Fibronectin Expression and Aortic Wall Elastic Modulus in Spontaneously Hypertensive Rats
Abstract—Recent studies have shown that large-artery wall remodeling per se does not reduce distensibility in hypertension, indicating qualitative or quantitative changes in arterial components. The aim of the study was to determine in 1-year-old spontaneously hypertensive rats (SHRs) the changes in the elastic properties of large arteries, as assessed by the incremental elastic modulus (Einc), and the changes in the extracellular matrix, including fibronectin (FN) and α5β1-integrin. The relationship between Einc and circumferential wall stress was calculated from in vivo pulsatile changes in blood pressure and arterial diameter by using a high-resolution echo-tracking system at the site of the abdominal aorta and in vitro medial cross-sectional area. Einc-stress curves and FN and integrin α5-subunit contents were determined for each animal. Mean stress and Einc were higher in SHRs than in Wistar rats. However, in a common range of stress, Einc-stress curves for SHRs were superimposable on those for Wistar rats, indicating that wall materials in both strains have equivalent mechanical behavior. Immunohistochemistry indicated that total FN, EIIIA FN isoform, and α5-integrin increased in the SHRs aortas without changes in elastin and collagen densities. Total FN was also increased in SHRs as determined by Western blot analysis. No differences in FN and α5-subunit mRNAs were detected between SHRs and Wistar rats. These results indicate that the aortic wall material of SHRs and Wistar rats have equivalent mechanical properties, although in SHRs it is subjected to a higher level of stress. By increasing cell-matrix attachment sites, FN may participate in the mechanical adaptation of both cellular and matrix components in SHRs.
- Received July 15, 1997.
- Accepted December 18, 1997.
The mechanical properties of large arteries play a major role in cardiovascular hemodynamics through the buffering of stroke volume and the propagation of the pressure pulse.1 2 It is well recognized that mechanical properties of large arteries are primarily determined by the composition of the arterial wall. The ECM proteins, mainly collagen and elastin, influence the “passive” mechanical properties of the arterial wall whereas its “active” properties depend on the activation of VSMCs.
It was generally accepted that hypertension produced an increase in large-artery stiffness.1 2 3 4 However, recent studies have shown that arterial stiffness is not increased, despite wall hypertrophy, in either hypertensive patients or SHRs.5 6 7 8 9 This finding suggests that sustained hypertension is associated with a rearrangement of the arterial wall material, implying qualitative or quantitative changes in arterial components leading to the mechanical adaptation of the arterial wall.
The elastic properties of the arterial wall material depend not only on the SMC, elastin, and collagen contents but also on the way these components are spatially organized within the media.3 10 11 Through an interaction with specific cellular integrin receptors, FN plays an important role in cell-matrix interactions. In addition, FN may also influence VSMC phenotype.12 13 14 The present study was undertaken to relate the changes in the elastic properties of the arterial wall material to its composition in the ECM and to focus on FN and its specific receptor, the α5β1-integrin.
The interaction of specific ECM proteins with their integrin receptors has been shown to play a central role in transmitting mechanical forces to VSMCs.15 When cyclic mechanical strain was applied to matrixes containing different adhesion proteins, FN produced one of the largest mitogenic responses in rat VSMCs.16 In addition, FN expression in the rat aorta increases with age and hypertension.17 18 19 We hypothesized that accumulation of FN and its receptor may play a role in the regulation of elastic properties of large arteries during chronic hypertension.
Our objectives were to determine (1) the intrinsic elastic properties of the arterial wall by evaluating the relationship between the Einc and circumferential wall stress under in vivo conditions and (2) the changes in expression of FN and the α5β1-integrin receptor in the AA of 1-year-old SHRs and Wistar rats. We discovered that SHRs and Wistar rats expressed equivalent elastic properties of the wall material associated with an increase in FN content and its receptors in the aortic media of SHRs. FN may participate in the passive and active mechanical properties of the arterial wall components of SHRs.
All procedures were in accordance with institutional guidelines for animal experimentation. One-year-old male Wistar rats (n=19) and SHRs (n=14) (Iffa-Credo, Fresnes, France) weighing 640±10 and 424±7 g (mean±SEM), respectively, were used in the experiments. For each animal we determined the Einc–wall stress curves, the composition of the AA, and the immunohistologic staining for FN and α5-subunits of integrin. The relation between Einc and EIIIA FN, a variant of FN synthesized by VSMCs,13 was studied in all but 3 SHRs and 4 Wistar rats, in which determination of all arterial parameters was unsuccessful owing to technical reasons.
