Institut National de la Santé et de la Recherche Médicale Unit 460, Centre Hospitalier Universitaire Xavier Bichat, 75870 Paris cedex 18, France
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ABSTRACT |
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After deendothelialization, the most
luminal smooth muscle cells of the neointima are in contact with blood
flow and express inducible nitric oxide synthase (iNOS) in vivo. We
hypothesized that shear stress may be a stimulus for this iNOS
overexpression. We have thus submitted smooth muscle cells to laminar
shear and measured the iNOS expression. Shear stress (20 dyn/cm2) induced iNOS mRNA and protein expression, whereas
brain NOS mRNA expression was decreased. Conversely, nitrite production was increased. This production was blocked by a selective iNOS inhibitor. Pyrrolidine dithiocarbamate, an antioxidant molecule, and
BXT-51072, a gluthation peroxidase mimic, both inhibited the shear-induced iNOS expression. Shear stress also increased the expression of both membrane subunits of NADPH oxidase
p22phox and Mox-1. Shear stress activated the
redox-sensitive nuclear translocation of the transcription nuclear
factor-B (NF-
B) and stimulated the degradation of both cytosolic
inhibitors
B
and
. These results show that shear
stress can induce iNOS expression and nitrite production in smooth
muscle cells and suggest that this regulation is probably mediated by
oxidative stress-induced NF-
B activation.
nuclear factor-B; nitric oxide synthase; NADPH oxidase
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INTRODUCTION |
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THE VASCULAR WALL of large arteries is organized into three different functional and structural compartments. This specific organization (compartmentalization of the vascular wall) modulates the response of the vascular wall to different stimuli. Smooth muscle cells constitute the media layer, whereas endothelial cells constitute the intima and physiologically perceive the shear stress at the interface between the flowing blood and the fixed arterial wall. In these endothelial cells, shear stress regulates the expression of numerous genes [angiotensin converting enzyme, platelet-derived growth factor, vascular cell adhesion molecule-1 (VCAM-1), etc.] (see Ref. 17 for review). In particular, increased shear stress intensity upregulates endothelial nitric oxide synthase (NOS) activity and leads to nitric oxide (NO) overproduction in endothelial cells (23, 24). In response to deendothelialization, smooth muscle cells migrate from the media to the lumen and form the neointima. During this process, the most luminal smooth muscle cells are in close contact with the blood flow and thus sense the shear stress. Therefore, smooth muscle cells undergo the shear stress that could modify the pattern of protein expression within the most luminal smooth muscle cells. In vivo, a gradient of inducible protein expression including inducible NO synthase (iNOS) (12) is usually observed from the most luminal to the deeper part of the neointimal proliferation (28). We have recently shown that shear stress induces angiotensin converting enzyme expression in smooth muscle cells (9). It would permit the shift of a constitutive endothelial function to smooth muscle cells in an intimal position. In this way, it has been demonstrated that iNOS expression is induced in the most luminal smooth muscle cells of the intimal layer in vivo (12).
The formation of the neointima is inhibited by high flow rate (35), and this phenomenon is partially mediated by NO production (6). Administration of L-arginine, the NOS substrate, was associated with a reduced neointimal hyperplasia (11) independent of any endothelial process (32), suggesting that NO may be produced by smooth muscle cells. Given that smooth muscle cells are exposed to flow in neointima formation, we hypothesized that shear could participate in iNOS induction and NO production in smooth muscle cells. The antiproliferative effects of NO on smooth muscle cells and its ability to induce expansive remodeling would be of interest to limit intimal proliferation and lumen stenosis in different pathophysiological situations.
A key component of the induction of iNOS is the nuclear factor-B
(NF-
B). NF-
B is a redox-sensitive factor that is activated by the
cytosolic release of the inhibitor
B (I
B) proteins and the
translocation of the active p50/p65 heterodimer to the nucleus. Increase in the production of radical oxygen species is a common pathway to a wide variety of NF-
B inducers (1).
