCs,

including MMP-1, MMP3, MMP-10, MMP-13 (Schonbeck et al., 1997;

Mao et al., 1999; Wu et al., 2003), we evaluated the expression of these

enzymes in WT-Ab1-40-treated VSMCs and showed that the expression of

MMP-3 and MMP-13 mRNA was induced by WT-Ab1-40 (Fig. 6A). Never-

theless, although WT-Ab1-40-induced MMP activity was inhibited by

GM6001 or Batimastat (two broad-spectrum hydroxamate inhibitors of

MMPs, Fig. 6B), these compounds had no effect on WT-Ab1-40-induced

cell death (Fig. 6C). These data clearly demonstrate that MMP

proteolytic activity is not the primary cause of VSMC apoptosis induced

by WT-Ab1-40.

Discussion

Here, we show that the WT-Ab1-40 peptide induces the expression of

MT1-MMP and MMP-2 in VSMCs, similarly to the Dutch mutant of Ab1-40

(Davis et al., 1999; Jung et al., 2003). In addition, we determine the

molecular mechanism of MMP-2 activation by demonstrating a MT1-

MMP–dependent MMP-2 activation (Fig. 3) through the recruitment of

TIMP-2 to the cell surface (Fig. 4). This is consistent with the mechanism

of pro-MMP-2 activation via MT1-MMP and TIMP-2 described by Strongin

et al., 1995, in which TIMP-2 bound to the MT1-MMP catalytic domain

acts as a cell surface ‘receptor’ for pro-MMP-2. The bound pro-MMP-2 is

then activated by an adjacent MT1-MMP molecule free of TIMP-2.

Ab-peptides upregulated MMP activity has been associated with hav-

ing either protective or deleterious effects. The protective effect, which

essentially translates into a reduction of cell degeneration in the blood

vessel wall, is mostly attributed to the ability of MMP to degrade the Ab

peptides (Miners et al., 2008). The deleterious role includes ECM degra-

dation, blood–brain-barrier disruption and, possibly, an induction of hem-

orrhagic phenotype (Rosenberg & Navratil, 1997; Rosell et al., 2006;

Hernandez-Guillamon et al., 2011). Consistent with a recent study high-

lighting the participation of caspase-mediated mechanisms in the pro-

apoptotic effect of Ab-peptides (Fossati et al., 2010), we showed that, in

addition to the induction of MMP-2, the WT-Ab1-40 peptide led to VSM

cell death, reinforcing the possible contribution of apoptotic mechanisms

in sporadic CAA. We also clearly demonstrated that there is no cause-to-

effect relationship between MMP activation and apoptosis. Interestingly

in VSMCs, plasmin (generated from the cleavage of circulating inactive

plasminogen by urokinase Plasminogen Activator and tissue Plaminogen

Activator) has been also shown to induce both MMP-2 activation and

apoptosis, while MMP inhibitors failed to rescue cell death (Meilhac et al.,

2003); these suggest that plasmin, but not plasmin-activated MMP, is

responsible for apoptosis. Besides, our data likely oppose Jung et al.

(2003) study where they suggested, using the Dutch mutant of Ab1-40, a

link between MMP-2 activity, smooth muscle cell mortality and degrada-

tion of specific matrix components. However, our result, pointing out an

MMP-independent VSM cell death, is consistent with the recent study

demonstrating that MMP-2 activity is not directly involved in cell toxicity

induced by Ab peptides (Hernandez-Guillamon et al., 2010).

Therefore, instead of supporting the controversy surrounding MMP-2’s

role in Ab-peptide induction of cell toxicity, we believe that this apparent

contradiction indicates that the ontogenesis of the Dutch familial and

sporadic forms of CAAs is different. ECM degradation and VSMC degen-

eration are tightly connected in the Dutch familial form while being

two independent processes in sporadic forms of CAA. Indeed, MMP

(A)

(B)

Fig. 3 Wild-type (WT)-Ab1-40 peptide increases MT1-matrix metalloprotease (MMP) expression, leading to MMP-2 activation in vascular smooth muscle cells. Serum-starved

cells were treated for 48 h with WT-Ab1-40 (50 lM), inverted Ab40-1 (50 lM) or vehicle (control). (A) Left Panel. Transcripts encoding MT1-MMP were assayed by RT–PCR. Data

represent the means ± SD of four independent experiments performed in triplicate, n.s, not significant, and ***P < 0.001 compare to control (Ctl). Right Panel. Total

proteins (20 lg) were separated by electrophoresis. MT1-MMP and GAPDH were immunodetected with appropriate antibodies. (B) Culture media samples from mock or inert

