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Am J Physiol Heart Circ Physiol 293: H2597-H2604, 2007. First published July 27, 2007; doi:10.1152/ajpheart.00299.2007
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Role of {alpha}1beta1-integrin in arterial stiffness and angiotensin-induced arterial wall hypertrophy in mice

Huguette Louis,1,2,3 Augustine Kakou,1,2 Veronique Regnault,2,4 Carlos Labat,1,2 Aude Bressenot,2 Jacqueline Gao-Li,5,6 Humphrey Gardner,7 Simon N. Thornton,1,2 Pascal Challande,6,8 Zhenlin Li,5,6 and Patrick Lacolley1,2

1Institut National de la Santé et de la Recherche Médicale, U684, 2Henri Poincare University, and 4Institut National de la Santé et de la Recherche Médicale, U734, Nancy; 5Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7079 and 6Pierre et Marie Curie University, Paris; 8Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7190, Saint-Cyr l'Ecole, France; 3Centre de Recherche Public-Santé, Laboratory of Cardiovascular Research, Luxembourg; and 7Biogen, Incorporated, Cambridge, Massachusetts

Submitted 9 March 2007 ; accepted in final form 21 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We examined the arterial phenotype of mice lacking {alpha}1-integrin ({alpha}1–/–) at baseline and after 4 wk of ANG II or norepinephrine (NE) administration. Arterial mechanical properties were determined in the carotid artery (CA). Integrin expression, MAPK kinases, and focal adhesion kinase (FAK) were assessed in the aorta. No change in arterial pressure was observed in {alpha}1–/– mice. Elastic modulus-wall stress curves were similar in {alpha}1–/– and {alpha}1+/+ animals, indicating no change in arterial stiffness. The rupture pressure was lower in {alpha}1–/– mice, demonstrating decreased mechanical strength. Lack of {alpha}1-integrin was accompanied by an increase in beta1-, {alpha}v-, and {alpha}5-integrins but no change in {alpha}2-integrin. ANG II increased medial cross-sectional area of the CA in {alpha}1+/+, but not {alpha}1–/–, mice, whereas equivalent pressor doses of NE did not produce a significant increase in either group. In {alpha}1+/+ mice, ANG II induced {alpha}1-integrin expression and smooth muscle cell (SMC) hypertrophy in the CA in association with increased aortic expression of {alpha}-smooth muscle actin and smooth muscle myosin heavy chain and phosphorylation of ERK1/2, p38 MAPK, and FAK. ANG II did not induce SMC hypertrophy or phosphorylation of p38 MAPK and FAK in {alpha}1–/– mice. A functional anti-{alpha}1-integrin antibody inhibited in vitro the ANG II-induced phosphorylation of FAK and p38 MAPK. In conclusion, {alpha}1–/– mice exhibit a reduced mechanical strength at baseline and a lack of ANG II-induced SMC hypertrophy. These results emphasize the importance of {alpha}1beta1-integrin in p38 MAPK and FAK phosphorylation during vascular hypertrophy in response to ANG II.

arterial mechanical properties; vascular hypertrophy; MAPK kinases; integrin expression; smooth muscle cells

CELL-MATRIX INTERACTIONS play a major role in vascular smooth muscle cell (SMC) function (hypertrophy, proliferation, and differentiation) and mechanical properties of the vascular wall. Integrins, transmembrane heterodimers consisting of an {alpha}- and a beta-subunit, are major receptors for extracellular matrix (ECM) ligands. Some studies reported that increased {alpha}5beta1- and {alpha}vbeta3-integrin expression may modulate functional adaptation and remodeling of large and resistance arteries in hypertension (2, 4, 15, 16). These studies suggested that integrins may play an important role in regulating arterial elasticity.

Among the integrins, {alpha}1beta1-integrin is a major receptor for collagens (3). In vivo, it is highly expressed in human aortic media, coronary arteries, and the microvascular endothelium. Expression of {alpha}1-integrin was also detected on SMCs in the aortic arch in mice (30).