Although several lines of evidence suggest that shear stress is an
inducer of NF-
B activation in endothelial cells (4,
19), there is no data available about the induction of NF-
B
in smooth muscle cells submitted to shear stress. The aim of our
present study was to evaluate whether shear stress regulates the
expression of iNOS in smooth muscle cells and whether the
redox-sensitive factor NF-
B was involved in this regulation.
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MATERIALS AND METHODS |
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Animals. Normotensive male Wistar rats (160-180 g) were obtained from Iffa Credo (Labresle, France). The procedure followed for the care and euthanasia of the studied animals was in accordance with the European Community standards on the care and use of laboratory animals (Ministère de l'Agriculture, France; authorization no. 00577).
Cell isolation and culture.
The smooth muscle cells from the aortic media were isolated and
cultured as described previously (2) and were used at
passage 3. The purity of the cultures was assessed by using
morphological and immunohistological criteria. Smooth muscle cells were
characterized with antibodies raised against smooth muscle cell
-actin (8).
Shear stress device. The cells were seeded on a rectangular plastic (cell culture-treated) plate previously coated with collagen (0.1% in hydroxy chloride, Sigma). Collagen was chosen as a substrate to increase the adherence forces of the cells submitted to shear stress. Cells were used 2 to 4 days after reaching confluence.
Cells were then exposed to a fluid-imposed shear stress with the use of a parallel plate channel flow device derived from the one described by Levesque and Nerem (15). The cell culture flow chamber was designed to provide a steady, uniform laminar flow. It was positioned in a closed continuous flow loop. The flow loop consisted of an elevated reservoir that provided the required pressure drop across the chamber and a roller pump to return the outflow from the collecting reservoir back to the feeding reservoir. The flow chamber and the entire apparatus were sonicated and sterilized before each experiment to avoid lipopolysaccharide (LPS) contamination. The upper reservoir was filled with 350 ml of Dulbecco's modified Eagle's medium (DMEM, Biomedia) at 37°C (equilibrated with 95% air-5% CO2) supplemented with 10% heat-inactivated fetal calf serum (Biomedia), 20 mM HEPES (Life Technologies), and 1% antibiotic-antimycotic solution (Sigma), with its pH, temperature, and flow rate monitored continuously. The experimental surface of shear-subjected cells was 18 cm2. As a control, unstressed cells were seeded in the same conditions without insertion in the flow chamber device and were incubated with shear stress-conditioned medium to test LPS contamination. After being submitted to shear, the cell plates were removed from the flow chambers under sterile conditions.Shear stress values. The shear stress intensity Y (dyn/cm2) was calculated as follows (15): Y = 6 µQ/ph2 where µ is the media viscosity (DMEM 0.0084 ± 0.08 poised at 37°C), Q the flow rate (ml/s), p the flow path (1.8 cm), and h the gap height over the cell layer (0.025 cm). The viscosity of the media and the flow chamber cross section were constant in the device. To change the intensity of shear stress, the induction flow rate had to be modified. For pharmacological experiments, cells were preincubated with the blocker for 15 min before being submitted to shear stress. The experiments were performed with the same concentration of compound in the medium.
Endotoxin measurement. The level of endotoxins (i.e., LPS) was measured in the supernatant of control cells, in the supernatant conditioned by a 20-dyn/cm2 shear stress, and in the freshly prepared culture medium using a commercial semiquantitative assay kit (Sigma) (22). One hundred microliters of the sample were incubated for 1 h at 37°C with 100 µl of amoebocyte lysate from Limulus polyphemus. The results were evaluated by comparison of the gelation of the sample and compared with a standard curve from 400 to 0.06 endotoxin units (EU)/ml of commercial endotoxin solution (Sigma).
Nitrite production.