MT1-MMP E240A-transfected cells were recovered after indicated treatment and the MMP activity was evaluated by gelatin zymography (left panel). The protein bands

corresponding to the pro- or active form of MMP-2 were visualized after Coomassie Blue staining and decoloration. The zymography image is a representative of three

independent experiments. Expression level of MT1-MMP E240A was evaluated by Western blot (right panel).



387

(A)

(C)

(D)

(B)

Fig. 4 Matrix metalloprotease (MMP)-2 activation is dependent on the accumulation of TIMP-2 at the cell surface induced by wild-type (WT)-Ab1-40 in vascular smooth

muscle cells. Serum-starved cells were treated for 48 h with WT-Ab1-40 (50 lM), inverted Ab40-1 (50 lM) or vehicle (control). (A) Culture media obtained from cells previously

transfected with control siRNA or TIMP-2 siRNA were collected after indicated treatment and MMP activities were evaluated by gelatin zymography (left panel). The gelatin

zymography image shown is representative of three independent experiments. TIMP-2 siRNA efficiency was evaluated by RT–PCR (right panel); data represent the

means ± SD of three independent experiments performed in triplicate, ***P < 0.001 compared with control siRNA related to each treatment. (B) Left Panel. Cell lysate

samples (20 lg) were separated by electrophoresis; TIMP-2 and GAPDH were immunodetected with the appropriate antibodies; the western blot shown is representative of

five independent experiments. Right Panel. Transcripts encoding TIMP-2 were assayed by RT–PCR (upper right panel). The data represent the means ± SD of four independent

experiments performed in triplicate, n.s, not significant compared with control (Ctl). TIMP-2 immunoblot was performed on 40 lL of conditioned culture medium separated

by electrophoresis (lower right panel). The western blot is representative of two independent experiments. (C) Immunostaining of TIMP-2 on PFA-fixed cells was performed

after 24 h-treatment using monoclonal antibody against TIMP-2 and a secondary antibody coupled to FITC (Green Stain, 20·). Cell nuclei were stained with Hoechst (blue,

20· same microscopic field as FITC). (D) Total proteins (20 lg) obtained from treated cells previously transfected with control or MT1-MMP siRNA were separated by

electrophoresis; TIMP-2 and GAPDH were immunodetected with appropriate antibodies. The western blot shown is representative of three independent experiments.



388

broad-range inhibitor GM6001 can significantly reduce the apoptosis

induced by a 48 h Ab1-40Dutch-treatment (Fig S3B), but did not have this

effect on WT-Ab1-40-treated cells (Fig. 6). A likely divergence between

WT, Dutch and Arctic Ab-peptides has already been reported regarding

anti-angiogenic effects in brain endothelium (Solito et al., 2009). How-

ever, although several data argue for distinct mechanisms between WT

and Dutch Ab1-40 peptides (including ours, demonstrating that the

WT-Ab1-40- but not the Ab1-40Dutch-induced apoptosis is inhibited by

fetal calf serum, see Fig S3C), one might be caution with comparing

these two peptide effects as they are differently solubilized and display

distinct secondary structures and oligomerization ⁄ fibrillization kinetics

(Solito et al., 2009; Fossati et al., 2010).