The principal functional consequences of deletion of {alpha}1beta1-integrin in mice are increased collagen and collagenase synthesis in the dermis and decreased proliferation of dermal fibroblasts (10, 29) due to failure of the {alpha}1-integrin-deficient fibroblasts in vitro to activate the Ras-Shc-MAPK pathway promoting cell proliferation. Mice lacking {alpha}1-integrin ({alpha}1–/–) show normal development of the vascular system (11) but exhibit a reduced tumor angiogenesis (28). Apolipoprotein E (ApoE)-{alpha}1-integrin double-knockout mice develop smaller advanced aortic plaques than ApoE-knockout mice, suggesting that {alpha}1-integrin plays a role in arteriosclerosis (30). However, the vascular phenotype of {alpha}1–/– mice has not been reported.

The ANG II pathway is implicated in the hypertrophic response of the vascular wall via MAPK kinases (ERK1/2, JNK, and p38 MAPK) and focal adhesion kinase (FAK) (23). Although ANG II enhances integrin expression in cardiac fibroblasts (7, 19), supporting a role for integrins in ANG II-induced cardiac remodeling (31), the influence of ANG II on SMC integrin expression (17), especially during vascular hypertrophy, has received little attention. In addition, it has been reported recently that concomitant administration of ANG II and the ANG type 1 receptor blocker losartan increased expression of {alpha}1beta1-integrin in rats, suggesting a role for ANG II in the regulation of {alpha}1beta1-integrin in SMCs (6).

Our objective was to examine the role of {alpha}1-integrin in arterial mechanical properties during hypertrophic remodeling. Thus, using high-resolution techniques that have been validated in the common carotid artery (CA) in vivo in mice (24), we have investigated arterial stiffness and mechanical strength in {alpha}1–/– and {alpha}1+/+ mice. We then examined modifications of carotid morphology and aortic SMC signaling molecules in response to ANG II. We report decreased arterial strength at baseline, decreased carotid wall hypertrophy, and changes in vascular signaling by p38 MAPK and FAK in ANG II-treated {alpha}1–/– mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Mice with a null mutation in {alpha}1-integrin have been described previously by Gardner et al. (11). Sixty-five {alpha}1–/– mice were used in these experiments together with age-matched (6–9 mo old) littermate wild-type ({alpha}1+/+) mice (n = 67). The CA was used for mechanical measurements, histomorphometry, and markers of cell proliferation. Integrin expression and SMC signaling molecule analysis were determined in the aorta. All procedures were carried out in accordance with institutional guidelines for animal experimentation and were approved by the Institut National de la Santé et de la Recherche Médicale U684 (Nancy).

Indirect tail-cuff sphygmomanometer blood pressure measurement. The noninvasive indirect tail-cuff sphygmomanometer method allowed measurement of baseline systolic arterial pressure (SAP) in conscious mice. The mouse was kept in a warmed black box, and an inflatable cuff was applied to the base of the tail. The tail was then placed on a piezoelectric sensor for analysis of the pressure waveforms. After inflation, SAP was determined as the moment when the waveform falls to a value indistinguishable from baseline noise.

In vivo arterial mechanical parameters. Using an ultrasonic echo-tracking device (model NIUS-01, Asulab), we recorded simultaneously intra-arterial diameter (left CA) and blood pressure (right CA) in pentobarbital sodium-anesthetized mice, as previously described (20, 24). The relationship between pressure and luminal cross-sectional area (CSA) was fitted using an arctangent function. CSA distensibility (Dist), a derivative of this function, was used to assess the global elastic behavior of the artery. Circumferential wall stress (WS) and incremental elastic modulus (Einc), which characterizes the intrinsic mechanical properties of the wall material, were calculated with the above-mentioned parameters and the medial CSA area (MCSA).

The mechanical strength of the intact CA was characterized by determination of the in vitro intraluminal pressure leading to vascular wall rupture, as previously described (8). This parameter provides additional information concerning fragility of the vascular wall material (22).

Histomorphometry and immunohistochemistry. Sections of formalin-fixed (under pressure in vivo), paraffin-embedded CA were stained with hematoxylin and eosin for nuclei and Sirius red F3B for collagen. Weigert's resorcin-fuchsin method was used for elastic fibers and an alcian blue pH 2.5 stain for proteoglycans. MCSA and matrix composition were determined by computer-directed color analysis.