To measure nitrite production, each cell plate was put into 10 ml of
DMEM without phenol red and 20 mM HEPES with or without 2.105 M of a selective iNOS inhibitor
[L-N6(1-iminoethyl)lysine
(L-NIL)] (20) for 4 h. The medium was
then removed and nitrites were detected with the use of a fluorescent assay (18). Nitrite production was measured with the use
of a fluorescent assay as described previously (18). In
this assay, nitrites, a degradation product of NO, interact with a
nonfluorescent substrate (diaminonaphthalene, Fluka) to form a
fluorescent component that is detectable at 450 nm
(1-[H]naphthotriazole). A standard curve from 10 nM to 10 µM was made using a commercial nitrite solution (Merck) and treated
in the same conditions to test the proportionality of the method. One
hundred microliters of diaminonaphthalene (0.05 mg/ml in HCl 0.62 N)
was added to 1 ml of the sample. After 10 min of incubation in a dark
room, 50 µl of NaOH 2.8 N was added to stop the reaction.
Fluorescence was read in a spectrofluorometer (Hitachi F2000).
The cells were scraped off and the total proteins were assayed using
the protein assay system (Bio-Rad). Results were expressed as nanomoles
of nitrites per milligram of proteins. Nitrite levels have been shown
to reflect >75% of the total NO produced by vascular smooth muscle
cells (31).
RT-PCR.
For RT-PCR analysis, cells were scraped from each plate into 1 ml of
TRIzol solution (GIBCO BRL). Total RNA was prepared using the
manufacturer's instructions. One microgram of total mRNA was reverse
transcripted using an oligo (dT) primer. PCR amplification of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), S14, iNOS, brain NOS
(bNOS), p22phox, and Mox-1 mRNA were
performed using the primers presented in Table
1.
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Western blot. Cells were scraped off into lysis buffer that contained protease inhibitors for Western blot experiments and total protein measurement. Cell proteins were denatured using Laemmli reagent.
For the IElectrophoretic mobility shift assay.
Nuclear proteins from cells were prepared as previously described
(26). Gel-shift assays were performed with a commercial kit according to the manufacturer's instructions (Promega). The NF-B oligonucleotide probe used (5'-AGT TGA GGG GAC TTT CCC AGG C-3') was labeled with [
-32P]ATP by using T4
polynucleotide kinase. Nuclear proteins (15 µg) were
incubated for 20 min with the labeled probe and migrated in a 4%
polyacrylamide gel. The specificity of the binding reaction was
determined by coincubating duplicate samples with 100-fold molar excess
of unlabeled oligonucleotide probe (competition).
Pharmacology signaling.
To test the role of NF-B in shear-induced iNOS overexpression, we
incubated smooth muscle cells with 10
5 M pyrrolidine
dithiocarbamate (PDTC), an NF-
B inhibitor (19), for
1 h. The cells were then submitted to a 20-dyn/cm2
shear stress for 24 h. To test the role of oxidative stress in the
observed phenomenon, the same experiments were performed with 10
5 M of BXT-50172 (21), a potent
antioxidant that mimics the gluthation-peroxidase activity.
Statistical method. Results were expressed as means ± SE. Significance was estimated by analysis of variance and the Bonferroni test or by the Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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Cell viability and endotoxin measurement.
Shear-stressed cells in primary culture for 24 h is a difficult
experimental condition that can lead to cell suffering and death. To
estimate the cell density, we assayed the levels of total proteins in
both control and treated plates. A 24-h exposure to shear stress (20 dyn/cm2) did not significantly change the cell density on
plates submitted to shear compared with the control, since the amount
of total proteins was not modified in the plates treated with
L-NIL and shear stress (Fig.
1A). Furthermore,
cell integrity was tested by estimating both the GAPDH and S14 (2 housekeeping genes) mRNA levels by RT-PCR. Neither shear stress nor
pharmacological treatments influenced the GAPDH nor the S14 mRNA signal
(Fig. 1B).
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Shear stress induced the expression of iNOS.
The expression of iNOS was dependent on the shear stress intensity
(Fig. 2A) and its duration
(Fig. 2B). The iNOS/GAPDH mRNA level was 0.02 ± 0.004 in control cells. No induction was observed at 10 dyn/cm2
for 24 h (0.05 ± 0.02). A shear value of 20 dyn/cm2 for 24 h induced a significant response
(3.42 ± 0.95, P < 0.001). Six hours of a
20-dyn/cm2 shear rate was insufficient to induce any iNOS
expression (0.02 ± 0.006). After a 20-dyn/cm2 shear
stress for 24 h, the level of iNOS protein was also significantly increased in smooth muscle cells (+170 ± 73%, P < 0.05; Fig. 2C).