It is not clear whether or not WT-Ab peptide-induced MMP activity has

a beneficial role (protecting the cells by degrading the WT-Ab peptides) in

our experimental conditions. If it was the case, inhibition of MMPs should

have translated into an increased Ab-induced apoptosis (Hernandez-

Guillamon et al., 2010). A possible explanation for not being able to

detect such an increase could be the dose of peptide used (50 lM). As a

matter of fact, such a high concentration could prevent the effect of a

minor Ab-proteolytic degradation. Consistent with this, degradation of

WT-Ab peptides in a cellular context was only observed at a low dose

(< 1 lM) using MMP overexpressing cells (Liao & Van Nostrand, 2010). In

addition, because our cell culture condition medium does not contain

plasminogen, it is also conceivable that the Ab-dependent MMP activa-

tion (and therefore Ab-peptide degradation) was limited. Indeed, plasmin

activates MMP in VSMCs (Galis & Khatri, 2002). Interestingly, plasmin

activators (uPA and tPA) are induced by Ab-peptides (Davis et al., 2003

and Fig. S4), whereas the expression of the tPA ⁄ uPA inhibitor, PAI-1, is

decreased (Fig. S4), suggesting that Ab-treated VSMCs could convert

plasminogen into plasmin. It is important to note that plasmin is also

capable of degrading Ab peptides (Tucker et al., 2000; Jacobsen et al.,

2008) similar to other proteases including neprisylin, insulin-degarding

enzyme, endothelin-converting enzyme, angiotensin-converting enzyme

(Miners et al., 2008). Besides, plasmin, similar to MMP-2, can have both

protective and deleterious effects, whereas plasmin activity can protect

Ab-peptide-treated VSMCs from apoptosis and can also lead to cell

(A)

(B)

(C)

(D)

Fig. 5 Effect of matrix metalloprotease (MMP)-2 and MT1-MMP silencing on wild-type (WT)-Ab1-40-induced cell death. Serum-starved cells were treated for 48 h with WT-

Ab1-40 (50 lM), or vehicle (control). (A,C) upper panel. siRNAs efficiency was evaluated by RT–PCR on transcripts obtained from cells transfected with control, MMP-2 siRNA

(A) or MT1-MMP siRNA (C). Data represents the means ± SD of three independent experiments performed in triplicate, ***P < 0.001 compared with vehicle (Ctl)-treated

cells transfected with control siRNA. ###P < 0.001 compared with WT-Ab1-40-treated cells transfected with control siRNA. (A,C) lower panel. Culture medium samples issued

from MMP-2 siRNA (A) or MT1-MMP siRNA (C) -transfected cells were collected after indicated treatment; MMP activities were evaluated by gelatin zymography. The

zymography shown are representative of three independent experiments. (B,D) the apoptosis of MMP-2 siRNA (B) or MT1-MMP siRNA (D) -transfected cells was determined

by Propidium Iodide staining. Data represents the means ± SD of three independent experiments performed in triplicate. n.s, not significant; ***P < 0.001; **P < 0.01 and

*P < 0.05 compared with related control (Ctl).



389

detachment (Davis et al., 2003), the process resulting in cell death by

anoıkis (Michel, 2003).

Although the expression of MMP-2 was upregulated by WT-Ab1-40

within a 72-h time frame, no effect could be detected on MMP-9. As

expected, the pro-inflammatory cytokine IL-1b triggered its expres-

sion ⁄ activity (Fig. 2). Taking into account that MMP-9 is an inflammatory

marker (del Zoppo, 2010), we suggest that it is unlikely that WT-Ab1-40

initiates VSMC inflammation directly. Interestingly, Ab1-40 peptides

(Dutch or WT) induce the expression of adhesion molecules and pro-

inflammatory cytokines in human arterial endothelial cells (Suo et al.,

1998) arguing for an inflammatory context present in CAA (Vukic et al.,

2009). Because MMP-9 (as well as MMP-2) has been extensively involved

in blood–brain barrier breakdown and intracerebral hemorrhage (Rosen-

berg & Navratil, 1997; Rosell et al., 2006; Hernandez-Guillamon et al.,

2011), two main features of CAA, it would be of interest to re-evaluate

the WT-Ab1-40 capacity of inducing MMP activity within the inflammatory

context of CAA. This could first be approached in vitro on IL-1b-treated

VSMCs.

In summary, our results tend to prove that the deposition of WT-Ab1-40

(present in sporadic CAA) induces VSMC apoptosis independently from

MMP activity. One might suggest that the WT-Ab1-40-induced MMPs

(because of Ab accumulation) should actively participate in the destruc-

tion of the blood–brain barrier and the alteration of vessel wall integrity

characteristics of the CAAs. Nevertheless, in vivo data should be obtained

on relevant CAA models to further support our hypothesis.

Materials and methods

Reagents

Dulbecco’s modified Eagle’s medium, type I collagen from calf skin, glu-

tamine, penicillin, streptomycin, fatty acid-free bovine serum albumin

were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France).