Immunohistochemical detection of {alpha}-smooth muscle actin ({alpha}-SMA) was carried out on formalin-fixed, paraffin-embedded CA sections with a non-species-specific mouse monoclonal antibody (Dakopath; 1:600 dilution). The fraction of proliferating SMCs in the media and phosphorylated p38 were measured on sections stained with a non-species-specific rabbit monoclonal antibody to the cell cycle-associated antigen Ki-67 (clone SP6, Labvision, Westinghouse; 1:100 dilution) and a rabbit polyclonal anti-phosphorylated p38 antibody (Cell Signaling; 1:50 dilution). Antibodies were applied to tissue sections that had been heated in a microwave oven for 5 min in 0.5 M EDTA, pH 8. Primary antibodies were applied for 16 h at 4°C. Tissue-bound primary antibodies were detected using the biotinylated antibody-streptavidin-peroxidase detection system. Bound peroxidase was identified using the Novared detection system (Vector).

Isolation of mRNA and gene expression. Total RNA was isolated from the aorta with the RNeasy fibrous tissue kit (Qiagen). First-strand cDNA was synthesized according to the manufacturer's instructions (Roche). Quantitative PCR analysis was then performed with SYBR green PCR technology (ABGene) with use of the following primers: 5'-aactttggcattgtggaagg-3' and 5'-ggatgcagggatgatgttct-3' for GAPDH, 5'-caaatgagcctggaaccaat-3' and 5'-ccatccacgttgaggtcttt-3' for {alpha}1-integrin, 5'-caaggtgacaggactcagca-3' and 5'-ggtctctggatccaactcca-3' for {alpha}5-integrin, 5'-gggacagggagaaaggagtc-3' and 5'-gattccacagcccaaagtgt-3' for {alpha}v-integrin, 5'-cgcaggtgtattttggaggt-3' and 5'-ttcaaggttgctgtgacgag-3' for {alpha}6-integrin, and 5'-tcacatgcaggtttggaaaa-3' and 5'-tgtgacctcagctgacaagg-3' for beta1-integrin. Relative quantification was achieved with the following equation: R = 2{Delta}Ct(target) (control – sample) – {Delta}Ct(Ref) (control – sample), where Ct is threshold cycle (www.gene-quantification.de) and GAPDH was used as the reference (Ref) transcript. Results from three independent RT-PCR analyses are expressed as the ratio of mutant to control samples.

Chronic administration of pressor doses of ANG II. Osmotic minipumps (model 2004, Alza) containing Val5-ANG II were implanted to permit subcutaneous infusion of ANG II for 28 days. Similar pumps were used to compare action of ANG II (200 ng·kg–1·min–1) with that of norepinephrine (NE, 1.73 µg·kg–1·min–1).

Western blot. Aortas from {alpha}1–/– and {alpha}1+/+ mice were ground in ice-cold lysis buffer containing 20 mM Tris·HCl, 5 mM EDTA, 0.5% NP-40, phosphatases, and protease inhibitors. Detergent-soluble fractions were retained, and protein concentrations in samples were equalized using a Bradford protein assay. For Western blot analysis, lysates containing 40 µg of protein were electrophoresed on polyacrylamide gels and transferred to Hybond-P polyvinylidene difluoride membranes (Amersham).

Arterial SMCs at passage 3–5 derived from thoracic aortas of 6-wk Wistar rats were treated with ANG II for 15 min–24 h. Cells were placed in 0.5% fetal bovine serum 2 days before ANG II treatment. We used a low dose (1–5 nM) of ANG II to approach our in vivo conditions, i.e., 200 ng·kg–1·min–1. Cells were lysed with a Kinexus buffer (www.kinexus.ca). To assess the effect of {alpha}1-integrin inhibition, cells were preincubated with an anti-{alpha}1-antibody (Pharmingen; 10 µg/ml) 30 min before ANG II treatment.

Membranes were probed with antibodies to {alpha}1-, {alpha}2-, {alpha}5-, {alpha}v-, beta1-, and beta3-integrins and {alpha}-SMA (Sigma); smooth muscle myosin heavy chain (SM-MHC; Biomedical Technologies); GAPDH (Santa Cruz Biotechnology); ERK1/2, phosphorylated ERK1/2, FAK, phosphorylated (Tyr576/577) FAK, MKK3/6, phosphorylated MKK3/6, p38, phosphorylated p38, JNK, and phosphorylated JNK (Cell Signaling); and phosphorylated (Tyr397) FAK (Upstate). An enhanced chemiluminescence system was used as the detection method (Amersham).