Determination of Mechanical Parameters
We performed simultaneous recording of arterial diameter and blood pressure in pentobarbital-anesthetized rats. Arterial diameter measurement was obtained by using an ultrasonic echo-tracking device (NIUS-01, Asulab SA).6 7 8 9 20 21 22 23 The relationship between P and the LCSA was fitted with the model of Tardy et al.21
Local arterial cross-sectional C, in the case of a cylindrical vessel, is defined by the change in LCSA for a given change in intravascular P. D is defined by the relative change in LCSA for a given change in intravascular P (see the Appendix for details).
The intrinsic mechanical behavior of the wall material should be assessed through the strain-stress relationship [σ(ε) or ε(σ)]. Because no reference state can be determined in vivo, the strain cannot be evaluated. Thus, we used a derivative expression to characterize the wall material through the Einc.24 25 Einc can be computed from the available parameters under several hypotheses: the arterial wall material is presumed to be homogeneous, isotropic, and incompressible. The circumferential wall stress (σ) was calculated according to Laplace’s law. The determination of Einc and σ required the value of arterial thickness or MCSA. Our echographic system, based on a 12-MHz probe, does not allow the measurement of arterial wall thickness in small animals in vivo (≈100 μm in rats). Therefore, MCSA was not measured in vivo but in vitro after tissue fixation. According to our hypotheses, MCSA is not influenced by blood pressure because of the incompressibility of the arterial wall (see the Appendix for details).
We determined MCSA and the composition of the AA in 4% formaldehyde–fixed arteries. Three successive sagittal sections (5 μm thick in series) were treated by specific staining to obtain a monochromatic color associated with the various structures studied in the aortic media. Sirius red was used for collagen staining, orcein for elastin, and hematoxylin after periodic acid oxidation for nuclear staining. As previously described,6 22 aortic thickness, MCSA, and composition were quantified with an automated image processor and software based on morphological principles.
Immunohistologic staining was performed in the same animals by using a technique whereby fresh, unfixed tissue is freeze-dried and then directly embedded in paraffin wax.26 In brief, a 10-mm segment from the AA was frozen in LN2 and stored at −70° until they were sectioned. On the day of use, frozen tissues were placed on the precooled plate of an Edwards-Pearce tissue dryer and freeze-dried at −45°C, 10-2 torr in the presence of phosphorous pentoxide for 24 hours. The tissue was then allowed to warm to room temperature and embedded in paraffin wax at 55°C to 56°C for 24 hours. Five-micrometer-thick sections were cut, and the paraffin blocks were stored at room temperature. Immediately before they were stained, sections were dewaxed by incubation in xylene for 1 minute followed by 10 minutes of incubation in acetone.
FN and α5-Subunit Immunoperoxidase Staining
The antibodies used were mouse mAbs reactive with an alternatively spliced form of FN, EIIIA (clone IST-9, Sera-Laboratory), all FN isoforms (Total FN, Valbiotech), and a rabbit anti-integrin α5-subunit polyclonal antibody (Valbiotech). The antibody characterization has already been described.27 28 FN mAbs and the α5-integrin polyclonal antibody were diluted 1:200 in Tris-buffered saline, and then we performed the indirect immunoperoxidase technique. In brief, samples were treated with the antibodies to be tested, followed by incubation with a biotinylated anti-mouse and anti-rabbit antibody (kit LSAB2, Dako Laboratories). After 3 extensive washes in Tris-buffered saline, specimens were incubated with streptavidin-peroxidase complex. The presence of peroxidase was revealed after incubation with diaminobenzidine (Sigma Chemical Co). Control sections were made and studied by omitting the first or second antibody. Tissue sections were counterstained with hematoxylin. The distribution and quantification of FN and the α5-integrin subunit were then determined by computer-directed color analysis performed with Quant’Image software (Quancoul). The number of positive pixels was determined as already described with minor modifications.29 To account for the color intensity as an index of the intensity of antigenic detection, we expressed the antigenic intensity in arbitrary units as the product of the mean percentage of positive-labeled surface and the hue intensity of the same positive pixels.