Fetal calf serum and collagenase were from Gibco BRL (Cergy Pontoise,

France). Elastase, protease inhibitors, LightCycler-DNA Master Plus SYBR

Green and Fugene HD transfection reagent were obtained from Roche

Diagnostics (Meylan, France). Oligonucleotides were sourced from

MWG Biotech AG (Courtaboeuf, France). Kits for RNA extraction

(RNeasy Mini kit) were obtained from Qiagen (Courtaboeuf, France).

RT-MMLV, RNAsin, Lipofectamine RNAi Max transfection reagent were

obtained from Invitrogen (Cergy Pontoise, France). siRNA were from

Ambion (Invitrogen, Cergy Pontoise, France) or Qiagen (Courtaboeuf,

France). Nitrocellulose membranes were from Schleicher and Schuell

(Dassel, Germany). ECL reagent kit was from Amersham Pharmacia

Biotech (les Ulis, France). Interleukin-1b (IL-1b) was from Santa Cruz

Biotechnology (Heildberg, Germany). GM6001 and Batimastat broad-

range MMP inhibitors were from Millipore (Molsheim, France) and

Santa cruz Biotechnology, respectively. The FRET MMPs substrate

Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH2 was purchased

from Bachem. Ethidium Homodimer III (ETHD-III) DNA dye was from

Promocell (Heildberg, Germany).

(A)

(B) (C)

Fig. 6 Effect of matrix metalloprotease (MMP) inhibitors on wild-type (WT)-Ab1-40-induced cell death. Serum-starved cells were treated for 48 h with WT-Ab1-40 (50 lM), or

vehicle (control), with or without MMP inhibitors (GM6001 or Batimastat). (A) Transcripts encoding MMP-3 (left panel) and MMP-13 (right panel) were assayed by RT–PCR.

Data represent the means ± SD of four independent experiments performed in triplicate, ***P < 0.001 and *P < 0.05 compared with control (Ctl). (B) Inhibition of

WT-Ab1-40-induced MMP activities by GM6001 (25 lM) or Batimastat (2.5 lM) treatment was evaluated by adding the MMP fluorescent substrate (40 lM) to the collected

culture medium. The fluorescence of MMP-cleaved substrate (kex = 320 nm, kem = 390 nm) was measured with a fluorescence multiplate reader. Data represent the

means ± SD of three independent experiments performed in triplicate, n.s, not significant; ***P < 0.001, ###P < 0.001 and ��P < 0.001 compared with related control (Ctl).

(C) Apoptosis of treated cells was determined by Eth-DIII staining. Data represent the means ± SD of three independent experiments performed in triplicate; n.s, not

significant and ***P < 0.001 compared with related control (Ctl). The fluorescence of DNA-bound EthD-III (kex = 515 nm, kem = 620 nm) was measured with a fluorescence

multiplate reader. The results are expressed in percentage of dead cells compared with cells treated by absolute ethanol (referred to as 100% dead cells). Data represent the

means ± SD of six independent experiments performed in triplicate; n.s, not significant; ***P < 0.001, ###P < 0.001 and ��P < 0.001 compared with related control (Ctl)-

treated cells.



390

Wild-type amyloid beta 1-40 (WT-Ab1-40) (DAEFRHDSGYEVHHQKL

VFFAEDVGSNKGAIIGLMVGGVV), Dutch mutant (E22Q) amyloid beta

peptide (Ab1-40 Dutch) (DAEFRHDSGYEVHHQKLVFFAQDVGSNKGAIIGL

MVGGVV), or control inverted 40-1 (Ab40-1) (VVGGVMLGIIAGKNSG

VDEAFFVLKQHHVEYGSDHRFEAD) peptides were synthesized by Gene-

Cust (Dudelange, Luxembourg) and analyzed by HPLC and mass spectrom-

etry with a purity ranged between 94% and 98%. WT-Ab1-40 and

scrambled Ab40-1 peptides were resuspended in ultrapure water at 2 mM

and immediately frozen. WT-Ab1-40 and scrambled Ab40-1 peptides were

directly added to culture medium at 50 lM. Ab1-40 Dutch was resuspended

at 1 mM in 1,1,1,3,3,3,-hexafluoro-2-propanol (Sigma-Aldrich) and incu-

bated at room temperature until obtaining a clear solution. Ab1-40 Dutch

peptides were aliquoted and allowed to evaporate over night in a fume

hood. Dried peptide films were stored at )20 �C. Prior to use, Ab1-40 Dutch

peptides films were resuspended at 5 mM into DMSO (Sigma-Aldrich) and

diluted to 1 mM with PBS before added to cell culture at 50 lM.