Statistical analysis. Values are means ± SE. Unpaired Student's t-tests or two-way analysis of variance was performed to compare the two strains with and without treatment. Dist and Einc were logarithmically transformed to generate linear relations (24). The quality of the transformation was checked by calculation of the R2 of the linear regression obtained with the new parameters for each individual. After this transformation, we calculated the mean slopes of the curves. If the slopes were not significantly different, we compared the curves by using the median values of the common ranges of either AP for Dist or Einc for WS. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Arterial phenotype at baseline. Figure 1 shows {alpha}1-integrin (a 380-bp amplified fragment) expression in the aorta of {alpha}1+/+ mice, but not {alpha}1–/– mice. The transcripts of {alpha}5-, {alpha}v-, and beta1-integrins in the aorta were increased by seven-, four-, and threefold, respectively, in {alpha}1–/– compared with {alpha}1+/+ mice. There was a parallel increase (1.5-, 1.5-, and 1.6-fold, respectively) in protein levels. No difference was detected in {alpha}6-integrin cDNA products between strains.


Figure 1
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  		Fig. 1. Quantitative RT-PCR and Western blot analysis of integrin expression in aorta from control (wild-type) mice ({alpha}1+/+) and mice lacking {alpha}1-integrin ({alpha}1–/–). A: {alpha}1-integrin from {alpha}1–/– and {alpha}1+/+ mice (n = 2 in each group). B and C: relative RNA and protein expression of {alpha}5-, {alpha}v-, and beta1-integrins in aorta of {alpha}1–/– and {alpha}1+/+ mice (n = 6 in each group). GAPDH was used as a loading control for protein levels.

 
Under basal conditions, SAP and heart rate were not different in conscious (measured by tail-cuff sphygmomanometry) and anesthetized {alpha}1–/– and {alpha}1+/+ mice (Table 1). There was no difference in diastolic arterial pressure, mean arterial pressure, and pulse pressure between anesthetized {alpha}1–/– and {alpha}1+/+ mice. CA luminal diameter, Dist, Einc, and WS at mean arterial pressure were similar in {alpha}1–/– and {alpha}1+/+ mice (Table 1). Dist-pressure curves did not differ between the two strains at baseline; i.e., the slopes were not different: Dist at 80 mmHg = 6.4 ± 0.9 x10–3 and 7.1 ± 1.0 x 10–3 mmHg–1 in {alpha}1+/+ and {alpha}1–/– mice, respectively [P = not significant (NS); Fig. 2A]. Einc-WS curves showed no difference in WS at 500-kPa Einc (WS500 = 153 ± 19 and 196 ± 40 kPa in {alpha}1+/+ and {alpha}1–/– mice, respectively, P = NS), indicating equivalent intrinsic elasticity (Fig. 2B). However, the pressure inducing CA wall rupture (Table 1) was significantly reduced in {alpha}1–/– mice, indicating a lower mechanical strength of the arterial wall.


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  		Table 1. Blood pressures and mechanical properties of the CA in {alpha}1+/+ and {alpha}1–/– mice

 

Figure 2
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  		Fig. 2. Distensibility (Dist)-arterial pressure (AP) curve (A) and incremental elastic modulus (Einc)-wall stress (WS) curve (B) in ANG II-treated carotid artery of {alpha}1+/+ [n = 9 for control, non-ANG II-treated (C) and n = 12 for ANG II-treated] and {alpha}1–/– (n = 7 for C and ANG II) mice. Values are means ± SE.

 
MCSA did not differ between {alpha}1–/– and {alpha}1+/+ mice (Table 2). There were no visible modifications of the elastic lamellae in {alpha}1–/– mice. Collagen and elastic fiber densities and contents and nuclear number and density were similar in both strains. A very low expression of Ki-67 proliferative index was detected on SMCs in both groups.


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  		Table 2. Effects of ANG II on matrix and SMCs in CA from {alpha}1+/+ and {alpha}1–/– mice

 
Effects of ANG II. ANG II or NE produced a significant increase in mean arterial pressure in {alpha}1–/– and {alpha}1+/+ mice (Fig. 3). There was no difference between the pressor effects of ANG II and NE in the two strains. In both strains, ANG II produced a significant increase in mean arterial luminal diameter (511 ± 29 and 395 ± 27 µm in ANG II-treated {alpha}1+/+ mice and 480 ± 22 and 365 ± 17 µm in ANG II-treated and -untreated {alpha}1–/– mice). Similar results were observed with NE treatment. ANG II induced vascular wall hypertrophy only in {alpha}1+/+ mice. MCSA was significantly lower in ANG II-treated {alpha}1–/– than ANG II-treated {alpha}1+/+ mice (Table 2). NE did not induce a significant increase in MCSA in {alpha}1–/– or {alpha}1+/+ mice (Fig. 3).