RNA Isolation and Northern Blot Analysis
To obtain a sufficient amount of total RNA for electrophoresis, tissue from the distal AA and the proximal thoracic aorta from each animal was pooled. Total RNA was extracted by the guanidinium thiocyanate–phenol-chloroform extraction method. Denatured total RNA (20 μg) was size fractionated by agarose gel electrophoresis and transferred to a nylon membrane (Hybond N+, Amersham). After RNA fixation by UV cross-linking (0.15 J/cm2), the membrane was prehybridized for 1 hour with Rapid hybridization buffer (Amersham) at 55°C or 65°C for oligonucleotide or cDNA probes, respectively. Hybridization was carried out at 65°C for 2 hours using cDNA probes and at 55°C using synthetic oligonucleotide probes. The hybridized membrane was washed under stringent conditions (2× SSC and 0.1% SDS) for 30 minutes at room temperature and then 1× SSC and 0.1 SDS for 10 minutes at 50°C and exposed to x-ray film (Kodak X-Omat) by use of intensifying screens (Cronex Lightning Plus) for 12 to 36 hours at −70°C. Laser densitometry (Instar) was used to quantify the relative signal intensity of the bands obtained as normalized to the hybridization signal obtained with an 18S RNA probe to correct for differences in loading or transfer efficiencies. The cDNA probes used (human FN and α5-chain) were purchased from Gibco BRL and labeled with the use of [α32-]dCTP and a random-primer kit (Amersham). Probes for the 18S RNA were end-labeled synthetic oligonucleotides.30 Each experiment was repeated twice with samples from the same animals.
Western Blot Analysis of FN Protein
To confirm the results of the immunohistochemistry experiments, a Western blot analysis of FN was performed on the remaining tissue from the distal part of the thoracic aorta. Arterial tissue from SHRs and Wistar rats was carefully cleaned and cut into rings by standard techniques that have described elsewhere.19 Total protein content was determined by the Bradford technique. Protein extracts were reconstituted in sample buffer containing 0.5 mol/L Tris HCl, 10% SDS, 10% glycerol, and 5% β-mercaptoethanol, and the mixture was boiled for 5 minutes. Equal amounts (50 μg) of the denatured proteins were loaded per lane, separated on a 4% to 15% SDS polyacrylamide gel (Mini Protean II, Bio-Rad), and transferred to a nitrocellulose membrane (Hybond-C Extra) in a 25 mmol/L Tris–192 mmol/L glycine buffer solution (pH 8.3) with 20% (vol/vol) methanol overnight at 20°C. The membrane was blocked with 10% evaporated milk (Carnation). Membranes were incubated with a mouse anti-human mAb to all FN isoforms at a dilution of 1:1000 (Valbiotech). Subsequent analysis utilized a biotinylated goat anti-rabbit IgG+ streptavidin peroxidase complex, diluted 1:5000, as the second antibody, and chemiluminescence emitted from the luminol oxidized by peroxidase was used as the detection method (ECL Western blotting detection system, Amersham).
All values were averaged and expressed as mean±SEM. Unpaired Student’s t tests were performed to compare SHRs with Wistar rats. Differences were considered significant for values of P<0.05. The different mechanical arterial parameters (internal diameter, D, Einc, and wall stress) of SHRs were compared with those of Wistar rats at MAP and for a given blood pressure level common to all animals (132 mm Hg: Einc132). Einc-stress curves were compared for a given circumferential wall stress common to both groups (150 kPa). Differences in morphology between SHRs and Wistar rats were evaluated by unpaired Student’s t tests.
Hemodynamics and Aortic Mechanics
The SHRs at 1 year of age were significantly lighter than Wistar rats. The MAP was significantly higher in SHRs, with no change in heart rate (Table 1⇓). The diameter-P, D-P, and Einc-P curves of SHRs were shifted to the right compared with those of Wistar rats (Figure 1A⇓ through 1C). At a common level of blood pressure (132 mm Hg), D values of SHRs were significantly higher (37%) and Einc significantly lower (−36%) than in Wistar rats. Circumferential stress at 132 mm Hg was not significantly reduced in SHRs. At MAP, which reflects the operational pressure level, D was significantly lower (−35%) and Einc and wall stress higher (47% and 37%, respectively) in SHRs than in Wistar rats.