Isolation and culture of smooth muscle cells

Rat vascular smooth muscle cells (VSMC) were isolated by enzymatic

digestion of aortic media from male Wistar rats and cultured as described

previously (Clement et al., 2006). All experiments were performed on cells

at passages ranging from two to six. Twenty-four hours before any treat-

ment, confluent cells were made quiescent by culturing them in a serum-

free medium. All experiments were performed in serum-free medium. Of

note, basal cell death 72 h after serum starvation is very similar to that of

measured in presence of 10% fetal calf serum within this time frame

(approximately 10%). Conditions of cell treatment are as indicated in the

figure legends. All incubations were performed at 37 �C, 5% CO2. Cells

were exposed to peptides diluted in culture medium at 50 lM for 24–72 h.

Real-time quantitative reverse transcription–polymerase

chain reaction (RT–PCR) assays

Total RNA was extracted from VSMCs using the RNeasy kit (Qiagen,

Hilden, Germany) according to the manufacturer’s instructions. Con-

centrations were determined spectrophotometrically. After annealing

oligodT (1 lM) to template RNAs (0.5 lg) at 70 �C for 5 min, primer

extension was initiated by adding the RT-MMLV enzyme plus 0.5 mM

dNTP, 1U RNAsin, and 10 mM dithiothreitol (DTT) and carried out for

45 min at 37 �C. Quantitative PCR was performed using the LightCycler

LC480 (Roche Diagnostics). The PCR mix included 5 lL of each reverse

transcriptase (diluted 1:25) and 300 nM of each primer in 1 · LightCy-

cler-DNA SYBR Green 1 Master Mix. Specific primers for complemen-

tary DNA (cDNA) were chosen with the LightCycler Probe Design2

program according to European Molecular Biology Laboratory accession

numbers. The forward and reverse primers used to selectively amplify

the cDNA encoding rat hypoxanthine phosphoribosyltransferase (HPRT),

MMP-2, MMP-3, MMP-9, MMP-13, MT1-MMP and TIMP-2 are pre-

sented in Table 1.

The PCRs were performed using the following thermal settings: dena-

turation and enzyme activation at 95 �C for 5 min, with cycles at 95 �Cfor 10 s, 60 �C for 15 s, and 72 �C for 10 s. Amplification was followed

up online, and the PCRs were stopped after the logarithmic phase. Melt-

ing curve analyses were also performed after PCR to check the reaction

specificity. Controls and water blanks were included in each run; they

were negative in all cases.

Real-time quantitative PCR data represent the amount of each tar-

get messenger RNA (mRNA) relative to the amount of a housekeeping

reference gene mRNA, the HPRT, estimated in the logarithmic phase

of the PCR. Serial dilutions were used to determine the fit coefficients

of the relative standard curve. When the PCR target efficiencies were

similar, individual cultures could be compared. If not, an internal cali-

brator was used.

Plasmid preparation and transient transfections

Expression plasmid for MT1-MMP mutant E240A was made as described

in Rozanov et al., 2001. For plasmid expression, cells were transfected at

70–80% confluency by adding 10 lg DNA and 15 lL Fugene HD (ratio

2:3) per 10-cm dish according to the manufacturer’s instructions (Roche

Diagnostics). Plasmids were inert MT1-MMP E240A mutant or control

(pCDNA3, invitrogen). Twenty-four to 48 h post-transfection, cells were

starved overnight and treated as indicated in the figure legends.

siRNA were transfected with Lipofectamine RNAiMAX reagent (Invitro-

gen) according to the manufacturer’s instructions. Briefly, 50% confluent

cells were cultured for 4 h in complete medium without antibiotics and

transfected by siRNA (5 nM final for 5 lL of reagent per well in 6-wells

plate) for 24–48 h. After 12-h serum starvation, the cells were treated as

indicated in the figure legends. The siRNA forward and reverse strand

sequences are, respectively, 5¢-CCAUCGAGACCAUGCGGAATT-3¢ and

5¢-UUCCGCAUGGUCUCGAUGGTG-3¢ for MMP-2, 5¢-CCAGUGUUUCA

UUUGCCUATT3¢ and 5¢- UAGGCAAAUGAAACACUGGTT-3¢ for TIMP-2;

5¢-CUUCUACAAAGGGAACAAATT-3¢ and 5¢-UUUGUUCCCUUUGUAGA

AGTA-3¢ for TIMP-2.