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Shear stress-induced nitrite production.
We estimated the production of NO of the stressed smooth muscle
cells by measuring the amount of nitrite, its main metabolite. The
nitrite production was enhanced in the medium of smooth muscle cells
submitted to a 20-dyn/cm2 shear stress for 24 h.
Nitrite production in control cells was 0.26 ± 0.017 nmol/mg
protein for 4 h, whereas stressed cells synthesized 0.69 ± 0.09 nmol/mg protein for 4 h (P < 0.001). This
increase in nitrite production was inhibited by 2 × 105 M L-NIL, a selective inhibitor for iNOS
(0.37 ± 0.08 nmol/mg protein for 4 h, P < 0.001), confirming that this production is mainly due to iNOS
expression (Fig. 3B).
Role of NF-B and oxidative stress on shear
stress-induced iNOS expression.
To test the implication that NF-
B plays a role in the shear
stress-induced iNOS expression, we incubated smooth muscle cells with
PDTC, an NF-
B blocker, and an antioxidant drug. PDTC at the
concentration of 10
5 M completely prevented the induction
of iNOS by 24-h shear stress at 20 dyn/cm2 (0.01 ± 0.003 vs. 3.4 ± 0.9, P < 0.001; Fig.
4A). Furthermore, BXT-50172
(10
5 M), an antioxidant that mimics gluthation peroxidase
activity, suppressed the shear-induced iNOS induction in smooth muscle
cells (Fig. 4B). These results suggest that oxidative stress
and NF-
B are involved in the response of smooth muscle cells to
shear stress. We then tested the effect of shear stress on both NF-
B
translocation to the nucleus and the expression of its I
B. In smooth
muscle cells submitted to shear stress, the amount of NF-
B
translocated to the nucleus was increased by 400 ± 30%
(P < 0.001; Fig.
5A). The coincubation of the
sample with an excess of unlabeled oligonucleotide probe suppressed the
gel shift, which showed the specificity of the binding (Fig.
5A, lane competition). Furthermore, in the
cytoplasm of the stressed cells, I
B
- and
-proteins were
reduced, respectively, by 86.56 ± 10.3% and 64.21 ± 10.2%
of control values (P < 0.001; Fig. 5B).
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Shear stress increased the NADPH oxidase expression.
The enzyme implicated in the production of reactive oxygen species
and thus in generation of oxidative stress in smooth muscle cells is
the NADPH oxidase. Because our experiments with BXT-51072 have
suggested that oxidative stress was implicated in the response to shear
stress, we estimated the expression of the two active membrane-associated subunits of the NADPH oxidase: p22phox
and Mox-1. Shear stress (20 dyn/cm2, 24 h)
significantly increased the p22phox and Mox-1 mRNA levels
in smooth muscle cells (from 5.47 ± 0.12 in the control cells to
7.03 ± 0.38 in the stressed ones, P < 0.01 for
p22phox; and from 0.331 ± 0.04 in the control cells
to 0.879 ± 0.19 in the stressed ones, P < 0.05 for Mox-1; Fig. 6).
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DISCUSSION |
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The present study shows that shear stress increased both iNOS mRNA
and protein expression in rat aortic smooth muscle cells. This
induction occurred only after prolonged exposure (24 h) to shear stress
and was accompanied by a rise in nitrite production in the conditioned
medium. This nitrite overproduction (+160 ± 26%) was more
important than that which we observed in smooth muscle cells treated
for 24 h with 40 µg/ml LPS (i.e., 20,000 EU/ml) plus 100 U/ml
interferon- (+75 ± 22%; data not shown), suggesting that this
production was physiologically relevant.