The Einc-stress curve of SHRs (Figure 2⇓) was shifted rightward and upward with respect to the curve for Wistar rats and appeared to be an of the curve for Wistar rats; within the common range of wall stress (140 to 160 kPa), however, Einc in SHRs was not significantly different from that of Wistar rats, indicating equivalent intrinsic mechanical behavior of wall materials in both groups. Thus, the higher Einc of the aortic wall in SHRs under physiological conditions was explained by the higher level of circumferential wall stress.
Composition of the AA
Table 2⇓ shows that aortic thickness, MCSA, and MCSA-to–body weight ratio were significantly greater in SHRs than in Wistar rats. Collagen and elastin densities were not significantly increased in SHRs. Collagen content, but not elastin content, was significantly increased in SHRs compared with Wistar rats. The number of nuclei of SMCs was similar in SHRs and Wistar rats, whereas the size of nuclei was significantly increased in SHRs.
Aortic FN and α5-Integrin Subunit Expression
Northern Blot Analysis
Aortic FN and α5-subunit mRNAs were similar in SHRs and Wistar rats (Figure 3⇓).
Western Blot Analysis
Total aortic FN content was higher in SHRs than in Wistar rats (Figure 4⇓). Densitometric quantification indicated that the amount of protein had increased by 2-fold in SHRs compared with Wistar rats.
In the AA of Wistar rats, cellular FN immunoreactivity for the isoform EIIIA was prominently seen in the internal part of the media (Figure 5⇓). In SHRs, EIIIA FN reactivity was detected diffusely in the media, and EIIIA FN–positive staining was significantly increased (by 3-fold, P<0.01) compared with Wistar rats (Table 3⇓). The quantification of the amount of positively labeled surface is not sufficiently accurate to use color intensity as an estimate of the antigenic amount. We expressed antigenic quantification in arbitrary units as the product of the amount of labeled surface and the mean hue density. This parameter, indicated in Table 3⇓ as antigenic content, was higher in SHRs than in Wistar rats. A significant increase was also observed for the total FN immunoreactivity.
In Wistar rats, α5-subunit–positive staining was relatively low throughout the aorta and appeared to have the same distribution within the media as the total FN. α5-Subunit–positive staining and α5-antigenic content were greater in SHRs than in Wistar rats (by 2- and 2.5-fold, respectively, P<0.05).
The aim of the present study was to determine concomitant changes in the intrinsic mechanical properties and the ECM of the AA in 1-year-old SHRs compared with those of Wistar rats. The main findings reported here are as follows: (1) In 1-year-old SHRs, the Einc-stress curves were superimposable, (2) the EIIIA FN isoform was markedly increased within the media in SHRs without changes in elastin and collagen densities, and (3) α5β1-integrin content was higher in SHRs than in Wistar rats.
Arterial Wall Mechanics
The method used to establish the in vivo diameter–P and D-P curves in rats has been considered in detail previously in both humans8 9 20 21 and rats.6 7 22 23 Whereas arterial D evaluates the elastic properties of the artery as a hollow structure, Einc evaluates the elastic properties of the wall material.10 25 The arterial wall is not homogeneous and is composed of various elements, including SMCs, collagen, elastin, and various components of ECM. All of these elements contribute to the mechanical behavior of the wall material through their own elastic modulus and the way in which they are arranged.1 3 10 Because the spatial arrangement of wall materials is dependent on the level of circumferential wall stress,3 we compared Einc of SHRs and Wistar rats within a common range of circumferential wall stress. To our knowledge, this is the first study in which the Einc-stress curve of large arteries has been established in rats under physiological conditions of pressure, flow, and innervation.
One of the main findings of the present study is that in SHRs, the Einc of the aortic wall material, determined for a given level of circumferential wall stress, was not significantly different from that of Wistar rats. This indicates that wall materials in both rat strains have similar mechanical properties and can be considered equivalent. These results are consistent with previous data obtained in humans and some in vitro work in animals. Indeed, Laurent et al9 have previously shown that despite increased wall thickness, the stiffness of the radial artery wall material, as assessed by the Einc-stress curve, was not increased in hypertensives.9 At the site of pial arterioles and small branches of the posterior cerebral arteries, Baumbach et al31 and Hajdu and Baumbach32 reported that the Einc determined in vivo at a given wall stress was lower in stroke-prone SHRs than in normotensive rats. In 6-month-old-SHRs, van Gorp et al33 reported that the value of Einc of the thoracic aorta, calculated at comparable low pressure, was not different between SHRs and Wistar-Kyoto rats. Thus, the present study extends, to large arteries of SHRs, the concept proposed for rat small arteries31 34 and human large arteries5 : arterial wall remodeling with hypertension is not necessarily associated with increased stiffness. This study also explains why under in vivo conditions, the intrinsic stiffness of the aortic wall material is higher in SHRs than in Wistar rats. Indeed, the physiological conditions of the two groups corresponded to two different points on the same Einc-stress curve, with a higher level of circumferential wall stress in SHRs. Thus, in SHRs, the mechanical adaptation of the arterial wall is incomplete, because equivalent wall material is subjected to a higher level of circumferential stress. Under these conditions, the important question is to determine how the arterial wall of SHRs can be protected against this higher level of mechanical stress.