Figure 3
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  		Fig. 3. Effects of ANG II and norepinephrine (NE) administration on mean arterial pressure (MAP), medial cross-sectional area (MCSA), and diameter (Diam) at MAP of carotid artery from {alpha}1+/+ (n = 9 for control, n = 12 for ANG II, and n = 6 for NE) and {alpha}1–/– (n = 7 for control, n = 7 for ANG II, and n = 6 for NE) mice. Values are means ± SE. *P < 0.05 vs. control of the same strain. {ddagger}P < 0.05 vs. the same treatment in {alpha}1+/+ mice.

 
ANG II induced a rightward shift of the Dist-pressure curves in both strains (Fig. 2). Dist did not differ between the two strains in response to ANG II; i.e., slopes were not different: Dist at 100 mmHg = 7.6 ± 1.4 x10–3 and 7.8 ± 0.9 x 10–3 mmHg–1 in {alpha}1+/+ and {alpha}1–/– mice, respectively (P = NS). To evaluate whether the lack of arterial wall hypertrophy in {alpha}1–/– mice might change the intrinsic elastic properties of the arterial wall, we studied the effects of ANG II on Einc-WS curves. ANG II did not induce a significant difference in elastic behavior in {alpha}1+/+ mice, since, in the common range of Einc, the two Einc-WS curves did not differ: WS500 = 153 ± 19 and 199 ± 23 kPa in untreated and ANG II-treated {alpha}1+/+ mice (P = NS). In {alpha}1–/– mice, results were similar: WS500 = 196 ± 40 and 246 ± 26 kPa in untreated and ANG II-treated {alpha}1–/– mice (P = NS). With ANG II treatment, WS at >600-kPa Einc was higher in {alpha}1–/– than in {alpha}1+/+ mice, indicating a higher elasticity in {alpha}1–/– mice.

The histological changes in ANG II-treated {alpha}1+/+ included an increase in {alpha}-SMA-positive areas associated with a parallel enlargement of the space between the elastic lamellae or their surrounding collagen fibers compared with untreated {alpha}1+/+ mice (Fig. 4). These changes were not associated with a reduplication of elastic laminae, nor were they associated with an increased density of collagen fibers. On alcian blue-stained sections, the ground substance appeared normal. The number of nuclei per unit surface was lower in ANG II-treated {alpha}1+/+ than untreated {alpha}1+/+ mice, but the total number of nuclei per MCSA was not different. Finally, on sections stained for Ki-67, no SMC nuclei were stained. These results indicate that thickening of the media in ANG II-treated {alpha}1+/+ mice was due to cellular hypertrophy. These effects of ANG II on immunostaining of {alpha}-SMA and collagen fibers were not observed in {alpha}1–/– mice (Fig. 4).


Figure 4
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  		Fig. 4. Histomorphometry of carotid artery from {alpha}1+/+ and {alpha}1–/– mice and effects of ANG II. Transverse sections were stained with an {alpha}-smooth muscle actin ({alpha}-SMA) antibody to show SMC hypertrophy and Sirius red to show collagen fibers.

 
ANG II significantly increased elastin and collagen contents in the CA from {alpha}1+/+ compared with {alpha}1–/– mice (Table 2). Elastin and collagen densities did not differ between strains. ANG II did not change the total number of nuclei per MCSA or Ki-67 expression in {alpha}1–/– mice.

ANG II increased {alpha}1-integrin levels in {alpha}1+/+ mice, whereas no signal was detected in {alpha}1–/– mice (Fig. 5). Thus we conclude that ANG II induces increased {alpha}1-integrin expression in the vascular wall in vivo. Expression of {alpha}2-integrin was not modified in either strain after ANG II treatment. ANG II increased expression of {alpha}5-, {alpha}v-, beta1-, and beta3-integrin, as well as {alpha}-SMA and SM-MHC, in both strains, but the increase was greater in {alpha}1+/+ than in {alpha}1–/– mice. ANG II significantly increased phosphorylation of ERK1/2 in {alpha}1+/+ and {alpha}1–/– mice (Fig. 5). It also significantly increased phosphorylation of p38 MAPK in aorta from {alpha}1+/+ mice. This response was not observed in {alpha}1–/– mice. The level of p38 MAPK was not altered by NE in either strain. These results are consistent with a specific effect of ANG II to enhance phosphorylated p38 expression. The phosphorylation of MAPK kinases and MKK3/6, which are upstream regulators of p38 MAPK, was also increased in ANG II-treated {alpha}1+/+, but not {alpha}1–/–, mice. The state of JNK phosphorylation was unaltered by ANG II stimulation in both strains. ANG II induces phosphorylation of FAK on Tyr397 and Tyr576/577 in aortas from {alpha}1+/+, but not {alpha}1–/–, mice.