Protein extraction and western blot analysis

Cells were washed with ice-cold phosphate-buffered saline (PBS) and

resuspended in lysis buffer (20 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1 mM

EDTA, 1% Triton, 1% sodium deoxycholate) plus Complete Protease

Inhibitor mixture (Roche Diagnostics), for 10 min on ice. The lysate was

centrifuged at 13 000 g for 10 min at 4 �C, and the supernatant (cell

lysate) was collected. Cell lysates (20–40 lg of protein) were analyzed by

Table 1 Summary of PCR primers

Target gene Forward primer (5¢-3¢) Reverse primer (5¢-3¢) Accession number

HPRT aggacctctcgaagtgt atccctgaagtgctcattata NM_012583.2

MMP-2 atcgcccatcatcaagttc catgttctcgatggtgttc NM_031054.2

MMP-3 atgtagatggtattcaatccctctat aaagcagagctacacatt NM_133523.2

MMP-9 ttcgagggagcgtcctat ttagtggtgcaggcagagta NM_031055.1

MMP-13 tggtcttctggcacacg gagtggtccagaccg NM_133530.1

MT1-MMP tgggaaggaatccctgagt ctctaccttcagcttctggtt NM_031056.1

TIMP-2 gacctgacaaggacatcgaa tcccagggcacaataaagt NM_021989.2

HPRT, hypoxanthine phosphoribosyltransferase; MMP, matrix metalloprotease.



391

sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 12%

resolving gels or 4–12% gradient gels (Invitrogen NuPAGE system) fol-

lowed by transfer to nitrocellulose membranes. After blocking 1 h at

room temperature with a Tris-buffered saline containing 0.1% Tween 20

(TBS-T) (20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) and 5%

fat-free milk or bovine serum albumin, membranes were incubated over-

night with primary antibodies in TBS-T with 5% of milk or 5% bovine

serum albumin, at 4 �C, washed in TBS-T, and incubated with horseradish

peroxidase (HRP)-conjugated secondary antibodies for 1 h at room tem-

perature. Signals were detected with the ECL detection system and

exposed to Fujifilm LAS-300 (Fujifilm Medical Systems, Stamford, CT,

USA). Equal protein loading and transfer efficiency were determined by

Ponceau Red staining after electrotransfer and GAPDH detection. The

densitometry patterns were analyzed with ImageGauge software (Sci-

ence Lab 2004; Fujifilm) and normalized to the density of GAPDH.

Antibodies

Anti-MMP-2 (mouse monoclonal; Santa Cruz), Anti-TIMP-2 (rabbit mono-

clonal; Cell signaling, Ozyme, St Quentin Yvelines, France), Anti MT1-

MMP (mouse monoclonal; Millipore), anti-GAPDH (goat polyclonal;

Sigma-Aldrich) were purchased. HRP-coupled secondary antibodies were

from P.A.R.I.S Ltd, Compiegne, France.

Determination of MMP-2 ⁄ -9 activity by

gelatin zymography assay

Zymography of conditioned media samples was performed as previously

described (Delbosc et al., 2008). Briefly, equal volume of sample issued

from conditioned medium was loaded onto an 10% polyacrylamide gel

containing 1% gelatin. After electrophoresis, gels were washed in a

2.5% Triton X-100 solution (2 · 30 min), incubated for 20 h in buffer

containing 50 mM Tris and 2.5 mM CaCl2, stained with Coomassie Blue,

and destained in methanol ⁄ acetic acid buffer until the bands could be

detected.