We found no differences between SHRs and Wistar rats concerning elastin and collagen densities. Because these two compounds play a major role in the mechanical properties of the arterial wall, this result is in agreement with the maintenance of the intrinsic elastic properties of SHRs compared with Wistar rats.
The second major finding of the present study is that FN at the protein level, and more specifically the EIIIA isoform, was increased in the aortic media of SHRs compared with Wistar rats. We hypothesize that accumulation of FN, through changes in cell-matrix interactions, may play a role in the adaptation of aortic wall material to the higher level of circumferential wall stress.
Aortic FN and α5β1-Integrins During Hypertension
Previous studies have shown that the expression of aortic FN is increased in SHRs17 18 19 and in vitro models with induced hypertension,35 36 with a very prominent induction of EIIIA FN.17 We studied the SHR because this animal is the most representative model of human essential hypertension and therefore the one in which arterial mechanics has been the best described. The α5β1-integrin is a higher-affinity receptor specific for FN.12 37 38 Vascular expression of this receptor has been shown to be downregulated during development.39 40 However, the effects of hypertension on vascular α5β1-integrin have not been studied, despite overexpression of FN. We studied α5-subunit expression because the α-subunit, by contrast to the β-subunit, is responsible for the binding specificity of integrin α5β1 with FN. In the present study, total aortic FN and α5 mRNAs were similar in both groups, suggesting that the observed increase in FN and α5 subunit contents in SHRs are unlikely caused by transcriptional factors.
At the protein level, we found that both cellular and total FN contents as determined by immunohistochemistry were significantly increased in SHRs compared with age-matched normotensive Wistar rats. These results were confirmed by Western blot analysis for total FN. To our knowledge, the only published immunohistochemical study of aortic EIIIA FN in hypertension is that by Contard et al,41 which did not show any change in EIIIA FN in stroke-prone SHRs compared with Wistar-Kyoto rats. The apparent discrepancy between that study and ours may originate from different genetic models of hypertension (SHRs versus stroke-prone SHRs), the age of the animals used, and the method used for immunohistologic staining (freeze-dried, paraffin-embedded sections versus immunofluorescence). Using an α5-integrin polyclonal antibody, we found that SMCs from SHRs expressed significantly greater amount of α5-integrin subunits. The functional relevance of a higher expression of α5β1-integrin in SMCs from SHRs is consistent with the changes in expression of its specific ligand, FN.
FN and Aortic Wall Mechanics
Whether FN accumulation, specifically EIIIA FN, plays a role in the mechanical properties of large-artery walls of SHRs implies that one must take into account the complex interactions between FN, cells, and ECM proteins. FN is a major cell attachment glycoprotein. FN binds to VSMCs via specific integrin receptors, including α5β1.12 The α5β1-integrin has also been implicated in the assembly of FN matrixes in relation to other ECM proteins, including collagen.42 43 Finally, the FN receptor is also able to bind to various intracellular components at adhesion plaques,12 44 suggesting that α5β1-integrins may be involved in the transduction of signals and mechanical forces mediated by the ECM.
In the present study, the parallel increase in EIIIA FN density and FN receptor α5β1-integrin may reflect an increased number of mechanical attachments between the ECM, cells, and collagen fibers within the media of SHRs. From a mechanical point of view, an increase in the number of cell-matrix attachments leads to an increase in the passive stiffness of the arterial wall material. However, modifications of tissue organization are associated with cellular phenotypic modulation, including changes in adhesive properties. Therefore, in vivo, the degree of activation of SMCs may influence the global stiffness of the wall material through variable “recruitment” of anchorage sites.