A preferential inhibitor of iNOS blocked this shear stress-induced nitrite production. Because this inhibitor is 30-fold more specific to iNOS than to bNOS (20), it is unlikely that the observed NO overproduction also resulted from the activity of the brain isoform. Furthermore, shear stress significantly decreased bNOS mRNA levels, confirming that bNOS was not involved in the observed phenomenon. The decrease in bNOS expression was not due to cell death during the experiment because the amounts of total proteins and housekeeping gene expression (GAPDH and S14) were not significantly diminished in stressed plates. These data confirm the observation reported by Papadaki and coworkers (25). In their study, they showed that two stages in nitrite production rate can be discerned in cultured smooth muscle cells exposed to flow. In the first stage (in the first hour of exposure to shear stress), nitrite production was quickly increased because of an upregulation of the calcium/calmodulin-sensitive bNOS activity. In the second stage (from 1 to 24 h of exposure to high shear stress levels), shear stress induced a stable production of nitrites that was not sensitive to a calmodulin inhibitor (25). These data suggest that the mechanism implicated in this NO overproduction in the late stage (enzyme expression) is different from that implicated in the first stage (enzyme activity). Our results confirm that although the initial burst in nitrite production in stressed cells could depend on the activity of constitutive NOS, a prolonged exposure to a physiological level of shear stress induces the expression of the iNOS. Its activity is not directly regulated and produces a large amount of NO (13, 24). A small quantity of protein is able to produce significant levels of NO, and this induction leads to a stable and long-term production of NO (12).
This expression can be regulated by numerous extracellular factors, including LPS from bacteria (10, 31) that could be present in our apparatus and then interfere with the effect of shear stress. The amount of LPS in the medium conditioned by a 20-dyn/cm2 shear stress for 24 h was not different from that which was measured in the supernatant of the control cells and was lower than 4 EU/ml. Furthermore, 10 dyn/cm2 shear stress and passive transfer of shear-conditioned medium failed to induce any change in iNOS expression. Thus LPS contamination could not be responsible for the induction we observed.
Our results clearly demonstrate that shear stress is a potent inductor of iNOS expression in smooth muscle cells. They agree with the in vivo data reported by Yan and coworkers (38) showing that iNOS expression is induced in the innermost layers of the neointima in rat carotid arteries after balloon-induced deendothelialization and intimal smooth muscle cell migration. In response to mechanical injury, smooth muscle cells migrate in the intima and proliferate in contact with the blood flow and are thus submitted to shear stress (~20 dyn/cm2) (38). The in vivo expression of the enzyme is localized in the smooth muscle cells that are closest to the lumen, suggesting that the blood flow contact could be necessary for the induction of iNOS expression. Furthermore, in this study, the phenomenon is delayed, occurring only 24 h after deendothelialization. The intensity of the applied shear stress and the delay necessary for iNOS induction in smooth muscle cells correspond to what we have observed in vitro and are consistent with an important role of shear stress in iNOS overexpression in vivo. Recently, Fukuda and coworkers (7) have demonstrated that iNOS expression is involved in the media layer of shear-induced cerebral aneurysm in rats. Their results strongly suggest that the increase in wall shear stress is responsible for the iNOS induction because the reduction of shear attenuated the iNOS immunoreactivity in the artery.
The cytokine-induced iNOS expression is under the control of the
NF-B (33). Because shear stress is able to induce
NF-
B activation in endothelial cells (14), we wondered
whether shear stress-induced iNOS expression in smooth muscle cells
could be mediated by NF-
B activation. One part of NF-
B activation
is due to the phosphorylation of I
B by I
B kinase. I
B kinase
activity is increased by shear stress in endothelial cells
(4) and leads to the dissociation of the cytosolic
I
B-NF-
B complex that is accompanied by the degradation of I
B
(37). Thus the degradation of I
B is considered to be a
marker of NF-
B translocation. Our results show that shear stress
caused a significant decrease in the cytosolic I
B
and
levels
and an increase in NF-
B translocation to the nucleus of smooth
muscle cells. Moreover, PDTC, a potent inhibitor of NF-
B
translocation that acts by scavenging the intracellular reactive oxygen
intermediates (30), abolished the iNOS induction in
response to shear stress. Thus the effect of shear stress on iNOS
expression was, at least in part, mediated by NF-
B.