Figure 5
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  		Fig. 5. Effects of ANG II on protein levels in aorta from {alpha}1+/+ and {alpha}1–/– mice. A: Western blot analyses of untreated aorta and aorta treated with ANG II 4 wk. GAPDH was used as loading control. B: protein levels of {alpha}1-, {alpha}2-, {alpha}5-, {alpha}v-, beta1-, and beta3-integrins, {alpha}-SMA, and smooth muscle myosin heavy chain (SM-MHC). Data are normalized to GAPDH. C: phosphorylation levels of ERK1/2, p38 MAPK, MKK3/6, JNK, and focal adhesion kinase (FAK). Data are expressed as ratio of phosphorylated to total protein content. Levels of total ERK1/2, p38, MKK3/6, JNK, and FAK were similar in treated and untreated {alpha}1+/+ and {alpha}1–/– mice after normalization to GAPDH (data not shown). Values are means ± SE of 5 experiments. Results are expressed as fold increase vs. {alpha}1+/+ mice. *P < 0.05 vs. untreated animals in the same strain. {ddagger}P < 0.05 vs. the same treatment in {alpha}1+/+ mice.

 
ANG II (5 nM) induced phosphorylation of ERK1/2 and JNK in rat SMCs at 15 min, whereas phosphorylation of p38 MAPK and FAK was observed at 24 h (Fig. 6). Expression of {alpha}1-integrin and {alpha}-SMA, but not {alpha}v-integrin, was increased at 2–24 h, and this increase occurred before activation of p38 MAPK and FAK. Incubation of SMCs with the anti-{alpha}1-integrin antibody 30 min before administration of ANG II reduced {alpha}1-integrin expression and phosphorylation levels of p38 MAPK and FAK by 50–70% at 24 h. No inhibition was observed for {alpha}v-integrin, {alpha}-SMA, ERK1/2, and JNK.


Figure 6
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  		Fig. 6. Effects of anti-{alpha}1-integrin antibody on ANG II-treated rat SMCs. A: Western blots of cells stimulated for 15 min–24 h with 5 nM ANG II. SMCs were preincubated with anti-{alpha}1-integrin antibody 30 min before ANG II administration. GAPDH was used as a loading control. B and C: {alpha}1-integrin (normalized to GAPDH) and phosphorylation levels of ERK1/2, p38 MAPK, JNK, and FAK after 15 min and 24 h of ANG II treatment. Results are expressed as ratio of phosphorylated to total protein content. Values are means ± SE of 5 experiments. *P < 0.05 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first study of an arterial phenotype in relation to invalidation of the {alpha}1-integrin gene. In {alpha}1–/– mice, mechanical strength of the CA is decreased at baseline and there is no hypertrophic response to ANG II. Inactivation of {alpha}1-integrin results in important changes in ANG II cell signaling, including lower levels of p38 MAPK and FAK phosphorylation.

Vascular {alpha}1-integrin has been detected in SMCs in mice (30), and we confirmed its expression in the vascular wall by RT-PCR and Western blot. We and others (4, 16) previously suggested that adhesion molecules and integrins may contribute to the determination of arterial wall stiffness, independently of elastin and collagen content; i.e., an increased number of attachments should lead to an increased stiffness. {alpha}1beta1-Integrin, which is known to bind collagen, may also be implicated in arterial stiffness. A reduction of cell attachments to ECM components induced by an increase in proteoglycans has been reported to decrease arterial stiffness (9). At baseline, we found no difference in mechanical parameters of the CA of {alpha}1–/– mice or in arterial pressure measured by the tail-cuff method, nor did we find any modifications of matrix components or proliferation or differentiation of SMCs in {alpha}1–/– mice. These findings indicate that interaction between {alpha}1-integrin and its ECM ligands is not necessary for maintenance of elasticity of large arteries.