Apoptosis detection

Cell death was determined as the percentage of hypodiploid nuclei (HN)

assessed by propidium iodide staining (Mateo et al., 2007). Briefly, cells

were resuspended in a hypotonic solution containing 0.1% sodium cit-

rate, 0.1% Triton X-100 (Sigma-Aldrich), and 50 lg mL)1 propidium

iodide (Sigma). Cell nuclei were analyzed on EPICS XL instrument using

Expo32 software (Becton Coulter). The percentage of induced apoptosis

was calculated as 100 · (percentage of experimental HN)percentage of

spontaneous HN) ⁄ (100)% of spontaneous HN). Cell death was also

determined by fluorescent ethidium homodimer (EthD-III) assay that pro-

duces a bright red fluorescence in dying cells. VSMCs were plated in black

with clear bottom 96-well microplates at 10 000 cells ⁄ well 2 days prior

treatment. After 48–72 h of treatment, the medium was removed and

replaced by a volume of 100 lL per well of PBS containing 5 lM of ethidi-

um homodimer III (EthD-III). After 30 min incubation, fluorescence was

evaluated on a microplate reader (Infinite F500; Tecan France S.A.S, Lyon,

France) at ex ⁄ em 520 nm ⁄ 630 nm. One hundred percent dead cells were

determined by measuring the fluorescence emitted by an absolute

ethanol-treated well. Percentage of cell death was determined as

100 · (Fluorescence of experimental condition)background fluorescence) ⁄(Fluorescence of ethanol-treated cells)background fluorescence). Broad-

range MMP inhibitors GM6001 (25 lM) or Batimastat (2.5 lM) were added

simultaneously to Ab-peptide and renewed after 24 h.

Immunocytochemistry and microscopy

Vascular smooth muscle cells were seeded directly onto glass coverslips

placed in 24-well culture plates. After treatment (always indicated in fig-

ure legends), cells were washed twice with PBS, fixed in 20 min at room

temperature in 4% paraformaldehyde (neutralized with 50 mM of NH4Cl

for 10 min), permeabilized in PBS+0.2% Triton X100, and incubated for

1 h in PBS+10% FCS with TIMP-2 monoclonal antibody. The incubation

with DyLight 488-Conjugated Goat Anti-Rabbit secondary antibody was

performed during 1 h and Hoechst staining for 5 min. Coverslips were

mounted with Dako fluorescent mounting medium (Dako, Carpinteria,

CA, USA). Cells were examined by phase contrast or fluorescent micros-

copy using a Nikon Diaphoto 300 microscope equipped with a mercury

lamp (Nikon, Tokyo, Japan).

Statistical analysis

The data are reported as the mean ± SD. The numbers of independent

experiments are reported in figure legends. Values were compared

between groups with the Welch’s unpaired, corrected t-test.

Acknowledgments

We thank Dr. Xavier Houard for helpful discussion and advice and

Dr. Alex Strongin for providing reagents. This project has been finan-

cially supported by ‘Pierre Fabre Innovation’.

Authors contribution

Isabelle Limon had full access to all of the data in the study and takes

responsibility for the integrity of the data and the accuracy of the

data analysis. All the authors approved the final manuscript version

to be published. Regis Blaise, Veronique Mateo, Vladislav Golubkov,

and Isabelle Limon contributed to the study conception and design.

Regis Blaise, Veronique Mateo, Clotilde Rouxel, Francois Zaccarini,

Martine Glorian, and Vladislav Golubkov contributed to the acquisi-

tion of data. Regis Blaise, Veronique Mateo, Francois Zaccarini, Gil-

bert Bereziat, Vladislav Golubkov, and Isabelle Limon carried out the

analysis and interpretation of data. Regis Blaise, Veronique Mateo,

Vladislav Golubkov, and Isabelle Limon contributed to the manu-

script preparation. Regis Blaise and Isabelle Limon performed the

statistical analysis.

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Supporting information

Additional supporting information may be found in the online version of

this article:

Fig. S1 TIMP-2 ⁄ MT1-MMP-dependent MMP-2 activation.

Fig. S2 WT-Ab1-40 induces cell death in MCF-7.

Fig. S3 WT-Ab1-40 and Ab1-40 Dutch-induced cell-death are differentially

affected by GM6001 and Serum.

Fig. S4 WT-Ab1-40 peptide increases tPA and decreases PAI-1 expression in

VSMCs.

As a service to our authors and readers, this journal provides supporting

information supplied by the authors. Such materials are peer-reviewed and

may be re-organized for online delivery, but are not copy-edited or typeset.

Technical support issues arising from supporting information (other than

missing files) should be addressed to the authors.



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Wild-type amyloid beta 1-40 peptide induces vascular smooth muscle cell death independently from matrix metalloprotease activity

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