Therefore, additional changes in SMC phenotypic characteristics should be evoked. Selective induction of EIIIA isoform expression has been shown to be correlated with a phenotypic shift to a less-differentiated phenotype in VSMCs.14 45 46 47 Moreover, several previous studies have demonstrated the presence of less-differentiated cells in the aortas of renovascular hypertensive rabbits and stroke-prone SHRs, as suggested by the increase in nonmuscle myosin heavy-chain content and medial hypertrophy.41 48 49 Our finding of an increase in EIIIA FN and its receptor in SHRs is in keeping with these data, showing that some degree of a dedifferentiation process may occur in the aortas of SHRs. The mechanical properties assessed by the in vivo Einc-stress relationship depend on both the passive MEC behavior and the phenotypic characteristics of VSMCs. We suggest that both phenotypic changes and increases in cell-matrix attachment sites contribute to the mechanical adaptation of the arterial wall in SHRs.
In conclusion, the present study demonstrates that the wall material of SHRs and Wistar rats has equivalent mechanical properties although in SHRs it is subjected to a higher level of stress. Our results also provide evidence for an increase in aortic EIIIA FN and α5β1-integrin immunostaining in SHRs. We suggest that FN may participate in the mechanical adaptation of the arterial wall in SHRs owing to an increase in the number of cell-matrix attachment sites associated with SMC phenotypic changes.
Selected Abbreviations and Acronyms
|Einc||=||incremental elastic modulus|
|LCSA||=||lumen cross-sectional area|
|MCSA||=||medial cross-sectional area|
|SHR||=||spontaneously hypertensive rat|
|(V)SMC||=||(vascular) smooth muscle cell|
Expression of Einc as a Function of Circumferential Stress
The relationship between P and LCSA was fitted with the model of Langewouters et al21 by using an arctangent function and three optimal-fit parameters (α, β, and γ): where d is the internal diameter (under the assumption that the vessel is cylindrical).
Due to the nonlinearity of the cross section–P curve, C decreases as blood pressure increases. To determine C for a given level of blood pressure, we established the C-P curve for the entire systolic-diastolic range. This was done by deriving the equation of the P -section curve. By using Equation (1), the following analytical form was obtained for local arterial C: Local arterial cross-sectional D is defined by the relative change in LCSA for a given change in intravascular P: The circumferential wall stress (σ) was calculated according to Laplace’s law with the following equation: The Einc is defined as the equivalent of Young’s modulus in an infinitesimal transform δσ(δε). The in vivo measurements provide the relationship between the arterial diameter and the arterial blood pressure over the systolic-diastolic range. The calculation of Einc was done under several hypotheses. We determined the mechanical properties of equivalent material that occupied the same space as the real one. This material is assumed to be homogeneous, isotropic, and incompressible. We also hypothesized that the artery was cylindrical.
Using these hypotheses, we were able to use a well-known method from mechanical engineering that calculates the parameters of interest for thick-walled pipes. The reference state is defined by rint and rext, the internal and external radii, respectively, and Pint and Pext, the internal and external Ps, respectively. A change δP in Pint leads to variations δε in strain and δσ in stress. The material state is described by the displacement field u⟶. Because of symmetry, the displacement is radial and depends only on the radius r: u⟶=u(r)e⟶r in cylindrical coordinates. The development gives a linear relationship: where the double overbar indicates that both stress and strain are tensors. λ and μ are the Lamé coefficients related to Einc and Poisson’s ratio ν: Stress can be expressed by the quasistatic equation divδσ̿=0. If one takes into account the boundary limits for r=rint and r=rext, straightforward computation yields the following: Because incompressible material is characterized by ν=1/2, The experimental data are the variations of dint (or r): δrint=u(rint); then If the values for LCSA and MCSA are introduced, where LCSA=πrint2 and MCSA=π(rext2−rint2) and the expression for D is given by the expression for Einc is given by With previous notation, circumferential stress δσθθ is given by and we can retrieve the mean stress value given by Laplace’s law: To obtain the Einc–wall stress relationship, we numerically combined Equations 5, and 13 for each individual animal. The mean curves relating Einc to circumferential wall stress curves were then calculated by averaging the values of Einc for a given level of stress.
This study was supported by grants from INSERM (494014) to Dr F. Iannascoli from Zeneca Pharma Laboratories. We thank M. Glukhova for comments on the manuscript; J. Lavallée for help in quantification of the immunochemistry results; and B. Lucet, K. Le Dudal, and C. Perret for technical assistance.
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