The threshold for vascular rupture was lower in {alpha}1–/– than {alpha}1+/+ mice, indicating a decreased mechanical strength of the vascular wall materials, which is revealed only under a high level of stress. This result does not reflect the physiological situation in the vascular wall but may reveal an excessive fragility of the vascular wall components over time, as previously suggested (22). In mice lacking desmin, in which the interactions between SMCs and ECM were drastically modified, arterial strength was reduced (20). We show here, for the first time, that an integrin is directly involved in mechanical strength. Other integrins or ECM ligands must also be involved in providing mechanical resistance, since the decrease in rupture pressure was small (–15%) in {alpha}1–/– mice. It was shown previously in human cultured SMCs that {alpha}2-integrin may replace {alpha}1-integrin as a collagen receptor (33), but we found no overexpression of the {alpha}2-subunit in vivo. However, we did find an overexpression of {alpha}5-, {alpha}v-, and beta1-integrins in {alpha}1–/– mice. It is highly unlikely that {alpha}5-integrin is implicated in mechanical resistance, since a previous report showed that an increase in {alpha}5beta1-integrin did not modify this parameter (8). We can suggest that upregulation of {alpha}v- and beta1-integrins, which may interact with noncollagenous ligands (25), may compensate partly for the absence of {alpha}1-integrin.

After ANG II administration, we showed that the MCSA increased in {alpha}1+/+, but not {alpha}1–/–, mice. The thickening of the media in ANG II-treated {alpha}1+/+ mice was due to cellular hypertrophy, not hyperplasia, as demonstrated by the increase in {alpha}-SMA and no change in the total number of SMC nuclei per MCSA, Ki-67, or collagen and elastin densities. This lack of vascular hypertrophy in {alpha}1–/– mice was not due to differences in arterial pressure between the two strains, since ANG II increased arterial pressure exactly to the same extent in {alpha}1+/+ and {alpha}1–/– mice. The response to NE indicates that a similar increase in arterial pressure was not able to produce a significant increase in MCSA in {alpha}1+/+ or {alpha}1–/– mice. Therefore, we can suggest that the development of carotid wall hypertrophy requires {alpha}1-integrin and that the synthesis of {alpha}1-integrin is further increased by administration of ANG II. Taken together, these results demonstrate that the lack of arterial hypertrophy in ANG II-treated {alpha}1–/– mice was not related to pressure mechanisms but, rather, involves specific ANG II-dependent pathways that are influenced by integrins.

Functional consequences have been studied on Dist-pressure curves. Dist was similar in ANG II-treated {alpha}1–/– and {alpha}1+/+ mice, despite different structure of the vascular wall. The only significant difference of elasticity between the two strains was observed in response to ANG II: the Einc-WS curve of {alpha}1–/– mice was shifted rightward compared with that of the {alpha}1+/+ mice, indicating less stiffness of the wall material in ANG II-treated {alpha}1–/– mice. Thus the absence of arterial wall hypertrophy in response to ANG II was associated with an increase of the intrinsic elastic properties in {alpha}1–/– mice. We found no difference in elastin and collagen contents between the two groups that could explain a difference in elasticity. However, elastin and collagen fibers are integrated in a very complex network within the media that is dependent on cell matrix attachments. These quantitative and qualitative structural modifications, which are revealed by ANG II, suggest that ANG II stimulates these attachments during the hypertrophic process. This hypothesis needs to be tested using in vitro systems. However, this view is supported by the observation that ANG II increased {alpha}1-integrin expression in {alpha}1+/+ mice.

Our data demonstrate that ANG II elicited vessel wall hypertrophy and increased {alpha}1-integrin expression in {alpha}1+/+ mice. ANG II did not induce an increase in {alpha}2-integrin expression, suggesting a role for ANG II in the regulation of {alpha}1-integrin, but not {alpha}2-integrin. ANG II increased {alpha}5-, {alpha}v-, beta1-, and beta3-integrin expression in both strains, but the increase was less in {alpha}1–/– than in {alpha}1+/+ mice. These results paralleled the {alpha}-SMA and SM-MHC results. Thus we can suggest that these integrins are involved in cellular hypertrophy in response to ANG II. The weaker integrin expression in {alpha}1–/– mice indicates that this strain does not compensate for ANG II-induced cellular hypertrophy.