Translocated NF-B is able to bind both the promoter sequence (called
shear stress responsive element) that is present in many genes in which
expression is modulated by shear stress (29) and an
NF-
B-specific responsive element. The rat iNOS gene promoter has
been cloned (30). No shear stress responsive element has been found, but several functional NF-
B responsive elements have been described and are implicated in the cytokine-induced iNOS expression. Thus shear stress-induced iNOS expression seems to be due,
more in part, to the binding of NF-
B on its specific responsive
element in the iNOS gene promoter than to a direct shear-dependent
response. These data fit well with the delayed character of the response.
The intracellular signaling pathway mechanisms that lead to NF-B
activation in response to shear are not yet identified. However,
regulation of the I
B-NF-
B system is considered to be, in part,
under the dependence of the redox state of the cell. Shear stress is
able to increase superoxide anion production in endothelial cells by
increasing NADPH oxidase activity (5). A similar
phenomenon could occur in stressed smooth muscle cells. It has been
shown that rat aortic smooth muscle cells use a NADH/NADPH oxidase to
generate superoxide anions (36). This enzyme is composed of two membrane-bound [p22phox (36) and Mox-1
(34)] and three cytosolic subunits (p40, p47, and p67) in
smooth muscle cells. The two membrane-associated subunits (p22phox and Mox-1) have been shown to be important for
free radical production. Transfection of smooth muscle cells with
antisense-p22phox inhibits angiotensin II-stimulated
O2
· production, showing that p22phox is
essential in free radical synthesis in these cells (36). Recently, Mox-1 has been described as the active subunit of the NADH/NADPH oxidase in smooth muscle cells (34). In our
experiments, 24 h of a 20-dyn/cm2 shear stress
increased both p22phox and Mox-1 mRNA expression in smooth
muscle cells. Because the transcriptional regulation of these genes has
not yet been described, the mechanism involved in this induction
remains to be determined. These results suggest that oxidative stress
is increased in smooth muscle cells submitted to shear stress. We then
tested the effect of BXT-51072, an efficient antioxidant with
gluthation peroxidase mimicking activity (21), on the iNOS
expression. We have recently shown that BXT-51072 inhibits oxidative
stress-induced VCAM-1 expression in endothelial cells
(26). Because 10
5 M of BXT-51072 abolished
the shear stress-induced iNOS expression, we can conclude that
oxidative stress is implicated in the delayed response of smooth muscle
cells to shear stress.
Our results clearly demonstrate that shear stress is one of the inducers of iNOS expression in aortic smooth muscle cells. This conclusion fits with numerous studies showing that flow inhibits neointima formation after angioplasty (6, 16) and that NO plays a crucial role in this phenomenon (6). Nevertheless, as NO production appeared to be accompanied by an increase in free radical production, it is likely that a part of the synthesized NO was converted into peroxynitrites (3). Because peroxynitrites have been shown to lead to cell disturbances, as well as to the development of atherosclerotic lesions (27), it is difficult to know whether or not iNOS induction in intimal smooth muscle cells is beneficial.
In conclusion, our study shows that shear stress increases oxidative
stress in smooth muscle cells and induces NF-B-dependent gene
expression such as iNOS. Therefore, shear stress could be responsible
for luminal iNOS expression in the neointima in vivo (38).
Conversely, our data suggest that in the response of smooth muscle
cells to arterial wall injury, the shear stress induced by blood flow
could be an important stimulus that follows their intimal migration and
proliferation. Nevertheless, further experiments need to be performed
to better understand the direct or indirect signaling pathways that
lead to the increase in oxidative stress in response to shear stress.
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FOOTNOTES |
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Address for reprint requests and other correspondence: W. Gosgnach, Institut National de la Santé et de la Recherche Médicale u460, CHU X. Bichat, 16, rue H. Huchard, 75870 Paris cedex 18, France (E-mail: gosgnach{at}bichat.inserm.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 June 2000; accepted in final form 14 July 2000.
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