A few studies have demonstrated that ANG II is able to increase directly integrin production in cardiac fibroblasts (7, 14, 19) and ventricular smooth muscle fibroblasts (5). Kappert et al. (17) reported that ANG II stimulates beta1-integrin-mediated adhesion and spreading, but not proliferation, of SMCs. Brassard et al. (6) recently showed in the rat that aortic {alpha}1-integrin expression was unaffected after 7 days of ANG II administration. The differences between these data and our observations may be related to the greater duration of treatment with a higher dose of ANG II in the present study, which largely contributes to the hypertrophic response, as previously described for ANG II (23).

Previous studies showed that ANG II induced cellular hypertrophy and contributed to the increased SMC differentiation in cultured aortic SMCs (18). In addition, ANG II has been shown to induce a rapid increase in the expression of transcription factors, MAPK kinases, tyrosine receptor kinases, and non-tyrosine receptor kinases (Src, Jak/Stat, and FAK) in SMCs before hypertrophy (23). Touyz et al. (34) and Govindarajan et al. (13) showed in vascular SMCs that ANG II induces activation of p38 MAPK and FAK phosphorylation, respectively.

In {alpha}1+/+ mice, our investigation showed that ANG II increased phosphorylation of ERK1/2, p38 MAPK and FAK, which are widely implicated in SMC hypertrophy (13, 23, 32), but did not change the state of JNK. These findings are consistent with the changes in morphology. The increase in {alpha}-SMA and SM-MHC indicates SMC differentiation, as previously described with ANG II administration (27).

Invalidation of the {alpha}1-integrin gene decreased the effects of ANG II on SMC differentiation and inhibited the phosphorylation of p38 MAPK and FAK induced by ANG II. There was no difference in response of other parameters (Ki-67, ERK1/2, and JNK) to ANG II between the two strains. The absence of phosphorylation of MKK3/6, which is an essential upstream regulator of the p38 MAPK pathway (26, 32, 35), may explain the lack of phosphorylation of p38 MAPK. The inhibition of p38 MAPK phosphorylation raises the possibility that p38 MAPK contributes to the lack of vascular hypertrophy during ANG II administration. It has been demonstrated in isolated SMCs that p38 MAPK is involved in collagen production (34), and the MKK3/6-p38 pathway has been reported to have a role in leptin-induced SMC hypertrophy (32). Our results showing less hypertrophy in SMC with an unchanged collagen content accompanying the defect of p38 phosphorylation in {alpha}1–/– mice after chronic exposure to ANG II confirm the conclusions of these previous studies that were performed under acute conditions. In addition, we have shown that inhibition of {alpha}1-integrin with a functional antibody in rat primary cultured SMCs reduced by ≥50% the ANG II-induced {alpha}1-integrin expression and phosphorylation of FAK and p38 MAPK. This provides a link between ANG II function and {alpha}1-integrin expression and reinforces our in vivo findings.

Integrins may activate MAPK via two major pathways, one involving FAK and the other the protein Shc (12). Because we did not detect any modification in ANG II-induced Shc phosphorylation, any major Shc dysfunction is unlikely (data not shown). The decreased levels of phosphorylation of FAK in {alpha}1–/– mice support the demonstration of Lehoux et al. (21) that FAK phosphorylation may be inhibited by the integrin-matrix interaction-blocking RGD peptide in vitro in the aorta. Our results suggest that {alpha}1-integrin is necessary for the ANG II-induced phosphorylation of FAK and activation of p38 MAPK previously suggested in cardiomyocytes (1, 19).

In conclusion, we have established that suppression of {alpha}1beta1-integrin produces important changes in the vascular phenotype, i.e., a reduced mechanical strength of the vascular wall and a lack of SMC hypertrophy in response to ANG II. This phenotype depends on p38 MAPK and FAK phosphorylation. These results emphasize the importance of {alpha}1beta1-integrin in signal transduction during vascular wall hypertrophy in response to ANG II.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a grant from the Association Française contre les Myopathies, the Agence Nationale de la Recherche, and la region Lorraine.


   ACKNOWLEDGMENTS
 
We thank Mary Osborne-Pellegrin for editing the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Lacolley, Unité INSERM U684, 9 Ave. de la Foret de Haye, BP 184, 54505 Vandoeuvre-les-Nancy, France (e-mail: patrick.lacolley{at}nancy.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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